Comparative analysis of soybean transcriptional profiles reveals defense mechanisms involved in resistance against Diaporthe caulivora.

Soybean stem canker (SSC) caused by the fungal pathogen Diaporthe caulivora is an important disease affecting soybean production worldwide. However, limited information related to the molecular mechanisms underlying soybean resistance to Diaporthe species is available. In the present work, we analyzed the defense responses to D. caulivora in the soybean genotypes Williams and Génesis 5601. The results showed that compared to Williams, Génesis 5601 is more resistant to fungal infection evidenced by significantly smaller lesion length, reduced disease severity and pathogen biomass. Transcriptional profiling was performed in untreated plants and in D. caulivora-inoculated and control-treated tissues at 8 and 48 h post inoculation (hpi). In total, 2.322 and 1.855 genes were differentially expressed in Génesis 5601 and Williams, respectively. Interestingly, Génesis 5601 exhibited a significantly higher number of upregulated genes compared to Williams at 8 hpi, 1.028 versus 434 genes. Resistance to D. caulivora was associated with defense activation through transcriptional reprogramming mediating perception of the pathogen by receptors, biosynthesis of phenylpropanoids, hormone signaling, small heat shock proteins and pathogenesis related (PR) genes. These findings provide novel insights into soybean defense mechanisms leading to host resistance against D. caulivora, and generate a foundation for the development of resistant SSC varieties within soybean breeding programs.

Fusarium Species and Mycotoxins Associated with Sorghum Grains in Uruguay.

Grain mold and stalk rot are among the fungal diseases that cause significant losses in sorghum worldwide and are caused by different Fusarium spp. The presence of Fusarium species in sorghum grains causes yield losses and mycotoxin contamination, which represents a risk to consumers. In this study, Fusarium graminearum species complex (FGSC) had a high incidence, followed by Fusarium fujikuroi species complex (FFSC) and F. incarnatum-equiseti species complex. Within FFSC, F. proliferatum, F. andiyazi, F. fujikuroi, F. thapsinum, F. verticillioides and F. subglutinans were identified, and this was the first report of F. fujikuroi in sorghum. The most frequent toxins found in sorghum samples were deoxynivalenol (DON) and zearalenone (ZEN). The presence of fumonisins and nivalenol (NIV) was detected at low levels. This study adds new knowledge about the occurrence of Fusarium species and mycotoxins in sorghum grains. Furthermore, this is the first report in Uruguay on fungicide sensitivity for Fusarium isolates from sorghum, which constitutes an important starting point for defining management practices to minimize fungal infection and mycotoxin contamination.

Comparative genomics of plant pathogenic Diaporthe species and transcriptomics of Diaporthe caulivora during host infection reveal insights into pathogenic strategies of the genus.

BACKGROUND: Diaporthe caulivora is a fungal pathogen causing stem canker in soybean worldwide. The generation of genomic and transcriptomic information of this ascomycete, together with a comparative genomic approach with other pathogens of this genus, will contribute to get insights into the molecular basis of pathogenicity strategies used by D. caulivora and other Diaporthe species. RESULTS: In the present work, the nuclear genome of D. caulivora isolate (D57) was resolved, and a comprehensive annotation based on gene expression and genomic analysis is provided. Diaporthe caulivora D57 has an estimated size of 57,86 Mb and contains 18,385 predicted protein-coding genes, from which 1501 encode predicted secreted proteins. A large array of D. caulivora genes encoding secreted pathogenicity-related proteins was identified, including carbohydrate-active enzymes (CAZymes), necrosis-inducing proteins, oxidoreductases, proteases and effector candidates. Comparative genomics with other plant pathogenic Diaporthe species revealed a core secretome present in all Diaporthe species as well as Diaporthe-specific and D. caulivora-specific secreted proteins. Transcriptional profiling during early soybean infection stages showed differential expression of 2659 D. caulivora genes. Expression patterns of upregulated genes and gene ontology enrichment analysis revealed that host infection strategies depends on plant cell wall degradation and modification, detoxification of compounds, transporter activities and toxin production. Increased expression of effectors candidates suggests that D. caulivora pathogenicity also rely on plant defense evasion. A high proportion of the upregulated genes correspond to the core secretome and are represented in the pathogen-host interaction (PHI) database, which is consistent with their potential roles in pathogenic strategies of the genus Diaporthe. CONCLUSIONS: Our findings give novel and relevant insights into the molecular traits involved in pathogenicity of D. caulivora towards soybean plants. Some of these traits are in common with other Diaporthe pathogens with different host specificity, while others are species-specific. Our analyses also highlight the importance to have a deeper understanding of pathogenicity functions among Diaporthe pathogens and their interference with plant defense activation.

Paenibacillus sp. Strain UY79, Isolated from a Root Nodule of Arachis villosa, Displays a Broad Spectrum of Antifungal Activity.

