GREENHOUSE EVALUATION OF SOYBEAN FOR RESISTANCE TO SCLEROTINIA STEM ROT AND QUANTITATIVE TRAIT LOCI STUDY IN RECOMBINANT INBRED LINES By Ramkrishna Kandel A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Plant Breeding, Genetics, and Biotechnology 2011 ABSTRACT GREENHOUSE EVALUATION OF SOYBEAN FOR RESISTANCE TO SCLEROTINIA STEM ROT AND QUANTITATIVE TRAIT LOCI STUDY IN RECOMBINANT INBRED LINES By Ramkrishna Kandel Sclerotinia stem rot [caused by Sclerotinia sclerotiorum (Lib) de Bary] is an economically important disease of soybean [Glycine max (L.) Merr] and no soybean cultivars show complete resistance to the disease. To screen soybean cultivars and lines for resistance to this disease, three related but independent studies were conducted in the greenhouse and laboratory. In the first study, 392 F4:6 recombinant inbred lines (RILs) from seven populations were evaluated for resistance to S. sclerotiorum by drop- and spray-mycelium methods under the greenhouse conditions. Individual lines in two of seven populations evaluated by drop-mycelium method were significantly different (P<0.0500). Parental polymorphism was tested with 132 simple sequence repeat (SSR) markers associated with Sclerotinia stem rot resistance in other studies and 97 polymorphic markers were used to test the progenies from the seven populations. Sixteen markers showed high correlations with the phenotypic data in the seven populations. In the second study, 66 plant introductions (PIs) were evaluated with the drop-mycelium method and significant (P < 0.0050) differences were found among the PIs for resistance to Sclerotinia stem rot. In the third study, drop-mycelium, spray-mycelium, and field evaluation methods were compared in terms of correlation of the data. The data from drop-mycelium inoculation had 2 strong correlations with that from spray-mycelium (R = 0.63, P< 0.0005) and field evaluations 2 (R = 0.40, P<0.0381) for resistance to Sclerotinia stem rot. ACKNOWLEDGMENTS I am very indebted to my major professor Dr. Dechun Wang for his constant academic guidance, financial support, and editorial assistance. I would like to appreciate my committee members Dr. James D. Kelly and Dr. Jianjun Hao for their thoughtful reviews, comments, and advice on my thesis. I would also like to thank Guorong Zhang, Menghan Liu, Cherry Wang, and the whole soybean lab at Michigan State University, East Lansing for their constant guidance and support during the course of my study. I would like to express my gratitude to all my friends and my family members for their constant support and love. iii TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................... v LIST OF FIGURES ...................................................................................................................... vii CHAPTER ONE: STUTY OF QUANTITATIVE TRAIT LOCI IN SOYBEAN FOR RESISTANCE TO SCLEROTINIA STEM ROT ................................................................. 1 ABSTRACT… ................................................................................................................................ 2 INTRODUCTION .......................................................................................................................... 3 MATERIALS AND METHODS .................................................................................................. 11 Phenotypic Analysis............................................................................................................ 11 Drop-mycelium method ...................................................................................................... 11 Inoculum preparation .......................................................................................................... 12 Spray-mycelium method .................................................................................................. 13 Tissue sampling and DNA extraction and SSR marker genotyping .................................... 14 RESULTS AND DISCUSSION ................................................................................................... 15 Drop-mycelium method ....................................................................................................... 15 Spray-mycelium method ...................................................................................................... 15 Genotypic result ................................................................................................................... 18 REFERENCES ............................................................................................................................. 37 CHAPTER TWO: GREENHOUSE SCREENING OF SOYBEAN GENOTYPES AND PLANT INTRODUCTIONS FOR RESISTANCE TO SCLEROTINIA STEM ROT ..................... 47 ABSTRACT… .............................................................................................................................. 48 INTRODUCTION ........................................................................................................................ 48 MATERIALS AND METHODS .................................................................................................. 53 RESULTS…… ............................................................................................................................. 55 DISCUSSION .. ………………………………………………………………………………….57 APPENDIX…. .............................................................................................................................. 69 REFERENCES ............................................................................................................................. 87 iv LIST OF TABLES Table 1.1- Listing of crosses used in this study and number of lines in each cross ..................... 23 Table 1.2- List of crosses and major events carried out in the greenhouse(drop-mycelium).…..24 Table 1.3- Complete list of major events occurring during screening of the seven populations (spray-mycelium method) ............................................................................................................. 24 Table 1.4- SAS GLM output for the seven populations evaluated by drop-mycelium method ... 25 Table 1.5- Results of variance analysis and broad-sense heritability estimates for resistance to sclerotinia stem rot ........................................................................................................................ 26 Table 1.6-Difference between lines in populations 1 and 7 shown by least significant difference (LSD) ............................................................................................................................................ 26 Table 1.7-Difference between lines within the seven populations by spray-mycelium method . 27 Table 1.8- Significant difference within lines of the 17 December, 2007 evaluation ................. 28 Table 1.9-Reported QTLs associated with partial resistance to Sclerotinia stem rot in soybean. 29 Table 1.10-List of polymorphic SSR markers across seven populations and their respective linkage groups ............................................................................................................................... 35 Table 2.1- Thirty-five soybean genotypes and their reactions to Sclerotinia stem rot (spraymycelium) ..................................................................................................................................... 61 Table 2.2- Reactions of 35 soybean genotypes to different method of inoculations ................... 63 Table 2.3- Pearson Correlation Coefficients, N = 26 Prob > |r| under H0: Rho=0, for 35 genotypes ...................................................................................................................................... 65 Table 2.4- Sixty-six soybean plant introductions and their reactions to Sclerotinia stem rot (dropmycelium method) ........................................................................................................................ 66 Table 2.5- Significant differences among the plant introductions screened for resistance to Sclerotinia stem rot (2008 and 2009 data) .................................................................................... 68 Table 2.6- Significan difference betweenplant introductions shown by least significant difference (LSD) test..................................................................................................................... 68 Table 2.7- Broad-sense heritabilities for 66 plant introductions evaluated in 2008 and 2009..... 69 Table A1- Sixty-six plant introductions and their maturity group, origin, and survival rate for 2009 greenhouse evaluation .......................................................................................................... 72 v Table A2- Sixty-six soybean plant introductions and their maturity groups, origins, and reactions to sclerotinia stem rot evaluated in 2008 ...................................................................................... 75 Table A3-Plant mortality for 392 lines evaluated by two different methods ............................... 79 vi LIST OF FIGURES Figure 1.1- Survival distributions of the seven populations obtained from spray-mycelium method. Populations 4, 5, and 6 represent both non-cup and cup modification results................ 19 Figure 1.2- SSR markers polymorphic and non-polymorphic in different parents ..................... 22 Figure 1.3- Polymorphic markers showing polymorphism in population 3. ............................... 22 Figure 2.1- Soybean plants before (a) and after (b) inoculation with S. sclerotiorum.......................56 Figure 2.2- Different levels of resistance shown by the 66 PIs in 2009 and 2008 greenhouse evaluations .................................................................................................................................... 59 Figure 2.3- Plant mortality for the 35 soybean genotypes evaluated in different environments.. 60 vii CHAPTER ONE STUTY OF QUANTITAT IVE TRAIT LOCI IN SOYBEAN FOR RESISTANCE TO SC LEROT INIA STEM ROT 1 ABSTRACT Sclerotinia stem rot [caused by Sclerotinia sclerotiorum (Lib) de Bary] is considered an economically important disease of soybean [Glycine max (L.) Merr]. Some soybean cultivars show partial resistance to Sclerotinia stem rot but no complete resistance to Sclerotinia stem rot has been reported. The objectives of this study were to evaluate seven populations for resistance to Sclerotinia stem rot under the greenhouse condition and validate the quantitative trait loci (QTLs) associated with Sclerotinia stem rot resistance in soybean. Seven populations with a total of 392 recombinant inbred lines (RILs) of soybean were developed by crossing Skylla, a partial resistant cultivar, and E00290, a susceptible cultivar with five plant introductions (PIs): PI 089001, PI 153259, PI 437764, PI 548404, and PI 548312 that exhibit partial resistance to Sclerotinia stem rot. The 392 F4:6 RILs from the seven populations were evaluated for resistance to S. sclerotiorum by drop and spray-mycelium methods in the greenhouse conditions. Individual lines in populations one and seven were significantly different (P<0.0235 and P<0.0019, respectively) in levels of resistance obtained with the drop-mycelium method. Parental polymorphism was tested with 132 simple sequence repeat (SSR) markers associated with Sclerotinia stem rot resistance found in previous studies and 97 polymorphic markers were used to screen the progenies from the seven populations. Sixteen markers were identified to highly correlate with phenotypic data in the seven populations. Markers such as Sat_267, Satt651, Satt571, Satt619, and Satt475 showed significant correlations in more than one population. Satt494, Satt154, Satt197, Satt481, Satt394, Satt197, Satt243, Satt153, Satt478, and Satt691 markers were significant in individual populations. 