Antifungal Metabolites Research Articles

Evaluation of Bacillus subtilis Czk1 Metabolites by LC–MS/MS and Their Antifungal Potential against Pyrrhoderma noxium Causing Brow Rot Disease

Brown rot disease caused by Pyrrhoderma noxium is a widespread disease that severely affects the roots of rubber trees (Hevea brasiliensis Muell. Arg.). The economic losses, along with environmental and health problems arising from the use of disease control chemicals, have raised the interest of scholars to explore the use of biological control agents for the effective control of fungal pathogen P. noxium. Here, the inhibition effect of the culture filtrate of B. subtilis Czk1 on P. noxium was demonstrated. The findings indicate that the antifungal activity of this strain is mediated wholly or partly by compounds produced in the culture filtrate. The combined use of liquid chromatography–tandem mass spectrometry and antifungal activity assays rapidly identified compounds produced by B. subtilis Czk1. Metabolic profiles were assessed and used to identify major metabolites based on the scores of variable importance in the projection and the plot scores of principal component analysis. A total of 296 differential metabolites were screened, including 208 in positive ion mode and 88 in negative ion mode. Two key metabolites, diacetyl and trans-2-octenoic acid, were screened from 29 metabolites by antifungal activity assays. The median effective concentration (EC50) of trans-2-octenoic acid and diacetyl were 0.9075 mg/mL and 4.8213 mg/mL, respectively. The antifungal metabolites can disrupt the internal structure of the pathogenic fungal mycelium, thereby impeding its growth. This study is expected to contribute to the existing knowledge of Czk1-produced metabolites and their future antifungal applications. This study is also expected to provide a new biopreservative perspective on unexplored antifungal metabolites produced by Czk1 as a biocontrol agent.

Biomass from phytopathogens and culture conditions improve Penicillium chrysogenum antimicrobial activity and antifungal compounds production

AbstractThe present study evaluated the effect of culture conditions and phytopathogenic strain co‐culture on the production of antimicrobial metabolites and antifungal activity of Penicillium chrysogenum R1, which PCR identified. Antimicrobial activity was determined using the Hunter‐Hunter experimental design with three factors (pH, incubation temperature, and inoculum, at two levels each). The antifungal metabolites, β 1‐3 glucanase and chitinase, produced in the presence of live and inactivated Fusarium oxysporum Fsox C11 biomass, were evaluated using HPLC‐MS and GC‐MS. Results showed that P. chrysogenum inhibited the growth of five phytopathogenic fungal strains, and the most significant inhibition was observed for F. oxysporum Fsox C11. The best conditions to achieve the highest antifungal activity of the cell‐free extract were pH 7, 28°C, 1 × 106 spores/mL, and 144 h of fermentation, observing 86% inhibition of F. oxysporum Fsox C11 growth. Production of antifungal metabolites such as 1,4‐benzoquinone imine, viridicatic acid, phenol‐5‐methyl‐2‐(1‐methyl ethyl), and hydrolytic enzymes β 1‐3 glucanase and chitinase was detected. The results define the perspective in designing new processes and products for biocontrol phytopathogens.

Investigation on Chemical Composition, Antioxidant, Antifungal and Herbicidal Activities of Volatile Constituents from Deverra tortuosa (Desf.).

This study aims to analyze the chemical composition of the essential oils (EOs) obtained from stems and umbels of D. tortuosa as well the assessment of their biological activity. EOs were extracted by hydrodistillation and analyzed by gas chromatography coupled to mass spectrometry (GC/MS). The antioxidant properties were determined by DPPH and ABTS assays. The phytotoxic potential was assessed against dicots weeds (Sinapis arvensis and Trifolium campestre), monocots weeds (Lolium rigidum) and the crop Lepidium sativum. The antifungal activity was evaluated against four target phytopathogenic fungal strains. High diversity of compounds was detected in D. tortuosa Eos, varying among plant parts and consisting mainly of α-pinene (24.47-28.56%), sabinene (16.2-18.6%), α-phellandrene (6.3-11.7%) and cis-ocimene (5.28-7.85%). D. tortuosa EOs exhibited remarkable antioxidant activity, as well as interesting variable antifungal activities depending on the dose and fungi strain. The herbicidal activity of EOs showed significant efficacy on the inhibition of germination and seedling growth of all tested herbs. These results suggest that the EOs of Deverra tortuosa represent a valuable source of antioxidant, antifungal and phytotoxic metabolites and could be potential candidates for pest management, contributing to the promotion of sustainable agriculture.

