First Report of Phytophthora pseudosyringae on Chestnut Nursery Stock in Spain

Phytophthora pseudosyringae causes stem necrosis and collar rot of deciduous tree species (Quercus spp., Fagus silvatica, and Alnus glutinosa) in several European countries (1,2). In November 2006, we received diseased Castanea sativa seedlings from a nursery in Galicia (northwest Spain). These plants had tongue-shaped necroses of the inner bark and cambium. Reddish, sunken lesions occurred on the surface of the bark, either in the stem base or higher on the stem. Tissue from the leading edge of the lesions was transferred to a selective V8 agar medium (4) and incubated for 7 days at 20°C in the dark. A Phytophthora sp. was isolated, transferred to cornmeal agar (CMA) and V8 agar, and incubated in the dark. Colonies were appressed with stellate to rosaceous growth patterns on CMA and stellate, limited aerial mycelium on V8 agar. Growth on V8 occurred from 2 to 25°C with an optimum at 20°C and a radial growth rate of 4.5 mm per day at 20°C. Chains of inflated spherical to deltoid hyphal swellings with radiating hyphae were abundantly produced in water (2). Chlamydospores were not observed on agar media. The deciduous, sympodial, semipapillate, rarely bipapillate sporangia with pedicels had a length/breadth average ratio of 1.55. Oogonia, antheridia, and oospores were produced within a single culture. Oogonia were spherical and smooth walled, antheridia were predominantly paraginous, but some were amphyginous, and oospores were plerotic that turned golden yellow with age (2). Internal transcribed spacer (ITS)-rDNA and mitochondrial DNA (mtDNA) regions were amplified by nested-PCR and sequenced with DNA extracted from mycelium. The amplicon sizes obtained were similar to those reported for P. pseudosyringae (2,3). DNA sequences showed 99 to 100% homology with those previously identified as P. pseudosyringae and deposited in GenBank. Pathogenicity of the isolate was confirmed by inoculating 10 C. sativa seedlings, as well as three detached leaves from each of another 10 young plants growing in containers. For the seedlings, one shallow cut was made into the bark on the main stem. A colonized agar plug was inserted beneath the flap that was sealed with Parafilm. Unwounded and wounded detached leaves of C. sativa were dipped into a zoospore aqueous suspension (1 × 105 zoospores ml–1) for 10 s., seedlings and leaves were incubated at 20°C and 95% humidity for 60 and 7 days, respectively. After 7 days, foliar lesions that developed exceeded 25 mm, and the pathogen was consistently reisolated. Leaves inoculated with sterile water did not develop symptoms. On inoculated seedlings, the external surface of the bark was reddish and sunken. Stem lesions progressed bidirectionally from the wound. P. pseudosyringae was recovered from inoculated seedlings but not from controls. On the basis of its unique combination of morphological and physiological characters, pathogenicity, and ITS and mtDNA sequences, the Phytophthora isolated from chestnut was identified as P. pseudosyringae. To our knowledge, this is the first report of P. pseudosyringae on C. sativa in Spain. References: (1) EPPO Reporting Service. Online publication. No. 10 2005/162, 2005. (2) T. Jung et al. Mycol. Res. 107:772, 2003. (3) F. N. Martin et al. Phytopathology 94:621, 2004. (4) C. Pintos Varela et al. Plant. Dis. 87:1396, 2003.

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First Report of Chestnut Blight Caused by Cryphonectria parasitica in a Chestnut Orchard in Andalusia (Southern Spain)

Plant Disease ◽

10.1094/pdis-07-13-0781-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 283-283 ◽

Cited By ~ 1

Author(s):

J. Bascón ◽

S. Castillo ◽

C. Borrero ◽

S. Orta ◽

A. Gata ◽

...

Keyword(s):

