Charcoal rot disease of soybean is a major threat to soybean harvests in the United States. This disease, caused by the fungal pathogen Macrophomina phaseolina (MP), cannot be economically treated in agronomic field conditions, and breeding of resistant cultivars has been largely unsuccessful due to multiple gene resistance. For difficult-to-treat phytopathogens, nascent research has emphasized the potential utility of endophyte modulation of disease symptoms. Most research into biocontrol of MP in soybeans has focused on in vitro or limited greenhouse/pot experiments, which may not emulate field conditions. Here, using a paired framework of diseased and asymptomatic soybeans exposed to MP in a production field setting, coupled with model-based community analyses, we identify several fungal endophytic taxa that apparently modulate disease pathogenicity. We use a multilevel analytical framework to provide evidence of taxa that respond to the disease state by querying plants across years and plant compartments. Additional work is needed to test the mechanisms by which these identified taxa may modulate MP, but this work provides the strongest evidence to date that endophytes may modulate charcoal rot disease levels in production field conditions. We posit that breeding for improved colonization of disease-modulating endophytes is a promising option to manage charcoal rot in soybean. [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 .

Aflatoxin is a secondary metabolite produced by Aspergillus fungi and presents a major food safety concern globally. Among the available methods for prevention and control of aflatoxin, the application of antifungal bacteria has gained favor in recent years. An endophytic bacterium MS455, isolated from soybean, exhibited broad-spectrum antifungal activity against economically important pathogens, including Aspergillus flavus. MS455 was identified as a strain of Burkholderia based on genomic analysis. Random and site-specific mutations were used in discovery of the genes that share high homology to the ocf gene cluster of Burkholderia contaminans strain MS14, which is responsible for production of the antifungal compound occidiofungin. RNA sequencing analysis demonstrated that ORF1, a homolog to the ambR1 LuxR-type regulatory gene, regulates occidiofungin biosynthesis in MS455. Additionally, 284 differentially expressed genes, including 138 upregulated and 146 downregulated genes, suggesting that, in addition to its role in occidiofungin production, ORF1 is involved in expression of multiple genes, especially those involved in ornibactin biosynthesis. Plate bioassays showed the growth of A. flavus was significantly inhibited by the wild-type strain MS455 as compared with the ORF1 mutant. Similarly, corn kernel assays showed that growth of A. flavus and aflatoxin production were reduced significantly by MS455 as compared with buffer control and the ORF1 mutant. Collectively, the results demonstrated that production of occidiofungin is essential for antifungal activity of the endophytic bacterium MS455. This research has provided insights about antifungal mechanisms of MS455 and development of biological approaches to prevent aflatoxin contamination in plant production.

Burkholderia sp. strain MS389, an endophytic bacterium, was isolated from a healthy soybean plant growing adjacent to a patch of plants affected by charcoal rot disease, caused by the fungal pathogen Macrophomina phaseolina. Preliminary studies demonstrated that strain MS389 possesses antimicrobial activities against multiple plant pathogens. Burkholderia sp. strain MS389 was found to have three circular chromosomes of 3,563,380 bp, 3,002,449 bp, and 1,180,421 bp in size, respectively. The 7,746,250-bp genome, with 66.73% G+C content, harbors 6,756 protein coding genes in the predicted 6,985 genes. In total, 18 rRNAs, 68 tRNAs, and four ncRNAs were identified and 139 pseudogenes were annotated as well. The findings of this study will provide valuable data to explore the antimicrobial mechanisms of the endophytic bacterial strain.

Burkholderia contaminans MS14, isolated from a soil sample in Mississippi, is known for producing the novel antifungal compound occidiofungin. In addition, MS14 exhibits a broad range of antibacterial activities against common plant pathogens. Random mutagenesis and gene complementation indicate that four genes are required for antibacterial activity of strain MS14 against the fire blight pathogen Erwinia amylovora. With the aim of finding the biosynthetic gene cluster for the unknown antibacterial compound, we used RNA-seq to analyze the transcriptome of MS14 wild type and mutants lacking antibacterial activity. The twofold lower expressed genes in all mutants were studied, and a polyketide synthase (PKS) gene cluster was predicted to be directly involved in MS14 antibacterial activities. The nptII-resistance cassette and CRISPR-Cas9 systems were used to mutate the PKS gene cluster. Plate bioassays showed that either insertion or frame-shifting one of the PKS genes resulted in a loss of antibacterial activity. Considering that the antibacterial-defective mutants maintain the same antifungal activities as the wild-type strain, the results suggest that this PKS gene cluster is highly likely to be involved in or directly responsible for the production of MS14 antibacterial activity. Purification efforts revealed that the antibacterial activity of the compound synthesized by the gene cluster is sensitive to UV radiation. Nevertheless, these findings have provided more insights to understand the antibacterial activity of strain MS14.