2 INTRODUCTION Soybean, [Glycine max (L.) Merr.] is the second most important crop in terms of area and production in the United States (US). It belongs to the genus, Glycine, which is divided into two subgenera; Glycine and Soja. The subgenus Soja, include the cultivated soybean, G. max, and the wild progenitor of G. max, G. soja. G.max and G. soja are cross-compatible. Sclerotinia stem rot, caused by necrotrophic homothallic fungal pathogen, Sclerotinia sclerotiorum (Lib.) De Bary, is a major soybean disease in the north-central areas of the United States (Hartman et al., 1998). Sclerotinia stem rot was first found in the US in 1946 and reported in 1951 but outbreaks of the disease became more frequent and more severe after 1990s (Yang et al., 1999). S. sclerotiorum is capable of colonizing over 400 species of plants including soybean (Boland and Hall, 1994). The pathogen requires wet soil and canopy conditions at flowering for infections to occur (Grau, 1988). Sclerotia are the primary long-term survival structures and play a major role in disease cycle (Willets and Wong, 1980). Sclerotia germinate carpogenically or myceliogenically depending on environmental conditions. Myceliogenic germination of sclerotia produces mycelia that can directly attack plant tissue (Le Tourneau, 1979) while carpogenic germination produces apothecia and subsequently ascospores (Bardin and Huang, 2001). Airborne ascospores are the primary inoculums for disease development and senescent flowers are the primary infection sites (Cline and Jacobsen, 1983; Abawi and Grogan, 1979). Infection of soybean plants occurs during the reproductive phase of soybean plant growth. Ascospores that land on flower petals germinate when free water is present on plant surfaces, utilizing the petal as a nutrient base (Kurle et al., 2001). Infection starts with colonization of petals and mycelium 3 spreads to pods, nodes, and stems and may result in premature plant death (Grau and Radke, 1984). The typical foliar symptoms of Sclerotinia stem rot include necrotic leaves, lesions on stem and pods, white fluffy mycelia, and black sclerotia present on the plant surface and internally in the stems and pods (Chen and Wang, 2005). Sclerotinia stem rot caused estimated yield loss of 235 kg/ha (Chun et al., 1987) and 147 to 370 kg ha-1 (2-5 bu acre-1) for every 10% increase in disease severity, depending on the environment and cultivar (Grau et al., 1982). Hoffman et al. (1998) reported that Sclerotinia stem rot of soybean caused a significant reduction in seed size, seed oil content, seed germination, and seed quality. Sclerotia are often harvested inadvertently along with the seed and can cause reduced seed quality as well as broader distribution of the pathogen (Grau et al., 2004; Danielson et al., 2004). Sclerotinia stem rot can cause as much yield loss as soybean cyst nematode ( Heterodera glycines Ichinohe) and Phytophthora root and stem rot ( Phytophthora sojae Kauffman and Gerdemann) when environmental conditions are conducive (Grau et al., 2004; Arahana et al., 2001). Sclerotinia stem rot ranks fifth after Soybean Cyst Nematode, Phytophthora root rot, seedling diseases, and brown stem rot (Wrather and Koernning, 2006). Sclerotinia stem rot in soybean is difficult to control due to pathogen’s wide host range in combination with its persistent resting structures, sclerotia (Phillips, 1989). Solarization reduced the populations of S. sclerotiorum and ability of the surviving sclerotia significantly at 10 and 15 cm depths (Philips, 1990). The prevalence of Sclerotinia stem rot was less in no-till than in minimum-till or conventional-till fields. In addition, the prevalence was greater in minimum-till than in conventional-till fields (Workneh and Yang, 2000). Cultural practices like use of narrow row spacing, higher plant density, and optimal fertilizer application create a dense plant canopy, 4 which in turn favors high humidity leading to fungal infection and disease outbreak (Mueller et al., 2004). Crop management practices such as use of clean seeds, early planting date, soil tillage, and adjustment of row width and plant density contribute to a reduction in Sclerotinia stem rot severity, but the effectiveness of these measures can be very limited (Steadman, 1979; Muller et al., 2002). These management practices recommended for controlling the Sclerotinia stem rot in soybean were found to be ineffective and contrary to the high yield potential of soybean (Kim and Diers, 2000). The widespread occurrence of Sclerotinia stem rot is due to changes in management practices, planting susceptible germplasm, and weather conditions that favor disease development (Kurle et al., 2001). Dann et al. (1999) found significant reductions in disease severity after treatment of soybean plants with lactofen at the R1 growth stage, and yields were higher after treatment with 0.07 and 0.11 kg a.i. ha-1 lactofen compared with water control. Foliar-applied fungicide benomyl aids in the control of Sclerotinia stem rot in dry beans when applied at 10 percent bloom, but this practice has not been thoroughly tested in soybeans (Scott et al., 1998). Benomyl, thiophanate methyl, and vinclozolin applied to soybean seedlings at V2 growth stage in greenhouse condition prevented S. sclerotiorum from expressing symptoms or signs on leaf tissue. Vinclozolin was the most effective in inhibiting S. sclerotiorum mycelia growth at 1.0 µg a.i. ml of potato dextrose agar (Mueller et al., 2004). The disease pressure must be sufficient to justify the application of fungicides indicating a little value in applying fungicides when fewer than 25 percent of the plants become infected (Venete, 1998). The effectiveness of fungicides to control Sclerotinia stem rot in soybean has been shown to be inconsistent (Grau et al., 1994; Mueller et al., 2002), due to difficulties in achieving good coverage with fungicides and timing of application with regard to ascospore release (Hunter et al., 1978; Steadman, 1979). Chemical 5 control is not economically viable for controlling Sclerotinia stem rot of soybean due to the requirement of many preventative and systemic treatments (Mueller et al., 2004). Oxalic acid is the main pathogenic factor of S. sclerotiorum (Cessna et al., 2000). Soybean plants inserted with transgene that produces oxalate oxidase (oxalic acid degrading enzyme) showed disease severity index (DSI) as low as resistant commercial cultivars, and in addition, showed very low DSI as compared to non-transgenic line in fields infested with S. sclerotiorum (Cober et al., 2003). Soybean plants transformed with a wheat germin gene (gf-2.8) greatly reduced the Sclerotinia stem rot, providing evidence that wheat germin gene (gf-2.8) degrades oxalic acid produced by S. sclerotiorum (Donaldson et al., 2001). Livingstone et al. (2005) transformed peanut plants with a barley oxalate oxidase gene. Transgenic peanut plants reduced the lesion size by 75% to 97% compared to non-transgenic plants, providing evidence that oxalate oxidase can confer resistance to Sclerotinia blight in peanut. Hu et al. (2003) found that sunflower plants transformed with a wheat OXO gene exhibited enhanced resistance against S. sclerotiorum. Dias et al. (2006) transformed lettuce (Lactuca sativa) with decarboxylase gene (oxdc) isolated from a Flammulina sp. The transgenic lettuce plants either had no symptoms or had slow disease development in comparison with a non-transgenic control line for resistance to S. sclerotiorum. However, transgenes have the potential risk of escaping into the environments (Burke and Rieseberg, 2003). Host resistance is the most economical and long-term strategy for controlling the Sclerotinia stem rot in soybean (Grau et al., 1982). But Current sources of resistance to Sclerotinia stem rot show only partial resistance, and are limited in number within soybean germplasm (Hoffman et al., 1998). Other researchers also have reported that soybean accessions 6 and cultivars do not show complete resistance to Sclerotinia stem rot (Hartman et al., 2000; Hoffman et al., 2002; Kim et al., 1999) but show partial resistance in the field, greenhouse (Nelson et al., 1991), and growth room evaluations (Boland and Hall, 1986). Use of partial resistance varieties is the most effective way to enhance the yield of soybean (Kim and Diers, 2000). Partial resistance to S. sclerotiorum is inherited as a quantitative trait in soybean (Kim and Diers, 2000; Vuong et al., 2008) and common bean (Miklas et al., 2004). Hoffman et al. (1999) suggested that inheritance of partial resistance is controlled by single recessive allele. Kim and Diers (2000) suggested a multi-locus model to define the genetics of soybean cultivars for showing differential susceptibility to Sclerotinia stem rot. Mestries et al. (1998) found that resistance to S. sclerotiorum in sunflower was polygenic and complex. Arahana et al. (2001) argues that genetic complexity of the trait and the variability in disease development in field evaluations make it difficult for breeding resistance to Sclerotinia stem rot. Partial resistance to S. sclerotiorum is composed of physiological resistance and disease escape mechanism in the field evaluations (Kim and Diers, 2000; Rousseau et al., 2004). Planting cultivars that are physiologically resistant to Sclerotinia stem rot is the most effective way to manage the disease due to difficulties in controlling the environmental conditions (Kurle et al., 2001). Molecular markers are powerful tools for breeders to find new sources of resistant QTLs or alleles (Song et al., 2004). Rongwen et al. (1995) argues that morphological and pigmentation markers have limited potential to distinguish the uniqueness of new soybean cultivars. SSR markers, composed of tandemly repeated 2-5 base pair DNA sequences, have flanking DNA sequences that are generally conserved allowing the selection of polymerase chain reaction (PCR) primers which amplify the SSR markers. Akkaya et al. (1992) reported that SSR markers are abundant and highly polymorphic in soybean. One soybean SSR locus has as many as 23 7 alleles, which provides the evidence of high level of polymorphism shown by SSR markers that helps in dissecting genetics of soybean (Cregan et al., 1994) and defining linkage group homology across mapping populations unambiguously (Cregan et al., 1999). Miklas et al. (2000) states that breeding for genetic resistance is complex since it is conditioned by both physiological and avoidance mechanisms. Thus developing varieties with partial resistance to S. sclerotiorum is a major goal of soybean breeding programs. Quantitative trait loci (QTLs) are the parts of DNA that are closely linked to the genes that underlie a quantitative trait. Quantitative trait loci analysis is a statistical method that attempts to explain genetic basis of complex traits (Lynch and Walsh, 1998). There have been many studies to identify the QTLs associated with resistance to Sclerotinia stem rot in soybean germplasm. Low lignin concentration in the stem of soybean is positively correlated