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Downy mildew caused by Peronospora sparsa is a major disease of blackberry (Rubus sp.) worldwide. The objectives of this research were a) to detect potential sources of primary inoculum of downy mildew of cultivated blackberry and b) to determine some components of resistance to P. sparsa in 4 wild blackberry species from Michoacan Mexico. A region-wide sampling of symptomatic and asymptomatic blackberry plants from nurseries, commercial plots, wild blackberries (R. adenotrichos) and rose plants (Rosa sp.), was conducted. Nested-PCR assays with PS3/PS1 primers in the first reaction followed by a second amplification with PR3/PR4 primers, amplifying a final fragment of 477 bp of the ITS region were used, and then sequenced in both directions. Detached leaflets of 4 common wild blackberry species were inoculated with P. sparsa sporangia under controlled conditions. Sporangiophores (SP) were recorded over a period of 18 days and oospores (OO) were counted 21 days after inoculation; the latent period (LP) and disease severity (DS) were also estimated. Phylogenetic reconstruction with Bayesian inference confirmed the presence of P. sparsa in all collected samples. Rubus adenotrichos, R. pringlei and R. sapidus were high inoculum producers, whereas R. cymosus allowed neither SP nor OO production and had the longest LP and lowest DS. Together, these results indicate that the primary source of inoculum for commercial plantings is mainly the blackberry plants from nurseries, and potentially 3 species of wild blackberries. Rubus cymosus has potential to be used in blackberry breeding programs as a source of resistance to P. sparsa.

Lychnothamnus barbatus (Meyen) Leonhardi is a rare, endangered charophyte species considered to be a reliable indicator of low water fertility, with its fossilized gyrogonites (calcified oospores) being applied as a biomarker of past oligotrophic conditions. This paper presents a case study from a lake in which L. barbatus occurs abundantly under eutrophic conditions (Lake Kuźnickie, central-western Poland). The species overwinters in this lake in the form of extensive meadows along a wide depth gradient and, in addition, produces fully developed gyrogonites. The occurrence of L. barbatus in Lake Kuźnickie was investigated at 1 m depth intervals at nine permanent transects in November 2015 and in April 2016. Spring thalli were observed microscopically to determine whether L. barbatus overwintered (with new outgrowths on old thalli) or grew anew in spring (from oospores). The species occurred at almost 50% of the surveyed sites in both sampling periods. At most sites L. barbatus formed compact carpets in which it was the only species or co-occurred with the charophyte Nitellopsis obtusa or the angiosperm Ceratophyllum demersum . L. barbatus occurred along a wide depth gradient: 1.5–6.5 m deep in autumn and 2–6.5 m after the winter period. The species coverages in April 2016 were comparable to those observed in November 2015 (Mann-Whitney U -test, P > 0.05). The thalli collected in April were well preserved after the winter, with numerous spring outgrowths. In addition, fully developed gyrogonites were found in April 2016. This study indicates wider fertility tolerance of L. barbatus and its ability to overwinter under eutrophic conditions.

Pythium myriotylum and P. zingiberis have both been implicated in soft rot of ginger (Zingiber spp.). The status of these two taxa as distinct species follows original descriptions of physiology, morphology and pathogenicity. However, their status has been questioned by phylogenetic analyses. In this study, putative P. zingiberis isolates recovered from edible ginger (Z. officinale) rhizomes with Pythium soft rot (PSR) disease sampled from Queensland, Australia, were compared with reference isolates of P. myriotylum from peanut in Israel (CBS254.70) and P. zingiberis from ginger in Japan (NBRC30817). All isolates differed slightly in temperature optima for growth and produced similar sizes of oogonia and oospores. The sequence homology of 20 gene fragments retrieved from nuclear and mitochondrial genomes ranges from over 99 to 100% to each other. In vitro pathogenicity assays were conducted on excised carrot, ginger, potato, radish and sweet potato tuber/root sections and on seeds and seedlings of cucumber, cauliflower, millet, rye, sweet corn, tomato and wheat with each of the isolates. The reference isolate P. zingiberis NBRC30817, which was previously believed to have a narrow host range, was pathogenic on a number of tested plant species. Analysis of this comprehensive set of data allowed us to assign all tested isolates, including the isolate P. zingiberis NBRC30817, to the taxon P. myriotylum, thus confirming that the causal pathogen of PSR disease of ginger in Australia is P. myriotylum.

Currently, forty-nine plant pathogenic species of the genus Pythium have been described and identified all over mainland China. Most of them cause epidemics of herbaceous Monocotyledonous crops, for instance seedling damping-off, soft rot of fruit/vegetables and stem/root rot. Moreover, it may also infect woody Dicotyledonous plants resulting in stem/root rot. P. porphyrae was reported to cause disease of the marine red alga known as Porphyra. Similarly, P. carolinianum and P. guiyangense were found to be parasitic on mosquito larvae, whereas new species like P. nunn and P. oligandrum are considered as mycoparasitic species. There are excellent opportunities to use P. carolinianum and P. guiyangense to control the mosquitoes which can transmit diseases in human beings and the mycoparasitic species to manage some diseases caused by Pythium and various other soil-borne fungi. Currently, chemical fungicides like metalaxyl and hymexazol have been utilized to control plant diseases caused by Pythium species. These chemicals might have upset the natural environment and stimulated tolerance to the fungicides, and good efforts were made towards biological control by integrating antagonistic microorganisms like Trichoderma species, Pseudomonas and Rhizobacteria as a seed dressing and soil amendments. Oospores of the mycoparasitic P. oligandrum have been suggested as a biofungicide: polysersum WP to control plant diseases caused by the genus Pythium and other fungal pathogens.

The Phytophthora genus is composed, mainly, of plant pathogens. This genus belongs to the Oomycete class, also known as “pseudo-fungi”, within the Chromista Kingdom. Phytophthora spp. is highlighted due to the signifcant plant diseases that they cause, which represents some of the most economically and cultural losses, such as European chestnut ink disease, which is caused by P. cinnamomi. Currently, there have been four genome assemblies placed at the National Center for Biotechnology Information (NCBI), although the progress to understand and elucidate the pathogenic process of P. cinnamomi by its genome is progressing slowly. In this review paper, we aim to report and discuss the recent fndings related to P. cinnamomi and its genomic information. Our research is based on paper databases that reported probable functions to P. cinnamomi proteins using sequence alignments, bioinformatics, and biotechnology approaches. Some of these proteins studied have functions that are proposed to be involved in the asexual sporulation and zoosporogenesis leading to the host colonization and consequently associated with pathogenicity. Some remarkable genes and proteins discussed here are related to oospore development, inhibition of sporangium formation and cleavage, inhibition of fagellar assembly, blockage of cyst germination and hyphal extension, and bioflm proteins. Lastly, we report some biotechnological approaches using biological control, studies with genome sequencing of P. cinnamomi resistant plants, and gene silencing through RNA interference (iRNA). The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO UID/AGR/00690/2019. info:eu-repo/semantics/publishedVersion

A study was conducted to investigate the potential of Aspergillus terreus obtained from Avicennia marina mangrove roots in inhibiting Pythium aphanidermatum and damping-off disease of cucumber. Aspergillus terreus exhibited in vitro inhibition of Pythium aphanidermatum growth. Electron microscope examination revealed that the antagonistic fungal isolate resulted in shrinking and groves in Pythium hypha. When Aspergillus terreus culture filtrate was added to Pythium aphanidermatum, it resulted in a significant increase (by 73%) in electrolyte leakage from Pythium hypha compared to the control, as well as significant reduction (by 71%) in oospore production. The Aspergillus terreus culture was also found to produce a cellulase enzyme, which is suggested to be involved in the antagonism against Pythium aphanidermatum. Adding Aspergillus terreus to soil infested with Pythium aphanidermatum significantly reduced percent mortality in cucumber seedlings by 70%. Aspergillus terreus, when applied alone on cucumber seedlings, did not show any suppressive effects on cucumber growth (length and fresh and dry weight). This appears to be the first report of isolation from mangrove of Aspergillus terreus with antagonistic activity against Pythium aphanidermatum-induced damping-off of cucumber. The study indicates that fungal isolates obtained from marine environments may serve as potential biocontrol agents against some plant pathogens.

Pythium-induced damping-off disease is a major disease limiting cucumber and tomato production in different parts of the world. The current study investigated the efficiency of Talaromyces variabilis and its bioactive metabolites in suppressing Pythium-induced damping-off of cucumbers and tomatoes. T. variabilis inhibited the in vitro growth of P. aphanidermatum in solid and liquid media. In addition, abnormalities in P. aphanidermatum hyphae were observed as a result of T. variabilis. Extracts from T. variabilis induced cellular leakage and suppressed oospore production of P. aphanidermatum. Biochemical analyses of T. variabilis metabolites showed that T. variabilis produces glucanase, cellulase and siderophores, suggesting the contribution of these metabolites in the inhibition of P. aphandermatum growth and in hyphal abnormalities. Treating cucumber seeds with spore and mycelial suspension of T. variabilis isolates led to a significant improvement in the seedling survival of P. aphanidermatum-inoculated seedlings from 18 to 52% (improvement by 34%) for isolate 48 P and from 30–66% (improvement by 36%) for isolate 28 R. Similarly, treating tomato seeds with spore and mycelial suspension of T. variabilis isolates led to a significant improvement in the seedling survival of P. aphanidermatum-inoculated seedlings from 7 to 36% (improvement by 29%) for isolate 28 R and from 20 to 64% (improvement by 44%) for isolate 48 P. Differences in the percent improvement in seedling survival between experiments may be related to difference in the efficacy of the two different isolates or their interaction with the hosts and pathogen. The use of T. variabilis in the biocontrol of Pythium-induced diseases may offer alternatives to the currently used chemical control.

