S. Swain

Composting is an effective treatment option for sanitization of Phytophthora ramorum-infected plant material

Aims:  To determine the effects of heat and composting treatments on the viability of the plant pathogen Phytophthora ramorum grown on both artificial and various natural substrates.

First report of foliar infection of Rosa gymnocarpa by Phytophthora ramorum.

In May 2003, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from leaflets of wood rose (Rosa gymnocarpa Nutt.), a native, low shrub of the Rosaceae family, at the Jack London State Park in Sonoma County, California. Affected leaflets had cream-to-brown lesions or spots, sometimes delimited by a chlorotic zone. Lesions coalesced with time and spread into the petiole and rachis. Lesions on the stems were not observed. Isolates were typical of P. ramorum with large chlamydospores and caduceus, semipapillate sporangia, and the sequence (GenBank Accession No. AY526571) of the internal transcribed spacer (ITS) region of the rDNA matched those published previously (4). The site was a mixed forest containing some confirmed P. ramorum-infected trees of coast redwood (Sequoia sempervirens), bay laurel (Umbellularia californica), and tanoak (Lithocarpus densiflorus) (3,4). These sites also contained California rose (R. californica Cham. & Schldl.); however, no symptoms were observed on this species. A terminal leaflet of asymptomatic, pesticide-free, potted-plants of California rose and wood rose (four plants each) was inoculated with zoospores of a P. ramorum isolate (American Type Culture Collection, Manassas, VA, ATCC MYA-3281; Centraal Bureau voor Schimmelcultures, Baarn, the Netherlands, CBS 114390) obtained from infected wood rose (2). A control leaflet of each plant was dipped in sterile deionized water. Branches containing the inoculated and control leaflets were placed in moist plastic bags, and plants were maintained at 21 to 22°C in the laboratory for 6 days. The inoculation experiment was repeated. In both inoculations, brown lesions (extending up to 8 mm from the leaflet tip) were observed on leaflets of both species 2 days after inoculation with P. ramorum. At 6 days after inoculation, lesions starting from the leaflet tip averaged 12.2 mm in length (range 10 to 16 mm) for wood rose and 9.6 mm (range 3 to 20 mm) for California rose. Some lesions extended into the petiole in both rose species. Sporangia were observed in washings of the lesions from four plants of California rose and one plant of wood rose, and P. ramorum was reisolated on Phytophthora-selective agar medium modified with 25 mg of pentachloronitrobenzene (PCNB) (4) from all lesions. Control leaflets had no lesions, and P. ramorum was not reisolated. To our knowledge, this is the first report of a species of Rosa as a natural host for P. ramorum, although R. sempervirens was identified as being susceptible in artificial inoculations of detached leaves (E. Moralejo and L. Hernández, personal communication). Toyon (Heteromeles arbutifolia) in California and salmon berry (Rubus spectabilis) in Oregon are the other known hosts from the family Rosaceae (1). Wood rose is popular in the horticultural industry and is readily available from native plant nurseries in California, Oregon, Washington, and British Columbia, Canada. California rose is also popular, primarily in California. The pathogen could be disseminated on these plants, especially since sporangia were obtained from inoculated leaflets of these two species. References: (1) J. M. Davidson et al. On-line publication. doi:10.1094/PHP-2003-0707-01-DG. Plant Health Progress, 2003. (2) D. Hüberli et al. Plant Dis. 87:599, 2003. (3) P. E. Maloney et al. Plant Dis. 86:1274, 2002. (4) D. M. Rizzo et al. Plant Dis. 86:205, 2002.

A new report of Phytophthora ramorum on Rhamnus purshiana in Northern California.

