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Foliar fungal endophytes are ubiquitous and hyperdiverse, and tend to be host-specific among dominant forest tree species. The fungal genus Tubakia sensu lato is comprised of foliar pathogens and endophytes that exhibit host preference for Quercus and other Fagaceae species. To clarify interspecific differences in ecological characteristics among Tubakia species, we examined the endophyte communities of seven evergreen Quercus spp. at three sites in eastern Japan during summer and winter. Host tree species was the most significant factor affecting endophyte community composition. Tubakia species found at the study sites were divided into five specialists and three generalists according to their relative abundance in each host species and their host ranges. Specialists were dominant on their own host in summer, and their abundance declined in winter. To test the hypothesis that generalists are more widely adapted to their environment than specialists, we compared their spore germination rates at different temperatures. Spores of generalist Tubakia species were more tolerant of colder temperatures than were spores of specialist Tubakia species, supporting our hypothesis. Seasonal and site variations among Tubakia species were also consistent with our hypothesis. Host identity and ecology were significantly associated with endophyte community structure.

The aim of our investigation is to elucidate the impact of ultrasonic treated Bacillus amyloliquefaciens (B. amyloliquefaciens) on production of peptides during solid-state fermentation of soybean meal and its mechanisms. There was a substantial enhancement in levels of peptides, biomass, and protease activity within fermented soybean meal by 14.61%, 65.96%, and 16.58%, respectively, relative to control group (p < 0.05). Nevertheless, omics analysis and qRT-PCR verification declared that ultrasonic treatment did not bring the growing of B. amyloliquefaciens or the protease production in single cell, but accelerate the germination of spores. Genes encoding spo0A, spo0B, spo0F, spo0J were all substantially down-regulated ranged from − 0.25 to − 1.75 (p < 0.05). Concurrently, the gene encoding sleB displayed a remarkable up-regulation with log2-fold change of + 3.07 (p < 0.05). Besides, their respective proteins performed the same change tendency. Moreover, after ultrasonic treatment, spores germination rate and DPA content significantly increased by 3.29 and 12.14 times, respectively. Meanwhile, OD600 of spores suspension obviously decreased by 37.81% (p < 0.05). Most importantly, Pearson’s correlation analysis revealed that peptides yield in fermented products was strongly correlated with spores germination (Pearson’s correlation coefficient r = 0.839, p < 0.01). In brief, ultrasonic treatment promoted the germination of spores in the inoculum, which resulted in microbial proliferation and protease activity raising in fermented products, and consequently improving peptides yield. The improvement of spore germination rate after ultrasonic treatment might be achieved by reducing bacterial aggregation and lowering viscosity.

The current study was aimed to assess the chemical compositions of Melia azedarach Linn (Chinaberry) leaves aqueous extracts. Additionally, the extracts were also tested to investigate its antifungal potentials against Aspergillus flavus and Aspergillus parasiticus. Leaf extract of M. azedarach was obtained by maceration technique, subsequently analyzed using UV-Visible Spectrophotometer, Fourier Transform Infrared (FTIR) and Gas Chromatography-Mass Spectrometry (GC-MS). The total phenolic and flavonoids contents were; 67.5 ± 0.4 mg GAE/g DW and 12.7 ± 0.2 mg QE /g DW respectively. The presence phytochemicals were confirmed from various functional groups recorded in FT-IR spectra. The results were further validated through GC-MS analysis where a total of 18 compounds were identified with seven major compounds; namely 1-Butanol, 3-methyl-, acetate (11.53%), followed by coumaran (10.04%), (R, S)-2-propyl-5-oxohexanal (7.07%), 10-octadecenoic acid, methyl ester (5.16%) and 5,7-Octadien-2-one, 3-acetyl (3.06%). The extract exhibited antifungal activities against two major aflatoxin-producing fungi, A. flavus and A. parasiticus. The aqueous extract (31.25 to 500 mg mL-1) was active to inhibit the spore germination, mycelial growth, biomass production and aflatoxin biosynthesis. Spore germination was significantly reduced, with maximum inhibition of 83% against A. flavus and 85% against A. parasiticus at 500 mg mL-1. Mycelial growth and fungal biomass were markedly declined with increasing trend in extract concentration. The recorded biomass inhibition was 73.2% and 76.9% respectively against A. flavus and A. parasiticus. The extract also significantly suppressed the aflatoxin production in the selected fungal strains at higher concentrations, exceeded from 75% with respect to aflatoxins B₁, B₂, G₁, and G₂. The findings suggest that M. azedarach leaves extract is a valuable source of bioactive compounds possessing strong antifungal and anti-aflatoxigenic properties and could be considered as a promising natural alternative for controlling aflatoxins contamination in agricultural food sectors.