International audience; Aphanomyces euteiches is a polyphagous, homothallic soilborne pathogen producing asexual (zoospores) and sexual (oospores) spores. Even if oospores are essential for disease development and survival, to date, no study has focused on the production rates of oospores or the quality of the offspring produced by oospores. In this study, a nonabrasive oospore extraction method from infected roots of leguminous species (pea, faba bean and vetch) was developed. This methodology includes steps of grinding and filtration. The quality of oospores (viable, dormant and dead) was assessed with tetrazolium bromide staining, and germination of oospores was tested using exudates of peas, faba bean and vetch. The average yield of the extraction method was approximately 21%. Staining revealed some differences between strains and between leguminous species. The germination percentage of oospores extracted from pea, faba bean and vetch was 25%, 62% and 70%, respectively, and a significant difference was observed according to the origin of A. euteiches‐inoculated strains. Application of exudates seems to stimulate the germination of oospores (2% for the control, 18% for pea exudates and 1% for vetch exudates). Differences observed between A. euteiches strains and leguminous species indicate that more knowledge concerning the biology of oospores is needed. This will help to better estimate evolution process of the pathogen and manage resistance and crop successions.

In 1996, commercial plantings of sage (Salvia officinalis) in the Salinas Valley in Monterey County, CA, were affected by a root and crown disease. Roots were necrotic, and crowns and lower stems turned black. Affected plants withered and died. A Phytophthora sp. was consistently isolated from roots and stems of the symptomatic plants. The species was identified as Phytophthora cryptogea based upon the formation of hyphal swellings, morphology of sporangia and oospores, and growth at cardinal temperatures (1). Pathogenicity of representative isolates was confirmed by applying 2 ml of a zoospore suspension (2.0 × 105 zoospores per ml) to roots and crowns of 3-month-old potted sage plants. After treatment, plants were placed for 24 h in shallow trays of water to saturate the root zone, then were removed from trays and incubated in a greenhouse. After 4 days, foliage of all inoculated plants began to wilt. After 7 days, plant crowns and stems turned black and the plants collapsed. P. cryptogea was reisolated and recharacterized from all plants. Control plants, which were treated with water and then handled in the same way as inoculated plants, did not develop any symptoms. The tests were repeated and the results were similar. This is the first report of P. cryptogea attacking commercial plantings of sage. The authors also detected this disease in experimental plantings of sage in Stanislaus County in 1990. Reference: (1) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society, St. Paul, MN.

Severe symptoms of potato late blight were observed in July 1996 on potato (Solanum tuberosum L.) cv. Desirée grown on a farm in western Hungary. Isolation was made directly from diseased leaf tissues onto selective pea-agar medium. A recovered isolate, H2a, was identified as Phytophthora infestans (Mont.) de Bary on the basis of Koch's postulates and morphological characteristics of the fungus. Pairing of H2a with isolates of known mating types, A1 and A2 from Germany, revealed that it represents the A2 mating type. After a 2-week incubation on pea-agar medium at 20°C, oospores formed in abundance in the region of contact between the colonies of H2a and the A1 mating type isolate. After extended incubation scattered formation of gametangia was observed when isolate H2a had been paired with itself or with the A2 mating type isolate. The same phenomenon of presumed self fertilization also took place when single, zoospore-derived colonies of H2a were combined with one another or with the known A2 isolate. Of an incomplete set of potato differentials, leaves of potato genotypes r, R2, R3, R4, R1.2.3.4, R7, R8, and R11 were all susceptible to infection with a zoospore suspension of the isolate H2a. The complex virulence phenotype of H2a, its tolerance to metalaxyl in agar cultures and on leaf disks (EC50 >100 mg liter-1), and its A2 mating type behavior collectively suggest that H2a represents a genotype that recently has been introduced into Hungary.

Phytophthora infestans (Mont.) de Bary, causing late blight on potatoes and considered to be a typical airborne disease, was found to be infective also by oospores in the soil. P. infestans is heterothallic, and is known to reproduce asexually in Sweden since only one mating type, A1, was present until 1986. Since the 1970s, the other mating type, A2, of P. infestans has migrated to most parts of the world from its original location in central Mexico (2). When A1 and A2 meet, they may form oospores, which are thick-walled, resting structures, giving the pathogen a possibility to recombine as well as survive without its host, for instance in the soil. The soil stages of the pathogen are now therefore under intense investigation. Oospores of P. infestans were produced from two Scandinavian A1 and A2 isolates in Rye A broth mixed with talcum powder and dried for 7 weeks. The inoculum was mixed with sterile, standardized soil in concentrations of 10, 150, 250, and 400 oospores per ml of soil. Cv. Bintje plants cultivated in vitro from nodal cuttings on Murashige and Skoog medium were transplanted to the soil after rooting. Brown discolorations were obtained on the underground stems and tubers on potato plants grown in the two highest concentrations of oospores for 1 month at 15°C and 16-h day length. After 3 days of incubation on P. infestans-selective medium (3), sporangia covered the tissue from plants grown in 250 and 400 oospores per ml of soil and the pathogen was reisolated. This shows that germinating oospores of P. infestans can infect underground stems and tubers of potatoes in soil and further explains the early attack of late blight as observed in a potato crop in Sweden 1996 and 1997 (1). References: (1) B. Andersson et al. Potato Res. 41:305, 1998. (2) D. Andrivon. Phytopathology 85:1053, 1995. (3) G. W. Griffith et al. Mycologist 9:87, 1995.

Damping-off was observed on experimentally planted seedlings of Ana-cardium excelsum (wild cashew), a timber tree, and Tetragastris panamensis, a canopy tree, within lowland tropical rain forest in Panama. Disease impact was greatest during the wet season (May through December). During the 1995 wet season, 40.7% (572/1,404) of T. panamensis seedlings died due to damping-off disease. Sixty-eight percent (703/1,034) of A. excelsum seed lings died due to damping-off during the 1996 wet season. Symptoms included leaf, cotyledon, and stem necrosis. Phytophthora heveae sporangia were observed on both host species, and oospores were found within stems of T. panamensis. Plating of diseased A. excelsum seedlings on potato dextrose agar with rifampicin (25 mg/ml) and pimaricin (10 mg/ml) produced cultures of Phytophthora heveae and Pythium from 27.4% (110/402) and 44.5% (179/402) of seedlings, respectively. Pythium isolates included P. vexans, P. splendens, and P. chamaehyphon species types, but P. vexans species types accounted for 70% of the Pythium isolates. Disease symptoms on experimental seedlings also were evident on naturally occurring seedlings. Mycelial plugs from six A. excelsum isolates of Phytophthora heveae were used to separately inoculate stems of three A. excelsum seedlings each. Of 18 seedlings inoculated, 88.8% developed characteristic symptoms and died in an average of 8.7 ± 1.0 (standard error [SE]) days. Nine Pythium isolates were used to separately inoculate stems of one to three A. excelsum seedlings each; three of these isolates were known to be P. vexans species types. All of the 20 seedlings inoculated with a Pythium isolate developed characteristic symptoms and died in an average of 6.1 ± 0.3 (SE) days. Both Phytophthora heveae and Pythium isolates were reisolated readily from diseased seedlings. Cotyledons and stems of seven to eight T. panamensis seedlings per isolate were inoculated with two Phytophthora heveae isolates originating from T. panamensis. Necrotic lesions on cotyledons consistent with field symptoms developed on 33.3% of 15 seedlings, but disease did not spread within the stem. Measurements of key morphological structures and cardinal temperatures of four Phytophthora heveae isolates from A. excelsum were consistent with published species descriptions (1), except (i) sporangia with two apices were present, although infrequent; (ii) chlamydospores were produced; and (iii) antheridia were narrower and often shorter than published measurements (7 to 12 m long; 2 to 6 m wide). Internal transcribed spacer (ITS) sequences from Phytophthora heveae isolates cultured from A. excelsum and T. panamensis were matched to reference sequences of Phytophthora heveae with only 3-bp differences (2). ITS sequences for isolates of Pythium vexans, P. splendens, and P. chamaehyphon species types clustered within clades of reference strains of these species (C. A. Lévesque, personal communication). Phytophthora heveae and Pythium spp. have been reported from the tropics. However, this is the first report of these pathogens on seedlings of A. excelsum and T. panamensis. Reference: (1) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, pp. 100–107, 336–337. (2) D. E. L. Cook et al. A molecular phylogeny of Phytophthora and related Oomy-cetes. Fungal Genet. & Biol. In press.