A nodule-inhabiting Paenibacillus sp. strain (UY79) isolated from wild peanut (Arachis villosa) was screened for its antagonistic activity against diverse fungi and oomycetes (Botrytis cinerea, Fusarium verticillioides, Fusarium oxysporum, Fusarium graminearum, Fusarium semitectum, Macrophomina phaseolina, Phomopsis longicolla, Pythium ultimum, Phytophthora sojae, Rhizoctonia solani, Sclerotium rolfsii, and Trichoderma atroviride). The results obtained show that Paenibacillus sp. UY79 was able to antagonize these fungi/oomycetes and that agar-diffusible compounds and volatile compounds (different from HCN) participate in the antagonism exerted. Acetoin, 2,3-butanediol, and 2-methyl-1-butanol were identified among the volatile compounds produced by strain UY79 with possible antagonistic activity against fungi/oomycetes. Paenibacillus sp. strain UY79 did not affect symbiotic association or growth promotion of alfalfa plants when coinoculated with rhizobia. By whole-genome sequence analysis, we determined that strain UY79 is a new species of Paenibacillus within the Paenibacillus polymyxa complex. Diverse genes putatively involved in biocontrol activity were identified in the UY79 genome. Furthermore, according to genome mining and antibiosis assays, strain UY79 would have the capability to modulate the growth of bacteria commonly found in soil/plant communities. IMPORTANCE Phytopathogenic fungi and oomycetes are responsible for causing devastating losses in agricultural crops. Therefore, there is enormous interest in the development of effective and complementary strategies that allow the control of the phytopathogens, reducing the input of agrochemicals in croplands. The discovery of new strains with expanded antifungal activities and with a broad spectrum of action is challenging and of great future impact. Diverse strains belonging to the P. polymyxa complex have been reported to be effective biocontrol agents. Results presented here show that the novel discovered strain of Paenibacillus sp. presents diverse traits involved in antagonistic activity against a broad spectrum of pathogens and is a potential and valuable strain to be further assessed for the development of biofungicides.

Soybean Stem Canker Caused by Diaporthe caulivora; Pathogen Diversity, Colonization Process, and Plant Defense Activation.

Soybean is an important crop in South America, and its production is limited by fungal diseases caused by species from the genus Diaporthe, including seed decay, pod and stem blight, and soybean stem canker (SSC). In this study, we focused on Diaporthe species isolated from soybean plants with SSC lesions in different parts of Uruguay. Diaporthe diversity was determined by sequencing the internal transcribed spacer (ITS) regions of ribosomal RNA and a partial region of the translation elongation factor 1-alpha gene (TEF1α). Phylogenetic analysis showed that the isolates belong to five defined groups of Diaporthe species, Diaporthe caulivora and Diaporthe longicolla being the most predominant species present in stem canker lesions. Due to the importance of D. caulivora as the causal agent of SSC in the region and other parts of the world, we further characterized the interaction of this pathogen with soybean. Based on genetic diversity of D. caulivora isolates evaluated with inter-sequence single repetition (ISSR), three different isolates were selected for pathogenicity assays. Differences in virulence were observed among the selected D. caulivora isolates on susceptible soybean plants. Further inspection of the infection and colonization process showed that D. caulivora hyphae are associated with trichomes in petioles, leaves, and stems, acting probably as physical adhesion sites of the hyphae. D. caulivora colonized the stem rapidly reaching the phloem and the xylem at 72 h post-inoculation (hpi), and after 96 hpi, the stem was heavily colonized. Infected soybean plants induce reinforcement of the cell walls, evidenced by incorporation of phenolic compounds. In addition, several defense genes were induced in D. caulivora-inoculated stems, including those encoding a pathogenesis-related protein-1 (PR-1), a PR-10, a ß-1,3-glucanase, two chitinases, two lipoxygenases, a basic peroxidase, a defensin, a phenylalanine-ammonia lyase, and a chalcone synthase. This study provides new insights into the interaction of soybean with D. caulivora, an important pathogen causing SSC, and provides information on the activation of plant defense responses.

Comparison of three microsatellite analysis methods for detecting genetic diversity in Phytophthora sojae (Stramenopila: Oomycete).

Analysis of an organism's genetic diversity requires a method that gives reliable, reproducible results. Microsatellites are robust markers, however, detection of allele sizes can be difficult with some systems as well as consistency among laboratories. In this study, our two laboratories used 219 isolates of Phytophthora sojae to compare three microsatellite methods. Two capillary electrophoresis methods, the Applied Biosystems 3730 Genetic Analyzer and the CEQ 8000 Genetic Analysis system, detected an average of 2.4-fold more alleles compared to gel electrophoresis with a mean of 8.8 and 3.6 alleles per locus using capillary and gel methods, respectively. The two capillary methods were comparable, although allele sizes differed consistently by an average of 3.2 bp across isolates. Differences between capillary methods could be overcome if reference standard DNA genotypes are shared between collaborating laboratories.