First Report of Phomopsis Stem Canker of Sunflower (Helianthus annuus) Caused by Diaporthe gulyae in Canada

During September 2012, Phomopsis stem canker was observed on sunflowers (Helianthus annuus L.) in a production field during seed filling with an average incidence of 15% in Morden, Manitoba (approximately 49°11′N and 98°09′W). The infected plants had elongated, brown-black lesions surrounding the leaf petiole, with numerous pycnidia, pith damage, and mid-stem lodging. Twenty sunflower plants were randomly sampled from the field. Isolations were made from the margins of the necrotic stems lesions by plating small pieces (5 mm) on potato dextrose agar (PDA) amended with 0.02% streptomycin sulfate. Plates were incubated at 25°C for 14 days under a 12-h photoperiod, and hyphal tips of white to grey colonies were transferred to PDA. Five isolates producing black pycnidia (occasionally with ostiolate beaks) and alpha conidia were tentatively identified as a Diaporthe sp. Alpha conidia were ellipsoidal, hyaline, and 6.5 to 8.5 × 2.5 to 3.5 μm. DNA was extracted from the mycelium of five isolates, and the ITS region was amplified and sequenced using primers ITS5 and ITS4 (4). BLASTn analysis of the 600-bp fragment (GenBank Accession Nos. KM391960 to KM391964) showed that the best match was Phomopsis sp. AJY-2011a strain T12505G (Diaporthe gulyae R.G. Shivas, S.M. Thompson & A.J. Young [3], Accession No. JF431299) from H. annuus with identities = 540/540 (100%) and gaps = 0/540 (0%). The five D. gulyae isolates were tested for pathogenicity on a sunflower confection inbred cv. HA 288 using the stem-wound method (2). Four-week-old sunflower plants (10 plants per isolate) were inoculated by wounding the stems on the second internode with a micropipette tip and placing a Diaporthe-infested mycelial plug on the wound. All plugs were attached to the wound with Parafilm. The pots were placed on the greenhouse benches at 25°C under a 16-h light/dark cycle. At 3 days after inoculation, dark brown lesions were observed on the stems extending upward from the inoculation site. Stem and leaves wilted, causing plant death 14 days after inoculation. Disease severity was calculated as a percentage of stem lesion (lesion length/stem length × 100%) at 14 days after inoculation. Significant differences (P ≤ 0.05) in disease severity were observed among D. gulyae isolates, which ranged from 34.9 to 100.0% (n = 5). Ten control plants similarly treated with sterile PDA plugs did not display symptoms. To complete Koch's postulates, D. gulyae was re-isolated from the inoculated stems, and the pathogen's identity was confirmed via sequencing of the ITS regions using primers ITS5 and ITS4 (4). The pathogen was not isolated from the control plants. D. gulyae was first reported as a pathogen on H. annuus in Australia and United States in 2011 (1,3). The pathogen was determined to be as or more aggressive than the other causal agents of Phomopsis stem canker (2,3), and its identification in both countries was circumstantially associated with increased incidence and yield loss in commercial production fields (1,3). In Canada, Phomopsis stem canker has been observed in sunflower fields over the last 10 years at low incidences, especially in years with above-normal temperatures during the sunflower growing season; however, the causal agent was not confirmed. To the best of our knowledge, this is the first report of D. gulyae causing Phomopsis stem canker on sunflowers in Canada. Since there is currently no known resistance to D. gulyae in sunflower, this newly discovered pathogen may become a threat to sunflower production in Canada. References: (1) F. Mathew et al. Phytopathology 101:S115, 2011. (2) F. Mathew et al. Phytopathology 103:S2.91, 2013. (3) S. M. Thompson et al. Persoonia 27:80, 2011. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

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First Report of Diaporthe australafricana Associated with Stem Canker on Blueberry in Chile

Plant Disease ◽

10.1094/pdis-12-11-1025-pdn ◽

2012 ◽

Vol 96 (5) ◽

pp. 768-768 ◽

Cited By ~ 3

Author(s):

B. A. Latorre ◽

K. Elfar ◽

J. G. Espinoza ◽

R. Torres ◽

G. A. Díaz

Keyword(s):

