3. Genetic diversity and host specificity of Botrytis spp.

The genus Botrytis comprises about 27 species, all of which are necrotrophic plant pathogens (Hyde et al., 2014). Based on gene sequence analyses, Botrytis has been divided into two phylogenetically separated clades (Staats et al., 2005). Clade 1 includes B. cinerea and three other species which infect only dicotyledonous plants. Clade 2 is phylogenetically more diverse and currently comprises 23 host-specific Botrytis species that infect predominantly monocotyledonous plants (Staats et al., 2005; Hyde et al., 2014).

B. pseudocinerea has been originally found as a minor sympatric species occurring together with B. cinerea in vineyards. PCR-based analyses of more than 1500 Botrytis isolates from diverse locations and plant species have confirmed that B. pseudocinerea has a wide host range similar to B. cinerea, and can occur as a major pathogen on several cultivated plants. However, in contrast to B. cinerea, B. pseudocinerea does not accumulate fungicide resistance mutations in the field. In fungicide treated strawberry fields, B. pseudocinerea disappeared and was replaced by fungicide-resistant B. cinerea strains, whereas both species coexisted over the whole season in an untreated field (Fig. 1). A partial explanation for the inability of B. pseudocinerea to develop fungicide resistance was found in the presence of an intron in the cytB gene in all tested B. pseudocinerea isolates, which prevents the most effective QoI resistance mutation (Plesken et al., submitted).

<br>Fig. 1: Seasonal changes in the occurrence of <i>B. pseudocinerea </i>(● black shading), <i>B. cinerea </i>N (● dark grey shading) and <i>B. cinerea </i>S (● light grey shading) in strawberry fields. One field was intensively fungicide-treated (8 treatments in early summer), whereas the other field remained untreated throughout the year. The pie charts show the relative frequencies of isolates, and the histograms show the frequencies of isolates carrying different numbers of resistances (0R: No resistance; 1R - 5R: One to five resistances). (Plesken et al., submitted).

The occurrence of B. calthae is restricted to its host species, Caltha palustris. Naturally growing C. palustris plants are colonized not only by B. calthae but also by B. pseudocinerea and B. cinerea (Fig. 1A). A phenotypic comparison of B. cinerea and B. calthae revealed similarities and differences in their growth behaviour and their sensitivity to SDHI fungicides. B. calthae was able to cause necrotic lesions on several plants after artificial inoculations, but to a lower extent than B. cinerea, except for its host C. palustris (Fig. 1B; Plesken et al., 2014).

<br>Fig. 2. A: Genetic diversity of <i>Botrytis </i>strains isolated from grey mould-infected <i>C. palustris </i>stems and leaves. B: Lesion formation by <i>B. cinerea </i>and <i>B. calthae </i>strains on different plant tissues: Leaves (<i>C. palustris</i>, tomato, broad bean, pea; 72 hours), flowers (Gerbera; 48 hours) and fruits (apples; 96 hours). Significant differences are indicated by asterisks. (Plesken et al., 2014).

The genome sequence of B. cinerea has been published (Amselem et al., 2011). The B. cinerea genome has a low GC content and encodes approx. 11,500 genes. Except for a large number of genes encoding pectinolytic enzymes, no special features were discovered so far that would explain the broad host range and necrotrophic life style of B. cinerea. We have recently determined the genome sequences from several B. cinerea strains and the other species of Botrytis clade 1 (B. pseudocinerea, B. fabae and B. calthae). All genomes share high sequence similarity (>90%; Plesken et al., unpublished). A phylogenetic analysis confirmed the known relationships and revealed genetically distinct lineages within B. cinerea (Fig. 3A; C. Plesken, unpublished). Comparative investigations of the gene contents in the sequenced genomes revealed differences in genes for secondary metabolite synthesis. The gene cluster for botrydial synthesis, a potential virulence factor, was found to be present in all B. cinerea genomes sequenced, but not in the other Botrytis species (Fig. 3B; C. Plesken, unpublished). These results, and ongoing transcriptomics and mutant analyses will lead to a better understanding of the molecular basis of host specificity in B. cinerea and related Botrytis spp.

<br>Fig. 3. A: Neighbour joining tree, based on 3602 genes, of <i>B. cinerea </i>strains and other <i>Botrytis </i>clade 1 species. B: Presence of genes for botrydial biosynthesis in the genome of <i>B. cinerea</i>, but not (except for BOT4 in <i>B. fabae</i>) in the other <i>Botrytis </i>clade 1 species. (C. Plesken, unpublished data).

Publications

Plesken C, Weber RWS, Rupp S, Leroch M, Hahn M 2015. Botrytis pseudocinerea is a significant pathogen of several crop plants but often displaced by fungicide-resistant B. cinerea strains. Submitted for publication.

Hahn M, Viaud M, van Kan J 2014. The genome of Botrytis cinera, a ubiquitous broad host range necrotroph. In: Genomics of Plant-Associated Fungi and Oomycetes: Dicot Pathogens, Dean RA et al. (eds.), Springer-Verlag Berlin Heidelberg, DOI: 10.1007/978-3-662-44056-8_2.

Plesken C, Westrich LD, Hahn M 2014. Genetic and phenotypic characterization of Botrytis calthae. Plant Pathol DOI: 10.1111/ppa.12240.

Hyde KD et al. (40 authors, including M. Hahn) 2014. One stop shop: backbones trees for important phytopathogenic genera. Fungal Divers 67: 21-125.

Leroch M, Plesken C, Weber RWS, Kauff F, Scalliet G, Hahn M 2013. Gray mold populations in German strawberry fields are resistant to multiple fungicides and dominated by a novel clade closely related to Botrytis cinerea. Appl Environ Microbiol 79: 159-167.

Amselem J et al (71 authors, incl. M. Hahn, M. Leroch, M. Kretschmer) 2011. Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet 7: e1002230.