Secondary Metabolites with Herbicidal and Antifungal Activities from Marine-Derived Fungus Alternaria iridiaustralis.

Weed and soil-borne pathogens could synergistically affect vegetable growth and result in serious losses. Investigation of agricultural bioactive metabolites from marine-derived fungus Alternaria iridiaustralis yielded polyketides (1-4), benzopyrones (5-7), meroterpenoid derivatives (8), and alkaloid (9). The structures and absolute configurations of new 1, 3, 5-6, and 8 were elucidated by extensive spectroscopic analyses, as well as comparisons between measured and calculated ECD and 13C NMR data. Compounds 1-4, 6, and 9 showed herbicidal potentials against the radicle growth of Echinochloa crusgalli seedlings. Especially 9 exhibited inhibition rates over 90% at concentrations of 20 and 40 μg/mL, even better than the commonly used chemical herbicide acetochlor. Furthermore, 9 also performed a wide herbicidal spectrum against the malignant weeds Digitaria sanguinalis, Portulaca oleracea, and Descurainia sophia. Compounds 5-8 showed antifungal activities against carbendazim-resistant strains of Botrytis cinerea, with minimum inhibitory concentration (MIC) values ranging from 32 to 128 μg/mL, which were better than those of carbendazim (MIC = 256 μg/mL). Especially 6 exhibited integrated effects against both soil-borne pathogens and weed. Overall, marine-derived fungus A. iridiaustralis, which produces herbicidal and antifungal metabolites 1-9, showed the potential for use as a microbial pesticide to control both weed and soil-borne pathogens.

Uji Aktivitas Antijamur Bacillus spp. Terhadap Colletrotrichum scovilei Penyebab Antraknosa Cabai Rawit

Anthracnose caused by the Colletotrichum scovillei is main diseases of cayenne pepper (Capsicum frutescens L.) that affect on plant productivity. The control of plant diseases caused by pathogenic fungi has been widely reported by utilizing of biological agents that applied in biofungicides. Bacillus spp. was reported to have antagonistic abilities against several pathogenic fungus. The purpose of this study was to determine the antagonistic ability of several Bacillus species against Colletotrichum scovillei. The begins was rejuvenatio of the C. scovillei and B. subtillis, B. cereus, B. coagulans, B. thuringiensis that have been identified. The inhibition of Bacillus spp. on the growth and biomass of C. scovillei was tested in vitro, as well as testing the inhibition of Bacillus spp. filtrates on C. scovillei colonies. The inhibitions of Bacillus spp. against the growth of C. Scovillei were ranged from 91.36% to 96.46% and a percentage inhibition on biomass of C. scovillei were ranged from 85.35% to 88.53% compared to the control. Bacillus spp. filtrates was tested at concentrations of 20%-90% were able to inhibit the growth of C. scovillei colonies with a percentage inhibition up to 99.84%. The good quality standard of biological agent must have >70% of inhibition in vitro. The inhibition that occurs by Bacillus spp. on the growth of C. scovillei is likely due to competition for nutrient and space. And other, the possibility of an antagonistic mechanism in the form of the presence of chitinase, which is one of the antifungal metabolite compounds.

Efficacy of Streptomyces murinus JKTJ-3 in Suppression of Pythium Damping-Off of Watermelon.