Abstract Strains MS586T and MS82, which are aerobic, Gram‐negative, rod‐shaped, and polar‐flagellated bacteria, were isolated from the soybean rhizosphere in Mississippi. Taxonomic positions of MS586T and MS82 were determined using a polyphasic approach. 16S rRNA gene sequence analyses of the two strains showed high pairwise sequence similarities (>98%) to some Pseudomonas species. Analysis of the concatenated 16S rRNA, rpoB, rpoD, and gyrB gene sequences indicated that the strains belonging to the Pseudomonas koreensis subgroup (SG) shared the highest similarity with Pseudomonas kribbensis strain 46‐2T. Analyses of average nucleotide identity (ANI), genome‐to‐genome distance, delineated MS586T and MS82 from other species within the genus Pseudomonas. The predominant quinone system of the strain was ubiquinone 9 (Q‐9), and the DNA G+C content was 60.48 mol%. The major fatty acids were C16:0, C17:0 cyclo, and the summed features 3 and 8 consisting of C16:1ω7c/C16:1ω6c and C18:1ω7c/C18:1ω6c, respectively. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. Based on these data, it is proposed that strains MS586T and MS82 represent a novel species within the genus Pseudomonas. The proposed name for the new species is Pseudomonas glycinae, and the type strain is MS586T (accession NRRL B‐65441 = accession LMG 30275).

The genomic sequence of Pseudomonas sp. strain MS82 isolated from the rhizosphere of a soybean plant is reported and analyzed in relation to its extensive antifungal activity. Broth media used for production of the antifungal extract from strain MS82 against the mushroom pathogen Trichoderma viride were optimized using the routine plate bioassays. Culture extract of strain 82 in the peptone–K2HPO4–MgSO4 medium (PKM; peptone 20 g/L, K2HPO4 1.5 g/L, MgSO4 1.5 g/L and sterilized water) showed the best antifungal activity with an inhibition rate of 88.69 ± 3.87% to the fungal pathogen. Control efficacy of the T. viride contamination was investigated in mushroom production compost. The disease severity index of P. ostreatus hyphae infected by T. viride of treatment mixed with MS82 supernatant (38.33 ± 5.20%) was lower than that of the compost mixed with non-inoculated broth (97.50 ± 2.50%). The multilocus sequence analysis, containing four partial sequences from the gyrB, rpoB, recA and rpoD, suggests that strain MS82 is a Pseudomonas strain. The strain MS82 genome consists of a circular chromosome of 6,207,556 bp that was predicted to encode 5401 proteins and 131 RNA genes. Genome analysis revealed the presence of the gene clusters for biosynthesis of antifungal compounds, such as phenazine, pyocyanin, pyoverdine, volatile HCN and cyclic lipopeptides (arthrofactin). Genome analysis presented in the report will provide insights into development of biological control for fungal contamination in mushroom cultivation.

Burkholderia contaminans MS14 was isolated from soil in Mississippi. When it is cultivated on nutrient broth-yeast extract agar, the colonies exhibit bactericidal activity against a wide range of plant-pathogenic bacteria. A bacteriostatic compound with siderophore activity was successfully purified and was determined by nuclear magnetic resonance spectroscopy to be ornibactin. Isolation of the bactericidal compound has not yet been achieved; therefore, the exact nature of the bactericidal compound is still unknown. During an attempt to isolate the bactericidal compound, an interesting relationship between the production of ornibactin and the bactericidal activity of MS14 was characterized. Transposon mutagenesis resulted in two strains that lost bactericidal activity, with insertional mutations in a nonribosomal peptide synthetase (NRPS) gene for ornibactin biosynthesis and a luxR family transcriptional regulatory gene. Coculture of these two mutant strains resulted in restoration of the bactericidal activity. Furthermore, the addition of ornibactin to the NRPS mutant restored the bactericidal phenotype. It has been demonstrated that, in MS14, ornibactin has an alternative function, aside from iron sequestration. Comparison of the ornibactin biosynthesis genes in Burkholderia species shows diversity among the regulatory elements, while the gene products for ornibactin synthesis are conserved. This is an interesting observation, given that ornibactin is thought to have the same defined function within Burkholderia species. Ornibactin is produced by most Burkholderia species, and its role in regulating the production of secondary metabolites should be investigated.IMPORTANCE Identification of the antibacterial product from strain MS14 is not the key feature of this study. We present a series of experiments that demonstrate that ornibactin is directly involved in the bactericidal phenotype of MS14. This observation provides evidence for an alternative function for ornibactin, aside from iron sequestration. Ornibactin should be further evaluated for its role in regulating the biosynthesis of secondary metabolites in other Burkholderia species.

