DEVELOPMENT IN COTTON SHILPI CHAWLA, B.S., M.S. A Dissertation AGRONOMY

Transcription

1 POPULATION DYNAMICS OF Verticillium dahliae AND Fusarium oxysporum f. sp. vasinfectum OVER TIME AND THEIR IMPLICATIONS FOR DISEASE DEVELOPMENT IN COTTON by SHILPI CHAWLA, B.S., M.S. A Dissertation In AGRONOMY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Approved Jason E. Woodward (Chair of Committee) Terry A. Wheeler Robert J. Wright Michael San Francisco Ronald D. French-Monar Peggy Miller Interim Dean of Graduate School August, 2011

2 2011 Shilpi Chawla All Rights Reserved

3 DEDICATION To My Respected Parents Mrs. Vinod Chawla and Mr. Mukand Lal Chawla And Loving Husband Siddharth

4 ACKNOWLEDGEMENTS Firstly I would like to thank the Almighty God before whom I offer my respectful obeisance who has led me down this path and has made it all possible. It is my privilege to express my heartfelt gratitude to Dr. Jason E. Woodward, chair to my advisory committee for his valuable guidance, constant support, enduring help, and encouragement throughout the course of my PhD and in preparation of this manuscript. This research effort would not have been complete without consistent inputs from Dr. Terry Wheeler and I am thankful for her guidance throughout the degree program. I would like to thank Dr. Robert Wright for giving me an opportunity to work in his lab and learn molecular techniques and expand my horizons. My heartfelt thanks are due to the members of my advisory committee Dr. Michael San Francisco and Dr. Ronald French-Monar for their valuable suggestions and support during the progress of my research work. I am highly thankful to Mr. Benjamin Mullinix for helping me with the statistical analyses. I would like to take this opportunity to extend my sincere regards to Dr. Thomas Thompson, Chair Department of Plant and Soil Science, Dr. Richard Zartman and Dr. Eric Hequet, Graduate Coordinators, for allowing me the opportunity of pursuing a career I enjoy. Debts are also owed to International Cotton Research Center at Texas Tech University, and Texas A & M University for providing funds and facilities for the present investigation. I would also like to extend my sincere thanks to all the faculty members of the Department of Plant and Soil Science for their guidance and help during the course of my study. I am deeply obliged to all the staff members of the Department of Plant of Soil Science as well as Texas AgriLife Research and Extension Center for their continuous ii

5 cooperation and timely help rendered during my graduate studies. I would also like to thank Mitchell Ratliff, Ira Yates and Justin Spradley for their outstanding technical help and Lindsey Thiessen, and Ellen Ryan for their prudent love, timely help and extended cooperation. My sincere thanks to my fellow graduate students for their friendship, support and advice. It is an immense pleasure to acknowledge the affection, inspiration and encouragement rendered by Dr. Sukant Misra and Mrs. Rina Misra during my stay in Lubbock. I am grateful to my best buddies Deepika, Ruchi, Simer, Michael, Angela, and Amelia for their joyous company and giving me the most vital moral support at the time when I needed it the most. Leaving everything behind, they always stood by me. A special gratitude is expressed towards my family especially my mother and father Mrs. Vinod Chawla and Mr. Mukand Lal Chawla. My sole existence is because of their boundless love, constant inspiration, moral support, sacrifices made and above all blessings. Thanks are also warranted for my brothers Archit and Gaurav Chawla for always being there for me when I needed to talk. I also express heartfelt respect to my mother and father in-law Ranjana and Pramod Bajpai for their love and support. I am most grateful to my sister and brother in-law Mridula and Satyarth Bajpai for their warm affection and constant support. No word will suffice to appreciate the patience, undivided love, support, compelling words of encouragement and constant help by my loving husband Siddharth throughout the duration of my graduate career. Last but not least, my sincere thanks to all beloved and respectful people who helped me directly or indirectly and could not find a separate mention. iii

6 TABLE OF CONTENTS ACKNOWLEDGEMENTS ii LIST OF TABLES...viii LIST OF FIGURES.....xi CHAPTER I INTRODUCTION AND LITERATURE REVIEW INTRODUCTION COTTON PRODUCTION COTTON DISEASES Verticillium Wilt Relationship between V. dahliae Inoculum Density and Wilt Development Management of Verticillium Wilt of Cotton Fusarium Wilt-Root-Knot Nematode Complex Management of Fusarium Wilt-Root-knot Nematode Disease Complex RESEARCH OBJECTIVES LITERATURE CITED CHAPTER II...40 INFLUENCE OF Verticillium dahliae INFESTED PEANUT RESIDUE ON INOCULUM RELEASE IN SOIL AND WILT DEVELOPMENT IN SUBSEQUENT COTTON ABSTRACT INTRODUCTION.. 42 iv

7 2.3 MATERIALS AND METHODS Microplot Experiment Soil Sampling and Data Collection Statistical Analysis RESULTS DISCUSSION LITERATURE CITED 51 CHAPTER III 57 EFFECT OF CULTIVAR SELECTION ON SOIL POPULATION DYNAMICS OF Verticillium dahliae OVER TIME AND IMPLICATIONS ON VERTICILLIUM WILT DEVELOPMENT IN COTTON ABSTRACT INTRODUCTION MATERIALS AND METHODS Microplot Experiment Soil Sampling and Data Collection Statistical Analysis RESULTS DISCUSSION LITERATURE CITED 73 v

8 CHAPTER IV...87 EFFECT OF INOCULUM DENSITY OF GENETICALLY DISTINCT Fusarium oxysporum f. sp. vasinfectum Race 1 ISOLATES, Meloidogyne incognita, AND CULTIVAR ON FUSARIUM WILT DEVELOPMENT IN COTTON ABSTRACT INTRODUCTION MATERIALS AND METHODS Greenhouse Experiment Data Collection and Statistical Analysis RESULTS AND DISCUSSION LITERATURE CITED CHAPTER V EFFECT OF COTTON CULTIVAR SELECTION ON POPULATION DYNAMICS OF Fusarium oxysporum f. sp. vasinfectum in SOIL OVER TIME ABSTRACT INTRODUCTION MATERIALS AND METHODS Microplot Experiment Soil Sampling and Data Collection Statistical Analysis RESULTS DISCUSSION LITERATURE CITED..128 vi

9 CHAPTER VI..141 SUMMARY AND CONCLUSION SUMMARY CONCLUSION APPENDIX Figure Figure Table Table Table Table Table Table Modified Sorensen s NP-10 Semi-Selective Medium.157 Komada s Semi-Selective Medium..158 vii

10 LIST OF TABLES TABLE 2.1. PARAMETERS FROM NON-LINEAR QUADRATIC REGRESSION OF PERCENT GERMINATION OF COTTON SEEDS, DISEASE INCIDENCE, AND INOCULUM DENSITY OF Verticillium dahliae IN SOIL DUE TO INFLUENCE OF INFESTED PEANUT RESIDUE TABLE 3.1. EFFECT OF CULTIVAR ROTATION AND TIME ON SOIL INOCULUM DENSITIES OF Verticillium dahliae...77 TABLE 3.2. PARAMETERS FROM LINEAR REGRESSION OF SOIL INOCULUM DENSITY OF Verticillium dahliae OVER TIME FOR SIX CULTIVAR ROTATIONS...78 TABLE 3.3. EFFECT OF CULTIVAR ROTATION ON PERCENT DISEASE INCIDENCE IN 2008, 2009, AND TABLE 3.4. TESTING THE SLOPES FROM REGRESSING PRE-PLANT SOIL INOCULUM DENSITY OF Verticillium dahliae ON DISEASE INCIDENCE IN 2008, 2009, AND TABLE 3.5. EFFECT OF CULTIVAR ROTATION SCHEME ON PLANT HEIGHT IN TABLE 3.6. EFFECT OF CULTIVAR ROTATION SCHEME ON LINT YIELD IN TABLE 3.7. EFFECT OF CULTIVAR AND PRE-PLANT SOIL INOCULUM DENSITY OF Verticillium dahliae ON LINT YIELD IN TABLE 4.1. EFFECT OF GENETICALLY DISTINCT ISOLATES OF Fusarium oxysporum f. sp. vasinfectum, Meloidogyne incognita DENSITY AND COTTON viii

11 CULTIVARS ON FUSARIUM WILT ROOT-KNOT NEMATODE DISEASE COMPLEX: p - VALUES OF THE MAIN EFFECTS AND THEIR INTERACTIONS TABLE 4.2. EFFECT OF INTERACTION BETWEEN INOCULUM DENSITY OF Fusarium oxysporum f. sp. vasinfectum Race 1, Meloidogyne incognita AND COTTON CULTIVAR ON SHOOT WEIGHT TABLE 4.3. EFFECT OF INTERACTION BETWEEN INOCULUM DENSITY OF Fusarium oxysporum f. sp. vasinfectum Race 1 AND Meloidogyne incognita ON TOTAL PLANT WEIGHT TABLE 4.4. EFFECT OF INOCULUM DENSITY OF Fusarium oxysporum f. sp. vasinfectum Race 1 AND COTTON CULTIVAR ON AREA UNDER THE DISEASE PROGRESS CURVE (AUDPC) TABLE 4.5. EFFECT OF INTERACTION BETWEEN INOCULUM DENSITY OF Fusarium oxysporum f. sp. vasinfectum Race 1 AND CULTIVAR ON PLANT HEIGHT AND TOTAL PLANT WEIGHT..105 TABLE 4.6. EFFECT OF INTERACTION BETWEEN Fusarium oxysporum f. sp. vasinfectum race 1 ISOLATES AND CULTIVARS ON PLANT GROWTH PARAMETERS TABLE 4.7. EFFECT OF Meloidogyne incognita DENSITY ON PLANT GROWTH PARAMETERS TABLE 4.8. EFFECT OF CULTIVAR SELECTION ON PLANT GROWTH PARAMETERS ix

12 TABLE 5.1. EFFECT OF CULTIVAR ROTATION AND TIME ON SOIL INOCULUM DENSITY OF fusarium oxysporum f. sp. vasinfectum TABLE 5.2. PARAMETERS FROM LINEAR REGRESSION OF SOIL DENSITY OF Fusarium oxysporum f. sp. vasinfectum OVER TIME FOR SIX CULTIVAR ROTATIONS TABLE 5.3. EFFECT OF CULTIVAR ROTATION ON PERCENT DISEASE INCIDENCE IN 2008, 2009, AND TABLE 5.4. TESTING THE SLOPES FROM REGRESSING PRE-PLANT SOIL INOCULUM DENSITY OF Fusarium oxysporum f. sp. vasinfectum ON DISEASE INCIDENCE IN 2008, 2009, AND TABLE 5.5. EFFECT OF CULTIVAR ROTATION ON PLANT HEIGHT IN TABLE 5.6. EFFECT OF CULTIVAR ROTATION ON LINT YIELD IN TABLE 5.7. EFFECT OF CULTIVAR AND PRE-PLANT SOIL INOCULUM DENSITY OF Fusarium oxysporum f. sp. vasinfectum ON LINT YIELD IN x

13 LIST OF FIGURES FIGURE EFFECT OF INFESTED PEANUT RESIDUE AMOUNT ON PERCENT GERMINATION OF COTTON SEEDS, DISEASE INCIDENCE, AND INOCULUM DENSITY OF Verticillium dahliae IN SOIL...55 FIGURE 3.1. EFFECT OF CULTIVAR ROTATION ON Verticillium dahliae INOCULUM DENSITY IN SOIL OVER TIME...84 FIGURE 3.2. EFFECT OF SOIL INOCULUM DENSITY OF Verticillium dahliae ON LINT YIELD IN FIGURE 3.3. EFFECT OF DISEASE INCIDENCE ON LINT YIELD IN FIGURE 4.1. GENETICALLY DISTINCT Fusarium oxysporum f. sp. vasinfectum Race 1 ISOLATES USED FOR PRESENT STUDY, SHOWING DISTINCT COLONY MORPHOLOGY IN CULTURE ON KOMODA S SELECTIVE MEDIA FIGURE 4.2. EFFECT OF INTERACTION OF GENETICALLY DISTINCT ISOLATES OF Fusarium oxysporum f. sp. vasinfectum Race 1, THEIR INOCULUM DENSITIES, Meloidogyne incognita AND COTTON CULTIVAR ON AREA UNDER THE DISEASE PROGRESS CURVE (AUDPC) FIGURE 4.3. EFFECT OF GENETICALLY DISTINCT ISOLATES OF Fusarium oxysporum f. sp. vasinfectum Race 1, Meloidogyne incogita, AND COTTON CULTIVAR ON AREA UNDER THE DISEASE PROGRESS CURVE (AUDPC) FIGURE 5.1. EFFECT OF CULTIVAR ROTATION ON Fusarium oxysporum f. sp. vasinfectum DENSITY IN SOIL OVER TIME xi

14 FIGURE 5.2. EFFECT OF SOIL INOCULUM DENSITY OF Fusarium oxysporum f. sp. vasinfectum ON LINT YIELD IN FIGURE 5.3. EFFECT OF DISEASE INCIDENCE ON LINT YIELD IN xii

15 CHAPTER I INTRODUCTION AND LITERATURE REVIEW 1.1 INTRODUCTION Cotton is one of the oldest natural fibers under human cultivation. Traces of cotton have been recovered from archaeological sites from more than 5,000 years ago. It is the single most important fiber crop worldwide, which grows in environments ranging from tropical to temperate. It is a major crop in parts of China, India, the United States of America, the African tropics, Australia, Egypt, Mexico, Pakistan, the Soviet Union, Sudan and warmer regions of Central and South America (Pegg and Brady, 2002; Smith and Cothren, 1999). Cotton belongs to the family Malvaceae and genus Gossypium, a genus that includes several wild perennial species of cotton, as well as four species (G. herbaceum, G. arboreum, G. hirsutum and G. barbadense) that are cultivated for lint. Millions of hectares are devoted to the production of cotton globally. The crop is broadly adapted to a range of soil types and temperatures. Cotton s perennial nature and indeterminate growth habit makes it one of the most complex agricultural crops to produce (Kirkpatrick and Rothrock, 2001). Cotton is valued primarily for its extraordinarily strong, fine, and durable fibers made up of cellulose, which are tubular extensions of epidermal cells of the seed coat. Cotton continues to be the most used natural fiber for an array of textile products. Cotton is also important for high quality vegetable oil for human consumption, seed cake as a 1

16 protein source for livestock and the base chemicals for a plethora of industrial products (Smith and Cothren, 1999). 1.2 COTTON PRODUCTION China ranks first in the world in cotton production with 33 Million bales, followed by India (25 Million bales), and the United States (16.7 Million bales) (USDA, 2010). Within the United States, cotton is grown in California, Arizona, New Mexico, Texas, Kansas, Oklahoma, Missouri, Arkansas, Louisiana, Mississippi, Tennessee, Alabama, Georgia, Florida, North Carolina, South Carolina, and Virginia (Cotton Council International, 2009). Texas ranks first in cotton production in the United States accounting for approximately one-half of the cotton hectares and roughly 40% of the total production in the United States (Smith and Anisco, 2000). Cotton is the leading cash crop in the state and it is grown across 2 Million hectares. This crop generates $1.6 billion for farmers and has a total economic impact of $5.2 billion for the state. According to the Crop Profile for Cotton in Texas (2009), more than 120 Texas counties in six different regions produce cotton. The High Plains region of Texas consists of 27 counties producing 64% of the state's cotton crop. There is a wide year-to-year variation in both total and seasonal rainfall. Fifty percent of the cotton is irrigated in this region. The average rainfall for the region is cm. The hot days and cool nights as well as loamy and sandy soil type make this region prone to several diseases (Crop Profile for Cotton in Texas, 2009). 2

17 1.3 COTTON DISEASES Cotton diseases present limitations to profitable production in every area where the crop is grown. The average annual cotton production loss due to diseases and nematodes in the United States over the last four decades has been estimated at about 12% (Blasingame and Patel, 2001). Major diseases of cotton are caused by pathogens such as fungi, bacteria, and nematodes with the importance of certain pathogens varying by region (DeVay, 2001) Verticillium Wilt Verticillium spp. cause vascular wilts of vegetables, flowers, field crops, perennial ornamentals, and fruit and forest trees. Two species, V. albo-atrum and V. dahliae attack hundreds of plant species causing wilts and losses of varying severity (Schnathorst, 1981). Verticillium wilt is a global production concern in most countries where cotton is cultivated especially in temperate climates (Pegg and Brady, 2002). The disease is responsible for annual yield reductions of approximately 1.5 million bales (Bell, 2001). Carpenter (1914) first identified the disease on G. hirsutum in Arlington, Virginia and later found that the pathogen was pathogenic on okra. Bewley (1922) found that isolates of V. dahliae obtained from tomato caused wilting when inoculated on G. herbaceum. The disease then spread to Tennessee and extensively in Mississippi, Arizona, and California. Thereafter, Verticillium wilt was found in all cotton growing areas of the United States (Pegg and Brady, 2002). In Texas Verticillium wilt is caused predominantly by Verticillium dahliae Kleb (Wooward, unpublished data). This soilborne fungus prefers slightly higher temperatures 3

18 than V. albo-atrum and also produces resting structure (Schnathorst, 1981). It colonizes the vascular tissues of the plants, causing wilt in many other economically important crops of more than 200 plant species including field crops, most vegetables, and forest trees (Bhat and Subbarao, 1999; McCain et al., 1981). In West Texas, cotton is grown in rotation with peanut (Arachis hypogaea L.) which also is a host crop for Verticillium dahliae. Farmers routinely leave peanut residues on the soil surface rather than incorporating them into the soil. This practice helps reduce soil erosion, conserve energy, maintain soil moisture, and improve soil fertility, which lead to increase crop yields. However, V. dahliae can survive in the previous year's crop residue, making diseases more problematic under reduced-tillage conditions by protecting the residue from microbial degradation, lowering soil temperature, and leaving soil undisturbed. Microsclerotia (ms), the survival structures of V. dahliae composed of masses of melanized hyphae, are considered as the principal source of inoculum for wilt development (Rowe and Powelson, 2002). Microsclerotia can survive for more than 20 years in the soil (Wilhelm, 1955), and root exudates stimulate germination of ms initiating infections (Mol, 1995; Schnathorst, 1981). In a study of tolerance physiology through comparison of root exudates constituents from Verticillium-tolerant and highly susceptible cotton, alanine content was found higher in exudates from the susceptible cotton line (Booth, 1968). In another study conducted by Wu et al. (2008), alanine was higher in root exudates of a susceptible cotton line while, argenine was higher in root exudates of a resistant cotton line. Alanine was found to stimulate ms germination, while argenine was found to inhibit ms germination. Microsclerotia are exposed to repeated cycles of drying and wetting. Menzies and Griebel (1967) found that ms which had 4

19 ceased multiplying for several months when the soil was dried would multiply again when rewetted. Farley et al., (1971) demonstrated that ms can germinate several times when alternate cycles of wet and dry periods occur. Hawke and Lazarovits (1994) found that larger ms (>75 µm) exhibited faster, more synchronous germination and produced larger colonies on agar plates than smaller ms. Existence of nitrogenous and carbonaceous sources in soil may also serve as an external food source for the germinating ms (Ben-Yephet and Pinkas, 1977). Klimes and Dobinson (2006) identified a differentially expressed class II hydrophobin gene (VDH1) involved in the molecular mechanism of the development of ms, the desiccation and cold-tolerant resting structures. VDH1 s functions are multi-faceted, and seem generally relevant to long-term survival in V. dahliae. Microsclerotia are formed depending on temperature and moisture availability with the decay of plant tissues. Microsclerotia are dispersed in the soil and only a single cycle of inoculum is produced during a growing season (Paplomatas et. al 1992). Microsclerotia of V. dahliae generally occur in clustered or aggregated patterns in naturally infested commercial fields (Xiao et al., 1997). This may affect the soil sampling scale for a survey in commercial fields to determine inoculum density and wilt relationships (Nicot and Rouse, 1987b). Cool, wet conditions favor disease development with temperatures between C being best suited for V. dahliae growth and survival (Rowe, et al., 1987). The fungus is capable of infecting plant roots directly or through wounds throughout the growing season. Verticillium dahliae primarily colonizes the rhizoplane of the host plant (Huisman, 1988). It penetrates roots early in season, and then infects the vascular system and grows systemically throughout the plant and produces ms within the xylem vessels. 5

20 The fungus is spread by contaminated seeds, vegetative cuttings and roots, wind, surface water, and by soil, which may contain ms. Initially, colonies of V. dahliae on media are hyaline, but later become black due to production of allomelanin pigment (Heale, 2000). The fungus produces conidia that are borne on hyaline conidiophores, having several whorls of three or four phialids ( x µm) arranged in verticillate form. The haploid conidia ( x µm) accumulate in a sphere of mucilage at the tip of the phialids aerially. The black colored colony is the result of the ms production (Bell, 200; Melouk, 2001; Pegg and Brady, 2002). Disease symptoms in cotton plants infected by V. dahliae are variable and often influenced by strains of the pathogen. Among the strains that infect cotton, two groups defoliating and non-defoliating types have been identified (Tzeng and DeVay, 1985). According to Wiese and DeVay (1970), strains that cause the most defoliation do so by inducing high amounts of ethylene in infected plants. At low temperature young plants show stunting, epinasty, and yellowing. Leaves exhibit interveinal chlorosis, necrosis, curling, and die from the margins inward (Schnathorst, 1981). Plants develop characteristic mosaic patterns on leaves, starting from the base of the plant and progressing towards the top. Chlorosis is caused by the occlusion of leaf veins in limited areas of the leaves (Misaghi et al., 1978). The first appearance of these leaf symptoms is associated with a cessation of both plant growth and fruit development (Gutierrez et al., 1983). Later, internal tissues become necrotic, leaves may shed, and young bolls may abscise or become malformed. Ramification of the fungus in the xylem vessels lead to a brown colored discoloration of the vascular system by decreasing hydraulic conductance and plants may eventually wilt and die (Schnathorst, 1981). In infested cotton plants, 6

21 bolls abscise or do not open (Pullman and DeVay, 1982b). Infection of cotton plants by V. dahliae causes impairment in the uptake and translocation of potassium that is often associated with the development of potassium deficiency symptoms in leaves of plants with large boll loads (DeVay et al., 1997b; Hafez et al., 1975; Mikkelsen et al., 1988). Less recognized symptoms mimic potassium deficiency symptoms in infected plants. These symptoms do not stop growth, although they may result in severe stunting and a reduction in lint yields (Tzeng and DeVay, 1985). Verticillium wilt of cotton reduces plant height, lateral branching, and dry matter accumulation in leaves, stems, roots, and bolls (Pullman and DeVay, 1982b). Several studies have shown that increased stomatal resistance in leaves is a good indicator of physiological responses to V. dahliae infection (Bowden et al., 1990; MacHardy et al., 1976; Tzeng and DeVay, 1985). Increased stomatal resistance reduces the concentration of CO 2 within leaves, and results in decreased net photosynthesis (Bowden et al., 1990; Bowden and Rouse, 1991). Disease incidence is usually assessed as percent foliar symptoms or vascular discoloration. Although, vascular discoloration is a positive indicator of plant infection in cotton, it has little or no correlation with lint yields (DeVay and Pullman, 1984; Pullman and DeVay, 1981). In contrast, the incidence of foliar symptoms of Verticillium wilt is directly related to lint and seed losses (Gutierrez et al., 1983; Pullman and DeVay, 1981). Paplomatas et al. in 1992 also found a significant correlation between inoculum density at planting time and disease incidence at the end of the cropping season for different field locations. 7

22 1.3.2 Relationship between V. dahliae Inoculum Density and Wilt Development Inoculum density of the pathogen in field soils at planting plays a critical role in the epidemiology of Verticillium wilt (Harris and Yang, 1996; Paplomatas et al., 1992; Xiao and Subbarao, 1998). Understanding the relationship between inoculum density in soil at planting and wilt development is essential for developing a disease risk assessment based on pre-plant soil assays (Frankl et al., 1987; Harris and Yang, 1996). Inoculum density can also be an important factor in determining the timing, nature, and duration of the management practices (Gamliel and Stapleton,, 1993; Powelson and Rowe, 1993). In general, the incidence of Verticillium wilt in herbaceous hosts is proportional to the pathogen inoculum density expressed as the number of viable ms per gram of soil. However, considerable variation occurs, with such a relationship depending upon crops and cultivars (Grogan et al., 1979; Jordan, 1974; Khan et al., 2000; Paplomatas et al. 1992; Pullman and DeVay 1982a; Xiao and Subbarao, 1998). In tomato, 0.5 ms per g of soil caused 50% wilt incidence and, 6 ms per g of soil caused 100% wilt incidence by the end of the growing season (Grogan et al., 1979). Whereas, 0.3 ms per g of soil can cause 5% wilt incidence in strawberry, and a small increase in ms numbers in soil can cause the death of a majority of plants (Harris and Yang, 1996). In California, the number of cotton plants (Acala cotton) infected by V. dahliae at the end of the crop season was found to be directly related to the density of ms in soil (Ashworth et al., 1979; Paplomatas et al., 1992; Pullman and DeVay, 1982a). This relationship has never been investigated for G. hirsutum grown in deficit irrigated cotton in the High Plains of West Texas. Very little is known about the dynamics of V. dahliae ms populations in the soil. Wheeler et al. (2000) found that densities of V. dahliae in Ohio potato fields were higher 8

