V8 Juice Research Articles

First Report of Root Rot Caused by Phytopythium helicoides on Pistachio Rootstock in California

HomePlant DiseaseVol. 100, No. 11First Report of Root Rot Caused by Phytopythium helicoides on Pistachio Rootstock in California PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Root Rot Caused by Phytopythium helicoides on Pistachio Rootstock in CaliforniaE. J. Fichtner, G. T. Browne, M. Mortaz, L. Ferguson, and C. L. BlomquistE. J. FichtnerSearch for more papers by this author, G. T. BrowneSearch for more papers by this author, M. MortazSearch for more papers by this author, L. FergusonSearch for more papers by this author, and C. L. BlomquistSearch for more papers by this authorAffiliationsAuthors and Affiliations E. J. Fichtner , University of California Cooperative Extension Tulare Co., Tulare, CA 93274 G. T. Browne , USDA-ARS, CPGRU, Department of Plant Pathology, University of California Davis, CA 95616 M. Mortaz L. Ferguson , Department of Plant Sciences, University of California Davis, CA 95616 C. L. Blomquist , California Department of Food and Agriculture, Plant Pest Diagnostics Laboratory (CDFA-PPDC), Sacramento, CA 95832. Published Online:16 Aug 2016https://doi.org/10.1094/PDIS-12-15-1424-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Approximately 25 potted 2-year-old pistachio rootstock trees in a Kern County, CA, research plot maintained outdoors and irrigated to container capacity exhibited wilting in June 2013. Symptomatic plants gradually declined and died through November 2013. Extensive root necrosis was observed on symptomatic plants. Necrotic roots of seedling UCB-1 rootstock (Pistacia atlantica × Pistacia integerrima) submerged in CMA-PARP selective medium (Jeffers and Martin 1986) yielded a Pythium-like organism. On V8 juice agar, a representative isolate produced rapidly growing colonies (188 mm/day, radial) that produced subglobose, papillate sporangia measuring 25 to 42.5 × 25.0 to 37.5 µm (33.4 × 30.4 µm average). Sexual structures were not observed. DNA was extracted from the isolate and amplified by PCR with primers ITS1 and ITS4 (White et al. 1990). BLAST query of the sequence, GenBank accession no. KT901799 matched HQ643382, Phytopythium helicoides, with 98% identity. Diseases caused by P. helicoides have been reported on several plants and include root and stem rot on begonia (e.g., Yang et al. 2013) and rhizome rot on lotus (Yin et al. 2016). Phytopythium is a recently established genus containing organisms formerly classified as Pythium Clade K species (de Cock et al. 2015). For completion of Koch’s postulates, two isolates of P. helicoides were grown for 14 days on V8-oat-vermiculite substrate. UCB-1 clonal rootstock plants were purchased from a wholesale nursery, acclimatized in the greenhouse, grown to approximately 12 cm in height, and then transplanted into potting medium mixed with either uninfested substrate (the control) at a rate of 5% (v/v), or P. helicoides-infested substrate (2.5% v/v of each isolate). To induce zoospore release, pots were submerged in water for 4 h with the water surface maintained 1 cm above the soil. Control plants were treated identically to those in infested soil. The soil was then allowed to drain, and plants were watered daily. Two experimental runs included eight and 12 replicate plants of each treatment, respectively. Plants challenged with P. helicoides wilted within 1 week of inoculation. By 2 weeks after inoculation, 100% and 43% of inoculated plants died in the first and second experimental runs conducted in summer and winter, respectively. Control plants remained asymptomatic. By 6 weeks after inoculation, root mass of inoculated plants was only 28% and 65% of that of control plants in respective experimental runs. P. helicoides was consistently isolated from symptomatic plants and never from control plants. To our knowledge, this is the first report of root rot caused by P. helicoides on pistachio. The results indicate that the pathogen can be aggressive on UCB-1 rootstock, which is used for approximately half the commercial pistachio acreage in California. The greater mortality observed in summer than in winter suggests that variation in host or environmental factors may modulate disease development. The source of primary inoculum in the original symptomatic plants is unknown. Future work is needed to assess the importance and epidemiology of P. helicoides in commercial pistachio orchards.

First Report of Neofusicoccum parvum Causing Shoot Blight and Stem Cankers on Pomegranate in Florida

