First Report and Genome Resource of Monilinia vaccinii-corymbosi, causal agent of Mummy Berry Disease of Black Huckleberry (Vaccinium membranaceum).

Monilinia vacccinii-corymbosi (phylum Ascomycota, family Sclerotiniaceae) causes fruit disease 'mummy berry' on berry crops and responsible for yield losses and quality of fruits. We reported mummy berry disease of black huckleberry (Vaccinium membranaceum) first time in British Columbia, Canada. We have performed sequencing and genome assembly of M. vacccinii-corymbosi from infected fruits of huckleberry. The resulting genome was 33.8 Mbp in size and consisted of 2,437 scaffolds with an N50 of 33,816 bp. To our best knowledge, this is the first report of resource announcement of whole genome sequence of mummy berry pathogen (M. vacccinii-corymbosi) infecting black huckleberry. The genome resource will be valuable for future studies to understand the genomic structure of pathogen, and mechanisms associated with black huckleberry-M. vacccinii-corymbosi interactions.

Genome Resource of Raspberry Root Rot Pathogen Phytophthora gonapodyides.

Phytophthora gonapodyides is a newly reported oomycetes pathogen associated with root rot of red raspberry. We generated high-quality whole genome resource for P. gonapodyides, which was pathogenic on red raspberry. The genome size was 88,717,598 bp with a BUSCO completeness score of 93.9%. This genome resource provides insight on pathogen biology of Phytophthora spp. causing root rot of raspberry. To our best knowledge, this is the first complete genome assembly of plant pathogenic P. gonapodyides.

Genetic structure and population diversity of Phytophthora infestans strains in Pacific western Canada.

Late blight caused by Phytophthora infestans is an economically important disease of potato and tomato worldwide. In Canada, an increase in late blight incidence and severity coincided with changes in genetic composition of P. infestans. We monitored late blight incidence on tomato and potato in Pacific western and eastern Canada between 2019 and 2022, identified genotypes of P. infestans, and examined their population genetic diversity. We identified four major existing genotypes US11, US17, US8, and US23 as well as 25 new genotypes. The US11 genotype was dominant in Pacific western Canada, accounting for 59% of the total population. We discovered the US17 genotype for the first time in Canada. We revealed a higher incidence of late blight and quite diverse genotypes of P. infestans in Pacific western Canada than in eastern Canada. We found high genetic diversity of P. infestans population from Pacific western Canada, as evidenced by the high number of multilocus genotypes, high values of genetic diversity indices, and emergence of 25 new genotypes. Considering the number of disease incidence, the detection of diverse known genotypes, the emergence of novel genotypes, and the high number of isolates resistant to metalaxyl-m (95%) from Pacific western Canada, the region could play a role in establishing sexual recombination and diverse populations, which could ultimately pose challenges for late blight management. Therefore, continuous monitoring of P. infestans populations in Pacific western region and across Canada is warranted. KEY POINTS: • Genotypes of P. infestans in Pacific western were quite diverse than in eastern Canada. • We discovered US17 genotype for the first time in Canada and identified 26 novel genotypes. • Approximately 95% of P. infestans isolates were resistant to metalaxyl-m.

Genome resources and whole genome resequencing of Phytophthora rubi isolates from red raspberry.

Phytophthora rubi is a primary causal agent of Phytophthora root rot and wilting of raspberry (Rubus idaeus L.) worldwide. The disease is a major concern for raspberry growers in Canada and USA. To date, no information is available on genomic diversity of P. rubi population from raspberry in Canada. Using a PCR-free library prep with dual-indexing for an Illumina HiSEQX running a 2x150 bp configuration, we generated whole genome sequence data of P. rubi isolates (n = 25) recovered during 2018 to 2020 from nine fields, four locations and four cultivars of raspberry growing areas of British Columbia, Canada. The assembled genome of 24 isolates of P. rubi averaged 8,541 scaffolds, 309× coverage, and 65,960,000 bp. We exploited single nucleotide polymorphisms (SNPs) obtained from whole genome sequence data to analyze the genome structure and genetic diversity of the P. rubi isolates. Low heterozygosity among the 72% of pathogen isolates and standardized index of association revealed that those isolates were clonal. Principal component analysis, discriminant analysis of principal component, and phylogenetic tree revealed that P. rubi isolates clustered with the raspberry specific cultivars. This study provides novel resources and insight into genome structure, genetic diversity, and reproductive biology of P rubi isolated from red raspberry. The availability of the P. rubi genomes also provides valuable resources for future comparative genomic and evolutionary studies for oomycetes pathogens.

First report of Phytophthora gonapodyides causing root rot on raspberry in Canada.

