Sylvain Lerat

A high-throughput sequencing-based grapevine metagenomic survey was conducted across all grape-growing Canadian provinces (British Columbia, Ontario, Nova Scotia, and Québec) with the objective of better understanding the grapevine virome composition. In total, 310 composite grapevine samples representing nine Vitis vinifera red; five V. vinifera white; seven American–French red; and five white hybrid cultivars were analyzed. dsRNA, enriched using two different methods, was used as the starting material and source of viral nucleic acids in HTS. The virome status on the distribution and incidence in different regions and grapevine cultivars is addressed. Results from this study revealed the presence of 20 viruses and 3 viroids in the samples tested. Twelve viruses, which are in the regulated viruses list under grapevine certification, were identified in this survey. The major viruses detected in this survey and their incidence rates are GRSPaV (26% to 100%), GLRaV-2 (1% to 18%), GLRaV-3 (15% to 63%), GRVFV (0% to 52%), GRGV (0% to 52%), GPGV (3.3% to 77%), GFkV (1.5% to 31.6%), and GRBV (0% to 19.4%). This survey is the first comprehensive virome study using viral dsRNA and a metagenomics approach on grapevine samples from the British Columbia, Ontario, Nova Scotia, and Quebec provinces in Canada. Results from this survey highlight the grapevine virome distribution across four major grapevine-growing regions and their cultivars. The outcome of this survey underlines the need for strengthening current management options to mitigate the impact of virus spread, and the implementation of a domestic grapevine clean plant program to improve the sanitary status of the grapevine ecosystem.

Herpotrichia needle browning (HNB) is a disease that affects several species of fir trees in Europe and North America. HNB was first described by Hartig in 1884, who isolated a fungal pathogenic agent identified as responsible for the disease. This fungus was later named Herpotrichia parasitica but is currently named Nematostoma parasiticum. However, the identity of the pathogens causing HNB is regularly questioned and, to date, the true causal agent of this disease has not been definitely established. The present study aimed to identify the fungal populations present in needles of Christmas fir trees (Abies balsamea) and to correlate them with needle health status using robust molecular methods. PCR primers specific to N. parasiticum allowed detection of the presence of this fungus in DNA samples from symptomatic needles. Furthermore, high-throughput sequencing (Illumina MiSeq) clearly showed that N. parasiticum was associated with symptomatic needles. However, high-throughput sequencing results revealed that the presence of other species such as Sydowia polyspora and Rhizoctonia sp. may also correlate with the development of HNB. A diagnostic tool, based on quantitative PCR using a probe, was then developed to detect and quantify N. parasiticum in DNA samples. The efficacy of this molecular approach was validated through the detection of the pathogenic agent in symptomatic needle samples as well as in nonsymptomatic needles collected in trees affected by HNB. In contrast, N. parasiticum could not be found in needles from healthy trees. The present study argues for the importance of N. parasiticum in causing HNB symptoms.

Suberin is a recalcitrant plant biopolymer composed of a polyphenolic and a polyaliphatic domain. Although suberin contributes to a significant portion of soil organic matter, the biological process of suberin degradation is poorly characterized. It has been suggested that Streptomyces scabiei, a plant pathogenic bacterium, can produce suberin-degrading enzymes. In this study, a comparative analysis of the S. scabiei secretome from culture media supplemented or not with potato suberin was carried out to identify enzymes that could be involved in suberin degradation.

Background: Thaxtomin A (TA), a phytotoxin produced by the phytopathogen Streptomyces scabies, is essential for the development of potato common scab disease. TA inhibits cellulose synthesis but its actual mode of action is unknown. Addition of TA to hybrid poplar (Populus trichocarpa x Populus deltoides) cell suspensions can activate a cellular program leading to cell death. In contrast, it is possible to habituate hybrid poplar cell cultures to grow in the presence of TA levels that would normally induce cell death. The purpose of this study is to characterize TA-habituated cells and the mechanisms that may be involved in enhancing resistance to TA. Results: Habituation to TA was performed by adding increasing levels of TA to cell cultures at the time of subculture over a period of 12 months. TA-habituated cells were then cultured in the absence of TA for more than three years. These cells displayed a reduced size and growth compared to control cells and had fragmented vacuoles filled with electron-dense material. Habituation to TA was associated with changes in the cell wall composition, with a reduction in cellulose and an increase in pectin levels. Remarkably, high level of resistance to TA was maintained in TA-habituated cells even after being cultured in the absence of TA. Moreover, these cells exhibited enhanced resistance to two other inhibitors of cellulose biosynthesis, dichlobenil and isoxaben. Analysis of gene expression in TA-habituated cells using an Affymetrix GeneChip Poplar Genome Array revealed that durable resistance to TA is associated with a major and complex reprogramming of gene expression implicating processes such as cell wall synthesis and modification, lignin and flavonoid synthesis, as well as DNA and chromatin modifications. Conclusions: We have shown that habituation to TA induced durable resistance to the bacterial toxin in poplar cells. TA-habituation also enhanced resistance to two other structurally different inhibitors of cellulose synthesis that were found to target different proteins. Enhanced resistance was associated with major changes in the expression of numerous genes, including some genes that are involved in DNA and chromatin modifications, suggesting that epigenetic changes might be involved in this process.

The biocontrol agent Streptomyces melanosporofaciens strain EF-76 produces geldanamycin, an ansamycin antibiotic which displays antagonistic activity towards several Gram-positive bacteria and fungi. Biopolymers [chitin, carboxymethylcellulose (CMC) and xylan] or N-acetylglucosamine were added to S. melanosporofaciens culture media and the effect of these additional carbon sources on geldanamycin production was analysed. Geldanamycin biosynthesis was only stimulated in the presence of 1 g L⁻¹ of chitin. Higher concentrations of chitin, as well as the presence of CMC or xylan, did not improve antibiotic production. N-acetylglucosamine, the final degradation product of chitin, did not stimulate geldanamycin production at all concentrations tested. Nevertheless, the addition of chitin in culture media had no or a negative impact on the antagonistic activity of EF-76 towards the pathogen Streptomyces scabies. Furthermore, the effect of chitin, CMC and xylan on the biocontrol ability of EF-76 against S. scabies was tested on radish seedlings. None of the polymers affected the biocontrol efficiency of EF-76 under gnotobiotic conditions. However, the addition of CMC or xylan to plant growth medium protected radish seedlings against S. scabies infection.