HomePlant DiseaseVol. 100, No. 8First Report of Neofusicoccum parvum Causing Shoot Blight and Stem Cankers on Pomegranate in Florida PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Neofusicoccum parvum Causing Shoot Blight and Stem Cankers on Pomegranate in FloridaA. N. KC and G. E. ValladA. N. KCSearch for more papers by this author and G. E. ValladSearch for more papers by this authorAffiliationsAuthors and Affiliations A. N. KC G. E. Vallad , University of Florida, Gulf Coast Research and Education Center, Wimauma, FL 33598. Published Online:9 May 2016https://doi.org/10.1094/PDIS-01-16-0067-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat A survey on prevalence of pomegranate (Punica granatum L.) diseases in central Florida was conducted during the spring and summer of 2014 and 2015. Approximately 30% of the trees had blighted shoots and 10% had stem canker leading to death of entire tree. Symptomatic tissues were collected, surface-disinfected with 1% chlorine for 1 min, and placed on V8 juice agar amended with rifampicin (0.01 mg liter−1) and ampicillin (0.25 mg liter−1). After 3 days of incubation at 21°C and 12 h continuous light, fast-growing white colonies with abundant aerial mycelia were observed. The reverse sides of the plates were initially white but turned dark to olive green after 1 week of incubation. Observed conidiomata were globose and lined with conidiogenous cells. Conidia were hyaline, unicellular, ellipsoidal with flat base and round apex, and ranged from 10 to 14 × 3 to 5 µm (average of 50 conidia, 12 × 4 µm). The causal pathogen was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (Crous et al. 2006; Palavouzis et al. 2015) based on morphological characteristics. For further confirmation, the rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF-1α) gene, and β-tubulin gene were amplified on two isolates using the respective primer pairs ITS1 and ITS4, EF-AF and EF-BR, and T1 and T2, and subjected to Sanger sequencing. The sequence data were deposited in GenBank (Accession Nos. KU530156, KU530155, and KU530157 for ITS, EF-1α, and β-tubulin, respectively). A BLAST analysis with corresponding GenBank sequences found that the ITS, EF-1α, and β-tubulin sequences exhibited 100% (522/522), 99% (318/320), and 99% (603/605) identity with N. parvum (CMW37263 and GenBank KF432951; CBS 111524 and GenBank FJ150709; CCF216 and GenBank KC507808), respectively. Pathogenicity tests were conducted on three replicated sets of 2-year-old containerized trees by applying a 1 × 105 spores/ml conidial suspension of an N. parvum isolate with a handheld sprayer, and covering branches with plastic bags for 48 h to maintain high humidity. Three additional trees sprayed with sterile distilled water served as controls. Branches of inoculated trees started to blight 3 months after inoculation and exhibited abundant black conidiomata on the surface of infected stems similar to the symptoms and signs observed on stems in the field. After 6 months, cankers developed on the main stem. No symptoms developed on any control treatments. The fungus, N. parvum, was reisolated from all symptomatic tissues, satisfying Koch’s postulates. N. parvum has been reported to cause shoot blight of pomegranate in northern Greece, causing 5% yield loss with 2% disease incidence (Palavouzis et al. 2015). A similar species, N. mediterraneum, has been reported to cause cankers on the trunks and branches of pomegranate in California (Morgan and Michailides 2012). However, N. parvum is morphologically and phylogenetically distinct from N. mediterraneum (Crous et al. 2006). To our knowledge, this is the first report of N. parvum causing shoot blight and stem canker on pomegranate in Florida. The pathogen is of significant importance due to its ability to cause severe losses on pomegranate, an emerging alternative crop in southeastern United States.

First report of tobacco root rot caused by Phytophthora nicotianae in Mexico

During 2014 and 2015, symptoms of root rot were observed in tobacco fields located in Veracruz, Mexico. Phytophthora colonies developing rosaceous pattern were consistently isolated on potato dextrose agar (PDA) and V8 juice agar media. Sporangia were papillate, noncaducous, measuring 40 to 49 x 33 to 37 μm, and with short pedicels. The sporangial shape was ovoid or ellipsoid. Chlamydospores were mostly solitary, intercalary, or terminal, measuring 29-33 μm in diameter. The oomycete was identified as Phytophthora nicotianae based on descriptions of Gallegly and Hong (2008). To confirm the identification, genomic DNA from a representative isolate was extracted, and the internal transcribed spacer (ITS) region was amplified by PCR and sequenced using the primers ITS5 and ITS4 (White et al., 1990). The resulting 896 bp sequence was deposited as GenBank Accession No. KR906059. BLASTn analysis showed a 97% similarity with those of P. nicotianae (GenBank Accession No. AB688370). Pathogenicity tests were performed twice on 20 healthy tobacco plants by immersion of roots in a zoospore suspension adjusted to 1 x 104 zoospores ml-1. Ten tobacco plants inoculated with sterile distilled water served as controls. Inoculated plants developed root rot symptoms after 10 days, whereas no symptoms were observed on the control plants. The oomycete present on inoculated plants was morphologically identical to that originally observed on diseased plants, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of tobacco root rot caused by Phytophthora nicotianae in Mexico.

First Report of Canker and Root Rot of Almond Caused by Phytophthora plurivora in Turkey

HomePlant DiseaseVol. 100, No. 7First Report of Canker and Root Rot of Almond Caused by Phytophthora plurivora in Turkey PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Canker and Root Rot of Almond Caused by Phytophthora plurivora in TurkeyO. Çiftçi, Ş. Türkölmez, S. Derviş, and Ç. U. SerçeO. ÇiftçiSearch for more papers by this author, Ş. TürkölmezSearch for more papers by this author, S. DervişSearch for more papers by this author, and Ç. U. SerçeSearch for more papers by this authorAffiliationsAuthors and Affiliations O. Çiftçi Ş. Türkölmez , Diyarbakır Plant Protection Research Station, 21110 Yenişehir, Diyarbakır, Turkey S. Derviş , Department of Plant Protection, Agriculture Faculty, Mustafa Kemal University, 31034 Antakya, Hatay, Turkey Ç. U. Serçe , Department of Plant Production and Technologies, Faculty of Agricultural Sciences and Technologies, Niğde University, 51240 Niğde, Turkey. Published Online:5 May 2016https://doi.org/10.1094/PDIS-12-15-1533-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Almond [Prunus dulcis (Miller) D.A. Webb.] is one of the most frequently consumed nut species grown in Turkey. In early March 2015, ∼20% of 13,000 one-year-old almond (P. dulcis cv. Ferragnes) seedlings in a nursery in Gaziantep showed sunken and blackened cankers on aboveground parts and root rot. Tissue samples taken from the margins of stem cankers and root lesions were placed on grated apple corn meal agar (Türkölmez et al. 2015) amended with P5ARPH per liter. Plates were incubated for 4 days at 20°C in the dark and a Phytophthora species was consistently isolated from the tissues. Isolates were homothallic with paragynous antheridia, smooth-walled spherical oogonia (24.7 to 29.6-μm diam), and both plerotic (1.3 to 1.4-μm wall diam) and aplerotic golden brown oospores (23.3 to 27.8 μm diam) on V8 juice agar (V8A) and produced semipapillate, noncaducous sporangia (35.5 to 53.2 × 26.3 to 38.4 μm; L:B ratio from 1.3 to 1.4 μm), which were usually obpyriform, ovoid, ellipsoid or irregular, and rarely possessed more than one apex, formed on simple or sympodial sporangiophores in nonsterile soil extracts. On V8 agar, colonies had a slight chrysanthemum growth pattern with limited aerial mycelium at the center of the colonies, whereas on PDA the colonies had a clear chrysanthemum growth pattern and aerial mycelia at the center of the colony. The minimum, optimum, and maximum temperatures for mycelium growth on V8A were 5, 25, and 32°C, respectively. On the basis of the morpho-physiological features, isolates were identified as P. plurivora T. Jung & T.I. Burgess from the ‘P. citricola complex’ (Jung and Burgess 2009). Genomic DNA was extracted from two representative isolates. The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS6/ITS4 primer pair and sequenced (GenBank Accession Nos. KU221328 and KU310937). BLAST searches showed a 100% identity with a type isolate of P. plurivora (FJ665225). The sequence also contained the polymorphic nucleotides that are distinctive of this species (Jung and Burgess 2009). Pathogenicity was tested on shoot segments from ten 1- to 2-year-old P. dulcis cv. Ferragnes (5 to 7 mm diam, 20 cm length) by replacing the 5-mm bark disks of shoots in the middle of each shoot segment with the same-sized mycelial disks of P. plurivora grown on V8 agar. Ten control segments were similarly inoculated with sterile V8 agar disks. The wounds were wrapped with Parafilm, and the shoots were incubated in moist chambers in 100% RH at 23°C. Phytophthora plurivora caused cankers on almond shoots within 5 days after inoculation, and the noninoculated control shoots remained symptomless. To test the pathogenicity on roots, primary roots of 10 P. dulcis cv. Ferragnes seedlings (1-year-old) were wounded with a scalpel, and mycelial disks (3 per plant) of P. plurivora grown on V8 agar were placed under the cortical tissue after removing 10 to 15 cm of soil around the root base. The inoculated and exposed roots were then covered with soil. Ten control seedlings were similarly inoculated with sterile disks. The soil was kept wet by watering plants daily as required. All seedlings were grown in a greenhouse at air temperatures from 24 to 30°C. No disease developed in the controls, but P. plurivora caused wilt and root rot after 2 months. Koch’s postulates were satisfied after reisolating P. plurivora from all inoculated shoots and roots. To our knowledge, this is the first report of P. plurivora causing canker and root rot on almond plants. In late April 2015, all naturally infected almond seedlings in the nursery were collapsed and destroyed by this oomycete, indicating that it might be a significant threat to almond plantations and orchards in the postnursery phase.

