Exploring the specialized metabolome of the plant pathogen Streptomyces sp. 11-1-2.

Streptomyces bacteria are notable for producing chemically diverse specialized metabolites that exhibit various bioactivities and mediate interactions with different organisms. Streptomyces sp. 11-1-2 is a plant pathogen that produces nigericin and geldanamycin, both of which display toxic effects against various plants. Here, the 'One Strain Many Compounds' approach was used to characterize the metabolic potential of Streptomyces sp. 11-1-2. Organic extracts were prepared from 11-1-2 cultures grown on six different agar media, and the extracts were tested in antimicrobial and plant bioassays and were subjected to untargeted metabolomics and molecular networking. Most extracts displayed strong bioactivity against Gram-positive bacteria and yeast, and they exhibited phytotoxic activity against potato tuber tissue and radish seedlings. Several known specialized metabolites, including musacin D, galbonolide B, guanidylfungin A, meridamycins and elaiophylin, were predicted to be present in the extracts along with closely related compounds with unknown structure and bioactivity. Targeted detection confirmed the presence of elaiophylin in the extracts, and bioassays using pure elaiophylin revealed that it enhances the phytotoxic effects of geldanamycin and nigericin on potato tuber tissue. Overall, this study reveals novel insights into the specialized metabolites that may mediate interactions between Streptomyces sp. 11-1-2 and other bacteria and eukaryotic organisms.

Changes in the phyllosphere and rhizosphere microbial communities of soybean in the presence of pathogens.

Soybean (Glycine max L.) is host to an array of foliar- and root-infecting pathogens that can cause significant yield losses. To provide insights into the roles of microorganisms in disease development, we evaluated the bacterial and fungal communities associated with the soybean rhizosphere and phyllosphere. For this, leaf and soil samples of healthy, Phytophthora sojae-infected and Septoria glycines-infected plants were sampled at three stages during the production cycle, and then subjected to 16S and Internal Transcribed Spacer (ITS) amplicon sequencing. The results indicated that biotic stresses did not have a significant impact on species richness and evenness regardless of growth stage. However, the structure and composition of soybean microbial communities were dramatically altered by biotic stresses, particularly for the fungal phyllosphere. Additionally, we cataloged a variety of microbial genera that were altered by biotic stresses and their associations with other genera, which could serve as biological indicators for disease development. In terms of soybean development, the rhizosphere and phyllosphere had distinct microbial communities, with the fungal phyllosphere most influenced by growth stage. Overall, this study characterized the phyllosphere and rhizosphere microbial communities of soybean, and described the impact of pathogen infection and plant development in shaping these bacterial and fungal communities.

Nigericin and Geldanamycin Are Phytotoxic Specialized Metabolites Produced by the Plant Pathogen Streptomyces sp. 11-1-2.

Streptomyces bacteria are a key source of microbial specialized metabolites with useful applications in medicine and agriculture. In addition, some species are important plant pathogens and cause diseases such as potato scab, which reduces the quality and market value of affected potato crops. Most scab-associated Streptomyces spp. produce the phytotoxic metabolite thaxtomin A as the principal pathogenicity factor. However, recent reports have described scab-causing strains that do not produce thaxtomin A, but instead produce other phytotoxins that are thought to contribute to plant host infection and symptom development. Streptomyces sp. 11-1-2 is a highly pathogenic strain that was originally isolated from a scab symptomatic potato tuber in Newfoundland, Canada. The strain secretes one or more phytotoxic compounds of unknown identity, and it is hypothesized that these compounds serve as virulence factors for this organism. We analyzed the genome sequence of Streptomyces sp. 11-1-2 and found biosynthetic gene clusters for producing the known herbicidal compounds nigericin and geldanamycin. Phytotoxic culture extracts were analyzed using liquid chromatography-coupled tandem mass spectrometry and molecular networking, and this confirmed the production of both compounds by Streptomyces sp. 11-1-2 along with other, potentially related metabolites. The biosynthesis of both metabolites was found to be suppressed by the addition of N-acetylglucosamine to the culture medium, and pure nigericin and geldanamycin were able to exhibit phytotoxic effects against both radish seedlings and potato tuber tissue. Furthermore, the coadministration of the two compounds produced greater phytotoxic effects against potato tuber tissue than administration of each compound alone. IMPORTANCE Plant pathogens use a variety of mechanisms, including the production of phytotoxic specialized metabolites, to establish an infection of host tissue. Although thaxtomin A is considered the key phytotoxin involved in the development of potato scab disease, there is increasing evidence that other phytotoxins can play a role in disease development in some instances. In this study, we show that the highly pathogenic Streptomyces sp. 11-1-2 is capable of producing nigericin and geldanamycin, which individually and combined can cause significant damage to potato tuber tissue and radish seedlings. Our results suggest that the pathogenic phenotype of Streptomyces sp. 11-1-2 is due in part to the production of these specialized metabolites. As the biological activity of nigericin and geldanamycin is vastly different from the proposed activity of thaxtomin A against plants, the secretion of these compounds may represent a novel mechanism of plant pathogenicity exhibited by some Streptomyces species.

