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By Kristin Ann Schneider A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences Plant Breeding and Genetics 1 999 ABSTRACT BREEDING FOR RESISTANCE TO FUSARIUM ROOT ROT IN BEAN (PHASEOL US VULGARIS L.) By Kristin A. Schneider Genetic resistance to the root rot causing organism, Fusarium solani f.sp. phaseoli, has been documented in bean (Phaseolus vulgaris) but progress towards introgression of this resistance into the highly susceptible large-seeded market classes has been limited. Resistance to this soilbome pathogen is complex and strongly influenced by environmental factors. As an approach to reduce effects of environmental variation and aid in the identification of physiological resistance, a simple, rapid, and inexpensive perlite- based greenhouse screen was developed. The procedure allowed for the evaluation of lateral roots, correlated well with field ratings and permitted evaluation of large populations. Correlations between root rot scores of two genotypic groups measured in field and greenhouse experiments were as high as 0.99. This protocol was subsequently used to facilitate the genetic characterization of root rot resistance in bean using two recombinant inbred populations derived from resistant X susceptible crosses (‘Montcalm’lFR266 and ‘Seafarer’lN203). Heritability estimates calculated on an entry mean basis ranged from 0.48 to 0.71 for Montcalm/FR266 and from 0.09 to 0.66 for Seafarer/N203 based on field and greenhouse experiments. Using greenhouse data from MF, DNA from five resistant and five susceptible genotypes was pooled to create two contrasting bulks for identification of marker associations. Forty-six RAPD markers identified by this method were significant in one-way analyses of variance using root rot data from greenhouse and field experiments. The amount of phenotypic variation explained by these markers ranged from 0.04 to 0.14. Those markers that were significantly associated with greenhouse ratings tended not to be associated with field ratings and vice versa. Mapmaker was used to determine linkage groups among 35 markers. Three of seven linkage blocks identified could be positioned on the integrated P. vulgaris linkage map. Based on these placements, several significantly associated markers mapped to genomic locations that corresponded to P. vulgaris pathogenesis-related proteins. Two RAPD markers, P7700 and G320“, were identified to be consistently and significantly associated with greenhouse and field ratings, respectively, and could be utilized for marker assisted selection. Multiple regression analyses using these two markers explained from 7 to 13 % of the phenotypic variation for root rot. Copyright by Kristin Ann Schneider 1999 To my friends and family. ACKNOWLEDGMENTS I would like to acknowledge the many people who have contributed to this project and my program. To Dr. James Kelly, my major advisor, I would like to express my overwhelming gratitude for his support and guidance. He has played an instrumental role in the development and success of my professional career and will always be remembered as an outstanding mentor. I would like to thank the other members of my committee, Drs. David Douches, Patrick Hart and Alvin Smucker for all their help and support through this process. And, a warm thanks to Ken Grafton for his collaboration and interest in this project. To the bean group, Halima Awale, Jerry Taylor, Norman Blakely and Dr. George Hosfield, I would like to express my deep appreciation for your help and suggestions. I would also like to acknowledge Michele Mance Bonner for her friendship and curiosity; we spent many an adventure researching bean root rot. Financial support for this Ph.D. project was provided by the USDA National Needs Fellowship program and the State of Michigan’s Project GREEEN (Generating Research and Extension to meet Economic and Environmental Needs). Their funding was greatly appreciated. I would finally like to acknowledge my friends and family who have supported me, unconditionally, through both my Masters and Ph.D. I am deeply indebted to all of them for my success and am extremely grateful that I had the Opportunity to spend this time with them. vi TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... x LIST OF FIGURES ...................................................................................................... xiii LIST OF ABBREVIATIONS ....................................................................................... xiv LITERATURE REVIEW ................................................................................................ 1 RESEARCH JUSTIFICATION ................................................................................... 1 DISEASE COMPLEX ................................................................................................. 2 Pythium spp. .......................................................................................................... 3 Rhizoctonia solani ............................................................................... . ..................... 4 Thielaviopsis basicola and Aphanomyces euteiches .................................................. 5 F USARI UM SOLANI F .SP.PHASEOLI ....................................................................... 5 Symptomotology ....................................................................................................... 6 Penetration ........................................................................................................... 7 Cortical invasion ................................................................................................... 8 Environment .......................................................................................................... 10 Germination ....................................................................................................... 10 Penetration ......................................................................................................... l4 Inoculum Density ....................... 19 Organic soil amendments .................................................................................... 22 PHASEOLUS VULGARIS .......................................................................................... 25 Environment .......................................................................................................... 25 vii Soil Compaction and Anoxia .............................................................................. 25 Tillage ................................................................................................................ 27 Organic soil amendments .................................................................................... 27 Resistance .............................................................................................................. 29 Gerrnplasm Sources ............................................................................................ 29 Inheritance ......................................................................................................... 3 1 CONCLUSION ......................................................................................................... 32 REFERENCES .......................................................................................................... 36 CHAPTER 1: A GREENHOUSE SCREENING PROTOCOL FOR THE IMPROVEMENT OF FUSARIUM ROOT ROT IN BEAN (PHASEOLUS VULGARIS L.) ....................................................................................... 42 ABSTRACT .............................................................................................................. 42 INTRODUCTION ..................................................................................................... 43 MATERIALS AND METHODS ............................................................................... 47 Fungal Isolation Procedure ................................................................................... 47 Greenhouse Screen ............................................................................... ' ................ .48 Field Trials ............................................................................................................ 51 Statistical Analysis ................................................................................................. 54 RESULTS ................................................................................................................. 54 DISCUSSION ........................................................................................................... 57 REFERENCES ....................... 63 CHAPTER 2: ANALYSIS OF RESISTANCE TO FUSARIUM ROOT ROT IN BEAN AND THE IDENTIFICATION OF ASSOCIATED QTL .............................................. 65 ABSTRACT .............................................................................................................. 65 viii INTRODUCTION ..................................................................................................... 66 Objectives .............................................................................................................. 69 MATERIALS AND METHODS ..................................................... ' .......................... 69 Germplasm and Population Development. ............................................................. 69 Root Rot Evaluation ............................................................................................... 70 Field ................................................................................................................... 70 Greenhouse ........................................................................................................ 71 Statistical Analysis ................................................................................................. 72 Marker analysis ..................................................................................................... 73 DNA Extraction ................................................................................................. 73 RAPD Protocol ................... .............................................................................. 74 RESULTS ................................................................................................................. 75 Root rot evaluations ............................................................................................... 75 Marker evaluation .................................................................................................. 82 DISCUSSION ........................................................................................................... 90 REFERENCES ........................................................................................................ 101 APPENDD< A ............................................................................................................. 105 GREENHOUSE PROTOCOL ................................................................................. 106 APPENDIX B ............................................................................................................. 109 GENETICS AND MARKER ANALYSIS ..................... 110 LIST OF TABLES Table 1. Description of disease rating scale used for all root rot experiments. ................. 49 Table 2. Seed type, greenhouse rating (GH96), first (RI) and second (RR2) field ratings, average root rot rating of the two field evaluations (AVG), seed yield, and 100 seed weight (SW) for 24 genotypes grown in Presque Isle County, MI in 1996. ........................................................................................... 52 Table 3. Seed type, greenhouse rating (GH97), first (RI) and second (RR2) field ratings, average root rot rating of the two field evaluations (AVG), seed yield, and 100 seed weight (SW) for 21 genotypes grown in Presque Isle County, MI In 1997. ........................................................................................... 53 Table 4. Pearson rank correlation coefficients for yield, 100 seed weight (SW), first (RRRl) and second field (RRR2) root rot ratings, averaged field root rot rating (AVG), and greenhouse (GI-I) evaluations for each group of genotypes, GH96 and GH97. GH96 values are presented in normal print in the upper right-hand diagonal whereas the GH97 values are printed in bold in the lower left-hand diagonal. ........................................................................... 56 Table 5. Fusarium solani f.sp. phaseoli isolates and location of origin used to inoculate greenhouse evaluations for three recombinant inbred populations, Seafarer/N203 (SN), Montcalm/FR266 (MF) and Isles/FR266 (IF). Two greenhouse screens were conducted using SN (SNGHl and SNGH2) and three were performed using MF (MFGHl, MFGH2, MFGH3) ............................ 73 Table 6. Analyses of variance for root rot ratings for the recombinant inbred populations, Seafarer/N203 (SN) and Montcalm/FR266 (MF). Four experiments are presented for each population. Two greenhouse experiments (SNGHI and SNGH2) for SN and three for MF (MFGHl, MFGH2 and MFGH3). A combined analysis over two locations in Presque Isle County, MI (PI) and Perham County, MN (PM) is presented for both populations. The SN population was evaluated in 1997 and the MP in 1998. An additional PI experiment was conducted for SN in 1998 and is presented ....................... 76 Table 7. Root rot ratings of the top and bottom five Montcalm/FR266 (MP) recombinant inbred lines based on the second MF greenhouse evaluation (MFGH2). Rankings of these genotypes in all other evaluations including two greenhouse screenings (MFGHI and MFGH3), two field trials conducted in Presque Isle County (1998 PI), MI and Perham County, MN (1998 PM) and the combined analysis over these two locations are given in parentheses. Scores for check genotypes evaluated for each experiment are also given for comparison. .................................................................................. 77 Table 8. Root rot ratings of the top and bottom five Seafarer/N203 (SN) recombinant inbred lines based on the first SN greenhouse evaluation (SNGHI). Rankings of these genotypes in all other evaluations including a second greenhouse screenings (SNGH2), two field trials conducted in Presque Isle County (1997 PI), MI and Perham County, MN (1997 PM) in 1997, the combined analysis over these two locations and another field trial conducted in 1998 in Presque Isle County, MI (1998 PI) are given in parentheses. Scores for check genotypes evaluated for each experiment are also given for comparison. .................................................................................. 78 Table 9. Heritability estimates (hz) and standard errors of Fusarium root rot ratings for the Montcalm/FR266 (MF) and Seafarer/N203 (SN) populations. Six greenhouse and field experiments were analyzed for each population. ................. 81 Table 10. Pearson correlation coefficients between means of Fusarium root rot ratings for 6 greenhouse and field experiments conducted using the Montcalm/FR266 population and days to flower (DTF). ..................................... 83 Table 11. Linkage groups (LG) generated by MAPMAKER/EXP from 35 RAPDS screened against the Montcalm/FR266 population. Markers not presented were independent ................................................................................................ 84 Table 12. Coefficients of determination (r2) from one-way analyses of variance for a select group of RAPDs significantly associated with mean root rot scores of 79 Montcalm/FR266 F4-derived RILS collected from three greenhouse experiments (MFGHl, MFGH2, MFGH3), two field trials located in Presque Isle County, MI (PI) and Perham County, MN (PM), and the combined analysis over locations. The linkage group (LG) is also given and corresponds to those linkage arrangements presented in Table 11. The r2 for RAPDs significantly associated with days to flower (DTF) is also presented. ........................................................................................................... 88 Table 13. A comparison of mean root rot scores of control treatment (inoculated with deionized water) and 5 different isolates isolated from Presque Isle County, MI (Rs. 12, Hawks 2a, 2b and 6d) and Huron County, MI (Huron 2a) inoculated on to three bean genotypes possessing differing levels of resistance .......................................................................................................... 106 Table 14. Analysis of variance including degrees of freedom (DF), sums of squares (SS) and error mean square (EMS) for the 24 entries planted in the 1996 Presque Isle County, MI root rot trial. .............................................................. 107 Table 15. Analysis of variance including degrees of freedom (DF), sums of squares (SS) and error mean square (EMS) for the twenty-four entries planted in the 1997 Presque Isle County, MI root rot trial ................................................. 108 Table 16. Analyses of variance including degrees of freedom (DF), sums of squares (SS), error mean square (EMS), F-value, experimental mean, LSD (a < 0.05) and coefficient of variation (CV) for indivdiual field ratings performed on the Montcalm/FR266 (MF) population in Presque Isle County, MI and Perham County, MN in 1998. The analyses of variance combined over locations and calculated for all greenhouse experiments are presented in Table 6 .......................................................................................... l 10 Table 17. Analyses of variance including degrees of freedom (DF), sums of squares (SS), error mean square (EMS), F-value, experimental mean, LSD (on < 0.05) and coefficient of variation (CV) for each of the two ratings (RI and RR2) performed on the Seafarer/N203 (SN) population in Presque Isle County, MI and Perham County, MN in 1997. The analyses of variance combined over locations and calculated for all greenhouse experiments are presented in Table 6 .......................................................................................... l 11 Table 18. Probability, coefficient of deterimination (r2), number of individuals (n), mean of the group of individuals lacking the RAPD (X1) and mean of the group of individuals possessing the RAPD band (X2) for all significant associations determined using one-way analyses of variance against root rot data collected from three greenhouse evaluations (MFGHl, MFGH2, MFGH3), two field trials conducted in Presque Isle County, MI (P198) and Perham County, MN (PM98), the combined analysis over these two locations (C98), days to flower (DTF) and days to maturity (MAT) .................. 112 LIST OF FIGURES Figure 1. Interaction between environment, fungal pathogen and host. Environmental aspects which fall into the Rs. f.sp. phaseoli circle have been shown to alter characteristics of the pathogen. Environmental aspects which fall into the Phaseolus vulgaris circle influence plant growth and disease susceptibility. .......................................................................................... 35 Figure 2. Visual representations of bean roots at corresponding disease severity ratings described in Table 1. ............................................................................... 50 Figure 3. Fusarium root rot infection of ‘Montcalrn’ dark red kidney (right) compared to a non- -inoculated control (left). ....................................................... 60 Figure 4. Representative distributions of root rot scores for Seafarer/N203 (SN) and ..................................................................................................................... 80 Figure 5. Comparative linkage maps of linkage group (LG) 2 and its corresponding position on linkage group BZ from the integrated bean linkage map and linkage group D2 from the Davis map. BZ and D2 are reproduced from Freyre et al. (1998). All markers on L62 were signficantly associated with root rot resistance (Table 10) .............................................................................. 86 Figure 6. Comparative linkage maps of linkage groups (LG) 7 and 3 and their corresponding positions on linkage group B3 from the integrated linkage map and linkage group D3 from the Davis map. B3 and D3 are reproduced from Freyre et al. (1998). Markers OPS8500 and OPIl8lsoo were both ‘ significantly associated with root rot resistance (Table 10). ................................. 87 Figure 7. Monthly precipitation totals for the weather stations in A. Presque Isle County, MI and B. Perham County, MN for 1997 and 1998. ............................ 