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

Fungal enzyme sets for plant polysaccharide degradation

Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families

Endo-xylogalacturonan hydrolase

A cDNA library of Aspergillus tubingensis was constructed in the yeast Kluyveromyces lactis, using a carbon source rich in xylogalacturonan. The library was screened using a hairy regions preparation from apple, and xylogalacturonan prepared from gum tragacanth as substrates. A novel endo-xylogalacturonase was found, XGH. The enzyme specifically degrades xylose-substituted galacturonic acid backbones. In lab scale filtration experiments, XGH was able to decrease membrane fouling caused by hairy regions from appl