Antimicrobial and antioxidant activities of silver nanoparticles synthesized by novel biogenic method using mixed reductants.

A novel method, for the synthesis of silver nanoparticles that are eco-friendly by means of mixed reductants method, has been developed. The combined extract of Mentha viridis plant and Prunus domestica gum were used as reducing agents for the synthesis of silver nanoparticles of the size less than 40 nm in diameter. The effect of time and concentration on the formation of silver nanoparticles were also monitored. The silver nanoparticles formed were verified by surface Plasmon spectra using single and double beam UV-Vis spectrophotometer. The XRD technique and scanning electron microscopy were performed to analyze the crystalline structure, crystallite size and morphology. The synthesized silver nanoparticles were tested against different bacterial and fungus strains. The silver nanoparticles showed good inhibition in antimicrobial study and low MIC for bacterial strains. The antioxidant assay was performed to check the scavenging activity. In DPPH, the silver nanoparticles showed good scavenging activity and were found close to that of ascorbic acid.

Evidence for the Role of CYP51A and Xenobiotic Detoxification in Differential Sensitivity to Azole Fungicides in Boxwood Blight Pathogens.

Boxwood blight, a fungal disease of ornamental plants (Buxus spp.), is caused by two sister species, Calonectria pseudonaviculata (Cps) and C. henricotiae (Che). Compared to Cps, Che is documented to display reduced sensitivity to fungicides, including the azole class of antifungals, which block synthesis of a key fungal membrane component, ergosterol. A previous study reported an ergosterol biosynthesis gene in Cps, CYP51A, to be a pseudogene, and RNA-Seq data confirm that a functional CYP51A is expressed only in Che. The lack of additional ergosterol biosynthesis genes showing significant differential expression suggests that the functional CYP51A in Che could contribute to reduced azole sensitivity when compared to Cps. RNA-Seq and bioinformatic analyses found that following azole treatment, 55 genes in Cps, belonging to diverse pathways, displayed a significant decrease in expression. Putative xenobiotic detoxification genes overexpressed in tetraconazole-treated Che encoded predicted monooxygenase and oxidoreductase enzymes. In summary, expression of a functional CYP51A gene and overexpression of predicted xenobiotic detoxification genes appear likely to contribute to differential fungicide sensitivity in these two sister taxa.

Isolation and Purification of a New Bacillus Subtilis Strain from Deer Dung with Anti-microbial and Anti-cancer Activities.

OBJECTIVE: Bacillus strains are well known for their natural bioactive products that have antimicrobial and/or anti-cancer activities. Many of Bacillus' structurally unique metabolites can combat human diseases, including cancers. However, because Bacillus' metabolites are so abundant, few have been studied extensively enough to fully characterize their chemical constitutions and biological functions. METHODS: In this study, we focused on the isolation and purification of a new Bacillus strain, and determined the effects of its metabolites on bacteria and cancer cells. Our study focused on a new strain of Bacillus isolated from deer dung. Based on BLAST results, this isolate belongs to Bacillus subtilis, and therefore we named the strain Bacillus subtilis NC16. Congo red assay was used to test the cellulase activity. The inhibition zone was measured to test the antimicrobial activity. CCK-8, wound healing and flow cytometry were used to test the anti-cancer activity. RESULTS: Metabolites from Bacillus subtilis NC16 have both antimicrobial and anti-cancer activities. They can both suppress the growth of Trichoderma vride and Staphylococcus aureus, and inhibit the proliferation and promote the apoptosis of non-small cell lung cancer cell lines. CONCLUSION: Our results suggest that Bacillus subtilis NC16 can not only degrade cellulose, but its metabolites may be sources of antibiotics and anti-cancer drugs.

Synergistic effect of ozone and chlorine on inactivating fungal spores: Influencing factors and mechanisms.

Effective control of fungal contamination in water is vital to provide healthy and safe drinking water for human beings. Although ozone was highly effective in inactivating fungi in water, it was limited by a lack of continuous disinfection ability in water supply system. In present study, the inactivation of fungal spores by combining ozone and chlorine was investigated. The synergistic effects of Aspergillus niger and Trichoderma harzianum spores reached 0.47- and 0.55-log within 10 min, respectively. The inactivation efficiency and the synergistic effect would be affected by disinfectant concentration, pH, and temperature. The combined inactivation caused more violent oxidative stimulation and more severe damage to the fungal spores than the individual inactivation based on the flow cytometry analysis and the scanning electron microscopy observation. The synergistic effect during the combined inactivation process was attributed to the generation of hydroxyl radicals by the reaction between ozone and chlorine and the promotion of chlorine penetration by the destruction of cell wall by ozone. The combined inactivation efficiency in natural water samples was reduced by 26.4-43.8% compared with that in PBS. The results of this study provided an efficient and feasible disinfection method for the control of fungi in drinking water.