Vitis Vinifera ◽

Vascular Tissue ◽

Its Region ◽

Pathogenic Fungi ◽

Stem Canker ◽

Vaccinium Corymbosum ◽

Academic Press ◽

First Report ◽

Potted Plants ◽

Blastn Analysis

Stem cankers of blueberry (Vaccinium corymbosum L.) have been observed on as much as 15% of the plants in plantations in central and southern Chile since 2006. Symptoms consisted of apical necrosis of the shoots and brown-to-reddish necrotic lesions on the stems. Internally, a brown-to-reddish discoloration of the vascular tissue can be observed. Twenty, single-plant samples were collected in 12 blueberry plantings (approximately 33°27′ to 40°53′S). Isolations from the margins of the necrotic lesions on the stems were made by plating small pieces (5 mm) on potato dextrose agar acidified with 0.5 μl/ml of 92% lactic acid (APDA). The plates were incubated at 20°C for 5 to 7 days, and hyphal tips of white colonies with septate and hyaline mycelium were transferred to APDA. Colonies were then transferred to autoclaved Pinus radiata needles on 2% water agar and incubated for 20 days at 20°C. Twelve isolates producing black pycnidia and alpha conidia were tentatively identified as a Phomopsis sp. (teleomoph Diaporthe Nitschke). Other fungi, including Botryosphaeriaceae spp. and Pestalotiopsis spp., were also isolated. Alpha conidia were smooth, unicellular, hyaline, fusoid, biguttulate, and 6.4 to 7.9 × 2.3 to 3.3 μm (n = 20). Beta conidia were not observed. The internal transcribed spacer (ITS) region of the rDNA was amplified using primers ITS1 and ITS2 (4) and sequenced. BLASTn analysis of the 473-bp fragment (GenBank Accession No. JQ045712) showed 100% identity to Diaporthe australafricana Crous & J.M. van Niekerk from Vitis vinifera (3). The pathogenicity of D. australafricana was studied on blueberry cv. O'Neal using detached stems (n = 4) in the laboratory, on 2-year-old potted plants (n = 4) in a greenhouse, and on attached stems of mature plants (n = 4) established in the ground. Inoculations were done by placing mycelial plugs taken from 7-day-old APDA cultures in a 7-mm long incision made on the stems. Inoculations with sterile mycelium plugs served as negative controls. Inoculation sites were wrapped with Parafilm to avoid rapid dehydration. Dark brown, necrotic lesions on the internal tissues were obtained on all inoculated stems 15 days after inoculation. Mean lesion lengths were 18.0 ± 7.4 mm on detached stems, 7.8 ± 6.9 mm on stems of 2-year-old plants, and 7.3 ± 2.5 mm on mature plants in the field. No symptoms developed on control stems. Reisolations were successful in 100% of the inoculated stems and D. australafricana was confirmed by the presence of pycnidia and alpha conidia. To our knowledge, this is the first report of D. australafricana causing stem canker in V. corymbosum. Previously, this pathogen has been reported to be affecting Vitis vinifera in Australia and South Africa (3). These results do not exclude that other plant-pathogenic fungi may be involved in this syndrome (1,2). References: (1) J. G. Espinoza et al. Plant Dis 92:1407, 2008. (2) J. G. Espinoza et al. Plant Dis. 93:1187, 2009. (3) J. M. van Niekerk et al. Australas. Plant Pathol. 34:27, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, NY, 1990.

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First Report of Phomopsis Stem Canker of Sunflower in Illinois Caused by Phomopsis helianthi

Plant Disease ◽

10.1094/pdis-93-7-0760a ◽

2009 ◽

Vol 93 (7) ◽

pp. 760-760 ◽

Cited By ~ 2

Author(s):

M. D. Meyer ◽

G. R. Zhang ◽

D. K. Pedersen ◽

C. A. Bradley

Keyword(s):

