Avocado /Pc Interactions

The HMF/FABI avocado research project at the University of Pretoria is almost 9 years old and has made a significant contribution to the international avocado community. Professor Noëlani van den Berg leads the research team that hosts the following researchers in 2015-2016: 3 Post-doctoral fellows, 3 PhDs, 5 MSc students and 1 BSc Hons student.  In line with the HMF vision, the majority of these students are South African. Four students are from previously disadvantaged groups and four are female.. During 2015 two BSc Hons and 1 PhD (Waheed Mahomed) graduated. Students in the programme are supported with bursaries from HMF and the NRF. In addition to HMF funding in 2013, the FTBP also leveraged funds from the NRF programme for Y-rated scientists and UP.  During 2015/6 two research papers were accepted for publication in ISI-rated journals (European Journal of Plant Pathology and BMC Plant Biology). Research results were presented as three papers and three posters at one international conference (World Avocado conference) and one local conference (Southern African Genetics Society) during 2015/6.

The FTBP’s approach to studying the avocado/Phtophthora cinnamomi interactions has been to study both host and pathogen genes involved in parthenogenesis.

Host genes:
A Ph.D. student, Robert Backer, has focused on studying the NPR family of genes in avocado Persia America.  Various plants overexpressing NPR1 show increased PR gene expression and pathogen resistance and mutants of this protein are more susceptible to virulent pathogens.

Similarly, a M.Sc. student, Tsakani Miyambo has chosen to focus on the identification and expression of avocado cell wall genes that encode, polygalacturonase inhibitory proteins (PGIPs) which aid in defense mechanisms against pathogens.  PGIPs, are proteins secreted by the host plant for defense, to reduce the hydrolytic activity of the polygalacuronases (PGs) secreted by the pathogen, P. cinnamomi.  Tsakani is also attempting to identify potential PGs.

Other studies are focussed on the identification of physiological resistance mechanisms to Phytophthora cinnamomi and the identification of physiological and genetic markers associated with those mechanisms (Michael Bufe, M.Sc. year 3) and the biological difference between tolerant and susceptible avocado upon infection with P. cinnamomi (Brittany Mitchell, M.Sc. year 2).

Pathogenesis related genes in P. cinnamomi:
A Ph.D. student, Buyani Ndlovu, is doing research on the functional characterisation of putative RxLR effector genes from Phytophthora cinnamomi.  The approach is to clone the putative effector genes and perform functional characterisation through transient expression in Nicotiana benthamiana leaves.  A M.Sc. student , Melissa Joubert, is focused on the functional characterisation of RxLR effector genes.

Similarly, a M.Sc. student, Mohamed Seedat is focusing on the identification and profiling of glucanase inhibitor and Crinkler (CRN) genes from P. cinnamomi.

A research technician, Juanita Engelbrecht, is focused on developing microsatellite markers for P. cinnamomi.  Molecular markers have been widely used to study plant pathogen populations. One of these markers which have been used intensively by various studies is simple sequence repeats (SSR) marker. SSR markers are short tandem repeats present in the genome of organisms, which are also referred to as microsatellite regions or microsats.

Student training remains a key priority of the project outcomes. Younger students in the group are mentored by the post-doctoral fellows and Juanita Engelbrecht. This ensures that we maintain continuity in terms of molecular techniques that are utilised in different projects. Students have attended workshops and training courses in statistical analysis, bio-informatics and quantitative RT-PCR data analysis.  Another highlight for 2016 is the launch of our HMF intern programme.  This programme aims to identify undergraduate students that have the academic ability and passion to excel in science. Three interns, Rachel Mokwasi, Lise Elliot and Leandri Bezuidenhout, are mentored by Juanita Engelbrecht whilst helping out in the laboratory. Interns receive a bursary of R20 000 per year and in return work a set number of hours as laboratory assistants.

Dr Nicky Taylor has joined the team and is the main supervisor of a MSc project focused on the physiological responses in avocado infected with P.  cinnamomi.

