R.A. Cramer 1, P.F. Byrne 2, M.A. Brick 2, L. Panella 3, E. Wickliffe 1, and H.F. Schwartz 1

Transcription

1 1 Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random Amplified Polymorphic DNA Markers R.A. Cramer 1, P.F. Byrne 2, M.A. Brick 2, L. Panella 3, E. Wickliffe 1, and H.F. Schwartz 1 Authors addresses: 1 Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO ; 2 Department of Soil and Crop Sciences, Colorado State University, Fort Collins, CO ; 3 United States Department of Agriculture-ARS Crop Research Center, Fort Collins, CO Abstract Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the United States, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. One hundred and sixty-six isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar UI 114. Only 4 of the 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non-pathogenic (Fo) isolates, were selected for RAPD analysis. Twelve RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages (UPGMA) dendrogram based on Jaccard s coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. Because RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study. Introduction Fusarium oxysporum f.sp. phaseoli Kendrick and Snyder (Fop) is the causal agent of Fusarium wilt of common bean (Kendrick and Snyder, 1942). Fop was first identified in the United States in 1929 by Harter (Harter and Weimer, 1929), and in Colorado in 1988 by Schwartz et al. (1989). Fusarium wilt of common bean has since been found in many other bean producing regions of the world, including Brazil, Colombia, Peru, Costa Rica, Italy, Spain, Greece, the Netherlands, and central Africa (Alves-Santos et al., 1999; Buruchara and Camacho, 2000; Diaz-Minguez et al., 1996; Pastor-Corrales and Abawi, 1987; Ribeiro and Hagedorn, 1979). In the Central High Plains (CHP) region of the United States (Colorado, Nebraska, and Wyoming), common dry bean, Phaseolus vulgaris L., and sugar beet, Beta vulgaris L., are two economically important crops susceptible to Fusarium wilt, also known as Fusarium yellows.

2 2 Under appropriate stressful conditions, such as soil compaction, poor soil drainage, and extremes in soil moisture levels and temperature, common bean yield losses in the CHP have been reported as high as 30% (Schwartz et al., 1996). To date, seven pathogenic races of Fop have been identified around the world by screening isolates of Fop with a set of differential cultivars (Alves-Santos et al., 2002a; Ribeiro and Hagedorn, 1979; Woo et al., 1996). However, identification of pathogenic strains by screening with differential cultivars can be problematic. Race identification using cultivar screening can be strongly influenced by cultivar selection and number, genetic purity of the cultivar, environmental conditions, pathogen strain virulence, and screening methodology (Windels, 1991). Because resistance to Fop has been shown to be race specific, understanding the genetic variability and race distribution of Fop is important for developing common bean cultivars with appropriate resistance genes (Cross et al., 2000; Ribeiro and Hagedorn, 1979; Salgado et al., 1995). Several methods have been used to characterize formae speciales of F. oxysporum including vegetative compatibility groups (VCGs) (Puhalla, 1985), isozyme analysis (Bosland and Williams, 1987), restriction fragment length polymorphisms (RFLPs) (Elias et al., 1993), and repetitive DNA sequences (Barve et al., 2001; Kistler et al., 1991). Random amplified polymorphic DNA (RAPD), however, provides investigators with a quick and cost-effective method to study genetic similarity (Welsh and McClelland, 1990; Williams et al., 1990). Genetic similarity has been successfully assessed with RAPD markers in several formae speciales of F. oxysporum (Assigbetse et al., 1994; Bently et al., 1995; Chiocchetti et al., 1999; Clark et al., 1998; Crowhurst et al., 1995; Grajal-Martin et al., 1993; Kelly et al., 1994; Manulis et al., 1994; Nelson et al., 1997; Vakalounakis and Fragkiadakis, 1999). The objectives of this study were to characterize isolates of F. oxysporum obtained from diseased common bean plants in the CHP using RAPD markers and pathogenicity assays to determine if RAPD banding patterns could be used to (i) determine the genetic similarity among Fop isolates collected in the CHP region of the United States; (ii) distinguish between CHP Fop isolates and non pathogenic (Fo) isolates; and (iii) distinguish between Fop and F. oxysporum f.sp. betae (Fob) isolates. Materials and Methods Naming protocol of Fusarium oxysporum isolates and races Isolates pathogenic on common bean are referred to as Fop and on sugar beet as Fob, and identifiers are as designated in the collection of H.F. Schwartz at Colorado State University. Isolates previously assigned a race designation by Woo et al. (1996) and Alves-Santos et al. (2002a) are referred to by their physiological race designation. These include isolate 8 (ATCC 90245) which is referred to as Fop race 4, Fop 32 (ATCC 18131) referred to as Fop race 1, Fop 24 referred to as Fop race 3, and Fop 58 (AB-6) referred to as Fop race 6. Isolates collected from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms, but upon subsequent greenhouse testing on common bean and sugar beet cultivars did not produce disease symptoms, are referred to as Fo, and considered to be non pathogenic isolates. Collection of fungal isolates and sampling strategy Commercial bean production regions, especially those along the South Platte River drainage in eastern Colorado and the North Platte River drainage in western Nebraska were