Rhamnus purshiana, or cascara, is a deciduous tall shrub or small tree as much as 9 m high with thin, smooth, silver-gray bark. It is often present in shady sites in redwood and mixed evergreen forests of the North Ameri-can west coast, from British Columbia to northern California. In July 2005, symptomatic leaves with irregular, black spots, 2 to 5 mm in diameter and concentrated toward the tips, were collected from four cascara plants in the Samuel P. Taylor State Park, Marin County, California. There was no evidence of defoliation. Pieces of necrotic tissue were plated on selective medium (PARP) and maintained at 19°C for 2 weeks. A Phytophthora sp. was consistently isolated and it was identified as P. ramorum on the basis of morphological and molecular traits published previously (3,4). The P. ramorum isolate Pr-418 has been deposited in the American Type Culture Collection (ATCC MYA-3676) and a portion of the internal transcribed sequence (ITS) of rDNA has been deposited in the NCBI database (GenBank Accession No. DQ168874). Kochs postulates were completed using the leaf-dip method (2) on detached leaves collected from three cascara plants growing at the University of California Botanical Garden at Berkeley. Zoospore inoculum was prepared by flooding a 2-week-old culture growing on V8 agar with sterile water for 4 days. The liquid was filtered after cold shocking at 4°C for 30 min and incubated at room temperature for 1 h. Fifteen leaves were dipped in the resulting zoospore suspension (1.6 × 104 zoospores per ml) for either 1 min (experiment 1) or overnight (experiment 2). Leaves used as negative controls were dipped in sterile water. After removal from the inoculum, excess liquid was allowed to drain. Leaves were maintained in a moist chamber at 19°C with 13 h of natural light for 1 week. After 3 days of incubation, necrotic spots similar to those observed in the field had developed on leaves in experiment 2, while no symptoms were observed in experiment 1. Necrotic lesions were observed on 12 and 15 of 15 leaves in experiments 1 and 2, respectively, after 7 days of incubation. For each leaf, the necrotic area and percent necrosis was determined by placing the leaves in a flatbed scanner and processing the images with Assess (Version 1.01; The American Phytopathological Society, St. Paul, MN). Lesions extended from the tip of the leaves and covered 3 ± 1% of the total leaf area in experiment 1 and 33 ± 3% in experiment 2. Reisolation of P. ramorum on PARP was successful for all inoculated leaves. P. ramorum was never isolated from negative controls and no symptoms of infection were observed. The leaf-dip inoculation method is a rapid and reliable indicator of host susceptibility to P. ramorum, with many species developing necrosis when exposed to high concentrations of zoospores (3). Our results show that exposure time to the pathogen can play an important role in the development of symptoms. R. purshiana has been previously reported as a host in Oregon (1,2), but to our knowledge, this is the first report of cascara as a natural host of P. ramorum in the state of California. Our results confirm those from Oregon (2). The impact of infection by P. ramorum on cascara is unknown. References: (1) J. M. Davidson et al. Plant Health Prog. DOI:10.1094/PHP-2003-0707-01-DG, 2003. (2) E. Hansen et al. Plant Dis. 89:63, 2005. (3) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (4) S. Werres et al. Mycol Res. 105:1155, 2001.

First Report of Twig Canker on Willow Caused by Colletotrichum acutatum in California

Following prolonged spring rains and cool summer weather in 2010, mature weeping willow trees (Salix babylonica L.) growing next to a manmade lake in Marin County, CA, showed symptoms of a previously undescribed disease. During summer, small branches developed dark brown to black, sunken cankers. Canker lengths ranged from 3 to 20 cm. Within the cankered areas, affected twigs, shoots, and leaves turned brown, collapsed, and died. The distal portions of infected branches also died, giving the trees a blighted appearance. Acervuli and pink sporulation were observed in the canker tissue. When placed on acidified potato dextrose agar (A-PDA), canker tissues consistently yielded one type of fungal organism. On A-PDA, isolates produced gray aerial mycelium, acervuli, and single-celled fusiform conidia. Two isolates were identified as Colletotrichum acutatum based on sequence analysis of the ITS region of the ribosomal DNA and the 1-kb intron of the glutamine synthase gene (1) and fungal morphology (2,3) (GenBank Accession Nos. JQ951597 and JQ951598). The willow isolates examined were identified as C. acutatum based on a 99% identity of the ITS sequence with accession FR716517 and a 98% identity of the 1-kb intron sequence with accession GQ387248 in GenBank. Interestingly, the isolates were confirmed to be homothallic producing perithecia from monoconidial cultures. To demonstrate Kochs postulates, inocula were prepared from 2-week-old colonies of each of four isolates grown on A-PDA. Using containerized weeping willow trees as test material, shallow slits were cut into the epidermis of small (1.5-cm diameter or less) branches; one colonized agar plug was placed within each cut area and the epidermis was resealed by wrapping the branch with Parafilm. Ten inoculations were made for each isolate and inoculated plants were maintained in a greenhouse. After 4 weeks, inoculated branches exhibited dark cankers and twig dieback. C. acutatum was reisolated from all symptomatic cankers and matched the characteristics of the original isolates. Control twigs, inoculated with sterile agar plugs, did not develop any blight symptoms. This experiment was repeated and the results were the same. To our knowledge, this is the first documentation of C. acutatum as a pathogen of weeping willow in California. The disease resulted in repeated defoliation of trees in the Santa Venetia area of Marin County. Badly infected trees declined as a result of repeated defoliation and twig loss. Discussions with parks personnel suggested that the disease may have been present at low levels in the area for some years, and that disease severity increased dramatically with weather that was atypically wet and cool (max. mean temps. 5.5°C cooler and 6 cm more total rainfall than the records of the previous two years) for the area during May and June 2010, when the disease was discovered. References: (1) J. C. Guerber et al. Mycologia 95:872, 2003. (2) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (3) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990.

