Broad-spectrum Antifungal Activity Research Articles

Microscopic and Transcriptomic Analyses to Elucidate Antifungal Mechanisms of Bacillus velezensis TCS001 Lipopeptides against Botrytis cinerea.

Botrytis cinerea is an important fungal pathogen that causes gray mold disease in plants. Previously, Bacillus velezensis TCS001 live culture presented broad-spectrum antifungal activity against various plant pathogenic fungi and oomycetes, particularly B. cinerea. Here, the bioactivity of lipopeptides produced by TCS001 against B. cinerea was investigated. The IC50 values of the crude lipopeptide extract (CLE) from TCS001 to suppress mycelial growth and conidial germination were 14.20 and 49.39 mg/L, respectively. SEM and TEM imaging revealed that CLE caused morphological deformities and ultrastructural changes in the mycelium. Transcriptomic analyses combined with ΔBcpsd mutant construction demonstrated that the CLE could confer antifungal activity via suppressing Bcpsd expression in the pathogen. In addition, the CLE activated the plant immune system by increasing the content of defense-related enzymes and the expression of marker genes in immunity signaling pathways in cucumber plants. Therefore, TCS001 CLE could be potentially developed into biopesticides for the biocontrol of gray mold disease.

MALDI-TOF MS profiling and antifungal activity of lactic acid bacteria from kunu aya, a tiger nut traditional beverage of Nigeria

The application of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) as a technique for high-throughput characterization of microbiota in African traditional foods is limited. Kunu aya is an unexplored reservoir of protective lactic acid bacteria (LAB) strains. A total of 26 LAB isolates from kunu aya were identified using MALDI-TOF MS as five species, including Lactiplantibacillus plantarum (14), Pediococcus pentosaceus (9), Levilactobacillus brevis (1), Lactiplantibacillus pentosus (1), and Weissella paramesenteroides (1). Antifungal activity screening of all LAB strains by agar-plate overlay assay revealed inhibition against Penicillium crustosum FOO5BA and/or Aspergillus flavus FOO8BA by six strains: L. plantarum (3), Pd. pentosaceus (1), L. brevis (1), and W. paramesenteroides (1). The six LAB strains demonstrated broad spectrum antifungal activity, when tested against eight molds from plant-based foods, inhibiting 4 to 7 molds, with average inhibition scores (AIS) of 0.75–2.75. L. brevis MKAIA1b showed the strongest antifungal activity (AIS; 2.75). Agar diffusion method revealed that antifungal activity against all target molds was retained in cell-free supernatants of L. brevis MKAIA1b, with the highest inhibition demonstrated against P. citrinum 3AS1 (15.34%). In challenged kunu aya fermented with L. brevis MKAIA1b or L. brevis MKAIA1b and L. plantarum TMP051, the growth of A. flavus F008BA, Fusarium langesthiae 13,014, and P. crustosum FOO5BA was delayed beyond 7 days at ambient temperature (25 °C). In this study, MALDI-TOF MS was an effective tool to reveal the species composition of LAB in kunu aya, which harbors LAB strains that displayed strong antifungal activity, while showing great potentials as a protective culture and for bio-control.

Complete genome of Pseudomonas putida strain WBB028 isolated from leaf litter.

We report the complete genome of Pseudomonas putida strain WBB028, which exhibits broad-spectrum antifungal activity. This strain was isolated from leaf litter collected at Walker Branch Watershed located on the Oak Ridge Reservation in eastern Tennessee (35.9614 N 84.2864 W). The genome is 6.3 Mbp with a 62.5% GC content.

Inhibitory Effect and Mechanism of Carvacrol against Black Mold Disease Agent Alternaria alternata in Goji Berries.

