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.

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.

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.

Cloning, expression, and substrate specificity of MeCPA, a zinc carboxypeptidase that is secreted into infected tissues by the fungal entomopathogen Metarhizium anisopliae.

To date zinc carboxypeptidases have only been found in animals and actinomycete bacteria. A cDNA clone (MeCPA) for a novel fungal (Metarhizium anisopliae) carboxypeptidase (MeCPA) was obtained by using reverse transcription differential display polymerase chain reaction to identify pathogenicity genes. MeCPA resembles pancreatic carboxypeptidases in being synthesized as a precursor species (418 amino acids) containing a large amino-terminal fragment (99 amino acids). The mature (secreted) form of MeCPA shows closest amino acid identity to human carboxypeptidases A1 (35%) and A2 (37%). MeCPA was expressed in an insect cell line yielding an enzyme with dual A1 + A2 specificity for branched aliphatic and aromatic COOH-terminal amino acids. However, in contrast to the very broad spectrum A + B-type bacterial enzymes, MeCPA lacks B-type activity against charged amino acids. This is predictable as key catalytic residues determining the specificity of MeCPA are conserved with those of mammalian A-type carboxypeptidases. Thus, in evolutionary terms the fungal enzyme is an intermediate between the divergence of A and B forms and the differentiation of the A form into A1 and A2 isoforms. Ultrastructural immunocytochemistry of infected host (Manduca sexta) cuticle demonstrated that MeCPA participates with the concurrently produced endoproteases in procuring nutrients; an equivalent function to digestive pancreatic enzymes.

Endopolygalacturonase genes from Colletotrichum lindemuthianum: cloning of CLPG2 and comparison of its expression to that of CLPG1 during saprophytic and parasitic growth of the fungus.

Following the previous isolation of CLPG1, a gene encoding an endopolygalacturonase (endoPG) secreted into the culture filtrate of Colletotrichum lindemuthianum, we have isolated and sequenced an additional endoPG gene, CLPG2. This gene is present as a single copy in the genome of the fungus. At the amino acid level, CLPG2 shows 61% identity to CLPG1 and between 37 to 59% identity to other fungal endoPGs. RNA blot analyses of endoPG gene expression were followed with specific probes during in vitro culture of the fungus. When conidia were used to inoculate a synthetic medium containing pectin as sole carbon source, only CLPG1 was found to be expressed after 3 days of culture. However, transferring the mycelium grown on glucose for 4 days to a pectin-containing medium allowed the detection of CLPG1 and CLPG2 transcripts as early as 12 h after transfer on this substrate. Expression of CLPG2 was transient while that of CLPG1 was more prolonged. Immunocytological localization of endoPG in C. lindemuthianum-infected bean tissues with antibodies against CLPG1 confirmed that the protein is produced in planta and is associated with extensive degradation of the host cell wall. Detection of endoPG transcripts by reverse transcription-polymerase chain reaction revealed that CLPG1, but not CLPG2, is expressed at the beginning of the necrotrophic stage of infection. These results show that the two endoPG genes are differentially expressed and that CLPG1 encodes the major secreted endoPG both during saprophytic growth and during plant infection.

Analysis of swainsonine and its early metabolic precursors in cultures of Metarhizium anisopliae.

The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 microg ml(-1). Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition.

Cloning and characterisation of glutamine synthetase from Colletotrichum gloeosporioides and demonstration of elevated expression during pathogenesis on Stylosanthes guianensis.

Experiments were designed to clone and identify genes of the fungal phytopathogen Colletotrichum gloeosporioides expressed at high levels during growth on the compatible host Stylosanthes guianensis when compared with expression in axenic culture. A cDNA clone (pCgGS) that hybridised preferentially to a cDNA probe prepared from infected leaves was isolated by the differential screening of a cDNA library from a nitrogen-starved axenic culture of C. gloeosporioides. The DNA sequence of pCgGS is highly homologous to genes for glutamine synthetase (GS) in other organisms. pCgGS contained all of the conserved regions assigned as catalytic domains in GS enzymes. Comparison with genomic sequences indicated that in C. gloeosporioides the GS gene is present as a single copy with three introns. To our knowledge this is the first report of the cloning of a GS from a filamentous fungus. A second clone (pCgRL1) was also isolated and represented a partial cDNA of the 25s rRNA of C. gloeosporioides. Because pCgRL1 did not hybridise to plant rRNA under high-stringency hybridisation conditions, it was used as a reference to quantify the expression of fungal GS mRNA during pathogenesis in S. guianensis compared to fungal growth in axenic culture. The results indicated that elevated expression of GS occurred during pathogenesis of C. gloeosporioides on S. guianensis, particularly at early stages of infection where expression was about six-times higher than during growth in rich culture media. This work also demonstrates that fungal-specific 25s rRNA fragments, such as pCgRL1, have considerable utility as a reference for quantifying pathogen gene expression in infected plants.

