Fusarium virguliforme, the causal agent of sudden death syndrome (SDS) in North America, and Heterodera glycines, soybean cyst nematode (SCN), are significant threats to the production of soybean. In 2014, two field trials were established to evaluate seed treatments and their efficacy in managing SDS and SCN. The locations were selected because each has a history of SDS and SCN. Plots were 3.04 meters wide by 6.1 meters in length with row spacing of 0.76 meters. Each plot received 2.45 grams of infested sorghum per 30.5 centimeters of row. At each location, two soybean varieties and ten different seed treatments were tested in 2014. In 2015, twelve seed treatments were tested on the same two varieties at the same two locations. Seed treatments were evaluated for phytotoxicity, vigor, stand count, SDS foliar ratings and soybean yield. Soil samples were collected at planting and at harvest to determine SCN reproduction. Root samples were collected from each plot to quantify the amount of F. virguliforme DNA in the soybean roots using a real-time quantitative polymerase chain reaction (qPCR) protocol. ANOVA and Fisher’s LSD Test were used to separate treatment means. Analysis of the qPCR was done by comparing the quantification cycle (Cq) values and separating means using ANOVA. There were significant differences between varieties for soybean yield and disease index. Treatments containing fluopyram had more phytotoxicity than treatments lacking fluopyram at both locations. Several treatments allowed for higher SCN reproduction than treatments with Clariva. At Ina, a fluopyram treatment and one of the Clariva treatments had higher soybean yield than the non-treated control. At Shawneetown, one fluopyram treatment had higher soybean yield than all other treatments. In the qPCR analysis, seed treatments with fluopyram had a lower amount of F. virguliforme DNA in the roots than the non-treated control.

Fusarium virguliforme is a soil-borne pathogen that causes sudden death syndrome (SDS) disease in soybean. SDS is one of the most significant diseases of soybean in the United States. Fungal infection results in root and crown rot as well as SDS typical foliar symptoms including chlorosis, necrosis and complete defoliation. The use of soybean cultivars tolerant to SDS is still the most effective way to overcome the disease. On the other hand, the fungal isolates are known to have varied levels of aggressiveness on soybean indicated by the field and greenhouse experiments. Understanding the pathogen and its defense mechanism is the first step in exploring the pathogen-plant interaction. Therefore, the primary aim of this research was to elucidate the mechanism behind F. virguliforme response to soybean defense mechanisms. We further attempted to identify chromosome length polymorphism among F. virguliforme isolates and characterize the possible relationship to their level of aggressiveness. In order to fulfill the first objective, a series of differentially expressed genes were identified in F. virguliforme in the presence of soybean phytoalexin, glyceollin. The Fvgrx2 gene, a Saccharomyces cerevisiae grx2homologue, was selected for further analysis. This study demonstrates for the first time the identification and characterization of dithiol glutaredoxin gene in F. virguliforme . The role of FvGRX2 in the fungal defense to phytoalexin, glyceollin and induced oxidative burst was also investigated by generating anFvΔgrx2 knockout. In order to establish a link between the fungal karyotype and the level of fungal aggressiveness, the chromosome length polymorphism (CLP) was assessed for twenty-two F. virguliforme isolates exhibiting different levels of aggressiveness on soybean. The findings are instrumental in identifying novel pathogenicity such as the ones involved in phytotoxin production, fungicide resistance and aggressiveness.

AN ABSTRACT OF THE THESIS OF JESSE MILLER, for the Master of Science degree in PLANT, SOIL, and AGRICULTURAL SYSTEMS, presented on May 13, 2016 at Southern Illinois University Carbondale. TITLE: THE EFFECTS OF STARTER FERTILIZER ON SOYBEAN INFESTED WITH FUSARIUM VIRULIFORME OR RHIZOCTONIA SOLANI MAJOR PROFESSOR: Dr. Jason Bond Fusarium virguliforme (Aoki), the fungus that causes sudden death syndrome of soybeans (SDS), is prevalent in most of the soybean (Glycine max L. Merr.) production regions throughout the United States. Sudden death syndrome management has been limited to cultural practices and host resistance. Rhizoctonia solani (Kühn) is a fungus responsible for pre-emergence and post emergence damping off. Control methods include seed treatments and cultural practices. Several companies have advocated the use of in-furrow starter fertilizers in soybean production. Promoting root growth and emergence are a couple of the alleged benefits. It is unknown if the increased fertility in the root zone may actually increase or decrease the severity of root or seedling diseases. An objective of this study is to determine if the starter fertilizers (2-6-16), (7-12-11), (3-10-13) Nachurs Alpine Solutions™ impacts seedling disease caused by Rhizoctonia solani and soybean yield. A second objective is to determine if starter-fertilizer influences the incidence and severity of SDS and soybean yield. One trial was infested with R. solani at the rate of 0.9 g of inoculum/30.5 centimeters of row. A second trial was infested with F. virguliforme at the rate of 2.25 g/30.5 centimeters of row. Inoculum consisted of sterilized white sorghum inoculated with either pathogen. Plots were 3.04 meters wide by 6.1 meters in length with row spacing of 0.76 meters. Trials took place during the growing season of 2014 and 2015. In 2014, a randomized complete block design consisted of 4 treatments that were replicated 6 times and planted into 4 row plots. Treatments consisted of treated (Metalaxl™, Fluxapyroxad™, Pyraclostrobin™, and Imidacloprid™) or non-treated seed (‘Asgrow 4730’) combined with either fertilizer (2-6-16) or non-fertilizer. Across both trials, there were no seed treatment and fertilizer rate interactions. In the R. solani trial, stand counts were similar between the fertilizer and non-fertilizer treatments. Stand counts were higher when the seed treatment was used. There was no significant difference in soybean yield regardless of treatment. In the F. virguliforme trial, stand counts were reduced in the fertilizer treatment when compared to the non-fertilizer treatment. Foliar symptoms of SDS and soybean yield were not affected by treatment. In 2015, there were changes in treatment structure due to additions of fertilizer treatments 7-12-11 and 3-10-13. Seed treatments and randomized complete block design remained for 2015. Stand counts were higher in plots that received fertilizer treatments in the R. solani trial. Stand counts were lower in R. solani plots with treated seed. Yield was not influenced by seed treatment but was increased by 3-10-13 and 7-12-11 fertilizer treatments. For the F. virguliforme trial, reduced stand counts were found in the plots with seed treatments. Seed treatments did not influence yield. Fertilizer did not impact stand or yield. Foliar symptoms of SDS were not influenced by seed treatment or fertilizer.

