Botrytis species on flower bulb crops: phylogeny, genetic variation and host specificity

Fungi of the genus Botrytis (teleomorph Botryotinia) can cause serious damage in a large range of ornamental crops. Except for B. cinerea, Botrytis species that are pathogenic on flower bulb crops are host-specific, i.e. each species is able to infect only one or a few closely related host species. This thesis mainly focuses on the economically important species B. elliptica and B. tulipae, the causal agents of 1eaf blight in lily and tulip, respectively.

Molecular markers were developed that allowed for the unambiguous identification of the Botrytis species of interest and to study their intraspecific variation (Chapter 2, 3 & 4). Chapter two describes a classification of the genus Botrytis based on DNA sequence data of three protein-coding genes {RPB2, GSPDB and HSP60). The phylogenetic analysis that encompassed all 22 species of the genus corroborated the classical species delineation. In addition, the hybrid status of B. allii (B. byssoidea X B. aclada) was confirmed. A comparison of Botrytis and angiosperm phylogenies showed that cospeciation of pathogens and their hosts have not occurred. Rather, it is proposed that host shifts have occurred during Botrytis speciation.

Chapter three describes three molecular typing methods to differentiate isolates of B. tulipae, B. elliptica and B. cinerea. Restriction analysis of the intergenic spacer (IGS) region was the least discriminatory, followed by DNA sequencing of five gene regions (G3PDH, RPB2, HSP60, H3 and EF-Ia) and the internal transcribed spacer (ITS) region. Amplified fragment length polymorphism (AFLP) analysis appeared the most discriminatory method. It was, therefore, used to assess the genotypic diversity among field isolates of B. elliptica and B. tulipae from the Netherlands (Chapter 4). In addition, analyses were performed to infer their modes of reproduction. Isolates of both species were sampled during successive growth seasons from experimental field plots in Lisse and from several other locations in the Netherlands. The genotypic diversity for B. elliptica was high and clonal genotypes were found only within growing seasons. Linkage disequilibrium analyses strongly supported the occurrence of genetic recombination in the field, most likely as a result of sexual reproduction. Multilocus association analyses, however, provided no evidence for random mating.

B. tulipae has a mainly clonal population structure, as evidenced by the low genotypic diversity, the repeated recovery of clonal genotypes over long distances and over different years, and the strong multilocus associations. However, the level of linkage disequilibrium is lower than expected for a strictly clonal organism. Therefore, it is possib]e that some level of recombination would have resulted in generating new genotypes.

In chapters 5 and 6 the roles of Nepl-like proteins {NLPs) as determinants of host specificity are investigated. Two NLF-encoding genes, designated NEPl and NEP2, were present in all Botrytis species (Chapter 5). Both NLPs may have different functions as their sequence similarity is low and because they contain different types of posttranslational modification motifs. While both NLPs are moderately conserved and are overall under purifying selection, a number of amino acid residues were predicted to be under positive selection based on inferences using maximum likelihood models. The functional effects of these amino acid replacements on the NEP protein activity remain to be resolved.

Chapter 6 describes the functional characterization of NLP genes from B, elliptica. Mutants of B. elliptica in which BeNEPl or BeNEP2 was replaced showed normal virulence on lily leaves. In addition, the B. elliptica NLPs that were produced in the yeast Pichia pastoris were not toxic to monocots, including lily. These results show that NLPs are not essential virulence factors and do not function as host-selective toxins for B. elliptica.

Chapter 7 presents a general discussion of the thesis and provides future directions to study the evolution, life cycles and pathogenicity of Botrytis species on flower bulb crops.