Destruxin production by the entomogenous fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> in insects and factors influencing their degradation

There is much public interest in the use of fungal biological control agents as alternatives to chemical pesticides. However, there are some concerns as to whether the metabolites produced by these fungi pose a risk to humans and the environment. Destruxins are the main metabolites produced by the insect pathogenic fungus Metarhizium anisopliae (Metsch.) Sorok. The production of these compounds in two different insect hosts and their subsequent fate in the soil were assessed as a case study. Destruxin profiling revealed that the amount and type of destruxin produced was dependant upon the fungal strain and insect host and that these compounds decomposed shortly after host death. Destruxin decomposition was presumably due to the activity of hydrolytic enzymes in the cadavers and appeared to be independent of host or soil type and biota. Temperature strongly influenced destruxin decomposition. Our studies are the first to show that the destruxins are essentially restricted to the host and pathogen and are unlikely to contaminate the environment or enter the food chain.     

Melanin production in Alternaria helianthi

Abstract

Most of the plant pathogenic fungi produce a dark phenolic polymer called melanin. The high performance liquid chromatography (HPLC) analysis of the mycelial extract of Alternaria helianthi revealed an accumulation of scytalone and a shunt metabolite 2-hydroxyjuglone which confirms the production of dihydroxynapthalene type of melanin. The growth and melanin of A. helianthi increased when grown in host extract broth at 6.5 pH and a temperature beyond 30°C had an inhibitory effect on the pathogen. The production and type of melanin produced in Alternaria helianthi is reported for the first time.     

Nonspecific Phytotoxic Effects of Sirodesmins on Host and Nonhost Plants of Leptosphaeria maculans

Sirodesmin PL, deacetylsirodesmin PL and sirodesmin C were isolated from cultures of an aggressive strain of Leptosphaeria maculans, purified by thin layer and column chromatography, and tested on leaves and roots of 14 host and non-host plants of the pathogen. The sirodesmins showed no host-specificity. However, various plant species developed symptoms of different severities. In both bioassay systems deacetylsirodesmin PL had a lower phytotoxicity than sirodesmin PL and sirodes-min C. Our results show that sirodesmins belong to the non-host-specific phytotoxins.     

Tomato resistance to Alternaria stem canker: localization in host genotypes and functional expression compared to non-host resistance

Summary The Alternaria stem canker resistance locus (Asc-locus), involved in resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici and in insensitivity to host-specific toxins (AAL-toxins) produced by the pathogen, was genetically mapped on the tomato genome. Susceptibility and resistance were assayed by testing a segregating F₂ population for sensitivity to AAL-toxins in leaf bioassays. Linkage was observed to phenotypic markers solanifolium and sunny, both on chromosome 3. For the Asc-locus, a distance of 18 centiMorgan to solanifolium was calculated, corresponding to position 93 on chromosome 3. This map position of the resistance locus turned out to be the same in three different resistant tomato accessions, one Dutch and two American, that are at least 40 years apart. AAL-toxin sensitivity in susceptible and resistant tomato genotypes was compared with AAL-toxin sensitivity in a non-host Nicotiana tabacum during different levels of plant cell development. In susceptible and resistant tomato genotypes, inhibitory effects were demonstrated at all levels, except for leaves of resistant genotypes. However, during pollen and root development, inhibitory effects on susceptible genotypes were larger than on resistant genotypes. In the non-host Nicotiana tabacum, hardly any effects of AAL-toxins were demonstrated. Apparently, a cellular target site is present in tomato, but not in Nicotiana tabacum. It was concluded that three levels of AAL-toxin sensitivity exist: (1) a susceptible host sensitivity, (2) a resistant host sensitivity, (3) a non-host sensitivity, and that the resistance mechanism operating in tomato is different from that operating in Nicotiana tabacum.     

