G. Q. Li

Intergeneric transfer of ribosomal genes between two fungi

BackgroundHorizontal gene transfer, also called lateral gene transfer, frequently occurs among prokaryotic organisms, and is considered an important force in their evolution. However, there are relatively few reports of transfer to or from fungi, with some notable exceptions in the acquisition of prokaryotic genes. Some fungal species have been found to contain sequences resembling those of bacterial genes, and with such sequences absent in other fungal species, this has been interpreted as horizontal gene transfer. Similarly, a few fungi have been found to contain genes absent in close relatives but present in more distantly related taxa, and horizontal gene transfer has been invoked as a parsimonious explanation. There is a paucity of direct experimental evidence demonstrating the occurrence of horizontal gene transfer in fungi.ResultsWe found a fungal field isolate from rice (Oryzae sativa) that contains ribosomal DNA sequences from two species of fungal rice pathogens (Thanatephorus cucumeris and Ceratobasidium oryzae-sativae). This field isolate has four types of ribosomal DNA internal transcribed spacers (ITS), namely pure ITS of C. oryzae-sativae, which was dominant in this field isolate, pure ITS of T. cucumeris, and two chimeric ITS, with ITS1 derived from C. oryzae-sativae and ITS2 from T. cucumeris, or ITS1 from T. cucumeri s and ITS2 from C. oryzae-sativae. The presence of chimeric forms indicates that the intergeneric hybrid was not merely composed of nuclei from the parental species, but that nuclear fusion and crossing over had taken place.ConclusionHyphae of T. cucumeris and C. oryzae-sativae are vegetatively incompatible, and do not successfully anastomose. However, they parasitize the same host, and perhaps under the influence of host enzymes targeted to weaken pathogen cells or in dying host plant tissue, the fungal hyphae lost their integrity, and normal vegetative incompatibility mechanisms were overcome, allowing the hyphae to fuse. Based on the presence of other similarly anomalous isolates from the field, we speculate that these types of intergeneric hybridization events and occurrences of horizontal gene transfer may not be so rare in the field.

Morphological and phylogenetic identification of Botrytis sinoviticola, a novel cryptic species causing gray mold disease of table grapes (Vitis vinifera) in China.

Seventy-five isolates of Botrytis collected from table grapes (Vitis vinifera) with gray mold symptoms in China were identified based on morpho-cultural characteristics on potato dextrose agar (20 C) and/or phylogenetic analysis using the sequences of three nuclear genes (G3PDH, HSP60, RPB2). Isolates of different species of Botrytis were compared with fenhexamid sensitivity, Bc-hch gene-RFLP haplotyping and pathogenicity to V. vinifera. The 75 isolates comprise two species, B. cinerea (63 isolates) and an undescribed Botrytis sp. (12 isolates) described here as Botrytis sinoviticola Zhang et al. sp., nov. Both B. sinoviticola (Bs) and B. cinerea (Bc) were found to have 20 C optimum for mycelial growth and 25 C for conidial germination. Sensitivity to fenhexamid was significantly greater (P < 0.05) for Bc (EC50 = 0.04 ± 0.01 μg mL−1) than for Bs (EC50 = 0.08 ± 0.02 μg mL−1). Digestion of the PCR amplicons of the Bc-hch gene with Hha I generated two haplotypes, Group I haplotype for Bs and Group II haplotype for Bc. Bs infected table grapes (leaves, berries) only through wounds, whereas Bc infected both injured and non-injured tissues of table grapes. This study suggests that Bs is a cryptic species sympatric with Bc on table grapes in China.

First report of Botrytis pseudocinerea causing gray mold on tomato (Lycopersicon esculentum) in central China.