Cloning and heterologous expression of a second (+)-delta-cadinene synthase from Gossypium arboreum.

Screening of a Gossypium arboreum L. cv. Nanking cDNA library resulted in the identification and cloning of a second (+)-delta-cadinene synthase. A probe for the screens was prepared by PCR using primers based on conserved sequences in farnesyl diphosphate cyclases and genomic DNA as a template. This second cDNA clone encodes a protein that is 80% identical to the recently described (+)-delta-cadinene synthases CAD1-C1 and C14 from G. arboreum and maintains a significant degree of homology to the other known mono-, sesqui-, and diterpene synthases. As in the case of CAD1-C1 (+)-delta-cadinene synthase from cultured cotton cells, the synthesis of the second CAD1-A mRNA was induced by treatment of cotton cell suspension cultures with a partially purified elicitor preparation from the phytopathogenic fungus Verticillium dahliae. Expression of CAD1-A mRNA was quantitated with reverse transcription PCR and showed that CAD1-A mRNA was maximally increased 8-fold, 6 h after addition of elicitor. Heterologous expression of this second cDNA produced a 64 kD protein that catalyzed the cyclization of farnesyl diphosphate to (+)-delta-cadinene, the identical product produced by CAD1-C1. The steady-state kinetic parameters of CAD1-A were similar to CAD1-C, showing a Km of 7 mM farnesyl diphosphate and kcat of 0.039 s-1 at 30 degrees C. However, the optimal pH and Mg2+ concentration for CAD1-A activity were significantly higher than those observed for CAD1-C.

Characterization of a human and murine gene (CLCN3) sharing similarities to voltage-gated chloride channels and to a yeast integral membrane protein.

We describe the isolation and characterization of a human gene (CLCN3) and its murine homologue (Clcn3) sharing significant sequence and structural similarities with all previously identified members of the voltage-gated chloride channel (ClC) family. This gene is expressed primarily in tissues derived from neuroectoderm. Within the brain, Clcn3 expression is particularly evident in the hippocampus, olfactory cortex, and olfactory bulb. CLCN3 encodes a 760-amino-acid protein that differs by only 2 amino acid residues from the protein encoded by Clcn3. CLCN3 protein also shows a high similarity with GEF1, an integral membrane protein of the yeast Saccharomyces cerevisiae known to be involved in respiration and iron-limited cell growth, and with the predicted protein product of a DNA sequence from the mold Septoria nodorum. This high degree of sequence conservation in very distantly related species such as human and yeast indicates that this gene has retained a fundamental function throughout evolution.

Isolation, characterization, and expression of a second beta-tubulin-encoding gene from Colletotrichum gloeosporioides f. sp. aeschynomene.

Colletotrichum gloeosporioides f. sp. aeschynomene is a fungal plant pathogen of Aeschynomene virginica. A beta-tubulin-encoding gene (TUB2) from this pathogen was cloned and sequenced. The deduced amino acid sequence of TUB2 had a high degree of homology to other fungal beta-tubulins. A portion of TUB2 from a benomyl-resistant C. gloeosporioides f. sp. aeschynomene mutant was also cloned and sequenced. A point mutation resulting in a glutamic acid-to-lysine substitution at amino acid 198 likely confers benomyl resistance. The mutation is relevant for use as a selectable marker in developing a gene transfer system in C. gloeosporioides f. sp. aeschynomene. Northern (RNA) hybridizations with C. gloeosporioides f. sp. aeschynomene TUB2 and another C. gloeosporioides f. sp. aeschynomene beta-tubulin-encoding gene (TUB1) as probes showed differential expression of these genes in different cell types.

Oligonucleotide fingerprinting detects genetic diversity among Ascochyta rabiei isolates from a single chickpea field in Tunisia.

