The Pennsylvania State University. The Graduate School. College of Agricultural Sciences FACTORS INFLUENCING THE DEVELOPMENT OF GRAY LEAF SPOT OF

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1 The Pennsylvania State University The Graduate School College of Agricultural Sciences FACTORS INFLUENCING THE DEVELOPMENT OF GRAY LEAF SPOT OF PERENNIAL RYEGRASS TURF AND SEASONAL AVAILABILITY OF THE INOCULUM A Dissertation in Plant Pathology by Yinfei Li Yinfei Li 2013 Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2013

2 ii The dissertation of Yinfei Li was reviewed and approved* by the following: Wakar Uddin Professor of Plant Pathology Dissertation Co-Advisor Co-Chair of Committee John Kaminski Associate Professor of Turfgrass Science Dissertation Co-Advisor Co-Chair of Committee Beth Gugino Assistant Professor of Plant Pathology Scott Isard Professor of Aerobiology James Rosenberger Professor of Statistics Frederick Gildow Professor of Plant Pathology Head of the Department of Plant Pathology and Environmental Microbiology *Signatures are on file in the Graduate School

3 iii ABSTRACT Gray leaf spot, caused by Magnaporthe oryzae Couch, is a devastating disease of perennial ryegrass turf. Severe disease epidemics have been reported in various regions of the United States, and it has been most prevalent in the northeastern region. Airborne conidia of M. oryzae serve as the major inoculum for the development of disease epidemic that can cause extensive damage on perennial ryegrass turf under favorable environmental conditions. However, the effects of environment on infection, fungal colonization, disease development and conidiation are not fully understood. Therefore, understanding the role of environmental factors on inoculum and disease development of gray leaf spot of perennial ryegrass turf is warranted. The objective of the first study was to determine the effects of intermittent and interrupted leaf wetness periods on incidence and severity of gray leaf spot of perennial ryegrass turf. Results indicated significant effects of wet and dry cycles on disease incidence and severity. Under optimum temperature (28 C), the highest disease incidence and severity were observed on plants exposed to 18h continuous leaf wetness. The frequencies and durations of dry periods significantly reduced gray leaf spot incidence and severity. Further, strong correlations were found between frequency and duration of dry periods and disease incidence or severity reduction rates. Interruption of leaf wetness durations with longer dry periods significantly reduced gray leaf spot incidence and severity. Additionally, there were negative correlations between the length of interrupted dry periods and gray leaf spot incidence or severity. The relationship was best described by a quadratic model for disease incidence and a linear model for disease severity.

4 iv Results of this study indicate that turfgrass canopy moisture management strategies may be employed as an important cultural practice as part of a gray leaf spot integrated disease management program in perennial ryegrass turf. In the second study, the effects of relative humidity (RH, 85% to 100%) on infection, colonization, and conidiation by M. oryzae were investigated at 28ºC in controlled environment chambers. Results of the study showed that the RH threshold for successful M. oryzae infection was 91% at 28 C. The fungal biomass from colonized leaf tissue increased with increasing levels of RH. Green fluorescent protein-tagged M. oryzae strains provided a rapid and accurate method for visual and quantitative determination of the fungal colonization. Further, daily conidiation rates were quantified at 88% to 100% RH. The most abundant conidiation of M. oryzae was found at eight days after inoculation at 100% RH. Reduced conidiation was associated with decreased RH, and no conidiation occurred at RH of 91% or lower. This study showed that relative humidity at >90% is required for infection of perennial ryegrass plants by M. oryzae and the subsequent disease development under the favorable temperature regime in the gray leaf spot pathosystem. The results from this research can be integrated to improve the current disease forecasting model. The objectives of the third study were to examine the availability and the dispersal pattern of conidia in the field, and to determine the relationship between certain environmental parameters and the concentration of airborne inoculum. The field monitoring of airborne conidia was conducted in Dillsburg and Leesport, Pennsylvania in 2010 and Analyses of the air samples indicated that airborne M. oryzae conidia were present at all sampling sites. Average hourly conidia counts indicated that the peak

5 v concentration was generally observed during the early part of the day (0500 to 0900 hour). Concurrent occurrence of both high concentration of airborne conidia (>1000 conidia/m 3 ) and favorable environmental condition (percent infection index>90) was always confirmed before the reported disease epidemics which is the important determinant for predicting disease epidemics. Correlation analyses between environmental parameters and conidia concentration indicated that the temperature may be used to forecast initial inoculum availability and humidity levels may be a critical indicator for the hourly conidia concentration. Results of this study will be employed as a component of the current gray leaf spot forecast system based on inoculum level.

6 vi TABLE OF CONTENTS List of Figures... viii List of Tables... x Acknowledgements... xi Chapter 1 Literature Review... 1 Introduction... 1 The causal agent, Magnaporthe oryzae... 2 The inoculum... 3 Propagules of Magnaporthe oryzae... 3 Conidiation of Magnaporthe oryzae... 4 The airborne pathogen: Magnaporthe oryzae... 5 Magnaporthe oryzae and host plant interaction... 7 Host attachment and pre-penetration process... 7 Penetration, infection and colonization... 9 Green fluorescent protein and its application in fungus-host interaction Epidemiology and management of gray leaf spot Epidemics of gray leaf spot Disease forecast system Disease management Chapter 2 Effects of Intermittent or Interrupted Leaf Wetness on Disease Development of Gray Leaf Spot of Perennial Ryegrass Turf Abstract Introduction Materials and Methods Growth of perennial ryegrass and inoculation with M. oryzae Intermittent leaf wetness study Interrupted leaf wetness study Disease assessment and data analysis Results Effects of intermittent leaf wetness Effects of interrupted leaf wetness Effects of intermittent or interrupted leaf wetness on incubation periods Discussion Chapter 3 Effects of Relative Humidity on Infection, Fungal Colonization and Conidiation of Magnaporthe oryzae on Perennial Ryegrass Turf Abstract Introduction... 42

7 vii Materials and Methods Growth of perennial ryegrass and preparation of detached leaf blades Transformation of M. oryzae and preparation of conidia suspension Preparation of humidity chambers Effect of relative humidity on fungal infection Effects of relative humidity on inoculum development Effect of relative humidity on colonization of perennial ryegrass leaf tissue by M. oryzae Effect of relative humidity on conidiation of M. oryzae on perennial ryegrass Results Infection of M. oryzae on perennial ryegrass Evaluation of advancement of inoculum on perennial ryegrass Quantitative assessment of fungal biomass from infected leaf tissues Conidiation at various levels of relative humidity Discussion Chapter 4 Seasonal and Daily Patterns of Magnaporthe oryzae Conidia Availability in the Gray Leaf Spot Pathosystem and Relationship between Conidia Concentration and Environmental Parameters Abstract Introduction Materials and Methods Sampling sites and weather data collection Air spore samplers and quantification of airborne conidia Data analyses Availability of conidia in sampling fields Concurrence of favorable environment conditions and high availability of inoculum Relationship between average hourly environmental parameters and average hourly conidia concentration Discussion Chapter 5 Summary and Future Directions References Appendix: Statistical analyses Chapter Chapter Chapter

8 viii List of Figures Figure 2.1. Schematic diagram for the design of intermittent leaf wetness with different combinations of wet (gray) and dry (black) periods Figure 2.2. Schematic diagram for the design of interrupted leaf wetness with increasing duration of drying periods from 3 h to 18 h Figure 2.3. Effects of intermittent leaf wetness periods (T m 1-T m 6) on incidence or severity of gray leaf spot. Disease incidence was represented in Experiment 1 (A) and Experiment 2 (B) and disease severity was represented in Experiment 1 (C) and Experiment 2 (D). Disease incidence and severity were evaluated 10 days after inoculation. Each column is the mean of 4 plants and error bars represent standard error of the mean. Significant differences found between treatments (P 0.05) according to Student-Newman-Keuls (SNK) test Figure 2.4. Effects of interrupted leaf wetness periods (T r 1-T r 6) on incidence or severity of gray leaf spot. Disease incidence was represented in Experiment 1 (A) and Experiment 2 (B) and disease severity was represented in Experiment 1 (C) and Experiment 2 (D). Disease incidence and severity were evaluated 10 days after inoculation. Each column is the mean of 4 plants and error bars represent standard error of the mean. Significant differences found between treatments (P 0.05) according to Student-Newman-Keuls (SNK) test Figure 2.5. The relationship between interrupted dry periods and disease incidence or severity of gray leaf spot. Disease incidence was represented in Experiment 1 (A) and Experiment 2 (B) and disease severity was represented in Experiment 1 (C) and Experiment 2 (D). Y Inc is the disease incidence, Y Sev is the disease severity and T d is the dry period in hours Figure 2.6. The relationship between incubation period and disease incidence or severity of gray leaf spot. Disease incidence was represented in Experiment 1 (A) and Experiment 2 (B) and disease severity was represented in Experiment 1 (C) and Experiment 2 (D). Y Inc is the disease incidence, Y Sev is the disease severity and T i is the incubation period for the initial symptom in days Figure 3.1. The humidity chamber with glycerol solution, devised for relative humidity experiment. A. Profile picture B. Each chamber contained 10 detached leaf blades Figure 3.2. Effects of relative humidity on infection and colonization of perennial ryegrass tissue by Magnaporthe oryzae. Microscopic images were taken 10 days after inoculation at RH from 85% to 100% at 3% intervals. A. No colonized mycelia observed at 85% RH. B. No colonized mycelia observed in the perennial ryegrass leaf tissues at 88% RH. C. Colonized mycelia (indicated by the red arrow) observed at 91% RH. D. Colonized mycelia