Pcr Amplification ◽

Its Region ◽

Stem Canker ◽

Small Subunit ◽

The State ◽

Lesion Length ◽

Growth Stages ◽

First Report ◽

Small Subunit Rdna ◽

Sunflower Production

Stem cankers were observed on confection sunflower (Helianthus annuus) plants growing in a field in Champaign County, Illinois in August 2008. Lesions were brown to reddish brown, elongated (approximately 10 to 15 cm long), and centered over the area where leaf petioles connected to the stems. Stem tissues underneath the lesions were degraded. Lesions from diseased stems were cut into 5- to 7-mm pieces and immersed in a 0.5% NaOCl solution for 1 min, rinsed with sterilized distilled water, and placed into petri dishes containing acidified potato dextrose agar (APDA; 4 ml of 25% lactic acid per liter). Fungal colonies that grew from the stem lesion pieces on APDA were white, floccose, and dense with dark colored substrate mycelia. On the basis of the symptoms on sunflower plants and the growth characteristics on APDA, the fungus was tentatively identified as Phomopsis helianthi (1). To confirm the identity of the fungus, PCR amplification of the small subunit rDNA and internal transcribed spacer (ITS) region with primers EF3RCNL and ITS4 was done (2). The PCR product was sequenced with these primers at the Keck Biotechnology Center at the University of Illinois, Urbana. The resulting nucleotide sequence was compared with small subunit rDNA and ITS sequences deposited in the nucleotide database ( http://www.ncbi.nlm.nih.gov ) and showed highest homology to sequences of Diaporthe helianthi, teleomorph of P. helianthi. To confirm pathogenicity of the fungus, sunflower plants (cv. Cargill 270) were grown in the greenhouse and inoculated with the isolated fungus. The stems of sunflower plants between the V2 and V4 growth stages (3) were excised just below the uppermost node. Mycelia plugs of the fungus were placed into the large end of disposable micropipette tips (200 μl). The micropipette tip containing the fungus was subsequently placed over a cut sunflower stem. The fungal isolate was used to inoculate five stems. To serve as controls, five cut sunflower stems were inoculated with micropipette tips containing plugs of noninfested PDA and five cut stems were not inoculated. Mean lesion length on the stem was measured from the inoculated tip toward the soil line 7 days after inoculation. The experiment was replicated over time. Mean lesion length over both replications averaged 24 mm on the fungus-inoculated plants, 2 mm on the noninfested PDA-inoculated control plants, and no lesions were present on the noninoculated control plants. The fungus was reisolated on PDA from the inoculated plants in the greenhouse. To our knowledge, this is the first report of P. helianthi causing a stem canker of sunflower in Illinois. Although commercial sunflower production in Illinois is currently limited, it is being evaluated as a potential crop to follow winter wheat in portions of the state. If sunflower production were to increase in the state, growers may have to monitor for and manage Phomopsis stem canker. References: (1) T. Gulya et al. Sunflower diseases. Page 263 in: Sunflower Technology and Production. American Society of Agronomy, Madison, WI, 1997. (2) N. S. Lord et al. FEMS Microbiol. Ecol. 42:327, 2002. (3) A. A. Schneiter and J. F. Miller, Crop Sci. 21:901, 1981.

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First Report of Sclerotinia sclerotiorum on Calceolaria integrifolia in Italy

Plant Disease ◽

10.1094/pdis-92-7-1133c ◽

2008 ◽

Vol 92 (7) ◽

pp. 1133-1133

Author(s):

A. Garibaldi ◽

A. Minuto ◽

M. L. Gullino

Keyword(s):

Sclerotinia Sclerotiorum ◽

Soil Surface ◽

Its Region ◽

The United States ◽

First Report ◽

Potted Plants ◽

Initial Symptoms ◽

Mycelial Plug ◽

Blastn Analysis ◽

White Mycelium

Calceolaria integrifolia L. is an ornamental species grown as a potted plant in Liguria, northern Italy. In the winter of 2006, extensive chlorosis was observed on approximately 10% of the 10-month-old potted plants in a commercial greenhouse. Initial symptoms included stem necrosis and darkening of leaves. As stem and foliar necrosis progressed, infected plants wilted and died. Wilt occurred on young plants within a few days after the initial appearance of symptoms. Infected plants were characterized by the presence of soft, watery tissues that became covered with white mycelium and dark sclerotia. The diseased stem tissue was surface sterilized for 1 min in 1% NaOCl and plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate. Sclerotinia sclerotiorum (Lib.) de Bary (3) was consistently recovered from infected stem pieces. Sclerotia observed on infected plants measured 0.7 to 1.0 × 2.8 to 4.4 mm (average 1.6 to 2.1 mm). Sclerotia produced on PDA measured 1.0 to 1.1 × 3.0 to 4.2 mm (average 1.7 to 2.3 mm). The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 522-bp amplicon resulted in 100% homology with the sequence of S. sclerotiorum. The nucleotide sequence has been assigned GenBank Accession No. EU 627004. Pathogenicity of two isolates obtained from infected plants was confirmed by inoculating 10 120-day-old plants grown in individual 14-cm-diameter pots maintained in a greenhouse under partial shade. Inoculum consisted of 1 cm2 of mycelial plugs excised from a 10-day-old PDA culture of each isolate. Plants were inoculated by placing a mycelial plug on the soil surface around the base of each plant. Ten plants were inoculated per isolate and an equal number of noninoculated plants served as controls. The trial was repeated once. All plants were kept at temperatures ranging between 8 and 17°C (average 12.5°C) and watered as needed. All inoculated plants developed leaf yellowing within 8 days after inoculation, soon followed by the appearance of white mycelium and sclerotia, and then by wilt. Control plants remained symptomless. S. sclerotiorum was reisolated from the stems of inoculated plants. S. sclerotiorum was reported previously on a Calceolaria sp. in the United States (2). To our knowledge, this is the first report of white mold on C. integrifolia in Italy. The economic importance of this disease is currently limited. References (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) Anonymous. USDA Agric. Handb. 165:441, 1960. (3) N. F. Buchwald. Den. Kgl. Veterin.er-og Landbohojskoles Aarsskrift 75, 1949.