117 SW AN OVA AVG BCMV C:N CBB CFU cM CV DAP DNA DRK DTF LOD LRK LSD MFGH l MFGH2 MFGH3 MI MN PCR PI PM PR PvPR l PvPR2 QTL r R2 r2 RAPD RRRl LIST OF ABBREVIATIONS 100 seed weight Analysis of variance average Bean common mosaic virus base pair Carbon Carbon to Nitrogen ratio Common bacterial blight Colony forming units Centimorgan Coefficient of variation Days after planting Deoxyribosenucleic acid Dark red kidney Days to flower Forma speciales Heritability estimate Horizontal resistance Isles/FR266 population Linkage group Likelihood of the odds ratio Light red kidney Least significant difference Montcalm/FR266 population Montcalm/FR266 greenhouse evaluation 1 Montcalm/FR266 greenhouse evaluation 2 Montcalm/FR266 greenhouse evaluation 3 Michigan Minnesota Nitrogen Polymerase chain reaction Presque Isle County, MI Perham County, MN Pathogenesis-related protein Phaseolus vulgaris pathogenesis-related protein 1 Phaseolus vulgaris pathogenesis-related protein 2 Quantitative trait loci Correlation coefficient Coefficient of determination Randomly amplified polymorphic DNA Recombinant inbred line Field root rot rating 1 xiv SN SNGHl SNGH2 VR Field root rot rating 2 Seafarer/N 203 population Seafarer/N203 greenhouse evaluation 1 Seafarer/N203 greenhouse evaluation 2 Vertical resistance XV LITERATURE REVIEW RESEARCH JUSTIFICATION Dry bean (Phaseolus vulgaris L.) is a major cash crOp for growers in the mid and northern regions of Michigan with an annual farm gate value of $100-150 million and an additional $20 million in revenues for handling, processing and marketing (Christenson and Kelly, 1992). The strength of the industry lies in the diversity of market classes, production of both tablestock and seed crop beans and the ability to niche certain seed types to specific ecogeographic regions of the state. Over the last quarter century, northern Michigan has successfully raised dark red kidney beans for certified seed production (Copeland, 1995). Climate, isolation and management have all helped to maintain a high level of productivity. Seed from this region is planted commercially in central Wisconsin and Minnesota but problems with root rots in all three states are forcing farmers to limit their planting of dark red kidney bean cultivars from Michigan. The lack of resistance to the major root rot causing pathogen, Fusarium solani (Mart) Sacc. f.sp. phaseoli (Burk.) Snyd. & Hans. in the larger bean seed types has reduced productivity and limited the market potential of Michigan dark red kidney cultivars. Since chemical control methods are expensive and inconsistent, resistant cultivars offer growers the only effective opportunity to maintain and expand kidney bean production both in Michigan and elsewhere. Without adequate levels of resistance to this persistent soil pathogen, Michigan could, potentially, loose the kidney bean seed industry just as it lost the pea (Pisum sativum L.) industry to similar problems with root pathogens some fifty years ago. Genetic resistance, identified in tropically adapted lines, needs to be characterized and incorporated into locally adapted kidney bean cultivars. Moreover, since disease ratings require destructive evaluation and are often confounded by climatic factors, indirect selection using linked markers would improve the efficiency of the breeding program. Although root rot is a special problem of kidney beans grown in northern Michigan, it reduces the yield potential of all bean types grown in Michigan and is particularly severe in wet and cool seasons. Understanding the inheritance of resistance and using indirect screening methods would permit the more rapid incorporation of resistance into a number of other commercial seed types such as navy beans. Without adequate resistance to root rot, the dark red kidney bean seed industry will continue to shrink, leaving growers in northern Michigan with few alternative crops to plant for revenue. The loss of a vital commodity, in which local growers have been effective global competitors for the last four decades, is a major concern. DISEASE COMPLEX Root rot in common bean is comprised of a complex of pathogens including F usarium solani f.sp. phaseoli, Rhizoctonia solani Kuhn, Pythium spp., Thielaviopsis basicola (Berk. & Br.) Ferr. and Aphanomyces euteiches f.sp. phaseoli Drechs. F usarium oxysporum Schlecht. is also a predominant component of the rhizosphere and is a serious problem in the inter mountain regions of the US, but in Midwestern production areas it is often considered to be non-pathogenic or of minor importance (Sippell and Hall, 1982b). Depending on environmental conditions and bean production region, any one of these pathogens in any combination can be of significant consequence (Reeleder and Hagedom, 1981). Root rot of snap beans is particularly severe in the central sands of Wisconsin where continuous cropping of bean is common. Reports of the predominant root rot inciting fungus in this production area have changed over time. Yang and Hagedom (1966) first classified F.s. f.sp. phaseoli as the major root rot pathogen in Wisconsin followed by Pythium spp. and R. solani. By 1975, Hoch et al. concluded that Pythium species were the predominant causal organism of root and hypocotyl rot of bean. Pythium species were most frequently isolated from bean hypocotyls and roots and disease symptoms on infected plants resembled those of Pythium infection. While F.s. f.sp. phaseoli and R. solani were also isolated, they were not considered major pathogens. Root rot in the central sands of Wisconsin is now considered to be predominantly caused by Pythium spp. and A. euteiches f.sp. phaseoli and breeding programs currently address developing resistance to these two pathogens (Rand et al., 1984). These results do not preclude F.s. f.sp. phaseoli as a major pathogen in Wisconsin in areas other than the central sands. Seed decay, pre-emergence damping-off and root rot caused by P. ultimum Trow. also substantially reduce yields of snap bean in New York State (Pieczarka and Abawi, 1978). Pythium spp. Pythium symptoms first occur 16 to 20 days after planting as water soaked lesions on hypocotyls that later become drier, tan to reddish-brown and sunken. In advanced cases hypocotyls appear papery and reddish brown and can resemble F.s. f.sp. phaseoli infection. Below-ground hypocotyl and fibrous roots can be completely destroyed in more severe infections. Pythium infection appears to be more severe at higher soil water potentials than F.s. f.sp. phaseoli infection which is considered to be a dry root rot (Hoch et al., 1975). Stunting of seedlings caused by Pythium infection reduces total plant weight and decreases number of pods per plant which ultimately affects yield (Sippell and Hall, 1982b). In this same study, F.s. f.sp. phaseoli did not affect plant size but reduced seed weight per pod. Sippell and Hall (1982a) suggest that reductions in different yield components caused by these two pathogens reflect differing times of infection. While symptoms of both diseases can occur early in plant development, negative effects on yield are Observed earlier for P. ultimum infection than Rs. f.sp. phaseoli infection. Stunting of plants caused by P. ultimum occurs before flowering which reduces total biomass and ultimately reduces number of pods per plant. In contrast, F.s. f.sp. phaseoli exerts negative effects on plant growth and development post-bloom resulting in reduced seed weight per pod. Rhizoctonia solani Another often cited member of the root rot complex is R. solani. Rhizoctonia solani is the causal organism of web blight (foliar disease) and contributes to root rot in bean. The fungus, by delaying emergence, ultimately delays maturity and reduces marketable yields (van Bruggen et al., 1986). Disease development occurs mainly under moderate to high moisture conditions and moderate temperatures. While root rot is most severe at 16 to 22° C for F.s. f.sp. phaseoli and between 14 to 21° C for Pythium, maximum infection by R. solani occurs between 21 and 27°C (Sippell and Hall, 1982a; van Brugen et al., 1986). Rhizoctonia solani and resulting root rot and web blight are major problems for bean growing regions of Latin America and Africa, but it is of minor importance compared to Pythium and ES. f.sp. phaseoli in the North American continent (Abawi and Pastor Corrales, 1990). Thielaviopsis basicola and Aphanomvces euteiches Other root pathogens of relative importance are T. basicola and A. euteiches. Thielaviopsis basicola is the causal organism of black root rot which has a wide host range including alfalfa, cotton, tobacco and sweet potato (Abawi and Pastor Corrales, 1990). Disease can be controlled in bean, however, using several fungicides as seed or soil treatments. High levels of resistance are also available and utilized in specific breeding programs. In contrast to T. basicola which can cause root rot on several hosts, A. euteiches consists of two formae speciales which are both pathogenic to bean, A.e. f.sp. phaseoli and A.e. f.sp. pisi. Aphanomyces euteiches f.sp. phaseoli is only pathogenic on bean whereas A.e f.sp. pisi infects both bean and pea. Symptoms of this root disease can resemble Pythium root rot infection including chlorosis, wilting, and premature defoliation. Recognition of A. euteiches as a serious root rot pathogen has occurred relatively recently and, as discussed above, is now believed to be the primary pathogen of bean production areas in the Wisconsin and Minnesota central sands (Rand et al., 1984). F USARIUM SOLANI F.SP.PHASEOLI Some of the most devastating root pathogenic fungi include Fusarium spp. (Kommedahl and Windels, 1979). Fusarium solani f.sp. phaseoli is 'a major root rot pathogen of bean in Nebraska, Wyoming, Colorado, California, Washington, Ontario, North Dakota and Michigan (Steadman et al., 1975; Keenan et al., 1974; Smith and Houston, 1960; Burke and Silbemagel, 1965; Saettler and Anderson, 1978; Sippell and 5 Hall, 1982a; Rackham and Vaughn, 1959; Estevez de Jensen et al., 1998). Steadman et al. (1975) isolated F.s. f.sp. phaseoli from bean roots and hypoctols in 71% of all plants collected in western Nebraska, 90% of which were pathogenic on bean. Hall (1979) surveyed thirty-one bean fields in Ontario and identified F.s. f.sp. phaseoli in 74% of all fields sampled. Yield reductions caused by Fusarium root rot have been reported as high as 84% for pinto production in Utah, Wyoming, Colorado, and New Mexico. Likewise, Fusarium root rot is considered one of the most serious diseases of irrigated beans in the Pacific Northwest after white mold (Sclerotinia sclerotiorum (Lib.) de Bary) (Dryden and Van Alfen, 1984; Miller and Burke, 1986). Synergistic effects between F.s. f.sp. phaseoli and Pythium that increase disease severity have been demonstrated in controlled greenhouse studies (Piecarka and Abawi, 1978) but effects of F.s. f.sp. phaseoli and other root rot components are generally considered additive (Sippell and Hall, 1982a). The widespread nature and importance of F.s. f.sp. phaseoli as a predominant root rot pathogen in bean emphasizes the need for effective control against this disease which will ultimately be achieved through a combination of proper cultural management and through the development of resistant cultivars. Symptomotology Typical symptoms of Fusarium root rot occur 1 to 2 weeks after planting where parallel red-brown streaks appear on the hypocotyl and tap root. These lesions become more pronounced with time and, in severe cases, will converge to form larger lesions that encompass the entire underground stem and root system. The primary and lateral root tissue can die and hypocotyl tissue can deteriorate and become hollow at which stage adventitious root growth proceeds from tissue above the site of infection. Above ground symptoms include premature defoliation, stunting, and chlorosis which leads to a reduction in number of pods and seed size (Abawi and Pastor Corrales, 1990). Christou and Snyder (1962) present an excellent description of F.s. f.sp. phaseoli infection of bean and will be summarized as follows: Penetration F usarium solani f.sp. phaseoli penetrates directly into healthy epidermis or proceeds through wound sites or hypocotyl stomata at soil surface level without the formation of an appresorium. Although F. s. f.sp. phaseoli is mainly an intercellular pathogen, it can invade dead or senescing cells intracellularly. Germinating spores in the vicinity of bean plant tissue possess l or 2 germ tubes per spore. Before penetration, germ tubes form a small thallus that attaches to the hypocotyl. Penetration through stomata proceeds directly through the stomatal opening often resulting in browning and collapse of the guard cells. Vigorous hyphal growth then proceeds in the stomatal chamber. Under optimum conditions hyphae in the stomatal chamber can re-emerge from the stomata to become sites of sporodochial formation at the soil level and sources of secondary infection. Hyphae from the thallus can also penetrate directly through the cuticle after which the fungus grows longitudinally following the long axis of epidermal cells. Hyphae will then travel through the middle lamella of epidermal cells and multiply profusely once they reach the first intercellular space of the cortical tissue. Infection proceeds intercellularly until cortical cells die or senesce. Another source of hypocotyl or root infection occurs through wound sites. Wounding is the result of one to many injured cells that provide entrance for fungal hyphae. Once established in the epidermal middle lamella of the hypocotyl, the fungus advances into the cortical tissue as previously and subsequently described. Wound sites include the broken bases of trichomes or points of emergence of adventitious roots along the hypocotyl and often exhibit marked browning of the epidermal or cortical cells surrounding the wound. Root penetration is similar to that observed for the hypocotyl where the fungus will invade directly through the epidermis or through wound sites. Penetration of root hairs was not observed. Contrary to direct or stomatal penetration, wound penetration begins with intracellular invasion of the damaged and/or dying cells. Once established, the fungus proceeds intercellularly. Cortical invasion Hyphae grow rapidly through the intercellular spaces of the cortex in a parallel fashion often filling the entire intercellular space. Hyphae can also proceed laterally from the aforementioned longitudinal hyphae, invading the cortex inwardly. The'perpendicular or oblique growing hyphae are very distinct exhibiting a characteristic digitate envelopment of the cortical cells. These hyphae can reorient themselves longitudinally to subsequently give rise to more inwardly-growing hyphae. Fungal invasion is stopped by the endodermis. Longitudinally growing hyphae develop more rapidly than the inward, lateral hyphae resulting in the characteristic reddish brown streaks observed on the hypocotyl. These lesions will ultimately coalesce and can completely encircle the hypocotyl. The reddish brown discoloration is the result of advanced infection and represents the accumulation of phenolic compounds from the host. The fungus will also invade cortical cells through rupture sites created by emerging laterals and can infect newly developed lateral roots by this route. Vascular infection by F.s. f.sp. phaseoli is believed to originate through this inlet. Infection was not observed in the pericycle but could be identified in the meristematic and procambial tissue of the root laterals which would be the source of infection observed in xylem and phloem vessels (Chatterjee, 1958). Christou and Snyder (1962) report that the primary mode of penetration is through the stomata. Consequently, F. solani should be regarded as a hypocotyl pathogen as Opposed to a root rot pathogen. The authors’ conclusions are based on the finding that fewer infections are observed on the roots compared to the hypocotyl and root lesions are smaller. Burke and Barker (1966), however, showed that yield reductions resulted only when severe root damage occurred. The authors grew seedlings in both F.s. f.sp. phaseoli infested “islands” of soil surrounded by non-infested soil and vice versa. Yield was only reduced in the latter where lateral roots grew out of non-infested “islands” and into infested soil. In the case where the hypocotyl and taproot were in contact with infested soil and laterals were allowed to grow out of this contaminated island, no yield reductions were observed. If the root system was functional, a severely rotted hypocotyl could still sustain a productive plant. Further evidence in support of lateral importance is provided by the lack of effective root rot control by fungicides applied to hypocotyl. - After tissue death occurs and the fungus has invaded cortical and vascular tissues, chlamydospores are produced within the senescing or dead tissue. Additionally, sporodocia are produced from mycelium in the substomatal stromatic tissue and release 9 macroconidia that form more chlamydospores or become sources of new infection (Christou and Snyder, 1962). The basic colony forming units (CFU) of F.s. f.sp. phaseoli is the chlamydospore which is predominantly produced from macroconidia. The canoe-shaped macroconidia of F.s. f.sp. phaseoli produce germ-tubes which form infection thalli or chlamydospores (Garrett, 1977). Chlamydospores are found in soil associated with plant tissue or humus (Nash and Snyder, 1962). Either chlamydospores or macroconidia can produce infection. Environment For foliar diseases, moisture is the determining factor in spore germination (Garrett, 1977). Soilbome pathogens, however, inhabit a more complicated environment and germination is influenced by other factors. Moisture in soil environments is usually not limiting. If spores of a soilbome pathogen germinated as a result of adequate moisture conditions, most would die of nutrient starvation. Thus, soilbome pathogens sense other factors and germinate under optimum conditions. Germination Chlamydospores of F.s. f.sp. phaseoli are uniformly distributed in the soil within the plow layer based on non-significant differences in spore counts obtained from soil core samples and are relatively immobile in the absence of the host (Nash and Snyder, 1962). Growth of F.s. f.sp. phaseoli in the presence of non-host species is negligible consisting mainly of a recycling of chlamydospores (Huber and Watson, 1970). While spore germination is observed under certain conditions in the absence of host and saprophytic growth of F. s. f.sp. phaseoli in contact with lettuce, maize, and tomato crops can result in 10 a doubling of the pathogen population, penetration and invasion of non-host does not occur (Garrett, 1977). It can be concluded, therefore, that a common set of environmental cues, not involved in host-pathogen recognition, stimulates spore germination. Mondel et al. (1996) concluded that there is no specific recognition of nutrient exudation of the host plant because Spores germinated equally well in root extracts from bean, pea, cotton, barley and tomato. Pea, bean and cotton root extracts, however, resulted in larger chlamydospore formation suggesting a greater store of nutrients which could enhance germination capability. Spores formed on pea, bean and cotton extracts correlated with increases in virulence while greater numbers of chlamydospores were produced on oat and barley root extracts. The smallest chlamydospores were formed from F.s. f.sp. phaseoli grown in tomato extract. The major constituents of these plant exudates are carbon (C) and nitrogen (N) and most hypotheses attempt to explain stimulation of spore germination in terms of C and N and the resulting ratio (C:N). Upon closer analysis of spore germination stimulated by host exudate, it was observed that glucose, alone, resulted in 16 to 20% germination while amino acids triggered from 40 to 50% germination. Glucose in combination with ammonium (NHH) nitrogen or potassium nitrate also triggered from 40 to 50% germination whereas zero germination was observed when N was added alone (Cook and Snyder, 1965). These results indicate that both N and C are required for spore germination (Toussoun, 1970). The minimal germination rates observed in glucose amended soil could be explained by the presence of background N in soils in which the spores were tested. It appears, therefore, that N requirements for spore germination are minor (Griffin, 1964). Low N requirements could provide F .s. f.sp. phaseoli a competitive advantage over other soil microorganisms 11 under N-lirniting conditions. Toussoun (1970) reports that as little as 10 to 20 ppm of N is required for Spore germination whereas 100 to 1000 ppm of glucose is necessary to achieve germination. It can be inferred, accordingly, that with respect to spore germination, carbon is the limiting nutrient. However, high C soil amendments such as barley straw (83:1) can reduce spore germination and Fusarium root rot (Griffin, 1964; Maier, 1959; Huber et al., 1965; Maurer and Baker, 1965). High C amendments effectively immobilize all available soil N below levels required for spore germination. This observed reduction in root rot is rapidly diminished with the addition of N in the form of ammonium or nitrate (N03) (Griffin, 1964). Griffin (1964) demonstrated that with the addition of as little as 5 ppm NH4N03 the suppressive effect of barley straw could be overcome. Stimulation and inhibition of spore germination are also strongly influenced by the microbial environment and its impact on nutrient availability. Cook and Snyder (1965) observed, in their studies on spore germination, that asparagine, added to a sandy loam soil, induced 30% germination of chlamydospores but within a short period the germ tubes and spores were disrupted by autolysis believed to be initiated by carbon starvation. Less than 4% of germinated chlamydospores survived under this treatment. In contrast, glucose amendments in the presence of background N resulted in 15% germination with no lysis. These results provide further evidence in support of the influence of carbon availability on spore germination. The following theory is presented to achieve a broader perspective. In soil, very high C:N ( > 30:1) will suppress spore germination because the presence of large amounts of C effectively immobilizes any available N required to support germination. When the C:N decreases to a more moderate level (25:1), enough N is present to initiate 12 germination. Fusarium solani f.sp. phaseoli, however, requires much less N for germination than many of the other, more competitive microorganisms in the soil (Lindsey, 1965; Griffin, 1964). Thus, while F.s. f.sp. phaseoli is germinating under a relatively minimal amount of N, the available C is not being consumed by other soil microorganisms that are active under higher amounts of N. Saprophytic growth of F.s. f.sp. phaseoli continues or, in the presence of host tissue, infection can ensue. At lower C:N (8:1), enough N is available to support active growth of more competitive soil microorganisms which subsequently consume available carbon, depleting this nutrient and enhancing autolysis of F. s. f.sp. phaseoli spores. The C:N content of soil appears to effect spore germination inasmuch as it influences soil microbiota and that F.s. f.sp. phaseoli persistence is a reflection of this process. While the nutrient stimulation of spore germination has been thoroughly addressed, recent research by Ruan et al. (1995) has identified a second stimulation mechanism of spore germination. The presence of the flavanone, naringenin, and the flavone, apigenin, have been identified in the rhizosphere of legumes and have been shown to induce nod gene expression of many Rhizobia species. nod gene expression is involved in the initiation of the symbiotic plant-bacteria interactions required for nodulation and Ng-fixation. F usarium solani f.sp. phaseoli spore germination appears to be stimulated by the isoflavone, genistein, and flavanone, naringenin, which are both inducers of nod genes in bean. Beans appear to exude adequate concentrations of these secondary metabolites to significantly enhance spore germination. The pterocarpan phytoalexins, rnedicarpin and maackiain from chickpea (Cicer arietinum L.) also stimulated spore germination of F.s. f.sp. phaseoli. Different formae speciales of F. solani exhibit different stimulatory l3 responses to different sets of flavonoids suggesting a mechanism for host recognition. Spore germination of the pea pathogen F.s. f.sp. pisi (Jones) Snyd & Hans was stimulated by pisatin, medicarpin, and maackiain; phytoalexins for which F.s. f.sp. pisi possesses a corresponding degradation enzyme. The bean phytoalexin, phaseollin, to which F.s. f.sp. pisi is sensitive did not stimulate spore germination of this fungus. These pathogens may have developed an ability to not only tolerate these toxins but to use them to their advantage for host recognition. Penetration Although spore germination is the initial step necessary for successful infection and disease development, the degree of germination above a certain threshold has little influence on the severity of Fusarium root rot in bean. After examining chlamydospore behavior in soil, Baker and Nash (1965) concluded that spore lysis does not significantly reduce root rot symptoms in bean. The deciding factor contributing to both disease incidence and severity is the ability of the fungus to penetrate and colonize host tissue (Toussoun, 1970). It appears, moreover, that the optimal nutrient requirements for fungal penetration are opposite those that enhance spore germination. Many researchers agree that abundant soil nitrogen enhances root rot severity especially when present as NH;+ (Weinke, 1962; Huber and Watson, 1970; Huber et al., 1965; Snyder et al., 1959; Maurer and Baker, 1964; Griffin, 1964; Toussoun and Patrick, 1963; Toussoun, 1970). Weinke (1962) observed that ammonium N amendments increased root rot severity but had little effect on the size of the pathogen population. The pathogen appears to become more aggressive in the presence of high levels of exogenous N as evidenced by an increase in 14 the number and size of hypocotyl lesions and an increase in thallus development. Using a double pot experiment where roots of individual bean plants were grown in both infested