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.

Metabolites Produced by Fungi against Fungal Phytopathogens: Review, Implementation and Perspectives

Many fungi, especially endophytes, have been found to produce multiple benefits in their plant hosts, with many of these benefits associated with the protection of plants against fungal diseases. This fact could be used in the development of new bio-products that could gradually reduce the need for chemical fungicides, which have been associated with multiple health and environmental problems. However, the utilization of the living organism may present several issues, such as an inconsistency in the results obtained and more complicated management and application, as fungal species are highly influenced by environmental conditions, the type of relationship with the plant host and interaction with other microorganisms. These issues could be addressed by using the bioactive compounds produced by the fungus, in cases where they were responsible for positive effects, instead of the living organism. Multiple bioactive compounds produced by fungal species, especially endophytes, with antifungal properties have been previously reported in the literature. However, despite the large amount of these metabolites and their potential, extensive in-field application on a large scale has not yet been implemented. In the present review, the main aspects explaining this limited implementation are analyzed, and the present and future perspectives for its development are discussed.

Evaluation of Antioxidant and Antifungal Activity of the Whole Part of Dactyloctenium aegyptium Weed on Bengal Gram

Background: This study was conducted about the effectiveness of weed Dactyloctenium aegyptium. Weedsare familiar dominant, redundant, adverse and weed that contend with sophisticated crop for water, nutrient and sunlight and another several reasons such as, high growth rate, high reproductive rate and produce harmful or beneficial allelopathical effect of cultivated crops. The stems of Dactyloctenium aegyptium are willowy, geniculate and leaves are found roughly. Methods: This Investigation was done in 2018-19 to 2020-21. Different part of Dactyloctenium aegyptium as leaves and seed was taken from the near area of Sardar Vallabh Bhai Patel Agriculture and Technology University Modipuram Meerut. The samples were shade dry for 24 to 48 hours and then grind in the powder form. The extract were prepare in different organic solvent as Methanol, Ethyl acetate, Butyl alcohol, Benzene and Water at 1:10 ratio. Antioxidant activity of weed extracts by three methods named DPPH FRAP and ABTS methods. Result: All part of this weed show effectiveness due to the presence of active compound, who responsible for the positive result. Extract mixed with media at a particular concentration i.e. 5%, 10% and 15% show effect on soil borne fungal phytopathogens and then over the surface of petriplate the growth was appear reduce when we increase the concentration of extract. At the end we conclude that the 15% extract concentration of both part of Dactyloctenium reduce the growth of all the soil borne fungal phytopathogens.

Marine Macroalgae, a Source of Natural Inhibitors of Fungal Phytopathogens

Fungal phytopathogens are a growing problem all over the world; their propagation causes significant crop losses, affecting the quality of fruits and vegetables, diminishing the availability of food, leading to the loss of billions of euros every year. To control fungal diseases, the use of synthetic chemical fungicides is widely applied; these substances are, however, environmentally damaging. Marine algae, one of the richest marine sources of compounds possessing a wide range of bioactivities, present an eco-friendly alternative in the search for diverse compounds with industrial applications. The synthesis of such bioactive compounds has been recognized as part of microalgal responsiveness to stress conditions, resulting in the production of polyphenols, polysaccharides, lipophilic compounds, and terpenoids, including halogenated compounds, already described as antimicrobial agents. Furthermore, many studies, in vitro or in planta, have demonstrated the inhibitory activity of these compounds with respect to fungal phytopathogens. This review aims to gather the maximum of information addressing macroalgae extracts with potential inhibition against fungal phytopathogens, including the best inhibitory results, while presenting some already reported mechanisms of action.

High performance liquid chromatography based characterization of Fengycin produced by Bacillus amyloliquefaciens against Fusarium graminearum and Rhizoctonia solani

A substantial loss of crop production worldwide is attributed to fungal phytopathogens. The most important economic pathogens among these fungal phytopathogens are Fusarium graminearum and Rhizoctonia solani, which cause a wide range of plant diseases. In the present study, Bacillus amyloliquefaciens secondary metabolite fengycin was identified by High performance liquid chromatography (HPLC) and in vitro screened against the Fusarium graminearum and Rhizoctonia solani. Based on the HPLC result, fengycin was identified at 215nm wavelength, retention time 5-7 minutes, and peak area was 3.914. The obtained results indicated that fengycin (1, 1/2, 1/4, and 1/8) concentrations have a significant effect (p<0.005) on the growth of F. graminearum and R. solani. The current study concluded that B. amyloliquefaciens secondary metabolites fengycin having a high potential to inhibit the growth of F. graminearum and R. solani.