Damping-off caused by Pythium aphanidermatum (Pa) is one of the most destructive diseases for watermelon seedlings. Application of biological control agents against Pa has attracted the attention of many researchers for a long time. In this study, the actinomycetous isolate JKTJ-3 with strong and broad-spectrum antifungal activity was screened from 23 bacterial isolates. Based on the morphological, cultural, physiological, and biochemical characteristics as well as the feature of 16S rDNA sequence, isolate JKTJ-3 was identified as Streptomyces murinus. We investigated the biocontrol efficacy of isolate JKTJ-3 and its metabolites. The results revealed that seed and substrate treatments with JKTJ-3 cultures showed a significant inhibitory effect on watermelon damping-off disease. Seed treatment with the JKTJ-3 cultural filtrates (CF) displayed higher control efficacy compared to the fermentation cultures (FC). Treatment of the seeding substrate with the wheat grain cultures (WGC) of JKTJ-3 exhibited better control efficacy than that of the seeding substrate with the JKTJ-3 CF. Moreover, the JKTJ-3 WGC showed the preventive effect on suppression of the disease, and the efficacy increased with increase in the inoculation interval between the WGC and Pa. Production of the antifungal metabolite actinomycin D by isolate JKTJ-3 and cell-wall-degrading enzymes such as β-1,3-glucanase and chitosanase were probably the mechanisms for effective control of watermelon damping-off. It was shown for the first time that S. murinus can produce anti-oomycete substances including chitinase and actinomycin D. This is the first report about S. murinus used as biocontrol agent against watermelon damping-off caused by Pa.

Antifungal activity of volatile and non-volatile metabolites of endophytes of Chloranthus elatior Sw.

Agriculture crops that have fungal infections suffer significant economic losses and reduced crop output. Chemical fungicides are used to tackle the problem, although this has additional detrimental side effects. There is an urgent need for safe and novel antifungals. Volatiles from plant-beneficial endophytic fungi are considered promising alternatives for the biological control of fungal pathogens as a sustainable approach in an agroecosystem. In the present investigation, a volatile-emitting sterile endophytic fungus, Diaporthe sp. CEL3 with bio-fumigation activity, was isolated from leaves of the ethnomedicinal plant Chloranthus elatior Sw., collected from the Passighat forest of North-East India. The camphor odor volatiles of CEL3 showed an inhibitory effect against eight fungal pathogens in vitro and minimized the infections of Monilinia fructicola, a causal agent of cherry fruit rot, in VOC-exposed cherry fruits. Rhizoctonia solani, Botrytis cinerea, Pythium ultimum, and M. fructicola were maximally inhibited up to 51.5%, 55.8%, 61.9%, and 78.5%, respectively, in comparison to control by the volatiles. Another isolate, CEL7, identified as Curvularia sp., synthesized non-volatile, soluble antifungal metabolites in its cell-free extracts and exhibited antifungal action. Bioassay-guided fractionation revealed the presence of imidazole compounds- (2-aminoethyl)-1H-imidazole-2-carbaldehyde, Pyrazole 4, 5 imidazole, 1-formyl 3-ethyl, phenol compounds-Phenol, 4-[2-(methylamino) ethyl]-, 6-Nitro-3-chlorophenol, Phenol, 2,4,6-tri-tert-butyl-, etc., in the cell-free extracts, with a MIC value of 250-2,000 µg ml-1. Optimum VOC emission was achieved in a modified PDA medium with instantly smashed potato (150 g L-1), dextrose (20 g L-1), wheat husk (20 g L-1), and yeast extract (20 g L-1), with additional salts. Interestingly, endophytic CEL3 emitted different types of volatiles, and trans-verbenol (32.25%), geraniol (30.32%), trans-ocimenol (12.90%), and mentha-4,8-diene (5.16%) were the prime ones. These VOCs cause lethal leakage of protein and necessary intracellular molecules from the fungal pathogens. Thus, CEL3 could potentially be used as a bio-fumigating agent to control post-harvest infections caused by fungal pathogens. This study opens a new approach to the use of endophytic fungi in biocontrol.

Antifungal effects and biocontrol potential of lipopeptide-producing Streptomyces against banana Fusarium wilt fungus Fusarium oxysporum f. sp. cubense.

