The continued success of New Zealand’s diverse primary sector relies on ongoing access to new plant genetics providing impetus for the importation of plant material. This importation is associated with biosecurity risk through the potential co-importation of pathogens as asymptomatic passengers on or in the plant material, necessitating robust quarantine procedures. To meet high health status, many plants are imported as tissue culture (TC), and these have historically been considered free of microbes. However, in the last decade improved understanding of plant microbiomes using deep sequencing platforms has shown that plants harbour a high diversity and abundance of microorganisms. Given the recognised importance of the plant microbiome to the growth, development and resilience of plants, it is unclear whether plants can ever truly be free of microorganisms. The frequency of established, asymptomatic TC plants carrying endophytes, including pathogens, beneficials or benign taxa, is not well understood.
In the absence of robust data on the TC microbiome, risk analyses have tended to default to worst-case scenarios. Thus, there is opportunity to produce experimental data that could strengthen the TC importation pathway so that a larger number and wider range of plants can be more quickly imported. The goal of this research was to understand the frequency with which asymptomatic TC plants carry endophytes. We examined explant derived tissue-cultured material of 21 hops (Humulus lupulus) and six blueberry (Vaccinium spp.) cultivars, a total of 108 samples (individual TC pottles). These lines were well-established, being >1 year old and with a minimum of four cycles of subculturing. The tissue was assessed by metabarcoding of DNA extracted from the samples and culturing of macerated tissue onto four types of agar media.
Sixteen out of the 108 samples contained bacterial and/or fungal sequences. Of the 108 samples, five blueberry and nine hop samples were positive for fungal detection by metabarcoding, with four confirmed by recovery of live cultures (Neofomitella hemitephra, Cladosporium sp., C. cladosporioides and Aureobasidium pullulans). For bacteria, it was more difficult to set a sequence detection threshold for presence of bacterial taxa because of the expected ubiquity of bacterial contamination during sample processing, such as residual DNA in extraction and PCR kits. The results indicated that 14 (seven blueberry and seven hop) of the 108 samples were positive for bacterial detection by metabarcoding; however, only Methylobacterium sp. was confirmed by a corresponding live culture.
Overall, few of the tested TC plants had detectable microbial colonisation using metabarcoding and this finding was supported by attempts to culture from the TC plants. We will discuss the application of metabarcoding to detection of asymptomatic microbial taxa and the setting of detection thresholds. These results provide greater understanding of specific taxa that can persist in well-established, asymptomatic TC material. Future work will determine the persistence over successive sub-cultures. This knowledge will support understanding of the magnitude of risk in the TC import pathway.