Peronospora sparsa biology and drivers of disease epidemics in boysenberry

Abstract

Downy mildew, caused by the biotrophic oomycetes, Peronospora sparsa, is a major disease of boysenberries, with crop loss from disease for conventional growers being up to 45% and for organic growers up to 100% in some years. Most boysenberry plant material (including tissue culture propagated plants) are systemically infected with the pathogen. Disease expression by sporulation of naturally infected leaves, stems/canes and calyxes at 15ºC under high humidity in the field was identified as the source of inoculum in the wider Nelson area in 2010 and 2011 with peak spore dispersal in mid-November. A strong relationship between rainfall pattern, humidity and temperature and P. sparsa spore dispersal was observed. Spore dispersal was triggered by the frequency (%) of rainy days, RH and warm temperatures (16-23ºC) in early spring where early wet periods with high moisture levels promoting sporulation and a subsequent dry period allowed spore release. In vitro, the optimum temperature for spore germination, infection, sporulation and lesion expansion were 20ºC (24 h darkness), 15 or 20ºC (12 h/12 h light/ dark), 15 or 20ºC (12 h/12 h light/ dark) under high humidity, respectively. The optimum spore numbers for infection was 200. Similarly in vivo evaluations showed that disease expression on young foliage, stems/canes, calyxes/sepals, petals and stamen by symptoms and sporulation was favoured at temperatures ranging from 5-15ºC, under high relative humidity (90-100%) in potted systemically infected boysenberry plants. An existing nested PCR for detection of P. sparsa was optimised and limits of detection determined in different plant tissues. The optimised Plant & Food Research (P&FR) protocol (using modified Aegerter buffer) extracted more P. sparsa DNA with a higher purity than the commercial PowerPlant® DNA isolation kit. The optimised nested PCR could detect as little as 0.4 pg of P. sparsa genomic spore DNA from a range of asymptomatic boysenberry tissues including primocane tips, leaves, leaf buds, canes/ stems, roots, flower buds, flowers and berries. This was approximately equivalent to 40 spores. The method was improved to a more rapid and robust one step nested PCR method with an equivalent sensitivity for detecting latent infection of P. sparsa. The results showed that the most robust tissues for reliable detection of latent infection were root or crown. In vitro and in vivo evaluations indicated that dryberry is caused by spore infection of flowers or berries by the spores produced on systemically infected canes/ stems, calyx, and petals under favourable environmental conditions. Accordingly two infection pathways resulting in dryberry were identified: 1) spores produced on petals and calyx may infect pollen followed by fertilisation of infected pollen then systemic infection of the drupelets of the developing berry initials, 2) spore infection of drupelets at the red partially ripe stage. Movement of the pathogen from the systemically infected cane to berries across the pedicel and calyx was not observed. Of the ten fungicides investigated in vitro, chlorothalonil, mandipropamid, fluazinam, azoxystrobin and dimethomorph were the most effective to inhibit P. sparsa spore germination and infection. In vivo evaluations on young disease-free boysenberry plants showed that dimethomorph, azoxystrobin and metalaxyl-M+mancozeb were the most effective at protecting leaves from infection. Three applications of phosphorous acid (PA) reduced both incidence and severity of dryberry whereas, acibenzolar-s-methyl reduced only incidence in systemically infected plants. A tissue culture protocol was developed for the production of P. sparsa pathogen free clean boysenberry propagation material by heat alone. As this method does not rely on treatment of infected plants with fungicides, there is no subsequent risk of fungicide resistant strains developing. Distribution of clean boysenberry planting material in new gardens or replanting areas in existing gardens in New Zealand is an important initial step to avoid disease epidemics in the field.