Master of Science / Department of Agronomy / William T. Schapaugh Jr / Fusarium virguliforme, the soilborne fungus which causes sudden death syndrome (SDS) of soybean, and Heterodera glycines Ichinohe, soybean cyst nematode (SCN), are two economically important pathogens in the Midwest. The pathogens are often found together in soybean (Glycine max (L.) Merr.) fields. This study was conducted to determine the effect of soybean genotype, F. virguliforme populations, and H. glycines populations have upon yield and to examine the interaction between the two pathogens. In 2008 and 2009, four genotypes with different levels of resistance to SDS and H. glycines were planted at seven environments. F. virguliforme and H. glycines soil populations were quantified at planting, midseason, and harvest. At the end of the growing season, area under the disease progress curves of SDS, F. virguliforme root populations, and H. glycines reproductive indices were determined and plots harvested for seed yield. Soil populations of F. virguliforme and H. glycines at planting, midseason, and harvest varied across environments. Within environments, generally, they were not significantly different. Seed yield varied within and across environments. As disease pressure increased, the performance of resistant genotypes increased compared to susceptible genotypes. Genotypes resistant to SDS yielded higher than susceptible genotypes. There were negative correlations between yield and disease rating and F. virguliforme root populations. F. virguliforme soil populations and H. glycines populations at planting were positively correlated. It is important to manage both SDS and H. glycines in fields with a history of the two diseases. This can be achieved through genetic resistance. Information in this study will improve decisions regarding genotype selection to minimize losses to SDS and H. glycines.

Fusarium virguliforme (Aoki, O’Donnell, Homma & Lattanzi), the causal agent of sudden death syndrome (SDS) of soybean (Glycine max [L.] Merrill), is responsible for major soybean yield losses in North America and South America. Despite the importance of SDS, few agronomic practices have been used to manage SDS successfully. Understanding the pathogen and the mechanisms it uses to cause disease is vital to devise effective disease control strategies. However, our knowledge of the pathogenicity mechanisms used by F. virguliforme is limited. The identification of pathogenic genes will shed light on the molecular basis of the interaction between F.virguliforme and soybean, which may ultimately lead to better management of SDS. Therefore, the studies presented in this thesis were aimed at identifying and characterizing candidate pathogenicity genes in F. virguliforme.To fulfill this objective, 40 candidate pathogenicity genes of F. virguliforme were identified based on a combined approach, which included hands-on literature and database mining, functional genomics as well as transcriptome analyses. From these genes, the FvSNF1gene (a sucrose non-fermenting protein kinase 1 ortholog), the Fvstr1 gene (a striatin protein ortholog) were functionally characterized through a gene knock-out strategy. Targeted disruption of the FvSNF1 locus in wild type F. virguliforme strain reduced virulence significantly on soybean and abolished galactose utilization. In addition, the FvΔSNF1 mutant displayed significant reduction in expression of several CWDEs genes and was defective in colonizing the vascular system of the roots. To identify putative target genes regulated by FvSNF1, transcriptome analyses were performed in the FvSNF1 deletion mutant and in the wild-type. Disruption of FvSNF1 affected the level of transcription of 393 genes and a majority of the genes were involved in carbohydrate metabolism, lignin degradation, and cellular signaling pathway. The disruption of Fvstr1, a striatin ortholog in F. virguliforme, resulted in a complete loss of virulence as well as impaired conidiation, conidiophore development and pigmentation in the fungus. The FvΔstr1 mutant also failed to colonize the vascular tissues of roots of inoculated soybean plants. The results suggest that FvSNF1and Fvstr1 have critical roles in pathogenicity. Another part of the study was to investigate the efficacy of ILeVO®, a succinate dehydrogenase inhibitor fungicide from Bayer CropScience, against F. virguliforme. Our results showed that ILeVO® was very active against F. virguliforme in vitro and was very effective in minimizing F. virguliforme infection thus providing yet another tool to combat SDS.

Soybean crops are vulnerable to a wide range of pathogens that reduce yield and cause extensive losses worldwide. In the United States, the soilborne pathogens Pythium spp., causing soybean damping-off, and Fusarium virguliforme, causing sudden death syndrome (SDS) of soybean, have been among the top diseases that most reduced soybean yields. This study demonstrated that biological control using native fungal antagonists could be a powerful tool to integrate with current management strategies for more efficient control of Pythium damping-off and SDS in soybean. Trichoderma spp. and Clonostachys rosea demonstrated the ability to mycoparasitize and antagonize the pathogens using different mechanisms and exhibited a protective effect on soybean in field conditions. The development of an efficient biological control program for disease management relies on a deep understanding of the BCA-pathogen interaction’s biology. This research also uncovered the molecular mechanisms involved in the F. virguliforme-T. afroharzianum interaction by using a dual RNAseq approach. Significant changes in both fungal organisms’ transcriptomes were discovered at different stages in their interaction. The results provided here can contribute to the future implementation of effective biological control programs for soybean. The benefits may also extend to other crops.