Antifungal Volatile Compounds Research Articles

Endophytic Fungi: A Treasure Trove of Antifungal Metabolites.

Emerging and reemerging fungal infections are very common in nosocomial and non-nosocomial settings in people having poor immunogenic profiles either due to hematopoietic stem cell transplants or are using immunomodulators to treat chronic inflammatory disease or autoimmune disorders, undergoing cancer therapy or suffering from an immune weakening disease like HIV. The refractory behavior of opportunistic fungi has necessitated the discovery of unconventional antifungals. The emergence of black fungus infection during COVID-19 also triggered the antifungal discovery program. Natural products are one of the alternative sources of antifungals. Endophytic fungi reside and co-evolve within their host plants and, therefore, offer a unique bioresource of novel chemical scaffolds with an array of bioactivities. Hence, immense possibilities exist that these unique chemical scaffolds expressed by the endophytic fungi may play a crucial role in overcoming the burgeoning antimicrobial resistance. These chemical scaffolds so expressed by these endophytic fungi comprise an array of chemical classes beginning from cyclic peptides, sesquiterpenoids, phenols, anthraquinones, coumarins, etc. In this study, endophytic fungi reported in the last six years (2018-2023) have been explored to document the antifungal entities they produce. Approximately 244 antifungal metabolites have been documented in this period by different groups of fungi existing as endophytes. Various aspects of these antifungal metabolites, such as antifungal potential and their chemical structures, have been presented. Yet another unique aspect of this review is the exploration of volatile antifungal compounds produced by these endophytic fungi. Further strategies like epigenetic modifications by chemical as well as biological methods and OSMAC to induce the silent gene clusters have also been presented to generate unprecedented bioactive compounds from these endophytic fungi.

Profiling Metabolites with Antifungal Activities from Endophytic Plant-Beneficial Strains of Pseudomonas chlororaphis Isolated from Chamaecytisus albus (Hack.) Rothm.

Fungal phytopathogens represent a large and economically significant challenge to food production worldwide. Thus, the application of biocontrol agents can be an alternative. In the present study, we carried out biological, metabolomic, and genetic analyses of three endophytic isolates from nodules of Chamaecytisus albus, classified as Pseudomonas chlororaphis acting as antifungal agents. The efficiency of production of their diffusible and volatile antifungal compounds (VOCs) was verified in antagonistic assays with the use of soil-borne phytopathogens: B. cinerea, F. oxysporum, and S. sclerotiorum. Diffusible metabolites were identified using chromatographic and spectrometric analyses (HPTLC, GC-MS, and LC-MS/MS). The phzF, phzO, and prnC genes in the genomes of bacterial strains were confirmed by PCR. In turn, the plant growth promotion (PGP) properties (production of HCN, auxins, siderophores, and hydrolytic enzymes, phosphate solubilization) of pseudomonads were bioassayed. The data analysis showed that all tested strains have broad-range antifungal activity with varying degrees of antagonism. The most abundant bioactive compounds were phenazine derivatives: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine, and diketopiperazine derivatives as well as ortho-dialkyl-aromatic acids, pyrrolnitrin, siderophores, and HCN. The results indicate that the tested P. chlororaphis isolates exhibit characteristics of biocontrol organisms; therefore, they have potential to be used in sustainable agriculture and as commercial postharvest fungicides to be used in fruits and vegetables.

Dynamics of microbial ecology and their bio-preservative compounds formed during the panettones elaboration using sourdough-isolated strains as starter cultures

The present study evaluated the application of Limosilactobacillus fermentum IAL 4541 and Wickerhamomyces anomalus IAL 4533 isolated from sourdoughs and their combinations, for bio-conservative action in panettones, as well as to identify the antifungal volatile compounds. Samples from different fermentation steps were submitted to physicochemical and microbiological evaluation, as to know: sourdough after 48 h (A), 96 h (B), and 144 h (C); dough of the first fermentation (D); final dough after second fermentation (E), and baked panettone (F). In all treatments, lactic bacteria and yeast counts demonstrated a mechanism of proto-cooperation. Panettones containing L. fermentum or W. anomalus in their composition had longer shelf life, which indicates their effect as potential biopreservatives. The genetic sequencing data demonstrated the dominance of the Lactobacilli throughout the process, corroborating the data obtained by cultivable methods. A total of 59 volatile organic compounds were found in this study, and a hierarchical cluster analysis presented the separation between the production stages (C, E) and the most recurrent compounds in the final product (F). Of these compounds, the following stood out (n = 26): acids (7.7%), alcohols (23.1%), aldehydes and ketones (34.6%), and esters (34.6%). The treatments containing L. fermentum presented more diversity of volatile organic compounds with potential antifungal effect. There was a higher production of acetic (1.17–8.85 mmol/kg), phenyllactic (5–10.4 mmol/kg), and propionic (3.5–4.3 mmol/kg) acids in the final product. This study demonstrates the feasibility of applying endogenous starter cultures from sourdoughs, with great biopreservative activity, enabling the production of healthier and more natural panettones.

