Gene regulation of Sclerotinia sclerotiorum during infection of Glycine max: on the road to pathogenesis.

BACKGROUND: Sclerotinia sclerotiorum is a broad-host range necrotrophic pathogen which is the causative agent of Sclerotinia stem rot (SSR), and a major disease of soybean (Glycine max). A time course transcriptomic analysis was performed in both compatible and incompatible soybean lines to identify pathogenicity and developmental factors utilized by S. sclerotiorum to achieve pathogenic success. RESULTS: A comparison of genes expressed during early infection identified the potential importance of toxin efflux and nitrogen metabolism during the early stages of disease establishment. The later stages of infection were characterized by an apparent shift to survival structure formation. Analysis of genes highly upregulated in-planta revealed a temporal regulation of hydrolytic and detoxification enzymes, putative secreted effectors, and secondary metabolite synthesis genes. Redox regulation also appears to play a key role during the course of infection, as suggested by the high expression of genes involved in reactive oxygen species production and scavenging. Finally, distinct differences in early gene expression were noted based on the comparison of S. sclerotiorum infection of resistant and susceptible soybean lines. CONCLUSIONS: Although many potential virulence factors have been noted in the S. sclerotiorum pathosystem, this study serves to highlight soybean specific processes most likely to be critical in successful infection. Functional studies of genes identified in this work are needed to confirm their importance to disease development, and may constitute valuable targets of RNAi approaches to improve resistance to SSR.

Resistance against Sclerotinia sclerotiorum in soybean involves a reprogramming of the phenylpropanoid pathway and up-regulation of antifungal activity targeting ergosterol biosynthesis.

Sclerotinia sclerotiorum, a predominately necrotrophic fungal pathogen with a broad host range, causes a significant yield-limiting disease of soybean called Sclerotinia stem rot. Resistance mechanisms against this pathogen in soybean are poorly understood, thus hindering the commercial deployment of resistant varieties. We used a multiomic approach utilizing RNA-sequencing, gas chromatography-mass spectrometry-based metabolomics and chemical genomics in yeast to decipher the molecular mechanisms governing resistance to S. sclerotiorum in soybean. Transcripts and metabolites of two soybean recombinant inbred lines, one resistant and one susceptible to S. sclerotiorum were analysed in a time course experiment. The combined results show that resistance to S. sclerotiorum in soybean is associated in part with an early accumulation of JA-Ile ((+)-7-iso-jasmonoyl-L-isoleucine), a bioactive jasmonate, increased ability to scavenge reactive oxygen species, and importantly, a reprogramming of the phenylpropanoid pathway leading to increased antifungal activities. Indeed, we noted that phenylpropanoid pathway intermediates, such as 4-hydroxybenzoate, cinnamic acid, ferulic acid and caffeic acid, were highly accumulated in the resistant line. In vitro assays show that these metabolites and total stem extracts from the resistant line clearly affect S. sclerotiorum growth and development. Using chemical genomics in yeast, we further show that this antifungal activity targets ergosterol biosynthesis in the fungus, by disrupting enzymes involved in lipid and sterol biosynthesis. Overall, our results are consistent with a model where resistance to S. sclerotiorum in soybean coincides with an early recognition of the pathogen, leading to the modulation of the redox capacity of the host and the production of antifungal metabolites.

Soybean Resistance to White Mold: Evaluation of Soybean Germplasm Under Different Conditions and Validation of QTL.

Soybean (Glycine max L. Merr.) white mold (SWM), caused by Sclerotinia sclerotiorum (Lib) de Barry), is a devastating fungal disease in the Upper Midwest of the United States and southern Canada. Various methods exist to evaluate for SWM resistance and many quantitative trait loci (QTL) with minor effect governing SWM resistance have been identified in prior studies. This study aimed to predict field resistance to SWM using low-cost and efficient greenhouse inoculation methods and to confirm the QTL reported in previous studies. Three related but independent studies were conducted in the field, greenhouse, and laboratory to evaluate for SWM resistance. The first study evaluated 66 soybean plant introductions (PIs) with known field resistance to SWM using the greenhouse drop-mycelium inoculation method. These 66 PIs were significantly (P < 0.043) different for resistance to SWM. However, year was highly significant (P < 0.00001), while PI x year interaction was not significant (P < 0.623). The second study compared plant mortality (PM) of 35 soybean breeding lines or varieties in greenhouse inoculation methods with disease severity index (DSI) in field evaluations. Moderate correlation (r) between PM under drop-mycelium method and DSI in field trials (r = 0.65, p < 0.0001) was obtained. The PM under spray-mycelium was also correlated significantly with DSI from field trials (r = 0.51, p < 0.0018). Likewise, significant correlation (r = 0.62, p < 0.0001) was obtained between PM across greenhouse inoculation methods and DSI across field trials. These findings suggest that greenhouse inoculation methods could predict the field resistance to SWM. The third study attempted to validate 33 QTL reported in prior studies using seven populations that comprised a total of 392 F4 : 6 lines derived from crosses involving a partially resistant cultivar "Skylla," five partially resistant PIs, and a known susceptible cultivar "E00290." The estimates of broad-sense heritability (h2) ranged from 0.39 to 0.66 in the populations. Of the seven populations, four had h2 estimates that were significantly different from zero (p < 0.05). Single marker analysis across populations and inoculation methods identified 11 significant SSRs (p < 0.05) corresponding to 10 QTL identified by prior studies. Thus, these five new PIs could be used as new sources of resistant alleles to develop SWM resistant commercial cultivars.