and non-infested soil, applications of N to the roots in non-infested soil did not enhance root rot severity in roots exposed to inoculum. The addition of N, however, to infested soils resulted in a marked increase in root rot severity suggesting that exposure to soil N, not plant N, is the source of the more aggressive pathogen behavior. Toussoun (1970) reported that maximum spore germination was achieved in soil solutions of glucose but rapid penetration of host tissue required an abundant source of N. In another study, phytotoxic compounds released through the decomposition of crop residue were shown to increase root rot severity (Toussoun and Patrick, 1963). Ether and aqueous extracts from several crop species were applied to hypocotyls of bean seedlings which were subsequently inoculated with F.s. f.sp. phaseoli. The application of these phytotoxic compounds elicited the release of nitrogenous compounds probably through the degradation of plant tissue and this source of N may have stimulated pathogenicity. The effect of N on root rot severity is further complicated by the observation that different forms of N have different effects. It was first observed that N amendments could nullify the beneficial effects of carbonaceous residues and that this was particularly apparent when using ammonium inputs (Huber et al., 1965). Maurer and Baker (1965) demonstrated that although the addition of nutrient amendments to soil in controlled experiments reduced root rot symptoms, disease severity was greatest when cellulose and (NI-I4)2SO4 was added as opposed to cellulose and KNOg. The process of biological oxidation of NH;~ to nitrate N03' is called nitrification and occurs readily in the soil. Without NHK fertilization, soil N is either tied up in microbial metabolism or present as 15 NO3' (Huber et al., 1965). Both N114+ and N03' are biologically available to plants but it appears that the presence of abundant NIL+ increases the aggressiveness of F.s. f.sp. phaseoli on bean. Although the form of nitrogen present in the soil appears to play a role in pathogen aggressiveness, this observation could be a secondary result of acidic soil conditions. When NIH is not limiting, nitrification is dependent on pH and tends to increase under less acidic conditions (Paavolainen and Smolander, 1998). Therefore acidic soil conditions tend to favor the presence of NH}. Furthermore, a decrease in F.s. f.sp. phaseoli virulence has been observed with increasing soil pH (Mondal and Hyakumachi, 1998). Oyarzun et al., (1998) also reported that variables which reflect acidic soil conditions tended to increase the soil receptivity of F.s. f.sp. pisi. Enhancing the nitrification capacity of the soil by increasing soil pH or by other means poses to be an effective control against Fusarium root rot by converting the root rot enhancing form of N, NHf, to the relatively less active form of NO;' without eliminating the beneficial effects of ammonium fertilizer on bean growth. Alternatively, in areas where Fusarium root rot is a problem, it may behoove the grower to seek alternative sources of N. Crop residues that inhibit nitrification have been shown to increase Fusarium root rot severity when NI-I4+ fertilizer is also applied. Likewise, the addition of N-serve, a product that inhibits nitrification, with N114+ amendments was also demonstrated to increase disease severity (Maurer and Baker, 1965; Huber et al., 1965). Clearly, there is compelling evidence supporting a connection between Fusarium root rot severity, nitrification and the abundance of NIH. It has been suggested that C:N ratios of various crop residues may influence disease severity inasmuch as they influence nitrification. 16 The enhanced aggressiveness of F.s. f.sp. phaseoli in the presence of abundant NIL;+ is believed to be due to changes in soil microorganism activity as evidenced by the lack of observable differences in pathogenicity when different N sources were used in experiments conducted under sterile laboratory conditions (Huber et al., 1965; Toussoun, 1970). The soil microorganism in question is believed to be an unclassified bacteria that is often found in intimate contact with F.s. f.sp. phaseoli spores (Huber et al., 1965). This bacterium was identified in roughly 12% of the soil samples assayed and appeared to induce fungal cell necrosis. In laboratory culture, the presence of the bacteria associated with spores was observed to prevent pathogenicity without affecting fungal population size. When the bacteria was absent, pathogenicity was restored (Huber and Watson, 1970). In the absence of F.s. f.sp. phaseoli, this particular bacteria utilized only ammonium N. In contact with the fungus, however the bacteria was capable of using nitrate. This finding may suggest that the increase in pathogenicity observed in the presence of NIL;+ may be due, in part, to the lack of association between the fungus and bacteria. Alternatively, the bacteria may only be associated with the fungus when pathogenicity is reduced and that a dirninishrnent in pathogenicity reflects the predominant form of available N in the soil. Pathogenicity of F .s. f.sp. phaseoli and disease severity is further determined at the host level and involves tolerance to plant defense mechanisms and host resistance. De novo phytoalexin synthesis is a frequently cited defense mechanism of bean. F usarium solani f.sp. phaseoli can metabolize many bean phytoalexins including kievitone, phaseollin, phaseollidin and phaseollinisoflavan and this detoxification is believed to be related to the pathogenicity ability of F.s. f.sp. phaseoli on bean (Li et al., 1995; Choi et 17 al., 1987). Keivitone detoxification by the hydratase enzyme, keivitone hydratase, has been correlated with F.s. f.sp. phaseoli virulence on bean (Li et al., 1995). Isolates pathogenic to bean always possess keivitone hydratase activity. The converse however is not true. Many isolates of F. solani not pathogenic or classified in a different forma speciales also possess keivitone hydratase activity. Cloning of the khs gene and transformation into non- pathogenic fungus resulted in the ability of the non-pathogenic fungi to hydrate keivitone hydratase but did not convert the fungus to a pathogen of bean. Additionally, keivitone hydratase enzyme activity was found to be independent and separate from phaseollin hydratase activity. These results suggest that although keivitone hydratase is required for F.s. f.sp. phaseoli virulence in bean, it is not sufficient to establish pathogenicity (Choi et al., 1987). Furthermore, Morris and Smith (1978) reported that although keivitone was strongly induced by biotic stresses caused by both R. solani and Pythium spp. (Morris and Smith, 1978; Li et al., 1995), a corresponding level of induction was not observed for F. s. f.sp. phaseoli. Other phytoalexins like phaseollin, however, did accumulate when beans were exposed to F. s. f.sp. phaseoli. These results could not be explained by" detoxification of keivitone but might reflect either the inability of the fungus to induce keivitone biosynthesis or the ability of the fungus to repress keivitone biosynthesis. It would be interesting to observe whether keivitone enhanced spore germination of F. s. f. sp. phaseoli and to determine if keivitone was, in any way, involved in host-pathogen recognition. In a related study, Mohr et al. (1998) observed a rapid induction of other defense- related enzymes after bean roots were inoculated with F.s. f.sp. phaseoli. Bean chitinase, B- l ,3-glucanase and phenylalanine ammonia-lyase activity increased four-fold in response to F. s. f. sp. phaseoli infection. These results were further confirmed at the transcript level. 18 In contrast, no corresponding accumulation of enzyme or product was isolated from bean roots inoculated with F.s. f.sp. pisi nor with the mychorrizal fungus Glomus mosseae. Inoculation of pea pods with F.s. f.sp. phaseoli has been demonstrated to induce class I chitinase activity, however, results from pod infection may not acurately reflect root infection by F.s. f.sp. phaseoli (Mauch et al., 1984). Apparently, defense-related genes including those involved in phytoalexin biosythenthesis are induced as a result of the compatible interaction between bean and F.s. f.sp. phaseoli. Glomus mosseae does not elicit nor does it suppress the host defense response suggesting that its interaction with the host is quite different from that of the pathogen. Clearly, bean roots recognize F.s. f.sp. phaseoli and mount a concerted defense response against the fungus which is not similarly observed in the presence of non-pathogenic F.s. f.sp. pisi. Fusarium solani f.sp. phaseoli possesses the capacity, in some form, to tolerate these antifungal products. Lange et al. (1996) observed that class I and class IV chitinases are proteolytically cleaved during infection of bean roots by F.s. f.sp. phaseoli. Detoxification of several plant defense products appears to be an effective strategy employed by the fungus to circumvent host resistance but is probably not the only factor involved in pathogenicity. Inoculum Density Fusarium root rot severity may be influenced by initial F.s. f.sp. phaseoli population density but, as has been implied in previous sections, is certainly not the deciding factor in determining disease severity. Hall (1996) provides an excellent review of F .s. f.sp. phaseoli population dynamics and its effect on root rot in bean and concludes that practices which reduce F .s. f.sp. phaseoli population densities should be a component 19 of any integrated effort towards reduced root rot. Population densities of F.s. f.sp. phaseoli range from 0 to 5,500 colony forming units (CFU) g'1 of soil but in controlled experiments only 5 CFU g‘1 of soil were required to infect 80% of plants. Field studies also confirm that low population densities can result in moderate root rot infection. Twenty to 40% of hypocotyl rot was achieved from population densities of 26 CFU g'l (Dryden and Van Alfen, 1984). Sippel and Hall (1982a) reported only an 8-fold increase in root rot severity with a 1,000 fold increase in population density. Furthermore, reports of high infection rates at low inoculum densities suggest that fungal population reductions may not be an effective means of control (Hall, 1996). Conversely, reports of reduced root rot in the presence of large pathogen populations support the conclusion that population density is not an important factor in determining Fusarium root rot severity (Huber et al., 1965). Abawi and Cobb (1984), on the other hand, demonstrated significant correlations between inoculum density and root weight (I = -0.54) and hypocotyl (r = 0.67) and root disease severity (r = 0.65) but not with yield reductions in small field plots. In contrast, Maloy and Burkholder (1959) found no significant relationship between F .s. f.sp. phaseoli density and disease severity although a trend towards this hypothesis was observed. Fusarium solani. f.sp. phaseoli is considered a minor pathogen when plants are grown under optimal conditions and it is only when environmental conditions conducive to disease occur that Fusarium root rot significantly reduces yield (Burke and Miller, 1983). It was not surprising, therefore, that F.s. f.sp. phaseoli populations as high as 4,000 CFU g'l did not result in significantly reduced yields under non-stress environments (Abawi and Cobb, 1984). Studies, however, assessing F.s. f.sp. phaseoli population densities as a 20 factor of hectare-years in Ontario concluded that the average density per county was positively correlated (r2 = 0.98) with the intensity of bean production but the author did not address the effect of population densities on root rot severity (Hall, 1996). In crop rotation studies on fungal population dynamics, Nash and Snyder (1962) reported a decline in pathogen population from 1,497 CFU g'I to 373 CFU g'1 in the same year. Furthermore, population densities fluctuated from 310 to 1,165 CFU g‘l measured in another field monocropped to bean (Hall and Phillips, 1992). Variations in population density suggest that conclusions based on fungal population densities can serve to identify infested fields and indicate disease potential but will not be an effective predictor of root rot severity nor will management practices directed towards decreasing pathogen populations necessarily reduce disease severity (McFadden et al., 1989). Seed treatments and other chemical controls to reduce soil inoculum or prevent disease have proven ineffective against Fusarium root rot (Silbemagel, 1990). Fungicides like Vorlex and methyl bromide have been effective as fumigants to control root rot severity but this method provides only temporary mitigation, is environmentally unsound and not cost-effective (Abawi and Edds, 1981). Soil fumigation can disrupt the microbial balance of the soil eliminating beneficial microorganisms as well as pathogens. Additionally, increased restrictions on pesticide use will preclude future fungicide fumigation as an effective control of root rot in bean. Chemical seed treatments have been effective for the control of damping-off caused by R. solani and Pythium but are ineffective as a control of Fusarium root rot (Miller et al., 1979). A recent report by Vogeli-Lange et al. (1995) demonstrated a developmental control of F.s. f.sp. phaseoli root rot susceptibility in bean. The authors suggested that emerging radicles lack a root 21 surface component required for the attachment of F.s. f.sp. phaseoli spores thereby excluding infection. Seedlings infected with the pathogen at stage four compared to stage ten had significantly fewer spores attached to the root as observed by low-temperature scanning electron microscopy. These results were paralleled with the pea/F.s. f.sp. pisi host/pathogen system and a correlation between spore adhesion and fungal virulence has also been demonstrated in the cucurbit pathogen F.s. f.sp. cucurbitae. The increasing susceptibility of bean with age to Fusarium spore adhesion may explain why seed treatments are ineffective. Organic soil amendments Crop rotation is one management practice that was believed to influence root rot severity by reducing pathogen populations. F usarium solani f.sp. phaseoli fungal populations increase with successive plantings of bean and can decrease in certain non- host crops. After fifteen years of monocropping to bean, F.s. f.sp. phaseoli was as high as 560 CFU g-l (Hall and Phillips, 1992). In crop rotation studies using repeated bean, com- soybean-com, wheat-soybean-wheat, repeated red clover and fallow, Hall (1996) demonstrated that population densities in fields under non-bean rotations declined to 60 to 70% of the repeated bean sequence but could not be eliminated entirely. Fusarium solani f.sp. phaseoli was not found in non-cultivated soils nor in soils never cropped to bean but can be introduced to new fields from seed dust or infested debris or through waterborne or airborne soil (Hall, 1996). Population density will increase with active growth of bean crops but may decrease in the absence of bean. However, a reduction in pathogen population does not necessarily translate to a reduction in root rot severity (Huber et al., 22 1965; Maloy and Burkholder, 1959; Lindsey, 1965; Huber and Watson, 1970; Oyarzun et al., 1998). This does not suggest, however, that a reduction in root rot due to cIOp rotation does not occur but that the observed decrease in severity may be due to factors other than a decline in pathogen population. Organic soil amendments have the potential of influencing disease in several ways. Crop residues alter the relative C and N content as well as the form of N available in the soil. The activity of soil microorganisms also fluctuates depending on many factors especially nutrient availability and can be enhanced or diminished by different crop residue. As has been discussed previously, C, N and microbiota play an important role in F.s. f.sp. phaseoli growth, development and pathogenicity. Beyond impact on fungal growth and development, crop residue can influence the health and vigor of the host plant through the modification of soil structure, release of phytotoxins, and nutrient availability. The combination of all of these factors ultimately influences the incidence and severity of disease. Several studies have been conducted to determine the impact of specific crop residues on Fusarium root rot in bean. Several authors have concluded that disease severity increases with decreasing C:N (Baker, 1970; Griffin, 1964; Maurer and Baker, 1965; Snyder et al., 1959; Hall, 1996). In one study, Fusarium root rot was reduced with the addition of barley straw (C:N 83:1), wheat straw (80:1), com stover (60:1) and pine shavings (400:1), and increased by soybean meal and alfalfa (7:1 and 21:1, respectively; Snyder et al., 1959). When green barley straw was used, or when ammonium nitrate was added with mature barley straw, root rot severity on bean hypocotyls increased. In a similar study, Maier (1959) reported that sorghum, soybean, com, and barley reduced root 23 rot severity; corn and cotton had no effect; and lettuce, alfalfa and tomato increased disease. Green barley, in this case, had no effect. The beneficial effect of residues with high C:N was nullified, however, with the addition of ammonium nitrate as was also found in other Studies (Griffin, 1964; Maurer and Baker, 1965). Observations that the beneficial effects of barley are nullified with the addition of as little as 5 ppm ammonium nitrate, that the form of N (N114+ versus N03) influences root rot severity and that reductions in disease can be achieved with residues of varying C:N, suggest that an inverse relationship between C:N and disease severity is not an adequate explanation (Huber et al., 1965). A more comprehensive hypothesis proposed by Huber et al. (1965) and Huber and Watson (1970) considers the effect of specific crop residues on nitrification. Alfalfa, Soybean, pea and corn residue, when added to plates containing Stephenson’s medium at a 2 ton acre"l equivalent, were demonstrated to enhance nitrification while wheat, oats, and cellulose had no effect, and barley and glucose inhibited nitrification. These results seem to coincide with previous field reports of crop residue influence on Fusarium root rot. The dichotomy with barley can then be explained by the capacity of barley to affect nitrification. In the absence of ammonium fertilizer, barley effectively irnmobilizes all available N thereby preventing infection. In contrast, when NHI’ is present, barley inhibits biological oxidation and NFL." remains available to exert its effect on pathogen aggressiveness. Further evidence in support of this hypothesis is demonstrated by the observation that N -serve added to non-amended soils significantly increases root rot severity (Maurer and Baker, 1965). Although it is not always the case, as is seen with barley, there is a general tendency for residues with high C:N to increase nitrification. It can be concluded, therefore, that C:N is an important component 24 influencing disease severity inasmuch as it influences nitrification (Huber and Watson, 1970). Considering the above conclusions, soil amendments which enhance nitrification may be an effective means in controlling Fusarium root rot of bean. PHASE OLUS VULGARIS Environment Environmental factors which impact the host plant also contribute to disease occurrence and severity. Root rot caused by F.s. f.sp. phaseoli is generally not economically significant unless compounded by environmental stresses that restrict root growth (Burke and Miller, 1983; Allrnaras et al., 1988). Root growth can be adversely affected by many environmental conditions including low soil temperatures, soil compaction, low soil fertility, drought, flooding resulting in anoxia, and plant competition. Low soil temperatures that do not favor healthy and rapid bean root growth aggravate Fusarium root rot incidence whereas warmer soil temperatures can reduce this effect (Burke, 1980). Planting later in the season when soil temperatures are higher can help reduce Fusarium root rot. Soil Compaction and Anoxia The effects of sub-optimal temperatures on disease are compounded by other root restricting environmental factors such as soil compaction and oxygen stress (Smucker and Erickson, 1987; Miller and Burke, 1985b; Allmaras et al., 1988). Soil compaction due to subsurface tillage pans and tillage methods to incorporate herbicides is a major contributor to root-limiting and disease predisposing environments. Soil compaction restricts root 25 growth to the plow layer where roots are in contact with 99% of soil inoculum. Furthermore, diseased roots infected with F.s. f.sp. phaseoli are less capable of penetrating compact soil (Dryden and Van Alfen, 1984; Miller and Burke, 1985b). In greenhouse experiments, Miller and Burke (1985b) demonstrated that plants can compensate for subsurface tillage pans by producing more root mass above the compaction layer but in Fusarium infested soils, this compensating ability is substantially reduced for root rot susceptible cultivars. In field studies, deep tillage significantly reduced root rot severity and increased plant dry weight and yield in Fusarium infested soils (Tan and Tu, 1995). An increase in dry weight and yield was also observed in non-infested soil but increases were more dramatic in the infested soils. In addition to physical obstruction, soil compaction also negatively restricts root growth and development by reducing soil aeration resulting in anoxia. This type of stress can also be achieved by excessive moisture. The most Fusarium resistant cultivars succumbed to infection under severe oxygen stress in greenhouse experiments (Smucker and Erikson, 1988; Miller and Burke, 1985a). Plant injury in the form of retarded growth incurred under severe oxygen stress in soils infested with of F.s. f.sp. phaseoli was found to be irreversible whereas Pythium, R. solani and T. basicola infected roots recovered from periods of poor soil aeration (Burke and Miller, 1983). Stress under excess moisture environments can be so severe as to predispose plants to infection by the pea pathogen (F.s. f.sp. pisi; Miller et al., 1980). Cochrane et al. (1963) demonstrated that ethanol can serve as the sole carbon source in the initiation of F.s. f.sp. phaseoli spore germination. Under anoxic conditions plant survival relies heavily on ethanol fermentation as an energy source. Ethanol fermentation is defined as the metabolic process which converts pyruvate 26 to ethanol to form ATP. If F.s. f.sp. phaseoli can utilize ethanol as a carbon source, this ability may provide the fungus with a competitive advantage over other soil microorganisms which cannot survive under oxygen stress conditions. A greater number of spores of F.s. f.sp. pisi were reported to germinate in the rhizosphere of stressed pea roots and anaerobic conditions were observed to increase root rot of pea by this pathogen by 400% (Smucker and Erikson, 1987). Clearly, implementing cultural practices that reduce oxygen stress will help reduce root rot damage in F. s. f.sp. phaseoli infested soils. Tillage While excessive moisture can have a devastating effect on root rot severity in bean, periods of intermittent drought also exacerbate the disease. Adequate inigatiomplanned to avoid periods of excessive or insufficient moisture may be a means to reduce plant stress and help decrease Fusarium root rot in infested fields. Subsoiling between bean rows before planting helps reduce the negative