Bacterial strain MS82, isolated from the rhizosphere of a soybean plant, belongs to the species Pseudomonas fluorescens. The most important feature of strain MS82 is the production of antifungal activity against the mushroom pathogenic fungus Mycogone perniciosa but not against the mushroom fungus Agaricus bisporus. In this study, the mutant MS82MT19 generated with the EZ-Tn5 transposon system completely lost the antifungal activity against M. perniciosa. An open-reading frame named as PafR and predicted to code for a sensory box GGDEF/EAL domain protein, was disrupted in MS82MT19. To further confirm the function of this gene, site-directed mutagenesis with insertion of the terminatorless nptII cassette into the PafR gene was used to generate the mutant MS82SD19. As expected, there was no detectable antifungal activity of mutant MS82SD19 against M. perniciosa. These results suggest that the PafR gene plays an important role in the production of antifungal activity of P. fluorescens strain MS82.

Burkholderia contaminans MS14 shows significant antimicrobial activities against plant and animal pathogenic fungi and bacteria. The antifungal agent occidiofungin produced by MS14 has great potential for development of biopesticides and pharmaceutical drugs. However, the use of Burkholderia species as biocontrol agent in agriculture is restricted due to the difficulties in distinguishing between plant growth‐promoting bacteria and the pathogenic bacteria. The complete MS14 genome was sequenced and analyzed to find what beneficial and virulence‐related genes it harbors. The phylogenetic relatedness of B. contaminans MS14 and other 17 Burkholderia species was also analyzed. To research MS14′s potential virulence, the gene regions related to the antibiotic production, antibiotic resistance, and virulence were compared between MS14 and other Burkholderia genomes. The genome of B. contaminans MS14 was sequenced and annotated. The genomic analyses reveal the presence of multiple gene sets for antimicrobial biosynthesis, which contribute to its antimicrobial activities. BLAST results indicate that the MS14 genome harbors a large number of unique regions. MS14 is closely related to another plant growth‐promoting Burkholderia strain B. lata 383 according to the average nucleotide identity data. Moreover, according to the phylogenetic analysis, plant growth‐promoting species isolated from soils and mammalian pathogenic species are clustered together, respectively. MS14 has multiple antimicrobial activity‐related genes identified from the genome, but it lacks key virulence‐related gene loci found in the pathogenic strains. Additionally, plant growth‐promoting Burkholderia species have one or more antimicrobial biosynthesis genes in their genomes as compared with nonplant growth‐promoting soil‐isolated Burkholderia species. On the other hand, pathogenic species harbor multiple virulence‐associated gene loci that are not present in nonpathogenic Burkholderia species. The MS14 genome as well as Burkholderia species genome show considerable diversity. Multiple antimicrobial agent biosynthesis genes were identified in the genome of plant growth‐promoting species of Burkholderia. In addition, by comparing to nonpathogenic Burkholderia species, pathogenic Burkholderia species have more characterized homologs of the gene loci known to contribute to pathogenicity and virulence to plant and animals.

Strain UFB2 was isolated from a soybean field soil in Mississippi and identified as a member of Pseudomonas chlororaphis . Strain UFB2 has a broad-spectrum antimicrobial activity against common soil-borne pathogens. Plate assays showed that strain UFB2 was especially efficient in inhibiting the growth of Clavibacter michiganensis 1–07, the causal agent of the devastating bacterial canker of tomato. Here, the complete genome sequence of P. chlororaphis strain UFB2 is reported and described. The strain UFB2 genome consists of a circular chromosome of 6,360,256 bp of which 87.86 % are protein-coding bases. Genome analysis revealed multiple gene islands encoding various secondary metabolites such as 2,4-diacetylphloroglucinol. Further genome analysis will provide more details about strain UFB2 antibacterial activities mechanisms and the use of this strain as a potential biocontrol agent.

