Yves Brygoo

The Endopolygalacturonase 1 from Botrytis cinerea Activates Grapevine Defense Reactions Unrelated to Its Enzymatic Activity

A purified glycoprotein from Botrytis cinerea (strain T4), identified as endopolygalacturonase 1 (T4BcPG1) by mass spectrometry analysis, has been shown to activate defense reactions in grapevine (Vitis vinifera cv. Gamay). These reactions include calcium influx, production of active oxygen species, activation of two mitogen-activated protein kinases, defense gene transcript accumulation, and phytoalexin production. Most of these defense reactions were also activated in grapevine in response to purified oligogalacturonides (OGA) with a degree of polymerization of 9 to 20. In vivo, these active OGA might be a part of the released products resulting from endopolygalacturonase activity on plant cell walls. Nevertheless, the intensity and kinetics of events triggered by OGA were very different when compared with T4BcPG1 effects. Moreover, chemical treatments of T4BcPG1 and desensitization assays have allowed us to discriminate enzymatic and elicitor activities, indicating that elicitor activity was not due to released oligogalacturonides. Thus, BcPG1 should be considered as both an avirulence and a virulence factor. The role of the secreted BcPG1 in the pathogenicity of Botrytis cinerea is discussed.

Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea

Calcineurin phosphatase and cyclophilin A are cellular components involved in fungal morphogenesis and virulence. Their roles were investigated in the phytopathogenic fungus Botrytis cinerea using gene inactivation, drug inhibition and cDNA macroarrays approaches. First, the BCP1 gene coding for cyclophilin A was identified and inactivated by homologous recombination. The bcp1Δ null mutant obtained was still able to develop infection structures but was altered in symptom development on bean and tomato leaves. Opposite to this, calcineurin inhibition using cyclosporin A (CsA) modified hyphal morphology and prevented infection structure formation. CsA drug pattern signature on macroarrays allowed the identification of 18 calcineurin‐dependent (CND) genes among 2839 B. cinerea genes. Among the co‐regulated CND genes, three were shown to be organized as a physical cluster that could be involved in secondary metabolism. The signature of BCP1 inactivation on macroarrays allowed the identification of only three BCP1 cyclophilin‐dependent (CPD) genes that were different from CND genes. Finally, no CsA drug pattern signature was observed in the bcp1Δ null mutant which provided a molecular target validation of the drug.

Partition of the Botrytis cinerea complex in France using multiple gene genealogies.

In micro-organisms biodiversity is often underestimated because relevant criteria for recognition of distinct evolutionary units are lacking. Phylogenetic approaches have been proved the most useful in fungi to address this issue. Botrytis cinerea, a generalist fungus causing gray mold, illustrates this problem. It long has been thought to be a single variable species. Recent population genetics studies have shown that B. cinerea is a species complex. However conflicting partitions were proposed. To identify the most relevant partitions within the B. cinerea complex we used a multiple-gene genealogies approach. We sequenced portions of four nuclear genes, of which genealogies congruently clustered into two well supported groups corresponding to Groups I and II previously described, indicating that they represent phylogenetic species. Estimates of migration rates and genetic differentiation showed that these groups had been isolated for a long time, without detectable gene flow. This was confirmed by the high number of polymorphic sites fixed within each group. The genetic diversity was lower within Group I, as revealed by DNA polymorphism and vegetative incompatibility tests. Groups I and II exhibited phenotypic differences in their phenology, host range, size of asexual spores and vegetative compatibility. All these morphological and molecular aspects suggest that B. cinerea Groups I and II may be different cryptic species, isolated for a long time. Phylogenies and molecular analyzes of variance revealed no genetic structure according to the other suggested partitions for the B. cinerea complex (i.e., among host plants, between strains with and without transposable elements, nor between strains responsible for noble rot and gray mold. This suggests that recombination regularly occurs, or occurred until recently, within B. cinerea Group II. This also was supported by recombination rates at each locus. Multiple-gene genealogies showed their utility by providing a relevant partition criterion for the B. cinerea complex.

Two Sibling Species of the Botrytis cinerea Complex, transposa and vacuma, Are Found in Sympatry on Numerous Host Plants

ABSTRACT Strains of Botrytis cinerea (the anamorph of Botryotinia fuckeliana) were collected from 21 different plant species around vineyards in the Champagne region (France). Strains were analyzed using three new polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) markers that were found by SWAPP (sequencing with arbitrary primer pairs), in addition to 15 other markers (PCR-RFLP, transposable elements, and resistance to fungicides). The markers revealed a high degree of genetic diversity and were used to investigate population structure. The two sympatric species transposa and vacuma, previously identified on grapes in these vineyards, were also detected on many of the plant species sampled. A new type of strain was also detected, having only the transposable element Boty. We did not detect any differentiation between strains from different organs or locations, but the prevalences of transposa and vacuma were significantly different on the different host plants. Fungicide resistance frequencies were significantly different in transposa and vacuma species. This study confirms that B. cinerea is a complex of sibling species and shows that the sibling species occur sympatrically on many host plants. However, the two species seemed to have different pathogenic behaviors. These findings contradict the traditional view of B. cinerea as a clonal population without specialization.