9 ix observed at 94% RH. E. Massive mycelial colonization observed at 97% RH. F. Massive mycelial colonization observed at 100% RH Figure 3.3. Confocal microscopic images of advancing Magnaporthe oryzae on perennial ryegrass leaf tissues. A. The intact conidium. B. The germinated conidium with germ tube only. C. The germinated conidium with germ tube and appressorium. D. Invasive hyphae. E. The conidium with degraded cell(s) Figure 3.4. Effects of relative humidity on growth and development of Magnaporthe oryzae during the infection process on perennial ryegrass leaf tissue. Percentage of inoculum development into various stages was recorded daily for the first 4 days after inoculation. A. Experiment 1. B. Experiment Figure 3.5. Effects of relative humidity on the biomass change of Magnaporthe oryzae during the infection on the perennial ryegrass leaf tissue using the measurement of GFP fluorescence intensity (mean ± SE). A. Experiment 1. B. Experiment Figure 3.6. Effects of relative humidity on conidiation (mean ± SE) of Magnaporthe oryzae during the infection process in gray leaf spot pathosystem. A. Experiment 1. B. Experiment Figure 4.1. Seasonal availability of conidia at perennial ryegrass fairways in Site 1 and Site 2 in 2010 and Figure 4.2. Summary plot of mean ± SE hourly conidia concentration compiling 2-year highest total conidia concentration in each sampling site Figure 4.3. The relationship between mean hourly conidia concentration and hourly temperature or hourly relative humidity at Site 1 in Figure 4.4. The relationship between mean hourly conidia concentration and hourly temperature or hourly relative humidity at Site 1 in Figure 4.5. The relationship between mean hourly conidia concentration and hourly temperature or hourly relative humidity at Site 2 in Figure 4.6. The relationship between mean hourly conidia concentration and hourly temperature or hourly relative humidity at Site 2 in

10 x List of Tables Table 2.1. Analysis of variance of disease incidence and severity of intermittent leaf wetness treatments Table 2.2. Analysis of variance of disease incidence and severity of interrupted leaf wetness treatments Table 2.3. Analysis of variance for reduction of disease incidence with various frequencies (F) and durations (D) of dry periods Table 2.4. Statistical model selection of goodness of fit for the relationship between duration of interrupted dry periods (T d ) and disease incidence or severity (α=0.05) Table 2.5. Statistical model selection of goodness of fit for the relationship between incubation periods (T i ) for the initial symptom development and disease incidence or severity (α=0.05) Table 3.1. Acquired relative humidity within humidity chambers controlled with varying glycerol solution concentrations and incubated at 28 C Table 4.1. Linear regression analysis with autoregressive errors of meteorological parameters with the average hourly conidia concentration from two sampling locations in Pennsylvania in 2010 and Table 4.2. Autoregressive analysis of temperature and relative humidity with the average hourly conidia concentration from two sampling locations in Pennsylvania in 2010 and

11 xi Acknowledgements I would like to acknowledge my committee members Dr. Wakar Uddin, Dr. John Kaminski, Dr. Scott Isard, Dr. James Rosenberger, and Dr. Beth Gugino for their support of my research: I especially thank my advisor, Dr. Wakar Uddin, who has provided me with this great opportunity to pursue my Ph.D. study at Penn State. Additionally, I thank the faculty, staff and students of the Department of Plant Pathology and Environmental Microbiology for their support and assistance throughout my time as a graduate student. Particularly, I thank all the members of Dr. Wakar Uddin s turfgrass disease research and education program, who have assisted me with my projects. My research could not be accomplished without the assistance from Dr. Nicholas Dufault, Dr. Joe Russo, Jay Schlegel, Brian Aynardi, Dr. Marcus Ludwig, Dr. Seogchan Kang, Dr. Hye-Seon Kim, Alamgir Rahman, Missy Hazen, Fei Gan, Dr. Gary Moorman, Pete Ramsey, Brian Hiester, Dr. Ran Liu, Dr. Deepak Jaiswal and Nancy Wenner. I also thank Gary Nolan, Brian Aynardi, Anna Testan, Sarah Bardsley and Dr. Jill Demers providing me with assistance during the prepararion of the draft of this dissertation. Personally, I would like to thank my family for their moral support and unconditional love throughout my life. Finally, I must thank Dr. Marcus Ludwig for his support over the past four years and in the future.

12 1 Chapter 1 Literature Review Introduction Gray leaf spot, caused by Magnaporthe oryzae Couch (anamorph: Pyricularia oryzae Cavara), is a foliar disease of many grass species (95). It was first reported in the southeastern United States on St. Augustinegrass (Stenotaphrum secundatum) in 1957 (29, 62) and on annual ryegrass (Lolium multiflorum) in the 1970s (4, 16). In 1985, gray leaf spot on perennial ryegrass was first reported in Maryland (22). Other known turfgrass hosts include fescues (Festuca species), bermudagrass (Cynodon dactylon), centipedegrass (Eremochloa ophiuroides) and kikuyugrass (Pennisetum clandestinum) (95, 125). The disease symptoms of gray leaf spot are characterized by initial water-soaked lesions which may become grayish or light brown often with a yellow halo. The lesions later turn light brown or gray when heavy conidiation occurs on the lesion surface. Finally, lesions on the leaf blade coalesce causing the entire grass blade to blight. Additionally, twisting or flagging of leaf tips occurs, as a result of the disruption of plant vascular tissue. Gray leaf spot of perennial ryegrass causes the most extensive damage and widest distribution primarily because of its widespread use on golf courses, athletic fields, and home lawns (31, 117). It has been subsequently reported in numerous regions

13 2 including the northeast, mid-atlantic, the Midwest, and some western regions in the United States (33, 51, 83, 92, 112, 124). Perennial ryegrass is a cool-season, bunch-type grass with many agronomic advantages including dark green color, rapid establishment, adaptation to close-mowing and toleration to soil compaction (31). All these attributes have enabled perennial ryegrass to become a widely-used turfgrass species since the 1980s, especially in the upper transition zone of the United States (117). However, perennial ryegrass cultivars currently lack resistance to gray leaf spot. Therefore, extensive damage and frequent epidemics can occur in perennial ryegrass fairways, athletic fields, and home lawns which has caused increased fungicide applications; thus increased turf management budgets. The causal agent, Magnaporthe oryzae Magnaporthe oryzae has been well known as the causal pathogen of rice blast disease, the earliest of which was recorded in the Ming Dynasty in China (79). To date, M. oryzae has been identified to be pathogenic to over 50 gramineous plants (79). Other reported non-gramineous hosts are mostly monocotyledons, including Giant Cavendish banana fruits and ornamental plants within the family Marantaceae, such as Ctenanthe spp. and one known dicotyledon, Arabidopsis thaliana (68, 80, 81). Isolates from various M. oryzae hosts are morphologically identical (89). Crosses between isolates may be successful if germinable ascospores are produced. Previously, the isolates of M. oryzae from rice plants have been considered to be genetically distinct from isolates from non-rice hosts. Therefore, two species names have been proposed:

14 3 Pyricularia oryzae for isolates from rice plants and Pyricularia grisea for isolates from non-rice host plants (27, 77, 91). However, the overlapping has been found between these two species from the aspects of conidial morphology and inter-fertility (127). Therefore, Rossman et al. have integrated Pyricularia oryzae Cav. under the name of Pyricularia grisea (Cooke) Sacc. (teleomorph Magnaporthe grisea (Hebert) Barr) (89). The latest phylogenetic analysis based on multilocus gene genealogy have indicated Magnaporthe oryzae as a new species name to describe the isolates causing rice blast and gray leaf spot on grasses except the genus Digitaria (21). Additionally, the isolates appear to exhibit host specificity. For example, isolates from perennial ryegrass contain AVR1-CO39 (a host specificity gene that confers avirulence to rice cultivar) fail to cause disease on rice cultivar CO39 (28, 84, 119). The inoculum Propagules of Magnaporthe oryzae Conidia of M. oryzae are the primary and secondary inoculum of gray leaf spot. The conidium is pyriform with a basal appendage at the point of attachment to the conidiophores. One-celled young conidium, produced on the conidiophores, first divides into two cells, then into a three-celled mature conidium that is µm in size (13, 27). In the mature conidium, each cellular compartment contains one nucleus. The perfect stage of M. oryzae has not been discovered in nature, but has been developed in vitro. Fusiform, hyaline and four-celled ascospores are produced inside cylindric to subclavate

15 4 asci which are found inside the long-beaked perithecia (35, 128). Although the fertility of the isolates from perennial ryegrass and St. Augustinegrass was confirmed in laboratory setting, the low level of fertility indicates that the role of ascospores in the disease cycle may be insignificant (118). Conidiation of Magnaporthe oryzae Conidia of M. oryzae are produced by the conidiogenous cells, which originate from pigmented hyphae, also commonly known as conidiophore mother hyphae (74). Other non-pigmented hyphae are known as vegetative hyphae. The conidiation initiates from the development of vesicle swellings on conidiophore stalks followed by formation of sympodial conidial production, and finally, the release and dispersal of conidia. A sympodial proliferation pattern is found to be closely related to the pathogenesis of M. oryzae, and acropetal mutants of M. oryzae have a 90% lower probability of appressorium formation and no spore tip mucilage production (54). Conidiation of M. oryzae has been researched in vitro using different substrata such as synthetic or non-synthetic media as well as sorghum seeds (49, 74). Abundance of conidia has been found on 3 to 4-day old hyphae in oatmeal agar, rice-polish, wheat, sorghum and oat-sorghum (38, 74). The conidial deposition on the agar plate from a rice blast leaf lesion is an oval circle with a bare center thus indicating the heaviest conidiation occurs at the lesion margin (5). Sporulation in the rice blast pathosystem only occurs from 9 C to 35 C with the optimum temperature C (38).