Amplicon Sequencing Reveals Extensive Coinfections of Foliar Pathogens in Soybean.

Soybean (Glycine max) is one of the most economically important crops grown in North America and in other regions worldwide. However, the plant is susceptible to a variety of foliar pathogenic microorganisms, some of which are a significant threat to production. Several molecular and serological approaches are currently available to diagnose plant pathogens, but all have limitations including their capability to accurately detect coinfections of individual plants. We therefore used 16S and internal transcribed spacer amplicon sequencing to identify the suite of bacterial and fungal organisms infecting 96 soybean leaf samples collected throughout southern Manitoba, Canada, at two growth stages (V2/3 and R6). We were able to confirm the presence of pathogens previously known to our sampling regions, such as Septoria glycines, Alternaria alternata, and Pseudomonas spp. Importantly, we found that most of plants were infected by more than one putative pathogen, with 64% of V2/3 and 26% of R6 plants infected by three or more pathogens. Amplicon sequencing also indicated the presence of residual pathogens that infect crops other than soybean, as well as nonfoliar pathogens and nonpathogenic microorganisms. We discuss some of the benefits and drawbacks of using amplicon sequencing to detect foliar pathogens of soybean.

Virulence mechanisms of plant-pathogenic Streptomyces species: an updated review.

Gram-positive Actinobacteria from the genus Streptomyces are best known for their morphological complexity and for their ability to produce numerous bioactive specialized metabolites with useful applications in human and veterinary medicine and in agriculture. In contrast, the ability to infect living plant tissues and to cause diseases of root and tuber crops such as potato common scab (CS) is a rare attribute among members of this genus. Research on the virulence mechanisms of plant-pathogenic Streptomyces spp. has revealed the importance of the thaxtomin phytotoxins as key pathogenicity determinants produced by several species. In addition, other phytotoxic specialized metabolites may contribute to the development or severity of disease caused by Streptomyces spp., along with the production of phytohormones and secreted proteins. A thorough understanding of the molecular mechanisms of plant pathogenicity will enable the development of better management procedures for controlling CS and other plant diseases caused by the Streptomyces.

Applications of Next-Generation Sequencing for Large-Scale Pathogen Diagnoses in Soybean.

Soybean (Glycine max) has become an important crop in Manitoba, Canada, with a 10-fold increase in dedicated acreage over the past decade. Given the rapid increase in production, scarce information about foliar diseases present in the province has been recorded. In order to describe the foliar pathogens affecting this legume, we harnessed next-generation sequencing (NGS) to carry out a comprehensive survey across Manitoba in 2016. Fields were sampled during the V2/3 (33 fields) and R6 (70 fields) growth stages, with at least three symptomatic leaves per field collected and subjected to RNA sequencing. We successfully detected several bacteria, fungi, and viruses known to infect soybean, including Pseudomonas savastanoi pv. glycinea, Septoria glycines, and Peronospora manshurica, as well as pathogens not previously identified in the province (e.g., Pseudomonas syringae pv. tabaci, Cercospora sojina, and Bean yellow mosaic virus). For some microorganisms, we were able to disentangle the different pathovars present and/or assemble their genome sequence. Since NGS generates data on the entire flora and fauna occupying a leaf sample, we also identified residual pathogens (i.e., pathogens of crops other than soybean) and multiple species of arthropod pests. Finally, the sequence information produced by NGS allowed for the development of polymerase chain reaction-based diagnostics for some of the most widespread and important pathogens. Although there are many benefits of using NGS for large-scale plant pathogen diagnoses, we also discuss some of the limitations of this technology.

First Complete Genome Sequence of Tobacco necrosis virus D Isolated from Soybean and from North America.

We present the first complete genome sequence of the tombusvirus Tobacco necrosis virus D (TNV-D) from North America, obtained from an infected soybean plant. Compared with the three other TNV-D genomes isolated from different geographic regions and host plants, its nucleotide identities were between 83% and 93%.