impact of F.s. f.sp. phaseoli on plant yield by eliminating root growth restrictions due to soil compaction, increasing water holding capacity of the soil and improving drainage (Burke and Miller, 1983). Burke and Miller (1983) demonstrated that subsoiling to a depth of 45 to 50 cm increased yield of bean in both infested and non-infested fields. Positive subsoiling effects, however, are negated once tillage is reapplied to the field after planting. Organic soil amendments Another cultural management practice that involves improving soil structure is crop rotation. As was previously mentioned, crop rotation can help reduce inoculum density, but more likely reduces Fusarium root rot by enhancing soil properties. Crop 27 sequences that involve alfalfa or small grains add large amounts of root and plant residue to the soil which can improve water holding capacity and reduce soil compaction. Soil channels developed from the deep rooting system of a previous crop of alfalfa allow access to deeper soil levels where excess moisture may be present or where Fusarium solani f.sp. phaseoli is not prevalent (Burke and Miller, 1983). Soil channels produced from a previous alfalfa crop may, however, serve to increase root rot. Roots grow and water flows along the path of least resistance. As a result, bean roots may commonly advance through channels left by decaying roots of the previous crop concentrating bean roots within this region (Rasse and Smucker, 1999). Inoculum potential of F.s. f.sp. phaseoli increased by saprophytic growth on decaying alfalfa will also be present in these channels and could potentially concentrate soil inoculum in the area of bean root growth. This may offer an explanation why some studies have reported conflicting results as to the effect of previous alfalfa crops on root rot severity. In addition to soil structure modification, some crop species may influence disease severity through the production of phytotoxic substances evolved during decomposition which can injure bean roots. It has been previously discussed that bean hypocotyls exposed to various phytotoxic plant extracts are more susceptible to infection (Toussoun and Patrick, 1963). Ether and aqueous extracts of rye, barley, broccoli and broad beans increased Fusarium root rot lesions when applied to hypocotyls before or with F.s. f.sp. phaseoli inoculation. Since disease severity was increased in spite of being inoculated after the toxic extracts had been removed, the authors concluded that wounding of plant tissue and its subsequent impact on Fusarium infection was important. N itrogenous compounds exuded from wounded plant tissue were implicated in enhancing fungal pathogenicity. It 28 was also observed that extract products obtained from material decomposing under cold, wet and anaerobic conditions were more toxic (Burke and Miller, 1983). Whether this toxicity enhances Fusarium root rot by providing a point of entry for the fungus, increasing pathogen aggressiveness, increasing host plant susceptibility or any of these factors in combination remains to be seen. If nitrogenous compounds exuded from damaged bean roots do, in fact, stimulate pathogenesis, this could help explain why stressed or damaged roots are predisposed to Fusarium root rot. Under drought, soil compaction and water stressed conditions, roots become weakened and finer roots and root hairs begin to decompose. This decomposition may provide the nitrogen needed to aggravate pathogenicity of F. s. f.sp. phaseoli. Resistance Germplasm Sources Traditional sources of genetic resistance for Fusarium root rot have been limited to P. coccineus gerrnplasm, and the P. vulgaris introduction PI 203958 (N203; Baggett and Frazier, 1959; Wallace and Wilkinson, 1966). NY—21 14-12, a small-seeded, tan colored breeding line developed by Wallace and Wilkinson (1965) is the best known source of resistance derived from P. coccineus and is often reported as possessing better resistance than N203 (Hassan et al., 1971; Beebe et al., 1981). Smith and Houston (1960) tested 12,000 bean accessions and identified seven that they considered Fusarium resistant. Each of these seven genotypes was crossed to ten commercial bean cultivars and only three of the 70 combinations resulted in progeny with superior root rot resistance. Of these three, two involved crosses with N203. After five years of selection from six different Fusarium 29 root rot resistance sources, Dickson (1973) identified five resistant genotypes all of which traced back to N203. It appears that N203 is often used as a source of genetic resistance to Fusarium root rot because it possesses good combining ability for resistance. Furthermore, although N203 is photoperiod sensitive, it is not as sensitive as many tropical genotypes and can mature in temperate regions in most years. A higher proportion of lines with acceptable maturity are available from crosses with N203 contributing to its success as a parent. Root rot resistance to several pathogens including F.s. f.sp. phaseoli, Rhizoctonia solani and Phythium spp. was evaluated for bean germplasm of Latin American origin (Beebe et al., 1981). Based on a scale from 1 to 9 (1 = Resistant; 9 = susceptible), F .s. f.sp. phaseoli ratings greater than 5.0 were not observed indicating a higher level of resistance than was expected. A list of other reportedly Fusarium root rot resistant germplasm is available in Abawi and Pastor Corrales (1990). In general, Fusarium root rot resistance has been identified in genotypes of Middle American origin. However, breeding efforts over the years have resulted in the release of several large-seeded breeding lines resistant to root rot. Wisconsin root rot resistant (RRR) lines 36, 46, 83, 77 were released in the late 1970s but were not widely adopted as parental germplasm (Hagedom and Rand, 1978; 1979). The Fusarium resistant snap bean breeding line, FR266, appears to have high levels of Fusarium root rot resistance and a large vigorous root system (Silbemagel, 1987). N203 was used five times in the background of this breeding line suggesting that resistance can be transferred from small-seeded to large-seeded genotypes. 30 Inheritance Several genetic studies have been conducted to characterize the resistance present in N203 (Smith and Houston, 1960; Bravo et al., 1969; Hassan et al., 1971). Based on F2, F3 and backcross (BC) data, Smith and Houston (1960) concluded that resistance derived from both N203 and PI 165435 was controlled by one dominant and one recessive gene as evidenced by a 13:3 ratio in the F2 generation of several populations. This report, however, may have been misleading since progress towards improved resistance in large- seeded bean has been limited (Bravo et al., 1969). A study using crosses between ‘Redkote’ light red kidney with five breeding lines and N203 estimated that from three to seven genes were involved in resistance. Conclusions were based on parental, F1 and F2 data collected from field experiments. In three of the crosses, the mean of F1 and F2 progeny was skewed towards the resistant side of the mid-parent value while the mean of progeny from the other three crosses occurred near the resistant parent. In only two of the six populations did the mean and distribution of the F2 progeny differ substantially from that of the F1. None of the progeny from any of the six populations exhibited roOt rot susceptibility similar to Redkote. These results are in contrast to Smith and Houston’s (1960) study that reported all F1 as susceptible and only a few F2 progeny as resistant. These contrasting results can be explained by the subjectivity involved in classifying root rot scores into one of two categories: resistant or susceptible. Smith and Houston (1960) reported that very few progeny scored less than the resistant parent and it is assumed, therefore, that the rest were considered susceptible. Care must be taken when attempting to compartmentalize disease scores into two classes for traits exhibiting a more quantitative nature since misleading conclusions can result. 31 Hassan et al. (1971), in another study, attempted to elucidate the nature of genetic resistance to F.s. f.sp. phaseoli in bean. Two resistant parents, N203 and NY-2ll4-l2, were compared by crossing each to the other and to Redkote light red kidney. Data from this study were collected from F1, F2 and BC progeny both in the field and greenhouse. Narrow sense heritability esitmates (hz) based on parent offspring regression for the Redkote/NY-2114-12 population were reported as 44.3 % in greenhouse experiments and ranged from 33.6 to 78.6 % for field studies involving several different scoring dates. Eight genes were estimated to be involved in resistance for this population. Heritabilities for the Redkote/N203 population ranged from 25.9 to 84.8 % for greenhouse and field experiments. Four genes were reported to be involved in the resistance derived from N203. No segregation was observed for progeny from the NY-2114-12/N203 cross indicating that some of the resistance genes present in N203 may be similar to those originating from P. coccineus. The authors concluded that Fusarium root rot resistance should be considered a complex trait for which quantitative breeding approaches would be the most appropriate improvement strategy. CONCLUSION A multitude of factors combine to result in severe Fusarium root rot in bean. The reader is referred to the classic disease triangle which describes disease in terms of interactions between pathogen, host and environment. For root rot caused by F.s. f.sp. phaseoli, the environment and its impact on both the host and pathogen appear to be the predominant determinant of disease severity. As a consequence, quantification of disease, evaluation of host resistance and consistent methods for control are difficult to achieve. 32 Figure 1 illustrates the different environmental aspects that affect the host and pathogen. It also lists aspects inherent to F.s. f.sp. phaseoli and P. vulgaris that interact with environmental conditions to cause disease. Saprophytic ability, spore germination, penetration, and infection are all factors that can influence the aggresiveness of the pathogen. These attributes are constant components of the fungus and can only be reduced or enhanced through manipulation of the environment or host. Disease control, therefore is obtained by altering environmental factors like soil C:N, nitrification and microbial activity which effect changes in germination, penetration, infection, etc. of the pathogen. Alternatively, aspects of host response can be manipulated to achieve greater control of root rot severity by influencing fungal pathogenicity. Cultural management practices that can help eliminate stress environments, such as irrigation, subsoiling, crop rotation, etc. have and will continue to be important for crop improvement and disease control. Some of these practices, however, may not be feasible in certain growing areas or may be uneconomical. Furthermore, environmental conditions are unpredictable making it difficult to establish consistent principals. Genetic irnprovement of traits inherent to. host genotype including seed type, disease resistance, stress resistance, growth habit and root morphology can potentially provide a durable, consistent and effective means of control against root rot. Improved genotypes and cultivars combined with established root rot- reducing cultural practices affecting both host and pathogen should become the predominant method for combating Fusarium root rot in bean. ' The ubiquitous presence of Fusarium root rot in bean production areas, its potential for substantial negative impact on yield and lack of adequate cultural control emphasizes the need to develop genetic resistance to Fusarium root rot in bean. Root rot 33 resistance combined with stress tolerance will ultimately improve the performance of bean in F.s. f.sp. phaseoli infested areas (Burke and Miller, 1983). Resistance has been identified in P. vulgaris and related species (P. coccineus) but has been difficult to transfer into adapted large-seeded germplasm. Conclusive evidence elucidating the genetic control of Fusarium root rot resistance has remained elusive in part because contemporary genetic research has neglected to emphasize this area in bean. The proceeding chapters of this research will address the inheritance of Fusarium root rot resistance using the traditional source of resistance, PI 203958. The development of a controlled greenhouse screen and the identification of molecular markers associated with Fusarium root rot resistance in common bean is discussed. 34 F usariunr solani f.s g phaseoli 0 Saprophtye ' CEN. . . Drought 0 Genotype . 0 Germination ’ Nrtnficatron . Compaction 0 Drsease resistance 0 penetration o Mrcrobrota . An oxia 0 Stress resistance 0 Infection . Temperature 0 Market class 0 Pathogenicity . Phytotoxins 0 Growth habit . Root regrowth . Figure 1. Interaction between environment, fungal pathogen and host. Environmental aspects which fall into the F.s. f.sp. phaseoli circle have been shown to alter characteristics of the pathogen. Environmental aspects which fall into the Phaseolus vulgaris circle influence plant growth and disease susceptibility. 35 REFERENCES Abawi, GS. and A.C. Cobb. 1984. Relating soil densities of Fusarium, Pythium, Rhizoctonia and Thielaviopsis to disease severity and yield of snap beans in field microplots. Phytopathology 74:813. 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Maurer, CL. and R. Baker. 1964. Ecology of plant pathogens in soil. 1. Influences of chitin and lignin amendments on development of bean root rot. Phytopathology 54: 1425-1426. Maurer, CL. and R. Baker. 1965. Ecology of plant pathogens in soil. 11. Influence of glucose, cellulose and inorganic nitrogen amendments on development of bean root rot. Phytopathology 55:69-72. 38 McFadden, W., R. Hall, and LG. Phillips. 1989. Relation of initial inoculum density to severity of Fusarium root rot of white bean in commercial fields. Can. J. of Plant. Path. 11:122-126. Miller, DE. and D.W. Burke. 1985a. Effects of soil physical factors on resistance in beans to Fusarium root rot. Plant Dis. 69:324-327. Miller, DE. and D.W. Burke. 1985b. Effects of low soil oxygen on Fusarium root rot of beans with respect to seedling age and soil temperature. Plant Dis. 69:328-330. Miller, DE. and D.W. Burke. 1986. Reduction of Fusarium root rot and sclerotinia wilt in beans with irrigation tillage and bean genotype. Plant Dis. 70: 163-166. Miller, D.E., D.W. Burke, and J .M. Kraft. 1980. Predisposition of bean roots to attack by the pea pathogen, Fusarium solani f.sp. pisi, due to temporary oxygen stress. Phytopathology 70: 1221- 1224. Miller, D.E., A.C. Cobb and GS. Abawi. 1979. Effect of seed treatment fungicides on bean yield and root-rot severit, 1979 bean (snap) (Phaseolus vulgaris ‘Early Gallatin’), root-rot complex; Fusarium solani f.sp. phaseoli, Pythium ultimum, Rhizoctonia solani, Thielaviopsis basicola. Fungic. Nematic. Tests Results Am. Phytopathol. Soc. 35:177. Mohr, U., J. Lange, T. Boller, A.‘ Wiemken and R. Vogeli-Lange. 1998. Plant defence genes are induced in the pathogenic interaction between bean roots and F.s. f.sp. phaseoli, but not in the symbiotic interaction with the arbuscular mycorrhizal fungus Glomus mosseae. New Phytol. 138:589-598. Mondel, S.N., K Kageyama and M. Hyakumachi. 1996. Chlamydospore formation in F usarium solani f.sp. phaseoli in root extracts of crop plants and their virulence to kidney beans (Phaseolus vulgaris L.). Soil Biol. Biochem. 28:539-543. Morris, A]. and D.A. Smith. 1978. Phytoalexin formation in bean hypocotyls induced by cell-free mycelial extracts of Rhizoctonia and Fusarium. Ann. Appl. Biol. 89:344- 347. Nash, S.N. and W.C. Snyder. 1962. Quantitative estimations by plate counts of propagules of the bean root rot Fusarium in field soils. Phytopathology 52:567- 572. Oyarzun, P.J., M. Gerlagh and J.C. Zadoks. 1998. Factors associated with soil receptivity to some fungal root rot pathogens of peas. Applied Soil Ecology 10:151-169. Paavolainen, L. and A. Smolander. 1998. Nitrification and denitrification in soil from a clear-cut Norway spruce (Picea abies) stand. Soil Biol. Biochem. 30:775-781. Pieczarka, DJ. and GS. Abawi. 1978. Effect of interaction between Fusarium, Pythium, and Rhizoctonia on severity of bean root rot. Phytopathology 68:403-408. Rackham, R.L. and J .R. Vaughn. 1959. The effects of gibberellin and fungicides on bean root rot. Phytopathology 43: 1023-1028. 39 Rand, R.E., W.F. Pfender and DJ. Hagedom. 1984. Evaluation of red kidney beans for resistance to Wisconsin’s bean root rot complex. Annu. Rep. Bean Improv. Coop. 27:64. Rasse, DP. and A.J.M. Smucker. 1999. Root recolonization of previous root channels in corn and alfalfa rotations. Plant and Soil 204:203-212. Reeleder, RD. and DJ. Hagedom. 1981. Pythium populations in Wisconsin bean fields. Can. J. Plant Pathol. 3:90-93. Ruan, Y., V. Kotraiah and DC. Straney. 1995. Flavonoids stimulate spore germination in Fusarium solani pathogenic on legumes in a manner sensitive to inhibitors of cAMP-depedent protein kinase. Mol. Plant-Microbe Interact. 6:929-938. Saettler, A.W. and AL. Andersen. 1978. pp. 172-179. Bean diseases and their control. In: L.S. Robertson and RD. Frazier’s (eds.) Dry Bean Production — Principles and Practice. Cooperative Extension Service and Agricultural Experiment Station of Michigan State University. Extension Bulletin E-1251. Silbemagel, M.J. 1987. Fusarium root rot-resistant snap bean breeding line FR266. HortScience 22: 1337-1338. Silbemagel, M.J. 1990. Genetic and cultural control of Fusarium root rot in bush snap beans. Plant Dis. 74:61-66. ' Sippell, D.W. and R. Hall. 1982a. Effects of pathogen species, inoculum concentration, temperature, and soil moisture on bean root rot and plant growth. Can. J. Plant Pathol. 4: 1-7. Sippell, D.W. and R. Hall. 1982b. Effects of Fusarium solani f.s. phaseoli, Pythium ultimum, and F. oxysporum on yield components of white bean. Can. J. Plant Pathol. 4:54-58. Smith, PL. and BK Houston. 1960. Root rot resistance in common beans sought in plant breeding program. California Agriculture Sept.:8. Smucker, A.J.M. and A.E. Erickson. 1987. Anaerobic stimulation of root exudates and disease of peas. Plant and Soil 99:423-433. Snyder, W.C., M.N. Schroth and T. Christou. 1959. Effect of plant residues on root rot of bean. Phytopathology 49:755-756. Steadman, J .R., E.D. Kerr and RF. Mumm. 1975. Root rot of bean in Nebraska: Primary pathogen and yield loss appraisal. Plant Dis. Rep. 59:305-308. Tan, CS. and J .C. Tu. 1995. Tillage effect on root rot severity, growth and yield of beans. Can. J. Plant Sci. 183-186. Toussoun, T.A. 1970. Nutrition and pathogenesis of Fusarium solani f.sp. phaseoli. pp. 95-97. In: T.A. Toussoun, R.V. Bega and PE. Nelson’s Root Diseases and Soil- bome pathogens. University of California Press, Berkley, Los Angeles, London. Toussoun, T.A. and Z.A. Patrick. 1963. Effect of phytotoxic substances from decomposing plant residues on root rot of bean. Phytopathology 53:265-270. 40 van Bruggen, A.H., C.H. Whalen and PA. Arneson. 1986. Emergence, growth, and development of dry bean seedlings in response to temperature, soil moisture, and Rhizoctonia solani. PythOpathology. 76:568-572. Vogeli-Lange, R., U. Mohr, A. Wiemken, M. Duggen, R. Guggenheim and T. Boller. 1995. Developmental regulation of susceptibility and tolerance to Fusarium root rot in beans. Bot. Acta 108:387-395. Wallace, DH. and RE. Wilkinson. 1965. Breeding for Fusarium root rot resistance in beans. Phytopathology 55: 1227- 123 l. Weinke, KB. 1962. The influence of nitrogen on the root disease of bean caused by F usarium solani f.sp. phaseoli. Phytopathology 52:757 (Abstr.). Yang, S. and DJ. Hagedom. 1966. Root rot of processing bean in Wisconsin. Plant Dis. Rep. 50:578-580. 41 CHAPTER 1 A GREENHOUSE SCREENING PROTOCOL FOR THE IMPROVEMENT OF FUSARIUM ROOT ROT IN BEAN (PHASEOLUS VULGARIS L.) ABSTRACT Root rot caused by F usarium solani f.sp. phaseoli is a devastating disease of bean for which successful control has been elusive. Resistance to this pathogen is considered quantitative and strongly influenced by environmental factors. To reduce environmental variation and facilitate selection in earlier generations, an accurate, consistent, and non- destructive greenhouse screen is desired for the evaluation of root rot resistance in bean. In this chapter, we propose a protocol which involves the germination of seedlings in perlite, inoculation of roots and hypocotyl 10 d after planting and evaluation within four weeks. The accuracy of this greenhouse screen was confirmed by demonstrating strong correlation between greenhouse and field ratings. Two genotypic groups which included 24 and 21 genotypes, respectively, were evaluated in the greenhouse and these ratings were significantly and positively correlated with field ratings. Correlation coefficients were as high as 0.99. Hundreds of genotypes can be evaluated within one month for relatively minimal costs and labor. Furthermore, once roots have been rated and dipped in benomyl, plants can be transplanted to pots for production of seed. This simple, rapid and inexpensive protocol reduces environmental variation inherent to field ratings thereby more accurately representing physiological resistance while maintaining a close association with observed field ratings. 