16 5 Conidiation of M. oryzae on rice seedlings is reportedly influenced by environmental factors. The optimum temperature (28 C) for M. oryzae conidiation on rice-polish agar and wheat and the maximum sporulation was observed nine days after inoculation (38, 102). The humidity level also determines conidiation capability. Conidiation was found to be profuse when the relative humidity was above 93% (37). Light generally induced the formation of aerial hyphae and conidiophores (57). Conidiation of M. oryzae has been stimulated by long exposures of artificial daylight from fluorescent lamps or incandescent lamps or from exposure to natural daylight. However, under darkness conidiation of colonies grown on the different media was significantly reduced (56). The airborne pathogen: Magnaporthe oryzae One hypothesis for release mechanism of M. oryzae conidia is that conidia are actively discharged by stalk-cell bursts (44). The dispersed conidia of M. oryzae have been observed at the distance of a fraction of a millimeter from the conidiophores with the upper half of the stalk cell attached (44). Environmental factors may also play an important role in the spore release process. High humidity conditions may be responsible for the turgor pressure buildup inside the stalk-cell, which explains that high availability of conidia under high humidity conditions (44, 68). The conidia release of M.oryzae was triggered by either increasing levels (from 24% to 100%) or decreasing levels (from 100% to 24%) of relative humidity; the maximum conidia release has been found when relative humidity levels increase from 76% to 100% (55). Another important factor for

17 6 release of M. oryzae conidia is light. Subdued light (10 µmol/s m 2 ) reportedly suppresses the release of M. oryzae conidia(57). Constant blue light and red light have been proven to induce higher conidia release compared to constant white light or darkness, and the transition from a light to a dark period also induces the release of conidia (57). The minimum wind speeds for spore release, however, have been reported to vary among different fungi (82). A minimum wind speed of 0.5 m/s was found for the spore release of Aspergillus fumigatus and Penicillium spp. from conidiophores. However, there is no evidence indicating that wind speed influences the conidia release of M. oryzae. Released spores can escape from the plant canopy and are dispersed or transported to sites at various distances. This is essential for the spread of the disease and perpetuation of the disease epidemic (3). Intermittent wind plays a major role in spore removal and canopy escape which shapes disease gradients for aerially dispersed fungal pathogens (3). Various air sampling techniques have been employed to estimate the concentration of M. oryzae conidia in rice production regions in Los Baños, Laguna, Philippines. Volumetric air sampling methods have been found to be more efficient than passive deposition methods, particularly when aerial conidia concentrations are relatively low (85). Through the use of a volumetric air sampler, a strong relationship between the number of conidia and rice blast disease progress has been reported in a multiple-year study (85). Increase of temperature may be one of the factors leading to the high airborne spore concentration of M. oryzae during the rice growing season (73). The relationship between spore release events and environmental parameters may vary based on fungal genera, crop management conditions, and host types (40, 104). Daily spore release patterns for M. oryzae have been well described in the rice blast

18 7 pathosystem. Among them is a nocturnal pattern of M. oryzae spore release observed under high concentration at night (5, 46, 55, 68). Also, bimodal distribution of conidia release has been discovered with changing humidity during the darkness (46, 55). Furthermore, peak periods of spore availability may vary under different environmental conditions. In one study a single peak from 0500 to 0700 hour on a clear day or multiple peaks (usually 2-3) from 0100 to 0900 hour on a cloudy day have been identified (46). In another study, spore concentration studies in the pathosystem of pitting disease of bananas indicate the diurnal pattern with the peak concentration at 0400 hour (68). Magnaporthe oryzae and host plant interaction Host attachment and pre-penetration process Host attachment is a requirement for infection by many phytopathogenic fungi (65). The conidium of M. oryzae develops an irreversible attachment mechanism on the hydrophobic waxy leaf surface (e.g. rice) (48). The adhesive periplasmic deposit (also known as spore tip mucilage) is secreted from the ruptured outer spore wall after the hydration of conidia (30). No common adhesive chemicals are confirmed for all the phytopathogenic fungi. The adhesion is usually contributed by water-insoluble glycoproteins, although lipids and polysaccharides are also found in adhesives (75, 76, 98, 126). Host attachment is affected by various environmental and host plant factors. Moisture, in form of dew, on the surface is required for hydration of the conidium (30).

19 8 Temperature and UV light may influence the respiration and metabolism of conidia which determines conidial adhesion-competency (67, 93). For example, incubation of Colletotrichum musae conidia at 24 C for 5 minutes had a similar attachment rate of incubation at 1 C for several hours (93). With the age of the conidia the attachment ability decreases due to the decline of adhesive compounds (66). The attachment surface is another influential factor. On artificial smooth surfaces, 90% of the conidia are resistant to removal 20 minutes after inoculation (30). However, on a rice leaf surface, conidia cannot attach to the surface directly due to the blocking of surface wart-like protuberances. Therefore, conidia develop their germ tubes first (at least 3 µm) and then attach to the surface. That reportedly results in relatively low and slow conidia attachment rate with only 5% attachment 1 hour after inoculation (48). After attaching on the leaf surface, conidia start germination, followed by the formation of appressoria (43). Most of conidia germinate from apical cells to form the germ tubes. During the germination process, the nucleus migrates to the germ tube and performs mitosis. When the growth tip of the germ tube stops, the appressorium starts to develop. One daughter nucleus moves into the newly-formed appressorium and the second daughter nucleus moves into the conidium. Only one nucleus is present in the appressorium and all other nuclei are subject to degradation via autophagic cell death within the conidium 24 hours after the infection (13). Conidia germination and appressorial formation can be inhibited by lipids from the lipophilic surface of conidia when the conidia are highly concentrated (> /ml) (36). The inhibition can be relieved by the hydrophobic epicuticular wax or wax-like components (36, 58). More differentiation of the appressoria has been observed on the rice surface compared to

20 9 artificial surfaces (45). This mechanism assists the survival of conidia until they can attach to the host surface under favorable conditions. Light plays a positive role on germ tube and appressorial differentiation, and the light irradiation with higher photon flux density induces a lower number of differentiated long germ tubes (45). Penetration, infection and colonization The penetration of M. oryzae during the infection process has been established as a direct pathogen penetration model (43). The melanization of the appressorium cell wall produces high osmotic pressure for the penetration process. A single penetration peg develops eccentrically with the appressorium pore which is the wall-less region close to the substrate. After the penetration peg appears, the infection hyphae grow inside the leaf tissue to colonize the host (9, 43). Hemibiotrophic infection of M. oryzae starts when hypha-like structures radiate from the point of the penetration peg. Hyphae invade host cells biotrophically moving from cell to cell via plasmadesmata (9). As long as they start to invade the surrounding cells, infection hyphae are non-biotrophically related with invaded cells (26). Once the hyphae are established inside the leaf tissue, conidiophores develop on the leaf surface and conidiation may be initiated. Upon conidiation, the conidia are dislodged and become airborne serving as the secondary inoculum furthering infection thus spreading the disease. Root infection by M. oryzae has also been observed in rice plants (94). After colonization of the roots by M. oryzae, the fungus continues the systemic invasion in the

21 10 host, causing development of the blast disease symptoms on the aerial parts of the plant. Specific root penetration structures, hyphopodia and microsclerotia, have been observed on the root surface of rice plants. However, melanization is reportedly not required for the pathogen penetration process compared to foliar infection (94). Environmental factors such as temperature and leaf wetness duration are vital in governing the development of gray leaf spot and interaction of M. oryzae and host plants (6, 70, 108). In the gray leaf spot pathosystem, 28 C was found to induce the highest disease incidence and severity of gray leaf spot. Under favorable temperature regimes, disease incidence and disease severity were observed most severe if the leaf wetness was continuous for 18 h or longer (108). M. oryzae infection on annual ryegrass showed the highest disease incidence and severity occurred at 26ºC and 24-hour leaf wetness duration (70). Green fluorescent protein and its application in fungus-host interaction Green fluorescent proteins (GFP) from jellyfish Aequorea victoria have been widely adapted for monitoring gene expression, protein localization, and vital visualization (63, 96, 105, 116). GFP can be expressed in bacterial, fungal, plant, and animal cells by various transformation systems. An Agrobacterium tumefaciens-mediated transformation (ATMT) system has been used for transforming several fungi by insertional mutagenesis (71, 72). A. tumefaciens can transform protoplasts, hyphae, spores and blocks of mushroom mycelia tissues (71). Recently, high level expression of GFP and bright fluorescence have been reported in the cytoplasm of M. oryzae, a rice

22 11 isolate, by using ATMT (88). The expressed GFP can produce a strong and stable green fluorescence and photobleaching only occurs when the cells are illuminated with 340 to 390 nm or 395 to 440 nm light (17). Visualization of fungal development and distribution is facilitated by GFP expressed in fungal cytoplasm (42, 60). Using GFP as a marker does not affect the examined plant tissue which always occurs during the traditional staining techniques caused by the substrate uptake. Therefore, GFP can provide real-time microscopic observation. Laser scanning confocal microscopy and conventional fluorescence microscopy are efficient tools to observe GFP-tagged fungus. Compared with conventional fluorescence microscopy, confocal laser scanning microscopy provides higher resolution and flexibility for image generation, processing and analysis. Furthermore, GFP fluorescence intensity is found directly proportional to fungal biomass, such as Cochliobolus heterostrophus inside maize leaves, which suggests a quick and accurate method to quantify the amount of colonized fungi (18, 63). Epidemiology and management of gray leaf spot Epidemics of gray leaf spot Generally, serious epidemics of gray leaf spot often occur on golf courses, athletic fields, and home lawns in a cyclic fashion (101, 107). Outbreaks of gray leaf spot on perennial ryegrass turf are usually reported during the late summer from late July to early September in the northeastern United States (113). The pathogen reportedly overwinters