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First Report of Potato Stem Canker Caused by Binucleate Rhizoctonia AG-A in Jilin Province, China

Plant Disease ◽

10.1094/pdis-10-12-0912-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1246-1246 ◽

Cited By ~ 6

Author(s):

Y. G. Yang ◽

X. H. Wu

Keyword(s):

Solanum Tuberosum L ◽

Its Region ◽

Stem Canker ◽

Jilin Province ◽

Molecular Characteristics ◽

Potato Seed ◽

First Report ◽

Binucleate Rhizoctonia ◽

Blastn Analysis ◽

Wheat Seeds

Potato (Solanum tuberosum L.) stem canker caused by Rhizoctonia solani occurs in potato-growing regions all over the world and can result in severe losses in crop yield and quality. In late July 2011, potato subterraneous stems with stem cankers composed of brownish, sunken lesions were observed at 15% incidence in seven sites in Jilin Province, northeast China. Samples were collected, and stem pieces (each 5 mm long) taken from the margins of the healthy and diseased tissues were surface-disinfested with 0.5% NaOCl for 2 min, rinsed with sterilized water, dried, then placed on potato dextrose agar at 25°C in the dark. Three (designated JL-3, JL-5-1, and JL-6) of seven Rhizoctonia isolates that developed from single hyphal tip transfers were identified preliminarily as binucleate Rhizoctonia (BNR) isolates (teleomorph Ceratobasidium Rogers). The colonies were white or light gray with fluffy aerial hyphae and no sclerotia after 14 days in culture. Hyphal cells were binucleate when stained with 4′-6-diamidino-2-phenylindole. Average hyphal diameters (mean ± standard deviation) of isolates JL-3, JL-5-1, and JL-6 were 4.8 ± 0.5 μm (range 4.1 to 5.6 μm), 4.4 ± 0.4 μm (range 3.9 to 5.2 μm), and 4.5 ± 0.3 μm (range 4.0 to 5.0 μm), respectively. The internal transcribed spacer (ITS) region of ribosomal DNA was amplified from genomic DNA with primers ITS1 and ITS4 and sequenced. BLASTn analysis indicated that the resulting sequences (GenBank Accession Nos. JX885459, JX885460, and JX885461 for JL-3, JL-5-1, and JL-6, respectively) were 100% identical to that of a Ceratobasidium sp. AG-A isolate CHR08-10 (HQ270171). So the three isolates were identified as BNR AG-A based on morphological and molecular characteristics. To determine pathogenicity of the BNR isolates, potato seed tubers (cv. Favorita), each with 3- to 5-mm-long sprouts, were inoculated with wheat seeds (sterilized by autoclaving twice at 121°C for 1 h with a 24-h interval between autoclavings) colonized with each isolate (1). One sprouted potato tuber was planted in a plastic pot with a single colonized wheat seed placed 10 mm above the uppermost sprout tip in a sand/sawdust mixture (1:2 v/v). Plants were incubated in a glasshouse at 25 to 27°C, and assessed after 21 days. The test was performed on 20 plants/isolate and the experiment was repeated. The incidence of plants inoculated with JL-3, JL-5-1, and JL-6 that developed stem canker symptoms averaged 11.1, 23.5, and 28.6%, respectively, whereas all control plants inoculated with sterilized wheat seeds remained asymptomatic. Rhizoctonia spp. were not reisolated from the control plants, whereas BNR isolates were reisolated consistently from symptomatic stems of the inoculated plants, and the identity confirmed by morphological and molecular characteristics as described above, fulfilling Koch's postulates. BNR AG-A has been reported to be pathogenic on soybean (Glycine max), pea (Pisum sativum), snap bean (Phaseolus vulgaris), and pak choi (Brassica chinensis) in China (4). Isolates of R. solani AG-3 are most often associated with potato stem canker (2), although unidentified BNR isolates were reported to cause mild symptoms on potato sprouts in Finland (1), and small lesions on potato roots and stems in the United Kingdom (3). To our knowledge, this is the first report of BNR AG-A causing potato stem canker in Jilin Province, one of the main potato-producing areas of China. References: (1) M. J. Lehtonen et al. Plant Pathol. 57:141, 2008. (2) L. Tsror. J. Phytopatology 158:649, 2010. (3) J. W. Woodhall et al. New Dis. Rep. 23:31, 2011. (4) G. H. Yang et al. J. Phytopathology 153:333, 2005.