Biological Control Activities of Rhizosphere Fungus Trichoderma virens T1-02 in Suppressing Flower Blight of Flamingo Flower (Anthurium andraeanum Lind.).

Flower blight caused by Neopestalotiopsis clavispora is an emerging disease of flamingo flower (Anthurium andraeanum Lind.) that negatively impacts flower production. The use of rhizosphere fungi as biocontrol agents is an alternative way to control this disease instead of using synthetic fungicides. This research aimed to screen the potential of rhizosphere fungi, Trichoderma spp., with diverse antifungal abilities to control N. clavispora and to reduce flower blight in flamingo flowers. A total of ten isolates were tested against N. clavispora by dual culture assay, and T1-02 was found to be the most effective isolate against N. clavispora, with inhibition of 78.21%. Morphology and molecular phylogeny of multiple DNA sequences of the genes, the internal transcribed spacer (ITS), translation elongation factor 1-α (tef1-α), and RNA polymerase 2 (rpb2) identified isolate T1-02 as Trichoderma virens. Sealed plate method revealed T. virens T1-02 produced volatile antifungal compounds (VOCs) against N. clavispora, with inhibition of 51.28%. Solid-phase microextraction (SPME) was applied to trap volatiles, and GC/MS profiling showed VOCs emitted from T. virens T1-02 contained a sesquiterpene antifungal compound-germacrene D. The pre-colonized plate method showed that T. virens T1-02 aggressively colonized in tested plates with inhibition of 100% against N. clavispora, and microscopy revealed direct parasitism onto fungal hyphae. Furthermore, the application of T. virens T1-02 spore suspension reduced the disease severity index (DSI) of flower blight in flamingo flowers. Based on the results from this study, T. virens T1-02 displays multiple antagonistic mechanisms and has the potential ability to control flower blight of flamingo flowers caused by N. clavispora.

A Rhizobacterium, Streptomyces albulus Z1-04-02, Displays Antifungal Activity against Sclerotium Rot in Mungbean.

Sclerotium rot causes damping-off and stem rot in seedlings and mature mungbeans, which negatively impacts cultivation. The use of a rhizobacterium to control soil-borne diseases is an alternative method to the excess use of synthetic fungicides; therefore, this study aims to screen rhizosphere actinobacteria with fungicidal activities against Sclerotium rolfsii, the pathogen that causes sclerotium rot in mungbeans. Primary screening showed that the Streptomyces sp. isolate Z1-04-02 displayed the highest effectiveness against S. rolfsii in dual culture plates, with a percentage inhibition of 74.28%. An assay containing enzymes that degrade cell walls, of the cell-free culture filtrate (CF) of Z1-04-02, showed that the activities of chitinase and β-1,3-glucanase were 0.0209 and 1.0210 U/mL, respectively, which was significantly higher than that of the control (media alone). The cell-free CF of Z1-04-02, incubated at 37 °C and 100 °C, using agar well diffusion, effectively inhibited the growth of S. rolfsii with inhibition percentages of 37.78% and 27.78%, respectively. Solid-phase microextraction (SPME) was applied to trap volatiles released from Z1-04-02 and gas chromatography–mass spectrometry (GC/MS); volatile antifungal compounds were tentatively identified as bicyclic monoterpene (1R)-(-)-myrtenal. The application of the cell-free CF, and the spore suspension of Z1-04-02, showed disease severity indexes (DSIs) of 12.5% and 8.25%, respectively, which were significantly lower than those showing inoculation by S. rolfsii alone. The identification of this strain by morphology, biochemistry tests, and 16s rDNA sequences revealed that Z1-04-02 was Streptomyces albulus. This finding revealed that S. albulus Z1-04-02 displayed diverse fungicidal activities against S. rolfsii, and it has the potential to act as a biological control agent in terms of inhibiting sclerotium rot in mungbeans.

