Endophytic Penicillium species secretes mycophenolic acid that inhibits the growth of phytopathogenic fungi.

The worldwide demand for reduced and restricted use of pesticides in agriculture due to serious environmental effects, health risks and the development of pathogen resistance calls for the discovery of new bioactive compounds. In the medical field, antibiotic-resistant microorganisms have become a major threat to man, increasing mortality. Endophytes are endosymbiotic microorganisms that inhabit plant tissues without causing any visible damage to their host. Many endophytes secrete secondary metabolites with biological activity against a broad range of pathogens, making them potential candidates for novel drugs and alternative pesticides of natural origin. We isolated endophytes from wild plants in Israel, focusing on endophytes that secrete secondary metabolites with biological activity. We isolated 302 different endophytes from 30 different wild plants; 70 of them exhibited biological activity against phytopathogens. One biologically active fungal endophyte from the genus Penicillium, isolated from a squill (Urginea maritima) leaf, was further examined. Chloroform-based extraction of its growth medium was similarly active against phytopathogens. High-performance liquid chromatography separation followed by gas chromatography/mass spectrometry analysis revealed a single compound-mycophenolic acid-as the main contributor to the biological activity of the organic extract.

trans-2-Octenal, a single compound of a fungal origin, controls Sclerotium rolfsii, both in vitro and in soil.

BACKGROUND: Sclerotium rolfsii is a soil-borne phytopathogenic fungus that causes diseases in economically important crops. Eradication of the fungus is hampered by its wide range of hosts, as well as its capacity to form sclerotia. Recently, we have shown that the endophytic fungus Daldinia cf. concentrica emits biologically active volatile organic compounds (VOCs); we also demonstrated that one VOC, trans-2-octenal, was the most effective against various phytopathogenic fungi. Thus, the aim of this study was to examine the potential of this compound to control hyphae and sclerotia of S. rolfsii, both in vitro and in soil. RESULTS: We found that in vitro exposure of S. rolfsii mycelium to trans-2-octenal in air fully inhibits and kills the fungus. Elimination of sclerotia viability occurred at the same concentration, but direct contact between the sclerotia and the compound was needed. trans-2-Octenal also affected the viability of both hyphae and sclerotia of S. rolfsii in small pots containing loam soil. CONCLUSION: We suggest the use of trans-2-octenal as a novel compound to control S. rolfsii. © 2020 Society of Chemical Industry.

Caenorhabditis elegans susceptibility to Daldinia cf. concentrica bioactive volatiles is coupled with expression activation of the stress-response transcription factor daf-16, a part of distinct nematicidal action.

The bionematicidal effect of a synthetic volatile mixture (SVM) of four volatile organic compounds (VOCs) emitted by the endophytic fungus Daldinia cf. concentrica against the devastating plant-parasitic root-knot nematode Meloidogyne javanica has been recently demonstrated in both in vitro and greenhouse experiments. However, the mode of action governing the observed irreversible paralysis of J2 larvae upon exposure to SVM is unknown. To unravel the mechanism underlying the anthelmintic and nematicidal activities, we used the tractable model worm Caenorhabditis elegans. C. elegans was also susceptible to both the fungal VOCs and SVM. Among compounds comprising SVM, 3-methyl-1-butanol, (±)-2-methyl-1-butanol, and 4-heptanone showed significant nematicidal activity toward L1, L4 and young adult stages. Egg hatching was only negatively affected by 4-heptanone. To determine the mechanism underlying this activity, we examined the response of C. elegans mutants for glutamate-gated chloride channel and acetylcholine transporter, targets of the nematicidal drugs ivermectin and aldicarb, respectively, to 4-heptanone and SVM. These aldicarb- and ivermectin-resistant mutants retained susceptibility upon exposure to 4-heptanone and SVM. Next, we used C. elegans TJ356 strain zIs356 (daf-16::GFP+rol-6), LD1 ldIs7 [skn-1B/C::GFP + pRF4(rol-6(su1006))], LD1171 ldIs3 [gcs-1p::gfp; rol-6(su1006))], CL2166 dvIs19 (gst-4p::GFP) and CF1553 muIs84 (sod-3p::GFP+rol-6), which have mutations in genes regulating multiple stress responses. Following exposure of L4 larvae to 4-heptanone or SVM, there was clear nuclear translocation of DAF-16::GFP, and SKN-1::GFP indicating that their susceptibility involves DAF-16 and SKN1 regulation. Application of 4-heptanone, but not SVM, induced increased expression of, gcs-1::GFP and gst-4::GFP compared to controls. In contrast, application of 4-heptanone or SVM to the sod-3::GFP line elicited a significant decline in overall fluorescence intensity compared to controls, indicating SOD-3 downregulation and therefore overall reduction in cellular redox machinery. Our data indicate that the mode of action of SVM and 4-heptanone from D. cf. concentrica differs from that of currently available nematicides, potentially offering new solutions for nematode management.

