A New Rice Disease, Black Sheath Spot, Caused by Curvularia fallax in China.

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.

Identification of WA-type three-line hybrid rice with real-time polymerase chain reaction (PCR) method.

A real-time fluorescent PCR (RTF-PCR) was developed to detect and quantify wild abortive (WA)-type three-line hybrid rice (Oryza sativa L.). The mitochondrial R2₋630 WA gene was reported to be closely related to male sterility in plants, and developed as a molecular maker to identify the cytoplasmic male sterility system of hybrid rice. First, we got the DNA sequence of R2₋630 WA gene in 17 rice species with traditional PCR. Then, a pair of specific primers (P3, P4) and TaqMan fluorescence probe (P3₋14) were designed based on the R2₋630 DNA sequence. The following RTF-PCR was performed on the 17 rice species finally. The results indicate that the probes used here are specific for three-line hybrid rice F1 and male sterile lines. We can even identify a single hybrid seed using the probes, which confirmed that the probes can be applied to the identification and quantification of the WA-type three-line hybrid rice. In addition, the RFT-PCR system can be optimized when the annealing temperature is 60 °C and the Mg²âº concentration is 3.5 mmol/L.

First Report of Artichoke Bacterial Stem Rot Caused by Pectobacterium carotovorum subsp. carotovorum in China.

A new disease on globe artichoke (Cynara scolymus L.) was observed in the springs of 2008 and 2009 and during the spring and fall seasons of 2010 in commercial fields (nearly 1,000 ha) in Changde, Hunan Province, China. Characteristic symptoms were wilting and necrosis of the outermost leaves and dark brown discoloration of the vascular tissue and pith of the stem base. Eventually, the plants wilted and died. Nearly 5, 35, and 4% (2008, 2009, and 2010, respectively) of the artichoke fields were destroyed because of the disease. Manual weeding and cuttings often led to the development of typical soft rot during propagation. To investigate the causal agent of the disease, isolations were made from rotted stems of field artichoke plants on nutrient agar (NA). Bacteria consistently isolated from the diseased tissues formed gray-white, glossy, convex, translucent, and round colonies on NA. The bacterial cells were gram-negative rods with two to eight peritrichous flagella. Ten isolates were negative for oxidase, arginine dehydrolase, H2S, gelatin liquefaction, and tryptophan ammonialyase. Isolates were positive for catalase, reduced NO3 to NO2, indole, glucuroide, galactosidase, Voges-Proskauer test, and ß-galactosidase, along with being facultatively anaerobic and insensitive to erythromycin (40 µg/ml). Negative results were obtained for utilization of maltose, gluconate, and phenylacetic acid, and positive results were obtained from arabinose, glucose, mannose, N-acetyl-glucosamine, mannitol, and sodium citrate for all isolates. Acid was produced from glucose, inositol, rhamnose, melibiose, arabinose, mannitol, sucrose, and amarogentin. All test results were similar to reference strain PCC1000 (GenBank Accession No. JF721959) of Pectobacterium carotovorum subsp. carotovorum. These isolates could also cause soft rot of Chinese cabbage stem, carrot slice, pepper, lettuce and artichoke stems, and tomato and potato slices within 48 h at 28°C in an artificial inoculation test (3). PCR amplification was carried out by utilizing universal 16S rDNA primer pair 16SF/16SR and pel gene primers Y1/Y2 (1). The 16S rDNA and pel gene sequences of isolate HNXDT002 (GenBank Accession Nos. JF721958 and JF721960, respectively) had 99 and 93% nucleotide identity with strains of P. carotovorum subsp. carotovorum (GenBank Accession Nos. U80197 and CP001657, respectively). Pathogenicity was confirmed by needle-stab inoculation (1 × 108 CFU/ml) at the stem on three healthy artichoke plants held at 28°C for 48 h. Sterile distilled water was used as a negative control. Within 72 h after inoculation, water-soaking and soft-rot symptoms were observed on all inoculated artichoke plants, while controls remained healthy. The bacterium was recovered only from rotted stems of inoculated plants. In recent years, P. carotovorum was reported on such plants as Pinellia ternata (4) and Chinese cabbage (2) in China. To our knowledge, this is the first report of bacterial rot disease caused by P. carotovorum subsp. carotovorum on artichoke in China. References: (1) D. J. Brenner et al. Bergey's Manual of Systematic Bacteriology. Vol. 2. Springer, NY, 2005. (2)Y. Fang et al. Acta Microbiol. Sinica 44:136, 2004. (3) H. Yi-Bo et al. Acta Phytopathol. Sinica 37:338, 2007. (4) F. X. Ying et al. Plant Dis. 91:1359, 2007.

First Report of Anthracnose Caused by Glomerella acutata on Chili Pepper in China.

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.