Detection of Phytophthora ramorum in Nurseries and Forest Lands in California in 2004 to 2009.

From December 2004 through May 2009, samples were collected from California nurseries and wild lands to survey for Phytophthora ramorum and comply with federal regulations of nursery stock. Samples were prescreened by an enzyme-linked immunosorbent assay (ELISA) that detects Phytophthora spp. and tested by culture, P. ramorum-specific real-time polymerase chain reaction (PCR), and nested PCR. Yearly percentages of infected samples ranged from 0.6 to 2.3%. Camellia spp., Rhododendron spp., Magnolia spp., Pieris spp., and Laurus nobilis tested positive the most frequently in the nurseries and Lithocarpus densiflorus, Umbellularia californica, and Quercus agrifolia tested positive most often from wild lands. Of the 118,410 samples isolated onto PARP media, 0.8% was identified as P. ramorum. Of 115,056 samples tested by ELISA, 5.9% tested positive for Phytophthora spp. Of the 6,520 samples tested by PCR, 12.4% tested positive for P. ramorum. The false-negative, positive, and internal control failure rates of the assays are discussed. After removing the seasonal effect of sampling strategy, isolation of the pathogen into culture was found to be seasonally dependent whereas detectability by PCR and ELISA was not. To our knowledge, this is the first evaluation of a regulatory testing program for a plant pathogen on this scale using standardized assays.

Incidence of Agrobacterium tumefaciens Biovar 1 in and on 'Paradox' (Juglans hindsii × Juglans regia) Walnut Seed Collected from Commercial Nurseries.

The walnut rootstock 'Paradox' (Juglans hindsii × J. regia) is susceptible to Agrobacterium tumefaciens, which often results in a high incidence of crown gall in nursery or walnut production orchards. Though A. tumefaciens is susceptible to the commonly used preplant soil fumigants, crown gall incidence can rise above acceptable levels. We examined the ability of Paradox seed to acquire A. tumefaciens as a function of harvest method used prior to planting. Over a 2-year period at two participating commercial nurseries, Paradox seed were collected directly from the mother tree without contacting the soil or gathered after sitting on the orchard floor for up to 28 days. A. tumefaciens was never detected in or on the 2,650 seeds collected directly from the mother tree. Both virulent and avirulent A. tumefaciens strains were detected in and on the husk of nuts incubated on the orchard floor at a frequency directly proportional to the time spent on the orchard floor. Regardless of A. tumefaciens contamination in or on the husk, A. tumefaciens was never detected in the seed interior. Avoiding soilborne populations of A. tumefaciens at the time of seed collection will play an important role in managing crown gall.

Cationic Surfactants: Potential Surface Disinfectants to Manage Agrobacterium tumefaciens Biovar 1 Contamination of Grafting Tools.

Nursery production of walnut seedlings is a 2-year process, during which crown gall, caused by Agrobacterium tumefaciens, often develops at grafting wounds. In this study, the spread of crown gall via contaminated tools and the efficacy of several disinfectants against A. tumefaciens were demonstrated. The cationic surfactants benzalkonium chloride (BC), cetyltrimethylammonium bromide (CTAB), and Physan 20 eliminated 100% of the A. tumefaciens population in water suspensions treated at 7, 5, and 2 ppm, respectively. Sodium hypochlorite eliminated 100% of the A. tumefaciens population at 0.5 ppm. Sodium hypochlorite efficacy, however, was reduced by 64% in the presence of total solids (0.7 g/ml) which are commonly found in field situations. At similar concentrations of total solids, the efficacy of cationic surfactants decreased, on average, by only 13%. The minimum effective treatment needed to eliminate A. tumefaciens on infested scalpels was a 5-s exposure to BC or CTAB at 5,000 ppm (0.5%). Infested scalpels treated with BC or CTAB at less than 5,000 ppm caused gall formation in 14 ± 7% of cuts made on Datura stramonium stems. This was significantly less than the tumor incidence (100%) in cuts made with inoculated blades not treated BC or CTAB.

Role of Systemic Agrobacterium tumefaciens Populations in Crown Gall Incidence on the Walnut Hybrid Rootstock 'Paradox'.

Greater than 75% of English walnut production in the United States occurs on the walnut rootstock Juglans hindsii × J. regia 'Paradox', which is highly susceptible to infection by Agrobacterium tumefaciens. When seed were germinated and grown in the presence of A. tumefaciens, in the absence of wounding, 94% of the seedlings exhibited tumors while 89% contained systemic A. tumefaciens populations. When seedlings were wound inoculated, A. tumefaciens established endophytic populations in stem tissue and often migrated from the site of infection. Distribution of A. tumefaciens in the stem was random and may exhibit seasonal variation. A. tumefaciens populations in root tissue were more readily detected than in stem tissue and may serve as a reservoir for subsequent infection of the aerial portions of the tree. Importantly, 7% of inoculated, asymptomatic seedlings contained endophytic populations of A. tumefaciens. In all, 17% of seedlings inoculated as seeds developed galls at secondary stem-wound sites. These results provide an ecological and epidemiological foundation upon which to modify existing tree-handling practices in both nursery and orchard production environments to manage crown gall incidence.

First Report of Leaf Spot Caused by Phytophthora taxon Pgchlamydo on Evergreen Nursery Stock in California.

