Predicting Cereal Root Disease in Western Australia Using Soil DNA and Environmental Parameters.

Root diseases have long been prevalent in Australian grain-growing regions, and most management decisions to reduce the risk of yield loss need to be implemented before the crop is sown. The levels of pathogens that cause the major root diseases can be measured using DNA-based services such as PreDicta B. Although these pathogens are often studied individually, in the field they often occur as mixed populations and their combined effect on crop production is likely to vary across diverse cropping environments. A 3-year survey was conducted covering most cropping regions in Western Australia, utilizing PreDicta B to determine soilborne pathogen levels and visual assessments to score root health and incidence of individual crop root diseases caused by the major root pathogens, including Rhizoctonia solani (anastomosis group [AG]-8), Gaeumannomyces graminis var. tritici (take-all), Fusarium pseudograminearum, and Pratylenchus spp. (root-lesion nematodes) on wheat roots for 115, 50, and 94 fields during 2010, 2011, and 2012, respectively. A predictive model was developed for root health utilizing autumn and summer rainfall and soil temperature parameters. The model showed that pathogen DNA explained 16, 5, and 2% of the variation in root health whereas environmental parameters explained 22, 11, and 1% of the variation in 2010, 2011, and 2012, respectively. Results showed that R. solani AG-8 soil pathogen DNA, environmental soil temperature, and rainfall parameters explained most of the variation in the root health. This research shows that interactions between environment and pathogen levels before seeding can be utilized in predictive models to improve assessment of risk from root diseases to assist growers to plan more profitable cropping programs.

Fungal Planet description sheets: 107-127.

Novel species of microfungi described in the present study include the following from Australia: Phytophthora amnicola from still water, Gnomoniopsis smithogilvyi from Castanea sp., Pseudoplagiostoma corymbiae from Corymbia sp., Diaporthe eucalyptorum from Eucalyptus sp., Sporisorium andrewmitchellii from Enneapogon aff. lindleyanus, Myrmecridium banksiae from Banksia, and Pilidiella wangiensis from Eucalyptus sp. Several species are also described from South Africa, namely: Gondwanamyces wingfieldii from Protea caffra, Montagnula aloes from Aloe sp., Diaporthe canthii from Canthium inerne, Phyllosticta ericarum from Erica gracilis, Coleophoma proteae from Protea caffra, Toxicocladosporium strelitziae from Strelitzia reginae, and Devriesia agapanthi from Agapanthus africanus. Other species include Phytophthora asparagi from Asparagus officinalis (USA), and Diaporthe passiflorae from Passiflora edulis (South America). Furthermore, novel genera of coelomycetes include Chrysocrypta corymbiae from Corymbia sp. (Australia), Trinosporium guianense, isolated as a contaminant (French Guiana), and Xenosonderhenia syzygii, from Syzygium cordatum (South Africa). Pseudopenidiella piceae from Picea abies (Czech Republic), and Phaeocercospora colophospermi from Colophospermum mopane (South Africa) represent novel genera of hyphomycetes. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

Fungal Planet description sheets: 69-91.

Novel species of microfungi described in the present study include the following from Australia: Bagadiella victoriae and Bagadiella koalae on Eucalyptus spp., Catenulostroma eucalyptorum on Eucalyptus laevopinea, Cercospora eremochloae on Eremochloa bimaculata, Devriesia queenslandica on Scaevola taccada, Diaporthe musigena on Musa sp., Diaporthe acaciigena on Acacia retinodes, Leptoxyphium kurandae on Eucalyptus sp., Neofusicoccum grevilleae on Grevillea aurea, Phytophthora fluvialis from water in native bushland, Pseudocercospora cyathicola on Cyathea australis, and Teratosphaeria mareebensis on Eucalyptus sp. Other species include Passalora leptophlebiae on Eucalyptus leptophlebia (Brazil), Exophiala tremulae on Populus tremuloides and Dictyosporium stellatum from submerged wood (Canada), Mycosphaerella valgourgensis on Yucca sp. (France), Sclerostagonospora cycadis on Cycas revoluta (Japan), Rachicladosporium pini on Pinus monophylla (Netherlands), Mycosphaerella wachendorfiae on Wachendorfia thyrsifolia and Diaporthe rhusicola on Rhus pendulina (South Africa). Novel genera of hyphomycetes include Noosia banksiae on Banksia aemula (Australia), Utrechtiana cibiessia on Phragmites australis (Netherlands), and Funbolia dimorpha on blackened stem bark of an unidentified tree (USA). Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

First Report on an Infestation of Phytophthora cinnamomi in Natural Oak Woodlands of California and its Differential Impact on Two Native Oak Species.