Fungal tolerance to Congo red, a cell wall integrity stress, as a promising indicator of ecological niche.

Differential sensitivities to the cell wall stress caused by Congo red (CR) have been observed in many fungal species. In this study, the tolerances and sensitivities to CR was studied with an assorted collection of fungal species from three phylogenetic classes: Sordariomycetes, Dothideomycetes, and Eurotiomycetes, three orders, and eight families. These grouped into different ecological niches, such as insect pathogens, plant pathogens, saprotrophs, and mycoparasitics. The saprotroph Aspergillus niger and the mycoparasite Trichoderma atroviride stood out as the most resistant species to cell wall stress caused by CR, followed by the plant pathogenic fungi, a mycoparasite, and other saprotrophs. The insect pathogens had low tolerance to CR. The insect pathogens Metarhizium acridum and Cordyceps fumosorosea were the most sensitive to CR. In conclusion, Congo red tolerance may reflect ecological niche, accordingly, the tolerances of the fungal species to Congo red were closely aligned with their ecology.

Establishment of a new and efficient Agrobacterium-mediated transformation system in the nematicidal fungus Purpureocillium lilacinum.

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is widely commercialized for controlling plant-parasitic nematodes and represents a potential cell factory for enzyme production. This nematicidal fungus is intrinsically resistant to common antifungal agents used for genetic transformation. Therefore, molecular investigations in P. lilacinum are still limited so far. In the present study, we have established a new Agrobacterium tumefaciens-mediated transformation (ATMT) system in P. lilacinum based on the uridine/uracil auxotrophic mechanism. Here, uridine/uracil auxotrophic mutants were simply generated via UV irradiation instead of a complicated genetic approach for the pyrG gene deletion. A stable uridine/uracil auxotrophic mutant was then selected as a recipient for fungal transformation. We further indicated that the pyrG gene from Aspergillus niger can be used as a selectable marker for genetic transformation of P. lilacinum. Under optimized conditions for ATMT, the transformation efficiency reached 2873 ± 224 transformants per 106 spores. Using the constructed ATMT system, we succeeded in expressing the DsRed reporter gene in P. lilacinum. Additionally, we have identified a very promising mutant for chitinase production from a collection of T-DNA insertion transformants. This mutant possesses a special phenotype of hyper-branching mycelium and produces more conidia in comparison to the wild strain. Conclusively, our ATMT system can be exploited for overexpression of target genes or for T-DNA insertion mutagenesis in the agriculturally important fungus P. lilacinum. The genetic approach in the present work may also be applied for developing similar ATMT systems in other fungi, especially for fungi that their genome databases are currently not available.

Evaluation of Seven Essential Oils as Seed Treatments against Seedborne Fungal Pathogens of Cucurbita maxima.

Essential oils are gaining interest as environmentally friendly alternatives to synthetic fungicides for management of seedborne pathogens. Here, seven essential oils were initially tested in vivo for disinfection of squash seeds (Cucurbita maxima) naturally contaminated by Stagonosporopsis cucurbitacearum, Alternaria alternata, Fusarium fujikuro, Fusarium solani, Paramyrothecium roridum, Albifimbria verrucaria, Curvularia spicifera, and Rhizopus stolonifer. The seeds were treated with essential oils from Cymbopogon citratus, Lavandula dentata, Lavandula hybrida, Melaleuca alternifolia, Laurus nobilis, and Origanum majorana (#1 and #2). Incidence of S. cucurbitacearum was reduced, representing a range between 67.0% in L. nobilis to 84.4% in O. majorana #2. Treatments at 0.5 mg/mL essential oils did not affect seed germination, although radicles were shorter than controls, except with C. citratus and O. majorana #1 essential oils. Four days after seeding, seedling emergence was 20%, 30%, and 10% for control seeds and seeds treated with C. citratus essential oil (0.5 mg/mL) and fungicides (25 g/L difenoconazole plus 25 g/L fludioxonil). S. cucurbitacearum incidence was reduced by ~40% for plantlets from seeds treated with C. citratus essential oil. These data show the effectiveness of this essential oil to control the transmission of S. cucurbitacearum from seeds to plantlets, and thus define their potential use for seed decontamination in integrated pest management and organic agriculture.