42 INTRODUCTION Fusarium root rot is caused by the soilbome fungal pathogen F usarium solani f.s. phaseoli (Burk.) Snyd. & Hans. and has been reported to reduce yield of bean (Phaseolus vulgaris L.) in California, Colorado, Wisconsin, Washington, Nebraska, North Dakota, New York, Minnesota, and Michigan (Smith and Houston, 1960; Burke and Silbemagel, 1965; Keenan et al., 1974; Steadman et al., 1975; Saettler and Anderson, 1978; Sippell and Hall, 1982; Estevez de Jensen et al., 1998). The pathogen invades underground roots and stems directly through the epidermis, stomates and wounds. Infection results in characteristic red streaks along the base of the hypocotyl and discoloration and deterioration of the main taproot and laterals. Severely diseased roots cannot sustain plant growth and development resulting in visible symptoms such as chlorosis, defoliation and stunting of the above ground plant (Abawi and Pastor Corrales, 1990). Fusarium root rot is a particular problem in dark red kidney beans due, in part, to the lack of any degree of genetic tolerance to this pathogen. Compounding this problem, drought conditions aggravated by the sandy soils on which this market class is produced inhibit root health and vigor which may ultimately lead to increased disease susceptibility. Environmental factors like drought stress play a large role in Fusarium root rot severity and can influence disease both at the pathogen and host levels. Soil factors such as carbon and nitrogen content of the soil affect fungal growth, development and pathogenicity by altering spore germination, penetration, and invasion (Christou and Snyder, 1962; Huber et al., 1965; Toussoun, 1970). Soil nutrient availability also exerts an indirect effect on fungal development through its impact on microbial activity (Huber et 43 al., 1965). Cultural management practices that do not favor pathogenicity can be an effective means of controlling disease but approaches which also involve enhancing host defenses will be more consistent and comprehensive. Host defense responses can be enhanced by eliminating stressful environmental conditions which affect root health and vigor. A robust root system with active defense mechanisms is more likely to avoid, tolerate or resist pathogen infection. Although root health and vigor are often determined by environmental conditions, they are also a function of genotype. By genetically improving disease resistance or stress tolerance, in combination with optimized cultural practices, yield reductions in bean caused by Fusarium root rot will be minimized. Resistance to F.s. f.sp. phaseoli is considered quantitative due to environmental variation and polygenic inheritance. While genetic variation for root rot resistance exists and root rot resistant P. vulgaris genotypes have been documented, progress towards improving root rot resistance has been limited. Polygenic inheritance compounded by strong environmental effects has made it difficult to achieve a complete understanding of physiological disease resistance mechanisms under field conditions (Smith and Houston, 1960; Wallace and Wilkinson, 1965; Bravo et al., 1969; Hassan et al., 1971; Boomstra et al., 1977; Beebe et al., 1981; Silbemagel, 1990; Tu and Park, 1993). Furthermore, evaluating roots, in general, is difficult and not amenable to large-scale population analysis. It was our objective therefore, to develop an efficient, controlled greenhouse screen to evaluate genotypes for Fusarium root rot resistance while eliminating environmental variation inherent to field experiments. The screening method presented will ultimately assist us in the identification and evaluation of physiological resistance mechanisms present in bean. Many studies examining Fusarium root rot resistance in bean have utilized greenhouse screening methods with variable results (Baggett and Frazier, 1959; Wallace and Wilkinson, 1965; Baggett et al., 1965; Hassan et al., 1971; Boomstra et al., 1977; Beebe et al., 1981; Tu and Park, 1993). In most cases, an association between greenhouse and field data was not established because a corresponding field trial including similar genotypes to those used in the greenhouse screen was not conducted. Wallace and Wilkinson (1965) proposed an elaborate greenhouse screening method whereby six inch pots, placed in wooden frames, heated by sub-surface coils and filled with field soil were used to evaluate seedlings. From six to 15 seeds were planted into collars and covered with sand to which liquid inoculum was added. Collars were subsequently removed upon evaluation to expose the infected hypocotyl. Field tests were also Conducted using the same genotypes but greenhouse results did not appear to correspond well with field ratings (Wallace and Wilkinson, 1965). Furthermore, ratings based on hypocotyl infection may not be an accurate indication of lateral root damage which is reported to be the predominant determinant of yield reduction caused by F.s. f.sp. phaseoli (Burke and Barker, 1966). The technique proposed by Wallace and Wilkinson (1965) was relatively cumbersome such that only 27 to 58 pots could be measured in a single test. The number of individual plants in 58 pots is sufficient to rate a large F2 population. For rating replicated trials of 100 or more recombinant inbred lines, however, this method does not appear feasible (Hassan et al., 1971). Another technique developed by Boomstra et al. (1977) attempted to compare two different greenhouse screens with field ratings recognizing that to be effective, data from any greenhouse evaluation must reflect observed field root rot severity. The first screen 45 involved a nutrient culture technique where seedlings were germinated in perlite, removed, dipped in inoculum and transferred to a hydroponic nutrient solution. The second greenhouse evaluation was a variation on Wallace and Wilkinson’s (1965) pot test. Ratings from the nutrient culture technique which were based on hypocotyl and foliar symptoms did not appear to correspond well with field ratings nor pot test results which were based on hypocotyl and root damage. The nutrient culture technique was, however, an improvement over Wallace and Wilkinson’s (1965) pot test Since many individuals could be evaluated using minimal resources. Other methods involve evaluating seedlings grown in infested soils Obtained from documented F.s. f.sp. phaseoli infested fields (Park and Tu, 1994). For evaluating resistance to the root rot complex in general, this technique is adequate. However, separating and confirming reactions to individual components of the root rot complex, such as F.s. f.sp. phaseoli, may be difficult. Evaluation of tap and lateral roots is further complicated by this method because soil particles adhere tightly to the root mass preventing clear observations. To prevent this problem, sand was adopted as the. soil medium of choice in two studies (Beebe et al., 1981; Baggett, 1969). Beebe et al. (1981) sowed seeds into flats containing a sand and inoculum mixture. The authors scored root rot resistance based on hypocotyl and root lesions separately and reported a non- significant correlation between these two ratings of 0.47. Type of soil media, form and concentration of inoculum, hypocotyl versus root ratings, number of ratings, labor and cost are all considerations that must enter into the development of a useful greenhouse screening method. It was our objective to choose the best aspects of all these methods to create a greenhouse screen that was inexpensive and relatively simple to allow evaluation 46 of lateral roots that correlated well with field ratings and would permit the evaluation of large populations. MATERIALS AND METHODS Fungal Isolation Procedure F. solani was isolated from Michigan dry bean production fields in Presque Isles, Gratiot, and Saginaw counties. Roots infected with F.s. f.s. phaseoli sampled from these fields were stored at 4° C until isolation. Stern and root sections were bisected longitudinally and then cut into 0.5 cm pieces. Plant segments from each plant were wrapped in cheesecloth and soaked under running deionized water for 24 h. Stem sections were then surface sterilized in 95% ethanol, soaked in 10% Clorox for five minutes, and dried on a paper towel moistened with 10% Clorox. Sections were placed in a petri dish containing water agar (15 g agar in l L of water) and left under continuous light. Four to five days later, mycelia emanating from plant sections was removed at the growing point and transferred to PDA media containing} 100 mg L" ampicillin. These pl isolates were grown under continuous light for four to five days and then replated on more PDA plus ampicillin. Isolates were stored at 4° C in sterilized soil. Ten ml of distilled water were added to spore cultures after four to five days of growth and mycelia and conidia were scraped into solution. Two ml of this solution was then added to a screw cap, 20 ml, test tube with 3 ml of sterilized potting soil. The test tubes were set under continuous light for four to five days and then transferred to the refrigerator. One ml of this inoculum was grown out on PDA plus arnpicillin when desired. Isolates were characterized by conidial shape, blue Spore color on PDA and pathogenicity on bean. Continued culture of the 47 fungus and verification of pathogenicity was performed by inoculating several plants with the specific isolate and reisolating the fungus using the above procedure. Greenhouse Screen Several greenhouse screening tests were attempted and the following protocol was adopted. Using perlite-filled 72-well greenhouse flats, a single seed was germinated in each well, with 3 to 6 seedlings per variety per replication. The perlite was saturated with half strength Hoagland’s solution at planting and flats were fertilized once every week thereafter with the same. Ten days after planting, 10 ml of 2 X 105 spore suspension of F .s. f.s. phaseoli macroconidia was applied over the base of hypocotyl using a 4 L hand pump sprayer. Inoculum was prepared by scraping PDA plates of F.s. f.s. phaseoli macroconidia into a distilled water solution which was then quantified by a hemocytometer and adjusted to the proper spore concentration. The isolate Hawks 2b was used for all inoculations (Table 13). Fourteen d after inoculation, seedlings were removed from flats, cleaned of excess Perlite and rated on a scale from 1 to 7 (Table 1; Figure 2). When seed was desired from inoculated seedlings, roots were dipped in a fungicidal solution of benomyl and transplanted to normal potting soil. The aforementioned greenhouse screen was used to evaluate two sets of genotypes. The first experiment (GH96) included 24 cultivars and advanced breeding lines some of which have been previously reported as possessing good root rot resistance. The 24 genotypes were replicated four times and three plants were sowed per replication (Table 2). The second greenhouse evaluation (GH97) consisted of 21 of the most popular 48 Table 1. Description of disease rating scale used for all root rot experiments. Score 1 2 Phenotypic Description healthy root system with no discoloration of root or hypocotyl tissue and no reduction in root mass; localized reddening at base of hypocotyl with majority of root mass size and appearance healthy; increase in intensity and size of localized root/hypocotyl lesion and some discoloration but no reduction in size of root mass; increasing intensity of discoloration in hypocotyl lesion with lesions becoming extended and very little reduction in root mass with some brown discoloration; increasingly discolored and extended hypocotyl lesions. Roots on these plants were brown and had reduced root mass; hypocotyl lesions encircled stem and become more extended, very little root mass was present and was highly discolored; and pithy or hollow hypocotyl with very extended lesions. Root was limited in abundance and functionally dead. 49 Figure 2. Visual representations of bean roots at corresponding disease severity ratings described in Table l. 50 Michigan-grown bean cultivars and advanced breeding lines. Six of the seven market classes (except cranberry) were represented in this experiment (Table 3). Greenhouse evaluation was similar to GH96 and consisted of four replications with three plants per replication. Field Trials Field trials were conducted on a Grace very fine sandy loam soil (coarse-silty, mixed, frigid, Typic, Hapludulfs) in Presque Isles County, MI where previous F. solani f.sp. phaseoli infections have been reported and where beans have been planted without sufficient rotation. The first field trial was conducted in the summer of 1996 and included the same twenty-four cultivars and advanced breeding lines described in GH96 (Table 2). Pre-plant incorporated Eptam herbicide treatment was applied. Two-row plots, 7.6 m long were hand planted on June 17, 1996 and replicated four times. Between-row spacing was 0.76 In Two weeks after planting, plots were thinned to 13 plants In". Plots were arranged in a randomized complete block design with four replications. Twice during the growing season at pod fill (51 days after planting (DAP) and 70 DAP), five random plants from each plot were carefully removed from soil using a shovel and rated for Fusarium root rot symptoms using the root rating scale described in Table 1 and Figure 2. Yield was recorded on a 6.6 m section of each plot for a total harvestable area of 10.00 m2. Seed moisture samples were also taken for each plot and yields were adjusted to 18% moisture (Table 2). Field root rot evaluations were averaged over the two ratings for each genotype to provide an average score. 51 Table 2. Seed type, greenhouse rating (GH96), first (RI) and second (RR2) field ratings, average root rot rating of the two field evaluations (AVG), seed yield, and 100 seed weight (SW) for 24 genotypes grown in Presque Isle County, MI in 1996. Field Yield SW Genotype Seed Type? GH96 Rm: RR2 AVG (kg rm“)' (g) B95219 Black 1.9 2.3 2.1 2.2 774 19.3 N203 Black 2.0 1.4 1.2 1.3 - - Newport Navy 2.2 2.8 2.8 2.8 1002 17.1 Huron Navy 2.3 1.9 1.7 1.8 1573 24.6 T39 Black 2.4 2.0 1.7 1.8 920 19.3 FR266 Snap 2.5 1.2 1.5 1.3 - - A300 Cream 2.5 1.7 1.7 1.7 823 19.2 N94080 Navy 2.5 2.3 2.1 2.2 1137 18.8 NW590 Pinto 2.6 2.4 2.3 2.4 699 28.7 Seafarer Navy 2.6 2.4 2.4 2.4 1075 19.9 Sierra Pinto 2.7 1.9 2.1 2.0 827 32.1 Mackinac Navy 2.8 2.7 2.9 2.8 955 20.2 Roza Pink 2.9 2.8 2.3 2.5 1165 31.7 UI-l l4 Pinto 3.0 2.1 2.0 2.0 635 37.7 NW 63 Small Red 3.0 2.1 2.6 2.3 1153 31.3 K93654 LRK 3.0 3.0 4.8 3.9 1605 59.8 Viva Pink 3.1 3.0 1.5 2.3 1134 28.0 Chinook LRK 3.4 2.8 4.3 3.5 1492 53.4 Redhawk DRK 3.6 4.6 5.8 5.2 1807 52.5 K94515 LRK 3.9 4.0 5.1 4.6 1321 55.6 K94803 WK 4.1 3.7 5.4 4.5 1407 52.5 Chinook 2000 LRK 4.2 3.0 4.3 3.6 1502 54.1 Montcalm DRK 4.5 3.1 4.7 3.9 1529 55.9 Isles DRK 4.8 4.7 5.4 5.0 1428 62.6 Mean 3.0 2.6 3.0 2.8 1182 36.4 LSD (0.05) 0.9 1.0 0.9 446 3.5 , CV (%) 17.0 27.4 20.3 26.3 6.7 TLRK. DRK and WK signify light red kidney, dark red kidney and white kidney seed types, respectively. 1 RI and RR2 are the root rot ratings taken 51 and 70 days after planting, respectively. § Yield data is unavailable for the genotypes marked with a “-“ due to problems with maturity. 52 Table 3. Seed type, greenhouse rating (GH97), first (RI) and second (RR2) field ratings, average root rot rating of the two field evaluations (AVG), seed yield, and 100 seed weight (SW) for 21 genotypes grown in Presque Isle County, MI in 1997. Field Yield SW Genotype Seed Type'l' GH97 RRli RR2 AVG -- kL " -- m g «- Avanti Navy 2.4 2.0 2.8 2.4 2410 19.0 Newport Navy 2.4 2.3 2.5 2.4 1849 19.3 T39 Black 2.4 2.6 2.4 2.5 953 18.0 A300 Cream 2.5 2.5 2.4 2.5 2267 20.1 B95204 Black 2.5 2.9 2.2 2.5 1739 18.0 Mackinac Navy 2.6 2.0 3.2 2.6 1740 19.1 N94080 Navy 2.6 2.7 2.6 2.6 1529 18.2 Mayflower Navy 2.8 3.2 2.4 2.8 1091 19.5 Vista Navy 2.9 2.7 3.0 2.9 1378 14.8 Alpine GN 3.2 2.9 3.5 3.2 997 29.5 Huron Navy 3.3 2.8 3.7 3.3 1635 20.5 Kodiak Pinto 3.4 3.7 3.2 3.4 997 30.2 Matterhorn GN 3.7 3.1 4.3 3.7 791 ' 30.1 K93629 LRK 4.1 3.8 4.5 4.2 740 55.8 Aztec Pinto 4.1 3.6 4.7 4.2 1153 33.7 Redhawk DRK 4.4 4.1 4.6 4.4 1565 55.1 Chinook 2000 LRK 4.5 3.8 5.1 4.5 1717 54.4 Isles DRK 4.5 4.1 4.9 4.5 638 61.8 K93613 LRK 4.7 4.0 5.3 4.7 1797 63.7 Chinook LRK 5.0 4.5 5.5 5.0 493 47.0 Montcalm DRK 5.1 4.6 5.5 5.1 1279 52.0 Mean 3.5 3.2 3.7 3.5 1369 33.2 LSD (0.05) 0.8 1.3 1.1 873 2.2 CV (%) 14.5 24.3 17.9 30.5 3.2 TLRK and DRK signify light red kidney and dark red kidney seed types, respectively. iRRl and RR2 are the root rot ratings taken 52 and 86 days after planting, respectively. 53 A second field trial consisting of the same 21 cultivars and advanced breeding lines described for GH97 was conducted in 1997 on the same field in Presque Isle County, Michigan. No herbicide treatment was applied. A randomized complete block design experiment with three replications was planted on June 13, 1997 with plot area and design similar to the 1996 trial. Plots were rated twice during pod fill, 52 DAP and 86 DAP, and the scoring procedure was identical to the 1996 experiment. Statistical Analysis Analyses of variance for all experiments were performed using SAS’s procedure GLM (SAS Institute, 1994). Pearson rank correlation coefficients between all ratings and yield data were calculated using SAS’s PROC CORR. RESULTS Significant genetic variation for root rot ratings was observed for all greenhouse and field experiments conducted. Significant genetic variation was also observed for yield and 100 seed weight (SW) for both field trials. Root rot ratings for GH96 ranged from 1.9 to 4.8 with a mean of 3.0 (Table 2). The black bean breeding line B95219 had the lowest root rot rating followed by N203, the traditional source of root rot resistance (Wallace and Wilkinson, 1966). FR266, which derives its resistance from N203, was the lowest rated large-seeded genotype with a score of 2.4 (Silbemagel, 1987). Black beans tended to be the most resistant followed by navy beans. Pinto and pink seed types scored moderately from 2.6 to 3.1 and the most susceptible seed types were the large-seeded dark red, light red and white kidneys. ‘Isles’ had the highest root rot rating of 4.8 followed bythe highly susceptible dark red kidney, Montcalm. 54 Field trial root rot ratings confirmed these tendencies and demonstrated a positive and significant correlation with greenhouse ratings (r = 0.73; Table 4). Field ratings ranged from 1.3 to 5.2 with a mean of 2.8 (Table 2). The dark and light red kidney genotypes tended to score the highest for root rot but also tended to be the highest yielding. Redhawk, a dark red kidney cultivar released from Michigan State University, was the highest yielding genotype but also received the highest root rot score followed by Isles. N203 and FR266 were the most highly resistant genotypes in this trial. B95219, which was the most resistant genotype in GH96 was the most susceptible black bean genotype in the field trials. However, black bean genotypes remained the most resistant followed by pinto and navy seed types. Dark red kidney genotypes scored, on average, from 3.9 to 5.2 and light red kidneys scored from 3.5 to 4.6. Mean root rot scores based on field evaluations were 2.6 and 3.0 for the first (RRl) and second (RR2) evaluations, respectively, and ratings were positively and significantly correlated (r = 0.87***; Table 4). The coefficients of variation (CV) were moderate to high for yield and field root rot ratings while the CV was lower for the greenhouse evaluation. Yields for the variety trial conducted in 1996 ranged from 910 to 2,592 kg ha" with a mean of 1,702 kg ha" (Table 2). Yield data were difficult to interpret for this trial because many genotypes lacked adequate adaptation to this northern environment. Yield was positively and significantly correlated with field and greenhouse root rot ratings and. was significantly correlated with seed size. The highest yielding genotypes were the regionally-adapted large-seeded kidney types. The tendency for certain market classes to cluster for root rot 55 Table 4. Pearson rank correlation coefficients for yield, 100 seed weight (SW), first (RRRl) and second field (RRR2) root rot ratings, averaged field root rot rating (AVG), and greenhouse (GH) evaluations for each group of genotypes, GH96 and GH97. GH96 values are presented in normal print in the upper right-hand diagonal whereas the GH97 values are printed in bold in the lower left-hand diagonal. Yield SW RRRl RRR2 AVG GH Yield O.69*** 0.63*** 0.74*** 0.72*** 061’” SW -0.33 0.72*** 0.89*** 0.85*** 0.87*** RRRl -0.38 0.86" 0.87*** 0.95*** 0.77*** RRR2 -0.54** 037*" 0.84*** 0.98*** 0.83*** AVG -0.47* 031*" 034*" 0.97 *** 0.73*** GH -0.46* 031*" 0.94" 0.97 *** 0.99" *, **; *** Significance at the P < 0.05. 0.01, 0.001 levels, respectively. ratings was reflected in a significant and positive correlation (r = 0.85***) between SW and average root rot score (Table 4). Average ratings for GH97 ranged from 2.4 to 5.1 with a mean of 3.5. Field ratings mirrored greenhouse ratings almost exactly (r = 0.99***; Table 4) so subsequent results can be applied to either field or greenhouse experiments. The dark red kidney cultivar Montcalm, demonstrated the highest greenhouse rating whereas ‘Avanti’ navy, ‘Newport’ navy and ‘T39’ black exhibited the highest levels of resistance with scores of 2.4 (Table 3). Navy genotypes clustered as the most resistant market class along with black bean cultivars ‘T 39’ and B95204 in contrast to the highly susceptible kidney cultivars. ‘Huron’ was the only navy bean that scored above a 3.0 for this trial. Medium seeded, race Durango genotypes scored from 3.2 to 4.1 and kidneys scored from 4.1 to 5.1. The average score for field evaluations was 3.5 similar to the mean of GH97 (3.5). The CV for 56 GH97 was comparable to that observed in GH96. Coefficients of variation for both greenhouse experiments ranged from 3 to 10% less than CVs for field ratings (Table 2 and 3). Repeatability was confirmed based on GH97 as a significant and positive correlation between repeated greenhouse experiments (data not shown). Yield for the 1997 field trial ranged from 493 to 2,410 kg ha‘1 with a mean of 1,369 (Table 3). Average yields were calculated on two replications only because of extreme weed pressure in the third replication. Weeds were controlled manually but were difficult to contain since no herbicide was applied. Avanti was the top yielding cultivar as well as one of the most resistant genotypes of this trial. Contrary to the positive correlation between yield and GH96 root rot ratings, yield was negatively correlated with average root rot score in 1997 (Table 4). 