23 in autumn than in spring of the same year. In a different study, inoculum density of soil sampled in May increased on the average of propagules per g per year cropped to cotton over seven years (Pullman and DeVay, 1982a). In studying the seasonal fluctuation in the number of V. dahliae ms in cotton fields, Evans et al. (1967) found that inoculum density generally decreased throughout the growing season but increased at harvest time with the release of ms from infected plants into soil. Similar trends have been observed in Artichoke (Berbegal et al., 2007). Huisman and Ashworth (1976) found that the inoculum usually increased rapidly following one year of a susceptible crop, with a higher inoculum density often occurring the second year regardless of whether the subsequent crop was a non-susceptible or a susceptible host. Tjamos (1981) and Zilberstein et al., (1983) also demonstrated that the pathogenicity of V. dahliae was related to the cropping history. Exponential or linear relationships between inoculum density and wilt incidence have been observed on several crops (Atibalentja and Eastburn, 1997; Grogan et al., 1979; Harris and Yang, 1996; Nicot and Rouse, 1987b; Paplomatas et al., 1992). Under field conditions, this relationship might vary from field to field and inconsistent relationships between inoculum density and disease incidence have been observed on cotton in commercial cotton fields (Bejarano-Alcazar et al., 1995; Davis et al., 1996a; DeVay et al., 1974; Pullman and DeVay, 1982a). In Britain, Verticillium wilt on strawberry was more severe on light, sandy soils than on heavier soil types (Harris and Yang, 1996). Removal of infested potato and field bean debris was found to reduce the number of ms in soil in subsequent years (Mol et al., 1995). Additional studies have shown that 9

24 the removal of infested aerial crop debris from the field is an effective means of preventing the accumulation of ms in the soil (Easton et al., 1975; Takeuchi, 1987). Microbial antagonism could be involved in the quick loss in the number of ms from soil (Evans et al., 1967). Microsclerotia would germinate near the plant roots, leading to a decline in the population of viable propagules in soil until the incorporation of new inoculum from infected tissues at the end of the crop. Davis et al. (1983) found a good correlation between ms population in soil and the number of ms in stem tissues. Mol et al. (1995) observed an increase of the ms population over more than 1 year after mixing the soil with colonized ground potato stems and removing potato debris led to a lower ms population in the following year. Also, Huisman and Ashworth (1976) and Joaquim et al. (1988) found a sharp increase in ms population in the second year after growth of a susceptible crop, despite host susceptibility. The apparent increase in ms population, a year after incorporation may be explained by the disaggregation of plant debris containing ms. Hoekstra (1989) also found that removing the debris of infested field bean resulted in a lower ms population in the next spring. Nicot and Rouse (1987a) compared three widely used techniques to quantitatively assay field soil for V. dahliae for precision, bias, and time required to assay a sample. These techniques were dilution plating, wet sieving, and the Andersen sampler technique. Among these techniques the dilution plating technique was the least biased with a recovery rate of nearly 100% in steamed silica sand amended with a known number of propagules. It was the least time consuming, had the highest recovery rate of the fungus in naturally infested soil, and was moderately variable. In this technique, a semi-selective media, Sorensen s NP-10 medium (NP-10) was used to quantify the number of 10

25 propagules of V. dahliae (Sorensen et al. 1991). Polygalacturonic acid (PGA) was identified as an important constituent in the semi-selective medium for the growth of V. dahliae ms more than three decades ago. The PGA from orange (P-3889) (Sigma- Aldrich, St. Louis) amended with N NaOH in NP-10 medium is best suited for recovery of V. dahliae from soil (Kabir et al., 2004) Management of Verticillium Wilt of Cotton Currently, the disease is partially managed by the use of wilt-tolerant or partially resistant cultivars, crop rotation, and cultural practices (Grimes and Yamada, 1982; Hafez et al., 1975; Pullman and DeVay, 1981). Reductions in soilborne ms are usually accomplished with a combination of chemical (Wilhelm and Paulus, 1980) and cultural methods (Davis et al., 1996a; Gutierrez and DeVay, 1986). Crop rotation is used to reduce the number of V. dahliae ms in soil and to reduce wilt incidence, the incidence being the number of plants with wilt symptoms expressed as a percentage of the total, in certain crops (Davis et al., 1996a; Rowe and Powelson, 2002; Tjamos 1989). Short-term crop rotations cannot successfully control V. dahliae because the number of ms after a short-term rotation usually remain above the threshold, resulting in significant crop losses (Davis et al., 1996a; Powelson and Rowe, 1993). Therefore, the length of rotation needed for effective Verticillium control depends on the inoculum level before rotation. The long term survival of V. dahliae in soil (Wilhelm, 1955) and broad host range, including some bridging hosts (Evans and Gleeson, 1973), are limiting factors for the effectiveness of crop rotation as a control strategy for Verticillium wilt in many crops (Khan et al., 2000; Rowe and Powelson, 2002). Crop rotation is rarely able to eradicate V. dahliae ms because of its wide host range and the persistence of ms in soil at levels that can cause 11

26 significant crop losses (Davis et al., 1996a; Powelson and Rowe, 1993). However, Xiao et al., (1998) found that rotating broccoli with cauliflower and incorporating broccoli residues into the soil is a novel means of managing Verticillium wilt on cauliflower and perhaps on other susceptible crops. Butterfield et al. (1978) found a significant decrease in disease incidence and increase in cotton lint yield from rotations with paddy rice, perennial ryegrass, and in some fields from safflower, grain sorghum, or frequent soil irrigation followed by grain sorghum. Cotton growers do not want to deviate much from cotton due to suitability of the crop for the region and also due to economic constraints of suitable rotation crops. Fumigants with Vapam can be very effective, but not at rates that cotton producers can afford (Woodward et al., 2011). In potato production, fumigation is used routinely to manage Verticillium wilt. Tenuta and Lazarovitis (2002) found that accumulation of nitrous acid from nitrogenous amendments kill the microsclerotia of V. dahliae and is a promising strategy in acidic soils. In recent years, greater regulation of agrochemicals and increasing costs of application are also motivating the development of alternative management strategies like use of resistant cultivars, amendment with green manure, and soil solarization (Berbegal et al., 2008; Ochiai et al., 2007). Broccoli residues reduce V. dahliae ms in soil and wilt of cauliflower as much as or more than chloropicrin and metam sodium. Quantitative information on the relationship between inoculum density in soil and wilt development can be used for determining the relative length of rotation with certain crops. Quantitative information about the inoculum density in soil and its relationship with Verticillium wilt development would be useful for assessing the efficacy of management strategies for upland cotton. 12

27 1.3.4 Fusarium Wilt-Root-Knot Nematode Complex Another increasingly important soilborne disease of cotton (G. hirsutum) in the High Plains of Texas is Fusarium wilt-root-knot nematode complex. There are two causal agents involved in it. The first one is a soilborne fungus Fusarium oxysporum Schlechtend.:Fr. f. sp. vasinfectum (Atk.) W. C. Snyder & H. N. Hans (Fov), and the second one is Southern root-knot nematode Meloidogyne incognita (Kofoid & White) Chitwood. The disease complex is a major problem in most cotton-growing regions of the world (DeVay, 1986; Smith et al., 1981). Since the recognition of Fusarium wilt of cotton in Alabama by Atkinson in 1892 (Atkinson, 1892), the disease complex has spread and increased in importance, especially in the southeastern United States (Smith et al., 1981). Annual production losses from Fusarium wilt declined as improved wilt-resistant cultivars were released for wide-scale production in the United States during the 1970s and 1980s (Colyer, 2001); but, with the introduction of new susceptible cultivars, Fusarium wilt has again started to cause severe losses in farmers fields. The disease is responsible for losses of $20 million each year across the cotton belt of the United States of America (Blasingame et al., 2008). Under conducive environmental conditions, extremely high losses occur when susceptible cultivars are grown on heavily infested soil. The root-knot nematode commonly associated with Fusarium wilt of upland cotton (G. hirsutum), is M. incognita (Martin et al., 1956; Smith et al., 1981). However, this species is not mentioned in relation to Fusarium wilt of cotton in Egypt or India. Infections with Rotylenchulus reniformis were found to increase Fusarium wilt in upland cotton in the United States (Neal, 1954) and in G. barbadense in Egypt (Khadr et al., 13

28 1972). Other nematodes implicated in association with wilt of upland cotton are Belonolaimus gracilis, B. longicaudatus, and Pratylenchus brachyurus (Cooper and Brodie, 1963; Holdeman and Graham, 1954). Meloidogyne incognita alone is an important pathogen of cotton. Orr and Robinson (1984) estimated that annual losses of cotton were 10% of potential yield due to the nematode in Texas. Starr and Veech (1986) demonstrated a negative, linear relation between cotton yield and initial nematode populations. Both pathogens can damage plants independently, but losses are more severe when they appear together (Kappelman and Sappenfield, 1973; Martin et al., 1956; Minton and Minton, 1966). The incidence, rate of development, and severity of Fusarium wilt in cotton may increase in the presence of M. incognita (Abawi and Chen, 1998; DeVay et al., 1997a; Garber et al., 1979). Symptoms of Fusarium wilt on cotton appeared sooner with an increase of population densities of both M. incognita and Fov and even at the lowest levels of Fov the height and fresh weight of plants were reduced if the nematode levels were high (Garber et al., 1979). At high nematode densities, plant height decreased with increasing fungus densities. The shortest plants were those exposed to the two highest densities of combined organisms. Yang et al., (1976) found that Fusarium wilt occurs with Fov alone at high spore numbers. There are few studies, however, that have examined the epidemiology of Fusarium wilt-root-knot nematode complex under field conditions. Roberts et al. (1985) reported that the negative slope parameter of the regression equation for the relationship between initial nematode populations and cotton yield was of greater magnitude when the fungal pathogen also was present. Thus, the 14

29 incremental effect of increasing nematode population was greater in the presence of Fusarium spp. In a study conducted in microplots by Starr et al. (1989), no interaction was observed at high levels of Fov or at the lowest levels of M. incognita, but a significant interaction was observed at intermediate populations of Fov and the higher populations of M. incognita. This interaction was evident in the time of occurrence of initial mortality and in the final levels of mortality. These observations revealed that the low nematode initial population densities stimulate cotton growth, whereas growth was stunted at the highest nematode population density. Cotton growth was not affected at the intermediate nematode population densities, and the presence of Fov tended to negate both of these plant responses to the nematode. In a greenhouse study conducted by Davis et al. (2006), submerging root systems of susceptible cultivars in inoculum of Fov did not result in visible above ground symptoms until spore concentrations in excess of 10 5 conidia per milliliter were used (Wang et al., 1999). Symptoms in more resistant cotton cultivars were not evident until concentrations of 10 6 conidia per milliliter were used to inoculate the plants. Hao et al., (2009) conducted a study with Fov Race 1 inoculum densities (0 to 10 6 conidia/g of potting mix) on cotton cultivars and found that in the susceptible cultivar Deltapine (DP) 744, symptoms of wilt and reductions in plant growth occurred at inoculum levels of 10 3 conidia/g of potting mix and higher, whereas plant growth of the resistant Pima cv. Phytogen (PHY) 800 was not affected by any soil inoculum densities. Cultivars DP 340, PHY 72, and UltEF all responded similarly, with a relatively moderate negative growth response to soil inoculum densities generally beginning at 10 4 conidia/g of potting mix. 15

30 The importance of interactions between M. incognita and Fov in the development of wilt diseases has been extensively studied (Mai and Abawi, 1987); however, the overall mechanism of interaction between both Meloidogyne species and Fusarium oxysporum pathotypes, is poorly defined (Mai and Abawi, 1987). Fungal hyphae do not appear to enter at the sites of nematode entry; rather instead penetrate the undamaged epidermis of the root tip at adjacent cells. Moreover, Khadr et al. (1972) found that the penetration of the epidermis and cortex of the young root was delayed when root tissues were damaged by penetration by root-knot nematodes. Smith et al. (1981) suspected that plants stunted by nematode infection may exude more and different nutrients from their roots than do healthy, vigorously growing plants, and those plants may be more readily infected. Infections with nematodes on the other hand, may also interfere with the water uptake and transpiration of a plant, thereby slowing the upward movement of a plant pathogen. However, in-a-date-of-planting study with little variability in inoculum densities of M. incognita and Fov, marked differences in the severity and incidence of Fusarium wilt occurred (Jeffers and Roberts, 1993). A correlation between Fov inoculum density and disease incidence has been found to exist (DeVay et al. 1997a; Hao et al. 2009; Starr et al. 1989). The genus Fusarium belongs to Deuteromycotina (Hawkworth, 1983) although, species of Fusarium belong to the Fungi Imperfecti, a number of species have teleomorphs in Hypocreales and Ascomycotina (Hawkworth, 1983) and contains numerous species. Fusarium is widely distributed in soil, on subterranean and aerial plant parts, plant debris, and other organic substrates (Burgess, 1981). In nature, sexual stages do not seem to be important because most Fusarium spp. complete their life cycle 16

31 asexually. All species of Fusarium have a saprophytic stage, and many are facultative parasites. Some are primarily root cortex invaders causing pre- and post-emergence damping-off, root rots, and cankers. According to Walker, (1950) the highly developed pathogens are those within the species F. oxysporum, which invade the xylem vessels of their hosts and are referred to as vascular wilt Fusaria. Cosmopolitan wilting agent F. oxysporum is frequently isolated from diseased cotton seedling roots, and has been often reported as pathogens of several species of the genus Gossypium as well as species of Leguminosae, Malvaceae and Solanaceae (Moricca et al., 1998). The combined effect of fungal metabolites and the production of lipoidal substances by the host in response to infection may lead to the occlusion of the vascular tissues, resulting in wilt of the cotton plant (Hillocks 1984; Shi et al., 1992). The pathogen is introduced into fields through infected seed or may be transported from infested fields on farm equipment or by workers. Losses due to Fusarium wilt of cotton vary depending upon host resistance, general plant health, inoculum potential, environmental factors, presence of nematodes, and use of chemical fertilizers (Smith et al., 1981). In Australia, substantial losses have been incurred since a new lineage of extremely virulent strains of Fov were found in These strains caused damage to Australian cotton despite the absence of root-knot nematodes (Davis et al., 1996b). In the San Joaquin Valley a virulent form of Fov was discovered in 2001 and has since spread to major Pima cotton (G. barbadense) fields even in the absence of root-knot nematodes (Bennett, et al. 2011). Pathogenicity tests were carried out on different cotton species including G. hirsutum, G. barbadense, and G. arboreum, and by pathogenicity tests on 17

32 alfalfa, soybean, and tobacco (Armstrong and Armstrong, 1958; 1978; Chen et al., 1985; Ibrahim, 1966). Phylogenetic analyses of three genes (β-tubulin, translation elongation factor, and phosphate permase), and restriction digests of the intergenic spacer of nuclear rdna revealed the causal agent to be Fov Race 4, a Race originally described from India in 1960 (Kim, et al. 2005). Screening trials conducted since 2003 have shown that nearly all of the commercially available G. barbadense varieties are highly susceptible, and that many varieties of G. hirsutum also exhibit significant disease symptoms under certain conditions. Diagnostic tools such as Race 4-specific PCR primers and macroarrays for the rapid identification of all known lineages of Fov have been developed (Bennett, et al. 2009; Maruthachallam et al., 2010). Isolates of Fov from the Ivory Coast were characterized using vegetative compatibility group (VCG), restriction fragment length polymorphism of the ribosomal intergenic spacer region (IGS), and mating type (MAT) idiomorph, and compared with a worldwide collection of the pathogen containing all available reference strains. Strains from the Ivory Coast were found in 7 of 11 groups detected, suggesting multiple sources for Fusarium wilt in the country (Abo, et al. 2005). Historically, different Races of the pathogen have been associated with specific areas of the world. Only Races 1 and 2 were identified in the United States, and Race 2 was restricted to a small area in South Carolina (Armstrong and Armstrong, 1960; DeVay, 1986; Kim et al., 2005; Skovgaard et al., 2001). Recent research has identified Races 1, 3, 4, and 8 in California, and described severe losses to Pima cottons caused by Race 4 (Kim et al., 2005). Races 1, 3, and 8 have also been identified from the mid-south of the United States and are mildly virulent causing little or no wilt symptoms in the absence of root-knot nematodes. Race 3 and Race 8 were identified in a limited number 18

33 of fields (Kim et al., 2005). Race 4 has now been confirmed in Alabama and Mississippi (Bennett et al., 2011). The Fusarium wilt pathogen Fov, is a diverse species that contains many pathogenic and saprophytic forms. Many isolates of F. oxysporum are non-pathogenic or moderately pathogenic on cotton roots but do not induce wilt. Fusarium wilt is favored by temperatures between C. Fov produces three types of spores in infested plant tissues which are macroconidia, microconidia, and chlamydospores. Macroconidia- These are formed in pale orange, sporodochia. Macroconidia (3-4.5 x µm), are short to medium in length, falcate to almost straight, thin walled and usually 3 septate. The apical cell is notched or foot-shaped. These are formed from monophialids on branched conidiophores in sporodochia and to a lesser extent from monophialids on hyphae (Barnett and Hunter, 2006; Leslie and Summerell, 2006b). Microconidia- These usually are non-septate, may be oval, elliptical or reniform (2-4 x 5-12 µm), and are formed abundantly in false heads on short monphialids (Leslie and Summerell, 2006b). Chlamydospores- These are formed in abundance terminally or intercalary in aerial, submerged, or surface hyphae. These are usually formed singly or in pairs, but may also be found in clusters or short chains with smooth or rough walls. Chlamydospores (7-13 µm in diameter), may be slow (4-6 weeks) to form in some isolates (Leslie and Summerell, 2006b). Morphological characteristics of these spores on media are used to differentiate Fusarium species. Carnation Leaf-piece Agar (CLA), Spzieller Nährstoffarmer Agar 19

34 (SNA), and Potato Dextrose Agar (PDA) are the standard media used in the identification of Fusarium species (Leslie and Summerell, 2006a). Colony morphology, pigmentation and growth rates of cultures of most Fusarium species on PDA are found to be reasonably consistent. A number of media have been developed for the specific isolation of Fusarium species; however, Komada s semi-selective medium is used for isolating Fov from plant tissue or soil (Komada, 1975; Nelson et al., 1983; Windels, 1993). This medium was developed for the selective isolation of F. oxysporum from soil. Colonies of F. oxysporum are distinctly pigmented on this medium, and usually separable from other Fusarium species on this basis (Burgess et al., 1981). Once a field is infested with Fov, the fungus usually persists indefinitely (Smith and Snyder, 1975). Survival of the fungus in the soil not planted to cotton for over 10 years has been documented (Smith et al., 2001). Longevity of Fov in soil and in decaying plant tissue, like most formae speciales of the fungus, is probably in the form of chlamydospores, the thick walled resting structures (Nelson, 1981; Smith et al., 1981; Smith and Snyder, 1975). Gin trash and other plant debris as well as cotton seed from fields with Fusarium wilt are also sources of Fov inoculum (Jeffers et al., 1984). Chlamydospores are under fungistasis and remain dormant in the soil until fresh organic matter or exudates and leachates from plant roots stimulate their germination (Mai and Abawi, 1987). The germinated chlamydospores produce hyphae that eventually form conidia and new chlamydospores if a suitable host is not found. Organic matter, exudates and leachates from roots are required to stimulate the germination of chlamydospores and subsequent colonization is not limited to cotton. The invasion of roots occurs via direct penetration or through wounds. The ability of Fov to colonize the roots of plants other 20

35 than cotton is significant for its long-term survival, since hyphae, conidia, and chlamydospores may be destroyed by soil microorganisms (Subramanian, 1950). Once the fungus enters the plant, it grows and reproduces, spreading throughout the plant in the vascular system (Colyer, 2001). Regardless of the mode of survival, the ability of the pathogen to survive in soils for long periods has important consequences on disease management. Meloidogyne incognita is a sedentary endo-parasitic nematode that deposits its eggs into a gelatinous matrix, which normally protrudes into the soil from the surface of the root gall. Eggs develop into first stage juvenile that molt within the egg shell to produce second stage juveniles (J2s) before hatching. The J2 is the only infective stage of this root parasite that emerges from the egg and penetrates cotton roots just above the root cap (Koenning, et al. 2004). The nematodes migrate through the root cortex and pierce developing vascular or adjacent parenchymatous cells with their stylets, delivering esophageal gland secretions that induce the formation of specialized feeding sites, known as giant cells. These enlarged, multinucleate cells become permanent feeding sites. As the giant cells enlarge and the root tissue proliferates, the feeding sites become visible as knots or galls on the roots. The life cycle may be completed in as few as 25 days at optimal temperatures of C (Koenning, et al. 2004). Symptoms of M. incognita are typically more severe in plants grown in soils containing more than 50% sand. These soils favor nematode interaction and enhance water stress on plants. Initial symptoms of Fusarium wilt- root-knot nematode disease complex include chlorosis, yellowing, and wilting of leaves. Chlorosis starts at the margins of the leaf between the main veins. These areas eventually turn necrotic. In seedling and young 21

36 plants, cotyledons and leaves wilt and drop, resulting in bare stems. Severely diseased plants often remain stunted (Lawrence and McLean, 2001). The vascular system of the plants is discolored brown to black. In the most severely affected plants, leaves wilt and drop and the plant may die (Colyer, 2001; Nelson et al., 1981). The time of infection determines the extent of any yield reduction. Plants that develop symptoms early usually die before producing any bolls, whereas plants that develop symptoms after the onset of flowering often survive but produce few bolls. In Verticillium wilt, infection of plant roots and disease development are progressive throughout the growing season (Pullman and DeVay, 1982a) but in Fusarium wilt, an early cycle of infection appears sufficient to induce slow growth and reductions in cotton seed yields for the remainder of the growing season. The most characteristic and definitive symptoms of M. incognita on cotton is the presence of the spindle-shaped or rounded galls on the plant roots. Galls are often small and are most numerous on lateral roots. Infected plants also may exhibit considerable proliferation of lateral roots (Koenning, et al. 2004). Infection disrupts normal development of root vascular tissue, resulting in reduced ability of the plant to utilize water and nutrients (Lawrence and McLean, 2001) Management of Fusarium Wilt-Root-knot Nematode Disease Complex There are very limited management options available for Fusarium wilt-root-knot nematode disease complex that include rotations with crops that are poor hosts for the nematode, planting cotton cultivars that are resistant to either the nematode or the fungus, and soil fumigation (DeVay, 1986; Hillocks, 1992; Kappelman and Smith, 1981). Use of rotations to reduce Fusarium wilt has been of limited value because Fov is a good saprophyte and is able to survive in soils for several years on the roots of non-host plants 22

37 (Smith and Snyder, 1975). In addition, M. incognita has a broad host range, and there are few economically viable alternative crops for use by cotton producers in a rotation program. Rotation with peanut is an excellent option to reduce Fusarium wilt in the subsequent cotton crop, since peanut is a non-host for M. incognita (Johnson, 1998; Kirkpatrick and Sasser, 1984) It is well known that control of root-knot nematodes result in a marked reduction in the incidence and severity of Fusarium wilt of cotton (Mai and Abawi, 1987; Starr et al., 1989). Control of nematodes with soil fumigants has been found to result in considerable decreases in Fusarium wilt and correspondent yield increases (Hyer et al., 1979; Jogenson et al., 1978; Jorgenson, 1979; Smith, 1948). Management of Fusarium wilt depends mainly on planting partially resistant cultivars or using other methods to reduce the population density of M. incognita. Other methods, such as solarization, are effective but too costly to use (Ben-Yephet et al., 1987; Katan, 1981; Pullman et al., 1979). Chemical control of root-knot nematodes utilizing fumigant or non-fumigant nematicides is the most frequently used management strategy. The profitability of this method of management is influenced by the nematode population density, the portion of the field affected, and a realistic estimation of the amount of nematode-induced yield loss likely to occur without nematode treatment (Hyer, et al., 1979; Jogenson et al., 1978; Jogenson, 1979; Smith, 1948). More restrictive uses of nematicides, due to environmental and human health concerns and economic considerations, have resulted in greater emphasis on the development of resistant cultivars for the management of nematodes and wilt disease. Resistance to M. incognita has proved to be an effective management 23

