Pathogenicity and virulence of south African isolates of Venturia inaequalis

Venturia inaequalis isolates (causing apple scab) vary in pathogenicity when infecting common susceptible apple cultivars. This quantitative effect may be due to variation in the pathogen's virulence factors. South African V. inaequalis isolates were collected in seasons 2012/13 and 2013/14 from various cultivars in the four principal apple growing regions and were characterized using phylogenetic analysis. A partial sequence of the candidate virulence factor ABC2-NB2 gene region was analysed; this gene region was highly polymorphic with six possible haplotypes, and non-synonymous changes were identified. Most of the SA isolates had the amino acid changes V1005I (41%) or S971P (47%) and 5% had two amino acid changes, namely serine to proline (S971P) and arginine to cysteine (R957C). None of the SA isolates were identical to the ABC-NB2 gene Genbank reference sequence. The ABC2-NB2 gene region was found to be under positive/diversifying selection pressure indicating its potential role as virulence factor, while other parts of the gene were under negative selection. The 15 V. inaequalis isolates differed with respect to their conidia production on the media and their germination rate. Isolates LOSL 206-1 and KCL 3-1 produced the most conidia, with 1.5 x 10(6) and 1 x 10(6) spores/mL, and they were among those of the highest viability, with germination rates of 92% and 82%, respectively. Pathogenicity testing with conidiospore inoculum from these single spore isolates on common cultivars 'Fuji', 'Golden Delicious', 'Royal Gala' and the rootstock 'M793' showed that not all isolates could infect and sporulate on all the cultivars. Some single spore isolates caused chlorotic spots without any sporulation and may have presented a delayed sporulation if incubated for 21 days. A qPCR method was evaluated as a molecular tool to quantify severity of apple scab infection. The amount of fungal DNA in the leaf tissue, indicating variation in virulence, was quantified using qPCR after single spore inoculations during 2015 on 'Golden Delicious', 'Royal Gala' and 'M793' at 7 and 14 days after inoculation. The fungal DNA concentration was lower in inoculations of 'Golden Delicious' and 'Royal Gala' and higher in 'M793'. The single spore isolate LOSL 206-1, from 'Cripps Red', was the most virulent isolate on 'Royal Gala' and isolate OBL 210-1, from 'Braeburn', the most virulent on 'M793'. Isolates that caused chlorosis also gave high amounts of fungal DNA. This study concludes that the single spore isolates differed in their virulence and infection ability with respect to different apple cultivars and the qPCR method reliably detected V. inaequalis DNA in asymptomatic Malus tissue.