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Commercial production of raspberry ( Rubus ideaus ) transplants is almost exclusively accomplished through clonal propagation. One system involves forcing young shoots to grow from roots. The shoots are cut and rooted in propagation trays and referred to as tray plants. Sanitation is important during tray plant production as this method carries some risk due to contamination by substrate pathogens. In May 2021, a new disease was observed on raspberry tray plant cuttings at one nursery location in California, and observed again in 2022 and 2023 but at a much lesser extent. Multiple cultivars were affected; however, up to 70% mortality was observed on cv. RH740.1. In less affected cultivars, mortality ranged from 5-20%. Symptoms included chlorotic leaves, lack of rooting, and blackening at the basal end of shoots, followed by death of the cutting. Affected propagation trays had inconsistent foliage and patchy growth. Chains of chlamydospores (two to eight spores in each chain) similar in morphology to Thielaviopsis species (Shew and Meyer 1992) were observed at the cut end of symptomatic tray plants using a microscope. Isolates were retrieved by incubating tissue on surface-disinfested (1% NaOCl) carrot discs in a humid chamber for 5 days until greyish black mycelium was observed (Yarwood 1946). Mycelium was transferred to acidified potato dextrose agar and formed a gray to black compact mycelial colony with both endoconidia and chlamydospores. Endoconidia were catenulate, single-celled with slightly rounded ends, colorless, and 10-20 µm x 3-5 µm in size; dark-colored chlamydospores were 10-15 µm x 5-8 µm in size. The ITS region of isolates 21-006 and 22-024 was amplified with ITS5 and ITS4 primers using a 48°C annealing temperature (White et al. 1990), Sanger sequenced (GenBank accession OQ359100) and yielded 100% match to Berkeleyomyces basicola accession MH855452. Pathogenicity was confirmed by dipping 80 grams of roots of cv. RH740.1 into a suspension of 10 6 conidia/mL of isolate 21-006 for 15 min. For the non-inoculated control, 80 grams of roots were dipped in water. Roots were then planted into trays of coir (Berger, Watsonville, CA). Six weeks after inoculation, twenty-four shoots were harvested from each treatment, stuck into propagation trays filled with coir and maintained in a humid chamber for 14-days to induce rooting. Tray plants were then harvested and assessed for root development, black basal shoot tips, and presence of chlamydospores. Forty-two percent of cuttings from the inoculated treatment had rotten basal tips and failed to root, in comparison to 8% of the cuttings from the non-inoculated control. Chlamydospores were visualized only on shoots that emerged from inoculated roots and B. basicola was isolated only from cuttings originating from inoculated roots. Post-inoculation isolates were confirmed as B. basicola using methods described above. To our knowledge, this is the first report of B. basicola infecting raspberry. Confirmation of this pathogen on tray plants is significant because of the potential impact this disease may have in commercial nursery production worldwide. In 2021, the value of the harvested raspberry crop in the U.S. totaled $531 M, of which California represented $421 M (USDA 2022). The value of the 2021 crop was highest in the U.S. ($531 M), followed by Russia ($512 M), Spain ($405 M) and Mexico ($332 M) (FAO 2021).

Microbial-root associations are important to help plants cope with abiotic and biotic stressors. Managing these interactions offers an opportunity for improving the efficiency and sustainability of agricultural production. By characterizing the bacterial and archaeal community (via 16S rRNA sequencing) associated with bulk and rhizosphere soil of sixteen strawberry cultivars in two controlled field studies, we explored the relationships between the soil microbiome and plant resistance to two soil-borne fungal pathogens (Verticillium dahliae and Macrophomina phaseolina). Overall, the plants had a distinctive and genotype-dependent rhizosphere microbiome with higher abundances of known beneficial bacteria such as Pseudomonads and Rhizobium. The rhizosphere microbiome played a significant role in the resistance to the two soil-borne pathogens as shown by the differences in microbiome between high and low resistance cultivars. Resistant cultivars were characterized by higher abundances of known biocontrol microorganisms including actinobacteria (Arthrobacter, Nocardioides and Gaiella) and unclassified acidobacteria (Gp6, Gp16 and Gp4), in both pathogen trials. Additionally, cultivars that were resistant to V. dahliae had higher rhizosphere abundances of Burkholderia and cultivars resistant to M. phaseolina had higher abundances of Pseudomonas. The mechanisms involved in these beneficial plant-microbial interactions and their plasticity in different environments should be studied further for the design of low-input disease management strategies.