In 1995 loquat trees (Eriobotrya japonica [Thunb.] Lindl.) in several orchards in central Taiwan suffered from a severe stemrot and rootrot (2). Two populations of Phytophthora spp. isolates were recovered from diseased trees by placing pieces of plant tissue in a petri dish containing selective medium (2). One group of isolates possessed papillate sporangia, appeared to be heterothallic with amphigynous antheridia, and had a maximum growth temperature of 36°C. Upon mating, isolates of this group produced oogonia and oospores measuring 26.1 and 21.4 μm in diameter, respectively. These isolates were identified as typical strains of P. parasitica (=P. nicotianae) (2). The second group of isolates showed papillate sporangia, was homothallic with predominantly amphigynous antheridia, and had a maximum growth temperature of 34.5°C. However, the sizes of oogonia and oospores were considerably larger (average 34.7 and 30.3 μm in diameter, respectively) than those of P. nicotianae. Amplification of genomic DNA of one isolate (95023) of the second group by polymerase chain reaction (PCR) using P. nicotianae species specific primers PAR1 and PAR2, produced a special 1,000 bp sequence, indicating that the atypical isolates of second group were closely related to P. nicotianae. Isozyme analysis of two isolates (95023 and 95034, provided by L. L. Chern, Taiwan) of the second group, using the dimeric enzymes malic enzyme (MDHP; EC 1.1.1.40) and malate dehydrogenase (MDH; EC 1.1.1.37), was applied as described before (1,3). These two isolates generated the same three banded patterns using either enzyme (genotype: MDHP: 92/100; MDH-2: 93/100); the Mdhp92 allele and the Mdh-293 allele are diagnostic for P. cactorum, whereas the Mdhp100 allele and the Mdh-2100 allele are diagnostic for P. nicotianae. These well-defined heterozygous band patterns were exactly identical to those generated in atypical isolates which were identified earlier as hybrids of P. nicotianae and P. cactorum (1,3). Based on their atypical morphology and isozyme genotyping, it was concluded that these two isolates of the second group represent hybrids of P. nicotianae and P. cactorum. One of the parents, P. nicotianae, was also found closely associated with diseased loquat trees; the other parental species, P. cactorum, was not isolated from loquat in this study. It is the first time that natural hybrids of P. nicotianae and P. cactorum were found on loquat in Asia. References: (1) P. J. M. Bonants et al. Phytopathology 90:867–874, 2000. (2) L. L. Chern et al. Plant Dis. 82:651–656, 1998. (3) W. A. Man in 't Veld et al. Phytopathology 88:922–929, 1998.

A technique was developed that allows prolific production, easy collection, and increased germination frequency of single oospores of Aphanomyces euteiches. The influence of root exudates and roots of various plant species, including pea, bean, alfalfa, oat, soybean, corn, and tomato, on germination of A. euteiches oospores also was studied. Compared with a sterile, deionized water control, root exudates from several hosts were only slightly effective in stimulating oospore germination, since only 0 to 11.1% of oospores exposed to various exudates germinated. By contrast, oospores placed directly on plant roots germinated at higher frequencies. Oospores of pea pathotype isolates P30 and P46 germinated at a greater frequency (30.6 to 61.1%) on pea, bean, and oat roots than on roots of any of the other plant species tested. Oospores of bean pathotype isolates GB33 and GB71 had a higher germination frequency (47.2 to 52.8%) on bean roots than on the roots of the other plant species tested. A higher percentage of oospores germinated if placed on lateral roots as opposed to taproots of pea and bean. A higher percentage of oospores of bean pathotype isolates and one pea pathotype isolate germinated on 10-day-old rather than on 20-day-old roots of bean. Therefore, pea and bean roots can be used effectively to germinate oospores of pea and bean isolates of A. euteiches, respectively. This technique will be valuable for studies of sexual reproduction and genetics of A. euteiches.

Oospores of Phytophthora infestans produced in vitro and in planta, from a cross between US-17 and US-8 genotypes, were exposed to a variety of environments and their survival was assessed. Additionally, the pathogenic characteristics of some resultant progeny isolates were assessed. Viability of oospores as measured by plasmolysis declined slightly over a period of 18 months whether they were stored in water at 4°C, in soil at 18°C, or in soil under natural field conditions. In comparison, viability as measured by germination was lower overall but appeared to increase after storage in soil. Oospores produced in planta were buried in the field in the fall of 1998, and were capable of infecting both tomato and potato leaflets when recovered in May 1999. Single oospore progeny (n = 53) from the in vitro cross were analyzed individually for genetic and pathogenicity characteristics. All 53 progeny tested for restriction fragment length polymorphisms with probe RG57 were hybrids. All but one progeny produced sporulating lesions on detached potato or tomato leaflets in growth chamber tests, but most lesions were smaller and developed more slowly than those produced by either parental isolate. In a further test of pathogenicity, under field conditions, none of a subset of 10 A2 progeny was capable of initiating a detectable epidemic in small plots of either potatoes or tomatoes.

Oospore formation by Phytophthora infestans in nature has been detected on potato leaflets in central Mexico (1), but there are no reports of oospore formation on tubers. A severe late blight epidemic occurred in Calimaya, Mexico, in fields where potato cv. Alpha was planted during the summer of 2000. Yield was reduced despite numerous applications of fungicide. Four hundred potato tubers left in the field were collected from the upper 10 cm of soil and examined for late blight symptoms. Tubers with soft and dry rot symptoms were observed, but symptoms of pink rot (Phytophthora erythroseptica) were not found. Four percent of the tubers showed late blight symptoms. Sections of 10 tubers with late blight symptoms were air-dried for 2 weeks in the laboratory and homogenized with a mortar and pestle. Glycerol was added to the homogenized tissue and observed microscopically. Aplerotic oospores (10 to 15 oospores per tuber) with amphyginous antheridia typical of P. infestans were observed. P. mirabilis morphologically similar to P. infestans is present in the area but it does not infect potato tubers. The number of oospores observed in our tuber sample was much lower than the number reported on leaflets (>1,000 oospores per leaflet) in the Toluca Valley. Low numbers of oospores have been reported on tubers artificially inoculated with P. infestans under field conditions (2). Infected tubers left in the field may act as a source of primary inoculum. To our knowledge, this is the first report of oospores of P. infestans found on tubers in Mexico under natural field conditions. References: (1) M. E. Gallegly and J. Galindo. Phytopathology 48:274, 1958. (2) A. Levin et al. Phytopathology 91:579, 2001.

In California, Digitalis purpurea (common foxglove) and D. grandiflora (yellow foxglove) are grown as cutflower, potted, and landscape plant commodities. In the spring of 2002, after seasonably wet and cool weather, severe downy mildew was observed on potted common foxglove plants in commercial nurseries in coastal California (Santa Cruz County). Initial symptoms on leaves consisted of light green, rectangular areas that were vein-delimited and measured 2 to 5 × 8 to 12 mm. Such spots later became chlorotic. As disease progressed, chlorotic spots coalesced and turned necrotic. The purple-gray sporulation of the pathogen could be seen primarily on abaxial leaf surfaces. However, in some cases, extensive fungal growth developed on adaxial surfaces of lower leaves. Conidiophores branched dichotomously and measured 278 to 520 μm long from the lower end to the first branches and 11 to 14 μm across at the widest part of the swollen base. Branch ends were slender with curved tips that measured 11 to 22 μm long. Conidia were hyaline, ellipsoid to ovoid, and measured 22 to 31 μm × 17 to 19 μm. Oospores were not observed. The pathogen was identified as Peronospora digitalidis (1,2). Pathogenicity tests were not conducted. However, the consistent association of sporulation with symptoms, the internal hyphal growth giving rise to conidiophores, and the obligate nature of Peronospora pathogens clearly indicated that P. digitalidis was the causal agent of this disease. Field observations indicated that D. purpurea cvs. Alba and Apricot and Foxy hybrids were very susceptible, D. × mertonensis ( = D. grandiflora × D. purpurea) appeared to be moderately susceptible, and D. grandiflora grown in the same area was symptomless. One planting of Foxy hybrid was 100% infected, and the entire lot of several hundred plants was discarded. The disease also was found on Foxy hybrid seedlings grown in propagation greenhouses. To our knowledge, this is the first report of downy mildew caused by P. digitalidis on cultivars of D. purpurea in California and the United States. This disease has been reported previously in Europe, Asia, and New Zealand (1,2). References: (1) G. Hall. Mycopathologia 126:47, 1994. (2) E. H. C. McKenzie and J. M. Dingley. N. Z. J. Bot. 34:263, 1996.