with lower disease severity and suggested that stem lignin concentration can be used as a biological marker for selection of soybean lines for resistance to Sclerotinia stem rot (Peltier et al., 2009). Open plant architecture, early maturity, and upright architecture of the soybean cultivars caused inconsistent disease ratings in the field (Kim et al., 2000). But reactions of soybean to Sclerotinia stem rot in the greenhouse or laboratory evaluations are due to physiological resistance with little chance of escape mechanisms (Grau and Bissionette, 1974; Nelson et al., 1991). Arahana et al. (2001) identified twenty-eight putative QTLs that confer partial resistance to Sclerotinia stem rot in five RIL populations encompassing 15 linkage groups but the amount of phenotypic variation explained by each QTL was less than 10%. Kim and Diers (1999) discovered three QTLs in 152 F3- derived soybean lines developed from a cross between a partially resistant cultivar, NKS19-90, and a susceptible cultivar, Williams 82, associated with 8 resistance to Sclerotinia stem rot with each QTL explaining less than 10% of the total phenotypic variation. Li et al. (2010) reported three QTLs on two linkage groups associated with partial resistance to Sclerotinia stem rot, each QTL explaining less than 16% of the total phenotypic variation. Guo et al. (2008) reported seven QTLs associated with resistance to S. sclerotiorum in two PIs 391589A and 391589B. Vuong et al. (2008) identified four QTLs on four linkage groups (LGs A2, B2, K, and L) associated with resistance to Sclerotinia stem rot, each QTL explaining less than 13% phenotypic variation. Quantitative trait loci mapped on LG A2 is located 12 cM from a QTL reported by Han et al. (2007) in the patent application. Huynh et al. (2010) identified three QTLs on two different linkage groups (LGs C2 and I) of soybean associated with resistance to Sclerotinia stem rot. Guo et al. (2008) argues that favorable alleles of QTLs identified in different studies that are associated with resistance to Sclerotinia stem rot can be used for resistance gene pyramiding. Diers et al. (2006) derived Soybean cultivar AxN-1-55 from a cross of two partially resistant cultivars Asgrow A2506 and NKS-1990. AxN-1-55 had lower disease ratings than A2506 or S19-90. Wang et al. (2006) developed a cultivar Skylla, partially resistant to Sclerotinia stem rot from the cross Dairylan ‘DSR-217’ x NKS19-90. Skylla was developed by advancing F1 plants to F4 using single-seed descent. It had disease severity index (DSI) ratings lower than resistant check cultivar NKS19-90 and higher than Dwight, a susceptible cultivar. Soybean plant introductions (PIs) are mostly used as sources of pest resistance in backcrossing breeding programs, but not as sources of genes for yield improvement programs. Over half of the genetic base of North American soybeans is derived from less than fifty plant 9 introductions (Delannay et. al., 1983). Shands and Wiesner (1991) pointed out that germplasm in major crops have been primarily used to identify single gene sources of resistance to diseases and insects or tolerance to abiotic stresses. In addition, germplasm have been introgressed to increase the genetic base and variability in adapted cultivars. A study has shown a linear increase in yield as the percentage of germplasm from PIs decrease in intermated populations, but greatest amount of genetic variability for yield was observed when the intermated populations had fifty percent PI germplasm (Schoener and Fehr, 1979). PIs of soybean may enhance crop genetics for yield improvement (Thorne and Fehr, 1970; Vello et al., 1984). Soybean germplasm collection may be a rich source of alternative alleles (Li et al., 2008). About 6,520 soybean PIs from maturity group 0 to IV were evaluated for resistance to Sclerotinia stem rot in the US and Canada both in the field and greenhouse conditions. Only sixty-eight PIs were selected as partially resistant PIs based on their reactions to S. sclerotiorum (Hoffman et al., 2002). Reactions of soybean cultivars to Sclerotinia stem rot in the field conditions are confounded by escape mechanisms posing difficulties in identifying physiological resistance (Boland and Hall, 1987) whereas reactions in the controlled conditions are largely due to physiological resistance (Nelson et al., 1991). Since the environment has a large role in the development of Sclerotinia stem rot in soybean, it is very important to control the environment when attempting to map QTL associated with physiological resistance (Kim and Diers, 2000; Vuong et al., 2008). Different inoculation methods have been developed to screen soybean cultivars for resistance to Sclerotinia stem rot in a greenhouse or laboratory including; cotyledon inoculation (Grau and Bissonette, 1974; Kull et al., 2003), excised stem or detached leaf assay (Chun et al., 1987, Steadman et al., 2001; Wegulo et al., 1997), cut-stem inoculation (Kull et al., 2003; Vuong et al., 2003), cut-petiole inoculation (del Rio et al., 2001), and drop- and spray10 mycelium method (Chen and Wang, 2005). The reaction of soybean cultivars to Sclerotinia stem rot showed significant correlation between greenhouse and field data (Kim et al., 2000). Chen and Wang (2005) suggested that drop and spray-mycelium are non-destructive, low cost, and efficient methods for evaluation of soybean germplasm and breeding lines for resistance to Sclerotinia stem rot in greenhouse or controlled conditions. Skylla, a partially resistant cultivar (Wang et al., 2006) and E00290, a susceptible cultivar, were crossed with 5 partially resistant Plant Introductions (PI 089001, PI 153259, PI 437764, PI 548404, and PI 548312) from Hoffman et al. (2002) to derive seven populations (Table 1) with a total of 392 recombinant inbred lines (RILs) and they were evaluated in the greenhouse for resistance to S. sclerotiorum. The locations of QTLs in these five PIs are crucial for future soybean breeding programs. If we could locate the position of QTLs in these sources, that knowledge can be used for pyramiding resistance genes in developing soybean cultivars with high level of resistance to Sclerotinia stem rot. If the QTLs in these resistance sources are not co-localized with any reported QTLs, it should carry new resistant QTLs. Our objectives were to a) evaluate a total of 392 RILs in greenhouse for resistance to Sclerotinia stem rot and b) validate the already reported QTLs associated with resistance to Sclerotinia stem rot in these PIs. MATERIALS AND METHODS PHENOTYPIC ANALYSIS DROP-MYCELIUM METHOD Seven soybeans F4:6 RIL populations were evaluated in the greenhouse conditions by drop-mycelium method as described by Chen and Wang (2005) for resistance to S. sclerotiorum. 11 A total of 392 lines were planted with a resistant check (NKS19-90) and a susceptible check (Olympus) in different dates (Table 2). Six seeds were planted in each 10cm x 10cm x 15cm plastic pot. The pots were arranged in a randomized complete block design with 3 replications in each population. Clear plastic 32-ounce PET cups with the bottoms removed were put upside down over each pot to keep the plant upright. Plants were allowed to germinate and reach to V-3 growth stage before inoculation was carried out. When the plant mortality of susceptible (Olympus) check was about 100%, data collection was performed. INOCULUM PREPARATION Fungal inoculums were prepared from the sclerotia obtained from the previous year. The sclerotia were surface-sterilized with 10% bleach. Sterilized sclerotia were grown in potato dextrose agar (PDA) medium for 3-4 days. The mycelia on the PDA plates were cut in small pieces and transferred into liquid potato dextrose broth medium. To facilitate quick and even mycelial growth, the liquid medium was shaken by a G10 GYROTORY shaker for 96 hours. The mycelium suspension was homogenized by blending in a household blender. The mycelium suspension (approximately 1 ml) was applied at the unfolded trifoliate leaves at V3 growth stage. The misting chamber was equipped with humidifiers, which constantly provided almost 100% humidity required for disease development. Seven to ten days after inoculation when the susceptible checks had a mortality of over 80%, the total number of dead plants per pot for each line was counted and plant mortality rate was calculated as follows; Plant mortality (PM) = number of dead plants/ total number of plants in pot The PROC GLM procedure of SAS (SAS, 2008) was used to calculate the significant difference between lines within the population. The broad-sense heritability for significantly 12 different populations was calculated with the variance component method described by Fehr (1987). The variance components were estimated with PROC GLM of SAS (SAS Institute, Cary, NC) using the statistical model: Yij = µ + Gi + Rj + GRij + ε ij where Yij is the observed th phenotypic value of ith genotype ( i = 1...., 59, and 324….., 392) in j replication ( j = 1, 2, 3), µ is the overall mean, Gi is the effect of genotype, Rj is the effect of replication, GRij is the interaction of genotype by replication, ε ij is the plant-to-plant variation within the replication. SPRAY-MYCELIUM METHOD For spray-mycelium method, the 392 lines and the resistant (NKS19-90) and susceptible (Olympus) checks were planted in 2 replications (Table 1.3). Each line had two pots in two replications. Six seeds per line were planted in 10cm x 10cm x 15 cm plastic pots filled with Baccto porous potting mix. Planting, spraying, and data collection dates are found in Table 1.2. Plants were inoculated at V3 growth stage. In order to keep plant upright in the pots, 32 ounce clear plastic PET cups with the bottoms removed were placed upside down over all pots. The pots were arranged in randomized complete block design. Two semi-opaque plastic chambers housed the two benches containing pots. The chambers remained open until inoculation. Each chamber had two humidifiers at the end of bench. Humidifiers were set to a 2-minute on, 3 minute off regime 24 hours a day. Inoculum was prepared with the same methodology as in drop-mycelium method. But inoculum suspension was applied by a battery operated hand sprayer. Plant mortality data were collected on day 14 after inoculation. 