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A straightforward and easy-to-apply semi-quantitative RT-PCR method was developed to study multigenic expression in the phytopathogenic fungus Botrytis cinerea. This procedure is based on the one-step reverse transcription-amplification of a specific transcript within total RNA and product amount determination by densitometric analysis of ethidium bromide fluorescence upon gel electrophoresis. The semi-quantitative analysis is achieved, at a fixed PCR cycle-number, within a range of total RNA concentrations that stays in the exponential phase of the PCR. Co-amplification of the transcript of interest with internal controls allowed comparison between different RNA samples. Using this method, we could demonstrate a differential regulation of chitin synthase genes during fungal growth and an effect of the culture carbon source on the expression of two pectin methylesterase genes in B. cinerea. Finally, the method was shown to be applicable to plant-infected tissue, making it a useful tool to detect pathogenicity genes in B. cinerea.

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.

Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B. cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B. cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.

A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain.

Random amplified polymorphic DNA (RAPD) assays were applied on 34 fungal strains isolated from strawberry and other host plants, in order to detect polymorphism to consequently identify and isolate molecular markers specific to Botrytis cinerea. Among the 26 10-mer primers tested, one primer mainly amplified a 750-bp product present in all the B. cinerea strains and absent in the other species and genera examined. This product was cloned and sequenced in order to design a specific 20-mer primer pair, which was tested on the 34 fungal isolates by polymerase chain reaction (PCR). A single 0.7-kb band was amplified in all the 13 strains of B. cinerea isolated from different host plants. Moreover, a 0.6-kb band was amplified in both Botrytis fabae strains. No band was observed for the nine other Botrytis species and 12 fungal genera isolated from strawberry plants. A comparison between the 0.7-kb B. cinerea sequence and the 0.6-kb B. fabae sequence revealed 98% homology and one 122-bp deletion for B. fabae, including an EcoRI site. Hybridization of Southern blots with RAPD and EcoRI-digested DNA confirmed the specificity of the marker. The limit of detection of B. cinerea genomic DNA was approximately 0.2 pg. The PCR procedure was able to amplify the 0.7-kb B. cinerea fragment form mixed samples of DNA as low as 2 pg B. cinerea genomic DNA and 1 microg plant DNA. Thus this PCR-based detection procedure is a powerful tool for diagnosis of B. cinerea in symptomless strawberry plants, and should allow infection and latency sites to be localized in order to improve knowledge of the epidemiology of the pathogen under field conditions.

We have cloned a novel ABC transporter gene PMR5 from the phytopathogenic fungus Penicillium digitatum by RT-PCR using degenerate primers. The deduced amino acid sequence of PMR5 showed 37% identity to PMR1 from the same fungus, 71% identity to AtrB from Aspergillus nidulans, and 65% identity to BcatrB from Botrytis cinerea. Disruption mutants for PMR5 were generated in two independent P. digitatum strains and their phenotypes were characterized. These mutants displayed increased sensitivity to thiabendazole (a benzimidazole), benomyl (a benzimidazole), dithianon (a quinone), resveratrol (the phytoalexin of grape), and camptothecin (an alkaloid). Delta pmr1 disruption mutants were previously reported to show resistance to demethylation inhibitors (DMIs). These mutants were found also to display increased sensitivity to phloretin (the phytoanticipin of apples), camptothecin and oligomycin (an antibiotic). Transcription of PMR1 and PMR5 was strongly induced in response to several toxicants, including DMIs that specifically induced PMR1. In contrast, dithianon and resveratrol specifically induced PMR5 transcription. These findings indicate that expression of the two ABC transporter genes is regulated differently, and that they have complementary roles in multidrug resistance, with each having different substrate-specificities.

The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol-producing host plant, but Bclcc2 replacement mutants were as virulent as the wild-type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant-pathogen interactions and the phytoalexin concept.

