Complexes of potentially pathogenic microscopic fungi in anthropogenic polluted soils.

This study was undertaken to investigate the species' diversity and structure of potentially pathogenic microscopic fungal complexes in podzolic soils polluted by fluorine, heavy metals (Cu, Ni, Co), oil products (diesel fuel, gas condensate, mazut). Lists of potentially pathogenic fungi isolated from soils are made specifically for north-western part of Russia (Kola Peninsula). The majority of studied fungus species belong to the following genera: Penicillium, Aspergillus, Mucor, Lecanicillium and Phoma. Penicillium miczynskii was identified as the most stable type of fungus with respect to all studied types of oil products. Mucor hiemalis was identified as the most sensitive type. An increase of 15% portion of potentially pathogenic fungi as compared to the background soil in zones of aluminum and copper-nickel plants was revealed. The results indicate an increase of 20-25% of potentially pathogenic fungi in pollution of soil with oil products. The structure of fungal complexes was observed to have changed in the polluted soils and the species number and frequency of occurrence of potentially pathogenic fungi were also increased.

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

In vitro antifungal activity of the tea tree (Melaleuca alternifolia) essential oil and its major components against plant pathogens.

AIMS: The aim of this study was to examine the effect of Melaleuca alternifolia essential oil (TTO) and its principal components on four cereal-pathogenic fungi. METHODS AND RESULTS: The antimycotic properties of TTO and of terpinen-4-ol, gamma-terpinen and 1,8-cineole (eucalyptol) were evaluated in vitro on Fusarium graminearum, Fusarium culmorum and Pyrenophora graminea. Moreover, barley leaves infected with Blumeria graminis were treated with whole TTO. All the tested fungi were susceptible to TTO and its components. CONCLUSIONS: TTO exerted a wide spectrum of antimycotic activity. Single TTO purified components were more active than the whole oil in reducing in vitro growth of fungal mycelium and, among the tested compounds, terpinen-4-ol was the most effective. SIGNIFICANCE AND IMPACT OF THE STUDY: TTO and its components can be considered potential alternative natural fungicides.

Exploiting the genetic diversity of Beauveria bassiana for improving the biological control of the coffee berry borer through the use of strain mixtures.

Beauveria bassiana is an entomopathogen widely used to control the coffee berry borer in Colombia, as part of an Integrated Pest Management strategy. Traditionally, the development of fungal insect pathogens as biocontrol agents in crop pests has been oriented towards the selection and formulation of elite clonal strains. Instead, we explored the potential application of genetic diversity in B. bassiana by determining the effect of strain mixtures on coffee berry borer mortality compared to clonal isolates. Genomic DNA from 11 strains was characterized using internal transcribed spacers and beta-tubulin sequences as well as amplified fragment length polymorphism markers. Cluster analysis produced three genetic groups and confirmed the low but significant intraspecific genetic diversity present among the strains. Single strain virulence towards the coffee berry borer under laboratory conditions, using 1x10(6) conidia ml(-1), ranged between 89.9 and 57.5%. All the inoculations with mixtures resulted in coinfection events. Combinations of genetically similar strains showed no significant differences when their virulences were compared. However, mixtures of genetically different strains led to both antagonism and synergism. The lowest virulence percentage (57%) was obtained by putting together the most virulent strain of each group, contrary to the highest virulence percentage (93%) that resulted from mixing the three least virulent strains. The results indicate the promising potential of designing strain mixtures as an alternative for the biocontrol of Hypothenemus hampei and other pests and provide tools for the understanding of the ecological dynamics of entomopathogen populations under natural conditions.

Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi.

The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco (Nicotiana tabacum var. Petit Havana) plants. Polymerase chain reaction products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Contrary to previous predictions, accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. This may be due to differences in the lipid composition of plastid membranes compared with the membranes of susceptible target microbes. In vitro assays with protein extracts from T(1) and T(2) plants confirmed that MSI-99 was expressed at high levels to provide 88% (T(1)) and 96% (T(2)) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. When germinated in the absence of spectinomycin selection, leaf extracts from T(2) generation plants showed 96% inhibition of growth against P. syringae pv tabaci. In addition, leaf extracts from transgenic plants (T(1)) inhibited the growth of pregerminated spores of three fungal species, Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae, by more than 95% compared with non-transformed control plant extracts. In planta assays with the bacterial pathogen P. syringae pv tabaci resulted in areas of necrosis around the point of inoculation in control leaves, whereas transformed leaves showed no signs of necrosis, demonstrating high-dose release of the peptide at the site of infection by chloroplast lysis. In planta assays with the fungal pathogen, Colletotrichum destructivum, showed necrotic anthracnose lesions in non-transformed control leaves, whereas transformed leaves showed no lesions. Genetically engineering crop plants for disease resistance via the chloroplast genome instead of the nuclear genome is desirable to achieve high levels of expression and to prevent pollen-mediated escape of transgenes.

