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 β-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.

Diseases or disease complexes caused by oomycetes, fungi, viruses, bacteria and nematodes are known to constrain raspberry production, which adversely impacts plant health, fruit yield, and quality. The root rot and wilting complex (RRWC) caused by Phytophthora spp. and other soil-borne pathogens are the most economically important raspberry disease worldwide, including in the Pacific Northwest (PNW) region of North America [3,7]. RRWC causes millions of dollars in losses due to plant decline and reduced raspberry fruit yields and quality in the PNW. Although several species of Phytophthora were reported as causal agents of RRWC, P. rubi (Wilcox and Duncan) Man in't Veld (synonym. P. fragariae Hickman var. rubi) is considered as the most important species associated with RRWC of raspberry in PNW of Canada and USA [3,8]. Earlier research in Washington indicated that disease complex can cause up to 100% plant mortality under the saturated field conditions [2]. In BC, control measures (such as, fumigation and fungicides application) for RRWC are the single largest non-labor expenses for raspberry growers [3]. However, comprehensive studies to identify causal agents associated with RRWC and their pathogenicity studies were not done previously from BC.
The above-ground symptoms on raspberry plants is the first step for diagnosing RRWC. These symptoms include scorching, chlorosis, reddening, and necrosis of leaves, and partial wilting of stems and the entire plant. Moderate to severe wilting occurs in advanced stages and plants can die. In the root and crown area, discoloration and decay occurs. Typical RRWC root symptoms include production of fewer and weaker rootlets and presence of dark-reddish brown lesion at the crown. To identify the causal agents (Phytophthora spp.), colony characteristics on selective media and examination of spore morphology are traditionally used. However, identifying Phytophthora using morphological features is challenging, as several species have similar colony or spore morphology. Morphology-based techniques are time and laborintensive and also require mycological expertise for accurate identification [9,10].
In recent years, molecular techniques that target specific DNA regions in the Phytophthora genome have been developed [10,11], especially sequencing of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA [12], cytochrome oxidase subunits (I, II), nad1, and nad9 of the mitochondrial genome [13], and nuclear loci (β-tubulin, HSP90) [14]. ITS-based sequencing has some limitations for accurately distinguishing among species of Phytophthora. For instance, P. rubi and P. fragariae have identical ITS sequences [10,11], making it difficult to distinguish these species without additional genetic data. The majority of available molecular assays were designed to target a single gene to detect a single species in one sample, not allowing for simultaneous identification of multiple species of Phytophthora [11]. Moreover, amplifying and sequencing single genetic regions in separate PCR assays is time-consuming and expensive. Therefore, molecular characterization assays providing genetic data on multiple housekeeping genes in one reaction would allow one to amplify and sequence multiple potential variant positions in a single reaction, providing a finer scale understanding of the species involved within a single infected tissue sample.
The main goal of this study was to develop and apply multiplex targeted-sequencing approaches to identify and characterize Phytophthora spp. associated with RRWC from diverse fields in major raspberry growing areas of BC. Isolations were made from diseased samples and isolates of Phytophthora spp. associated with RRWC were identified and pathogenicity on red raspberry was confirmed.