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First Report of Leaf Blight and Stem Canker of Pachysandra terminalis Caused by Pseudonectria pachysandricola in Korea

Plant Disease ◽

10.1094/pdis-09-11-0813 ◽

2012 ◽

Vol 96 (2) ◽

pp. 287-287

Author(s):

K. S. Han ◽

J. H. Park ◽

S. E. Cho ◽

H. D. Shin

Keyword(s):

United States ◽

Its Region ◽

Stem Canker ◽

The United States ◽

Culture Collection ◽

Leaf Blight ◽

First Report ◽

Cornell University ◽

Plastic Bags ◽

Young Leaves

Pachysandra terminalis Siebold & Zucc., known as Japanese pachysandra, is a creeping evergreen perennial belonging to the family Buxaceae. In April 2011, hundreds of plants showing symptoms of leaf blight and stem canker with nearly 100% incidence were found in a private garden in Suwon, Korea. Plants with the same symptoms were found in Seoul in May and Hongcheon in August. Affected leaves contained tan-to-yellow brown blotches. Stem and stolon cankers first appeared as water soaked and developed into necrotic lesions. Sporodochia were solitary, erumpent, circular, 50 to 150 μm in diameter, salmon-colored, pink-orange when wet, and with or without setae. Setae were hyaline, acicular, 60 to 100 μm long, and had a base that was 4 to 6 μm wide. Conidiophores were in a dense fascicle, not branched, hyaline, aseptate or uniseptate, and 8 to 20 × 2 to 3.5 μm. Conidia were long, ellipsoid to cylindric, fusiform, rounded at the apex, subtruncate at the base, straight to slightly bent, guttulate, hyaline, aseptate, 11 to 26 × 2.5 to 4.0 μm. A single-conidial isolate formed cream-colored colonies that turned into salmon-colored colonies on potato dextrose agar (PDA). Morphological and cultural characteristics of the fungus were consistent with previous reports of Pseudonectria pachysandricola B.O. Dodge (1,3,4). Voucher specimens were housed at Korea University (KUS). Two isolates, KACC46110 (ex KUS-F25663) and KACC46111 (ex KUS-F25683), were accessioned in the Korean Agricultural Culture Collection. Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced using ABI Prism 337 automatic DNA sequencer (Applied Biosystems, Foster, CA). The resulting sequence of 487 bp was deposited in GenBank (Accession No. JN797821). This showed 100% similarity with a sequence of P. pachysandricola from the United States (HQ897807). Isolate KACC46110 was used in pathogenicity tests. Inoculum was prepared by harvesting conidia from 2-week-old cultures on PDA. Ten young leaves wounded with needles were sprayed with conidial suspensions (~1 × 106 conidia/ml). Ten young leaves that served as the control were treated with sterile distilled water. Plants were covered with plastic bags to maintain a relative humidity of 100% at 25 ± 2°C for 24 h. Typical symptoms of brown spots appeared on the inoculated leaves 4 days after inoculation and were identical to the ones observed in the field. P. pachysandricola was reisolated from 10 symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in the United States, Britain, Japan, and the Czech Republic (2,3), but not in Korea. To our knowledge, this is the first report of P. pachysandricola on Pachysandra terminalis in Korea. Since this plant is popular and widely planted in Korea, this disease could cause significant damage to nurseries and the landscape. References: (1) B. O. Dodge. Mycologia 36:532, 1944. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , September 24, 2011. (3) I. Safrankova. Plant Prot. Sci. 43:10, 2007. (4) W. A. Sinclair and H. H. Lyon. Disease of Trees and Shrubs. 2nd ed. Cornell University Press, Ithaca, NY, 2005.

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First Report of Stem Canker Disease of Pitaya (Hylocereus undatus and H. polyrhizus) Caused by Neoscytalidium dimidiatum in Taiwan

Plant Disease ◽

10.1094/pdis-08-11-0689-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 906-906 ◽

Cited By ~ 18

Author(s):

M. F. Chuang ◽

H. F. Ni ◽

H. R. Yang ◽

S. L. Shu ◽

S. Y. Lai ◽

...

Keyword(s):