Phytoalexin Accumulation in Arabidopsis thaliana during the Hypersensitive Reaction to Pseudomonas syringae pv syringae

Inoculation of leaves of Arabidopsis thaliana (L.) Heynh. with the wheat pathogen, Pseudomonas syringae pv syringae, resulted in the expression of the hypersensitive reaction and in phytoalexin accumulation. No phytoalexin accumulation was detected after infiltration of leaves with a mutant of P. s. syringae deficient in the ability to elicit a hypersensitive reaction; with the crucifer pathogen, Xanthomonas campestris pv campestris; or with 10 millimolar potassium phosphate buffer (pH 6.9). Phytoalexin accumulation was correlated with the restricted in vivo growth of P. s. syringae. A phytoalexin was purified by a combination of reverse phase flash chromatography, thin layer chromatography, followed by reverse phase high performance liquid chromatography. The Arabidopsis phytoalexin was identified as 3-thiazol-2′-yl-indole on the basis of ultraviolet, infrared, mass spectral, ¹H-nuclear magnetic resonance, and ¹³C-nuclear magnetic resonance data.     

The phytopathogenic fungus Alternaria brassicicola: Phytotoxin production and phytoalexin elicitation

The metabolites and phytotoxins produced by the phytopathogenic fungus Alternaria brassicicola (Schwein.) Wiltshire, as well as the phytoalexins induced in host plants, were investigated. Brassicicolin A emerged as the most selective phytotoxic metabolite produced in liquid cultures of A. brassicicola and spirobrassinin as the major phytoalexin produced in infected leaves of Brassica juncea (whole plants). In detached infected leaves of B. juncea, the main component was N′-acetyl-3-indolylmethanamine, the product of detoxification of the phytoalexin brassinin by A. brassicicola. In addition, the structure elucidation of three hitherto unknown metabolites having a fusicoccane skeleton was carried out and the antifungal activity of several plant defenses against A. brassicicola was determined.     

Toxicity of Essential Oil from a New Strain of Ocimum gratissimum (Clocimum) against Betelvine Pathogenic Fungi

The leaves of Ocimum gratissimum (Clocimum) exhibited strong volatile fungitoxicity against betelvine (Piper betle L.) pathogens—Alternaria alternata, Colletotrichum capsici and Sclerotium rolfsii. Fifteen compounds could be identified from the fungitoxic constituents—the essential oil. The oil at its minimum inhibitory concentrations of 50, 250 and 500 ppm against S. rolfsii, A. alternata and C. capsici, respectively, was fungistatic, although, fungicidal at higher concentrations. Eugenol was found to be the major fungitoxic principle in the oil. The oil was either equally effective or superior to synthetic commercial fungicides and was non-phytotoxic to the host plants. Thus, the oil can be used as a valuable indigenous and biodegradable agent against fungi that cause losses to the betelvine industry.     

Alternaria cassiae Alters Phenylpropanoid Metabolism in Sicklepod (Cassia obtusifolia)

Phenylpropaniod metabolism has been implicated in plant defence mechanism(s) against pathogen attack. In this study, phenylpropanoid metabolism was examined over a 72 h time course in the weed sicklepod (Cassia obtusifolia) in relation to pathogenic effects of the fungus Alternaria cassiae. When 3- to 4-week old seedlings were challenged by the pathogen, extrable phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) activity was dramatically increased above that in uninfected plants severalhours after inoculation and exposure to dew. Greatest increases of enzyme activity (3-fold, specific activity basis) occurred at ca 15–23 after treatment with fungal spores. After this peak of activity, PAL activity declined with time in infectedtissue, but remained greater than in uninfected plants through 65 h after treatment. Total methanol-soluble hydroxyphenolic compound levels (PAL products) were higher in shoots (stems and leaves) of infected plants at 48–72 h. Leaves contained a higherconcentration (per gram fresh weight) of hydroxyphenolic compounds than did stems, and infected leaves exhibited a phenolic content greater than that of uninfected leaves at ca 27–72 h. Increased soluble phenolic compound production correlated with the appearance of lesions and necrotic spots on leaves and stems. UV irradiation examination and spectrofluorometric analysis of thin layer chromatographic separations of methanolic exatracts revealed a substantial increase of several components ininfected tissue 48 h after inoculation. Results support the view that PAL activity increases correlate with increased phenolic compound production in this host/pathogen interaction.     