23 12 as mycelia in plant debris in the soil or in live plant tissue (100). Under a laboratory setting, M. oryzae could maintain its viability years at -18ºC if it is sub-cultured regularly (53). However, mutation and virulence loss may occur during multiple transfers on artificial media such as potato dextrose agar and oatmeal agar. Therefore, the primary inoculum of gray leaf spot could first develop from overwintering mycelia in high-cut perennial ryegrass rough areas in golf courses. These areas are not as intensely managed as fairways and prolonged leaf wetness periods always occur due to the higher mowing height of the turf. Under favorable environmental conditions, production of conidia may be initiated on the necrotic lesions or blighted leaf blades building up inoculum for secondary infection. Possible means of inoculum dispersal are by equipment, irrigation, wind, rain water-splash or windblown rain. Additionally, the conidia can also be tracked by human and animal traffic on golf courses. Transmission of the pathogen through seeds has not been reported. Disease forecast system Plant disease forecast systems can assist growers in making economic decisions with regard to management costs and chemical applications (1). Predictive models are commonly developed for disease forecasting which take into account both environmental and biological factors. Most forecasting systems focus on disease symptom development by monitoring certain favorable environmental conditions conducive to disease infection. These disease forecasts can usually provide hourly or daily information available for growers, which provide background information on the disease, real-time disease

24 13 epidemics and management recommendations. Currently, only two major weather determinants have been identified for the gray leaf spot forecast system which include temperature and leaf wetness duration (108). Gray leaf spot can develop during periods of moderate to warm temperatures ranging from 20ºC to 32ºC. However, the most favorable temperature is 28ºC. An extended leaf wetness period is required for disease development. Furthermore, disease incidence or severity increases with the increase of leaf wetness duration. Moreover, information on availability of initial inoculum, favorable weather conditions for production of secondary inoculum, or both initial and secondary inoculum may also contribute to development of disease forecast systems (19, 24). However, such information is currently not available for the gray leaf spot forecast system. Disease management The study of plant disease epidemiology generates numerous disease management strategies. In the gray leaf spot of perennial ryegrass pathosystem, management approaches are generally based on cultural and chemical strategies due to the lack of truly resistant cultivars in the market (7). Avoiding excessive nitrogen fertilization is commonly recommended (12, 111). Common cultural practices include scheduling early morning irrigation and proper mowing height and timing to keep turf canopy open and dry (12). Although it is costly to control gray leaf spot by fungicide applications, it is still the most effective means of managing the disease. The most commonly used fungicides in managing gray leaf spot include contact and systemic fungicides such as chlorothalonil

25 14 and strobilurin (103, 109, 122). Due to the heavy reliance on chemical applications, there are reports of field resistance development to azoxystrobin within the pathogen population (122). Therefore, further investigation of disease epidemiology to develop innovative and sustainable integrated management strategies for managing gray leaf spot of perennial ryegrass are necessary.

26 15 Chapter 2 Effects of Intermittent or Interrupted Leaf Wetness on Disease Development of Gray Leaf Spot of Perennial Ryegrass Turf Abstract Gray leaf spot, caused by Magnaporthe oryzae Couch, is a devastating disease of perennial ryegrass (Lolium perenne L.) turf. Severe disease outbreaks have been reported from various regions of the United States. The effects of intermittent and interrupted leaf wetness on incidence and severity of gray leaf spot were investigated in the controlled environment chambers. The intermittent leaf wetness study was devised with treatments including different wet and dry periods. There were significant effects of intermittent leaf wetness duration on disease incidence and severity (P<0.0001). Additionally, the frequency and duration of dry periods for intermittent leaf wetness significantly influenced the reduction of disease incidence and severity (P<0.0001). Further, given the same duration of initial and final wet periods, the length of interrupted dry periods significantly influenced gray leaf spot incidence and severity (P<0.0001). The negative relationship between the length of interrupted dry periods (T d ) and gray leaf spot 2 incidence or severity was best described by a quadratic model (Y = T d +0.05T d ; r 2 =0.92, P<0.0001), and a linear model (Y= T d ; r 2 =0.92, P<0.0001), respectively.

27 16 Results of this study indicate that turfgrass canopy moisture management strategies may be employed as part of the integrated management program in the gray leaf spot pathosystem of perennial ryegrass turf.

28 17 Introduction Gray leaf spot, caused by Magnaporthe oryzae Couch (anamorph: Pyricularia oryzae Cavara), is a devastating foliar disease of perennial ryegrass (Lolium perenne L.) (34). Due to the extensive damage to perennial ryegrass fairways and rapid development of the disease in various regions of the United States, the disease has drawn serious attention in the turfgrass industry over the past two decades (113, 120). Perennial ryegrass is generally a desirable turfgrass species in the golf industry due to its select agronomic attributes such as quick germination and establishment, tolerance to closemowing, rapid tillering, high density, upright growth, and dark green color (31). Therefore, it is a common turfgrass species on golf course fairways and roughs as well as athletic fields and home lawns. However, perennial ryegrass is not being utilized to its full potential due to its susceptibility to M. oryzae (86, 95, 106). Gray leaf spot is prevalent on golf courses in the United States partly due to few resistant perennial ryegrass cultivars available in the market (7, 8). Management of gray leaf spot is chiefly achieved by fungicide programs including broad spectrum contact and systemic fungicides. Effective fungicides, such as azoxystrobin, have been extensively applied which has resulted in the development of field resistance by M. oryzae (61, 122). Therefore, integration of all possible management practices is suggested to develop more sustainable gray leaf spot management strategies. Cultural practices such as the utilization of appropriate nitrogen sources and rates as well as proper irrigation schedules are instrumental in reducing the severity of gray leaf spot (12, 64, 111).

29 18 Identifying novel cultural practices and improving the timing of fungicide applications are critical for accurate and economical gray leaf spot management programs. Management of plant diseases is often more effective when predictive models are available to forecast the disease outbreaks (113). In the gray leaf spot pathosystem, 28 C was found to induce the highest disease incidence and severity of gray leaf spot (108). Under favorable temperature regimes, the greatest disease incidence and severity occur when leaf wetness is present for 18 h or longer (108). The current gray leaf spot forecasting model provides turfgrass professionals with an index of disease potential which is based on temperature and the duration of continuous leaf wetness. Therefore, turfgrass professionals can time fungicide applications more accurately and achieve greater disease control. The occurrence of continuous leaf wetness, however, may not always reach 18 h in nature or in practice. As a late summer disease, gray leaf spot development may be influenced by intermittent leaf wetness in an alternate fashion of wet and dry events or varying lengths of interruption of continuous leaf wetness duration on the leaf surface. This could be caused by individual or combined periods of morning dew, rain events, or irrigation. Effects of intermittent or interrupted leaf wetness on disease development have not been well described in the gray leaf spot pathosystem. The influence of wet and dry cycles has been documented in other crops. For example, the dry periods can significantly reduce disease severity of Ascochyta blight in chickpea if the dry period was imposed at 8 h after inoculation or earlier (2). The duration and relative humidity levels

30 during dry periods have also been shown to influence lesion development of Cerospore blight of carrot and olive leaf spot respectively (14, 78). 19 Investigating gray leaf spot development under alternate wet and dry periods or interrupted dry periods could provide insights into the development of innovative practices for gray leaf spot management. Additionally, the influence of intermittent or interrupted leaf wetness on disease incidence and severity could improve the current disease forecasting system. Therefore, the objective of this study was to research the quantitative relationship between intermittent or interrupted leaf wetness durations and disease incidence or severity of gray leaf spot of perennial ryegrass turf. Materials and Methods Growth of perennial ryegrass and inoculation with M. oryzae Legacy II perennial ryegrass seeds were surface sterilized for 30 seconds in 0.5% NaOCl (6.15% Clorox, Oakland, CA), rinsed twice in distilled water and then grown in 4-cm diameter 20-cm plastic cone-tainers (Stuewe & Sons, Corvallis, OR) filled with a sterilized 50: 50 mixture of peat (Sun Gro's Metro-Mix, Bellevue, WA) and sand. Two weeks after seeding, the plants were fertilized with water-soluble N-P-K fertilizer and trimmed weekly after the plants were 3-week old to maintain the grass at 5- cm height. A monoconidial isolate of M. oryzae, collected from a perennial ryegrass fairway at Marple Township, Broomall, PA, was used for the experiments. The fungus was

31 20 collected and preserved on nitrocellulose filter paper disks at -80 C (114). Prior to inoculation, the filter paper disks were removed from storage, placed on potato dextrose agar plates, and incubated at 28 C at 12 h: 12 h light/dark cycle and 177 µmol/s m 2 light intensity for 5 to 7 days. Agar blocks (2 2 mm) containing actively growing mycelia of M. oryzae were transferred to oatmeal decoction agar plates (74) and incubated at the same conditions stated above. After 4 days of growth of M. oryzae on the oatmeal agar, sterilized alfalfa stems were placed on the oatmeal agar to induce profuse production of conidia. After 8 to 10 days of incubation at 28 C, the conidia were harvested by scraping the surface of colony with a sterilized needle. The conidial suspension was then filtered through 4 layers of Graphic Arts Cheesecloth (Fiberweb, Old Hickory, TN). The conidial suspension with Tween 20 (0.1%) was adjusted to approximately conidia/ml using a haemocytometer. Six-week-old perennial ryegrass plants were inoculated by spraying the plants with the aqueous suspension of M. oryzae conidia using an atomizer until runoff. Non-inoculated plants were sprayed with sterile distilled water and served as the control. Intermittent leaf wetness study Six treatments were designed by alternating wet and dry periods to investigate the effects of frequency and duration of dry periods (Fig.1). Inoculated plants were placed into either the dew chamber or the environment chamber following the wet and dry cycles in each treatment. The continuous free moisture on the leaf surface was achieved by adjusting a built-in misting system in the dew chamber at 28ºC (Percival Scientific,