During an intense survey of natural woodlands around Lake Hodges (33°N, 117°W) in June 2001, symptoms typical of root and collar rot caused by Phytophthora spp. were observed on 27% of 474 coast live oaks (Quercus agrifolia Nee.) and on none of 86 Engelmann oaks (Q. engelmannii Greene), in spite of complete spatial intermixing of the two species. Symptoms on coast live oaks included viscous exudates emerging through intact bark matched by underbark dark lesions with irregular margins. Lesions were delineated by a dark line and present on the root collar or the buttress of symptomatic trees. Crowns of trees with lesions ranged from completely healthy to declining or dead. All symptomatic trees were in proximity of the lake or streams. Phytophthora cinnamomi Rands was isolated from four trees in three distinct sites by plating tissues from lesion margins on PARP selective medium and from four soil samples by using standard pear baiting and plating lesions from pear tissue onto PARP. Identification of the isolates was obtained from microscopic observations and direct sequencing of the internal transcribed spacer region of the rDNA (Genbank Accession Nos. AY302148, MC2 and AY302149, MC3). P. citricola Sawada was also isolated once. Pathogenicity tests were completed to compare the susceptibility of the two species of oaks growing in the Lake Hodges region with P. cinnamomi. Two P. cinnamomi isolates from Lake Hodges (MC2, ATCC MYA-3711; MC3) and one isolate from an avocado orchard in San Diego County (MC6) were used to inoculate separately 10 5-year-old trees each of Q. agrifolia and Q. engelmannii grown in 5-gallon containers. Inoculations were performed in two lath-house experiments during February and September 2002 by placing an 8-mm diameter V8-agar plug from the margin of a P. cinnamomi colony underbark and sealing the wound with Parafilm and grafting wax. Lesion lengths were measured 2 months after inoculation, and the presence of the pathogen confirmed by reisolation on PARP. Mean average, maximum, and minimum temperatures were 14, 19, and 9°C and 21, 24, and 18°C for the February and September inoculations, respectively. The February inoculation resulted in small lesions only on Q. agrifolia (26 ± 15 mm, SD). The September inoculation resulted in 135 ± 68 mm (SD) lesions on Q. agrifolia and 49 ± 35 mm (SD) lesions on Q. engelmannii. Controls did not show any lesions. The length of lesions was significantly different between the two hosts (P < 0.0001) and significant differences were observed among the three isolates (P = 0.0018). Although Q. agrifolia is a known host for P. cinnamomi in California (2,3), to our knowledge, this is the first report of widespread infestation of P. cinnamomi in natural oak woodlands in the western United States. Survey and inoculation results indicated Q. engelmannii to be less susceptible to infection. Inoculation results confirm previous research that cold temperatures are unfavorable to this pathogen and isolates differed in pathogenicity toward Q. agrifolia. Decline of oaks infected by P. cinnamomi was observed only in conjunction with other factors, in particular with the presence of the oak twig girdler, Agrilus angelicus Horn., an insect favored by stress conditions such as drought. Similar effects have been reported for Mediterranean oaks infected by the same pathogen (1). References: (1) C. M. Brasier. Nature 360:539, 1992. (2) P. A. Miller. Western Shade Tree Conf. Proc. 8:39, 1941. (3) S. M. Mircetich et al. Plant Dis. Rep. 61:66, 1977.

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). Koch's 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 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.

First Report of Foliar Infection of Maianthemum racemosum by Phytophthora ramorum.