Isolation of Trichoderma Spp. from Desert Soil, Biocontrol Potential Evaluation and Liquid Culture Production of Conidia Using Agricultural Fertilizers

Three Trichoderma isolates were obtained from sandy soils collected at the “Gran Desierto de Altar” in the northwest of Mexico and characterized by morphologic and molecular analyses as Trichoderma harzianum 8.4, Trichoderma asperellum 12-2 and Trichoderma asperellum BP60. Isolate T. asperellum BP60 inhibited Setophoma terrestris, grew above 50°C, and produced chitinases and siderophores, therefore it was chosen to obtain enough biomass and conidia for field applications. Conidia production was intended in liquid culture fermentation using food grade ingredients and agricultural fertilizers. Assays were done using baffled Erlenmeyer flasks containing 75 mL of culture media, kept under constant agitation at 150 RPM, with initial pH adjusted to 6.5 (NaOH 1N) at 28 ± 2°C and evaluated at 3, 6, 9 and 12 days after inoculation (DAI). Among the carbon sources, sucrose and vinaze; the former induced higher yields of biomass and conidia. Regarding nitrogen sources, the fertilizer (NH4) NO3 induced higher conidia yield. V8 juice (V8) induced the highest effect on production of both biomass and conidia. Therefore, maximum yield was 1.06 × 109 conidia.mL-1, with the formulation with 5 g of KH2PO4 (MKP, Greenhow®), 1.3 g of MgSO4•7H2O (Sul-Mag, Penoles®), 20 mg of FeCl3•6H2O (Fermont®), 150 ml of V8, 10 ml of vinaze and 2.5 g.L-1 of (NH4)NO3. Results presented here prove the potential for using an alternative, low cost, liquid media to produce conidia of T. asperellum.

A unique species in Phytophthora clade 10, Phytophthora intercalaris sp. nov., recovered from stream and irrigation water in the eastern USA.

A novel species of the genus Phytophthora was recovered during surveys of stream and nursery irrigation water in Maryland, Massachusetts, North Carolina, Virginia and West Virginia in the USA. The novel species is heterothallic, and all examined isolates were A1 mating type. It produced rare ornamented oogonia and amphigynous antheridia when paired with A2 mating type testers of Phytophthora cinnamomi and Phytophthora cryptogea. Sporangia of this novel species were non-papillate and non-caducous. Thin-walled intercalary chlamydospores were abundant in hemp seed agar and carrot agar, while they were produced only rarely in aged cultures grown in clarified V8 juice agar. Phylogenetic analyses based on sequences of the internal transcribed spacer region and the β-tubulin and mitochondrial cytochrome-c oxidase 1 (cox1) genes indicated that the novel species is phylogenetically close to Phytophthora gallica in Phytophthora clade 10. The novel species has morphological and molecular features that are distinct from those of other species in Phytophthora clade 10. It is formally described here as Phytophthora intercalaris sp. nov. Description of this unique clade-10 species is important for understanding the phylogeny and evolution of Phytophthora clade 10.

Phenotypic and phylogenetic studies associated with the crucifer white leaf spot pathogen, Pseudocercosporella capsellae, in Western Australia

Pseudocercosporella capsellae (white leaf spot disease) is an important disease on crucifers. Fifty‐four single‐conidial isolates collected from Brassica juncea (Indian mustard), B. napus (oilseed rape), B. rapa (turnip), and Raphanus raphanistrum (wild radish) across Western Australia were investigated for differences in pathogenicity and virulence using cotyledon screening tests, genetic differences using internal transcribed spacer (ITS) sequencing and phylogenetic analysis, and growth rates on potato dextrose, V8 juice and malt extract agars. All isolates from the four crucifer hosts were pathogenic on the three test species: B. juncea, B. napus and R. raphanistrum, but showed differences in levels of virulence. Overall, isolates from B. juncea, B. napus and B. rapa showed greatest virulence on B. juncea, least on R. raphanistrum and intermediate virulence on B. napus. Isolates from R. raphanistrum showed greatest virulence on B. juncea, least on B. napus and intermediate virulence on R. raphanistrum. Growth and production of a purple‐pink pigment indicative of cercosporin was greatest on malt extract agar and cercosporin production on V8 juice agar was positively correlated with virulence of isolates on B. juncea and B. napus. ITS sequencing and phylogenetic analysis showed that isolates collected from B. napus, B. juncea and B. rapa, in general and with few exceptions, had a high degree of genetic similarity. In contrast, isolates from R. raphanistrum were clearly differentiated from isolate groups collected from Brassica hosts. Pseudocercosporella capsellae reference isolates from other countries generally grouped into a single separate cluster, highlighting the genetic distinctiveness of Western Australian isolates.

