WINETECH Technical Yearbook 2019

The objectives of the study were: • To adapt existing molecular quantification methods to directly quantify BFD and CRR pathogen DNA in grapevine nursery soils over a three-year period, and • To i so l ate these pathogens f rom grapevines, weeds and rotation crops in these nurseries over the same three-year period. METHODOLOGY AND RESULTS In 2013, soil and grafted grapevine cuttings were collected from five nurseries. In 2014, rotation crops and weeds were collected from the same nurseries at approximately the same sites within the nursery. In 2015, soil and grapevine samples were collected again from the five nurseries. The soil samples were taken at two depths (0-30 cm and 30-60 cm), 10 cm away from the grapevines that were sampled (Photo 1). Soil analyses were conducted on the soil sampled. OBJECTIVE 1: QUANTIFY BFD AND CRR PATHOGEN DNA IN GRAPEVINE NURSERY SOILS Total DNA extractions were done on the soil samples. The pathogen DNA in these total soil DNA samples were then quantified using optimised Quantitative Real-Time Polymerase Chain Reaction (qPCR) protocols for the detection of Phytophthora species, Pythium irregulare , and Dactylonectria and Ilyonectria species. qPCR is a molecular technique used to detect and quantify

target DNA in a sample (soil), while the DNA amplification can be viewed in real- time. Dactylonectria and Ilyonectria species, Py. irregulare and Phytophthora DNA was successfully detected, and quantified from grapevine nursery soil. The Dactylonectria and Ilyonectria DNA was detected in all nursery soil samples, except for one site in one nursery, and ranged from 0.04 pg.μL -1 to 37.14 pg.μL -1 . The Py. irregulare was detected in all nurseries (though not for all sites with 22 of the sites testing negative over the three-year period of sampling) with the DNA concentration in the soil that ranged from 0.01 pg.μL -1 to 3.77 pg.μL -1 . Phytophthora DNA was detected in all samples except for three sites in one nursery in 2013. The Phytophthora DNA concentrations ranged from 0.01 pg.μL -1 to 29.53 pg.μL -1 . Pathogen DNA concentrations increased over the three years for four of the nurseries. OBJECTIVE 2: ISOLATE BFD AND CRR PATHOGENS FROM GRAPEVINES, WEEDS AND ROTATION CROPS FROM NURSERIES Pathogen (fungal and Oomycete) isolations were done from the grapevine cuttings, weeds and rotation crops. Isolations were made from the roots and vascular tissue of the grapevines onto potato dextrose agar, and Pythium and Phytophthora selective media. The fungal and Oomycete cultures were identified using Polymerase Chain Reaction (PCR), and sequencing of two gene regions (ITS-rDNA and histone H3).

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PHOTO 2. Isolations were made from visually healthy vines. A longitudinal cut through the base of the vine clearly shows the typical brown discolouration due to infection of black foot pathogens (a). A transverse cut through the rootstock shows brown water soaked discolouration associated with black foot (b).

has been reported that some Phytophthora spp. may even infect the crowns and cause cankers which may girdle grapevine trunks. This girdling may then lead to plant collapse. The infected vines may be isolated or occur in small groups. CRR pathogens can produce sexual oospores that can persist in the soil. Nursery soils in South Africa are known as an inoculum source for BFD and CRR pathogens. The treatment of BFD is limited to cultural

practices, such as hot water treatment, as there are no fungicides registered against this disease. However, the treatment for CRR involves cultural practices, as well as the use of phosphonate fungicides. The extent to which BFD and CRR pathogens are present in nursery soils is not known. Also the influence of rotation crops on the occurrence of BFD and CRR has not been investigated, and could hold potential in an integrated control strategy.