38 strategy for the Fusarium wilt-root-knot nematode complex (Hillocks, 1992; Mai and Abawi, 1987; Ogallo, et al., 1997; Shepherd, 1986). High levels of resistance to M. incognita reduced wilt severity more effectively than tolerance to wilt or moderate resistance to the nematode (Hyer et al., 1979; Shepherd, 1982). Field observations suggest that repeated use of a M. incognita partially resistant cultivar (Stoneville 5599BR) led to dramatic decrease in Fusarium wilt incidence over a 2-3 year period (Wheeler, unpublished data). Resistance to M. incognita has proved to be an effective management strategy for the Fusarium wilt-root-knot nematode complex (Ogallo, et al., 1997). Some studies, however, indicate that the nematodes may not be able to infest the cultivars possessing high levels of resistance to the Fusarium species (Abawi and Barker, 1984). Shepherd (1975) suggested that damage due to Fusarium wilt-root-knot nematode complex could be largely avoided if cultivars with greater resistance to both root-knot and Fusarium wilt were utilized. The general consensus is that the cultivars with partial resistance to M. incognita suffer much less wilt than cultivars with resistance to Fov alone. Furthermore, nematode resistance combined with intermediate wilt resistance can be more effective than nematode resistance alone in protecting plants from the disease (Bertrand et. al., 2000; Castillo et. al., 2003; Roberts et. al., 1995; Wang and Roberts, 2006). Management of the above mentioned soilborne pathogens of upland cotton are important to avoid the losses caused by these economically important diseases. It is very crucial to understand the relationship between inoculum density in soil at planting and its effect on upland cotton cultivars. With the release of transgenic cotton cultivars having true or partial resistance to soilborne pathogens, there is a need to screen them against 24

39 different levels of inoculum pressure. Additionally, this information needs to be communicated to the growers of the West Texas region so that they can remain economically competitive. 25

40 1.4 RESEARCH OBJECTIVES The specific objectives of this research are following: 1. To examine the influence of Verticillium dahliae infested peanut residue amount on inoculum release in soil and wilt development in subsequent cotton. Hypothesis: Peanut residue infested with Verticillium dahliae will increase microsclerotia density in soil and Verticillium wilt on subsequent cotton will also increase. 2. To examine the consequence of cultivar selection on soil population dynamics of Verticillium dahliae over time and implications for Verticillium wilt development in cotton. Hypothesis: The choice of cultivar will affect soil inoculum density of Verticillium dahliae and disease incidence. 3. To examine the impact of inoculum densities of genetically distinct Fusarium oxysporum f. sp. vasinfectum Race 1 isolates, Meloidogyne incognita, and cultivar on Fusarium wilt development in cotton. Hypothesis: Isolates of Fusarium oxysporum f. sp. vasinfectum collected from Texas will demonstrate variability in aggressiveness. 4. To investigate the effect of cotton cultivar selection on population dynamics of Fusarium oxysporum f. sp. vasinfectum in soil over time. Hypothesis: Due to the use of resistant cultivar, soil inoculum densities of Fov and disease incidence will decrease. 26

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42 Bell, A.A Verticillium Wilt. Compendium of Cotton Diseases 2 nd Edition. T.L. Kirkpatrick and C.S. Rothrock. eds. APS Press, pp Bennett, R. S., Bell, A. A., Woodward, J. E., Lawrence, K. S., Rothrock, C. S., Kirkpatrick, T. L., Lawrence, G. W., Colyer, P. D., and Davis, R. M Progress report on a contemporary survey of the Fusarium wilt fungus in the United States. Proc. Beltwide Cotton Conference, 5-7 Jan., Atlanta, GA. In Press. Bennett, R., Davis, R., Hutmacher, R. B Fusarium oxysporum f. sp. vasinfectum Race 4 in California. World Cotton Research Conferences - 4 Proceedings; paper #1383, CD- ROM. Ben-Yephet, Y., and Pinkas, Y Germination of individual microsclerotia of Verticillium dahliae. Phytoparasitica 5: Ben-Yephet, Y., Stapleton, J. J., Wakeman, R. J., and DeVay, J. E Comparative effects of soil solarization with single and double layers of polyethylene film on survival of Fusarium oxysporum f. sp. vasinfectum. Phytoparasitica 15: Berbegal, M., García-Jiménez, J., and Armengol, J Effect of cauliflower residue amendments and soil solarization on Verticillium wilt control in artichoke. Plant Dis. 92: Berbegal, M., Ortega, A., García-Jiménez, J., and Armengol, J Inoculum densitydisease development relationship in Verticillium wilt of artichoke caused by Verticilluim dahliae. Plant Dis. 91: Bertrand, B., Nunez, C., and Sarah, J. L Disease complex in coffee involving Meloidogyne arabicida and Fusarium oxysporum. Plant Pathol. 49: Bewley, W. F Sleepy disease of the tomato. Ann. Appl. Biol. 9: Bhat, R. G., and Subbarao, K. V Host range specificity in Verticillium dahliae. Phytopathology 89: Blasingame, D. J., and Patel, M. V., Cotton diseases and their causal agents. Compendium of Cotton Diseases, 2 nd Edition. T.L. Kirkpatrick and C.S. Rothrock. eds. APS Press, pp Blasingame, D., Banks, J. C., Colyer, P. D., Davis, R. M., Gazaway, W. S., Goldburg, N., Kemerait, R. C., Kirkpatrick, T. L., Koenning, S. R., Muller, J., Newman, M. A., Olsen, M., Phipps, P. M., Sciumbato, G. L., Sprenkel, R., Woodward, J. E., Wrather, A. and Patel, M. V Beltwide Cotton Conference Cotton disease loss estimate committee report Proceedings, pp

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52 Starr, J. L., and Veech, J. A Susceptibility to root-knot nematodes in cotton lines resistant to the Fusarium wilt-root-knot complex. Crop Sci. 26: Starr, J. L., Jeger, M. J., Martyn, R. D., and Schilling, K Effects of Meloidogyne incognita and Fusarium oxysporum f. sp. vasinfectum on plant mortality and yield of cotton. Phytopathology 79: Subramanian, C. V Soil conditions and wilt diseases in plants with special reference to Fusarium vasinfectum on cotton. Proc. Indian Acad. Sci., Section B. 31: Takeuchi, S Importance and problems of disposal of crop residues containing pathogens of plant diseases. Japan Agricultural Research Quaterly 21: Tenuta, M., and Lazarovits, G Ammonia and nitrous acid from nitrogenous amendments kill the microsclerotia of Verticillium dahliae. Phytopathology 92: Tjamos, E. C., Problems and prospects in controlling Verticillium wilt. In: Vascular Wilt Diseases of Plants. E. C. Tjamos and C. Beckman, eds. Springer-Verlag, Berlin, pp Tjamos, E. C., Virulence of Verticillium dahliae and V. albo-atrum isolates in tomato seedlings in relation to their host of origin and the applied cropping system. Phytopathology 71: Tzeng, D. D., and DeVay, J. E Physiological responses of Gossypium hirsutum L. to infection by defoliating and nondefoliating pathotypes of Verticillium dahliae Kleb. Physiol. Plant Pathol. 26: United States Department of Agriculture, Foreign Agricultural Service World Agricultural Production. Verified on 2/7/11. Walker, J. C The Fusarium diseases. In: Plant Pathology. McGraw-Hill Book Company Inc., New York, pp Wang, B., Dale, M. L., and Kochman, J. K Studies on pathogenicity assay for screening cotton germplasmsfor resistance to Fusarium oxysporum f. sp. vasinfectum in the glasshouse. Aust. J. Exp. Agric. 39: Wang, C., and Roberts, P. A A fusarium wilt resistance gene in Gossypium barbadense and its effect on root-knot nematode-wilt disease complex. Phytopathology 96: Wheeler, T. A., Madden, L. V., Rowe, R. C., and Riedel, R. M Effects of quadrat size and time of year for sampling Verticillium dahliae and lesion nematodes in potato fields. Plant Dis. 84:

53 Wiese, M. V., and DeVay, J. E Growth regulator changes in cotton associated with defoliation caused by Verticillium albo-atrum. Plant Physiol. 45: Wilhelm, S Longevity of the Verticillium wilt fungus in the laboratory and field. Phytopathology 45: Wilhelm, S., and Paulus, A. O How soil fumigation benefits California strawberry industry. Plant Dis. 64: Windels, C. E Fusarium. In: Methods for Research on Soilborne Phytopathogenic Fungi. L. L. Singleton, J. D. Mihail, and C. M. Rush, eds. American Phytopathological Society, St. Paul, MN, pp Woodward, J. E., T. A. Wheeler, M. G. Cattaneo, S. A. Russell, and T. A. Baughman Evaluation of soil fumigants for management of Verticillium wilt of peanut in Texas. Online. Plant Health Progress doi: /php rs. Wu, Y., Fang, W., Zhu, S., Jin, K., and Ji, D The effects of cotton root exudates on the growth and development of Verticillium dahliae. Front. Agric. China 2: Xiao, C. L., and Subbarao, K. V Relationship between Verticillium dahliae inoculum density and wilt incidence, severity, and growth of cauliflower. Phytopathology 88: Xiao, C. L., Hao, J. J., and Subbarao, K. V Spatial patterns of microsclerotia of Verticillium dahliae in soil and Verticillium wilt of cauliflower. Phytopathology 87: Xiao, C. L., Subbarao, K. V., Schulbach, K. F., and Koike, S. T Effects of crop rotation and irrigation on Verticillium dahliae microsclerotia in soil and wilt in cauliflower. Phytopathology 88: Yang, H., Powell, N. T., and Barker, K. R Interactions of concomitant species of nematodes and Fusarium oxysporum f. sp. vasinfectum on cotton. J. Nematol. 8: Zilberstein, Y., Chet, I., and Henis, Y Influence of microsclerotia source of Verticillium dahliae on inoculum quality. Trans. British Mycol. Soc. 81:

54 CHAPTER II INFLUENCE OF Verticillium dahliae INFESTED PEANUT RESIDUE ON INOCULUM RELEASE IN SOIL AND WILT DEVELOPMENT IN SUBSEQUENT COTTON 2.1 ABSTRACT A microplot study was conducted in 2008 and 2009 to investigate the impact of peanut residue infested with Verticillium dahliae Kleb. on inoculum production in soil and Verticillium wilt development in cotton. The hypothesis was that Peanut residue infested with V. dahliae will increase microsclerotia density in soil and Verticillium wilt on subsequent cotton will also increase. The effects of infested peanut residue amount on percent germination of cotton seeds and on Verticillium wilt incidence were monitored in both cropping seasons. Microsclerotia density in soil was also quantified to investigate the release of inoculum from infested peanut residue over time. Infested peanut residue was collected from a field with a history of Verticillium wilt and used to artificially infest microplots with the amount of 370, 925, 1850, 2775, 3700, 18,495, and 37,000 kg/ha. Non-infested microplots served as a control. Treatments were arranged in a randomized complete block design with nine replications. Microplots were planted with a cotton cultivar, Stoneville (ST) 4554B2RF, susceptible to Verticillium wilt. Increasing infested peanut residue amount had a negative effect on percent germination of cotton seeds with a slope of (R 2 = 0.90) in 2008, and (R 2 = 0.98) in A positive effect was found between increasing infested peanut residue amount and Verticillium wilt incidence 40

55 in cotton, with a slope of 3.07 (R 2 = 0.97) in 2008, and 6.43 (R 2 = 0.99) in Soil samples collected before incorporation of infested peanut residue artificially were void of V. dahliae inoculum for all the microplots. Densities of microsclerotia in the soil were found to increase significantly with increasing rates of V. dahliae infested peanut residue over time, with a slope of 0.42 (R 2 = 0.99) in April 2009, 0.85 (R 2 = 0.89) in November 2009, and 1.32 (R 2 = 0.89) in April Results show the importance of removing infested peanut residue which otherwise may serve as a source of inoculum for subsequent cotton crops. Additional keywords: Arachis hypogaea, Gossypium hirsutum, inoculum density, residue management. 41

56 2.2 INTRODUCTION Verticillium wilt, caused by the soilborne fungus Verticillium dahliae Kleb., is an economically important disease of cotton (Gossypium hirsutum L.) and peanut (Arachis hypogaea L.). The pathogen has a broad host range of more than 400 plant species including field crops, most vegetables, and forest trees (McCain et al., 1981). Several factors, including cultivar selection, pathogen aggressiveness, inoculum density and environmental conditions influence Verticillium wilt development (Bhat and Subbarao, 1999; Markakis et al., 2010). Verticillium wilt affected plants show stunting, epinasty, and yellowing (Smith, 1960). Leaves exhibit interveinal chlorosis, necrosis, curling, and die back from the margins inward. Plants develop characteristic mosaic patterns on leaves, starting from the base of the plant and progressing towards the top (Misaghi et al., 1978). Ramification of the fungus in the xylem vessels lead to a tan to brown colored discoloration of the vascular system by decreasing hydraulic conductance and plants may eventually wilt and die (Schnathorst, 1981). In infested cotton plants, bolls abscise or do not open (Pullman and DeVay, 1982); whereas, in peanut, pegs are formed in less numbers and have fewer seeds (Melouk et al., 1983). Cool, wet conditions favor disease development with temperatures between C being best suited for V. dahliae growth and survival (Rowe et al., 1987). The fungus is capable of infecting plant roots directly or through wounds throughout the growing season. V. dahliae primarily colonizes the rhizoplane of the host plant (Huisman, 1988). It penetrates roots early in the growing season, and then infects the vascular system and grows systemically throughout the plant. Microsclerotia (ms), the survival 42

57 structures of V. dahliae, are produced once the plant dies. Microsclerotia are composed of masses of melanized hyphae and are considered as the principal source of inoculum for Verticillium wilt development. Microsclerotia can survive for more than 20 years in the soil (Wilhelm, 1955), and root exudates stimulate germination of ms initiating infections (Mol, 1995; Schnathorst, 1981). Microsclerotia are formed depending on temperature and moisture availability with the decay of plant tissues. Microsclerotia are dispersed in the soil and only a single cycle of inoculum is produced during a growing season (Paplomatas et al., 1992). Thus, inoculum density in field soils at planting plays a critical role in disease development. Microsclerotia of V. dahliae are produced in large numbers on senescing parts of host plants and the formation mainly occurs in the aerial parts of the crop (Mol and Scholte, 1995) and therefore, removal of the aerial crop debris from the field is potentially an effective measure to prevent the accumulation of ms in the soil (Mol et al., 1995). In parts of West Texas, cotton and peanut are commonly grown in rotation. Farmers of this region routinely leave peanut residues on the soil surface rather than removing them. Typical rates of peanut residue left in the field after harvest is approximately 3700 kg/ha (Woodward, unpublished data). This practice help reduce soil erosion, conserve energy, maintain soil moisture, improve organic matter content, and soil fertility (Balkcom et al., 2004). However, V. dahliae can survive in the crop residue and disease problems may be more severe by protecting the residue from microbial degradation and lowering soil temperature. Both peanut and cotton are suitable hosts for V. dahliae allowing for a continued increase in ms production. Currently, there is no quantitative data available regarding the 43

58 influence of V. dahliae infested peanut residue on wilt development in cotton. The objective of this study was to determine the effect of V. dahliae infested peanut residue amount on release of ms in the soil and its implications for Verticillium wilt development in cotton over time. 2.3 MATERIALS AND METHODS Microplot Experiment A microplot experiment was conducted in 2008 and 2009 to examine the effect of increasing amount of peanut residue infested with V. dahliae on ms production and disease development in cotton over time. Microplots were constructed out of cylindrical galvanized aluminum rings (90 cm diameter and 60 cm height), and buried at the depth of 50 cm. Treatments (0, 370, 925, 1850, 2775, 3700, 18,495, and 37,000 kg/ha) were arranged in a randomized complete block design with nine replications. Peanut residue was collected from a field that was infested with V. dahliae and had experienced severe Verticillium wilt for several growing seasons. Residues were incorporated by hand tilling two months prior to planting in Microplots were planted with a susceptible cotton cultivar, Stoneville (ST) 4554B2RF at the rate of 25 seeds per microplot in a circular pattern. Irrigation, fertilizer and weeding practices were conducted as needed, according to local extension recommendations for both seasons Soil Sampling and Data Collection All microplots were sampled in February 2008, prior to the assigning of residue treatments, to determine baseline populations of V. dahliae within the soil. Subsequent soil samples were taken in April and November 2009, and April A 2.5 cm 44

59 diameter auger to a depth of 20 cm was used in taking soil samples from each microplot. Each sample consisted of four cores and had a total soil weight of approximately 250 g. The samples were air-dried at room temperature for 14 days. A soil dilution plating technique (Isaac et al., 1971), utilizing Sorensen s NP-10 semi-selective medium (Sorensen et al. 1991) amended with N NaOH as suggested by Kabir et al. (2004) was used for enumeration of ms in soil. Air-dried soil was ground with a roller pin and a 20 cm 3 soil sample was combined with 80 ml of de-ionized water, and then stirred using a magnetic stir plate. A 1-ml aliquot of the soil solution was distributed on each Petri dish (10 replications) containing Sorensen s NP-10 semi-selective medium, and was spread with a glass rod. After 14 days of incubation at room temperature in the dark, the soil was rinsed from the Petri dishes by gently rubbing, and then air dried for 2 hours prior to counting. The numbers of colonies of V. dahliae were counted under a stereo dissecting microscope and expressed as the number of ms/cm 3 of dry soil. Percent germination of cotton seed was recorded in June and disease incidence was assessed in September as percent symptomatic plants in each microplot for both the seasons Statistical Analysis Data for percent germination of cotton seeds, percent disease incidence and inoculum density of V. dahliae in soil (ms/cm 3 ) were analyzed using Proc MIXED (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). Infested peanut residue amounts (treatments) were log transformed to make their distribution smooth. Data were analyzed as a split-plot in time where the sub-plots were four sampling dates as described in Steel and Torrie (1960). The method used to adjust the degrees of freedom (df) to match adjustments in the sums of square was the Satterthwaite option in the LSMEANS 45

60 statement in Proc MIXED. Standard error and LSD were determined from the PDIFF (probability of difference of two means) option. Regression analysis was carried out for percent germination, percent disease incidence and inoculum density of V. dahliae in soil (ms/cm 3 ) with log 10 transformed infested peanut residue rates using Proc MIXED. 2.4 RESULTS Increasing peanut residue rates had a negative effect on percent germination of cotton seeds (Fig. 2.1.a), with a non-linear quadratic curvature of (R 2 = 0.90) in June 2008, and 4.0 (R 2 = 0.98) in June 2009 (Table 2.1). Cotton germination in microplots amended with the lowest rate of residue averaged 93.8 and 94.2 % for 2008 and 2009, respectively; whereas, microplots amended with the highest rate of infested peanut residue resulted in 64.4 and 50.7 % over the two years (Fig. 2.1.a). Germination of cotton seeds in the non-amended controls averaged 99.6 %; whereas, germination for all other treatments was intermediate (Fig. 2.1.a). A positive correlation was found to exist between increasing peanut residue amounts and Verticillium wilt incidence on cotton (Fig. 2.1.b), with a slope of 3.07 (R 2 = 0.97) in September 2008, and 6.43 (R 2 = 0.99) in September 2009 (Table 2.1). Overall, percent disease incidence was observed to be increased significantly with increasing amounts of infested peanut residue in both the growing seasons, 2008 and 2009 (Fig. 2.1.b). Disease incidence was increased from 1.9 % to 27.8 % when comparing the lowest and highest residue amounts, respectively (Fig. 2.1.b). A similar trend was observed for the 2009 growing season. Disease incidence increased substantially between years in microplots amended with 1850 kg/ha residue amount or greater (Fig. 2.1.b). 46

61 Soil samples collected in February 2008 were void of V. dahliae inoculum in all the microplots prior to artificial incorporation of infested peanut residue (Fig. 2.1.c). There was a positive correlation between increasing infested peanut residue amounts and ms densities in soil (Fig. 2.1.c), with a slope of 0.42 (R 2 = 0.99) in April 2009, 0.85 (R 2 = 0.89) in November 2009, and 1.32 (R 2 = 0.89) in April 2010 (Table 2.1). Overall, ms densities were found to increase with higher amounts of V. dahliae infested peanut residue and with time (Fig. 2.1.c). 2.5 DISCUSSION Microsclerotia of V. dahliae are produced in large numbers on senescing parts of host plants and may remain viable in the soil for many years (Mol, and Scholte, 1995). Peanut is commonly used as a rotation crop with cotton in West Texas and, both crops are suitable hosts for V. dahliae. This study demonstrates that ms can survive in infested peanut residue, and then can infect susceptible cotton in the next season. Root exudates stimulate germination of ms leading to a decline in the population of viable propagules in soil until the incorporation of new inoculum from infected tissues at the end of the crop (Mol, 1995; Mol et al., 1995). Inoculum potential may be related to the density and distribution of infested residues as well as to the susceptibility of the host crop (Bhat and Subbarao, 1999). Being a monocyclic disease, disease incidence in Verticillium wilt is positively related to the amount of primary inoculum. Understanding the relationship between ms density in soil at planting and wilt development is therefore essential for developing a disease risk assessment based on pre-plant soil assays and also for disease management (Paplomatas et. al 1992). 47

62 The beneficial effects of leaving crop residue are frequently offset by the negative effects as a nutrient source and shelter for survival, growth, and reproduction of plant pathogens and raise concerns over the role of crop residue in epidemics caused by soilborne pathogens. Crop residue at the soil surface is a principal source of inoculum. Infested crop residue was found to increase soil inoculum density and disease incidence in this and also for other pathosystems (Adee et al., 1997). Tjamos (1981) and Zilberstein et al., (1983) found that the degree of pathogenicity of V. dahliae was related to the plant species from which the isolate was obtained, and was also dependent on the previous cropping history. Continual uses of susceptible host cultivars and cultural practices that leave abundant infested residue on the soil surface have been observed to increase the damage caused by other soilborne pathogens in subsequent crops (Pereyra and Dill- Macky, 2008). Microsclerotia mainly occur in the aerial parts of the crop (Mol and Scholte 1995) and therefore, removal of the aerial crop debris from the field is potentially an effective measure to prevent the accumulation of ms in the soil. In the present study, lower ms/cm 3 of soil in the treatments had lower rates of infested peanut residue, which agrees with results of Hoekstra (1989), who found that many ms can be produced in plant residue of field bean and, removing the debris of field bean resulted in a lower ms population in the next spring. The apparent increase in ms population, a year after incorporation of infested peanut residue may be explained by the disintegration of plant debris containing ms. Higher numbers of V. dahliae ms were found on cauliflower roots eight weeks after harvest (Xiao et al., 1998). These ms may serve as a source of inoculum for the next crop. Davis et al. (1983) found a strong correlation between ms population in the soil and the 48

63 number of ms in stem tissue, which can further contribute to soil inoculum for the next season. Huisman and Ashworth (1976) and Joaquim et al. (1988) found a sharp increase in ms population in the second year after growth of a susceptible crop, despite host susceptibility. In Texas, peanut residue is often used as a feed supplement for cattle. Peanut residue is composed of the vines and leaves of peanut plants after the peanuts are harvested and serve as a good source of nutrition (Parish, 2006). Cost of feed represents the largest single cost item in most beef operations (Darrell, 2004). This is an additional way to add economic benefits to removing peanut residue from the field. Balkcom et al. (2004) found that peanut residue does not contribute significant amounts of nitrogen to the subsequent cotton crop; however, these interactions need to be investigated further under the arid conditions of West Texas. While removing residue may remove nitrogen from the soil, the potential benefits are two-fold reducing soil populations of V. dahliae and sale of hay for feed-stock. Results from the present study suggest the importance of removing the peanut residue infested with V. dahliae, for managing the Verticillium wilt in the subsequent cotton crop. Due to the lack of effective fungicides and truly resistant cultivars, disease management will likely rely on an integrated management program using a number of management options. Reducing inoculum of V. dahliae in host debris and other reservoirs may be a key to Verticillium wilt management. Cropping systems have a changing dynamics of disease and soilborne pathogens that are influenced by cultural practices such as residue management. The importance of primary inoculum in Verticillium wilt development, justifies the use of cultural management practices, including the use of 49

64 partially resistant cotton cultivars and destruction of infested peanut residues for effective disease management. 50

65 2.6 LITERATURE CITED Adee, E. A., Grau, C. R., and Oplinger, E. S Population density of Phialophora gregata in soybean residue. Plant Dis. 81: Balkcom, K. S., Wood, C. W., Adams, J. F., and Wood, B. H Composition and decomposition of peanut residue in Georgia. Peanut Sci. 31:6-11. Bhat, R. G., and Subbarao, K. V Host range specificity in Verticillium dahliae. Phytopathology 89: Darell, L. R. Jr By-product feeds for Alabama beef cattle. Verified on 2/13/2011. Davis, J. R., Pavek, J. J., Corsim, D. L A sensitive method for quantifying Verticillium dahliae colonization in plant tissue and evaluating resistance among potato genotypes. Phytopathology 73: Harris, D. C., and Yang, J.R The relationship between the amount of Verticillium dahliae in soil and the incidence of strawberry wilt as a basis for disease risk prediction. Plant Pathol. 45: Hoekstra, O Effects of leguminous crops on potato production and on incidence of Verticillium dahliae in various crop rotations with potatoes. In: Vos, J., Van Loon C. D., Bollen, G. J. eds. Effects of Crop Rotation on Potato Production in the Temperate Zones. Dordrecht, The Netherlands: Kluwer Academic Publishers, pp Huisman, O. C Colonization of field-grown cotton roots by pathogenic and saprophytic soilborne fungi. Phytopathology 78: Huisman, O. C., and Ashworth, Jr. L. J Influence of crop rotation on survival of Verticillium albo-atrum in soils. Phytopathology 66: Isaac I., Fletcher P., and Harrison J.A.C., Quantitative isolation of Verticillium spp. from soil and moribund potato haulm. Ann. Appl. Biol. 67: Joaquim, T. R., Smith, V. L., and Rowe, R. C Seasonal variation and effects of wheat rotation on populations of Verticillium dahliae Kleb. in Ohio potato field soils. Am. Potato J. 65: Kabir, Z., Bhat, R. G., and Subbarao, K. V Comparison of media for recovery of Verticillium dahliae from soil. Plant Dis. 88:

66 Markakis, E. A., Tjamos, S. E. and Antoniou, P. P., Roussos, P. A., Paplomatas, E. J., and Tjamos, E. C Phenolic responses of resistant and susceptible olive cultivars induced by defoliating and non-defoliating Verticillium dahliae pathotypes. Plant Dis. 94: McCain, A. H., Raabe, R. D., and Wilhelm, S Plants Resistant or Susceptible to Verticillium Wilt. Cooperative Extension, U.S. Department of Agriculture, University of California, Berkeley, pp Melouk, H. A., Wadsworth, D. F., and Sherwood, J. L Effect of Verticillium wilt on root and top weight of peanut cultivar Tamnut 74. Plant Dis. 67: Misaghi, I. J., DeVay, J. E., and Duniway, J. M Relationship between occlusion of xylem elements and disease symptoms in leaves of cotton plants infected with Verticillium dahliae. Can. J. Bot. 56: Mol, L Effect of plant roots on the germination of microsclerotia of Verticillium dahliae. II. Quantitative analysis of the luring effect of crops. Eur. J. Plant Pathol. 101: Mol, L., and Scholte, K Formation of microsclerotia of Verticillium dahliae on various plant parts of two potato cultivars. Potato Res. 38: Mol, L., Scholte, K., and Vos, J Effects of crop rotation and removal of crop debris on the soil population of two isolates of Verticillium dahliae. Plant Pathol. 44: Paplomatas, E.J., Bassett, D.M., Broome, J.C., and DeVay, J.E Incidence of Verticillium wilt and yield losses of cotton cultivars (Gossypium hirsutum) based on soil inoculum density of Verticillium dahliae. Phytopathology 82: Parish, J Alternative feedstuffs for beef cattle operations - Part II. Verified on 2/13/2011. Pereyra, S. A., and Dill-Macky, R Colonization of the residues of diverse plant species by Gibberella zeae and their contribution to Fusarium head blight inoculum. Plant Dis. 92: Pullman, G. S., and DeVay, J. E Epidemiology of Verticillium wilt of cotton: Effects of disease development on plant phenology and lint yield. Phytopathology 72: Rowe, R. C., and Powelson, M. L Potato early dying: Management challenges in a changing production environment. Plant Dis. 86: Rowe, R. C., Davis, J. R., Powelson, M. L., and Rouse, D. I Potato early dying: Causal agents and management strategies. Plant dis. 71:

67 Schnathorst, W. C., Life cycle and epidemiology of Verticillium. In: Fungal Wilt Diseases of Plants. M. E. Mace, A. A. Bell, and C. H. Beckman, eds. Academic Press, New York, pp Smith, T. E Occurrence of Verticillium wilt on peanuts. Plant Dis. Rep. 44:435. Sorensen, L. H., Scheider, A. T., and Davis, J.R Influence of sodium polygalacturonate sources and improved recovery of Verticillium spp. from soil (Abstr.) Phytopathology 81:1347. Steel, R. G. D. and Torrie, J. H Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., pp Tjamos, E. C., Virulence of Verticillium dahliae and V. albo-atrum isolates in tomato seedlings in relation to their host of origin and the applied cropping system. Phytopathology 71: Wilhelm, S Longevity of the Verticillium wilt fungus in the laboratory and field. Phytopathology 45: Xiao, C. L., Subbarao, K. V., Schulbach, K. F., and Koike, S. T Effects of crop rotation and irrigation on Verticillium dahliae microsclerotia in soil and wilt in cauliflower. Phytopathology 88: Zilberstein, Y., Chet, I., and Henis, Y Influence of microsclerotia source of Verticillium dahliae on inoculum quality. T Brit Mycol Soc 81:

68 Table 2.1. Parameters from non-linear quadratic regression of percent germination of cotton seeds, disease incidence, and inoculum density of Verticillium dahliae in soil due to influence of infested peanut residue a Parameters Time Intercept (y 0 ) SE Slope (a) SE Curvature (b) SE R 2 Germination of cotton seeds (%) Disease incidence (%) Inoculum density (ms/cm 3 ) February na 0.00 na 0.00 na na April November April a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Non-linear quadratic equation f = y 0 + ax + bx 2 was used to calculate intercept (y 0 ), slope (a) and curvature (b). Soil samples collected in February 2008 were void of V. dahliae inoculum prior to artificial incorporation of infested peanut residue. 54

69 a Germination (%) Jun 08, R 2 = 0.90 Jun 09, R 2 = Log 10 infested peanut residue rate (kg/ha) b Disease incidence (%) Sep 08, R 2 = 0.97 Sep 09, R 2 = Log 10 infested peanut residue rate (kg/ha) Inoculum density of V. dahliae in soil (ms/cm 3 ) Feb Apr 09, R 2 = 0.99 Nov 09, R 2 = 0.89 Apr 10, R 2 = Log 10 infested peanut residue rate (kg/ha) c Fig Effect of infested peanut residue amounts on a. germination of cotton seeds (%), b. disease incidence (%), and c. inoculum density of Verticillium dahliae in soil (ms/cm 3 ). Infested peanut residue amounts 0, 370, 925, 1850, 2775, 3700, 18495, and kg/ha were Log 10 transformed and were expressed as 0, 2.6, 3.0, 3.3, 3.4, 3.6, 4.3, and 4.6 on X-axis. 55

70 CHAPTER III EFFECT OF CULTIVAR SELECTION ON SOIL POPULATION DYNAMICS OF Verticillium dahliae OVER TIME AND IMPLICATIONS ON VERTICILLIUM WILT DEVELOPMENT IN COTTON 3.1 ABSTRACT Verticillium wilt is an economically important disease of cotton worldwide. A microplot study was conducted over the 2008 to 2010 growing seasons to investigate the influence of planting combinations of susceptible and/or partially resistant cotton cultivars on soil population density of Verticillium dahliae. The hypothesis tested was that the choice of cultivar will affect soil inoculum density of V. dahliae and disease incidence over time. Stoneville (ST) 4554B2RF was used throughout the test as a susceptible cultivar and either AFD 5065B2F or an advanced breeding line was used as the partially resistant cultivar. Microplots were augmented with field soil naturally infested with V. dahliae. Stoneville 4554B2RF when planted in three sequential seasons increased V. dahliae populations in soil from 1.3 to 11.1 microsclerotia (ms)/cm 3 ; however, V. dahliae populations in microplots planted to the partially resistant cultivars over three seasons increased from 1.4 to 3.0 ms/cm 3. Disease incidence increased from 8% to 58% over 3 yrs for ST 4554B2RF and from 0% to 5% for AFD 5065B2F or advanced breeding line over the same period. Yield was highest after 3 yrs of AFD 5065B2F or a breeding line and lowest after 3 yrs of ST 4554B2RF. Yield was related primarily by the current year cultivar, pre-plant V. dahliae densities, and disease 56

71 incidence. Adoption of a resistant cultivar for at least 2 years was necessary to maintain a low ms density in soil. Results from this study indicate that cultivar selection can impact ms density and incidence of wilt in cotton and should be considered when developing management strategies. Additional keywords: Gossypium hirsutum L., inoculum density. 57

72 3.2 INTRODUCTION Verticillium wilt, caused by a soilborne fungus Verticillium dahliae Kleb., is a concern in most countries where cotton (Gossypium hirsutum L.) is cultivated. Yield losses in cotton due to Verticillium wilt are as high of 1.5 million bales worldwide (Bell, 2001). The fungus has a broad host range of more than 400 plant species (McCain et al., 1981), though within a vegetative compatibility group (VCG), the host range is more limited (Daayf et al., 1995). Isolates from cotton were found in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B (Collado-Romero et al., 2006). Carpenter (1914) first identified V. dahliae on cotton in Virginia. The disease was then observed in Tennessee and extensively in Mississippi, Arizona, and California. Verticillium wilt has been found in all cotton growing areas of the United States (Pegg and Brady, 2002). Verticillium wilt has been confirmed all over the world where cotton is grown, with the disease being more important in temperate than in subtropical or tropical regions (Pegg, 1984). Integrated management systems can effectively minimize losses due to Verticillium wilt. The most effective management option is achieved by growing adapted partially resistant cultivars and using cultural management practices known to reduce disease severity (El-Zik, 1985). The benefits derived from different management options depend largely on the pathotype of the causal agent and the amount of inoculum in the soil. Disease development and symptom expression are influenced profoundly by environmental factors, including temperature, soil moisture, soil nutrients, and cultivar selection (Bhat and Subbarao, 1999; El-Zik, 1985; Markakis et al., 2010). The disease 58

73 was responsible for significant losses throughout the 1970 s and 1980 s in cotton producing states of the United States (Halloin, 1983). The widespread, long term use of partially resistant cultivars is believed to have disrupted soil populations of V. dahliae; however, the introduction and rapid adoption of picker type cultivars resulted in a resurgence of the disease (Woodward, personal communication). To date, Verticillium wilt has become an economically important disease in Texas causing loss of $20.5 million each year (Blasingame, et al., 2008). Substantial yield losses and reductions in fiber quality may result from severe infections (Pegg, 1984). Such conditions, can lead to reductions in net returns to growers. Symptoms of Verticillium wilt are typified by yellowing, interveinal chlorosis, and necrosis of leaves starting from the base of the plant and progressing towards the top (Gutierrez et al., 1983; Misaghi et al., 1978). Later, plants may become stunted, and young bolls may abscise or become malformed. Ramification of fungus in the xylem vessels leads to a tan to brown discoloration of the vascular system; plants may eventually wilt and die (Schnathorst, 1981). Symptoms of disease in cotton plants infected by V. dahliae are variable and often influenced by strains of the pathogen. Among the strains that infect cotton two major groups, those that defoliate plants and those that do not defoliate have been recognized in pathogenicity tests (Tzeng and DeVay, 1985). Strains that cause the most defoliation do so by inducing high amounts of ethylene in infected plants (Wiese and DeVay, 1970). The pathogen is capable of infecting plant roots throughout the growing season (Pullman and DeVay, 1981). Microsclerotia (ms), the survival structures of V. dahliae are produced once the plant dies. Microsclerotia are composed of masses of melanized 59

74 hyphae and are considered the principal source of inoculum for development of Verticillium wilt. Microsclerotia can survive for more than 20 years in the soil (Wilhelm, 1955), and root exudates stimulate germination of ms initiating infections (Mol, 1995; Schnathorst, 1981). V. dahliae primarily colonizes the rhizoplane of the host plant (Huisman, 1988) early in the season. Hyphae penetrate deep into the root cortex, and enter the xylem vessels by direct penetration or through wounds. Microsclerotia are formed depending on temperature and moisture availability with the decay of plant tissues. The fungus is favored by cool, wet conditions for disease development, and temperature range of C is best suited for its growth (Rowe et al., 1987). Being a monocyclic disease, the inoculum density of the pathogen in the field soils at planting plays a critical role in the epidemiology of Verticillum wilt (Paplomatas et al., 1992). According to Grogan et al. (1979), the incidence of Verticillium wilt in herbaceous hosts is proportional to the pathogen inoculum density expressed as the number of viable ms per cm 3 of soil. However, considerable variation occurs, with such a relationship depending upon crops and cultivars (Grogan et al., 1979). Inoculum density (ms) of V. dahliae decreased throughout the growing season, but increased at harvest time with the release of ms into soil from damaged tissues of infected cotton plants (Evans et al., 1967). Understanding the relationship between inoculum density in soil at planting and wilt development is essential for developing a disease risk assessment based on pre-plant soil assays (Francl et al., 1987; Harris and Yang, 1996). Inoculum density can also be an important factor in determining the timing, nature, and duration of the management practices (Powelson and Rowe, 1993). The objective of this study was to determine the 60

75 effect of cotton cultivar selection on soil populations of V. dahliae over time and its implications for Verticillium wilt development. 3.3 MATERIALS AND METHODS Microplot Experiment A microplot study was conducted over the 2008 to 2010 growing seasons at the Texas Tech University, Quaker Research Farm located in Lubbock, Texas. Microplots were constructed out of cylindrical galvanized aluminum rings (90 cm diameter and 60 cm height), and buried at the depth of 50 cm. In the year 2008 the microplots were augmented with soil naturally infested with V. dahliae before planting. There were six treatments consisting of cultivar rotation schemes over three years utilizing all possible combinations of a susceptible cultivar (Stoneville (ST) 4554B2RF), and/or a partially resistant cultivar (Associated Farming Delinting (AFD) 5065B2F (2008) or an advanced breeding line (2009 and 2010)). The partially resistant advanced breeding line had been verified in both Verticillium wilt field nurseries and in inoculated greenhouse tests (Wheeler, unpublished data). In 2009, the resistant cultivar AFD 5065B2F was replaced with a breeding line, as AFD 5065B2F was no longer available commercially. Cultivars were selected based on the results of cotton cultivar performance trials conducted in 2006 and 2007 (Wheeler, 2007; Wheeler and Woodward, 2008). Microplots were planted at the rate of 25 seeds per microplot in a circular pattern on May of 2008, 2009 and 2010 resulting in planting density of 200,000/ha. Microplots were irrigated using drip irrigation system and fertilized with urea ammonium nitrate as needed (32-0-0, N-P-K). 61

76 Weeds were controlled throughout the study using both pre- and post-emergence herbicides according to local extension recommendations and hand hoeing Soil Sampling and Data Collection In April 2008, microplots were sampled to determine baseline inoculum densities of V. dahliae. Subsequently, soil samples were taken in February, August, and December 2009 and April, August, and December 2010 to enumerate the inoculum density (ms/cm 3 soil) over time. A 2.5-cm diam. auger to a depth of 20-cm was used for taking soil samples from each microplot. Each sample consisted of four cores and had a total weight of approximately 250-g of air dry soil. Soil cores were mixed together and air dried at room temperature for 14 days. Air-dried soil was grounded with roller pin and a 20 cm 3 soil sample was combined with 80 ml of de-ionized water, and then stirred well using a magnetic stir plate. A 1-ml aliquot of the soil solution was distributed on each Petri dishes (10 replications) containing Sorensen s NP-10 semi-selective medium. Enumeration of inoculum density was done using dilution plating technique (Isaac and Harrison, 1971; Nicot and Rouse, 1987) utilizing Sorenson s NP-10 semi selective medium (Sorensen et al., 1991), amended with N NaOH as suggested by Kabir et al. (2004). After 14 days of incubation at room temperature and dark conditions, the soil was rinsed from the Petri dishes, and then air dried for about 2 hours prior to counting. The numbers of V. dahliae colonies were counted under a dissecting microscope and expressed as the number of ms/cm 3 of dry soil. Colonies of ms were identified based on their distinctive starburst shaped colony morphology and black pigmentation. Microsclerotia densities were recorded for three years. Stand counts were taken in mid-june and disease incidence was 62

77 assessed in late September as percent symptomatic plants in each microplot for each season and disease incidence was expressed as (No. of wilted plants in a microplot/stand count in that microplot) 100. Plant height (cm) in each microplot was measured before harvesting on 22 nd December Cotton was hand harvested and weighed to determine lint yields for each plot. Lint samples were sent to Texas Tech University, Fiber and Biopolymer Research Institute (FBRI) for fiber quality analysis using High Volume Instrument (HVI) Statistical Analysis Six treatments were arranged in a randomized complete block design with seven replications. Percent germination, disease incidence, inoculum density, plant height, and lint yield data were analyzed using Proc MIXED (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). Data were analyzed as a split-plot in time where the sub-plots were seven sampling dates as described in Steel and Torrie (1960). Linear model was fitted for V. dahliae inoculum densities in soil (ms/cm 3 ) over time (month value). Linear change was on month basis with replication and treatment as random effects. Center of the month values (0, 10, 16, 20, 24, 28 and 32) was determined as described by Draper and Smith (1981) and intercepts came out at center of data. Error was natural error of the experiment. Regression analysis for pre-plant soil inoculum density of V. dahliae (ms/cm 3 ), and disease incidence (%) was done on yield in 2010 using Proc MIXED with replication as the random effect. The method used to adjust the degrees of freedom (df) to match adjustments in the sums of square was the Satterthwaite option in the LSMEANS statement in Proc MIXED (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). Standard error and LSD were determined from the PDIFF option. Slopes for Stoneville 4554B2RF 63

78 and AFD 5065B2F/advanced breeding line were tested by regressing pre-plant soil inoculum density of V. dahliae (ms/cm 3 ) on disease incidence (%) over the three year period using Proc Reg (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). 3.4 RESULTS At the beginning of the experiment, there were 1.3 ± ms/cm 3 of soil in microplots, and V. dahliae soil densities (ms/cm 3 ) were similar across all rotations. Stoneville 4554B2RF planted for three sequential years increased ms densities in soil significantly from 1.3 to 11.1 ms/cm 3 soil; whereas, ms densities changed from 1.4 to 3.0 ms/cm 3 soil only with planting a partially resistant cultivar over the same period (Table 3.1). Soil ms densities in the microplots initially planted with a partially resistant cultivar followed by a susceptible cultivar for next two years (RSS) were not different (11.1 ms/cm 3 ) from those planted to ST 4554B2RF for three years (SSS) (11.1 ms/cm 3 ) (Table 3.1). Microplots initially planted with a susceptible cultivar followed by a resistant cultivar for the next two years (SRR) had similar ms densities (3.0 ms/cm 3 ) from those planted to AFD/Advance breeding line for three years (RRR) (3.0 ms/cm 3 ) (Table 3.1). The dynamics of V. dahliae population densities over the three years could be fitted with a linear model for the cultivar rotations (Fig. 3.1). In general, the soil inoculum densities increased over time when there were at least two years of a susceptible cultivar, and the ms densities were much lower when there were at least two years of a partially resistant cultivar (Fig. 3.1). A linear model was significant (p < 0.01) for cultivar rotation RRS, RSS, SSS, and SSR with positive slopes (Table 3.2). Microsclerotia densities over time for the RRR and SRR rotations were only slightly increased and linear model for both 64

79 rotations were not significant at p = 0.05 level (Table 3.2 & Fig. 3.1). Microsclerotia densities over time with the highest increases occurred with the RSS and SSS rotations. The models describing the dynamics of ms for these rotations were best fitted with linear models, and their slopes were not significantly different from each other, but were significantly higher than the slopes associated with the RRR and SRR models (Table 3.2). Microsclerotia densities for RRS were relatively low for the first two years and then increased dramatically in 2010 with the planting of a susceptible cultivar (Fig. 3.1). Microsclerotia densities for SSR increased rapidly for 2008 and 2009, but slowed in 2010 when a resistant cultivar was planted (Fig. 3.1). A quadratic model was significant only for RRS and had a concave shape due to the rotation of cultivars (Fig. 3.1). Both RRS and SSR had intermediate slopes (Table 3.2). Germination was similar (98%) between ST 4554B2RF and AFD 5065B2F in In 2009, ST 4554B2RF had slightly lower germination than the advanced breeding line. In 2010, germination was similar between ST 4554B2RF and the advanced breeding line (Data not shown) In 2008, incidence of wilt was higher for microplots planted with ST 4554B2RF (10%) than for microplots planted with AFD 5065B2F (0%) (Table 3.3). In 2009, when a susceptible cultivar was planted for two consecutive years (SS), the disease incidence averaged 32%. When partially resistant cultivars were planted for two consecutive years in 2009 (RR), then disease incidence averaged 2% (Table 3.3). When cultivars were rotated so that the partially resistant cultivar was planted in 2008 and the susceptible in 2009 (RS), then incidence of wilt was as high as two consecutive years of susceptible cultivars (31%) (Table 3.3). However, when a susceptible cultivar was planted in 2008, 65

80 followed by a partially resistant cultivar in 2009 (SR), the disease incidence was intermediate (10%) (Table 3.3). In 2010, disease incidence was lowest for the RRR rotation scheme (4.9%) as compared the SSS rotation scheme (58.0%) (Table 3.3). Overall, it was observed that in the rotations planted with a resistant cultivar (AFD/advanced breeding line) for at least 2 consecutive years, there was significantly lower disease incidence as compared to the rotations planted with a susceptible cultivar for two or more years (Table 3.3). An increase in disease incidence was found to be negligible (0% to 5% only) for the treatment RRR; whereas, the percent disease incidence was found to have significantly increased from 8% to 59% with the treatment SSS for the same period and other treatments had intermediate effects (Table 3.3). Two years of a susceptible cultivar followed by a partially resistant cultivar (SSR) resulted in more wilt in 2010, than two years of a partially resistant cultivar followed by a susceptible cultivar (RRS) (Table 3.3). So, planting a partially resistant cultivar could have more disease than a susceptible cultivar, if higher ms had occurred because of previous susceptible rotations. Slopes obtained by regressing pre-plant V. dahliae inoculums densities in soil with disease incidence in three years were significantly different (p = ) for AFD 5065B2F/advanced breeding line (3.07) and ST 4554B2RF (6.2) (Table 3.4). The different slope values indicate that disease incidence would increase faster with per unit V. dahliae inoculum for ST 4554B2RF than for AFD 5065B2F/advanced breeding line (Table 3.4). Significant differences were observed in plant height with partially resistant and susceptible cultivar rotations in 2010 (Table 3.5). When a partially resistant cultivar was grown for three consecutive years then plants were taller (45%) than any other rotation 66

81 combinations (Table 3.5). When a susceptible cultivar was grown for three consecutive years or for two of the three years, then plants were shorter (27%) than any other rotation combinations (Table 3.5). In 2010, rotation schemes planted to a partially resistant cultivar had tallest plants (69.7 cm) with RRR rotation followed by SRR (57.5 cm) and SSR (45.6 cm); whereas, rotation schemes planted to a susceptible cultivar had shortest plants (42.4 cm) with rotation SSS followed by RSS (43.1 cm) and RRS (51.3 cm) (Table 3.5). Lint yield was significantly different among rotations in 2010 (Table 3.6). In 2010, lint yield for the rotation schemes planted to a partially resistant cultivar was highest with rotaion RRR (3454 kg/ha) followed by SRR (3031 kg/ha) and SSR (1962 kg/ha); whereas, rotation schemes planted to a susceptible cultivar had lowest lint with rotation SSS (932 kg/ha) followed by RSS (1206 kg/ha) and RRS (1408 kg/ha) (Table 3.6) suggesting, that lint yield was affected by the current year cultivar and cultivar history. In 2010, for the advanced breeding line, pre-plant V. dahliae densities with rotation RRR and SRR were similar (averaged 2.1 ms/cm 3 ); whereas, lint yield was significantly higher for rotation RRR (3454 kg/ha) than for rotation SRR (3031 kg/ha) (Fig. 3.2.A) indicating, the importance of cultivar history for lint yield as the rotation SRR had a susceptible cultivar in the year Pre-plant V. dahliae densities were significantly higher (averaged 9.0 ms/cm 3 ) and corresponding lint yield was low (1962 kg/ha) (Fig. 3.2.A) for SSR which implies that yield in 2010 was affected by pre-plant ms densities. For cultivar ST 4554B2RF pre-plant V. dahliae densities with rotation SSS were high (averaged 8.9 ms/cm 3 ) with low lint yield (932 kg/ha) and rotation RSS also 67

82 had high soil density (averaged 8.8 ms/cm 3 ) with low lint yield (1206 kg/ha); whereas, with rotation RRS inoculum density was low (averaged 2.1 ms/cm 3 ) with 1408 kg/ha lint yield (Fig 3.2.B) suggesting, that the lint yield was affected by pre-plant V. dahliae density in soil. Additionally, the lint yield was inversely related to mid season V. dahliae density in soil. Rotation RRR had lowest soil inoculum densities (averaged 2.7 ms/cm 3 ) with highest lint yield (3454 kg/ha) (Fig. 3.3.C) and rotation SSS had highest soil inoculum density (averaged 10 ms/cm 3 ) with lowest lint yield (932 kg/ha) (Fig 3.2.D) indicating, that V. dahliae densities in soil play an important role in determining lint yield. Rotation SRR had lower yield than RRR that may be due to V. dahliae densities in soil and/or cultivar history (SRR rotation had a susceptible cultivar in 2008) (Fig. 3.2.C). Resistant rotations (RRR, SRR, SSR) resulted in significantly higher lint yield in 2010 (Fig. 3.2.A & C) than susceptible rotations (SSS, RSS, RRS) (Fig. 3.2.B & D) suggesting, that lint yield was primarily affected by the cultivar of the current season. The lint yield with rotation SRR was significantly higher than rotation SSR (Fig. 3.2.A & C) and yield for RSS was significantly lower than RRS (Fig. 3.2.B & D) which implies that cultivar history is also an important factor in determining the lint yield. There was negative relationship between pre-plant V. dahliae density in soil (ms/cm 3 ) and lint yield in 2010 for both the cultivars (Table 3.7). With the advanced breeding line the slope for pre-plant ms density and lint yield was ; whereas, the slope was for the ST 4554B2F (Table 3.7). At low ms density (1.9 ms/cm 3 ), yield for the advanced breeding line was 3279 kg/ha; whereas, yield was 1908 kg/ha with high ms density in soil (9.3 ms/cm 3 ) (Table 3.7). With ST 4554B2F, at low ms density (1.8 68