A 3.48-kb DNA region containing the gdhA gene, which codifies the NADP-dependent glutamate dehydrogenase enzyme from Botrytis cinerea, has been cloned and characterized. A fragment of 2351 nucleotides was sequenced and found to contain an ORF of 1350 bp that encodes a protein of 450 amino acids. The gene, containing two introns that showed polymorphic size between them, was located by pulsed-field gel electrophoresis in chromosome X in seven strains, which were isolated from several hosts and had different levels of pathogenesis. The protein was similar to the gdhA of various other organisms, with nine highly conserved motifs that included the known active site sequence. The cloned gene was proven to be functional since it complemented two different Aspergillus nidulans gdhA mutants, restoring high levels of NADP-dependent glutamate dehydrogenase activity to the transformants. gdhA was transcribed as a monocistronic transcript of 1.7 kb starting at an A or a T, located 40 or 47 bp, respectively, upstream from the initial ATG codon of the ORF. Transcription levels of the gdhA gene were high during the rapid growth phase. Very high expression levels of the gdhA gene were observed in media with asparagine as the nitrogen source, whereas glutamic acid repressed transcription of the gdhA gene. Similarly high levels of gdhA gene transcription were observed in media with acetate as the carbon source, while glycerol strongly repressed gdhA gene transcription. These results indicate that expression of the gdhA gene is subject to strong nitrogen and carbon regulation at the transcriptional level., (Copyright 2001 Academic Press.)

To identify signal transduction pathways of the gray mold fungus Botrytis cinerea involved in host infection, we used heterologous hybridization and a polymerase chain reaction (PCR)-based approach to isolate two genes (bcg1 and bcg2) encoding alpha subunits of heterotrimeric GTP-binding proteins. Both genes have homologues in other fungi: bcg1 is a member of the G alpha(i) class, whereas bcg2 has similarities to the magC gene of Magnaporthe grisea and the gna-2 gene of Neurospora crassa. Reverse-transcription (RT)-PCR experiments showed clearly that both genes are expressed at very early stages in infected bean leaves. Gene replacement experiments were performed for both genes. bcg1 null mutants differ in colony morphology from the wild-type strain, do not secrete extracellular proteases, and show clearly reduced pathogenicity on bean and tomato. Conidia germination and penetration of plant tissue is not disturbed in bcg1 mutants, but the infection process stops after formation of primary lesions. In contrast, bcg2 mutants show wild-type colony morphology in axenic culture and are only slightly reduced in pathogenicity. Complementation of bcg1 mutants with the wild-type gene copy led to the full recovery of colony morphology, protease secretion, and pathogenicity on both host plants. Application of exogenous cyclic AMP restored the wild-type growth pattern of bcg1 mutants, but not the protease secretion, implicating an essential role of BCG1 in different signaling pathways.

Botrytis cinerea, the causal agent of blight, rot, and gray mold on many plant species, secretes various endopolygalacturonases during all stages of infection. The expression pattern of the encoding genes (Bcpg 1-6) was studied on four hosts: tomato, broad bean, apple, and courgette (also known as zucchini). All gene family members are differentially expressed, depending on the stage of infection and the host. Bcpg1 is expressed in all tissues tested although differences in transcript levels occur. Bcpg2 expression is detected early in the infection of three of four plant tissues tested. Bcpg3 and Bcpg5 are expressed in apple fruit tissue, although probably as a result of different regulatory mechanisms. The expression patterns of Bcpg4 and 6 are in agreement with their inducibility by monogalacturonic acid. The pattern of Bcpg gene expression indicates that B. cinerea is equipped with a flexible enzymatic pectate degradation machinery. The studies pinpoint new targets for gene disruption studies., (Copyright 2001 Academic Press.)