32 21 Inc., Perry, IA). The dry period was accomplished by air-drying with a hair-dryer (Conair Corp., East Windsor, NJ) set at warm temperature and medium air emission for 2 minutes and then plants were maintained in the controlled environment chamber (Environmental Growth Chambers, Chagrin Falls, OH) with relative humidity of 65 to 75% at 28ºC (108). The temperature and percent relative humidity in each chamber were monitored using a data logger (ESCORT Data Loggers Inc, Buchanan, VA). After 18 h, all plants were transferred to a controlled environment chamber (Environmental Growth Chambers, Chagrin Falls, OH) set at 28 C, high level of relative humidity (91 to 97%), and a 12 h: 12 h light/dark cycle. The incubation periods of the first visible lesion were also recorded. The study was repeated (Experiment 2) following the same procedure. Interrupted leaf wetness study Based on the results from the intermittent leaf wetness study, a study was designed to determine the effect of incremental increases of dry periods between two given wet periods on disease development. A continuous 6 h wet period was followed by continuous dry periods of 3, 6, 9, 12, 15, or 18 h followed by a 6 h final wet period (Figure 2.2). The plants were subjected to wet and dry periods after the inoculation. The wet and dry periods and post treatment periods were conducted as described previously. The incubation periods for the first visible lesion were also recorded. The study was repeated (Experiment 2) following the same procedure.

33 22 Both studies were conducted as randomized complete block designs. Each treatment had 4 plants as replicates and each replicate was used as a block factor. Control plants were treated with sterilized distilled water. Disease assessment and data analysis Ten days after inoculation, disease incidence (DI) (percent symptomatic leaf blades) and disease severity (DS) (index 0 to 10; 0 = plants asymptomatic; 10= >90% of the leaf area necrotic) were assessed. Data from both studies were subjected to Analysis of Variance (ANOVA) and Student-Newman-Keuls (SNK) multiple comparison test (P 0.05). Generalized linear model analysis was conducted to determine the effect of frequency and duration of dry periods on the reduction of DI and DS based on the data from the intermittent leaf wetness study. The frequency was defined as the number of times of interruption of dry periods, and the duration was defined by the hour of each dry period of interruption. The reduction of DI and DS was calculated by comparing the difference between each intermittent leaf wetness treatment (T m 2-T m 5) with T m 1 (continuous 18 h wet period). The relationship between the durations of dry period (independent variables) and DI or DS (dependent variables) was analyzed by multiple linear regression. Further, the relationship between incubation periods for the initial symptom and DI or DS was analyzed by multiple linear regression. The criteria for selecting the best fit model was compared using residuals, r 2 (coefficient of determination), AIC (the Akaike Information Criterion) and BIC (the Bayesian

34 Information Criterion). All statistical procedures were performed using Statistical Analysis System software 9.2 (SAS Institute, Cary, NC). 23 Results Effects of intermittent leaf wetness Based on ANOVA analysis, significant differences in DI and DS were found among all the treatments (P<0.0001) (Table 1). Highest DI and DS (88.3 and 9.8, respectively) were found when plants were subjected to continuous 18 h leaf wetness (T m 1) (Figure 2.3). Lowest DI and DS (0.4 and 0.4, respectively) were observed in plants subjected to a continuous dry period (T m 6), which showed no significant difference from the untreated control plants. Compared to DI and DS of plants exposed to continuous 18 h leaf wetness (T m 1), treatments with intermittent leaf wetness showed significantly lower levels of DI and DS (P 0.05) (Figure 2.3). Among all treatments with intermittent leaf wetness, the treatment with a 3 h wet period alternated by a 6 h dry period (T m 3) demonstrated the lowest DI and DS (24.5 and 5.6, respectively). The treatment with a 3 h wet period alternated by a 3 h dry period (T m 2) had the second lowest DI and DS (31.5 and 5.9, respectively). Treatments with 6 h wet periods alternated with a 3 h dry period (T m 4) or a 6 h dry period (T m 5) had significantly higher DI and DS when compared to treatments subjected to 3 h wet periods. DI and DS of gray leaf spot were consistently higher in plants subjected to a 3 h dry period (T m 4; 74.0 and 8.4, respectively) when compared to plants subjected to a 6 h dry period (T m 5; 61.5 and 7.3, respectively). In

35 24 Experiment 2, a similar trend among the treatments was found but with lower overall DI and DS and reduced levels of differences among the six treatments (P 0.05) (Figure 2.3). Although T m 1 (18 h continuous leaf wetness) had the highest DI and DS (72.7 and 8.1, respectively), it was not significantly (P 0.05) higher than DI and DS of treatments received a 3 h dry period (T m 4; 60.5 and 7.6, respectively) (Figure 2.3). Both effects of frequency and duration of dry periods for intermittent leaf wetness were significant (P<0.0001) on the reduction of DI or DS. The effect of interaction of frequency and duration was also found significant (P= for DI and P< for DS, respectively) (Table 3). Effects of interrupted leaf wetness Significant (P<0.0001) differences were observed among DI and DS of the plants exposed to various durations of interrupted dry periods (Table 2). In Experiment 1, DI and DS decreased significantly with every 3 h increase of dry periods (T r 1-T r 6) (Figure 2.4). DI and DS of the treatment with an 18 h dry period (T r 6; 25.8 and 3.8, respectively) were 48.25% less than those of treatment with a 3 h dry period (T r 1; 74.0 and 8.4, respectively). Similar trends in the decrease of the disease with the increase of dry periods were found in Experiment 2 (Figure 2.4). DI also had 49.5% reduction from 60.5 (T r 1) to 11.1 (T r 6) but differences in DS were not pronounced (from 7.3 to 4.5). Further, negative relationships were found between the duration of dry period and DI or DS (Figures 2.4 and 2.5).

36 25 Residuals plotted against predicted values exhibited a random scatter and all studentized residuals fell inside -2 and +2. The AIC, BIC and r 2 (Table 4) all indicated that the relationship between duration of dry periods and DI was best described by a 2 quadratic model: Y Inc = T d +0.05T d (r 2 =0.92, P<0.0001), and the relationship between duration of dry period and DS data was best described by a linear model: Y Sev = T d (r 2 =0.92, P<0.0001), where Y Inc is the disease incidence, Y Sev is the disease severity and T d is the dry period in hours (Figure 2.6). In Experiment 2, the negative relationships between interrupted dry period and DI or DS were also best described by a quadratic model (P<0.0001) and a linear model respectively (P<0.0001) (Figure 2.6). Effects of intermittent or interrupted leaf wetness on incubation periods Monitoring the expression of symptoms indicated that treatments with continuous leaf wetness (T m 1), treatments with 6 h wet periods alternated with 3 h dry period (T m 4) or treatments with 3-h interrupted dry period (T r 1) always had the shortest incubation period for initial symptoms 3 days after inoculation. The expression of symptoms for treatments with a 3 h wet period alternated by a 3 h dry period (T m 2) or treatments with a 3 h wet period alternated by a 6 h dry period (T m 3) didn t develop until 5 or 6 days after inoculation. Symptom development for T r 5 (15 h interrupted dry period) and T r 6 (18 h interrupted dry period) required the longest incubation periods with symptoms generally developing after 6 or 7 days. Further observation showed that plants with shorter incubation periods always had higher levels of DI and DS. Similar observations were obtained in the repeated experiment.

37 26 The relationships between incubation period and DI and DS were both best described by quadratic models: Y Inc = T i +4.1T 2 i (r 2 =0.86, P<0.0001) and Y Sev = T i +0.3T i (r 2 =0.79, P<0.0001), where Y Inc is the disease incidence, Y Sev is the disease severity and T i is the incubation period for the initial symptom (Figure 2.6). A similar trend was found in the repeated experiment (Figure 2.6). Goodness-of-fit of the statistical model selection followed the same procedure as in the interrupted leaf wetness experiment (Table 5). Discussion To our knowledge, this is the first study to demonstrate the effects of intermittent and interrupted leaf wetness on the development of gray leaf spot of perennial ryegrass. Leaf wetness of the turfgrass canopy in perennial ryegrass fairways and roughs occurs due to dew formation, rain events, or irrigation water. Previous studies have shown that temperature and leaf wetness duration are two major determinants of gray leaf spot development in annual and perennial ryegrass (70, 108). The effect of leaf wetness duration was evaluated by exposing the plants to continuous leaf wetness periods up to 36 h (108). The duration of continuous leaf wetness can serve as a predictor for the development of gray leaf spot especially under moderately warm conditions (28 to 32 C). However, a prolonged leaf wetness period may become fragmented or interrupted as a result of environmental conditions, inadequate irrigation scheduling, syringing, or physical removal. Therefore, intermittent leaf wetness may be a common phenomenon in real field situations found on golf courses, athletic fields, and home lawns.