Characterization of SNP1, a cell wall-degrading trypsin, produced during infection by Stagonospora nodorum.

Stagonospora (= Septoria) nodorum when grown in liquid culture with wheat cell walls as the sole carbon and nitrogen source secretes numerous extracellular depolymerases, including a rapidly produced, alkaline, trypsin-like protease (SNP1). The enzyme was purified 417-fold by cation exchange chromatography and has a molecular mass of 25 kDa on sodium dodecyl sulfate gels, pI 8.7, and pH optimum of 8.5. It cleaved peptide bonds on the carboxyl side of lysine or arginine, was strongly inhibited by the trypsin inhibitors aprotinin and leupeptin and weakly by phenylmethylsulfonyl fluoride, and its activity was stimulated by calcium. SNP1 has the characteristic, conserved, fungal, trypsin N terminus. Polymerase chain reaction (PCR) primers based on this sequence and the conserved trypsin active site were used to amplify a DNA fragment that facilitated isolation of the corresponding genomic clone from a lambda library of S. nodorum. The full-length sequence confirmed its identity as a trypsin-like protease containing the N-terminal sequence of the previously purified enzyme. Infected leaf tissue contained a protease, not present in controls, that coeluted with the fungal trypsin from cation exchange, and had properties (pI and inhibitor characteristics) similar to those of the fungal trypsin. SNP1 expression in planta was detected by Northern (RNA) blotting, reverse transcription PCR, and green fluorescent protein confocal microscopy. SNP1 released hydroxyproline from wheat cell walls. The release of hydroxyproline, together with its early expression in planta, suggests that SNP1 participates in the degradation of host cell walls during infection.

Avirulent mutants of Macrophomina phaseolina and Aspergillus fumigatus initiate infection in Phaseolus mungo in the presence of phaseolinone; levamisole gives protection.

To evaluate the role of phaseolinone, a phytotoxin produced by Macrophomina phaseolina, in disease initiation, three nontoxigenic avirulent mutants of the fungus were generated by UV-mutagenesis. Two of them were able to initiate infection in germinating Phaseolus mungo seeds only in the presence of phaseolinone. The minimum dose of phaseoli-none required for infection in 30% seedlings was 2 5 mg/ml. A human pathogen, Aspergillus fumigatus was also able to infect germinating seeds of P. mungo in the presence of 5 mg/ml concentration of phaseolinone. Phaseolinone seemed to facilitate infection by A. fumigatus, which is not normally phytopathogenic, by reducing the immunity of germinating seedlings in a nonspecific way. Levamisole, a non-specific immunopotentiator gave protection against infection induced by A. fumigatus at an optimum dose of 50 mg/ml. Sodium malonate prevented the effects of levamisole.

clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean.

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.

Characterization and cloning of cutinase from Ascochyta rabiei.

Ascochyta rabiei, the causal agent of Ascochyta blight on chickpea plants, secretes a cutinase in the culture filtrate when it is induced by cutin or hydroxylated fatty acids. This cutinase is the main esterase in the culture fluids. The enzyme was purified to homogeneity by three successive chromatographic steps. It showed an apparent molecular weight of 22 kD in SDS-PAGE and cleaved ester bonds of 3H-labelled cutin or p-nitrophenylbutyrate with maximal activities around pH 8. As a serine esterase, cutinase is strongly inhibited by organophosphorous compounds and the most effective inhibitor 2,3,5-trichloropyridine-6-(O-methyl-O-n-butyl)-phosphateester++ + (MAT 9564) shows a Ki value of 0.8 nM. The cutinase gene was cloned from a genomic cosmid library by screening with two oligonucleotides directed against cutinase consensus peptides. The gene was subcloned to a 1.7 Kb SaII/HindIII-insert and sequenced. The cutinase gene codes for a 223 amino acid protein with strong homology to other fungal cutinase sequences. The purified cutinase is encoded by a single copy gene.