Anthracnose disease, caused by the Colletotrichum gloeosporioides species, affects vegetables, fruits, pulses, and cereals, leading to significant economic losses worldwide. Although many synthetic fungicides are used to control this pathogen, eco-friendly biological alternatives are gaining popularity. This study focuses on isolating and purifying chitinase ( Af Chi)from a marine bacterium and testing its antifungal efficacy against C. gloeosporioides spore germination by targeting the chitin in the fungal cell wall. The chitinase was purified from a marine bacterium A. faecalis from the Arabian Sea and had a molecular mass of 45 kDa and a specific activity of 84.6 U/mg. Af Chi worked best at 50 °C and pH 7.0 in Tris HCl buffer. Na+ ion was the highest cofactor, highlighting the halophilic nature of this chitinase. K+, Ca2+, Cu2+, Mg2+, Mn2+, and EDTA also increased activity, while Fe3+, Zn2+, Co2+, and Pb2+ decreased it. The Km and Vmax values were 1.87 µg/mL and 17.45 U/mL, respectively. Purified Af Chi at 10 mg/mL completely inhibited spore germination within 8 h and reduced the size of the spores.Key pointsThe chitinase remain stable at high temperature.The chitinase from A. faecalis shown positive result in development of natural bio-fungicide.

Arbuscular mycorrhizal fungi (AMF) are mutualistic symbionts considered a key group in soil systems involved in the provision of several ecosystem services. Recently they have been listed by EFSA as organisms to be included in the test battery for the risk assessment of plant protection product (PPPs). This study aimed to contribute to improve the ISO Protocol (ISO 10832: 2009) by assessing the feasibility of using other AMF species under different test conditions. Overall, results showed that AMF species Gigaspora albida and Rhizophagus clarus (selected out of five AMF species) are suitable to be used in spore germination tests using the ISO protocol (14 days incubation with sand or artificial soil as substrate) to test PPPs. However, several modifications to the protocol were made in order to accommodate the use of the tested isolates, namely the incubation temperature (28 °C instead of 24 °C) and the change of reference substance (boric acid instead of cadmium nitrate). The need for these changes, plus the results obtained with the three fungicides tested (chlorothalonil, mancozeb and metalaxyl-M) and comparisons made with literature on the relevance of the origin of AMF isolates in dictating the adequate test conditions, emphasize the importance of adjusting test conditions (AMF species/isolates and test temperature) when assessing effects for prospective risk assessment targeting different climatic zones. So, further studies should be conducted with different AMF species and isolates from different climatic regions, in order to better define which species/isolate and test conditions should be used to assess effects of a particular PPP targeting a given climatic zone.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04–6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.

Phosphate (P)-solubilizing microorganisms as a group form an important part of the microorganisms, which benefit plant growth and development. Growth promotion and increased uptake of phosphate are not the only mechanisms by which these microorganisms exert a positive effect on plants. Microbially mediated solubilization of insoluble phosphates through release of organic acids is often combined with production of other metabolites, which take part in biological control against soilborne phytopathogens. In vitro studies show the potential of P-solubilizing microorganisms for the simultaneous synthesis and release of pathogen-suppressing metabolites, mainly siderophores, phytohormones, and lytic enzymes. Further trends in this field are discussed, suggesting a number of biotechnological approaches through physiological and biochemical studies using various microorganisms.