First Report of Infection of Maiden-Hair Fern (Adiantum jordanii and A. aleuticum) by Phytophthora ramorum in California

During July 2005, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from nine native Adiantum jordanii plants growing at two forest sites (Samuel P. Taylor State Park, Marin County and Peachland Road, Mendocino County) and from seven A. aleuticum plants at one forest site (Peachland Road) in California. At both locations, symptomatic plants were distributed close to rivers and roads and in association with infected bay laurel trees (Umbellularia californica), toyon (Heteromeles arbutifolia), and tanoaks (Lithocarpus densiflorus). Symptomatic leaflets showed brown spots that sometimes coalesced, killing entire leaves, but the disease did not appear to be fatal to the ferns. Necrotic tissues were plated on PARP and maintained in the dark at 18°C for 1 to 2 weeks. Isolates were identified as P. ramorum on the basis of colony morphology, the presence of chlamydospores and caducous, semipapillate sporangia, and the internal transcribed spacer (ITS) rDNA sequences (1,2). The P. ramorum isolates, Pr-419 from A. jordanii and Pr-422 from A. aleuticum, have been deposited in the American Type Culture Collection (ATCC MYA-3677 and MYA-3679, respectively) and a region of the ITS rDNA sequence deposited in the NCBI database (GenBank Accession No. DQ173082 and DQ219821, respectively). To test the pathogenicity, the tips of freshly detached leaves of A. jordanii and A. aleuticum were dipped into a solution of 1 × 103 zoospores per ml of Pr-419 and Pr-422 for 1 min. The wounded end of the leaves was not exposed to the inoculum. The zoospores were produced by flooding agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar with sterile deionized water. After 3 days of incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then at room temperature for 60 min. For each Adiantum species and P. ramorum isolate, 15 leaves collected from five potted nursery plants were tested. Control leaves were dipped in sterile deionized water. Leaves were maintained in a moist chamber at 19°C with 13 h of natural light for 9 days. Brown lesions similar to those seen in the forest developed on approximately 60 and 33% of the A. jordanii and A. aleuticum leaves, respectively, inoculated with Pr-419 and on approximately 73 and 40% of the leaves inoculated with Pr-422. Under these experimental conditions, A. aleuticum appeared to be slightly more susceptible than the A. jordanii, with a necrotic leaf area of approximately 38% compared with 20%. The pathogen was reisolated on PARP after surface sterilization from all symptomatic leaves. Control leaves did not develop symptoms and P. ramorum was not recovered. A. jordanii and A. aleuticum have already been listed as associated hosts for P. ramorum on the APHIS (USDA Animal and Plant Health Inspection Service) website ( http://www.aphis.usda.gov/ ). To our knowledge, this is the first report of ferns as natural hosts of P. ramorum. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol Res. 105:1155, 2001.