Generation of superoxide anion and induction of superoxide dismutase and peroxidase in bean leaves infected with pathogenic fungi.

Generation of superoxide anion (O2-.) and peroxidase activity were significantly increased in bean leaves infected with incompatible and compatible pathogens: Botrytis fabae and Botrytis cinerea, respectively, but the induction was greater on direct inoculation with B. fabae, than with B. cinerea. A slightly higher O2-. level was also detected in the parts of leaves surrounding the inoculation side. Overproduction of O2-. was observed earlier than the increase in peroxidase activity. Pretreatment of the leaves with methyl jasmonate enhanced both O2-. production and peroxidase activity following inoculation with B. cinerea. Induction of superoxide dismutase activity after the infection was less pronounced than changes in O2-. level. The differences in the rate of NADH oxidation in the extracts from control and inoculated leaves, correlated with the differences in the rate of O2-. production. The results indicate that O2-. level is one of the essential factors responsible for the difference in the interactions between bean plant and compatible and incompatible pathogens.

Lupinus albus L. pathogenesis-related proteins that show similarity to PR-10 proteins.

We describe a group of three acidic proteins, pathogenesis-related (PR)-p16.5a, PR-p16.5b, and PR-p16.5c, that accumulate in the leaves of Lupinus albus L. cv Rio Maior plants when infected with the fungus Colletotrichum gloeosporioides Penz. These proteins co-migrate in sodium dodecyl sulfate-polyacrylamide gels as a single band of 16.5 kD, behaving as charge isomers, and are related to several members of the defense-related PR-10 protein family. Localization of the proteins was investigated by techniques of tissue printing and immunogold electron microscopy; they are predominantly associated with the vascular system and are localized extracellularly. The accumulation of PR-p16.5a, PR-p16.5b, and PR-p16.5c also seems to be induced by cucumber mosaic virus and by two forms of abiotic stress, salicylic acid and ultraviolet, suggesting a general defense role for these proteins.

Elicitors and suppressors of hydroxyproline-rich glycoprotein accumulation are solubilized from plant cell walls by endopolygalacturonase.

Treatment of bean cell walls with a pure endopolygalacturonase of the bean pathogen Colletotrichum lindemuthianum race beta released oligogalacturonides and pectic fragments which were separated according to their charge and size. Among galacturonic-acid-containing components, elicitors and suppressors of the plant cell wall hydroxyproline-rich glycoprotein (HRGP) were recovered. Two active small oligogalacturonides with degrees of polymerization of 2 and 3 were characterized by high-performance anion-exchange-chromatography pulsed amperometric detection and fast-atom-bombardment mass spectrometry; they elicited 40-70% hydroxyproline increase within 48 hours at 450 nmol/bean cutting. In contrast, pectic fragments of higher molecular mass, predominantly composed of galacturonic acid and containing sugars typical of the rhamnogalacturonan II domain of pectic polysaccharides, had the ability to substantially suppress hydroxyproline deposition. Maximum suppressor activity, 30-40% below the activity of the control, occurred in 48 hours. In view of the low one-cycle turnover of these proteins in the cell wall and of their structural role, these changes might significantly affect cell wall properties. Elicitation and/or suppression of hydroxyproline were correlated to modifications of HRGP-extensin gene expression. Northern-blot analysis of RNA showed that changes in the transcript intensity became clearly visible within the first 12 hours after the start of either treatment. The results show that pectic components of the plant extracellular matrix have the potential to regulate wall matrix biogenesis. Implications of this finding in plant defense and development are discussed.

Mutagenic and membranal effect of a phytotoxic molecule isolated from olive leaves parasitized by the fungus Cycloconium oleaginum Cast.

A phytotoxic substance (C23H44O3) which is named 'Substance A', was purified from olive leaves infected with Cycloconium oleaginum Cast. The mutagenic effect of this substance was detected using TA 100 and TA 102 strains of Salmonella in the Ames test using Bacillus subtilis strains M45 rec-, H17 rec+ in the rec assay. Another substance manifesting the mutagenic effect was found in the extract from the Cycloconium oleaginum culture. This substance was not detected in the extract from contaminated olive leaves. Substance A increased electrolytes leakage from tissue of olive leaves, thus manifesting its phytotoxicity.