3 3 visited annually during the course of this study ( ). In addition, county extension agents and field scouts were asked to submit diseased plant samples to our laboratory. The objective was to obtain representative samples from both production areas and drainages. Symptomatic plants (expressing chlorosis, wilting, and/or necrosis) during pod fill to maturation were most often observed near edges of fields where soil compaction and poor soil drainage were most apparent. Three or more random symptomatic plants per field were carefully extracted from the soil to retain most of the root system, the stem was cut above the first node, and the tissue samples were placed in a plastic bag, sealed, and transported in a cooler with ice to the laboratory within hrs. Plant stems were cut in 0.3 cm cross-sections and placed in a 5% ethanol, 10% NaOH, and 85% sterile distilled water (DI) solution for 2-3 minutes to kill saprophytic fungal and bacterial contaminants (Windels, 1992). Disinfested pieces of tissue were transferred to petri dishes containing ½ strength potato dextrose agar (PDA) (Sigma Chemical Co., St. Louis, MO) and placed in the dark at 22ºC. Four days later, F. oxysporum isolates were single spored, identified, and stored in 20% glycerol at -80ºC for future studies. A total of 241 F. oxysporum isolates were collected from common bean plants exhibiting Fusarium wilt symptoms. Of these, 166 were selected based on cultural characteristics for further analysis. A total of 14 F. oxysporum isolates collected from sugar beet were used in this study. Additional Fop and Fob isolates not collected from the CHP were donated by other Fusarium investigators (Table 1). Pathogenicity screenings The set of 166 isolates collected from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar UI 114. Pathogenicity screenings were performed using the modified root-dip technique previously described by Pastor-Corrales and Abawi (1987) and Salgado and Schwartz (1993). Disease reactions were assessed 21 days after inoculation using a modified CIAT disease severity index based on foliar symptoms and internal examination of vascular tissues (Fall et al., 2001; Pastor-Corrales and Abawi, 1987). Plants are scored on a 1 to 9 scale with 1 exhibiting no Fusarium wilt symptoms and 9, plant death. Based on this scale, plants scored 1 to 3 are classified as resistant, 4 to 6 as intermediate, and 7 to 9 as susceptible. Isolates that caused susceptible reactions were considered pathogenic, and were further screened on the common bean cultivars Fisher, Diacol Calima, Viva, and B40. The donated Fop races and Fop 31 from Holland were also included in these evaluations. All screenings were repeated a minimum of two times. Pathogenicity of isolates collected from sugar beet was tested at the USDA-ARS sugar beet pathology program at the USDA-ARS Crop Research Center, Fort Collins, CO, using a root-dip technique on the susceptible cultivar FC 716 (Panella et al., 1995). Inoculated sugar beet plants were evaluated on a 0 to 4 rating scale where 0 = no disease evident; 1 = plants slightly stunted to extremely stunted, leaves may be wilted; 2 = chlorotic leaves with necrosis at the edges; 3 = tap root becoming dried and brown to black in color, leaves dying; and 4 = plant death (L. Panella, unpublished). A total of 34 isolates were then selected to compose a core collection for RAPD analysis based on pathogenicity screening results and geographic location. These 34 isolates, listed in Table 1, consisted of all pathogenic Fop isolates collected in the CHP, samples of Fop races and Fop 31 donated from other Fusarium investigators, 11 non-pathogenic isolates collected from bean, all isolates collected and donated from sugar beet, and one isolate of Fusarium solani