Mid-Term Progress Report May 2017

Avocado-Phytophthora cinnamomi interation

  1. Molecular cloning and functional characterisation of NPR1-like genes from Persea americana (Mill.) – Robert Backer
  • Cloning of NPR1 genes are being optimized.
  • Trials are underway to complete the replicate trials for the benthamiana-Pc system
  • Avocado clonal plants are acclimatizing for the final Pc infection for the confirmation of the expression of NPR1-like genes
  1. Functional characterization of putative RxLR effector genes from Phytophthora cinnamomi. – Buyani Ndlovu
  • 192 RxLR genes were identified from the Pc genome of which 15 have been selected, primers designed and amplified from Pc-infected avocado plants
  • Cloning of the GFP-FLAG construct has been successful
  • Currently we are optimizing the insertion of the RxLR genes into the plasmid via the GATEWAY cloning strategy
  1. Identification of CRN effector genes in Phytophthora cinnamomi during avocado root infection. – Mohamed Seedat
  • Dual RNA sequencing of both tolerant and susceptible avocado infected with Pc has been completed and the data arrived in February 2017.
  • Bio-informatic analysis of the data is underway under the supervision of Dr. Sarah Mwangi. CRN genes will be analyzed for their expression
  • CRN genes have also been identified from public databases to compare to those found in our data
  1. Early physiological responses of three avocado rootstocks to infection with Phytophthora cinnamomi. – Michael Bufe
  • Plant trial for the assessment of callose, ROS and lignin has been completed and plant material harvested and stored
  • The callose assay has been optimized
  • The MDA assay for the quantification of ROS has been optimized
  1. In silico identification and characterization of polygalacturonases (PGs) in Phytophthora cinnamomi and polygalacturonase-inhibiting proteins (PGIPs) in Persea americana during the PG-PGIP interaction. – Tsakani Miyambo
  • 18 full-length PGs from Pc and 2 full-length PGIPs from avocado have been identified using bio-informatic tools
  • an additional 156 PGs from other oomycetes and fungi were identified and used along with the 18 PGs from Pc to conduct a phylogenetic tree to aid in the final selection of candidates for downstream application
  • PGIP1 and PGIP2 were successfully amplified from avocado material and preliminary sequencing confirmed the identities
  • PGIP1 was then cloned and sequenced and the cloning and sequencing of PGIP2 is underway 
  1. Developing a novel patho-system for the molecular study of Phytophthora cinnamomi using Nicotiana benthamiana. – Melissa Joubert
  • Trials infecting both the roots and leaves of young plants with Pc are currently underway
  1. Evaluating the susceptibility of avocado rootstocks to Rosellinia necatrix. – Jesse Hartley
  • White fungal growth of avocado trees was isolated, morphologically identified and confirmed by sequencing to be Rosellinia necatrix
  • Growth studies on different media have shown that optimal growth occurs on PDA. Similar temperature studies are underway.
  • We have optimized an inoculation method using sterilized chop sticks that were co-cultivated with necatrix. These were then placed in the soil around the avocado stem.  Disease symptoms and fungal growth were present and Koch’s postulates were confirmed
  • Other necatrix isolates were sourced from Spain, Israel and Stellenbosch and DNA was extracted.
  1. Determining the cinnamomi load in N. benthamiana leaves at two time points after inoculation. – Michael du Toit
  • DNA has been extracted from the lab strain of Pc
  • Pc was assessed under the microscope to confirm the identity
  • LPV3 PCR amplification was conducted to verify the size of this Pc specific gene
  • Other Phytophthora species from clade 7 were sourced from CBS and were plated out for DNA extraction
  1. Proteomics analysis of a tolerant avocado inoculated with cinnamomi. ­– Dr. Ashok Prabhu
  • Avocado samples infected with Pc have been used to optimize the protein extraction method for down-stream application in label-free quantitative proteomic analysis. This will be done in collaboration with Dr. Stoyan Stoychev from the CSIR
  • Avocado clonal plants have been re-planted and will be used to generate the samples for the quantitative proteomics study
  1. Development of SSR markers for Phytophthora cinnamomi – Juanita Engelbrecht
  • 16 SSR markers were used to analyse the genetic diversity if 220 Pc isolates from South Africa
  • cloning and sequencing if the different alleles are currently underway 
  1. Dual-RNA Sequencing analysis of the avocado- cinnamomi interaction. – Dr. Sarah Mwangi
  • The data has been received from Novogene and basic bio-informatics is underway to assess sequencing quality.
  • CRN genes, PGs, PGIPs, RxLRs and aquaporins are the specific transcripts that are currently being focused on.

Outputs for 2017

  • Melissa Joubert obtained her BSc (Hons) Cum Laude and was also awarded the prize for the best honours student in the Department of Microbiology
  • Ashok Prabhu and Tsakani Miyambo presented their research as posters at the SASPP conference in January 2017
  • Juanita Engelbrecht presented a paper at the IUFRO working group on Phytophthora in natural forests and ecosystems in Vietnam in March 2017

Publications submitted:

  • Van den Berg, N., Christie, J.B, Engelbrecht, J. & Aveling, T.A.S. Callose and reactive oxygen species combat Phytophthora cinnamomi in a tolerant avocado rootstock.

The FTBP has also undertaken to perform genomic DNA sequencing of important avocado strains as proposed below.