Fusarium wilt of banana (FWB), caused by Fusarium oxysporum f. sp. cubense (Foc), especially tropical race 4 (TR4), presents the foremost menace to the global banana production. Extensive efforts have been made to search for efficient biological control agents for disease management. Our previous study showed that Streptomyces sp. XY006 exhibited a strong inhibitory activity against several phytopathogenic fungi, including F. oxysporum. Here, the corresponding antifungal metabolites were purified and determined to be two cyclic lipopeptide homologs, lipopeptin A and lipopeptin B. Combined treatment with lipopeptin complex antagonized Foc TR4 by inhibiting mycelial growth and conidial sporulation, suppressing the synthesis of ergosterol and fatty acids and lowering the production of fusaric acid. Electron microscopy observation showed that lipopeptide treatment induced a severe disruption of the plasma membrane, leading to cell leakage. Lipopeptin A displayed a more pronounced antifungal activity against Foc TR4 than lipopeptin B. In pot experiments, strain XY006 successfully colonized banana plantlets and suppressed the incidence of FWB, with a biocontrol efficacy of up to 87.7%. Additionally, XY006 fermentation culture application improved plant growth parameters and induced peroxidase activity in treated plantlets, suggesting a possible role in induced resistance. Our findings highlight the potential of strain XY006 as a biological agent for FWB, and further research is needed to enhance its efficacy and mode of action in planta.

Bioactive metabolites of Streptomyces misakiensis display broad-spectrum antimicrobial activity against multidrug-resistant bacteria and fungi.

Antimicrobial resistance is a serious threat to public health globally. It is a slower-moving pandemic than COVID-19, so we are fast running out of treatment options. Thus, this study was designed to search for an alternative biomaterial with broad-spectrum activity for the treatment of multidrug-resistant (MDR) bacterial and fungal pathogen-related infections. We isolated Streptomyces species from soil samples and identified the most active strains with antimicrobial activity. The culture filtrates of active species were purified, and the bioactive metabolite extracts were identified by thin-layer chromatography (TLC), preparative high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). The minimum inhibitory concentrations (MICs) of the bioactive metabolites against MDR bacteria and fungi were determined using the broth microdilution method. Preliminary screening revealed that Streptomyces misakiensis and S. coeruleorubidus exhibited antimicrobial potential. The MIC50 and MIC90 of S. misakiensis antibacterial bioactive metabolite (ursolic acid methyl ester) and antifungal metabolite (tetradecamethylcycloheptasiloxane) against all tested bacteria and fungi were 0.5 μg/ml and 1 μg/mL, respectively, versus S. coeruleorubidus metabolites: thiocarbamic acid, N,N-dimethyl, S-1,3-diphenyl-2-butenyl ester against bacteria (MIC50: 2 μg/ml and MIC90: 4 μg/mL) and fungi (MIC50: 4 μg/ml and MIC90: 8 μg/mL). Ursolic acid methyl ester was active against ciprofloxacin-resistant strains of Streptococcus pyogenes, S. agalactiae, Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica serovars, colistin-resistant Aeromonas hydrophila and K. pneumoniae, and vancomycin-resistant Staphylococcus aureus. Tetradecamethylcycloheptasiloxane was active against azole- and amphotericin B-resistant Candida albicans, Cryptococcus neoformans, C. gattii, Aspergillus flavus, A. niger, and A. fumigatus. Ursolic acid methyl ester was applied in vivo for treating S. aureus septicemia and K. pneumoniae pneumonia models in mice. In the septicemia model, the ursolic acid methyl ester-treated group had a significant 4.00 and 3.98 log CFU/g decrease (P < 0.05) in liver and spleen tissue compared to the infected, untreated control group. Lung tissue in the pneumonia model showed a 2.20 log CFU/g significant decrease in the ursolic acid methyl ester-treated group in comparison to the control group. The haematological and biochemical markers in the ursolic acid methyl ester-treated group did not change in a statistically significant way. Moreover, no abnormalities were found in the histopathology of the liver, kidneys, lungs, and spleen of ursolic acid methyl ester-treated mice in comparison with the control group. S. misakiensis metabolite extracts are broad-spectrum antimicrobial biomaterials that can be further investigated for the potential against MDR pathogen infections. Hence, it opens up new horizons for exploring alternative drugs for current and reemerging diseases.

