2019-11_18-006 – Understanding the underlying biology of citrus black spot for improved disease management

Understanding the underlying biology of citrus black spot for improved disease management

Report Date: 12/11/2019
Project: 18-006   Year: 2019
Category: Other
Author: Megan Dewdney
Sponsor: Citrus Research and Development Foundation, Inc.

Sept 2015
Objective 1: Evaluate the optimal spray timing for Florida and investigate if tree skirting or alternative products improves fungicidal control of citrus black spot.
Objective 3: A MAT-1-1 isolate may enter Florida and allow for the production of ascospores. The industry needs to know if this happens, as it will affect management practices. Additionally, the existing asexual population may be more diverse than currently measured. If multiple clonal linages exist, then there may be different sensitivities to fungicides or other phenotypic traits. We also need to determine whether P. paracitricarpa or P. paracapitalensis are present in Florida for regulatory concerns due to misidentification. We plan to survey for the MAT-1-1 mating type, unique clonal lineages, and two closely related Phyllosticta spp.

We are still waiting for symptom experssion in the fungicide and skirting trials. As explained in the previous quarterly report, we expect symptom expression in early 2020 as Valencia, a late harvested cultivar, start to ripen. We have marked off products that should not be harvested and have occassionally visited the trials to make sure that no symptoms have started to express unexpected. None have been observed so far.

The subcontract with CRI is finally complete. Despite this, 23 isolates were sequenced (6 from South Africa and 17 from the USA) using the Ion Torrent System. The genomes of all the isolates have been successfully assembled and analysed using a customised bioinformatics pipeline. Previous genotypes obtained with SSR primers were confirmed and new SSR primers were developed in silico. To date, mapping and SNP variant statistics as well as in silico genotyping data revealed significantly less variation between the USA isolates than between the isolates from South Africa.

To investigate the fine-scale genetic differences within the USA P. citricarpa population, the assembled genomes were annotated by mapping the reference genes to the assembled contig sets, using GMAP. The variant calling results together with the annotations were further analysed using SNPeff, to detect putative variable genes. In silico detection of mating types were also performed, and confirmed that only one mating type is present in the USA.

Five more South African isolates is in the process of being sequenced. For some of the isolates selected for the next round of sequencing, the available DNA are not of high enough quality, and new cultures of these isolates are grown and DNA will be extracted.

New fungicide products are being tested for the management of CBS in South Africa. Products being evaluated include Enable (copper hydroxide) sprayed in alternation with Amistar Top, Luna Sensation or Headline, which is also tested in alternation with copper hydroxide. Fungicides are applied every 4 weeks from October 2019 until March 2020. Additional fungicides including Miravis, Miravis Top, Ph-D, Priaxor and Luna experience could not be tested due to their unavailability in South Africa. The fungicides are being tested in a ‘Valencia’ orange orchard with a history of CBS.

Within Objective 3 (Survey for the MAT-1-1 mating type and two closely related Phyllosticta spp.), the culture collection of P. citricarpa isolates from Florida which contains more than 125 single-speed isolates has been plated and individual isolates cultured for DNA extraction. Thirty-six isolates have been extracted and high quality DNA purified. PCR conditions for amplifying the tef1 sequence from Phylosticta spp. are currently under evaluation and optimization. Amplicons will be sequenced and compared to determine if the cryptic P. paracitricarpa and P. paracapitalensis species exist within our collections. In regards to screening for matting type, all isolates from our current collection have been screened and determined to be of the MAT1-2 mating type. Screens are ongoing at the Division of Plant Industry to evaluate mating types of new isolates coming from outside of the current quarantine zone and we are coordinating with Dr. Hector Urbina and colleagues to monitor the findings. Beyond Florida, permits have been approved from both the US and Cuba to receive extracted DNA samples from the Cuban collection of P. citricarpa isolates. DNA representing these isolates will be screened for mating type and used later for analysis of global P. citricarpa population structure. Additional efforts have been focused on determining the role of fruit developmental etiology on susceptibility to CBS. A quarantine greenhouse experiment has been established with fruit-bearing Myer lemon trees. Eighty-six fruit of varying developmental stages as well as controls have been inoculated. We are currently monitoring for symptom development and collecting data on environmental parameters and fruit development.

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