38 27 The findings of our research indicate the importance of frequency and duration of dry periods on the reduction of disease development in the gray leaf spot pathosystem. Compared to treatments with continuous leaf wetness, both disease incidence and severity were significantly reduced due to the integration of dry periods at various levels of frequency or duration (Figure 2.3). Regression analysis indicated a possible linear relationship between frequency and duration of dry periods and reductions in disease incidence or severity. Both frequency and duration had significant effects on the reduction of disease incidence and disease severity (P<0.0001). Parameter estimates from the models indicated the larger contribution to disease incidence and severity reduction is from the frequency of dry periods, which almost contributed twice the effects of duration of dry periods. These results could be applied to the development of preventive novel cultural practices. For example, dew removal practices with certain frequency can be integrated when prolonged leaf wetness is predicted. As the results indicate, the reduction of disease incidence and disease severity can reach 50 to 60% when dry periods prevail. Longer duration of dry periods caused more disease reduction at the same frequency of dry periods. Furthermore, results of intermittent leaf wetness demonstrated the cumulative effects of wet periods on gray leaf spot incidence and severity. Treatments with longer cumulative duration of wet periods had higher disease incidence and severity. However, the duration of dry periods also influenced the disease development significantly. In the interrupted leaf wetness study, the treatments with longer interrupted dry periods had a significantly lower level of disease incidence and severity, although all the treatments had the same duration of wet periods. The results showed the importance of extending the dry

39 28 period in preventing the progress of the disease once the dry period is initiated. The quantitative data from both studies could be incorporated to the current gray leaf spot forecast model which is only based on the effect of continuous leaf wetness. The knowledge and quantitative data of the effects of dry periods on gray leaf spot development provides a more accurate predictive model for disease incidence and severity in the gray leaf spot forecasting system. Studies on the leaf spot of olive caused by Spilocaea oleagina have indicated the significance of a dry period in reducing the disease development, especially when relative humidity is at a lower range (78). A similar study on pear scab has also indicated the suppressive effects of dry periods on lesion development. The fungal mortality has been described to be negatively related to the length of a dry period for the infection process in pear leaves by Venturia nashicola (59). In our study, the dry periods were maintained at relative humidity of 65 to 75%, which was suggested to be unfavorable for the infection and colonization of M. oryzae on perennial ryegrass (Y. Li, and W. Uddin, unpublished). The survival rate and infection potential of M. oryzae may be reduced during the dry periods that will result in reduced disease incidence and severity. In our research, the occurrence of dry periods in either intermittent or interrupted patterns could suppress disease development. Innovative gray leaf spot management strategies from the aspect of turf canopy moisture management may be successfully integrated into a broader turfgrass cultural management program, including fertility management, mowing practices, herbicide and plant growth regulator applications as well as fungicide applications (12, 103, 109, 121). Further, the accuracy of fungicide application timing may also be improved with the quantitative data incorporation from

40 29 this research into the current gray leaf spot predictive model. Therefore, based on the results of this work, further studies on sustainable management strategies, particularly on irrigation scheduling and moisture management may be developed.

41 Figure 2.1. Schematic diagram for the design of intermittent leaf wetness with different combinations of wet (gray) and dry (black) periods. 30

42 Figure 2.2. Schematic diagram for the design of interrupted leaf wetness with increasing duration of drying periods from 3 h to 18 h. 31

43 32 Figure 2.3. Effects of intermittent leaf wetness periods (T m 1-T m 6) on incidence or severity of gray leaf spot. Disease incidence was represented in Experiment 1 (A) and Experiment 2 (B) and disease severity was represented in Experiment 1 (C) and Experiment 2 (D). Disease incidence and severity were evaluated 10 days after inoculation. Each column is the mean of 4 plants and error bars represent standard error of the mean. Significant differences found between treatments (P 0.05) according to Student-Newman-Keuls (SNK) test.

44 33 Figure 2.4. Effects of interrupted leaf wetness periods (T r 1-T r 6) on incidence or severity of gray leaf spot. Disease incidence was represented in Experiment 1 (A) and Experiment 2 (B) and disease severity was represented in Experiment 1 (C) and Experiment 2 (D). Disease incidence and severity were evaluated 10 days after inoculation. Each column is the mean of 4 plants and error bars represent standard error of the mean. Significant differences found between treatments (P 0.05) according to Student-Newman-Keuls (SNK) test.

45 34 Figure 2.5. The relationship between interrupted dry periods and disease incidence or severity of gray leaf spot. Disease incidence was represented in Experiment 1 (A) and Experiment 2 (B) and disease severity was represented in Experiment 1 (C) and Experiment 2 (D). Y Inc is the disease incidence, Y Sev is the disease severity and T d is the dry period in hours.

46 35 Figure 2.6. The relationship between incubation period and disease incidence or severity of gray leaf spot. Disease incidence was represented in Experiment 1 (A) and Experiment 2 (B) and disease severity was represented in Experiment 1 (C) and Experiment 2 (D). Y Inc is the disease incidence, Y Sev is the disease severity and T i is the incubation period for the initial symptom in days.

47 Table 2.1. Analysis of variance of disease incidence and severity of intermittent leaf wetness treatments. Source DF Mean Square F Pr > F Disease incidence Block <.0001 Treatment <.0001 Error Total 27 Disease severity Block <.0001 Treatment <.0001 Error Total 27 36

48 Table 2.2. Analysis of variance of disease incidence and severity of interrupted leaf wetness treatments. Source DF Mean Square F Pr > F Disease incidence Block <.0001 Treatment <.0001 Error Total 27 Disease severity Block <.0001 Treatment <.0001 Error Total 27 37

49 Table 2.3. Analysis of variance for reduction of disease incidence with various frequencies (F) and durations (D) of dry periods. Source DF Mean Square F Value Pr > F Disease incidence Model <.0001 F <.0001 D <.0001 F D Error Total 15 Disease severity Model <.0001 F <.0001 D <.0001 F D <.0001 Error Total 15 38

50 Table 2.4. Statistical model selection of goodness of fit for the relationship between duration of interrupted dry periods (T d ) and disease incidence or severity (α=0.05). Variables in Model Adjusted r 2 r 2 AIC BIC Disease Incidence 2 T d T d T d T d Disease severity T d T d T d T d

51 Table 2.5. Statistical model selection of goodness of fit for the relationship between incubation periods (T i ) for the initial symptom development and disease incidence or severity (α=0.05). Variables in Model Adjusted r 2 r 2 AIC BIC Disease Incidence 2 T i T i T i T i Disease severity 2 T i T i T i T i

52 41 Chapter 3 Effects of Relative Humidity on Infection, Fungal Colonization and Conidiation of Magnaporthe oryzae on Perennial Ryegrass Turf Abstract Gray leaf spot, caused by Magnaporthe oryzae Couch, causes severe damage on perennial ryegrass (Lolium perenne L.) turf. In this study, the effects of relative humidity (RH, 85% to 100% at 28ºC) on infection, colonization, and conidiation of M. oryzae on perennial ryegrass were investigated in the controlled humidity chambers. Results showed that the RH threshold for successful M. oryzae infection was 91% or greater at 28 C. The advancement of inoculum on the leaf tissue was further examined with a green fluorescent protein (GFP)-tagged M. oryzae strain. No appressorium formation was found when the inoculum was incubated at RH 88% or greater. Additionally, the GFP-tagged straining provided a rapid method to quantitatively compare the fungal biomass from colonized leaf tissue at different levels of RH. Conidiation was only observed when RH was at 96% or greater with the most abundant conidiation occurring 8 days after inoculation. Reduced conidiation was associated with decreasing RH, and no conidiation occurred at the RH of 92% or lower. This study indicates that infection and conidiation of M. oryzae on perennial ryegrass required different thresholds. The thresholds for

53 42 infection and conidiation of M. oryzae on perennial ryegrass were RH of 91% and 96%, respectively. The quantitative data from this research will assist in gray leaf spot disease outbreaks and secondary infections of perennial ryegrass. Introduction Gray leaf spot, caused by Magnaporthe oryzae Couch (anamorph: Pyricularia oryzae Cavara), is a devastating foliar disease on perennial ryegrass (Lolium perenne L.) turf (22, 106, 113). The disease has drawn vast attention in the turfgrass industry due to severe outbreaks on golf courses in various regions of the United States during the past two decades (33, 51, 83, 92, 110, 112, 124). The host plant, perennial ryegrass, has been widely cultivated due to its desirable agronomic attributes, such as rapid germination, tolerance to close-mowing, rapid tillering, high density, up-right growth habit, and dark green turf color (31). However, most perennial ryegrass cultivars available in the market are highly susceptible to M. oryzae (7). Therefore, gray leaf spot management is heavily dependent on expensive and extensive fungicide applications which have caused the development of field resistance strains to azoxystrobin (34, 47, 122). Gray leaf spot epidemics are particularly prevalent in the northeastern United States, usually reported on golf course fairways during the late summer, from late July to early September (113, 120). The pathogen overwinters as mycelia in plant debris or in live plant tissue (100). Environmental conditions are important determinants of gray leaf spot epidemics in perennial ryegrass fairways and roughs. Gray leaf spot usually first develops in the canopy of high-cut perennial ryegrass where prolonged leaf wetness is

54 43 present (108, 110). Leaf wetness and temperature have been identified as influential environmental factors for gray leaf spot development (108). Periods of leaf wetness may be roughly estimated by the duration of high levels of relative humidity (RH 90%) (99). As a measurable and forecastable weather parameter, RH has been widely studied or applied as a predictor for disease development in various pathosystems at different stages including fungal growth and sporulation of numerous plant pathogens (14, 37, 69). Critical cytological changes of M. oryzae, including germ tube development, appressorium formation and invasive hyphae, have been observed during its interaction with artificial (i.e. cellophane) and rice leaf surfaces (43, 48). A conidiation study of M. oryzae on rice seedlings indicates that conidia can be scarcely produced at the RH of 90%, while the favorable RH for production occurs at 93% or higher at temperatures ranging from 16ºC to 34ºC (37). However, little information is available regarding the effects of RH on the development of gray leaf spot of perennial ryegrass. To our knowledge, no research has emphasized the influence of various humidity levels on infection, colonization or conidiation by M. oryzae on perennial ryegrass. The research for fungi and plant interaction has been facilitated by green fluorescent protein (GFP) expressed in fungal cytoplasm (42, 60). High level expression of GFP and bright fluorescence have been recently developed in the cytoplasm of M. oryzae (from the rice plant strain) by using Agrobacterium tumefaciens-mediated transformation (ATMT) system (88). The expressed GFP can produce a strong and stable green fluorescence and photobleaching only occurs when the cells are illuminated with 340 to 390 nm or 395 to 440 nm light (17). Additionally, GFP can provide real-time

