University of Pretoria

Elucidating the Avocado / Phytophthora cinnamomi Interactions Project

Introduction
The Avocado Research Programme was established at the University of Pretoria in 2008 under the auspices of the Hans Merensky Foundation (HMF). This industrial–academic research partnership has become an integral part of the R&D Strategy of HMF and has benefitted both the university – by providing funds for world-class research and post-graduate student training – and the industry, through gaining access to new research trends and novel experimental techniques that facilitate enhancements in the avocado-breeding programme.

The HMF/FABI avocado research programme has to date graduated two PhD students, eight MSc and six BSc (Hons) students and published six peer-reviewed papers in ISI journals.  In 2015, the programme hosted four PhD, five MSc and two BSc (Hons) students, a research assistant and three post-doctoral fellows.  The team is culturally diverse with a good team spirit and work ethic.  Post-doctoral fellows play an important role in the mentorship of younger post-graduate students and this contributes to a positive learning environment.

The Hans Merensky Foundation has been the primary benefactor of the research and has invested R 8.47 million in two phases (2008 to 2011 and 2012 to 2016). To our knowledge this is the world’s largest research programme focussing on the avocado – Phytophthora cinnamomi interaction.

Research Programme Overview

Research Focus
Avocado is an important agricultural crop gaining increasing popularity in fresh produce markets throughout the world. Phytophthora cinnamomi is regarded as one of the most serious diseases of the fruit crop and has a large financial impact on the South African and worldwide avocado industry. Undoubtedly the most significant problem is the lack of total resistance against the disease and severe crop losses are reported in South Africa, Latin America, California and Australia.  This oomycete is notoriously difficult to control and strategies in avocado rely on an integrated approach of tolerant/resistant plant material, the use of phosphites, mulching and managing irrigation and soil moisture.

The research is aimed at understanding the basis of disease tolerance in avocado rootstocks against PRR.  The objectives are:

  • Understanding the avocado tolerance/resistance to PRR by identifying the host defence mechanisms in various rootstocks;
  • Elucidating flooding and salinity in avocado rootstocks and the effects these abiotic factors have in PRR;
  • Investigating Phytophthora cinnamomi, the causal agent of root rot in avocado; and
  • Establishing a platform to elucidate the avocado transcriptome.

Research has generated transcriptome sequence data for avocado infected with P. cinnamomi   as well as RNA-sequencing data for the zoospores and germinating cysts of P. cinnamomi.  The research programme has mined the sequence data for transcripts involved in the host defence response or pathogen virulence. Expression analyses of selected candidates are studied using quantitative RT-PCR and micro-array analysis and will also be complimented by protein profiling.  It however remains essential to conduct functional studies to validate the role of the selected transcript in either the host defence response or pathogen virulence and pathogenicity.

Research highlights for 2015 include:

Avocado:

  • Identification and characterisation of five putative NPR1 genes
  • Avocado microarray used to profile gene expression (i) in avocado infected with Rosellinia necatrix, (ii) tolerant and susceptible avocado infected with cinnamomi; (iii) avocado treated with salicylic acid, jasmoic acid and P. Cinnamomi, and (iv) P. cinnamomi-tolerant avocado exposed to flooding and infection.

Phytophthora cinnamomi:

  • Expression profiles for RxLR genes;
  • Identification of Crinkler protein inducing genes, and Glutanase inhibitor genes;
  • Microarray data for a set of 500 cinnamomi genes expressed during avocado infection; and
  • The cinnamomi / strawberry pathosystem as a tool for the functional characterisation of P. cinnamomi pathogenesis genes

Avocado genome sequencing project

Avogenome Consortium
In October 2016 a number of avocado researchers initiated a SKYPE working group to discuss important advances in avocado genomics research.  As a result of these positive discussions, mutual respect and previous collaborations amongst all we have now established the Avogenome sequencing project.  Members of this group will all share in the data that we generate under the Fort Lauderdale agreement.

The following researchers are involved in this initiative:

  • David Kuhn, USDA, Florida
  • Patricia Manosalva, UCR, California
  • Noëlani van den Berg, UP, South Africa
  • Sarah Mwangi, UP, South Africa
  • Antonio Javier Matas Arroyo, Departamento de Biología Vegetal, University of Malaga, Spain
  • Aureliano Bombarley Gomez, Virginia Tech Horticulture, USA
  • Randy Ploetz, University of Florida, USA
  • Alan Chambers, University of Florida, USA

Scope of the project
After several discussion and financial considerations we have decided to join forces to sequence the avocado genome. Four PIs from the group (Dr David Kuhn, Dr Patricia Manosalva, Prof Aureliano Bombarely Gomez and Prof Noëlani van den Berg) will form the core of this sequencing initiative.  Dr Sarah Mwangi, as our bioinformatician, will extensively be involved in the annotation of the genome alongside Prof Bombarely.  Based on Dr Mwangi’s assessment of the avocado genome data that we have from the Mexicans the group feels that the draft assembly is not of sufficient quality and we therefore propose to sequence a homozygous avocado, followed by the re-sequencing of two rootstocks, one from the UCR breeding programme and one from WTS.

We propose the re-sequencing of the genome of R0.06, a Phytophthora – tolerant avocado rootstock selected by Westfalia Technological Services.  The rootstock was selected as target because of its commercial importance to Westfalia Fruit and due to its performance in the field.  We propose a hybrid whole genome sequencing approach using Illumina and PacBio. The sequence data will be assembled and annotated by Prof Aureliano Bombarely Gomez and Dr Sarah Mwangi.

To facilitate the process of developing molecular markers for avocado we suggest that an avocado half-sibling breeding population be established on the Westfalia Fruit Estate, Tzaneen as well as in at least one other location in South Africa.  We also suggest that Dr Kuhn be involved in this process, as he has done a similar population in Florida for avocado fruit cultivars.

The second objective is to genotype avocado rootstocks and to determine parentage of valuable material.  A selection of 91 rootstocks from the WTS genepool block has been collected and will be analysed with the use of the Illumina SNP chip designed by Dr Kuhn, USDA, Florida.  An MSc student, Juanita Hanneman, has joined the group and will be supervised by Prof Noëlani van den Berg, Dr Sarah Mwangi, Dr David Kuhn and Prof Zander Myburg.

Finally, we would like to use the genome data of both avocado and Phytophthora cinnamomi for in depth bioinformatic analyses to aid in the identification of avocado defence targets and pathogen effectors.  Three P. cinnamomi genomes are now available through the Joint Genomes Institute and we feel that it is not necessary to sequence another genome.  The current amount of genome data is sufficient for our purposes.