Diversity of soil fungi studied by PCR–RFLP of ITS

Ribosomal DNA sequences provide new molecular approaches to characterise fungal species from the environment. This study presents a culture-independent approach to assessing fungal diversity by direct amplification, cloning, restriction digestion and sequencing of internal transcribed spacers (ITS). We have assessed this novel approach both with culturable soil fungi and with total environmental DNA extracted from the same soil sample. Sixty-seven colonies and 51 cloned amplicons from total DNA were compared on the basis of their ITS restriction patterns. Then, 58 representative ITS sequences were determined and classified by comparing their sequences with ITS sequences of known origin. All culturable fungi were ascomycetes with one basidiomycete exception. In contrast, cloned amplicons from total environmental DNA were identified to ascomycetes, one basidiomycete, one zygomycete and to several fungi belonging to the kingdoms Chromista and Protozoa. The results show that PCR-RFLP of ITS provides an efficient culture-independent molecular tool for ecological studies and to assess fungal diversity.

Genetic characterisation of Botrytis cinerea populations in Chile

This work aimed to evaluate the genetic diversity of Botrytis cinerea in Chile and to determine whether the two genetically different groups transposa and vacuma , described in France, are present in the country. Isolates collected in Chile from grapes, tomato, kiwifruit and blueberry were analysed using molecular markers. We developed a PCR test to identify the two groups of B. cinerea, transposa (with the transposable elements Boty and Flipper ) and vacuma (with neither). As described in France, both kind of isolates were found to be present and sympatric in Chile. Isolates containing the transposable element Boty alone ( boty isolates) were also detected. The frequencies of transposa, boty and vacuma isolates were significantly different on kiwifruit compared to grapes, tomato and blueberry. RAPD analysis revealed a high degree of genetic diversity, with no widespread clonal lineages. Dendrograms, analysis of molecular variance and Fst values revealed the existence of genetic differentiation in our sample between isolates from the different host plants. PCR-RFLP markers also showed that isolates sampled from grapes and tomato were genetically differentiated. Additional sampling is required to confirm these findings.

FLIPPER, A MOBILE FOT1-LIKE TRANSPOSABLE ELEMENT IN BOTRYTIS CINEREA

Abstract A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.

Multiple recent horizontal transfers of a large genomic region in cheese making fungi

While the extent and impact of horizontal transfers in prokaryotes are widely acknowledged, their importance to the eukaryotic kingdom is unclear and thought by many to be anecdotal. Here we report multiple recent transfers of a huge genomic island between Penicillium spp. found in the food environment. Sequencing of the two leading filamentous fungi used in cheese making, P. roqueforti and P. camemberti, and comparison with the penicillin producer P. rubens reveals a 575 kb long genomic island in P. roqueforti—called Wallaby—present as identical fragments at non-homologous loci in P. camemberti and P. rubens. Wallaby is detected in Penicillium collections exclusively in strains from food environments. Wallaby encompasses about 250 predicted genes, some of which are probably involved in competition with microorganisms. The occurrence of multiple recent eukaryotic transfers in the food environment provides strong evidence for the importance of this understudied and probably underestimated phenomenon in eukaryotes.

Characterization of Bc-hch, the Botrytis cinerea homolog of the Neurospora crassa het-c vegetative incompatibility locus, and its use as a population marker

The Botrytis cinerea homolog (Bc-hch) of Nc-het-c and Pa-hch (vegetative incompatibility loci of Neurospora crassa and Podospora anserina respectively) was cloned and sequenced. The gene structure of Bc-hch is very close to those of Nc-het-c and Pa-hch. A PCR-RFLP approach on a 1171 bp fragment was used to screen polymorphism at this locus among 117 natural isolates of B. cinerea. Restriction patterns by the restriction enzyme HhaI fell into two allelic types. Moreover, haplotypes at the Bc-hch strictly corresponded to the resistance phenotypes to fenhexamid, a novel Botryticide. The use of Bc-hch as a population marker thus reveals a new structuring of B. cinerea natural populations into two groups (I and II). This result was confirmed by genic differentiation tests performed with five other markers on a sample of 132 B. cinerea isolates from the French region of Champagne.

Ribosomal RNA sequence divergence within the Pythiaceae

Partial sequences of 28S RNA genes from 49 strains belonging to 23 species of the Pythiaceae were compared to assess their phylogenetic relationships. A high level of diversity was found within Pythium and the eight species studied were classified in four distinct subgroups. In contrast to Pythium, Phytophthora appeared to be very homogeneous, with small phylogenetic distances among all 15 species investigated, including representatives of the six commonly accepted taxonomic groups. Finally, we found within Pythium a strict correlation between groups of species defined by molecular taxonomy and groups defined on the basis of a morphological criterion which has been traditionally considered of secondary importance for classification: the sporangial form.