First Report of Gummosis Disease of Japanese Apricot Caused by Botryosphaeria dothidea in Taiwan.

Japanese apricot (Prunus mume Sieb. et Zucc.) is an economically important fruit crop grown on more than 10,000 ha in Taiwan. During May 2008, twigs of Japanese apricot trees in the commercial farms of Renai Region (Nantou County) showed symptoms of gummosis disease, with 12 to 18% of the trees affected. The disease was more severe on trees weakened by drought stress. Limb and twig infections began around lenticles as small, sunken, discolored lesions at the margins of wounds. Following infection, cortical cells collapsed, bark became depressed, and blisters developed, which were often cracked with whitish gummy exudation. Necrotic areas were seen on the cortical tissues. Leaves showed yellowing and drooping. In winter months, numerous black pycnidia or perithecia formed on infected twigs. Single conidial isolates of the pathogen were obtained from diseased twigs on acidified potato dextrose agar (PDA) incubated at 25 ± 1°C for 3 days. On the basis of morphological characteristics, the fungus was identified as Botryosphaeria dothidea (3). Conidia (17 to 22.6 × 4.3 to 6.0 µm) were hyaline, unicellular, and spindle shaped. Asci (78 to 125 × 15 to 17 µm) were hyaline, bitunicate, clavate, and eight spored. Ascospores (18 to 22 × 7.0 to 8.2 µm) were hyaline and spindle shaped or fusoid. The pathogen identity was further confirmed by PCR amplification and sequencing of ribosomal DNA internal transcribed spacer from the fungus with the primers ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (4), and a representative sequence was deposited in NCBI GenBank (Accession No. GU594225). The sequence showed 99 to 100% homology with previously characterized strains of B. dothidea (GenBank Accession Nos. EU441944, DQ177876, and AY786320). Pathogenicity tests were conducted with inoculum prepared by culturing the fungus on PDA under a continuous photoperiod of 128 ± 25 µE·m-2·s-1 at 25°C for 3 days. Shallow cuts (3 × 3 × 3 mm) were made on 12- to 15-month-old healthy twigs with a scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of conidial suspension (105 conidia/ml) of the fungus. Two twigs on each of six trees were inoculated. Inoculated areas were covered with moist, sterile cotton and the entire twigs were enclosed in plastic bags. Twigs were inoculated with 5-mm PDA discs or sterile water for controls. The symptoms described above were observed on all inoculated twigs 14 days after inoculation, whereas control twigs remained healthy. Reisolation from the inoculated twigs consistently yielded B. dothidea. In Taiwan, B. dothidea has been reported as the causal agent of gummosis of peach (1) and fruit ring rot of pear (2); however, to our knowledge, this is the first report of B. dothidea causing gummosis on Japanese apricot. References: (1) Y. Ko et al. Plant Pathol. Bull. 1:70, 1992. (2) Y. Ko et al. Plant Prot. Bull. (Taiwan) 35:211, 1993. (3) B. Slippers et al. Mycologia 96:83, 2004. (4) T. J. White et al. In: Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. Academic Press. San Diego, CA, 1990.

First Report of Fruit Rot of Loquat Caused by an Alternaria sp. in Taiwan.

