First report of 'Candidatus Phytoplasma pruni' associated with X-disease on sweet cherry (Prunus avium L.) in Canada.

British Columbia (BC) is the lead producer of sweet cherries in Canada with more than 2,000 ha in production and a farm gate value of over CAD$100 million annually. Since 2010, an outbreak of little cherry disease caused by Little cherry virus 1 (LChV1) and Little cherry virus 2 (LChV2), as well as X-disease (XD) caused by 'Candidatus Phytoplasma pruni' has caused significant economic losses in neighboring Washington State (WA), USA. LChV1 and LChV2 have long been known to occur in BC (Theilmann et al. 2002); however, 'Ca. P. pruni' has not yet been reported in BC. Due to its geographical proximity to WA State, the BC cherry industry expressed significant concerns about the possible presence of the phytoplasma in cherry orchards. Accordingly, the main objective of this study was to survey cherry orchards to determine whether 'Ca. P. pruni' was present in symptomatic trees in BC. A total of 118 samples of leaves and fruit stems from individual symptomatic trees were collected prior to harvest from nine cherry orchards and one nectarine orchard in the Okanagan and Similkameen Valleys in BC. Characteristic symptoms included small and misshapen fruit with poor color development. Samples were submitted to AGNEMA, LLC (Pasco, WA) for testing using qPCR TaqMan assays for LChV1 (Katsiani et al. 2018), LChV2 (Shires et al. 2022) and 'Ca. P. pruni' (Kogej et al. 2020). Test results showed 21 samples (17.8%) from three cherry orchards positive for LChV2 and 2 samples (1.7%) from one cherry orchard positive for 'Ca. P. pruni'. In order to confirm the identification of 'Ca. P. pruni', part of the 16S ribosomal RNA gene was amplified by nested PCR using the P1/P7 followed by R16F2n/R2 primer sets (Gundersen and Lee 1996) and Sanger sequenced. BC-XD-Pa-1 (GenBank Acc. No. OR539920) and BC-XD-Pa-2 (OR537699) were identical to one another and showed 99.92% identity to the 'Ca. P. pruni' reference strain CX-95 (JQ044397). Analysis using iPhyClassifier (Zhou et al. 2009) indicated that they were 16SrIII-A strains. Interestingly, the two partial 16S sequences showed 100% nucleotide identity to strain 10324 (MH810016) and others from WA. For additional confirmation, partial secA (Hodgetts et al. 2008) and secY (Lee et al. 2010) translocases were amplified and sequenced. As with the 16S sequences, secY sequences (OR542980, OR542981) showed 99.92% nucleotide identity to strain CX-95 (JQ268249), and 100% to strain 10324 (MH810035). The secA sequences (OR542978, OR542979) had nucleotide identities of 99.77% to strain CX (MW547067), and 100% to the Green Valley strain from California (EU168733). Accordingly, 'Ca. P. Pruni' was confirmed to be present in sweet cherry samples from BC. 'Ca. P. Pruni'-related strains have been previously reported to occur in Canada in commercial poinsettias (Euphorbia pulcherrima) (Arocha-Rosete et al. 2021). To our knowledge, this is the first report of 'Ca. P. Pruni' in sweet cherry in Canada. Due to the important economic value of sweet cherries in BC, these findings are highly significant and represent the first steps towards the development of a surveillance system for early detection of XD, and consequent implementation of management strategies, including vector control. As required by federal and provincial regulations, cherry trees infected with LChV2 and 'Ca. P. Pruni' found in the survey were removed by the growers.

Health Status of Ready-to-Plant Grapevine Nursery Material in Canada Regarding Young Vine Decline Fungi.

Young vine decline (YVD), caused by several taxonomically different fungi, results in the decline and death of grapevines within a few years after planting. Infection can occur in nursery mother blocks and/or at several stages in the nursery propagation process, but the final plant material may remain asymptomatic. Four nurseries that sell ready-to-plant grapevines in Canada were sampled to evaluate the health status with regard to YVD fungi, including Botryosphaeriaceae spp., Cadophora luteo-olivacea, Dactylonectria macrodidyma, Dactylonectria torresensis, Phaeoacremonium minimum, and Phaeomoniella chlamydospora. Plants representing three cultivars, 'Chardonnay', 'Merlot', and 'Pinot noir', either grafted onto '3309C' rootstock or self-rooted, were provided by the nurseries. Samples from the roots, base of the rootstock or self-rooted cultivar, graft-union, and scion were collected from each plant. DNA was extracted, and the total abundance of each fungus was quantified using Droplet Digital PCR. Results revealed that 99% of plants harbored at least one of the fungi studied, with a mean of three different fungal species that were present per grapevine. Droplet Digital PCR results showed that the abundance of the different fungi significantly varied between different sections of each plant, individual plants for each cultivar, and cultivars from the same nursery. Necrosis measurements were recorded from the base of the rootstock or self-rooted cultivars and did not correlate with fungal abundance recorded in that section for each grapevine, but necrosis was consistent across cultivars within nurseries. Five different rootstocks were compared from one nursery, and results showed no differences between rootstocks and their health status. Among all nurseries, C. luteo-olivacea was the most prevalent fungus (97% of the plants), while D. macrodidyma was the least commonly found (13% of the plants). This study shows that ready-to-plant nursery material sold in Canada is likely to be infected with several YVD fungi and that presence and abundance of fungi vary significantly among individual grapevines and nurseries.

