Inner plant space - bacterial endophytes of Leptospermum scoparium (mānuka)

Abstract

Leptospermum scoparium or mānuka is a New Zealand native medicinal plant that produces an essential oil with antimicrobial properties. In other plants endophytic bacteria have key roles in plant growth, health and metabolite production. However, studies to define the community structure and bioactivity of endophytic bacteria in L. scoparium have not been carried out. The aim of this study was to i) describe the community structure of endophytic bacteria in L. scoparium and identify its core members, ii) investigate the bioactivity of cultured endophytic bacteria and investigate their ability to modify essential oil composition and iii) determine whether endophytic bacteria with in vitro biocontrol activity against fungal and bacterial pathogens could be transmitted to agriculturally important plants. Denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq were used to characterise bacteria inhabiting L. scoparium. This study used propidium monoazide (PMA) to enrich amplification of endophyte DNA. Plant materials from three diverse sites were analysed by DGGE and this showed that plant tissue was the main factor that influenced endophytic bacterial communities followed by plant maturity and plant location. It showed that the communities became more stable and uniform as plant maturity increased. The Gammaproteobacteria were the class most affected by location. Analysis of a larger number of sampling sites (n=11) across New Zealand using Illumina MiSeq confirmed that plant tissue affected the endophytic bacterial communities and one OTU that belonged to Pseudomonas was defined as a member of the core endomicrobiome. A representative collection of 330 bacteria were recovered into culture from 32 plant samples taken from five sites across New Zealand. The majority of bacteria (70%) were recovered from roots followed by stems (23%) and leaves (7%). Seven in vitro assays were used to assess the bioactivity of bacterial isolates. A high proportion of endophytic bacteria produced siderophores (66%) and solubilized tricalcium phosphate and hydroxyapatite (68% and 72%) in vitro. Three, ten and five isolates showed strong in vitro antagonism against two fungal pathogens, Ilyonectria liriodendri and Neofusicoccum luteum and a bacterial pathogen, Pseudomonas syringae pv. actinidiae (Psa) respectively. Based on the 16S rRNA gene, 89% of bacteria with bioactivity belonged to Gammaproteobacteria. This is the first study to investigate the effect of bacteria, isolated from L. scoparium on the growth and essential oil production by one regional plant chemotype. Erwinia sp. T4MS11P and Pseudomonas sp. M3R43 increased shoot and root dry weight by 170-210% compared to the control (P=0.02 and P=0.04, respectively). Plants inoculated with Erwinia sp. T4MS11P had similar grandiflorone concentration as control plants, whereas, plants inoculated with Pseudomonas sp. M3R43 had lower grandiflorone concentrations (P=0.01). In contrast, inoculation with a bacterial consortium isolated from the West Coast did not increase plant growth, but gave higher grandiflorone concentration (>160%) compared to control plants, indicating that modification in essential oil composition was not related to plant growth. This work demonstrated that plant chemistry can also be modulated by the bacteria either as endophytes or within the rhizosphere. The antagonism in vitro against Neofusicoccum spp., Ilyonectria spp. and Psa suggested that endophytic bacteria from L. scoparium may be new biocontrol candidates. Two bacteria antagonistic to botryosphaeriaceous species, i.e Pseudomonas sp. I2R21 and Pseudomonas sp. W1R33, were transmissible to grapevine as a new host. Once resident in the host they inhibited colonization by N. parvum and N. luteum and reduced lesion length 35-52% compared to controls. Three endophytic bacteria with in vitro antagonism against Psa were able to colonize kiwifruit and exert their inhibitory activity by reducing disease severity by 48-75% compared to controls. All bacteria with strong antagonism against either botryosphaeriaceous species or Psa contained at least one antibiotic producing gene detected by PCR. The antagonism against the pathogen was likely mediated through antibiosis although this was not directly proven. Overall this study has revealed the structure and bioactivity of the endophytic bacteria in L. scoparium and suggested a role in growth and essential oil composition. This study identified the Gammaproteobacteria as an important group in L. scoparium. Endophytic bacteria with biocontrol activity could be transmitted to new hosts where they inhibited pathogens in planta. Thus, this study indicated that L. scoparium provides a new source of microorganisms for use in sustainable agriculture.