A tomato field in Qianjiang County, Hubei Province, China, was surveyed for gray mold in April 2013. Diseased leaves with V-shaped lesions along the margin and masses of grayish hyphae and conidia on the surface were collected from different plants. Eight Botrytis isolates were obtained from eight symptomatic leaves by plating the conidia from each leaf onto potato dextrose agar (PDA). A representative isolate (No. 116) was compared to two reference isolates, B. cinerea B05.10 (from Z. H. Ma, Zhejiang University, China) and B. pseudocinerea 10091 (from A. S. Walker, INRA, France) for morpho-cultural and molecular features. On PDA at 20°C, isolate 116 grew 13.8 mm/day (n = 9), which was similar to those of isolates 10091 (13.7 mm/day), and B05.10 (14.6 mm/day). The isolates all formed black sclerotia of similar shape and size (2 to 13 × 1 to 7 mm). To induce conidia production, the isolates each were inoculated onto tomato fruit (cv. Hezuo 903, Jiangsu Seed Co., China) using colonized agar plugs (each 6 mm in diameter), with four plugs per fruit and four fruits tested per isolate. After incubation of the fruit for 10 days (20°C), abundant conidia were produced on the fruit surface. The conidial size of isolate 116 (6.8 to 14.3 × 6.1 to 10.2 μm) was similar to that of isolates 10091 (7.7 to 12.2 × 7.0 to 9.8 μm) and B05.10 (7.0 to 14 × 6.6 to 10.5 μm). The three isolates were indistinguishable morphologically. The sequences of each of four nuclear genes (Bc-hch, G3PDH, HSP60, and MS547) and the microsatellite Bc6 locus (1,4) were determined and analyzed for each isolate. DNA was extracted from mycelium of each isolate and used as a template to amplify each gene by PCR using specific primers (1,2,4). Bc-hch-RFLP genotyping of the 1,171-bp amplicon (2,4) showed that isolates 116 and 10091 had a 601-bp DNA product, whereas B05.10 had a 517-bp product. The G3PDH, HSP60, and MS547 sequences of isolate 116 (GenBank Accession Nos. KJ534270, KJ534271, and KJ534273, respectively) and those of B. aclada, B. calthae, B. cinerea, B. pseudocinerea, and Sclerotinia sclerotiorum (3) were used for phylogenetic analysis. Isolate 116 and eight B. pseudocinerea isolates formed a subclade with 100% bootstrap support. Furthermore, two DNA markers, 86 bp for isolates 116 and 10091 vs. 170 bp for B05.10 were identified at the Bc6 locus. These results suggest that isolate 116 belongs to B. pseudocinerea (1,4). Pathogenicity of each isolate was tested by inoculation of each of five newly expanded tomato leaves on a 50-day-old plant (cv. Hezuo 903, Jiangsu Seed Co) with a 20-μl droplet of a conidial suspension (1 × 105 conidia/ml), using a pipette. Five noninoculated control leaves were treated similarly with water. The plants were all maintained at 20°C and 100% RH for 72 h, and lesion diameter was then measured. While control leaves remained asymptomatic, leaves inoculated with isolates 116, 10091, and B05.10 developed necrotic lesions averaging 19 to 20 mm in diameter. A fungus re-isolated from the lesions on isolate-116-inoculated leaves formed colonies with morphology identical to that of the original isolate 116. To our knowledge, this is the first report of B. pseudocinerea on tomato in China. The remaining seven isolates were identified as B. cinerea based on Bc-hch-RFLP genotyping (data not shown), suggesting that B. pseudocinerea may infect tomato plants at a low frequency in this region of China. References: (1) E. Fournier et al. Mol. Ecol. Notes 2:253, 2002. (2) E. Fournier et al. Mycologia 95:251, 2003. (3) P. R. Johnston et al. Plant Pathol. 63:888, 2014. (4) A. S. Walker et al. Phytopathology 101:1433, 2011.

Botrytis pyriformis sp. nov., a novel and likely saprophytic species of Botrytis.

A novel species of Botrytis from Sedum sarmentosum was described based on morphology and analyses of DNA sequences of nuc rDNA ITS regions and three nuclear genes (G3PDH, HSP60, RPB2). Meanwhile pathogenicity in 32 plant species, response to temperature for growth and conidial germination for the species were determined. The Botrytis species was named Botrytis pyriformis sp. nov. It was characterized by formation of grayish mycelia, brownish conidia and melanized sclerotia on PDA. The conidia are pear-shaped, melanized and covered with abundant villiform appendages on the conidial surface. Comparison of the ITS sequences confirmed its placement in the genus Botrytis. Phylogenetic analysis based on DNA sequences of G3PDH, HSP60 and RPB2 genes indicated that B. pyriformis and other 30 Botrytis species form a monophyletic clade, which was further divided into three subclades. Subclade I comprised B. pyriformis alone, whereas subclades II and III comprised six and 24 Botrytis species, respectively. Botrytis pyriformis could not infect 32 plant species including S. sarmentosum, possibly due to deficiency in formation of infection cushions. This study presents a formal description and illustrations for B. pyriformis and provides experimental evidence, indicating that B. pyriformis might be a saprophytic species.