Phylogenetic and Evolutionary Studies of Grapevine Pinot Gris Virus Isolates from Canada.

This study investigated the phylogenetic relationship of grapevine Pinot gris virus (GPGV) isolates from Canada with GPGV isolates reported worldwide. Full-length genomes of 25 GPGV isolates representing the main four grape-growing regions in Canada (British Columbia, Ontario, Nova Scotia and Quebec) were sequenced and compared to genomes of 43 GPGV isolates representing eight countries and three continents. Phylogenetic analysis based on full genome sequences revealed an unambiguous separation of North American GPGV isolates with isolates from Europe and Asia. Within the North American clade, GPGV isolates from the USA segregated into a distinct subclade, whereas the relationships amongst GPGV isolates from different regions of Canada were not clearly defined. The phylogenetic analysis of the overlapping regions of MP and CP genes involving 169 isolates from 14 countries resulted in two distinctive clades, which were seemingly independent of their country of origin. Clade 1 included the majority of asymptomatic isolates (81% asymptomatic), whereas clade 2 was predominantly formed of symptomatic isolates (78% symptomatic). This research is the first study focused on the genetic variability and origin of GPGV in Canada.

Biocontrol Activity of Trichoderma Species Isolated from Grapevines in British Columbia against Botryosphaeria Dieback Fungal Pathogens.

Botryosphaeria dieback (BD) is a grapevine trunk disease (GTD) causing significant yield losses and limiting the lifespan of vineyards worldwide. Fungi responsible for BD infect grapevines primarily through pruning wounds, and thus pruning wound protection, using either synthetic chemicals or biological control agents (BCAs), is the main available management strategy. However, no products to control GTDs are currently registered in Canada. With a focus on more sustainable grapevine production, there is an increasing demand for alternatives to chemical products to manage GTDs. Accordingly, the objective of this study was to identify Trichoderma species from grapevines in British Columbia (BC) and evaluate their potential biocontrol activity against BD fungi Diplodia seriata and Neofusicoccum parvum. Phylogenetic analyses identified seven species, including T. asperelloides, T. atroviride, T. harzianum, T. koningii, T. tomentosum, and two novel species, T. canadense and T. viticola. In vitro dual culture antagonistic assays showed several isolates to inhibit fungal pathogen mycelial growth by up to 75%. In planta detached cane assays under controlled greenhouse conditions identified T. asperelloides, T. atroviride and T. canadense isolates from BC as providing 70% to 100% pruning wound protection against BD fungi for up to 21 days after treatment. In addition, these isolates were shown to provide similar or better control when compared against commercial chemical and biocontrol products. This study demonstrates the potential that locally sourced Trichoderma species can have for pruning wound protection against BD fungi, and further supports the evaluation of these isolates under natural field conditions.

Identification of Putative SDHI Target Site Mutations in the SDHB, SDHC, and SDHD Subunits of the Grape Powdery Mildew Pathogen Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are fungicides used in control of numerous fungal plant pathogens, including Erysiphe necator, the causal agent of grapevine powdery mildew (GPM). Here, the sdhb, sdhc, and sdhd genes of E. necator were screened for mutations that may be associated with SDHI resistance. GPM samples were collected from 2017 to 2020 from the U.S. states of California, Oregon, Washington, and Michigan, and the Canadian province of British Columbia. Forty-five polymorphisms were identified in the three sdh genes, 17 of which caused missense mutations. Of these, the SDHC-p.I244V substitution was shown in this study to reduce sensitivity of E. necator to boscalid and fluopyram, whereas the SDHC-p.G25R substitution did not affect SDHI sensitivity. Of the other 15 missense mutations, the SDHC-p.H242R substitution was shown in previous studies to reduce sensitivity of E. necator toward boscalid, whereas the equivalents of the SDHB-p.H242L, SDHC-p.A83V, and SDHD-p.I71F substitutions were shown to reduce sensitivity to SDHIs in other fungi. Generally, only a single amino acid substitution was present in the SDHB, SDHC, or SDHD subunit of E. necator isolates, but missense mutations putatively associated with SDHI resistance were widely distributed in the sampled areas and increased in frequency over time. Finally, isolates that had decreased sensitivity to boscalid or fluopyram were identified but with no or only the SDHC-p.G25R amino acid substitution present in SDHB, SDHC, and SDHD subunits. This suggests that target site mutations probably are not the only mechanism conferring resistance to SDHIs in E. necator.

