Isolation and Characterization of Extracellular Vesicles from the Fungal Phytopathogen Colletotrichum higginsianum

Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14-3-3 protein (Bmh1) as potential EV markers and generated transgenic lines expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV inter-kingdom communication between plants and fungi.

In vitro antifungal potential of surfactin isolated from rhizospheric Bacillus thuringiensis Berliner 1915 against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe

<p><em>Fusarium graminearum</em> fungus cause significant loss in maize (<em>Zea mays</em> L.) and other cereal crops all over the world. The usage of chemical agents cause severe environmental problems. <em>Bacillus</em> species and other plant growth-promoting bacteria (PGPR) play key role in biopesticide development. A wide range of environmentally safe antimicrobial agents are already being manufactured. The current investigation was focused on exploring the antifungal activity of <em>Bacillus thuringiensis</em> lipopeptide surfactin against fungal phytopathogen <em>Fusarium graminearum</em>. <em>B. thuringensis</em> was isolated from the rhizosphere of maize crop and cultivated to produce lipopeptides. Surfactin was identified by high-performance liquid chromatography (HPLC) from the extract at 210 nm, retention time 3-5 minutes and the obtained peaks area was 3.990. The growth of <em>F. graminearum </em>was successfully inhibited by surfactin at different concentrations<em>.</em> Among these, 80<em> </em>% concentration showed the highest zone of inhibition in comparison to 60<em> </em>%, 40<em> </em>% and 20<em> </em>% concentrations (<em>p</em> &lt; 0.005), respectively. The current study concludes <em>B. thuringensis </em>lipopeptide surfactin has a high potential to inhibit the growth of <em>F. graminearum</em>.</p>

Current Perspectives of Biocontrol Agents for Management of Fusarium verticillioides and Its Fumonisin in Cereals—A Review

Fusarium verticillioides is the most predominant fungal phytopathogen of cereals and it is posing great concern from a global perspective. The fungus is mainly associated with maize, rice, sorghum, wheat, sugarcane, banana, and asparagus and causes cob, stalk, ear, root, crown, top, and foot rot. F. verticillioides produces fumonisins as the major secondary metabolite along with trace levels of beauvericin, fusaric acid, fusarin C, gibberiliformin, and moniliformin. Being a potential carcinogen, fumonisins continue to receive major attention as they are common contaminants in cereals and its processed food products. The importance of elimination of F. verticillioides growth and its associated fumonisin from cereals cannot be overemphasized considering the significant health hazards associated with its consumption. Physical and chemical approaches have been shown to reduce fumonisin B1 concentrations among feeds and food products but have proved to be ineffective during the production process. Hence, biological control methods using microorganisms, plant extracts, antioxidants, essential oils, phenolic compounds, and other advanced technologies such as growing disease-resistant crops by applying genetic engineering, have become an effective alternative for managing F. verticillioides and its toxin. The different methods, challenges, and concerns regarding the biocontrol of F. verticillioides and production of fumonisin B1 have been addressed in the present review.

Fungal phytopathogen modulates plant and insect responses to promote its dissemination

Vector-borne plant pathogens often change host traits to manipulate vector behavior in a way that favors their spread. By contrast, infection by opportunistic fungi does not depend on vectors, although damage caused by an herbivore may facilitate infection. Manipulation of hosts and vectors, such as insect herbivores, has not been demonstrated in interactions with fungal pathogens. Herein, we establish a new paradigm for the plant-insect-fungus association in sugarcane. It has long been assumed that Fusarium verticillioides is an opportunistic fungus, where it takes advantage of the openings left by Diatraea saccharalis caterpillar attack to infect the plant. In this work, we show that volatile emissions from F. verticillioides attract D. saccharalis caterpillars. Once they become adults, the fungus is transmitted vertically to their offspring, which continues the cycle by inoculating the fungus into healthy plants. Females not carrying the fungus prefer to lay their eggs on fungus-infected plants than mock plants, while females carrying the fungus prefer to lay their eggs on mock plants than fungus-infected plants. Even though the fungus impacts D. saccharalis sex behavior, larval weight and reproduction rate, most individuals complete their development. Our data demonstrate that the fungus manipulates both the host plant and insect herbivore across life cycle to promote its infection and dissemination.

