Thiosulfinate Tolerance Gene Clusters Are Common Features of Burkholderia Onion Pathogens.

Burkholderia gladioli pv. alliicola, B. cepacia, and B. orbicola are common bacterial pathogens of onion. Onions produce organosulfur thiosulfinate defensive compounds after cellular decompartmentalization. Using whole-genome sequencing and in silico analysis, we identified putative thiosulfinate tolerance gene (TTG) clusters in multiple onion-associated Burkholderia species similar to those characterized in other Allium-associated bacterial endophytes and pathogens. Sequence analysis revealed the presence of three Burkholderia TTG cluster types, with both Type A and Type B being broadly distributed in B. gladioli, B. cepacia, and B. orbicola in both the chromosome and plasmids. Based on isolate natural variation and generation of isogenic strains, we determined the in vitro and in vivo contribution of TTG clusters in B. gladioli, B. cepacia, and B. orbicola. The Burkholderia TTG clusters contributed to enhanced allicin tolerance and improved growth in filtered onion extracts by all three species. TTG clusters also made clear contributions to B. gladioli foliar necrosis symptoms and bacterial populations. Surprisingly, the TTG cluster did not contribute to bacterial populations in onion bulb scales by these three species. Based on our findings, we hypothesize onion-associated Burkholderia may evade or inhibit the production of thiosulfinates in onion bulb tissues. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Linkage Mapping and Genome-Wide Association Study Identified Two Peanut Late Leaf Spot Resistance Loci, PLLSR-1 and PLLSR-2, Using Nested Association Mapping.

Identification of candidate genes and molecular markers for late leaf spot (LLS) disease resistance in peanut (Arachis hypogaea) has been a focus of molecular breeding for the U.S. industry-funded peanut genome project. Efforts have been hindered by limited mapping resolution due to low levels of genetic recombination and marker density available in traditional biparental mapping populations. To address this, a multi-parental nested association mapping population has been genotyped with the peanut 58K single-nucleotide polymorphism (SNP) array and phenotyped for LLS severity in the field for 3 years. Joint linkage-based quantitative trait locus (QTL) mapping identified nine QTLs for LLS resistance with significant phenotypic variance explained up to 47.7%. A genome-wide association study identified 13 SNPs consistently associated with LLS resistance. Two genomic regions harboring the consistent QTLs and SNPs were identified from 1,336 to 1,520 kb (184 kb) on chromosome B02 and from 1,026.9 to 1,793.2 kb (767 kb) on chromosome B03, designated as peanut LLS resistance loci, PLLSR-1 and PLLSR-2, respectively. PLLSR-1 contains 10 nucleotide-binding site leucine-rich repeat disease resistance genes. A nucleotide-binding site leucine-rich repeat disease resistance gene, Arahy.VKVT6A, was also identified on homoeologous chromosome A02. PLLSR-2 contains five significant SNPs associated with five different genes encoding callose synthase, pollen defective in guidance protein, pentatricopeptide repeat, acyl-activating enzyme, and C2 GRAM domains-containing protein. This study highlights the power of multi-parent populations such as nested association mapping for genetic mapping and marker-trait association studies in peanuts. Validation of these two LLS resistance loci will be needed for marker-assisted breeding.

Characterization of Seed-to-Seedling Transmission of Alternaria brassicicola in Broccoli.