4 4 obtained from a common bean plant which exhibited root-rot symptoms. The F. solani isolate was included as an out-group for dendrogram construction. DNA extraction Genomic DNA was extracted from each isolate using a modified method of Nelson et al. (1997). Briefly, mycelial tissue plugs from 7 day old cultures grown on ½ strength potato dextrose agar (PDA) were transferred to 50 ml of potato dextrose broth (PDB) (Sigma Chemical Co., St. Louis, MO) and incubated at 24ºC with shaking at 100 RPM. Approximately 1-2 grams of mycelial tissue was collected from liquid media after 14 days, washed with sterile water, dried with sterile cheese cloth, ground in liquid nitrogen, and subjected for 1 hour to 1-2 ml of an extraction buffer containing 25 mm tris-hcl ph 8.0, 25 mm EDTA, 50 mm NaCl, and 1% SDS. Samples were then extracted with an equal volume of a phenol/chloroform/isoamyl alcohol (25:24:1) mixture. DNA was precipitated with 4ºC isopropanol, washed in 70% ethanol, air dried, suspended in 1X TE buffer (10 mm Tris-HCl ph 7.6 and 0.1 mm EDTA), and quantified using a spectrophotometer. RAPD amplification RAPD reactions were performed in 25 l reactions containing 50 ng of genomic DNA and a 20 l master mix which contained 1x PCR buffer (Promega, Madison, WI), 2.5 mm MgCl 2, 2 mm dntps, 2 M of primer, and 1 unit of Taq polymerase (Promega, Madison, WI). Reactions were amplified in a MJ PTC 200 thermocycler (M.J. Research, Inc., Watertown, MA) for a 1 minute pre-dwell at 94 C then 40 cycles of: 20 seconds at 94 C, 1 minute ramp to 36 C, 1 minute dwell at 36 C, 3 minute ramp to 72 C, and 1 minute extension at 72 C. As a control, sterile water was used in place of fungal DNA to test for primer dimerization and contamination. Amplified DNA was separated on 1.5% agarose gels in 1X Tris-borate-EDTA (TBE) and stained with ethidium bromide. All RAPD reactions were repeated a minimum of 2 times to check for reproducibility. A total of mer RAPD primers were screened. Selection of the 12 primers used in the final analysis was based on the number of markers generated by each primer, quality of markers (intensity and reproducibility of the marker), and degree of polymorphism among the markers generated. A list of primers and their sequences and sources is presented in Table 3. Analysis of genetic similarity The numerical taxonomy package NTSYS 2.0 (Rohlf, 1998) was used in all statistical analyses in this study. A binary matrix of RAPD marker data was subjected to the SIMQUAL module in NTSYS using Jaccard s coefficient of similarity to generate a genetic distance matrix. Jaccard s coefficient was selected because it does not include conjoint absences in its calculation of similarity. Absence of a band can be due to many factors and thus does not necessarily imply genetic similarity. The genetic distance matrix was subjected to the SAHN module to create a dendrogram based on an unweighted paired group method with arithmetic averages (UPGMA). To provide statistical support for the dendrogram created, the COPH module was used to compute a matrix of cophenetic (ultra metric) values. This matrix was then used in the MXCOMP module to compute the correlation between the cophenetic matrix values and the original matrix being clustered (produced by the SIMQUAL module). This analysis provided an estimate of the goodness of fit of the original cluster analysis by generating a cophenetic correlation value, r.

5 5 Results Pathogenicity screenings Of the 166 CHP F. oxysporum isolates collected in this study and screened on common bean, only 4 isolates (Fop 16, Fop 52, Fop 53, and Fop 308c2) from the CHP (2.4%) were pathogenic on the universal susceptible common bean cultivar UI 114 (Table 2). Of the donated non-chp isolates, Fop races 1 and 6, and Fop 31 were pathogenic, while the Fop race 3 isolate from Colombia did not produce symptoms on any of the cultivars examined (Table 2). The Fop race 4 isolate used in this study was originally collected from the CHP in 1988 (Schwartz et al., 1989). Fop races 1 and 4 had identical reactions on the cultivars examined. Fop race 6 was able to cause wilt symptoms on the cultivars Fisher and B40, whereas Fop races 1 and 4 did not. The 4 newly collected pathogenic isolates from the CHP had identical reactions with Fop races 1 and 4 on the cultivars tested. Fop 31, an isolate from the Netherlands with no race designation, had an identical reaction with Fop race 6 from Spain to the cultivars examined. Results from pathogenicity tests of Fusarium isolates collected from sugar beets revealed 10 of 14 isolates were pathogenic on susceptible sugar beet cultivar FC 716 and were classified as Fob (data not shown). RAPD amplifications Amplification of F. oxysporum DNA using the RAPD technique produced clear, reproducible, and polymorphic bands (Fig. 1) that allowed the characterization of isolates examined in this study. Twelve primers, which generated 105 RAPD markers, were selected and used to compare the genetic diversity between fungal isolates. No amplification products were seen in the water control using the 12 primers selected for analysis. Polymorphisms were seen between Fop race 4 and races 3 and 6, and between all Fop races tested and non-pathogenic Fo isolates (Fig. 1A). No polymorphisms in RAPD banding patterns were observed among Fop isolates collected in the CHP (Fop 16, Fop 52, Fop 53, Fop 308c2) (Fig. 1A). These isolates had identical RAPD banding patterns with the previously characterized Fop race 4 isolate (ATCC 90425) from Colorado and Fop race 1 isolate (ATCC 18131) from South Carolina with all 12 primers examined, as well as with the other 64 primers not included in the cluster analysis (data not shown). RAPD markers readily distinguished the pathogenic CHP isolates from Fop races 3 and 6 (Fig. 1A). The pathogenic isolate from the Netherlands, Fop 31, was differentiated from both the CHP group and from races 3 and 6 by RAPD banding patterns. Polymorphisms in RAPD banding patterns were also found among non-pathogenic isolates collected in this study (Fig. 1A, 1B and 1C). Non-pathogenic isolates from the same county, such as the isolates from Scotts Bluff, Nebraska, displayed polymorphisms in RAPD banding patterns. A substantial degree of polymorphism can be seen between the non-pathogenic isolate collected from sugar beet, Fo 220d, and pathogenic Fob isolates (Fig. 1C). Pathogenic isolates from sugar beet Fob 216b, 216c, and 216d collected from the same location, had different RAPD banding patterns (Fig. 1C). Further, polymorphism in RAPD banding patterns was also seen among Fop and Fob isolates (compare Fig. 1A and Fig. 1C). UPGMA cluster analysis of RAPD banding patterns