Biosynthesis of Sinapigladioside, an Antifungal Isothiocyanate from Burkholderia Symbionts.

Sinapigladioside is a rare isothiocyanate-bearing natural product from beetle-associated bacteria (Burkholderia gladioli) that might protect beetle offspring against entomopathogenic fungi. The biosynthetic origin of sinapigladioside has been elusive, and little is known about bacterial isothiocyanate biosynthesis in general. On the basis of stable-isotope labeling, bioinformatics, and mutagenesis, we identified the sinapigladioside biosynthesis gene cluster in the symbiont and found that an isonitrile synthase plays a key role in the biosynthetic pathway. Genome mining and network analyses indicate that related gene clusters are distributed across various bacterial phyla including producers of both nitriles and isothiocyanates. Our findings support a model for bacterial isothiocyanate biosynthesis by sulfur transfer into isonitrile precursors.

Transcriptomic studies on Purpureocillium lilacinum reveal molecular mechanisms of response to fluconazole and itraconazole.

Filamentous fungus Purpureocillium lilacinum is an emerging pathogen that infects immunocompromised and immunocompetent individuals and is resistant to several azole molecules. Although azole resistance mechanisms are well studied in Aspergillus sp. and Candida sp., there are no studies to date reporting P. lilacinum molecular response to these molecules. The aim of this study was to describe P. lilacinum molecular mechanisms involved in antifungal response against fluconazole and itraconazole. Transcriptomic analyses showed that gene expression modulation takes place when P. lilacinum is challenged for 12 h with fluconazole (64 µg/mL) or itraconazole (16 µg/mL). The antifungals acted on the ergosterol biosynthesis pathway, and two homologous genes coding for cytochrome P450 51 enzymes were upregulated. Genes coding for efflux pumps, such as the major facilitator superfamily transporter, also displayed increased expression in the treated samples. We propose that P. lilacinum develops antifungal responses by raising the expression levels of cytochrome P450 enzymes and efflux pumps. Such modulation could confer P. lilacinum high levels of target enzymes and could lead to the constant withdrawal of antifungals, which would force an increase in the administration of antifungal medications to achieve fungal morbidity or mortality. The findings in this work could aid in the decision-making for treatment strategies in cases of P. lilacinum infection.

Influence of the capping of biogenic silver nanoparticles on their toxicity and mechanism of action towards Sclerotinia sclerotiorum.

BACKGROUND: Biogenic nanoparticles possess a capping of biomolecules derived from the organism employed in the synthesis, which contributes to their stability and biological activity. These nanoparticles have been highlighted for the control of phytopathogens, so there is a need to understand their composition, mechanisms of action, and toxicity. This study aimed to investigate the importance of the capping and compare the effects of capped and uncapped biogenic silver nanoparticles synthesized using the filtrate of Trichoderma harzianum against the phytopathogenic fungus Sclerotinia sclerotiorum. Capping removal, investigation of the composition of the capping and physico-chemical characterization of the capped and uncapped nanoparticles were performed. The effects of the nanoparticles on S. sclerotiorum were evaluated in vitro. Cytotoxicity and genotoxicity of the nanoparticles on different cell lines and its effects on nontarget microorganisms were also investigated. RESULTS: The capped and uncapped nanoparticles showed spherical morphology, with greater diameter of the uncapped ones. Functional groups of biomolecules, protein bands and the hydrolytic enzymes NAGase, ß-1,3-glucanase, chitinase and acid protease from T. harzianum were detected in the capping. The capped nanoparticles showed great inhibitory potential against S. sclerotiorum, while the uncapped nanoparticles were ineffective. There was no difference in cytotoxicity comparing capped and uncapped nanoparticles, however higher genotoxicity of the uncapped nanoparticles was observed towards the cell lines. Regarding the effects on nontarget microorganisms, in the minimal inhibitory concentration assay only the capped nanoparticles inhibited microorganisms of agricultural importance, while in the molecular analysis of the soil microbiota there were major changes in the soils exposed to the uncapped nanoparticles. CONCLUSIONS: The results suggest that the capping played an important role in controlling nanoparticle size and contributed to the biological activity of the nanoparticles against S. sclerotiorum. This study opens perspectives for investigations concerning the application of these nanoparticles for the control of phytopathogens.

Enhanced resistance of Trichoderma harzianum LZDX-32-08 to hygromycin B induced by sea salt.