Antagonistic and growth enhancement activities of native Pseudomonas spp. against soil and tuber-borne diseases of potato (Solanum tuberosum L.)

BackgroundSoil and tuber-borne diseases are serious threat for potato cultivation worldwide which causes severe economical losses in terms of yield, quantity and quality. Generally, these diseases managed by chemical pesticide, are a major concern for human health and environment. Therefore, finding an eco-friendly alternative management strategies are necessary. The native bacterial isolates collected from different crops rhizosphere soil were evaluated against Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii and Fusarium spp and were also tested for their growth enhancement attributes on potato crop and phosphate solubilising efficiency.ResultsBased on the morphological and phenotypic characters, most of the isolates were identified as Pseudomonas spp (18 isolates). A total of twenty-two bacterial isolates were screened for bio-control activity in dual culture assay. Isolate Pf14 showed the highest mycelial inhibitory potential (ranged from 62.2 to 59.3%) against R. solani, S. sclerotiorum, S. rolfsii and Fusarium spp. In sealed plate assay, Pf14 produced antifungal volatile compounds that significantly inhibited mycelial growth (ranged from > 80 to > 50%). Maximum reduction in fungal biomass (ranged from > 80%) was observed in King’s broth in shake liquid culture in all the pathogens. Cell-free culture filtrate of the selected isolate inhibited mycelial growth ranged from 68.9 to 42.6% of the tested pathogens with 48-h old culture filtrate. Additionally, the isolates exhibited higher phosphorus solubilizing efficiency on PVK media. Under field conditions, talc based formulation of Pf14 showed enhanced agronomical characters and inhibits black scurf severity up to 67.59%. This treatment also recorded a highest tuber yield (21.90 t/ha) with increase of 15.38% in comparison to untreated control.ConclusionOverall, antagonistic bacterium Pf14 can be recommended as bio fertilizers for eco-friendly management of major potato fungal diseases as well as increasing marketable yield and used as an alternative to the pesticides and chemical fertilizers.

Chitosan films as pH-responsive sustained release systems of naturally occurring antifungal volatile compounds

Reversible imine bonds have been used as a strategy to develop pH-dependent antifungal systems based on grafting benzaldehyde and citral onto the surface of chitosan films. Formation of imine bonds was confirmed by ATR-FTIR and XPS. Aldehyde unit incorporation respect to glucosamine units of chitosan polymer was estimated by elemental analysis. The rate and extent of imine bond hydrolysis depended on the pH of the media and the chemical structure of the aldehyde. The release of the aldehydes was monitored by gas chromatography observing acidic media favours the release. Imine bond obtained from benzaldehyde was more prone to be hydrolysed than citral. Chitosan films grafted with benzaldehyde and triggered at acidic pH controlled in vitro growth of common fruit and vegetable spoilage and pathogenic fungi. The films developed could be applied in the design of food packages intended to prevent postharvest fungal spoilage.

Biocontrol Potential of Endophytic Actinobacteria against Fusarium solani, the Causal Agent of Sudden Decline Syndrome on Date Palm in the UAE.

Thirty-one endophytic streptomycete and non-streptomycete actinobacteria were isolated from healthy date palm root tissues. In vitro screening revealed that the antifungal action of isolate #16 was associated with the production of cell-wall degrading enzymes, whereas with diffusible antifungal metabolites in isolate #28, albeit their production of volatile antifungal compounds. According to the 16S rRNA gene sequencing, isolates #16 and #28 were identified as Streptomyces polychromogenes UAE2 (Sp; GenBank Accession #: OK560620) and Streptomyces coeruleoprunus UAE1 (Sc; OK560621), respectively. The two antagonists were recovered from root tissues until 12 weeks after inoculation, efficiently colonized root cortex and xylem vessels, indicating that the date palm roots are a suitable habitat for these endophytic isolates. At the end of the greenhouse experiments, the development of sudden decline syndrome (SDS) was markedly suppressed by 53% with the application of Sp and 86% with Sc, confirming their potential in disease management. Results showed that the estimated disease severity indices in diseased seedlings were significantly (p < 0.05) reduced from 4.75 (scale of 5) to 2.25 or 0.67 by either Sp or Sc, respectively. In addition, conidial numbers of the pathogen significantly (p < 0.05) dropped by 38% and 76% with Sp and Sc, respectively, compared to infected seedlings with F. solani (control). Thus, the suppression of disease symptoms was superior in seedlings pre-inoculated with S. coeruleoprunus, indicating that the diffusible antifungal metabolites were responsible for F. solani retardation in these plants. This is the first report of actinobacteria naturally existing in date palm tissues acting as microbial antagonists against SDS on date palm.

