High-throughput sequencing (HTS) technologies may be a useful tool for testing imported plant germplasm for multiple pathogens present in a sample, offering strain-generic detection not offered by most PCR-based assays. Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) were tested as HTS-based techniques to detect viruses present in low titres. Strawberry mottle virus (SMoV), an RNA virus, and strawberry vein banding virus (SVBV), a DNA virus, were selected for comparison of RNAseq and TA-PCR with quantitative PCR assays. RNAseq of plant ribosomal RNA-depleted samples of low viral titre was used to obtain datasets from 3 M to 120 M paired-end (PE) reads. RNAseq demonstrated PCR-like sensitivity, able to detect as few as 10 viral copies/µL when 60 million (M) PE reads were generated. The custom TA-PCR primer panels designed for each virus were successfully used to recover most of the reference genomes for each virus. Single- and multiple-target TA-PCR allowed the detection of viruses in samples with around 10 viral copies/µL with a minimum continuous sequence length recovery of 500 bp. The limit of detection of the HTS-based protocols described here is comparable to that of quantitative PCR assays. This work lays the groundwork for an increased flexibility in HTS detection of plant viruses.

The draft genome sequences of three “ Candidatus Symbiopectobacterium” isolates that were collected from New Zealand-grown potato tubers represent the first report of this proposed taxon in the Southern Hemisphere. Their symbiosis with insects and nematodes and their presence on plants may lead to new strategies for pest control and crop management.

Phytophthora pluvialis is a pathogen present in the United States, New Zealand, the United Kingdom, and Belgian forests. Reported hosts include Douglas-fir in the United States, New Zealand, the United Kingdom, and Belgium, as well as tanoak in the United States, radiata pine in New Zealand, and Japanese larch and western hemlock in the United Kingdom. Disease symptoms range from needle lesions and casting on radiata pine through to twig and stem cankers and crown dieback on western hemlock, Douglas-fir, and Japanese larch. Current detection methods rely on isolation and culture or PCR targeting a single-copy gene ( ypt1). A qPCR assay targeting a multiple-copy mitochondrial gene (Cytochrome c oxidase subunit 2 [ cox2]) was designed to increase the sensitivity of P. pluvialis detection, critical for early diagnostics. The resulting assay has a detection limit of 12.8 fg of mycelial DNA, detecting the pathogen on average 6.12 qPCR cycles before the ypt1 assay. In New Zealand samples, the assay was found to consistently detect P. pluvialis in all stages of needle disease symptoms, from early asymptomatic infection to fully cast needles. The new assay allowed for asymptomatic detection of P. pluvialis in pine needles 4 weeks before visual symptoms of disease were observed. The availability of a highly sensitive assay has enabled diagnostic support of the biosecurity response in the United Kingdom during recent detection of P. pluvialis. The assay has been used in applications requiring detection at low pathogen titer levels, including asymptomatic infection, stream baiting, cast needles, and biosecurity response, demonstrating its efficacy for early detection of P. pluvialis in affected forests.

Phytophthora pluvialis is a forest tree pathogen present in the USA, New Zealand, UK and Belgium. Reported hosts include Douglas-fir in the USA, New Zealand, UK and Belgium, as well as tanoak in the USA, radiata pine in New Zealand, Japanese larch and western hemlock in the UK. Disease symptoms range from needle lesions and casting on radiata pine through to twig and stem cankers, and crown dieback on western hemlock, Douglas-fir and Japanese larch. Current detection methods rely on isolation and culture, or PCR using a single-copy gene target with limited sensitivity at low pathogen titre. A qPCR assay targeting a multiple-copy mitochondrial gene was designed to increase sensitivity of P. pluvialis detection in forest samples, critical for informing biosecurity, long term disease management and ongoing research. The resulting assay has a detection limit of 12.8 fg mycelial DNA and can detect the pathogen on average 6.12 qPCR cycles before the single-copy target assay. In New Zealand forest samples, the assay was found to consistently detect P. pluvialis in all stages of radiata pine needle disease symptoms from early asymptomatic infection through to fully cast needles. The new assay allowed for asymptomatic detection of P. pluvialis in radiata pine needle samples four weeks before visual symptoms of disease were observed. The availability of a highly sensitive assay has also enabled rapid and confident diagnostic support of the biosecurity response in the UK during recent detection of P. pluvialis . The assay has been used in applications requiring detection at low pathogen titre levels including asymptomatic infection, stream baiting, cast needles and during biosecurity responses, making it a useful tool for effective early detection and management of P. pluvialis in affected forests.

Cyclaneusma needle cast, caused by Cyclaneusma minus, affects Pinus species world wide. Previous studies suggested the presence of two distinct morphotypes in New Zealand, ‘verum’ and ‘simile’. Traditional mycological analyses revealed a third morphotype with clear differences in colony morphology and cardinal growth rates at varying temperatures. Genome sequencing of eight C. minus isolates provided further evidence of the existence of a third morphotype, named as ‘novus’ in this study. To further analyse these morphotypes, we predicted candidate effector proteins for all eight isolates, and also characterized a cell-death eliciting effector family, Ecp32, which is present in other pine phytopathogens. In concordance with their distinct classification into three different morphotypes, the number of Ecp32 family members differed, with patterns of pseudogenization in the ‘simile’ morphotype, and some members being found exclusively either in the ‘simile’ or ‘verum’ morphotypes. We also showed that the Ecp32 family proteins trigger cell death in non-host Nicotiana species, and, as previously demonstrated in other plant pathogens, the Ecp32 family proteins in C. minus adopt a β-trefoil fold. These analyses provide further evidence that the three morphotypes might be distinct species that need formal descriptions. Understanding the geographical range of different Cyclaneusma species and variations in virulence and pathogenicity will provide a better understanding of pine needle diseases and enable the development of more durable methods to control this disease.