Biocontrol Methods for the Management of Sclerotinia sclerotiorum in Legumes: A Review.

Sclerotinia sclerotiorum is an economically damaging fungal pathogen that causes Sclerotinia stem rot in legumes, producing enormous yield losses. This pathogen is difficult to control due to its wide host spectrum and ability to produce sclerotia, which are resistant bodies that can remain active for long periods under harsh environmental conditions. Here, the biocontrol methods for the management of S. sclerotiorum in legumes are reviewed. Bacillus strains, which synthesized lipopeptides and volatile organic compounds, showed high efficacies in soybean plants, whereas the highest efficacies for the control of the pathogen in alfalfa and common bean were observed when using Coniothyrium minitans and Streptomyces spp., respectively. The biocontrol efficacies in fields were under 65%, highlighting the lack of strategies to achieve a complete control. Overall, although most studies involved extensive screenings using different biocontrol agent concentrations and application conditions, there is a lack of knowledge regarding the specific antifungal mechanisms, which limits the optimization of the reported methods.

Screening of Diverse Lupinus spp. Highlights New Resistances to Sclerotinia sclerotiorum.

Stem rot caused by Sclerotinia sclerotiorum is a serious and sometimes devastating disease of lupin (Lupinus spp.). A total of 236 lupin accessions from across 12 Lupinus species were screened against the prevalent S. sclerotiorum isolate MBRS-1 (pathotype 76). L. angustifolius accession 21655 and L. albus var. albus accession 20589 showed immune and "near-immune" responses, respectively. Thirteen accessions of L. angustifolius, three accessions each of L. albus and L. albus var. albus, and a single accession each of L. albus var. graecus, L. mutabilis, L. palaestinus, and L. pilosus (totaling ∼4%) showed a highly resistant (HR) response. A further 19 accessions of L. angustifolius, 2 accessions each of L. albus and L. pilosus, and a single accession of L. mutabilis (totaling ∼10%) showed a resistant (R) response. The reactions of 16 (15 L. angustifolius, 1 L. digitatus) of these 236 accessions were also compared with their reactions to a different isolate, Walkaway-3 (WW-3; pathotype 10). Against this isolate, five L. angustifolius accessions showed an HR response and four showed an R response, and the L. digitatus accession showed a moderate resistance response. Overall, isolate WW-3 caused significantly (P < 0.05) smaller lesions than MBRS-1 across tested accessions in common. In addition, 328 plants in a "wild" naturalized field population of L. cosentinii were screened in situ in the field against isolate MBRS-1. Five (∼1.5%) of the 328 plants of wild lupin showed an immune response, 63 (∼19%) showed an HR response, and 146 (∼45%) showed an R response. We believe this is the first examination of diverse Lupinus spp. germplasm responses to a prevalent pathotype of S. sclerotiorum. Lupin genotypes exhibiting high-level resistance to Sclerotinia stem rot identified in this study can be used as parental lines for crosses in lupin breeding programs and/or directly as improved cultivars to reduce the adverse impact of this disease on lupin crops.

Variation in Pathogenicity and Subsequent Production of Sclerotia of Sclerotinia sclerotiorum Isolates in Different Cover Crops, Flower Strips, and Weeds.

Cover crops and flower strips are used in agricultural fields as part of integrated pest management strategies. However, their potential as secondary hosts of soilborne pathogens such as Sclerotinia sclerotiorum in oilseed rape cultivation is not fully comprehended. In the current study, we evaluated the effect of pathogen virulence on the development of Sclerotinia stem/leaf rot and sclerotia production in 33 plant species from 11 botanical families using two S. sclerotiorum isolates. Furthermore, the effect of sclerotial size on carpogenic germination was studied. Results showed that the pathogen's virulence significantly affected the occurrence and development of Sclerotinia stem/leaf rot and the subsequent production of sclerotia. Among all plant species tested, 26 were more susceptible to the highly aggressive S. sclerotiorum isolate, which produced more and bigger sclerotia in 17 species than the less aggressive isolate. Moreover, a stronger positive correlation was found between the relative lesion length of plants inoculated with the highly aggressive isolate and the number of sclerotia produced by this isolate (Spearman's rank correlation coefficient [rs] = 0.572; P = 0.004). Additionally, we found that larger and heavier sclerotia produced stipes and apothecia earlier and at a greater rate than smaller ones. The heavyweight class had the highest carpogenic germination rate (82.4%), followed by the average (67.2%) and lightweight classes (59.5%). Our findings highlight the need for further investigation into the potential risks associated with cover crops, weeds, and flower strips as secondary hosts of soilborne pathogens in agricultural fields.

Association Mapping Combined with Whole Genome Sequencing Data Reveals Candidate Causal Variants for Sclerotinia Stem Rot Resistance in Brassica napus.