New York ◽

Aerial Mycelium ◽

Its Region ◽

Stem Canker ◽

Canker Disease ◽

First Report ◽

Neoscytalidium Dimidiatum ◽

Succulent Plant ◽

Sterile Medium ◽

Hylocereus Undatus

Pitaya (Hylocereus undatus and H. polyrhizus Britt. & Rose), a perennial succulent plant grown in the tropics, is becoming an emerging and important fruit plant in Taiwan. In September of 2009 and 2010, a number of pitaya plants were found to have a distinctive canker on stems. The disease expanded quickly to most commercial planting areas in Taiwan (e.g., Pintung, Chiayi, and Chunghua). Symptoms on the stem were small, circular, sunken, orange spots that developed into cankers. Pycnidia were erumpent from the surface of the cankers and the stems subsequently rotted. After surface disinfestation with 0.1% sodium hypochloride, tissues adjacent to cankers were placed on acidified potato dextrose agar (PDA) and incubated at room temperature for 1 week, after which colonies with dark gray-to-black aerial mycelium grew. Hyphae were branched, septate, and brown and disarticulated into 0- to 1-septate arthrospores. Sporulation was induced by culturing on sterile horsetail tree (Casuarina equisetifolia) leaves. Conidia (12.79 ± 0.72 × 5.14 ± 0.30 μm) from pycnidia were one-celled, hyaline, and ovate. The internal transcribed spacer (ITS) region of ribosomal DNA was PCR amplified with primers ITS1 and ITS4 (2) and sequenced. The sequence (GenBank Accession No. HQ439174) showed 99% identity to Neoscytalidium dimidiatum (Penz.) Crous & Slippers (GenBank Accession No. GQ330903). On the basis of morphology and nucleotide-sequence identity, the isolates were identified as N. dimidiatum (1). Pathogenicity tests were conducted in two replicates by inoculating six surface-sterilized detached stems of pitaya with either mycelium or conidia. Mycelial plugs from 2-day-old cultures (incubated at 25°C under near UV) were inoculated to the detached stems after wounding with a sterile needle. Conidial suspensions (103 conidia/ml in 200 μl) were inoculated to nonwounded stems. Noninoculated controls were treated with sterile medium or water. Stems were then incubated in a plastic box at 100% relative humidity and darkness at 30°C for 2 days. The symptoms described above were observed on inoculated stems at 6 to 14 days postinoculation, whereas control stems did not develop any symptoms. N. dimidiatum was reisolated from symptomatic tissues. To our knowledge, this is the first report of N. dimidiatum causing stem canker of pitaya. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

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First Report of Phytophthora citrophthora on Penstemon barbatus in Italy

Plant Disease ◽

10.1094/pd-90-1260a ◽

2006 ◽

Vol 90 (9) ◽

pp. 1260-1260 ◽

Cited By ~ 1

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

D. Minerdi ◽

M. L. Gullino

Keyword(s):

Its Region ◽

The United States ◽

Phytophthora Species ◽

Crown Rot ◽

Pathogenicity Test ◽

Phytophthora Citrophthora ◽

First Report ◽

Blastn Analysis ◽

Root And Crown Rot ◽

Pathogenicity Tests

Penstemon barbatus (Cav.) Roth (synonym Chelone barbata), used in parks and gardens and sometimes grown in pots, is a plant belonging to the Scrophulariaceae family. During the summers of 2004 and 2005, symptoms of a root rot were observed in some private gardens located in Biella Province (northern Italy). The first symptoms resulted in stunting, leaf discoloration followed by wilt, root and crown rot, and eventually, plant death. The diseased tissue was disinfested for 1 min in 1% NaOCl and plated on a semiselective medium for Oomycetes (4). The microorganism consistently isolated from infected tissues, grown on V8 agar at 22°C, produced hyphae with a diameter ranging from 4.7 to 5.2 μm. Sporangia were papillate, hyaline, measuring 43.3 to 54.4 × 26.7 to 27.7 μm (average 47.8 × 27.4 μm). The papilla measured from 8.8 to 10.9 μm. These characteristics were indicative of a Phytophthora species. The ITS region (internal transcribed spacer) of rDNA was amplified using primers ITS4/ITS6 (3) and sequenced. BLASTn analysis (1) of the 800 bp obtained showed a 100% homology with Phytophthora citrophthora (R. & E. Sm.) Leonian. The nucleotide sequence has been assigned GenBank Accession No. DQ384611. For pathogenicity tests, the inoculum of P. citrophthora was prepared by growing the pathogen on autoclaved wheat and hemp kernels (2:1) at 25°C for 20 days. Healthy plants of P. barbatus cv. Nano Rondo, 6 months old, were grown in 3-liter pots (one plant per pot) using a steam disinfested substrate (peat/pomix/pine bark/clay 5:2:2:1) in which 200 g of kernels per liter of substrate were mixed. Noninoculated plants served as control treatments. Three replicates were used. Plants were maintained at 15 to 20°C in a glasshouse. The first symptoms, similar to those observed in the gardens, developed 21 days after inoculation, and P. citrophthora was consistently reisolated from infected plants. Noninoculated plants remained healthy. The pathogenicity test was carried out twice with similar results. A nonspecified root and crown rot of Penstemon spp. has been reported in the United States. (2). To our knowledge, this is the first report of P. citrophthora on P. barbatus in Italy as well as in Europe. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997 (2) F. E. Brooks and D. M. Ferrin. Plant Dis. 79:212, 1995. (3) D. E. L. Cooke and J. M. Duncan. Mycol. Res. 101:667, 1997. (4) H. Masago et al. Phytopathology 67:425, 1977.