Two strains of airborne Nocardiopsis alba producing different volatile organic compounds (VOCs) as biofungicide for Ganoderma boninense.

Nocardiopsis are actinobacteria which produce active compounds, such as antifungals and volatile compounds. Ganoderma boninense is a pathogenic and aggressive fungus that decreases palm oil yield during production. In this study, we isolated two strains of Nocardia (GME01 and GME22) from airborne contaminants on the actinobacteria culture collection in the laboratory. The aim of this study is to identify two strains of Nocardiopsis and to obtain the antifungal potency of volatile organic compounds (VOCs) against G. boninese. We characterized the morphology using Scanning Electrone Microscope (SEM), molecular properties and whole-cell protein spectra using Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS), antifungal assay on G. boninense and VOCs analysis of Nocardia using solid phase micro extraction/gas chromatography (SPME/GC). The two Nocardiopsis strains had the similar characteristic such as white aerial mycelium and spores, aerobic, grow well on ISP-2, TSA and NA medium without diffusible pigment and had the highest similarity with Nocardiopsis alba DSM 43377 (99.63% and 99.55% similarity for GME01 and GME22, respectively), Different morphological feature was found in aerial mycelium and spores. GME22 has a clearly fragmented mycelium whereas GME01 has none. Other features also showed different on the whole-cell protein spectra, antifungal activity and VOCs profiles. Antifungal activity assay on G. boninense showed that N. alba GME22 has higher antifungal activity than GME01 related with the VOCs abundance in two strains. Almost 38.3% (18 VOCs) of N. alba GME22 and 25.5% (12 VOCs) of N. alba GME01 were found specifically in each strain, and 36.2% (the 17 same VOCs) produced by both. The known volatile antifungal compounds S-methyl ethanethioate, 1,2-dimethyldisulfane, acetic acid, 2-methyl propanoic acid, 3-methyl-butanoic acid, nonan-2-one, undecan-2-one and 2-isopropyl-5-methylcyclohexan-1-ol only produced by N. alba GME22 and 1,3-dimethyltrisulfane only produced by N. alba GME01. A total of two known antifungal compounds 1,2-dimethyldisulfane and 6-methylheptan-2-one were produced by both N. alba. The abundance of antifungal VOCs produced by these bacteria is potentially to be used as biocontrol agent for pathogenic fungi in plants.

Application methods and modes of action of Pantoea agglomerans and Paenibacillus sp., to control the grapevine trunk disease-pathogen, Neofusicoccum parvum

Despite an increasing number of studies being carried out on the biocontrol of grapevine trunk diseases (GTDs), no commercial bacterial products have yet been developed to control GTDs. Knowledge of the precise modes of action (MOA) and the different application methods (AM) for biocontrol agents is crucial if they are to be successful in the field. In light of this, the present study aimed at selecting the most appropriate AM for eight bacterial strains with high potential for controlling Neofusicoccum parvum. These strains were applied on one-year-old grapevine in pots (grown from cuttings) using three methods: co-inoculation at stem level, preventive inoculation at stem level and preventive inoculation at the soil surface. The inhibitory activity of the bacterial strain against N. parvum was significantly dependent on the AM. Application of bacterial strains to stems, especially in a preventive way, was much more efficient than inoculation in the soil. When performing preventive inoculation on stems, the inhibition of N. parvumwood necrosis reached 50 and 65 % for Pantoea agglomerans (S1) and Paenibacillus sp. (S19) respectively. To decipher the underlying processes linked to fungal inhibition, the way in which several MOA affected the antagonistic capacity of these two strains was studied via in vitro and in planta assays. While P. agglomerans (S1) inhibited N. parvum by the secretion of antifungal volatile compounds, Paenibacillus sp. (S19) mainly inhibited this pathogen by antibiosis. In addition, both bacterial strains induced systemic defenses in grapevine. However, this affect tended to be higher at 15-dpi after inoculation with P. agglomerans (S1) than after innoculation with Paenibacillus sp. (S19) (three defense genes repressed versus five respectively). Finally, P. agglomerans (S1) and Paenibacillus sp. (S19) were shown to be potential biocontrol candidates for fighting N. parvum in grapevine, due to the combination of direct control via their antifungal activity and indirect control via their ability to activate the grapevine defense system.