13 TISSUE SAMPLING, DNA EXTRACTION AND SSR MARKER GENOTYPING Tender leaves from 392 lines were collected and stored at -80 degree Celsius for two days before lypholization. The lyophilized tissue was ground by vigorous shaking with glass beads in 15-ml tubes with a paint shaker. The DNA was extracted with the CTAB (hexadecyltrimethyl ammonium bromide) method as described by Kisha et al. (1997) and the DNA concentration was measured with a ND-1000 Spectrophotometer (NanoDrop Technologies, Inc, Wilmington, Delware). The PCR was performed in MJ TetradTM thermal cycler (MJ Research, Waltham, MA). the PCR products were separated on 6% non-denaturing polyacrylamide gels using an electrophoresis unit DASG-400-50 (C.B.S. Scientific Co. DelMar, CA) as described by Wang et al. (2003). Ethidium bromide was used to stain the gel and PCR products were visualized under UV light, and photographed. A total of 132 simple sequence repeat (SSR) primer pairs (SOYBASE) were selected for the parental polymorphism flanking already reported 33 QTLs from the integrated soybean linkage map (Song et al., 2004; Choi et al., 2007). Genotyping with SSR markers was carried out as described by Wang et al. (2003). These SSR markers (Table 9) were tested for polymorphism between seven parental combinations and about 97 polymorphic markers were scored on the seven populations. For each polymorphic marker, the DNA bands of each RIL were scored as ‘a’, ‘b’ or ‘h’, where ‘a’ means only band of the resistant parent present, ‘b’ means only band of the susceptible parent present, and ‘h’ means band of the both parents present. Polymerase Chain Reactions (PCR) were performed for DNA amplification. The populations as shown in the Table 1.1 were genotyped with polymorphic SSR markers from regions containing 32 reported QTLs. The selected SSR were tested with the 14 parent DNA of each population for polymorphism according to Wang et al. (2003). The markers which show polymorphism between the two parents were then used to genotype the entire populations (Appendix, Table 3). The phenotypic data obtained from the greenhouse experiment were analyzed with the genotypic data obtained from marker analysis to determine if the DNA markers are associated with resistance to the disease in these seven populations. Single marker analysis was carried out to determine the marker-resistance association. RESULTS AND DISCUSSION DROP-MYCELIUM METHOD Among the seven populations studied, soybean lines in population one and population seven showed significant difference (P< 0.0235 and P<0.0019 respectively) among one another (Table 1.6). Soybean lines in the other populations were not significantly different. The mortality rate of soybean lines in seven populations varied from 0 to 100 percent. NKS 19-90 and Olympus were used as resistant and susceptible checks, respectively. Average plant mortality for seven populations ranged from 18.7 % to 57.4 %. The variation in plant mortality among the populations is expected due to differences in genetic contributions by different parents, different planting dates, and varying ambient temperature. SPRAY-MYCELIUM METHOD For the spray-mycelium method, the effect of evaluation date, individual population, and individual line were accounted. Evaluation date and population were significant (P<0.001). Individual lines had no significance (P<0.2633). The significance in evaluation date signifies that 15 there was marked difference in the percentage of survival between evaluation dates. Since each evaluation date had different populations, this difference was as expected. A significant difference in survival between populations is also expected due to different genetic backgrounds of their parents. Plant mortality distributions of all populations are displayed in the appendix. Average plant mortality for individual lines ranged from 29.1% in population 7 to 79.8% in population 3. Variation in mortality distribution is show in Fig. 1.2. Populations 4, 5, and 6 were evaluated twice, once with cup modification 25 February evaluation, represented by bars in charts) and once without the cup modification (17 December evaluation, represented by the line in charts) shown in Fig. 1.3. Plants fell over after sprayed allowing a more aggressive spread of Sclerotinia stem rot since infections occurred in multiple places on the plant in without cup modification evaluation. Data collection was performed at ten days after inoculation because disease developed rapidly and caused plants to die early. Since true resistance response elicited by the plant is better measured if the disease progressed downward from infection point, we used cup to keep the plant upright. The rest of the evaluations were carried out by cup modifications. Evaluation with the cup modification was done fourteen days after inoculation as described by Chen and Wang (2005). Further analysis (Table 1.7) was performed to look at the variance between replications of a single population in only one evaluation date. Under this analysis, populations 2 and 4 in the Dec, 2007 evaluation were significant for survival variation (P<.0189, P<.0212 respectively). Since the lines were derived from same parents, the variation is explained by the effects of the genotype. These populations were also grouped by the Duncan method to determine the division of significant differences between lines (Table 1.8). Since all of the lines of a population were not planted during the same evaluation date. This explains why only twenty lines in population 2 and eight lines in population 4 considered in the Duncan groupings. Seven 16 of the twenty lines in population 2 were not significantly different and only two groups had individuals that were independently significant from all other groups. Population 4 had one line that was significantly different than the remaining seven lines. It is interesting to note that the without cup evaluation is the only one giving significant difference between lines. This further provides evidence that allowing the infection to grow for 14 days is too long to get good results. The spray-mycelium method is effective to identify the significant differences between lines but it has some demerits too. During the spraying process, pieces of mycelia often clogged the spray nozzle, increasing the time to apply the inoculums. Because there was high error rate with this method, additional studies should be conducted taking the data multiple times throughout the disease growth period to determine the best growth period. Number of plants with higher plant mortality is more for spray-mycelium method (Fig. 1.2). Plant mortality is normally or near normally distributed for populations 2, 4, 5, and 6. Variation in plant mortality distribution for drop-mycelium method is less than spray-mycelium method. This also indicates that drop-mycelium method is more uniform and more reliable. Kim and Diers (2000) estimated the broad-sense heritability of Sclerotinia stem rot resistance at 0.59 in a 152 F3- derived lines from S19-90 crossed with Williams 82. Miklas and Grafton (1992) estimated the broad-sense heritability in three populations of common bean for resistance to Sclerotinia stem rot that ranged from 0.58 to 0.77. The broad-sense heritability in our populations 1 and 7 were 0.59 and 0.60, respectively (Table 1.5). Grau et al. (1982) concluded that field resistance to soybean Sclerotinia stem rot is a heritable trait. Previous studies of broad-sense heritability of Sclerotinia stem rot resistance trait in soybean along with our study suggest that partial resistance to Sclerotinia stem rot in soybean is heritable trait. 17 GENOTYPIC RESULT Altogether 97 SSR markers (Table 1. 10) were polymorphic for seven populations covering 15 linkage groups of soybean consensus map (Song et al., 2004). A total of 5 markers were polymorphic across all seven populations. Some markers are polymorphic in two or more populations. Since there were few markers per population per linkage group, increasing the marker density in + 20 cM region of the polymorphic markers would help better construct the linkage map and detect the QTLs associated with it. The correlation coefficients between population and polymorphic markers identified in this study are depicted in the Table 1.8. It is difficult to conclude whether seven populations used in this study possess the already reported QTLs, but this study gave us some insights on which region of chromosome our future study should concentrate. 18 Number of Observations Population 1 (E00290 X PI 089001) 12 10 8 6 4 2 0 % Survival Number of Observations Population 2 (E00290 X PI 437764) 12 10 8 6 4 2 0 % Survival FIGURE 1.1- SURVIVAL DISTRIBUTIONS OF THE 7 POPULATIONS OBTAINED FROM SPRAY-MYCELIUM. POPULATIONS 4, 5, AND 6 REPRESENT BOTH NON-CUP AND CUP MODIFICATION RESULTS. (FOR INTERPRETATION OF THE REFERENCES TO COLOR IN THIS AND ALL OTHER FIGURES, THE READER IS REFERRED TO THE ELECTRONIC VERSION OF THIS THESIS.) 19 FIGURE 1.1(CONT'D) Number of Observations Popoulation 3 (E00290 X PI 548312) 25 20 15 10 5 0 % Survival Number of Observations Population 4 (Skylla X PI 089001) 18 16 14 12 10 8 6 4 2 0 With cup W/O cup % Survival 20 FIGURE 1.1 (CONT'D) Population 6 (Skylla X PI 437764) With cup W/O cup % Survival Number of Observations 12 10 8 6 4 2 0 30 25 20 15 10 5 0 With cup W/O cup % Survival Population 7 (Skylla X PI 548404) Number of Observations Number of Observations Population 5 (Skylla X PI 153259) 16 14 12 10 8 6 4 2 0 % Survival 21 FIGURE 1.2- SSR MARKERS POLYMORPHIC AND NON-POLYMORPHIC IN DIFFERENT PARENTS FIGURE 1.3- MARKERS SHOWING POLYMORPHISMS IN POPULATION 22 TABLE 1.1- LISTING OF CROSSES USED IN THIS STUDY AND NUMBER OF LINES IN EACH CROSS Population female parent male parent 1 E00290 PI89001 59 2 E00290 PI437764 50 3 E00290 PI548312 4 Skylla PI89001 5 Skylla PI153259 6 Skylla PI437764 7 Skylla PI548404 1 3 2 1 2 3 Number of lines 3 3 3 3 63 62 51 38 69 Susceptible to Sclerotinia stem rot carries resistance to Sclerotinia stem rot from NKS19-90 partially resistant to Sclerotinia stem rot obtained from Hoffman et al., (2002) 23 TABLE 1.2- LIST OF CROSSES AND MAJOR EVENTS CARRIED OUT IN THE GREENHOUSE (DROP-MYCELIUM METHOD) Population 1 Crosses a) Skylla × PI 153259 b) Skylla × PI437764 c) E00290 × PI 437764 Planting Dec 12, 2008 Inoculation Jan 4, 2009 Data taken Jan 14, 2009 2 a)Skylla × PI 089001 b)PI 548404 × E00290 Dec 27, 2008 Jan 23, 2009 No data taken 3 a)E00290 × PI548312 b)E00290 × PI089001 Jan 11, 2009 Feb 7, 2009 Feb 15, 2009 4 a) Skylla × PI08900124 b) Skylla × PI 548404 Oct 16, 2009 Nov 12, 2009 Nov 20 and Nov 21 TABLE 1.3- COMPLETE LIST OF MAJOR EVENTS OCCURRING DURING SCREENING OF THE 7 POPULATIONS (SPRAY-MYCELIUM METHOD) Plant date Spray date Data Collection 9 Nov, 2007 6 Dec, 2007 17 Dec, 2007 27 Nov, 2007 24 Dec, 2007 7 Jan, 2008 14 Dec, 2007 14 Jan, 2007 28 Jan, 2008 12 Jan, 2008 11 Feb, 2008 25 Feb, 2008 24 TABLE 1.4- GLM PROCEDURE OF SAS OUTPUT FOR SEVEN POPULATIONS EVALUATED BY DROPMYCELIUM METHOD Populations source DF Type III SS Mean Square F- Value Pr > F 1 Pid 58 62726.36306 1058.95270 1.49 0.0345 2 Pid 50 39118.35659 782.36713 0.87 0.7038 3 pid 63 55724.46742 884.51536 0.95 0.5878 4 pid 61 64193.02086 1052.34460 1.13 0.2764 5 pid 50 42167.21149 843.34423 0.97 0.5311 6 pid 37 47313.59417 1278.74579 1.38 0.1202 7 pid 68 103734.8964 1525.5132 1.51 0.0216 25 TABLE 1.5- RESULTS OF VARIANCE ANALYSIS AND BROAD-SENSE HERITABILITY ESTIMATES FOR RESISTANCE TO SCLEROTINIA STEM ROT Source of variation Mean Square Population 1 population 7 Genotype 1081.4890 1525.5132 Error 724.0448 1011.2664 0.59 0.60 Heritability TABLE 1.6- SIGNIFICANCE DIFFERENCE BETWEEN LINES IN POPULATION 1AND 7 SHOWN BY LEAST SIGNIFICANT DIFFERENCE (LSD) t-grouping at α=0.05 A Ab Abc Abcd Bcdefg Cdegfh Defghi Fghij pop 7 mean 95.2 93.3 68.8 67.7 42.1 41.6 16.6 5.5 Pid 359 361 343 352 324 376 364 337 t-grouping at α=0.05 A Ab Ab Ab Abc Bc Bc C C 26 pop 1 mean 84.1 60.0 55.6 55.6 48.9 33.3 33.3 0.0 0.0 pid 8 7 1 6 45 59 29 53 42 TABLE 1.7- SIGNIFICANCE DIFFERENCE BETWEEN LINES WITHIN 7 POPULATIONS BY SPRAY-MYCELIUM METHOD Dec 17, 2007 Jan 7, 2007 Jan 28, 2007 Feb 25, 2007 Population P P P P 1 .5520 .2266 .7795 2 .0189 .7952 .5027 3 .6797 .7055 .4526 4 .0212 .6091 .2110 .5425 5 .8124 .0685 6 .1942 .3560 7 .3946 .3435 27 .6280 TABLE 1.8- SIGNIFICANT DIFFERENCE WITHIN LINES OF 17 DEC, 2007 EVALUATION Population 4 2 ID 184 178 186 183 179 185 180 187 Population 2 Mean Duncan group ID Mean Duncan group 0.6365 0.1805 0.1750 0.0910 0.0000 0.0000 0.0000 0.0000 A B B B B B B B 91 82 93 88 102 89 61 94 77 76 62 83 87 60 85 73 75 67 65 64 0.4745 0.4575 0.4320 0.2865 0.2535 0.2265 0.2080 0.1820 0.1780 0.1705 0.1540 