This study demonstrates that the ATP-binding cassette (ABC) transporter BcatrB from Botrytis cinerea influences the activity of phenlpyrrole fungicides against the pathogen. This conclusion is based on toxicity assays and northern analysis experiments which show that BcatrB replacement mutants, which do not express the BcatrB gene, show an increased sensitivity to the phenylpyrrole fungicides fludioxonil and fenpiclonil. Mutants overexpressing BcatrB exhibit a decreased sensitivity to these fungicides. In addition, accumulation of fludioxonil by BcatrB replacement mutants was higher than by wild-type isolates. For mutants overexpressing BcatrB the reverse was observed. Additional ABC and major facilitator superfamily (MFS) transporter genes were identified in an expressed sequence tag (EST) database, suggesting that B cinerea has gene families of ABC and MFS transporters. Corresponding fragments of ten ABC (BcatrC-BcatrN) and three MFS transporter genes (Bcmfs1-4) were cloned and characterised. Fludioxonil affected the transcript level of some members of these gene families in germlings during a short treatment with the fungicide at sub-lethal concentrations. Hence, other ABC and MFS transporters may affect the activity of phenylpyrrole fungicides as well. Other fungicides such as the anilinopyrimidine fungicide cyprodinil, the azole fungicide tebuconazole, the dicarboximide fungicide iprodione and the strobilurin fungicide trifloxystrobin also induced transcription of some of the ABC and MFS transporter genes identified. Therefore, we propose that various ABC and MFS transporters function in protection of the fungus against fungicides and are involved in multi-drug resistance development.

We report the isolation and expression profiles of a single-copy gene from the mycoparasitic fungus Trichoderma hamatum encoding a 60 S cytoplasmic ribosomal protein. The gene, named RPL36, was cloned through its nutrient-mediated expression, using mRNA differential screening. Its predicted ORF, interrupted by two introns, encoded a 105-aa polypeptide. The deduced rpL36 protein showed high overall homologies with other L36-type ribosomal proteins isolated from yeast, rat and human. Analysis of the promoter region of RPL36 revealed the presence of two ribosomal protein gene (RPG) boxes and a T-rich region known to be involved in the regulation of most ribosomal protein genes. Expression of RPL36 was tightly regulated by carbon and nitrogen availability. The mRNA levels of this gene decreased upon exposure of the mycelium to different stresses, whereas the addition of cycloheximide resulted in a super-induction. Levels of RPL36 transcripts also increased during mycoparasitic interaction between T. hamatum and Botrytis cinerea.

In Magnaporthe grisea, a well-conserved mitogen-activated protein (MAP) kinase gene, PMK1, is essential for fungal pathogenesis. In this study, we tested whether the same MAP kinase is essential for plant infection in the gray mold fungus Botrytis cinerea, a necrotrophic pathogen that employs infection mechanisms different from those of M. grisea. We used a polymerase chain reaction-based approach to isolate MAP kinase homologues from B. cinerea. The Botrytis MAP kinase required for pathogenesis (BMP) MAP kinase gene is highly homologous to the M. grisea PMK1. BMP1 is a single-copy gene. bmp1 gene replacement mutants produced normal conidia and mycelia but were reduced in growth rate on nutrient-rich medium. bmp1 mutants were nonpathogenic on carnation flowers and tomato leaves. Re-introduction of the wild-type BMP1 allele into the bmp1 mutant restored both normal growth rate and pathogenicity. Further studies indicated that conidia from bmp1 mutants germinated on plant surfaces but failed to penetrate and macerate plant tissues. bmp1 mutants also appeared to be defective in infecting through wounds. These results indicated that BMP1 is essential for plant infection in B. cinerea, and this MAP kinase pathway may be widely conserved in pathogenic fungi for regulating infection processes.

The creA genes of two plant pathogenic fungi, the gibberellin-producing rice pathogen Gibberella fujikuroi and the gray mold Botrytis cinerea, were isolated and characterized. The deduced amino acid sequences of both glucose repressors are 64% identical to each other and 59% (G. fujikuroi) and 61% (B. cinerea) identical to the CreA protein of Aspergillus nidulans. The zinc finger regions of the Gibberella and Botrytis CreA proteins shared 98% identity with the corresponding zinc finger region of the A. nidulans protein, and studies by complementation of a creA null mutant of A. nidulans showed that the proteins are functional homologues of A. nidulans CreA. Northern blot analysis revealed that creA transcript levels are independent of the carbon source in both fungi.