Fifty isolates of Ascochyta rabiei (Pass.) Labr. were hierarchically sampled from four well-separated locations of a single chickpea field in Beja (Tunisia), and single-spored. DNA was isolated from in-vitro-grown mycelia, digested with HinfI or RsaI, and hybridized to a set of synthetic oligonucleotides complementary to simple repetitive sequences. According to the fingerprint patterns derived from the probes (CA)8, (CAA)5, (CAT)5 and (GATA)4, 12 different fungal haplotypes were found at various frequencies within the investigated field. Seven haplotypes were confined to one location only, four occurred at two, one at three, and none at all four locations. Most of the genetic variability originated from diversity within, rather than between, locations. In some cases, more than one haplotype was isolated from the same lesion of a single host plant. Genetic distances between isolates, as calculated from band-sharing data, varied between 0.05 and 0.22. Relatedness between the different haplotypes was evaluated by cluster analysis using UPGMA.

DNA addition or deletion is associated with a major karyotype polymorphism in the fungal phytopathogen Colletotrichum gloeosporioides.

A 1.2 Mb minichromosome resolved by pulsed-field electrophoresis was present in two independent race 3 isolates of Colletotrichum gloeosporioides causing Type B anthracnose specifically on Stylosanthes guianensis cv. Graham in Australia. This chromosome was absent in duplicate isolates representing races 1, 2 and 4 which infect other S. guianensis cultivars. A gene library was prepared specifically from the 1.2 Mb minichromosome and ten independent DNA clones unique to this chromosome were identified by differential hybridisation to whole chromosome probes. All of the ten selected probes hybridised only to the 1.2 Mb minichromosome unique to the race 3 isolates but not to any chromosome in isolates of the other races. These ten probes also hybridised only to restriction-digested DNA of race 3 and were thus both chromosome- and strain-specific for Type B C. gloeosporioides. Hybridisation analysis of NotI fragments of the 1.2 Mb minichromosome with these sequences indicated that they were not tightly clustered on the chromosome. These data demonstrate that the variation in the occurrence of the 1.2 Mb minichromosome did not arise by rearrangement of the genome of a progenitor strain but involved either large scale deletion or addition of DNA. The 1.2 Mb minichromosome did not contain a cloned high-copy-number repeat sequence present on all other mini- and maxichromosomes, suggesting addition from a genetically distinct strain. All ten chromosome-specific DNA probes hybridised to a 2.0 Mb chromosome in all races of C. gloeosporioides causing Type A anthracnose on Stylosanthes spp. including S. guianensis.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Selenocysteyl-tRNAs recognize UGA in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus.

Selenocysteyl-tRNAs that decode UGA were previously identified in representatives of three of the five life kingdoms which were the monera, animal and protist kingdoms. In the present study, we show that these tRNAs also occur in representatives of the two remaining kingdoms, plants and fungi; i.e., selenocysteyl-tRNAs which code for UGA occur in Beta vulgaris, a higher plant, and in Gliocladium virens, a filamentous fungus. The fact that selenocysteyl-tRNAs are present in all five life kingdoms strongly suggests that UGA, in addition to dictating the cessation of protein synthesis, also codes for selenocysteine in the universal genetic code.

Oligonucleotide fingerprinting of plant and fungal genomes: a comparison of radioactive, colorigenic and chemiluminescent detection methods.

Digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences were introduced into nonradioactive fingerprint analysis of plant and fungal DNA. The fragment patterns, obtained by blot hybridization of TaqI-restricted DNA from chickpea (Cicer arietinum) and its fungal pathogen Ascochyta rabiei with digoxigenated probes and either a colorigenic or a chemiluminescent detection method, were compared to those obtained with 32P-labeled probes. In combination with alkaline phosphatase and its chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) digoxigenated oligonucleotides yielded clear-cut fingerprints with high signal-to-background ratios within several minutes of exposure to X-ray films. The chemiluminescence reaction remained stable for at least two weeks. A comparison of banding patterns obtained by radioactive versus digoxigenin-based hybridization and detection techniques revealed substantial differences in the relative signal intensities of bands. Both nonradioactive techniques show a tendency to "equalize" band intensity differences. Whereas 32P-labeled oligonucleotides are also applicable to in situ hybridization with DNA immobilized in dried agarose gels, gel hybridization did not work efficiently with digoxigenated probes and either substrate.