First Report of Lasiodiplodia theobromae Associated with Stem Canker of Cassia fistula in Guangxi, South China.

Cassia fistula, a member of the Fabaceae, known as the golden shower tree, is native to South Asia. It is now distributed worldwide and is popular as an ornamental plant as well as being used in herbal medicine. In October 2013, symptoms of stem canker were observed on C. fistula in a nursery (108°38' E, 22°87' N) in Nanning, Guangxi, China. The symptoms began as small brown lesions, which enlarged over several months to long, striped, slightly sunken lesions, 1 to 9 cm in width and 16 to 135 cm in length. The conspicuous cankers had vertical cracks outlining the canker and evenly spaced horizontal cracks, eventually resulting in whole plants dying back. The cankers were found on 90% of six-year-old plants in this nursery and were also observed in other plantings. On potato dextrose agar (PDA), isolates with similar morphological characteristics were consistently recovered from symptomatic plant tissues after surface sterilization in 75% ethanol for 30 sec and then in 0.1% mercuric chloride for 2 min. Over 100 conidia were examined from three isolates and were found to be elliptical and hyaline when immature, becoming dark brown, one-septate, and longitudinally striate when mature and ranging from 20 to 31 × 11 to 16 µm (average 25.5 × 13.6 µm). The rDNA internal transcribed spacer (ITS) region of isolate LC-1 was sequenced (GenBank Accession No. KM387285), and it showed 100% identity to Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (GenBank KC964548), confirming the morphological identification (2) as L. theobromae (also known as Botryosphaeria rhodina (Cooke) Arx). A culture of this isolate has been preserved in the Guangxi Academy of Agricultural Sciences fungal collection. The pathogenicity of the isolate was tested on healthy twigs and branches of C. fistula trees in a field setting at Guangxi Agricultural Vocational-Technical College, Nanning, Guangxi, in June and August 2014. For each treatment, five green twigs and five 2-year-old branches were used. Five adjacent needle punctures were made on each branch with a sterilized needle. A mycelial plug was then placed on the wound of each branch and wrapped with Parafilm. Control twigs were treated with sterile PDA plugs. One week later, typical lesions were observed on the inoculated branches, with symptoms becoming more extensive after two weeks, but no symptoms were seen on the controls. Koch's postulates were fulfilled by re-isolation of L. theobromae from diseased branches. L. theobromae is recognized as an important wood pathogen and has been reported to cause cankers, dieback, and fruit and root rots in over 500 different hosts, including perennial fruit and nut trees, vegetable crops, and ornamental plants (2). The fungus has been reported on C. fistula in India since the 1970s (1); however, to our knowledge, this is the first report of L. theobromae infecting C. fistula in China. References: (1) R. S. Mathur. The Coelomycetes of India. Bishen Singh Mahendra Pal Singh, Delhi, India, 1979. (2) J. R. Úrbez-Torres et al. Plant Dis. 92:519, 2008.

First Report of Leaf Spot Disease Caused by Glomerella magna on Lobelia chinensis in China.