6 6 Cluster analysis of RAPD banding patterns revealed a high degree of genetic diversity among the isolates examined in this study (Fig. 2). The dendrogram created in this study had a cophenetic correlation value of r > (1 = best possible fit), indicating the goodness of fit of the cluster with the original binary RAPD banding pattern data. Only one clear group (referred to as the CHP group) formed a cluster consistent with the pathogenicity screenings and geographic location. This group contained all 4 pathogenic isolates collected from the CHP (Fop 16, 52, 53 and 308c2), as well as the Fop race 4 isolate collected in a previous study (Fig. 2). Interestingly, this group also contained the Fop race 1 isolate collected from South Carolina. No other clear groups could be distinguished that correlated with geographic location or pathogenicity. However, Fob isolates (with the exception of Fob isolates 21 I and 216d) were clustered together in a group at the bottom of the dendrogram separate from any Fop isolates. This Fob group also contained the non-pathogenic isolate Fo 204b collected from sugar beet. Isolates Fob 21 I and 216d, from Montana and Colorado respectively, were more closely related to the CHP group than other isolates pathogenic on sugar beet. Fop 31, from the Netherlands, was approximately 88% similar to the CHP group and only 39% similar to Fop race 6 according to the cluster analysis. However, according to the differential cultivar screen, Fop 31 and Fop race 6 had identical reactions. Fop races 3 and 6 were more closely related to each other (46%) than to the CHP group (39%). Unlike the Fop isolates collected in the same location, Fob isolates collected from the same location (Logan County, Colorado, Fob 216b, 216c, 216d, and 220a) did not cluster together. Fob 216b from Logan County, Colorado was less similar to the other F. oxysporum isolates than the intended out-group F. solani. Fob 216c was the isolate most closely related to the F. solani isolate (16%). In general, non-pathogenic isolates exhibited a wide range of genetic diversity with some more closely related to pathogenic isolates than to other non-pathogenic isolates. For instance, Fo 120a, 112a and 126a from Scotts Bluff County, Nebraska were more closely related to the CHP clade (77%) than to other non-pathogenic isolates (Fig. 2). Interestingly, the nonpathogenic isolate Fo 48, collected from the same field as the pathogenic isolates Fop 16, 52 and 53, was quite distinct from the CHP group and most closely related to isolate Fo 103a from Morgan County, Colorado. Isolates Fo 127bl and Fo 127bd, from the same plant, were identical according to this analysis even though in culture they display different pigmentation (data not shown). Nearly all non-pathogenic isolates collected from sugar beet were genetically distinct from each other as well as from non-pathogenic isolates collected from bean. An exception was isolate Fo 204a (collected from common bean), which appeared to be more closely related to the sugar beet isolates than the bean isolates. Discussion In this study we used pathogenicity assays and RAPDs to examine the relationships among F. oxysporum isolates collected from common bean and sugar beet plants that exhibited Fusarium wilt symptoms. Specific RAPD banding patterns were able to distinguish among some races of Fop, non-pathogenic Fo isolates, and Fob isolates. Cluster analysis of the RAPD banding pattern data, however, revealed a substantial amount of genetic diversity among all the isolates examined. The high amount of genetic diversity resulted in virtually no clusters that clearly identified geographic origin or pathogenicity of the isolates. One exception was the CHP group, which had identical RAPD banding patterns (Fig. 2). This group also contained the Fop race 1 and 4 isolates. The CHP Fop group was separated from isolates of Fob collected in this study. Despite this, the results of this study suggest that RAPD banding patterns are not suitable