Disease monitoring, sampling and pathogen isolation
Eleven, 20, and 16 fields from raspberry growing areas of BC were monitored between April and December of 2018, 2019, and 2020 ( Fig 1) and a total of 57, 67, and 76 samples with RRWC infected root and cane as well soil samples were collected in 2018, 2019, and 2020 respectively (Table 1). Each sample was kept in a plastic bag and bags were stored at 4˚C until processed for pathogen isolation. The presence of Phytophthora spp. on plant tissues was first confirmed using Agdia ImmunoStrip assay (Agdia Inc., Elkhart, IN, USA) following the manufacturer's standard protocol. Positive samples were further processed in the laboratory to isolate the pathogen either directly from root and cane tissues near the crown or from soil and tissue using baiting techniques. For pathogen isolation from plant tissues, tissues were washed in running water and small pieces (~5 mm 2 ) from the margin of the symptomatic and healthy sections of root and cane tissues were obtained. The root tissues were surface sterilized in a 25% Ivory dish soap (Procter and Gamble, Ohio, USA) solution for 2 minutes followed by three rinses in sterile water for 1 minute. The canes pieces were sterilized in 10% commercial bleach (6.25% Javex) for 50 s, blotted dry and sprayed once with 70% ethanol, blotted dry, followed by three rinses in sterile water for 30 sec. Rhododendron leaves and pears were used in baiting techniques to isolate the pathogen from soil and root samples. Briefly, buckets (~2 L) were sterilized using 70% ethanol and filled with~500 cm 3 of a mixture of field soil and fine roots. Sterile water was added to the bucket until the soil was completely flooded and the water level maintained at~3 to 5 cm above the soil layer. The rhododendron leaves and unripe pears were sterilized using 70% ethanol and dried in sterile paper towels. Two pieces of leaves (cut into half) and a pear fruit were placed on the soil in each bucket. The buckets were placed in a Conviron growth chamber at 12˚C for 10 to 14 days. For pathogen isolation, rhododendron leaves and pear tissues with dark brown lesions were cut into small pieces (~5 mm 2 ) from the edge of infected and healthy sections of the tissue. The tissue pieces were sterilized as described above for processing cane tissues. The tissue pieces were air-dried on sterile paper towels then placed onto corn meal agar (CMA) amended with 200 μl pimaricin (P), 250 mL ampicillin (A), 400 μl rifampicin (R), 100 mg pentachloronitrobenzene (P), and 50 mg hymexazol (H) per liter; PARPH) and incubated at 16˚C for~2 weeks and colony growth was monitored. Once sparse colony growth was observed on a plate, a small plug of mycelia was transferred from the edge of the colony onto V8 agar medium amended with 400 μl pimaricin, 250 mg ampicillin, 400 μl rifampicin (V8PAR) per liter. These plates were incubated at 16˚C for 1 to 2 weeks in darkness. Once the pathogen was fully grown, plates were stored at 10˚C for short-term. For long-term storage, each isolate was grown on V8PAR media with popcorn and rye grains placed on the surface. After 2 weeks, the colonized grains containing mycelia were transferred to 30 mL French square vials. Each vial received 8 to 10 rye or popcorn grains,~15 mL sterile water and four 5-mm V8A plugs. Also, isolates were grown on oatmeal agar slants in 30 mL French square vials for 2 weeks. All isolates of Phytophthora spp. were then stored at 10˚C in darkness for further use. Colony morphology of all isolates of both species were observed by culturing them on V8A and CMA media. In addition, sporangial morphology of few representative isolates of P. rubi (n = 4) and P. gonapodyides (n = 2) were also examined. To induce the pathogen sporulation, 8-day old isolates grown on V8A were transferred onto Petri dishes con-taining~15 mL suspension of non-sterile soil extract (1.5 g of soil per liter of sterile water) and were incubated at 18˚C under continuous fluorescent light for 2 to 4 days.