83 ms/cm 3 ) yield averaged 1425 kg/ha; whereas, at high V. dahliae density (9.5 ms/cm 3 ) yield was averaged 1035 kg/ha (Table 3.7). A negative relationship was also found between disease incidence taken during the same growing season and lint yield for both the cultivars (Fig. 3.3). Slope for disease incidence and lint yield was for ST 4554B2F and it was for the advanced breeding line (Fig. 3.3). Disease incidence and yield could be explained through cultivar choice and preplant V. dahliae density in soil. Fiber properties such as micronaire, uniformity, elongation, degree of reflectance, and yellowness in general were affected by cultivar more than by the rotation history, with the exception of fiber length. Fibers were longer when the partially resistant cultivar was grown in microplots that had the least amount of wilt (RRR rotation) than for a susceptible cultivar grown in microplots where wilt was the highest (SSS rotation) (data not shown). 3.5 DISCUSSION There is a correlation between inoculum density at planting and disease incidence at the end of the cropping season (Paplomatas et al., 1992). In Acala cotton fields of California, the number of plants infected by V. dahliae at the end of the crop season was found to be directly related to the density of ms in soil (Ashworth et al., 1979; Pullman and DeVay, 1981). In cotton, the role of the inoculum density on vascular infections or development of foliar symptoms changes with the cultivar tolerance and pathotypes of V. dahliae (Bejarano-Alcázar et al., 1995; DeVay et al., 1974). Only a single cycle of inoculum is produced during a growing season because with the decay of tissue the ms 69

84 again disperse in the soil (Paplomatas et al., 1992). Microsclerotia populations in the soil can further contribute to soil inoculum for the next season (Davis et al., 1983). Management options are limited for Verticillium wilt of cotton, due to longevity of ms (Wilhelm, 1955) and its broad host range (Evans and Gleeson, 1973.). In other systems, reducing ms is usually accomplished with a combination of chemical (Wilhelm, 1955) and cultural methods (Davis et al., 1996; Grogan et al., 1979), such as use of resistant cultivars and crop rotations (Khan et al., 2000; Rowe and Powelson, 2002). However, it is not economically feasible for the cotton growers to rotate for many years with other crops (non-hosts to V. dahliae) in this region. There are limited economically viable chemical options available due to lack of efficacy at economical rates (Woodward et al., 2011). Management tactics to prevent Verticillium wilt should be aimed at preventing the increase of initial inoculum. The choice of cultivar is probably the single most important decision growers can make in an integrated crop management system. The cultivar sets the framework for the level of susceptibility to disease, the tactics applied to manage the crop, and production costs. Differences in the resistance of cotton cultivars to Verticillium wilt have been identified (Wheeler, 2007). Thus, cultivar selection seems to be the cornerstone to manage the V. dahliae inoculum density in the soil. Planting susceptible cotton cultivars is believed to increase inoculum of V. dahliae in soil. The use of partially resistant cotton cultivars appears to be a promising management option. The importance of primary inoculum in Verticillium wilt development, justifies the use of partially resistant cotton cultivars for effective disease management. 70

85 In the present microplot study, planting a partially resistant cultivar for three consecutive years did not increase ms density significantly; whereas, planting a susceptible cultivar significantly increased ms density (1.3 to 11.1 ms/cm 3 ). These results suggest that if susceptible cotton cultivar is planted in soil infested with V. dahliae, then ms density and wilt incidence will increase and yield losses will subsequently increase. Vallad and Subbarao (2008) found a 159-fold difference in inoculum density of V. dahliae per g root tissue between resistant and susceptible cultivars of lettuce. Huisman and Ashworth (1976) and Joaquim et al. (1988) found a sharp increase in ms population in the second year after growth of a susceptible crop, despite host susceptibility. While there were distinct differences in the rotation schemes when there were at least two consecutive years of resistant or susceptible cultivars, there were also more subtle differences due to the cultivar grown in the last year of the rotation study. This suggests, that any gains made by two years of growing resistant cultivars, would probably be lost using susceptible cultivars in subsequent years. There was negative relationship between pre-plant V. dahliae density and lint yield for both the cultivars. Though the loss in yield per unit ms was greater for the partially resistant cultivar than the susceptible cultivar, the more important factor was the greater sensitivity to losses at low ms densities found with ST 4554B2RF relative to the partially resistant cultivar. A negative relationship was also found between disease incidence taken during the same growing season and lint yield for both the cultivars. Again as noted above, the important factor is the low yields associated with ST 4554B2RF even at low disease incidence of Verticillium wilt. The yield loss was close to maximum even at < 10% 71

86 incidence wilt. That suggests that plants of ST 4554B2RF not exhibiting symptoms of wilt were still heavily damaged by the pathogen. Lint yield was better when the 2010 cultivar was partially resistant, than when it was susceptible, regardless of rotation of the previous years. Given that the advanced breeding line was not considered good enough to release as a cultivar, and that ST 4554B2RF is a high yielding commercial cultivar under non Verticillium wilt conditions, these yield differences emphasize how much Verticillium wilt reduces yield. With respect to the plots with a partially resistant cultivar in 2010, yield was reduced as the previous years under a susceptible cultivar was increased (3454, 3031, and 1962 kg lint/ha, for 3, 2, and 1 year of a partially resistant cultivar. A similar trend was seen with ST 4554B2RF planted in 2010, except the differences were less consistent (1408, 1206, and 932 kg/ha for 1, 2, and 3 years of a susceptible cultivar). Results suggest that lint yield was affected by cultivar of the current season, pre-plant soil inoculum densities of V. dahliae, and disease incidence. This study demonstrates the importance of planting partially resistant cultivars for managing Verticillium wilt in cotton. If V. dahliae densities are low in field resistant cultivar can help keep inoculum levels flat; but, if inoculum levels in soil are already high then it may take several years to bring inoculum density down. Adoption of a resistant cultivar for at least 2 years was required to negatively impact V. dahliae inoculum density in soil, wilt incidence, plant stunting, and low lint yield. 72

87 3.6 LITERATURE CITED Ashworth, L. J., Jr., Huisman, O. C., Harper, D. M., Stromberg, L. K., and Bassett, D. M Verticillium wilt disease of cotton: Influence of inoculum density in the field. Phytopathology 69: Bejarano-Alcázar, J., Melero-Vara, J. M., Blanco-López, M. A., Jiménez-Díaz, J Influence of inoculum density of defoliating and nondefoliating pathotypes of Verticillium dahliae on epidemics of Verticillium wilt of cotton in southern Spain. Phytopathology 85: Bell, A.A Verticillium Wilt. Compendium of Cotton Diseases 2 nd Edition. T.L. Kirkpatrick and C.S. Rothrock. eds. APS Press, pp Bhat, R. G., and Subbarao, K. V Host range specificity in Verticillium dahliae. Phytopathology 89: Blasingame, D., Banks, J. C., Colyer, P. D., Davis, R. M., Gazaway, W. S., Goldburg, N., Kemerait, R. C., Kirkpatrick, T. L., Koenning, S. R., Muller, J., Newman, M. A., Olsen, M., Phipps, P. M., Sciumbato, G. L., Sprenkel, R., Woodward, J. E., Wrather, A. and Patel, M. V Beltwide Cotton Conference Cotton Disease Loss Estimate Committee Report Proceedings, pp Carpenter, C. W The Verticillium wilt Problem (Abstr). Phytopathology 4:393. Collado-Romero, M., Mercado-Blanco, J., Olivares-García, C., Valverde-Corredor, A., and Jiménez-Díaz, R. M Molecular variability within and among Verticillium dahliae vegetative compatibility groups determined by fluorescent amplified fragment length polymorphism and polymerase chain reaction markers. Phytopathology 96: Daayf, F., Nicole, M., and Geiger, J. P Differentiation of Verticillium dahliae populations on the basis of vegetative compatibility and pathogenicity on cotton. Eur. J. Plant Pathol. 101: Davis, J. R. Huisman, O. C., Westerman, D. T., Hafez, S. L., Everson, D. O., Sorensen, L. H., and Schneider, A. T Effects of green manures on Verticillium wilt of potato. Phytopathology 86: Davis, J. R., Pavek, J. J., and Corsim, D. L A sensitive method for quantifying Verticillium dahliae colonization in plant tissue and evaluating resistance among potato genotypes. Phytopathology 73:

88 DeVay, J. E., Forrester, L. L., Garber, R. H., and Butterfield, E. J Characteristics and concentration of propagules of Verticillium dahliae in air-dried field soils in relation to the prevalence of Verticillium wilt in cotton. Phytopathology 64: Draper, N. R., and Smith, H Applied Regression Analysis 2 nd edition. New York: Wiley, pp El-Zik, K. M Integrated control of Verticillium wilt of cotton. Plant Dis. 12: Evans, G., and Gleeson, A. C Observations on the origin and nature of Verticillium dahliae colonizing plant roots. Aust. J. Biol. Sci. 26: Evans, G., Wilhelm, S., and Snyder, W. C Quantitative studies by plate counts of propagules of the Verticillium wilt fungus in cotton field soils. Phytopathology 57: Francl, L. J., Madden, L.V., Rowe, R. C., and Riedel, R. M Potato yield loss prediction and discrimination using pre-plant densities, of Verticillium dahliae and Pratylenchus pentrans. Phytopathology 77: Grogan. R. G., Ioannouu, D. Schneider, R. W., Sall, M.A., and Kimble, K.A Verticillium wilt on resistant tomato cultivars in California: Virulence of isolates from plants and soil and relationship of inoculum density to disease incidence. Phytopathology 69: Gutierrez, A. P., and DeVay, J. E Studies on plant-pathogen-weather interactions: Cotton and Verticillium wilt. In: Plant Disease Epidemiology: Population Dynamics and Management. Vol 1. K. J. Leonard and W. E. Fry. Eds. Macmillan Publishing Co., New York, pp Gutierrez, A. P., DeVay, J. E., Pullman, G. S., and Friebertshauser, G. E A model of Verticillium wilt in relation to cotton growth and development. Phytopathology 73: Halloin, J. M Thirty year summary of cotton disease loss estimates: Crop years In: Proc. Beltwide Cotton Prod. Res. Conf. National Cotton Council, Memphis, TN, pp Harris, D. C., and Yang, J.R The relationship between the amount of Verticillium dahliae in soil and the incidence of strawberry wilt as a basis for disease risk prediction. Plant Pathol. 45: Huisman, O. C Colonization of field-grown cotton roots by pathogenic and saprophytic soilborne fungi. Phytopathology 78: Huisman, O. C., and Ashworth, L. J Influence of crop rotation on survival of Verticillium albo-atrum in soils. Phytopathology 66: Isaac I., Fletcher P., and Harrison J.A.C., Quantitative isolation of Verticillium spp. from soil and moribund potato haulm. Ann. Appl Biol 67:

89 Joaquim, T. R., Smith, V. L., and Rowe, R. C Seasonal variation and effects of wheat rotation on populations of Verticillium dahliae Kleb. in Ohio potato field soils. Am. Potato J. 65: Kabir, Z., Bhat, R. G., and Subbarao, K. V Comparison of media for recovery of Verticillium dahliae from soil. Plant Dis. 88: Khan, N., Atibalentja, N., and Eastburn, D. M Influence of inoculum density of Verticillium dahliae on root discoloration of horseradish. Plant Dis. 84: Markakis, E. A., Tjamos, S. E. and Antoniou, P. P., Roussos, P. A., Paplomatas, E. J., and Tjamos, E. C Phenolic responses of resistant and susceptible olive cultivars induced by defoliating and non-defoliating Verticillium dahliae pathotypes. Plant Dis. 94: McCain, A. H., Raabe, R. D., and Wilhelm, S Plants Resistant or Susceptible to Verticillium Wilt. Cooperative Extension, U.S. Department of Agriculture, University of California, Berkeley, pp Misaghi, I. J., DeVay, J. E., and Duniway, J. M Relationship between occlusion of xylem elements and disease symptoms in leaves of cotton plants infected with Verticillium dahliae. Can. J. Bot. 56: Mol, L Effect of plant roots on the germination of microsclerotia of Verticillium dahliae. II. Quantitative analysis of the luring effect of crops. Eur. J. Plant Pathol. 101: Nicot, P. C., and Rouse, D. I Precision and bias of three quantitative soil assays for Verticillium dahliae. Phytopathology 77: Paplomatas, E. J., Bassett, D. M., Broome, J. C., and DeVay, J. E Incidence of Verticillium wilt and yield losses of cotton cultivars (Gossypium hirsutum) based on soil inoculum density of Verticillium dahliae. Pythopathology 82: Pegg, G. F The impact of Verticillium diseases in agriculture. Phytopathol. Mediterr. 23: Pegg, G. F., and Brady, B. L Verticillium Wilts, CAB International Publishing, Wallingford (UK), New York (USA), pp Powelson, M. L., and Rowe, R. C Biology and management of early dying of potatoes. Annu. Rev. Phytopathol. 31: Pullman, G. S., and DeVay, J. E Effect of soil flooding and paddy rice culture on the survival of Verticillium dahliae and incidence of Verticillium wilt in cotton. Phytopathology 71:

90 Pullman, G. S., and DeVay, J. E Epidemiology of Verticillium wilt of cotton: A relationship between inoculum density and disease progression. Phytopathology 72: Rowe, R. C., and Powelson, M. L Potato early dying: management challenges in a changing production environment. Plant Dis. 86: Rowe, R. C., Davis, J. R., Powelson, M. L., and Rouse, D. I Potato early dying: Causal agents and management strategies. Plant Dis. 71: Schnathorst, W. C., Life cycle and epidemiology of Verticillium. In: Fungal Wilt Diseases of Plants. M. E. Mace, A. A. Bell, and C. H. Beckman, eds. Academic Press, New York, pp Sorensen, L. H., Scheider, A. T., and Davis, J. R Influence of sodium polygalacturonate sources and improved recovery of Verticillium spp. from soil (Abstr.) Phytopathology 81:1347. Steel, R. G. D. and Torrie, J. H Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., pp Tzeng, D. D., and DeVay, J. E Physiological responses of Gossypium hirsutum L. to infection by defoliating and nondefoliating pathotypes of Verticillium dahliae Kleb. Physiol. Plant Pathol. 26: Vallad, G. E., and Subbarao, K. V Colonization of resistant and susceptible lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahliae. Phytopathology 98: Wheeler, T. A Variety performance in fields infested with Verticillium dahliae in the High Plains of Texas. In: Beltwide Cotton Conferences, New Orleans, LA Jan 9-12, pp Wheeler, T. A. and Woodward, J. E Effect of Verticillium wilt on cotton varieties in Texas. In Beltwide Cotton Conferences, Nashville, TN, 8-11 Jan CD format., pp Wiese, M. V., and DeVay, J. E Growth regulator changes in cotton associated with defoliation caused by Verticillium albo-atrum. Plant Physiol. 45: Wilhelm, S Longevity of the Verticillium wilt fungus in the laboratory and field. Phytopathology 45: Wilhelm, S., and Paulus, A. O How soil fumigation benefits California strawberry industry. Plant Dis. 64: Woodward, J. E., T. A. Wheeler, M. G. Cattaneo, S. A. Russell, and T. A. Baughman Evaluation of soil fumigants for management of Verticillium wilt of peanut in Texas. Online. Plant Health Progress doi: /php rs. 76

91 Table 3.1. Effect of cultivar rotation and time on soil inoculum densities of Verticillium dahliae a V. dahliae density in soil (ms/cm 3 ) Cultivar rotation schemes f Time (month value) RRR RRS RSS SSS SSR SRR April 2008 (0) 1.4 c b, A c 1.3 d, A 1.2 e, A 1.3 g, A 1.3 e, A 1.3 e, A LSD e = 0.3 February 2009 (10) 1.2 c, B 1.1 d, B 1.2 e, B 3.3 f, A 3.3 d, A 3.2 a, A August 2009 (16) 2.1 b, BC 2.0 c, C 6.5 d, A 6.6 d, A 6.5 c, A 2.3 c, B December 2009 (20) 1.8 b, B 1.8 c, B 6.2 d, A 6.2 e, A 6.2 c, A 1.8 d, B df = 197 April 2010 (24) 2.1 b, B 2.1 c, B 8.8 c, A 8.9 c, A 9.0 a, A 2.1 cd, B August 2010 (28) 2.7 a, D 5.7 b, C 9.9 b, A 10.0 b, A 7.7 b, B 2.8 b, D December 2010 (32) 3.0 a, D 6.4 a, C 11.1 a, A 11.1 a, A 7.5 b, B 3.0 ab, D t-value = 1.97 LSD d 0.4 df 67 t-value 2.0 a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same lower case letter in the same column are not significantly different at p 0.05 level according to Fisher's LSD. c Means followed by the same upper case letter in the same row are not significantly different at p 0.05 level. d LSD for comparing time. e LSD for comparing cultivar rotation schemes. f R = Resistant (AFD 5065B2F/advanced breeding line), S = Susceptible (Stoneville 4554B2RF). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letters refer to the cultivar grown in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in N = 7 for all cultivar rotations. 77

92 Table 3.2. Parameters from linear regression of soil inoculum density of Verticillium dahliae over time for six cultivar rotations a Cultivar rotation Inoculum density of V. dahliae in soil (ms/cm 3 ) schemes c Intercept Slope p-value RRR 2.05 b b 0.05 de p > 0.10 RRS 2.91 b 0.16 cd p < 0.01 RSS 6.42 a 0.34 a p < 0.01 SSS 6.75 a 0.32 ab p < 0.01 SSR 5.92 a 0.22 bc p < 0.01 SRR 2.36 b 0.03 e p > 0.10 SE LSD df t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same letter in the same column are not significantly different according to Fisher's LSD. Quadratic regression was only significant for RRS rotation. c R = Resistant (AFD 5065B2F/advanced breeding line), S = Susceptible (Stoneville 4554B2RF). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar grown in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in N = 49 for all cultivar rotations. 78

93 Table 3.3. Effect of cultivar rotation on percent disease incidence in 2008, 2009, and 2010 a Disease incidence (%) Cultivar rotation schemes f RRR 0.0 c b, A c 1.4 c, A 4.9 e, A LSD e = 5.4 RRS 0.0 c, B 2.4 c, B 17.8 d, A RSS 0.0 c, C 31.1 a, B 39.2 b, A df = 90 SSS 8.3 b, C 32.5 a, B 58.0 a, A SSR 10.3 ab, B 31.9 a, A 30.2 c, A SRR 11.2 a, A 10.3 b, A 9.3 e, A t-value = 1.99 LSD d df 30 t-value 2.04 a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same lower case letter in the same column are not significantly different at p 0.05 level according to Fisher's LSD. c Means followed by the same upper case letter in the same row are not significantly different at p = 0.05 level. d LSD for comparing cultivar rotations. e LSD for comparing years and it was constructed as follows: LSD = sqrt (( )/3). f R = Resistant (AFD 5065B2F/advanced breeding line), S = Susceptible (Stoneville 4554B2RF). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar grown in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in N = 7 for all cultivar rotations. 79

94 Table 3.4. Testing the slopes from regressing pre-plant soil inoculum density of Verticillium dahliae on disease incidence in 2008, 2009, and 2010 a Disease Incidence (%) AFD 5065B2F/advanced breeding line Slope 3.07 SE Stoneville 4554B2RF Slope 6.20 SE a Data were analyzed using Proc Reg (SAS, Ver. 9.2, 2008). Comparision of slopes for AFD 5065B2F/advanced breeding line and Stoneville 4554B2RF had an observed t-value of that was computed using method described by Steel and Torrie (1960) as (( )/( ) 0.5 ). The tabular t-value was at p = and 124 df. AFD 5065B2F/Advanced breeding line was partially resistant and Stoneville 4554B2RF was susceptible to Verticillium dahliae. 80

95 Table 3.5. Effect of cultivar rotation scheme on plant height in 2010 a Cultivar rotation schemes c Plant height (cm) Partially resistant cultivar RRR 69.7 a b SRR 57.5 b SSR 45.6 c Susceptible cultivar RRS 51.3 a b RSS 43.1 a SSS 42.4 a LSD 10.0 df 36 t-value 2.03 a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same letter for partially resistant cultivar and susceptible cultivar are not significantly different at p 0.05 level according to Fisher's LSD. c R = Resistant (AFD 5065B2F/Advanced breeding line), S=Susceptible (Stoneville 4554B2RF). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar grown in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in N = 7 for all cultivar rotations. 81

96 Table 3.6. Effect of cultivar rotation scheme on lint yield in 2010 a Cultivar rotation schemes c Lint yield (kg/ha) Partially resistant cultivar RRR a b SRR b SSR c Susceptible cultivar RRS a b RSS a SSS a LSD df 30 t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same letter for partially resistant cultivar and susceptible cultivar are not significantly different at p 0.05 level according to Fisher's LSD. c R = Resistant (AFD 5065B2F/Advanced breeding line), S = Susceptible (Stoneville 4554B2RF). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar grown in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in Planting density was 200,000/ha. N = 7 for all cultivar rotations. 82

97 Table 3.7. Effect of cultivar and pre-plant soil density of Verticillium dahliae on lint yield in 2010 a Inoculum level in soil Advanced breeding line Pre-plant V. dahliae density (ms/cm 3 ) Lint yield (kg/ha) Stoneville 4554B2RF Pre-plant V. dahliae density (ms/cm 3 ) Lint yield (kg/ha) Low High Average 4.4 (± 0.7) (± 154.2) 6.6 (± 0.7) (± 60.3) Slope = Slope = a Regression analysis for pre-plant soil inoculum density was done on yield using Proc MIXED (SAS 2008, Ver. 9.2) and df were determined using the Satterthwaite option. Observed t-value to compare slopes between Advanced breeding line and Stoneville was and was found using method from Steel and Torrie (1960). Tabular t-value was at p 0.05 and df = 26. Advanced breeding line was partially resistant and Stoneville 4554B2RF was susceptible to Verticillium dahliae. Planting density was 200,000/ha. 83

98 Verticillium dahliae density in soil (ms/cm 3 ) RRR RRS RSS SSS SSR SRR Time (month value) Figure 3.1. Effect of cultivar rotation on Verticillium dahliae inoculum density in soil over time. Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Linear model was significant (p < 0.01) for cultivar rotation RRS, RSS, SSS, and SSR; whereas, quadratic regression was significant only for RRS rotation. R = Partially resistant (AFD 5065B2F/Advanced breeding line), S = Susceptible (Stoneville 4554B2RF). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar grown in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in LSD 0.05 =

99 RRR SRR SSR SSS RSS RRS Lint yield (kg/ha) A. B RRR SRR SSR SSS RSS RRS Lint yield (kg/ha) C V. dahliae inoculum density (ms/cm 3 ) D V. dahliae inoculum density (ms/cm 3 ) Figure 3.2. Effect of soil inoculum density of Verticillium dahliae on lint yield in Lint yield due to pre-plant (April-2010) soil inoculum density for A. Advanced breeding line B. Stoneville 4545B2RF. Lint yield due to mid-season (August-2010) soil inoculum density for C. Advanced breeding line D. Stoneville 4545B2RF. Advanced breeding line was partially resistant and Stoneville 4554B2RF was susceptible to Verticillium dahliae. RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar grown in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in

100 4000 Advanced breeding line Stoneville 4554B2RF 3500 Lint yield (kg/ha) in y = x , R 2 = y = x , R 2 = Disease incidence (%) Figure 3.3. Effect of disease incidence on lint yield in Data were analyzed using Proc Reg (SAS Ver. 9.2, 2008). Advanced breeding line was partially resistant and Stoneville 4554B2RF was susceptible to Verticillium dahliae. Slope for the Advanced breeding line (-52.95) was significantly different from slope for Stoneville 4554B2RF (-10.44) at p = 0.05 level. Tabular t-value was 2.055; whereas observed t-value was