Lobelia chinensis is a perennial herbaceous plant in the family Campanulaceae that is native to China, where it grows well in moist to wet soils. It is commonly used as a Chinese herbal medicine. In May 2012, symptoms of leaf spot were observed on leaves of L. chinensis in Nanning, Guangxi Zhuang Autonomous Region, China. The leaf lesions began as small, water-soaked, pale greenish to grayish spots, which enlarged to gray to pale yellowish spots, 4 to 6 mm in diameter. At later stages, numerous acervuli appeared on the lesions. Acervuli were mostly epiphyllous, and 40 to 196 µm in diameter. On potato dextrose agar (PDA), a fungus was consistently recovered from symptomatic leaf samples, with a 93% isolation rate from 60 leaf pieces that were surface sterilized in 75% ethanol for 30 s and then in 0.1% mercuric chloride for 45 s. Three single-spore isolates were used to evaluate cultural and morphological characteristics of the pathogen. Setae were two to three septate, dark brown at the base, acicular, and up to 90 µm long. Conidia were long oblong-elliptical, guttulate, hyaline, and 11 to 20 × 4.1 to 6.3 µm (mean 15.2 × 5.1 µm). These morphological characteristics of the fungus were consistent with the description of Colletotrichum magna (teleomorph Glomerella magna Jenkins & Winstead) (1). The rDNA internal transcribed spacer (ITS) region of one isolate, LC-1, was sequenced (GenBank Accession No. KC815123), and it showed 100% identity to G. magna, GenBank HM163187.1, an isolate from Brazil cultured from papaya (2). Although KC815123 was identified as G. magna, it shows 99% identity to GenBank sequences from isolates of C. magna, and more research is needed to elucidate the relationships between these taxa, especially with consideration to host specificity. Pathogenicity tests were performed with each of the three isolates by spraying conidial suspensions (1 × 106 conidia/ml) containing 0.1% Tween 20 onto the surfaces of leaves of 30-day-old and 6- to 8-cm-high plants. For each isolate, 30 leaves from five replicate plants were treated. Control plants were treated with sterilized water containing 0.1% Tween 20. All plants were incubated for 36 h at 25°C and 90% relative humidity in an artificial climate chamber, and then moved into a greenhouse. Seven days after inoculation, gray spots typical of field symptoms were observed on all inoculated leaves, but no symptoms were seen on water-treated control plants. Koch's postulates were fulfilled by reisolation of G. magna from diseased leaves. To our knowledge, this is the first report of G. magna infecting L. chinensis worldwide. References: (1) M. Z. Du et al. Mycologia 97:641, 2005. (2) R. J. Nascimento et al. Plant Dis. 94:1506, 2010.

First Report of Leaf Spot Caused by Corynespora cassiicola on Baphicacanthus cusia in China.

Baphicacanthus cusia is a perennial herbaceous plant in the family Acanthaceae that is native to China, where it grows in warm temperate mountainous or hilly regions. It is commonly used as a Chinese herbal medicine. In March 2012, symptoms of leaf spot were observed on leaves of B. cusia in Long'an County, Guangxi, China, where this plant is extensively cultivated. Symptoms were initially small brown dots which developed into irregular to circular leaf spots. These spots enlarged and overlapped, extending until the 7- to 9-cm-long and 3- to 4-cm-wide leaves withered entirely, mostly within 2 months. On potato dextrose agar (PDA), the same fungus was cultured from 92% of 75 symptomatic leaf samples that had been surface sterilized in a 45-second dip in 0.1% mercuric chloride. Fungal structures were observed on diseased leaves: conidiophores (85 to 460 × 4 to 8 µm) were erect, brown, single or in clusters, and conidia (36 to 90 × 5 to 16 µm) were single or in chains of two to four, brown, cylindrical or obclavate, straight or slightly curved, with 3 to 18 pseudosepta and a conspicuous hilum. Three single-spore isolates were identified as Corynespora cassiicola (Berk & Curt.) Wei based on morphological and cultural characteristics (1). The rDNA internal transcribed spacer (ITS) region of one isolate, ZY-1, was sequenced (GenBank Accession No. JX908713), and it showed 100% identity to C. cassiicola, GenBank FJ852716, an isolate from Micronesia cultured from Ipomoea batatas (2). Pathogenicity tests were performed with each of the three isolates by spraying conidial suspensions (5 × 104 conidia/ml) containing 0.1% Tween 20 onto the surfaces of leaves of 60-day-old, 20-cm tall plants. For each isolate, 30 leaves from five replicate plants were treated. Control plants were treated with sterilized water containing 0.1% Tween 20. All plants were incubated for 36 h at 25°C and 90% relative humidity in an artificial climate chamber, and then moved into a greenhouse. Seven days after inoculation, dark brown spots typical of field symptoms were observed on all inoculated leaves, but no symptoms were seen on water-treated control plants. Koch's postulates were fulfilled by reisolation of C. cassiicola from diseased leaves. To our knowledge, this is the first report of C. cassiicola infecting B. cusia worldwide. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew, Surrey, England, 1971. (2) L. J. Dixon et al. Phytopathology 99:1015, 2009.