Alternaria alternata, as a main decay fungus of goji berry, can produce mycotoxins such as alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA). Carvacrol (CVR) has exhibited a broad-spectrum antifungal activity in vitro. We assumed that CVR can also be applied to control Alternaria rot on goji berries and mycotoxins produced by the pathogens. To investigate whether CVR impacts the accumulation of mycotoxins and cell membrane damage of A. alternata, the antifungal activity of CVR on the fungal growth and mycotoxin production was evaluated in this study. The results showed that the minimum inhibitory concentration (MIC) of CVR against A. alternata was 0.12 µL/mL. Meanwhile, the destruction of plasma membrane integrity, cytoplasmic leakage, intracellular oxidative damage, and inhibitory effect in vivo were also observed in A. alternata treated with CVR. Moreover, CVR significantly reduced the accumulation of AOH, AME, and TeA. Transcriptomic profiling was performed by means of comparative RNA-Seq analysis to research the gene expression level of A. alternata, which attested to significant changes in nitrogen metabolism, carbon utilization, fatty acid oxidation, and antioxidant enzymes in CVR-treated A. alternata. This study suggests a new understanding of the molecular mechanism of response to CVR treatment in A. alternata, indicating that CVR is a novel antifungal agent with the potential to be applied to various fungi.

Design, Synthesis, and Antifungal Evaluation of Diverse Heterocyclic Hydrazide Derivatives as Potential Succinate Dehydrogenase Inhibitors.

Plant pathogenic fungi pose a significant threat to agricultural production, necessitating the development of new and more effective fungicides. The ring replacement strategy has emerged as a highly successful approach in molecular design. In this study, we employed the ring replacement strategy to successfully design and synthesize 32 novel hydrazide derivatives containing diverse heterocycles, such as thiazole, isoxazole, pyrazole, thiadiazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, thiophene, pyridine, and pyrazine. Their antifungal activities were evaluated in vitro and in vivo. Bioassay results revealed that most of the title compounds displayed remarkable antifungal activities in vitro against four tested phytopathogenic fungi, including Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, and Rhizoctonia solani. Especially, compound 5aa displayed a broad spectrum of antifungal activity against F. graminearum, B. cinerea, S. sclerotiorum, and R. solani, with the corresponding EC50 values of 0.12, 4.48, 0.33, and 0.15 μg/mL, respectively. In the antifungal growth assay, compound 5aa displayed a protection efficacy of 75.5% against Fusarium head blight (FHB) at a concentration of 200 μg/mL. In another in vivo antifungal activity evaluation, compound 5aa exhibited a noteworthy protective efficacy of 92.0% against rape Sclerotinia rot (RSR) at a concentration of 100 μg/mL, which was comparable to the positive control tebuconazole (97.5%). The existing results suggest that compound 5aa has a broad-spectrum antifungal activity. Electron microscopy observations showed that compound 5aa might cause mycelial abnormalities and organelle damage in F. graminearum. Moreover, in the in vitro enzyme assay, we found that the target compounds 5aa, 5ab, and 5ca displayed significant inhibitory effects toward succinate dehydrogenase, with the corresponding IC50 values of 1.62, 1.74, and 1.96 μM, respectively, which were superior to that of boscalid (IC50 = 2.38 μM). Additionally, molecular docking and molecular dynamics simulation results revealed that compounds 5aa, 5ab, and 5ca have the capacity to bind in the active pocket of succinate dehydrogenase (SDH), establishing hydrogen-bonding interactions with neighboring amino acid residues.

Insights into the biocontrol and plant growth promotion functions of Bacillus altitudinis strain KRS010 against Verticillium dahliae

BackgroundVerticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar “Zhongzhimian No. 2.”ResultsThe strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth.ConclusionsThis study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

Antifungal activity of bio-active cell-free culture extracts and volatile organic compounds (VOCs) synthesised by endophytic fungal isolates of Garden Nasturtium

