{"created":"2023-05-15T13:37:52.510722+00:00","id":1586,"links":{},"metadata":{"_buckets":{"deposit":"c20bbca3-e21d-4489-a60e-9a09a53ddf0c"},"_deposit":{"created_by":12,"id":"1586","owners":[12],"pid":{"revision_id":0,"type":"depid","value":"1586"},"status":"published"},"_oai":{"id":"oai:repository.naro.go.jp:00001586","sets":["87:599:601:127:207"]},"author_link":["5714"],"item_10002_biblio_info_7":{"attribute_name":"書誌情報","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2009-03-25","bibliographicIssueDateType":"Issued"},"bibliographicPageEnd":"45","bibliographicPageStart":"1","bibliographicVolumeNumber":"13","bibliographic_titles":[{"bibliographic_title":"中央農業総合研究センター研究報告"},{"bibliographic_title":"Bulletin of the National Agricultural Research Center","bibliographic_titleLang":"en"}]}]},"item_10002_description_5":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"1.    Introduction Fire blight, caused by Erwinia amylovora, is the world's most important bacterial disease of pear and apple. It was first reported in the eastern area of the United States of America (Hudson Valley, New York State) in the 1780s. Thereafter the disease spread south and westward in the USA. In early 1900s, the disease was confirmed all over the USA. Furthermore, the first occurrence of fire blight was mentioned as early as the 1840s in Canada. Since 1924, the disease has spread to all pear and apple growing areas of Canada. On the other hand, the reports of fire blight came from New Zealand in 1919, from England in 1958, and from Egypt in 1964. Fire blight has been spreading in European and Middle Easetern countries and occasionally causes severe damage. There are about 40 countries known with fire blight worldwide at present. Erwinia amylovora has not been isolated in Japan. However, bacterial shoot blight of pear with symptoms resembling fire blight was reported and the causal pathogen closely related to E. amylovora was assigned to Erwinia sp. This disease was eradicated by Japanese governmental emergency control measures and has not been reported since eradication. Erwinia amylovora also has not been isolated in South Korea. In 1999, a new disease with symptoms resembling fire blight was reported on Asian pears in South Korea. The causal agent was named E. pyrifoliae. In this study, the phylogenetic relationships among E. amylovora, Erwinia sp. (the pathogen of bacterial shoot blight of pear), and E. pyrifoliae were revealed on the basis of their common nucleotide sequences. For the detection and identification of each bacterium, PCR primers were designed using the nucleotide sequences. Epiphytic bacteria on Japanese pear flowers, which may cause misidentification in the diagnosis of fire blight using direct PCR, were investigated. The sensitivity and suitable sites for direct PCR were also investigated. 2. Phylogenetic relationships among E. amylovora, Erwinia sp. (the pathogen of bacterial shoot blight of pear), and E. pyrifoliae Phylogenetic trees were made based on the sequences of 16S rRNA, gyrB, and rpoD genes of E. amylovora (28 isolates), Erwinia sp. (the pathogen of bacterial shoot blight of pear 9 isolates), and E. pyrifoliae (2 isolates). The gyrB genes encode the subunit B protein of DNA gyrase which is the enzyme responsible for introducing negative supercoils into bacterial chromosomes and plays a crucial role in the replication of chromosomes. The rpoD genes encode the σ70 factor which is one of the sigma factors that confers promoter-specific transcription initiation on RNA polymerase. The tree based on 16S rRNA gene sequences demonstrated low reliability. On the other hand, the trees based on gyrB and rpoD demonstrated very high reliability. In each tree, the 39 isolates formed two groups: \"group 1\" containing fire blight pathogen isolates and \"group 2\" containing bacterial shoot blight pathogen and Asian pear pathogen isolates. Furthermore, the tree based on combined 16S rRNA, gyrB, and rpoD sequences was similar to the gyrB-based and rpoD-based trees. Thus, phylogenetic analyses suggested that isolates from the three diseases could be divided into two groups. The gyrB and rpoD sequences of the 25 E. amylovora isolates, except for isolates from Rubus sp., were almost identical. Meanwhile, the gyrB and rpoD sequences of the 9 isolates of bacterial shoot blight of pear pathogen were slightly different from one another. The 25 E. amylovora isolates had been isolated from different geographical areas, different host plants, and at different times. The 9 isolates of bacterial shoot blight of pear had been isolated only from the Hokkaido region in Japan, only from pear plants, and only during a period of several years. These results suggested that E. amylovora, except for Rubus strains, was a homogeneous group which had been spread worldwide, and that the pathogen of bacterial shoot blight of pear which was a heterogeneous group, existed in a limited region. 3. Design of primers for the detection and identification of bacteria belonging to group 1 and 2 The primers which can distinguish bacteria belonging to group 1 and 2 based on the rpoD gene sequences were designed. The specificity of these primers was investigated using 71 isolates of E. amylovora, 14 isolates of bacterial shoot blight of pear pathogen, 3 isolates of E. pyrifoliae, and 11 isolates of closely related bacteria. The group 1-specific primer set amplified 375 bp of DNA from all of the 71 E. amylovora isolates, but not from other isolates. The group 2-specific primer set amplified 375 bp of DNA from all of the 14 isolates of bacterial shoot blight of pear pathogen and E. pyrifoliae, but not from any of the 71 E. amylovora isolates, the only other size of amplification was recognized in 4 isolates of closely related bacteria. The detection limits of these primers were 10^4-5cfu/ml. 4. Epiphytic bacteria