Identification and characterisation of Phytophthora spp. in New Zealand apple orchards : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

Apples, a prominent and widely favoured fruit crop on a global scale, are traded internationally and carry substantial economic importance worldwide. They play a vital role in New Zealand’s agriculture sector, with the World Apple Review consistently ranking the New Zealand apple industry as the most competitive in the world. Apple production encounters various challenges notably the adverse effects of pests and diseases, with Phytophthora crown and root rot being an important soil borne disease in all apple growing regions. Worldwide the disease is primarily caused by Phytophthora cactorum, although other species of Phytophthora have also been associated with crown rot of apple. The aim of this study was to determine the identity of Phytophthora species associated with apple orchards in New Zealand and explore their genetic structure and relative pathogenicity. Furthermore, the impact of two commercial microbial inoculants, arbuscular mycorrhizal fungi and Trichoderma species on the susceptibility of two apple rootstocks to P. cactorum infection in a whole-plant pathogenicity assay was evaluated. In Chapter 2 a survey was conducted to determine the Phytophthora species in the soils of apple orchards. The survey sampled soils from 12 different apple orchards of the five main apple growing regions in New Zealand: Hawke’s Bay (5 orchards), Tasman (4 orchards), Waikato (1 orchard), Otago (1 orchard) and Canterbury (1 orchard). Soil samples were obtained from blocks of the main orchard sites, with apple trees representing different cultivars and ages groups (young, medium and mature). The sampling included 11 orchard blocks from Hawke’s Bay, 13 in Tasman and 2 each from Otago, Waikato and Canterbury, resulting in a total of 30 blocks. The Phytophthora species were recovered by baiting using healthy Himalayan cedar needles (Cedrus deodara), lupin radicles (Lupinus polyphyllus) and apple cotyledons as baits. Lupin was the most effective bait, recovering 147 Phytophthora spp. isolates compared to apple cotyledon and cedar needle recovering 72 and 52 Phytophthora spp. isolates, respectively from the Hawke’s Bay, Tasman, Otago and Waikato samples (Canterbury samples baited with pine and rose). The 280 Phytophthora isolates recovered were identified based on combined results of morphology and DNA sequencing, with a total of seven species identified. These were Phytophthora cactorum, P. cambivora, P. megasperma, P. plurivora, P. rosacearum, P. chlamydospora, P. cryptogea and isolates with unresolved identity (Phytophthora spp.). The most prevalent species was P. cactorum. In Chapter 3, the genetic diversity within the P. cactorum population was determined using random amplified microsatellites (RAMS) and universally primed polymerase chain reaction (UP-PCR) primers and relative aggressiveness of the recovered Phytophthora species assessed using detached tissue assays. The 59 P. cactorum isolates were placed into five major groups of 3, 16, 9, 9 and 20 isolates each with two isolates each placed in groups by themselves, with the P. cactorum population in the orchards dispersed in the different groups. From the genetic diversity analysis isolates of P. cactorum and representative isolates of the different species recovered were selected and their aggressiveness assessed used a developed detached root assay and an existing shoot assay. All Phytophthora species and P. cactorum isolates tested were virulent on the apple rootstocks tested, with rootstock MM106 being more susceptible than M9 and M26. There was no correlation between the genetic diversity and aggressiveness. In Chapter 4 the pathogenicity of four P. cactorum isolates on whole apple plants was determined on MM106, M26 and M9 rootstocks. The rootstocks were inoculated by dipping in 1 × 105zoospores per mL or using 50 g colonized oats grain into the planting hole. All four P. cactorum isolates caused disease on all three apple rootstocks, with there being no difference in the disease score between rootstocks or isolates irrespective of the type of propagule used. In Chapter 5 the effect of inoculation with AMF and/or Trichoderma on susceptibility to P. cactorum infection and overall growth of young rootstock plants was investigated. Rootstocks M9 and MM106 were inoculated with AMF, Trichoderma and combined AMF and Trichoderma, and, after 60 days growth, inoculated with P. cactorum. At harvest, the AMF and/or Trichoderma inocula had no effect on P. cactorum disease level on either rootstock. However, in the absence of P. cactorum, inoculation with both Trichoderma and AMF tended to enhance growth compared to the control. Overall, this study has identified the Phytophthora diversity in New Zealand apple orchards and confirmed the pathogenicity of the recovered species, with particular emphasis on the dominant species, P. cactorum. The findings highlight that further studies are warranted including investigation of management strategies to reduce the potential threats posed by these pathogens.