The plant pathogen Phytophthora infestans causes a destructive blight of potato tubers and foliage. A rapid polymerase chain reaction (PCR) assay has been developed for detection of P. infestans in potato tubers. In this study, the effect of method of DNA extraction on different propagule types and the minimal number of propagules of P. infestans detectable by PCR were assessed using the PINF and internal transcribed spacer (ITS)5 primers. Sensitivity of the primers for PCR was high, and DNA was detectable at concentrations as low as 10 pg/ml. Zoospores and oospores responded differently to different extraction methods, whereas all extraction methods worked equally well for sporangia. Freeze-thaw DNA lysis, in which propagules were frozen at -80°C and thawed at 65°C three times for 15 min each, or direct PCR, in which propagules were placed directly in the reaction mix, were effective methods for PCR detection of sporangia or zoospores but were not effective methods for PCR detection of DNA in oospores of P. infestans. DNA from a single sporangium or oospore could be amplified by PCR after hexadecyltrimethyl-ammonium bromide (CTAB) or NaOH lysis extraction methods, whereas DNA from a single zoospore could be amplified by CTAB or direct PCR methods. “IsoCode” Stixs, used in forensic applications, were used to collect the pathogen from leaf and tuber lesions and provided another simple method to extract template DNA. PCR detection of the pathogen in infected tubers using PINF and ITS5 primers was compared to tissue isolation or visual observation. The probability of detection of P. infestans in infected tubers at 7 days post inoculation using the PCR assay, tissue isolation, or visual observation was 0.90, 0.80, and 0.75, respectively. The PINF and ITS5 primers provide a powerful tool for rapid and sensitive detection of zoospores, sporangia, and oospores of P. infestans when used with appropriate extraction methods, and could easily be deployed to reduce spread of the pathogen in potato tubers.

Iceland poppy (Papaver nudicaule L.) is increasingly grown on the Italian Riviera for export as a cut flower. During the spring of 2003, leaves with irregular, brown, angular spots were collected from a commercial crop grown outdoors near Ventimiglia (northern Italy) with temperatures ranging from 3 to 14°C. Leaves of infected plants appeared curled and blistered; the infected portions of the leaves turned chlorotic. On both surfaces of infected leaves, a characteristic gray, furry growth was evident, particularly at the center of the necrotic areas. Infected leaves eventually died without dropping. Basal leaves with poor air circulation were the most severely affected by the disease. Microscopic observations revealed conidiophores branching dichotomically at least five times. Conidiophores ended with sterigmata bearing single conidia. Conidia measured 19 to 24 × 16 to 18 μm and were elliptical to near spherical and hyaline. Oospores were not present. The pathogen was identified as Peronospora arborescens based on the morphological characteristics (2). Pathogenicity was confirmed by inoculating 60-day-old healthy P. nudicaule plants with a conidial suspension (1 × 105 conidia per ml). Five plants were used as replicates. Inoculated and noninoculated plants were maintained in a growth chamber at 15°C and 90 to 95% relative humidity. After 7 to 10 days, typical symptoms of downy mildew developed oninoculated plants. Peronospora arborescens was observed on infected leaves. Noninoculated plants did not show symptoms. To our knowledge, this is the first report of Peronospora arborescens on P. nudicaule in Italy. Peronospora arborescens was previously reported on P. nudicaule and on many other species of Papaver (P. somniferum, P. dubium, P. caucasicum, P. rhoeas, P. setigerum, and P. argemone) in everal countries (1,2). References: (1) P. J. Cotteril and I. G. Pascoe. Australas. Plant Pathol. 27:263, 1998. (2) S. M. Francis. No. 686 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, U.K., 1981.

In late June and early July 2002, stunted, chlorotic, and partially defoliated lentils (Lens culinaris Medik.) were observed throughout the lentil-growing areas of eastern Washington. These symptoms were investigated in two fields near Garfield, WA and one field near Genesee, ID. Cv. Mason was more affected than cv. Brewer. Roots were dry and brittle with black discoloration in some cases. Isolates of Fusarium oxysporum and F. solani were obtained from washed roots plated on water agar, but they were nonpathogenic in greenhouse testing in pasteurized field soil and peat-based growing mixes. On 21 April 2003, volunteer lentils growing in the same fields showed symptoms of root rot, and Pythium oospores were observed in the roots. Pythium spp. were isolated by using a selective medium (2). Oospores were aplerotic, intercalary, 12.6 to 21 μm long × 11.2 to 18.2 μm wide, mostly smooth, and often formed in chains. Isolates resembled P. paroecandrum Drechs. and P. irregulare Buisman on the basis of morphological characters (3), but DNA sequences of the internal transcribed spacer region were closer to P. irregulare on the basis of a comparison with a worldwide database of Pythium sequences (C. A. Lévesque, personal communication). Isolates were deposited with the USDA-ARS Western Regional Plant Introduction Station, Pullman, WA. Four hyphal-tip isolates were tested in the greenhouse with inoculum grown in autoclaved sandy loam amended with 1% ground rolled oats. Experiments were performed twice in Thatuna silt loam, first in pasteurized and then in nonpasteurized soil. Inoculum was added to the soil at 500 CFU/g, and seeds were planted on the same day. Each isolate was tested on cvs. Brewer and Mason, with five replicates per treatment. Plants were grown in 4- × 20.5-cm plastic tubes (two plants per tube) for 1 month at 16 to 22°C and supplemented with 14 h of light per day. P. irregulare was reisolated from infected roots in both experiments. Damping-off, stunting, chlorosis, and root rot were observed in the Pythium-inoculated treatments, which corresponded to symptoms observed in the field in 2002. In pasteurized soil, only one isolate reduced the whole, dry, plant weight of Brewer, but the other three isolates reduced the dry weight of Mason. All isolates reduced the root dry weight of Mason in natural soil, but only two isolates reduced the root dry weight of Brewer. To our knowledge, Pythium spp., but not P. irregulare, have been reported previously from lentils (1). P. irregulare also causes root rot on winter wheat, which is rotated with lentils, and this pathogen likely causes yield reduction in both crops. References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (2) S. M. Mircetich and J. M. Kraft. Mycopathol. Mycol. Appl. 50:151, 1973. (3) A. J. van der Plaats-Niterink. Stud. Mycol. 21:1, 1981.

Islam, S. Z., Babadoost, M., Lambert, K. N., Ndeme, A., and Fouly, H. M. 2004. Characterization of Phytophthora capsici isolates from processing pumpkin in Illinois. Plant Dis. 89:191197. This study was conducted to investigate pathogenic, morphologic, and genetic variations among Phytophthora capsici isolates from processing pumpkin (Cucurbita moschata) fields in Illinois. Random amplified polymorphic DNA (RAPD) markers were employed to assess genetic variation among 24 isolates of P. capsici from 10 individual fields at six locations. Unweighted mean pair group analysis clustered isolates into six groups. The genetic distances ranged from 0.03 to 0.45. Inoculation of pumpkin seedlings in the greenhouse revealed that the isolates belonged to six distinct genetic groups differing significantly (P = 0.05) in virulence. Isolates tested exhibited four growth patterns in culture: cottony, rosaceous, petaloid, and stellate. P. capsici isolates, including an ATCC isolate (ATCC-15427), with cottony growth pattern did not grow at 36°C. The mean oospore diameter of A1 mating type isolates was greater than that of A2 mating type isolates. Nine of 24 isolates tested produced chlamydospores in V8-CaCO3 liquid medium.

During August 2003, we conducted a statewide survey of rhododendrons to determine if Phytophthora ramorum was present in Ohio ornamental nurseries. In total, 240 samples were randomly collected in 12 nurseries throughout Ohio from rhododendrons showing foliar necrotic lesions and twig dieback symptoms. The samples yielded 51 Phytophthora spp. isolates on PARP-V8 agar. The internal transcribed spacer (ITS) region of all isolates was amplified using the universal primers ITS1 and ITS4 and was sequenced. Consensus sequences from sense and antisense were then blasted against the GenBank database, allowing for the identification to species of ˜80% of all isolates. These identifications, and the ˜20% unknowns, were confirmed using blind morphological tests on the basis of the following parameters: colony morphology; shape and dimensions of sporangia and type of papillae; dimensions of oogonia and oospores; type and position of antheridia; presence or absence of chlamydospores; presence or absence and morphology of hyphal swellings; and growth rate at 35°C according to the Revisited Tabular Key of the species of Phytophthora (1). No P. ramorum was detected among the isolates; however, P. cactorum, P. citricola, P. citrophthora, and P. nicotianae were detected. We also found two occurrences of P. inflata Caros & Tucker and one of P. insolita Ann & Ko. (P. inflata: e-value ≤e-179, identities ≥95%; P. insolita: e-value = 0.0; identities = 95%.) P. inflata was isolated from two tissue types, a dead twig and a necrotic leaf tip. P. insolita was isolated from a necrotic leaf tip. Identity of the two species was confirmed morphologically using the parameters listed above as well as the following measurements (N = 40; all in μm) (1): P. inflata - sporangia: 40 × 24 ([24 to 68] × [18 to 34]); oogonia: 34.6 (28 to 40); oospores: 30.8 (25 to 38); P. insolita - sporangia: 42 × 28 ([34 to 56] × [22 to 38]); oogonia: 32 (26 to 36); oospores: 26 (22 to 30). Koch's postulates were satisfied by inoculating two rhododendron plants (cvs. PJM and Nova Zembla) with the putative pathogens. On each plant, each of three leaves was pierced with a dissecting needle and was inoculated by placing a 0.5-cm-diameter plug of mycelium that was taken from the margin of a colony actively growing on PARP-V8 agar on the wound. The inoculum was retained using clear adhesive tape. A similar procedure was used for twigs. Controls consisted of inoculations with sterile PARP-V8 agar medium. Both cultures of P. inflata and P. insolita produced necrotic lesions in all inoculations on both tissue types within 1 week, and they were reisolated from the margins of lesions on PARP-V8. The lesion margin was at least 2 cm away from the inoculum plug in leaf inoculations and several centimeters in twig inoculations. To our knowledge, this is the first report of P. inflata and P. insolita occurring on rhododendron and the first time P. insolita has been reported outside of Southeast Asia where it has been recovered only from soil. Reference: (1) D. J. Stamps et al. Mycol. Pap. No. 162. CAB Int. Mycol. Inst. Wallingford, UK, 1990.