55 44 microscopic observation. Laser scanning confocal microscopy and conventional fluorescence microscopy are efficient tools to observe GFP-tagged fungi. Compared with conventional fluorescence microscopy, confocal laser scanning microscopy provides higher resolution and flexibility for image generation, processing and analysis (20). Furthermore, GFP fluorescence intensity is found directly proportional to fungal biomass, such as Cochliobolus heterostrophus inside maize leaves which suggests a quick and accurate method to quantify the colonized fungi (18, 63). Therefore, the objectives of this study were to 1) determine the threshold humidity levels for critical events in disease development such as infection, colonization, and conidiation in the gray leaf spot pathosystem and 2) compare fungal biomass change at various levels of RH and assess the conidiation of M. oryzae on perennial ryegrass. Results of this study may be useful for integrating RH into the current gray leaf spot forecasting program and assist turf managers in developing an integrated diseases management strategy. Materials and Methods Growth of perennial ryegrass and preparation of detached leaf blades Legacy II perennial ryegrass seeds were surface sterilized for 30 seconds in 0.5% NaOCl (Clorox, Oakland, CA), rinsed twice in distilled water and then grown in 4-cm diameter 20-cm plastic cone-tainers (Stuewe & Sons, Corvallis, OR) that were filled with a sterilized 50:50 (v/v) mixture of peat (Sun Gro's Metro-Mix, Bellevue, WA) and

56 45 sand. Two weeks after seeding, the plants were fertilized weekly with water-soluble N-P-K fertilizer. Detached leaves were obtained from 6-week old plants. Leaf blades were cut into 4-cm sections, surface sterilized with 0.5% NaOCl (Clorox, Oakland, CA) for 90 seconds and rinsed with sterile distilled water for 10 seconds 3 times after surface sterilization. Transformation of M. oryzae and preparation of conidia suspension A monoconidial isolate of M. oryzae, collected from a perennial ryegrass fairway at Marple Township, Broomall, PA, was used for all experiments. The fungus was collected and maintained on nitrocellulose filter paper disks at -80 C for long term storage (114). The filter paper disks were removed from storage, placed on potato dextrose agar plates, and incubated at 28 C at 12 h:12 h light/dark cycles (177 µmol/s m 2 ) for 5 to 7 days. Agar blocks (2 2 mm 2 ) containing actively growing mycelia of M. oryzae were transferred to oatmeal agar (OMA) plates (74) and incubated at the same conditions mentioned above. Conidia were harvested from the plates following 7 to 10 days incubation at 28 C. A strain of Agrobacterium tumefaciens with pbmt2 vector (obtained from Dr. Seogchan Kang s Laboratory, The Pennsylvania State University, University Park) was grown at 28 C for 48 h in a minimal medium (MM) supplemented with kanamycin (Kan) (50 µg/ml). The bacterial culture (2 ml aliquot) was harvested and incubated with induction medium (IM) containing Kan (50 µg/ml) and acetosyringone (AS). Bacterial cells were subsequently suspended with 5 ml of IM, both in the presence and absence of 200 µm AS. Bacterial cells were grown for an additional 6 h before

57 46 mixing them with an equal volume of conidia suspension of the M. oryzae ( conidia/ml). This mixture (200 µl) was placed on nitrocellulose filters (0.45 µm-pore and 45-mm diameter, Whatman) on a co-cultivation medium (same as IM except it contains 5 mm of glucose instead of 10 mm glucose) in the presence or absence of 200 µm AS. Following co-cultivation at 25 C for 24, 36, and 48 h, the fungal and bacterial cells on the filter paper were harvested with 2 ml of MM with 200 µg/ml hygromycin B. Individual transformants were transferred into 24-well plates (Costar, USA) that contained 1.5 ml of OMA with hygromycin B (200 µg/ml) and incubated until conidiation occurred (88). One conidium from each transformant was transferred to OMA to create monoconidial cultures. The transformed conidia were harvested by scraping the surface of the fungal colony in each plate with a sterilized needle. A volume of 5 ml Tween 20 (0.1%) was added to each plate and the suspension was then filtered through 4 layers of Veratec Graphic Arts Cheesecloth (Fiberweb, Old Hickory, TN). Preparation of humidity chambers The influence of RH on the development of gray leaf spot of perennial ryegrass was investigated using mini humidity control chambers. Each chamber consisted of a 90 mm diameter by 50 mm height glass dish, a mm glass petri dish lid, and a 75 mm diameter acrylic plastic stand as described by Dufault et al. (25). Glass dishes were autoclaved for 45 minutes and plastic stands were sterilized using 0.5% NaOCl (Clorox, Oakland, CA) for 10 minutes, air dried and then rinsed with sterilized distilled water. Filter papers (125 mm) (VWR International, Radnor, PA) were cut in a way that there

58 47 were four appendages extending from four different sides and autoclaved for 45 min (Figure 3.1). They were then placed on the plastic stand with four appendages that were soaked by submerging the tips into the solution (Figure 3.1). Glycerol-water solutions with different glycerol concentrations were prepared with a magnetic stirrer and 30 ml solution was added in each chamber (23) (Table 3.1). Detached ryegrass leaf blades were placed on the soaked filter paper supported by the plastic stand. Humidity control chambers were sealed with parafilm (Fisher Scientific, Pittsburg, PA) to maintain consistent RH levels. Temperature and RH in the chambers were monitored and recorded using ilog data loggers (Escort Data Loggers Inc, Buchanan, VA). Effect of relative humidity on fungal infection Each leaf blade was inoculated by placing 100 µl of conidial suspension ( conidia/ml) on each leaf blade using a pipette. Inoculated detached leaf blades were incubated inside humidity chambers with RH (85% to 100% at 3% intervals). Each treatment (level of RH) was replicated in 3 separate humidity chambers and each chamber contained 10 detached leaf blades. All humidity chambers were placed in a Revco incubator (Thermo Fisher Scientific Inc., Asheville, NC) at 28 C. Incandescent light was utilized to maintain a 12 h: 12 h dark/light (177 µmol/s m 2 ) cycle. Infection of M. oryzae was evaluated 10 days after inoculation using a compound bright field microscope (Nikon Eclipse E600; Nikon Instrument group, Melville, NY). Histological examination with a bright field microscope (10 eyepiece) was facilitated by staining

59 tissues with Chlorazol black E (87). The experiment was repeated following the same procedure. 48 Effects of relative humidity on inoculum development Plant tissues and humidity chambers were prepared as previously described. Three replicate chambers were utilized for each RH level (88% to 100% RH at 4% intervals). Leaf tissues were inoculated with the transformed M. oryzae strain ( conidia/ml). Three pieces of detached leaf blades from each humidity chamber were observed every 24 h for the first 4 days after inoculation by using a FV-1000 laser scanning confocal microscope (Excitation and emission wavelengths of 488 nm and 508 nm respectively; Olympus, Hamburg, Germany). Observation of inoculum development was based on five developmental stages: intact conidia, germinated conidia with only germ tubes, germinated conidia with appressoria, invasive hyphae, and conidia with collapsed cells. The experiment was repeated in Experiment 2 following the same procedure. Effect of relative humidity on colonization of perennial ryegrass leaf tissue by M. oryzae Fungal biomass was evaluated by measuring fluorescence intensity (FI) of GFP produced in the cytoplasm of transformed M. oryzae. Leaf tissues inoculated with GFPtagged M. oryzae were incubated at RH ranging from 88% to 100% at 4% intervals. The FI of GFP produced from the colonized mycelia was measured daily for 14 days after

60 49 inoculation. Soluble GFP from each replicate at each RH was extracted by thoroughly homogenizing the colonized plant samples using a TissueLyser (Qiagen, USA) with 5 mm glass beads (Fisher Scientific, USA) in 200 µl of pre-chilled extraction buffer (30 mm Tris HCl, 10 mm EDTA ph 8, 10 mm NaCl, 5 mm DTT) (18). TissueLyser parameters were set at 30 Hz for 5 min, and centrifugations were performed at 14000g for 15 min. Supernatant (800µl) with soluble GFP was measured for green fluorescence using a SLM-Aminco 8100 Spectrofluorometer (SLM Instruments Inc., Bogart, GA) with an excitation wavelength of 485 nm and emission wavelength of 508 nm. The experiment was repeated in the experiment 2 following the same procedure. Effect of relative humidity on conidiation of M. oryzae on perennial ryegrass Inoculated detached leaves were incubated in humidity chambers ranging from 88% to 100% at 4% intervals at 28 C as previously described. Three leaf blades from each humidity chamber were evaluated daily by quantifying the conidiation amount. Three replicate chambers were utilized for each RH level. For quantification of conidia, leaves were immersed in 1.5 ml centrifuge tubes (VWR International, Radnor, PA) with 500 µl Tween 20 (0.1%) solution and agitated for 30 seconds with a Vortex-Genie (Scientific Industries, Inc., Bohemia, New York). The conidia density was then determined using a haemocytometer (Hausser Scientific, Horsham, PA). The daily quantification of conidia density continued every 24 h until conidia were no longer observed. The experiment was repeated (Experiment 2) following the same procedure.