First Report of Leptosphaeria biglobosa Causing Black Leg on Raphanus sativus in Central China

Chinese radish (Raphanus sativus) is an important vegetable grown widely in China. In 2010 to 2013, surveys for Leptosphaeria spp. on cruciferous vegetables were conducted in 17 counties in Hubei Province, China. Black leg symptoms on tuber roots and basal stems of radish were observed in Hanchuan, Jingmen, and Macheng counties. Disease incidence ranged from 2 to 25% in 10 surveyed radish fields. Five fungal isolates were obtained from diseased radish plants by surface-sterilizing radish tissue (5% NaOCl for 90 s, and then rinsed in sterilized water three times) and plating onto potato dextrose agar (PDA) plates incubated at 20°C. The isolations produced fluffy white colonies with a yellow pigment, and black-brown, globose pycnidia with pink conidial ooze formed after 10 days. Pycnidia were 150 to 200 × 80 to 100 μm. Conidia were hyaline, cylindrical, and 4 to 5 × 2 μm. The cultural and morphological characteristics of the isolates matched the description for Phoma lingam, anamorph of Leptosphaeria maculans and L. biglobosa (3). All five isolates were identified by PCR assay using the species-specific primers LbigF, LmacF, and LmacR (1), and isolate HCLB-1 was identified further by PCR cloning and analysis of the sequences coding for actin, β-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (3,4). Genomic DNA was extracted from mycelium of each isolate harvested from 7-day-old cultures in V8 broth using the CTAB method (5). A 444-bp DNA fragment was detected by PCR assay, suggesting that all five isolates belonged to L. biglobosa rather than L. maculans as the latter generates a 331-bp DNA fragment (1). The HCLB-1 sequences for ITS (587 bp, GenBank Accession. No. KC880981), actin (899 bp, KF307762), and β-tubulin (432 bp, KF220296) genes were 99 to 100% identical to those of L. biglobosa isolates in GenBank. All five isolates were tested for pathogenicity on R. sativus cultivars Duan Ye 13 and Qi Ye Hong. Cotyledons of 10-day-old radish seedlings and post-harvest mature roots were wounded using a sterilized needle, and 10 μl of a conidial suspension (1 × 107 conidia/ml) of each isolate was pipetted onto the wounded area on each cotyledon or root, respectively, with 12 cotyledons (= 24 wounded sites) and 1 root (= 6 wounded sites) inoculated/isolate. One wounded root and 12 wounded cotyledons inoculated with water were used as control treatments. Treated roots and seedlings were incubated at 20°C and 100% RH in the dark for 7 days, and under a 12 h light/12 h dark cycle for 12 days, respectively. While the control roots and cotyledons remained asymptomatic, the roots and cotyledons inoculated with all five test isolates formed black cankers and necrotic lesions, respectively, in the inoculated wounds. A fungus re-isolated from symptomatic roots and cotyledons resembled the original isolates in colony morphology and the 444-bp DNA fragment detected by PCR assay. No fungus was isolated from control seedlings or roots. Thus, L. biglobosa appears to be the causal agent of black leg observed on radish in Hubei, China. L. biglobosa was reported to infect wild radish (R. raphanistrum) (2). To our knowledge, this is the first report of L. biglobosa causing black leg on R. sativus. References: (1) S. Y. Liu et al. Plant Pathol. 55:401, 2006. (2) A. Maxwell and J. K. Scott. Australas. Plant Pathol. 37:523, 2008. (3) L. Vincenot et al. Phytopathology 98:321, 2008. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990. (5) J. Zhang et al. Mycologia 102:1114, 2010.

First Report of Sclerotinia minor on Brassica rapa subsp. pekinensis in Central China