OBJECTIVES: To determine the effect of sea salt on the resistance of Trichoderma harzianum LZDX-32-08 to hygromycin B and speculate the possible mechanisms involved via transcriptome analysis. RESULTS: Sea salt addition in media to simulate marine environment significantly increased the tolerance of marine-derived fungus Trichoderma harzianum LZDX-32-08 to hygromycin B from 40 to 500 µg/ml. Meanwhile, sea salt addition also elicited the hygromycin B resistance of 5 other marine or terrestrial fungi. Transcriptomic analyses of T. harzianum cultivated on PDA, PDA supplemented with sea salt and PDA with both sea salt and hygromycin B revealed that genes coding for P-type ATPases, multidrug resistance related transporters and acetyltransferases were up-regulated, while genes coding for Ca2+/H+ antiporter and 1,3-glucosidase were down-regulated, indicating probable increased efflux and inactivation of hygromycin B as well as enhanced biofilm formation, which could jointly contribute to the drug resistance. CONCLUSIONS: Marine environment or high ion concentration in the environment could be an importance inducer for antifungal resistance. Possible mechanisms and related key genes were proposed for understanding the molecular basis and overcoming this resistance.

Functional characterization of the AGL1 aegerolysin in the mycoparasitic fungus Trichoderma atroviride reveals a role in conidiation and antagonism.

Aegerolysins are small secreted pore-forming proteins that are found in both prokaryotes and eukaryotes. The role of aegerolysins in sporulation, fruit body formation, and in lysis of cellular membrane is suggested in fungi. The aim of the present study was to characterize the biological function of the aegerolysin gene agl1 in the mycoparasitic fungus Trichoderma atroviride, used for biological control of plant diseases. Gene expression analysis showed higher expression of agl1 during conidiation and during growth in medium supplemented with cell wall material from the plant pathogenic fungus Rhizoctonia solani as the sole carbon source. Expression of agl1 was supressed under iron-limiting condition, while agl1 transcript was not detected during T. atroviride interactions with the prey fungi Botrytis cinerea or R. solani. Phenotypic analysis of agl1 deletion strains (Δagl1) showed reduced conidiation compared to T. atroviride wild type, thus suggesting the involvement of AGL1 in conidiation. Furthermore, the Δagl1 strains display reduced antagonism towards B. cinerea and R. solani based on a secretion assay, although no difference was detected during direct interactions. These data demonstrate the role of AGL1 in conidiation and antagonism in the mycoparasitic fungus T. atroviride.

Purpureocillium roseum sp. nov. A new ocular pathogen for humans and mice resistant to antifungals.

BACKGROUND: Infectious keratitis is the main cause of preventable blindness worldwide, with about 1.5-2.0 million new cases occurring per year. This inflammatory response may be due to infections caused by bacteria, fungi, viruses or parasites. Fungal keratitis is a poorly studied health problem. OBJECTIVES: This study aimed to identify a new fungal species by molecular methods and to explore the possible efficacy of the three most common antifungals used in human keratitis in Mexico by performing in vitro analysis. The capacity of this pathogen to cause corneal infection in a murine model was also evaluated. METHODS: The fungal strain was isolated from a patient with a corneal ulcer. To identify the fungus, taxonomic and phylogenetic analyses (nrDNA ITS and LSU data set) were performed. An antifungal susceptibility assay for amphotericin B, itraconazole and voriconazole was carried out. The fungal isolate was used to develop a keratitis model in BALB/c mice; entire eyes and ocular tissues were preserved and processed for histopathologic examination. RESULTS AND CONCLUSION: This fungal genus has hitherto not been reported with human keratitis in Mexico. We described a new species Purpurecillium roseum isolated from corneal infection. P roseum showed resistance to amphotericin B and itraconazole and was sensitive to voriconazole. In vivo study demonstrated that P roseum had capacity to developed corneal infection and to penetrate deeper corneal tissue. The global change in fungal infections has emphasised the need to develop better diagnostic mycology laboratories and to recognise the group of potential fungal pathogens.

The Copper-Controlled RNA Interference System in Trichoderma reesei.

Trichoderma reesei is capable of secreting large amounts of lignocellulose-degrading enzymes. Although the genome sequence of T. reesei has been available, the molecular mechanisms of the hyper-production of cellulases, including the transcriptional regulation and the protein secretion, have not been completely elucidated yet. This is partially due to the lack of genetic manipulation approaches. RNA interference (RNAi) is a powerful tool for functional genomic studies in eukaryotes. Some successful examples of RNAi have already been reported; however, these systems were either uncontrolled or relied on a nutrient source inducible promoter. Here, we present a copper-controlled RNAi system in T. reesei for reversible silencing of different target genes. As the proof of concept, T.reesei xyr1, the key transcriptional activator of cellulase genes, has been knocked down using this method.