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First Report of Powdery Mildew Caused by Podosphaera xanthii on Calendula officinalis in Italy

Plant Disease ◽

10.1094/pdis-92-1-0174c ◽

2008 ◽

Vol 92 (1) ◽

pp. 174-174 ◽

Cited By ~ 1

Author(s):

A. Garibaldi ◽

G. Gilardi ◽

M. L. Gullino

Keyword(s):

Powdery Mildew ◽

Its Region ◽

Pathogenicity Test ◽

Podosphaera Xanthii ◽

Calendula Officinalis ◽

First Report ◽

Potted Plants ◽

Blastn Analysis ◽

Voucher Specimens ◽

Pot Marigold

Calendula officinalis L. (Asteraceae) (pot marigold or English marigold) is an ornamental species grown in gardens and as potted plants for the production of cut flower. It was also used in ancient Greek, Roman, Arabic, and Indian cultures as a medicinal herb as well as a dye for fabrics, foods, and cosmetics. During the summer of 2007, severe outbreaks of a previously unknown powdery mildew were observed on plants in several gardens near Biella (northern Italy). Both surfaces of leaves of infected plants were covered with dense, white mycelia and conidia. As the disease progressed, infected leaves turned yellow and died. Mycelia and conidia also were observed on stems and flower calyxes. Conidia were hyaline, ellipsoid, born in short chains (four to six conidia per chain), and measured 27.0 to 32.1 (31.4) × 12.9 to 18.4 (18.2) μm. Conidiophores measured 49 to 77.3 (67.2) × 8 to 13.3 (10.8) μm and showed a foot cell measuring 44 to 59 (51.9) × 9.3 to 12.6 (11.3) μm followed by one shorter cell measuring 15.6 to 18.9 (17.6) × 10.4 to 13.6 (12.2) μm. Fibrosin bodies were present. Chasmothecia were spherical, amber colored, with a diameter of 89 to 100 (94.5) μm. Each chasmothecium contained one ascus with eight ascospores. On the basis of its morphology, the causal agent was determined to be a Podosphaera sp. (2). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 588 bp showed a 100% homology with the sequence of Podosphaera xanthii (2). The nucleotide sequence has been assigned GenBank Accession No. EU100973. Pathogenicity was confirmed through inoculations by gently pressing diseased leaves onto leaves of healthy C. officinalis plants. Five plants were inoculated. Five noninoculated plants served as control. Plants were maintained in a greenhouse at temperatures ranging from 20 to 26°C. Eleven days after inoculation, typical symptoms of powdery mildew developed on inoculated plants. Noninoculated plants did not show symptoms. The pathogenicity test was carried out twice. To our knowledge, this is the first report of powdery mildew on C. officinalis in Italy. C. officinalis was previously described as a host to Sphaerotheca fuliginea (synonym S. fusca) in Great Britain (4) as well as in Romania (3). Voucher specimens are available at the AGROINNOVA Collection, University of Torino. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000. (3) E. Eliade. Rev. Appl. Mycol. 39:710, 1960. (4) F. J. Moore. Rev. Appl. Mycol. 32:380, 1953.

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First Report of Powdery Mildew Caused by Golovinomyces cichoracearum on English Daisy (Bellis perennis) in Italy

Plant Disease ◽

10.1094/pdis-92-3-0484a ◽

2008 ◽

Vol 92 (3) ◽

pp. 484-484 ◽

Cited By ~ 1

Author(s):

A. Garibaldi ◽

A. Minuto ◽

M. L. Gullino

Keyword(s):

Powdery Mildew ◽

Its Region ◽

Northern Italy ◽

Flowering Plant ◽

Pathogenicity Test ◽

First Report ◽

Golovinomyces Cichoracearum ◽

Blastn Analysis ◽

Commercial Farms ◽

Voucher Specimens

Bellis perennis (English daisy) is a flowering plant belonging to the Asteraceae and is increasingly grown as a potted plant in Liguria (northern Italy). In February 2007, severe outbreaks of a previously unknown powdery mildew were observed on plants in commercial farms at Albenga (northern Italy). Both surfaces of leaves of affected plants were covered with white mycelia and conidia. As the disease progressed, infected leaves turned yellow. Mycelia and conidia also were observed on stems and flower calyxes. Conidia were hyaline, ellipsoid, borne in chains (as many as three conidia per chain), and measured 27.7 × 16.9 (15.0 to 45.0 × 10.0 to 30.0) μm. Conidiophores measured 114.0 × 12.0 (109.0 to 117.0 × 11.0 to 13.0) μm and showed a foot cell measuring 78.0 × 11.0 (72.0 to 80.0 × 11.0 to 12.0) μm followed by two shorter cells. Fibrosin bodies were absent. Chasmothecia were not observed in the collected samples. The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 415 bp obtained showed an E-value of 7e–155 with Golovinomyces cichoracearum (3). The nucleotide sequence has been assigned the GenBank Accession No. AB077627.1 Pathogenicity was confirmed through inoculations by gently pressing diseased leaves onto leaves of healthy B. perennis plants. Twenty plants were inoculated. Fifteen noninoculated plants served as a control. Plants were maintained in a greenhouse at temperatures ranging from 10 to 30°C. Seven days after inoculation, typical symptoms of powdery mildew developed on inoculated plants. The fungus observed on inoculated plants was morphologically identical to that originally observed. Noninoculated plants did not show symptoms. The pathogenicity test was carried out twice. To our knowledge, this is the first report of powdery mildew on B. perennis in Italy. The disease was already reported in other European countries (2). Voucher specimens are available at the AGROINNOVA Collection, University of Torino. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) U. Braun The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena, Germany, 1995. (3) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000.