Production of phytotoxic spore germination liquids by Alternaria brassicae and A. brassicicola and their effect on species of the family Brassicaceae

Experiments assessed the susceptibility of Brassica spp. and non-Brassica spp. in the family Brassicaceae to infection by Alternaria brassicae and A. brassicicola, and determined the sensitivity of the host species to spore germination liquids (SGLs) produced by the pathogens on B. napus leaves. There was a wide range of sensitivity to the pathogens. Brassica spp. were generally more susceptible, and some non-Brassica spp. (Barbarea vulgaris and Capsella bursa-pastoris) were immune to A. brassicicola. Measurable damage was caused by SGLs but with significant variation between host species. Non-hosts and weak hosts also showed necrosis. It was concluded that, in the case of both pathogens, the toxic factors in these SGLs were host-selective. Selectivity in toxin production was also demonstrated in relation to the host surface or growing medium in which spores germinated. A substantial amount of toxin was produced on all Brassicaceae tested but not on unrelated species (Triticum aestivum, Pisum sativum and Lycopersicon esculentum). Neither pathogen produced measurable amounts of toxin when cultured in Czapek (Dox) broth.     

Optimization and production of novel antimicrobial agents from sponge associated marine actinomycetes <Emphasis Type="Italic">Nocardiopsis dassonvillei</Emphasis> MAD08

The sponge-associated actinomycetes were isolated from the marine sponge Dendrilla nigra, collected from the southwest coast of India. Eleven actinomycetes were isolated depending upon the heterogeneity and stability in subculturing. Among these, Nocardiopsis dassonvillei MAD08 showed 100% activity against the multidrug resistant pathogens tested. The culture conditions of N. dassonvillei MAD08 was optimized under submerged fermentation conditions for enhanced antimicrobial production. The unique feature of MAD08 includes extracellular amylase, cellulase, lipase, and protease production. These enzymes ultimately increase the scope of optimization using broad range of raw materials which might be efficiently utilized. The extraction of the cell free supernatant with ethyl acetate yielded bioactive crude extract that displayed activity against a panel of pathogens tested. Analysis of the active thin layer chromatography fraction by Fourier transform infrared and gas chromatography-mass spectrometry evidenced 11 compounds with antimicrobial activity. The ammonium sulfate precipitation of the culture supernatant at 80% saturation yielded an anticandidal protein of molecular weight 87.12 kDa. This is the first strain that produces both organic solvent and water soluble antimicrobial compounds. The active extract was non-hemolytic and showed surface active property envisaging its probable role in inhibiting the attachment of pathogens to host tissues, thus, blocking host–pathogen interaction at an earlier stage of pathogenesis.     

The Influence of Plant Extracts on Phytotoxin Production and Growth Rate of Alternaria helianthi

When liquid cultures of Alternaria helianthi were supplemented with aqueous extracts of leaf tissue of its host plant (sunflower), pronounced effects on both growth and production of the toxin deoxyradicinin were apparent. Very low levels of leaf extract stimulated toxin production but did not significantly affect growth, while higher levels markedly stimulated mycelial growth and at the same time toxin production was greatly suppressed. Leaf extracts of non-host plant species were also stimulatory to phytotoxin biosynthesis.     

Inhibition of Rust Diseases of Cereals by Metabolic Products of Verticillium chlamydosporium

Verticillium chlamydosporium produced in submers culture several antifungal and/or phytotoxic compounds which were detected in a bioassay by using the pathogen-host system Puccinia coronata and oat seedlings. The antifungal compounds were also tested against P. recondita on wheat and P. sorghi on corn seedlings. The production of the active metabolic compounds highly depended on the nutrient solution (peptone-Czapek [PC] and malt extract [ME]) and on the fermentation times. Cell-free filtrates of PC-cultures of the fungus were highly phytotoxic; the fungitoxic and phytotoxic compounds were heat-labile and dialyzable. The ethyl acetate extracts of the PC-culture filtrates contained only the antifungal active substances. The antifungal compounds in ME-culture filtrates proved to be heat-stable, could be dialyzed and extracted with ethyl acetate. Ethyl acetate extracts of PC- and ME-culture filtrates at concentrations of 500 μg/ml reduced rust disease incidence by up to 80 % compared to the control treatment. Further studies with extracts of ME-culture filtrates displayed a distinct protective but no systemic activity. The extract interfered with the development of several infection structures of the rust fungi, mostly with the growth of germ tubes as well as with the formation of the aappressoria and haustorial mother cells. Three rust-active fractions were obtained by preparative layer chromatography on silica gel. One of these fractions exhibited phytotoxic activity. The most active antifungal fraction is identical with the macrolid antibiotic monorden which caused a desorientated spiral growth in P. coronata germlings on oat leaves.     

