Genetics and ecology of Pseudomonas syringae pathovars in New Zealand cherry orchards : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

The research presented in this thesis investigated the genetic diversity, pathogenicity and ecology of Pseudomonas spp. isolates through a population study in sweet cherry orchards within New Zealand. This is the first detailed study of the P. syringae species complex focusing only on sweet cherry in New Zealand. The research is the first worldwide to look at the dynamics of P. syringae pathovars in cherry buds in commercial cherry orchards using qPCR. A total of 250 P. spp. isolates were collected from symptomatic and asymptomatic tissue from commercial sweet cherry orchards in Central Otago, New Zealand in 2015. Isolates were classified into strains belonging to three different taxonomic groups by determining the phylogeny of the gltA gene for all the strains and the Multi Locus Sequence Analysis (MLSA) of four housekeeping genes for 35 strains. Pathogenicity tests on different plant tissues were carried out and virulence associated factors determined to further characterise the strains. The two main taxonomic groups were P. s. pv. syringae (Pss) and P. s. pv. morsprunorum race 1 (Psm1), in Phylogroup 2 and Phylogroup 3 respectively (Berger, 2014). The third group comprised non-pathogenic strains classified as P. spp. It was concluded that Pss is the predominant pathovar in Central Otago sweet cherry orchards. Pss inoculated on immature cherry fruit produced black, necrotic lesions, whilst those produced by Psm1 were water-soaked. Lesions on fruit inoculated with Pss strains increased rapidly in size, having a larger area under the disease progress curve (AUDPC) at 10 days post inoculation (dpi), compared with fruit inoculated with Psm1 strains. Symptoms on leaves infiltrated with Pss appeared within 1-dpi. At 7-dpi, the necrosis caused by most Pss strains had extended considerably from the inoculation site and into the leaf veins. Comparatively, necrosis caused by Psm1 strains remained restricted to the inoculation site. On 1-year-old cherry shoots, the smallest lesions occurred with strains classified as non-pathogenic P. spp. The largest lesions were associated with Pss and Psm1 strains, which did not statistically (p>0.05) differ. Pss and Psm1-specific PCR and qPCR protocols were developed and standardised to detect each pathovar from sweet cherry tissue obtained from commercial orchards. The specificity of each protocol was assessed for Pss and Psm1 strains and from closely related P. spp. from Prunus species and other hosts. The sensitivity of the protocol was evaluated with inoculated cherry leaf and bud tissue, and naturally infected cherry buds. Both PCR and qPCR were highly specific and detected Pss and Psm1 from sweet cherry. The specific Pss and Psm1-qPCR reactions were able to detect 102 cfu/per reaction from inoculated and naturally infected cherry buds. The optimized qPCR protocols provided a robust method to detect and quantify each pathovar from naturally infected asymptomatic cherry buds. The developed duplex PCR method was effective at detecting both Pss and Psm1 pathovars within the one reaction, providing a fast and cost effective detection method. The optimized protocols were used to study the disease epidemiology in cherry orchards. This study provided evidence that the colonization of cherry buds by Pss and Psm1 in Central Otago orchards occurs mainly through external sources soon after buds form and long before leaf fall. Cherry buds were shown to be colonised by Pss and Psm1 soon after they had formed, and the bacterial population had established by the time the bud scales lignified, which defined the overall bud infection for the season (i.e. there was not subsequent infection of the buds). The predominant pathovar on cherry buds was Pss. Both pathovars occurred commonly together in cherry buds in 2017, but not in 2018 and 2019. The knowledge generated in this study will inform the development of sustainable strategies, to determine optimal timing for control and to help with the development of much-needed new control tools for plant diseases caused by P. syringae.