Raspberry (Rubus idaeus L.) is an economically important fruit crop in Canada and about 80% of red raspberries are cultivated in British Columbia. In 2018, foliar symptoms associated with root rot and wilting complex disease were observed in raspberry field of Fraser Valley areas of British Columbia. Plants were stunted with reduced numbers of primocanes. Chlorosis and necrosis on leaves and partial wilting of branches were observed. When plants were uprooted, necrosis and browning on roots were observed. Two isolates of oomycetes pathogen were isolated using baiting with rhododendron leaves and pear fruit as described in Sapkota et al. 2022. Using FastDNA Spin kit (MP Biomedical, Burlingame, CA), genomic DNA of pathogen isolates was extracted from mycelia cultured on 20% clarified V8 agar medium amended with 10 mg pimaricin, 250 mg ampicillin, 10 mg rifampicin (V8PAR) per liter following the manufacturer's standard protocol. Pathogens were identified using colony morphology on 20% clarified V8 PAR as well as internal transcribed spacer (ITS) sequencing with ITS1 primers (White et al. 1990) and multiplex targeted-sequencing with degenerate primers of three nuclear genes: heat shock protein90 (HSP90), elongation factor 1 alpha (EF1α) and beta tubulin (ßtub). BLAST searches of ITS sequences of isolates of this study (accession nos. OP180065, OP180066) in NCBI GenBank showed 98.5 to 99.6% identity with the ITS sequence of P. gonapodyides (accession nos. MN513238.1, MG753496.1). Multiplex targeted sequencing also identified both isolates as a P. gonapodyides (accession nos. SRR20227809, SRR20227807) when mapped with the reference sequences (accession nos. HSP90: KX251233.1, EF1α: KX251231.1, ß-tub: KX639710.1). Pathogenicity was confirmed by inoculating mycelial suspension of one isolate of P. gonapodyides on root of intact plants and mycelial plugs of two isolates on detached stems of the raspberry plants, 'Chemainus' in the greenhouse using methods described in Sapkota et al. 2022. Two experiments were conducted with three replicates in each test. Experiments were arranged using completely randomized design. In detached stem assays, distinct dark-lesion symptom appeared at 7 to 9 days after inoculation while uninoculated control stems remained asymptomatic. Intact plants showed wilting and foliar symptoms 15 days after inoculation and progressed higher at 4 to 5 weeks after inoculation. Root infection with dark brown to black color was observed when roots were assessed at 5 weeks after inoculation. The diseased root and crown tissues tested positive for Phytophthora in Agdia ImmunoStrip and P. gonapodyides was re-isolated and confirmed with multiplex-targeted sequencing. Phytophthora gonapodyides was previously reported from raspberry in Chile (Wilcox and Latorre 2002). To our best knowledge, this is the first report of P. gonapodyides infecting red raspberry in British Columbia, Canada. The detection of new Phytophthora species on raspberry may become a new potential problem to growers in addition to P. rubi, which is already a major cause of raspberry decline in the region.

Development of Screening Assays for Pathogen Virulence and Resistance to Phytophthora Root Rot and Wilting Complex in Raspberry.

Phytophthora root rot and wilting complex (PRRW) of red raspberry, caused primarily by Phytophthora rubi, is an economically important disease in British Columbia (BC) and in raspberry producing regions globally. Reliable, rapid, and efficient screening methods are lacking for evaluating germplasm for potential disease resistance in raspberry breeding programs as well as for screening pathogen isolates for virulence. The objective of this study was to compare various screening methods for efficiency and rapidity in inducing symptoms of disease to identify the most suitable approach. We compared several intact plant root inoculation (IPRI) assays, detached stem assays, and an intact plant stem inoculation (IPSI) assay. A virulent isolate of P. rubi was inoculated in two commercial cultivars: 'Chemainus' (susceptible to PRRW) and 'Cascade Bounty' (moderately resistant to PRRW). For IPRI assays, days to first symptom development, plant wilt progression, and root assessment were recorded. For detached stem tissue and IPSI assays, days to first visible lesions and lesion size were assessed. Experiments were arranged in a completely randomized design with three replications in each experiment. Three IPRI assays produced reliable symptoms in both cultivars. Among the detached stem assays, a node inoculation method performed better than other methods. Detached stem assays are useful for rapid pathogenicity testing of P. rubi, whereas IPRI assays are better for screening germplasm for disease resistance. Overall, this study identified several assays that can be used for conducting studies on pathogen phenotypic diversity (pathogenicity and virulence tests) and screening raspberry cultivars, germplasm, and breeding materials for response to PRRW.

Development and application of multiplex targeted-sequencing approaches to identify Phytophthora species associated with root rot and wilting complex of red raspberry.

Phytophthora species are primary causal agents of raspberry root rot and wilting complex (RRWC), a disease complex that is of major concern to raspberry producers worldwide. Accurate identification of the causal agents is a first step for effective disease management. Advancements in molecular diagnostics can facilitate the detection of multiple pathogen species associated with this disease complex. We developed multiplex targeted-sequencing methods using degenerate primers for heat shock protein 90, elongation factor 1α and ß-tubulin genes to identify Phytophthora species causing RRWC. One hundred and twenty-eight isolates recovered during 2018 to 2020 from diverse fields in major raspberry growing areas of British Columbia (BC) were sequenced and identified by comparing with known reference sequences of 142 Phytophthora species, 111 Pythium species, and nine Phytopythium species in the NCBI database. This multiplex targeted-sequencing method was highly specific and identified two species of Phytophthora associated with RRWC. These were P. rubi (85% of isolates) and P. gonapodyides (15% of isolates). Phytophthora rubi was predominantly isolated from the cultivars 'Chemainus' (51%), 'Rudi' (27%) and 'Meeker' (15%), whereas P. gonapodyides was predominately isolated from the moderately resistant cultivar 'Cascade Bounty'. Pathogenicity studies on intact plants and detached leaves confirmed that P. rubi and P. gonapodyides can cause symptoms of RRWC on raspberry, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. gonapodyides as a causal agent of RRWC on raspberry in BC. This study provides novel insights into the identification and species composition of Phytophthora associated with RRWC in raspberry production systems.