Antimicrobial resistance in fungal pathogens (both human and plant) is increasing alarmingly, leading to massive economic crises. The existing anti-fungal agents are becoming ineffective, and the situation worsens on a logarithmic scale. Novel antifungals from unique natural sources are highly sought to cope sustainably with the situation. Metabolites from endophytic microbes are the best-fitted alternatives in this case. Endophytes are the untapped sources of ‘plants’ internal microbial population’ and are promising sources of effective bio-therapeutic agents. Fungal endophytes were isolated from Tropaeolum majus and checked for antifungal activity against selected plant and human pathogens. Bioactive metabolites were identified through chromatographic techniques. The mode of action of those metabolites was evaluated through various spectroscopic techniques. The production of antifungal metabolite was optimized also. In particular VOCs (volatile organic compounds) of TML9 were tested in vitro for their anti-phytopathogenic activity. Ethyl acetate (EA) extract of cell-free culture components of Colletotrichum aenigma TML3 exhibited broad-spectrum antifungal activity against four species of Candida and the major constituents reported were 6-pentyl-2H-pyran-2-one, 2-Nonanone, 1 propanol 2-amino. The volatile metabolites, trans-ocimene, geraniol, and 4-terpinyl acetate, produced from Curvularia lunata TML9, inhibited the growth of some selected phyto pathogens. EA extract hampered the biofilm formation, minimised the haemolytic effect, and blocked the transformation of Candida albicans (MTCC 4748) from yeast to hyphal form with a Minimum Fungicidal Concentration (MFC) of 200–600 µg mL−1. Central carbohydrate metabolism, ergosterol synthesis, and membrane permeability were adversely affected and caused the lethal leakage of necessary macromolecules of C. albicans. Volatile metabolites inhibited the growth of phytopathogens i.e., Rhizoctonia solani, Alternaria alternata, Botrytis cinerea, Cercospora beticola, Penicillium digitatum, Aspergillus fumigatus, Ceratocystis ulmi, Pythium ultimum up to 89% with an IC50 value of 21.3–69.6 µL 50 mL−1 and caused leakage of soluble proteins and other intracellular molecules. Citrusy sweet odor volatiles of TML9 cultured in wheat-husk minimised the infections of Penicillium digitatum (green mold), in VOC-exposed sweet oranges (Citrus sinensis). Volatile and non-volatile antifungal metabolites of these two T. majus endophytes hold agricultural and pharmaceutical interests. Metabolites of TML3 have strong anti-Candida activity and require further assessment for therapeutic applications. Also, volatile metabolites of TML9 can be further studied as a source of antifungals. The present investigational outcomes bio-prospects the efficacy of fungal endophytes of Garden Nasturtium.

Esporotricose felina: Sinais clínicos e prevenção em animais e humanos

Sporotrichosis is a mycosis caused by the fungus Sporothrix sp, which is found in plants, trees, and soil, associated with decomposing plant remains. The disease can affect various species, especially felines which, in turn, are the main transmitters of sporotrichosis to humans. Infection by Sporothrix sp. occurs through contact between injured skin and surfaces contaminated with the fungus, or scratching or biting by diseased animals. In addition to the skin manifestation, sporotrichosis can also occur in extracutaneous form, affecting multiple organs such as the lungs, lymph nodes, liver, spleen and kidneys. There is no preventive vaccine for the disease and it is treated with synthetic fungistatic agents with a broad spectrum of antifungal activity. Sporotrichosis is considered one of the most relevant mycoses in veterinary medicine and an underdiagnosed zoonosis, mainly due to the lack of control and prevention initiatives, which consequently leads to a lack of public awareness. In the states of Pernambuco, Rio de Janeiro and in some cities in states of São Paulo, Minas Gerais Bahia, Rio Grande do Norte and Paraíba have compulsory notification of cases. Since December 1, 2020, the notification of sporotrichosis in dogs and cats has become mandatory in the city of São Paulo. The purpose of this review is to emphasize that sporotrichosis is an emerging zoonosis that is underdiagnosed in Brazil. Information and publicity campaigns are needed to reinforce the importance of treatment and the potential for a cure, as well as effective educational measures related to public health programs for the control of human and animal sporotrichosis. Responsible pet ownership campaigns, which emphasize the importance of neutering and restricting street access, are of paramount importance for the epidemiological control of the disease. Veterinarians have a fundamental role to play in guiding the guardian, emphasizing that the lack of treatment, in addition to causing great suffering to the affected animal, can contribute to the spread of the disease, including to humans.