Lycopene Dietary Intervention

Heart failure (HF) is a condition of chronic exacerbations and injury resulting from an intricate relationship between biochemical and biological mechanisms. Inflammation can be a significant contributor in the pathophysiology of HF. Antioxidants may slow the progression of HF because of their ability to inhibit damaging inflammatory processes. The purpose of this study was to test a dietary intervention in patients with HF to assess the impact of lycopene on biomarkers of inflammation. Forty participants with HF were randomly assigned to 1 of 2 groups: lycopene intervention and usual care. The lycopene intervention group received 29.4 mg of lycopene intake per day by drinking an 11.5 oz serving of V8 100% vegetable juice for 30 days. We obtained serum lycopene, uric acid, C-reactive protein (CRP), and b-type natriuretic peptide to determine the impact of the intervention. Plasma lycopene levels increased in the intervention group compared with the usual care group (0.51 μmol/L to 0.76 μmol/L, P = .002; 0.56 μmol/L to 0.58 μmol/L). C-reactive protein levels decreased significantly in the intervention group in women and but not in men (P = .04). The preintervention CRP level for women was 5.9 ± 3.7 mg/dL and for men was 2.2 ± 2.1 mg/dL. The postintervention CRP level for women was 4.5 ± 3.6 mg/dL and for men was 2.4 ± 2.1 mg/dL. These findings suggest that the antioxidants in a 30-day intervention of V8 juice affect CRP levels in a sample of female patients with HF.

Morphological, physiological and molecular characterization of Phytophthora alni isolates from Western Spain

During extensive surveys for Phytophthora associated with alder mortality in western Spain, isolates were consistently recovered from necrotic bark of the collar and lower stem of Alnus glutinosa. Morphological and molecular characteristics of the selected isolates together with their physiology were examined. Morphologically, the isolates were homothallic and characterized by abundant production of terminal oogonia predominantly with two-celled amphigynous antheridia exhibiting ornamentation. Simple sporangiophores were observed bearing terminal, non-papillate, ellipsoid to ovoid sporangia. Colony growth patterns developed on V8 juice agar (V8A), carrot agar (CA), corn-meal agar (CMA) and potato-dextrose agar (PDA) showed uniform, radial or irregular growth patterns with appressed and/or woolly morphology. Temperature (2 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 27 °C, 30 °C and 32 °C), pH (5, 7, 9 and 11) and osmotic potential (−0.6, −1.2, −1.8 and −2.4 MPa) significantly influenced radial mycelial growth of the isolates under laboratory conditions. Molecular analyses including ITS DNA region sequencing and subspecies specific primers amplification confirmed identification of the isolates as Phytophthora alni ssp. alni. Further research is under way to carry out more surveys in order to determine the extent of damage and severity of the disease in different regions of Spain.

First Report of Stem Rot of Konjac Caused by Phytophthora nicotianae in China.

Konjac (Amorphophallus) is an important economic crop widely used in health products and biomaterials in Asia (2). A serious foliage disease of Konjac was observed in Fuyuan County, Yunnan Province, China, in July 2012. The symptoms began with leaf color changes from light green to yellow, followed by discoloration on the stem base, plant wilting, bulb rotting, and ultimately plant death. Symptomatic tissues were cut into small pieces, surface-sterilized, and cultured on 20% V8 juice agar at 28°C. Five days after incubation, white fluffy colonies with the typical sporangium of Phytophthora sp. were observed from root and stem pieces. Isolates were identified as P. nicotianae based on morphological characteristics and DNA analysis. The growth rate of the colonies was 16 mm/d at 28°C. Sporangia were pyriform, ovoid to spherical, and papillate, and the dimensions of the 80 sporangia measured ranged from 23.7 to 60.4 × 19.4 to 45.7 μm (avg. 42.4 × 31.5 μm) with length-to-breadth ratios of 1.19 to 1.44 (avg. 1.34). The chlamydospores were spherical with a smooth surface, and their dimensions ranged from 20.3 to 47.3 × 18.9 to 45.9 μm (avg. 32.7 × 30.4 μm) (3). DNA was extracted from one colony containing spores and hyphae of the isolated P. nicotianae, and the nuclear ribosomal DNA internal transcribed spacer (ITS) region was amplified with primers ITS6 and ITS4 (4). The obtained 854-bp amplicon was purified and sequenced. NCBI BLAST retrieved a 100% identity with P. nicotianae (GenBank Accession No. KJ506732). A pathogenicity test of the isolated P. nicotianae was conducted in a greenhouse. After 7 days in a humidity-controlled greenhouse, all 10 inoculated plants showed similar symptoms as observed initially in the field, while control plants were symptomless. P. nicotianae was re-isolated from the inoculated stems, thus successfully completing Koch's postulates (1). To our knowledge, this is the first report of P. nicotianae as a pathogen of Konjac in China.

First Report of Black Leaf Mold of Tomato Caused by Pseudocercospora fuligena in Ohio.