7 to differentiate isolates based on their pathogenicity or geographic origin. These results confirm those of Woo et al. (1996), who also found that RAPDs were not useful in differentiating Fop pathogenic types. Characterization of Fob isolates from sugar beet has been less thorough than in other formae speciales (Harveson and Rush, 1997). Fob isolates tested for vegetative compatibility revealed a considerable amount of genetic diversity (Harveson and Rush, 1997), and RAPD analysis of Fob isolates from California, Montana and Oregon showed distinct RAPD banding patterns for isolates among these regions (Fisher and Gerik, 1994). Yet, no physiological races of Fob have been determined to date. Our RAPD results confirm previous reports (Fisher and Gerik, 1994; Harveson and Rush, 1997) of substantial genetic variability in Fob, and indicate the need for further research in this important forma specialis. The two distinct groups of Fob which were present in this study (Fig. 2) seem to suggest a polyphyletic origin for forma specialis betae, and bring into question whether RAPDs truly represent this forma specialis. This is exemplified in Fig. 2 where one group of Fob isolates (Fob 21 I and Fob 216d) was more similar to Fop isolates (the CHP group) than the rest of the Fob isolates, which were more similar to each other. It is also important to note that common bean and sugar beet are often part of the same crop rotations in the CHP. Fop is known to colonize a range of non-host species (Dhingra and Netto, 2001), but whether the same F. oxysporum isolates colonize both bean and sugar beet in the CHP is not known. Future studies will involve pathogenicity screening of Fop isolates on sugar beet cultivars, and Fob isolates on common bean cultivars. A potential application of the results obtained in this study is the development of a molecular marker, such as a sequenced characterized amplified region (SCAR), to distinguish Fop, Fo, and Fob isolates. If our results with a limited set of CHP Fop isolates are verified in a larger sample, it should be possible to develop a marker specific to Fop in the CHP region. However, the development of markers that can be used to differentiate all origins of Fop, Fob, and Fo may be hindered by the large degree of genetic diversity found in this study. Recently, Alves-Santos et al. (2002a) were able to develop a SCAR marker from a RAPD band that appears capable of differentiating Fop from Fo isolates. Though the RAPD band was present in both Fop and Fo isolates, specific regions of the RAPD fragment were polymorphic between Fop and Fo isolates, allowing them to design primers to differentiate between pathogenic and nonpathogenic isolates. However, this is an example of a marker based on a specific gene (Ramos et al., 2001), and it is questionable given the polyphyletic nature of the formae speciales in question, whether a neutral marker such as a RAPD could be used for diagnostic purposes. Thus, more research is needed to identify polymorphic genes with alleles specific to individual formae speciales, which should lead to a greater understanding of the F. oxysporum complex. Perhaps as the cost of sequencing entire genomes becomes less prohibitive, genome comparisons between formae speciales of F. oxysporum may become possible, and lead to potential diagnostic markers for these two important formae speciales. Despite the fact that the isolates collected in this study from common bean were all from plants exhibiting Fusarium wilt symptoms, only four isolates were pathogenic on common bean based on further greenhouse testing, and all were from Colorado. Given the prevalence of Fusarium wilt in the CHP and the number of isolates examined in this study, this result may reflect the difficulty in obtaining pathogenic isolates of F. oxysporum from diseased common bean plants. It has been speculated that part of the difficulty in collecting Fop isolates arises from the shear abundance of opportunistic non-pathogenic isolates in the roots of diseased plants 7