Alternaria brassicicola is part of a complex of Alternaria species that causes leaf blight and head rot in brassica crops such as broccoli, kale, cabbage, cauliflower, and collards. Seed can serve as a potential source of inoculum for the transmission of A. brassicicola in broccoli as demonstrated earlier; however, seed-to-seedling transmission of pathogen was never characterized empirically. So, the objectives of this study were to (i) re-evaluate the effect of artificial seed infestation on seed germination and seed-to-seedling transmission of A. brassicicola in broccoli; (ii) determine the effect of A. brassicicola-seed inoculum levels on seed-to-seedling transmission; (iii) evaluate if variations in A. brassicicola aggressiveness affect A. brassicicola seed-to-seedling transmission; and (iv) evaluate seed treatments that can reduce seed-to-seedling transmission of A. brassicicola in broccoli. Artificially infested seedlots were generated by inoculating broccoli seeds with a spore suspension of 1 × 105 conidia/ml of A. brassicicola using the vacuum infiltration method. Inoculated (n = 10 seedlots; 300 seeds/seedlot) or control seedlots in three replicates were planted on two layers of sterile blotter paper saturated with sterile water in transparent plastic boxes and incubated at 20°C and >90% relative humidity (RH) under continuous fluorescent light. Percent seed germination and percent seed-to-seedling transmission were recorded every other day for 21 days. Percent seed germination was significantly affected with artificial pathogen inoculation. One hundred percent of the seedlots transmitted the pathogen to broccoli seedlings, and seed-to-seedling percentages of the seedlots varied considerably. A strong linear and significant relationship between A. brassicicola inoculum level and seed-to-seedling transmission (%) within each seedlot was observed. Interestingly, variations in aggressiveness of A. brassicicola isolates did not affect seed-to-seedling transmission, as 100% of the seedlots were able to transmit the pathogen. Seed treatment with Miravis (a.i. pydiflumetofen 18.3%) significantly increased seed germination and reduced seed-to-seedling transmission percentages in A. brassicicola-inoculated seedlots. These results indicate that artificial seed inoculation with A. brassicicola can result in consistent seed-to-seedling transmission with significant impact on seed germination. Seed inoculum density of ≥104 conidia/ml is necessary for reliable transmission of A. brassicicola. Further seed-to-seedling transmission is not dependent on aggressiveness of A. brassicicola isolates and seed treatment with Miravis can significantly reduce pathogen transmission in broccoli seedings. Overall, this study provides detailed characterization of seed-to-seedling transmission of A. brassicicola in broccoli that can be further used to determine inoculum threshold, which has potential applications in seed-health testing and sample size determination. Furthermore, we also provide options for effective seed treatments that can significantly reduce A. brassicicola seed-to-seedling transmission and may potentially aid in managing seedborne fungal infection.

Aggressive Alternaria brassicicola with Reduced Fungicide Sensitivity Can Be Associated with Naturally Infested Broccoli Seeds.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures (n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent (n = 133) of the isolates formed a cluster with a known A. brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates (n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates (n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola. We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields.

First Report of Erwinia aphidicola Causing Bulb Rot of Onion in Chile.

During the 2021-22 and 2022-23 seasons (December to February), onion plants (Allium cepa L.) showing decay, leaf blight, chlorosis and water soak lesions were collected in Central Chile. Five symptomatic plants were sampled from 20 different onion fields. Brown rot of the external scales was observed in bulbs from two fields: one planted with the cv. Campero (20 ha; O'Higgins Region), and another with cv. Marenge (2 ha; Metropolitan Region). The disease incidence in these fields ranged from 2% to 5%. Isolations were carried out from symptomatic leaves and bulbs from these fields on King's B medium, resulting in small white colonies with smooth margin. Three isolates were selected, two from first field (QCJ3A & QCJ2B), and one from second field (EPB1). A preliminary identification based on 16S rRNA sequences was conducted. BLAST analyses of strains QCJ3A, QCJ2B and EPB1 (GenBank Accession No. PP345601 to PP345603) against the NCBI Database resulted in a match with strains (GenBank Accession No. ON255770.1 and ON255825.1) isolated from infected bulbs in Texas, USA identified as Erwinia spp. (Khanal et al. 2023), with 100% coverage and 100% identity (707 bp out of 707). To evaluate the pathogenicity of these three strains, onion bulbs were inoculated (Guajardo et al. 2023). Toothpicks previously immersed in a bacterial suspension at ~ 108 colony forming units (CFU)/mL were pricked at a 4 cm depth into the shoulders of onion bulbs bought from commercial store and incubated at room temperature. Bulbs inoculated with sterile water served as negative control. A known onion bulb rotting bacterial strain of Dickeya sp. was used as a positive control. At the end of the incubation period (20 days), bulbs were opened longitudinally across their inoculation site, showing that the external scales had a brown color. Negative control remained asymptomatic. Strains were re-isolated from damaged tissue and identified as Erwinia sp. This assay was repeated three times with the same results. For further identification, genomic DNA extraction was carried out using the Blood & Cell Culture DNA Kit (Qiagen), and genome sequencing was performed in the Illumina HiSeq 2500 platform. The Whole Genome Shotgun project for strains QCJ3A, QCJ2B and EPB1 have been deposited at DDBJ/ENA/GenBank under the accession JBANEI010000000, JBANEJ010000000 and JBANEK010000000. The average nucleotide identity (ANI) values were 99.6% (EPB1), 98.2% (QCJ2B), and 99.6% (QCJ3A) and DNA-DNA hybridization (dDDH) values were 96.9% (EPB1), 83.7% (QCJ2B), and 97.1% (QCJ3A), when compared with the type strain Erwinia aphidicola JCM 21238 (GenBank accession No. GCF_014773485.1). The three strains were deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM). Erwinia aphidicola has been previously described causing diseases in common bean (Phaseolus vulgaris) and pea (Pisum sativum), in Spain (Santos et al. 2009) and in pepper (Capsicum annuum) in China (Luo et al. 2018). Its close relative E. persicina has been reported causing bulb rot in onion in Korea (Cho et al. 2019) and garlic in Europe (Galvez et al. 2015). To our knowledge, this is the first report of E. aphidicola causing a bulb rot of onion in Chile. Although the distribution and prevalence of this bacterium in Chilean agroecosystems is not known, it can be a potential cause of losses in onions and other crops such as beans, peas, and peppers. Additional studies should be conducted to determine the host range of Chilean Erwinia aphidicola strains.