Streptomyces is a model bacterium to study multicellular differentiation and the major reservoir for antibiotics discovery. However, the cellular‐level lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools that can mimic their natural conditions. In this study, we developed a simple microfluidic chip for the cultivation and observation of the entire lifecycle of Streptomyces development from the single‐cell perspective. The chip consists of channels for loading samples and supplying nutrients, microwell arrays for the seeding and growth of single spores, and air chambers beside the microwells that facilitate the development of aerial hyphae and spores. A unique feature of this chip is that each microwell is surrounded by a 1.5 µm nanogap connected to an air chamber, which provides a stabilized water–air interface. We used this chip to observe the lifecycle development of Streptomyces coelicolor and Streptomyces griseus germinated from single spores, which revealed differentiation of aerial hyphae with progeny spores at micron‐scale water–air interfaces and air chambers. Finally, we demonstrated the applicability of this chip in phenotypic assays by showing that the microbial hormone A‐Factor is involved in the regulatory pathways of aerial hyphae and spore formation. The microfluidic chip could become a robust tool for studying multicellular differentiation, single‐spore heterogeneity, and secondary metabolism of single‐spore germinated Streptomyces. Impact statement We describe a microfluidic chip that mimics the natural porous environment for the growth and development of Streptomyces, a central paradigm for the study of bacterial multicellularity and natural product discovery. We uesd a microfluidic chip for single‐celled spore germination, cultivation, and observation of the whole lifecycle of Streptomyces, including the development of aerial hyphae and chains of spores. In addition, we also observed the early growth, opportunistic transformation, and anastomosis of aerial hyphae of Streptomyces with precise time and stimulation control. We believe this microfluidic‐based approach will play an essential role in understanding the complex multicellular differentiation and the regulation of secondary metabolites of Streptomyces.

In vitro studies were undertaken to determine the effects of five fungicide and disinfectant treatments [propiconazole (Tilt 250EC), azoxystrobin (Amistar 250EC), didecyldimethyl ammonium chloride (Sporekill), alkali metal salts of alkylbenzene sulfonic acid and coconut diethanolamide (Farmcleanse), and potassium peroxymonosulfate (Virkon)] in preventing the germination of spores of Puccinia graminis f. sp. tritici, Kabatiella caulivora, Leptosphaeria maculans and Magnaporthe oryzae. Germination was inhibited by all fungicides and disinfectants, with maximum reductions at the manufacturer’s recommended concentration. Overall, azoxystrobin was the most effective, reducing germination of M. oryzae by 89%, L. maculans by 78% and P. graminis f. sp. tritici by 77%. Propiconazole was the most effective in reducing germination of K. caulivora by 72%. The extent of inhibition of germination was dependent on the pathogen; for example, alkali metal salts of alkylbenzene sulfonic acid and coconut diethanolamide, and potassium peroxymonosulfate were more effective on M. oryzae and P. graminis f. sp. tritici compared with L. maculans or K. caulivora. Studies undertaken to define the effectiveness of the fungicides/disinfectants reducing germination of the pathogens on five inert carrier materials (steel, fabric, wood, paper, and rubber) showed azoxystrobin and propiconazole to be the most effective, having 12–15% spore germination following decontamination treatment of carrier materials. The results demonstrate the potential for increased use of fungicides, particularly demethylation inhibitor and QoI fungicides, to decontaminate carrier materials to address the critical need to implement a practical commercial solution for dealing with threats posed by the long-term viability of these and other plant pathogens on inert materials associated with movement of humans, farming equipment, and commodities nationally and internationally.

There are increasing environmental risks associated with extensive use of fungicides for crop protection. Hence, the use of new approaches using natural plant defense mechanisms, including application of plant antimicrobial peptides (AMPs), is of great interest. Recently, we studied the structural–function relationships between antifungal activity and five hevein-like AMPs from the WAMP (wheat AMP) family of Triticum kiharae Dorof. et Migush. We first discovered that short peptides derived from the central, N-, and C-terminal regions of one of the WAMPs (WAMP-2) were able to augment the inhibitory effect of Folicur® EC 250, a triazole fungicide, on spore germination of the wheat pathogenic fungi, including Fusarium spp. and Alternaria alternata. In this research, we explored the ability of chemically synthesized WAMP-2-derived peptides for enhancing the sensitivity of two other Fusarium and Alternaria species, F. oxysporum and A. solani, causing wilt and early blight of tomato, respectively, to Folicur®. The synthesized WAMP-2-derived peptides synergistically interacted with the fungicide and significantly increased its efficacy, inhibiting conidial germination at much lower Folicur® concentrations than required for the same efficiency using the fungicide alone. The experiments on co-applications of some of WAMP-2-fragments and the fungicide on tomato leaves and seedlings, which confirmed the results obtained in vitro, are described.