Diseased tomato (Solanum lycopersicum L. cvs. Geronimo, Rebelski, and Big Dena) plants were received for diagnosis from a home gardener in Wayne County, Ohio, in August 2013 and from a 0.14-ha greenhouse in Brown County, Ohio, in September 2013. Approximately 10 and 60% of leaf area was diseased in the home garden and greenhouse, respectively. One or more lesions, each with an indistinct border, were observed on the leaves. Black fungal growth was observed on both sides of the leaf in association with the lesions. Microscopic examination revealed Cercospora-like conidia (2). Three symptomatic leaves from each location were surface-sterilized with 0.5% NaClO for 1 min and cultured on V8 juice agar medium at room temperature under continuous fluorescent lighting. One isolate was selected from each of Wayne Co. (SAM33-13) and Brown Co. (SAM34-13). The fungus produced small, dark-brown colonies within 2 weeks of plating. Mycelium was olive brown and septate, producing fascicles of conidiophores. Conidia were cylindrical, 2 to 14 septate, and 25.8 to 109.7 × 6.5 μm. Genomic DNA was extracted from colonies of isolate SAM33-13 grown on V8 juice agar medium using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI). The internal transcribed spacer (ITS) region of rDNA was amplified by PCR using primer pair ITS1 and ITS4 (5), and the purified amplicon was sequenced (OARDC Molecular and Cellular Imaging Center, Wooster, OH). The ITS sequence was 99% identical to those of GenBank accessions of Pseudocercospora fuligena from Korea (JX290079) and Thailand (GU214675). The sequence was deposited in GenBank (KF931141). Based on morphology (4) and sequence analysis, the fungus was identified as P. fuligena (Roldan) Deighton (basionym Cercospora fuligena). To satisfy Koch's postulates, three 4-week-old tomato plants each of the cultivars L390 (AVRDC, Taiwan) and Mountain Spring (Siegers Seed Co., Holland, MI) were sprayed with a suspension of 1 × 103 conidia/ml of isolates SAM33-13 or SAM34-13 prepared from 3-week-old cultures growing on V8 juice agar medium. Three non-inoculated control plants were sprayed with sterilized water. Plants were maintained in a growth chamber at 25 to 30°C, 80% RH, and a 12 h/12 h day/night cycle. The first symptoms appeared 3 weeks after inoculation as light yellow foliar lesions. The lesions enlarged and turned black due to fungal growth, and the infected leaves dried. Disease severity was 70 and 10% of leaf area for cvs. L390 and Mountain Spring, respectively, for each isolate. Non-inoculated control plants were symptomless, and no fungus was re-isolated from the leaves. P. fuligena was isolated from symptomatic leaves of inoculated plants as described above, and the identity was confirmed based on morphology. In the United States, C. fuligena has not been reported infecting tomato since the first report in Florida in 1974 (1). To our knowledge, this is the first report of black leaf mold of tomato caused by P. fuligena in Ohio. Resistant cultivars, crop sanitation, and fungicides are recommended to manage the disease (3).

First Report in Chile of Mycocentrospora acerina, Causal Agent of Peony (Paeonialactiflora) Red Spot.

A survey to identify pathogens causing peony diseases in southern Chile (37°95' to 40°58'S) was conducted from 2008 to 2011. A noticeable symptom consisted of small red spots on leaves, stems, sepals, and first petals of the bud. The central part of the lesions (1 cm) enlarged, darkened, and became necrotic, coincident with rainy and cold weather, including frosts. Lesions grew but never coalesced completely. At the edges of leaves, the infected tissues cracked and produced a twisted and corrugated appearance. Rhizome symptoms were also observed and consisted of black spots, which enlarged to elongated lesions. Small pieces (~1 × 1 mm) of symptomatic leaves, previously washed with 2% NaOCl for 2 min were cultured on potato dextrose agar (PDA) and V8 juice agar (20%) media. The cultures were incubated in growth chambers at 20°C (dark) and 5°C (dark)/20°C (light) for 12/12 h (dark/light). Sporulation was obtained only with V8 under the dark and light regime. Mycelia growth was superficial and immersed in the media; hyphae were septate with thick black walls. Conidiophores were flexuous, not branched, and produced a single spore. Spores were elongate, multiseptate, with a long, strongly curved beak. After one week, sporulation decreased and thick-walled, round black clamydospores formed in the media. Spores ranged from 125 to 235 μm long and most frequently were 170 to 220 μm. The number of septa ranged from 5 to 12. Morphological and cultural characteristics fit the description of Mycocentrospora acerina (Harting) Deighton (1,2). DNA was obtained from fungal culture. The internal transcribed spacer (ITS) region was amplified using ITS1/ITS4 primers (3), and part of the amplicon (502 of 550 pb) was sequenced. The sequence was deposited in GeneBank (Accession No. KF015599) and showed 100% identity values with sequences of similar regions from M. acerina (strain ATCC 16259, Accession No. KF278454). Healthy leaves and rhizome pieces were washed with sterile distilled water and placed on sterile moist paper towel. A small agar disc with mycelium was placed on the leaves and rhizomes with and without wounding before inoculation. The inoculated materials were kept in closed boxes with high humidity at 5 to 20°C. Tests were positive in leaves and rhizomes with and without wounds. Seven days after leaf inoculations, necrotic symptoms developed. A second inoculation test in peony leaves of plants bagged with plastic under field conditions corroborated the in vitro test. Inoculated rhizomes developed dark orange lesions initially, then turned black with a watery consistency, similarly to affected rhizomes in the field. In both tests, controls showed no symptoms. To our knowledge, this is the first report of M. acerina on P. lactiflora globally (4). In an evaluation at Carillanca (38°41'S, 72°25'W), using a scale from 0 (no symptoms) to 7 (necrotic lesions covering >60% of foliage, with death of stems), 11 out of 31 peony varieties scored 0-1. As a reference, a score of 3 is the maximum damage allowed on peonies for export. These varieties might help to keep Chilean peonies in the market without an economic impact.

First Report of Cherry Stem Rot and Leaf Necrosis Disease Caused by Phytophthora nicotianae in Yantai, China.