Biocontrol Stem-end Rot Disease Agent, Alternaria alternata YZU, on Pitaya by Soil Phosphate Solubilizing Bacteria

Biocontrol of stem end disease agent, Alternaria alternata YZU, on pitaya is more interested in altering the application of chemical pesticides. This study was conducted to characterize antagonistic phosphate solubilizing bacteria (PSB) from rhizosphere soil for their biocontrol activities against A. alternata YZU under laboratory and greenhouse conditions. Six PSB isolated from 31 rhizosphere soil samples were tested for inhibiting the mycelial growth of A. alternata YZU in dual cultures. Six isolates were determined to inhibit the mycelial growth of A. alternata YZU. Among them, PSB31 presented the highest level of antagonistic activity against A. alternata YZU with a mean inhibition diameter of 0.64 ± 0.02 cm, while the other strains, including PSB11, PSB21, PSB41, PSB51, and PSB61 presented a weaker inhibition. The results also showed that the strain PSB31 was identified as Bacillus sp. strain PSB31 (Accession number: ON422095) and could control the mycelial growth of A. alternata YZU by secreting antifungal metabolites. Moreover, an in vivo antagonistic experiment of PSB31 on pitaya twigs showed a significant reduction of lesions on twigs than the control. The results suggested that the isolated PSB31 is a potential biological control agent. Further studies should be done to identify the biochemical basis of their activity against A. alternata YZU.

Application of antifungal metabolites from Streptomyces philanthi RL-1-178 for maize grain coating formulations and their efficacy as biofungicide during storage

The major safety risk of maize grain is contamination with mycotoxins. In this study, a maize-coating formulation containing freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 (DCF RL-1-178) was developed and evaluated to prevent the growth of mycotoxins during maize grain storage. In vitro studies using confrontation tests on PDA plates indicated that S. philanthi RL-1-178 inhibited the growth of Aspergillus parasiticus TISTR 3276 (89.0%) and A. flavus PSRDC-4 (95.0%). The maize grain coating formulations containing the DCF RL-1-178 (0, 5, 10, and 15% (v/v)) and the polymer polyvinylpyrrolidone (PVP-K90, 4.0% (w/v)) were tested for their efficacy in In vitro and during 5 months storage. In In vitro assay, maize coating formular containing the optimum concentration (15.0%, v/v) of the DCF RL-1-178 exhibited 54.80% and 54.17% inhibition on the growth of A. parasiticus TISTR 3276 and A. flavus PSRDC-4 respectively. The inhibition was also illustrated by the microstructures of interactions between the coated maize grains with or without the DCF RL-1-178 and the fungal pathogens observed under microscope and SEM. Incorporating the DCF RL-1-178 or fungicidal Metalaxyl® into the polymer PVP-K90 maize grains coating resulted in the complete inhibition of the production of aflatoxin B1 (analysed by HPLC) by the two aflatoxigenic pathogens after 5 months storage at room temperature. However, the shelf-life was shortened to only 3 months during storage at room temperature with 90% relative humidity. Overall, the application of the 10-15% DCF RL-1-178 into the maize grain coating formular provides a new alternative measure to control the mycotoxins during storage for at least 5 months. The In vitro cell cytotoxicity study showed that a concentration of 15% (v/v) or 1000μg/mL of the DCF RL-1-178 had a strong cytotoxic effect on Vero cells. These findings indicate that DCF RL-1-178 is a potential biofungicide for controlling mycotoxins contamination in maize seed storage for planting, but not maize grain storage for animal feed.