Assessment of Prickly Sida as a Potential Inoculum Source for Sida Golden Mosaic Virus in Commercial Snap Bean Farms in Georgia, United States.

Sida golden mosaic virus (SiGMV), an obligate pathogen that infects snap beans (Phaseolus vulgaris), is known to infect prickly sida (Sida spinosa L.), which is a common weed in agricultural farms in Georgia. Prickly sida has also been reported as a suitable host of sweetpotato whitefly (Bemisia tabaci), the vector of SiGMV. Despite being a host for both SiGMV and its vector, the role of prickly sida as a reservoir and inoculum source for SiGMV in snap bean farms has not been evaluated. This study was conducted to document the occurrence of SiGMV-infected prickly sida plants and to assess its potential role as a source of SiGMV inoculum in snap bean farms. A survey of 17 commercial snap bean farms conducted in spring 2021 confirmed the presence of SiGMV-infected prickly sida in southern Georgia. In fall 2021 and 2022, on-farm field trials were conducted in four commercial farms where SiGMV-infected prickly sida plants were documented earlier as a part of survey in spring 2021. The spatial distribution and temporal patterns of adult whiteflies and SiGMV on snap bean were compared between macroplots (13.7 × 30.5 m) "with prickly sida" or "without prickly sida" that were at least 232 m apart from each other. We did not observe any consistent differences in counts of adult whiteflies between macroplots with or without prickly sida in the four commercial farms. SiGMV infection was detected earlier and with higher incidences in snap bean macroplots "with prickly sida" compared with macroplots "without prickly sida." An apparent disease gradient was observed in two of the four farms assessed. Higher SiGMV incidences were observed on the edges of macroplots "with prickly sida." These findings indicate prickly sida as a potential natural reservoir and a source for SiGMV spread in snap bean farms in southern Georgia.

From weeds to natural enemies: implications of weed cultivation and biopesticides for organic onion production.

Weed management is challenging for vegetable crops that are highly sensitive to weed competition, such as onions. Thrips (Thysanoptera: Thripidae) are major insect pests of onions, causing damage through feeding, and vectoring bacterial pathogens causing bulb rot. Both thrips and their associated pathogens are known to survive on many weed species in onion growing regions. Combining weeding with biopesticides may synergistically manage thrips and reduce disease prevalence. However, disturbances from weeding may negatively impact natural enemies. We estimated the effects of organic weed management and biopesticides on weed density, thrips and natural enemy activity, disease severity, and yield. The experiment was a randomized complete block design, with 4 replications of each weeding (control, tine-weeded twice, tine-weeded 4 times, and hand-weeded) and biopesticide (control, OxiDate 2.0, Serenade) combination. Arthropods were monitored using yellow sticky cards, and weed counts, marketable yield, and bulb rot prevalence were estimated. Hand-weeding resulted in the lowest weed density and thrips abundance. Additionally, hand-weeding produced a 9× higher yield compared to all other treatments. Significant interactions were observed between tine-weeding and biopesticide treatments on the prevalence of bulb rot. Natural enemy abundance was slightly negatively impacted by weeding, dependent on the year. DNA metabarcoding results showed high parasitoid diversity in this onion system and high numbers of reads for multiple genera containing important known biological control agents. Our study suggests hand-weeding is necessary in the southeast for maximum onion yield. Future research should focus on exploring the impact of management on natural enemy communities in onion systems on a large scale.