Bioactive Volatiles from an Endophytic Daldinia cf. concentrica Isolate Affect the Viability of the Plant Parasitic Nematode Meloidogyne javanica.

Plant-parasitic nematodes form one of the largest sources of biotic stress imposed on plants, and are very difficult to control; among them are the obligate parasites, the sedentary root-knot nematodes (RKNs)-Meloidogyne spp.-which are extremely polyphagous and exploit a very wide range of hosts. Endophytic fungi are organisms that spend most of their life cycle within plant tissue without causing visible damage to the host plant. Many endophytes secrete specialized metabolites and/or emit volatile organic compounds (VOCs) that exhibit biological activity. Recently, we demonstrated that the endophytic fungus Daldinia cf. concentrica secrets biologically active VOCs. Here we examined the ability of the fungus and its VOCs to control the RKN M. javanica both in vitro and greenhouse experiments. The D. cf. concentrica VOCs showed bionematicidal activity against the second-stage juveniles (J2s) of M. javanica. We found that exposure of J2s to fungal volatiles caused 67% reduction in viability, and that application of a synthetic volatile mixture (SVM), comprising 3-methyl-1-butanol, (±)-2-methyl-1-butanol, 4-heptanone, and isoamyl acetate, in volumetric ratio of 1:1:2:1 further reduced J2s viability by 99%. We demonstrated that, although each of the four VOCs significantly reduced the viability of J2s relative to the control, only 4-heptanone elicited the same effect as the whole mixture, with nematicidal activity of 90% reduction in viability of the J2s. Study of the effect of the SVM on egg hatching demonstrated that it decreased eggs hatching by 87%. Finally, application of the SVM to soil inoculated with M. javanica eggs or J2s prior to planting susceptible tomato plants resulted in a significantly reduced galling index and fewer eggs produced on each root system, with no effect on root weight. Thus, D. cf. concentrica and/or SVM based on fungal VOCs may be considered as a novel alternative approach to controlling the RKN M. javanica.

Use of the Endophytic Fungus Daldinia cf. concentrica and Its Volatiles as Bio-Control Agents.

Endophytic fungi are organisms that spend most of their life cycle within plant tissues without causing any visible damage to the host plant. Many endophytes were found to secrete specialized metabolites and/or emit volatile organic compounds (VOCs), which may be biologically active and assist fungal survival inside the plant as well as benefit their hosts. We report on the isolation and characterization of a VOCs-emitting endophytic fungus, isolated from an olive tree (Olea europaea L.) growing in Israel; the isolate was identified as Daldinia cf. concentrica. We found that the emitted VOCs were active against various fungi from diverse phyla. Results from postharvest experiments demonstrated that D. cf. concentrica prevented development of molds on organic dried fruits, and eliminated Aspergillus niger infection in peanuts. Gas chromatography-mass spectrometry analysis of the volatiles led to identification of 27 VOCs. On the basis of these VOCs we prepared two mixtures that displayed a broad spectrum of antifungal activity. In postharvest experiments these mixtures prevented development of molds on wheat grains, and fully eliminated A. niger infection in peanuts. In light of these findings, we suggest use of D. cf. concentrica and/or its volatiles as an alternative approach to controlling phytopathogenic fungi in the food industry and in agriculture.

Characterization of Alternaria alternata Causing Black Spot Disease of Pomegranate in Israel Using a Molecular Marker.