Role of Sertaconazole in the Treatment of Dermatophytosis

Dermatophytosis is a major health burden worldwide and is now increasing day by day. Dermatophytosis is also becoming increasingly unresponsive to topical conventional antifungals now a day. Newer topical antifungals may be more effective in these patients. Sertaconazole is a new, broad spectrum, fungicidal and fungistatic imidazole with added antipruritic and anti-inflammatory activity that may be effective and beneficial in improving the quality of life for the patient with dermatophytoses. It is indicated in the European Union(EU) for the treatment of superficial skin mycoses such as dermatophytosis (including tinea corporis, tinea cruris, tinea manus, tinea barbae and tinea pedis), cutaneous candidiasis, pityriasis versicolor and seborrhoeic dermatitis of the scalp, and in the US for tinea pedis only. Sertaconazole has broad-spectrum antifungal activity against dermatophytes of the Trichophyton, Epidermophyton and Microsporum genera, and yeasts of the genera Candida and Cryptococcus; additionally, it is effective against opportunistic filamentous fungi and Gram-positive bacteria. Moreover, the antifungal activity of sertaconazole is maintained in clinical isolates of dermatophytes that show reduced susceptibility to other azoles. While the drug has good dermal penetration, this is not associated with systemic absorption. In clinical trials in patients with superficial mycoses, 2% sertaconazole cream applied twice daily was effective in the eradication of a range of dermatophytoses, and a significantly greater proportion of patients were cured compared with those receiving 2% miconazole cream twice-daily treatment. Both as a topical cream and suppository preparation, sertaconazole was generally well tolerated. Sertaconazole is a well-established antifungal agent, which is now available in a variety of formulations, and remains a useful treatment option particularly in patients with fungal infections resistant to other azoles. Medicine Today 2023 Vol.36 (1): 62-66

A novel antimicrobial peptide Spasin141-165 identified from Scylla paramamosain exhibiting protection against Aeromonas hydrophila infection

Aeromonas hydrophila is a widely distributed Gram-negative waterborne pathogen responsible for large-scale outbreaks of hemorrhagic septicemia in fish, causing severe economic losses to the global aquaculture industry. In view of few therapeutic drugs currently available for use in aquaculture and the emergence of multidrug-resistant A. hydrophila, there is an urgent need to develop new antimicrobial agents as an alternative to antibiotics. Antimicrobial peptides (AMPs) have strong antimicrobial effects and unique machanisms of action, and are not easy to induce bacterial resistance, making them an ideal alternative to antibiotics. In this study, we identified a new functional gene from marine crab Scylla paramamosain, named Spasin, which encodes a mature peptide of 240 amino acids and expressed widely in various tissues of crabs. Notably, a truncated peptide Spasin141–165 derived from Spasin, showed potent and broad-spectrum antibacterial and antifungal activity at minimum inhibitory concentrations (MICs) ranged from 0.75 μM–48 μM, which is a novel AMP. It effectively killed S. aureus and A. baumannii within 2 h and 90 min, respectively. Importantly, it is not toxic to HEK-293 T, RAW 264.7, and ZF4 cell lines. Mechanistic studies showed that addition of 8 μg mL−1 lipoteichoic acid or lipopolysaccharides decreased bacterial susceptibility to Spasin141–165. In addition, Spasin141–165 altered the permeability of the inner and outer cell membranes, and resulted in the release of ATP and the accumulation of intracellular reactive oxygen species. Notably, Spasin141–165 significantly increased the survival rate of zebrafish infected with A. hydrophila, redcued the bacterial load in the spleen, and effectively inhibited the expression of inflammation-associated factors (e.g., IL-1β, TNF-α, etc.). Taken together, Spasin141–165 is a potential antibacterial agent for the treatment of A. hydrophila infections in aquaculture and a promising antibiotic alternative.

Seriphidium herba-alba (Asso): A comprehensive study of essential oils, extracts, and their antimicrobial properties.

Seriphidium herba-alba (Asso), a plant celebrated for its therapeutic qualities, is widely used in traditional medicinal practices throughout the Middle East and North Africa. In a detailed study of Seriphidium herba-alba (Asso), essential oils and extracts were analyzed for their chemical composition and antimicrobial properties. The essential oil, characterized using mass spectrometry and retention index methods, revealed a complex blend of 52 compounds, with santolina alcohol, α-thujone, β-thujone, and chrysanthenone as major constituents. Extraction yields varied significantly, depending on the plant part and method used; notably, methanol soaking of aerial parts yielded the most extract at 17.75%. The antimicrobial analysis showed that the extracts had selective antibacterial activity, particularly against Staphylococcus aureus, and broad-spectrum antifungal activity against organisms such as Candida albicans and Aspergillus spp. The methanol-soaked extract demonstrated the strongest antimicrobial properties, indicating its potential as a natural antimicrobial source. This study not only underscores the therapeutic potential of Seriphidium herba-alba (Asso) in pharmaceutical applications but also sets a foundation for future research focused on isolating specific bioactive compounds and in vivo testing.