Cherry (Cerasus avium (Linn.) Moench) is the third most economically important fruit in Yantai, Shandong Province, China. In August 2012, brown spots or necrosis on cherry seedling leaves, with an incidence of 8.2 to 34.3%, were observed in some fields of cherry seedlings in Yantai. Our survey indicated that the economic losses could reach up to 15.3% if disease conditions, such as a cool rainy summer season, were favorable. Conspicuous watery lesions on the stems turned to brown streaks; the leaves all wilted; and finally the plants collapsed. Diseased stem and leaf samples were surface-disinfected in 1% sodium hypochlorite for 1 min, rinsed three times in sterile water, which was absorbed with filter paper, and then transferred to 10% V8 juice agar medium containing 50 μg/ml ampicillin and 5 μg/ml carbendazim (1). The plates were incubated at 22°C in the dark for 5 days. The colonies consisted of white, loose, fluffy aerial mycelia. Eight isolates were obtained, and all were identified as Phytophthora nicotianae based on morphological characteristics and the sequence of the internal transcribed spacer (ITS) region of rDNA. The sporangia were ovoid/spherical, obturbinate with rounded bases and prominent papillae that were 37.5 to 62.5 × 30 to 50 μm (average 46.4 × 37.8 μm, n = 100) in size, with an average length-to-breadth ratio of 1.2. Chlamydospores were terminal, intercalary, and measured 19 to 42 μm (average 30.4 μm), which is typical of P. nicotianae (2). The genomic DNA of the eight isolates was extracted from mycelia. The ITS region of all eight isolates was amplified using primers ITS1 and ITS4, producing specific products that were directly sequenced. The sequence of a representative isolate P1401 (895 bp) was submitted to GenBank (Accession No. KJ754387). It was 100% similar to P. nicotianae strains NV-20T and TARI 22073 (KC768775 and GU111667). To confirm the pathogenicity, at least 10 cherry leaves and new stems were inoculated with mycelial plugs (5 × 5 mm) from each isolate. Necrosis of leaves and stems was observed 4 and 7 days after inoculation, respectively. No symptoms were observed on the control leaves and stems that were inoculated with blank agar plugs. P. nicotianae was re-isolated from the infected leaves, and the ITS sequence was analyzed to confirm its identity. Phytophthora species, such as P. cambivora, P. megasperma, and P. drechsleri, had been previously isolated from cherry (3), but to the best of our knowledge this is the first report of stem rot and leaf necrosis disease caused by P. nicotianae on cherry. Since the economic loss caused by this disease could reach 15% if an outbreak occurred in a rainy summer, control measures should be implemented.

First Report of Resistance to QoI Fungicides in Alternaria alternata Isolates from Potato in South Africa.

Since the first report of brown spot of potatoes (Solanum tuberosum) caused by Alternaria alternata (Fr.) Keissl in South Africa (3), disease intensity has steadily increased. No fungicides are registered for control of brown spot of potatoes in South Africa but growers attempt to control the disease with products registered for early blight, which include various QoI fungicides. Failure to control brown spot with QoI fungicides led to an investigation on putative development of resistance among A. alternata populations. In the summer of 2012, diseased leaves were collected from five potato growing regions. Isolations were made from the margin of brown spot lesions by plating surface disinfested tissue on V8 agar medium (200 ml V8 juice, 3 g CaCO3, 20 g agar). Plates were incubated at 25°C in darkness for 7 days, purified, and single-spore cultures transferred to fresh potato dextrose agar (PDA) plates. Identity of isolates was confirmed using conidial morphology and PCR amplification using species-specific primers AAF2 and AAR3 (1). Eight A. alternata isolates (PPRI 13607, 13608, 13609, 13610, 13611, 13612, 13613, and 13614) were obtained and screened for sensitivity to azoxystrobin in vitro by evaluating relative conidial germination on media amended with 0, 1.0, 2.5, 5.0, 10, 25, 50, 75, 85, and 100 μg of azoxystrobin per ml of media. The dose effect of the fungicide on germination and the EC50 of each isolate were computed using the probit analysis. Isolates were subjected to DNA extraction and the partial cytochrome b (cyt b) was amplified using primer pair CBF1 and CBR2 (2). PCR products were transformed into DH5α competent cells using a pGEM-T Easy vector. Both strands of the cloned fragments were sequenced using primers T7 and SP6 (4). Isolates PPRI 13611 and 13614 had low EC50 values of 0.11 and 0.23 μg/ml, respectively, and a mean EC50 of 0.17 μg/ml, showing their relative sensitivity to azoxystrobin. The other six isolates had EC50 values ranging from 51.88 to 114.92 μg/ml, and a mean EC50 of 71.60 μg/ml, showing their resistance to azoxystrobin. Sequence analysis of the partial cyt b gene showed strong association of resistance in isolates PPRI 13607, 13608, 13609, 13610, 13612, and 13613 to a base substitution resulting in an amino acid substitution at position 143 (G143A). Isolates PPRI 13611 and 13614 did not exhibit this mutation. Although resistance has been reported on other crops where QoI fungicides, including azoxystrobin, have been used to control different pathogens, this is the first report of resistance to a QoI fungicide in field isolates of A. alternata from potatoes in South Africa. Identification of resistance will help to explain failure to control this disease using QoI fungicides. Further monitoring of resistance to azoxystrobin and other QoI fungicides is warranted.

Improved production of phleichrome from the phytopathogenic fungus Cladosporium phlei using synthetic inducers and photodynamic ROS production by phleichrome

Two different diketopiperazines, cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe), which were isolated from the culture filtrate of Epichloe typhina and found to be inducers of phleichrome production, were chemically synthesized and evaluated for use in the improved production of phleichrome from wild-type and UV-mutagenized strains (M0035) of Cladosporium phlei. When supplemented with PDA and V8 juice agar media, both inducers showed significant increases in the production of phleichrome. Phleichrome production was increased in a dose-dependent manner up to a concentration of maximum yield for both inducers. No further significant induction was observed by supplementing inducers over the concentration of maximum yield. Among the two inducers, cyclo-(L-Pro-L-Phe) showed better inducing capability than cyclo-(L-Pro-L-Leu). The maximum yield was observed from the M0035 strain grown on V8 juice media supplemented with 150 μM cyclo-(L-Pro-L-Phe), which was estimated to be 232.6 mg of phleichrome per gram of mycelia and 10.2 mg of secreted phleichrome per 20 agar-plugs. Interestingly, growth inhibition was observed on V8 juice agar media with 100, 150, and 200 μM cyclo-(L-Pro-L-Phe) but not on PDA with the same amount of inducer, which suggests that the inhibitory effect might be through the overproduction of phleichrome rather than the toxic effect of the inducer itself. Superoxide production by purified phleichrome was dramatically stimulated upon illumination, thus demonstrating photodynamic production of superoxide in vitro by phleichrome.

