Deciphering genetic factors contributing to enhanced resistance against Cercospora leaf blight in soybean (Glycine max L.) using GWAS analysis.

Cercospora leaf blight (CLB), caused by Cercospora cf. flagellaris, C. kikuchii, and C. cf. sigesbeckiae, is a significant soybean [Glycine max (L.) Merr.] disease in regions with hot and humid conditions causing yield loss in the United States and Canada. There is limited information regarding resistant soybean cultivars, and there have been marginal efforts to identify the genomic regions underlying resistance to CLB. A Genome-Wide Association Study was conducted using a diverse panel of 460 soybean accessions from maturity groups III to VII to identify the genomic regions associated to the CLB disease. These accessions were evaluated for CLB in different regions of the southeastern United States over 3 years. In total, the study identified 99 Single Nucleotide Polymorphism (SNPs) associated with the disease severity and 85 SNPs associated with disease incidence. Across multiple environments, 47 disease severity SNPs and 23 incidence SNPs were common. Candidate genes within 10 kb of these SNPs were involved in biotic and abiotic stress pathways. This information will contribute to the development of resistant soybean germplasm. Further research is warranted to study the effect of pyramiding desirable genomic regions and investigate the role of identified genes in soybean CLB resistance.

Characterization of QoI-Fungicide Resistance in Cercospora Isolates Associated with Cercospora Leaf Blight of Soybean from the Southern United States.

Cercospora leaf blight (CLB) of soybean, caused by Cercospora cf. flagellaris, C. kikuchii, and C. cf. sigesbeckiae, is an economically important disease in the southern United States. Cultivar resistance to CLB is inconsistent; therefore, fungicides in the quinone outside inhibitor (QoI) class have been relied on to manage the disease. Approximately 620 isolates from plants exhibiting CLB were collected between 2018 and 2021 from 19 locations in eight southern states. A novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on two genes, calmodulin and histone h3, was developed to differentiate between the dominant species of Cercospora, C. cf. flagellaris, and C. cf. sigesbeckiae. A multilocus phylogenetic analysis of actin, calmodulin, histone h3, ITS rDNA, and transcription elongation factor 1-α was used to confirm PCR-RFLP results and identify remaining isolates. Approximately 80% of the isolates collected were identified as C. cf. flagellaris, while 15% classified as C. cf. sigesbeckiae, 2% as C. kikuchii, and 3% as previously unreported Cercospora species associated with CLB in the United States. PCR-RFLP of cytochrome b (cytb) identified QoI-resistance conferred by the G143A substitution. Approximately 64 to 83% of isolates were determined to be QoI-resistant, and all contained the G143A substitution. Results of discriminatory dose assays using azoxystrobin (1 ppm) were 100% consistent with PCR-RFLP results. To our knowledge, this constitutes the first report of QoI resistance in CLB pathogen populations from Alabama, Arkansas, Kentucky, Mississippi, Missouri, Tennessee, and Texas. In areas where high frequencies of resistance have been identified, QoI fungicides should be avoided, and fungicide products with alternative modes-of-action should be utilized in the absence of CLB-resistant soybean cultivars.

Identification of new regulatory genes involved in the pathogenic functions of the rice-pathogenic bacterium Burkholderia glumae.

Burkholderia glumae is an emerging plant-pathogenic bacterium that causes disease in rice in several of the major rice-producing areas throughout the world. In the southern United States, B. glumae is the major causal agent of bacterial panicle blight of rice and has caused severe yield losses in recent decades. Despite its importance, few management options are available for diseases caused by B. glumae, and knowledge of how this pathogen causes disease is limited. In an effort to identify novel factors that contribute to the pathogenicity of B. glumae, random mutagenesis using the miniTn5gus transposon was performed on two strains of B. glumae. Resultant mutants were screened in the laboratory for altered phenotypes in various known or putative virulence factors, including toxoflavin, lipase and extracellular polysaccharides. Mutants that exhibited altered phenotypes compared to their parent strain were selected and subsequently characterized using a PCR-based method to identify the approximate location of the transposon insertion. Altogether, approximately 20 000 random mutants were screened and 51 different genes were identified as having potential involvement in the production of toxoflavin, lipase and/or extracellular polysaccharide. Especially, two regulatory genes, ntpR and tepR, encoding a LysR-type transcriptional regulator and a σ54-dependent response regulator, respectively, were discovered in this study as new negative regulatory factors for the production of toxoflavin, the major phytotoxin synthesized by B. glumae and involved in bacterial pathogenesis.

Biological Control Activities of Rice-Associated Bacillus sp. Strains against Sheath Blight and Bacterial Panicle Blight of Rice.

Potential biological control agents for two major rice diseases, sheath blight and bacterial panicle blight, were isolated from rice plants in this study. Rice-associated bacteria (RABs) isolated from rice plants grown in the field were tested for their antagonistic activities against the rice pathogens, Rhizoctonia solani and Burkholderia glumae, which cause sheath blight and bacterial panicle blight, respectively. Twenty-nine RABs were initially screened based on their antagonistic activities against both R. solani and B. glumae. In follow-up retests, 26 RABs of the 29 RABs were confirmed to have antimicrobial activities, but the rest three RABs did not reproduce any observable antagonistic activity against R. solani or B. glumae. According to16S rDNA sequence identity, 12 of the 26 antagonistic RABs were closest to Bacillus amyloliquefaciens, while seven RABs were to B. methylotrophicus and B, subtilis, respectively. The 16S rDNA sequences of the three non-antagonistic RABs were closest to Lysinibacillus sphaericus (RAB1 and RAB12) and Lysinibacillus macroides (RAB5). The five selected RABs showing highest antimicrobial activities (RAB6, RAB9, RAB16, RAB17S, and RAB18) were closest to B. amyloliquefaciens in DNA sequence of 16S rDNA and gyrB, but to B. subtilis in that of recA. These RABs were observed to inhibit the sclerotial germination of R. solani on potato dextrose agar and the lesion development on detached rice leaves by artificial inoculation of R. solani. These antagonistic RABs also significantly suppressed the disease development of sheath blight and bacterial panicle blight in a field condition, suggesting that they can be potential biological control agents for these rice diseases. However, these antagonistic RABs showed diminished disease suppression activities in the repeated field trial conducted in the following year probably due to their reduced antagonistic activities to the pathogens during the long-term storage in -70C, suggesting that development of proper storage methods to maintain antagonistic activity is as crucial as identification of new biological control agents.

Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae.

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.