Dna Sequences ◽

Castanea Sativa ◽

Cryphonectria Parasitica ◽

Southern Spain ◽

Chestnut Blight ◽

Commonwealth Mycological Institute ◽

Species Identity ◽

First Report ◽

Sweet Chestnut ◽

Agar Plug

In Europe, chestnut blight caused by Cryphonectria parasitica (Murrill) Barr was first seen in Italy in 1938 (1). In Spain, the disease was first detected in Basque country in 1947 and later in other areas of northern Spain: Galicia, León, Navarra, and Catalonia, and in Trás-os-Montes in Portugal (2). In November 2012, in an orchard (2 ha) in Almonaster la Real (Huelva, Spain), approximately 20 cankered Castanea sativa (sweet chestnut) trees cv. Vazqueño, 40 to 50 years old, were observed. The trees were grafted 2 years before. In May and June 2013, six new disease focuses were detected near the first one. Five focuses were located in the same village and the other in Jabugo (a neighboring village). Diseased trees exhibited sunken cankers, cracked bark with mycelial fan spreads under the bark, and in some cases, orange fungal sporulation was visible on the bark. Samples were collected from two affected trees and symptom-bearing bark pieces were then placed in moist chambers at 20°C for up to 8 days to induce fungal sporulation. Cultures were made from spore masses extruding from the cankered bark and from the edge of necrotic lesions visible in the phloem of cankered bark tissue onto potato dextrose agar (PDA). Monoconidial fungal isolates were obtained from both trees. The morphological structure of two isolated fungi was identical to that described as C. parasitica (3). Species identity was confirmed by analysis of nucleotide sequences of the internal transcribed spacer (ITS) rDNA, using ITS1-ITS4 (4) as primer pairs, respectively. BLAST searches showed a high similarity between collected isolates' DNA sequences and C. parasitica sequences found on GenBank (96% coverage, 99% identity). Our isolates have been included in GenBank as KF220298 and KF220299. The pathogenicity assay of these two isolates was conducted using two cultivars of sweet chestnut (seedlings from Huelva and Granada nurseries). Isolate pathogenicity was tested on 3-year-old chestnut seedlings in a growth chamber at 25°C (day) and 20°C (night) with a 14-h photoperiod. The isolates were cultured on PDA at 25°C for 7 days. Stems were wounded at 10 cm height with a drill. Each isolate was inoculated to 25 replicates per cultivar by placing a mycelia agar plug (4 to 5 mm diameter) in the hole and wrapping the stem with Parafilm. Plants treated identically with sterile agar plugs were used as controls. Plants were then maintained at 100% relative humidity for 2 h. Both isolates induced diseases symptoms and death of seedlings of both cultivars at a mean time of 37.5 days after inoculation. No significant differences between isolates or between cultivars were detected. Twenty control plants similarly treated with sterile PDA discs did not display symptoms. C. parasitica was re-isolated from lesions, confirming Koch's postulates. Andalusia has 14,000 ha of chestnut crops with high commercial value due to their precocity. Dispersion of chestnut blight in this zone can reduce crop productivity. To our knowledge, this is the first report of C. parasitica causing chestnut blight in Andalusia (southern Spain), one of the few areas left in southwestern Europe free of chestnut blight. References: (1) A. Biraghi. Italia Agricola 7:1, 1946. (2) G. González-Varela et al. Eur. J. Plant Pathol. 131:67, 2011. (3) A. Sivanesan and P. Holliday. Cryphonectria parasitica. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 704, Set. 71. Commonwealth Mycological Institute, Kew, UK, 1981. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Amplifications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

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First Report of Dieback on Hybrid Rhododendrons Caused by Neofusicoccum luteum and N. parvum in Spain

Plant Disease ◽

10.1094/pdis-10-10-0723 ◽

2011 ◽

Vol 95 (2) ◽

pp. 221-221 ◽

Cited By ~ 6

Author(s):

C. Pintos Varela ◽

V. Redondo Fernández ◽

J. P. Mansilla Vázquez ◽

O. Aguín Casal

Keyword(s):

