Chitinase as a molecular bioindicator of resistance to Fusarium oxysporum in Acacia koa

Abstract

Acacia koa, a tree legume native to Hawaii, serves as a vital resource to the islands both culturally and economically. However, its population is currently at risk due to infection by the fungal pathogen, Fusarium oxysporum, which causes koa wilt. Therefore, a means by which resistance to F. oxysporum can easily be screened for is highly desirable. The objectives of this research are to isolate cDNA clones for chitinases from koa and to determine their expression levels using quantitative reverse transcription PCR. Chitinases catalyze the hydrolysis of chitin, a structural component of fungal cell walls. We hypothesize that chitinase expression is directly related to F. oxysporum resistance and can therefore be used as a bioindicator to screen for resistance. Degenerate primers were used to screen for chitinase genes based on highly conserved regions characteristic of individual classes of chitinases. The resulting cDNA fragments were expanded to their full length sequences using 3'-and 5'-RACE. These methods resulted in the successful isolation of four cDNA ORFs corresponding to chitinase genes of classes I through IV, which were designated Akchit1, Akchit3, Akchit3, and Akchit4, respectively. Quantitative RT-PCR was used to monitor the expression of these chitinases in Fusarium-wilt resistant and susceptible koa seedlings following inoculation with F. oxysporum. Significant differences (p<0.05) between resistant and susceptible seedlings were observed for Akchit1 and Akchit4 at 48, 72, and 96 hours post inoculation, in which chitinases were up-regulated among resistant seedlings. However, differences between the two groups for Akchit2 and Akchit3 were less pronounced. This research will aid in the development of an effective screening method for determining resistance to F. oxysporum in koa, one which can be easily implemented into Hawaii's forestry industry.