Complete genome sequencing ofBacillus subtilis(CWTS 5), a siderophore-producing bacterium triggers antagonistic potential againstRalstonia solanacearum

The aims of this study were to explore the antagonistic potential of siderophore-producing Bacillus subtilis (CWTS 5) for the suppression of Ralstonia solanacearum and to explore the mechanisms of inhibition by FTIR, LC-MS, and whole genome analysis. A siderophore-producing B. subtilis (CWTS 5) possessing several plant growth-promoting properties such as IAA and ACC deaminase production, phosphate solubilization, and nitrogen fixation was assessed for its inhibitory effect against R. solanacearum, and its mechanisms were explored by in vitro and in vivo analyses. The active secondary metabolites in the siderophore extracts were identified as 2-deoxystreptamine, miserotoxin, fumitremorgin C, pipercide, pipernonaline, gingerone A, and deoxyvasicinone by LC-MS analysis. The Arnow's test and antiSMASH analysis confirmed the presence of catecholate siderophores, and the functional groups determined by FTIR spectroscopy confirmed the presence of secondary metabolites in the siderophore extract possessing antagonistic effect. The complete genome sequence of CWTS 5 revealed the gene clusters responsible for siderophore, antibiotics, secondary metabolite production, and antibacterial and antifungal metabolites. Furthermore, the evaluation of CWTS 5 against R. solanacearum in pot studies demonstrated 40.0% reduced disease severity index (DSI) by CWTS 5, methanolic extract (DSI-26.6%), ethyl acetate extract (DSI-20.0%), and increased plant growth such as root and shoot length, wet weight and dry weight of Solanum lycopersicum L. owing to its antagonistic potential. This genomic insight will support future studies on the application of B. subtilis as a plant growth promoter and biocontrol agent against R. solanacearum for bacterial wilt management. The results of this study revealed that B. subtilis (CWTS 5) possesses multiple mechanisms that control R. solanacearum, reduce disease incidence, and improve S. lycopersicum growth.

The antifungal metabolites from coculture of Aspergillus fumigatus and Alternaria alternata associated with Coffea arabica

One new cyclohexenone derivative, asperfumtone A (1) along with six known compounds were obtained from the coculture of Aspergillus fumigatus and Alternaria alternata associated with Coffea arabica. The configuration of 2 was first reported in the research. The structures were determined by extensive spectroscopic analyses, and ECD calculation. Compounds 3, 4 and 7 showed significant antifungal activities against coffee phytopathogens A. alternata and Fusarium incarnatum with MICs of 1 μg/mL. Compounds 1 and 2 showed weak antifungal activities against A. alternata and F. incarnatum with MICs of 32–64 μg/mL.

Bacillus amyloliquefaciens FG14 as a potential biocontrol strain against rusty root rot of Panax ginseng, and its impact on the rhizosphere microbial community

Rusty root rot is the most destructive disease in ginseng production. The bacterial strain FG14 isolated from the rhizosphere of Panax ginseng was characterised as Bacillus amyloliquefaciens. The inhibition rates of the FG14 bacterial suspension against the mycelial growth, spore production and spore germination of Ilyonectria robusta which causes ginseng rusty root rot were all more than 90% at a concentration of 108 cfu/mL. The sterile fermentation broth of strain FG14 also had significant inhibitory ability. The results of plantgrowth promotion showed that the fresh weight and root length of ginseng seedlings treated with 3 × 108 cfu/mL FG14 bacterial suspension were 4.49 times and 2.12 times greater than the seedlings treated with I. robusta. The soil enzyme activities of urease, phosphatase, invertase, and catalase of strain FG14 and the expression of seven lipopeptide biosynthesis genes were up-regulated, and antifungal metabolite production was stable. The diversity and abundance of bacterial and fungal communities in rhizosphere soil changed during different growth stages of ginseng after inoculation with strain FG14. The relative abundance of the Ilyonectria genus was significantly lower than the water control at the leaf spreading stage of P. ginseng. These results indicate the great potential of B. amyloliquefaciens FG14 for use as a biological agent for the green control of ginseng rusty root rot.