0.1415 0.1130 0.0555 0.0415 0.0415 0.0000 0.0000 0.0000 0.0000 a ab abc abcd abcd abcd abcd abcd abcd abcd cbd cbd Cd D D D D D D D 2 Plant ID 28 TABLE 1.9- REPORTED QTLS ASSOCIATED WITH PARTIAL RESISTANCE TO SCLEROTINIA STEM ROT IN SOYBEAN SSR locus LG cM Position in LG Satt619 A1 69.21 CC453983 31044813 (ATT)11 Satt545* A1 71.39 BH126713 14970216 (ATT)19 Sat_267 A1 78.45 CC453802 31044632 (AT)32 Satt424* A2 60.59 BH126603 14970106 (ATT)52 Satt212 E 32.27 BH126418 14969921 (ATT)10 Satt341 A2 77.7 BH126532 14970035 (ATT)17 Satt197* B1 46.39 Satt638 B1 37.8 CC453997 31044827 (ATT)13 Sat_247 B1 49.73 CC453785 31044615 (AT)21 Satt070* B2 72.81 BH126318 14969821 (ATT)24 Sat_189 B2 72.92 CC453730 31044560 (AT)10 Satt122 B2 72.46 BH126336 14969839 (ATT)8 Satt147* D1a 108.89 BH126359 14969862 (ATT)14 Satt129 D1a 109.67 BH126343 14969846 (ATT)26 BH126404 GenBank Accession GenBank GI Number 14969907 29 Repeat motif (ATT)20 TABLE 1.9(CONT'D) SSR Locus LG cM Position in LG GenBank Accession GenBank GI Number Repeat motif Satt459* D1b 118.62 BH126632 14970135 (ATT)13 Satt274 D1b 116.35 BH126470 14969973 (ATT)18 Sat_202 D1b 118.86 CC453743 31044573 (AT)17 Satt256* D2 124.31 BH126454 14969957 (ATT)10 Sat_022 D2 120.3 BH126254 14969757 (AT)27 Satt386 D2 125 BH126571 14970074 (ATT)15 Satt720* E 20.8 CC454064 31044894 (ATT)19 Satt651 E 32.1 CC454006 31044836 (ATT)10 Satt691 E 19.7 CC454043 31044873 (ATT)17 Sat_317* F 72.97 CC453848 31044678 (AT)24 Satt510 F 71.41 BH126681 14970184 (ATT)21 Sat_120 F 75.97 BH126290 14969793 (AT)31 Satt191* G 96.57 BH126398 14969901 (ATT)18 Sat_117 G 100 BH126287 14969790 (CT)6(CA)8'(AT)9 Satt472 G 94.84 BH126644 14970147 (ATT)37 * reported QTLs 30 TABLE 1.9 (CONT'D) SSR locus LG cM Position in LG GenBank Accession GenBank GI Number Repeat motif Satt451* I 20.34 BH126625 14970128 (ATT)10 Satt419 I 21.9 BH126598 14970101 (ATT)22 Satt571 I 18.5 BH126737 14970240 (ATT)14 Satt588* K 117.02 BH126754 14970257 (ATT)18(AT)10(CT)14 Sat_126 K 108.2 BH126296 14969799 (AT)17 Satt481* L 54.57 BH126653 14970156 (ATT)14 Sat_340 L 55.51 CC453866 31044696 (AT)31 Sat_150 L 53.67 CC453702 31044532 (AT)24 Satt494* M 71.71 BH126665 14970168 (ATT)13 Sct_147 M 73.88 BH126779 14970282 (CT)10 Satt175 M 66.99 BH126384 14969887 (ATT)16 Satt387* N 53.25 BH126572 14970075 (ATT)10 Satt549 N 70.6 BH126716 14970219 (ATT)29 Sat_266 N 47.28 CC453801 31044631 (AT)30 Sat_109* O 127.5 CC453690 31044520 (AT)28 Sat_231 O 128.44 CC453770 31044600 (AT)22 31 TABLE 1.9 (CONT'D) SSR locus LG cM Position in LG GenBank Accession GenBank GI Number Repeat motif Sat_307 O 123.43 CC453839 31044669 (AT)34 Sat_233* A2 86.42 CC453772 31044602 (AT)14 Satt301* D2 93.71 BH126492 31044710 (ATT)24 Satt458* D2 24.52 BH126631 14970134 (ATT)31 Satt154* D2 57.07 BH126366 14969869 (ATT)20 Sat_092 D2 57.51 CC453687 31044517 (AT)31 Satt582 D2 53.85 BH126748 14970251 (ATT)16 Satt114* F 63.69 BH126332 14969835 (ATT)17 Sat_234 F 66.55 CC453773 31044603 (AT)22 Sat_229 F 62.79 CC453768 31044598 (AT)21 Satt394* G 43.38 BH126577 14970080 (ATT)31 Satt115 G 43.78 BH126333 14969836 (ATT)18 Satt273* K 56.62 BH126469 14969972 (ATT)13 Satt725 K 56.85 CC454067 31044897 (ATT/ATT)25 Sat_111 K 55.7 BH126281 14969784 (AT)16 Satt260* K 80.12 BH126458 14969961 (ATT)22 Sat_167 K 85.19 CC453714 31044544 (AT)23 32 TABLE 1.9 (CONT'D) SSR locus LG cM Position in LG GenBank Accession GenBank GI Number Repeat motif Satt475 K 78.68 BH126647 14970150 (ATT)16 Sat_134* L 28.27 BH126304 14969807 (AT)35 Sat_405 L 29.62 CC453929 31044759 (AT)33 Satt523 L 27.92 BH126693 14970196 (ATT)15 Satt009* N 28.52 BH146212 15243078 (ATT)14 Satt478* O 71.1 BH126650 14970153 (ATT)17 Sat_242 O 74.05 CC453780 31044610 (AT)18 Satt563 O 68.39 BH126729 14970232 (ATT)18 Satt243* O 119.5 BH126444 14969947 (ATT)17 Sat_307 O 123.43 CC453839 31044669 (AT)34 Sat_109 O 127.5 CC453690 31044520 (AT)28 Satt172* D1b 100.89 BH126381 14969884 (ATT)9 33 TABLE 1.10- LIST OF POLYMORPHIC SSR MARKERS ACROSS SEVEN POPULATIONS AND THEIR RESPECTIVE LINKAGE GROUPS Across all populations E00290 × PI89001 E00290× PI437764 SSR markers Sat_267 Satt619 Satt651 Satt571 Sat_244 Satt153 Satt243 Sat_109 Satt478 Sat_242 Satt494 Sat_256 Sat_092 Sat_229 Satt154 Sat_234 Satt475 Satt641 Sat_340 Satt481 Sat_199 Satt186 Sat_199 Satt186 Linkage Groups A1 A1 E I M O O O O O M M D2 F D2 F K N L L A2 D2 A2 D2 34 cM position 78.45 69.21 32.1 18.5 48.86 118.4 119.5 127.5 71.1 74.05 71.71 74.53 57.51 62.79 57.07 66.55 78.68 29.28 55.51 54.57 84.09 105.45 84.09 105.45 TABLE 1.10 (CONT'D) E00290×Pi548312 Skylla × PI89001 SSR markers Sat_340 Satt641 Sat_092 Satt494 Sat_256 Sat_236 Sat_109 Satt153 Satt243 Sat_022 Satt691 Satt451 Satt243 Sat_109 Sat_236 Sat_234 Satt260 Satt641 Satt159 Sat_340 Satt481 Satt523 Satt394 Satt147 Satt197 Sat_247 Linkage Groups L N D2 M M N O O O D2 E I O O N F K N N L L L G D1a B1 B1 35 cM position 55.51 29.28 57.51 71.71 74.53 57.59 127.5 118.14 119.5 120.3 19.7 20.34 119.5 127.5 57.59 66.55 80.12 29.28 27.13 55.51 54.57 27.92 43.38 108.89 46.39 39.73 TABLE 1.10 (CONT'D) Skylla × PI 153259 Skylla × PI437764 SSR markers Sat_340 Satt154 Sat_234 Sat_092 Satt494 Sat_236 Satt243 Satt153 Satt691 Satt691 Satt472 Satt153 Satt478 Sat_342 Sat_256 Satt175 Satt197 Satt481 Sat_199 Satt301 Satt691 Satt153 Satt243 Sat_236 Sat_342 Satt494 Satt175 Linkage Groups L D2 F D2 M N O O E E G O O B2 M M B1 L A2 D2 E O O N B2 M M 36 cM position 55.51 57.07 66.55 57.51 71.71 57.59 119.5 118.4 19.7 19.7 94.84 118.4 71.1 20.31 74.53 66.99 46.39 54.57 84.09 93.71 19.7 118.4 119.5 57.59 20.31 71.71 66.99 TABLE 1.10 (CONT'D) Skylla × PI548404 SSR markers Sat_092 Satt154 Satt475 Satt197 Sat_340 Satt301 Satt598 Satt197 Sat_247 Sat_340 Satt154 Sat_234 Sat_092 Satt494 Sat_342 Sat_236 Satt243 Satt153 Satt691 Linkage Groups D2 D2 K B1 L D2 E B1 B1 L D2 F D2 M B2 N O O E 37 cM position 57.51 57.07 78.68 46.39 55.51 93.71 34.2 46.39 49.73 55.51 57.07 66.55 57.51 71.71 20.31 57.59 119.5 118.4 19.7 REFERENCES 38 REFERENCES Abawi, G. 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Registration of 'Skylla' Soybean. Crop Science, 46(2), 974-a-975. Wang, D., Shi, J., Carlson, S. R., Cregan, P. B., Ward, R. W., & Diers, B. W. (2003). A low-cost, high-throughput polyacrylamide gel electrophoresis system for genotyping with microsatellite DNA markers. Crop Science, 43(5), 1828-1832. Wang, J. L., Liu, C. Y., Wang, J., Qi, Z. M., Li, H., Hu, G. H., et al. (2010). An integrated QTL map of fungal disease resistance in soybean (glycine max l. merr): a method of metaanalysis for mining R genes. Agricultural Sciences in China, 9(2), 223-232. Willetts, H. J., & Wong, J. A. L. (1980). The biology of Sclerotinia sclerotiorum, Sclerotinia trifoliorum, and Sclerotinia minor with emphasis on specific nomenclature. [Review]. Botanical Review, 46(2), 101-165. Workneh, F., & Yang, X. B. (2000). Prevalence of sclerotinia stem rot of soybeans in the northcentral United States in relation to tillage, climate, and latitudinal positions. Phytopathology, 90(12), 1375-1382. Wrather, J. A., & Koenning, S. R. (2006). Estimates of disease effects on soybean yields in the United States 2003 to 2005. Journal of Nematolology, 38(2), 173-180. Yang, X. B., Lundeen, P., & Uphoff, M. D. (1999). Soybean varietal response and yield loss caused by Sclerotinia sclerotiorum. Plant Disease, 83(5), 456-461. Zeng, Z. B. (1994). Precision mapping of quantitative trait loci. Genetics, 136(4), 1457-1468. 46 CHAPTER TWO GREENHOUSE SCREENING OF SOYBEAN GENOTYPES AND PLANT INTRODUCTIONS FOR RESISTANCE TO SCLEROTINIA STEM ROT 47 ABSTRACT Two related but independent studies were conducted in the greenhouse. In the first study, 66 soybean plant introductions (PIs) were evaluated in the greenhouse for resistance to Sclerotinia stem rot in the winter of 2008 and 2009. All the 66 PIs, which were selected from more than six thousands PIs, were inoculated with S. sclerotiorum mycelia by drop-mycelium method. All the 66 PIs showed significant (p < 0.005) differences between lines for resistance to Sclerotinia stem rot. In the second study, 35 soybean genotypes were evaluated in the greenhouse and field conditions to predict the resistance levels of the lines for resistance to Sclerotinia stem rot. Greenhouse and field data had strong correlations with field data for resistance to Sclerotinia stem rot. They also showed different levels of resistance to Sclerotinia stem rot both in the field and the greenhouse studies. The data from drop-mycelium method of inoculation showed strong correlation of 0.63 (P < 0.0005) and 0.40 (P< 0.0300) with the data from spray-mycelium and Iowa field data respectively. This study showed that drop- and spray-mycelium methods are viable greenhouse methods to predict the field reaction of soybean to S. sclerotiorum infection. 48 INTRODUCTION Sclerotinia stem rot of soybean caused by Sclerotinia sclerotiorum (Lib) de Bary is a major soybean disease in north-central regions of the United States and southern Canada. The disease caused the total yield loss of 59,275,000 bushels in the United States in 2009, which ranked after Soybean cyst nematode (http://www.aes.missouri.edu/delta/research/soyloss.stm) in terms of total yield loss in soybean. Yang et al. (1999) estimated yield loss of soybean due to Sclerotinia stem rot ranging from 170 to 335 kg ha-1 for each 10% increase in disease incidence. S. sclerotiorum overwinters in the soil and debris as sclerotial bodies (Yang, 1997). The Sclerotinia stem rot was more prevalent when yearly temperatures were below normal (60-70°F) than when they were above normal. The prevalence of disease was less in no-till than in minimum-till fields (Workneh and Yang, 2000). The sporadic occurrence of Sclerotinia stem rot in soybean is due to the sensitivity of S. sclerotiorum to environmental factors. The soybean shows environmental-sensitivity to S. sclerotiorum pathogen. The response of soybean to S. sclerotiorum was studied with respect to light intensity and temperature in the greenhouse. Lightsensitive cultivars had decreased disease ratings as the photosynthetically active radiation (PAR) increased but light-insensitive cultivars had constant disease ratings with increased PAR (Pennypacker and Risium, 1999). Different management practices have been applied to reduce the yield loss by Sclerotinia stem rot in soybean. Lactofen-treated soybean plants showed significant lesion size reduction when S. sclerotiorum were inoculated at V3 or R1 growth stages (Dann et al., 1999). Soybean tolerance to Sclerotinia stem rot was not related to glyphosate-resistance in soybean cultivars: 'S12-49', 'S14-M7' Roundup Ready ® (RR), 'S19-90', and 'S20-B9' (RR). Glyphosate did not 49 affect soybean growth and development or the incidence of Sclerotinia stem rot in glyphosateresistant soybean (Nelson, 2000). Totir (2000) studied the effectiveness of seed treatment fungicides for controlling seed borne infections of soybean by S. sclerotiorum. Carboxim + thiram fungicides inoculated seeds showed reduced expression of fungus by 99% and PCNB + thiabendazole inoculated seeds showed 89% reduction in fungus expression. Biological control agents such as Pseudomonas chlororaphis, Bacillus amyloliquefaciens, and Pseudomonas species have been used to control S. sclerotiorum both in greenhouse and field conditions in canola (Fernando et al., 2007). Similarly, Zeng et al. (2008) studied the effectiveness of biocontrol agents like Coniothyrium minitans, Bacillus subtilis, and Trichoderma harzianum to control S. sclerotiorum