Identification and Survey of Banana bract mosaic potyvirus (BBrMV)－A New Banana Virus in Taiwan

• Main Authors: Ya-Chih Feng, 馮雅智
• Other Authors: Hong-Ji Su
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/32885242424550910664

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 94 === Banana bract mosaic disease was first discovered in Davao of the Philippines in 1979. It is a newly rising banana disease but it has caused a great impact to the banana industry in several southeastern Asian countries including the Philippines, India, Sri Lanka, Western Samoa and Vietnam. Based on the dark spindle to mosaic discoloration on the bract of the inflorescence, this disease was therefore named “bract mosaic”. It is caused by Banana bract mosaic virus (BBrMV), a positive-sense RNA virus which is a member of Potyvirus, Potyviridae. BBrMV may be transmitted by the vegetative propagating materials such as suckers and tissue-cultured plantlets, and it is also transmitted by the aphids in the field in non-persistent manner. The One-step RT-PCR assay with “total nucleic acid preparation” using a BBrMV-specific primer pair (BBrMV 902) was devised for the accurate detection of BBrMV in this study. Flexuous rod-shaped virus particles (c. 750 nm long) could be observed under a transmitting electron microscope through the PEG purification method of BBrMV from Latundan (PHI-1 isolate, Latundan, AAB). PHI-1 was transmitted to 14 Cavendish banana plantlets by banana aphids (Pentalonia nigronervosa). Eight plantlets were positive for the BBrMV RT-PCR assay 1.5 months later, 11 plantlets were positive 3 months later, and only 3 plantlets were positive 14 months later. All test plantlets did not show the typical or apparent symptoms for banana bract mosaic disease through whole experimental period. On the other hand, the field survey was conducted in the thesis to identify whether BBrMV existed in Taiwan. Totally 50 banana cultivars were collected from “banana germplasm orchard” in Taiwan Banana Research Institute. Cultivar Cocos (serial No. 21), San Chi Chiao (81) and Pei Chiao (85) tested positive in the RT-PCR assay for BBrMV, but they did not show symptoms. Their amplified 902-bp fragments (coat protein gene) of RT-PCR were further cloned and sequenced. The nucleotide sequences of three BBrMV isolates from Taiwan were about 94% identical to foreign published sequences of coat protein gene, and their translated amino acid sequences were about 95.7% identical. These domestic BBrMV isolates were then aphid-transmitted to Saba (BBB), Kluai Khai (AA), Nam Wa (ABB) and Cavendish (AAA) cultivars. Only 2 plantlets (one Saba and one Kluai Khai) tested positive for BBrMV 6 months after inoculation, and they did not show apparent symptoms. In addition, lots of banana foliar samples were collected for another survey from the fields of Pingtung, Kaohsiung, Tainan and Nantou counties. Only one sample of the San Chi Chiao (San-1) cultivar collected from southern Pingtung tested BBrMV-positive. The results of field survey demonstrated that few cases showed positive for BBrMV. It seems that the most banana cultivars grown in Taiwan are tolerant or resistant to BBrMV, or ecological factors in Taiwan are not suitable for multiplication and spreading of BBrMV. The antiserum against BBrMV was also prepared in the thesis for more economic and efficient BBrMV detection. The E. coli expressed coat proteins of BBrMV were prepared and used for the antigens of immunization. A BBrMV-specific antiserum (named PcAb-BBrMV-1) was obtained for the application in ELISA tests, and PcAb-BBrMV-1 showed good results in the BBrMV detection. An ideal extraction buffer (0.5 M borate buffer with 0.5％ skim milk，pH 6.8) was selected from the buffer tests, and this buffer can give the best result in ELISA with PcAb-BBrMV-1. The devised RT-PCR and immunological assays can provide different detection methods for BBrMV detections, which should be helpful to inspection and quarantine of BBrMV in Taiwan.