Occurrence of Phytophthora citrophthora on Syringa vulgaris in Poland

<i>Phytophora citrophora</i> was isolated from dieback shoots, base of diseascd stems and top parts of lilac plants during 3 vegetation periods. The species was detected from about 75% of analysed plant parts. On V8 juice agar and potato-dextrose agar the quickest growth of the species was observed at 25·30°C but development did not occur at 5° and 35°C. Zoosporangia were mostly papillate from spherica1 to ellipsoidal with average dimensions 45.6 x 31.5 μm. <i>P. citrophthora</i> from 5 host plants caused necrosis development of leaves and stem pars of lilac but the quickest spread of disease occurred when plants or their parts were inoculated with isolates from <i>Syringa vulgaris</i> and <i>Pieris japonica</i>. It indicates that the pathogen may be transmissed in nurseries from diseased pieris grown near lilac.

First Report of Pythium Root and Stalk Rot of Forage Corn Caused by Pythium arrhenomanes in Japan.

Corn (Zea mays L.) is the most important forage crop in Japan. It was cultivated on 92,000 ha in 2011 and was mainly used as whole crop silage for cattle feed. In September 2009, a root and stalk rot disease was detected on corn plants cultivated in Tochigi, located in the central region of Japan. The symptoms of the disease included wilting of whole plants after the R5 (dent) stage (2) with drooping ears. Roots turned black and their number decreased. Further, the stalks became hollow and soft and harbored white hyphae. This tissue deterioration made machine harvest difficult. We obtained seven isolates of a Pythium-like organism by single hypha isolation from surface-sterilized pieces of diseased roots and stems on water agar and deposited one of the isolates at the NIAS genebank, Japan, under the accession no. MAFF511547. The isolate was grown in the dark on V8 juice agar medium for 10 days to produce oogonia. The oogonia were globose, light brown to yellow, smooth, 23.9 to 30.5 μm in size, and had 1 to 8 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick. The morphology of the isolates was similar to that of Pythium arrhenomanes Drechsler and consistent with the species description (3). We analyzed the rDNA-ITS region sequences of the isolate as described by Kageyama et al. (1). The sequence (GenBank Accession No. AB903904) showed 99.1% (783/790 bp) similarity with that of P. arrhenomanes (AY598628). On the basis of morphological and rDNA sequence similarities, we identified the isolates obtained from corn as P. arrhenomanes. The pathogenicity of the isolate was confirmed by planting corn seedlings of the commercial Pioneer Brand hybrid 36B08 immediately after germination in five replicate pots containing soil mixed with 5% boiled barley grain by weight, incubated with or without the isolate for 7 days. After 10 days of incubation in a greenhouse at 20 to 25°C, only the inoculated plants exhibited symptoms of root and stalk rot. Since the inoculated organism was readily re-isolated from the diseased stems and roots, the pathogenicity of the isolate was confirmed. For field observation, the same hybrid of forage corn was sown in the fields in Nasushiobara, Tochigi, on 16 May 2011. The hybrid was sown in a row of 2 m, with 20 seeds planted at a distance of 10 cm with two replicates. For inoculum, the isolate was cultured on 5-cm-long wooden toothpicks, previously soaked in potato dextrose broth and placed on a V8 agar plate for 7 days at 25°C in the dark until covered by hyphae. The toothpicks were pierced into wounds made on the stems of corn plants, approximately 10 cm above the ground, using a thin iron needle. The wounds were about 2 mm in diameter and 2 cm deep. Field inoculation was conducted in late July at the R1 (silking) growth stage. Disease symptoms were observed in mid-September at R5, and only those plants that were inoculated with the toothpicks harboring the hyphae exhibited the typical stem rot symptoms. To our knowledge, this is the first report of root and stalk rot caused by P. arrhenomanes in forage corn in Japan.

First Report of Root and Stem Rot Caused by Phytophthora tentaculata on Mimulus aurantiacus in North America.