绿针假单胞菌YL-1对植物多种重要病原细菌和病原真菌有较强的抑制作用.采用转座子插入突变技术电转化绿针假单胞菌YL-1,构建随机插入突变体库.通过平板抑菌试验从突变体库中筛选对3种病原细菌荚壳伯克霍尔德菌Burkholderia glumae、解淀粉欧文氏菌Erwinia amylovora和胡萝卜果胶杆菌Pectobacterium carotovora抑菌效果显著下降的突变体,并采用PCR和Southern Blot验证转座子是否成功插入到YL-1染色体基因组上.利用质粒拯救技术克隆转座子插入位点基因、测序和生物信息学分析.结果表明:本研究成功构建了绿针假单胞菌YL-1随机插入突变体库,筛选出7株对3种病原细菌抑制效果明显下降的突变体,但其对立枯丝核菌的抑制效果与野生型菌株YL-1相同;PCR和Southern Blot试验结果表明绿针假单胞菌YL-1的7株突变体均是单拷贝;克隆到7个突变体插入位点的基因序列并测序,生物信息学分析结果表明其中6个突变位点是嗜铁素合成基因簇,1个突变位点是蛋白分泌基因secY.

Strain YL23 was isolated from soybean root tips and identified to be Pseudomonas sp. This strain showed broad-spectrum antibacterial activity against bacterial pathogens that are economically important in agriculture. To characterize the genes dedicated to antibacterial activities against microbial phytopathogens, a Tn5-mutation library of YL23 was constructed. Plate bioassays revealed that the mutant YL23-93 lost its antibacterial activities against Erwinia amylovora and Dickeya chrysanthemi as compared with its wild type strain. Genetic and sequencing analyses localized the transposon in a homolog of the secG gene in the mutant YL23-93. Constitutive expression plasmid pUCP26-secG was constructed and electroporated into the mutant YL23-93. Introduction of the plasmid pUCP26-secG restored antibacterial activities of the mutant YL23-93 to E. amylovora and D. chrysanthemi. As expected, empty plasmid pUCP26 could not complement the phenotype of the antibacterial activity in the mutant. Thus the secG gene, belonging to the Sec protein translocation system, is required for antibacterial activity of strain YL23 against E. amylovora and D. chrysanthemi.

Strain YL23 was isolated from soybean root tips and identified to be Pseudomonas sp. This strain showed broad-spectrum antibacterial activity against bacterial pathogens that are economically important in agriculture. To characterize the genes dedicated to antibacterial activities against microbial phytopathogens, a Tn5-mutation library of YL23 was constructed. Plate bioassays revealed that the mutant YL23-93 lost its antibacterial activities against Erwinia amylovora and Dickeya chrysanthemi as compared with its wild type strain. Genetic and sequencing analyses localized the transposon in a homolog of the secG gene in the mutant YL23-93. Constitutive expression plasmid pUCP26-secG was constructed and electroporated into the mutant YL23-93. Introduction of the plasmid pUCP26-secG restored antibacterial activities of the mutant YL23-93 to E. amylovora and D. chrysanthemi. As expected, empty plasmid pUCP26 could not complement the phenotype of the antibacterial activity in the mutant. Thus the secG gene, belonging to the Sec protein translocation system, is required for antibacterial activity of strain YL23 against E. amylovora and D. chrysanthemi.

Strain JX22, exhibiting a broad range of antimicrobial activities to fungal pathogens, was isolated and classified as representing Pseudomonas kilonensis. In this study, the mutant JX22MT1 was obtained by the EZ-Tn5 transposon mutation and showed no antifungal activity against Fusarium oxysporum f. sp. lycopersici as compared with wild-type strain JX22. The pqqC gene was disrupted in the mutant. Antifungal activity at the wild-type level was restored from the mutant JX22MT1 with the introduction of the functional pqqC gene, which encodes pyrroloquinoline–quinone synthesis protein C. The results suggest that pqqC is essential for antifungal activity of P. kilonensis JX22 against F. oxysporum f. sp. lycopersici.

Pseudomonas chlororaphis YL-1 was isolated from soybean root tips and showed a broad range of antagonistic activities to microbial plant pathogens. Here, we report the high-quality draft genome sequence of YL-1, which consists of a chromosome with an estimated size of 6.8 Mb with a G+C value of 63.09%.

Strain JX22, exhibiting a broad range of antimicrobial activities to fungal pathogens, was isolated and classified as representing Pseudomonas kilonensis. In this study, the mutant JX22MT1 was obtained by the EZ-Tn5 transposon mutation and showed no antifungal activity against Fusarium oxysporum f. sp. lycopersici as compared with wild-type strain JX22. The pqqC gene was disrupted in the mutant. Antifungal activity at the wild-type level was restored from the mutant JX22MT1 with the introduction of the functional pqqC gene, which encodes pyrroloquinoline-quinone synthesis protein C. The results suggest that pqqC is essential for antifungal activity of P. kilonensis JX22 against F. oxysporum f. sp. lycopersici.