Pythium drechsleri Paul was described as a new species from soil samples taken in a salt-marsh of Arzew, Algeria [Paul, B. (1988) Une nouvelle espèce de Pythium isolée d'une saline de l'ouest Algérien. Cryptogam. Mycol. 9, 325-333]. The name of the fungus, P. drechsleri, is a nomen invalidum, as it is a later homonym of P. drechsleri Rajgopalan and Ramakrishnan [Rajagopalan, S. and Ramakrishnan, K. (1971) Phycomycetes in agricultural soils with special reference to the Pythiaceae. Madras Univ. J. Sect. B 37,38, 100-117]. A new name, Pythium contiguanum is now being given to P. drechsleri Paul. This species is characterised by its contiguous inflated type of sporangia, smooth-walled oogonia and mostly monoclinous antheridia. Although the fungus is not known to be a pathogen, it has a very well developed appressorial system comprised of both sickle shaped and coiled appressoria. Morphological features, its antagonism towards the grape-vine pathogen Botrytis cinerea, together with the sequences of the ITS1 region of its nuclear ribosomal DNA and its comparison with related species are given here.

The phytopathogenic fungus Botrytis cinerea produces a set of endopolygalacturonases (endoPGs) which are involved in the enzymatic degradation of pectin in plant cell walls. The endoPG-encoding genes of B. cinerea are differentially expressed when the fungus is grown in liquid culture on different carbon sources. A basic constitutive expression level was observed for two genes, Bcpg1 and Bcpg2, which encode basic isozymes. Galacturonic acid was shown to induce the expression of Bcpg4 and Bcpg6. Low pH of the culture medium resulted in induced expression of the Bcpg3 gene. Expression of the Bcpg5 gene was inducible; however the inducing factors could not be identified. Finally, galacturonic acid-induced expression of the Bcpg4 gene was repressed by the presence of more-favourable carbon sources, such as glucose.

A study was conducted to assess genetic variation within and among populations of Beauveria bassiana (Deuteromycotina: Hyphomycetes) associated with the darkling beetle, Alphitobius diaperinus (Coleoptera: Tenebrionidae), using RAPD markers. A hierarchical collection of samples (strains from the same insect specimen, from insects from the same location, and from insects from different locations) was obtained from infected beetles from North Carolina (NC) and West Virginia (WV), USA. Ten primers resolved 81 strains into 80 distinct multiband phenotypes reflecting the substantial amount of variation that was present. Variation present within populations was evident not only in the separation of each strain as a distinct multiband phenotype but also in the separation of strains within a population into separate clusters. Among populations, a group sharing more than 89% similarity was observed among all the strains from Martin Co. and Greene Co., NC and 61% of the strains collected from WV. Some genetic differentiation was present among the other populations but the separation was not distinct with a few strains from some populations showing greater affinity to strains from other collection sites., (Copyright 1999 Academic Press.)

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.

A minisatellite was identified in the intron of the ATP synthase of the filamentous fungus Botrytis cinerea, and it was named MSB1. This is the second fungal minisatellite described to date. Its 37-bp repeat unit is AT-rich, and it is found at only one locus in the genome. The introns of 47 isolates of Botrytis species were sequenced. The number of tandem repeats varied only from 5 to 11, but there were many repeat variants. The structure of MSB1 is peculiar: the variants are in the same physical order in all individuals, and this order follows the most parsimonious tree. These original characteristics, together with a total lack of recombination between alleles of the flanking regions, suggest that MSB1 probably mutates by slippage. MSB1 was found in the intron of the ATP synthase of all of the Botrytis species analyzed, but the repeat unit was not found in any other genus examined, including Sclerotinia, which is the genus closest to Botrytis.

Botrytis cinerea, a fungus that causes diseases in over 200 plant species, secretes a number of endopolygalacturonases that have been suggested to be involved in pathogenesis. However, so far the corresponding genes have not been isolated from this fungus. We cloned Bcpg1, encoding endopolygalacturonase, with the pgaII gene from Aspergillus niger as a heterologous probe. The Bcpg1 gene is expressed to similar levels in liquid cultures of B. cinerea containing either 1% polygalacturonic acid or 1% sucrose, and is expressed during infection of tomato leaves. The Bcpg1 gene was eliminated by partial gene replacement, and the resulting mutants were tested for virulence on tomato leaves and fruits, as well as on apple fruits. Although the mutants were still pathogenic and displayed similar primary infections when compared with control strains, a significant decrease in secondary infection, i.e., growth of the lesion beyond the inoculation spot, was observed on all three host tissues. These results indicate that the Bcpg1 gene is required for full virulence.