100 SW was significantly and positively correlated with field root rot scores and GH97 (Table 4). Pearson rank correlation coefficients (r) for root rot ratings of 11 common genotypes grown in both GH96 and GH97 were also calculated. AnDr value of 0.83" resulted between average 1996 and 1997 field scores. GH97 was correlated with 1996 field scores (0.77") and GH96 was positively correlated with 1997 field ratings (0.92***). GH96 and GH97 scores were also positively correlated (r = 0.86“). DISCUSSION Our objectives were to develop a greenhouse screen that was inexpensive and relatively simple to allow evaluation of lateral roots that correlated well with field ratings and would permit the evaluation of large populations. The current perlite-based greenhouse screen was inexpensive and required little maintenance. From 12 to 24 57 gentoypes can be grown in one 54x27x6 cm flat. Minimal requirements for greenhouse space ensure that large populations can be evaluated with limited resources. Time and labor constraints are also minimized since the time from planting to evaluation takes four weeks and only daily watering and weekly fertilization is necessary. Using perlite, an inexpensive soil medium, roots could be cleared of adhering particles with relative ease so that ratings were not based on hypocotyl symptoms alone. Hypocotyl ratings are not adequate indicators of root rot damage as evidenced by Burke and Barker’s (1966) observation that lateral root damage was the more important contributor to yield reductions. The authors further demonstrated using infested and non-infested islands of soil surrounding the hypocotyl and taproot, that severely diseased hypocotyls. could sustain adequate plant growth and development. The greenhouse screening presented in this paper provides a means to evaluate both hypocotyl and lateral root damage. Figure 3 shows typical root symptoms of a severely infected Montcalm seedling in contrast to a non-inoculated control. The reddish discoloration typical of F.s. f.s. phaseoli root infection is striking and easily scorable using a visual rating system. Positive and highly significant correlations between greenhouse and field ratings provide further evidence supporting the advocacy of this screening technique. Correlations between field root rot ratings and greenhouse scores were strongly positive for both trials and were as high as 0.99 between GH97 and corresponding field ratings (Table 4). Interrelationships between 11 genotypes common to both trials also demonstrated a significant and positive correlation. The positive and significant relationship achieved between the proposed greenhouse screening method and replicated field ratings prove that 58 a controlled experiment can reflect field tolerance while reducing environmental variation and reducing resource limitations. Reductions in environmental variation using this greenhouse screen are confirmed by a decrease in CVs between field and greenhouse evaluations. The CVs for GH96 and GH97 were from 3 — 10% less than the CVs from corresponding field evaluations (Table 2 and 3). Coefficients of variation are a means to compare the variability of two experiments with different means and express the standard deviation as a percentage of the mean. A decrease in CV can reflect an increase in mean value, a decrease in the standard deviation or both. A comparison of mean scores between field and greenhouse ratings were similar for both groups of genotypes indicating that the reduction in CV reflects a decrease in the variability present for greenhouse trials thereby rmeting our main objective to develop a greenhouse screening method which could reduce the influence of environmental factors. Considering the aforementioned criteria for an inexpensive, accurate, consistent, simple and rapid greenhouse screen to evaluate bean resistance to F.s. f.sp. phaseoli, the causal organism of root rot in Michigan, the proposed protocol accurately addresses these issues. Furthermore, once rated, plants can be dipped in benomyl fungicide solution and transplanted. The non-destructive nature of this protocol allows for the testing of early generation material for which advanced generation seed is desired. Simple inoculation and plant culture procedures, positive and significant correlation for greenhouse and field evaluations, minimal space and resource requirements combine to make this an effective procedure to further the improvement of root rot resistance in bean. 59 Figure 3. Fusarium root rot infection of ‘Montcalm’ dark red kidney (right) compared to a non-inoculated control (left). o) .v~ Economic damage from any disease is ultimately manifested by its effect on yield. However, correlation between field and root rot using the genotypes described in this study exemplifies the potential problems which can arise when measuring disease severity based on yield potential. For the genotypes in GH96, yield was significantly and positively correlated with field and greenhouse root rot ratings which means that an increase in disease severity corresponds to an increase in yield (Table 4). This counterintuitive result can be explained by the lack of adaptation for many of the more root rot resistant genotypes. Cultivars UI-114, ‘Viva’, ‘Roza’ are reported as being root rot resistant according to Abawi and Pastor Corrales (1990) but were not adapted to northern Michigan and consequently did not mature nor achieve optimum yield. Cultivars NW 590, NW 63 and ‘Sierra’, all of which scored, on average, below 3.0, also lacked adequate adaptation to this Northern latitude (>45°N). In contrast, the large-seeded genotypes were developed specifically for Michigan kidney production areas and were highly adapted to the Presque Isle County, MI environment. These same large-seeded genotypes possess high levels of susceptibility and all scored, on average, 3.0 or greater for root rot infection (Table 2). It is not surprising, therefore, to observe a significant positive correlation between root rot resistance and yield for these genotypes and any further conclusions based on these correlations must be made with caution. Contrary to the positive correlation observed between root rot ratings and yield for the GH96 genotypes, yield and root rot ratings for GH97 genotypes were significantly and negatively correlated (Table 4). These results are not surprising considering differences in the genotypes tested and the environmental conditions encountered in each year. One of the objectives of GH97 was to evaluate levels of root rot resistance in advanced breeding . 61 lines and popular cultivars developed for Michigan. All of the genotypes in this experiment were adapted to Michigan and would, therefore, be expected to perform well in Presque Isle County, MI. Conditions at planting, however, were considerably drier than optimum (Figure 7) and problems with emergence resulted, especially for some of the large-seeded genotypes. Many of the large-seeded genotypes like Isles and Chinook struggled to germinate under the dry conditions present in 1997 thereby delaying maturity. Many green pods and stems were observed at harvest. Although yield data reflect problems with seasonal conditions, root rot ratings confirm levels of resistance reported in the 1996 experiment for those lines which were duplicated in 1997. Furthermore, significant correlation between field root rot ratings and greenhouse evaluations verify that resistance to root rot can be visually measured but, depending on environmental conditions will not be good indicators of yield reductions caused by this disease. To more accurately define yield reductions caused by Fusarium root rot, control experiments in non-infested soils should be conducted simultaneously with Fusarium-infested treatments in the same location. This will, however, be difficult to achieve with this ubiquitous pathogen. 62 REFERENCES Abawi, GS and M.A. Pastor Pastor Corrales. 1990. Root rots of beans in Latin American a Africa: Diagnosis, research methodologies and management strategies. p. 114. Centro Intemacional de Agricultura Tropical (CIAT), Cali, Colombia. Baggett, JR. and W.A. Frazier 1959. Disease resistance in the runner bean, Phaseolus coccineus L. Plant Dis. Rep. 43:137-143. Baggett, J.R., W.A. Frazier and E.K. Vaughn. 1965. Tests of Phaseolus species for resistance to Fusarium root rot. Plant Dis. Rep. 492630-633 Beebe, S.E., F.A. Bliss, and HF. Schwartz. 1981. Root rot resistance in common bean germ plasm of Latin American origin. Plant Dis. 65:485-489. Boomstra, A.G., F.A. Bliss and SE. Beebe. 1977. New sources of Fusarium root rot resistance in Phaseolus vulgaris. J. Amer. Soc. Hort. Sci. 102: 182-185. Bravo, A., D.H. Wallace, and RE. Wilkinson. 1969. Inheritance of resistance to Fusarium root rot of beans. Phytopathology 59: 1930-1933. Burke, D.W. and A.W. Barker. 1966. Importance of lateral roots in Fusarium root rot of beans. Phytopathology 56:293-294. Burke, D.W. and M.J. Silbemagel. 1965. Testing beans for field tolerance to Fusarium root rot. Phytopathology 55: 1045- 1046. Christou, T. and W.C. Snyder. 1962. Penetration and host-parasite relationships of Fusarium solani f. phaseoli in the bean plant. Phytopathology 52:219-226. Estevez de Jensen, C., R. Meronuck and J.A. Percich. 1998. Etiology and control of kidney bean root rot in Minnesota. Annu. Rep. Bean Improv. Coop. 41:55-56. Hassan, A.A., D.H. Wallace and RE. Wilkinson. 1971. Genetics and heritability of resistance to Fusarium solani f. phaseoli in beans. J. Amer. Soc. Hort. Sci. 96:623-627. Huber, D.M., R.D. Watson and G.W. Steiner. 1965. Crop residues, nitrogen, and plant disease. Soil Sci. 100:302-308. Keenan, J.G., H.D. Moore, N. Oshima and LE. Jenkins. 1974. Effect of bean root rot on dryland pinto bean production in southwestern Colorado. Plant Dis. Rep. 58:890- 892. SAS Institute. 1994. The SAS system for Windows. Release 6.10. SAS Inst., Cary, NC. Saettler, A.W. and AL. Andersen. 1978. pp. 172-179. Bean diseases and their control. In: L.S. Robertson and RD. Frazier’s (eds.) Dry Bean Production - Principles and Practice. Cooperative Extension Service and Agricultural Experiment Station of Michigan State University. Extension Bulletin E-1251. 63 Silbemagel, M.J. 1987. Fusarium root rot-resistant snap bean breeding line FR266. HortScience 22:1337-1338. Silbemagel, M.J. 1990. Genetic and cultural control of Fusarium root rot in bush snap beans. Plant Dis. 74:61-66. Sippell, D.W. and R. Hall. 1982. Effects of pathogen species, inoculum concentration, temperature, and soil moisture on bean root rot and plant growth. Can. J. Plant Pathol. 4: 1-7. Smith, PL. and B.R. Houston. 1960. Root rot resistance in common beans sought in plant breeding program. California Agriculture Sept.:8. Steadman, J .R., ED. Kerr and RF. Mumm. 1975. Root rot of bean in Nebraska: Primary pathogen and yield loss appraisal. Plant Dis. Rep. 59:305-308. Toussoun, T.A. 1970. Nutrition and pathogenesis of Fusarium solani f.sp. phaseoli. pp 95-99. In: T.A. Toussoun, R.V. Bega and RE. Nelson’s Root Diseases and Soil- bome pathogens. University of California Press, Berkley, Los Angeles, London. Tu, J.C. and 8.]. Park. 1993. Root-rot resistance in common bean. Can. J. Plant Sci. 73:365-367. Wallace, DH. and 12.13. Wilkinson. 1965. Breeding for Fusarium root rot resistance in beans. Phytopathology 55: 1227- 123 1. Wallace, DH. and RE. Wilkinson. 1966. Origin of N203 (PI 203958). Annu. Rep. Bean Improv. Coop. 9:38. CHAPTER 2 ANALYSIS OF RESISTANCE TO FUSARIUM ROOT ROT IN BEAN AND THE IDENTIFICATION OF ASSOCIATED QTL ABSTRACT Using recombinant inbred populations, genetic analysis of the nature of resistance to the bean root rot pathogen, F usarium solani f.sp. phaseoli (Burk.) Snyd. & Hans, was conducted and RAPD markers associated with QTL controlling this trait were identified. Several markers demonstrated significant associations with root rot resistance determined from both greenhouse and field evaluations. Markers associated with field root rot ratings tended not to be associated with greenhouse and vice versa except for the RAPD P700 which was significantly associated with greenhouse and field data. Markers identified in this study did not explain more than 15% of the phenotypic variation for root rot resistance but a combination of 4 markers explained 29% of the phenotypic variation for field root rot ratings. Based on these results, it was concluded that genetic resistance to F. s. f.sp. phaseoli derived from PI 203958 (N203) is polygenically controlled and strongly influenced by environmental factors. Supporting this conclusion were moderate heritability estimates (hz) that ranged from 0.48 to 0.71 for one population and from 0.09 to 0.66 for another population. We identified two regions of the genome associated with root rot resistance that also corresponded to locations of Phaseolus vulgaris pathogenesis-related proteins. It appears that mechanisms associated with host defense responses may be involved in Fusarium root rot resistance and selection directed towards enhancing these 65 traits may play a role in the future improvement of root rot resistance caused by F.s. f.sp. phaseoli in bean. INTRODUCTION Root rot caused by F.s. f.sp. phaseoli negatively affects all large-seeded bean production areas of the US and is often considered one of the most serious diseases of irrigated beans in the Pacific Northwest after white mold (Dryden and Van Alfen, 1984; Miller and Burke, 1986). The disease is particularly severe on large-seeded genotypes due to a lack of genetic resistance in these market classes. Montcalm, a popular dark red kidney cultivar in Michigan, Minnesota and Wisconsin exemplifies the high degree of susceptibility inherent to this market class. An overemphasis on quality traits in both dark red kidney and snap market classes and consequent reduction in genetic variability may have contributed to the lack of resistance in these seed and pod types. Small-seeded genotypes, although not immune to Fusarium root rot, do not appear as susceptible as the larger seed types. The large-seeded (Andean) are distinguished from small-seeded (Middle American) by morphological, biochemical and molecular characteristics (Gepts, 1988; Haley et al., 1994). Genetic aspects like these, largely unknown, and unique to Andean genotypes may enhance sensitivity to this pathogen. Likewise, soil types and environmental conditions specific to large-seeded production areas can increase disease severity. Several attempts have been made to elucidate the genetic control of resistance to F. s. f.sp. phaseoli but results from these studies have failed to rapidly advance improvement of Fusarium root rot resistance, especially in large seed and pod types 66 (Smith and Houston, 1960; Wallace and Wilkinson, 1965; Hassan et al., 1971). Conclusions based on these studies may be misleading for several reasons one of which concerns the population structure employed. The studies of Smith and Houston (1960), Wallace and Wilkinson (1965) and Hassan et al. (1971) were based on methods and populations appropriate for a more qualitative type of inheritance. Evidence supporting quantitative control of Fusarium root rot include the lack of immunity to F.s. f.sp. phaseoli in P. vulgaris and observable differences in levels of susceptibility which suggest polygenic control (Baggett et al., 1965). Furthermore, F .s. f.sp. phaseoli disease incidence and severity are strongly influenced by the environment. These two observations define Fusarium root rot resistance as a quantitative trait. Conclusions from previous genetic studies were based on unreplicated F1, F2 and BC p0pulation structures. Scoring of individual plants in unreplicated experiments can be problematic for traits strongly influenced by environmental factors and an average score for a particular genotype is preferred. While the need to control environmental variation through replicated field trials is important for analyzing Fusarium root rot resistance, the actual scoring method is equally significant. Evaluation for root rot in the Hassan et al. (1971) study was based on hypocotyl reaction in both field and greenhouse studies (rating systems in the other two studies were not documented). As has been previously discussed (Literature Review), hypocotyl ratings are not indicative of root damage which has been implicated as a more accurate indicator of yield reductions caused by F .s. f.sp. phaseoli (Burke and Barker, 1966). A method for evaluation that includes assessment of lateral root infection will provide more relevant results. 67 The third limitation of the aforementioned genetic studies involves the utilization of wide, inter gene pool crosses. While recombination between Andean and Middle American gene pools occurs readily, hybrid lethality can result (Koinange and Gepts, 1992). Skewed segregation as a result of this phenomenon is common and may lead to misinterpretation. More recent development of root rot resistant, Andean breeding lines provide an opportunity to study inheritance patterns within the same gene pool. Molecular markers may prove valuable as another tool that can be employed to facilitate genetic analysis of root rot resistance in bean. RAPD markers associated with quantitative trait loci (QTL) controlling resistance to common bacterial blight (CBB; Xanthomonas campestris pv. phaseolicola (Smith) Dye) and white mold (Sclerotinia sclerotiorum) in bean have been identified (Miklas et al. 1996; Miklas et al., 1999; Park et al., 1999). Marker assisted selection used to indirectly select for resistant genotypes could facilitate improvement of disease resistance for traits like Fusarium root rot resistance where field selection is laborious and demanding. While the practical application of marker assisted selection for quantitative traits has yet to be realized, many studies recognize its potential to facilitate the improvement of quantitative traits (Pilet et al., 1998; Mangin et al., 1999; Schechert et al., 1999; Miklas et al., 1998; Faris et al., 1999; Lubberstedt et al., 1998). QTL-marker associations may also provide a basis for a greater understanding of quantitative disease resistance through the identification of loci that influence resistance to more than one disease. The opportunity, for cloning QTL controlling. complex disease resistance, and identification of syntenic areas in other species may result from this type of research. 68 Objectives The objective of this study was to i) define the inheritance of root rot resistance in bean using recombinant inbred populations with replicated field and greenhouse trials; and ii) identify significant QTL-marker associations which could be used to facilitate indirect selection of Fusarium root rot resistance. MATERIALS AND METHODS Germplasm and Population Development. Five parental genotypes were used in this study. Montcalm dark red kidney, ‘Isles’ dark red kidney and Seafarer navy cultivars were utilized as susceptible parents whereas FR266 and PI 203958 (N203) obtained from the USDA Germplasm Bank (Prosser, WA) were used as resistant parents. Montcalm and Isles are determinate, dark red kidney cultivars developed at Michigan State University and adapted to kidney bean production areas in Michigan. Both are full season cultivars and highly susceptible to root rot caused by F .s. f.sp. phaseoli. Seafarer, a determinate, early season navy bean cultivar developed at Michigan State University is also reported as being susceptible to Fusarium root rot. N 203 is a small, viny, photoperiod sensitive black bean from Mexico identified by plant collector, explorer, Oliver Norvell, as resistant to Fusarium root rot (Wallace and Wilkinson, 1966). FR266 is a Fusarium resistant snap bean breeding line possessing a large root system, white seed, and snap bean-like pods (Silbemagel, 1987). It tends to flower earlier but mature later than other cultivars in the study. Three populations were developed using these five parents in intra gene pool, resistant by susceptible crosses. Montcalm and Isles were crossed to FR266 to create MF 69 and IF recombinant inbred Andean populations, respectively, and Seafarer was crossed to N203 to deve10p SN Middle American recombinant inbred population. Progeny from ME, SN, and IF were advanced by single seed descent to the F4 generation where seed was bulked and advanced to create 79, 93 and 68 Fa-derived recombinant inbred lines (RILs), respectively. Seed from individual F2 plants in SN was bulked to create 105 F24 RILs which were used in field experiments conducted in 1997. An individual plant from each Fm RIL was selected and advanced to create an Fa-derived RIL population. Root Rot Evaluation Field Field trials were conducted in both Presque Isle County, MI (PI) and Perham County, MN (PM) in 1997 and 1998. Selected fields were previously identified as infested with F.s. f.sp. phaseoli. All field experiments were planted in a randomized complete block design with three to four replications. A combination of pre-emergence herbicide and mechanical and hand cultivation was used to control weeds. Seed was treated with a combination fungicide and insecticide containing thiophanate methyl, captan and lorsban. Agronomic practices were applied to insure good crop growth and development. Single- row plots consisting of 10 seeds each were 1.2 m long and 0.76 m wide. During mid and late pod-fill, five plants per plot were uprooted with a shovel taking care not to disturb the main portion of the root system. Roots were cleaned of debris and rated-on a scale from 1 to 7 (Table 1; Figure 2). One hundred and two SN Fm RILs, seven checks and both parents were planted in P1 on June, 13, 1997 and replicated four times. Ratings were taken 52 and 86 DAP. 70 Ninety-four SN F24 RILs were planted in three replications on June 1, 1997 in PM and rated 53 and 71 d after planting. An experiment consisting of 92 SN F45 RILs, two parents and four checks replicated three times was also planted on June 4, 1998 in P1 using the same experimental design and plants were rated 91 DAP. Only one rating was conducted for this experiment due to problems with emergence. Field trials for MF were planted on June 4, 1998 in P1 and on May 21, 1998 in PM. Eighty FM, RILs from ME, two parents and 10 checks, replicated four times, were planted in P1 and plots were rated 78 DAP. Seventy-eight MF FM RILs, two parents and four checks, replicated three times were planted in PM and plots were rated 74 DAP. Only one rating, recorded during late pod-fill, was taken for these 1998 MP experiments due to problems with plant emergence. In addition to root rot evaluation experiments, all RIL for both MF and SN populations were increased in four- and two-row unreplicated plots, respectively, in Ingham County, MI in 1998. Data for days to flower (DTF) and days to maturity were recorded based on this experiment. Greenhouse The MP, SN and IF populations were evaluated for root rot resistance in the greenhouse using the perlite screen described in the previous chapter. Two SN greenhouse evaluations were conducted using and F45 (SNGHl) and F4;7 (SNGH2) SN recombinant inbred populations. The genotypes were the same as those reported for the 1997 and 1998 field trials, respectively. Three replications with three plants per genotypic entry were planted and scored. Three MF greenhouse screenings were conducted using F45 (MFGHI) 71 and Fan (MFGH2, MFGH3) MF recombinant inbred populations using four replications and three plants per genotypic entry. Genotypes included in these screenings were the same as those used in the 1998 PI trial. A single unreplicated greenhouse screen consisting of six plants each of 68 F45 IF recombinant inbred population, parents and six checks was also conducted. Fusarium solani f.sp. phaseoli isolates used for each greenhouse evaluation of SN, MF and IF and corresponding county from which the isolates were collected are given in Table 5. Statistical Analysis All experiments conducted in the field and greenhouse involving MF and SN were analyzed as one-way analyses of variance (ANOVA) using the PROC GLM and PROC MIXED programs of SAS (SAS Institute, 1994). Root rot ratings from the five plants scored from each plot were averaged to give a plot mean which was subsequently used for ANOVAs. Mean scores, averaged over all plants rated for the 1997 SN experiments conducted in P1 and PM, were used in a combined analysis over locations. Due to uneven plant numbers per plot, analysis of MP and SN experiments conducted in P1 and PM in 1998 included a subsampling error. Heritability estimates were calculated on an entry mean basis for all greenhouse and field trials according to Hallauer and Miranda (1981) except for the IF population which was an unreplicated greenhouse screen. Pearson rank correlation coefficients were calculated using SAS PROC CORR to compare means of root rot ratings for RILs within MP for all greenhouse and field evaluations conducted for this population (SAS Institute, 1994). 