Multiplex targeted-sequencing
Suspected oomycete isolates were grown on cellophane placed on top of V8 agar for 10-13 days. The mycelia were then harvested, freeze-dried for 2 to 3 days, and ground to fine powder with magnetic MixerMill bead beating device (Qiagen) and genomic DNA was extracted using the MagMAX kit (ThermoFisher Scientific Inc.) following the manufacture's protocols. The DNA from representative samples was visualized on 1% gel and quantified using a high sensitivity QuBit device (ThermoFisher Scientific Inc.). Degenerate PCR primers of Phytophthora (S1 Fig) were created using Batch Primer 3 (https://probes.pw.usda.gov/batchprimer3/). Primer pairs were developed to target beta tubulin (β-tub), elongation factor 1 alpha (EF1α), and heat shock protein 90 (HSP90) genes by alignment of a representative set of Phytophthora species in CLC Genomics Workbench v.9.5.2 (Qiagen) and use of the redundant consensus sequence for primer designs (S1 Table). Each unknown isolate was barcoded and the targeted loci were amplified in a single multiplexed PCR reaction using an optimized Hi-plex PCR approach [15]. The resulting sample-specific barcoded amplicons were then sequenced on an Illumina HiSEQX next-generation DNA sequencing device following the manufacturer's directions at Admera Health LLC (Plainfield, NJ). The reference sequences (n = 499) of all published targeted pathogens (Phytophthora spp. n = 142; Pythium spp. n = 111; and Phytopythium spp. n = 9) for three nuclear genes (β-tub, EF1α, and HSP90) were obtained from National Center for Biotechnology Information (NCBI) GenBank database and European Molecular Biology Laboratory (EMBL) as of June 2021 [14,[16][17][18], and used as reference sequences mapping the raw data. The sample-specific raw reads were quality checked using FastQC 0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)), then imported into Geneious 2020.2.4 (Biomatter, Auckland, New Zealand; https://www.geneious.com/), where they were trimmed using BBDuk and aligned to reference sequences using BBMap [19] with a minimum identity of 98%, maximum mismatch per reads of 2%, and exclusion of multiple map reads. The species identification for all unknown samples was determined based on the species of the reference sequences with the highest number of mapped reads across all three target nuclear genes. The multiplex targeted-sequencing steps used in this study to identify Phytophthora species associated with root rot and wilting of red raspberry is illustrated in

ITS sequencing
To compare the results from ITS sequencing and multiplex targeted sequencing, selected isolates (n = 14) were sequenced using universal primers for ITS1 [20]. Pathogen genomic DNA was extracted from 10 to 12-day-old mycelial cultures (~20 mg) grown on V8PAR using Fas-tDNA Spin kit (MP Biomedical, Burlingame, CA) following the manufacturer's standard protocol. One μl pathogen DNA was used for PCR in a 20 μl reaction volume consisting of 10 μl of 2× GoTaq Green Master Mix (Promega, Madison, WI), 0.5 μl of 10 mM forward and reverse primers, and 8 μl deionized water. PCR amplification was performed in a Bio-Rad thermal cycler. The thermal cycles were 3 min at 94˚C; 35 cycles, of 10 s at 94˚C, 45 s at 58˚C; and 10 min final extension at 72˚C. Five μl of amplified PCR product of each sample was separated on 1% agarose gels to confirm the amplification and remaining 15 μl product of each sample were sent to MCLAB (http://www.mclab.com) for sequencing. For species identity, the resulting ITS sequence reads were compared to NCBI GenBank database using Basic Local Alignment Search tool (BLAST) in Geneious Prime 2022.0.2.

Pathogenicity studies
To confirm the ability of species of Phytophthora to cause root rot of raspberry, pathogenicity trials were conducted by inoculating strains on detached rhododendron leaves in the laboratory and on roots of 'Chemainus' raspberry seedlings in the greenhouse at the Agassiz Research and Development Center, Agassiz, BC. Raspberry seedlings were propagated using tissue cultures from Agriforest Bio-Technologies Ltd. (www.agriforestbiotech.com, Kelowna, BC). In detached rhododendron leaf assays, three isolates each of P. rubi and P. gonapodyides were assessed. Rhododendron leaves were surface sterilized with 70% ethanol for 1 minute, rinsed three times with sterile water, then dried on sterile paper towels, and placed in a 9-cm Petri dish lined with moist sterile filter papers. Leaves were scratched in the center using a sterile needle. A 5 mm mycelial plug of a 12-day old isolate grown on V8PAR was placed on the wounded section of leaves and the petri dishes were sealed with Parafilm. Dishes were incubated at 16 to 18˚C in the dark for 3 to 4 weeks. Leaves treated with V8PAR plugs served as negative controls. For each isolate, three leaves were inoculated using mycelia plug and repeated independently. Once a visible lesion appeared, the lesion length was measured 3 to 4 times at 4 to 6 days interval for lesion progress. The area under the disease progress curve (AUDPC) was calculated for lesion length using the equation by Madden et al. [21].
