Development of an in vitro assay to screen Agathis australis (kauri) for resistance to Phytophthora agathidicida : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University, Manawatū, New Zealand

Abstract

The iconic 𝘈𝘨𝘢𝘵𝘩𝘪𝘴 𝘢𝘶𝘴𝘵𝘳𝘢𝘭𝘪𝘴 (kauri) of New Zealand, is under serious threat from kauri dieback disease caused by the soil-borne pathogen 𝘗𝘩𝘺𝘵𝘰𝘱𝘩𝘵𝘩𝘰𝘳𝘢 𝘢𝘨𝘢𝘵𝘩𝘪𝘥𝘪𝘤𝘪𝘥𝘢. Infected kauri express symptoms of root and collar rot, bleeding resins at the base of the trunk, yellowing of foliage, canopy thinning, and tree mortality. 𝘗𝘩𝘺𝘵𝘰𝘱𝘩𝘵𝘩𝘰𝘳𝘢 𝘢𝘨𝘢𝘵𝘩𝘪𝘥𝘪𝘤𝘪𝘥𝘢 was first associated with kauri decline in 1972, where it was initially identified as 𝘗. 𝘩𝘦𝘷𝘦𝘢𝘦 however, there was some uncertainty about its significance and taxonomy. The pathogen was officially identified as a new organism in 2008 and was called 𝘗𝘩𝘺𝘵𝘰𝘱𝘩𝘵𝘩𝘰𝘳𝘢 taxon Agathis until its formal description as 𝘗𝘩𝘺𝘵𝘰𝘱𝘩𝘵𝘩𝘰𝘳𝘢 𝘢𝘨𝘢𝘵𝘩𝘪𝘥𝘪𝘤𝘪𝘥𝘢 in 2015. This pathogen is easily vectored through root to root contact and mobile zoospores. Management and research has focused on mapping pathogen distribution, reducing spread, improving detection, 𝘦𝘹 𝘴𝘪𝘵𝘶 conservation and clonal production using tissue culture techniques.

In order to gain better understanding of the disease epidemiology and to develop better breeding programmes, a reliable 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 resistance screening assay is required. This research focused on the development of a screening assay using detached leaves from tissue culture material as a means of accelerating screening assays compared to the more labour-intensive root inoculation assays.

Foliar inoculations and assessment techniques were initially optimised on kauri leaves from tissue culture lines. The most successful inoculation method involved placing 𝘗. 𝘢𝘨𝘢𝘵𝘩𝘪𝘥𝘪𝘤𝘪𝘥𝘢-colonised agar plugs on wounded detached leaves. The assay was further tested on 2 year old kauri seedlings. Variation in susceptibility across kauri genotypes and leaf age, and variation in virulence among 𝘗. 𝘢𝘨𝘢𝘵𝘩𝘪𝘥𝘪𝘤𝘪𝘥𝘢 isolates was observed. To further investigate the impact of leaf age on lesion extension, an assay was conducted on detached leaves from six rooted kauri saplings over 5 years of age, across three leaf age groups with 𝘗. 𝘢𝘨𝘢𝘵𝘩𝘪𝘥𝘪𝘤𝘪𝘥𝘢, 𝘗. 𝘮𝘶𝘭𝘵𝘪𝘷𝘰𝘳𝘢, and 𝘗. 𝘤𝘪𝘯𝘯𝘢𝘮𝘰𝘮𝘪. Variation in virulence among these 𝘗𝘩𝘺𝘵𝘰𝘱𝘩𝘵𝘩𝘰𝘳𝘢 species was observed. Leaf necrosis was most severe with young tissue and susceptibility tended to decrease with increasing leaf age. Preliminary studies with 50 kauri clones identified different levels of susceptibility and tolerance across the different genotypes to 𝘗. 𝘢𝘨𝘢𝘵𝘩𝘪𝘥𝘪𝘤𝘪𝘥𝘢.

The methods developed within this study have increased our understanding of the overall response of kauri to 𝘗. 𝘢𝘨𝘢𝘵𝘩𝘪𝘥𝘪𝘤𝘪𝘥𝘢 foliar inoculations. This study demonstrated variation in the susceptability of kauri foliage to 𝘗𝘩𝘺𝘵𝘰𝘱𝘩𝘵𝘩𝘰𝘳𝘢 inoculation, although no complete resistance was observed. Further work is required to determine if there is a relationship between root and leaf responses which will help establish if 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 genotypic variation can accurately predict natural genotypic variation seen within kauri forests.