72 Table 5. Fusarium solani f.sp. phaseoli isolates and location of origin used to inoculate greenhouse evaluations for three recombinant inbred populations, Seafarer/N203 (SN), Montcalm/FR266 (MF) and Isles/FR266 (IF). Two greenhouse screens were conducted using SN (SNGHl and SNGH2) and three were performed using MF (MFGHl, MFGH2, MFGH3). Evaluation Isolate Origin SNGHl Huron 2a Huron County, MI SNGH2 Hawks 2b Presue Isle County, MI MFGHl F .s. 12 Presque Isle County, MI MFGH2 Huron 2a Huron County, MI MFGH3 Hawks 2b Presque Isle County, MI IF * Hawks 2b Presque Isle County, MI Marker analysis DNA Extraction DNA was extracted from SN and IF F2 individuals using a mini-extraction protocol described by Schneider et al. (1997). DNA was extracted from tissue sampled from 6 plants of each FM MF RIL and all parents using a larger DNA extraction method (Kisha et al. 1998). Each of the F2 individuals sampled for DNA extraction in these two p0pulations was the progenitor of a corresponding RIL. 73 RAPD Protocol RAPDs were generated using the protocol outlined by Schneider et al. (1997) with slight modification. PCR products and repeatability was enhanced by doubling the amount of enzyme used in each reaction. RAPDs greater than 1200 bp were amplified using GIBCO BRL® brand Taq polymerase (Life Technologies, MD) and RAPDs less than 1200 bp were amplified by AmpliTaq® DNA polymerase, Stoffel Fragment (Perkin Elmer, CT). Seven hundred random 10-mer primers (Operon Technology, CA) were used to amplify random regions of the genome. DNA was amplified using a Perkin Elmer Cetus DNA Thermal Cycler 480 (Perkin Elmer, Cetus, Norwalk, CT) using the following cycles: 3 cycles of l min. at 94 °C, 1 min. at 35 °C, and 2 min. at 72 °C, 34 cycles of l min. at 94 °C, 1 min. at 40 °C, and 2 min. at 72 °C (final step extended by 1 sec for each of the 34 cycles), and a final extension cycle of 5 min. at 72 °C (Haley et al., 1994a). Approximately 20 111 of amplified DNA from each sample was run on a 1.4% agarose gel containing ethidium bromide (0.5 ug ml"), 40 mM Tris-acetate, and 1 mM EDTA. DNA was viewed under ultraviolet light and photographed for permanent record. While populations were being developed, primers were screened against Seafarer, N 203, Montcalm and FR266 parents to identify polymorphisms which would subsequently be utilized in the population analyses. A method of selective genotyping proposed by Lander and Botstein (1989) and utilized by Miklas et al. (1996) was employed to facilitate the identification of markers associated with QTL controlling Fusarium root rot resistance in MF. DNA from the five lowest scoring or most resistant RIL of MF based ondata from MFGH2 was pooled to create a resistant bulk (R). Likewise, DNA from the 5 highest 74 scoring or most susceptible RIL was pooled to create a susceptible bulk (S). RAPD markers polymorphic between R and S bulks were subsequently screened against the individuals. If the RAPD continued to segregate according to R and S classifications of the individuals used to make the bulks, the primer was screened against the entire population. RAPDs reported on an integrated P. vulgaris linkage map were also used for screening MF although many were not polymorphic between Montcalm and FR266 and, thus, uninforrnative (Freyre et al., 1998). Significant associations between root rot resistance and marker genotype were determined by one-way ANOVAs using SAS’s PROC GLM (SAS Institute, 1994). Marker-QTL associations were considered significant at P < 0.05. Multiple regression analyses using combinations of significant markers were also performed using PROC GLM. RAPDs which did not exhibit a 1:1 segregation were disregarded. Linkage relationships among these same markers were determined using MAPMAKER/EXP group command with minimum LOD of 4.0 and maximum distance of 50 cM (Lincoln et al., 1987). RESULTS Root rot evaluations Significant variation for root rot scores was observed in all tests of MF (Table 6). Results for SN were more variable. Significant genetic variation for root rot ratings was observed for one of the two greenhouse experiments (SNGHl) and the 1998 PI trial (Table 6). Genetic variation for the 1997 field trial combined over two locations, PI and 75 Table 6. Analyses of variance for root rot ratings for the recombinant inbred populations, Seafarer/N 203 (SN) and Montcalm/FR266 (MF). Four experiments are presented for each population. Two greenhouse experiments (SNGHl and SNGH2) for SN and three for MF (MFGHl, MFGH2 and MFGH3). A combined analysis over two locations in Presque Isle County, MI (PI) and Perham County, MN (PM) is presented for both populations. The SN population was evaluated in 1997 and the MF in 1998. An additional PI experiment was conducted for SN in 1998 and is presented. MF SN Source df MS F Test df MS F-Test ----MFGH1---- ----SNGH1---- Repil: 1 1.99 2 1.23 Genotype l 1.59 2. 14*" 92 0.41 196" Error 83 0.74 174 0.21 ----MFGH2---- ----SNGH2---- Rep 3 1.25 2 2.88 Genotype 78 0.97 2.1 1*** 93 0.36 1.22 Error 225 0.46 179 0.30 ----MFGH3---- ----1998 SN PIT-m Rep 3 9.30 2 0.07 Genotype 77 0.57 1.97*** 92 2.58 2.60*** Rep*Genotype 170 0.99 242*" Error 228 0.30 862 0.41 ----1998 MF Combined---- ----1997 SN Combined---- Loc. 1 916.05 1 212.45 Rep within Loc 5 27.85 4.41 Rep 3 25.32 5 0.43 Genotype 79 4.53 3.23*** 101 0.47 0.92 Loc*Genotype 91 0.21 2.22*** Rep*Genotype 227 1 .40 2. 10* ** Error 1 894 0.67 48 1 0.5 *, **; *** Significance at the P < 0.05. 0.01, 0.001 levels, respectively. TN] 1998 field trials were analyzed using a subsampling term for individuals scores per plot. iRep = replication; Loc = location 76 Table 7. Root rot ratings of the top and bottom five Montcalm/FR266 (MF) recombinant inbred lines based on the second MF greenhouse evaluation (MFGH2). Rankings of these genotypes in all other evaluations including two greenhouse screenings (MFGHl and MFGH3), two field trials conducted in Presque Isle County (1998 PI), MI and Perham County, MN (1998 PM) and the combined analysis over these two locations are given in parentheses. Scores for check genotypes evaluated for each experiment are also given for comparison. Genotype MFGH2 MFGH] MFGH3 1998 PI 1998 PM Combined MF33 2.8 (l) 1.9 (1) 3.0 (9) 4.1 (43) 5.1 (19) 4.5 (23) MF8 3.0 (2) 2 (2) 2.6 (2) 4.4 (62) 4.9 (11) 4.7 (41) MF21 3.0 (3) 2.3 (8) 2.9 (6) 4.2 (47) 6.2 (65) 5.3 (68) MF17 3.0 (4) 2.3 (9) 3.1 (10) 3.7 (25) 5.2 (27) 4.6 (28) MF26 3.0 (5) 2.3 (10) 3.5 (47) 2.4 (1) 5.4 (37) 3.9 (8) MF4 4.6 (74) 3.1 (57) 4.0 (48) 4.3 (59) 6.1 (36) 5.1 (50) MF20 4.6 (75) 3.1 (55) 3.5 (72) 4.4 (52) 5.3 (63) 4.9 (60) MF13 4.7 (76) 3.3 (75) 3.3 (30) 4.6 (71) 6.4 (73) 5.5 (74) MF9 5.3 (77) 4.0 (78) 4.5 (77) 4.2 (51) 6.5 (74) 5.3 (72) MF77 4.6 (84) 2.8 (38) 3.3 (27) 4.4 (63) 6.5 (75) 5.6 (77) FR266 2.5 2.1 2.4 3.7 4.5 4.2 Montcalm 5.3 4.2 5.2 4.4 6.2 5.4 N203 2.5 - 2.6 - - - Seafarer 3.1 - 3.3 2.8 - 3 .5 Redhawk 5.3 - 5.3 4.8 6.0 5.3 Isles - 4.2 5.1 4.9 - 5.5 Foxfire - - - - 5.4 4.7 [(95757 4.3 - 4.6 4.6 - 5.3 K93613 4.5 - 4.6 4.7 - 5.4 Tendergreen Imp 3.2 - 3.1 4.4 - 5.2 WIRRR36 3.2 - 3.2 - 2.7 2.0 G122 4.0 3.9 3.6 3.8 5.0 4.1 Taylor Hort. - - - 3.9 - . 4.7 Mean 3.8 3.4 3.5 4.2 5.4 7 4.7 LSD ( " = 0.05) 1.0 1.7 0.8 1.0 1.1 0.7 CV (%) 18.5 25.6 15.9 14.6 17.4 16.6 77 Table 8. Root rot ratings of the top and bottom five Seafarer/N203 (SN) recombinant inbred lines based on the first SN greenhouse evaluation (SNGHl). Rankings of these genotypes in all other evaluations including a second greenhouse screenings (SNGH2), two field trials conducted in Presque Isle County (1997 PI), MI and Perham County, MN (1997 PM) in 1997, the combined analysis over these two locations and another field trial conducted in 1998 in Presque Isle County, MI (1998 PI) are given in parentheses. Scores for check genotypes evaluated for each experiment are also given for comparison. Genotype SNGHl SNGH2 1997 PI 1997 PM Combined 1998 PI SN03-220 1.1 (l) 2.9 (64) 1.5 (14) 2.8 (42) 2.1 (25) 2.4 (38) SN08-240 1.3 (2) 2.8 (58) 1.3 (l) 2.9 (54) 2.1 (22) 2.7 (51) SN09-249 1.5 (3) 2.8 (47) 1.5 (16) 5.1 (82) 3.3 (90) 1.8 (5) SN28-3l4 1.7 (4) 2.3 (16) 1.7 (12) 2.9 (55) 2.3 (15) 1.9 (55) SN33-330 1.7 (5) 2.3 (65) - - - 2.0 (56) SN15-270 2.9 (89) 2.8 (54) 2.0 (78) 2.4 (9) 2.2 (29) 3.4 (89) SN07-235 3.0 (90) 3.0 (75) 1.6 (27) 2.4 (6) 2.0 (5) 2.7 (49) SN08-241 3.1 (91) - 1.6 (23) 3.0 (56) 2.3 (48) - SN09-247 3.2 (92) 2.6 (36) 1.8 (66) 2.5 (19) 2.2 (30) 3.0 (72) SN29-312 3.3 (93) 2.9 (66) 1.7 (44) 2.5 (14) 2.1 (21) 2.4 (36) N203 1.6 2.1 1.9 2.1 1.7 1.5 Seafarer 2.4 3.6 3.0 4.1 3.0 4.0 Mackinac - 2.9 ~ - - - 4.1 Newport - 3.4 - - - 3.1 A300 2.9 - 3.5 - 3.5 - T39 2.6 3.6 2.9 - 2.9 3.0 FR266 2.0 - - - 2.7 - WI RRR36 2.7 2.8 1.7 - 2.6 - Montcalm 5.3 - 5.6 5.2 5.4 - Isles 5.4 - 6.4 4.9 6.4 - Redhawk 3.8 - 6.3 - 6.3 - Foxfire - - - 3.7 3.7 - Mean 2.3 2.7 2.9 2.7 2.4 2.7 LSD (" = 0.05) 0.8 0.8 0.9 1.0 0.6 1.0 CV (%) 20.3 20.3 21.0 23.8 20.2 23.8 78 PM was not significant for SN nor was genetic variation significant for SNGH2. Location by genotype interactions were significant for the combined analyses of variance over two locations in both populations. Means, LSDs and CVs are presented in Tables 7 and 8 for all experiments conducted in both populations. In general, the mean score of ME was higher than the mean of SN, and MF had a wider distribution of scores than SN (Figure 4). The CVs remained relatively constant for each population and ranged from 14.9 to 25.6 % for ME (Table 7) and from 20.0 to 24.5 % for SN (Table 8). Analysis of MFGHl resulted in a higher than expected CV than the rest of the MF tests because it comprised only two replications due to limitations on seed and was not chosen to establish ranked order. Narrow sense h2 was consistent and significantly different from zero for MF. Heritability estimates ranged from 0.48 to 0.71 suggesting a moderate to high heritability for root rot resistance in this population (Table 9). In five of the six experiments presented for MF, h2 remained at approximately 50%. The one exception was the 1998 PI trial which resulted in h2 of 0.71. The h2 for SN varied with experiment and ranged from 0.09 to 0.66 (Table 9). As expected, h2 significantly different from zero occurred only in those experiments which demonstrated significant genetic variation. Moderate to low heritability for this population was suggested by these results. Representative frequency distributions are illustrated in Figure 4. Continuous variation was observed over the range of root rot scores for both populations but the maximum score for SN rarely exceeded a score of 4.0 although susceptible check genotypes scored as high as 6.0 (Table 8). Seafarer, the susceptible parent for this 79 1 1.5 2 25 3 35 4 45 5 55 6 65 L _ 1 15 2 25 3 35 4 45 5 55 6 r, I I eeeaeee ‘0., ,. . , I 11.522533544555563 I I I I {Tam-2T; ”FT—T “I :20 I I I I I L ! ,_. ,,_,,-_#_I 1 15 2 25 3 35 4 45 5 55 6 65 Figure 4. Representative distributions of root rot scores for Seafarer/N203 (SN) and Montcalm/FR266 populations (MF). A) Distribution of root rot scores for the 1998 SN experiment conducted in Presque Isle County, MI; B) Distribution of root rot scores for the second SN greenhouse evaluation (SNGH2); C) Distribution of root rot scores for the 1998 MP experiment conducted in P1; D) Distribution of root rot scores for the first MF greenhouse evaluation (MFGHl). 80 Table 9. Heritability estimates (hz) and standard errors of Fusarium root rot ratings for the Montcalm/FR266 (MF) and Seafarer/N203 (SN) populations. Six greenhouse and field experiments were analyzed for each population. ExperimentT h2 and SE MF Population MFGHl 0.53 :t: 0.17 MFGH2 0.49 :t 0.17 MFGH3 0.48 :1: 0.17 1998 PI 0.71 1:018 1998 PM 0.52 :1: 0.18 1998 Comb. 0.48 :1: 0.21 SN Population SNGHl 0.48 :1: 0.16 SNGH2 0.17 :i:0.17 1997 PI 0.09 :1: 0.16 1997 PM 0.22 :t 0.16 1997 Comb. 0.20 :1: 0.17 1998 PI 0.66 :t 0.16 1' MFGH and SNGH refer to greenhouse experiments in ME and SN, respectively; PI and PM refer to field trials conducted in Presque Isle County, MI and Perham County, MN, respectively; Comb. refers to the combined analysis over both PI and PM locations. 81 population, generally did not score above a 3.0. While this is a significantly higher score than that observed for N203, the range of values for SN was not large enough to achieve consistent genetic variation especially for experiments involving the F23 lines. For purposes of this study, SN was not subsequently used for genetic analysis. MF distributions were much broader as evidenced by the significant genetic variation observed in all experiments involving this population (Table 6, Table 8 and Figure 4). FR266 scored from 2.0 to 4.5 whereas Montcalm scored from 1 to 2 points more than FR266 in all experiments. Transgressive segregation towards susceptibility was observed. Correlations between greenhouse and field evaluations were positive and significant for all comparisons except between MFGH2 and 1998 PI and between MFGH2 and 1998 combined analysis (Table 10). Greenhouse trials were positively and significantly correlated with each other and correlation coefficients (r) ranged from 0.40 to 0.67. Data from individual field locations were also positively and significantly correlated with an r value of 0.41. Significant negative correlations between field ratings and days to flower (DTF) were observed as well as between MFGH] and DTF. Data collected on days. to maturity was not reported because severe drought conditions during the 1998 growing season prevented accurate determination of this measurement. Marker evaluation Of the 330 primers analyzed against the parents, 50% demonstrated at least one polymorphic band between both sets of parents. A total of 680 primers which included polymorphic primers identified in the preliminary parental screen were analyzed against 82 $8382 33 :8 E 53 8>e math—:8 8:588 05 2 Echo: .nEoU 503838: .22 5:80 858m :8 =2 5580 03 2.085 :m 888:8 m3: Bow 9 8:818: SE :8 E £2 E 388830 omsofiooem 9 flame.— 2052 H 52888. .225. 58 :8 .85 .26 v a 2: a 858:5me a: m..... .L annmwd SE maa— nnnehd 2.... :6 E maa— smme .53 {no 80:: e2. 33 N3 585 $82 ammo .35 33 rate :23 50:: 52.5- $.52- :93- _ 3- 2.? :2. :5 250 M32 2: was: a :2 $82 $0.3 522 SEC ass: 9 98: e8 832:8: Sag—8:82 05 gm: 888:8 $888qu 22.: :8 88::on o :8 $58 .2 88 8:88.”— .«0 SEE: 8953 386508 8:22.80 3.8.— 8988 .2 28,—. 83 Table 11. Linkage groups (LG) generated by MAPMAKER/EXP from 35 RAPDS screened against the Montcalm/FR266 population. Markers not presented were independent LG RAPDS Distance (CM) 1 133600“ (B6) 1 1.2 P71550 2 P7700* (B2) 19.4 P101600 9.0 G6iioo* (132) 3 [181800 21.6 1181700* (B3) 4 AG2800 14.7 (317900 5 63300 16.0 632000 1.4 P91550 6 Y1 1600 1.4 7 S8500* (B3) 7.6 V121ioo *Previously mapped RAPDs reported on the bean integrated map (Freyre et al., 1998). The number in parentheses corresponds to the linkage group on which these RAPDs reside. R and S bulks in MF and 46 RAPDs polymorphic between bulks were analyzed against the entire population. Eleven were discarded due to skewed segregation ratios. The remaining 35 were analyzed by MAPMAKER/EXP to determine linkage relationships. Seven linkage groups resulted in none with more than three markers (Table 11). Five of the 35 RAPDs analyzed corresponded to markers previously reported on the P. vulgaris integrated linkage map (Freyre et al., 1998). Twenty-five of the markers identified and screened against MF significantly associated with at least one of the 6 experiments conducted on this population. Coefficients of determination (r2) which reflect the amount of variation 84 explained by a given marker were low and ranged from 5 - 15% (Table 12). The three markers in linkage group (LG) 2 appeared to associate consistently with greenhouse ratings. RAPD P7700 was also significantly associated with 1998 PM and the combined analysis. In this study, P7700 and G612“) were 28.4 cM apart which confirms the linkage relationship between these two markers on linkage group B2 (Figure 5; Freyre et al. 1998). Located in the vicinity of these two markers is the locus for the pathogenicity- related protein, PvPR2, perhaps implicating a role for this protein in Fusarium root rot resistance. The alleles associated with lower root rot scores for all three of these markers originated from the FR266 parent. A different set of markers, those from LG5, were consistently associated with field root rot ratings in MF and 632000 explained as much as 13 % of the variation for field root rot resistance (Table 12). No corresponding area representing these markers was found on the P. vulgaris linkage map. Alleles associated with reduced root rot scores for the markers in LG5 originated from Montcalm and one of the RAPDs, G3300 was also associated with DTF. Generally, RAPDs were either significantly associated With greenhouse ratings or with field ratings and those associated with field ratings were also associated with DTF. This is expected considering the correlation observed between field ratings and DTF. P7700 was an exception to this generalization since it was significantly associated with both greenhouse and field ratings and not with DTF (Table 12). 1181700 on LG3 is located on chromosome B3 of the P. vulgaris map suggesting a position for both 1181700 and 1181300 on this chromosome (Figure 6). 1181300 was positively and significantly associated with greenhouse and field ratings and DTF. 88500 was also reported to be on 85 32 1200191490 019.1500 «III P Romeo-.. '32 ”'BI--—-—--I- {1.01 1300350.”- 107.550 _._ _._01135 Acmm “I” «‘63.:7 012.900 .. . . _ " 01207 P077oo — I’07.7w__l,I - DIM ' 110401 #533 "" '1 335:; P10 - "m wswofl: 211.2 angm . 012.1600 : ___ Cd ”7am" I ......01054 [“550 I "-0100 Amusoo 1 ___:: 519103, 019.000 I Nos 41100000 013.1000 as ”11100 “.0159: 101.2000 __ / 01595!‘ 100900 .. 441107.300 :IP was: .. Figure 5. Comparative linkage maps of linkage group (LG) 2 and its corresponding position on linkage group B2 from the integrated bean linkage map and linkage group D2 from the Davis map. B2 and D2 are reproduced from Freyre et al. (1998). All markers on LG2 were signficantly associated with root rot resistance (Table 10). 86 as 03 01000.2 .4. M,“ 01096 01132 _.L 110 01132 0.000 \ 01120 009.1200 100.400 " ., . -0103: M1000 I L - I _Dlmo 110021300 . . _._1’1003 100 700 ,_ :5 21002 111103.050 011104 01020 ' 010.350 I. anus: I. 170.900 .. . -01009 00.1000 f“ 01009 _ . _png 1120.700 _, -1- 33 1111101050 0.21:: -= -......... 1.67 020350 _,_hr1t.1, 0110.403, 01012 010000 '\-- - ---01377 mm. 1900.700 "i 3 58..., — 000.500 55 --—sm01739 L63 017.000 __ 1181 700 .. 03010 "81700 / 711-350 :1; : : £01131 01377 N- " ' \013300 101.1300 811:!) 003.1150 122+!!!” 118m. 110111000 01151 132 Figure 6. Comparative linkage maps of linkage groups (LG) 7 and 3 and their corresponding positions on linkage group B3 from the integrated linkage map and linkage group D3 from the Davis map. B3 and D3 are reproduced from Freyre et al. (1998). Markers OPSSsoo and OPIl8rgoo were both significantly associated with root rot resistance (Table 10). 87 8882: 2: 833088 885:»: 82.. 828 N._ 5.5 H 2.3880: .226. 88 .8... .85 v a as a 8:85.05 :1 r. .. nwod need Mod .390 mod 5 Exam nood vod need 0506 wed m 82w 3 1:..— ~.o nhod *mod n 8020 aimed *mod n 8nNO< 3.326 nmod 000:6 0 82am 0506 *mod . 000d 0 2.me slim—d need nee—_.o o 8ome and need tic ~ .o shod nine—d N 82.?— vod emod :1 —.o N 8_ 60 1:86 “1:35 N 825: Dag who @800 EL E mIOmE NIOmZ _IOQE Os— .88088 02: 8 She .530: 9 38 53> 8.88%: 388580 man—<1 8.2 N: 05. .: 038,—. 8 8808.8 $88888: 0308: 085 9 8:008:28 .8: :38 02: mm A05 95% omeg— 05. .8038:— 8>o max—:8 8:388 2: :8 A21: 22 £8on 880m :8 an: :2 5850 2m— 2585 8 8:80— mfira 20m 02: @3me .NZOmE JmOm—zv 0.808898 88::on 8:: 80¢ 880:8 32 83885 chMEEEB8—z an .20 8:80. :8 .08 :88 53> 82808:: 388$:w6 max—é .20 macaw 80.3 e :8 8:58; t8 women—m8 830:0 80¢ AN: :omugmgoaou .3 3835er .m. 28H 88 chromosome B3 linked to 118 1700 (Freyre et al., 1998). Although these two markers were not linked in the present study, 88500 was significantly associated with greenhouse and field ratings suggesting that this region of chromosome B3 may also influence Fusarium root rot resistance. In close linkage with S8500 and 1181700 on the integrated P. vulgaris map (Figure 6) is PvPRl implicating another PR protein acting in response to Fusarium root rot. S8500 was also significantly associated with DTF explaining 8% of the phenotypic variation for root rot resistance. Multiple regression analyses using combinations of significant markers and their interactions revealed that epistatic interactions were not significant. Up to 14% and 29% of the phenotypic variation for greenhouse and field ratings, respectively, was explained by a set of markers including G320“), 617900, U121 500 and P7700. Interestingly, this same set of markers explained 24% of the variation for DTF. When U125.» and 017900 were eliminated because both were strongly associated with DTF, the remaining two markers G320“) and P7700 accounted for 7 to 13% of the phenotypic variation for root rot ratings in both field and greenhouse with no significant association observed With DTF. Primers GB, P7 and G6 were screened against the 68 F2 individuals from IF. RAPDS 66300 and 63300 were linked at 7.6 cM. 66300 was not a polymorphic loci in MF. 032000 was unscorable in IF, 661100 and 63300 were not associated with F45 root rot scores whereas P7700 was significantly associated (P < 0.05) with root rot ratings for this population confirming previously reported associations for this marker. 89 DISCUSSION The predominant constraint to realizing maximum yield potential in bean is disease (Kelly et al., 1998). Bean is a host to many bacterial, viral and fungal pathogens and is often used as a model system for studying host/pathogen interactions (Lamb et al., 1989). Genetic resistance to anthracnose (Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.-Scrib.), bean common mosaic virus and rust (Uromyces appendiculatus (Pers.:Pers.) Unger) are classic bean examples of Flor’s (1956) gene-for-gene disease interaction model (Christ and Groth, 1982; Kelly and Miklas, 1998). All three of these diseases involve monogenic, vertical (VR) resistance for which the knowledge base is highly advanced. Germplasm sources are well characterized, the incorporation of single- gene resistance is common practice for disease control, tightly-linked molecular markers have been identified, gene-pyramiding for more durable resistance is being conducted, and cloning of resistance genes is underway (Rivkin et al., 1999; Creusot et al., 1999). The classification of genetic resistance into discrete qualitative units for many other complex bean diseases has not, however, been as forthcoming. Vertical resistance is often criticized because of its numerous failures in the face of pathogen co—evolution (Parlevliet, 1977). A single disease resistance gene which confers immunity imposes an intense selection pressure on the pathogen in favor of novel avirulence genes which are not recognized by the host. Failure of monogenic resistance during a given growing season can have disastrous consequences. complex horizontal resistance (HR), on the other hand, appears to be more durable. Vanderplank (1963) described HR as independent of pathogen genotype or pathotype in contrast to VR as resistance. Resistance of this type is usually characterized by a continuous distribution and 90 polygenic control. Horizontal resistance is generally manifested as a reduction in disease as opposed to complete immunity which can be the result of host resistance to infection, delay of infection, reduced development of pathogen after infection or reduced reproductive capacity of the pathogen (Simmonds, 1991). Horizontal resistance is regarded as more stable than VR because the likelihood that a pathogen can defeat several resistance genes at once is minimal. Furthermore, the lack of immunity allows for pathogen reproduction in the absence of strong selective pressure (Simmonds, 1991). In practice, VR has remained a preferred form of resistance, when available, due to its relative genetic simplicity and ease of use but most breeders recognize the need for more stable, HR especially for diseases for which no VR has been documented. The inability to classify root rot scores from either greenhouse or field trials into discrete categories suggests that root rot resistance in bean is under polygenic control and resistance should be treated as a quantitative trait (Figure 4). Consistently moderate h2 over greenhouse and field experiments support the idea that improvement of genetic resistance to F.s. f.sp. phaseoli can be achieved but progress may be slow (Table 9). However, since HR is more durable than qualitative resistance, improving resistance to F .s. f.sp. phaseoli could, perhaps, provide protection against more than one pathogen if a general strengthening of host defense responses is involved (Simmonds, 1991). The disease reaction of a Fusarium resistant genotype mirrors that of a susceptible genotype in symptomotology except that disease progress in the resistant genotype is substantially reduced. An incompatible reaction involving the hypersensitive response is not observed for resistant genotypes inoculated with this pathogen. Under severe disease 91 pressure, resistance can be overcome as evidenced by root rot ratings greater than 4.0 for FR266 (Table 7). Susceptible genotypes, however, scored consistently more than the resistant parents supporting the conclusion that rate of progress of disease is substantially reduced in the resistant genotypes and that environmental factors play a large role in disease severity (Table 7; Figure 4). Fusarium solani f.sp. phaseoli infection elicits host defense responses which include the induction of phytoalexin biosynthesis, chitinase, 13,1-3 glucanase, PR proteins, phenylalanine ammonia lyase and the release of phenolic compounds (Morris and Smith, 1978; Mohr et al., 1998). The fungus, however, appears to tolerate these defense products and is known to possess enzymes for the detoxification of the phytoalexins, phaseollin and keivitone (Choi et al., 1987; Li et al., 1995). Although F.s. f.sp. phaseoli can metabolize keivitone, the fungus does not appear to induce the production of this particular phytoalexin or, alternatively, it may repress the biosynthesis of keivitone as a means for enhanced pathogenesis (Morris and Smith, 1978). Furthermore, it was demonstrated that the flavanone, naringenin, and the isoflavanone, genistein, and phytoalexins, medicarpin and maackiain, from chickpea (Cicer arietinum L.) stimulate spore germination of F.s. f.sp. phaseoli. Studies examining bean defense responses to F.s. f.sp. phaseoli, generally concern the response of cultivars highly susceptible to F .s. f.sp. phaseoli but do not document the responses of resistant genotypes. Clearly the interaction between host defenses and pathogen infection is complicated. Differences in the expression of genes involved in plant defense response may exist between genotypes and could possibly correspond to differing levels of resistance to F .s. f.sp. phaseoli. 