Population structure analysis of phytophthora species infecting black pepper

Abstract

Black pepper is among the highly valued spice crops worldwide, but several diseasesravage its cultivation. Foot rot is one of the catastrophic diseases caused by adevastating pathogen, Phytophthora, which leads to a severe outbreak if leftunnoticed. In the present study, the population structure of P. capsici and P. tropicalisinfecting black pepper from black pepper growing areas in Kerala, Karnataka, TamilNadu and Goa were studied by characterizing the isolates viz., species identification,distribution and mating type. The isolates obtained were analyzed by PCR assay usingYpt1 gene-based species-specific and ITS primers for species identification. Theresults showed that P. capsici and P. tropicalis were the species that were mostassociated with the disease, though other species were also observed infrequently. Themating type analysis further revealed that all the isolates belong to A1 mating type.Genome size analysis ensures the robustness of the genetic variation that exists withinthe population. An extensive comprehension on the genome size would unravel theadaptive ability of the pathogen to extreme environmental conditions, increasedpathogenicity and development of fungicide resistance, which would help instreamlining the management approaches. Here, we adopted one of the most reliabletechniques, i.e., flow cytometry. It was observed that P. capsici had slightly greatergenome content than that of P. tropicalis. Further, some of the phenotypic characterswere analyzed and correlation with the genome size was studied, and it was observedthat although there was no significant positive correlation between the variables, aslight negative correlation was observed between the mycelial growth and virulence.However, the pathogens were sensitive to the commonly recommended fungicides todate.The genetic diversity of Phytophthora isolates was resolved using RAMS (RandomAmplified Microsatellites) and REP (Repetitive Extragenic Palindromic)-PCRfingerprinting. The population parameters were studied using POPGENE version 1.32software, which showed the extent of variation that existed within the population.Based on the scoring data of the band produced by RAMS and REP-PCR, UPGMAdendrogram was plotted, which broadly divided the isolates into four clusters. P.capsici and P. tropicalis were grouped in separate clusters, i.e., in sub-clusters I, II andIII, and IV, respectively. This was also confirmed by PCoA analysis. The isolatesdisplayed varied morphology irrespective of the clusters generated, and further, thecross-infectivity analysis showed that the black pepper isolates were capable ofinfecting crops like tomato, cucumber, pumpkin, nutmeg and chilli and a few isolatesinfected cardamom in vitro. No symptoms were observed in vanilla, coconut andarecanut.Haplotype analysis was carried out using mitochondrial (Cox1, Cox2, Nad1 andNad5) and nuclear genes (β-tubulin, EF-1α, Enolase, HSP90, TigA and Ura3). Themorphological characterization of isolates based on species was found to be unreliabledue to the presence of coinciding characters between the species. Various geneticdiversity parameters and demographic studies were performed using DnaSP v6.12.03.Among the isolates studied, the greater number of haplotypes was observed for EF1-αand that of Nad1 and Ura3 were less. The haplotype network analysis performed withthe black pepper isolates and P. capsici infecting diverse hosts from Hawaii and someof the contiguous United States showed that the Indian population is rooted in the USpopulation. Further, the population bottleneck event and its restoration were revealedby the demographic analysis.This study also attempted to study the expression of various RxLR effectors by real-time PCR and two of the highly expressed RxLR genes during the infection, i.e.,RxLR29 and RxLR132, were selected for further studies. The three-dimensionalstructures of two of the RxLR proteins were resolved using homology modelling.Moreover, its interacting counterpart was selected based on earlier reports andprotein-protein docking studies were performed. The results showed the existence ofbiologically feasible interactions between the proteins, i.e., RxLR29 and DRB4 andRxLR132 and CMPG1.