Chinese cabbage (Brassica rapa subsp. pekinensis Hanelt) is a leafy vegetable widely grown in China. In December 2012 to March 2013, a leaf rot disease was observed on the lower part of cabbage leaves in a field in Xianning, Hubei Province, China, with the incidence of 6.3% in that field. The diseased leaves showed water-soaked rot and brown symptoms at the top surface. White fluffy mycelia and small black sclerotia were produced on the lesion surface. Cabbage leaf tissues from the disease/healthy-bordering areas and the sclerotia from the lesions were separately surface-sterilized in 75% ethanol (v/v) for 30 s, followed by rinsing three times in sterilized water. The surface-sterilized tissues and sclerotia were placed on potato dextrose agar (PDA) and incubated at 22°C. Individual emerging fungal colonies from the leaf tissue pieces and the sclerotia were transferred to new 9-cm-diameter PDA plates and incubated for 15 days. A total of 40 isolates (20 from diseased tissue and 20 from sclerotia) were obtained. All the isolates grew rapidly on PDA with an average growth rate of 2.2 cm/day and produced abundant sclerotia on the colony surface (1,179 sclerotia/plate on average). None of the isolates produced conidia and any other spores in the PDA cultures. Mature sclerotia were black, irregular, spherical or elliptical, had a diameter of 0.5 to 1 mm, and easily detached from the colonies. The cultural and morphological characteristics of the isolates matched the description for Sclerotinia minor Jagger (3). Two isolates, A1 (from leaf tissue) and S2 (from a sclerotium), were further identified by analysis of the ITS region (ITS1-5.8S rDNA-ITS2) using the primer pair ITS1/ITS4. The resulting 540-bp DNA sequences (GenBank Accession Nos. KC836493 for A1 and KC836494 for S2) shared 100% identity S. minor isolate 62907 (JF279880). Pathogenicity of the isolates A1 and S2 was tested by inoculating detached cabbage leaves with mycelial agar plugs removed from the colony margin of the 3-day-old cultures. Isolates A1 and S2 were each inoculated on three leaves with three plugs per leaf. Three cabbage leaves inoculated with PDA plugs were treated as a control. The treated leaves were covered with plastic films to maintain high humidity (>90% RH) and incubated at 22°C for 72 h under the regime of 12 h light/12 h dark. Results showed that while the control leaves remained healthy, brown and water-soaked lesions appeared around the mycelial agar plugs of each isolate. Average lesion diameters were 47.5 mm for A1 and 47.8 mm for S2. Abundant small sclerotia were produced on necrotic leaf lesions after 7 days. The fungus in diseased leaf tissues was re-isolated and the morphological characteristics of the resulting fungus were the same as S. minor isolated from infected field-grown cabbages. Therefore, S. minor is the causal agent for the leaf rot disease on Chinese cabbage. S. minor has been reported to infect a few plant species in the genus Brassica, including B. rapa subsp. oleifera (3), B. oleracea var. gemmifera (3), B. napus (2), B. oleracea var. capitata (3), B. oleracea var. botrytis (3), and B. rapa (3). It was found on B. rapa subsp. pekinensis in Korea (1). To our knowledge, this is the first report of S. minor on B. rapa subsp. pekinensis in China. References: (1) W. D. Cho and H. D. Shin. Page 779 in: List of Plant Diseases in Korea, 4th ed. Korean Society of Plant Pathology, 2004. (2) S. A. Gaetán and M. Madia. Plant Dis. 92:172, 2008. (3) M. S. Melzer et al. Can. J. Plant Pathol. 19:272, 1997.

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

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.

First Report of Garlic Leaf Blight Caused by Botrytis porri in China

In the spring of each year from 2007 to 2009, a leaf blight of garlic (Allium sativum L.) was observed in more than 50 fields in Zhushan County of Hubei Province, China. Gray mold was observed on many of the blighted garlic leaves. The percentage of garlic plants with blight and gray mold symptoms ranged from 10 to 50% with one to three blighted leaves on each plant, which severely reduced the yield of young garlic plants (produced as a green vegetable). Ten strains of a Botrytis sp. were isolated from symptomatic garlic leaves collected from 10 different fields. These strains were inoculated onto potato dextrose agar (PDA) in petri dishes and incubated at 20°C for 3 to 15 days for observation of colony characteristics and morphology of sclerotia and conidia. All 10 Botrytis strains formed flat and ropy mycelia (mycelial strands) on PDA. Abundant sporulation with a gray powdery appearance was observed on the colonies after 6 days. Conidiophores were erect with alternate branches at the top and ranged from 907 to 1,256 μm high. Conidia were borne in botryose clusters on conidiophores, obovate, and 10.4 to 17.6 × 7.6 to 13.1 μm with an average length/width ratio of 1.36. Discrete sclerotia were produced on each colony after 15 days. Mature sclerotia were black, cerebriform and convoluted, and 1.9 to 9.1 × 1.6 to 6.5 mm. Morphological characteristics of the colonies, conidia, and sclerotia of these Botrytis strains were similar to Botrytis porri Buchwald (1,2). Strain GarlicBC-16 was selected as a representative for molecular identification. Genomic DNA was extracted from mycelia of this strain and used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA using primer pair ITS1/ITS4. A 539-bp amplicon was obtained and sequenced (GenBank Accession No. EU519206). Excluding the flanking regions, the amplicon contained a 453-bp ITS sequence (ITS1 + 5.8S rDNA + ITS2) 100% identical to the ITS sequence of strain MUCL3234 of B. porri (GenBank Accession No. AJ716292). Pathogenicity of strain GarlicBC-16 was tested by inoculation of 10 young and fully expanded garlic leaves taken from 100-day-old garlic plants with mycelial agar plugs (three plugs per leaf and spaced by 5 cm). Ten garlic leaves inoculated with agar plugs of PDA alone served as controls. Inoculated garlic leaves were covered with a plastic film (0.1 mm thick; Gold Mine Plastic Industrial Ltd. Jiangmen, China) and incubated at 20°C with 12-h light/12-h dark. Control leaves remained healthy after 48 to 120 h, but gray, water-soaked lesions appeared on leaves inoculated with strain GarlicBC-16 after 48 h. The average lesion length reached 27.3 mm after 90 h and abundant sporulation was produced on necrotic leaf lesions after 120 h. Microscopic examination showed the shape and size of conidia that formed on garlic leaf lesions were similar to those formed by strain GarlicBC-16 on PDA. On the basis of the isolation, identification, and pathogenicity tests, B. porri was determined to be the causal agent of garlic leaf blight in Zhushan County. B. porri has been reported to cause neck rot of leek (A. porrum) (1) and clove rot of garlic (2), and has been isolated from asymptomatic foliage and seeds of A. cepa (3). To our knowledge, this is the first report of garlic leaf blight caused by B. porri in China. References: (1) S. K. Asiedu et al. Plant Dis. 70:259, 1986. (2) F. M. Dugan et al. J. Phytopathol. 155:437. 2007. (3) L. J. du Toit et al. Plant Dis. 86:1178, 2002.