Production and identification of two antifungal terpenoids from the Posidonia oceanica epiphytic Ascomycota Mariannaea humicola IG100.

BACKGROUND: Marine fungi are an important repository of bioactive molecules with great potential in different technological fields, the annual number of new compounds isolated from marine fungi is impressive and the general trend indicates that it is still on the rise. In this context, the antifungal and antimicrobial activity of the marine strain Mariannaea humicola IG100 was evaluated and two active terpenoids were isolated and characterized. METHODS: Preliminary screening of activity of marine strain IG100 was carried out by agar plug diffusion methods against fungal (Penicillium griseofulvum TSF04) and bacterial (Bacillus pumilus KB66 and Escherichia coli JM109) strains. Subsequently, inhibition tests were done by using the cultural broth and the organic extract (ethyl acetate, EtOAc) by the agar well diffusion methods. The main active fractions were identified and tested for their antifungal activity against P. griseofulvum TSF04 in a 24 wells microplate at different concentrations (1000, 100, 10 and 1.0 µg/mL). Two active compounds were characterized and their relative MIC measured by the broth micro-dilution methods in a 96-well microplate against Aspergillus flavus IG133, P. griseofulvum TSF04, and Trichoderma pleuroticola IG137. RESULTS: Marine strain IG100 presented significant antifungal activity associated with two active compounds, the terpenoids terperstacin 1 and 19-acetyl-4-hydroxydictyodiol 2. Their MIC values were measured for A. flavus (MIC of 7.9 µg/mL and 31.3 µg/mL for 1 and 2, respectively), P. griseofulvum (MIC of 25 µg/mL and 100 µg/mL for 1 and 2, respectively) and T. pleuroticola (MIC > 500 µg/mL and 125 µg/mL for 1 and 2, respectively). They showed a rather good fungistatic effect. CONCLUSIONS: In this study, the first marine strain of M. humicola (IG100) was investigated for the production of bioactive molecules. Strain IG100 produced significant amounts of two bioactive terpenoids, terperstacin 1 and 19-acetyl-4-hydroxydictyodiol 2. The two compounds showed significant antifungal activities against A. flavus IG133, T. pleuroticola IG137 and P. griseofulvum TSF04. Compound 2 was identified for the first time in fungi.

Characterization of a novel dsRNA mycovirus of Trichoderma atroviride NFCF377 reveals a member of "Fusagraviridae" with changes in antifungal activity of the host fungus.

Trichoderma atroviride is a common fungus found in various ecosystems that shows mycoparasitic ability on other fungi. A novel dsRNA virus was isolated from T. atroviride NFCF377 strain and its molecular features were analyzed. The viral genome consists of a single segmented double-stranded RNA and is 9,584 bp in length, with two discontinuous open reading frames (ORF1 and ORF2). A mycoviral structural protein and an RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively, between which is found a canonical shifty heptameric signal motif (AAAAAAC) followed by an RNA pseudoknot. Analysis of sequence similarity and phylogeny showed that it is closely related to members of the proposed family "Fusagraviridae", with a highest similarity to the Trichoderma atroviride mycovirus 1 (TaMV1). Although the sequence similarity of deduced amino acid to TaMV1 was evident, sequence deviations were distinctive at untranslated regions (UTRs) due to the extended size. Thus, we inferred this dsRNA to be a different strain of Trichoderma atroviride mycovirus 1 (TaMV1-NFCF377). Electron microscopy image exhibited an icosahedral viral particle of 40 nm diameter. Virus-cured isogenic isolates were generated and no differences in growth rate, colony morphology, or conidia production were observed between virus-infected and virus-cured strains. However, culture filtrates of TaMV1-NFCF377-infected strain showed enhanced antifungal activity against the plant pathogen Rhizoctonia solani but not to edible mushroom Pleurotus ostreatus. These results suggested that TaMV1-NFCF377 affected the metabolism of the fungal host to potentiate antifungal compounds against a plant pahogen, but this enhanced antifungal activity appeared to be species-specific.

Sarocladium and Acremonium infections: New faces of an old opportunistic fungus.