Where Wi represents the percentage wilting index at the i th observation, Ti the time (days) at the i th observation, and n the total number of observations. Finally, the relative area under disease progress curve (rAUDPC) was calculated as described by Forbes et al. [22]. For root inoculation on seedlings, a mycelial suspension of three isolates of P. rubi were inoculated onto 3 to 4-week-old seedlings of raspberry cultivar 'Chemainus'. Plants were grown in 36-well trays containing a 3:1 ratio of autoclaved potting mix (Sumas Gro-Media Ltd, Chilliwack, BC) and vermiculite (Sun Gro Horticulture Distribution Inc. Washington, USA) and watered and fertilized as needed with a hydroponic solution (0.4 g/L Plant Prod 1 20-20-20 classic, Terralink, Abbotsford, Canada). Plants were inoculated using mycelial suspension inoculum in 36-well tray method as previously described in Sapkota et al. [23]. Inoculated plants were then placed on a saucer and flooded with distilled water for 24-hour to facilitate zoospore production and their attachment to plant roots. Inoculated plants were then drained and maintained in the greenhouse at 18 ± 2˚C, with a 14-h day/night cycle, and 70 ± 5% relative humidity. Nine raspberry plants received sterile water, flooded with distilled water as described above and used as negative controls. For each isolate, experiments were arranged in a completely randomized design with three replicates. Each treatment had three plants in each replicate. Two independent experiments were carried out. Once first symptoms appeared, plants were assessed 3 to 4 times at 5 to 7 days intervals to record wilting progress using a 0 to 4 rating scale, where the scale 0 = healthy, vigorous growth with no wilting, 1 = wilting (slight) in one or a few leaves with slight foliage symptoms (scorching, yellowing, necrosis), 2 = wilting (slight) started in whole plants, stem lesion present from stem base, 3 = moderate wilting in leaves and stem, stem lesion progressing towards stem tip, and 4 = severe to complete wilting, dead (whole plant dead including leaves, stem). Three to four weeks after inoculation, plants were uprooted, roots were washed, and visually assessed for root rot using a 0 to 4 rating scale, where 0 = 0% infection, healthy roots, 1 = 1 to 12% infection, 2 = 13 to 25% infection, 3 = 26 to 50% infection, 4 = 51 to 75% infection, and 5 = � 76% infection. For whole plant inoculation, the percent wilting index (PWI) was calculated using disease severity of plant wilting progress according to the formula by Cooke [24]: � 4ÞÞ=ðtotal numbers of plants rated � maximum score on scaleÞÞ � 100 where P0 to P4 are number of plants with rating scale of 0 to 4. The AUDPC was calculated for PWI using the equation by Madden et al. [21]. Finally, the rAUDPC was calculated as described by Forbes et al. [22]. In addition, root rot rating was converted to percent root rot index (PRAI) using similar formula used to calculate PWI. The pathogen was re-isolated from infected tissues of both pathogenicity assays by culturing on CMA-PARPH medium. Species of Phytophthora were confirmed by morphological observation and the HiPlex targeted sequencing approach.
Statistical analyses. Pathogenicity data were analyzed using SAS v. 9.4 (SAS Institute, Cary NC). The data were tested for normality using Shapiro-wilk test and non-normal data were transformed using square root and log transformation prior to further statistical analyses. Brown-Forsythe test was used to determine significance of variance homogeneity among data sets to decide whether or not data from two or more experiments could be combined for analyses. Analysis of variance was performed for disease assessment and other traits recorded in both detached and intact pathogenicity assays. Treatment means were separated using Fisher's protected least significant difference (LSD) at α = 0.05.