101 CHAPTER IV EFFECT OF INOCULUM DENSITY OF GENETICALLY DISTINCT Fusarium oxysporum f. sp. vasinfectum Race 1 ISOLATES, Meloidogyne incognita, AND CULTIVAR ON FUSARIUM WILT DEVELOPMENT IN COTTON 4.1 ABSTRACT The Fusarium wilt Root-knot nematode complex is an economically important disease of cotton. Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1 populations in Texas are genetically diverse. To examine the impact of inoculum densities of genetically distinct Fov Race 1 isolates, Meloidogyne incognita, and cultivar on Fusarium wilt development in cotton an experiment was conducted with 12 Fov isolates at four densities (0 to cfu/cm 3 soil), Meloidogyne incognita densities (0 and 1,000 eggs/pot), and two cultivars (partially resistant Stoneville (ST) 4554B2RF and susceptible Fibermax (FM) 9058F) in the greenhouse. The hypothesis was that the isolates of Fov collected from Texas will demonstrate variability in aggressiveness. FM 9058F had a significantly (p = ) higher area under the disease progress curve (AUDPC) than ST 4554B2RF for all the Fov isolates tested with the four-way interaction of Fov Race 1 isolates, their inoculum densities, M. incognita densities, and cotton cultivars. Fusarium oxysporum f. sp. vasinfectum isolates 3, 4, 5, 6, 7, 10, 11, and 12 exhibited significantly (p 0.05) higher AUDPC than isolates 1, 2, 8, and 9 suggesting, that variability in aggressiveness exist among Fov isolates. Isolates 3, 4, 6, and 7 with 87

102 cultivar FM 9058F showed significantly higher AUDPC in the absence of M. incognita at higher inoculum densities of Fov ( cfu/cm 3 ) compared to other Fov isolates tested which implies that disease incidence may be higher even in the absence of M. incognita when inoculum densities of Fov in soil are high. Fusarium oxysporum f. sp. vasinfectum isolates 5 and 11 of Fov had significantly (p 0.05) higher AUDPC in the presence of M. incognita (1000 eggs/pot) at lower inoculum density ( cfu/cm 3 ) than other Fov isolates tested suggesting, the root-knot nematodes were required to cause significant wilt incidence when inoculum densities of Fov were low in soil. Plant growth and symptom expressions were affected by the susceptibility of cultivar. Susceptible cultivar, FM 9058F had significantly stunted plants, decreased root weight, shoot weight and total plant weight as compare to partially resistant cultivar, ST 4554B2RF. Plants inoculated with M. incognita (1000 eggs/pot) had root galls and were stunted with reduced shoot weight and total plant weight. Additional keywords: Gossypium hirsutum L. 88

103 4.2 INTRODUCTION The Fusarium wilt Root-knot nematode complex is an economically important disease of cotton (Gossypium hirsutum L.) in most cotton-growing regions of the world (Colyer et al., 1997). The disease complex is caused by a soilborne fungus, Fusarium oxysporum Schlechtend.:Fr. f. sp. vasinfectum (Atk.) W. C. Snyder and H. N. Hans (Fov), and the root-knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood (DeVay et al., 1997; Martin et al., 1956). The disease is responsible for losses of $20 million each year across the cotton belt of the United States of America (Blasingame et al. 2008). Since the first report of Fusarium wilt of cotton, in Alabama (Atkinson, 1892), the disease has increased in importance (Smith et al., 1981). Under conducive environmental conditions, high losses occur when susceptible cultivars are grown on heavily infested soil. Losses are greatest on sandy soils that are infested with M. incognita (DeVay et al. 1997; Koenning et al., 2004; Martin et al., 1956). Losses due to Fusarium wilt of cotton vary depending upon the virulence of Fov, host resistance, environmental factors, soil type and fertility, and interactions with nematodes (Hao et al., 2009; Smith and Snyder, 1975). Symptoms of Fusarium wilt appear earlier when densities of both M. incognita and Fov are increased (Garber et al., 1979). There are eight Races of Fov that have been described throughout the world, with Race 1 and Race 2 historically being most prevalent in the United States (Kim et al. 2005; Skovgaard et al., 2001). Recent studies have found that Races 1, 3, and 8 are mildly virulent and cause wilt symptoms in the presence of M. incognita; however, Race 89

104 4 of Fov, which was identified in California (Kim et al., 2005) is capable of causing severe wilt symptoms and economic loss in the absence of nematodes. Fusarium oxysporum f. sp. vasinfectum density in soil and wilt incidence are correlated (DeVay et al., 1997; Starr et al., 1989). It is important to understand the effect of Fov inoculum density on incidence of wilt for cultivars that differ in their susceptibility to the disease. It is also important to determine how isolates of Fov may differ in aggressiveness to different genotypes of cotton, or more importantly, are some genotypes relatively resistant to a collection of isolates, or is there a cultivar isolate interaction. Initial symptoms of Fusarium wilt include chlorosis and necrosis of the leaf margins. Severely diseased plants often remain stunted. Fov invades the host through the taproots behind the root tip. The combined effect of fungal metabolites and the production of lipoidal substances by the host in response to infection may lead to the occlusion of the vascular tissues (Shi et al., 1992). The vascular system of plants exhibits discoloration due to systemic infection of the fungus. In most severely affected plants, leaves wilt and drop and the plants may die (Colyer, 2001; Nelson, 1981). Plants that develop symptoms early usually die before producing any bolls, whereas plants that develop symptoms after the onset of flowering often survive but produce fewer bolls. Chlamydospores are thick walled specialized resting structures that remain dormant in the soil until exudates or leachates from plant roots stimulate their germination (Mai and Abawi, 1987). The germinated chlamydospores produce hyphae that eventually form conidia and new chlamydospores if a suitable host is not found. Once a field is infested with Fov, the fungus usually persists indefinitely in the decaying plant tissues and soil as chlamydospores (Nelson, 1981; Smith and Snyder, 1975). The 90

105 fungus is capable of surviving for over 10 years in the soil not planted to cotton (Smith et al., 2001). The ability of the pathogen to survive in soils for long periods has important consequences on disease management. Management options available for Fusarium wilt are typically aimed at control of M. incognita, rather than Fov. These options include rotations with non-host crops for M. incognita, nematicides, and planting of nematode resistant cultivars (Abawi and Baker, 1984; DeVay, 1986). Use of rotations to reduce Fov has been of limited value because of its saprophytic ability (Smith and Snyder, 1975). There are few economically viable crops for use by cotton producers in a rotation program due to the broad host range of M. incognita. Control of nematodes with nematicides has caused considerable decrease in Fusarium wilt and increase in yield (Hyer et al., 1979; Jorgenson, 1979; Smith, 1948). Field observations indicate that use of a partially nematode resistant cultivar (Stoneville 5599BR) over a period of three years led to a substantial decrease in disease incidence (Wheeler, personal communication). A recent survey found that extensive genetic diversity exists in Fov populations in West Texas; however, all isolates collected to date were found to be characterized as Race 1 (Woodward, unpublished). The purpose of this study was to characterize the effects of genetically distinct isolates of Fov Race 1, their interaction with M. incognita, and cotton cultivars with varying levels of resistance to Fov on Fusarium wilt development. 91

106 4.3 MATERIALS AND METHODS Greenhouse Experiment An experiment was conducted in the greenhouse during spring and fall of The experiment was designed as a split-split-split plot with four replications. Twelve genetically distinct isolates of Fov Race 1 served as the main plot, Fov inoculum densities (0, , , colony forming units (cfu)/cm 3 of potting mix) served as sub-plots, two cultivars (Stoneville 4554B2RF (partially resistant), and FiberMax 9058F (susceptible)) served as sub-sub-plots and root-knot nematode densities (0 and 1000 eggs/pot) served as sub-sub-subplots. Plastic containers (Stuewe & Sons, Tangent, OR) (30 cm height and 6 cm diameter at top) were filled with 70 % sand, 25% top soil, and 5% peat moss Fov inoculum was prepared from 3 week old cultures (Figure 4.1) on Komada s Selective Medium (Komada, 1975) and maintained at room temperature under continuous light. Petri dishes were flooded with water and conidia were scraped off the culture with a rubber spatula. The conidial suspension was then filtered through four layers of cheesecloth, quantified with the aid of a hemacytometer, and diluted with water to make the desired density for each Fov isolate. Fov inoculum was delivered using pipette inoculation technique (Latin and Snell, 1986) into a potting mixture. Root-knot nematodes were reared in a greenhouse on a susceptible tomato cultivar Homestead and inoculum was extracted according to methods described by Hussey and Barker (1973). Soil was inoculated with M. incognita eggs (1,000/pot) to ensure root-knot nematode infestation and cotton (three seeds per cone) was planted. Plant densities were thinned to two plants per cone after three weeks. 92

107 4.3.2 Data Collection and Statistical Analysis Plant height (cm) and disease incidence (%) was measured four weeks after planting. Disease incidence was observed every five days after appearance of first disease symptom. Percent disease incidence was rated on the following scale: 0 % - no symptoms, 50 % - chlorosis, necrosis, and wilting of 1 plant, 100 % - chlorosis, necrosis, and wilting of both plants. After 12 weeks of planting, plant growth was scored by measuring height, fresh root weight, fresh shoot weight, and total fresh plant weight before termination of the experiment in each season. Root galling was also noted at this time to confirm the presence or absence M. incognita. Vascular discoloration was examined from cross and longitudinal section of the stem at the soil line and then positive samples were placed on to Petri dishes containing potato dextrose agar media to confirm Fov presence. Area under the disease progress curve (AUDPC) was calculated for quantitative disease assessment using repeated disease incidence as described by Shaner and Finey (1977). Plant height (cm) at the 5th week, plant height (cm) at the 13 th week, AUDPC, root weight (g), shoot weight (g), and total plant weight (g) were analyzed using Proc MIXED (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). The method used to adjust the degrees of freedom (df) to match adjustments in the sums of square was the Satterthwaite option in the LSMEANS statement in Proc MIXED (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). Standard error and LSD were determined from the PDIFF option. 93

108 4.4 RESULTS AND DISCUSSION Interaction of Fov Race 1 isolates, their inoculum densities, M. incognita densities and cotton cultivars was significant (p = ) for AUDPC (Table 4.1). FM 9058F had significantly (p = 0.05) higher AUDPC than ST 4554B2RF (Fig 4.2) indicating the importance of planting a resistant cultivar. Fov isolates 3, 4, 5, 6, 7, 10, 11, and 12 had significantly (p 0.05) higher AUDPC with FM 9058F than isolates 1, 2, 8, and 9 (Fig. 4.2) suggesting, that variability in aggressiveness occur among Fov isolates. Isolates 3, 4, 6, and 7 showed significantly higher AUDPC in the absence of M. incognita at higher inoculum densities of Fov ( cfu/cm 3 ) with cultivar FM 9058F compared to other Fov isolates tested (Fig. 4.2) which implies that disease incidence may be higher even in the absence of M. incognita when Fov inoculum density in soil is high. Isolates 5 and 11 of Fov Race 1 had significantly (p 0.05) higher AUDPC in the presence of M. incognita (1000 eggs/pot) at lower inoculum density ( cfu/cm 3 ) compared to other Fov isolates tested (Fig. 4.2) indicating that root-knot nematodes were required to cause significant wilt incidence when inoculum densities of Fov were low in soil. Isolates 4, 7, 10, and 11 showed significantly higher AUDPC than rest of Fov isolates tested in the presence of M. incognita (1000 eggs/pot) at Fov inoculum density of cfu/cm 3. Isolates 2, 4, 6, 7 and 10 resulted in higher AUDPC at highest Fov inoculum density used for the study ( cfu/cm 3 ) in the presence of M. incognita (1000 eggs/pot) compared to other Fov isolates tested (Fig. 4.2). Interaction between inoculum densities of Fov, M. incognita, and cultivar was significant (p = ) for shoot weight (g) (Table 4.1). Partially resistant cultivar, ST 4554B2RF had higher shoot weight than susceptible cultivar, FM 9058F with or without 94

109 root-knot nematode at all four Fov inoculum densities tested (Table 4.2). AUDPC was significantly higher (p 0.05) for FM 9058F than ST 4554B2RF for all genetically distinct Fov isolates tested with or without M. incognita (Fig. 4.4). Total plant weight was significantly higher for plants not inoculated with M. incognita at all four Fov inoculum densities tested (Table 4.3) showing that root-knot nematode affects plant growth and development. The plants inoculated with M. incognita (1000 eggs/pot) showed low total plant weight at higher Fov inoculum densities ( cfu/cm 3 and cfu/cm 3 ) than low Fov inoculum density ( cfu/cm 3 ) or non-inoculated plants (Table 4.3). High Fov inoculum density ( cfu/cm 3 ) resulted in significantly higher AUDPC with both the cultivars tested and AUDPC was significantly higher for FM 9058F than ST 4554B2RF (Table 4.4). Interaction between inoculum densities of Fov and cultivars tested was significant for plant height (p 0.05) and total plant weight (p = ) (Table 4.1). ST 4554B2RF had higher plant height and total plant weight than FM 9058F and high inoculum density of Fov had higher plant heights and total plant weight than low inoculum densities (Table 4.5). Interaction between Fov Race 1 isolates and cultivars tested was significant for plant height (cm) (at 5th th and 13 th week) at p = 0.05 level, root weight, shoot weight and total plant weight at p = level (Table 4.1). All these parameters were significantly higher for partially resistant cultivar, ST 4554B2RF than susceptible cultivar, FM 9058F (Table 4.6). Root galling was found to be associated with M. incognita presence and plants that exhibited galls were stunted, had reduced shoot and total plant weight; whereas, root weight was not affected by the presence of root galls (Table 4.7) because roots infested with M. incognita were small and fibrous than roots without galls. Plant growth and 95

110 symptoms expressions were affected by the susceptibility of cultivar. Susceptible cultivar, FM 9058F had significantly stunted plants, decreased root weight, shoot weight and total plant weight compared to partially resistant cultivar ST 4554B2RF (Table 4.8) suggesting, the importance of planting a resistant cultivar in infested fields. There is a positive correlation between Fov inoculum density and disease incidence (DeVay et al. 1997; Hao et al. 2009; Starr et al. 1989). Fov Race 1, which resulted in significant damage when plants were co-infected with M. incognita, caused significant symptom expression with isolates 3, 4, 6, and 7 in the absence of nematodes on FiberMax 9058F at cfu/cm 3 inoculum density (Fig. 4.2), suggesting that variability exists in aggressiveness among Fov Race 1 isolates at high inoculum density especially with planting a susceptible cultivar. Garber et al. (1979) and Yang et al. (1976) also found that Fusarium wilt occurs with Fov alone at high spore numbers ( 77,000 propagules per g soil) or in combination with root-knot nematodes at much lower densities (< 650 propagules per g soil with 50 second-stage juveniles) on a susceptible cultivar (Acala SJ-2). Kim et al. (2005) found that virulent Australian Fov isolates in controlled experiments, which, like Fov Race 4, do not require damage from nematodes to cause disease, and caused increasingly severe symptoms at higher aqueous suspensions of conidia. However, the effect of Fov inoculum density in soil varied with the resistance of the cotton cultivar. Indeed, results of this greenhouse study may not reflect the field responses of cotton cultivars because field grown plants are frequently under environmental or biotic stresses not present in the greenhouse. Another inherent difference between the conditions of this greenhouse study and those of the field is the inoculum itself. Fov 96

111 overwinters in field soil primarily as chlamydospores (Nelson, 1981), but micro- and macro-conidia were used in this study to infest the potting mixture. Conidia are not as well suited for long term survival in the soil as are the thick-walled chlamydospores but may germinate in pot cultures (Nelson, 1981). Despite the differences between this experiment and field conditions, disease development in the field likely follows the general trends observed here. Further research is necessary in order to determine the soil inoculum threshold for disease development in the field with varying levels of Fov virulence. The relationship between Fov inoculum density, M. incognita, and severity of Fusarium wilt-root-knot nematode complex is important for the development of management strategies because populations of Fov propagules in the soil may be affected by the cotton cultivar selection. This information is critical for managing Fusarium wilt- Root-knot nematode complex, especially with the pending loss of aldicarb, and unavailability of highly resistant cultivars. 97

112 4.5 LITERATURE CITED Abawi, G. S., and Baker, K. R Effects of cultivar, soil, temperature, and levels of Meloidogyne incognita on root necrosis and Fusarium wilt of tomatoes. Phytopathology 74: Atkinson, G. F Some diseases of cotton. III, Frenching. Alabama Agric. Exp. Stn. Bull. 41: Blasingame, D., Banks, J. C., Colyer, P. D., Davis, R. M., Gazaway, W. S., Goldburg, N., Kemerait, R. C., Kirkpatrick, T. L., Koenning, S. R., Muller, J., Newman, M. A., Olsen, M., Phipps, P. M., Sciumbato, G. L., Sprenkel, R., Woodward, J. E., Wrather, A. and Patel, M. V Beltwide Cotton Conference Cotton Disease Loss Estimate Committee Report Proceedings, pp Colyer, P. D Fusarium Wilt. In: Compendium of cotton Diseases, 2 nd edition, T. L. Kirkpatrick, and C. S. Rothrock, APS Press, pp Colyer, P. D., Kirkpatrick, T. L., Caldwell, W. D., and Vernon, P. R Influence of nematicide application on the severity of the root-knot nematode Fusarium wilt disease complex in cotton. Plant Dis. 81: DeVay, J. E Half a century dynamics and control of cotton disesases: Fusarium and Verticillium wilts. In: 1986 Proc. Beltwide Cotton Conf. J. Brown, ed. National Cotton Council of America, Memphis, TN, pp DeVay, J. E., Gutierrez, A. P., Pullman, G. S., Wakeman, R. J., Garber, R. H., Jeffers, D. P., Smith. S. N., Goodell, P. B., and Roberts, P. A Inoculum densitites of Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita in relation to the development of Fusarium wilt and the phenology of cotton plants (Gossypium hirsutum). Phytopathology 87: Garber, R. H., Jorgenson, E. C., Smith, S., and Hyer, A. H Interaction of population levels of Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita on cotton. J. Nematology, Vol 11, No. 2, pp Hao, J. J., Yang, M. E., and Davis, R. M Effect of soil inoculum density of Fusarium oxysporum f. sp. vasinfectum Race 4 on disease development in cotton. Plant Dis. 93: Hussy, R. S., and Barker, K. R A comparison of methods of collecting inocula of Meloidogyne spp., including a new technique. Plant Dis. Rep. 57:

113 Hyer, A. H., Jorgenson, E. C., Garber, R. H., and Smith, S Resistance to root-knot nematode in control of root-knot nematode-fusarium wilt disease complex in cotton. Crop Sci. 19: Jorgenson, E. C Granular nematicides as adjuncts to fumigants for control of rootknot nematodes. J. Nematol. 11: Kim, Y., Hutmacher, R. B., and Davis, R. M Characterization of California isolates of Fusarium oxysporum f. sp. vasinfectum. Plant Dis. 89: Koenning, S. R., Wrather, J. A, Kirkpatrick, T. L., Walker, N. R., Starr, J. R., and Mueller, J. D Plant-parasitic nematodes attacking cotton in the United States: Old and emerging production challenges. Plant Dis. 88: Komada, H Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Review of Plant Protection Research 8: Latin, R. X., and Snell, S. J Comparison of methods for inoculation of muskmelon with Fusarium oxysporum f. sp. melonis. Plant Dis. 70: Mai, W. F., and Abawi, G. S Interactions among root-knot nematodes and Fusarium wilt fungi on host plants. Annu. Rev. Phytopathol. 25: Martin, W. J., Newsom, L. D., and Jones, J. E Relationship of nematodes to the development of Fusarium wilt in cotton. Phytopathology 46: Nelson, P. E Life cycle and epidemiology of Fusarium oxysporum. In: Fungal wilt diseases of plants. M. E. Mace, A. A. Bell, and C. H. Beckman, eds. Academic Press, New York, pp Shaner, G. and R.E. Finney The effect of nitrogen fertilization on the expression of slow-mildewing resistance in Knox wheat. Phytopathology. 25: Shi, J., Mueller, W. C., and Beckman, C. H Vessel occlusion and secretory activities of vessel contact cells in resistant or susceptible cotton plants infected with Fusarium oxysporum f. sp. vasinfectum. Physiol. Mol. Plant Pathol. 40: Skovgaard, K., Nirenberg, H. I., O Donnell, K., and Rosendahl, S Evolution of Fusarium oxysporum f.sp. vasinfectum Races inferred from multigene genealogies. Phytopathology 91: Smith, A. L Control of cotton wilt and nematodes with a soil fumigant. Phytopathology 38: Smith, S. N., and Snyder, W. C., Persistence of Fusarium oxysporum f. sp. vasinfectum in fields in the absence of cotton. Phytopathology 65:

114 Smith, S. N., DeVay, J. E., Hsieh, W. H., and Lee, H. J Soil-borne populations of Fusarium oxysporum f. sp. vasinfectum, a cotton wilt fungus in California fields. Mycologia 93: Smith, S. N., Ebbels, D. L., Garber, R. H., and Kappelman, Jr. A. J Fusarium wilt of cotton, In: Fusarium: Diseases, Biology, and Taxonomy, P. E. Nelson, T. A. Toussoun, and R. J. Cook, The Pennsylvania State University Press, University Park, pp Starr, J. L., Jeger, M. J., Martyn, R. D., and Schilling, K Effects of Meloidogyne incognita and Fusarium oxysporum f. sp. vasinfectum on plant mortality and yield of cotton. Phytopathology 79: Yang, H., Powell, N. T., and Barker, K. R Interactions of concomitant species of nematodes and Fusarium oxysporum f. sp. vasinfectum on cotton. J. Nematol. 8:

115 Table 4.1. Effect of genetically distinct isolates of Fusarium oxysporum f. sp. vasinfectum, Meloidogyne incognita density and cotton cultivars on Fusarium wilt Root-knot nematode disease complex: p-values of the main effects and their interactions a Plant height (cm) Main Effects At 5 th week At 13 th week Area under the disease progress curve (AUDPC) Root weight (g) Shoot weight (g) Total plant weight (g) Isolate (I) ns ns Ns ns ns ns Inoculum density (Id) ns ns Ns ns ns ns Nematode (N) **** **** ** ns *** * Cultivar (Cv) ns **** * ** *** * Interactions I Id N Cv ns ns **** ns ns Ns Id N Cv ns ns Ns ns **** Ns I N Cv ns ns **** ns ns Ns I Id N ns ns Ns ns ns Ns I Id Cv ns ns Ns ns ns Ns Id N ns ns Ns ns ns ** I Cv * * Ns **** **** **** Id Cv * ns **** ns ns **** I Id ns ns Ns ns ns Ns I N ns ns Ns Ns ns Ns N Cv ns ns Ns Ns ns Ns Plants missing a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Significant interactions are from the reduced model. Full model was examined first to determine corrected reduced model. *, **, ***, **** denote significance levels (p-value) at 0.05, 0.01, 0.001, and , respectively. Twelve genetically distinct isolates of Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1 with four inoculum densities (0, , , & ) two Meloidogyne incognita densities (0 & 1000 eggs/pot) and two cotton cultivars (Stoneville 4554B2RF (partially resistant to Fov) & FiberMax 9058F (susceptible to Fov) were used in the study. A few seeds did not germinate and in a few cones, the soil mixture came out after planting resulting in missing plants. 101

116 Table 4.2. Effect of interaction between inoculum density of Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1, Meloidogyne incognita and cotton cultivar on shoot weight a Shoot weight (g) Inoculum density of M. incognita (0 eggs/pot) M. incognita (1000 eggs/pot) Fov Race 1 FiberMax Stoneville FiberMax Stoneville B b,a c 8.9 A,b c 7.3 B b,a 8.5 A,a c LSD e = B,a 9.3 A,ab 7.3 B,a 8.2 A,a df = B,a 9.3 A,ab 6.7 B,b 8.2 A,a t-value = B,a 9.5 A,a 5.6 B,c 8.5 A,a LSD d 0.56 df 1110 t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Means followed by the same letter in the same column are not significantly different at p 0.05 levels according to Fisher s LSD. b Upper case letters are between cultivar FiberMax and Stoneville of each nematode level. c Lower case letters are for between inoculum densities of each cultivar. d LSD for comparing means between cultivars FiberMax and Stoneville of each nematode level. e LSD for comparing means between Fov inoculum densities of each cultivar. FiberMax 9058F was susceptible and Stoneville 4554B2RF was partially resistant to Fov. N=

117 Table 4.3. Effect of interaction between inoculum density of Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1 and Meloidogyne incognita on total plant weight a Inoculum density Total plant weight (g) of Fov in soil (cfu/cm 3 ) M. incognita (0 eggs/pot) M. incognita (1000 eggs/pot) A b,a c 10.2 B,a c LSD e = A,a 10.1 B,a A,a 9.7 B,ab A,a 9.3 B,b LSD d 0.50 df 1117 df = 724 t-value = t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Means followed by the same letter in the same column are not significantly different at p 0.05 levels according to Fisher s LSD. b Upper case letters are for comparing means between nematode densities of each inoculum density. c Lower case letters are for comparing means between inoculum densities at each nematode level. d LSD for comparing means between nematode levels. e LSD for comparing means between inoculum densities. N =