Application methods and modes of action of &lt;i&gt;Pantoea agglomerans&lt;/i&gt; and &lt;i&gt;Paenibacillus&lt;/i&gt; sp. to control the grapevine trunk disease-pathogen, &lt;i&gt;Neofusicoccum parvum&lt;/i&gt;

Despite an increasing number of studies being carried out on the biocontrol of grapevine trunk diseases (GTDs), no commercial bacterial products have yet been developed to control GTDs. Knowledge of the precise modes of action (MOA) and the different application methods (AM) for biocontrol agents is crucial if they are to be successful in the field. In light of this, the present study aimed at selecting the most appropriate AM for eight bacterial strains with high potential for controlling Neofusicoccum parvum. These strains were applied on one-year-old grapevine in pots (grown from cuttings) using three methods: co-inoculation at stem level, preventive inoculation at stem level and preventive inoculation at the soil surface. The inhibitory activity of the bacterial strain against N. parvum was significantly dependent on the AM. Application of bacterial strains to stems, especially in a preventive way, was much more efficient than inoculation in the soil. When performing preventive inoculation on stems, the inhibition of N. parvumwood necrosis reached 50 and 65 % for Pantoea agglomerans (S1) and Paenibacillus sp. (S19) respectively. To decipher the underlying processes linked to fungal inhibition, the way in which several MOA affected the antagonistic capacity of these two strains was studied via in vitro and in planta assays. While P. agglomerans (S1) inhibited N. parvum by the secretion of antifungal volatile compounds, Paenibacillus sp. (S19) mainly inhibited this pathogen by antibiosis. In addition, both bacterial strains induced systemic defenses in grapevine. However, this affect tended to be higher at 15-dpi after inoculation with P. agglomerans (S1) than after innoculation with Paenibacillus sp. (S19) (three defense genes repressed versus five respectively). Finally, P. agglomerans (S1) and Paenibacillus sp. (S19) were shown to be potential biocontrol candidates for fighting N. parvum in grapevine, due to the combination of direct control via their antifungal activity and indirect control via their ability to activate the grapevine defense system.

Endophytic bacteria from strawberry plants control gray mold in fruits via production of antifungal compounds against Botrytis cinerea L.

Botrytis cinerea causes the gray mold disease in a wide range of plant hosts, especially in post-harvest periods. The control of this phytopathogen has been accomplished through the application of fungicides. However, this practice can cause environmental problems and increase fruit production costs. In addition, this fungus species has developed resistance to conventional synthetic fungicides. In this context, plant growth-promoting bacteria have shown potential for application in agricultural production because they are able to stimulate plant growth through different mechanisms, including the biological control of phytopathogens (indirect growth promotion mechanism). The aim of this work was to evaluate in vitro and in fruits the potential for indirect plant growth-promotion of bacteria isolated from strawberry leaves and roots. Dual plate method and inverted plate method were used to verify the ability of controlling in vitro the growth of Botrytis cinerea via the production of diffusible and volatile antifungal compounds, respectively. The effect of six bacterial isolates that showed greater potential for biological control in vitro was evaluated by scanning electron microscopy. Antifungal compounds produced by these bacterial isolates were identified by liquid chromatography coupled with mass spectrometry. Six bacterial strains were tested on strawberry pseudofruits. Five selected strains belong to the genus Bacillus and one to the genus Pantoea sp. Selected strains were able to inhibit more than 80 % of the mycelial growth of B. cinerea by the production of diffusible compounds and 90 % by volatile antifungal compounds production. Scanning electron microscopy showed the intense degradation of fungal hyphae caused by the presence of all bacterial strains. Bioactive compounds (salycilamide, maculosin, herniarin, lauroyl diethanolamide, baptifoline, undecanedioic acid, botrydial, 8 3-butylidene-7-hydroxyphthalide and N-(3-oxo-henoyl)-homoserine lactone) were obtained from liquid culture of these strains and extraction with ethyl acetate. All six isolates tested in vivo reduced the incidence of gray mold in strawberry pseudofruits in postharvest. It is concluded that isolates 26, 29, 65, 69, 132 (Bacillus sp.) and MQT16M1 (Pantoea sp.) have potential application for the biological control of Botrytis cinerea in strawberry via the production of diffusible and volatile antifungal compounds.