Accumulation of antifungal compounds in tea leaf tissue infected with Bipolaris carbonum.

Varietal resistance of tea towards Bipolaris carbonum was tested following detached leaf inoculation technique. Among the fourteen varieties tested, three were found to be highly susceptible, while other three were resistant. Leaf exudates and diffusates collected from the resistant varieties were more fungitoxic than those from the susceptible ones. Two antifungal compounds isolated from healthy and B. carbonum-infected tea leaves exhibited clear inhibition zones at RF 0.8 and 0.65, respectively, in a chromatographic bioassay. On the basis of their color reaction on TLC and UV-spectra these were identified to be catechin and pyrocatechol. Resistant and susceptible varieties accumulated 439-510 and 187-212 micrograms/g fresh mass tissue of pyrocatechol, respectively, 2 d after inoculation with B. carbonum, while a low concentration (45-58 micrograms/g) of this compound was detected in healthy leaf tissue.

The function of vacuolar beta-1,3-glucanase investigated by antisense transformation. Susceptibility of transgenic Nicotiana sylvestris plants to Cercospora nicotianae infection.

Vacuolar class I beta-1,3-glucanases (EC 3.2.1.39) are believed to be important in the induced defense reaction of plants to fungal infection. We used antisense transformation to test this hypothesis and to identify other possible physiological functions of this enzyme. Nicotiana sylvestris plants were transformed with antisense constructions containing the region from position 27 to 608 of the coding sequence of the basic, vacuolar beta-1,3-glucanase gene GLA of tobacco regulated by cauliflower mosaic virus 35S RNA expression signals. Plants homozygous for this transgene showed a marked, ca. 20-fold reduction in the constitutive expression of class I beta-1,3-glucanase antigen in their leaves. RNA blot analysis indicated that the antisense plants expressed low levels of the sense transcript of the host beta-1,3-glucanase gene and the antisense transcript of the transgene. Immune blot analysis of plant extracts indicated that only expression of the N. sylvestris homologue of class I tobacco beta-1,3-glucanase and not the acidic, class II isoforms of the enzyme was blocked in the antisense plants. Class I isoforms of beta-1,3-glucanase and chitinase were coordinately induced in leaves of untransformed and empty-vector-transformed N. sylvestris plants treated with ethylene or infected with the fungal leaf pathogen Cercospora nicotianae. In antisense plants, chitinase but not beta-1,3-glucanase was induced under these conditions indicating that antisense transformation effectively blocks constitutive as well as induced expression of class I beta-1,3-glucanase. Under greenhouse conditions, antisense plants developed normally and were fertile. The plants did not exhibit increased susceptibility to C. nicotianae infection. These results suggest that expression of the beta-1,3-glucanase isoform blocked by antisense transformation is not necessary for 'housekeeping' functions of N. sylvestris nor defense against the fungal pathogen tested.

Purification and characterization of two basic beta-1,3-glucanases induced in Colletotrichum lindemuthianum-infected bean seedlings.

Two beta-1,3-glucanases which are rapidly induced in the incompatible interaction between bean (cv. Processor) and Colletotrichum lindemuthianum race beta were purified to homogeneity. Characterization of the two enzymes, GE1 and GE2, showed that they both had a basic isolectric point and a similar molecular weight (36,500 for GE1 and 36,000 for GE2), but differed in their pH optimum, thermal stability, and specific activity. GE2 was present in higher amounts but was shown to be less active than GE1 against laminarin and fungal cell walls isolated from race beta of the fungus. Both enzymes were specific for beta-1,3 linkages and showed a strict endolytic mode of action. Further characterization of GE2 was achieved by amino acid sequence analysis of tryptic peptides; the degree of homology shared with other basic beta-1,3-glucanases depended on the plant source. A time-course study showed that GE1 and GE2 were increased during infection. They were also induced by fungal elicitors, thereby indicating that they originate from the host.

Fatal encephalitis caused by Dactylaria constricta var. gallopava in a snowy owl chick (Nyctea scandiaca).