In May 2003, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from the leaf tips of a single plant of false Solomon's seal (Maianthemum racemosum (L.) Link, formely known as Smilacina racemosa (L.) Desf.), a native, herbaceous perennial of the Liliaceae family, at the Jack London State Park in Sonoma County, California. Affected leaves had cream-to-brown lesions on the tips that were delimited by a yellow chlorotic zone. Lesions on the stems were not observed. The isolate (American Type Culture Collection [ATCC], Manassas, VA, MYA-3280; Centraal Bureau voor Schimmelcultures, Baarn, the Netherlands, CBS 114391) was typical of P. ramorum with large chlamydospores and caduceus, semipapillate sporangia, and the sequence (GenBank Accession No. AY526570) of the internal transcribed spacer region of the rDNA matched those published previously (4). The site, from which wood rose (Rosa gymnocarpa) was recently identified as a host, is a mixed forest containing confirmed P. ramorum-infected coast redwood (Sequoia sempervirens), California bay laurel (Umbellularia californica), and tanoak (Lithocarpus densiflora) trees (2,3). Two leaves per asymptomatic, pesticide free, potted plant of false Solomon's seal were inoculated with zoospores of the P. ramorum isolate obtained from infected false Solomon's seal (1). Five plants were inoculated in trial 1, and the following day, three plants were inoculated in trial 2. A control leaf of each plant was dipped in sterile deionized water. Plants were enclosed in plastic bags, misted regularly with sterile distilled water, and maintained at 16 to 21°C in the greenhouse. In both trials, plants did not have lesions on the leaves after 16 days and were reinoculated on separate days for each trial with higher concentrations of zoospores (1 × 105 [trial 1] and 2 × 105 [trial 2] zoospores/ml). Cream-colored lesions, similar to those observed in the field, were evident 1 week after the second inoculation and stopped progressing in both trials by 17 days. Lesions starting from the leaf tips averaged 13 mm (range 8 to 24 mm) long, and P. ramorum was reisolated on Phytophthora-selective agar medium modified with 25 mg of pentachloronitrobenzene from 44% (trial 1) and 83% (trial 2) of all lesions (4). Control leaves had no lesions, and P. ramorum was not reisolated. Sporangia were not observed on any leaves when examined with the dissecting microscope. The fact that lesions developed only after a second inoculation with higher concentrations of zoospores, and these lesions stopped progressing after 17 days, suggests that false Solomon's seal is much less susceptible than other hosts such as western starflower (Trientalis latifolia) (1) and wood rose (2). To our knowledge, this is the first report of a plant from the Liliaceae as a natural host for P. ramorum, although Smilax aspersa was identified as being susceptible in artificial inoculations of detached leaves (E. Moralejo and L. Hernández, personal communication). False Solomon's seal is popular in the horticultural industry. References: (1) D. Hüberli et al. Plant Dis. 87:599, 2003. (2) D. Hüberli et al. Plant Dis. 88:430, 2004. (3) P. E. Maloney et al. Plant Dis. 86:1274, 2002. (4) D. M. Rizzo et al. Plant Dis. 86:205, 2002.

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.

First Report of Foliar Infection of Starflower by Phytophthora ramorum.

In March 2002, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from pacific or western starflower (Trientalis latifolia Hook.), an herbaceous perennial of the Primulaceae family, at Castro Canyon in Big Sur, Monterey County, California. Affected leaves had numerous necrotic lesions >5 mm in diameter surrounded by a yellow halo, and the lesions coalesced with time. Isolates were identified as P. ramorum by the large chlamydospores, caduceus, semipapillate sporangia, and sequences of the internal transcribed spacer (ITS) region of the rDNA (1,2). The same symptoms were observed on starflower in a second location at the Soquel Demonstration Forest, Santa Cruz County. Although P. ramorum was not isolated from symptomatic leaves on the plants in Santa Cruz County, the ITS region of the pathogen was amplified and sequenced using P. ramorum-specific primers. Both sites were mixed forests of coast redwood (Sequoia sempervirens), bay laurel (Umbellularia californica), and tanoak (Lithocarpus densiflorus), which are confirmed hosts of P. ramorum. To test for pathogenicity to starflower, asymptomatic plants were carefully excavated from the two forest locations, replanted in 15-cm paper cups in the original forest soil, and the foliage was inoculated with zoospores of P. ramorum isolate Pr-52, an isolate used in previous inoculations. The zoospores were produced by placing agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar into 20 to 30 ml of sterile deionized water in petri dishes. After 2 days incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then to room temperature for 45 to 60 min. Three hundred µl of the zoospore suspension (approximately 2 × 104 zoospores/ml) was poured into 500-µl modified microcentrifuge tubes in which tips of leaves of starflower were submerged. Control leaves were dipped in sterile deionized water. Plants were placed in a humid-chamber consisting of moist paper towels placed on the tray and covered with a clear-plastic lid that was sprayed with sterile water. The chambers were maintained at 20 to 24°C in the laboratory. Two or three leaves were inoculated, and one leaf was left as the control on each of seven or eight plants in two separate trials. In both trials, water-soaked lesions were observed on the leaves 12 h after inoculation with P. ramorum. At 8 or 11 days after inoculation, necrotic lesions were present on all inoculated leaves starting from the leaf tips. Lesions averaged 29 mm (range 13 to 39 mm) and 45 mm (range 31 to 56 mm) in length in the respective trials. Some lesions covered entire leaves. P. ramorum was reisolated on Phytophthora-selective agar medium (1) from the lesions in both trials. Control leaves had no lesions, and P. ramorum was not reisolated. Infection of starflower and other understory species appears to occur under infested tree hosts such as bay laurel, which is known as a source of inoculum for P. ramorum. To our knowledge, this is the first report of an herbaceous host for P. ramorum and the first report of the disease on the Primulaceae. Previously, only woody hosts were known. Starflower is unlikely to play a major role in the natural spread of the disease, but the pathogen may be spread via movement of plants through the horticultural industry. Furthermore, Trientalis spp. in Europe where P. ramorum is present may also be potential hosts. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.