Functional analysis of diacylglycerol O-acyl transferase 2 gene to decipher its role in virulence of Botrytis cinerea.

Gray mold disease inflicted by Botrytis cinerea is a serious menace responsible for significant economic loss worldwide. Due to its polyphagous nature, the pathogen has enthused inquisitiveness in researchers to unravel its complexity. Agrobacterium tumefaciens-mediated transformation was used to generate insertional mutants of Botrytis cinerea. A mutant (BCM-55) with disruption in a gene (BcDGAT2) that encodes for diacylglycerol O-acyl transferase 2 (DGAT2), showed enervated virulence on various hosts' tissues. Enzyme DGAT2 is crucial in the final step of synthesis of triacylglycerol (TAG) that plays an important role in homeostasis of membrane and cellular processes. However, the role of DGAT2 has never been reported in a phytopathogenic fungus. In this study, BCM-55 was characterized to ascertain the role of DGAT2 in virulence of B. cinerea. The insertional mutant was defective in spore production and lacked sclerotia formation as a consequence of lower accumulation of TAG. A significant delay in spore germination in BCM-55 was accompanied with a low penetration potential. Hyphae of the mutant formed swollen endings with considerable impairment in penetration. Deletion of BcDGAT2 also led to increased sensitivity towards cell wall and membrane-disturbing agents. Furthermore, BCM-55 was deficient in the production of oxalic acid and showed lower activity of a cell wall-degrading enzyme, polygalacturonase. The role of BcDGAT2 in virulence was further confirmed by targeted deletion and complementation of the gene. The results insinuate a crucial role of BcDGAT2 in penetration and consequently virulence of B. cinerea. The study provides novel insights into plant-pathogen interactions that can be exploited to develop suitable disease management strategies.

A Botrytis cinerea KLP-7 Kinesin acts as a Virulence Determinant during Plant Infection.

Botrytis cinerea is a necrotrophic pathogen that infects many important crops. In an attempt to unravel some novel factors that govern pathogenicity in B. cinerea, Agrobacterium tumefaciens mediated transformation (ATMT) was deployed, and a number of tagged transformants were generated. Among these, a mutant, BCM-29 exhibited slower growth rate, reduced conidia size, conidiation and penetration. The mutant was also defective in secretion of oxalic acid (OA) and exhibited reduced activities of polygalacturonase (PG) and pectin methyl esterases (PME). TAIL-PCR followed by BLAST search identified the tagged gene as KLP-7 that encodes for kinesin. Targeted deletion of KLP-7 resulted in several folds decrease in virulence of mutants as compared to WT, while complementation of the gene helped in rescue of virulence traits. This is the first time when a unique kinesin KLP-7 that is mainly found in the phylum Pezizomycotina has been linked to virulence in B. cinerea.

Analyses of genetic and pathogenic variability among Botrytis cinerea isolates.

Seventy nine isolates of Botrytis cinerea were collected from different host plants and different locations of India and Nepal. All the isolates were identified as B. cinerea based on morphological features and were confirmed using B. cinerea specific primers. Differentiation among the isolates was assessed using morphological, genetic and biochemical approaches. To analyze morphological variability, differences in conidial size, presence or absence of sclerotia and their arrangement were observed. Genetic variability was characterized using RAPD analysis, presence or absence of transposons and mating type genes. Cluster analysis based on RAPD markers was used for defining groups on the basis of geographical region and host. The biochemical approach included determining differences in concentration of oxalic acid and activity of lytic enzymes. All the isolates were categorized into different pathogenic groups on the basis their variable reaction towards chickpea plants. Isolates with higher concentration of oxalic acid and greater activity of lytic enzymes were generally more pathogenic. Pathogenicity was also correlated to transposons. Isolates containing transposa group showed some degree of correlation with pathogenic behavior. However, isolates could not be grouped on the basis of a single approach which provides evidence of their wide diversity and high evolution potential. Sensitivity of sampled isolates was also tested against five botryticides. Most of the isolates from same region were inhibited by a particular fungicide. This feature provided interesting cues and would assist in devising novel and more effective measures for managing the disease.