Genetic transformation of Colletotrichum truncatum associated with anthracnose disease of chili by random insertional mutagenesis

Abstract

An Agrobacterium tumefaciens -mediated transformation (ATMT) system was successfully developed for Colletotrichum truncatum, the causal agent of chili anthracnose. A. tumefaciens carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (gfp) gene was used to transform the conidiospores of two C. truncatum pathotypes F8-3B and BRIP26974. Optimum transformation efficiency was obtained when equal volumes of A. tumefaciens strain AGL1 carrying either pJF1 or pPK2 binary vector was used to transform C. truncatum conidiospores at 10(6) /ml and co-cultivated at 24 °C for three days. Southern blot analysis indicated that 87.5% of the transformants contained randomly inserted, single copies of the T-DNA. Infection and colonisation of chili fruit at the mature red stage with F8-3B-GFP and BRIP26974-GFP confirmed the maintenance of virulence within these transformed pathotypes. In situ studies of infection and colonisation of the susceptible genotype fruit using fluorescent microscopy and transformed isolates of C. truncatum expressing GFP revealed that the pathogen was able to colonise healthy fruit tissue intercellularly in an endophytic manner without producing secondary biotrophic infection structures. The developed transformation system will be used to study the function of pathogenicity genes in C. truncatum using both forward and reverse genetics approaches.

Diacylglycerol acyl transferase: A pathogenicity related gene in Colletotrichum gloeosporioides

“…The single-copy percentage obtained in the present study is similar to already reported frequency with Agrobacterium-mediated transformation in Colletotrichum lindemuthianum [20] and Magnaporthe grisea [21]. However, Maruthachalam and coworkers and Auyong and coworkers reported 16% frequency of single gene integration for Agrobacterium-mediated transformation of C. graminicola and 65% frequency of single gene integration for other Colletotrichum species [22,23].…”

“…Genes and proteins identified as key components of the pathogenic process by large scale mutagenesis experiments [6,11,13,14,17,[20][21][22][23]28], acts as targets in the fungus for chemical control (fungicides or antifungal drugs). Similarly, defense related genes identified in the hosts can be modified to reduce their susceptibility to the pathogen e.g., through the use of genetically modified crop genotypes or viral therapeutics [34].…”

Chili anthracnose: Colletotrichum taxonomy and pathogenicity

“…Hemibiotrophy followed by a very short endophytic phase was identified as the main infection and colonization process for C. truncatum infection of chili fruit. Auyong et al (2012) studied colonization of chili fruit with a genetically modified strain of C. truncatum that expressed green fluorescent protein (gfp) to enable visualization of infection in situ. The C. truncatum::gfp transformant's growth revealed that after initial infection, the hyphae colonized intramurally within the parenchyma tissue of healthy chili fruit without further development of the secondary biotrophic structures.…”

A new anthracnose disease of pyrethrum caused byColletotrichum tanacetisp. nov

“…Colletotrichum species are among the most important plant pathogenic fungi worldwide and, depending on the species-host interactions, the initial host infection strategy has been reported as either intracellular hemibiotrophic, subcuticular intramural necrotrophic or a combination of both strategies . Some species such as Colletotrichum truncatum have been shown to exist as an endophyte because, during infection and early colonization, the pathogen is not recognized by the host and thus no symptoms are produced (Auyong et al, 2012). Traditionally, Colletotrichum species were identified and characterized based on morphological and cultural characteristics (Bailey & Jeger, 1992).…”