In a study of the Phytophthora infestans population in Taiwan, samples with symptoms typical of late blight were collected from field crops in an important potato- (Solanum tuberosum) and tomato-(Lycopersicon esculentum) production area in the central highlands region. Isolates were obtained by surface disinfecting leaf sections and plating them onto antibiotic-amended rye A agar (1). After subculturing, the pathogen was confirmed as P. infestans on the basis of morphological characters (2). Mating type was determined by co-inoculating unamended rye agar plates with mycelial plugs of the test isolate and a reference P. infestans isolate of either the A1 or A2 mating type (four plates per test isolate, two with different A1, and two with different A2 reference isolates). After incubation (15°C darkness, 7 to 14 days), plates were examined microscopically for the presence of oospores where the colonies interacted. In 2004, one isolate of 200 tested, and in 2006, one isolate of 102 tested, produced oospores only with A1 reference isolates and were concluded to be A2 mating type. In vitro testing showed the two A2 isolates were metalaxyl-resistant (ED50 values >100 mg of metalaxyl per liter on rye grain agar), which is typical of recent P. infestans isolates from potato and tomato in this area (2). Twenty-one single-sporangial isolates from each of the two A2 strains were tested for mating type against two different A1 isolates of P. infestans and confirmed as A2. These isolates were characterized using the techniques described by Deahl et al. (1) and had the allozyme genotype 100/100/111, 100/100 at the loci coding for glucose-6-phosphate isomerase and peptidase, respectively, and were mitochondrial haplotype IIb. This multi-locus genotype is characteristic of recent P. infestans isolates from tomato and potato in Taiwan, but all previous such isolates were A1 mating type and attributed to the US-11 clonal lineage (1). When evaluated on differential hosts, both A2 isolates were tomato race PH-1 and complex potato race R 0,1,2,3,4,7,9,11. RG57 fingerprinting showed that the A2 isolates had fingerprints identical to each other and to A1 P. infestans isolates of the US-11 clonal lineage from tomato in Taiwan (101 011 100 100 110 101 011 001 1). Koch's postulates were completed and the two A2 isolates were found to be highly aggressive on cultivars of potato and tomato. To our knowledge, this is the first report of A2 mating type strains of P. infestans in the field in Taiwan, but currently, their incidence is very low ( References: (1) K. L. Deahl et al. Pest Manag. Sci. 58:951, 2002. (2). D. C. Erwin and O. K. Ribeiro, Page 346 in: Phytophthora Diseases Worldwide. The American Phytopathological Society. St. Paul, MN, 1996.

Phytophthora citricola and P. cactorum cause bleeding cankers that lead to the death of mature European beech (Fagus sylvatica) in the northeastern United States. The effects of two fungicides and a plant growth regulator on in vitro pathogen characteristics and on canker expansion were investigated. In the first experiment, 16 single-spore isolates (11 P. citricola and 5 P. cactorum) were grown on clarified V8 juice agar amended with (i) 0 to 20 μg a.i./ml of mefenoxam, (ii) 0 to 301,429 μg a.i./ml phosphonate either with or without a bark-penetrating surfactant at 0.5 mg a.i./ml, or (iii) 0 to 25 mg a.i./ml of the surfactant alone. Radial growth, oospore production, and zoospore germination were observed to be dependent on isolate and treatment. A species effect on growth was also observed, as P. cactorum isolates were 2.5- to sevenfold less sensitive to phosphonate, but 2- to 150-fold more sensitive to mefenoxam than P. citricola isolates (based on 50% inhibition of growth). In the second experiment, bark and soil drenches of mefenoxam (50 mg a.i./ml and 19 μg a.i./ml, respectively), phosphonate (301,429 and 101 μg a.i./ml, respectively), and a soil drench of paclobutrazol (21 mg a.i./ml) were evaluated for their efficacy as curative or preventive treatments against bleeding canker. None of the treatments (curative or preventive) were able to stop canker expansion or prevent infection. However, saplings inoculated with P. citricola and treated with the phosphonate bark drench as either a curative or preventive treatment had cankers that were 36 to 82% shorter than those of inoculated control stems treated with water. For saplings inoculated with P. cactorum, the phosphonate bark drench was only effective when applied as a preventive (38% shorter than inoculated control stems treated with water), and not as a curative treatment. No other treatment was effective at limiting canker expansion.

Phytophthora alni, a soil- and waterborne pathogen, causes aggressive root and collar rot on riparian alder populations (1,2,4). The disease has been described from several European countries with a destructive impact in Great Britain (1,2). All European alder species and the red alder (Alnus rubra) are highly susceptible. P. alni has multiple variants that have been placed in three subspecies: P. alni subsp. alni, P. alni subsp. uniformis, and P. alni subsp. multiformis (1). In July 2009, a survey of symptoms of Phytophthora rot from A. glutinosa at 20 riparian stands along the Avia River in Galicia (northwest Spain) was conducted. Affected trees showed symptoms of Phytophthora rot including abnormally small, sparse, and yellowish foliage, dieback in the canopy, necroses of the inner bark and cambium, and bleeding cankers on the trunks (2,4). Phytophthora spp. were baited from saturated rhizosphere soil and watercourses using oak leaflets (4). Roots and tissue from fresh active inner bark lesions were transferred to selective medium V8-PARPH agar (4) and incubated for 7 days at 22°C in the dark. A Phytophthora sp. was isolated, transferred to carrot agar (CA), and incubated in the dark. Colonies were appressed, often irregular in outline, and with limited aerial mycelium (1). Growth on CA occurred from 4 to 31°C with optimum growth at 23 to 25°C. Chlamydospores were not observed. Ellipsoid, nonpapillate, noncaducous sporangia had a length/breadth average ratio of 1.4. Nesting and extended internal proliferation occurred. Oogonia, antheridia, and oospores were abundantly produced in a single culture. Oogonia with tapered stalks were spherical (mature oogonia 38 to 50 μm in diameter) and some had ornamented walls or bullate protuberances (1,2). Antheridia were large, amphigynous, and predominantly two-celled (23 to 37 × 16 to 23 μm). Oospores were plerotic. Distorted comma-shaped or smaller oogonia and aborted oospores were observed (1). Amplification of DNA was accomplished by using sequence-characterized amplification region-PCR primers (3). The amplicon sizes obtained were identical to P. alni subsp. alni (3). Internal transcribed spacer (ITS)-DNA and nadh1 mitochondrial gene were also amplified. DNA sequences of ITS and mt-DNA regions were deposited in GenBank (Nos. GU108602 and GU108603). Comparison of the sequences showed 100% homology with P. alni subsp. alni (GenBank Nos. FJ746679 and DQ202490). P. alni subsp. alni was recovered from trees at 3 of 20 riparian alder stands with symptoms. Pathogenicity of one representative isolate was confirmed by inoculating 10 3-year-old A. glutinosa seedlings grown in pots. One shallow cut was made into the bark at the collar level. A colonized agar plug, from the margin of an actively growing colony of P. alni subsp. alni, was inserted beneath the flap that was sealed with Parafilm. Five controls seedlings received only sterile CA agar plugs. Plants were incubated at 24°C and 95% humidity for 30 days. On inoculated plants, necroses progressed bidirectionally from the wound, and dead leaves and wilting of shoots were observed. P. alni subsp. alni was recovered from inoculated seedlings, but not from controls. To our knowledge, this is the first report of Phytophthora rot on alder caused by P. alni subsp. alni in Spain. References: (1) C. M. Brasier et al. Mycol. Res. 108:1172, 2004. (2) J. Gibbs et al. For. Comm. Bull. 126, 2003 (3) R. Ioos et al. Eur. J. Plant Pathol. 112:323, 2005. (4) T. Jung et al. Plant Pathol. 53:197, 2004.

Four A1 field isolates and one A2 field isolate of Phytophthora infestans were crossed to produce oospores in tomato leaves. The oospores were extracted and mixed with perlite and water, and healthy tomato leaves were used as bait for oospore-progeny infection. Twenty-nine lesions were obtained from the four crosses and 283 single-sporangium isolates were recovered and tested on four tomato differential lines carrying different major genes (Ph-0, Ph-1, Ph-2, and 3707) for late blight resistance. The pathogenic fitness (number of sporangia per unit leaf area) of parental and progeny isolates was strongly dependent on the host genotype; it decreased in the order Ph-0 > Ph-1 > Ph-2 > 3707. The A2 parent had a higher pathogenic fitness than the A1 parents on Ph-0 and Ph-1 but similar, lower fitness on Ph-2. Different levels of pathogenic fitness were observed across all isolates on Ph-0, although Ph-0 lacks resistance genes. Pathogenic fitness on one tomato genotype was not related to the pathogenic fitness on the other tomato genotypes. Some isolates exhibited reduced pathogenic fitness relative to the respective A1 parent, whereas others demonstrated a higher pathogenic fitness compared with the A2 parent. The tomato genotype Solanum pimpinellifolium L3707/5 was resistant to all five parental isolates of P. infestans. However, 37 of the 283 progeny isolates from 11 different lesions had compatible reactions with this line, producing up to 31 × 103 sporangia/cm2. Overall, reduced fitness was more frequent among the progeny isolates than increased fitness.