A new Phytophthora species was found associated with gummosis in black wattle plantations in the subtropical, humid, south of Brazil. The new species Phytophthora acaciae is formally named herein based on phylogenetic and morphological analyses. This is the fourth Phytophthora species found from this pathogen complex in black wattle plantations causing gummosis in Brazil. The other three species are P. nicotianae, P. boehmeriae , and P. frigida. Phytophthora acaciae is heterothallic with amphigynous antheridia, noncaducous, papillate sporangia and is placed in the Phytophthora clade 2 based on nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) sequences. Maximum parsimony and maximum likelihood phylogenetic analyses of P. acaciae isolates based on multigene sequences, including partial DNA sequences of three nuclear protein-coding genes (β-tubulin, translation elongation factor-1α, and ras-related protein), two mitochondrial protein-coding genes (cytochrome c oxidase subunits I and II), in addition to ITS sequence data, support the delimitation of this new species on Acacia mearnsii from the other previously described clade 2 Phytophthora species. Pathogenicity trial confirmed that the new species causes necrotic lesions on the plant stem, with either the presence or absence of gum.

Sensitivity of Botrytis cinerea to seven fungicide chemical classes was determined for 888 isolates collected in 2016 from 47 California strawberry fields. Isolates were collected early season (minimum fungicide exposure) and late season (maximum fungicide exposure) from the same planting block in each field. Resistance was determined using a mycelial growth assay, and variable frequencies of resistance were observed to each fungicide at both sampling times (early season %, late season %): boscalid (12, 35), cyprodinil (12, 46), fenhexamid (53, 91), fludioxonil (1, 4), fluopyram (2, 7), iprodione (25, 8), isofetamid (0, 1), penthiopyrad (8, 25), pyraclostrobin (77, 98), and thiophanate-methyl (81, 96). Analysis of number of chemical class resistances (CCRs) revealed an increasing shift in CCR from the early to late season. Phenotypes of 40 isolates that were resistant or sensitive to different chemical classes were associated with presence or absence of mutations in target genes. Fungicide-resistance phenotypes determined in the mycelial growth assay closely matched (93.8%) the genotype observed. Previously described resistance-conferring mutations were found for each gene. A survey of fungicide use from 32 of the sampled fields revealed an average of 15 applications of gray mold-labeled fungicides per season at an average interval of 12 days. The most frequently applied fungicides (average number of applications during the 2016 season) were captan (7.3), pyraclostrobin (2.5), cyprodinil (2.3), fludioxonil (2.3), boscalid (1.8), and fenhexamid (1.4). Multifungicide resistance is widespread in California. Resistance management tactics that reduce selection pressure by limiting fungicide use, rotating among Fungicide Resistance Action Committee codes, and mixing/rotating site-specific fungicides with multisite fungicides need to be improved and implemented.

Anthracnose crown rot of strawberry, caused by Colletotrichum acutatum, is an important disease affecting California nursery and fruit production. Preplant dip treatments of transplants with fludioxonil-cyprodinil or azoxystrobin are industry standards for managing the disease and have been used extensively. Following reports of reduced efficacy of azoxystrobin in the field, high levels of quinone outside inhibitor (QoI) resistance were detected in California isolates of the pathogen. Resistance was associated with the G143A mutation in the cytochrome b gene, similar to a previous report from Florida, and there were no detected fitness penalties in pathogenicity or virulence. Therefore, several alternative fungicides were investigated in laboratory and field studies. Subsequently, the new biofungicide natamycin was identified. Baseline sensitivities of 74 isolates of C. acutatum to natamycin were determined to be unimodal, with a range from 0.526 to 1.996 μg/ml (mean 0.973 μg/ml). Although this toxicity was considerably lower than that of azoxystrobin (using sensitive isolates), fludioxonil, or cyprodinil, dip treatments of transplants with natamycin (at 500 or 1000 mg/liter) were highly effective. Disease severity and plant mortality in field studies with inoculated transplants were reduced to similarly low levels as treatments containing fludioxonil, whereas azoxystrobin failed in inoculations with QoI-resistant isolates of C. acutatum. Fruit yield was also significantly increased by natamycin as compared with the inoculated control. Differences in disease susceptibility were observed among cultivars evaluated, with Monterey and Portola more susceptible than Fronteras. Natamycin has a unique mode of action that is different from other fungicides registered on strawberry and, based on this research, was registered in the United States as a preplant, biofungicide dip treatment of strawberry transplants for management of anthracnose crown rot.