An Outbreak of Gummosis of Mango Trees Caused by Lasiodiplodia theobromae in Guangxi, South China.

Mango (Mangifera indica L.) is an economically important fruit in southern provinces of China. In May 2012, field surveys including 2,250 mango trees were done in nine orchards of five different counties in Guangxi Province. An outbreak of gummosis was observed in the province involving over 30,000 ha with an average of 50% disease incidence (DI) and a maximum of 70% in some orchards. Until then, gummosis had been considered a common but not serious disease. In 2012, high temperatures in April and extensive rain in May favored increased disease development. Infected plants showed abundant gum secretion from branches, stems, and main trunks. Some branches died from the disease. During the early stages of infection, branches or stems turned brown followed by xylem necrosis and exudation of a milky sap. The sap turned yellow and finally formed amber gum within several days. Initially, the gum appeared as small droplets, increasing in number, and covering most of the branches and the trunk with bark cracking under severe conditions. On potato dextrose agar (PDA), isolates with similar morphology were consistently recovered from symptomatic plant tissues after surface sterilization in 75% ethanol for 30 seconds and then in 0.1% mercuric chloride for 1 min. Five single-spore isolates from five different locations in Guangxi Province were used to evaluate characteristics of the pathogen. On PDA, cultures were gray with an irregularly distributed, fast-growing, and fluffy aerial mycelium, showing a dark underside as the colony changed from greenish to black after 5 days at 28°C. After 1 month, cultures produced globose pycnidia. Conidia were elliptical and hyaline when immature, becoming dark brown and one-septate, longitudinally striate when mature, and ranged from 20.0 to 28.0 × 10.5 to 16.0 µm (average 23.3 × 13.7 µm). Paraphyses produced within the tissues of pycnidia were hyaline, cylindrical, nonseptate, and up to 61 µm long. The fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (=Botryosphaeria rhodina (Cooke) Arx) based on morphological and cultural characteristics (1,2). The rDNA internal transcribed spacer region of one isolate showed 100% identity to L. theobromae (GenBank HM346876.2) and was deposited in GenBank (JX982240). Pathogenicity of the five isolates was tested in the field on healthy tissues in June 2012. Five green twigs and five 3-year-old branches were used. Three wounds were made on each twig or branch with a sterilized needle. Mycelial plugs were placed on wounds and covered with Parafilm. Uncolonized PDA plugs were used as controls. Two weeks later, typical brown lesions were observed on inoculated branches, and gum exuded from infected wounds. No symptoms were seen on the controls. Koch's postulates were fulfilled by reisolation of L. theobromae from diseased branches. L. theobromae is well documented as a pathogen of mango. In China, the disease was observed in the 1990s in Hainan Province, and the causal agents were identified as L. theobromae and Colletotrichum gloeosporioides Penz. & Sacc based on morphological observation (3). To our knowledge, this is the most severe outbreak reported from China. References: (1) V. S. de Oliveira Costa et al. Eur. J. Plant Pathol. 127:509, 2010. (2) F. Wang et al. Plant Dis. 95:1378, 2011. (3) Q. C. Xiao et al. Tropical Crops Research (in Chinese) 2:25, 1995.

[A fusion protein of rotavirus VP6 and cholera toxin B subunit: expression in Escherichia coli and analysis of biological activities].

Rotavirus infection is a major cause of dehydrating diarrhea in infants worldwide. The non-toxic cholera toxin B subunit(CTB), known as an immunomodulatory carrier, might help to stimulate mucosal immune response when coupled to rotavirus antigens in oral immunization. Here we report for the first time the construction of a translational fusion of CTB gene 5' to the VP6 gene of a human rotavirus A(field strain T114), and expression of the CTB-VP6 fusion protein in E. coli BL21(DE3). The expressed fusion protein has a molecular weight of 56 kD, as expected, and accounts for about 15% of the total E. coli protein. Western blottings with the hyperimmune serum against rotavirus strain WA and the antibody against cholera toxin indicated that the fusion protein retains the antigenicity identical to the native CTB and VP6. The GM1-ELISA analysis proves that the renatured CTB-VP6 has strong affinity for GM1 ganglioside.