118 Table 4.4. Effect of inoculum density of Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1 and cotton cultivar on area under the disease progress curve (AUDPC) a Inoculum density of AUDPC Fov in soil (cfu/cm 3 ) FiberMax Stoneville A b,d c 0.3 A,c c LSDe = A,c 2.3 B,c A,b 9.4, B,b df = 672 t-value = A,a 16.7 B,a LSD d 6.48 df 996 t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Means followed by the same letter in the same column are not significantly different at p 0.05 levels according to Fisher s LSD. b Upper case letters are for comparing means between cultivar FiberMax and Stoneville at each inoculum density. c Lower case letters are for comparing means between inoculum densities of each cultivar. FiberMax 9058F was susceptible and Stoneville 4554B2RF was partially resistant to Fov. N =

119 Table 4.5. Effect of interaction between inoculum density of Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1 and cultivar on plant height and total plant weight a Fov inoculum Plant height at 5th week (cm) Total plant weight (g) density in soil (cfu/cm 3 ) FiberMax Stoneville FiberMax Stoneville A b, ab c 16.2 A b, ab c 10.0 B b, a c 11.0 A b, a c B, b 16.8 A, a 10.0 B, a 11.2 A, a B, a 16.8 A, a 9.7 B, a 11.1 A, a A, ab 16.4 A, ab 8.9 B, b 11.4 A, a LSD d Df t-value LSD e df t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Means followed by the same letter in the same column are not significantly different at p 0.05 levels according to Fisher s LSD. b Upper case letters are for comparing means between cultivars FiberMax and Stoneville for plant height and total plant weight at each inoculum density. c Lower case letters are for comparing means between Fov inoculum densities for each cultivar. d LSD for comparing means between cultivars for plant height and total plant weight at each inoculum density. e LSD for comparing means between Fov inoculum densities for each cultivar. FiberMax 9058F was susceptible and Stoneville 4554B2RF was partially resistant to Fov. N =

120 Table 4.6. Effect of interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1 isolates and cultivars on plant growth parameters a Fov Plant height at 5 th Plant height at 13 th Root weight (g) Shoot weight (g) Total plant weight (g) Race 1 week (cm) week (cm) isolate FM ST FM ST FM ST FM ST FM ST B b,b c 16.2 A,ab c 22.6 B b,bc c 24.1 A,ab c 2.0 B b,b c 2.3 A,b c 6.7 B b,b c 8.6 A,ab c 8.7 B b,b c 10.9 A,ab c A,ab 16.1 A,ab 24.8 A,a 25.2 A,a 2.2 B,ab 2.5 A,ab 7.1 B,ab 8.9 A,ab 9.3 B,ab 11.4 A,ab B,ab 17.1 A,a 21.9 B,c 24.6 A,ab 2.1 B,ab 2.7 A,a 6.8 B,b 9.6 A,a 8.9 B,ab 12.3 A,a A,a 16.5 A,a 22.8 B,bc 24.6 A,ab 2.2 B,ab 2.5 A,ab 7.6 B,ab 9.1 A,ab 9.8 B,ab 11.6 A,ab A,ab 16.3 A,ab 22.9 B,bc 24.5 A,ab 2.2 A,ab 2.4 A,ab 7.4 B,ab 8.8 A,ab 9.6 B,ab 11.2 A,ab A,a 15.8 A,ab 22.4 B,bc 23.4 A,b 2.4 A,a 2.1 B,b 7.6 A,ab 8.2 A,b 10.0 A,ab 10.3 A,b A,ab 16.1 A,ab 22.9 A,bc 23.5 A,b 2.3 A,ab 2.3 A,b 8.0 B,a 8.7 A,ab 10.3 A,a 11.0 A,ab A,ab 16.6 A,a 22.6 B,bc 24.2 A,ab 2.1 A,ab 2.3 A,b 7.6 A,ab 8.2 A,b 9.7 A,ab 10.5 A,b B,a 17.4 A,a 23.3 B,b 25.3 A,a 2.0 B,b 2.6 A,ab 7.2 B,ab 9.6 A,a 9.2 B,ab 12.1 A,a B,a 17.6 A,a 22.4 B,bc 23.7 A,b 2.1 A,ab 2.3 A,b 8.0 B,a 9.5 A,a 10.1 B,ab 11.8 A,ab A,ab 16.1 A,ab 23.4 A,ab 24.0 A,ab 2.2 A,ab 2.3 A,b 7.8 A,ab 8.2 A,b 10.0 A,ab 10.5 A,b A,a 16.6 A,a 23.8 A,ab 24.6 A,ab 2.2 A,ab 2.3 A,b 8.1 A,a 8.3 A,b 10.3 A,a 10.6 A,b LSD d df t-value LSD e df t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Means followed by the same letter in the same column are not significantly different at p 0.05 levels according to Fisher s LSD. b Upper case letters are for cultivar FM (FiberMax 9058F) and ST (Stoneville 4554B2RF) of each isolate. c Lower case letters are for isolates of each cultivar. d LSD for comparing means between cultivar. e LSD for comparing means between Fov isolates. N=

121 Table 4.7. Effect of Meloidogyne incognita density on plant growth parameters a M. incognita Plant height (cm) Root weight Shoot weight Total plant density (eggs/pot) At 5 th week At 13 th week (g) (g) weight (g) a b 25.4 a b 2.3 a b 8.7 a b 11.0 a b b 21.9 b 2.3 a 7.5 b 9.8 b LSD c df t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same letter in the same column are not significantly different at p 0.05 level according to Fisher s LSD. c LSD for comparing M. incognita densities. N=

122 Table 4.8. Effect of cultivar selection on plant growth parameters a Plant height (cm) Cultivar At 5 th week At 13 th week Root weight (g) Shoot weight (g) Total plant weight (g) FiberMax 9058F 15.9 b b 23.0 b b 2.2 b b 7.5 b b 9.6 b b Stoneville 4554B2RF 16.5 a 24.3 a 2.4 a 8.8 a 11.2 a LSD c df t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same letter in the same column are not significantly different at p 0.05 level according to Fisher s LSD. c LSD for comparing cotton cultivar. FiberMax 9058F was susceptible and Stoneville 4554B2RF was partially resistant to Fusarium oxysporum f. sp. vasinfectum. N =

123 Figure 4.1. Genetically distinct Fusarium oxysporum f. sp. vasinfectum Race 1 isolates used for present study, showing distinct colony morphology in culture on Komoda s media. 109

124 Figure 4.2. Effect of interaction of genetically distinct isolates of Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1, their inoculum densities, Meloidogyne incognita and cotton cultivar on area under the disease progress curve (AUDPC). Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. LSD for comparing means between Fov isolates was 20.76, for cultivars was 20.31, for nematode levels was and for inoculum densities was at p 0.05 levels according to Fisher s LSD. FiberMax 9058F was susceptible and Stoneville 4554B2RF was partially resistant to Fov. Four inoculum densities of Fov used in the study were 0, , , and colony forming units/cubic centimeter of soil. Nema (-) and Nema (+) represent Meloidogyne incognita levels 0 and 1000 eggs/pot, respectively. 110

125 Figure 4.3. Effect of genetically distinct isolates of Fusarium oxysporum f. sp. vasinfectum (Fov) Race 1, Meloidogyne incogita, and cotton cultivar on area under the disease progress curve (AUDPC). Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. Level of significance was determined at p 0.05 according to Fisher s LSD. LSD = for comparing means between cultivars FiberMax and Stoneville at each nematode level for each Fov isolate. LSD = for comparing means between Fov isolates of each cultivar at each nematode level. FiberMax 9058F was susceptible and Stoneville 4554B2RF was partially resistant to Fov. N =

126 CHAPTER V EFFECT OF COTTON CULTIVAR SELECTION ON POPULATION DYNAMICS OF Fusarium oxysporum f. sp. vasinfectum in SOIL OVER TIME 5.1 ABSTRACT The Fusarium wilt Root-knot nematode complex is an economically important disease of cotton worldwide. A microplot study was conducted over the 2008 to 2010 growing seasons to investigate the impact of planting combinations of susceptible (S) (Fibermax (FM) 9058F) and/or partially resistant (R) (Stoneville (ST) 4554B2RF) cotton cultivars, on soil population density of Fusarium oxysporum f. sp. vasinfectum (Fov). The hypothesis was that due to the use of resistant cultivar, soil inoculum densities of Fov and disease incidence will decrease over time. Field soil naturally infested with Fov was added to microplots in 2008 and Meloidogyne incognita density was augmented each spring before planting. Fov density increased to cfu/cm 3 after 3 yrs of FM 9058F(SSS); however, Fov density decreased to cfu/cm 3 after 3 yrs of ST 4554B2RF (RRR). Soil inoculum density in the microplots initially planted with ST 4554B2RF followed by FM 9058F planted for next two years (RSS) was not different ( cfu/cm 3 ) from those planted to FM 9058F for three years (SSS) ( cfu/cm 3 ). Microplots planted with a SRR rotation had similar Fov densities ( cfu/cm 3 ) from those planted to RRR rotation ( cfu/cm 3 ). Disease incidence increased from 18% in 2008 to 69% in 2010 with FM 9058F. Disease incidence was a 112

127 function of both the cultivar grown in that season and pre-plant Fov soil density. Yield was highest after 3 yrs of ST 4554B2RF and lowest after 3 yrs of FM 9058F. Yield was related primarily by the current year cultivar, pre-plant Fov density in soil and cultivar history. Results from this study indicate that cultivar selection can impact Fusarium wilt management in cotton and should be considered when developing management strategies. Additional keywords: Gossypium hirsutum. 113

128 5.2 INTRODUCTION Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum Schlechtend.:Fr. f. sp. vasinfectum (Atk.) W. C. Snyder & H. N. Hans (Fov), is an economically important disease of cotton (Gossypium hirsutum L.) in most cottongrowing regions of the world (Colyer et al., 2001). Losses are greatest on sandy soils that are infested with the root-knot nematode Meloidogyne incognita (Kofoid & White) Chitwood (DeVay et al., 1997; Koenning et al., 2004; Martin et al., 1956). There are eight Races of Fov that have been described throughout the world, with Race 1 and Race 2 historically being most prevalent in the United States (Kim et al., 2005; Skovgaard et al., 2001). Recent studies have found that Races 1, 3, and 8 are mildly virulent and cause wilt symptoms in the presence of M. incognita; however, Race 4 of Fov, which was identified in California, is capable of causing severe wilt symptoms and economic loss in the absence of nematodes (Kim et al., 2005). The disease is responsible for losses of $20 million each year across the cotton belt of the United States of America (Blasingame et al., 2008). Since the first report of Fusarium wilt of cotton, in Alabama (Atkinson, 1892) the disease has spread and increased in importance (Smith et al., 1981). Under conducive environmental conditions, extremely high losses occur when susceptible cultivars are grown on heavily infested soil. Losses due to Fusarium wilt of cotton vary depending upon the virulence of Fov, host resistance, environmental factors, soil type and fertility, and interactions with nematodes (Abawi and Barker, 1984; Hao et al., 2009; Smith and Snyder, 1975). 114

129 Chlamydospores are thick walled specialized resting structures that remain dormant in the soil until exudates or leachates from plant roots stimulate their germination (Mai and Abawi, 1987). The germinated chlamydospores produce hyphae that eventually form conidia and new chlamydospores if a suitable host is not found. Once a field is infested with Fov, the fungus usually persists indefinitely in the soil as chlamydospores (Smith and Snyder, 1975.). Survival of Fov in the soil not planted to cotton for over 10 years has been documented (Smith et al., 2001). Fusarium wilt occurs with Fov alone at high spore numbers ( 77,000 propagules per g soil) or in combination with root-knot nematodes at much lower densities (< 650 propagules per g soil with 50 second-stage juveniles) on a susceptible cultivar (Acala SJ- 2) (Garber et al., 1979; Yang et al., 1976). Disease symptoms appear sooner with an increase of population levels of both M. incognita and Fov (Garber et al., 1979). In a microplot study conducted by Starr et al. (1989), no interaction was observed at high levels of Fov or at the lowest levels of M. incognita, but a significant interaction was observed at intermediate populations of Fov (2.3 to cfu/g of soil) and higher populations of M. incognita (10-50 eggs and second-stage juveniles/cm 3 ). Davis et al. (2006) in a greenhouse study found that visible symptoms occurred at > 10 5 conidia/ml in susceptible cultivars and symptoms in resistant cotton cultivars were evident at > 10 6 conidia/ml. In a greenhouse study, Hao et al. (2009) tested soil inoculum densities of Fov Race 4 (0 to 10 6 conidia/g soil) for plant growth, disease symptoms and number of cfu/g of stem tissues on cotton and found that Fusarium wilt is an inoculum density dependent disease. Susceptible cultivar Deltapine 744, showed symptoms at inoculum levels of 10 3 conidia/g of soil and higher; whereas, plant growth of the resistant Pima cultivar 115

130 Phytogen 800, was not affected by any soil inoculum densities. The fungus was rarely recovered from stems at inoculum levels less than 10 4 conidia per g of soil. There are few studies, however, that have examined the epidemiology of Fusarium wilt-root-knot nematode complex over multiple years under field conditions. Initial symptoms of Fusarium wilt include chlorosis and necrosis of the leaf margins. Severely diseased plants often remain stunted throughout the growing season or die. Fov invades the host through the taproots behind the root tip. The combined effect of fungal metabolites and the production of lipoidal substances by the host in response to infection may lead to the occlusion of the vascular tissues (Shi et al., 1992). The vascular system of the plants is discolored brown to black due to systemic infection of the fungus. In the most severely affected plants, leaves wilt and drop and the plants may die (Colyer, 2001; Nelson, 1981). Plants that develop symptoms early usually die before producing any bolls, whereas plants that develop symptoms after the onset of flowering often survive but produce fewer bolls. There are limited management options available for Fusarium wilt-root-knot nematode disease complex. Resistance to M. incognita has proved to be an effective management strategy for the Fusarium wilt-root-knot nematode complex (Ogallo et al., 1997). Shepherd (Shepherd, 1975) suggested that damage due to Fusarium wilt-root-knot nematode complex could be largely avoided if cultivars with greater resistance to both root-knot and Fusarium wilt were utilized. It is crucial to understand the relationship between inoculum density in soil at planting and its effect on upland cotton cultivars. The objective of this study was to 116

131 determine the effect of cotton cultivar selection on release of Fov inoculum in soil over time and its impact on wilt development in cotton. 5.3 MATERIALS AND METHODS Microplot Experiment A microplot study was conducted over the 2008 to 2010 growing seasons at the Texas Tech University, Quaker Research Farm located in Lubbock, Texas. Microplots were constructed out of cylindrical aluminum rings of 90 cm diameter and 60 cm height, and buried in the soil at the depth of 50 cm and were augmented with field soil naturally infested with Fov and M. incognita. There were six rotation schemes over three years utilizing all possible combinations of a susceptible cultivar, FiberMax (FM) 9058F and a partially resistant cultivar, Stoneville (ST) 4554B2RF. Cultivars were selected based on the results of cotton cultivar performance trials conducted prior to 2008 (Woodward, unpublished data). Both of these cultivars were susceptible to M. incognita (Gannaway et al., 2007). The susceptible cultivar was referred to as S and the partially resistant cultivar as R. The three year rotations were: RRR, RRS, RSS, SSS, SSR, and SRR where the first letter refers to the cultivar in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in To maintain adequate M. incognita population microplots were inoculated with 28,000 eggs in 2009 and Inoculum was reared on the susceptible tomato cultivar Homested in the greenhouse and extracted according to methods described by Hussey and Barker (1973). Microplots were planted at the rate of 25 seeds per microplot in a circular pattern on May of 2008, 2009 and 2010 resulting in planting density of 200,000/ha. Irrigation, applied via a 117

132 drip irrigation system, and fertilizer (in the form of urea ammonium nitrate) was applied as needed. Weeds were controlled throughout the study using both pre- and postemergence herbicides using local extension recommendations, or via hand weeding Soil Sampling and Data Collection In April 2008, microplots were sampled to determine baseline Fov inoculum densities. Subsequent soil samples were taken in February, August and, December 2009, and April, August, and December 2010 to enumerate Fov inoculum densities over time. A 2.5 cm diameter auger to a depth of 20 cm was used taking soil samples from each microplot. Each sample consisted of four cores and had a total weight of approximately 250 g air-dry soil. Enumeration of inoculum was done using dilution plating technique (Nash and Snyder, 1962) utilizing Komada s selective medium (Komada, 1975). The cores were mixed together, air dried for 7 days, and ground with a rolling pin. A 20 cm 3 sub-sample was combined with 80 ml of de-ionized water. This solution was diluted four times (for the ease of counting the number of colonies on media) and stirred using a magnetic stir plate. A 1-ml aliquot of the soil solution was distributed on each Petri dish (10 replicates) containing the selective medium. After 72 hours of incubation at room temperature and light, numbers of Fov colonies were counted for each Petri dish and multiplied by four to get the number of colonies per cm 3 soil. Colonies of Fov were identified based on their distinctive colony morphology with respect to the density and architecture of aerial mycelium, the pigmentation evident from the colony and shape and size of microconidia and macroconidia. Three Petri dishes from each rotation and three colonies from those Petri dishes were selected for enumeration of colony forming units (cfu) per cubic centimeter (cm 3 ) of soil with a hemocytometer under a compound 118

133 microscope. Percent germination was recorded on 20 th, 22 nd, and 22 nd June of 2008, 2009 and 2010, respectively. Disease incidence was assessed on 28 th, 27 th, and 27 th August as percent symptomatic plants in each microplot for each season and disease incidence was expressed as (No. of wilted plants in a microplot/stand count in that microplot) 100. Plant height and lint yields were measured in Lint samples were sent to Texas Tech University, Fiber and Biopolymer Research Institute (FBRI) for fiber quality analysis using High Volume Instrument (HVI) Statistical Analysis The experiment was designed as a randomized complete block design with six treatments and nine replications. Percent germination, disease incidence, inoculum density, plant height, and lint yield data were analyzed using Proc MIXED (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). Data were analyzed as a split-plot in time where the subplots were seven sampling dates as described by Steel and Torrie (1960). Linear model was fitted for Fov inoculum densities in soil (cfu/cm 3 ) over time (month value). Linear change was on month basis with replication and treatment as random effects. Center of the month values (0, 10, 16, 20, 24, 28 and 32) was determined as described by Draper and Smith (1981) and intercepts came out at center of data. Error was natural error of the experiment. Regression analysis for pre-plant soil inoculum density of Fov (cfu/cm 3 ) and disease incidence (%) was done on yield in 2010 using Proc MIXED with replication as the random effect. The method used to adjust the degrees of freedom (df) to match adjustments in the sums of square was the Satterthwaite option in the LSMEANS statement in Proc MIXED (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). Standard error and LSD were determined from the PDIFF option. Slopes for Stoneville 4554B2RF 119

134 and FiberMax 9058F were tested by regressing pre-plant soil inoculum density of Fov (cfu/cm 3 ) on disease incidence (%) over the three year period using Proc Reg (SAS Institute Inc., 2008, Ver. 9.2, Cary, NC, USA). 5.4 RESULTS Baseline inoculum density of Fov at the start of the experiment was 4.6 ± cfu/cm 3 of soil among all rotation treatments (data not shown). Rotation schemes planted to ST 4554B2RF for at least two years (RRR, RRS, and SRR) resulted in lower inoculum densities (p 0.05) when compared to rotation schemes planted with FM 9058F over the same period (SSS, SSR, RSS) (Table 5.1). Planting FM 9058F for three sequential years (SSS) was found to increase Fov inoculum densities in soil from to cfu/cm 3 ; whereas, planting ST 4554B2RF over the same period (RRR) was found to decrease Fov populations in soil from to cfu/cm 3 (p 0.05) (Table 5.1). Soil inoculum density in the microplots initially planted with ST 4554B2RF followed by FM 9058F planted for next two years (RSS) was not different ( cfu/cm 3 ) from those planted to FM 9058F for three years (SSS) (Table 5.1). Microplots initially planted with FM 9058F followed by two years of ST 4554B2RF (SRR) had similar Fov densities ( cfu/cm 3 ) from those planted to ST 4554B2RF for three years (RRR) (Table 5.1). The dynamics of Fov population densities over the three years could be fitted with linear models for the cultivar rotations (Fig. 5.1). The linear model was significant (p < 0.01) for cultivar rotation SSS, SSR, and RSS with positive slopes (Table 5.2). In general, as discussed previously, the soil inoculum densities increased over time when there were 120

135 at least two years of a susceptible cultivar, and the densities were much lower when there were at least two years of a partially resistant cultivar (Fig. 5.1). Disease incidence for ST 4554B2RF when planted for at least two consecutive years was less (p 0.05) than disease incidence with FM 9058F planted for two or more consecutive years (Table 5.3). Disease incidence in the RRR rotation was similar in all three years, ranging from 4 to 6%; whereas, disease incidence increased from 18% in 2008 to 69% in 2010 in SSS (Table 5.3). In 2008, where Fov densities were all very similar at planting, disease incidence averaged 5% for ST 4554B2RF and 17% for FM 9058F (Table 5.3). In 2009, disease incidence averaged 6% for RR, 9% for SR, 28% for RS, and 33% for SS. Slopes obtained by regressing pre-plant Fov densities in soil with disease incidence in three years were significantly different (p = ) for ST 4554B2RF (0.19) and FM 9058F (0.58) (Table 5.4). The different slope values indicate that disease incidence would increase faster with per unit Fov inoculum for FM 9058F than for ST 4554B2RF, as was seen in the 2008 disease incidence ratings (Table 5.4). Differences (p 0.05) in plant heights were observed among rotation schemes in 2010 (Table 5.5). In 2010, rotation schemes planted with a partially resistant cultivar had tallest plants with rotation RRR (54.1 cm) followed by SRR (52.5 cm) and SSR (34.7 cm); whereas, rotation schemes planted with a susceptible cultivar had shortest plants with rotation SSS (21.7) followed by RSS (22.9 cm) and RRS (41.2 cm) (Table 5.5). Lint yields in 2010 were found to be significantly different (p 0.05) among cultivar rotation schemes (Table 5.6). In 2010, rotation schemes planted with a partially resistant cultivar had highest lint yield with rotation RRR (3816 kg/ha) followed by SRR (2240 kg/ha) and SSR (1171 kg/ha); whereas, rotation schemes planted with a susceptible 121

136 cultivar had lowest lint yield with rotation SSS (170 kg/ha) followed by RSS (342 kg/ha) and RRS (970 kg/ha) (Table 5.6) suggesting, that lint yield was affected by current year cultivar and cultivar history. In 2010, for the cultivar ST 4554B2RF pre-plant Fov inoculum densities with rotation RRR and SRR were low (averaging cfu/cm 3 ) and were marginally different from each other (Fov density was 3.2% lower for RRR); whereas, yield was significantly higher for RRR than for SRR rotation (Fig. 5.2.A). With the SSR rotation pre-plant Fov inoculum densities were significantly higher (averaging cfu/cm 3 ) and yield was low (Fig. 5.2.A). For the cultivar FM 9058F, pre-plant Fov inoculum densities with the rotation schemes SSS and RSS were higher (averaging cfu/cm 3 ) and both rotations resulted in significantly lower yields; however, with rotation RRS Fov density was low (averaging cfu/cm 3 ) and corresponding yield was also low (Fig 5.2.B) indicating that the lint yield was very sensitive to even low pre-plant Fov inoculum density. Additionally, lint yield was inversely related to mid season Fov inoculum densities in soil. The RRR rotation scheme had lowest Fov inoculum densities (averaging cfu/cm 3 ) with highest lint yield (Fig. 5.2.C) and SSS had highest Fov inoculum densities ( cfu/cm 3 ) with lowest lint yield (Fig. 5.2.D); whereas, the cultivar rotation SRR had lower lint yield than RRR, but only slightly higher mid-season Fov inoculum densities (Fig. 5.2.C). Utilization of ST 4554B2RF in 2009 and 2010 resulted in significantly higher lint yield when compared to FM 9058B2F planted in both the years 2009 and 2010 (Fig. 5.2.A, B, C, D). However, ST 4554B2RF and FM 9058F 122

137 yielded similarly for the combinations of SSR and RRS, indicating the importance of using resistant cultivars for multiple years. Pre-plant Fov inoculum densities in 2010 were similar to mid-season Fov inoculum densities, so much of the inoculum shift due to cultivar did not occur during the 2010 growing season (Table 5.1, Fig. 5.2.A, B, C, D). There was negative relationship between pre-plant Fov inoculum density in soil and lint yield during the same season for both the cultivars (Table 5.7). With the cultivar FM 9058F slope for pre-plant Fov inoculum density in soil and lint yield was and the slope was for cultivar ST 4554B2F (Table 5.7). At low Fov inoculum density in soil (averaging cfu/cm 3 ), yield for ST 4554B2RF was 3242 kg/ha; whereas, yield was 1074 kg/ha with high Fov inoculum density in soil ( cfu/cm 3 ) (Table 5.7). With FM 9058F, at low Fov inoculum density in soil ( cfu/cm 3 ) yield averaged 982 kg/ha; whereas, at high Fov inoculum density in soil ( cfu/cm 3 ) yield was averaged 220 kg/ha (Table 5.7), suggesting, that yield was primarily affected by cultivar grown in that year and pre-plant soil inoculum densities of Fov. A negative relationship was also found between disease incidence taken during the same growing season and lint yield with significantly different slopes (p 0.05) for both the cultivars (Fig. 5.3). Slope for disease incidence and yield was for cultivar FM 9058F and it was for cultivar ST 4554B2F (Fig. 5.3). There were no differences in fiber quality among the treatments evaluated; however, higher micronaire, uniformity, elongation, yellowness (+b), and leaf values were observed for FM 9058F (data not shown) than for ST 4554B2RF. 123