8 8 (Alves-Santos et al., 2002a). Perhaps due to the number of non-pathogenic isolates within infected common bean tissue, the single spore process used in this study was biased toward isolating non-pathogenic isolates. A high level of polymorphism was found among nonpathogenic isolates of Fo in this study confirming previous reports (Alves-Santos et al., 1999; Appel and Gordon, 1994), but how this apparent genetic diversity affects the fitness of nonpathogenic isolates is not known. Because of the small number of pathogenic Fop isolates found in this study, it may be premature to make any generalizations regarding the F. oxysporum population in the CHP. However, the fact that all the CHP Fop isolates we examined seem to be genetically identical has direct relevance to common bean breeders, plant pathologists, and growers. If only one race of Fop exists in the CHP, breeders can focus their efforts on incorporation of genetic resistance to Fop race 4 in common bean cultivars adapted to that region. A lack of genetic diversity among these CHP pathogenic isolates could suggest a recent introduction of Fop race 4 to this region by pathogen dispersal. However, the number and diversity of non-pathogenic isolates suggest that pathogenic forms could be derived from endemic non-pathogenic inhabitants (Gordon and Martyn, 1997). Future studies are needed to identify the potential origin of Fop race 4 in the CHP, perhaps by isolation techniques that somehow select for pathogenic isolates in infected common bean plants. Analyzing more pathogenic isolates from the CHP will allow firmer conclusions to be made regarding the Fop population in Colorado, Nebraska, and Wyoming. In conclusion, we feel that RAPDs are an efficient, cost effective method as a first step in analysis of F. oxysporum populations. RAPD banding patterns and cluster analysis of F. oxysporum isolates collected in the CHP revealed a substantial amount of genetic diversity among Fop, non-pathogenic Fo isolates, and pathogenic Fob isolates. Because the CHP Fop isolates had identical banding patterns and were separated from Fob isolates in the dendrogram, it may be possible to develop a SCAR marker specific for Fop in the CHP. However, due to the large degree of genetic diversity found in this study, RAPD markers were unable to clearly identify isolates based on their pathogenicity or geographic origin. A technique other than RAPDs is needed to differentiate physiological races and/or formae speciales of F. oxysporum. Finally, we have also shown that endemic non-pathogenic isolates in the CHP appear to colonize common bean plants infected with pathogenic F. oxysporum isolates. More research is required to develop a new isolation technique that can preferentially isolate pathogenic isolates from plants exhibiting Fusarium wilt symptoms. We suggest future research on F. oxysporum focus on the identification and isolation of genes with alleles unique to formae speciales and perhaps even physiological races. Acknowledgments This research was supported in part by the Western Regional IPM and Colorado State University Agricultural Experiment Station. The authors would like to thank Drs. Robert Harveson, J.M. Diaz-Minguez, and Barry Jacobsen for providing fungal isolates; and Amy Hill for help with DNA extractions and the RAPD technique. R.A. Cramer was supported by a fellowship in the Department of Soil and Crop Sciences, Colorado State University from the Litzenberger family. Thanks to Drs. Carol Ishimaru, Sarah Ward, and Linda Hanson for critically reviewing this manuscript.

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10 mapping of a major locus for Fusarium wilt resistance in common bean. Crop Sci. 41, Fisher, G.A., J.S. Gerik (1994): Genetic diversity of Fusarium oxysporum isolates pathogenic to sugar beets. (Abstr.) Phytopathology 84, Gordon, T.R., R.D. Martyn (1997): The evolutionary biology of Fusarium oxysporum. Annu. Rev. Phytopathol. 35, Grajal-Martin, M.J., C.J. Simon, F.J. Muehlbauer (1993): Use of random amplified polymorphic DNA (RAPD) to characterize race 2 of Fusarium oxysporum f.sp. pisi. Phytopathology 83, Harter, L.L., J.L.Weimer (1929): A monographic study of sweet-potato diseases and their control. US Dep. of Agr. Tech. Sci. Serv. Bull. 99, 118 pp. Harveson, R.M, C.M. Rush (1997): Genetic variability among Fusarium oxysporum isolates from sugar beet as determined by vegetative compatibility. Plant Dis. 81, Kelly, A., R. Alcala-Jimenez, W. Bainbridge, J.B. Heale, E. Perez-Artes, R.M. Jimenez-Diaz (1994): Use of genetic fingerprinting and random amplified polymorphic DNA to characterize pathotypes of Fusarium oxysporum f.sp. ciceris infecting chickpea. Phytopathology 84, Kendrick, J.B., W.C. Snyder (1942): Fusarium yellows of beans. Phytopathology 32, Kistler, H.C., E.A. Momol, U. Benny (1991): Repetitive genomic sequences for determining the relatedness among strains of Fusarium oxysporum. Phytopathology 81, Manulis, S., N. Kogan, M. Reuven, Y. Ben-Yephet (1994): Use of the RAPD technique for identification of Fusarium oxysporum f. sp. dianthi from carnation. Phytopathology 84, McDonald, B.A. (1997): The population genetics of fungi: tools and techniques. Phytopathology 87, Nelson, A.J., K.S. Elias, E. Arevalo, L.C. Darlington, B.A. Bailey (1997): Genetic characterization by RAPD analysis of isolates of Fusarium oxysporum f.sp. erythroxyli associated with an emerging epidemic in Peru. Phytopathology 87, Panella, L. W., E.G. Ruppel, R.J. Hecker R (1995): Registration of four multigerm sugarbeet germplasm resistant to Rhizoctonia root rot: FC716, FC717, FC718, and FC719. Crop Sci. 35, Pastor-Corrales, M.A., G.S. Abawi (1987): Reactions of selected bean germplasm to infection by Fusarium oxysporum f.sp. phaseoli. Plant Dis. 71, Puhalla, J.E. (1985): Classification of strains of Fusarium oxysporum on the basis of vegetative compatibility. Can. J. Bot. 63, Ramos, B., F.M. Alves-Santos, M. Perlin, E.A. Iturriaga, R. Martin-Dominguez, M.A. Garcia- Sanchez, A.P. Eslava, J.M. Diaz-Minguez (2001): Fopta1, a transcriptional activator specific to highly virulent strains of Fusarium oxysporum f.sp. phaseoli. Fungal Genet. Newsl. 48 (Suppl), 132. Rholf, J.F. (1998): Numerical Taxonomy and Multivariate Analysis System - version 2.0 (NTSYS-pc). Exeter Publishing, Setauket, NY. Ribeiro, R. de L. D., D.J. Hagedorn (1979): Screening for resistance to and pathogenic specialization of Fusarium oxysporum f.sp. phaseoli, the causal agent of bean yellows. Phytopathology 69, Salgado, M.O., H.F. Schwartz (1993): Physiological specialization and effects of inoculum concentration of Fusarium oxysporum f. sp. phaseoli on common beans. Plant Dis. 77,

