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Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 μmol liter -1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 10 2 CFU ml -1 and 11.86 CFU g -1 , respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 10 2 to 10 4 CFU g -1 . The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.

Zantedeschia aethiopica (L.) Spreng. (calla lily), belonging to family Araceae, is a popular ornamental plant in China. In the summer of 2010, leaves of calla lily with typical symptoms of necrotic lesions were observed in a commercial glasshouse in Beijing, China (116°20' E, 39°44' N). The initial symptoms were circular to subcircular, 1 to 3 mm, and dark brown lesions on the leaf lamina. Under high humidity, lesions expanded rapidly to 5 to 10 mm with distinct concentric zones and produced black sporodochia, especially on the backs of leaves. Later, the infected leaves were developing a combination of leaf lesions, yellowing, and falling off; as a result, the aesthetic value of the plant was significantly impacted. Leaf samples were used in pathogen isolation. Symptomatic leaf tissues were cut into small pieces and surface sterilized with 70% ethanol for 30 s and then in 0.1% mercuric chloride solution for 1 to 3 min. After being washed in sterile distilled water three times, the pieces were plated on potato dextrose agar (PDA) and incubated at 25°C in darkness for 7 days (5). Initial colonies of isolates were white, floccose mycelium and developed dark green to black concentric rings that were sporodochia bearing viscid spore masses after incubating 5 days. Conidiophores branched repeatedly. Conidiogenous cells were hyaline, clavate, and 10.0 to 16.0 × 1.4 to 2.0 μm. Conidia were hyaline, cylindrical, both rounded ends, and 6.0 to 8.2 × 1.9 to 2.4 μm. Morphological characteristics of the fungus were consistent with the description of Myrothecium roridum Tode ex Fr. (3,4). To confirm the pathogenicity, three healthy plants of calla lily were inoculated with a conidial suspension (1 × 10 6 conidia per ml) brushed from a 7-day-old culture of the fungus. Control plants were sprayed with sterile water. The inoculated plants were individual with clear plastic bags and placed in a glass cabinet at 25°C. After 7 days, all inoculated leaves developed symptoms similar to the original samples, but control plants remained disease free. Re-isolation and identification confirmed Koch's postulates. For molecular identification, genomic DNA of a representative isolate (MTL07081001) was extracted by modified CTAB method (1), and the rDNA-ITS region was amplified by using primers ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3). The 465-bp amplicon (GenBank Accession No. KF761293) was 100% identity to the sequence of M. roridum (JF724158.1) from GenBank. M. roridum has an extensive host range, covering 294 host plants (2). To our knowledge, this is the first record of leaf spot caused by M. roridum on calla lily in China. References: (1) F. M. Ausubel et al. Current Protocols in Molecular Biology. John Wiley & Sons Inc, New York, 1994. (2) D. F. Farr and A. Y. Rossman, Fungal Databases. Syst. Mycol. Microbiol. Lab., ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , October 2013. (3) M. T. Mmbaga et al. Plant Dis. 94:1266, 2010. (4) Y. X. Zhang et al. Plant Dis. 95:1030, 2011. (5) L. Zhu et al. J. Phytopathol. 161:59, 2013.

