Reproductive modes of Botrytis species in the field and in the lab

The genus Botrytis comprises 22 species, for most of which a teleomorph was described, named Botryotinia. For virtually all the Botryotinia species, it is unknown how frequently sexual reproduction occurs in nature. Occurrence of the teleomorph stage of Botrytis cinerea (Botryotinia fuckeliana) in the field has only been reported five times in literature since its first description in 1865. Nevertheless, studies on genetic diversity in France have suggested that B. cinerea shows abundant sexual reproduction. For other Botrytis / Botryotinia species, such studies have barely been performed. One objective of our study was to assess the genetic diversity in two host-specialised species, i.e. Botrytis elliptica and B. tulipae, and to infer from the diversity data whether these species reproduce sexually or asexually in bulb fields in the Netherlands. AFLP fingerprinting provided the most efficient method to differentiate isolates within each species and therefore this method was used for population analyses of B. elliptica and B. tulipae. Isolates of both species were sampled during successive growing seasons in experimental field plots in various locations in the Netherlands. Among 174 B. elliptica isolates, 105 genotypes could be discriminated and 87 genotypes were found only once, reflecting high genotypic variation. Clonal genotypes were found only within growing seasons and in the same location. We conclude that sexual recombination occurs in the B. elliptica population. Among the 170 B. tulipae isolates, 25 genotypes could be discriminated and 4 genotypes were found only once, reflecting a low genotypic variation. Clonal genotypes were frequently found in different growing seasons and different locations. We conclude that the B. tulipae population is mainly clonal with occasional recombination. A second, rather different study that has been ongoing for more than a decade, deals with the unusal observation that certain natural B. cinerea isolates behave as dual maters, i.e. they are able to mate successfully with two reference strains for the mating type alleles MAT1-1 (strain SAS56) and MAT1-2 (strain SAS405). Dual maters even occur (at low frequency) in progeny originating from a cross between SAS56 and SAS405. The mechanism underlying this behaviour could not be studied because the DNA sequences of the MAT alleles were not available until recently. The genome sequences of strains B05.10 (MAT1-1) and T4 (MAT1-2) have provided insight in the configuration of both MAT alleles. Compared to other ascomycetes, the B. cinerea MAT locus has some unusual features. We have started to analyse the MAT locus configuration in dual mater strains by PCR and sequencing analyses. The first results suggest that dual mater strains possess a MAT1-2 locus, however, we were not able to detect any MAT1-1 like sequence as yet. These results suggest that the dual mater behaviour is not the consequence of a conversion from heterothallism to homothallism, i.e. the presence of both MAT alleles/genes in a single thallus. An alternative mechanism for the phenomenon may need to be proposed.