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12 12 TABLE 1. Characteristics of Fusarium oxysporum isolates used in the RAPD analysis Identification a Host plant Source b Geographical origin c Race d Fop 8/ATCC Bean HFS Sedgwick, Colorado, USA 4 Fop 16 Bean HFS Sedgwick, Colorado, USA NR Fop 24/Fop CL-25 Bean HFS Colombia 3 Fop 308c2 Bean HFS Weld, Colorado, USA NR Fop 31 Bean HFS Netherlands NR Fop 32/ATCC Bean ATCC South Carolina, USA 1 Fop 52 Bean HFS Sedgwick, Colorado, USA NR Fop 53 Bean HFS Sedgwick, Colorado, USA NR Fop 58/AB-6 Bean JMDM Spain 6 Fob 13 Sugarbeet BH Oregon, USA NR Fob 19 Sugarbeet MSU Oregon, USA NR Fob 21 I Sugarbeet BJ Montana, USA NR Fob 21 II Sugarbeet BJ Montana, USA NR Fob 216b Sugarbeet HFS Logan, Colorado, USA NR Fob 216c Sugarbeet HFS Logan, Colorado, USA NR Fob 216d Sugarbeet HFS Logan, Colorado, USA NR Fob 220a Sugarbeet HFS Logan, Colorado, USA NR Fob 257a Sugarbeet HFS Morgan, Colorado, USA NR Fob 257b Sugarbeet HFS Morgan, Colorado, USA NR Fo 5 Sugarbeet BH Texas, USA NP Fo 48 Bean HFS Sedgwick, Colorado, USA NP Fo 103a Bean HFS Morgan, Colorado, USA NP Fo 112a Bean HFS Fo 115a Bean HFS Scotts Bluff, Nebraska, USA Scotts Bluff, Nebraska, USA NP NP

13 13 Fo 115b Bean HFS Scotts Bluff, Nebraska, NP USA Fo 120a Bean HFS Scotts Bluff, Nebraska, NP USA Fo 126a Bean HFS Scotts Bluff, Nebraska, NP USA Fo 127bd Bean HFS Scotts Bluff, Nebraska, NP USA Fo 127bl Bean HFS Scotts Bluff, Nebraska, NP USA Fo 144b e Bean HFS Scotts Bluff, Nebraska, NP USA Fo 201b Sugarbeet HFS Weld, Colorado, USA NP Fo 204a Bean HFS Weld, Colorado, USA NP Fo 204b Sugarbeet HFS Weld, Colorado, USA NP Fo 220d Sugarbeet HFS Logan, Colorado, USA NP a As designated in collection of Howard F. Schwartz, Colorado State University. Fop = Fusarium oxysporum f. sp. phaseoli, Fob = Fusarium oxysporum f. sp. betae, and Fo = Fusarium oxysporum. b Sources of isolates: ATCC, American Type Culture Collection; HFS, Howard F. Schwartz; JMDM, J.M. Diaz-Minguez; BH, Bob Harveson; MSU, Michigan State University; BJ, Barry Jacobsen. c Colorado and Nebraska locations include county of collection. d Pathogenic races as determined by Woo et al. (1996) on a set of differential cultivars. NR = no race designation; NP = non pathogenic isolate as determined by pathogenicity tests conducted in this study. e Fo 144b was not used in RAPD analysis after determination that it was a contaminated culture. However, this isolate does appear on the gel in Figure 1A and thus is listed in this table.