Black shank disease of tobacco, caused by the oomycete Phytophthora nicotianae, is a major threat to production in the United States and tobacco-producing areas worldwide. In a statewide survey of North Carolina, the rapid shift from race 0 to race 1 was documented. Collected pathogen isolates were characterized phenotypically for mating type and mefenoxam sensitivity, and genotypically by comparing sequences from three cytoplasmic and two nuclear regions. Both the A1 and A2 mating types were found throughout the state. When both mating types were recovered from the same field, pairings of isolates yielded viable oospores, indicating for the first time the potential for sexual sporulation by P. nicotianae in natural populations. Because the loss of complete resistance required a renewed use of the fungicide mefenoxam, a subset of the survey isolates was screened for sensitivity to the fungicide. All isolates were sensitive, with a mean effective concentration to inhibit 50% of hyphal growth of 0.4 μg/ml that was similar across mating types and races. Molecular characterization of 226 isolates revealed that the pathogen exists as multiple clonal types within the state. Genetic diversity among the pathogen population and the potential for sexual recombination may help explain the ability of the pathogen to rapidly adapt to host resistance genes.

Isolates of Phytophthora infestans (n = 178) were collected in 2002 to 2009 from the eastern United States, Midwestern United States, and eastern Canada. Multilocus genotypes were defined using allozyme genotyping, and DNA fingerprinting with the RG-57 probe. Several previously described and three new mulitilocus genotypes were detected. The US-8 genotype was found commonly on commercial potato crops but not on tomato. US-20 was found on tomato in North Carolina from 2002 through 2007 and in Florida in 2005. US-21 was found on tomato in North Carolina in 2005 and Florida in 2006 and 2007. US-22 was detected on tomato in 2007 in Tennessee and New York and became widespread in 2009. US-22 was found in 12 states on tomato and potato and was spread on tomato transplants. This genotype accounted for about 60% of all the isolates genotyped. The US-23 genotype was found in Maryland, Virginia, Pennsylvania, and Delaware on both tomato and potato in 2009. The US-24 genotype was found only in North Dakota in 2009. A1 and A2 mating types were found in close proximity on potato and tomato crops in Pennsylvania and Virginia; therefore, the possibility of sexual reproduction should be monitored. Whereas most individuals of US-8 and US-20 were resistant to mefenoxam, US-21 appeared to be intermediately sensitive, and isolates of US-22, US-23, and US-24 were largely sensitive to mefenoxam. On the basis of sequence analysis of the ras gene, these latter three genotypes appear to have been derived from a common ancestor. Further field and laboratory studies are underway using simple sequence repeat genotyping to monitor current changes in the population structure of P. infestans causing late blight in North America.

Background: The Southern Nursery Integrated Pest Management (SNIPM) working group surveyed ornamental nursery crop growers in the southeastern United States to determine their pest management practices. Respondents answered questions about monitoring practices for insects, diseases and weeds, prevention techniques, intervention decisions, concerns about IPM and educational opportunities. Survey respondents were categorized into three groups based on IPM knowledge and pest management practices adopted., Results: The three groups differed in the use of standardized sampling plans for scouting pests, in monitoring techniques, e.g. sticky cards, phenology and growing degree days, in record-keeping, in the use of spot-spraying and in the number of samples sent to a diagnostic clinic for identification and management recommendation., Conclusions: Stronger emphasis is needed on deliberate scouting techniques and tools to monitor pest populations to provide earlier pest detection and greater flexibility of management options. Most respondents thought that IPM was effective and beneficial for both the environment and employees, but had concerns about the ability of natural enemies to control insect pests, and about the availability and effectiveness of alternatives to chemical controls. Research and field demonstration is needed for selecting appropriate natural enemies for augmentative biological control. Two groups utilized cooperative extension almost exclusively, which would be an avenue for educating those respondents., (Copyright © 2012 Society of Chemical Industry.)