Dna Sequences ◽

Uv Light ◽

Morphological Characteristics ◽

Phytophthora Ramorum ◽

Malt Extract ◽

First Report ◽

Agar Plug ◽

Thin Walled ◽

Neofusicoccum Luteum

During the conducting of Phytophthora ramorum surveys at Galician public parks (northwestern Spain) in 2010, established Rhododendron spp. plants were observed to be exhibiting leaf spots and necrosis, shoot blight, and cankers and dieback of shoots and branches. Branches and leaves of affected rhododendrons contained pseudothecia with bitunicate asci and hyaline pseudoparaphyses, and pycnidia were observed within the same stromatic masses. Symptomatic samples were disinfested in 0.5% sodium hypochlorite for 3 min. Tissues were cut from the margin of lesions, placed onto malt extract agar amended with streptomycin (25 μg ml–1), and incubated at 25°C in the dark. Cultures displaying morphological characteristics associated with Botryosphaeriaceae species were subcultured on 2% water agar with sterilized Pinus pinaster needles as a substrate and incubated at 25°C under near-UV light to encourage pycnidial production (1). Single conidial cultures gave rise to two distinct colonies on potato dextrose agar (PDA) at 25°C. In type 1, isolates produced a sparse, aerial mycelium and a characteristic yellow pigment that was more intense after 3 days, thereafter becoming violaceous and gradually turning dark gray. Growth occurred in the range of 4 to 38°C with an optimum at 29°C. Conidia were hyaline, fusiform, aseptate, thin walled, and averaged 21.1 (14.3 to 25.0) × 5.7 (4.3 to 6.8) μm with a length/width (L/W) ratio of 3.7 ± 0.4 (n = 100). On the basis of these characteristics, isolates were identified as Neofusicoccum luteum (1,3). Colonies of type 2 produced a dense, white-to-yellowish mycelium that rapidly became gray followed by marked diurnal zonation. Mycelial growth occurred in the range of 6 to 38°C with an optimum at 29 to 30°C. Conidia were hyaline, elliptical or fusiform, aseptate, thin walled, and averaging 18.3 (14.1 to 20.7) × 5.8 (4.6 to 7.0) μm with a L/W ratio of 3.2 ± 0.4 (n = 100). These isolates were identified as N. parvum (1,2). Identity was confirmed by DNA sequences analysis of internal transcribed spacer (ITS) regions. Comparison of the sequences of type 1 and 2 showed 100% homology with N. luteum and N. parvum (GenBank Accession Nos. EU673311 and GU251146, respectively). Representative sequences were deposited at GenBank (Accession Nos. HQ197352 and HQ197351). Pathogenicity of each isolate of N. luteum and N. parvum was confirmed by inoculating four 3-year-old Rhododendron spp. seedlings grown in pots. Shallow cuts were made in three branches of each plant. A colonized 6-mm agar plug, removed from the margin of an actively growing colony, was inserted beneath the flap and sealed with Parafilm. Four control seedlings received only sterile PDA agar plugs. Plants were maintained at 26°C and 70% humidity for 21 days. Inoculated plants began showing symptoms after 3 days. Necrosis progressed quickly and bidirectionally from the wound, resulting in death of leaves and wilting of shoots. N. luteum and N. parvum were reisolated from all inoculated plants but not from the controls. To our knowledge, this is the first report of N. luteum and N. parvum on Rhododendron spp. in Spain. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) S. R. Pennycook et al. Mycotaxon 24:445, 1985. (3) A .J. L. Phillips et al. Sydowia 54:59, 2002.

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First Report of Grapevine Cankers Caused by Lasiodiplodia crassispora and Neofusicoccum mediterraneum in California

Plant Disease ◽

10.1094/pdis-94-6-0785b ◽

2010 ◽

Vol 94 (6) ◽

pp. 785-785 ◽

Cited By ~ 10

Author(s):

J. R. Úrbez-Torres ◽

F. Peduto ◽

W. D. Gubler

Keyword(s):

Dna Sequences ◽

Vascular Tissue ◽

Elongation Factor ◽

Fungal Culture ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Consensus Sequences ◽

Fast Growing ◽

Agar Plug ◽

Vascular Discoloration

Several species in the Botryosphaeriaceae family cause perennial cankers in the vascular tissue of grapevines and are responsible for the disease known as bot canker in California (3). Tissue from grapevine vascular cankers from samples submitted to our laboratory in the summer of 2009 were plated onto potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride. Lasiodiplodia crassispora (Burgess & Barber) and Neofusicoccum mediterraneum (Crous, M.J. Wingf. & A.J.L. Phillips) were identified based on morphological and cultural characters as well as analyses of nucleotide sequences. L. crassispora isolates were characterized by a fast-growing, white mycelium that turned dark olivaceous with age on PDA. Conidia from pycnidia formed in cultures were thick walled and pigmented with one septum and vertical striations when mature. Conidia measured (25.8–) 27.5 to 30.5 (–33.4) × (12.1) 14.3 to 16.8 (–18.2) μm (n = 60). Pycnidia contained septate paraphyses. N. mediterraneum was characterized as having moderately fast-growing, light green mycelia on PDA. Pycnidia formation was induced with pine needles placed on 2% water agar. Conidia from pycnidia were hyaline, ellipsoidal, thin walled, unicellular, and measured (18.2–) 20.5 to 27.8 (–29) × (5.1) 5.9 to 6.5 (–7.2) μm (n = 60). DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2), part of the β-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from L. crassispora (UCD23Co, UCD24Co, and UCD27Co) and N. mediterraneum (UCD695SJ, UCD719SJ, UCD720SJ, UCD749St, and UCD796St) grapevine isolates from California were amplified and sequenced. Consensus sequences from L. crassispora and N. mediterraneum from California showed 99 to 100% homology with L. crassispora and N. mediterraneum isolates previously identified and deposited in GenBank (1,2). Sequences from the examined DNA regions of all isolates were deposited at GenBank (GU799450 to GU799457 and GU799473 to GU799488). Pathogenicity tests using three isolates per species were conducted on detached dormant canes of cv. Red Globe. Ten canes per isolate were inoculated by placing a 7-day-old 5-mm-diameter agar plug from each fungal culture into a wound made with a drill on the internode (4). Twenty shoots were inoculated with noncolonized PDA plugs for negative controls. Six weeks after inoculations, necrosis was measured from the point of inoculation in both directions. One-way analysis of variance was performed to assess differences in the extent of vascular discoloration and means were compared using Tukey's test. L. crassispora isolates caused an average necrotic length of 21.1 mm, which was significantly lower (P < 0.05) than the average necrotic length of 35.6 mm caused by the N. mediterraneum isolates. Reisolation of L. crassispora and N. mediterraneum from necrotic tissue was 100% for each species. The extent of vascular discoloration in infected canes was significantly greater (P < 0.05) than in control inoculations (8 mm) from which no fungi were reisolated from the slightly discolored tissue. To our knowledge, this is the first report of L. crassispora and N. mediterraneum as pathogens of Vitis vinifera and as a cause of grapevine cankers in California. References: (1) T. I. Burgess et al. Mycologia 98:423, 2006. (2) P. W. Crous et al. Fungal Planet. No. 19, 2007. (3) J. R. Úrbez-Torres and W. D. Gubler. Plant Dis. 93:584, 2009. (4) J. R. Úrbez-Torres et al. Am. J. Enol. Vitic. 60:497, 2009.

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First Report of Phytophthora pseudosyringae Associated with Ink Disease of Castanea sativa in Italy

Plant Disease ◽

10.1094/pdis-94-8-1068b ◽

2010 ◽

Vol 94 (8) ◽

pp. 1068-1068 ◽

Cited By ~ 8

Author(s):

B. Scanu ◽

B. T. Linaldeddu ◽

A. Franceschini

Keyword(s):

Plant Protection ◽

Castanea Sativa ◽

Pond Water ◽

Culture Collection ◽

Selective Medium ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Representative Sequence ◽

Agar Plug ◽

Ink Disease

Since December 2008, a severe outbreak of ink disease has been observed in a chestnut grove in the Sardinia Region in Italy (40°01′N, 9°13′E, 1,200 m above sea level). Trees have shown symptoms such as microphylly and yellowish foliage as well as necrosis on the main roots and collar. Isolations were made from infected roots and soil using green apples as baits. Small pulp pieces were cut from the lesions that developed in the apples and plated on Phytophthora selective medium (1). In addition to Phytophthora cambivora, another Phytophthora sp. was detected from 60% of 25 symptomatic trees sampled. Colonies subcultured onto carrot agar (CA) were generally appressed and stellate. Growth occurred from 2 to 26°C with an optimum at 20°C (mean radial growth rate of 4.5 mm/day). Sporangia were produced abundantly in unsterile pond water; they were semipapillate, rarely bipapillate, limoniform or ovoid, occasionally caducous with short pedicels (<5 μm), and 35.2 to 58.1 (46.3) × 22.1 to 35.3 (31.9) μm, with a length/breadth ratio of 1.5:1. Catenulate hyphal swellings were frequently present, whereas no chlamydospores were observed. Isolates produced numerous homothallic oogonia with diameters from 23.7 to 31.7 (27.3) μm. Antheridia were predominantly paragynous. Cultural and morphological features were in close agreement with those described for P. pseudosyringae (2). Identity was confirmed by analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA. BLAST searches at GenBank showed 100% identity with reference sequences of P. pseudosyringae (Accession Nos. AY230190 and EU074793). The representative sequence of one P. pseudosyringae strain (CST2A), stored in the culture collection of the Department of Plant Protection-University of Sassari, was submitted to GenBank (Accession No. GU460375). Koch's postulates were fulfilled by inoculating 10 5-month-old chestnut seedlings grown in pots. One shallow cut was made into the bark on the main stem and an agar plug colonized by P. pseudosyringae was inserted beneath the flap. Seedlings were kept at the laboratory at temperatures varying from 16 to 22°C and watered as necessary. After 20 days, extensive, sunken, necrotic lesions measuring 27.2 ± 1.9 mm (mean + standard error) developed around the inoculation sites. Control plants inoculated with sterile CA plugs did not show any disease symptoms. The pathogen was consistently reisolated from infected tissues. P. pseudosyringae has recently been reported as the causal agent of stem necroses on chestnut seedlings in a nursery in Spain (3). To our knowledge, this is the first report of P. pseudosyringae on Castanea sativa in Italy. References: (1) C. M. Brasier and S. A. Kirk. Plant Pathol. 50:218, 2001. (2) T. Jung et al. Mycol. Res. 107:772, 2003. (3) C. Pintos Varela et al. Plant Dis. 91:1517, 2007.

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First Report of Amphobotrys ricini Causing Gray Mold Disease on Acalypha australis in Central China

Plant Disease ◽

10.1094/pdis-11-11-0929 ◽

2012 ◽

Vol 96 (3) ◽

pp. 460-460 ◽

Cited By ~ 3

Author(s):

L. Yu ◽

J. Zhang ◽

F. Xu ◽

L. Yang ◽

G. Q. Li

Keyword(s):

Dna Sequences ◽

Castor Bean ◽

Disease Incidence ◽

Average Diameter ◽

Gray Mold ◽

Potato Dextrose Agar ◽

Central China ◽

Colony Morphology ◽

First Report ◽

Detached Leaves

In 2008, we isolated a strain of an Amphobotrys sp., CotAr-12, from a cotton plant showing Verticillium wilt symptoms in Hubei Province, China. In October 2010 and 2011, surveys for Amphobotrys sp. on cotton were conducted in 14 counties in Hubei. No signs of an Amphobotrys sp. was observed on cotton. However, a gray mold disease was found on the weed, Acalypha australis in 40 of 51 cotton fields with an average disease incidence of 18.6%. The disease started on the inflorescences at the top of stems or branches and then spread downward to the main stems, branches, and leaves. Abundant sporulation on the necrotic tissues and formation of sclerotia underneath the epidermis or in the stem pith were evident. A total of 128 isolates, including CopAr-5, were obtained from A. australis (1 to 10 isolates per field). All isolates from A. australis and CotAr-12 appeared similar in colony morphology; ragged colony margins, erect conidiophores with long stipes and dichotomous branches at the top, globose and subhyaline conidia with an average diameter of 5.3 to 8.5 μm, and black, oval sclerotia of 0.6 to 26.2 × 0.5 to 19.0 mm. These characteristics matched the description for Amphobotrys ricini (Buchw.) Hennebert (teleomorph Botryotinia ricini [Godfrey] Whetzel) (2). Strains CopAr-5 and CotAr-12 were selected for molecular identification. DNA was extracted from mycelia and used for cloning of three nuclear genes (e.g., G3PDH, HSP60, and RPB2) using the procedures described by Staats et al. (4). The resulting DNA sequences were used for phylogenetic analysis with the corresponding sequences for Botrytis spp. (4). Results showed that strains CopAr-5 (GenBank Accession Nos. JN681883, JN681881, and JN6981879), CotAr-12 (GenBank Accession Nos. JN681882, JN681880, and JN6981878), and Botrytis (teleomorph Botrytinia) ricini (GenBank Accession Nos. GQ860998, GQ860996, and GQ860997) formed a separate clade that was distinct from Botrytis spp., supporting the distinction of Amphobotrys from Botrytis and suggesting that the two strains were Amphobotrys ricini. Pathogenicity was determined by placing mycelia of strains CopAr-5 and CotAr-12 on 10 detached leaves of A. australis, castor bean, and cotton. Control leaves of these plants were inoculated with potato dextrose agar alone. After incubation at 20°C under moist conditions (>90% relative humidity) for 60 h, the control leaves remained healthy, while the leaves of A. australis and castor bean inoculated with both strains formed extensively expanded lesions of 20 and 27 mm in diameter on average, respectively. Both strains also caused disease on cotton leaves with discontinuous and localized lesions of 18 mm in average diameter. A fungus was reisolated from the leaf lesions on these plant species and were identical to Amphobotrys ricini in colony morphology and conidial characteristics. We conclude that Amphobotrys ricini is a major pathogen on A. australis in central China. It is an important pathogen of castor bean (1) and has been reported to infect several euphorbiaceous plants, including A. hispida (3). To our knowledge, this is the first report of Amphobotrys ricini on A. australis. References: (1) G. H. Godfrey. J. Agric. Res. 23:679, 1923, (2) G. L. Hennebert. Persoonia 7:183, 1973. (3) B. V. Lima et al. Australas. Plant Dis. Notes 3:5, 2008. (4) M. Staats et al. Mol. Biol. Evol. 22:333, 2005.

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First Report of Leaf Spot Caused by Colletotrichum cf. linicola on Field Bindweed in Turkey

Plant Disease ◽

10.1094/pdis-92-2-0316a ◽

2008 ◽

Vol 92 (2) ◽

pp. 316-316 ◽

Cited By ~ 2

Author(s):

B. Tunali ◽

D. K. Berner ◽

H. J. Dubin

Keyword(s):

Dna Sequences ◽

Aqueous Suspension ◽

Severe Disease ◽

Leaf Tissue ◽

Malt Extract ◽

Voucher Specimen ◽

First Report ◽

Wheat Field ◽

Leaf Spots ◽

Field Bindweed

Field bindweed (Convolvulus arvensis L., Convolvulaceae) is one of the most problematic weeds in the world (1) and a target of biological control efforts (2). In the summer of 2006, dying field bindweed plants were found in a wheat field near Bafra, Turkey (41°21.197′N, 36°12.524′E). Plants had water-soaked lesions that developed into necrotic leaf spots on most of the leaves, particularly along the leaf margins, and on some stems. In most cases, the leaf spots coalesced, causing the leaves and later plants to wilt and die. Diseased leaves and stems were taken to the Phytopathology Laboratory of the Faculty of Agriculture, Ondokuz Mayis University, Samsun, Turkey. Diseased tissue was surface disinfested and placed on moist filter paper in petri dishes. Numerous acervuli with setae and conidia typical of a Colletotrichum sp. were observed after 2 to 5 days. A fungus, designated 06-01, was isolated from the diseased leaves. Stems and leaves of seven 12-week-old plants were spray inoculated in the laboratory with an aqueous suspension of conidia (106 spores per ml; 10 ml per plant) harvested from 6- to 8-day-old cultures grown on malt extract agar. The plants and two noninoculated checks were placed in a dew chamber at 22°C in darkness and continuous dew. After 48 h, plants from the dew chamber were moved to a greenhouse bench. All plants were watered twice daily. Symptoms were observed 5 days after inoculation. No symptoms were observed on noninoculated plants. Isolate 06-01 was reisolated from all inoculated plants. In the field, 20 inoculated plants became diseased after 20 days with approximately 36% diseased leaf tissue from which 06-01 was consistently reisolated. Diseased tissue and cultures of the fungus were sent to the Foreign Disease-Weed Science Research Unit, USDA/ARS, Fort Detrick, MD. The fungus conformed to the description of Colletotrichum linicola Pethybr. & Laff., which was noted as distinct from C. lini (3). The original description is also different than the description of C. lini (Westerdijk) Tochinai by Sutton (4). Acervuli were sparse, subepidermal, and erumpent. Conidia were hyaline, oblong or cylindrical or somewhat spindle-shaped with dull-pointed ends, guttulate, and 14 to 19 × 4 to 5 μm (mean 17 × 4 μm). Conidiophores were short, simple, hyaline, and emerged from subepidermal stroma. Setae were simple, erect, 3-septate, and dark with hyaline tips. DNA sequences were obtained for the internal transcribed spacer regions (GenBank Accession No. EU000060) and compared with other sequences in GenBank. Sequences from 06-01 matched 100% with one isolate of C. linicola and 99% with two other isolates of C. linicola. These isolates formed a unique clade. However, 06-01 was also 99% identical to other species of Colletotrichum. Thus, species identification is inconclusive. Isolate 06-01 is a destructive pathogen on field bindweed, and severe disease can be produced by inoculation of foliage with an aqueous suspension of conidia. To our knowledge, this is the first report of Colletotrichum on field bindweed. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 878174). References: (1) L. Holm et al. The World's Worst Weeds. University Hawaii Press, Honolulu, Hawaii, 1977. (2) G. Defago et al. BioControl 46:157, 2001. (3) G. H. Pethybridge and H. A. Lafferty. Sci. Proc. R. Dublin Soc. 15:359, 1918. (4) B. C. Sutton. The Coelomycetes. Commonw. Mycol. Inst., Kew, England, 1980.

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First Report of Branch Dieback of Walnut Caused by Neofusicoccum parvum in Korea

Plant Disease ◽

10.1094/pdis-12-12-1137-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1114-1114 ◽

Cited By ~ 5

Author(s):

W. Cheon ◽

Y. S. Kim ◽

S. G. Lee ◽

Y. H. Jeon ◽

I.-J. Chun

Keyword(s):

Dna Sequences ◽

Nucleotide Sequences ◽

Chitin Synthase ◽

Omega 3 ◽

Razor Blade ◽

Neofusicoccum Parvum ◽

First Report ◽

Fungal Isolates ◽

Agar Plug ◽

Pcr Product

Walnut (Juglans sinensis Dode) is an economically important tree in the world, both for its wood and its fruit. Walnut fruits, as rich sources of omega-3 essential fatty acid, are valuable nutritionally. Consumer interest in Korea for walnuts has increased in recent years, and production has increased to 1,042 ha with the Kyoungbuk region consisting of 402 ha (2). In May 2012, lethal dieback disease of walnut tree was detected in two orchards in Andong, Kyoungbuk region, Korea, each with an incidence of 25 to 30%. Disease symptoms included blight and dieback of the stems, flowing resin, dark decay inside the bark of dead twigs, and defoliation. The bark of dead twigs was removed and sliced thinly using a razor blade, and water-mounted, without staining, for observation of fungal structures, if present. Pycnidia were found embedded within the bark of dead twigs and conidia were mostly characterized by fusoid, hyaline, smooth, thin-walled, unicellular and 16.25 to 21.25 μm long and 4.37 to 6.87 μm wide. These characteristics are consistent with those reported previously for Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (1). Diseased branch tissues collected from the two locations were surface sterilized with 1% NaOCl, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA). The fungal isolates, recovered from the two different orchards, produced white, aerial mycelium and became light gray within a week after incubating plates at 25°C. To confirm the identities of the isolates, the complete internal transcribed spacer (ITS) rDNA of the fungi was amplified and sequenced using PCR. The sequences were compared with other DNA sequences in the GenBank database, using a BLAST search. BLAST analysis of the PCR product showed that the sequence had 99% identity with the nucleotide sequences for N. parvum (JQ411396.1 and GU997688.1). Additionally, the chitin synthase 1 gene was sequenced and analyzed using the BLAST server. The sequence of PCR product had 100% identity with the nucleotide sequences of N. parvum strain CMW9080 chitin synthase 1 gene (EU339501). Thus, both morphological and molecular characters confirmed this species as N. parvum. Pathogenecity tests were performed by inoculating 2-year-old J. sinensis trees. Inoculations consisted of inserting 5-mm-diamter agar plug bearing fresh mycelium of the fungal isolates into the wounds. Within 2 weeks, black lesions appeared on all inoculated plants accompanied by defoliation, whereas no symptoms were observed in the control plants. N. parvum has been reported a member of Botryosphaeriaceae, commonly associated with dieback and cankers of woody plants (1). To our knowledge, this study is the first report of N. parvum as a pathogen of Juglans sinensis in Korea. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) Statistics Korea. Forest Households by Growing Area of Walnut/Total Area. 2010.

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First Report of Root Rot of Pedunculate Oak and Other Forest Tree Species Caused by Phytophthora plurivora in the Czech Republic

Plant Disease ◽

10.1094/pdis-94-2-0272b ◽

2010 ◽

Vol 94 (2) ◽

pp. 272-272 ◽

Cited By ~ 2

Author(s):

M. Mrazkova ◽

K. Cerny ◽

M. Tomsovsky ◽

V. Holub ◽

V. Strnadova ◽

...

Keyword(s):

Czech Republic ◽

Dna Sequences ◽

Tree Species ◽

Root Rot ◽

Root Hairs ◽

Aerial Mycelium ◽

Pedunculate Oak ◽

The Czech Republic ◽

First Report ◽

Agar Plug

From 2006 to 2008, several similar Phytophthora isolates were obtained from roots of mature Quercus robur and other tree species (Acer platanoides, Fraxinus excelsior, Q. rubra, and Tilia cordata) in forests and parks in several areas in the Czech Republic. The trees were characterized by chlorotic and reduced foliage, crown dieback, and reduced root hairs. Several isolates of Phytophthora were obtained from necrotic roots of these trees and identified as Phytophthora plurivora Jung & Burgess (1). Isolated colonies grown on V8A medium were radiate to slightly chrysanthemum shaped with limited aerial mycelium in the center. Optimum growth was at 25°C, minimum at 5°C and maximum at 32°C. Radial growth of colonies averaged 6.4 mm/day at 20°C. The isolates were homothallic and produced abundant smooth-walled, spherical oogonia (23.3 to 29.1 μm in diameter), oospores were nearly plerotic or plerotic (21.8 to 26.9 μm in diameter), and the oospore wall was 1.2 to 1.4 μm thick. Antheridia were usually paragynous and measured 8.4 to 12 × 6.5 to 8 μm, but amphigynous antheridia were occasionally observed. Noncaducous, semipapillate sporangia formed on simple or sympodial sporangiophores, were obpyriform, ovoid, ellipsoid or irregular in shape, and occasionally distorted with more than one apex. Sporangia dimensions were 33 to 65 × 24 to 33 μm; L/B ratio 1.2 to 1.6 (–2.1). Comparison of DNA sequences of internal transcribed spacer (ITS) regions of isolates (representative strain GenBank Accession No. FJ952382) confirmed the 100% identity of P. plurivora (1). The soil infestation test was conducted using a P. plurivora isolate acquired from roots of Q. robur and 20 3-year-old plants of Q. robur. Sterilized millet seeds colonized by pathogen with the method as described (2) were used as inoculation medium and added into sterilized peat substrate at the rate of 0.5% (vol/vol). The plants were cultivated in 5.8-liter pots in a greenhouse (20°C, 16-h/8-h photoperiod). After 4 months, the roots of all plants were washed, dried, and weighed. The root biomass of 20 infected plants was significantly reduced by approximately 25% on average compared with the control 20 plants (P < 0.05, t-test, Statistica 7.1). The pathogen was consistently reisolated from the roots of infected plants but not from control plants. Stem inoculation tests were conducted with 20 replicates in each group of 2-year-old plants of oak, maple, ash, and lime and isolates acquired from the hosts. On each seedling, a 5-mm-diameter bark plug was removed 5 cm above the collar. The inoculum (5-mm-diameter V8A agar plug with actively growing mycelium) was applied to the exposed substrate. The wounds were sealed with Parafilm. Stem necrosis developed in all cases after 1 to 2 weeks, whereas control plants remained healthy. The pathogen was successfully reisolated from necrotic stem tissues. To our knowledge, this is the first report of P. plurivora causing root rot on oak, maple, ash, and lime in the Czech Republic. On the basis of the host range and distribution of P. plurivora in the Czech Republic, it can be assumed that, as elsewhere in Europe (1), this pathogen is widespread and is a common cause of decline of many tree species. References: (1) T. Jung and T. I. Burgess. Persoonia 22:95, 2009. (2) C. Robin et al. Plant Pathol. 50:708, 2001.

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First Report of Phytophthora Rot on Alders Caused by Phytophthora alni subsp. alni in Spain

Plant Disease ◽

10.1094/pdis-94-2-0273a ◽

2010 ◽

Vol 94 (2) ◽

pp. 273-273 ◽

Cited By ~ 5

Author(s):

C. Pintos Varela ◽

C. Rial Martínez ◽

J. P. Mansilla Vázquez ◽

O. Aguín Casal

Keyword(s):

Dna Sequences ◽

Mitochondrial Gene ◽

Aerial Mycelium ◽

Selective Medium ◽

Mt Dna ◽

First Report ◽

Inner Bark ◽

Agar Plug ◽

Plant Pathol ◽

Phytophthora Rot

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.

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First Report of Leaf Blight of Japanese Yew Caused by Pestalotiopsis microspora in Korea

Plant Disease ◽

10.1094/pdis-08-13-0821-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 691-691 ◽

Cited By ~ 2

Author(s):

Y. H. Jeon ◽

W. Cheon

Keyword(s):

Dna Sequences ◽

Apical Cell ◽

Morphological Characteristics ◽

Leaf Blight ◽

Economic Damage ◽

Leaf Margin ◽

Conidial Suspension ◽

First Report ◽

Pestalotiopsis Microspora ◽

Large Trees

Worldwide, Japanese yew (Taxus cuspidata Sieb. & Zucc.) is a popular garden tree, with large trees also being used for timber. In July 2012, leaf blight was observed on 10% of Japanese yew seedling leaves planted in a 500-m2 field in Andong, Gyeongsangbuk-do Province, South Korea. Typical symptoms included small, brown lesions that were first visible on the leaf margin, which enlarged and coalesced into the leaf becoming brown and blighted. To isolate potential pathogens from infected leaves, small sections of leaf tissue (5 to 10 mm2) were excised from lesion margins. Eight fungi were isolated from eight symptomatic trees, respectively. These fungi were hyphal tipped twice and transferred to potato dextrose agar (PDA) plates for incubation at 25°C. After 7 days, the fungi produced circular mats of white aerial mycelia. After 12 days, black acervuli containing slimy spore masses formed over the mycelial mats. Two representative isolates were further characterized. Their conidia were straight or slightly curved, fusiform to clavate, five-celled with constrictions at the septa, and 17.4 to 28.5 × 5.8 to 7.1 μm. Two to four 19.8- to 30.7-μm-long hyaline filamentous appendages (mostly three appendages) were attached to each apical cell, whereas one 3.7- to 7.1-μm-long hyaline appendage was attached to each basal cell, matching the description for Pestalotiopsis microspora (2). The pathogenicity of the two isolates was tested using 2-year-old plants (T. cuspidata var. nana Rehder; three plants per isolate) in 30-cm-diameter pots filled with soil under greenhouse conditions. The plants were inoculated by spraying the leaves with an atomizer with a conidial suspension (105 conidia/ml; ~50 ml on each plant) cultured for 10 days on PDA. As a control, three plants were inoculated with sterilized water. The plants were covered with plastic bags for 72 h to maintain high relative humidity (24 to 28°C). At 20 days after inoculation, small dark lesions enlarged into brown blight similar to that observed on naturally infected leaves. P. microspora was isolated from all inoculated plants, but not the controls. The fungus was confirmed by molecular analysis of the 5.8S subunit and flanking internal transcribed spaces (ITS1 and ITS2) of rDNA amplified from DNA extracted from single-spore cultures, and amplified with the ITS1/ITS4 primers and sequenced as previously described (4). Sequences were compared with other DNA sequences in GenBank using a BLASTN search. The P. microspora isolates were 99% homologous to other P. microspora (DQ456865, EU279435, FJ459951, and FJ459950). The morphological characteristics, pathogenicity, and molecular data assimilated in this study corresponded with the fungus P. microspora (2). This fungus has been previously reported as the causal agent of scab disease of Psidium guajava in Hawaii, the decline of Torreya taxifolia in Florida, and the leaf blight of Reineckea carnea in China (1,3). Therefore, this study presents the first report of P. microspora as a pathogen on T. cuspidata in Korea. The degree of pathogenicity of P. microspora to the Korean garden evergreen T. cuspidata requires quantification to determine its potential economic damage and to establish effective management practices. References: (1) D. F. Farr and A. Y. Rossman, Fungal Databases, Syst. Mycol. Microbiol. Lab. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ (2) L. M. Keith et al. Plant Dis. 90:16, 2006. (3) S. S. N. Maharachchikumbura. Fungal Diversity 50:167, 2011. (4) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990.

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