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

[摘要] Preface: Avocado (Persea americana Mill.) is a popular and nutritious crop and of great importance tothe global fruit industry. One of the major problems encountered is the susceptibility ofavocado trees to Phytophthora root rot (PRR) caused by the soil-born oomycetePhytophthora cinnamomi Rands. Control is achieved through an integrated control strategythat consists of mulching, chemical control in the form of phosphite injections and the use ofresistant rootstocks. Emphasis is now placed on the use of resistant rootstock varieties tocombat this devastating disease. Avocado breeding programmes around the world haveprovided 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 certainrootstocks display high levels of tolerance against P. cinnamomi and others not. Selectingresistant rootstocks is a time consuming process that can take up to 25 years. Due to thelack 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) toinvestigate the expression profiles of selected defense-associated genes from five avocadorootstocks 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 avocadovarieties.Chapter 1 entitled Plant defense mechanisms against Phytophthora provides an overview ofplant defense responses against Phytophthora and where possible specifically against P.cinnamomi. General plant defense concepts including pathogen triggered immunity andspecific effector triggered immunity is discussed. Both these immune responses are linked toother key players that regulate specific signalling pathways in order to achieve an effectivedefense response. Mechanisms involved in defense that are discussed include cell wallreinforcements, production of ion fluxes and ROS species, MAPK and phosphorylationcascades, rapid induction of defense genes, accumulation of defense-related proteins including phytoalexins and PR proteins which all negatively affect colonization of potentialpathogens.Chapter 2 reports on the establishment of two reliable small plant inoculation systems forstudying plant disease development in the greenhouse followed by subsequent molecularstudies. Briefly a hydroponics system was evaluated to allow easy access to root material forRNA extractions followed by quantitative PCR and the second solid system was evaluatedusing perlite and vermiculite as growth substrates to assess disease development andseverity 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 avocadorootstocks after infection with P. cinnamomi. The expression of each individual gene wasassessed 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 withrespect 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 toleranceagainst this soil-borne oomycete. A nested primer set was developed for the Lpv gene thatresulted in a P. cinnamomi – specific, sensitive assay that can be utilized to assess rootstocktolerance.The thesis concludes with a discussion (Chapter 5) on all the data generated during thecourse of this study, our findings and recommendations.

[发布机构] University of Pretoria