inhabiting Japanese pear flowers Epiphytic bacteria were isolated from Japanese pear flowers in Japan, and were investigated for the relationship to E. amylovora, the pathogen of bacterial shoot blight of pear, or E. pyrifoliae. A total of 278 bacteria were isolated from Japanese pear flowers in three areas (Ibaraki, Nagano, and Tottori prefectures) using LB medium. A total of 60 bacteria were also isolated using a semi-selective medium for E. amylovora. Partial 16S rRNA gene sequence similarity and colony count revealed that Pseudomonas spp. and Enterobacteriaceae dominated on Japanese pear flowers. These bacteria were not closely related to E. amylovora, the pathogen of bacterial shoot blight of pear, or E. pyrifoliae. All of the 338 bacterial isolates were checked for the false positive reaction using the present and newly designed primers. Thus, the newly designed primers were considered to be practical for the detection and identification of E. amylovora even from Japanese pear flowers. 5. PCR detection of E. amylovora in infected plant material It was necessary to identify suitable sites on diseased plants for PCR and to remove inhibitors from them for PCR. Therefore, an E. amylovora isolate tagged with bioluminescence genes was used to examine the behavior and movement of the pathogen in inoculated apple shoots. The sections of shoots with or without symptoms and luminescence were used for isolation and PCR detection. DNA was extracted from the sections using a simple grinding tube and glass fiber filter paper. From symptomatic sections, 10^5-7cfu of E. amylovora tagged with bioluminescence genes were isolated, and the 375bp DNA fragment was mostly detected by PCR. However, from one symptomatic section, neither E. amylovora nor the fragment was detected. On the other hand, from asymptomatic sections, 10^2-4cfu of the pathogen were isolated, but the fragment was not detected by PCR. The border of healthy and diseased tissue was a suitable site for detecting the pathogen and the fragment by PCR. Apple shoots inoculated with a wild E. amylovora isolate were also investigated. Eleven out of all 12 inoculated shoots showed the typical symptoms. From the symptomatic shoots, 10^5-6cfu of wild E. amylovora were isolated, and the fragment was detected by PCR. On the other hand, from the one symptomless shoot, E. amylovora was isolated, but the fragment was not detected. 6. Conclusion In this study, the phylogenetic relationships among E. amylovora (the pathogen of fire blight), Erwinia sp. (the pathogen of bacterial shoot blight of pear), and E. pyrifoliae (the pathogen of Asian pear blight) were investigated using the sequences of 16S rRNA, gyrB, and rpoD genes. Thirty-nine isolates belonging to the species formed two distinct monophyletic groups in the phylogenetic trees constructed based on the gyrB gene, rpoD gene, or a combination of the three genes, \"group 1\" containing the pathogen of fire blight and \"group 2\" containing the pathogen of bacterial shoot blight of pear and the pathogen of Asian pear blight. The pathogen of fire blight, except for isolates from Rubus sp., was homogeneous for sequences of gyrB and rpoD. The primers which could amplify the rpoD gene sequences specific to group 1 and 2 were designed. These primers were demonstrated only for each group containing isolates amplifying a unique DNA fragment. The suitable site for direct PCR detection of fire blight pathogen from the infected plant was revealed. The border of healthy and diseased tissue was very suitable for PCR. These results will contribute to the detection and identification of the pathogen of fire blight from infected plants if the pathogen enters into Japan. Furthermore, these methods should also be applicable to other plant pathogenic bacteria. \n","subitem_description_type":"Abstract"}]},"item_10002_identifier_registration":{"attribute_name":"ID登録","attribute_value_mlt":[{"subitem_identifier_reg_text":"10.24514/00001543","subitem_identifier_reg_type":"JaLC"}]},"item_10002_publisher_8":{"attribute_name":"出版者","attribute_value_mlt":[{"subitem_publisher":" 独立行政法人 農業・食品産業技術総合研究機構 中央農業総合研究センター"}]},"item_10002_relation_14":{"attribute_name":"DOI","attribute_value_mlt":[{"subitem_relation_type":"isIdenticalTo","subitem_relation_type_id":{"subitem_relation_type_id_text":"10.24514/00001543","subitem_relation_type_select":"DOI"}}]},"item_10002_source_id_9":{"attribute_name":"ISSN","attribute_value_mlt":[{"subitem_source_identifier":"1881-6738","subitem_source_identifier_type":"ISSN"}]},"item_10002_version_type_20":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"松浦, 貴之"},{"creatorName":"マツウラ, タカユキ","creatorNameLang":"ja-Kana"},{"creatorName":"MATSUURA, Takayuki","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2019-03-16"}],"displaytype":"detail","filename":"narc_report_No13p1-45p.pdf","filesize":[{"value":"2.3 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"narc_report_No13p1-45p.pdf","url":"https://repository.naro.go.jp/record/1586/files/narc_report_No13p1-45p.pdf"},"version_id":"dd20b10c-6996-4dbd-8e94-83dc9a867b84"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"departmental bulletin paper","resourceuri":"http://purl.org/coar/resource_type/c_6501"}]},"item_title":"火傷病菌及び類縁細菌の系統解析と検出方法に関する研究","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"火傷病菌及び類縁細菌の系統解析と検出方法に関する研究"},{"subitem_title":"Studies on Phylogenetic Analyses and Detection Method of Fire Blight Pathogen and its Closely Related Bacteria","subitem_title_language":"en"}]},"item_type_id":"10002","owner":"12","path":["207"],"pubdate":{"attribute_name":"公開日","attribute_value":"2019-03-22"},"publish_date":"2019-03-22","publish_status":"0","recid":"1586","relation_version_is_last":true,"title":["火傷病菌及び類縁細菌の系統解析と検出方法に関する研究"],"weko_creator_id":"12","weko_shared_id":-1},"updated":"2023-05-15T16:35:01.376743+00:00"}