Development and Evaluation of Glycine max Germplasm Lines with Quantitative Resistance to Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot, is a devastating fungal pathogen of soybean that can cause significant yield losses to growers when environmental conditions are favorable for the disease. The development of resistant varieties has proven difficult. However, poor resistance in commercial cultivars can be improved through additional breeding efforts and understanding the genetic basis of resistance. The objective of this project was to develop soybean germplasm lines that have a high level of Sclerotinia stem rot resistance to be used directly as cultivars or in breeding programs as a source of improved Sclerotinia stem rot resistance. Sclerotinia stem rot-resistant soybean germplasm was developed by crossing two sources of resistance, W04-1002 and AxN-1-55, with lines exhibiting resistance to Heterodera glycines and Cadophora gregata in addition to favorable agronomic traits. Following greenhouse evaluations of 1,076 inbred lines derived from these crosses, 31 lines were evaluated for resistance in field tests during the 2014 field season. Subsequently, 11 Sclerotinia stem rot resistant breeding lines were moved forward for field evaluation in 2015, and seven elite breeding lines were selected and evaluated in the 2016 field season. To better understand resistance mechanisms, a marker analysis was conducted to identify quantitative trait loci linked to resistance. Thirteen markers associated with Sclerotinia stem rot resistance were identified on chromosomes 15, 16, 17, 18, and 19. Our markers confirm previously reported chromosomal regions associated with Sclerotinia stem rot resistance as well as a novel region of chromosome 16. The seven elite germplasm lines were also re-evaluated within a greenhouse setting using a cut petiole technique with multiple S. sclerotiorum isolates to test the durability of physiological resistance of the lines in a controlled environment. This work presents a novel and comprehensive classical breeding method for selecting lines with physiological resistance to Sclerotinia stem rot and a range of agronomic traits. In these studies, we identify four germplasm lines; 91-38, 51-23, SSR51-70, and 52-82B exhibiting a high level of Sclerotinia stem rot resistance combined with desirable agronomic traits, including high protein and oil contents. The germplasm identified in this study will serve as a valuable source of physiological resistance to Sclerotinia stem rot that could be improved through further breeding to generate high-yielding commercial soybean cultivars.

Main and epistatic loci studies in soybean for Sclerotinia sclerotiorum resistance reveal multiple modes of resistance in multi-environments.

Genome-wide association (GWAS) and epistatic (GWES) studies along with expression studies in soybean [Glycine max (L.) Merr.] were leveraged to dissect the genetics of Sclerotinia stem rot (SSR) [caused by Sclerotinia sclerotiorum (Lib.) de Bary], a significant fungal disease causing yield and quality losses. A large association panel of 466 diverse plant introduction accessions were phenotyped in multiple field and controlled environments to: (1) discover sources of resistance, (2) identify SNPs associated with resistance, and (3) determine putative candidate genes to elucidate the mode of resistance. We report 58 significant main effect loci and 24 significant epistatic interactions associated with SSR resistance, with candidate genes involved in a wide range of processes including cell wall structure, hormone signaling, and sugar allocation related to plant immunity, revealing the complex nature of SSR resistance. Putative candidate genes [for example, PHYTOALEXIN DEFFICIENT 4 (PAD4), ETHYLENE-INSENSITIVE 3-LIKE 1 (EIL3), and ETHYLENE RESPONSE FACTOR 1 (ERF1)] clustered into salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways suggest the involvement of a complex hormonal network typically activated by both necrotrophic (ET/JA) and biotrophic (SA) pathogens supporting that S. sclerotiorum is a hemibiotrophic plant pathogen.

Molecular Identification of Sclerotinia trifoliorum and Sclerotinia sclerotiorum Isolates from the United States and Poland.