Olive Twig and Branch Dieback in California Caused by Cytospora oleicola and the Newly Described Species Cytospora olivarum sp. nov.

Field surveys conducted throughout California olive-growing regions in 2008 and 2009 resulted in a collection of 101 Cytospora-like isolates from olive twig and branch dieback symptoms. Cytospora isolates were isolated from multiple cvs. in different olive orchards in Fresno, Madera, Merced, Napa, Riverside, Santa Barbara, Sonoma, Tulare, and Ventura counties. Taxonomic studies of macro- and microscopic structures along with multigene phylogenetic analyses of the internal transcribed spacer region, including the 5.8S rDNA (ITS1-5.8S-ITS2), and fragments of the translation elongation factor 1-α, beta-tubulin, and actin genes identified two species, Cytospora oleicola and C. olivarum sp. nov. Pathogenicity studies conducted in mature olive trees cvs. Manzanillo and Sevillano showed both species to be pathogenic and able to cause vascular necrosis and cankers in olive branches. This study adds to the current knowledge on the etiology of olive twig and branch dieback and provides new important information for the development of effective control strategies against canker diseases affecting olive in California.

Comparative genomic analysis of Erwinia amylovora reveals novel insights in phylogenetic arrangement, plasmid diversity, and streptomycin resistance.

Erwinia amylovora is a destructive pathogen of Rosaceous plants and an economic concern worldwide. Herein, we report 93 new E. amylovora genomes from North America, Europe, the Mediterranean, and New Zealand. This new genomic information demonstrates the existence of three primary clades of Amygdaloideae (apple and pear) infecting E. amylovora and suggests all three independently originate from North America. The comprehensive sequencing also identified and confirmed the presence of 7 novel plasmids ranging in size from 2.9 to 34.7 kbp. While the function of the novel plasmids is unknown, the plasmids pEAR27, pEAR28, and pEAR35 encoded for type IV secretion systems. The strA-strB gene pair and the K43R point mutation at codon 43 of the rpsL gene have been previously documented to confer streptomycin resistance. Of the sequenced isolates, rpsL-based streptomycin resistance was more common and was found with the highest frequency in the Western North American clade.

Molecular Methods to Detect and Quantify Botryosphaeriaceae Inocula Associated With Grapevine Dieback in Australia.

Botryosphaeria dieback, caused by species of Botryosphaeriaceae, is an important grapevine trunk disease in Australia. Inocula produced by the pathogens are primarily dispersed by rain splash and wind and infect pruning wounds leading to cankers, dieback, and eventually death of vines. The objective of this study was to develop molecular tools to detect and quantify Botryosphaeriaceae inocula from the environment. These tools are essential for investigating spore dispersal patterns of Botryosphaeriaceae pathogens in Australian vineyards. DNA extraction protocols were evaluated and one modified protocol was found suitable for extracting Botryosphaeriaceae DNA from artificially and naturally inoculated Burkard volumetric spore sampler tapes. Multispecies primers and a hydrolysis probe for quantitative PCR (qPCR) were further developed to detect and quantify Botryosphaeriaceae inocula from environmental samples. Specificity tests showed that the multispecies primers were able to amplify the DNA of 10 Botryosphaeriaceae species (58 isolates) found in Australia while none of the 27 nontarget fungal species (90 isolates) tested were amplified. The qPCR assay was suitable for amplifying purified DNA, synthetic DNA fragments (gBlocks), and mixed DNA from spore trap tapes. The qPCR method developed in this study was shown to be rapid and sensitive in detecting Botryosphaeriaceae inocula from the environment using spore traps.

Managing Grapevine Trunk Diseases With Respect to Etiology and Epidemiology: Current Strategies and Future Prospects.

Fungal trunk diseases are some of the most destructive diseases of grapevine in all grape growing areas of the world. Management of GTDs has been intensively studied for decades with some great advances made in our understanding of the causal pathogens, their epidemiology, impact, and control. However, due to the breadth and complexity of the problem, no single effective control measure has been developed. Management of GTD must be holistic and integrated, with an interdisciplinary approach conducted in both nurseries and vineyards that integrates plant pathology, agronomy, viticulture, microbiology, epidemiology, biochemistry, physiology, and genetics. In this review, we identify a number of areas of future prospect for effective management of GTDs worldwide, which, if addressed, will provide a positive outlook on the longevity of vineyards in the future.