Written by Scott A. Lawrence, Elaine J. Burgess, Chris Pairama, Amanda Black, Wayne M. Patrick, Ian Mitchell, Nigel B. Perry & Monica L. Gerth (2019) Mātauranga-guided screening of New Zealand native plants reveals flavonoids from kānuka (Kunzea Robusta) with anti-Phytophthora activity, Journal of the Royal Society of New Zealand

Kauri is an ecologically important and culturally treasured tree species in Aotearoa New Zealand. It is under threat from the pathogenic oomycete Phytophthora agathidicida, which causes kauri dieback disease. We hypothesised that mātauranga Māori (Māori knowledge) of kauri forest health could be used to identify native plants that produce anti-Phytophthora compounds. We tested this hypothesis by using knowledge descended from Te Whare Wananga o Ngāpuhi to select and screen four native plants for activity against P. agathidicida and also P. cinnamomi (a broad host-range pathogen). Extracts of kānuka (Kunzea robusta) were active against various life cycle stages. Bioassay-directed isolation led to three flavanones, previously unreported from New Zealand Kunzea, as the main bioactives. These compounds have not previously been reported as having anti-Phytophthora activities. They inhibited P. agathidicida zoospore germination with IC50 values of 1.4–6.5 µg/mL, making them the most potent inhibitors reported against this stage of the life cycle. The three flavanones also inhibited zoospore motility at 2.5–5.0 µg/mL, and showed some inhibition of mycelial growth at 100 µg/mL. They were generally less active against P. cinnamomi. Overall, the results from this study emphasise the value of using mātauranga Māori in the response to kauri dieback.

Kauri (Agathis australis) is an important endemic species in New Zealand. It is a foundation species that has a profound influence on the surrounding soil, canopy, and biodiversity (Waipara et al. 2013; Wyse et al. 2013; Wyse et al. 2014). Kauri is also one of the longest-lived species known, with individual trees reported to be more than 1500 years old (Steward and Beveridge 2010). Massive trees with heights of up to 60 m and trunk diameters exceeding 7 m have been recorded (Ecroyd 1982; Steward and Beveridge 2010). However, land clearance and a century of unregulated logging (circa 1830–1930) has dramatically reduced the area of virgin primary kauri forest in New Zealand to less than 1% of that at the time of European settlement (Steward and Beveridge 2010). Kauri trees are now also threatened by the pathogen Phytophthora agathidicida, which causes kauri dieback disease (Beever et al. 2009; Weir et al. 2015). P. agathidicida is a member of the oomycete genus Phytophthora, other members of which cause diseases in thousands of economically and ecologically important plants worldwide. Often referred to as ‘fungus-like’, Phytophthora are actually more closely related to diatoms and brown algae (Baldauf et al. 2000). Practically speaking, this means Phytophthora are unaffected by most agrichemical fungicides. Phytophthora lack many of the common fungicide targets, such as the ergosterol biosynthesis pathway and chitin-based cell walls (Judelson and Blanco 2005; Oliver and Hewitt 2014). Phytophthora also have several life cycle stages that are not found in most true fungi. In addition to a mycelial growth phase (similar to fungi), P. agathidicida produces two key types of spores: oospores and zoospores (Weir et al. 2015). Oospores are non-motile ‘survival’ spores, which are generally known to persist in plant tissues or the surrounding soil for years after the host plant dies (Collins et al. 2012; Crone et al. 2013). Zoospores are motile ‘dispersal’ spores. They are key to the epidemic spread of disease, facilitating host-to-host transmission (Carlile 1985; Tyler 2002). Once a zoospore locates a host plant, it encysts and initiates infection (Judelson and Blanco 2005). When a kauri tree is infected by P. agathidicida, several symptoms typically occur including root and collar rot, trunk lesions, canopy thinning, and ultimately tree death (Beever et al. 2009; Bellgard et al. 2016). Trees of all ages are susceptible. Therefore this disease poses a significant threat to the long-term survival of kauri (Beever et al. 2009; Waipara et al. 2013). Globally, there are few anti-oomycete formulations available for controlling Phytophthora diseases. Phosphite is the only treatment currently being used to control P. agathidicida (Horner 2013). It is primarily applied via injections into the trunks of trees that are already showing visible signs of infection. Furthermore, the use of existing agrichemicals, including phosphite, is jeopardised by increasing anti-microbial resistance around the world (Parra and Ristaino 2001; Dobrowolski et al. 2008; Gisi and Sierotzki 2008; Miaoetal.2016). There is an urgent need to discover and develop novel compounds that target the growth, survival and dispersal of Phytophthora in general, and of P. agathidicida in the particular case of controlling kauri dieback. Plants are rich sources of known and potential anti-microbial compounds (Bennettand Wallsgrove 1994; Cowan 1999; Abreu etal. 2012; Pusztahelyi etal. 2015). In this study, we have begun to explore the anti-Phytophthora potential of New Zealand native plants. There are over 2,300 vascular plants native to New Zealand (de Lange et al. 2018) and ∼80% of these are endemic. Many are already known to be useful in rongoā (indigenous plant-based medicine practices). Therefore, we hypothesised that mātauranga Māori (Māori knowledge) could be used to select native plants that were most likely to produce anti-Phytophthora compounds. Here we report the screening of four plants that were selected based on mātauranga Māori for potential activity against P. agathidicida. In order to assess whether activity was specific toP. agathidicida, or generalisable to other Phytophthora, a second species (P. cinnamomi) was also tested. P. cinnamomi is a broad host-range pathogen capable of infecting thousands of different plant species worldwide (Kamoun et al. 2015; Hardham and Blackman 2018). In New Zealand, P. cinnamomi is found across native ecosystems, exotic forests, nurseries and both agricultural and horticultural settings (Scott and Williams 2014). P. cinnamomi has also been linked to ill-thrift of kauri (Waipara et al. 2013). However its overall impact in New Zealand remains poorly understood. The four plants screened for anti-Phytophthora activities were harvested from Waima, New Zealand. Root and leaf extracts of each plant were screened in vitro for inhibition of three key steps of the Phytophthora lifecycle process: zoospore motility, zoospore germination, and mycelial growth. The results of this screening, as well as identification and further characterisation of three compounds purified from kānuka (Kunzea robusta), are reported.

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