First Report of Leptosphaeria biglobosa Causing Black Leg on Brassica campestris ssp. chinensis var. purpurea in Central China

Purple cai-tai (Brassica campestris ssp. chinensis var. purpurea) is a traditional vegetable widely grown in southern China. In 2012 and 2013, black leg disease was observed on purple cai-tai in three surveyed cities (Jingzhou, Qianjiang, and Huanggang) in Hubei Province of China. Disease incidence ranged from 5 to 88% in eight surveyed fields. White cankers occurred on basal stems and numerous black pycnidia and pink conidia were present on the stem surface. Surface-sterilized (5% NaOCl for 90 s, rinsed in sterilized water three times) stem pieces were plated on potato dextrose agar (PDA) and incubated at 20°C and 12 h light/12 h dark for 7 days. A total of 22 isolates were obtained. All of the isolates appeared similar in colony morphology on PDA (20°C, 7 to 10 days), producing yellow pigment and black-brown, globose pycnidia containing cylindrical hyaline conidia (4 to 5 × 2 μm). These characteristics matched the description for Phoma lingam, the anamorph Leptosphaeria maculans and L. biglobosa (2). Species-specific primers LbigF, LmacF, and LmacR (1) were used in PCR-based identification of the isolates. A 444-bp DNA fragment characteristic of L. biglobosa was amplified from DNA extracted from all of the collected isolates. DNA amplification from the isolate UK-1 of L. maculans from B. napus in Hertfordshire of the United Kingdom yielded a 331-bp fragment. Two isolates, HGHCT2-1 and HGHCT2-2, were further identified by cloning and analysis of the ITS sequences and the partial sequences encoding β-tubulin and actin (3,4). The ITS sequences (586 bp, GenBank Accession. Nos. KF371660 and KF371661) were 100% identical to L. biglobosa brassicae strain UK28 (DQ133893). The DNA sequences for β-tubulin (479 bp, KF307760 and KF307761) and actin (899 bp, KF307758 and KF307759) were 99 and 100% identical to the partial β-tubulin gene sequence (AY748997) and the partial actin gene sequence (AY748949) of the L. biglobosa brassicae strain 2379-4, respectively. Pathogenicity of six randomly selected isolates was determined on two purple cai-tai cvs. Wanzi Qianhong and Jiu Yue Xian. Cotyledons of 10-day-old seedlings grown in potting mix in pots were pricked with a sterilized needle, and each wound was inoculated with 10 μl of conidial suspension (1 × 107 conidia/ml) of an isolate or 10 μl sterilized water (control). There were 12 cotyledons for each isolate and control. The experiment was repeated once. The treated seedlings were incubated at 20°C in an incubator under 12 h light/12 h dark for 12 days. The control cotyledons were healthy, but necrotic lesions were developed on the cotyledons that were inoculated with L. biglobosa and formation of pycnidia was observed on some lesions. Fungi re-isolated from the lesions were similar to the original L. biglobosa isolates both in colony morphology on PDA and in species-specific PCR testing. No fungi were isolated from the control cotyledons. This is the first report of L. biglobosa causing black leg on B. campestris ssp. chinensis var. purpurea in central China. The finding will be useful for understanding of the epidemiology of black leg on cruciferous crops and for management of this disease. References: (1) S. Y. Liu et al. Plant Pathol. 55:401, 2006. (2) R. A. Shoemaker and H. Burn. Can J Bot. 79:412, 2001. (3) L. Vincenot et al. Phytopathology 98:321, 2008. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.