A suspect bacterial leaf spot on vegetable sponge gourd (Luffa cylindrical (L.) Roem.) was found in a commercial greenhouse in Pi County, Chengdu City, Sichuan Province, China, in February 2011. Approximately 20 to 30% of plants were affected, causing serious economic loss. Symptoms occurred only on seedlings and consisted of water-soaked, irregularly shaped, black lesions on the surface and margins of cotyledons. A bacterium was consistently isolated on nutrient agar from diseased leaf tissues that had been surface disinfected in 70% ethyl alcohol for 30 s. The bacterium produced small gray colonies with smooth margins, was gram negative, fluoresced on King's B medium, and showed pectolytic activity when inoculated on potato slices. The partial sequences of 16SrRNA gene (1,377 bp) of the bacterium (GenBank Accession No. KC762217), amplified by using universal PCR primers 16SF (5'-AGAGTTTGATCCTGGCTCAG-3') and 16SR (5'-GGTTACCTTGTTACGACTT-3'), shared 100% similarity with that of Pseudomonas cichorii (GenBank Accession No. HM190228). The vegetable sponge gourd isolate was also identified by using the Biolog Microbial Identification System (version 4.2, Biolog Inc., Hayward, CA) as P. cichorii with the following characteristics (1): negative for arginine dihydrolase, gelatin liquefaction, and N 2 production. Positive reactions were obtained in tests for catalase, oxidase, potato rot, utilization of melibiose, and mannitol. Tests were negative for utilization of sucrose, trehalose, D-arabinose, raffinose, cellobiose, and rhamnose. A pathogenicity test was conducted on 4-week-old vegetable sponge gourd plants by spray-inoculation with 10 8 CFU/ml sterile distilled water on the leaves of 15 vegetable sponge gourd plants and by needle puncture on the stems of 15 other plants with P. cichorii, respectively. Control plants were misted with sterile distilled water or punctured on the stem with a clean needle. Plants were placed in a greenhouse maintained at 28 ± 2°C with relative humidity of 80 to 85%. Symptoms, the same as seen on the original diseased plants, developed after 7 to 10 days on inoculated plants. Control plants remained healthy. The bacterium was readily re-isolated from inoculated plants and identified as P. cichorii using P. cichorii-specific primer hrpla/hrp2a (1). To our knowledge, this is the first report of P. cichorii causing disease on commercially grown vegetable sponge gourd in China. This new finding will provide the basis for developing resources for diagnostics and management, including screening varieties for resistance. References: (1) S. Mazurier et al. J. FEMS Microbiol. Ecol. 49:455, 2004. (2) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. APS Press, St. Paul, MN, 2001.

Grafting has been widely and effectively used in cucumber (Cucumis sativus) cultivation for approximately 30 years in China to avoid Fusarium wilt caused by Fusarium oxysporum Schl. f. sp. cucumerinum Owen. In greenhouses, 90% of cucumbers are grafted onto pumpkin (Cucurbita moschata) rootstock. However, in March 2009, a severe crown rot causing yellowing and wilting of the leaves was observed on grafted cucumber in a large number of greenhouses in Lingyuan, western Liaoning Province in China. Symptoms consisted of dark brown, water-soaked lesions and a dense, white mycelial mat at the base of the stem. Lingyuan is the largest district for cucumber cultivation using grafting techniques in solar greenhouses in China. In 30 surveyed greenhouses in Sanshijiazi Village in the city of Lingyuan, the incidence of affected plants ranged from 10 to 40%, which caused serious economic losses. Fusarium spp. were isolated from the surface-sterilized basal stems of symptomatic plants on potato dextrose agar and incubated on potato sucrose agar for 4 days at 25°C. Colonies of the isolates produced a brown pigmentation and sparse, aerial mycelia, with a cream color on the underside. Conidiophores were elongated and branched or unbranched. Microconidia were abundant, hyaline, ellipsoid to ovoid, and 6 to 14 × 2.5 to 3.5 μm. Macroconidia were cylindrical, abundant, mostly two to six septate, 22 to 63 × 3.2 to 5.0 μm, with the apical cell rounded and blunt, and the basal cell rounded. On the basis of morphological characteristics, the fungus was identified as F. solani (C. Booth). For confirmation, the internal transcribed spacer region of rDNA was amplified and sequenced. A 449-bp sequence shared 99% homology with that of a F. solani GenBank accession previously reported from Japan (No. AF150473.1). The new sequence was deposited in GenBank (Accession No. HM015882). Pathogenicity of three isolates was determined in two experiments using different methods of inoculation. In one, 30 seedlings of pumpkin (C. moschata) with one true leaf each were inoculated by dipping their roots in a suspension of 10 6 spores ml -1 , while control plants were mock inoculated with sterile water. Plants were then potted in a sterile mix of peat moss and vermiculite (2:1 vol/vol). In the other, pregerminated pumpkin seeds were sown in the same medium with a conidial suspension added at a rate of 10 6 spores ml -1 , while other seeds were sown in sterile soil as controls. Plants for both experiments were maintained in a greenhouse at 25°C. Twelve days after inoculation, inoculated plants in both experiments showed a cortical rot on the crown and stem base with a brown, water-soaked appearance. Twenty-one days later, inoculated plants developed wilting and yellowed leaves. Disease incidence was 100%. No symptoms occurred on the control plants. Both experiments were repeated once with the same results. The pathogen was recovered from symptomatic tissue, confirming Koch's postulates. F. solani has been previously reported to cause root rot on cucurbit in California (2) and crown rot on grafted cucumber in the Netherlands (1). To our knowledge, this is the first report of crown rot of grafted cucumber caused by F. solani in China. References: (1) L. C. P. Kerling and L. Bravenboer. Neth. J. Plant Pathol. 73:15, 1967. (2) T. A. Tousson and W. C. Snyder. Phytopathology 51:17, 1961.