Disease distribution, symptoms, and pathogen isolation
A total of 11, 20, and 16 raspberry fields were monitored in 2018, 2019, and 2020, respectively, from diverse growing areas of BC (Table 1). RRWC disease was observed in all three years; however, disease and symptom expression varied among years and fields. Incidence of RRWC in raspberry fields was higher in 2020 compared to the other two years. Typical symptoms of RRWC were observed on plants infected with P. rubi (Fig 3A to 3F). Diseased plants exhibited symptoms of wilting of cane and leaves, leaf chlorosis, scorching, reddening, and necrosis ( Fig  3A to 3C). Plants in severely infected fields were stunted and had very poor vigor with yellowing, necrosis and moderate to severe wilting (Fig 3D). When plants were uprooted, dark reddishbrown lesions at roots and crown were observed and most had few rootlets (Fig 3E and 3F).
Colony morphology of isolates of P. rubi was similar, as sparse colonies were on CMA ( Fig  5A), and white colonies with relatively abundant aerial fluffy mycelia at the center on V8A ( Fig  5B). Phytophthora rubi sporangia were terminal, non-papillate, and ovoid to ellipsoid in shape ( Fig 5C) with an average size of 40.1 ± 1.5 μm long and 27.7 ± 1.1 μm wide. Colony pattern of isolates of P. gonapodyides on CMA was radiate sparse (Fig 5D) and on V8A was stellate to rosette with relatively less mycelia at the edge (Fig 5E). Phytophthora gonapodyides produced non-papillate, ellipsoid to ovoid sporangia (Fig 5F) with an average size of 33.3 ± 1.0 μm long and 19.7 ± 0.8 μm wide.

Species identification and composition
The HiPlex targeted-sequencing approach, as illustrated in Fig 2, amplified and produced sequences across three nuclear genes (HSP90, EF1α, and βtub) and was able to identify the species of Phytophthora infecting raspberry (S2 Table). The species ID for all unknown samples was determined based on the species of the reference sequences with the highest number of mapped reads across all three target nuclear genes (S2 Table). The multiplex targeted-sequencing method was highly specific and generally showed greater differences in total number of mapped reads between top species. An example of an exact alignment of targeted region of P. rubi and P. gonapodyides with multiplex targeted Illumina sequences for unknown raspberry samples can been seen in S2 Fig. Using representative isolates recovered from infected raspberry, we compared multiplex-targeted sequencing results with ITS sequencing. Based on BLAST search, ITS sequencing results showed the identical percentage identity (99 to 100%) for both P. rubi and P. fragariae. Further, ITS-based sequencing could not distinguish the majority of P. gonapodyides isolates from P. megasperma (percentage identity was similar (99.60 to 99.85%) for four isolates. ITS showed less than a 0.5% percent identity difference between P. gonapodyides and P. megasperma for remaining three isolates.
Out of 260 isolates of suspected Oomycete isolate, 109 and 19 isolates were identified as P. rubi and P. gonapodyides, respectively, from multiplex targeted-sequencing approach ( Table 1). All P. gonapodyides isolates, except one, were recovered from the baiting isolation method. Seventy-four percent of P. gonapodyides isolates were recovered from 2018 and the remainder were from 2019. About 57% of isolates of P. rubi were isolated from roots and 42% isolates were from canes and 1% of isolates were recovered from baiting techniques (Fig 6A). The majority of P. rubi isolates (85%) were recovered in 2020, while 9 and 6% of isolates were from 2018 and 2019, respectively (Fig 6B; Table 1). In 2018, P. rubi was most frequently recovered from the cultivar 'Chemainus' (51%), followed by 'Rudi' (27%) and 'Meeker' (15%) ( Fig  6C; Table 1), whereas P. gonapodyides was predominately isolated from moderately resistant cultivar 'Cascade Bounty'.

Pathogenicity studies
Isolates of P. rubi and P. gonapodyides produced distinct dark expanding lesions on rhododendron leaves (Fig 7A and 7B) which did not develop on control leaves (Fig 7C). For P. rubi, the first visible lesion was observed at 11 to 12 days after inoculation (DAI) and 8 to 10 DAI for P. gonapodyides. The mean rAUDPC of lesion length in P. rubi isolates ranged from 0.11 to 0.13 and 0.14 to 0.17 for P. gonopodyides.