Revalorization by lactic acid bacterial fermentation of goat whey from cheese industry as a potential antifungal agent

The industry is continuously searching for new methods to reduce the use of chemical preservatives while still produce safer foods for the consumer. Goat whey is a waste from the cheese industry which is primarily treated as a residue. It is rich in different nutrients and has the potential to be used as a matrix for lactic acid bacteria fermentation. This work aims to find a revalorization of whey for the production of a natural origin antifungal agent by a fermentation that can be used as food ingredient. First different whey culture mediums were developed and fermented. Quantification of different antifungal molecules in whey culture mediums in comparation to a fermentation of De Man, Rogosa and Sharpe (MRS) culture medium evidenced that the production of this antifungal metabolites was similar. The in vitro qualitative and quantitative assays evidenced that the whey-based mediums increased the antifungal activity of the CFS in comparation to the MRS. The fermented whey produced bigger inhibition halos (above 1 cm) in the agar diffusion test in comparation to the fermented MRS (bellow 0.6 cm). Alike, in the minimal fungicidal concentration test the lowest antifungal concentrations were detected on the fermented whey mediums, averaging antifungal concentrations lower than 130 g/L. Finally, a multivariable data analysis determined that more antifungal metabolites yet to be detected were affecting in the antifungal activity from the whey samples. The fermented whey has the potential to be a tool in reducing the use of chemical preservatives in foods.

Comparison of the colonization ability of Burkholderia strain B23 in the citrus rhizoplane and rhizosphere and assessment of the underlying mechanisms using full-length 16S rDNA amplicon and metatranscriptomic analyses.

The characterization of bacterial strains with efficient root colonization ability and the mechanisms responsible for their efficient colonization is critical for the identification and application of beneficial bacteria. In this study, we found that Burkholderia strain B23 exhibited a strong niche differentiation between the rhizosphere and rhizoplane (a niche with more abundant easy-to-use nutrients but stronger selective pressures compared with the tightly adjacent rhizosphere) when inoculated into the field-grown citrus trees. Full-length 16S rDNA amplicon analysis demonstrated that the relative abundance of B23 in the rhizoplane microbiome at 3, 5, and 9 days post-inoculation (dpi) was always higher than that at 1 dpi, whereas its relative abundance in the rhizosphere microbiome was decreased continuously, as demonstrated by a 3.18-fold decrease at 9 dpi compared to 1 dpi. Time-series comparative expression profiling of B23 between the rhizoplane and rhizosphere was performed at representative time points (1, 3, and 9 dpi) through metatranscriptomic analysis, and the results demonstrated that multiple genes involved in the uptake and utilization of easy-to-use carbohydrates and amino acids and those involved in metabolism, energy production, replication, and translation were upregulated in the rhizoplane compared with the rhizosphere at 1 dpi and 3 dpi. Several genes involved in resistance to plant- and microbial competitor-derived stresses exhibited higher expression activities in the rhizoplane compared with the rhizosphere. Furthermore, gene loci responsible for the biosynthesis of the key antifungal and antibacterial metabolites occidiofungin and ornibactin were induced, and their expression levels remained relatively stable from 3 dpi to 9 dpi in the rhizoplane but not in the rhizosphere. Collectively, our findings provide novel lights into the mechanisms underlying the root colonization of the inoculated bacterial strains and serve as a basis for the identification of strains with efficient colonization ability, thus contributing to the development of beneficial bacteria applications.

Deciphering the mechanism of Trichoderma spp. consortia possessing volatile organic compounds and antifungal metabolites in the suppression of Sclerotium rolfsii in groundnut

Groundnut, one of the oilseed crops is severely infected with necrotropic pathogen Sclerotium rolfsii, incurring huge economic loss to farmers. In the present study, the microbial volatiles (mVOCs) produced by six different Trichoderma spp. was tested for its in vitro efficacy against S. rolfsii IS (BDI)-8 infecting groundnut. Among the six isolates, Trichoderma longibrachiatum T (SP)-20 exhibited the maximum percentage inhibition (84.44%) followed by T (AR)-10 (75.44%) and TNAU TA (75.33%) in dual culture assay against S. rolfsii. Besides, volatiles of T. longibrachiatum T (SP)-20 and T. asperellum T (AR)-10 effectively reduced the mycelial growth of S. rolfsii by 61.11 and 54.44% respectively in paired plate assay. Co culturing of Trichoderma spp. with S. rolfsii produced specific antimicrobial mVOCs viz., Isolongifolan-7-ol and Trans-sesquisabinene hydrate, which could induce plant growth promotion and inhibited the growth of S. rolfsii. Application of consortia of Trichoderma longibrachiatum T (SP)-20 + Trichoderma asperellum T (AR)-10 as seed treatment at 4 g/kg and soil application at 50 g/pot before sowing and at 30 DAS (Days After Sowing), along with mahua oil cake as soil application at 100 g/pot recorded least stem rot disease incidence of 11.34% in pot culture. Besides, upregulation of a novel antifungal metabolite 6-pentyl-2H-pyran-2-one (6-PP) was also noticed in groundnut through GC-MS analysis. Thus, it is concluded that microbial volatiles and metabolites from Trichoderma spp. play an important role in resistance to S. rolfsii in groundnut.