As part of the Phytophthora ramorum testing program from 2005 through 2007, a Phytophthora sp. was isolated on PARP-CMA medium (4) at the CDFA lab in Sacramento, CA, from the margin of necrotic spots and tissue suffering from dieback on Arctostaphylos sp. (manzanita), Camellia spp., Laurus nobilis (bay), Buxus sempervirens (boxwood), Rhododendron sp., Arbutus unedo (strawberry tree), and Sequoia sempervirens (coast redwood). Isolates were collected from Shasta, Contra Costa, San Diego, Solano, Santa Cruz, Alameda, Sacramento, San Joaquin, Monterey, and Los Angeles Counties. Isolates from A. unedo tissue on PARP medium produced apapillate, obovate sporangia 25 to 80 × 15 to 40 µm (48.0 × 26.9 µm average) and a few isolates produced intercalary and terminal chlamydospores at 22°C (30 to 46 µm diameter, 38.9 µm average). The internal transcribed spacer region (ITS) of rDNA was amplified from four isolates using ITS1 and ITS4 primers as described by White et al. (3) and the amplicons sequenced (GenBank Accession Nos. JQ307188 through JQ307191). BLAST analysis of the amplicons showed 99 to 100% identity with the ITS sequence of Phytophthora taxon Pgchlamydo from forest streams in Oregon (GenBank Accession No. HM004224) (1). Pathogenicity tests were performed on B. sempervirens, C. sasanqua, L. nobilis, and A. unedo. Five plants of each species were inoculated with 6-mm plugs taken from the margin of a 7- to 10-day-old culture grown on V8 juice agar. Plant leaves were wounded with a sterile pushpin and two agar plugs were covered with a freezer tube cap filled with sterile dH2O and clipped to the underside of the leaves with a sterile pin-curl clip (4). Inoculated plants were sprayed with water, covered with plastic bags, and incubated for 2 days, when bags and plugs were removed. Five leaves of each isolate plus five control plugs using V8 juice agar alone were inoculated on each plant. Plants were incubated for 12 days at 18°C (16-h photoperiod). Lesions formed on all inoculated plants, ranging in size from approx. 1 mm on B. sempervirens to 9.2 × 10.9 mm average on A. unedo. The lesions on A. unedo grew into and caused the mid-vein to blacken. The lesion sizes on camellia and bay were larger than those formed on B. sempervirens and smaller than those formed on A. unedo, with most lesions surrounded by a dark ring. Phytophthora taxon Pgchlamydo is associated with leaf lesions on rhododendron and dieback of yew in Minnesota (2). To our knowledge, this is the first report of Phytophthora taxon Pgchlamydo causing disease in camellia, bay, strawberry tree, and boxwood in California. Phytophthora taxon Pgchlamydo causes damage that is indistinguishable from the quarantine pest, P. ramorum (4). References: (1) P. W. Reeser et al. Mycologia 103:22, 2011. (2) B. W. Schwingle and R. A. Blanchette. Plant Dis. 92:642, 2008. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds., Academic Press, San Diego, 1990. (4) L. E. Yakabe et al. Plant Dis. 93:883, 2009.

Soil Treatments for the Potential Elimination of Phytophthora ramorum in Ornamental Nursery Beds.

Ramorum leaf blight, caused by Phytophthora ramorum, has reemerged at several California nurseries after removal of infested material. In many cases, reemergence was not associated with reintroduction of the pathogen and may be attributed to inoculum surviving in soil beds because P. ramorum propagules can survive for over a year in soil. Using artificially infested soil in microcosms, fumigation and heat treatments were examined as potential eradicants of P. ramorum from soil. Treatments with chloropicrin, Vapam, and iodomethane were effective in reducing P. ramorum propagules below detection limits. Basamid was consistently effective only when fully incorporated into the soil. Application of Basamid (392 kg/ha) at infested ornamental nursery sites mirrored results from microcosm experiments, indicating that a tarp cover over treated soil is necessary for reliable efficacy. Dimethyldisulfide, 1,3-dichloropropene, and two formulations of hydrogen dioxide were less effective, resulting in only partial reduction of propagules. In heat treatments, P. ramorum in soil microcosms remained detectable 42 days after microcosms were incubated at 30 and 22°C but was not detectable in soil heated above 40°C for 3 days. Results from a solarized field plot indicate that prolonged sublethal temperatures, between 35 and 40°C for 42 days, can be effective in eliminating detectable propagules of P. ramorum.

Identification and Frequency of Phytophthora Species Associated with Foliar Diseases in California Ornamental Nurseries.

Numerous ornamental nurseries in 32 California counties were surveyed for leaf spots as part of the California Department of Food and Agriculture mandated surveys targeting Phytophthora ramorum. Tissue collected during the 2005 and 2006 surveys was initially screened by a Phytophthora-specific enzyme-linked immunosorbent assay. All positives samples were further tested using polymerase chain reaction to determine if P. ramorum was present. P. ramorum was detected in 1% of the total number of samples taken during the surveys. A total of 377 isolates were identified as species of Phytophthora other than P. ramorum, and their identity was determined by internal transcriber spacer (ITS) sequences. Subsets of the putative ITS-species were further verified using accepted morphological characters. Thirteen species of Phytophthora were found: P. cactorum, P. cambivora, P. citricola, P. citrophthora, P. cryptogea, P. foliorum, P. gonapodyides, P. hibernalis, P. nemorosa, P. 'Pgchlamydo', P. pseudosyringae, P. syringae, and P. tropicalis. P. syringae and P. citricola made up 55% of the total number of isolates. Species thought to be strictly forest pathogens, P. nemorosa and P. pseudosyringae, each made up less than 4% of the isolates. To test pathogenicity of acquired isolates, subsets of different species of Phytophthora were inoculated onto leaves of selected host plant genera. Of the 66 pathogen-host genera combinations tested, 44 resulted in lesion formation. Disease symptoms appeared as dark, water-soaked lesions with irregular margins and were similar among Phytophthora species.