14 14 TABLE 2. Reaction of pathogenic Fusarium oxysporum f.sp. phaseoli isolates used in this study to five common bean cultivars. Common bean cultivars Isolates a UI114 B40 Fisher Viva Diacol Calima Fop race 1 S b I R S S Fop race 3 c R R R R R Fop race 4 S I R S S Fop race 6 S S S S S Fop 16 S I R S S Fop 31 S S S S S Fop 52 S I R S S Fop 53 S I R S S Fop 308 c2 S I R S S a b Origin and characteristics of the isolates are listed in Table 1. S = susceptible reaction (7 to 9 on CIAT severity scale), I = intermediate reaction (4 to 6 on CIAT severity scale), R = resistant reaction (1 to 3 on CIAT severity scale) (Pastor-Corrales and Abawi, 1987). c Previously shown to be pathogenic on Diacol Calima (Woo et al., 1996). We cannot rule out the possibility that this isolate has lost virulence in culture. What effect this has on RAPD banding patterns is not known.

15 15 TABLE 3. RAPD primers used in creating binary matrixes Primer 5 to 3 sequence No. of RAPD bands CS 16 a,b CGT TGG ATG C 12 CS 19 TAC GGC TGG C 7 CS 24 GCG GCA TTG T 8 CS 27 AGT GGT CGC G 7 CS 29 CCA GAC AAG C 12 CS 34 GAT AGC CGA C 10 FUS 01 b CCG CAT CTA C 10 FUS 02 CAG GCC CTT C 2 UBC 6 c CCT GGG CCT A 12 UBC 53 CTC CCT GAG C 5 UBC 188 GCT GGA CAT C 12 UBC 195 GAT CTC AGC G 8 a Primers with a CS prefix were previously used by Grajal-Martin et al. (1993). b Primers with a CS or FUS prefix were synthesized by Integrated DNA Technologies, Coralville, IA. c UBC primers were synthesized at the University of British Columbia

16 16 Figure 1. Ethidium bromide stained agarose gels showing random amplified polymorphic DNA products obtained from amplified Fusarium oxysporum DNA generated from RAPD primer CS 24. A. Gel of CHP Fop isolates, plus Fop races 1, 3, 4, and 6, and Fo isolates 48, 103a, 204a, and 144b: Lane 1 = 1 kb ladder, Lane 2 = Fop race 4, Lane 3 = Fop 16, Lane 4 = Fo 48, Lane 5 = Fop 52, Lane 6 = Fop 53 Lane 7 = Fo 103a, Lane 8 = Fo 204a, Lane 9 = Fo 144b, Lane 10 = Fop race 1, Lane 11 = Fop race 3, Lane 12 = Fop race 6, Lane 13 = Fop 31, Lane 14 = Fop 308 c2. B. Gel of non-pathogenic Fo isolates: Lane 1 = 1 kb ladder, Lane 2 = Fo 120a, Lane 3 = Fo 126a, Lane 4 = Fo 127b lt, Lane 5 = Fo 127b dk, Lane 6 = Fop 112a, Lane 7 = Fo 115a, Lane 8 = Fo 115b. C. Gel of Fob isolates and 2 Fo isolates from sugar beet: Lane 1 = 1 kb ladder, Lane 2 = Fob 257a, Lane 3 = Fob 257b, Lane 4 = Fob 216c, Lane 5 = Fob 216b, Lane 6 = Fob 216d, Lane 7 = Fob 21 I, Lane 8 = Fob 21 II, Lane 9 = Fob 13, Lane 10 = Fob 19, Lane 11 = Fob 220a, Lane 12 = Fo 220d, Lane 13 = Fo 201b. Figure 2. Genetic similarity among Fusarium oxysporum isolates collected from the Central High Plains and donated from other Fusarium researchers. The dendrogram is an UPGMA tree based on analysis of 12 RAPD primers that generated 105 RAPD markers using Jaccard s coefficient of similarity. Cophenetic correlation value: r = 0.980

17 17 Figure 1: Top A B C

18 18 Figure 2 Top Jaccard's Coefficient of Similarity Jaccard s Coefficient of Similarity Foprace4 Fop16 Fop52 Fop53 Fop308c2 Foprace1 Fop31 Fo120a Fo112a Fo126a Fob21I Fob216d Fo48 Fo103a Fo127bl Fo127bd Fo115a Fo115b Fo5 Foprace3 Foprace6 Fo220d Fo204a Fo201b Fo204b Fob19 Fob220a Fob21II Fob13 Fob257b Fob257a Fob216c F.solani Fob216b