Suppression of Alternaria brassicicola infection by volatile compounds from spent mushroom substrates

Most commercially circulating mushrooms are produced via cultivation using artificially produced mushroom substrates. However, after mushroom harvesting, the disposal of spent mushroom substrates (SMSs) is a serious problem for the mushroom industry owing to the need for a disposal site and the cost involved. Thus, in view of the possibility of recycling SMSs as a soil modifier, we examined the effect of soil mixed with SMSs on the infection of Arabidopsis leaves by Alternaria brassicicola, the causal agent of cabbage leaf spot. The mixing of SMSs used for Hypsizygus marmoreus, Pholiota microspora, Lyophyllum decastes, and Auricularia polytricha into culture soil suppressed the lesion formation caused by A. brassicicola. The defense responses of Arabidopsis were not induced by the culturing of these seedlings in soils containing SMSs. Suppressed lesion formation was observed after the seedlings were treated with volatiles emitted from SMSs that were incubated with soil for 7 days and used for H.marmoreus, P. microspora, L. decastes, A. polytricha, Lentinula edodes, and Cyclocybe cylindracea. The volatiles from the SMSs reduced the elongation of A. brassicicola hyphae. GC-MS analyses of extracts from the SMS containing soils led to the detection of various volatile compounds; among these, skatole, 2,4-di-tert-butylphenol, γ-dodecalactone, butyric acid, guaiacol, 6-amyl-2-pyrone, and 1-octen-3-ol were examined for inhibitory activity on A. brassicicola and found to suppress hyphae elongation. These findings indicate that the antifungal volatile compounds emitted by the SMSs suppress A. brassicicola infection.

Volatile Organic Compound from Trichoderma asperelloides TSU1: Impact on Plant Pathogenic Fungi.

Soil microorganisms are well studied for their beneficial effects on plant growth and their impact on biocontrol agents. The production of volatile antifungal compounds emitted from soil fungi is considered to be an effective ability that can be applied in biofumigants in the control of plant diseases. A soil fungus, Trichoderma asperelloides TSU1, was isolated from flamingo flower cultivated soil and identified on the basis of the morphology and molecular analysis of the internal transcribed spacer (ITS), rpb2, and tef1-α genes. To test T. asperelloides TSU1-produced volatile organic compounds (VOCs) with antifungal activity, the sealed plate method was used. The VOCs of T. asperelloides TSU1 inhibited the mycelial growth of fungal pathogens that were recently reported as emerging diseases in Thailand, namely, Corynespora cassiicola, Fusarium incarnatum, Neopestalotiopsis clavispora, N. cubana, and Sclerotium rolfsii, with a percentage inhibition range of 38.88–68.33%. Solid-phase microextraction (SPME) was applied to trap VOCs from T. asperelloides TSU1 and tentatively identify them through gas chromatography–mass spectrometry (GC/MS). A total of 17 compounds were detected in the VOCs of T. asperelloides TSU1, and the dominant compounds were identified as fluoro(trinitro)methane (18.192% peak area) and 2-phenylethanol (9.803% peak area). Interestingly, the commercial 2-phenyethanol showed antifungal activity against fungal pathogens that were similar to the VOCs of T. asperelloides TSU1 by bioassay. On the basis of our study’s results, T. asperelloides TSU1 isolated from soil displayed antifungal abilities via the production of VOCs responsible for restricting pathogen growth.

Trichoderma asperellum T76-14 Released Volatile Organic Compounds against Postharvest Fruit Rot in Muskmelons (Cucumis melo) Caused by Fusarium incarnatum.

Postharvest fruit rot caused by Fusarium incarnatum is a destructive postharvest disease of muskmelon (Cucumis melo). Biocontrol by antagonistic microorganisms is considered an alternative to synthetic fungicide application. The aim of this study was to investigate the mechanisms of action involved in the biocontrol of postharvest fruit rot in muskmelons by Trichoderma species. Seven Trichoderma spp. isolates were selected for in vitro testing against F. incarnatum in potato dextrose agar (PDA) by dual culture assay. In other relevant works, Trichoderma asperellum T76-14 showed a significantly higher percentage of inhibition (81%) than other isolates. Through the sealed plate method, volatile organic compounds (VOCs) emitted from T. asperellum T76-14 proved effective at inhibiting the fungal growth of F. incarnatum by 62.5%. Solid-phase microextraction GC/MS analysis revealed several VOCs emitted from T. asperellum T76-14, whereas the dominant compound was tentatively identified as phenylethyl alcohol (PEA). We have tested commercial volatile (PEA) against in vitro growth of F. incarnatum; the result showed PEA at a concentration of 1.5 mg mL−1 suppressed fungal growth with 56% inhibition. Both VOCs and PEA caused abnormal changes in the fungal mycelia. In vivo testing showed that the lesion size of muskmelons exposed to VOCs from T. asperellum T76-14 was significantly smaller than that of the control. Muskmelons exposed to VOCs from T. asperellum T76-14 showed no fruit rot after incubation at seven days compared to fruit rot in the control. This study demonstrated the ability of T. asperellum T76-14 to produce volatile antifungal compounds, showing that it can be a major mechanism involved in and responsible for the successful inhibition of F. incarnatum and control of postharvest fruit rot in muskmelons.