Phytophthora sansomeana E.M. Hansen & Reeser is a newly described species and infects Douglas-fir, alfalfa, and soybean (1). Soybean production is an important part of the local economy in Yili State in Xinjiang Uygur Autonomous Region, northwest China. Unfortunately since 2005, root and stem rot disease has emerged on a number of farms. To identify the causal agent, plant samples with symptoms, including whole plant wilting or yellowing and stunting, were collected from fields during 2005 and 2008. Tissue from the edges of stem lesions was placed on selective lima bean agar (LBA) at 20°C for 3 to 4 days (2,3). Four single zoospore isolates of Phytophthora were obtained and maintained on LBA or 10% V8 juice liquid medium for examination of morphological and physiological characteristics. The colonies on LBA were aerial and rosaceous. The isolates were homothallic, and oogonia and oospores were readily produced in culture after 7 days on LBA plates. Oogonia averaged 38 μm and oospore width ranged from 23 to 48 μm and averaged 31 μm. Antheridia were approximately 15 × 12 μm and predominantly amphigynous in V8 juice. Sporangia were terminal or paragynous on persistent sporangiophores, nonpapillate, ovoid to obpyriform, and measured 52 × 35 μm with an average length/breadth ratio of 1.5. Hyphal swellings were produced in V8 juice 2 days after inoculation. The optimum temperature for growth was approximately 25°C and none occurred at 0 or 35°C. The internal transcribed spacer (ITS) sequence of this Phytophthora species (GenBank FJ966880) agreed 100% with sequences of P. sansomeana isolates deposited in GenBank (GQ853880 and EU925375). Pathogenicity tests were performed by hypocotyl inoculation method (2) using isolate Yili71 and potted soybean cv. Williams. Plants were grown in a growth chamber for 10 days before inoculation in 16-cm-diameter pots (2). Plants were inoculated with 2- × 2-mm plugs of mycelium grown for 4 days on LBA at 25°C, the plugs were adhered to the sides of wounded lower hypocotyls. As controls, plants were inoculated with LBA agar plugs without mycelium (2). Inoculated plants were maintained in a growth chamber at approximately 25°C with a 10-h dark/14-h light cycle and 50% relative humidity and symptom development was monitored daily for 1 week. Wounded stems inoculated with mycelium developed water-soaked lesions, which were similar to those seen on naturally infected plants. A Phytophthora sp. was reisolated from the margins of expanding lesions on wounded stems. To our knowledge, this is the first report of P. sansomeana infection of soybean in China and the threat it may pose to soybean production is unclear. References: (1) E. M. Hansen et al. Mycologia 101:129. 2009. (2) Z. Y. Wang et al. Fungal Genet. Biol. 43:826, 2006. (3) X. B. Zheng. Methods in Phytophthora. Chinese Agriculture Press. Beijing, China, 1995.

Water survey for Phytophthora spp. by baiting with rhododendron leaves in April 2006 at a perennial ornamental plant nursery in Virginia detected five isolates showing a unique, previously unknown single-strand conformation polymorphism (SSCP) fingerprint (1). These cultures were isolated from two reservoirs at different depths of water column from surface to 2 m. They were homothallic and produced smooth-surfaced spherical oogonia with an average diameter of 27 μm on 10% V8 agar. Oospores were aplerotic. The paragynous antheridia were averaging 12 μm in diameter. Sporangia were papillate, spherical to ovoid, averaging 39 by 28 μm (length by width). They were caducous with short (2 mycelial plug from a 7-day-old culture and securing with Parafilm. V8 agar was used instead of mycelial plugs on control plants. After inoculation, each plant was enclosed in a plastic bag for 1 day and then incubated at 22°C with a 12-h photoperiod. Distilled water was sprayed daily for 5 days postinoculation (dpi) until disease symptoms were observed. At 15 dpi, 3 of the 10 inoculated rhododendron leaves and 6 of the 10 stems showed leaf lesions, wilting, dieback, and cankers, eventually leading to rhododendron death. Two of the 10 viburnum leaves and 4 of the 10 stems showed similar symptoms. Leaf lesions were approximately 3 to 5 cm in diameter. P. hedraiandra was recovered from diseased tissues and all resulting cultures showed an identical SSCP fingerprint to tested isolates as well as a P. hedraiandra isolate from Minnesota (3). No symptom developed on control plants. To our knowledge, this is the first report of P. hedraiandra in Virginia. Considering neither host plant has been grown or bought for resale by this nursery, this study indicates that P. hedraiandra may have a wider host range than is currently known. This possibility and the importance of water dispersal for P. hedraiandra in disease epidemiology warrant further investigation. References: (1) P. Kong et al. Fungal Genet. Biol. 39:238, 2003. (2) W. A. Man in't Veld et al. Eur. J. Plant Pathol. 117:25, 2007. (3) B. W. Schwingle et al. Plant Dis. 90:109, 2006.

During an extensive survey carried out in Piedmont (northern Italy) aimed at identifying the emerging soilborne diseases affecting tomato in commercial fields where alternatives to methyl bromide have been implemented in response to national and international regulations, sudden collapse of tomato plants, cv. Tomahawk, grafted on cv. Beaufort, were repeatedly observed in a commercial plastic tunnel operation. Affected plants suddenly collapsed 60 days after transplant during the month of May 2010. Symptoms included chlorosis, stunting, and severe root and crown rot, leading to sudden collapse of approximately 25% of the plants within 60 days of transplant. Symptomatic tissues from the root and collar of infected plants were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in water, and submerged in selective medium based on corn meal agar. A Phytophthora-like organism (2) with characteristic coenocytic hyphae was consistently isolated and transferred to V8 agar. The sporangia were spherical to ovoid, papillate, and 40 to 77 × 23 to 34 (average 55.1 × 30.3) μm. Oospores were globose and 22.2 to 30.8 μm. The internal transcribed spacer (ITS) region of rDNA of a single isolate was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 750-bp segment showed a 100% homology with the sequence of Phytophthora capsici JN382543.1. The nucleotide sequence has been assigned the GenBank Accession No. JX090306. Pathogenicity tests were performed on healthy 30-day-old tomato plants cv. Beaufort by using one strain of P. capsici grown for 15 days at 22 to 25°C on a mixture of 2:1 wheat/hemp kernels, and then 1 g per L of the inoculum was mixed into a substrate based on peat blonde/peat black (15:85 v/v). Two plants were transplanted into 3-L pots, with five replicates. Ten non-inoculated plants represented the control treatment; the trial was repeated once. All plants were kept in a greenhouse at temperatures ranging from 22 to 25°C. Inoculated plants became chlorotic 7 days after inoculation and root and crown rot developed 30 days after inoculation. Control plants remained symptomless. P. capsici consistently was reisolated from inoculated plants. In Italy, the presence of P. nicotianae on hybrids of Solanum lycopersicum × S. hirsutum is known (3), while, to the best of our knowledge, this is the first report of P. capsici on the hybrid S. lycopersicum × S. hirsutum in Italy. The economic importance of the disease can increase due to the expanding use of grafted tomato plants. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. APS Press, St Paul, MN, 1996. (3) A. Garibaldi and M. L. Gullino. Acta Hortic. 833:35, 2010. (4) H. M. Masago et al. Phytopathology 67:425, 1977.

Polymerase chain reaction-based detection of plant-associated microbes depends on physical disruption of tissues of the host and microorganism in order to liberate nucleic acids during extraction. Using six types of plant tissues as well as an oospore preparation of Phytophthora capsici, we evaluated the use of pressure-cycling technology (PCT) compared with several common techniques for physical tissue disruption. With all tissues tested, bead-beating provided excellent yields of amplifiable nucleic acid, with a few inconsistent exceptions. The use of PCT did not consistently improve nucleic acid yields or “amplifiability”. The use of a mortar and pestle to physically disrupt plant tissue also provided good results at low cost, though it was not consistently as effective as the bead-beater. Furthermore, handling of ground tissues in an open mortar may present more challenges in minimizing cross-contamination than working with tissues pulverized in a bead-beater tube.