92 In the current study, RAPD markers associated with Fusarium root rot resistance in bean were identified using a method of selective genotyping. Several regions and independent markers were identified which significantly associated with field and greenhouse root rot ratings but none explained over 15% of the phenotypic variation for this trait (Table 12). In multiple regression analyses, only 29% of the phenotypic variation could be accounted for by a subset of four markers. QTL analysis for resistance to CBB and bean golden mosaic virus (BGMV) revealed four and five QTL for BGMV and CBB, respectively (Miklas et al., 1996). A single QTL identified as associated with CBB in this study explained up to 60% of the variation for this trait while two QTL associated with BGMV accounted for as much as 60% of the variation for this resistance. In another study involving quantitative resistance to the soilbome pathogen, Macrophomina phaseolina (Tassi) Goidanich, the causal agent of ashy stem blight which infects bean roots and stems, Miklas et al (1999) identified RAPD markers associated with this trait that, individually, could account for no more than 19% of the phenotypic variation for resistance. Only three of the eight RAPDs significantly associated in the Macrophomina study were consistently significant across two locations and multiple regression analyses using four selected markers explained from 28 to 47 % of the phenotypic variation in this population. Soilbome pathogens appear to have a complex life cycle which is strongly influenced by environmental factors. The large amount of environmental variation observed for diseases caused by soilbome pathogens makes quantification difficult and could explain why QTL associated with these traits are so difficult to resolve. In the current study, markers which were significantly associated with field root rot ratings were not significantly associated with greenhouse root rot ratings and vice versa. 93 Furthermore, those RAPDs associated with field ratings tended to be associated with DTF (Table 12). These results suggest independent mechanisms involved in resistance to Fusarium root rot between natural and artificial inoculations and that natural infections may reflect differences in maturity. Data from greenhouse experiments was generally significantly and positively associated with field trials for MF except for the non-significant but positive correlation between MFGH2 and 1998 PI trial (Table 10). In the previous chapter, the perlite greenhouse screen was successfully used to discriminate between resistant and susceptible individuals and should be indicative of physiological resistance. Data from repeated greenhouse screenings of MF were positively correlated among experiments and reflect segregation of physiological resistance in this population. The observation that the same QTL associated with greenhouse ratings were not associated with field experiments and vice versa is not surprising. Genetic variation for physiological resistance present in MF may have been masked by strong environmental factors present in field trials such that QTL associated in greenhouse trials could not be resolved from field data. Furthermore, environmental effects influencing disease development present in field trials were absent in greenhouse trials and QTL associated with these factors were, therefore, not identified. An example of an environmental factor present in field trials and not in greenhouse is DTF. Greenhouse plants were rated for disease severity before flowering but field trial ratings were conducted post-bloom The significant negative relationship between DTF and field root rot ratings could also correspond to differences in developmental stages at the time of rating (Table 8). All RILs were rated at the same time regardless of development. Thus, those genotypes which were more mature also had more disease. This 94 association between maturity and disease has been observed for other pathogens (Miklas et al., 1996; Pilet, 1998; Schechert et al., 1999) and has been reported previously for F. s. f.sp. phaseoli (Abawi, 1989). A negative association between root rot resistance. and DTF is not unexpected, however, especially when considering genotypes with determinate growth habit. In these genotypes, vegetative growth ceases at flowering and the plant partitions its resources to the developing seed resulting in less available energy for secondary processes like host defense. Furthermore, subsequent root decay may tend to aggravate the pathogen through the release of nitrogenous compounds which can stimulate aggressiveness of the fungus (Toussoun and Patrick, 1970). Considering the negative correlation between DTF and root rot resistance, breeders intending to improve Fusarium root rot resistance would benefit from selection methods like greenhouse screens and marker assisted selection that avoid concomitantly increasing DTF and associated late maturity. Two linkage groups (LG2 and LG5) for which all markers were consistently associated with either greenhouse or-field ratings were identified. LGZ containing P7700, P101600 and G6noo were all significantly associated with root rot in at least one of the greenhouse trials conducted and P700 was also significantly associated with root rot ratings in the 1998 PM trial and combined analysis (Table 12). Markers on LG5 were all positively and significantly associated with field root rot ratings but not greenhouse ratings. P700 and G6uoo from LGZ are located on chromosome BZ (Figure 5) and span a region that encompasses the PvPR2 locus suggesting a role for this PR protein in Fusarium root rot resistance. PvPR2 and its counterpart PvPRl are low molecular weight acidic proteins induced during fiingal elicitation (Walter et al., 1990). These bean PR 95 proteins share similarities with PR proteins in pea, potato and parsley. The role of PvPR proteins is further confirmed by the significant association observed between 8300 from LG7 and 1181300 of LG3 which map to chromosome B3 in the region of PvPRl (Figure 6). Differences in PvPR gene arrangements were detected between anthracnose resistant and susceptible genotypes indicating that polymorphism between PvPR as well as other defense response-related genes may contribute to our understanding of HR (Walter et al., 1990). QTL associated with resistance to the late blight fungus (Phytophthora infestans (Mont) de Bary) of potato (Solanum tuberosum L.)) have also been reported to associate with two potato PR proteins (Gephardt et al., 1991). To capitalize on the assumption that defense proteins may be associated with quantitative resistance, a method of candidate gene analysis, whereby genes known to be involved in host defense response are used as markers to identify potential QTL associated with disease resistance, has been applied in other crops and could be employed for Fusarium root rot resistance in bean (Goldman et al., 1993; Causse et al., 1995; Byme et al., 1996; Paris et al., 1999). RAPDs G32ooo and P7700 were used in a multiple regression analyses, and demonstrated significant associations with all greenhouse and field trials explaining between 7 to 19% of the genetic variation for root rot scores in MF. Selection based on these markers will be useful but not sufficient to improve Fusarium root rot resistance. Neither of these markers were significantly associated with DTF. Analysis of IF F2 DNA and root rot data based on corresponding F45 RIL indicated that P700. was significantly associated with greenhouse root rot scores in this population confirming the association observed in MF. G6noo, however, was not significantly associated with root rot ratings. 632000 was unscorable in this population and G3soo was not significantly associated root 96 rot ratings. This is not surprising since G3300 was only associated with field ratings and not with greenhouse ratings. The IF population was constructed as a means to verify marker associations in a population other than the one in which the marker was identified. DNA from F2 individuals of IF was used to confirm the effectiveness of early generation selection based on these markers. The observation that the P700 allele associated with improved root rot resistance was also present in N203 and not in Seafarer, further supports the usefulness of this marker. These results indicate that P700 will be a useful marker for early generation selection for root rot resistance when using FR266 as a resistance source. One-way analyses of variance using P7700 as marker genotype for analysis against SN root rot data failed to identify significant associations. The lack of genetic variation and adequate score distribution in this population prevented the identification of significant differences between marker genotype. These results however do not preclude the usefulness of this marker for selection in populations derived from N203 provided that polymorphism between the two parents of a given population is present. The insignificant associations for P7700 in SN can be attributed to higher levels of resistance in Seafarer than was expected which ultimately contributed to the lack of adquate distribution of root rot scores in this population. Fusarium root rot in bean involves a complex interaction between host, pathogen and environment. We have concluded that resistance to this disease should be considered quantitative and approaches which can reduce environmental variation, like greenhouse marker assisted selection, may accelerate progress towards the screening and incorporation of enhanced physiological resistance. The consistently moderate h2 which 97 was observed for root rot resistance in MF indicates that progress made by conventional selection will be limited (Table 9). Heritability estimates reflect the proportion of the total phenotypic variation of a population that is inherited. An h2 of approximately 0.50, like that observed for MF, suggests that even for greenhouse evaluations a large proportion of the phenotypic variation is due to environment. It might be expected, however, that h2 derived from greenhouse experimental data should be substantially higher than field h2 for a given population since the objective for developing greenhouse evaluations was to reduce environmental variation. But, in addition to reducing environmental variation, greenhouse screening also reduced the total amount of genetic variation by eliminating the contribution made by other traits like DTF and stress tolerance to the observed levels of field resistance. Marker assisted selection using P700 and 632000 combined with later generation conventional selection has the potential to enhance progress towards incorporating Fusarium root rot resistance in dark red kidney bean genotypes. Specific applications for the use of markers in improving Fusarium root rot resistance in the dark red kidney market class are presented. The introgression of Fusarium root rot resistance into large-seeded kidney germplasm will necessarily involve the utilization of resistance sources from other market classes. As such, agronomically acceptable dark red kidney genotypes possessing root rot resistance will not be achieved through single-cross breeding approaches. Systems involving recurrent selection and backcross breeding are the preferred alternatives for the improvement of this trait. Utilizing markers and greenhouse seedling evaluations to facilitate selection provides an opportunity for the breeder to make educated decisions about which progeny should be intennated or backcrossed before crosses are performed. 98 This additional information can save the breeder valuable time and resources. Ideally, selection for progeny to be involved in subsequent backcrosses should be selected prior to flowering to ensure that superior genotypes are included. In the case of quantitative traits like Fusarium root rot resistance, phenotypic selection, performed on a single plant basis, is not reliable. In the absence of accurate selection, backcross progeny are essentially selected at random which may substantially delay progress. The RAPD markers associated with root rot resistance identified in this study provide a means to genotypically select for Fusarium resistant progeny that could then be utilized in subsequent backcrosses. The added information afforded by these markers increases the likelihood that a desirable dark red kidney genotype possessing root rot resistance can be achieved. Resistance levels of inbred backcross progeny selected based on marker genotype can then be confinned using greenhouse evaluations before the line is advanced to costly and laborious field trials. Alternatively, RAPD markers associated with QTL controlling Fusarium root rot resistance can be used to select progeny possessing complementary QTL for intennating. A genotype possessing the P7700 allele can be recombined with another genotype possessing the G320“) allele with the ultimate goal of combining these two resistance QTL into a single genotype that will possess higher levels of resistance than either genotype alone. The markers identified in this chapter that were associated with Fusarium root rot resistance, individually, explained only a small amount of variation for this trait. The usefulness of marker assisted selection involving these markers as. a substitute for conventional selection approaches is, therefore, questionable. However, these markers can be utilized to enhance conventional breeding approaches by providing information that the 99 breeder can use to make educated decisions and choices of putatively linked resistance loci. One of the initial intentions for this study was to classify the resistance present in N203 using an intra gene pool cross. The SN population was abandoned because consistent significant genetic variation for root rot resistance was not observed. It appears that Seafarer is not as susceptible as was previously thought and that small-seeded genotypes, as a whole, are not as susceptible as the large-seeded kidney beans. An overemphasis on the improvement of quality traits in the kidney and snap market classes - may be responsible for the absence of genetic resistance to F.s. f.sp. phaseoli. 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A key for identification of different growth habits of Phaseolus vulgaris L. Annu. Rep. Bean Improv. Coop. 25:92-95. Smith, PL. and ER. Houston. 1960. Root rot resistance in common beans sought in plant breeding program. California Agriculture Sept.:8. Sonnante, G., T. Stockton, R.O. Nodari, B.L. Becerra Velasquez, and P. Gepts. 1994. Evolution of genetic diversity during the domestication of common bean (Phaseolus vulgaris L.). Theor. Appl. Genet. 89:629-635. Toussoun, T.A. 1970. Nutrition and pathogenesis of Fusarium solani f.sp. phaseoli. pp 95-99. In: T.A. Toussoun, R.V. Bega and RE. Nelson’s Root Diseases and Soil- bome pathogens. University of California Press, Berkley, Los Angeles, London. Vanderplank, J.E. 1963. Plant Diseases. Epidemics and Control. Academic Press, New York and London. Wallace, DH. and RE. Wilkinson. 1965. Breeding for Fusarium root rot resistance in beans. Phytopathology 55: 1227-123 1. Wallace, DH. and RE. Wilkinson. 1966. Origin of N203 (PI 203958). Annu. Rep. Bean Improv. Coop. 9:38. Walter, M.H., J. Liu, C. Grand, C.J. Lamb and D. Hess. 1990. Bean pathogenesis-related (PR) proteins deduced from elicitor-induced transcripts are members of a ubiquitous new class of conserved PR proteins including pollen allergens. Mol. Gen. Genet. 222:353-360. 104 APPENDIX A 105 APPENDIX A GREENHOUSE PROTOCOL Table 13. A comparison of mean root rot scores of control treatment (inoculated with deionized water) and 5 different isolates isolated from Presque Isle County, MI (F.s. 12, Hawks 2a, 2b and 6d) and Huron County, MI (Huron 2a) inoculated on to three bean genotypes possessing differing levels of resistance. Isolate Root Rot Score Montcalm (S) Control 1.7 F.s. 12 4.6 Hawks 2a 3.5 Hawks 2b 5.2 Hawks 6d 4.6 Huron 2a 4.4 N203 (R) Control 1.5 F.s. 12 2.1 Hawks 2a 1.4 Hawks 2b 1.5 Hawks 6d 1.9 Huron 2a 1.9 Seafarer (D Control 1.1 F.s. 12 3.2 Hawks 2a 1.8 Hawks 2b 2.6 Hawks 6d 2.7 Huron 2a 2.9 Mean 2.7 LSD (0.05) 0.3 CV (%) 13.2 TS, susceptible; R, resistant; 1, intermediate. 106 Table 14. Analysis of variance including degrees of freedom (DF), sums of squares (SS) and error mean square (EMS) for the 24 entries planted in the 1996 Presque Isle County. MI root rot trial. ' Source DF SS EMS F Value ---- Yield ---- Rep 3 302700.545 100900. 182 Genotype 21 9147338847 435587.564 4.51*** Error 61 5889042832 96541.686 ---- 100 Seed Weight ---- Rep 3 45.219 15.073 Genotype 21 21754575 1035.93 174.24*** Error 61 362.675 5.945 ---- Root Rot Rating 51 Days After Planting---- Rep 3 1.751 0.584 Genotype 23 75.122 3.266 629*“ Error 69 35.816 0.519 ---- Root Rot Rating 70 Days After Planting-«- Rep 3 0.090 0.030 Genotype 26 201.348 8.754 2345*“ Error 69 25.756 0.373 ---- Greenhouse Screen ---- Rep 2 3.221 1.610 Genotype 23 41.827 1.801 688*“ Error 45 1 1.895 0.264 *, **; *** Significance at the P < 0.05. 0.01, 0.001 levels, respectively. 107 Table 15. Analysis of variance including degrees of freedom (DF), sums of squares (SS) and error mean square (EMS) for the twenty-four entries planted in the 1997 Presque Isle County, MI root rot trial. Source DF SS EMS F Value ---- Yield ----1‘ Rep 1 1432642942 1432642942 Genotype 20 5185207627 259260.381 3.1 1** Error 20 1667050924 83352.546 ---- 100 Seed Weight ---- Rep 1 3.149 3.149 Genotype 20 1 1805.969 590.298 512.72*** Error 20 23.023 1.151 ---- Root Rot Rating 52 Days After P1anting---- Rep 2 17.548 8.774 Genotype 20 36.950 1 .848 298“ Error 62 24.825 0.621 ---- Root Rot Rating 86 Days After Planting---- Rep 2 2.561 1.281 Genotype 20 81.811 4.091 9. 14*" Error 62 17.892 0.447 ---- Greenhouse Screen 1---- Rep 2 8.263 4.132 Genotype 20 53.977 2.699 10.48*** Error 40 10.303 0.258 ---- Greenhouse Screen 2---- Rep 2 10.730 5.365 Genotype 20 17.153 0.858 2.56“ Error 40 41.279 0.335 *, **; *** Significance at the P < 0.05. 0.01, 0.001 levels, respectively. TThe third replication’s measurements for yield and 100 seed weight were abandoned due to extreme weed pressure. 108 APPENDIX B 109 APPENDIX B GENETICS AND MARKER ANALYSIS Table 16. Analyses of variance including degrees of freedom (DF), sums of squares (SS), error mean square (EMS), F-value, experimental mean, LSD (0t < 0.05) and coefficient of variation (CV) for indivdiual field ratings performed on the Montcalm/FR266 (MF) population in Presque Isle County, MI and Perham County, MN in 1998. The analyses of variance combined over locations and calculated for all greenhouse experiments are presented in Table 6. Source DF SS EMS F Value Perham County, MN, 1998 Rep 2 0.719 0.360 Genotype 77 244.060 3. 170 1.92*** Rep*Genotype 144 237.951 1.652 1.87*** Error 693 613.857 0.886 Mean, CV, LSD 5.44 17.3 1.1 Presque Isle County, MI, 1998 Rep 3 127.233 42.410 Genotype 78 273.419 3.506 2.97*** Rep*Genotype 201 237.242 1.180 3.07*** Error 1030 395.916 0.384 Mean, CV, LSD 4.16 14.9 1.0 110 Table 17. Analyses of variance including degrees of freedom (DF), sums of squares (SS), error mean square (EMS), F-value, experimental mean, LSD (or < 0.05) and coefficient of variation (CV) for each of the two ratings (RRl and RR2) performed on the Seafarer/N203 (SN) population in Presque Isle County, MI and Perham County, MN in 1997. The analyses of variance combined over locations and calculated for all greenhouse experiments are presented in Table 6. Source DF SS EMS F Value ---- Presque Isle County, MI (RRl) ----T Rep 3 16.150 5.383 Genotype 100 37.319 0.373 1.10 Error 298 101.405 0.340 Mean, CV, LSD 1.8 33.2 0.5 0.8 ---- Presque Isle County, MI (RR2) ----T Rep 3 3.650 1.217 Genotype 100 20.140 0.201 0.81 Error 298 73.221 0.247 Mean, CV, LSD 1.7 29.9 0.7 ---- Perham County, MN (RRl) ---- Rep 2 24.518 12.259 Genotype 93 63 .479 0.683 1 .29 Error 186 98.149 0.528 Mean, CV, LSD 3.1 23.2 1.2 ----1997 Perham County, MN (RR2) ---- Rep 2 10.020 5.010 Genotype 93 126.459 1.360 3.26*** Error 186 77.472 0.417 Mean, CV, LSD 2.6 24.6 1.0 *, **; *** Significance at the P < 0.05. 0.01, 0.001 levels, respectively. 111 :. 80.0 «x 8 8.0 _x E 2 . a 03.0 0000.0 4. 020.0 0000.0 . v0 .880 a. 0x :. _x E a 33.0 x 003.0 V0 3.200 0.0 0.0 «x 3 0.0 _x P 00 = 0000.0 88.0 N_ 2 8.0 008.0 X. 86.00 00.0 00.0 «x 8.0 3.0 _x Q E = 88... 3.00.0 a. $8.0 4.20.0 v0 _ :2 4.0.. 2.... «x 3.. 8.0 _x 2 $3.0 a $8.0 «$4.0 a. 003.0 080.0 v0 +2.03 :2 8.0 08 002.. 00: 0:00: 80...: 8002 0.3. $.35 £888 00 080 080 She 0030¢ 00 0.80 .308 0005002 030 0005 00>0 00.2050 8588 as 032.: z: .0550 828 05 02.0 .2 .0550 02 08$... 5 B888 was 22.. 23 .5802 .NEOn—E 4:936 0:088:80 0000:0008 00.80 80¢ 00002—00 800 00.— 0000 .080? 00:08; ¢0 0000800 83000 9:00 008800000 0000200000 8005080 :0 00¢ A93 083 92% 05 80000000 0800368 ¢0 anew 05 ¢0 0008 080 :5 as 05 mac—00. 0302308 ¢0 009% 05 ¢0 0008 55 0.002308 ¢0 .0088 AN: 088885000 .«0 8005000 52530.5 .3 030,—. 112 00.0 00.0 0.00 .00 00.0 0.00 00 00 0000.0 .0000 0000.0 0000.0 0000.0 0000.0 2.00 00.0 00.0 00 0000.0 38.0 8. .00 .00. 0.00 00.0 00.0 0.0. . 0.00 00.0 .00 00 00 00 00 0000.0 0000.0 0000.0 0000.0 0000.0 00.0.0 0.00.0 0000.0 8.00 0.00. 00.0 00.0 0..0 0.0. . 00.0 00.0 00.0 00 00 00 00 000.0 000.0 0000.0 00. ..0 00.0.0 0.00.0 0000.0 0000.0 8200 00.0 .00 0.00 00.0 00.0 0.00 00 00 00 00 .0000 0000.0 0000.0 0000.0 0000.0 0000.0 0000.0 00.0.0 820.0 0.00 .00 00.0 00.000 and. 0.9m 2.00 2.0... 0. #52 0000.0 0000.0 ”Wk.“ Ikk as: 0... )1/ 8. / 50... /s 0.3. 0.0.0 .0. 000 113 0.00 .0.0 «x 0. .0 00.0 .X 00 00 .0 0000.0 0000.0 0. :00... 0000.0 . 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