in soybean in controlled and field conditions. C. minitans and T. harzianum but B. subtilis significantly reduced the number and viability of sclerotia in both conditions. In other study, Sporidesmium sclerotivorum was used as biocontrol agent to determine its effectiveness in controlling Sclerotinia stem rot in soybean. Soybean plants were infested with macro conidia of S. sclerotivorum at a rate of 0, 2, 20, and 100 spores per cm2. Plots infested with 20 and 100 spores per cm2 had 56 to 100 percent less disease than control plots (del Rio et al, 2001). Soybean Sclerotinia stem rot was significantly reduced by the application of B. subtilis under control conditions. But the effectiveness of the biocontrol agent decreases if applied after 24 hours of S. sclerotiorum inoculation (Zhang and Xue, 2010). Use of partial resistant varieties is the most effective method for controlling Sclerotinia stem rot in soybean (Kurle et al., 2001). Controlled environment screening is required to identify soybean cultivars that are partially resistant to Sclerotinia stem rot. But using greenhouse evaluation methods to determine the field response of soybean cultivars has been difficult. There 50 have been several studies carried out to predict the field response of soybean lines from the greenhouse and laboratory evaluation methods such as excised stem or detached leaf assay (Chun et al., 1987; Kull et al., 2003; Nelson et al.,1991; Wegulo et al., 1997), or cut stem inoculation method (Vuong and Hartman, 2003, and Kull et al., 2003). These methods are very time-consuming and tedious to carry out and results between greenhouse and field evaluations were poorly correlated (Kim et al., 2000; Nelson et al., 1991; Boland et al., 1987; Chun et al., 1987). Differences in reaction to Sclerotinia stem rot were reported among soybean cultivars (McLaren and Craven, 2008). Otto-Hanson et al. (2009) screened soybean germplasm for resistance to S. sclerotiorum by different inoculation methods. Instead of using F-test or root mean square error or coefficient of variation, sensitivity ratio was used to compare the power of plant and pathogen screening tests. Nelson et al. (1991) concluded that excised stem technique performed in laboratory for screening commercial soybean cultivars did not show any correlation with field data. The reaction data from cut stem inoculation method showed significant correlation (P <0.05) with field data (Vuong et al., 2003). Cut stem inoculation method showed better result than detached-leaf and cotyledon methods when soybean and dry bean were screened for resistance to S. sclerotiorum in controlled environments (Kull et al., 2003). Ten cultivars of soybean were inoculated with S. sclerotiorum in laboratory and the disease reaction data were correlated with that of field data. The correlation coefficients between laboratory and field data varied in accordance with the inoculation methods used in laboratory for screening (Chun et al., 1987). Wegulo et al. (1998) studied different inoculation methods for screening soybean cultivars for resistance to S. sclerotiorum both in controlled and field environments. There were varied correlation coefficients between the data from controlled and field environments. Teran and Singh (2009) studied the efficacy of three greenhouse screening 51 methods for identifying physiological resistance to Sclerotinia stem rot in dry bean. Cut stem and infected bean flower methods were the most effective to identify physiological resistance to Sclerotinia stem rot in dry bean. Drop- and spray- mycelium methods are the two convenient methods of evaluating soybean lines in the greenhouse conditions that predict the soybean reactions in the field conditions for resistance to Sclerotinia stem rot. In addition, these methods are cost effective, less time consuming, reliable, and convenient for large-scale evaluations (Chen and Wang, 2005). Plant introductions (PIs) are important sources of genetic resistance to disease and pests. The narrow genetic-base of soybeans in the United States is due to the limited use of PIs in cultivar development. Only about eleven PIs were the major sources of current soybean cultivars (NAS, 1972; Gizlice et al., 1993). Shoener and Fehr (1978) argue that crosses involving PIs generally do not produce high yielding cultivars, though they are major sources of pest resistance genes. Thorne and Fehr (1970) discovered that the frequency of superior lines is greater in soybean populations derived from seventy-five percent adapted germplasm and twenty-five percent PIs. Exotic parentage needs to be introgressed with elite parentage to get high yield potential (Thompson and Nelson, 1998; Cornelious and Sneller, 2002; Vello et al., 1984). Similarly, Sneller (1999) concludes that soybean breeding is directed by extensive use of the parents derived from diverse crosses, which lead to significant yield increases. Narvel et al. (2000) found that the introgression of PIs germplasm into elite soybean cultivars depend on the amount of polymorphisms that exists between elite genotypes and PIs. Simple sequence repeat 52 (SSR) markers study showed that genetic diversity is more among the PIs than among the elite lines. The objectives of this study were to a) screen 35 soybean genotypes for resistance to Sclerotinia stem rot in controlled (greenhouse) and field environments and assess the correlation among different inoculation methods and b) evaluate the 66 soybean plant introductions, which are partially resistant to Sclerotinia stem rot, using drop-mycelium method. MATERIALS AND METHODS Thirty-five soybean genotypes were chosen from different north-central soybean breeding programs based on the availability of their phenotypic data for reactions to Sclerotinia stem rot for this study (Table 2.1). Field experiments were carried out in Iowa and Wisconsin during the summer of 2004. The experiments were arranged in randomized complete block design (RCBD) with three replications for both locations. In Iowa, single row plots of 4.5-meter long were used. Corn was used as a wind barrier around the soybean field. The plants were inoculated with sorghum seeds infested with S. sclerotiorum. The misting system equipped with a sensor was used to maintain leaf wetness from the day of inoculation to the end of flowering. In Wisconsin, disease nursery plots had dimension of 5.9 × .38 m. Row spacing was 76 cm between outer and experimental rows. Common susceptible accession (Golden Harvest H2627RR) was planted in the two outer rows and an experimental accession was planted in the middle three rows. The plant canopy was almost complete when apothecia were applied. Air temperature was normal and rainfall was slightly above normal during most of the season. For field experiments, disease scoring was done according the disease severity index (DSI) described by Grau et al. (1982) at the R7 growth stage. Ten consecutive plants from each of the three 53 experimental rows were rated on a 0-4 scale: 0 = no symptoms; 1 = lesions on lateral branches only; 2 = lesions on main stem, no wilt, and normal pod development; 3 = lesions on main stem resulting in plant death and poor pod fill; 4 = lesions on main stem resulting in plant death and no yielding pods. A DSI was calculated as: 100 * [(sum of ratings for a plot)/ [5(number of ratings classes) * 30 (number of plants rated/plot)]]. The spray-mycelium method was carried out in December of 2009 at Michigan State University. The experimental design was a randomized complete block design with three replications and a minimum of 6 plants per line. Sterilized sclerotia were grown in potato dextrose agar medium and transferred into liquid potato dextrose broth. Potato dextrose broth was homogenized by constantly shaking in the shaker for four nights. The mycelium suspension was homogenized by blending in household blender. The blended mycelium suspension was sprayed on plants at the V3 growth stage. The inoculated plants were placed in plastic chambers and humidifiers were used to maintain a near 100% humidity inside the chambers. Approximately ten days after inoculation the total number of diseased plants were counted, and the percentage of plant mortality was calculated. Similarly, the drop-mycelium method was performed as described by Chen and Wang (2005) for sixty-six PIs (Table 2.4). The pots were arranged as in spray-mycelium method, and mycelium suspension was also prepared as in spray-mycelium method. One ml of mycelium suspension was dropped on the top unfolding leaves of main stems. The plant mortality was calculated as described in spray-mycelium method. The GLM procedure of SAS (SAS, 2008) was used to analyze the data from field and greenhouse experiments. Fisher’s Protected Least Significant Differences (LSD) at a 5% 54 significance level was used to test the significance differences among genotypes in both greenhouse and field experiments. Replication 4 for 35 genotypes was not used for analysis purpose since the data had a lot of escapes (Table 2.1). Similarly, only two replications were used for analytical purpose in case of 66 PIs for the same purpose. Pearson’s correlation coefficients between plant mortality of greenhouse and DSIs of the field experiments were calculated by the CORR procedure of SAS (Table 2.3). The broad-sense heritability was calculated using the same method used in Chapter 1. RESULTS All the genotypes inoculated in the greenhouse showed typical symptoms and signs of Sclerotinia stem rot (Fig. 2.1). The disease developed at multiple points in the plants for spraymycelium method whereas disease progressed downward from apex for drop-mycelium method. In susceptible plants, disease progressed very fast but was arrested on the apical meristem in highly resistant lines. Necrotic lesions and white fluffy mycelia were visible on apical meristem and main stems. NKS-1990, a resistant check, reacted as expected, but BSR101, a susceptible check showed different level of resistance. AXN-1-68 consistently showed high level of resistance for different locations and evaluation methods while E99250 and LP02-240 consistently showed low level of resistance at all locations and for different evaluation methods (Table 2.2). Plant mortality of the genotypes evaluated in greenhouse ranged from 11.1% to 73% and 9.1% to 100% for drop-mycelium and spray-mycelium method, respectively. Data from 2009 spray-mycelium method is distorted because there was uneven spray of inocula (Fig. 2.3). Thus 2009 data were not included for calculating correlation. The DSI ranged from 20 to 94 and 55 from 25 to 72 for Wisconsin and Iowa, respectively. The correlation coefficient between the plant mortality from two greenhouse methods was 0.63 (P< 0.0005), implying that they are highly correlated (Table 2.3). The correlation coefficient between the DSI obtained from Iowa and the plant mortality obtained from drop and spray-mycelium method were 0.42 (P< 0.03) and 0.40 (P<0.03) respectively. In the field tests, correlation coefficient was 0.38 (P < 0.05) between Wisconsin and Iowa. The 66 PIs showed different levels of resistance to S. sclerotiorum. The accessions; PI 506654, PI 506728, and PI 506733A, which belong to maturity group IV, showed consistently high level of resistance to S. sclerotiorum in both years. The accessions PI 189861, PI 417507, PI 548354, and PI 153316, which belong to maturity group 0, showed low level of resistance to S. sclerotiorum for both years. The other PIs did not show consistent result across years 2008 and 2009 in our study. The correlation coefficient between the plant mortality for 2008 and 2009 was 0.18. Fig. 2.1 (a) Fig. 2.1 (b) FIGURE 2.1-SOYBEAN PLANTS BEFORE (A) AND AFTER (B) INOCULATION WITH S. SCLEROTIORUM. 