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First Report of Internal Black Rot Caused byNeoscytalidium dimidiatumonHylocereus undatus(Pitahaya) Fruit in Israel

Plant Disease ◽

10.1094/pdis-05-13-0535-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1513-1513 ◽

Cited By ~ 9

Author(s):

D. Ezra ◽

O. Liarzi ◽

T. Gat ◽

M. Hershcovich ◽

M. Dudai

Keyword(s):

Its Region ◽

Stem Canker ◽

Brown Spot ◽

Black Rot ◽

Post Inoculation ◽

Single Spore ◽

Flower Opening ◽

Disease Symptoms ◽

First Report ◽

Β Tubulin

Pitahaya (Hylocereus undatus [Haw.] Britton & Rose) was introduced to Israel in 1994, and is grown throughout the country. In the summer of 2009, fruit with internal black rot was collected from a field in central Israel. Symptomatic tissue from the black rot was placed on potato dextrose agar (PDA) plates amended with 12 μg/ml tetracycline and incubated at 25°C for 3 days. A dark, gray to black, fast-growing fungus was isolated from all samples (10 fruits). For identification, single-spore cultures were grown on PDA at 25°C for 5 days, and colonies with gray to black, wooly mycelium were formed. The mycelia were branched and septate (4 to 8 μm wide). The arthroconidia were dark brown, thick-walled, and one-celled, 6.3 to 14.2 × 2.0 to 4.5 μm (n = 5), and ovate to rectangular. Based on these characteristics, the fungus was identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (1). The internal transcribed spacer (ITS) region of rDNA and β-tubulin gene were amplified using ITS1 and ITS4, T121 (2), and Bt1b (3) primers, respectively, and then sequenced (GenBank Accessions KF000372 and KF020895, respectively). Both sequences were identical to sequences previously deposited in GenBank. The ITS (561 bp) and β-tubulin (488 bp) sequences exhibited 99% and 100% identity, and 100% and 84% coverage, respectively, to N. dimidiatum (JX524168 and FM211185, respectively). Thus, the results of the molecular identifications confirmed the morphological characterization. To establish fungal pathogenicity and the mechanism of infection, 60 flowers in a disease-free orchard were marked to form three different treatments (15 flowers per treatment): inoculations of the flower tube by inserting PDA plugs (0.5 × 0.5 cm) from a 5-day-old culture to the base of the flower, inoculations of the flower stigma by placing the fungus plug on intact, or pre-wounded flower stigma. The wounds were made by scratching the stigma with a sterile scalpel. For each treatment, five additional flowers were used as negative controls in which the PDA plugs did not contain any fungus. All flowers were hand-pollinated and left to grow for a month until the fruit had ripened. Only flowers inoculated by insertion of the fungus into the flower tube developed black rot in the fruit (8 of 15 fruit) 3 to 4 weeks post inoculation, suggesting involvement of the flower tube in the mechanism of infection. All other treatments and controls failed to develop any detectable disease symptoms. N. dimidiatum was reisolated from the rot, fulfilling Koch's postulates. Flowers with wounded stigma developed significantly smaller fruit. Interestingly, diseased fruit changed color about a week before ripening from the flower opening downwards, whereas healthy fruit changed color from the attachment point to the stem upwards. These results indicate that N. dimidiatum is the pathogen of pitahaya internal black rot disease. Recently, this pathogen was reported to cause brown spot disease and stem canker disease of pitahaya in China (4) and Taiwan (5), respectively. To date, the disease can be detected in all orchards in Israel, with up to 50% of the fruit being infected. Since the disease symptoms of the Israeli isolate are located in the fruit, the commercial loss due to pathogen attack is significant. To our knowledge, this is the first report of internal black rot caused by N. dimidiatum on pitahaya fruit in Israel.References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylo, Evol. 7:103, 1997. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microiol. 61:1323, 1995. (4) G. B. Lan and Z. F. He. Plant Dis. 96:1702, 2012. (5) M. F. Chuang et al. Plant Dis. 96:906, 2012.

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