In seedling assays inoculated with isolates of P. rubi, wilting of leaves and cane symptoms started at 9 to 12 DAI (Fig 8A to 8D). After 3 to 4 weeks following inoculation, severe wilting and root rot occurred (Fig 8E and 8F) in both experiments. The mean rAUDPC of wilting index ranged from 0.35 to 0.55. In addition, the PRAI values (92 to 100%) were very high among isolates of P. rubi. Infected leaves of rhododendron and raspberry roots tested positive for Phytophthora in the Agdia ImmunoStrip assay and Phytophthora was re-isolated from the infected tissues, confirming Koch's postulates.

Discussion
This study showed that RRWC was present in diverse fields of major raspberry growing areas in BC during 2018 to 2020. Foliage symptoms and wilting occurred in several fields across three years. Foliage symptoms and initial wilting began in late spring or early summer and severe wilting and dying of plants occurred in mid-summer and through the fall season. Higher disease severity and plant death occurred in the cultivars 'Chemainus', 'Meeker', and 'Squamish' than 'Cascade Bounty' and 'Cascade Harvest'. Isolation of Phytophthora spp. associated with RRWC is difficult because it can co-occur with other soil-borne pathogens and grows slowly in culture media. In 2018 and 2019, we used baiting techniques and direct isolation from infected roots, crowns and stem bases, since direct isolation from infected tissues was not very successful. However, isolation of P. rubi directly from infected tissue was successful in 2020, which may be favoured by frequent rainfall in spring and summer months. In previous studies, recovery of Phytophthora species directly from infected tissues was reported for P. rubi from cane and root tissues of red raspberry in western USA [8], and for P. cinnamomi and P. citrophthora from roots of blueberry in Chile [25]. Recovery of P. gonopodyides was more difficult directly from diseased tissues but not when a baiting method was used.
In this study, we developed and applied an Illumina-based multiplex targeted-sequencing approach to accurately identify Phytophthora species associated with RRWC. Our approach used degenerate primers designed to amplify and sequence multiple potential variant positions within three nuclear genes (HSP90, EF1α and βtub), which are unique to different Phytophthora species. Most previous studies used single genes or genetic regions for pathogen identification which may not be robust for accurate species identification or detect the presence of multiple species. Moreover, amplifying and sequencing single genes (one set of primers

PLOS ONE
per reaction per sample) is time consuming and expensive. We also compared multiplex-targeted sequencing results with ITS sequencing using representative isolates recovered from infected raspberry. However, the ITS sequencing results were not able to distinguish among isolates of P. rubi and P. fragariae, and P. megasperma and P. gonapodyides. Other researchers also reported that P. rubi shares identical ITS sequences with P. fragariae [10,11]. In addition with molecular characterization, colony and sporangial morphology of P. rubi and P. gonapodyides could be supportive for pathogen characterization, though similarity in morphological features can be observed among the species of Phytophthora. However, traditional methods including the use of morphological features for identifying species of Phytophthora can be slow and labor-intensive, and in the case of isolates with poor spore formation, can be inconclusive [3,10]. Given the limitations of existing identification methods, the method described here was highly specific, and clearly distinguished among species of Phytophthora and could be used to monitor the introduction of previously undescribed species. Also, this technique successfully identified Phytophthora species that are known to attack blueberry, for example P. cinnamomi (unpublished data), suggesting the potential of this method to identify different Phytophthora pathogens infecting different berry hosts. A recent study also reported that multiplex targeted-sequencing effectively identified common fungal, bacterial and other pests associated with Megachile rotundata brood cells [26]. Targeted sequencing is now often less expensive and will likely a powerful tool in species detection.
