Studying the interaction between Phytophthora cinnamomi and Persea americana using gene expression profiling and pathogen quantification

Abstract

Preface: Avocado (Persea americana Mill.) is a popular and nutritious crop and of great importance to the global fruit industry. One of the major problems encountered is the susceptibility of avocado trees to Phytophthora root rot (PRR) caused by the soil-born oomycete Phytophthora cinnamomi Rands. Control is achieved through an integrated control strategy that consists of mulching, chemical control in the form of phosphite injections and the use of resistant rootstocks. Emphasis is now placed on the use of resistant rootstock varieties to combat this devastating disease. Avocado breeding programmes around the world have provided growers with a selection of highly tolerant rootstocks such as Duke 7 and Dusa®. Despite the availability of tolerant material it has not yet been established why certain rootstocks display high levels of tolerance against P. cinnamomi and others not. Selecting resistant rootstocks is a time consuming process that can take up to 25 years. Due to the lack of research on the avocado/Phytophthora interaction, the aim of this dissertation was i) to establish a pathogenicity system that could be used to study the interaction ii) to investigate the expression profiles of selected defense-associated genes from five avocado rootstocks upon P. cinnamomi infection and lastly to develop an assay that could detect P. cinnamomi in planta that could be applied to aid in the selection process of tolerant avocado varieties. Chapter 1 entitled Plant defense mechanisms against Phytophthora provides an overview of plant defense responses against Phytophthora and where possible specifically against P. cinnamomi. General plant defense concepts including pathogen triggered immunity and specific effector triggered immunity is discussed. Both these immune responses are linked to other key players that regulate specific signalling pathways in order to achieve an effective defense response. Mechanisms involved in defense that are discussed include cell wall reinforcements, production of ion fluxes and ROS species, MAPK and phosphorylation cascades, rapid induction of defense genes, accumulation of defense-related proteins including phytoalexins and PR proteins which all negatively affect colonization of potential pathogens. Chapter 2 reports on the establishment of two reliable small plant inoculation systems for studying plant disease development in the greenhouse followed by subsequent molecular studies. Briefly a hydroponics system was evaluated to allow easy access to root material for RNA extractions followed by quantitative PCR and the second solid system was evaluated using perlite and vermiculite as growth substrates to assess disease development and severity in three avocado rootstocks with varying levels of resistance to PRR. In Chapter 3 the role of seven defense related genes were investigated in five avocado rootstocks after infection with P. cinnamomi. The expression of each individual gene was assessed over seven time points ranging from 0 to 72 hours using quantitative RT-PCR. Data were analyzed statistically to highlight differences amongst the five rootstocks with respect to their gene expression against the pathogen. Chapter 4 describes the development of a nested quantitative PCR that quantifies P. cinnamomi in planta in two avocado rootstocks displaying different levels of tolerance against this soil-borne oomycete. A nested primer set was developed for the Lpv gene that resulted in a P. cinnamomi – specific, sensitive assay that can be utilized to assess rootstock tolerance. The thesis concludes with a discussion (Chapter 5) on all the data generated during the course of this study, our findings and recommendations.