Fungal foliar diseases in Withania somnifera and its effect on secondary metabolites

Withania somnifera is a promising revitalizing medicinal herb. The plant is affected by foliar diseases in Lakkavalli forest region of Bhadra Wildlife Sanctuary. The symptomatology of foliar fungal disease incidence, severity and distribution in the study area was examined during 2006–2009. The seedborne nature and transmission of the causal pathogen and its management with seed dressing fungicides were studied. The results of the study indicated that Alternaria alternata caused severe leaf spot disease, while Myrothecium roridum and Fusarium oxysporum caused minor diseases. Based on the internal transcribed spacer (ITS1 and ITS2) regions of rDNA, the major pathogen was identified as A. alternata. The disease is homogeneously distributed in Lakkavalli forest region and high severity is recorded during November. Alternaria alternata and Fusarium oxysporum were the dominant seedborne pathogens that are transmitted to seedlings. Among the seed dressing fungicides used, Hyzeb was the most effective, followed by Captra, Antracol and Bavistin, in reducing the incidence of A. alternata and other seedborne fungi. The infected W. somnifera foliages had decreased steroids and alkaloids and increased phenolics and flavonoids. Analysis of alkaloids in diseased foliages by high performance thin layer chromatography indicated the occurrence of transformed compounds at R_f = 0.1, 0.77 (254 nm) and 0.2 (366 nm).     

Cell wall integrity and high osmolarity glycerol pathways are required for adaptation of Alternaria brassicicola to cell wall stress caused by brassicaceous indolic phytoalexins

Camalexin, the characteristic phytoalexin of Arabidopsis thaliana, inhibits growth of the fungal necrotroph Alternaria brassicicola. This plant metabolite probably exerts its antifungal toxicity by causing cell membrane damage. Here we observed that activation of a cellular response to this damage requires cell wall integrity (CWI) and the high osmolarity glycerol (HOG) pathways. Camalexin was found to activate both AbHog1 and AbSlt2 MAP kinases, and activation of the latter was abrogated in a AbHog1 deficient strain. Mutant strains lacking functional MAP kinases showed hypersensitivity to camalexin and brassinin, a structurally related phytoalexin produced by several cultivated Brassica species. Enhanced susceptibility to the membrane permeabilization activity of camalexin was observed for MAP kinase deficient mutants. These results suggest that the two signalling pathways have a pivotal role in regulating a cellular compensatory response to preserve cell integrity during exposure to camalexin. AbHog1 and AbSlt2 deficient mutants had reduced virulence on host plants that may, at least for the latter mutants, partially result from their inability to cope with defence metabolites such as indolic phytoalexins. This constitutes the first evidence that a phytoalexin activates fungal MAP kinases and that outputs of activated cascades contribute to protecting the fungus against antimicrobial plant metabolites.     

Investigations on fungicides. XX. The fungitoxicity of analogues of the phy toalexin 2-(2' -methoxy-4' -hydroxyphenyl)-6-methoxybenzofuran(vignafuran)

A series of hydroxy-and methoxy-2-phenyl benzofurans, structurally related to the phytoalexin vignafuran, have been synthesised and their antifungal activity assessed both as inhibitors of spore germination in vitro and as protective foliar sprays against three pathogens. All fungitoxic compounds have a hydroxy group in the molecule although most di- and tri-hydroxy compounds were inactive. Methoxy compounds were inactive but substituting methoxy groups into the molecule of monohydroxy compounds sometimes enhanced their fungitoxicity. No close correlation was established between antifungal activity and either Hansen n values or the n.m.r. chemical shift of the phenolic protons.     