The genera Acremonium and Sarocladium comprise a high diversity of morphologically and genetically related fungi generally found in the environment, although a few species, mainly Sarocladium kiliense and Acremonium egyptiacum, can also be involved in many human infections. Clinical management of opportunistic infections caused by these fungi is very complex, since their correct identification is unreliable, and they generally show poor antifungal response. More than 300 clinical cases involving a broad range of Acremonium/Sarocladium infections have so far been published, and with this review we aim to compile and provide a detailed overview of the current knowledge on Acremonium/Sarocladium human infections in terms of presentation, diagnosis, treatments and prognoses. We also aim to summarise and discuss the data currently available on their antifungal susceptibility, emphasising the promising results obtained with voriconazole as well as their impact in terms of animal infections.

Combined chitosan and Cympobogon citratus (D.C. ex Nees) Stapf. essential oil to inhibit the fungal phytopathogen Paramyrothecium roridum and control crater rot in melon (Cucumis melo L.).

This study evaluated the efficacy of combined chitosan (Chi) and Cymbopogon citratus (DC) Stapf. essential oil (CCEO) to inhibit the fungal phytopathogen Paramyrothecium roridum L. Lombard & Crous and control crater rot in melon (Cucumis melo L.). Effects of several Chi and CCEO concentrations to inhibit the growth of four P. roridum isolates in vitro, as well as the type of interaction of some combined concentrations of Chi and CCEO was evaluated. Effects of coatings with combined concentrations of Chi and CCEO on development of crater rot lesions in melon artificially inoculated with P. roridum during storage (15 days, 25 °C) were measured. Chi (2.5, 3.75, 5, and 6.75 mg/mL) and CCEO (0.3 and 0.6 µL/mL) led to growth inhibition of the four examined P. roridum isolates. Combinations of Chi (5 mg/mL) and CCEO (0.15 and 0.3 µL/mL) had additive interaction to inhibit P. roridum. Coatings with additive combined concentrations of Chi and CCEO decreased the development and severity of carter rot lesions in melon during room storage regardless of the inoculated P. roridum isolate. Therefore, application of coatings formulated with combined concentrations of Chi and CCEO could be alternative strategies to control crater rot caused by P. roridum in melon and decrease synthetic fungicide use in this fruit.

Antifungal Activity of Volatile Organic Compounds Produced by Bacillus methylotrophicus and Bacillus thuringiensis against Five Common Spoilage Fungi on Loquats.

Loquat fruit is one of the most perishable fruits in China, and has a very limited shelf life because of mechanical injury and microbial decay. Due to an increasing concern about human health and environmental security, antagonistic microorganisms have been a potential alternative for fungicides to control postharvest diseases. In this work, the antifungal effect of volatile organic compounds (VOCs) produced by Bacillus methylotrophicus BCN2 and Bacillus thuringiensis BCN10 against five postharvest pathogens isolated from loquat fruit, Fusarium oxysporum, Botryosphaeria sp., Trichoderma atroviride, Colletotrichum gloeosporioides, and Penicillium expansum were evaluated by in vitro and in vivo experiments. As a result, the VOCs released by BCN2 and BCN10 were able to suppress the mycelial growth of all targeted pathogens according to inhibition ratio in the double petri-dish assay as well as disease incidence and disease diameter on loquat fruits. The main volatile compounds were identified by solid-phase microextraction (SPME)-gas chromatography. These VOCs produced by the two strains played complementary roles in controlling these five molds and enabled loquat fruits to keep fresh for ten days, significantly. This research will provide a theoretic foundation and technical support for exploring the functional components of VOCs applicable in loquat fruit preservation.

Insect-Associated Bacteria Assemble the Antifungal Butenolide Gladiofungin by Non-Canonical Polyketide Chain Termination.

Genome mining of one of the protective symbionts (Burkholderia gladioli) of the invasive beetle Lagria villosa revealed a cryptic gene cluster that codes for the biosynthesis of a novel antifungal polyketide with a glutarimide pharmacophore. Targeted gene inactivation, metabolic profiling, and bioassays led to the discovery of the gladiofungins as previously-overlooked components of the antimicrobial armory of the beetle symbiont, which are highly active against the entomopathogenic fungus Purpureocillium lilacinum. By mutational analyses, isotope labeling, and computational analyses of the modular polyketide synthase, we found that the rare butenolide moiety of gladiofungins derives from an unprecedented polyketide chain termination reaction involving a glycerol-derived C3 building block. The key role of an A-factor synthase (AfsA)-like offloading domain was corroborated by CRISPR-Cas-mediated gene editing, which facilitated precise excision within a PKS domain.