Canola (Brassica napus) yield can be significantly reduced by the disease sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum, a necrotrophic fungal pathogen with an unusually large host range. Breeding cultivars that are physiologically resistant to SSR is desirable to enhance crop productivity. However, the development of resistant varieties has proved challenging due to the highly polygenic nature of S. sclerotiorum resistance. Here, we identified regions of the B. napus genome associated with SSR resistance using data from a previous study by association mapping. We then validated their contribution to resistance in a follow-up screen. This follow-up screen also confirmed high levels of SSR resistance in several genotypes from the previous study. Using publicly available whole genome sequencing data for a panel of 83 B. napus genotypes, we identified nonsynonymous polymorphisms linked to the SSR resistance loci. A qPCR analysis showed that two of the genes containing these polymorphisms were transcriptionally responsive to S. sclerotiorum infection. In addition, we provide evidence that homologues of three of the candidate genes contribute to resistance in the model Brassicaceae species Arabidopsis thaliana. The identification of resistant germplasm and candidate genomic loci associated with resistance are important findings that can be exploited by breeders to improve the genetic resistance of canola varieties.

Integration of Soybean (Glycine max) Resistance Levels to Sclerotinia Stem Rot into Predictive Sclerotinia sclerotiorum Apothecial Models.

Sclerotinia stem rot (SSR) is a major disease of soybean across the Upper Midwest region of the United States. Management of this disease has relied on fungicide applications, but due to the environmental conditions necessary for SSR to develop, many of these applications are unnecessary. To mitigate this, predictive models have been developed using localized weather data for predicting the formation of Sclerotinia sclerotiorum apothecia, the inoculum source of SSR, and these models were integrated into a decision support system called Sporecaster. However, these models do not account for the soybean resistance levels to SSR. In this study, fungicide trials were performed across seven site-years in Wisconsin between 2020 and 2022 examining fungicide applications applied at one of three action thresholds (low, moderate, and high) following Sporecaster recommendations in combination with four soybean varieties representing three SSR resistance levels (susceptible, moderately resistant, and resistant). From these trials, the low and moderate action thresholds resulted in similarly low disease severity index (DIX) levels comparable to the standard across all varieties. However, the low action threshold was most accurate for predicting SSR development in the susceptible variety, and the high action threshold was most accurate for predicting SSR development for the three resistant varieties. Both the susceptible soybean and a moderately resistant line yielded similarly high results. Additionally, the use of all fungicide applications led to similar partial profits at grain sale prices of either $0.44 or $0.55 kg-1. Overall, this study uncovered relationships between soybean resistance levels to SSR and Sporecaster, allowing for improved recommendations for fungicide applications.

Managing White Mold on Common Bean with Type III Growth Habit by Integrating Partial Resistance, Plant Density, and Fungicide.

The combination of plant density, fungicide, and levels of genotype resistance to white mold (WM) has not been studied for its effect on performance of common bean with type III growth habit under WM pressure. We established four sprinkler-irrigated trials in the fall to winter season in Brazil, combining density (8, 14, 20, or 26 plants/m2), genotype (susceptible or partially resistant to WM), and fungicide to manage WM (two fluazinam applications or without fluazinam) at 0.5 m row spacing in fields infested with sclerotia. One trial was established in a warm region, aiming to obtain low WM pressure. For three trials, the linear mixed model was used. WM pressure in the trials ranged from low to high. Genotype × density × fungicide and genotype × density interactions were not significant for incidence, severity, and yield for all trials. In the three trials, severity was 29% lower at eight than at 26 plants/m2, and yield at 14 plants/m2 (3,294 kg/ha) was 14% higher than yield at eight plants/m2 (P = 0.035) where fluazinam was applied. However, without fluazinam, density did not affect yield. In conclusion, choices of plant density for type III bean cultivars do not need to take into account their resistance levels to WM in the fall to winter season. However, fungicide treatment to manage WM affects the choice of density, with 8 to 12 plants/m2 recommended if fungicide is not an option to manage WM (as in organic systems), and 10 to 14 plants/m2 recommended if fungicide applications are likely.

Quantitative Inheritance of Sclerotinia Stem Rot Resistance in Brassica napus and Relationship to Cotyledon and Leaf Resistances.