The majority of isolates (85%) collected from major raspberry growing areas of BC were P. rubi. Previous studies also reported that P. rubi is a major causal agent associated with the RRWC and is widespread in most raspberry producing fields in the Pacific Northwest of USA [8,27]. Phytophthora rubi has been reported as a major pathogen from raspberry-growing regions worldwide [2,3,5,7,28]. However, we also identified isolates of P. gonapodyides in our study, the majority of which were predominately recovered from the cultivar 'Cascade Bounty' in one raspberry field. This cultivar was reported as moderately resistant to P. rubi at the time of release [29]. Isolation of P. gonapodyides from this cultivar suggests that the resistance to one species of Phytophthora may not be effective against another species. Wilcox and Latorre [5] also reported that some red raspberry cultivars with resistance to P. rubi showed susceptibility to P. megasperma. Therefore, knowledge of species composition of Phytophthora populations associated with RRWC is critical while identifying the sources of resistance and release of resistant cultivars. Several other species of Phytophthora have been sporadically reported to attack raspberry plants, such as P. gonapodyides (Petersen) Buisman and P. megasperma Drechs in Chile [5] and New York state [7], P. cryptogea Pethyb. and Leth., in New York state [7], Chile [5], Australia [30] and New Zealand [31]. In addition, P. citricola Sawada was reported from New York state [7], Chile [4,5] and Bulgaria [32] and P. citrophthora (R.E. Sm. and E. H. Sm.) Leonian from Chile [4]. This is the first report of recovery of P. gonapodyides from red raspberry in BC.
Compared to 2018 and 2019, the frequency of recovery of P. rubi isolates was higher in 2020 and isolation was successful from several fields throughout the growing season. In contrast, the recovery of Phytophthora isolates was only successful either in late spring or late fall in 2018 and 2019. The availability of high soil moisture levels and optimal soil and ambient temperature in Pacific Northwest including Fraser Valley areas of BC in 2020 (Fig 9) may have greatly favored pathogen spread, rapid multiplication in host tissues, and disease symptoms development of P. rubi, which is favored by high rainfall or soil moisture and average soil temperatures of 15 to 20˚C [9,33].
The comparison of species composition among raspberry cultivars indicated that the frequency of P. rubi was highest in 'Chemainus' followed by 'Rudi' and 'Meeker'. These cultivars are commonly grown in BC. We also found severe wilting and foliage symptoms on the cultivar 'Cascade Delight' from which P. rubi was isolated. This cultivar was reported as moderately tolerant to RRWC [34]. Our findings suggest that resistance levels of raspberry cultivars may be declining over time. Cultivars 'Chilliwack' and 'Meeker' were considered to have some level of resistance to root rot when they were released but they are now considered susceptible [35,36]. There is the possibility that P. rubi populations in BC could be different among different sites where the cultivar was evaluated for resistance. It is therefore critical to study the temporal dynamics of pathogen populations collected from diverse fields and cultivars.
We also demonstrated that representative isolates of P. rubi and P. gonapodyides were pathogenic on rhododendron leaves, producing dark brown lesions. Moreover, P. rubi isolates caused severe wilting and root rot on 'Chemainus' raspberry roots, suggesting that P. rubi isolates were highly virulent to raspberry. Additional studies are in progress to evaluate additional isolates of P. rubi and P. gonapodyides obtained from diverse fields/regions to understand the virulence diversity of the pathogen population.
In conclusion, this study is the first to investigate a multiplex targeted-sequencing approach using different nuclear genes for accurate identification and characterization of Phytophthora spp. associated with RRWC from diverse fields of major raspberry growing areas of BC. The detection capability of different Phytophthora species in a single reaction can reduce the overall costs and significantly facilitate species detection over current ITS-based identification approaches. P. rubi was identified as the main Phytophthora species associated with RRWC of raspberry in BC. We also identified P. gonapodyides associated with the disease complex in BC. This species was isolated from moderately resistant raspberry cultivars, indicating that the current sources of resistance to RRWC may not be adequate against all species of Phytophthora.
Supporting information S1 Fig. Example alignment of HSP90 gene region for representative Phytophthora species. Note: each sequence is slightly different (unique to that species). (TIF)  Table. Summary data for multiplex targeted-sequencing of unknown isolates obtained from red raspberry infected with RRWC. (DOCX)