Symptoms of clover rot caused by Sclerotinia trifoliorum or S. sclerotiorum are identical, making differentiation and identification of the causal species difficult and time consuming. Polymerase chain reaction (PCR) amplification and nucleotide sequencing were used to examine 40 isolates of S. trifoliorum (29 from Poland, 11 from the United States) and 55 isolates of S. sclerotiorum (26 from Poland, 29 from the United States). We determined that amplification of the ß-tubulin and calmodulin genes with TU1/TU2/TU3 and SscadF1/SscadR1 PCR primers and the presence of introns and single-nucleotide polymorphisms (SNP) within the ribosomal DNA (rDNA) as detected with NS1/NS8 and internal transcribed spacer (ITS)1/ITS4 PCR primers are effective for rapidly and accurately differentiating between the two species of Sclerotinia. In addition, our research revealed a lack of intraspecies variation within S. sclerotiorum isolates from the United States and Poland using these same molecular markers. We detected a relatively high degree of intraspecies variability among isolates of S. trifoliorum from the United States and Poland using the presence of introns and SNP within the rDNA. SNP and nuclear small-subunit rDNA analyses revealed distinct groups of S. trifoliorum among the isolates used in this study. The results of this study provide useful information for clover breeders and pathologists looking to develop clover varieties with durable resistance.

Thiophanate-Methyl and Propiconazole Sensitivity in Sclerotinia homoeocarpa Populations from Golf Courses in Wisconsin and Massachusetts.

Management of dollar spot, caused by the fungus Sclerotinia homoeocarpa, is dependent upon repeated fungicide applications in intensively managed turfgrass such as golf course putting greens and fairways. Repeated fungicide applications could potentially select for fungicide-resistant isolates and result in a reduction of disease control. The objectives of this study were to determine the degree of S. homoeocarpa in vitro sensitivity to the fungicides thiophanate-methyl and propiconazole using isolates collected from golf course putting greens, fairways, and roughs; and to determine the relationships of golf course age and fungicide history to the frequency of fungicide-insensitive isolates within the population. More than 1,400 S. homoeocarpa isolates were collected from putting greens, fairways, and roughs at six Wisconsin golf courses and one Massachusetts golf course and subjected to in vitro fungicide sensitivity assays with single discriminatory concentrations of thiophanate-methyl and propiconazole. Five of seven pathogen populations from rough areas were not significantly different from one another in propiconazole sensitivity. These populations were collectively the most sensitive to both fungicides and therefore, served as baseline populations for comparison with fungicide-exposed populations from putting greens and fairways. Greater propiconazole insensitivity was observed in populations collected from fairways and putting greens that received more frequent applications of the fungicide than those isolated from the roughs. In nearly all the golf courses, the frequency of thiophanate-methyl insensitivity was higher among isolates of S. homoeocarpa collected from fairways than from roughs regardless of the age of the golf course or history of benzimidazole use. Thus, while the development of resistance to propiconazole can be predicted in part by the relative frequency of demethylation inhibitor fungicide applications, the occurrence of populations resistant to thiophanate-methyl appears to be unrelated to recent use of the benzimidazole class of fungicides.

A Strain of Clover yellow vein virus that Causes Severe Pod Necrosis Disease in Snap Bean.

Soybean aphid (Aphis glycines) outbreaks occurring since 2000 have been associated with severe virus epidemics in snap bean (Phaseolus vulgaris) production in the Great Lakes region. Our objective was to identify specific viruses associated with the disease complex observed in the region and to survey bean germplasm for sources of resistance to the causal agents. The principle causal agent of the disease complex associated with extensive pod necrosis was identified as Clover yellow vein virus (ClYVV), designated ClYVV-WI. The virus alone caused severe mosaic, apical necrosis, and stunting. Putative coat protein amino acid sequence from clones of amplicons generated by reverse-transcription polymerase chain reaction was 98% identical to ClYVV strain no. 30 identified in Japan that has not been reported to cause pod necrosis. ClYVV-WI amplicons were 96% identical to a mild strain of ClYVV from Oregon. A distinguishing feature of this new strain is that it does not react with Potyvirus broad-spectrum monoclonal antibody PTY 1. A survey of common bean lines and cultivars revealed that, in addition to UI-31 and US1140 with known resistance to ClYVV, lines with the bc-3 gene for resistance to Bean common mosaic necrosis virus also were resistant to ClYVV-WI. An evaluation of 63 snap bean cultivars and breeding lines revealed just one, Roma 442, with a moderate level of tolerance to ClYVV-WI. Introgression of the bc-3 gene and resistances from UI-31 and US1140 into snap bean may offer a high level of resistance to extensive pod necrosis disease caused by ClYVV in the Great Lakes region.