<Emphasis Type="Italic">Streptomyces scabiei</Emphasis> and its toxin thaxtomin A induce scopoletin biosynthesis in tobacco and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Streptomyces scabiei is the predominant causal agent of common scab of potato in North America. The virulence of common scab-causing streptomycetes relies on their capacity to synthesize thaxtomins. In this study, the effects of S. scabiei infection and of thaxtomin A, the main toxin produced by S. scabiei, were tested for the elicitation of plant defense molecules in the model plants tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Tobacco leaves infected with spores of S. scabiei strain EF-35 or infiltrated with purified thaxtomin A produced a blue fluorescent compound that was not detected in leaves infiltrated with spores of a S. scabiei mutant deficient in thaxtomin A biosynthesis. Thin layer chromatography and high performance liquid chromatography identified this fluorescent compound as scopoletin, a plant defense phytoalexin. Arabidopsis seedlings grown in liquid medium also excreted scopoletin as a reaction to S. scabiei and thaxtomin A. The effects of the presence of scopoletin on S. scabiei were also investigated. The phytoalexin scopoletin caused a slight reduction of bacterial growth and a severe decrease of thaxtomin A production. Scopoletin was shown to inhibit thaxtomin A production by repression of a gene involved in the toxin biosynthesis.     

Assessment of aflatoxin and cyclopiazonic acid production by Aspergillus flavus isolates from Hungary

Thirty-two isolates of Aspergillus flavus were obtained from various sources in Hungary. All isolates were morphologically identified as A. flavus and three atypical variants were confirmed as A. flavus by comparing their DNA with an ex type culture of A. flavus. None of these isolates produced aflatoxins when tested on coconut agar or grown on rice medium and culture extracts examined by thin layer chromatography. Also, none of the isolates converted sterigmatocystin, O-methyl sterigmatocystin, norsolorinic acid, or sodium acetate to aflatoxin. However, 59% of the isolates produced cyclopiazonic acid based on thin layer chromatographic analysis of culture extracts. The isolates that lack the ability to produce both aflatoxin and cyclopiazonic acid are potential candidates for use in bicontrol studies.     

Jasmonoyl-l-isoleucine is required for the production of a flavonoid phytoalexin but not diterpenoid phytoalexins in ultraviolet-irradiated rice leaves

Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl₂-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves.     

Ultrastructural Changes in Brassica Leaves Caused by Alternaria brassicae and Destruxin B

Alternaria blight in Brassica spp (caused by Alternaria brassicae) is characterized by dark brown to blackish necrotic lesions surrounded by chlorotic areas on the leaves. Similar chlorotic lesions were mimicked by a chlorotic toxin (destruxin B) purified from the culture filtrate. Ultrastructural studies were performed to study and compare the changes caused by A. brassicae inoculation in vivo and its host selective toxin under in vitro conditions. Electron microscopy of healthy, chlorotic and necrotic portions of B. campestris leaves naturally infected with A. brassicae revealed considerable differences at ultrastructural level. The necrotic lesions showed plasmolysis with total disruption of cell organelles. The chlorotic lesions had normal plasma membrane but swollen mitochondria with reduced number of cristae and vesiculation of the envelope. Chloroplasts showed degeneration of granal fretwork with an increase in the number of plastoglobuli. Chlorotic lesions due to foliar application of destruxin B induced indentical canges in leaves.     

Production of a Chlorosis-Inducing, Host-Specific, Low-Molecular Weight Toxin by Isolates of Pyrenophora tritici-repentis Cause of Tan Spot of Wheat

An extraction procedure was developed to isolate toxin from cell-free culture filtrates of Pyrenophora tritici-repentis, cause of tan spot disease of wheat (Triticum aestivum). Designated Ptrtoxin, this low-molecular weight toxin (MW 800–1800) induces the characteristic chlororis associated with tan spot disease when inoculated to healthy wheat leaves and differentially inhibits the elongation of wheat seedling coleoptiles. The sensitivity of seven wheat, one barley, and non-host (corn) cultivars to Ptr-toxin corresponds with reported field reactions to the pathogen. Seven isolates of P. triticirepentis from wheat in Oklahoma and four from smooth bromegrass Bromus inermis, an alternative host of P. tritici-repentis), in North Dakota were evaluated for production of Ptr-toxin in vitro. Results indicate that Ptr-toxin may be involved in the expression of disease symptoms in tan spot of wheat, but is not the sole determinant of P. tritici-repentis pathogenicity. Instead, toxin production may contribute to the virulence of individual isolates of the pathogen. Isolates from the alternative host, smooth bromegrass, appear at least as toxigenic as those from wheat, with the exception of one which appeared more toxigenic than four of the wheat isolates tested. Possible relationships of Ptr-toxin to high-molecular weight toxins reported to be produced by P. triticirepentis in vitro (Ptr-necrosis-toxin) are not understood.