Impact of Nitric Oxide-Release Kinetics on Antifungal Activity.

Pathogenic fungi are an increasing health threat due to the rise in drug resistance. The limited number of antifungals currently available and growing incidence of multi-drug-resistant fungi has caused rising healthcare costs and a decreased quality of life for patients with fungal infections. Nitric oxide (NO) has previously been shown to act as an antimicrobial agent, albeit with a limited understanding of the effects of the NO-release kinetics against pathogenic fungi. Herein, the antifungal effects of four nitric oxide-releasing small molecules were studied against the pathogenic fungi Candida albicans, Candida auris, Cryptococcus neoformans, and Aspergillus fumigatus, to demonstrate the broad-spectrum antifungal activity of NO. A bolus dose of NO was found to eradicate fungi after 24 h, where nitric oxide donors with shorter half-lives achieved antifungal activity at lower concentrations and thus had wider selectivity indexes. Each NO donor was found to cause a severe surface destruction of fungi, and all NO donors exhibited compatibility with currently prescribed antifungals against several different fungi species.

An Effective and Promising Strategy for Plant Protection: Synthesis of L-Carvone-Based Thiazolinone-Hydrazone/Nanochitosan Complexes with Antifungal Activity and Sustained Releasing Performance.

The development of novel natural product-derived nano-pesticide systems with loading capacity and sustained releasing performance of bioactive compounds is considered an effective and promising plant protection strategy. In this work, 25 L-carvone-based thiazolinone-hydrazone compounds 4a~4y were synthesized by the multi-step modification of L-carvone and structurally confirmed. Compound 4h was found to show favorable and broad-spectrum antifungal activity through the in vitro antifungal activity evaluation of compounds 4a~4y against eight phytopathogenic fungi. Thus, it could serve as a leading compound for new antifungal agents in agriculture. Moreover, the L-carvone-based nanochitosan carrier 7 bearing the 1,3,4-thiadiazole-amide group was rationally designed for the loading and sustained releasing applications of compound 4h, synthesized, and characterized. It was proven that carrier 7 had good thermal stability below 200 °C, dispersed well in the aqueous phase to form numerous nanoparticles with a size of~20 nm, and exhibited an unconsolidated and multi-aperture micro-structure. Finally, L-carvone-based thiazolinone-hydrazone/nanochitosan complexes were fabricated and investigated for their sustained releasing behaviors. Among them, complex 7/4h-2 with a well-distributed, compact, and columnar micro-structure displayed the highest encapsulation efficiency and desirable sustained releasing property for compound 4h and thus showed great potential as an antifungal nano-pesticide for further studies.

Biocontrol efficiency and potential mechanism of streptomyces distatochromogenes XT34 against postharvest anthracnose caused by colletotrichum musae on banana fruit

Banana anthracnose caused by Colletotrichum musae is a serious postharvest disease. Use of fungicides has potential adverse effects on the environment safety and human health. Application of biological control agents (BCA) is a promising strategy for managing postharvest fruit diseases. In this study, Streptomyces sp. XT34 was isolated from the banana rhizosphere soil and exhibited a strong antifungal activity against C. musae. Combining the phenotypic and whole-genomic alignment, strain XT34 was identified as Streptomyces diastatochromogenes. Strain XT34 extracts reduced the disease incidence of anthracnose and maintained the fruit quality of banana. Treatment of extracts reduced the colonization of C. musae on fruit surface. The activity inhibition of cell wall-degrading enzymes maintained the cell-wall strength of fruit. The activity increase of defense enzymes contributed to the low disease symptom. It was supported by the differentially expressed genes (DEGs) related to supramolecular fiber organization and flavonoid biosynthesis. In addition, spore germination of C. musae was effectively inhibited by extracts. The morphology of mycelia and spore become wrinkled and ruptured. The treatment of extracts increased the optical density of A260 and soluble sugar and protein contents of C. musae. The integrity of cell membrane was also destroyed, reflecting on an increase in electrical conductivity and malondialdehyde. Several metabolites were found in the genome of strain XT34 using antiSMASH. Eleven volatile compounds were identified by gas chromatography-mass spectrometer (GC-MS). Notably, strain XT34 and extracts exhibited a broad-spectrum antifungal activity against eight phytopathogenic fungi. Hence, S. diastatochromogenes XT34 provides a potential BCA to control banana anthracnose.