Sticky monkey flower plant, Mimulus aurantiacus (Phrymaceae), is a small, perennial shrub that is widely distributed throughout California, especially in coastal and disturbed habitats. It is also found in native plantings in parks and landscapes. In October 2012, nearly all the M. aurantiacus plants grown in a Monterey County, CA nursery for a restoration project were stunted and had dull, yellowish leaves. Roots and stem collars had necrotic, sunken lesions with few feeder roots. Thirty percent of the plants had died. Samples of diseased plants were sent to the CDFA-PPDC Lab and tested positive for Phytophthora sp. using the Agdia ELISA Phytophthora kit (Agdia, Elkhart, IN). A Phytophthora sp. was consistently isolated from the tissue on corn meal agar-PARP (CMA-PARP) (2). Sporangia were spherical to ovoid, papillate to bipapillate and 17 to 42.5 (avg. 27.5) × 12 to 35 (avg. 22.9) μm, with a length/breadth ratio of 1.2:1. Chlamydospores, which were spherical, terminal to intercalary, thin walled and 27.5 to 40 μm, and hyphal swellings formed on CMA-PARP. Spherical oospores, 25 to 36 μm, with primarily paragynous antheridia formed readily on V8 juice agar. rDNA sequences of the internal transcribed spacer (ITS) region of the isolates (GenBank KF667505), amplified using primers ITS1 and ITS4, were 100% identical to Phytophthora tentaculata (CBS 552.96, GenBank AF266775) by a BLAST query (1,3). To assess pathogenicity, exposed root crowns of three 3.78-liter potted M. aurantiacus plants were inoculated with 20 ml of zoospore suspension (2 × 104 ml-1). Plants were maintained in a 23°C growth chamber with a 12-h photoperiod and watered daily. Sterile water was applied to the exposed crowns of three control plants. At 2 weeks, all inoculated plants were wilted with chlorotic foliage. After 3 weeks, the cortical tissue of the crowns and roots was discolored and sloughing and P. tentaculata was recovered on CMA-PARP. P. tentaculata did not grow from the asymptomatic control plants. Inoculations were repeated with similar results. P. tentaculata is a homothallic species in Phytophthora clade 1 that causes crown, root, and stalk rot of nursery plants in Europe and China (1,4). A USDA PERAL analysis lists it as one of the top 5 Phytophthora species of concern to the United States (4). Genera infected with P. tentaculata include Apium, Aucklandia, Chicorium, Chrysanthemum, Delphinium, Gerbera, Lavandula, Santolina, Origanum, and Verbena (4). To our knowledge, this is the first report of P. tentaculata in North America. The source of inoculum of P. tentaculata in California remains unknown. The nursery used seed and cuttings of M. aurantiacus from nearby native areas for propagation, and P. tentaculata was not found in neighboring plant hosts or by baiting soil and water at the nursery. All infected M. aurantiacus material was destroyed. The presence of P. tentaculata in California nurseries could have serious economic impacts on the nursery industry and environmental impacts on susceptible native hosts, if spread into the wildlands.

First Report of Foliar Blight Caused by Phytophthora capsici on Citrus reticulata Blanco cv. Nian Ju in Guangdong, China.

Citrus reticulata Blanco cv. Nian Ju, an important ornamental plant, is traditionally displayed during the Chinese Spring Festival because its golden fruits are a symbol of auspiciousness. In the spring of 2012, foliar blight was observed on 10 to 30% of the Nian Ju plants at four nurseries in Yangjiang, Guangdong Province, China. Initial symptoms appeared as brown to black foliar lesions, followed by expansion of spots into blight. Some young branches also had necrosis. During frequent rainfall and prolonged wet periods at 22°C to 30°C, white and dense mycelia and sporangia were observed on the infected seedlings. To isolate the causal organism, leaves and stems were cut into sections. Each section included some partial lesion and adjacent asymptomatic tissues. They were surface-disinfested in 1% sodium hypochlorite for 60 s, rinsed three times with sterile water, and placed on V8 juice agar (V8A) at 25°C. After 3 days, 10 isolates were obtained and purified by single-zoospore method. These isolates were identified to species level by sequencing the rRNA internal transcribed spacer (ITS) region. Four representative isolates had an identical ITS sequence (GenBank Accession No. KF750568), which had 99% homology with Phytophthora capsici sequences in GenBank. In addition, all recovered isolates were identical in morphological characteristics. They produced caducous, papillate, and ovoid to ellipsoid sporangia (Length × width = 46.2 ± 7.7 × 23.6 ± 11.3 μm), often with a tapered base. The average length of pedicels was 33.3 ± 4.5 μm. All isolates are A2 mating type. They produced gametangia when paired with an A1 tester of P. capsici isolated from pepper on V8A. Plerotic oospores were 25.3 ± 2.1 μm in diameter. Amphigynous antheridia were 13.6 ± 2.8 μm long and 11.2 ± 0.9 μm wide. Oogonia were 27.4 ± 3.2 μm in diameter. To determine the pathogenicity, three 3-year-old potted C. reticulata cv. Nian Ju plants were sprayed with 20 ml of zoospore suspension from one representative isolate at 105 per ml. Two control plants were sprayed with 20 ml distilled water. All plants were then maintained at 90% relative humidity at 25°C with a 12-h photoperiod. Symptoms similar to those observed in the nurseries developed on all inoculated plants but not on any control plants after 10 days. The pathogenicity test was repeated once and similar results were obtained. P. capsici was recovered from all inoculated plants and resultant isolates had identical morphology to that of the isolates used for inoculation. P. capsici has a relatively broad host range including pumpkins, cucumbers, peppers, beans, squashes, and spinach (1,2). To our knowledge, this is the first report of foliar blight of C. reticulata cv. Nian Ju caused by P. capsici. This study indicates that P. capsici is potentially an important pathogen of C. reticulata cv. Nian Ju plants and further investigations into its epidemiology and development of site-specific integrated management programs for this new disease are warranted.

Successful Agrobacterium mediated transformation of Thielaviopsis basicola by optimizing multiple conditions

Thielaviopsis basicola is a hemibiotrophic root pathogen causing black root rot in a wide range of economically important crops. Our initial attempts to transform T. basicola using standard Agrobacterium tumefaciens–mediated transformation (ATMT) protocols were unsuccessful. Successful transformation required the addition of V8 juice (to induce germination of T. basicola chlamydospores) and higher concentrations of acetosyringone in the co-cultivation medium, and of chlamydospores/endoconidia, A. tumefaciens cells during co-cultivation. With these modifications, two T. basicola strains were successfully transformed with the green (egfp) or red (AsRed) fluorescent protein genes. Chlamydospores/endoconidia transformed with the egfp gene exhibited strong green fluorescence, but their fluorescence became weaker as the germ tubes emerged. Transformants harbouring the AsRed gene displayed strong red fluorescence in both chlamydospores/endoconidia and germ tubes. Fluorescent microscopic observations of an AsRed-labelled strain colonizing roots of transgenic Nicotiana benthamiana plants, which express the actin filaments labelled with EGFP, at 24 hours post inoculation showed varying levels of fungal germination and penetration. At this stage, the infection appeared to be biotrophic with the EGFP-labelled host actin filaments not being visibly degraded, even in host root cells in close contact with the hyphae. This is the first report of ATMT of T. basicola, and the use of an AsRed-labelled strain to directly observe the root infection process.