Novel Antifungal and Antifeedant Metabolites from Penicillium chrysogenum Co-Cultured with Nemania primolutea and Aspergillus fumigatus.

The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A-B (1-2), and penigenumin (3) from N. primolutea, penemin (4) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites (13-16) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A-B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites (13-16) were the culture driven. Compounds 4, 6, 8, 10, 11, 14, and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MICS of 1-8 μg/mL, and compounds 7, 9, and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities.

Bioprospecting of desert actinobacteria with special emphases on griseoviridin, mitomycin C and a new bacterial metabolite producing Streptomyces sp. PU-KB10–4

BackgroundBioprospecting of actinobacteria isolated from Kubuqi desert, China for antibacterial, antifungal and cytotoxic metabolites production and their structure elucidation.ResultsA total of 100 actinobacteria strains were selectively isolated from Kubuqi desert, Inner Mongolia, China. The taxonomic characterization revealed Streptomyces as the predominant genus comprising 37 different species, along with the rare actinobacterial genus Lentzea. The methanolic extracts of 60.8% of strains exhibited potent antimicrobial activities against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and high to mild in vitro cytotoxicity against PC3 (prostate cancer) and A549 (lung carcinoma) cell lines. The metabolomics analysis by TLC, HPLC-UV/vis, HPLC-MS and NMR showed the presence of compounds with molecular weights ranging from 100 to 1000 Da. The scale-up fermentation of the prioritized anti-Gram-negative strain PU-KB10–4 (Streptomyces griseoviridis), yielded three pure compounds including; griseoviridin (1; 42.0 mgL− 1) with 20 fold increased production as compared to previous reports and its crystal structure as monohydrate form is herein reported for the first time, mitomycin C (2; 0.3 mgL− 1) and a new bacterial metabolite 4-hydroxycinnamide (3; 0.59 mgL− 1).ConclusionsThis is the first report of the bioprospecting and exploration of actinobacteria from Kubuqi desert and the metabolite 4-hydroxycinnamide (3) is first time isolated from a bacterial source. This study demonstrated that actinobacteria from Kubuqi desert are a potential source of novel bioactive natural products. Underexplored harsh environments like the Kubuqi desert may harbor a wider diversity of actinobacteria, particularly Streptomyces, which produce unique metabolites and are an intriguing source to develop medicinally valuable natural products.

Biochar for the Control of Plant Pathogens

Biochar, the solid product of pyrolysis of biomass at thermal degradation temperatures, is useful in agriculture as manure for enhancing plant growth through the supply of nutrients. It is used in protected cultivation practices of vegetables and flower crops in the pot culture and grow bags especially to improve soil physicochemical properties, and in hydroponics to remove pollutants like heavy metals in the water. The usage of biochar as a potential soil amendment for plant growth promotion, improving soil fertility and plant disease suppression are being explored in recent years. Biochar made from many of the agro waste materials was found to suppress the plant pathogens in the soil and also effective in controlling the pathogens affecting aerial parts of the plants. Although direct antifungal or antibacterial effects and metabolites of biochar are poorly understood, induced systemic resistance in plants through signal transduction and expression of defence chemicals and metabolites have been studied. In addition, microbiome analyses through metagenome sequencing revealed an increase in the population of beneficial microbes (antagonistic to plant pathogens) in the rhizosphere soils applied with biochar.