With the increased attention given to the genus Phytophthora in the last decade in response to the ecological and economic impact of several invasive species (such as P. ramorum, P. kernoviae, and P. alni), there has been a significant increase in the number of described species. In part, this is due to the extensive surveys in historically underexplored ecosystems (e.g., forest and stream ecosystems) undertaken to determine the spread of invasive species and the involvement of Phytophthora species in forest decline worldwide (e.g., oak decline). The past decade has seen an approximate doubling in the number of described species within the genus Phytophthora, and the number will likely continue to increase as more surveys are completed and greater attention is devoted to clarifying phylogenetic relationships and delineating boundaries in species complexes. The development of molecular resources, the availability of credible sequence databases to simplify identification of new species, and the sequencing of several genomes have provided a solid framework to gain a better understanding of the biology, diversity, and taxonomic relationships within the genus. This information is much needed considering the impact invasive or exotic Phytophthora species have had on natural ecosystems and the regulatory issues associated with their management. While this work is improving our ability to identify species based on phylogenetic grouping, it has also revealed that the genus has a much greater diversity than previously appreciated.

A root rot disease of cultivated tobacco called yellow stunt has been observed in the burley tobacco production areas of Brazil since the early 1990s. Root infecting fungi and straminipiles were isolated from the roots of diseased tobacco plants, including a semi-papillate, homothallic, slow growing Phytophthora species. Pathogenicity trials confirmed that Phytophthora sp. caused root rot and stunting of burley and flue-cured tobaccos. Morphological characteristics of the asexual and sexual stages of this organism did not match any reported Phytophthora species and were very different from the widely known tobacco black shank pathogen P. nicotianae. Phylogenetic analysis based on sequences of the internal transcribed spacer rDNA, β-tubulin and translation elongation factor 1-α regions indicated that this organism represents a previously unreported Phytophthora species that is significantly supported in clade 2 and most closely related to P. capsici. However P. glovera differs from P. capsici in a number of morphological characters, most significantly P. glovera is homothallic and produces both paragynous and amphigynous antheridia while P. capsici is heterothallic and produces only amphigynous antheridia. In this paper we confirmed pathogenicity of this species on tobacco and describe the morphological and molecular characteristics of Phytophthora glovera sp. nov.

Wood-based mulches are used in avocado production and are being tested on Fraser fir for reduction of Phytophthora root rot, caused by Phytophthora cinnamomi. Research with avocado has suggested a role of microbial cellulase enzymes in pathogen suppression through effects on the cellulosic cell walls of Phytophthora. This work was conducted to determine whether cellulase activity could account for disease suppression in mulch systems. A standard curve was developed to correlate cellulase activity in mulches with concentrations of a cellulase product. Based on this curve, cellulase activity in mulch samples was equivalent to a cellulase enzyme concentration of 25 U ml(-1) or greater of product. Sustained exposure of P. cinnamomi to cellulase at 10 to 50 U ml(-1) significantly reduced sporangia production, but biomass was only reduced with concentrations over 100 U ml(-1). In a lupine bioassay, cellulase was applied to infested soil at 100 or 1,000 U ml(-1) with three timings. Cellulase activity diminished by 47% between 1 and 15 days after application. Cellulase applied at 100 U ml(-1) 2 weeks before planting yielded activity of 20.08 μmol glucose equivalents per gram of soil water (GE g(-1) aq) at planting, a level equivalent to mulch samples. Cellulase activity at planting ranged from 3.35 to 48.67 μmol GE g(-1) aq, but no treatment significantly affected disease progress. Based on in vitro assays, cellulase activity in mulch was sufficient to impair sporangia production of P. cinnamomi, but not always sufficient to impact vegetative biomass.

A tomato (Solanum lycopersicum) foliar blight disease of unknown etiology was observed in North Carolina (NC) during 2005 to 2006. Symptoms included necrotic lesions and blighted leaves, with signs of white mycelial growth on abaxial leaf surfaces. The morphology of isolates from symptomatic leaves was consistent with that of Rhizoctonia solani. Because the pattern of symptom expression suggested that basidiospores were the primary inoculum source, Koch's postulates were fulfilled using a method to generate basidiospores in planta. Isolates were characterized by morphology, DNA sequence analysis, hyphal anastomosis, and somatic hyphal interactions. Phylogenetic analyses and hyphal anastomosis criteria support placement of the isolates in R. solani anastomosis group 3 (AG-3). Tomato foliar blight isolates from NC form a single phylogenetic group with tomato isolates of R. solani AG-3 from Japan and are more closely related to R. solani AG-3 isolates from potato than tobacco. Isolates exhibited both compatible and incompatible hyphal interactions when paired in vitro. To our knowledge, this is the first detailed report of tomato foliar blight caused by R. solani AG-3 in North America. A comprehensive description of the technique employed for producing basidiospores is presented with potential utility for understanding foliar disease etiology in other Rhizoctonia pathosystems.