56 DISCUSSION An inoculation method is used to evaluate soybean germplasm for reaction to S. sclerotiorum in a controlled environment for two primary reasons. The reaction of soybean to Sclerotinia stem rot in the field nurseries can be inconsistent due to the quantitative mode of the partial resistance trait and unpredictable nature of the weather conditions for the same location in different years. True physiological resistance is difficult to identify because of disease escape in the field conditions. In case of the sixty-six PIs, those lines with higher partial resistance can be used as parents to develop partial resistance mapping populations and can be studied for QTLs associated with Sclerotinia stem rot resistance. In our study, data from drop-mycelium method showed high correlation (0.63) with spray-mycelium method, which were conducted in similar controlled conditions in different years. Chen and Wang (2005) argued that drop- and spraymycelium methods are low-cost and high-efficiency greenhouse inoculation methods that give consistent and reproducible results. Our study validated that argument, and further argues that these methods can predict the field performance of soybean genotypes for resistance to Sclerotinia stem rot. The correlations between data from controlled environments with field performance ranged from 0.29 to 0.42, which signifies that for quantitative disease like Sclerotinia stem rot, these correlation coefficients are promising. Peltier and Grau (2008) found that Light intensity in controlled environments not only affect the Sclerotinia stem rot development in soybeans and but also affect the prediction of disease in field conditions. Since we controlled relative humidity and temperature inside the greenhouse, the intensity and duration of light hours might have some influence on the development of disease in the greenhouse. Kim and Diers (2000) estimated the broad-sense heritability of Sclerotinia stem rot resistance at 0.59 in a 152 F3- derived lines from S19-90 crossed with Williams 82. The broad57 sense heritability for resistance to Sclerotinia stem rot ranged from 0.58 to 0.77 in common bean (Miklas and Grafton, 1992). Guo et al. (2008) estimated broad-sense heritability of 0.29 and 0.44 for BC1F4:5 and BC1F4:6 soybean lines, respectively. The broad-sense heritability for soybean PIs in our study were 0.58 and 0.62 for 2008 and 2009 greenhouse evaluations respectively, which closely agrees with Kim and Diers (2000). Our study approves the argument made by Grau et al. (1982) that resistance to Sclerotinia stem rot in soybean is a heritable trait. The 66 plant introductions (PIs) were selected based on the fact that they showed partial resistance to Sclerotinia stem rot in different field locations and controlled environment conditions (Hoffman et al., 2002). Those PIs were evaluated in the greenhouse to narrow down to few PIs which would show promising resistance to Sclerotinia stem rot. Our study showed that those 66 PIs are significantly different (P<0.0511 and P< 0.0182 for 2008 and 2009 evaluations, respectively) from each other for reactions to Sclerotinia stem rot (Table 2.5). Significantly different PIs based on least significant difference at α = 0.05 is shown by Table 2.6. PI 427143, PI 506728, PI 506733A, PI 358318A, FC 030233, PI 132207, and PI 361059B showed consistently high level of resistance to Sclerotinia stem rot. These PIs range from early maturity (0) through late maturity group (IV). So these PIs can be used as sources of resistance in breeding for Sclerotinia stem rot resistance. 58 number of observations 20 18 16 14 12 10 8 6 4 2 0 2008 2009 10 20 30 40 50 60 70 80 90 100 survival rate FIGURE 1.2- DIFFERENT LEVELS OF RESISTANCE SHOWN BY 66 PIS IN 2009 AND 2008 GREENHOUSE STUDY 59 drop Wisconsin spray Iowa 120 plant mortality 100 80 60 40 20 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 genotypes FIGURE 2.3- PLANT MORTALITY FOR 35 SOYBEAN GENOTYPES EVALUATED IN DIFFERENT ENVIRONMENTS 60 TABLE 2.1- THIRTY-FIVE SOYBEAN GENOTYPES AND THEIR REACTIONS TO SCLEROTINIA STEM ROT (SPRAY-MYCELIUM) Genotypes rep1 rep2 rep3 rep4 01SSD-106 83.0 83.0 80.0 0.0 Mean mortality (%) 61.5 01SSD-119 100.0 100.0 0.0 0.0 50.0 01SSD-150 50.0 50.0 25.0 20.0 36.3 01SSD-177 33.3 33.3 75.0 0.0 35.4 01SSD-20 50.0 50.0 85.7 0.0 46.4 01SSD-36 0.0 0.0 50.0 33.3 20.8 01SSD-61 85.7 85.7 50.0 0.0 55.4 AXN-1-55 60.0 60.0 25.0 0.0 36.3 AXN-1-68 33.3 33.3 0.0 16.7 20.8 AXN-2-55 60.0 60.0 33.3 60.0 53.3 A2506 66.7 66.7 0.0 0.0 33.3 BSR101 71.4 71.4 16.7 42.9 50.6 Dwight 50.0 50.0 28.6 83.3 53.0 E99279 66.7 66.7 0.0 14.3 36.9 HSO-3243 33.3 33.3 50.0 40.0 39.2 61 TABLE 2.1 (CONT'D) Genotype rep1 rep2 rep3 rep4 Mean mortality (%) LD00-1938 50.0 50.0 0.0 50.0 LD00-497 50.0 50.0 57.1 0.0 LP02-221 71.4 71.4 42.9 66.7 LP02-222 57.1 57.1 0.0 66.7 45.2 LP02-240 57.1 57.1 116.7 66.7 74.4 LP02-250 66.7 66.7 0.0 71.4 51.2 LP02-253 83.3 83.3 16.7 25.0 52.1 Ohio FG3 100.0 100.0 0.0 42.9 60.7 NKS19-90 16.7 16.7 0.0 16.7 12.5 83.0 83.0 0.0 0.0 41.5 U409006 100.0 100.0 66.7 0.0 66.7 U409014 16.7 16.7 83.3 100.0 54.2 U419020 66.7 66.7 20.0 50.0 50.8 U423040 20.0 20.0 50.0 100.0 47.5 U412014 85.7 85.7 83.3 85.7 85.1 NE3303 100.0 100.0 40.0 0.0 60.0 Skylla 62 37.5 39.3 63.1 TABLE 2.2- REACTIONS OF 35 SOYBEAN GENOTYPES TO DIFFERENT METHOD OF INOCULATIONS 3 4 4 3 Genotypes 001SSD-106 Drop 25.0 01SSD-119 10.0 28.0 38.9 01SSD-150 40.0 44.0 29.8 01SSD-177 36.4 32.0 41.7 01SSD-20 18.2 45.0 67.5 01SSD-36 9.1 39.0 27.8 01SSD-61 18.2 35.0 50.0 AXN-1-55 36.4 25.6 32.0 45.0 AXN-1-68 30.0 36.5 25.0 11.1 AXN-2-55 60.0 43.7 43.0 31.1 A2506 81.8 54.5 61.0 22.2 BSR101 54.5 66.0 29.4 Dwight 100.0 70.7 79.0 37.3 E99279 63.6 54.9 43.0 46.0 HSO-3243 83.3 62.9 71.0 61.1 LD00-1938 50.0 51.0 50.0 LD00-497 100.0 94.0 55.7 Iowa DSI 3 4 WisconsinDSI 27.0 Greenhouse evaluation methods (plant mortality percentage) data from the fields (Disease Severe Index) 63 Spray10 61.0 TABLE 2.2 (CONT'D) 3 4 4 3 Genotypes Drop Iowa DSI Wisconsin DSI Spray10 LP02-221 40.0 62.2 77.0 38.1 LP02-222 45.5 72.3 76.0 19.0 LP02-240 72.7 58.4 78.0 57.9 LP02-250 80.0 66.4 84.0 28.9 LP02-253 75.0 68.4 89.0 47.6 Ohio FG3 55.6 54.2 20.0 50.0 NKS19-90 16.7 49.2 27.0 11.1 Skylla 10.0 53.3 57.0 37.2 U409006 70.0 55.6 64.0 61.1 U409014 58.3 66.3 51.0 33.3 U419020 57.1 38.3 55.0 28.9 U423040 62.5 45.6 U412014 60.0 34.8 NE3303 63.6 54.7 46.7 U413038 44.4 36.6 46.8 U425043 75.0 36.7 64.0 U416019 45.5 35.8 36.0 23.3 36.0 64 73.0 TABLE 2.2 (CONT'D) 3 4 4 3 Drop Iowa DSI Wisconsin DSI Spray10 E99250 71.4 49.9 52.0 72.2 Mean 52.0 51.5 52.7 42.3 STDEV 24.9 12.9 21.1 16.4 RMSE 4.2 2.6 3.9 2.8 LSD 26.8 20.7 22.8 49.5 Genotypes TABLE 2.3- PEARSON CORRELATION COEFFICIENTS, N = 26 PROB > |R| UNDER H0: RHO=0, FOR 35 GENOTYPES Drop Iowa 0.30162(0.1343) Spray10 0.63379(0.0005) Wisconsin 0.42073(0.0323) Wisconsin Iowa 0.38577(0.0516) 0.40883(0.0381) Correlations are followed by their respective p-values at α = 0.05 65 0.29231(0.1473) TABLE 2.4- SIXTY-SIX SOYBEAN PIS AND THEIR REACTIONS TO SCLEROTINIA STEM ROT (DROPMYCELIUM METHOD) Survival rate (%) Survival rate (%) Survival rate (%) PID PIs 2008 2009 PID PIs 2008 2009 PID PIs 2008 2009 1 PI 132207 57.8 60.0 23 PI 091733 55.0 47.2 45 PI 507352 73.3 35.0 2 PI 153259 50.0 54.4 24 PI 153282 64.3 34.3 46 PI 507353 71.4 45.6 3 PI 189861 33.3 33.3 25 PI 153316 40.0 41.7 47 PI 196157 90.5 34.4 4 PI 189899 50.0 38.9 26 PI 184042 88.9 32.8 48 PI 229324 58.1 13.3 5 PI 232996 63.9 45.8 27 PI 189896 66.7 16.7 49 PI 398637 69.1 31.7 6 PI 243547 67.8 40.0 28 PI 189919 83.3 42.2 50 PI 404180 62.0 39.5 7 PI 291319B 36.1 24.4 29 PI 391589B 65.7 13.3 51 PI 417201 93.3 44.4 8 PI 361059B 81.1 58.9 30 PI 416776 66.7 41.7 52 PI 423818 61.1 0.0 9 PI 417449 70.0 11.1 31 PI 416805 91.7 36.1 53 PI 417245 73.3 16.7 10 PI 417507 30.0 25.0 32 PI 427143 68.9 58.3 54 PI 506519 33.3 49.4 11 PI 417533 33.3 50.0 33 PI 504502 94.4 62.2 55 PI 506652 73.5 30.0 12 PI 437072 42.1 27.8 34 PI 548312 62.2 35.6 56 PI 506654 94.4 52.8 13 PI 437527 46.8 16.7 35 PI 548380 58.3 31.1 57 PI 506728 77.1 70.0 14 PI 437764 54.0 23.6 36 PI 548407 75.6 34.4 58 PI 506733A 91.7 69.4 66 TABLE 2.4 (CONT'D) Survival rate (%) PID PIs 2008 16 PI 548354 37.2 17 PI 548404 58.3 18 PI 548539 19 2009 Survival rate (%) Survival rate (%) PID PIs 2008 2009 PID PIs 38 PI 561284 70.0 5.6 60 PI 506868 66.7 16.7 33.3 39 PI 561331 45.2 13.3 61 PI 506892 90.5 24.4 72.2 47.2 40 PI 561345 47.8 50.0 62 PI 507222 73.8 50.0 PI 567157A 93.3 11.1 41 PI 561353 63.3 36.7 63 PI 567650B 66.7 18.9 20 PI 578501 50.0 38.9 42 PI 561367 86.7 25.0 64 PI 567721 36.7 5.6 21 FC 030233 73.3 58.3 43 PI 189931 40.0 33.3 65 PI 594286 100.0 35.5 22 PI 081775 64.7 44 PI 358318A 81.0 58.3 66 PI 594289 71.7 19.4 Mean 65.3 34.8 STDEV 18.0 16.9 LSD 67 2008 2009 46.3 TABLE 2.5- SIGNIFICANT DIFFERENCES AMONG THE PLANT INTRODUCTIONS RESISTANCE TO SCLEROTINIA STEM ROT (2008 AND 2009 DATA, RESPECTIVELY) Source SCREENED DF Type III SS Mean Square F-Value Pr > F pid 65 69668. 37889 1071.82121 1.41 0.0511 pid 65 63756.53076 980.86970 1.68 FOR 0.0182 ‘pid’ means plant ID TABLE 2.6- SIGNIFICANCE DIFFERENCE BETWEEN PIS SHOWN BY LEAST SIGNIFICANT DIFFERENCE (LSD) TEST t Grouping at α=0.05 Mean pid (PI) A ab abc bc abcd abcdefg cdefg cdefg efg g g 87.50 75.00 71.67 70.83 63.33 39.29 26.67 25.00 12.50 0.00 0.00 59 (PI 506784) 33 (PI 504502) 58 (PI 506733A) 19 (PI 567157A) 9 (PI 417449) 51 (PI 417201) 36 (PI 548407) 28 (PI 189919) 68 (PI 594289) 38 (PI 561284) 14 (PI 437764) 68 TABLE 2.7- BROAD-SENSE HERITABILITIES FOR 66 PIS EVALUATED IN 2008 AND 2009 Sources of variation Mean Square 2008 2009 Genotype 1071.8212 980.86970 Error 762.1222 582.4342 Heritability 0.58 0.62 69 APPENDIX 70 TABLE A1- 66 PIS AND THEIR MATURITY GROUP, ORIGIN, AND SURVIVAL RATE FOR 2009 GREENHOUSE EVALUATION Plant Introductions Maturity group Origin rep 1 rep 2 rep 3 Mean survival (%) WP02 PI 132207 0 Netherlands 80.0 0.0 100.0 60.0 WP03 PI 153259 0 Belgium 50.0 33.3 80.0 54.4 WP04 PI 189861 0 Germany 0.0 0.0 100.0 33.3 WP05 PI 189899 0 France 66.7 50.0 0.0 38.9 WP06 PI 232996 0 Germany 37.5 80.0 20.0 45.8 WP07 PI 243547 0 Japan 20.0 0.0 100.0 40.0 WP08 PI 291319B 0 China 33.3 0.0 40.0 24.4 WP09 PI 361059B 0 China 60.0 66.7 50.0 58.9 WP10 PI 417449 0 Japan 0.0 0.0 33.3 11.1 WP11 PI 417507 0 Germany 25.0 50.0 0.0 25.0 WP12 PI 417533 0 Germany 50.0 50.0 50.0 50.0 Wp13 PI 437072 0 Russian federation 50.0 0.0 WP14 PI 437527 0 Ukraine 0.0 0.0 50.0 16.7 WP15 PI 437764 0 China 14.3 40.0 16.7 23.6 WP16 PI 438267 0 China 0.0 100.0 33.3 44.4 WP17 PI 548354 0 China 0.0 0.0 0.0 0.0 71 33.0 27.7 TABLE A1 (CONT'D) Plant Introductions Maturity group Origin rep 1 rep 2 rep 3 Mean survival (%) WP19 PI 548539 0 Canada 66.7 75.0 0.0 47.2 WP20 PI567157A 0 China 0.0 33.3 0.0 11.1 WP21 PI 578501 0 China 0.0 16.7 100.0 38.9 WP22 FC 030233 I Canada 50.0 25.0 100.0 58.3 WP23 PI 081775 I Japan 33.3 20.0 80.0 44.4 WP24 PI 091733 I China 25.0 50.0 66.7 47.2 WP25 PI 153282 I Belgium 60.0 42.9 0.0 34.3 WP26 PI 153316 I France 25.0 0.0 100.0 41.7 WP27 PI 184042 I Yugoslavia 40.0 33.3 25.0 32.8 WP28 PI 189896 I Germany 0.0 50.0 0.0 16.7 WP29 PI 189919 I France 60.0 0.0 66.7 42.2 WP30 PI 391589B I China 0.0 0.0 40.0 13.3 WP31 PI 416776 I Japan 100.0 25.0 0.0 41.7 WP32 