Burkholderia pyrrocinia strain Lyc2 was isolated from the tobacco rhizosphere in China. This bacterium exhibits a remarkable capacity to inhibit the growth of multiple pathogens and shows strong suppression of cotton seedling damping-off. Here, we present the draft genome sequence of Burkholderia pyrrocinia strain Lyc2.

ABSTRACT Burkholderia contaminans strain MS14 produces the antifungal compound occidiofungin, which is responsible for significant antifungal activities against a broad range of plant and animal fungal pathogens. Occidiofungin is a cyclic glycolipopeptide made up of eight amino acids and one xylose. A 56-kb ocf gene cluster was determined to be essential for occidiofungin production. In this study, the ocfC gene, which is located downstream of ocfD and upstream of the ocfB gene in the ocf gene cluster, was examined. Antifungal activity of the ocfC gene mutant MS14KC1 was reduced against the indicator fungus Geotrichum candidum compared with that of the wild-type strain. Furthermore, the analysis of the protein sequence suggests that the ocfC gene encodes a glycosyltransferase. Biochemical analyses using nuclear magnetic resonance (NMR) and mass spectroscopy revealed that the ocfC mutant produced the occidiofungin without the xylose. The purified ocfC mutant MS14KC1 product had a level of bioactivity similar to that of the wild-type product. The revertant MS14KC1-R of the ocfC mutant produced the same antifungal activity level on plate assays and the same antifungal compound based on high-performance liquid chromatography (HPLC) and mass spectroscopy analysis as wild-type strain MS14. Collectively, the study demonstrates that the ocfC gene encodes a glycosyltransferase responsible to add a xylose to the occidiofungin molecule and that the presence of the xylose is not important for antifungal activity against Candida species. The finding provides a novel variant for future studies aimed at evaluating its use for inhibiting clinical and agricultural fungi, and the finding could also simplify the chemical synthesis of occidiofungin variants.

A polymerase chain reaction (PCR) based diagnostic assay was used to develop markers for detection of Fusarium verticillioides (=F. moniliforme), a fumonisin producing fungus in maize tissues. Species-specific primers were designed based on sequence data from the polyketide synthase (PKS) gene (FUM1- previously FUM5) responsible for fumonisin production in fungi. Four sets of oligonucleotide primers were tested for their specificity using 24 strains of F. verticillioides, 10 F. proliferatum, and 12 of other Fusarium species. In addition, 13 species of other fungal genera, from four phyla, were tested as negative controls. Among the four sets, primer set B consistently amplified a 419- bp fragment from the DNA 96% of all F. verticillioides strains and 83% of F. proliferatum. All other fungi tested were negative using primer set B. A total of 38% of the F. verticillioides strains grown on a selective liquid medium produced fumonisin and 92% formed the toxin on standard rice medium. When fumonisin formed in culture, PCR assay using primer set B detected every strain of F. verticillioides, but only amplified 80% of F. proliferatum strains that produced the toxin. PCR detection was consistent at 100 pg/μl concentration of genomic DNA from 4 F. verticillioides strains, but varied at 10 pg/μl. Two duplicate greenhouse tests using artificially inoculated maize plants, had greater levels of F. verticillioides detected after re-evaluting using primer set B than from culturing of the tissues. The molecular protocols described in this study requires only 1 day for completion compared to approximately 10 days for cultural work and morphological determination. In conclusion, conventional PCR assay using primer set B provides a sensitive and accurate detection assay that can be used as a primary or secondary confirmation method for identification and occurrence of F. verticillioides within the maize tissues. However, studies using primer set B for fumonisin production determined by strains of F. verticillioides and F. proliferatum will require further verification.

Scanning and transmission electron microscopy were used to study the morphology and formation of the eggshell in the tarnished plant bug, Lygus lineolaris. Eggs are bean-shaped, with an operculum at the anterior end surrounded by a row of 36–40 respiratory horns. Three micropylar openings are on the operculum, and are sealed in oviposited eggs. The chorion consists of the chorion proper and the innermost chorionic layer. An air layer composed of colonnades is present in the chorion. The innermost chorionic layer is homogeneous and electron lucent. The follicle cells secrete electron dense materials that later coalesced into the reticulated vitelline membrane. This is followed by the deposition of the innermost chorionic layer by the follicle cells. After the primordial innermost chorionic layer is formed, follicle cells at the anterior pole of the oocyte secrete the scaffold for the colonnades in the air layer. Later, the primordial scaffold matrix is redistributed and localized at the lateral and posterior end of the oocyte where it becomes secondarily modified. At the end of choriogenesis, follicle cells at the anterior pole secrete the operculum and respiratory horns.