Phenylacetic Acid and Methylphenyl Acetate From the Biocontrol Bacterium Bacillus mycoides BM02 Suppress Spore Germination in Fusarium oxysporum f. sp. lycopersici

Bacillus mycoides strain BM02 originally isolated from the tomato rhizosphere was found to have beneficial functions in tomato by promoting plant growth and reducing the severity of Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol). Cytological experiments demonstrated B. mycoides BM02 reduced Fol invasion by reducing spore attachment and increasing hyphal deformation in hydroponics-grown tomato root tissues. Two volatile antifungal compounds, phenylacetic acid (PAA) and methylphenyl acetate (MPA), were identified from the culture filtrates of B. mycoides BM02 by GC-MS analysis. Chemically synthesized PAA, and to a lower extent MPA, suppressed spore germination but have no effects on the hyphal growth of Fol. Our results indicated that the biocontrol agent B. mycoides BM02 produced an array of bioactive compounds including PAA and MPA to suppress plant diseases caused by Fol and other pathogenic microorganisms.

Biological control of Fusarium wilt and growth promotion in pigeon pea (Cajanus cajan) by antagonistic rhizobacteria, displaying multiple modes of pathogen inhibition

Fusarium udum is one of the most important pathogens impacting the production of pigeon pea (Cajanus cajan) legume. Legume growers depend heavily on pesticides; however, toxicity danger to farmers, consumers, non-target organisms as well as environmental health cause huge concern. Besides, the rise of fungicide resistance is problematic. The objectives of this study were to decipher the multiple mechanisms of biological control in previously identified two promising rhizobacteria (Pseudomonas sp. NS 1 and Bacillus sp. NS 22) in vitro and evaluate their disease suppression and plant growth promotion ability in pigeon pea in vivo. These rhizobacteria inhibited F. udum mycelia growth and biomass in vitro and reduced wilt disease severity in plants grown in Fusarium-infested soil in controlled environmental conditions. Fungicidal action of these rhizobacteria was due to the production of numerous biocidal compounds including different antifungal metabolites, chitinolytic (endochitinase, exochitinase, chitobiase) and other cell wall degrading lytic enzymes (proteinase, cellulose, amylase, pectinase, lipase), siderophores, and antifungal volatile compounds such as ammonia and cyanide. In planta assay, these rhizobacteria made the plant more resistant to fungal attacks through the induction of systemic defense in host tissue by improving the status of different defense enzymes (phenylalanine ammonia lyase, polyphenol oxidase, and peroxidase) and phenolics. Furthermore, these two bacteria significantly reduced disease severity, root mortality, and at the same time, promoted shoot and root elongation in pigeon pea seedlings as compared to the absolute, pathogen, and chemical control. In nutshell, Pseudomonas sp. NS 1 and Bacillus sp. NS 22 displayed the potential as biofungicides and biofertilizers to prevent F. udum induced disease incidence and promote plant growth in an integrative disease and nutrient management approach of sustainable agriculture practices.

Control of postharvest grey spot rot of loquat fruit with Metschnikowia pulcherrima E1 and potential mechanisms of action