During spring 2012, potted impatiens (Impatiens walleriana Hook.f.) plants with symptoms of a foliar disease were found in several commercial greenhouses in Mobile County, Alabama. Symptomatic leaves were chlorotic with no distinct lesions, and quickly wilted and abscised from erect green stems. In summer 2012 and 2013, numerous landscape impatiens plants with similar symptoms were observed in a large area from Mobile County north to Lee County, Alabama. A downy mildew was observed on the lower surfaces of symptomatic and abscised leaves from all locations. It consisted of hyaline, monopodial sporangiophores and ovoid, hyaline sporangia. Sporangiophores, which emerged from stomata, consisted of apical branches arranged at right angles to the supporting branches; they measured 69 to 90 μm long with individual branches measuring 7 to 14 μm long. Sporangia were borne on the tips of sporangiophore branches and measured 10 to 16.5 × 17 to 22.5 μm. No oospores were observed. In 2013, symptomatic plants were obtained from two separate locations in Alabama (Mobile and Tallapoosa counties). Total genomic DNA was extracted directly from symptomatic plant tissue and the large ribosomal subunit DNA was amplified by PCR using primers NL-1 and NL-4 (1). From both isolates, amplicons of 600 and 775 bp were obtained. DNA from each amplicon of both isolates was purified, sequenced, and the sequences were deposited in GenBank (Accession Nos. KF956518 to 21). The sequences of the 600-bp amplicons were 99% similar to that of I. walleriana (JX142135); the sequences of the 775-bp amplicons were 99% similar to Plasmopara obducens isolates from Florida (JX217746), Ohio (JX142134), Serbia (HQ246451), and the United Kingdom (AY587558). In pathogenicity tests, 10 potted impatiens plants, I. walleriana‘Super Elfin,’ were inoculated with a sporangial suspension (1 × 105 sporangia/ml washed from infected leaves) from the Mobile County isolate, by spraying until runoff. Controls were inoculated with sterile water. Plants were incubated in a moist chamber at 21°C for 48 h and then maintained in a greenhouse at 22 to 25°C until symptom development. All inoculated plants developed symptoms of downy mildew within 10 days. Microscopic examination of the symptomatic tissue revealed sporangiophores and sporangia similar to those observed in naturally infected plants. Control plants showed no symptoms. To our knowledge, this is the first report of downy mildew caused by P. obducens on impatiens in Alabama. This disease has been reported to have a significant economic impact for growers throughout the United States (2,3). Impatiens downy mildew is likely to be a recurring problem in Alabama. References: (1) K. O'Donnell. Curr. Genet. 22:213, 1992. (2) A. Palmateer et al. Plant Dis. 97:687, 2013. (3) S. Wegulo et al. Plant Dis. 88:909, 2004.

This study assessed survival of Phytophthora capsici oospores in soil in Illinois. Soils differing in texture and other characteristics were collected from four Illinois Counties (Champaign, Gallatin, Madison, and Tazewell), equilibrated to –0.3 MPa, and infested with oospores of P. capsici at a density of 5 × 103 oospores/g of dry soil. Samples (25 g) of the infested soil were placed in 15-μm mesh polyester bags, which were sealed and placed at 2-, 10-, and 25-cm depths in 15.3-cm-diameter PVC tubes containing the same field soil as the infested bags. Tubes were buried vertically in the ground at the University of Illinois Vegetable Research Farm in Champaign in October 2004. Soil samples were assayed for recovery and germination of oospores 1 day and 3, 6, 12, 24, 30, 36, and 48 months after incorporation of oospores into the soil. Overall, the percentage of oospore recovery and the percentage of germination of oospores were not affected significantly by soil source and burial depth but both the oospore recovery and oospore germination were significantly (P = 0.001) affected by the duration of oospore burial. The rate of oospore recovery from soil samples was 61.06, 16.69, 10.28, 1.05, 0.30, 0.06, 0.05, and 0.004% after 1 day and 3, 6, 12, 24, 30, 36, and 48 months, respectively, following incorporation of oospores into the soil; and mean oospore germination was 47.17, 30.53, 21.33, 15.64, 7.42, 2.67, 2.61, and 0.00%, respectively. Survival of P. capsici oospores was compared in soil samples stored in a laboratory at 22°C versus on the soil surface or buried 2, 10, or 25 cm deep in a field. Oospores were recovered 1, 3, 6, 12, and 24 months after incorporation for both storage locations. The percentage of oospores recovered from samples stored in the laboratory was significantly (P = 0.004) greater than recovery from samples stored in the field, regardless of the depth of burial. Twenty-four months after incorporation of oospores, 26.52% of oospores were recovered from soil samples in the laboratory, whereas only 0.12% of oospores were recovered from soil samples in the field. Overall, the percentages of germination of oospores recovered from samples in the laboratory and field over 24 months were not significantly different. In both experiments, germinated oospores produced mycelia, sporangia, and zoospores, and were virulent on ‘California Wonder’ bell pepper. This study showed that oospores of P. capsici can survive and remain virulent in Illinois soils for more than 36 months but oospores were no longer viable after 48 months in soil in a field environment.

Camelina or false flax (Camelina sativa), of the Brassicaceae, is an annual flowering plant native to Europe and Central Asia where it is grown commercially as an oilseed crop. At the end of May 2012, symptoms of downy mildew were observed on camelina plants grown in the Savinja Valley in Slovenia. The disease was found in four monitored fields (total area 3 ha), and the incidence ranged from 2 to 38% depending on the variety. Symptomatic plants showed whitish, abundant, and fluffy mycelia covering the stems, flowers, seed pods, and undersides of the leaves. The disease mainly affected the upper half of the plants, and the stems were reduced and distorted. During disease progression, the mycelium turned from gray to black. Microscopic observations revealed hyaline, straight conidiophores that were branched monopodially (3 to 4 times) with 6 to 12 re-curved tips/branch, and measured 140 to 300 × 12 to 20 μm. Conidia were hyaline, oval to broadly ellipsoidal, 24 to 29 × 18 to 24 μm. Oospores formed in necrotic stem and leaf tissues were dark brown and measured 30 to 38 μm in diameter. Based on these morphological characteristics, the causal agent was identified as Hyaloperonospora camelinae (1,3,4,5). DNA was extracted from mycelium and conidia collected from infected plants in two fields in the Savinja Valley (1HpC and 2HpC). Nuclear internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) were amplified by PCR assay from two isolates using the universal primers ITS4 and ITS5, and sequenced. Both samples yielded a 781-bp sequence, which showed 100% identity to H. camelinae ITS sequence JX445136 in GenBank. The nucleotide sequence was assigned to GenBank Accession No. KJ768405. Pathogenicity was confirmed by spraying 25 3-week-old plants of C. sativa cv. Ligena planted in pots (5 plants/pot) with a conidial suspension (105 conidia/ml) obtained from 10 infected plants of the same variety collected from the field 1HpC. Inoculated plants were covered with polyethylene bags for 2 days to maintain high humidity, and incubated at 20°C with a 12-h photoperiod/day in a growth chamber. Downy mildew symptoms first developed on leaves 6 days after inoculation. An additional 25 control plants sprayed with sterilized distilled water and otherwise treated similarly to the inoculated plants developed no symptoms. The identity of the pathogen on the inoculated plants as H. camelinae was confirmed based on the morphological features described above. Downy mildew of false flax caused by H. camelinae has been reported in Europe from Austria, Bulgaria, Germany, Poland, Portugal, Spain, and Switzerland (2); and in the United States from Florida, Oregon, Minnesota, Montana, Nebraska, and Washington (1,3,4,5). To the best of our knowledge, this is the first report of downy mildew caused by H. camelinae on C. sativa in Slovenia. The representative samples were deposited in the phytopatological herbarium of the Slovenian Institute of Hop Research and Brewing. References: (1) E. M. Babiker et al. Plant Dis. 96:1670, 2012. (2) D. F. Farr and A. Y. Rossman, Fungal Databases, Syst. Mycol. Microbiol. Lab. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ . (3) R. M. Harveson et al. Plant Health Progress. doi: 10.1094/PHP-2011-1014-01-BR, 2011. (4) M. L. Putnam et al. Plant Health Progress. doi: 10.1094/PHP-2009-0910-01-BR, 2009. (5) P. Srivastava et al. Plant Dis. 96:1692, 2012.

Roots of stunted broccoli plants (Brassica oleracea) from the Palo Verde Valley, CA, were observed at various stages of decay. A species of Pythium with large spiny oogonia was microscopically observed and consistently isolated from decayed roots. Isolates produced spherical, intercalary sporangia (average 34.5 μm in diameter) and aplerotic oospores (average 37.3 μm in diameter) in oogonia (average 47.4 μm in diameter) bearing numerous conical spines (average 8.5 μm in height and 5.5 μm basal width) with blunt apices. A representative broccoli isolate (P1) had a 99% internal transcribed spacer (ITS) sequence similarity with Pythium jasmonium (nom. inval., GenBank AF216654.1), a species which has not been formally described. Three other accessions in GenBank also carry the specific epithet of P. jasmonium and were originally isolated from diseased plants in the family Brassicaceae. In our study, these isolates were pathogenic on broccoli and morphologically similar to P1. P1 was pathogenic to 10 cultivated and 12 wild plants in the family Brassicaceae but not to 18 species of cultivated plants belonging to nine other plant families. Mycelial growth of our isolates occurred between

In Japan, rice seedlings grown in nurseries and used for transplanting are subject to a damping-off disease caused by Pythium spp. In this study, 148 isolates of Pythium spp. were obtained from rice seedlings in 39 locations of northern Japan. Among the isolates, 137 were identified as Pythium arrhenomanes using polymerase chain reaction (PCR) with species-specific primers, DNA sequencing analyses of the internal transcribed regions of ribosomal DNA, and the morphologies of oogonia, antheridia, oospores, and zoosporangia. Inoculation tests showed that the isolates identified as P. arrhenomanes were pathogenic to rice seedlings and parasitic to southern crabgrass with only minor damage. P. arrhenomanes was reisolated from the roots of both rice seedlings and southern crabgrass. Poaceae weeds, hosts of Pythium spp., grow in and around nurseries and in ridges surrounding rice fields. To detect Pythium spp., 188 Poaceae weeds were collected from 37 locations in Akita Prefecture. P. arrhenomanes was frequently detected in 164 weed roots from all locations by PCR using species-specific primers. Thus, we determined that P. arrhenomanes exists in and around rice seedling nurseries and rice fields, and that it is much more widely distributed than previously recognized in northern Japan.