Phytophthora spp. represent a serious threat to agricultural and ecological systems. Many novel Phytophthora spp. have been reported in recent years, which is indicative of our limited understanding of the ecology and diversity of Phytophthora spp. in nature. Systematic cataloging of genotypic and phenotypic information on isolates of previously described species serves as a baseline for identification, classification, and risk assessment of new Phytophthora isolates. The Phytophthora Database (PD) was established to catalog such data in a web-accessible and searchable format. To support the identification of new Phytophthora isolates via comparison of their sequences at one or more loci with the corresponding sequences derived from the isolates archived in the PD, we generated and deposited sequence data from more than 1,500 isolates representing the known diversity in the genus. Data search and analysis tools in the PD include BLAST, Phyloviewer (a program for building phylogenetic trees using sequences of selected isolates), and Virtual Gel (a program for generating expected restriction patterns for given sequences). The PD also provides a customized means of storing and sharing data via the web. The PD serves as a model that easily can be adopted to develop databases for other important pathogen groups.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum. Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

ABSTRACT Plant pathogen culture collections are essential resources in our fight against plant disease and for connecting discoveries of the present with established knowledge of the past. However, available infrastructure in support of culture collections is in serious need of improvement, and we continually face the risk of losing many of these collections. As novel and reemerging plant pathogens threaten agriculture, their timely identification and monitoring depends on rapid access to cultures representing the known diversity of plant pathogens along with genotypic, phenotypic, and epidemiological data associated with them. Archiving such data in a format that can be easily accessed and searched is essential for rapid assessment of potential risk and can help track the change and movement of pathogens. The underexplored pathogen diversity in nature further underscores the importance of cataloguing pathogen cultures. Realizing the potential of pathogen genomics as a foundation for developing effective disease control also hinges on how effectively we use the sequenced isolate as a reference to understand the genetic and phenotypic diversity within a pathogen species. In this letter, we propose a number of measures for improving pathogen culture collections.

ABSTRACT The choice of detection method for phytopathogens can be critically important in determining the success or failure of pest regulation systems. We present an assay for Phytophthora ramorum that uses 5' fluorogenic exonuclease (TaqMan) chemistry to detect and quantify the pathogen from diseased tissue, and include a universal primer and probe set for an internal positive control. This method is sensitive, detecting as little as 15 fg of target DNA when used in a nested design or 50 fg when used in a single round of polymerase chain reaction. None of the 17 other Phytophthora spp. tested was amplified by this assay. A comparison of the nested and non-nested TaqMan assays, and of one other nested assay, showed nested methods to be significantly more sensitive than nonnested and showed that host substrate significantly affected sensitivity of all assays. The nested TaqMan protocol was successfully field-tested; P. ramorum was detected in 255 of 874 plants in California woodlands, whereas the single-round TaqMan protocol detected significantly fewer positive samples. Finally, we documented increases in the quantity of pathogen DNA in Umbellularia californica leaves in initial stages of infection.

The genetic structure within and between USA and European populations of the emerging phytopathogen Phytophthora ramorum was examined. Four primer combinations were used for amplified fragment length polymorphism (AFLP) fingerprinting of 67 USA isolates from California and Oregon, and 18 European isolates from Belgium, Germany, The Netherlands, Spain and the UK. In addition, three DNA regions (ITS, cox II, and nad 5) of additional Phytophthora species were amplified by polymerase chain reaction, sequenced, and analysed to provide better phylogenetic understanding of P. ramorum within the genus Phytophthora. AFLP banding patterns indicate that the 85 isolates form two distinct lineages within a monophyletic group, distinct from the closely related outgroup species P. lateralis. With the exception of two isolates from an Oregon nursery, European and USA isolates clustered separately within individual clades. The AFLP profiles also indicate that a single clonal lineage dominates the North American population, while the European population consists of an array of mainly unique, closely related AFLP types. Sequences from the three DNA regions were identical among all P. ramorum isolates, and phylogenetic analysis indicates that P. ramorum is closely related to P. lateralis and P. hibernalis.