Analysis of gene expression profiles in response to Sclerotinia sclerotiorum in Brassica napus.

Sclerotinia sclerotiorum is a necrotrophic plant pathogen which causes serious disease in agronomically important crop species. The molecular basis of plant defense to this pathogen is poorly understood. We investigated gene expression changes associated with S. sclerotiorum infection in a partially resistant and a susceptible genotype of oilseed Brassica napus using a whole genome microarray from Arabidopsis. A total of 686 and 1,547 genes were found to be differentially expressed after infection in the resistant and susceptible genotypes, respectively. The number of differentially expressed genes increased over infection time with the majority being up-regulated in both genotypes. The putative functions of the differentially expressed genes included pathogenesis-related (PR) proteins, proteins involved in the oxidative burst, protein kinase, molecule transporters, cell maintenance and development, abiotic stress, as well as proteins with unknown functions. The gene regulation patterns indicated that a large part of the defense response exhibited as a temporal and quantitative difference between the two genotypes. Genes associated with jasmonic acid (JA) and ethylene signal transduction pathways were induced, but no salicylic acid (SA) responsive genes were identified. Candidate defense genes were identified by integration of the early response genes in the partially resistant line with previously mapped quantitative trait loci (QTL). Expression levels of these genes were verified by Northern blot analyses. These results indicate that genes encoding various proteins involved in diverse roles, particularly WRKY transcription factors and plant cell wall related proteins may play an important role in the defense response to S. sclerotiorum disease.

Seasonal Progression, Symptom Development, and Yield Effects of Alfalfa mosaic virus Epidemics on Soybean in Wisconsin.

The occurrence of Alfalfa mosaic virus (AMV) has increased in Wisconsin soybean fields in conjunction with the establishment of the soybean aphid (Aphis glycines). Field studies were conducted to determine the seasonal incidence of AMV-infected plants, progression of symptom severity caused by AMV, and the effect of AMV on soybean yield and seed quality. An isolate of AMV, collected from field-grown soybean, was introduced into plots by mechanical inoculation of plants at the V1 growth stage. The goal of the experiment was to achieve an incidence of AMV-infected plants of 0, 50, and 100% in 2002, and 0, 10, 25, 50, 75, and 100% in 2003. Severity of foliar symptoms was rated, and leaf samples were collected for serological assay (enzyme-linked immunosorbent assay [ELISA]) to estimate the incidence of AMV-infected plants from growth stages VC to R5. The maximum incidence of AMV-infected plants was 49% at growth stage R5, yet detection of the virus by ELISA dropped dramatically thereafter in both years. Incidence of AMV-infected plants accounted for 53 to 67% of the variability observed for severity of foliar symptoms in 2002 and 2003, respectively. Maximum yield loss ranged from 32% in 2002 to 48% in 2003 based on the difference in yield between noninoculated plots and plots with the highest incidence of AMV-infected plants. Incidence of AMV-infected plants explained 31% of the variation in yield in 2002 and 26% in 2003. An AMV incidence of 30% or greater was required for yield loss caused by AMV. Results of this study suggest that AMV has the potential to lower soybean yield and warrants further study.

Quantitative trait loci for resistance to Sclerotinia sclerotiorum and its association with a homeologous non-reciprocal transposition in Brassica napus L.

Sclerotinia stem rot, caused by fungus Sclerotinia sclerotiorum, is one of the most devastating diseases in rapeseed (Brassica napus L.). We report the identification of Quantitative trait loci (QTL) involved in the resistance to S. sclerotiorum in two segregating populations of DH lines: the HUA population, derived from a cross between a partially resistant Chinese winter line (Hua dbl2) and a susceptible European spring line (P1804); and the MS population, derived from a partially resistant French winter cultivar (Major) and a susceptible Canadian spring cultivar (Stellar). A petiole inoculation technique and two scoring methods, days to wilt (DW) and stem lesion length (SLL), were used for the resistance assessment. A total of eight genomic regions affecting resistance were detected in the HUA population, with four of these regions affecting both measures of resistance. Only one region, which affected both measurements, was detected in the MS population. Individual QTL explained 6-22% of the variance. At five of the QTL from both populations, alleles from the resistant parent contributed to the resistance. QTL on N2 from the HUA population had the highest LOD score and R (2) value and was detected for SLL in the first evaluation. The N12 resistance allele in Hua dbl2 was detected in a region containing a homeologous non-reciprocal transposition (HNRT) from the resistance-containing portion of N2. This result suggests that QTL in the N12.N2 HNRT enhanced the resistance of Hua dbl2 by increasing the dosage of resistance genes. The relationship of QTL from different genetic backgrounds and their associations with other agronomic traits are discussed.