First Report of Anthracnose of Mile-a-Minute (Persicaria perfoliata) Caused by Colletotrichum cf. gloeosporioides in Turkey.

Mile-a-minute (Persicaria perfoliata (L.) H. Gross; family: Polygonaceae) is an exotic annual barbed vine that has invaded the northeastern USA and Oregon (2). In July of 2010, in a search for potential biological control pathogens (3), diseased P. perfoliata plants were found along the Firtina River near Ardesen, Turkey. Symptoms were irregular dark necrotic lesions along leaf margins and smaller irregular reddish lesions on the lamellae of leaves. Symptomatic leaves were sent to the quarantine facility of FDWSRU, USDA, ARS in Ft. Detrick, MD, for pathogen isolation and testing. Symptomatic leaves were excised, surface disinfested in 0.615% NaOCl, and then incubated for 2 to 3 days in sterile moist chambers at 20 to 25°C. Numerous waxy sub-epidermal acervuli with 84-µm-long (mean) black setae were observed in all of the lesions after 2 to 3 days of incubation. Conidiophores within acervuli were simple, short, and erect. Conidia were one-celled, hyaline, guttulate, subcylindrical, straight, 12.3 to 18.9 × 3.0 to 4.6 µm (mean 14.3 × 3.7 µm). Pure cultures were obtained by transferring conidia onto 20% V-8 juice agar. Appressoria, formed 24 h after placing conidia on dialysis membrane over V-8 juice agar, were smooth, clavate, aseptate, regular in outline, and 6.4 to 10.0 × 5.1 to 7.2 µm (mean 7.5 × 6.6 µm). These characters conformed to the description of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (1). A voucher specimen was deposited in the U.S. National Fungus Collections (BPI 882461). Nucleotide sequences for the internal transcribed spacers (ITS 1 and 2), directly sequenced from ITS 1 and ITS 4 standard primers (4), were deposited in GenBank (JN887693). A comparison of these sequences with ITS 1 and 2 sequences of the C. gloeosporioides epitype IMI 356878 (GenBank EU 371022) (1) using BLAST found 479 of 482 identities with no gaps. Conidia from 14-day-old cultures, in an aqueous suspension of 1.0 × 106 conidia ml-1, were spray-inoculated onto healthy stems and leaves of twenty 30-day-old P. perfoliata plants. Another 10 plants were not inoculated. All plants were placed in a dew chamber at 25°C for 16 h with no lighting. They were then placed in a 20 to 25°C greenhouse with a 14-h photoperiod. Light was generated using 400W sodium vapor lights. Lesions developed on leaves and stems of all inoculated plants after 7 days, and symptoms were the same as observed in the field. Each plant was rated weekly for disease severity on a 0 to 10 rating scale where 0 = no disease symptoms and 10 = 100% symptomatic tissue. After 28 days, the average disease rating of inoculated plants was 3.95 ± 0.94. No disease developed on noninoculated plants. C. gloeosporioides was reisolated from all inoculated plants. Host range tests will determine the potential of this isolate as a biological control agent for P. perfoliata. To our knowledge, this is the first report of anthracnose caused by C. gloeosporioides on P. perfoliata. References: (1) P. F. Cannon et al. Mycotaxon 104:189, 2008. (2) J. T. Kartesz and C. A. Meacham. Synthesis of the North American Flora, Version 1.0., North Carolina Botanical Garden, Chapel Hill, N.C. 1999. (3) D. L. Price et al. Environ. Entomol. 32:229, 2003. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Exserohilum monoceras, Newly Reported on Late Watergrass in Turkey.

Echinochloa species are major weeds in rice-cropping systems and are among the most noxious weeds in the world. Throughout the world, Echinochloa oryzicola Vasing, (late watergrass) is one of the most important and serious weed species of this genus. In September 2010, punctiform, purplish dark brown leaf spots were observed on leaves and sheaths of Echinochloa oryzicola in a rice field in Terme, Turkey (41°13.412'N, 36°56.248'E). Individual lesions ranged from 1 to 3 mm in diameter. Infected leaf and sheaths were surface disinfected for 1 min in 1% NaOCl, plated on potato dextrose agar (PDA), and incubated at 25°C. Colonies of pure cultures on PDA turned to dark green colonies with increasing age. Conidia were 87 to 147 (120) × 15 to 21 (19) µm (n = 50), 6 to 10 pseudoseptate, straight or slightly curved, fusiform, tapering gradually toward the base, pale-to-dark straw colored, smooth, with a small protruding plenum-type hilum. The fungus was identified as Exserohilum monoceras (Drechsler) Leonard & Suggs based on its micromorphology and cultural features (1,2). Conidia were harvested from 3-week-old cultures grown on PDA by brushing the surface of the colonies with a small paint brush, suspending the conidia in sterile distilled water and filtering through cheesecloth for pathogenicity tests. Conidia were then diluted in sterile distilled water plus 0.1% polysorbate 20 to a concentration of 1 × 106 conidia/ml. Leaves and stems of Echinochloa oryzicola at the three-leaf stage were spray inoculated with 10 ml of this aqueous suspension per plant. Three inoculated plants and three noninoculated plants were placed in a dew chamber at 18 to 22°C with continuous dew, and after 48 h, plants were moved to a greenhouse bench. Symptoms, similar to those originally observed in the field, began to appear on the leaf and sheaths approximately 10 days later and E. monoceras was reisolated, successfully completing Koch's postulates. No symptoms developed on the control plants. E. monoceras has also been reported on Echinochloa oryzicola in Japan (3). To our knowledge, this is the first report of leaf spot on Echinochloa oryzicola caused by E. monoceras in Turkey where the fungus may have potential as a biological control agent. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute. Kew, Surrey, England, 1971. (2) M. Sisterna and R. Bezus. Plant Dis. 85:803, 2001. (3) H. Tsukamoton et al. Ann. Phytopathol. Soc. Jpn. 64:526, 1998.

First Report of Cylindrocarpon liriodendri on Kiwifruit in Turkey.

During a routine survey of diseases of kiwifruit (Actinidia chinensis Planch.) cv. Hayward conducted in autumn of 2009 in Ardesen, Rize Province (eastern Black Sea Region, Turkey), symptoms of a new disease were observed in five locations. Affected trees showed leaf wilting that frequently led to the death of the trees. Symptoms at ground level included necrotic lesions on woody tissues of both the rootstock and roots. Small pieces from necrotic wood and root tissues were surface disinfested and plated onto potato dextrose agar (PDA) medium amended with 0.5 g liter-1 of streptomycin sulfate and incubated for 7 days at 25°C in the dark. Isolates were transferred to PDA and presumptively identified as a Cylindrocarpon sp. by morphology and conidial characteristics. The isolates were transferred to PDA and Spezieller Nährstoffarmer Agar (SNA) and then incubated at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed floccose to felted mycelium, which varied in color from brown-yellow to sepia. On SNA, all isolates produced microconidia measuring 6.25 to 15 (9.6) × 2.5 to 5 (3.02) µm and macroconidia of one-septate measuring 7.5 to 20 (13.3) × 2.5 to 5 (3.8) µm, two-septate measuring 12.5 to 25 (20.7) × 3.25 to 5 (4.58) µm, and three-septate measuring 16.3 to 30 (11.04) × 3.75 to 5 (4.82) µm. Chlamydospores 7.5 to 11.3 (9.78) µm were intercalary or terminal in the mycelium, single or occasionally in chains. Identity of these isolates was determined by a multiplex PCR system using a set of three pairs of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a product size of 253 bp, which is characteristic of Cylindrocarpon liriodendri J.D. MacDonald and E.E. Butler, in agreement with morphological features (2). Additionally, the internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolates 10K-TR1 and 10K-TR2 and deposited in GenBank (Accession Nos. HQ113122 and HQ113123). These sequences showed high similarity (98%) with the sequence of C. liriodendri (GenBank Accession No. DQ718166). A pathogenicity test was conducted using isolate 10K-TR1 and repeated twice. Six 8-month-old callused and rooted cuttings of kiwifruit cv. Hayward were surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, washed twice with sterile distilled water (SDW), and inoculated by dipping their roots for 30 min in a spore suspension of the fungus (1 × 106 conidia ml-1) obtained from 30-day-old colonies grown on PDA. Six control cuttings were dipped in SDW. Two weeks later, cuttings were drench inoculated with 50 ml of the designated spore suspension to guarantee root infection and controls were drenched again with SDW. Plants were maintained in a greenhouse with a temperature range of 25 to 30°C. Four months after inoculation, the inoculated plants developed wilting and root symptoms similar to those observed in natural infections and C. liriodendri was reisolated, completing successfully Koch's postulates. No symptoms were observed on the control plants. To our knowledge, this is the first report of C. liriodendri on kiwifruit trees in Turkey. References: (1) S. Alaniz et al. Plant Dis. 93:821, 2009. (2) F. Halleen et al. Stud. Mycol. 55:227, 2006.

First Report of Leaf Spot on Horseweed (Conyza canadensis) Caused by Septoria erigerontis in Turkey.

Horseweed (Conyza canadensis (L).Cronq., Asteraceae) is an invasive exotic weed in Turkey and a problematic native weed in the United States where glyphosate-resistant populations of the weed have developed (2). These characteristics make horseweed a target for biological control efforts. In September 2009, small, brown leaf spots were observed on leaves of C. canadensis in Taflan, Turkey (41°25.398'N, 36°08.352'E). Globose, dark-walled pycnidia were also observed in brown spots on leaves. Diseased tissue was surface disinfested and placed on moist filter paper in petri plates. A fungus designated 09-Y-TR1 was isolated from the diseased leaves. Single-spore isolations were grown on potato dextrose agar (PDA). Cultures on PDA formed dark green-to-black colonies. Pycnidia matured after 3 to 4 weeks when plates were incubated at 23°C with a 12-h photoperiod (black light and cool white fluorescent light). Pycnidia were separate, immersed, and dark brown with a single apical ostiole. Matured conidia were one to three septate, filiform, straight to slightly curved, rounded at the apex, smooth walled, hyaline, and 22 to 40 × 1.4 to 2.5 µm. Morphology was consistent with Septoria erigerontis Peck (3). Comparison of the internal transcribed spacer (ITS) 1 and 2 sequence with available sequences of vouchered S. erigerontis specimens (GenBank EF535638.1, AY489273.1; KACC 42355, CBS 109094) showed 447 of 450 and 446 of 450 identities, respectively. Nucleotide sequences for the ribosomal ITS regions (ITS 1 and 2, including 5.8S rDNA) were deposited in GenBank (GU952666). For pathogenicity tests conidia were harvested from 3-week-old cultures grown on PDA, by brushing the surface of the colonies with a small paint brush, suspended in sterile distilled water, and filtered through cheese cloth. Conidia were then diluted in sterile distilled water plus 0.1% polysorbate 20 to a concentration of 5 × 106 conidia/ml. Stems and leaves of seven 5-month-old seedlings were spray inoculated with 10 ml of this aqueous suspension per plant. Inoculated plants and three noninoculated plants were placed in a dew chamber at 23°C in darkness and continuous dew, and after 48 h, plants were moved to a greenhouse bench. Symptoms were observed 2 days after inoculation. Disease severity was evaluated 2 weeks after inoculation by a rating system with a scale of 0 to 6 based on percentage of plant tissue necrosis, in which 0 = no symptoms, 1 = 1 to 5%, 2 = 6 to 25%, 3 = 26 to 75%, 4 = 76 to 95%, 5 = >95%, and 6 = dead plant. The average disease rating on inoculated plants was 3.55. No disease was observed on noninoculated plants. S. erigerontis was reisolated from all inoculated plants. To our knowledge, this is the first report of leaf spot on horseweed caused by S. erigerontis in Turkey where the fungus may have potential as a classical biological control agent. S. erigerontis has also been reported on C. canadensis in Korea and Portugal (1). In the United States, S. erigerontis has been reported on horseweed in several states (1) and these isolates may have potential as biological control agents of horseweed, particularly glyphosate-resistant horseweed, in the United States. References: (1) D. F. Farr et al. Fungal Databases. Systematic Mycology and Microbiology Laboratory, Online publication. ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , March 2010. (2) I. Heap. www.weedscience.org , 2006. (3) M. J. Priest. Fungi of Australia: Septoria. ABRS/CSIRO Publishing. Melbourne, 2006.

First Report of Root Rot of Bean and Soybean Caused by Rhizoctonia zeae in Turkey.

To determine the species of Rhizoctonia on bean and soybean plants grown in Samsun (Turkey), field surveys were performed at 104 locations during 2001 and 2002. Rhizoctonia spp. were obtained from isolations from the necrotic lesions on the hypocotyl and rhizosphere soils. Species were identified according to Ogoshi (3) on the basis of hyphal and colony morphology and anastomosis reaction with known tester isolates (provided by M. Hyakumachi, Gifu University, Japan). Fifty Rhizoctonia spp. isolates obtained from these locations were identified as Rhizoctonia zeae (teleomorph Waitea circinata var. zeae). Nine of the 27 bean isolates and 8 of the 23 soybean isolates were recovered from plant tissues. These isolates had optimum temperature (32°C) for growth. Colonies were orange when young, becoming salmon colored with age. Sclerotia formed both on the agar surface or submerged in the medium. Superficial sclerotia were more uniform and nearly spherical, mostly 0.2 to 0.5 mm in diameter, and they were first orange and then turned brown. Pathogenicity was tested with three R. zeae isolates grown on sterile oat seeds at 25°C for 10 days. Bean and soybean seedlings grown in 1-liter plastic pots containing sterile potting mix (field soil/composted manure/sand 2:2:1 [v/v]) at true-leaf stage were inoculated by placing five infested oat seeds adjacent to the roots. Sterile oat seeds were used for controls. After 3 to 4 weeks of incubation at 17 to 25°C in a glasshouse, roots of the plants were cleaned with tap water and evaluated for disease severity. Four replicate pots were used for each isolate/plant combination. All isolates produced superficial brown lesions on roots and hypocotyls similar to those observed on plants used for isolations and root growth declined. R. zeae was reisolated from the lesions on all bean and soybean plants used for the pathogenicity test. While R. zeae was previously reported from Johnsongrass roots (1) and corn kernels (2), to our knowledge, this is the first report of R. zeae isolated from bean and soybean plants and rhizosphere soils in Turkey. References: (1) E. Demirci, and C. Eken. Plant Dis. 83:200, 1999. (2) E. Demirci and S. Kordali. Plant Dis. 83:879, 1999. (3) A. Ogoshi. Rev. Plant. Prot. Res. 8:93, 1975.