61 50 Results Infection of M. oryzae on perennial ryegrass Microscopic examination of perennial ryegrass leaf tissues revealed different extents of fungal colonization at RH levels from 85% to 100% at 10 days after inoculation (Figure 3.2). No successful colonization of leaf tissue by the mycelia was observed at RH of 88% and below (Figures 3.2. A and B). Colonization of tissue by M. oryzae was only observed at RH of 91% and above (Figure 3.2.C, D, E and F). Visual assessment of inoculated leaf tissue samples indicated that increased colonization by mycelia was associated with increasing RH. Dense mycelial mass were observed at RH of 96% or higher (Figure 3.2.E and F). Evaluation of advancement of inoculum on perennial ryegrass Five developmental stages of M. oryzae were used to describe the progression of the pathogen (Figure 3.3). In Experiment 1, all inocula remained as intact conidia for the first 3 days after inoculation at 88% RH (Figure 3.4.A). Four days after inoculation, 15.3% of inocula still remained as intact conidia, 67.7% germinated and produced relatively short germ tubes (less than 10 µm) and 17.0% of the conidia had collapsed cells (Figure 3.4.A). At 92% RH, 85.9% of inocula germinated (57.3% with only germ tubes and 28.6% with appressoria) 1 day after inoculation (Figure 3.4.A). At 4 days after inoculation, 82.9% developed into invasive hyphae (Figure 3.4.A). At 96% RH, more than 60% of the inocula were observed as invasive hyphae, 1 day after inoculation, which

62 51 increased to 100% 4 days after inoculation (Figure 3.4.A). At 100% RH, 91.0% of inocula were observed as invasive hyphae, 1 day after inoculation, and reached 100% within 2 days after inoculation (Figure 3.4.A). Conidiophores and conidiation were observed at 4 days after inoculation at 100% RH. The results in Experiment 2 followed a similar pattern; however, the percentage of the collapsed cell was somewhat higher (28.0% compared to 17% in Experiment 1) 4 days after inoculation at 88% RH (Figure 3.4.B). Additionally, there were also some minor differences in percentage of intact conidia, germination of conidia with germ tube or appressorium, invasive hyphae, and collapse cells at the various levels of humidity in Experiment 1 and Experiment 2 (Figure 3.4.B). Quantitative assessment of fungal biomass from infected leaf tissues In Experiment 1, no fluorescence intensity (FI) was detectable from the inoculated leaves when RH was at 88% (Figure 3.5.A). At 92% RH, FI was first detected 10 days after inoculation (Figure 3.5.A). The incubation period for the first detectable FI level was 3 days after inoculation at 100% RH (Figure 3.5.A). The highest FI value occurred at 100% RH 11 days after inoculation but decreased sharply at 12 days after inoculation (Figure 3.5.A). Both FI values increased at 96% and 92% RH until 14 days after inoculation (Figure 3.5.A). The peak FI value at 96% RH was comparable with the FI value at 100% RH although it was observed 3 days after the peak at 100% RH (Figure 3.5.A).

63 52 The results in Experiment 2 followed a similar pattern. However, in this experiment, FI was first detected 11 days after inoculation (Figure 3.5.B) at 92% RH compared to 10 days after inoculation in Experiment 1. At 96% RH, the level of detectable FI was found 9 days after inoculation (Figure 3.5.B) compared to 6 days after inoculation in Experiment 1. Conidiation at various levels of relative humidity In Experiment 1, no conidiation was observed when RH was 92% (Figure 3.6.A). Conidiation was first observed at 4 days after inoculation at both 96% and 100% RH by a haemocytometer (Figure 3.6.A). The amount of conidiation was greatest at 8 days after inoculation at both 96% and 100% RH (Figure 3.6.A). Compared with the first quantified conidiation rates, the peak amount increased from 62 to 1700 conidia µl -1 at 100% RH and increased from 8 to 191 conidia µl -1 at 96% RH (Figure 3.6.A). After peak conidiation, conidiation dropped to 10% of the peak amount at 96% RH and 50% of the peak amount at 100% RH within 24 hours (Figure 3.6.A). The conidiation quantity fluctuated over time but was never observed to reach the peak rates found at 8 days after inoculation (Figure 3.6.A). After 21 days after inoculation, no additional conidia were produced (Figure 3.6.A). A similar pattern of conidiation was observed in Experiment 2, although the amount of peak conidiation was found lower with 134 conidia µl -1 and 1481 conidia µl -1 at 96% RH and 100% RH, respectively (Figure 3.6.B).

64 53 Discussion To our knowledge, this is the first documentation of the effects of humidity levels on the major events during M. oryzae-perennial ryegrass interaction, including infection, colonization and conidiation. Additionally, this research has demonstrated the effective application of GFP-tagged M. oryzae and its related procedures such as confocal microscopy and fluorescence intensity measurement to study the influence of relative humidity on the interaction of M. oryzae and perennial ryegrass. Our results indicate that specific relative humidity ranges after inoculation are critical for these major events during gray leaf spot development. High humidity levels (RH 91% at 28ºC) were required for successful infection by M. oryzae on perennial ryegrass which was further illustrated by microscopic examinations. A high percentage of inocula developed into invasive hyphae with shorter incubation periods at higher relative humidity. For example, over 80% of inocula developed into invasive hyphae within 24 hours after inoculation at 100% RH while it required at least 2 days to reach the similar percentage at 96% RH or even longer at 92% RH. Low humidity levels (RH 88% at 28ºC) only induced the germination of inoculum with short germ tube (<10µm) without the formation of appressoria. Failure of appressorium formation resulted in the failure of M. oryzae infection on the perennial ryegrass. At the low relative humidity ranges, inocula also remained as intact conidia or resulted in collapse of the cells. Therefore, we concluded that relative humidity of at least 91% is required for the gray leaf spot development of perennial ryegrass if favorable temperatures for disease development are present.

65 54 The high moisture levels in the atmosphere may not only induce the formation of appressorium but also favor the building of turgor pressure in the appressorium of M. oryzae which has been reportedly responsible for the penetration process (43). Once the penetration is successful, invasive hyphae can be observed. Shorter incubation time was required for the germinated conidia with appressorium to develop into invasive hyphae at higher relative humidity (Figures 3.4.A and B). In a previous study on M. oryzae causing rice blast disease, free moisture on the surface has been determined to be required for M. oryzae development, including germination and surface attachment (30). In our study, we further confirmed the importance of moisture level on inoculum development which is not only contributed from the surface moisture but also from the atmospheric moisture. Visual assessment from microscopic images indicated that heavy mycelial colonization on the leaf tissues was observed at near saturated humidity levels ( 96% RH at 28 ºC). However, in the comparison of the fluorescence intensity of GFP from infected leaf tissues at different levels of relative humidity, a higher mycelia growth rate was found to be associated with higher relative humidity. Compared with 100% RH, the incubation period to reach the peak mycelial amount at 96% RH required 3 more days. The mycelial growth rate at 92% RH was slower compared to that at RH 96%. Although the peak of the fluorescence intensity at 100% RH was similar to that at 96% RH during the observation period, it was observed 3 days earlier than that at 96%. This delay in producing fungal biomass under 96% RH is evidently a direct effect of reduced relative humidity level. Additionally, the sudden decrease of fluorescence intensity after the peak amount may be explained by the ceasing of fungal vegetative growth due to the deficiency of plant tissue nutrients and the collapse of colonized fungi.

66 55 Although nutrient limitation restricts the maximum amount of fungal colonization, it may play a positive role in the induction of conidiation of M. oryzae on perennial ryegrass. Vegetative growth such as mycelial colonization requires nutrient absorption from infected plant tissues. At the same time, the conidiation was induced as the colonization progresses. Other fungi such as those within the genus Alternaria and Aspergillus, also followed similar sporulation and mycelia colonization trends (10). Conidiation of M. oryzae on perennial ryegrass only occurred at near saturated RH ( 96% at 28 ºC) levels which required higher RH ranges than conidiation on the rice seedlings ( 93% RH) (37). Moreover, the substrates with higher moisture content also favor the conidiation process (38). High moisture levels may either occur continuously or intermittently to affect the fungal sporulation. For example, sporulation of Alternaria porri on potato and tomato plants, has been considerably higher during intermittent wet/dry periods than under continuously moist conditions (90). In conclusion, the infection, colonization, and conidiation of M. oryzae on perennial ryegrass were highly dependent on the atmospheric moisture levels. Nearsaturated humidity conditions are most favorable for M. oryzae infection and conidiation. Results of this research could integrate relative humidity into the current gray leaf spot forecasting system (108) which could provide critical infection and conidiation thresholds to assist the evaluation of disease outbreaks and the risk for secondary infections.

67 56 Figure 3.1. The humidity chamber with glycerol solution, devised for relative humidity experiment. A. Profile picture B. Each chamber contained 10 detached leaf blades.

68 57 Figure 3.2. Effects of relative humidity on infection and colonization of perennial ryegrass tissue by Magnaporthe oryzae. Microscopic images were taken 10 days after inoculation at RH from 85% to 100% at 3% intervals. A. No colonized mycelia observed at 85% RH. B. No colonized mycelia observed in the perennial ryegrass leaf tissues at 88% RH. C. Colonized mycelia (indicated by the red arrow) observed at 91% RH. D. Colonized mycelia observed at 94% RH. E. Massive mycelial colonization observed at 97% RH. F. Massive mycelial colonization observed at 100% RH.

69 58 Figure 3.3. Confocal microscopic images of advancing Magnaporthe oryzae on perennial ryegrass leaf tissues. A. The intact conidium. B. The germinated conidium with germ tube only. C. The germinated conidium with germ tube and appressorium. D. Invasive hyphae. E. The conidium with degraded cell(s).

70 59 Figure 3.4. Effects of relative humidity on growth and development of Magnaporthe oryzae during the infection process on perennial ryegrass leaf tissue. Percentage of inoculum development into various stages was recorded daily for the first 4 days after inoculation. A. Experiment 1. B. Experiment 2.