During March 2007, a fruit rot disease was observed in several loquat (Eriobotrya japonica (Thunberg) Lindley) fields located in Taichung, Nantou, and Miaoli counties. Loquat is a valuable fruit crop grown predominantly in central Taiwan, and hence, even a minor yield loss by this new disease is economically significant. Symptoms on fruits initially appeared as small lesions (<1 mm) that later developed into light-to-dark brown, circular, larger (7 mm), sunken lesions, indicating invasion of a pathogen into the fruit. Pieces of rotted fruit tissue (1 × 1 × 1 mm) were immersed for 1 min in 3% commercial bleach, followed by 70% ethanol, cultured on potato dextrose agar (PDA), and incubated under constant fluorescent light (185 ± 35 µE·m-2·s-1) at 24°C for 2 days. Three single conidial isolates (AS1 to AS3) were selected and used in morphological and pathogenicity studies. All three isolates were identified as an Alternaria sp. (1-3) and formed abundant, dark brown mycelium when cultured on PDA with light at 24°C. Conidiophores were 60 to 89 × 3 to 5 µm, densely fasciculate, cylindrical, simple or branched, and had distinct conidial scars. Conidia were 12 to 74 × 6 to 14 µm, golden brown, straight or curved, obclavate with beaks measuring half the length of the conidium, and observed in chains of 10 or more spores with four to seven transverse septa and several longitudinal septa. Pathogenicity tests were conducted twice by inoculating eight surface-sterilized wounded or unwounded fruits with each of the three isolates in each experiment. Two cuts (1 × 1 × 1 mm) were made on each fruit 3 cm apart with a sterile scalpel, and a 300-µl spore suspension (2 × 105 conidia per ml) was placed on each wound. Similarly, a 300-µl spore suspension was placed on unwounded fruits and air dried for 5 min. Control fruits were similarly treated with sterile water. Inoculated fruits were enclosed in a plastic bag and kept at 24 ± 1°C. Symptoms of soft rot were observed on 60% (unwounded) and 100% (wounded) of inoculated fruits 5 days after inoculation, while control fruits did not develop disease symptoms. Reisolation from the symptomatic fruits consistently yielded an Alternaria sp. This fungus previously has been reported as the causal agent of fruit rot or black spot of papaya, mango, kiwifruit, pear, and carambola from Australia, India, Malaysia, South Africa, and the United States (1-3). To our knowledge, this is the first report of fruit rot of loquat caused by an Alternaria sp. in Taiwan. To manage this disease, growers may resort to fungicidal sprays followed by bagging of fruits to reduce pre- and postharvest losses. References: (1) A. L. Jones and H. S. Aldwinckle. Compendium of Apple and Pear Diseases. The American Phytopathological Society. St. Paul, MN, 1990. (2) R. C. Ploetz. Diseases of Tropical Fruit Crops. CABI Publishing. Wallingford, Oxfordshire, UK, 2003. (3) R. C. Ploetz et al. Compendium of Tropical Fruit Diseases. The American Phytopathological Society. St. Paul, MN, 1994.

Transgenic resistance by N gene of a Peanut bud necrosis virus isolate of characteristic phylogeny.

The nucleocapsid protein (N) gene of a Tospovirus devastating tomato crop in the south Indian state of Tamil Nadu was cloned and characterized. The high identity of the cloned sequence to a Peanut bud necrosis virus (PBNV) tomato isolate (97.8/99.6% nucleotide/amino acid) and a PBNV peanut isolate (94.4/96.3% nucleotide/amino acid) identified the Tospovirus as an isolate of PBNV, designated PBNV Coimbatore tomato (PBNV CT) isolate. Phylogenetic analysis of PBNV CT N gene provided useful insights into the movement and evolution of PBNV within Indian Territory. The characteristic phylogeny of PBNV CT N gene implied its potential to be an efficient transgene to confer effective PBNV resistance on crop plants. The efficacy of PBNV CT N gene in conferring PBNV resistance was studied by generating tobacco (Nicotiana tabacum L. cv Wisconsin) lines transgenic to the sense or antisense version of the gene. Several transgenic lines showed transgenic mRNA and/or protein accumulation, ranging from very high to undetectable levels, accompanied by different degrees of PBNV resistance. The undetectable or very low levels of transgene transcripts in certain PBNV-resistant sense or antisense N gene transgenic lines suggested RNA-mediated resistance by post-transcriptional gene silencing (PTGS) mechanism. However, PBNV resistance of certain transgenic lines with high levels of N gene transcripts was suggestive of possible operation of RNA-mediated non-PTGS mechanism(s) of resistance in those lines. Moreover, the high levels of N protein in certain PBNV-resistant sense N gene transgenic lines suggested protein-mediated resistance. The results predict the potential of PBNV CT N gene to confer effective PBNV resistance on tomato and other economically important crops.

First Report of Stem-End Rot of Mango Caused by Phomopsis mangiferae in Taiwan.

