Determination of genetic diversity of Pseudomonas syringae pathovar garcae and its reaction to selected coffee varieties in Kenya

Abstract

Bacterial blight of coffee (BBC) caused by Pseudomonas syringae pathovar garcae is a major concern in Kenya and other coffee growing countries due to high incidence and severity in Arabica coffee. Previous work to develop disease resistant coffee varieties in Kenya in order to reduce the cost of production with safe environmental management did not take resistance to BBC into consideration. The commercial coffee varieties that are resistant to the major coffee diseases in Kenya such as Ruiru 11 and Batian were not selected for BBC resistance posing a great economic threat to coffee farming in Kenya especially in the BBC hot spots of Nakuru, Nyeri and Kapsabet. The diversity of the causative agent in Kenya had not been documented. Determination of genetic diversity of Pseudomonas syringae pv. garcae and its reaction to selected coffee varieties in Kenya was therefore the objective of this research. Phenotypic characterization of 12 isolates of P.s. pv. garcae collected from different regions in Kenya was done by inoculating them on four coffee varieties to test their virulence. Twenty four coffee genotypes were further screened for BBC resistance using the 3 most virulent and the least virulent BBC isolates by inoculation with a drop of approximately 10 µl of the isolates suspension (109 CFU/ml) using the injection method at the shoots of 4 months old seedlings from potting. The experiment was carried out in a complete randomized design. Disease symptoms were scored using a scale of 1 to 5 and the data was subjected to analysis of variance and effects declared significant at 5% level. Molecular characterization via PCR and sequencing of the 12 isolates was done using 16S ribosomal RNA primers 8 F and 1492 R (l). The sequences were then retrieved for alignment using vector NTI software and phylogenetic analysis done by MEGA 6 using clustalW. Eleven SSR primers were used to characterize the 24 genotypes using PCR and the bands’ data was used to estimate genetic distances using XLSTAT version 2014. The 12 isolates were found to have significant levels (P>0.05) of virulence to coffee which was closely linked with the molecular characterization using 16S gene showing the diverse nature of the pathogen and possible existence of different races of P. s. pv. garcae. Rume Sudan and Hibrido de Timor (HDT) were found to be the most promising donor candidates for resistance to BBC and diversity was observed among the coffee genotypes via SSR molecular characterisation. This study provides new knowledge on the nature of virulence of isolates collected from different areas in Kenya and possible sources of resistance to BBC. Further research to pyramid genes related to resistance to BBC in a view of developing resistant variety while genotyping of all existing BBC isolates is recommended.