Black spot disease of pomegranate is a relatively new disease in Israel that is caused by Alternaria alternata. The symptoms include black spots on leaves and fruit. Only the outer part of the fruit is damaged; the edible tissue remains unaffected. In this study, we obtained 50 isolates of A. alternata from infected pomegranate plants that were classified based on pathogenicity tests using detached leaves. Using an arbitrarily primed polymerase chain reaction, we identified one primer (primer CAG) that reacted only with DNA of isolates that induced the most severe disease symptoms. Based on the sequence of the amplified fragment, we generated a specific primer (primer C) that recognizes these highly virulent isolates. Therefore, we suggest that primer C can be utilized as a molecular marker for the detection of A. alternata isolates that cause black spot disease of pomegranate.

Hypoxylon sp., an endophyte of Persea indica, producing 1,8-cineole and other bioactive volatiles with fuel potential.

An endophytic fungus of Persea indica was identified, on the basis of its anamorphic stage, as Nodulosporium sp. by SEM. Partial sequence analysis of ITS rDNA revealed the identity of the teleomorphic stage of the fungus as Hypoxylon sp. It produces an impressive spectrum of volatile organic compounds (VOCs), most notably 1,8-cineole, 1-methyl-1,4-cyclohexadiene, and tentatively identified (+)-.alpha.-methylene-.alpha.-fenchocamphorone, among many others, most of which are unidentified. Six-day-old cultures of Hypoxylon sp. displayed maximal VOC-antimicrobial activity against Botrytis cinerea, Phytophthora cinnamomi, Cercospora beticola, and Sclerotinia sclerotiorum suggesting that the VOCs may play some role in the biology of the fungus and its survival in its host plant. Media containing starch- or sugar-related substrates best supported VOC production by the fungus. Direct on-line quantification of VOCs was measured by proton transfer mass spectrometry covering a continuous range with optimum VOC production occurred at 6 days at 145 ppmv with a rate of production of 7.65 ppmv/h. This report unequivocally demonstrates that 1,8-cineole (a monoterpene) is produced by a microorganism, which represents a novel and important source of this compound. This monoterpene is an octane derivative and has potential use as a fuel additive as do the other VOCs of this organism. Thus, fungal sourcing of this compound and other VOCs as produced by Hypoxylon sp. greatly expands their potential applications in medicine, industry, and energy production.

Acetylation represses the binding of CheY to its target proteins.

The ability of CheY, the response regulator of bacterial chemotaxis, to generate clockwise rotation is regulated by two covalent modifications - phosphorylation and acetylation. While the function and signal propagation of the former are widely understood, the mechanism and role of the latter are still obscure. To obtain information on the function of this acetylation, we non-enzymatically acetylated CheY to a level similar to that found in vivo, and examined its binding to its kinase CheA, its phosphatase CheZ and the switch protein FliM - its target at the flagellar switch complex. Acetylation repressed the binding to all three proteins. These results suggest that both phosphorylation and acetylation determine CheY's ability to bind to its target proteins, thus providing two levels of regulation, fast and slow respectively. The fast level is modulated by environmental signals (e.g. chemotactic and thermotactic stimuli). The slow one is regulated by the metabolic state of the cell and it determines, at each metabolic state, the fraction of CheY molecules that can participate in signalling.

In vivo acetylation of CheY, a response regulator in chemotaxis of Escherichia coli.

CheY, the excitatory response regulator in the chemotaxis system of Escherichia coli, can be modulated by two covalent modifications: phosphorylation and acetylation. Both modifications have been detected in vitro only. The role of CheY acetylation is still obscure, although it is known to be involved in chemotaxis and to occur in vitro by two mechanisms--acetyl-CoA synthetase-catalyzed transfer of acetyl groups from acetate to CheY and autocatalyzed transfer from AcCoA. Here, we succeeded in detecting CheY acetylation in vivo by three means--Western blotting with a specific anti-acetyl-lysine antibody, mass spectrometry, and radiolabeling with [(14)C]acetate in the presence of protein-synthesis inhibitor. Unexpectedly, the level and rate of CheY acetylation in vivo were much higher than that in vitro. Thus, before any treatment, 9-13% of the lysine residues were found acetylated, depending on the growth phase, meaning that, on average, essentially every CheY molecule was acetylated in vivo. This high level was mainly the outcome of autoacetylation. Addition of acetate caused an incremental increase in the acetylation level, in which acetyl-CoA synthetase was involved too. These findings may have far-reaching implications for the structure-function relationship of CheY.