Mango (Mangifera indica L.) is grown on approximately 20,000 ha in Taiwan. It is an economically important crop and the income of many fruit farmers comes primarily from mango production. During 2006 and 2007, a stem-end rot disease was observed 1 week after harvest on 28 to 36% of stored mangoes picked from six orchards in the Pingtung, Tainan, and Kaoshiung regions. Two popular mango cultivars, Keitt and Irwin, showed greater susceptibility to this disease, while 'Haden' was found to be moderately susceptible. In storage, symptoms initially appeared as light-to-dark brown lesions surrounding peduncles. Rot symptoms advanced slowly but eventually penetrated the mesocarp, which consequently reduced the commercial value of fruits. The fungus formed abundant pycnidia (0.1 to 0.6 mm in diameter) on infected fruits in advanced stages of symptom development. Pieces of symptomatic fruits plated on acidified potato dextrose agar (PDA) and incubated at 25 ± 1°C consistently yielded the same fungus. A single conidial isolate was cultured. Pycnidia developed on PDA after continuous exposure to light for 9 to 14 days. On the basis of morphological characteristics, the fungus was identified as Phomopsis mangiferae L. (2,3). Pycnidia released two types of conidia: α-conidia (5 to 10 × 2.3 to 4.0 µm) were hyaline and oval to fusoid; and ß-conidia (15.0 to 37.5 × 1.3 to 2.5 µm) were hyaline and filiform with characteristic curves. Conidiophores were hyaline, filiform, simple or branched, septate, and 15 to 75 µm long. Cultures incubated under continuous fluorescent light (185 ± 35 µE·m-2·s-1) at 25°C for 3 days were used as inoculum for pathogenicity tests. Five fruits from 'Keitt' were wounded with a sterilized scalpel and each wound (2 × 2 × 2 mm) was inoculated with either a 5-mm mycelium agar plug or a 0.5-ml spore suspension (105 conidia per ml) of the fungus. Five wounded fruits inoculated with 5-mm PDA plugs or sterile water alone served as controls. Inoculated areas were covered with moist, sterile cotton. Fruits were enclosed in plastic bags and incubated at 24°C for 3 days. The test was performed three times. The same symptoms were observed on all inoculated fruits, whereas no decay was observed on control fruits. Reisolations from the inoculated fruits consistently yielded P. mangiferae, thus fulfilling Koch's postulates. This disease has previously been reported in Australia, Brazil, China, Cuba, India, Malaysia, and the United States (1). To our knowledge, this is the first report of P. mangiferae causing stem-end rot disease on mangoes in Taiwan. Our report necessitates taking preventive strategies in the field, prior to or after harvest, to contain postharvest losses in mangoes. References: (1) G. I. Johnson. Page 39 in: Compendium of Tropical Fruit Diseases. R. C. Ploetz et al., eds. The American Phytopathological Society. St. Paul, MN, 1994. (2) R. C. Ploetz, ed. Page 354 in: Diseases of Tropical Fruit Crops. CABI Publishing. Wallingford, UK, 2003. (3) E. Punithalingam. No. 1168 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1993.

First Report of Downy Mildew Caused by Pseudoperonospora cubensis on Chayote (Sechium edule) in Taiwan.

Chayote (Sechium edule) is cultivated on more than 500 ha in Taiwan for its edible shoots and fruit. In August 2005 and later, 40 to 75% of the chayote plants cultivated in the Taichung District Agricultural Improvement Station in Puli developed pale yellow, irregular spots on the upper leaf surfaces with corresponding sporulation on the lower leaf surfaces. The lesions eventually became necrotic and spread over the entire leaf surface, leading to defoliation. Pseudoperonospora cubensis, which was previously reported as the cause of downy mildew on squash (Cucurbita moschata), muskmelon (Cucumis melo), cucumber (Cucumis sativus), and sponge gourd (Luffa cylindrica) in Taiwan, was identified (1). Sporangiophores were 182 to 410 µm long, 4.8 to 7.2 µm wide, and dichotomously branched. Sporangia were grayish, ovoid to ellipsoidal, 18.2 to 38.6 µm long, and 13.5 to 25.2 µm wide. Biflagellate zoospores were 9.5 to 12.6 µm in diameter. Pathogenicity tests were conducted four times with six 2-week-old plants in each trial. A sporangial suspension (1 × 105 spores per ml) prepared from infected leaves (5 to 6 weeks after infection) was sprayed on all leaves until runoff. The plants were then covered with polythene bags and incubated for 48 h at 18 ± 1°C in a growth chamber. Control plants were sprayed with sterile water. Characteristic symptoms developed on all inoculated plants after 20 days, while control plants remained symptomless. Microscopic observation of leaf tissues of symptomatic plants confirmed the presence of P. cubensis. To our knowledge, this is the first report of P. cubensis causing downy mildew on chayote in Taiwan. References: (1) S. T. Hsu et al. List of Plant Diseases in Taiwan. The Phytopathological Society of the Republic of China, 2002.

First Report of Fruit Rot of Strawberry Caused by an Alternaria sp. in Taiwan.