PI 416805 I Japan 33.3 25.0 50.0 36.1 72 TABLE A1 (CONT'D) Plant Introductions Maturity group Origin rep 1 rep 2 rep 3 Mean survival (%) WP33 PI 427143 I South Korea 50.0 100.0 25.0 58.3 WP34 PI 504502 I Taiwan 20.0 66.7 100.0 62.2 WP35 PI 548312 I China 50.0 16.7 WP36 PI 548380 I China 20.0 33.3 40.0 31.1 WP37 PI 548407 I Japan 20.0 16.7 66.7 34.4 WP38 PI 549066 I Japan 0.0 0.0 75.0 25.0 WP39 PI 561284 I China 0.0 16.7 0.0 5.6 WP40 PI 561331 I China 20.0 0.0 20.0 13.3 WP41 PI 561345 I China 16.7 33.3 100.0 50.0 WP42 PI 561353 I China 60.0 0.0 50.0 36.7 WP43 PI 561367 I China 0.0 25.0 50.0 25.0 WP44 PI 189931 II France 0.0 50.0 50.0 33.3 WP45 PI358318A II Japan 100.0 25.0 50.0 58.3 WP46 PI 507352 II Japan 25.0 80.0 0.0 35.0 WP47 PI 507353 II Japan 16.7 80.0 40.0 45.6 WP48 PI 196157 III Japan 20.0 0.0 83.3 34.4 73 33.3 TABLE A1 (CONT'D) Plant Introductions Maturity group Origin rep 1 rep 2 rep 3 Mean survival (%) WP54 PI 417245 IV Japan 0.0 0.0 50.0 16.7 WP55 PI 506519 IV Japan 33.3 40.0 75.0 49.4 WP56 PI 506652 IV Japan 40.0 50.0 0.0 30.0 WP57 PI 506654 IV Japan 25.0 50.0 83.3 52.8 WP58 PI 506728 IV Japan 60.0 83.3 66.7 70.0 WP59 PI506733A IV Japan 100.0 75.0 33.3 69.4 WP60 PI 506784 IV Japan 0.0 0.0 50.0 16.7 WP61 PI 506868 IV Japan 50.0 0.0 0.0 16.7 WP62 PI 506892 IV Japan 0.0 40.0 33.3 24.4 WP63 PI 507222 IV Japan 16.7 33.3 100.0 50.0 WP65 PI567650B IV China 16.7 0.0 40.0 18.9 WP66 PI 567721 IV China 0.0 0.0 16.6 5.5 WP67 PI 594286 IV Japan 0.0 40.0 66.6 35.5 WP68 PI 594289 IV Japan 0.0 25.0 33.3 19.4 75 TABLE A2- SIXTY-SIX SOYBEAN PIS AND THEIR MATURITY GROUPS, ORIGINS, AND REACTIONS TO SCLEROTINIA STEM ROT EVALUATED IN 2008 Test Number PIs Maturity Group WP02 PI132007 0 WP03 PI 153259 0 WP04 PI 189861 WP05 Mean Survival (%) Origin rep1 Netherlands rep2 rep3 100.0 40.0 33.3 57.8 Belgium 100.0 0.0 50.0 50.0 0 Germany 50.0 33.3 16.7 33.3 PI 189899 0 France 50.0 50.0 60.0 50.0 WP06 PI 232996 0 Germany 100.0 16.7 75.0 63.9 WP07 PI 243547 0 Japan 83.3 80.0 40.0 67.8 WP08 PI 291319B 0 China 16.7 16.7 75.0 36.1 WP09 PI 361059B 0 China 100.0 60.0 83.3 81.1 WP10 PI 417449 0 Japan 100.0 50.0 60.0 70.0 WP11 PI 417507 0 Germany 40.0 50.0 0.0 30.0 WP12 PI 417533 0 Germany 0.0 50.0 50.0 33.3 WP13 PI 437072 0 Russian Federation 16.7 42.9 66.7 42.1 WP14 PI 437527 0 Ukraine 50.0 57.2 33.3 46.8 WP15 PI 437764 0 China 33.3 28.6 100.0 54.0 WP16 PI 438267 0 China 80.0 80.0 33.3 64.4 WP17 PI 548354 0 China 66.7 25.0 20.0 37.2 WP18 PI 548404 0 Canada 75.0 25.0 75.0 58.3 WP19 PI 548539 0 Canada 66.7 75.0 75.0 72.2 75 TABLE A2 (CONT'D) Test Number PIs Maturity Group Origin Rep1 Rep2 Rep3 Mean Survival (%) WP22 FC 030233 I Canada 20.0 100.0 100.0 73.3 WP23 PI 081775 I Japan 25.0 83.3 85.7 64.7 WP24 PI 091733 I China 40.0 50.0 75.0 55.0 WP25 PI 153282 I Belgium 57.2 85.7 50.0 64.3 WP26 PI 153316 I France 0.0 100.0 20.0 40.0 WP27 PI 184042 I Yugoslavia 83.3 100.0 83.3 88.9 WP28 PI 189896 I Germany 60.0 80.0 60.0 66.7 WP29 PI 189919 I France WP30 PI 391589B I China 57.2 60.0 80.0 65.7 WP31 PI 416776 I Japan 50.0 66.7 83.3 66.7 WP32 PI 416805 I Japan 100.0 75.0 100.0 91.7 WP33 PI 427143 I South Korea 80.0 66.7 60.0 68.9 WP34 PI 504502 I Taiwan 100.0 83.3 100.0 94.4 WP35 PI 548312 I China 66.7 100.0 20.0 62.2 WP36 PI 548380 I China 100.0 75.0 0.0 58.3 WP37 PI 548407 I Japan 66.7 60.0 100.0 75.6 WP38 PI 549066 I Japan 100.0 83.3 60.0 81.1 WP39 PI 561284 I China 60.0 75.0 75.0 70.0 WP40 PI 561331 I China 85.7 16.7 33.3 45.2 WP41 PI 561345 I China 50.0 33.3 60.0 47.8 76 83.3 83.3 TABLE A2 (CONT'D) Test Number WP43 PIs Maturity Group PI 561367 I Origin China rep1 100.0 rep2 60.0 rep3 100.0 WP44 PI 189931 II France 33.0 20.0 0.0 40.0 WP45 PI 358318A II Japan 100.0 100.0 42.9 81.0 WP46 PI 507352 II Japan 40.0 100.0 80.0 73.3 WP47 PI 507353 II Japan 100.0 57.2 57.2 71.4 WP48 PI 196157 III Japan 71.4 100.0 100.0 90.5 WP49 PI 229324 III Japan 14.3 100.0 60.0 58.1 WP50 PI 398637 III 57.2 50.0 100.0 69.1 WP51 PI 404180 III China 16.7 85.7 83.3 62.0 WP52 PI 417201 III Japan 100.0 80.0 100.0 93.3 WP53 PI 423818 III 50.0 50.0 83.3 61.1 WP54 PI 417245 IV Japan 20.0 100.0 100.0 73.3 WP55 PI 506519 IV Japan 0.0 0.0 100.0 33.3 WP56 PI 506652 IV Japan 83.3 57.2 80.0 73.5 WP57 PI 506654 IV Japan 83.3 100.0 100.0 94.4 WP58 PI 506728 IV Japan 60.0 100.0 71.4 77.1 WP59 PI 506733A IV Japan 100.0 75.0 100.0 91.7 WP60 PI 506784 IV Japan 83.3 100.0 100.0 94.4 WP61 PI 506868 IV Japan 57.1 42.9 100.0 66.7 WP62 PI 506892 IV Japan 100.0 71.4 100.0 90.5 South Korea South Korea 77 Mean survival (%) 86.7 TABLE A2 (CONT'D) Mean Test Number PIs Maturity Group Origin rep1 rep2 rep3 survival (%) WP65 PI 567650B IV China 66.7 50.0 83.3 66.7 WP66 PI 567721 IV China 50.0 0.0 100.0 36.7 WP67 PI 594286 IV Japan 100.0 100.0 60.0 100.0 WP68 PI 594289 IV Japan 75.0 100.0 40.0 71.7 Checks S19-90 75.0 100.0 87.5 Olympus 33.3 33.3 33.3 78 TABLE A3 PLANT MORTALITY FOR 392 LINES EVALUATED BY TWO DIFFERENT METHODS Plant Mortality (%) Plant ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Population 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 spray 67.0 73.0 58.2 77.6 79.2 63.1 67.9 80.8 69.1 91.3 91.3 74.3 72.9 81.3 88.6 75.0 75.6 81.6 75.0 78.4 100.0 9.5 14.2 Drop 55.6 22.2 35.7 36.7 38.9 55.6 60.0 84.1 23.8 50.0 24.5 0.0 11.1 33.3 0.0 5.6 5.6 18.9 16.7 0.0 8.3 9.5 0.0 Plant Mortality (%) Plant ID 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 79 Population spray 4 33.3 4 29.2 4 21.2 4 54.2 4 30.3 4 40.0 4 45.5 4 29.2 4 12.5 4 25.3 4 34.1 4 8.3 4 36.3 4 47.5 4 4.2 4 34.1 4 50.7 4 50.0 4 16.7 4 41.7 4 54.2 4 38.1 4 53.6 Drop 70.8 52.4 75.9 14.3 66.7 65.5 60.7 45.8 70.8 55.7 30.6 50.5 63.9 41.7 54.6 72.2 33.3 26.1 33.3 62.5 57.1 79.4 54.2 TABLE A3 (CONT'D) Plant ID 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Population 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Plant Mortality (%) Spray Drop 25.8 0.0 76.1 22.2 86.0 10.3 73.3 11.1 84.6 6.7 68.3 33.3 85.0 0.0 0.0 42.9 75.7 27.8 81.3 15.1 82.0 16.7 78.4 0.0 72.7 11.1 48.9 26.7 12.7 0.0 76.3 11.4 77.5 40.0 95.8 22.2 85.3 0.0 67.6 0.0 72.1 9.5 62.5 48.9 88.6 19.1 80.9 20.0 87.5 16.7 89.6 24.6 76.9 0.0 80 Plant ID 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 Plant Mortality (%) Population Spray Drop 4 35.2 36.7 4 40.0 72.2 4 60.0 46.7 4 50.0 22.2 4 54.8 29.1 4 52.5 40.0 4 32.3 33.3 4 44.3 72.2 4 43.3 83.3 4 25.0 73.6 4 100.0 91.7 4 45.5 68.1 4 65.3 60.0 4 64.0 48.2 4 9.1 35.7 5 47.0 65.1 5 52.8 48.9 5 41.7 66.7 5 47.3 57.1 5 37.5 52.8 5 36.0 25.6 5 8.3 55.6 5 70.8 45.6 5 56.1 22.2 5 50.0 54.4 5 39.1 41.1 5 68.2 67.8 TABLE A3 (CONT'D) Plant ID 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 Population 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Plant Mortality (%) Spray Drop 86.7 0.0 79.7 0.0 54.7 0.0 86.4 6.7 81.3 0.0 95.4 22.2 48.9 11.1 70.6 0.0 80.3 33.3 81.7 6.7 51.7 72.2 70.1 22.2 72.9 27.8 91.3 41.1 93.8 58.3 44.3 63.3 100.0 35.6 14.3 30.6 13.4 53.3 0.0 51.1 45.0 50.0 51.2 66.7 86.8 46.7 32.2 36.7 100.0 41.7 76.1 18.9 Plant ID 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 81 Plant Mortality (%) Population Spray Drop 5 63.6 33.3 5 60.7 17.8 5 18.2 42.2 5 12.5 53.3 5 45.5 41.7 5 12.5 48.4 5 34.8 23.3 5 28.6 50.0 5 50.0 50.8 5 45.8 26.7 5 29.2 5.6 5 31.1 17.8 5 45.8 11.1 5 87.5 16.7 5 45.8 11.1 5 47.0 18.9 5 52.7 61.1 5 52.8 25.0 5 30.6 30.0 5 51.5 61.1 5 31.3 32.9 5 43.2 34.4 5 56.8 44.4 5 68.2 18.7 5 50.0 65.6 5 41.4 25.0 TABLE A3 (CONT'D) Plant ID 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 Population 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Spray 79.0 23.3 33.3 29.2 31.8 48.1 83.2 20.8 81.8 27.7 91.6 63.0 69.2 45.0 66.3 43.6 68.4 75.9 40.7 48.6 42.3 34.9 45.5 72.7 26.7 76.9 Plant Mortality (%) Drop 25.6 40.0 38.9 11.1 16.7 66.7 34.4 21.7 27.8 63.3 34.4 55.6 61.1 25.6 40.0 13.3 28.1 22.2 38.9 33.3 38.9 38.9 42.8 36.1 41.7 68.9 Plant ID 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 82 Population Spray 5 33.3 5 42.4 5 26.7 5 58.3 5 0.0 5 21.0 5 34.8 5 41.1 5 56.8 5 63.6 5 50.0 5 40.0 5 11.1 6 61.0 6 68.9 6 40.1 6 16.7 6 17.8 6 43.3 6 14.3 6 18.2 6 35.6 6 30.3 6 50.0 6 61.4 6 67.7 Plant Mortality (%) Drop 25.0 19.8 58.9 22.2 43.3 41.1 30.0 44.4 22.2 55.6 42.2 38.9 28.6 30.6 8.3 13.3 47.8 43.9 50.0 25.0 27.6 21.7 66.1 33.3 74.3 80.6 TABLE A3 (CONT'D) Plant ID 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 Plant Mortality (%) Population Spray Drop 2 40.9 11.1 2 39.3 30.0 2 15.0 27.8 2 34.1 38.9 2 40.0 26.7 2 25.0 38.9 2 45.5 46.7 3 77.1 49.6 3 79.9 74.6 3 93.6 62.7 3 88.9 45.6 3 88.5 36.0 3 66.7 61.1 3 57.5 66.7 3 78.8 60.2 3 85.4 44.4 3 72.5 55.0 3 89.6 49.7 3 81.7 44.4 3 82.1 88.9 3 64.6 46.0 3 88.8 11.1 3 90.0 18.9 3 87.1 59.0 3 57.5 47.8 3 75.0 68.9 Plant ID 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 83 Plant Mortality (%) Population Spray Drop 6 23.2 66.7 6 21.2 66.7 6 27.3 33.3 6 21.6 61.1 6 33.3 58.7 6 58.3 43.3 6 32.5 70.2 6 37.5 72.7 6 91.7 77.8 6 29.2 53.3 6 25.0 69.1 6 32.5 83.0 6 32.6 50.0 6 83.3 68.9 6 50.0 87.5 6 14.5 68.9 6 50.0 58.9 6 51.4 80.0 6 4.5 73.3 6 46.4 72.2 6 48.6 61.1 6 37.5 77.8 6 42.5 75.0 6 40.0 59.1 6 62.8 70.5 7 48.5 42.1 TABLE A3 (CONT'D) Plant ID 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 Population 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Plant Mortality (%) Spray Drop 42.3 48.0 69.7 46.3 83.6 26.2 87.1 25.0 86.9 33.3 23.3 66.7 84.5 56.7 90.8 16.7 42.9 25.0 88.0 81.5 74.4 47.5 77.7 55.4 97.9 49.5 46.7 58.3 83.0 36.1 72.7 38.4 83.0 54.4 87.5 37.8 70.9 8.3 80.9 70.2 86.6 48.5 90.2 31.7 72.9 51.6 83.4 56.3 91.7 31.7 93.2 36.9 Plant ID 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 84 Plant Mortality (%) Population Spray Drop 7 13.6 52.4 7 46.1 30.2 7 37.2 16.7 7 58.3 0.0 7 36.0 19.1 7 26.1 37.8 7 52.0 0.0 7 47.0 15.9 7 14.5 44.1 7 41.7 65.5 7 63.6 26.2 7 13.9 46.7 7 29.2 5.6 7 12.5 0.0 7 49.6 9.7 7 39.5 33.3 7 44.4 39.0 7 32.7 50.0 7 47.3 68.9 7 37.5 16.3 7 51.5 4.8 7 52.7 20.0 7 12.5 50.0 7 16.1 11.1 7 25.0 0.0 7 18.2 34.5 TABLE A3 (CONT'D) Plant ID 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 Plant Mortality (%) Population Spray Drop 3 87.5 33.3 3 90.1 62.4 3 93.8 47.8 3 95.5 33.3 3 80.7 58.2 3 87.5 63.5 3 78.1 40.0 3 56.1 40.7 3 97.9 51.9 3 83.3 80.0 3 81.9 44.4 3 86.7 51.9 3 97.9 29.3 3 90.9 33.3 3 68.6 65.5 3 80.7 72.2 3 85.7 76.4 3 95.5 31.7 4 65.0 68.1 4 37.5 66.1 4 31.8 56.6 4 9.1 70.7 4 25.4 62.7 4 81.9 53.3 4 87.5 59.1 4 78.5 22.2 Plant ID 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 85 Plant Mortality (%) Population Spray Drop 7 34.1 30.7 7 47.3 67.8 7 23.7 44.5 7 15.6 39.0 7 5.6 0.0 7 36.4 0.0 7 18.6 6.7 7 48.2 5.7 7 41.2 95.2 7 14.9 38.1 7 19.4 93.3 7 60.2 41.7 7 4.2 5.7 7 31.8 16.7 7 31.1 16.7 7 18.2 20.6 7 25.5 5.6 7 17.7 5.7 7 14.1 44.6 7 4.5 57.2 7 22.7 34.2 7 37.8 0.0 7 38.2 13.3 7 25.4 59.1 7 0.0 0.0 7 18.2 41.7 TABLE A3 (CONT'D) Plant ID 181 182 183 184 185 186 187 188 190 191 192 193 194 195 196 Plant Mortality (%) Population Spray Drop 4 58.9 33.3 4 61.9 46.2 4 74.2 25.0 4 43.2 33.3 4 86.9 83.3 4 88.5 72.2 4 81.3 79.1 4 10.0 63.0 4 58.3 22.2 4 50.0 45.6 4 22.0 35.1 4 32.1 69.1 4 9.1 33.3 4 30.7 31.6 4 21.4 33.4 Plant ID 377 378 379 380 381 382 383 384 386 387 388 389 390 391 392 NKS19-90 Olympus Mean STDEV 86 Plant Mortality (%) Population Spray Drop 7 32.5 16.7 7 39.4 33.4 7 19.4 52.4 7 54.2 22.2 7 13.3 40.0 7 12.5 11.1 7 25.8 38.9 7 20.0 21.0 7 11.1 26.4 7 35.0 63.3 7 17.0 33.3 7 44.5 59.6 7 29.2 9.7 7 24.3 38.1 7 0.0 38.1 74.4 27.1 43.5 51.9 39.6 26.3 22.4 REFERENCES 87 REFERENCES Boland, G. 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