Downy mildew in soybean plants is caused by the fungus Peronosporamanshurica. This fungus is an obligate parasite and its existence in Indonesia is still limited. This study aims to determine the pathogenicity of the fungus P. manshurica carried on imported seeds of soybeans from Malaysia. The Oospora from imported soybean seeds from the Class I Agricultural Quarantine Center was tested for its pathogenicity on soybean cultivars Anjasmoro, Grobogan, and Wilis in the greenhouses of the Jambi Class I Agricultural Quarantine Center. This test used a completely randomized design (CRD), using 4 treatments; 3 superior soybean cultivars (Anjasmoro, Grobogan, and Wilis),and 1 imported soybean cultivar. Each treatment was repeated 6 times so that there were 24 experimental units and each experimental unit consisted of 5 plants so that the total number was 120 plants. The results showed that the pathogenicity of the Oospore ofP.manshurica from imported soybean seeds still caused downy disease in the three soybean cultivars tested. There was no difference in the incubation period of downy mildew caused by the Oospora ofP.manshurica on Anjasmoro, Grobogan, and Wilis cultivars, which were 5.5, 8.0, and 7.0 days after inoculation, respectively. The highest disease severity was found in the Anjasmoro cultivar (8.91%) and the lowest was in the Wilis cultivar (2.66%), but it was not different from the disease severity in the Grobagan cultivar (4.28%).

This work proposes the use of a biomaterial sodium alginate for microencapsulating stonewort oospores. The microencapsulation method has been adopted and optimized to improve research work carried out on the oospores. A detailed protocol for the production of microcapsules with stonewort oospores has been presented, with particular attention paid to the critical stages and factors interfering with the efficient production process. The advantages and disadvantages of this method have also been indicated. Microcapsules with oospores can be successfully used in various laboratory (observation of the germination phases) and field experiments (reintroduction of rare species of stonewort). Alginate microcapsule may consist of water taken directly from the habitat of the stonewort, a medium, as well as specific additives (antibiotics or fungicides). The hardness of the encasement is regulated by the concentration of the sodium alginate. The optimum alginate concentration for oospores is between 2 and 3%. Encasements produced with less than 2% sodium alginate concentration do not form circular microcapsules during gelation. On the other hand, concentration >3% may limit the oospores' ability to germinate (higher density of the microcapsule). In such a dense environment, the germinating primary cells may encounter difficulties in outgrowing the microcapsule. It has been shown that alginate encasement creates an effective protective layer for the oospores inside it. The oospore is protected from mechanical damage as well as from direct exposure to the external environment; especially from pathogens and other microorganisms that can inhibit its germination and limit the growth of the protonemal and primary rhizoidal cell. The main application of oospore microencapsulation using sodium alginate is facilitating further mechanical manipulations of oospores, which by nature are very small structures (max. length 1 mm), while a microcapsule with an oospore embedded inside it can have a diameter from 3 to 5 mm (as required).

Phytophthora palmivora (Butl.) is a major obstacle in cocoa cultivation worldwide. The study aimed to determine the morphological character and pathogenicity of P. palmivora Pakuwon isolate. The study was carried out from November 2020 to January 2021, which consisted of 3 activities e.g. (1) pathogen isolation, tests on growth rate and colony formation, and stimulation of sporangia, zoospore, and clamydospore formation, which was performed in CMA, PDA, and mung bean media. The parameters observed were colony shape, growth rate, shape and size of sporangia, papillae, and sporangiophores, also chlamydospore and hyphal diameters; (2) To determine the possibility of different mating types, two isolates were grown on CMA medium in one petri dish; (3) The pathogenicity test, using a detached fruit assay by inoculating the cocoa pods with mycelium plug and zoospores. The patogenicity test was conducted in a completely randomized design with 4 replications, and the parameter observed was the area of the spot. The isolation obtained 3 isolates, the mycelium of all three isolates showed white in color, cottony, and no pattern in shape. The diameters of hyphae and chlamydospores were 5.6 - 6.3 mm and 39.0 - 43.2 mm, respectively. The sporangia were ovoid, ellipsoid, subsperic, obpyriform, and irregular. Sporangia measured 5.6 - 60.1 mm in length and 30.4 - 32.9 mm in width. Papillae were obviously visible with an average length of 6.8 mm. Test on mating types showed no oospores formed. PPkw1 isolates showed higher growth rates and more pathogenic than PPkw2 and PPkw3. The difference of pathogenicity of each P. palmivora isolates originated from Pakuwon can be used for selection of cocoa resistance in the future.

American ginseng (Panax quinquefolius) is a perennial herb whose dried roots are used for health care products, medicine, and food in China (Yuan et al. 2010). Shandong Province is the main area growing American ginseng and contributes more than 50% of the production in China. Wendeng city, located in the east of Shandong Peninsula, is the primary production area of American ginseng in Shandong Province since it has four distinct seasons, sufficient light, loose soil (pH 5.5～7.0), and with thus a similar geographical environment and climate conditions to the American ginseng production area of the United States and Canada. In March 2016, 2-year old American ginseng plants that were planted directly into the ground in the greenhouses in Wendeng city, contained up to 6-10% stunted plants. Water-soaked lesions were observed on the crowns and the tips of fine roots. The leaves of the infected plants became scalded, dark green starting at the top of the plants and gradually move downward. Moreover, the leaves and petioles gradually curled withered and drooped, and the whole plant collapsed. Tissue samples, 10 mm in size, were excised from the water-soaked roots and crowns of diseased plants, rinsed under running water for 24 hours, dipped in a 0.2% calcium hypochlorite solution for 10 minutes, placed on sterile filter paper to dry and then placed on V8 medium (200 mL V8 Campbell Soup, 15 g agar, 0.2 g CaCO3, and 1 L distilled water) and incubated in the dark at 28 °C for 5 days. Five Pythium-like isolates which were arachnoid-cottony on cornmeal agar were isolated and they all produced hyphal swellings, oogonia, antheridia and oospores. Oospores were globose, smooth and plerotic, with some being aplerotic. The dimensions of hyphal swellings, oogonia and oospores respectively ranged from 9.0 to 21.3 (average 14.1) µm, 12.9 to 22.5 (average 18.2) µm, and 12.5 to 20.5 (average 16.7) µm. Finger-like projections were uniformly distributed on the walls of the oogonia and the antheridia were curved rods. The five Pythium-like isolates were identified as P. spinosum based on morphological characteristics (van der Plaats-Niterink, 1981). Genomic DNA was extracted from the isolates of the Pythium sp. using a DNA extraction kit (OMEGA, U.S.A.). The cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer (ITS) region rDNA were amplified and sequenced using primers FM55/FM52R (Long et al. 2012) and ITS1/ITS4, respectively (White et al.1990). The five COI sequences were aligned and were identical for all five isolates, as well as the five ITS sequences. BLASTn analysis of the 538-bp COI sequence (accession no. MT822775) resulted in a 99% identity with that of the P. spinosum strain CBS122663 (accession no. HQ708832.1), and the 916-bp ITS sequence (accession no. MN847595) showed 100% identity with Genbank accession number AB217665 belonging to P. spinosum. Koch's postulates were confirmed. Corn kernels that had been soaked in water for 24 hours in water, autoclaved for 2 hours at 121˚C and allowed to cool were inoculated with agar plugs of P. spinosum grown on corn meal agar medium (CMA) for 10 days. The inoculated corn kernels were incubated at 28 ℃ for 13～15 days, until the corn kernels were covered with white hypha of P. spinosum. Ten healthy approximately 2-years old American ginseng plants growing in Wengdeng greenhouses were transplanted into a sterilized potting soil that was artificially infested with the corn inoculum (3 g inoculum per 100 g loam mixture). Inoculated and non-inoculated control plants were maintained in a greenhouse with a roof covered with sunshade net at 28 °C and 100% relative humidity. The experiment was repeated once. Four days after inoculation (DAI), the crown of inoculated plants developed water-soaked symptoms similar to those observed in field. No symptoms developed on the control plants. By 7 DAI, the inoculated fine roots and crowns showed water-soaked lesions identical to those observed in field, whereas control plants remained symptomless. The re-isolated isolate of P. spinosum was identical morphologically and by DNA sequence analysis to the original isolate. To our knowledge, this is the first report of root rot on American ginseng caused by P. spinosum in China and worldwide. Identification of the pathogen will assist in devising strategies to protect this important medicine plant from the pathogen, and to prevent yield losses.