1H NMR spectroscopic analysis of metabolite changes in kauri leaf apoplastic washing fluid following inoculation with Phytophthora agathidicida

Abstract

Phytophthora agathidicida is responsible for a devastating dieback disease that threatens the survival of Agathis australis (kauri), an ancient conifer species endemic to New Zealand. To develop durable control strategies against kauri dieback disease, a better understanding of the host metabolites necessary for the growth and survival of P. agathidicida during in planta growth, particularly during colonisation of the apoplastic environment, where early contact between host and pathogen cells is made, is required. As a starting point to address this knowledge gap, we investigated changes in the metabolite profile of apoplastic washing fluid (AWF) samples harvested from kauri leaves following either mock inoculation or inoculation with P. agathidicida. AWF was extracted from leaves of kauri saplings and inoculated with P. agathidicida on cellophane membranes or cellophane membranes without the pathogen as a control. The metabolite profile of the AWF samples was then analysed via proton nuclear magnetic resonance (1H NMR) spectroscopy at 24 hours and 10 days post-inoculation, and changes investigated relative to the control. Some changes in the metabolite profile of kauri AWF samples following P. agathidicida inoculation were observed using 1H NMR spectroscopy, including a decrease in sucrose and an increase in glucose resulting from the breakdown of more complex carbohydrates. Our results suggest that P. agathidicida modifies or utilises metabolites present in the leaf apoplast of kauri, including carbohydrates that serve as a source of nutrition. These results provide possible new insights into the nutritional requirements of P. agathidicida during apoplastic colonisation of kauri.