Genetic variation and pathogenicity of Botrytis cinerea.

Botrytis cinerea is a fungal pathogen of more than 200 hosts including a wide variety of economically important crops. Although many ecological and physiological studies on this destructive pathogen have been reported, not much is known about the molecular basis of the interaction of this pathogen with its various host plants. This thesis describes the use of molecular techniques to study the genetic variation and pathogenicity of B. cinerea.Genetic variation among ten strains of B. cinerea was studied by RAPID analysis. Strains appeared to be highly similar and could not be grouped according to their host, suggesting that host specialization does not occur in B. cinerea . Segregation analysis of RAPID markers in generative progeny collected from ordered ascospore octads, predominantly revealed segregation ratios of 1:1. Occasionally, a 1:3 segregation ratio, or the appearance and disapperance of RAPID markers were observed. These unexpected observations were explained by the heterokaryotic association of presumably polyploid nuclei.An important role for cutinase was suggested in penetration of undamaged host tissue. To investigate the relevance of cutinase in more detail, cloning of the cutinase gene was an essential step. Molecular strategies such as heterologous screening and immunoscreening of libraries and a PCR based cloning strategy were employed without success. Purification of the enzyme, partial determination of its amino acid sequence and the design of cutinase specific primers finally led to the cloning and identification of the corresponding gene ( cutA ). A second putative esterase was co-purified and the encoding gene (ekdA) was isolated as well.Using a cutA promoter - GUS reporter gene fusion, expression of the cutinase gene was studied in planta . Conidia of transformants harbouring the reporter construct and germinating on gerbera flowers and tomato fruits, contained GUS activity, indicating that the cutinase gene is expressed during penetration of host tissue by B. cinerea . However, disruption of the single copy cutA gene in a haploid strain of B. cinerea , showed that this cutinase is not required for successful infection of gerbera flowers and tomatoes. The ability of cutinase A-deficient mutants to infect and to develop disease was unaltered compared to the wild type strain. These results suggest that B. cinerea employs other strategies to penetrate undamaged host tissue. Production of other cutinases, generation of oxygen radicals by glucose oxidase or mechanical penetration by formation of appressoria might be involved