Plumbagin controls fungal postharvest pathogens by affecting metabolism and inducing autophagy

Botrytis cinerea is a devastating postharvest pathogenic fungus causing severe postharvest decay in a wide range of fruit and vegetables. Controlling postharvest pathogens is essential to the postharvest industry. Plumbagin is a plant-derived naphthoquinone compound with various pharmacological activities, such as antioxidant, anti-inflammatory, and antimicrobial effects. However, its antifungal effects on B. cinerea have not been fully elucidated. In this study, we investigated the antifungal activity of plumbagin against postharvest fungal pathogens and elucidated its potential mechanism on B. cinerea through transcriptomics. Plumbagin exhibited broad-spectrum antifungal activity and completely inhibited the growth of various postharvest pathogens at a concentration of 20 mg L−1in vitro. Moreover, plumbagin effectively inhibited the incidence of gray mold in grape, sweet cherry, and strawberry fruit. The spore germination and viability of B. cinerea were also suppressed by plumbagin. Transcriptomic analysis indicated that plumbagin significantly affected the metabolic processes and induced autophagy in B. cinerea. Our results suggest that plumbagin is a promising broad-spectrum plant-derived fungicide that suppresses postharvest gray mold by affecting the metabolism of B. cinerea and inducing autophagy. These findings provide a theoretical basis for applying plumbagin to control postharvest pathogens.

In Vitro and Ex Vivo Antifungal Activities of Metconazole against the Rice Blast Fungus Pyricularia oryzae.

Rice blast, caused by the filamentous fungus Pyricularia oryzae, has long been one of the major threats to almost all rice-growing areas worldwide. Metconazole, 5-(4-chlorobenzyl)-2, 2-dimethyl-1-(1H-1, 2, 4-triazol-1-ylmethyl) cyclopentanol, is a lipophilic, highly active triazole fungicide that has been applied in the control of various fungal pathogens of crops (cereals, barley, wheat), such as the Fusarium and Alternaria species. However, the antifungal activity of metconazole against P. oryzae is unknown. In this study, metconazole exhibited broad spectrum antifungal activities against seven P. oryzae strains collected from rice paddy fields and the wild type strain P131. Scanning electron microscopic analysis and fluorescein diacetate staining assays revealed that metconazole treatment damaged the cell wall integrity, cell membrane permeability and even cell viability of P. oryzae, resulting in deformed and shrunken hyphae. The supplementation of metconazole in vitro increased fungal sensitivity to different stresses, such as sodium dodecyl sulfate, congo red, sodium chloride, sorbitol and oxidative stress (H2O2). Metconazole could inhibit key virulence processes of P. oryzae, including conidial germination, germ tube elongation and appressorium formation. Furthermore, this chemical prevented P. oryzae from infecting barley epidermal cells by disturbing appressorium penetration and subsequent invasive hyphae development. Pathogenicity assays indicated a reduction of over 75% in the length of blast lesions in both barley and rice leaves when 10 μg/mL of metconazole was applied. This study provides evidence to understand the antifungal effects of metconazole against P. oryzae and demonstrates its potential in rice blast management.

Comprehensive genomic analysis of Bacillus subtilis and Bacillus paralicheniformis associated with the pearl millet panicle reveals their antimicrobial potential against important plant pathogens

BackgroundPlant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles.ResultsTwo potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens (Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis. Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin.ConclusionThe combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.

Isolating Antipathogenic Fungal Coumarins from Coriaria nepalensis and Determining Their Primary Mechanism In Vitro.