In March 2005, a fruit rot disease was found in several commercial strawberry (Fragaria × ananassa Duchesne) fields at Fongyuan, 24.25°N, 120.72°E, in Taichung County in central Taiwan. The disease was rare and was negligible in most cultivated areas. However, disease incidence has increased by 4 to 5% over the last 2 years and causes significant postharvest losses. In storage, symptoms on berries include light brown-to-black, sunken, irregularly shaped lesions. The lesions gradually enlarge and become firm with a dark green-to-black, velvety surface composed of mycelia, conidiophores, and conidia. Twelve single conidial isolates (AF-1 to AF-12) of a fungus were isolated by placing portions of symptomatic fruit from four locations onto acidified potato dextrose agar (PDA) and incubating at 24 ± 1°C. One isolate from each of the four locations, AF-2, 6, 9, and 12, was selected for identification and pathogenicity studies. The fungus was identified as an Alternaria sp. according to the morphological descriptions of A. tenuissima (2,3). Conidiophores were simple or branched, straight or flexuous, septate, pale to light brown, 3.0 to 5.0 µm in diameter, and bore two to six conidia in a chain. Conidia were dark brown, obclavate or oval, and multicellular with seven transverse (in most cases) and numerous longitudinal septa. Conidia were 15.5 to 56.5 µm (average 35.0 µm) long × 6.0 to 15.0 µm (average 11.0 µm) wide at the broadest point. The pathogen was consistently isolated from berries in the field or in storage. Pathogenicity tests were conducted by inoculating 12 surface-sterilized berries with each of the four isolates. Approximately 300 µl of a spore suspension (2 × 105 conidia per ml) was placed at two points on the uninjured surface of each fruit and allowed to dry for 5 min. Control fruits were treated with sterile water. The berries were then enclosed in a plastic bag and incubated at 24 ± 1°C for 2 days. Disease symptoms similar to those described above were observed on 95% of inoculated berries 3 days after inoculation, while no symptoms developed in control berries. Reisolation from the inoculated berries consistently yielded the Alternaria sp. described above. Pathogenicity tests were performed three times. Previously, strawberry fruit rot caused by A. tenuissima was reported from Florida (2) and Malaysia (1), however, to our knowledge, this is the first report of fruit rot of strawberry caused by a species of Alternaria in Taiwan. References: (1) W. D. Cho et al. List of Plant Diseases in Korea. Korean Society of Plant Pathology, 2004. (2) C. M. Howard and E. E. Albregts. Phytopathology 63:938, 1973. (3) R. D. Milholland. Phytopathology 63:1395, 1973.

First Report of Gummosis Disease of Plum (Prunus salicina) Caused by a Botryosphaeria sp. in Taiwan.

Plum (Prunus salicina Lindell) is grown on more than 3,870 ha in Taiwan. In 2004, a gummosis disease was observed on plum in the Ming Jian Region of Nantou County (120.675°E, 23.919°N), with 15% of the trees affected. Infections started on the current year's growth, primarily through lenticels, and formed small, sunken, discolored lesions. At later stages, white gum exuded from the lesions. Circular to oval, brown, necrotic areas were seen on the inner bark. Severely infected twigs showed defoliation and dieback. During the winter months, numerous black pycnidia or perithecia formed on infected twigs. Single conidial isolates of the pathogen were obtained from diseased twigs on acidified potato dextrose agar (PDA) and incubated at 25 ± 1°C for 3 days. On the basis of morphological traits, the fungus was identified as a Botryosphaeria sp. according to the CMI descriptions of Botryosphaeria ribis (3). Conidia (14.2 to 26.8 × 4.3 to 7.2 µm) were single celled, hyaline, and spindle shaped. Asci (105 to 135 × 12.5 to 15.5 µm) were hyaline, clavate, and bitunicate. Ascospores (18 to 22 × 7.0 to 8.2 µm) were hyaline and spindle shaped or fusoid. For pathogenicity tests, inoculum was prepared by culturing the fungus on PDA under continuous fluorescent light (128 ± 25 µE·m-2·s-1) at 25°C for 3 days. Two twigs on each of six trees were inoculated. Sharp incisions (3 × 3 × 3 mm) were made on healthy twigs (12 to 15 months old) with a sterilized scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of a conidial suspension (105 conidia/ml) of the fungus. Inoculated areas were covered with moist, sterile cotton and the entire twigs were enclosed in plastic bags. Twigs inoculated with 5-mm PDA discs or sterile water alone served as controls. The symptoms described above were observed on all inoculated twigs 14 days after inoculation, whereas control twigs did not develop any disease symptoms. Reisolation from the inoculated twigs consistently yielded the Botryosphaeria sp., thus fulfilling Koch's postulates. Botryosphaeria spp. have been reported to cause stem blight of many plants in temperate and tropical regions of the world (4). In Taiwan, B. dothidea has been reported as the causal agent of gummosis disease of peach (1) and fruit ring rot of pear (2); however, to our knowledge, this is the first report of a Botryosphaeria sp. causing gummosis of plum. References: (1). Y. Ko et al. Plant Pathol. Bull. 1:70, 1992. (2) Y. Ko et al. Plant Prot. Bull. (Taiwan) 35:211, 1993. (3) E. Punithalingam and P. Holliday. No. 395 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1973. (4) W. A. Sinclair et al. Diseases of Trees and Shrubs. Cornell University Press, Ithaca, NY, 1987.

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight.

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight.