Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China is the biggest single tobacco market and accounts for more than 40% of the global tobacco consumption (1). Tobacco seed harvested in Guiyang, Guizhou Province, China, are commonly contaminated or infected by various fungal pathogens, which can cause abnormal seedlings with dark brown lesions and stunting of roots and decayed seeds. In 2013, five samples of 500 seeds from tobacco cv. Guiyan 4 were tested for germination on moistened paper on petri dishes. On average, 35% of the seeds from all five samples developed into abnormal seedlings or were decayed and were plated onto potato dextrose agar media and grown for 5 days at 25°C in darkness to confirm the presence of a pathogen. However, one fungus was isolated from an average of 10% of the 500 seeds sampled. It was identified morphologically as Cladosporium cladosporioides (Fresen.) de Vries based on the velvety olive-brown with almost black reverse colony color and dimensions and color of conidia and conidiophores. Conidia formed in long branched chains that readily disarticulate, single celled, elliptical to limoniform, 2 to 8 (avg. 4.3) × 2 to 3 (avg. 2.1) μm. Conidia were pale to olive brown and smooth to verruculose. Ramoconidia were 0 to 1 septate, 7 to 14 (avg. 9.2) × 2 to 4 (avg. 2.6) μm, smooth or sometimes minutely verruculose. Conidiophores were pale to olive brown, macro- and micronemateus, smooth or sometimes verruculose, and of various lengths up to 320 μm long and 2 to 5 μm wide. Primer pair ITS1 and ITS4 was employed to amplify the regions of ITS1-5.8s-ITS2 of the pathogens. Sequences of all three isolates (G3, G10, and G18) (Accession Nos. KF841547, KF841554, and KF841560) were identical to each other and to four sequences in GenBank (JX230994.1, JQ768317.1, JQ768322.1, and AB763555.1). Pathogenicity of the three isolates of C. cladosporioides was verified on tobacco seedlings of 3-week-old grown on wet filter paper in the petri dishes (9 cm in diameter). For each isolate, 20 seedlings incubated in one plate were inoculated with 0.5 ml of a suspension of 105 conidia/ml. Twenty seedlings were treated with sterile water as control treatment. After inoculation, the petri dishes were incubated at 25°C, 100 to 120 μEm−2 S−1, RH > 80%, and 16 h light per day for disease development. At 96 h after inoculation, symptoms comprising medium brown to black lesions on the roots were clearly visible on inoculated plants but not on the control plants. All seedlings inoculated died 9 days after inoculation whereas control seedlings remained symptomless. Re-isolation attempts on PDA from roots demonstrated C. cladosporioides to be present in symptomatic seedlings but not in roots of the control plants. Moreover, the characteristics of the cultured fungi were exactly the same as those originally isolated. Isolates G3, G10, and G18 (KF841547, KF841554, and KF841560) were deposited with the Tobacco Diseased Fungi, Guizhou Academy of Tobacco Sciences, Guizhou, China. Previously, C. cladosporioides has also been isolated from macadamia (Macadamia integrifolia Maiden & Betche) racemes in South Africa (4), from diseased papaya (Carica papaya L.) in Taiwan province of China (2), and from seeds of Amaranthus spp. in Poland (3). To the best of our knowledge, this is the first report of C. cladosporioides causing seed disease on tobacco in China and the disease should be considered in existing disease management practices. References: (1) British American Tobacco Annual Report, 8, 2012. (2) R. S. Chen, et al. Plant Dis. 93:426, 2009. (3) W. Pusz. Phytopathologia 54:15, 2009. (4) N. van den Berg et al. Plant Dis. 92:484, 2008.

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China accounts for more than 39.6% of total global tobacco production (3). In May 2012, seedlings of tobacco cv. Honghuadajinyuan in a Guiyang tobacco commercial field (Guizhou, China, 26.35° N, 106.42° E) developed symptoms of severe wilting, chlorosis, and stunting. The main stem and taproot exhibited reddish to light brown vascular discoloration; further progression of these symptoms eventually caused mortality of infected seedlings. To isolate the causal agent, necrotic tissues from the symptomatic root were placed on potato dextrose agar (PDA) and incubated at 25°C in darkness. Colonies with white to rose mycelia and red-brown colony colors developed on PDA after 5 days of incubation. Microconidia were abundant, straight or slightly curved, clavate, 0- to 3-septate, and 7.5 to 20.0 × 2.5 to 5.0 μm. Macroconidia were straight or slightly curved, slender, 3- to 5-septate, and 25.0 to 45.0 × 3.3 to 5.0 μm. Based on the observed colony attributes, growth patterns, absence of chlamydospores, micro- and macro-spore attributes (1), and PCR amplification (using primers ITS1/4) combined with translation elongation factor primers (EF1/2) (2), the fungus was identified as F. kyushuense O'Donnell & T. Aoki. Sequence of ITS1-5.8s-ITS2 region of rDNA (GenBank Accession No. JX235957) exactly matched the sequences of F. kyushuense accession AB587020.1 (100% similarity). Analysis of the elongation factor (EF-1alpha) gene of the fungus (JX658565) resulted in a 99% match for F. kyushuense accession AB674297.1. Pathogenicity of the fungus was confirmed by performing Koch's postulate as follows. Pure cultures of the fungus F. kyushuense obtained from symptomatic tissues of tobacco seedlings were grown on PDA for 6 days. Tobacco plants to be used in pathogenicity tests were germinated and grown on potting soils in a plastic container. Additional fertilization was supplied by adding 0.2 g/L of 20-20-20 (N-P-K) in the float water. When seedlings got 6-leaf stage, they were ready for pathogenicity tests. Spores harvested from these culture plates were suspended in sterile distilled water, adjusted to a concentration of 1 × 104 conidia/ml, and inoculated by irrigating 10 ml of the conidia suspension onto roots of each of the 12 tobacco seedlings with 6-leaf stage. A group of 12 seedlings of the same age treated with sterile water served as control. Inoculated seedlings were maintained at 25°C, 100 μE m–2.s–1, relative humidity >70%, and 16 h light per day, and monitored for 9 days for symptom development. Seedlings inoculated with conidia developed disease symptoms with roots with vascular discoloration of roots whereas control seedlings remained symptomless. F. kyushuense was reisolated from the symptomatic seedlings 9 days after inoculation. F. kyushuense has also been isolated from rice seeds in China (4), and from diseased wheat in Japan (1). The common tobacco Fusarium disease reported in China was caused by F. oxysporium f. sp. nicotianae. However, to the best of our knowledge, this is the first report of F. kyushuense causing wilt on tobacco in China and the disease must be considered in existing disease management practices. References: (1) T. Aoki and K. O'Donnell. Mycoscience. 39:1, 1998. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) US Census Bureau. Foreign Trade Statistics. Washington DC, 2005. (4) Z. H. Zhao and G. Z. Lu. Mycotaxon. 102:119, 2007.

A new anthracnose disease on chili pepper (cayenne pepper cv. Hongxiu 2003, fruiting type pepper) was found in Zhijiang County, Hunan, China in 2009. The disease was observed only on the fruits. Lesions were generally elongated, on which dark acervuli were arranged concentrically. Later, cracking of older lesions was observed. With a microscope, fungal conidia were observed to be 15.8 × 4.1 μm, fusiform or oval with one end acute, and single celled with two to seven oil globules. No setae were found on the acervuli. Eight isolates (HNZJ001–HNZJ008) showed no difference in colony feature when cultured on potato dextrose agar. All the isolates showed white growth at the early stages, but colonies turned pink when they produced powdery spores and then finally became red gray. The average colony diameter was 68.5 to 72.3 mm after 7 days with obvious gray black concentric rings because of the development of aerial and substrate mycelia. After a needle-prick inoculation with a suspension of 1 × 106 spores per ml of HNZJ001 on 30 chili pepper fruits with three repeats, the same symptoms were observed and the same fungus was recovered. In bioassays, HNZJ001 caused lesions on both mature and immature fruits, while Glomerella cingulata strain LSQ1 (GenBank Accession No. HQ607386) used as a control did not infect immature fruits. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5. Sequencing of the PCR products of HNZJ001 (GenBank Accession No. GU059863) showed 100% identity to G. acutata (GenBank Accession No. EU008863) and Colletotrichum acutatum (GenBank Accession No. AF207794) after a BLAST search. The pathogen was identified as G. acutata (asexual stage: C. acutatum) on the basis of morphological characters and rDNA-ITS sequence analysis. Worldwide, it has been reported that pepper anthracnose might be caused by up to five species of Glomerella (Colletotrichum): G. cingulata, C. coccodes, C. capsici, C. dematium, and G. acutata (2), among which only the first three were previously reported in China. In recent years, G. acutata was reported on such plants as apple (3) and strawberry (1) in China, but not on pepper. To our knowledge, this is the first report of G. acutata on chili pepper in China. References: (1) X.-J. Ren et al. Acta Phytopathol. Sin. 38:325, 2008. (2) P. P. Than et al. Zhejiang Univ. Sci. B 9:764, 2008. (3) R. Zhang et al. Plant Dis. 92:1474, 2008.

In July 2011, we observed a new rice disease, black sheath spot, on tillering rice plants in Huayuan County, Hunan Province, China. Field surveys indicated that this disease covered a ~1,000-ha paddy field, mainly on Y series 2-line hybrid rice cultivars, especially Y Liangyou 7 and Y Liangyou 302, in hills of which 10 to 70% infection was observed (average of 45%), causing estimated damages up to $1.2 million (US). The diseased rice plants were cultivated with standard practices. Weather, flooding, herbicide damage, and fertilizer application did not appear to account for the symptoms. Typical symptoms of the disease included elliptical spots, about 10 × 5 mm, dark brown to black and with a diffuse yellow-brown margin. The leaf blades with diseased sheaths became yellow and blighted. After 7 days of growth on PDA, groups of cylindrical and branched stromata scattered over dark green colony. Conidia averaged 30.4 × l2.5 μm and contained five cells with three dark central cells. The morphology of spores was consistent with that of C. fallax (1). We inoculated 8-cm healthy rice sheath segments with culture disk of an isolate HNAH001. Sheath segments were divided into four groups: I) disks on wounded sheath surfaces; II) disks on non-wounded sheath surfaces; III) disks between the leaf sheath and stem, and IV) a sterilized water control. The segments were maintained in moist, covered plates in a 25°C incubator after inoculation. After another 24 h, circular brown lesions, less than 10 mm long, appeared on all inoculated segments in treatments I,II, and III. Treatment I induced the most serious symptoms. No lesions developed on control segments. For further verification of pathogenicity, we sprayed a spore suspension of HNHY001 on healthy rice plants at the boot stage. Black spots reappeared on the sheaths after 5 days. No lesions appeared on the stems or the leaf blades of inoculated rice plants. We recovered HNHY001 from the spots on inoculated plants and completed all steps of Koch's postulates. For molecular identification of the fungus, DNA was extracted from mycelia and used as a template for PCR with a primer pair of ITS 5 and ITS 4 targeting the rDNA-ITS. The sequence of the PCR product (Accession No. JQ360963) had 100% identity with rDNA-ITS of Cochliobolus geniculatus (teleomorphic state of C. geniculata) and C. affinis in GenBank after a BLAST search and clustered with them after a phylogenetic analysis. There was no sequence of C. fallax on the BLAST list because ITS sequence of C. fallax had not yet been submitted to any nucleotide databank. Hosokawa et al. concluded that C. fallax and C. affinis are synonyms for C. geniculata (2), which is supported by our results from BLAST and phylogenetic analysis. In view of its relative straight conidia and branching stromata, we suggest that the causal agent of black sheath spot of rice be C. fallax. Although Curvularia spp. were reported as pathogens responsible for black kernel of rice, there is no report of sheath spot of rice caused by Curvularia (3). To our knowledge, this is the first report of a rice sheath disease caused by C. fallax. It seems likely the disease exists in areas beyond Huayuan County. Further field inspection and molecular identification are necessary. References: (1) K. B. Boedijn. Bull. Jard. Bot. Buitenzorg, ser. 3 13:129, 1933. (2) M. Hosokawa et al. Mycoscience 44:227, 2003. (3) S. H. Ou. Page 317 in: Rice Diseases, 2nd Edition, Commonwealth Mycological Institute, Kew, UK, 1985.