Sclerotinia sclerotiorum is a necrotrophic fungus causing devastating stem rot and associated yield losses of canola/rapeseed (Brassica napus) worldwide, including in Australia. Developing host resistance against Sclerotinia stem rot is critical if this disease in canola/rapeseed is to be successfully managed, as cultural or chemical control options provide only partial or sporadic control. Three B. napus breeding populations, C2, C5 and C6, including the parents, F1, F2, BC1P1, and BC2P2, were used in a field study with an objective of exploring the inheritance pattern of disease resistance (based on stem lesion length [SLL]) and the genetic relationships of disease with stem diameter (SD) or days to first flowering (DTF), and to compare these new adult plant stem resistances against S. sclerotiorum with those of seedling (cotyledon and leaf) resistances in earlier studies. Heritability (broad sense) of SLL was 0.57 and 0.73 for population C2 at 3 and 5 weeks postinoculation and 0.21 for population C5 at 5 weeks postinoculation. Additive genetic variance was evident within all 3 populations for DTF but not for SD. Narrow-sense heritability for DTF was 0.48 (C2), 0.42 (C5), and 0.32 (C6). SD, DTF, and SLL were all inherited independently, with no significant genetic covariance between traits in bivariate analysis. Genetic variance for SLL in populations C2 and C5 was entirely nonadditive, and there was significant nonadditive genetic covariance of SLL at 3 and 5 weeks postinoculation. Generation means analysis in population C2 supported the conclusion that complex epistatic interactions controlled SLL. Several C2 and C5 progeny showed high adult plant stem resistance, which may be critical in developing enhanced stem resistance in canola/rapeseed. Although population C6 showed no genetic variation for SLL resistance in this study, it showed significant nonadditive genetic variance at the cotyledon and leaf stages in earlier studies. We conclude that host resistance varies across different plant growth stages, and breeding must be targeted for resistance at each growth stage. In populations C2, C5, and C6, resistance to S. sclerotiorum in stem, leaf, and cotyledon was always controlled by nonadditive effects such as complex epistasis or dominance. Overall, our findings in relation to the quantitative inheritance of Sclerotinia stem rot resistance, together with the new high-level resistances identified, will enable breeders to select/develop genotypes with enhanced resistances to S. sclerotiorum.

Genetic dissection of soybean partial resistance to sclerotinia stem rot through genome wide association study and high throughout single nucleotide polymorphisms.

Sclerotinia stem rot (SSR) is a disease of soybean [Glycine max (L.) Merr] that causes severe yield losses. We studied 185 representative soybean accessions to evaluate partial SSR resistance and sequenced these by the specific-locus amplified fragment sequencing method. In total, 22,048 single-nucleotide polymorphisms (SNPs), with minor allele frequencies (MAF) ≥5% and missing data <3%, were developed and applied to genome-wide association study of SSR responsiveness and assess linkage disequilibrium (LD) level for candidate gene selection. We identified 18 association signals related to SSR partial resistance. Among them, six overlapped the regions of previous quantitative trait loci, and twelve were novel. We identified 243 candidate genes located in the 200 kb genomic region of these peak SNPs. Based on quantitative real-time polymerase chain reaction and haplotype analysis, Glyma.03G196000 and Glyma.20G095100, encoding pentatricopeptide repeat proteins, might be important factors in the resistance response of soybean to SSR.

MicroRNA319a regulates plant resistance to Sclerotinia stem rot.

MicroRNA319a (miR319a) controls cell division arrest in plant leaves by inhibiting the expression of TCP (TEOSINTE BRANCHED 1/CYCLOIDEA/PCF) family genes. However, it is unclear whether miR319a influences infection by necrotrophic pathogens and host susceptibility. In this study, we revealed that miR319a affects plant resistance to stem rot disease caused by Sclerotinia sclerotiorum. In Brassica rapa plants infected with S. sclerotiorum, miR319a levels increased while the expression levels of several BraTCP genes significantly decreased compared with those of uninfected plants. Overexpression of BraMIR319a in B. rapa increased the susceptibility of the plants to S. sclerotiorum and aggravated stem rot disease, whereas overexpression of BraTCP4-1 promoted plant resistance. RNA sequencing data revealed a potential relationship between miR319a and pathogen-related WRKY genes. Chromatin immunoprecipitation, electrophoretic mobility shift, and reporter transaction assays showed that BraTCP4-1 could bind to the promoters of WRKY75, WRKY70, and WRKY33 and directly activate these pathogen-related genes. Moreover, the expression levels of WRKY75, WRKY70, and WRKY33 in plants overexpressing BraMIR319a decreased significantly, whereas those of plants overexpressing BraTCP4-1 increased significantly, relative to the wild type. These results suggest that miR319a and its target gene BraTCP4 control stem rot resistance through pathways of WRKY genes.

Expression of barley oxalate oxidase confers resistance against Sclerotinia sclerotiorum in transgenic Brassica juncea cv Varuna.

Sclerotinia Stem Rot (SSR) caused by the oxalic acid (OA)-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, causes significant yields losses in the crop Brassica sps. Oxalate oxidase (OxO) can metabolize OA to CO2 and H2O2. Degradation of OA during the early phase of fungal-host interaction can interfere with the fungal infection and establishment processes. The present study demonstrates the potential of barley oxalate oxidase (BOxO) gene in conferring stable resistance against stem rot in a productive and highly susceptible Brassica juncea cv Varuna under field conditions. Four stable, independent, single-copy transgenic lines (B16, B17, B18, and B53) exhibited a significant reduction in the rate of lesion expansion i.e. 11-26%, 39-47%, and 24-35% reproducibly over the three-generation i.e. T2, T3, and T4 respectively. The enhanced resistance in the transgenic lines correlated with high OxO activity, accumulation of higher levels of H2O2, and robust activation of defense responsive genes upon infection by S. sclerotiorum.