Through bioassay-guided isolation, eight undescribed coumarins (1-8), along with six reported coumarins (9-14), were obtained from Coriaria nepalensis. The new structures were determined by using IR, UV, NMR, HRESIMS, and ECD calculations. The results of the biological activity assays showed that compound 9 exhibited broad spectrum antifungal activities against all tested fungi in vitro and a significant inhibitory effect on Phytophthora nicotianae with an EC50 value of 3.00 μg/mL. Notably, compound 9 demonstrated greater curative and protective effects against tobacco balack shank than those of osthol in vivo. Thus, 9 was structurally modified to obtain new promising antifungal agents, and the novel derivatives (17b, 17j, and 17k) exhibited better effects on Sclerotinia sclerotiorum than did lead compound 9. Preliminary mechanistic exploration illustrated that 9 could enhance cell membrane permeability, destroy the morphology and ultrastructure of cells, and reduce the exopolysaccharide content of P. nicotianae mycelia. Furthermore, the cytotoxicity results revealed that compound 9 exhibited relatively low cytotoxicity against HEK293 cell lines with an inhibition rate of 33.54% at 30 μg/mL. This research is promising for the discovery of new fungicides from natural coumarins with satisfactory ecological compatibility.

Antagonistic activity and mechanism of Bacillus subtilis CG-6 suppression of root rot and growth promotion in Alfalfa

Root rot is a common disease, that severely affects the yield and quality of alfalfa. Biocontrol is widely used to control plant diseases caused by pathogenic fungi, however, biocontrol strains for alfalfa root rot are very limited. In this study, a Bacillus subtilis CG-6 strain with a significant biocontrol effect on alfalfa root rot was isolated. CG-6 secretes antibacterial enzymes and siderophore, phosphate solubilization and indoleacetic acid (IAA). The inhibition rate of strain CG-6 against Fusarium oxysporum was 87.33%, and it showed broad-spectrum antifungal activity. Inoculation with CG-6 significantly reduced the incidence of alfalfa root rot, the control effect of greenhouse cultivation reached 58.12%, and CG-6 treatment significantly increased alfalfa plant height, root length, fresh weight, and dry weight. The treatment with CG-6 significantly increased the levels of antioxidant enzymes (catalase, peroxidase, superoxide dismutase, and lipoxygenase) in alfalfa leaves by 15.52%–34.03%. Defensive enzymes (chitinase and β-1,3-glucanase) increased by 24.37% and 28.08%, respectively. The expression levels of regulatory enzyme genes (MsCAT, MsPOD, MsCu, Zn-SOD1, MsCu, Zn-SOD2, MsCu, Zn-SOD3, and MsLOX2) and systemic resistance genes (MsPR1, MsPDF1.2, and MsVSP2) increased by 0.50–2.85 fold, which were higher than those in the pathogen treatment group. Therefore, CG-6 could be used as a potential strain to develop biopesticides against alfalfa root rot.

Design, synthesis and antifungal activity of novel α-methylene-γ-butyrolactone derivatives containing benzothiophene moiety.

The discovery of agricultural fungicide candidates from natural products is one of the key strategies for developing environment friendly agricultural fungicides with high efficiency, high selectivity and unique modes-of-action. Based on previous work, a series of novel α-methylene-γ-butyrolactone (MBL) derivatives containing benzothiophene moiety were designed and synthesized. The majority of the proposed compounds displayed moderate to considerable antifungal efficacy against the tested pathogenic fungi and oomycetes, some exhibiting broad spectrum antifungal activity. Notably, compounds 2 (3-F-Ph) and 7 (4-Cl-Ph) showed excellent antifungal activity against Rhizoctonia with half maximal effective concentration (EC50) values of 0.94 and 0.99 mg L-1, respectively, comparable to the commercial fungicide tebuconazole (EC50 = 0.96 mg L-1), and also displayed significant inhibitory effects against V alsa mali with EC50 values of 2.26 and 1.67 mg L-1, respectively - better than famoxadone and carabrone. The in vivo protective and curative effects against R. solani of compound 2 were 57.2% and 53.7% at 100 mg L-1, respectively, which were equivalent to tebuconazole (51.6% and 52.4%). Further investigations found that compound 2 altered the ultrastructure of R. solani cell, significantly increased the relative conductivity of the cells, and reduced the activity of complex III in a dose-dependent manner. Molecular docking results showed that compound 2 matched well with the Qo pocket. The results revealed that MBL derivatives containing benzothiophene moiety are promising antifungal candidates and provide a new backbone structure for further optimization of novel fungicides. © 2024 Society of Chemical Industry.