Computational structural genomics unravels common folds and predicted functions in the secretome of fungal phytopathogen Magnaporthe oryzae

Magnaporthe oryzae relies on a diverse collection of secreted effector proteins to reprogram the host metabolic and immune responses for the pathogen’s benefit. Characterization of the effectors is thus critical for understanding the biology and host infection mechanisms of this phytopathogen. In rapid, divergent effector evolution, structural information has the potential to illuminate the unknown aspects of effectors that sequence analyses alone cannot reveal. It has recently become feasible to reliably predict the protein structures without depending on homologous templates. In this study, we tested structure modeling on 1854 secreted proteins from M. oryzae and evaluated success and obstacles involved in effector structure prediction. With sensitive homology search and structure-based clustering, we defined both distantly related homologous groups and structurally related analogous groups. With this dataset, we propose sequence-unrelated, structurally similar effectors are a common theme in M. oryzae and possibly in other phytopathogens. We incorporated the predicted models for structure-based annotations, molecular docking and evolutionary analyses to demonstrate how the predicted structures can deepen our understanding of effector biology. We also provide new experimentally testable structure-derived hypotheses of effector functions. Collectively, we propose that computational structural genomic approaches can now be an integral part of studying effector biology and provide valuable resources that were inaccessible before the advent of reliable, machine learning-based structure prediction.

Pangenome analysis of the soil-borne fungal phytopathogen Rhizoctonia solani and development of a comprehensive web resource: RsolaniDB

Rhizoctonia solani is a collective group of genetically and pathologically diverse basidiomycetous fungus that damages economically important crops. Its isolates are classified into 13 Anastomosis Groups (AGs) and subgroups having distinctive morphology and host range. The genetic factors driving the unique features of R. solani pathology are not well characterized due to the limited availability of its annotated genomes. Therefore, we performed genome sequencing, assembly, annotation and functional analysis of 12 R. solani isolates covering 7 AGs and selected subgroups (AG1-IA, AG1-IB, AG1-IC, AG2-2IIIB, AG3-PT (isolates Rhs 1AP and the hypovirulent Rhs1A1), AG3-TB, AG4-HG-I (isolates Rs23 and R118-11), AG5, AG6, and AG8), in which six genomes are reported for the first time, wherein we discovered unique and shared secretomes, CAZymes, and effectors across the AGs. Using a pangenome comparative analysis of 12 R. solani isolates and 15 other basidiomycetes, we also elucidated the molecular factors potentially involved in determining the AG-specific host preference, and the attributes distinguishing them from other Basidiomycetes. Finally, we present the largest repertoire of R. solani genomes and their annotated components as a comprehensive database, viz. RsolaniDB, with tools for large-scale data mining, functional enrichment and sequence analysis not available with other state-of-the-art platforms, to assist mycologists in formulating new hypotheses.

Selecting Bacillus spp. Antagonist of Fungal Phytopathogen Phytophthora infestans Causing Tomato Late Blight

The late blight caused by phytopathogen Phytophthora infestans has been one of the serious disease-causing yields and quality losses of tomato production in Vietnam. To control the fungal disease, chemical fungicides have been overused causing concerns about the ecological risks and human health, especially fungal resistance. Therefore, using natural products from beneficial microorganisms as a safer strategy is getting attention. The present study focused on the isolation of indigenous Bacillus sp. with potential antifungal activity against Phytophthora infestans with the aim to contribute to the diversification and improving the quality of biological control products Bacillus spp. in Vietnam. From 21 strains Bacillus spp. (marked BV1 - BV21) being isolated from different tomato farms in Danang City, Vietnam, Bacillus velezensis BV 16 was selected based on the most potential antagonistic strain in controlling fungal plant pathogen Phytophthora infestans attacking the tomato tree. The strongest inhibition of mycelial growth on P. infestans of Bacillus velezensis BV16 was recorded with 88.89% of growth inhibition percentage. The results also showed that strong activity of chitinase, protease and cellulase in the BV16 strain are consistent with the novel growth control of Phytophthora infestans.