Common bean (Phaseolus vulgaris L.) is an economically important crop in China. In June 2008, a new foliar disease was observed on beans in Shunyi District, Beijing, China. The disease occurred in approximately 15% of the plants in a commercial field. Leaf spots were circular to irregular, reddish brown, zonate, and 8 to 20 mm in diameter. Black sporodochia with white tuffs were present on older lesions and black spore masses were present in moist weather. Ten isolates recovered from lesions produced white, floccose colonies and spore masses after 4 days on potato dextrose agar. The rod-shaped, hyaline conidia had rounded ends and averaged 6.8 × 2.5 μm. All characteristics were consistent with the description of Myrothecium roridum Tode ex Fr. (1). The internal transcribed spacer regions of one isolate were sequenced and deposited in GenBank (Accession No. GQ 381291). Sequences of the isolate from bean in China were 98% similar to sequences of M. roridum in GenBank. To determine pathogenicity, 30 healthy seedlings of common bean were inoculated by spraying a 1 × 10 5 conidia ml -1 suspension of M. roridum onto the foliage. Ten seedlings were sprayed with sterile water and served as controls. Plants were kept in a humid chamber at 27°C overnight and then placed in a growth chamber. After 6 days, the symptoms described above were observed on leaves in all inoculated plants, whereas symptoms did not develop on the control plants. The pathogen was reisolated from inoculated leaves, fulfilling Koch's postulates. There is one report of M. roridum on soybean in Korea (2). To our knowledge, this is the first report of Myrothecium leaf spot on common bean in China. References: (1) M. Fitton et al. CMI Mycol. Pap. No. 253, 1970. (2) K. J. Yum et al. Plant Pathol. J. 6:313, 1990.

Balsam pear (Momordica charantia L.) is an economically important vegetable in China with increasing interest as a medicinal plant. In December of 2006, a new foliar disease caused by Corynespora cassiicola was observed on balsam pear growing in greenhouses in Shouguang City, Shandong Province, China. The disease occurred on 35% or less of the plants. Leaves of affected plants developed off-white halos surrounding circular lesions that were 1 to 5 mm broad. The lesions became dark brown, necrotic with concentric rings, and up to 15 mm in diameter. Severely affected plants eventually wilted and defoliated. Pieces of tissue from the leading edges of lesions were disinfected in 1% NaOCl for 1 min, rinsed in sterile water, and plated on potato dextrose agar. Colonies of the fungus were gray to dark green. Conidiophores were erect and simple, pale brown to brown, and 100 to 450 μm long and 3 to 8 μm wide. Conidia were obclavate to cylindrical, pale olivaceous brown to dark brown, smooth, 35 to 100 × 8 to 12 μm, and were produced in chains. On the basis of these characteristics, the fungus was identified as Corynespora cassiicola (1). The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and deposited in GenBank (Accession No. GQ381292). It was an exact match for a sequence of C. cassiicola previously deposited (Accession No. EU364555). To confirm pathogenicity, 30 1-month-old healthy seedlings of balsam pear were inoculated by spraying a suspension of conidia (1 × 10 5 conidia per ml) of one isolate of C. cassiicola until runoff. Ten seedlings were sprayed with sterile water as controls. Plants were kept in a humidity chamber at 27°C overnight and then placed in a growth chamber at 27°C. After 7 days, symptoms identical to those described above were observed, while no symptoms developed on the control plants. The pathogen was reisolated from inoculated leaves. C. cassiicola causes foliar diseases on many plants, including tomato, eggplant, soybean, and cucumber (2). There is one report on balsam pear in Korea (3). To our knowledge, this is the first report of target leaf spot caused by C. cassiicola on balsam pear in China. References: (1) M. B. Ellis. CMI Mycol. Pap. No. 65, 1957. (2) M. B. Ellis et al. CMI Mycol. Pap. No. 303, 1971. (3) J. H. Kwon et al. Plant Pathol. J. 21:164, 2005.