138 5.5 DISCUSSION Annual production losses from Fusarium wilt has declined as improved wilt resistant cultivars were released for wide scale production in the United States during the 1970s and 1980s (Colyer, 2001); however, with the introduction of new susceptible cultivars of cotton, Fusarium wilt has again started to cause severe losses in grower s fields (Woodward, personal communication). A correlation is known to exist between disease incidence and Fov inoculum density, where increasing populations of Fov results in higher levels of disease especially in the presence of the M. incognita (DeVay et al., 1997; Hao et al., 2009; Starr et al., 1989). In the present study, we observed that Fov inoculum density in soil was higher in the rotation schemes showing higher disease incidence in the presence of M. incognita. Field observations indicate a substantial reduction in wilt incidence following years where resistant/tolerant cultivars (primarily with Stoneville 5599BR) were grown (Wheeler, unpublished data). Damage due to Fusarium wilt-root-knot nematode complex can be largely avoided by planting cultivars with at least partial resistance to either pathogen. In this study, two cultivars were chosen which have no resistance to M. incognita, but did differ in their ability to increase reproduction of Fov. However, while ST 4554B2RF is not considered resistant to M. incognita, it typically does have lower population densities of M. incognita than FM 9058F (Gannaway et al., 2007). Disease severity is dependent upon pre-plant inoculum density of Fov in soil. Yield appeared to be partially related to pre-plant Fov inoculum density in soil and disease incidence. This relationship did not hold true when resistant and susceptible cultivars were rotated from 124

139 the previous year. It was necessary to have at least two years of a resistant cultivar to benefit with higher yield. Starr et al. (1989) observed a significant interaction between Fov at inoculum density of cfu/g of soil, and M. incognita at populations of eggs and second-stage juveniles/cm 3. No interaction was observed at lower populations of nematode or at higher populations of Fov. No significant difference in mortality and yield responses were observed between two cotton cultivars Tamcot SP37 and Tamcot CAB- CS, susceptible and resistant to Fusarium wilt-root-knot nematode complex, respectively (Starr et al., 1989). In the present study there was an upward trend of counts of Fov for RRS with a positive slope probably due to the susceptible cultivar planted in While there were distinct differences in the rotation schemes when there were at least two consecutive years of resistant or susceptible cultivars, there were also more subtle differences due to the cultivar grown in the last year of the rotation study. This suggests, that any gains made by two years of growing resistant cultivars, would probably be lost using susceptible cultivars. As the cultivar rotation scheme becomes more complex in 2010, then the impact of cultivar on disease incidence was a function of the cultivar planted in the current season, but also was related to the pre-plant Fov inoculum density in the soil. The different slope values obtained by regressing pre-plant Fov inoculum densities in soil with disease incidence in three years indicate that disease incidence would increase faster per unit Fov for susceptible cultivar than for partially resistant cultivar. Three year 125

140 rotation effects had some sort of cumulative effect on lint yield, where shorter rotations or susceptible or partially resistant cultivars resulted in intermediate yield responses. Utilization of ST 4554B2RF in 2010 resulted in significantly higher lint yield when compared to FM 9058F which suggests that lint yield was primarily affected by the cultivar of the current season; however, yield for the rotation SRR was significantly higher than the rotation SSR; whereas, yield for RSS was significantly lower than RRS which suggests that cultivar history is an important factor in determining lint yield. There was negative relationship between pre-plant Fov inoculum density in soil (cfu/cm 3 ) and lint yield in 2010 for both the cultivars. Though the loss in yield per unit cfu was greater for the partially resistant cultivar than the susceptible cultivar, the more important factor was the greater sensitivity to losses at low Fov inoculum densities found with FM 9058F relative to the partially resistant cultivar. A negative slope was obtained by regressing disease incidence and yield of the same growing season for both the cultivars. The important factor is the low yields associated with FM 9058F even at low disease incidence of Fusarium wilt. That suggests that plants of FM 9058F not exhibiting symptoms of wilt were still heavily damaged by the pathogen. Disease incidence and yield could be explained through cultivar choice and preplant Fov inoculum density in soil. With the SSR rotation pre-plant Fov inoculum densities were significantly higher and yield was low which implies that yield in 2010 was affected by pre-plant Fov inoculum densities. Additionally, lint yield was inversely related to mid-season soil inoculum densities of Fov. The RRR rotation scheme had lowest mid-season Fov inoculum densities with highest lint yield and SSS rotation had 126

141 highest Fov inoculum densities with lowest lint yield indicating, that the Fov inoculum densities in soil play an important role in determining lint yield. Lint yield was higher for RRR than for SRR rotation which could be due to cultivar history because SRR rotation had a susceptible cultivar in the year The cultivar rotation SRR had lower lint yield than RRR which may be due to Fov inoculum density in soil and/or cultivar history. ST 4554B2RF demonstrated resistance to Fov by not increasing the inoculum density, but the severely negative slope between disease incidence and yield, indicated that this cultivar is not tolerant of the disease, but will decline substantially as wilt symptoms increase. FM 9058F had poor yields even a lot incidence of wilt. The present study suggests that the adoption of resistant cultivars can negatively impact inoculum density of Fov in soil of over time, thus reducing Fusarium wilt incidence. Adoption of a partially resistant cultivar (ST 4554B2RF) for at least two sequential years was required to negatively impact Fov inoculum density in soil; however, three sequential years was required to maximize yield. This strategy can be implemented into integrated programs for sustaining the production of cotton in the fields infested with Fov and M. incognita. With the release of cotton cultivars having true or partial resistance to Fov and/or M. incognita, there is a need to screen them against different levels of inoculum pressure. Additionally, this information needs to be communicated to the growers so that they can remain economically competitive. 127

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144 Smith, S. N., DeVay, J. E., Hsieh, W. H., and Lee, H. J Soil-borne populations of Fusarium oxysporum f. sp. vasinfectum, a cotton wilt fungus in California fields. Mycologia 93: Smith, S. N., Ebbels, D. L., Garber, R. H., and Kappelman, Jr. A. J Fusarium wilt of cotton. In: Fusarium: Diseases, Biology, and Taxonomy, P. E. Nelson, T. A. Toussoun, and R. J. Cook, The Pennsylvania State University Press, University Park, pp Starr, J. L., Jeger, M. J., Martyn, R. D., and Schilling, K Effects of Meloidogyne incognita and Fusarium oxysporum f. sp. vasinfectum on plant mortality and yield of cotton. Phytopathology 79: Steel, R. G. D. and Torrie, J. H Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., pp Yang, H., Powell, N. T., and Barker, K. R Interactions of concomitant species of nematodes and Fusarium oxysporum f. sp. vasinfectum on cotton. J. Nematol. 8:

145 Table 5.1. Effect of cultivar rotation and time on soil inoculum density of Fusarium oxysporum f. sp. vasinfectum a Fov density in soil ( 10 5 cfu/cm 3 ) Cultivar rotation schemes f Time (month value) RRR RRS RSS SSS SSR SRR April 2008 (0) 4.4 a b, C c 4.8 a, A 4.7 e, AB 4.6 e, ABC 4.6 f, ABC 4.4 a, BC February 2009 (10) 2.8 d, B 2.7 d, B 2.7 f, B 4.7 e, A 4.7 f, A 4.7 a, A August 2009 (16) 3.6 b, B 3.6 c, B 6.8 d, A 6.8 d, A 6.8 e, A 3.8 bc, B December 2009 (20) 3.5 b, B 3.6 c, B 7.3 c, A 7.4 c, A 7.4 d, A 3.6 c, B April 2010 (24) 3.8 b, B 3.9 b, B 9.5 a, A 9.5 a, A 9.6 a, A 3.9 b, B August 2010 (28) 3.2 c, E 4.2 b, C 9.3 ab, A 9.3 ab, A 9.0 b, B 3.6 c, D December 2010 (32) 2.9 cd, D 4.6 a, C 9.2 b, A 9.1 b, A 8.5 c, B 3.0 d, D LSD d 0.31 LSD e 0.28 df 285 t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis as described previously and df were determined using the Satterthwaite option. b Means followed by the same lower case letter in the same column are not significantly different at p 0.05 level according to Fisher's LSD. c Means followed by the same upper case letter in the same row are not significantly different at p 0.05 level. d LSD for comparing time. e LSD for comparing cultivar rotation schemes. f R = Resistant (Stoneville 4554B2RF), S = Susceptible (FiberMax 9058F). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in N = 9 for all rotation schemes. 131

146 Table 5.2. Parameters from linear regression of soil inoculum density of Fusarium oxysporum f. sp. vasinfectum over time ( ) for six cultivar rotations a Inoculum density of Fov in soil ( 10 5 cfu/cm 3 ) Cultivar rotation schemes c Intercept Slope p-value RRR 3.5 b b b p > 0.20 RRS 3.9 b b p > 0.20 RSS 7.1 a a p < 0.01 SSS 7.3 a a p < 0.01 SSR 7.2 a a p < 0.01 SRR 3.9 b b p > 0.20 SE LSD df a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same letter in the same column are not significantly different at p 0.05 level according to Fisher's LSD. c R = Resistant (Stoneville 4554B2RF), S = Susceptible (FiberMax 9058F). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in N = 63 for all rotation schemes. 132

147 Table 5.3. Effect of cultivar rotation on percent disease incidence in 2008, Cultivar rotation Disease incidence (%) 2009, and 2010 a schemes f RRR 5.6 c b, A c 5.9 d, A 4.3 e, A LSD e = 3.9 RRS 4.7 c, B 5.0 d, B 17.5 d, A RSS 5.0 c, C 27.7 b, B 48.3 b, A df = 120 SSS 17.9 ab, C 33.9 a, B 69.0 a, A SSR 15.2 b, C 31.6 a, A 25.3 c, B t-value = 1.98 SRR 18.5 a, A 9.4 c, B 7.2 e, B LSD d df 40 t-value 2.02 a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same lower case letter in the same column are not significantly different at p 0.05 level according to Fisher's LSD. c Means followed by the same upper case letter in the same row are not significantly different at p 0.05 level. d LSD for comparing cultivar rotations. e LSD for comparing years and it was constructed as follows: LSD = sqrt (( )/3)*1.98. f R = Resistant (Stoneville 4554B2RF), S = Susceptible (FiberMax 9058F). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in N = 9 for all rotation schemes. 133

148 Table 5.4. Testing the slopes from regressing pre-plant soil inoculum density of Fusarium oxysporum f. sp. vasinfectum on disease incidence in 2008, 2009, and 2010 a Disease Incidence (%) Stoneville 4554B2RF Slope 1.92 SE FiberMax 9058F Slope 5.78 SE a Data were analyzed using Proc Reg (SAS, Ver. 9.2, 2008). Comparision of slopes for Stoneville and FiberMax had an observed t-value of that was computed using method described by Steel and Torrie (1960) as (( )/( ) 0.5 ). The tabular t-value was at p = and 160 df. FiberMax 9058F was susceptible to Fusarium and Stoneville 4554B2RF was partially resistant to Fusarium; whereas, both the cultivars were susceptible to root-knot nematode. 134

149 Table 5.5. Effect of cultivar rotation on plant height in 2010 a Cultivar rotation schemes c Plant height (cm) Partially resistant cultivar RRR 54.1 a b SRR 52.5 a SSR 34.7 b Susceptible cultivar RRS 41.2 a b RSS 22.9 b SSS 21.7 b LSD 7.1 df 44 t-value 2.02 a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same letter for partially resistant cultivar and susceptible cultivar are not significantly different at p 0.05 level according to Fisher's LSD. c R = Partially resistant (Stoneville 4554B2RF), S = Susceptible (FiberMax 9058F). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in N = 45 for the rotation schemes. 135

150 Table 5.6. Effect of cultivar rotation on lint yield in 2010 a Lint yield Cultivar rotation schemes c (kg/ha) Partially resistant cultivar RRR a b SRR b SSR c Susceptible cultivar RRS a b RSS b SSS b LSD df 40 t-value a Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. b Means followed by the same letter for partially resistant cultivar and susceptible cultivar are not significantly different at p 0.05 level according to Fisher's LSD. c R = Partially resistant (Stoneville 4554B2RF), S = Susceptible (FiberMax 9058F). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in Planting density was 200,000/ha. N = 9 for all the rotation schemes. 136

151 Table 5.7. Effect of cultivar and pre-plant soil inoculum density of Fusarium oxysporum f. sp. vasinfectum on lint yield in 2010 a Inoculum level in soil Stoneville 4554B2RF Pre-plant Fov density (cfu/cm 3 ) Lint yield (kg/ha) FiberMax 9058F Pre-plant Fov density (cfu/cm 3 ) Lint yield (kg/ha) Low High Average (± ) (± 239.9) (± ) (± 95.1) Slope = Slope = a Regression analysis for preplant soil inoculum density was done on yield using Proc MIXED (SAS 2008, Ver. 9.2) and df were determined using the Satterthwaite option. Observed t-value to compare slopes between Stoneville and FiberMax was and was found using method from Steel and Torrie (1960). Tabular t-value was at p 0.05 and df = 34. FiberMax 9058F was susceptible to Fusarium and Stoneville 4554B2RF was partially resistant to Fusarium; whereas, both the cultivars were susceptible to root-knot nematode. Planting density was 200,000/ha. 137

152 Fusarium (Fov) density in soil (x 10 5 cfu/cm 3 ) RRR RRS SRR RSS SSS SSR a b Time (month value) Figure 5.1. Effect of cultivar rotation on Fusarium oxysporum f. sp. vasinfectum density (cfu/cm 3 ) in soil over time. Data were analyzed using Proc MIXED (SAS 2008, Ver. 9.2) using mixed model analysis and df were determined using the Satterthwaite option. a. Linear model was significant (p < 0.01) for cultivar rotation SSS, SSR, and RSS with positive slopes. b. RRR, RRS, and SRR had non-significant slopes. LSD 0.05 = 2.8. R = Partially resistant (Stoneville 4554B2RF), S = Susceptible (FiberMax 9058F). RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in

153 4000 RRR SRR SSR SSS RSS RRS Lint yield (kg/ha) A. B RRR SRR SSR SSS RSS RRS Lint yield (kg/ha) C. D Fov inoculum density (x 10 5 cfu/cm 3 ) Fov inoculum density (x 10 5 cfu/cm 3 ) Figure 5.2. Effect of soil inoculum density of Fusarium oxysporum f. sp. vasinfectum on lint yield in Lint yield due to pre-plant (April-2010) soil inoculum density for A. Stoneville 4545B2RF B. FiberMax 9058F. Lint yield due to mid-season (August-2010) soil inoculum density for C. Stoneville 4545B2RF D. FiberMax 9058F. Stoneville 4554B2RF = Partially resistant cultivar, FiberMax 9058F = Susceptible cultivar. RRR, RRS, RSS, SSS, SSR, and SRR represent three year rotations where the first letter refers to the cultivar grown in 2008, the second letter to the cultivar grown in 2009 and the third letter to the cultivar grown in

154 4000 Stoneville 4554B2RF FiberMax 9058RF Lint yield (kg/ha) in y = x , R 2 = 0.54 y = x , R 2 = Disease incidence (%) Figure 5.3. Effect of disease incidence on lint yield in Data were analyzed using Proc Reg (SAS Ver. 9.2, 2008). Stoneville 4554B2RF = Partially resistant cultivar, FiberMax 9058F = Susceptible cultivar. Slope for Stoneville 4554B2RF ( ) was significantly different than slope for FiberMax 9058F ( ) at p 0.05 level. Observed t-value = that was found using method from Steel and Torrie (1960). Tabular t-value = at p 0.05 and df =

155 CHAPTER VI SUMMARY AND CONCLUSION 6.1 SUMMARY Verticillium wilt and the Fusarium wilt-root-knot nematode complex are economically important diseases of cotton. Verticillium wilt is caused by a soilborne fungus Verticillium dahliae Kleb. The fungus can survive in soil and/or in plant debris for more than 20 years as microsclerotia (ms). Verticillium dahliae has a broad host range. Fusarium wilt of cotton is caused by a soilborne fungus Fusarium oxysporum f. sp. vasinfectum Atk. (Fov). In West Texas the occurrence of Fusarium wilt is in conjunction with the root-knot nematode, Meloidogyne incognita. Fov can survive in soil for over 10 years in the form of chlamydospores. Management options are limited for both the diseases. Strategies that influence soil inoculum density may help manage these diseases. This study focuses on understanding the relationship between inoculum density in soil at planting and wilt development in cotton. To examine the influence of Verticillium dahliae infested peanut residue amount on inoculum release in soil and Verticillium wilt development in subsequent cotton, a microplot study was conducted in 2008 and The hypothesis was that Peanut residue infested with V. dahliae will increase microsclerotia density in soil and Verticillium wilt on subsequent cotton will also increase. The effects of infested peanut residue amount on percent germination of cotton and on Verticillium wilt incidence were monitored in both cropping seasons. Microsclerotia density in soil was also quantified to investigate the 141

156 release of inoculum from infested peanut residue over time. Infested peanut residue was collected from a field with a history of Verticillium wilt and used to artificially infest microplots at the rates of 370, 925, 1850, 2775, 3700, 18,495, and 37,000 kg/ha. Noninfested microplots served as a control. Treatments were arranged in a randomized complete block design with nine replications. Microplots were planted with a cotton cultivar, Stoneville (ST) 4554B2RF, susceptible to Verticillium wilt. Increasing infested peanut residue amount had a negative effect on percent germination of cotton seed with a slope of (R 2 = 0.90) in 2008, and (R 2 = 0.98) in A positive effect was found between increasing infested peanut residue amount and Verticillium wilt incidence in cotton, with a slope of 3.07 (R 2 = 0.97) in 2008, and 6.43 (R 2 = 0.99) in Soil samples collected before incorporation of infested peanut residue artificially were void of V. dahliae inoculum for all the microplots. Densities of ms in the soil were found to increase significantly with increasing amount of V. dahliae infested peanut residue over time, with a slope of 0.42 (R 2 = 0.99) in April 2009, 0.85 (R 2 = 0.89) in November 2009, and 1.32 (R 2 = 0.89) in April Results show the importance of removing infested peanut residue which otherwise may serve as a source of inoculum for subsequent cotton crops. To examine the consequence of cultivar selection on soil population dynamics of Verticillium dahliae over time and implications for Verticillium wilt development in cotton, a microplot study was conducted over the 2008 to 2010 growing seasons. The hypothesis tested was that the choice of cultivar will affect soil inoculum density of V. dahliae and disease incidence. ST 4554B2RF was used throughout the test as a susceptible cultivar and either (AFD) 5065B2F or an advanced breeding line was used as 142

157 the partially resistant cultivar. Microplots were augmented with field soil naturally infested with V. dahliae. ST 4554B2RF when planted in three sequential seasons increased V. dahliae populations in soil from 1.3 to 11 ms/cm 3 ; however, V. dahliae populations in microplots planted to the partially resistant cultivars over three seasons increased from 1.4 to 3 ms/cm 3. Disease incidence increased from 8% to 58% over 3 yrs for ST 4554B2RF and from 0% to 5% for AFD 5065B2F or advanced breeding line over the same period. Yield was highest after 3 yrs of AFD 5065B2F or a breeding line and lowest after 3 yrs of ST 4554B2RF. Yield was related primarily by the current year cultivar, pre-plant V. dahliae densities, and disease incidence. Adoption of a resistant cultivar for at least 2 years was necessary to maintain a low ms density in soil. Results from this study indicate that cultivar selection can impact ms density and incidence of wilt in cotton and should be considered when developing management strategies. Fusarium oxysporum f. sp. vasinfectum Race 1 populations in Texas are genetically diverse. To examine the impact of inoculum densities of genetically distinct Fusarium oxysporum f. sp. vasinfectum Race 1 isolates, Meloidogyne incognita, and cultivar on Fusarium wilt development in cotton an experiment was conducted with 12 Fov isolates at four densities (0 to cfu/cm 3 soil), Meloidogyne incognita densities (0 and 1,000 eggs/pot), and two cultivars (partially resistant ST 4554B2RF and susceptible Fibermax (FM) 9058F) in the greenhouse. The hypothesis was that the isolates of Fov collected from Texas will demonstrate variability in aggressiveness. FM 9058F had significantly (p = ) higher area under the disease progress curve (AUDPC) than ST 4554B2RF for all the Fov isolates tested with the interaction of Fov Race 1 isolates, their inoculum densities, M. incognita densities, and cotton cultivars. 143

158 Fusarium oxysporum f. sp. vasinfectum isolates 3, 4, 5, 6, 7, 10, 11, and 12 exhibited significantly (p = 0.05) higher AUDPC than isolates 1, 2, 8, and 9 suggesting, that variability in aggressiveness exist among Fov isolates. Isolates 3, 4, 6, and 7 with cultivar FM 9058F showed significantly higher AUDPC in the absence of M. incognita at higher inoculum densities of Fov ( cfu/cm 3 ) compared to other Fov isolates tested which implies that disease incidence may be higher even in the absence of M. incognita when Fov inoculum densities in soil are high. Isolates 5 and 11 of Fov had significantly (p = 0.05) higher AUDPC in the presence of M. incognita (1000 eggs/pot) at lower inoculum density ( cfu/cm 3 ) than other Fov isolates tested suggesting, the rootknot nematodes were required to cause significant wilt incidence when inoculum densities of Fov were low in soil. Plant growth and symptoms expressions were affected by the susceptibility of cultivar. Susceptible cultivar, FM 9058F had significantly stunted plants, decreased root weight, shoot weight and total plant weight as compare to partially resistant cultivar, ST 4554B2RF. Plants inoculated with M. incognita (1000 eggs/pot) had root galls and were stunted with reduced shoot weight and total plant weight. Results from this study show that variability in aggressiveness exists among Fov isolates of West Texas. To examine the impact of planting combinations of susceptible (FM 9058F) and/or partially resistant (ST 4554B2RF) cotton cultivars, on soil inoculum density of Fov a microplot study was conducted over the 2008 to 2010 growing seasons. The hypothesis was that due to the use of resistant cultivar, soil inoculum densities of Fov and disease incidence will decrease over time. Field soil naturally infested with Fov was added to microplots in 2008 and M. incognita density was augmented each spring before 144

159 planting. Inoculum density of Fov increased to cfu/cm 3 after 3 yrs of FM 9058F (SSS indicates 3 years of a susceptible cultivar); however, Fov density decreased to cfu/cm 3 after 3 yrs of ST 4554B2RF(RRR indicates 3 yrs of a partially resistant cultivar). Soil inoculum density in the microplots initially planted with ST 4554B2RF followed by FM 9058F planted for the next two years (RSS) was not different ( cfu/cm 3 ) from those planted to FM 9058F for three years (SSS) ( cfu/cm 3 ). Microplots planted with a SRR rotation had similar Fov densities ( cfu/cm 3 ) from those planted to a RRR rotation ( cfu/cm 3 ). Disease incidence increased from 18% in 2008 to 69% in 2010 with FM 9058F. Disease incidence was a function of both the cultivar grown in that season and preplant Fov soil density. Yield was highest after 3 yrs of ST 4554B2RF and lowest after 3 yrs of FM 9058F. Yield was related primarily by the current year cultivar, pre-plant Fov density in soil and cultivar history. Results from this study indicate that cultivar selection is the cornerstone for Fusarium wilt management in the fields infested with Fov and M. incognita. 6.2 CONCLUSION Verticillium and Fusarium wilt of cotton are not easily distinguishable under field conditions; thus, proper diagnosis is required for their management. Pre-plant soil inoculum densities of Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum play a critical role in disease development and lint yield. Therefore, strategies that influence soil inoculum densities help in managing both the diseases. Removal of V. dahliae infested residue help to prevent the accumulation of microsclerotia in soil. In Texas, variability in aggressiveness exists among Fov Race 1 isolates. Planting of 145

160 resistant cultivar is the cornerstone for managing both the diseases. The choice of cultivar is the single most important decision growers can make in an integrated crop management system to reduce the production costs in the fields infested with V. dahliae and/or Fov and M. incognita for sustaining the cotton production. 146

161 APPENDIX 147

162 Figure 1.1. Microsclerotial colony of Verticillium dahliae on semiselective media under disecting microscope. 148

163 A B C Figure 1.2. A. Morphology of Microconidia, B. Macroconidia, and C. Chlamydospores of Fusarium oxysporum f. sp. vasinfectum under compound microscope at 100X magnification. 149