In the present study, the biocontrol efficacy of Metschnikowia pulcherrima E1 against grey spot rot (Pestalotiopsis vismiae) on loquat fruits was assessed and along with potential mechanisms of action. Results indicated that decay incidence and lesion diameter were both significantly reduced by treatment of fruits with the yeast, M. pulcherrima E1. M. pulcherrima E1 quickly colonized wounds in loquat fruit stored at either 20 °C or 4 °C. Scanning electron microscopy revealed that the yeast formed a biofilm on the wound surface in loquat fruit which inhibited the invasion of pathogenic fungi. M. pulcherrima E1 also competed with the pathogen for nutrients (iron) and space. Results of dual-culture petri dish assay indicated that M. pulcherrima E1 produces volatile antifungal compounds that inhibit the growth of P. vismiae. SPME-GC–MS analysis revealed that 3-methyl-1-butanol, phenylethyl alcohol and 1-Hexanol are the main volatile organic compounds produced by M. pulcherrima E1 and that had a significant inhibitory effect on the growth of fungal mycelia. M. pulcherrima E1 induced a higher level of defense-related enzyme activity in loquat fruit, including polyphenol oxidase, peroxidase, catalase, ascorbate peroxidase, and phenylalanine ammonia-lyase. Collectively, the results of the present study indicate that M. pulcherrima E1 has good potential to be used as a biocontrol agent against postharvest grey spot rot on loquats. The different mechanisms of action exhibited by M. pulcherrima E1 enhances its ability to control P. vismiae.

EndophyticBbacteria origin of healthy rice plants produce antifungal volatile compound inhibited the growth of Pyricularia oryzae Cav., the causal agent of rice blast disease

Endophytic bacteria have been known as producers for bioactive compound in which widely used in the field of crop protection. Several isolates of endophytic bacteria have demonstrated their potential ability as biocontrol agents by inhibiting the development of major rice pathogens. However, their ability to produce bioactive compounds has not been explored. The study reported the ability of the endophytic bacteria isolates in producing antifungal volatile compounds to inhibit the growth of Pyricularia oryzae Cav., the causal agent of rice blast disease. The experiment was conducted by dual culture method using bipartite compartments in which the bacterial isolates and P. oryzae were grown separately and placed by facing to each other. The bacterial isolates were grown on ISP2 agar media, whereas P. oryzae was grown on PDA media. Nine isolates were tested for their ability in producing antifungal volatile compound. The potential of antifungal volatile compound was observed by the P. oryzae growth compared to control. Furthermore, its effect on the P. oryzae mycelia was observed under microscope. The result demonstrated that all of the tested isolates were able to produce volatile compound which were able to inhibit the growth of P. oryzae . In addition, the compounds were also causing mycelial alteration.

Phyllosphere Fungal Communities of Plum and Antifungal Activity of Indigenous Phenazine-Producing Pseudomonas synxantha Against Monilinia laxa.

European plum (Prunus domestica L.) is a significant commercial crop in Serbia in terms of total fruit production, and is traditionally processed into slivovitz brandy. The brown rot disease caused by Monilinia laxa drastically reduces plum yield almost every year. Fungal communities associated with leaves and fruits of four local Serbian plum cultivars (Požegača, Ranka, Čačanska Lepotica and Čačanska Rodna) were investigated in two phenological stages during early (May) and late (July) fruit maturation. Alpha diversity indices showed that fungal communities were heterogeneous and Beta diversity indicated that autochthonous fungal communities depended upon seasonal changes and the cultivars themselves. The phylum Ascomycota was the most abundant in all samples, with relative abundance (RA) between 46% in the Požegača cultivar (May) and 89% in the Lepotica cultivar (July). The most abundant genus for all plum cultivars in May was Aureobasidium, with RA from 19.27 to 33.69%, followed by Cryptococcus, with 4.8 to 48.80%. In July, besides Cryptococcus, different genera (Metschnikowia, Fusarium, and Hanseniaspora) were dominant on particular cultivars. Among all cultivable fungi, molecular identification of eleven M. laxa isolates from four plum cultivars was performed simultaneously. Bacterial isolates from the plum phyllosphere were tested for their potential antifungal activity against indigenous M. laxa isolates. The most potent antagonist P4/16_1, which significantly reduced mycelial growth of M. laxa, was identified as Pseudomonas synxantha. Further characterization of P4/16_1 revealed the production of volatile organic compounds and phenazine-1-carboxylic acid (PCA). Crude benzene extract of PCA exhibited 57–63% mycelial growth inhibition of M. laxa. LC/MS analysis of the crude extract confirmed the presence of phenazine derivatives amongst other compounds. Scanning electron microscopy revealed morpho-physiological changes in the hyphae of M. laxa isolates caused by the cell culture and the P. synxantha P4/16_1 crude benzene extract. This is the first report of antagonistic activity of P. synxantha against M. laxa induced by diffusible and volatile antifungal compounds, and it appears to be a promising candidate for further investigation for potential use as a biocontrol agent against brown rot-causing fungi.