Dactylaria constricta var. gallopava (Cooke) Salkin et Dixon was found to cause fatal encephalitis in a 28-day-old, captivity-bred snowy owl chick (Nyctea scandiaca). The previously healthy bird suddenly developed ataxia, severe torticollis, and extensor rigidity of the legs. Since the animal did not improve with antibiotic or vitamin-mineral supplement therapy, the chick was euthanized 5 days after the onset of neurologic signs. At necropsy, all tissues except the brain were grossly normal. Cultures inoculated with blood from the brain and heart yielded a dematiaceous mould that subsequently proved to be D. constricta var. gallopava. This is the first report of natural central nervous system infection caused by D. constricta var. gallopava in a snowy owl.

Incidencia de Alternaria crassa (Sacc) Rands en 3 especies herbáceas del género Datura L. (Comunicación) / Incidences of Alternaria crassa Runds on three herbaceous of genus Datura L. (Counication)

Se realizó un estudio de la incidencia del hongo Alternaria crassa (Sacc.) Rands en 3 especies herbáceas del género Datura L.; D. inoxia Mill., D. stramonium L. var stramonium y D. velutinosa Fuentes, durante los meses de junio, julio y agosto de 1978 en la Estación Experimental de Plantas Medicinales "Dr.Juan T. Roig", San Antonio de los Baños, La Habana. Se observaron diferencias significativas entre el valor de la enfermedad en las especies y entre las fechas, e interacción entre ambos, se encontró que en D. inoxia y D. stramonium, la enfermedad alcanzó su valor máximo (100), mientras que D. velutinosa resultó menos susceptible

Incidencia de Alternaria crassa (Sacc) Rands en 3 especies herbáceas del género Datura L. (Comunicación) / Incidences of Alternaria crassa Runds on three herbaceous of genus Datura L. (Comunication)

Se realizó un estudio de la incidencia del hongo Alternaria crassa (Sacc.) Rands en 3 especies herbáceas del género Datura L.; D. inoxia Mill., D. stramonium L. var stramonium y D. velutinosa Fuentes, durante los meses de junio, julio y agosto de 1978 en la Estación Experimental de Plantas Medicinales "Dr.Juan T. Roig", San Antonio de los Baños, La Habana. Se observaron diferencias significativas entre el valor de la enfermedad en las especies y entre las fechas, e interacción entre ambos, se encontró que en D. inoxia y D. stramonium, la enfermedad alcanzó su valor máximo (100), mientras que D. velutinosa resultó menos susceptible

Photosensitivity in South Africa. II. The experimental production of the ovine hepatogenous photosensitivity disease geeldikkop (Tribulosis ovis) by the simultaneous ingestion of Tribulus terrestris plants and cultures of Pithomyces chartarum containing the mycotoxin sporidesmin.

The mycoflora of toxic pastures were surveyed during a number of outbreaks of ovine hepatogenous photosensitivity in South Africa. Pure cultures of several isolates were dosed to sheep, but only those of Pithomyces chartarum and Myrothecium verrucaria proved to be toxic. Photosensitization was induced in sheep by dosing them with cultures of a P. chartarum isolate (GA10) obtained from Tribulus terrestris plants collected during an outbreak of geeldikkop in the Karoo. Thus for the first time a mechanism whereby T. terrestris plants can contribute to the causation of ovine hepatogenous photosensitivity was demonstrated. When cultures of GA10 equivalent to approximately 0,75--4,0 mg/kg sporidesmin were dosed at Onderstepoort Veterinary Research Institute to Highveld and Karoo sheep on a diet of lucerne, facial eczema was produced. Dosing the same cultures at levels equivalent to c. 1,0 mg/kg of sporidesmin in the Karoo resulted in lesions characteristic of both facial eczema and geeldikkop. Typical hepatic lesions of geeldikkop could be elicited by dosing GA10 at levels equivalent to c. 0,25--0,7 mg/kg of sporidesmin to Karoo sheep grazing on predominantly T. terrestris pastures in the Karoo. In the latter experiment geeldikkop was induced in the sheep on T. terrestris pastures, while those receiving identical doses on veld with little T. terrestris developed facial eczema. Geeldikkop, therefore, can be brought about by the ingestion of T. terrestris plants together with toxic cultures of P. chartarum. The plant appears not only to act as a vehicle for ingestion of spores, but also to interact with sporidesmin to induce lesions typical of geeldikkop, whereas sporidesmin alone results in facial eczema. Indications are that it can enhance the ability of sporidesmin to cause photosensitivity or, possibly, vice versa. The histopathological findings of these experiments are described in detail.