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

Effective In Vitro Control of Two Phytopathogens of Agricultural Interest Using Cell-Free Extracts of Pseudomonas fluorescens and Chitosan

A biofungicide is a natural product that can be derived from various sources such as, among others, microorganisms, higher plants, animal products, phytochemicals, semiochemicals, and antagonist microorganisms. One of the most important approaches for the production of biofungicides is the combination of biocontrol agents. This study showed the inhibition growth of Alternaria alternata and Fusarium solani treated with cell-free extracts of P. fluorescens. Using thin-layer chromatography and plate assays it was also demonstrated that the cell-free extracts of P. fluorescens contained siderophores and derivates of 4-diacetylphloroglucinol and phenazine. Moreover, the combination of cell-free extracts of P. fluorescens and chitosan [50–1.5% (v/v)] had a synergistic effect since they notably inhibited the mycelial growth of A. altenata and F. solani. Various morphological alterations to the mycelia and conidia of the treated fungi as a result of this combination were also observed. The present study could be a starting point to control other fungal phytopathogens using different cell-free extracts and chitosan as biocontrol agents.

In vitro antagonistic activity of Trichoderma harzianum and T. viride strains compared to carbendazim fungicide against the fungal phytopathogens of Sorghum bicolor (L.) Moench

Background High losses of sorghum crops due to fungal diseases, such as grain mold and stalk rot, are economically harmful and cause increased use of environmentally damaging chemical fungicides, which also are detrimental to human health. Hence, finding safe and effective ways to manage fungal diseases of sorghum is urgently needed. Results In the present study, the antagonistic activities of Trichoderma viride and T. harzianum against different pathogenic fungal strains were evaluated in vitro using a dual culture assay. Furthermore, the anti-mycotic activity of Trichoderma spp. culture filtrates was evaluated against different fungal strains using a food poisoning technique. Additionally, the antifungal activities of ethyl acetate extracts of T. viride and T. harzianum against different pathogens were evaluated using a disk diffusion method. As indicated by the dual culture assay, T. harzianum suppressed 66.8, 69.5, 68.7, 54.6, 84.12, and 71.39% of the mycelial growth of Curvularia lunata, Exserohilum rostratum, Fusarium chlamydosporum, F. incarnatum, F. proliferatum, and Macrophomina phaseolina, respectively. T. viride was more effective for controlling the growth of these pathogens, inhibiting 81.0, 89.0, 63.0, 70.7, 84.4, and 71.8% of mycelial growth, respectively. Both E. rostratum and M. phaseolina showed resistance to carbendazim fungicide at all tested concentrations, whereas the fungicidal concentrations of carbendazim against C. lunata, F. chlamydosporum, and F. incarnatum strains were 2.50, 1.50, and 2.00 ppm, respectively. Furthermore, F. proliferatum was sensitive to carbendazim fungicide at all tested concentrations. Antifungal assays of the ethyl acetate extracts of T. viride and T. harzianum indicated the potent activity of these extracts against fungal phytopathogens with different susceptibility patterns. F. chlamydosporum was the most sensitive to the extracts of T. viride and T. harzianum with minimum inhibitory concentrations of 0.5 and 1.0 mg/disk, respectively. Conclusion The potent suppression of sorghum phytopathogens by T. viride and T. harzianum makes them potential sources of safe and effective natural fungicides compared to carbendazim fungicide.

A Novel Antifungal Actinomycete Streptomyces sp. Strain H3-2 Effectively Controls Banana Fusarium Wilt

Banana Fusarium wilt disease caused by Fusarium oxyspoum f. sp. cubense (Foc) seriously threatens the banana industry. Foc tropical race 4 (Foc TR4) can infect almost all banana cultivars. Compared with traditional physical and chemical practices, biocontrol strategy using beneficial microbes is considered as an environmentally sound option to manage fungal disease. In this study, a strain, H3-2, isolated from a non-infected banana orchard, exhibited high antifungal activity against Foc TR4. According to its morphological, physiological, and biochemical characteristics, the strain H3-2 was identified as Streptomyces sp. and convinced by the polymorphic phylogenic analysis of 16S rRNA sequences. Extracts of the strain H3-2 suppressed the growth and spore germination of Foc TR4 in vitro by destroying cell membrane integrity and mycelial ultrastructure. Notably, the strain and its extracts showed broad-spectrum antifungal activity against the selected seven fungal phytopathogens. Fourteen chemical compounds in the extracts were identified by gas chromatography–mass spectrometer (GC-MS), primarily phenolic compounds. Additional pot inoculation experiment demonstrated that the fermentation broth of the strain H3-2 promoted the growth of banana seedlings by efficiently inhibiting the spread of banana Fusarium wilt disease. This study demonstrated the potential application of the novel Streptomyces sp. H3-2 for the management of banana Fusarium wilt.