The Philippines is an agricultural country that provides a rich source of microbial diversity. One agriculturally important group of microorganisms that exists in the country is the nitrogen-fixing bacteria or rhizobia which are beneficial in enhancing soil fertility and plant production. However, many of these indigenous rhizobial isolates have not been fully characterized and described. The great diversity of bacteria and leguminous plants, environmental differences and sometimes the exclusive interaction between plants and rhizobia provide a window of opportunity that new or novel species of bacteria can still be isolated. In this regard, taxonomic works on these indigenous isolates are deemed important, not only to orderly place these microorganisms in appropriate taxa, but also as basis of comparison for new microbes. Thus, the primary objective of this study is to isolate, identify and determine the taxonomic position of the novel species of rhizobia based on polyphasic taxonomy.

The symbiotic relationship of certain bacteria with the root nodules of leguminous plants (pod-bearing) paved the way to the identification and classification of several nitrogen fixing-nodulating and non-nitrogen fixing bacteria under the order Rhizobiales (class Alphaproteobacteria). To date, this order comprised of 12 families namely, Rhizobiaceae, 'Aurantimonadaceae', Bartonellaceae, Brucellaceae, Phyllobacteriaceae, Methylocystaceae, Beijerinkiaceae, Bradyrhizobiaceae, Hyphomicrobiaceae, Methylobacteriaceae, Rhodobacteriaceae and Xanthobacteraceae1.

The bacterial strains used in this study were obtained mostly from the root nodules of several leguminous plants locally found in the Philippines for the three year sampling periods (2004, 2006, & 2007), lyophilized rhizobial isolates were provided by the Microbial Culture Collection of BIOTECH-Philippines and from the root nodules of leguminous plants in the coastlines of Japan. Root nodules were superficially disinfected with 10% sodium hyphochlorite and 70% ethyl alcohol. The nodules were aseptically crushed and the aliquots were serially diluted and plated on the rhizobium medium supplemented with antifungal agent (Kabicidin) and congo red. Colonies that grew on the plates were randomly selected, purified and maintained in agar slants. The universal primer for the amplification of the 16S rRNA gene was used to partially identify all the axenic cultures. Isolates that showed 98% or lower BLAST sequence similarities were further investigated to clarify their taxonomic position. The polyphasic approach used in this study involved molecular techniques (like the 16S rRNA sequencing, DNA-DNA hybridization, amplification of the nitrogen fixing gene (nifH) and nodulation gene (nodD)) in conjunction with in silico analyses (Bioedit, Mega4 and ClustalX programs), chemical and physiological assays namely fatty acid profiles, G+C content, respiratory quinone type, API 50 CH (bioMerieux), API ZYM(TM) (bioMerieux) strips, Biolog GN2 Microplate (Biolog), antibiotic sensitivity tests and infection/nodulation test. Light microscopy was also employed to observe the motility and to determine the size and shape of the isolates.

A total of 411 bacterial isolates were obtained from 48 different species of leguminous plants. Among these leguminous plants, Leucaena leucocephala and Desmodium scorpiurus were found to be the promiscuous plants since several species of rhizobia (Bradyrhizobium, Rhizobium, Mesorhizobium and Ensifer) were isolated from their root nodules sampled from different localities. On the other hand, leguminous plants like Sesbania sesban and Mimosa species prefer only the species of Rhizobium. Among the species of rhizobia, the Rhizobium and Bradyrhizobium species were isolated from several species of plants suggesting their wide host range. Other groups of bacteria were isolated as contaminants because the medium used in this study is not highly selective to rhizobia.

The almost complete sequences of the 16S rRNA gene of the isolates were used to construct the phylogenetic tree (Fig.1) to reveal that there were 7 probable new species of rhizobia that intermingled within the members of family Rhizobiaceae and Hypomicrobiaceae. Strain Yak96B isolated from the root nodule of kudzu plant (Pueraria lobata) growing from the coastline of Yakushima Island, Kagoshima, Japan was identified as Devosia yakushimensis2 (family Hyphomicrobiaceae). The said strain is closely related to Devosia neptuniae J1T, the type strain of the genus Devosia. However, DNA-DNA hybridization and the significant differences in the physiological tests and the fatty acid contents proved that strain Yak96B is a novel species of Devosia.

Strains P5b, M30a, T25a, M9cR1, 56b, P-Ab and ELS-4 isolated from the root nodules of indigenous leguminous plants in the Philippines were clustered under the heterogeneous family of Rhizobiaceae. All of the aforementioned Philippine isolates have high G+C contents (mol%) and possessed ubiquinone 10 (Q10) as their major respiratory quinone. Strain P5b was isolated from the national tree of the Philippines, the narra tree (Pterocarpus indicus). This sodium chloride sensitive strain was identified as Shinella philippinensis3 which is closely related to Shinella granulli ChO6T. Strain ELS-4 isolated from Desmodium styracifolium is closely related with several Rhizobium species but DNA-DNA reassociation values proved that it is a novel strain and proposed as Rhizobium orientalis.

Strains T25a and M9cR1 isolated from Vigna radiata and Cajanus cajan belong to the same species and proposed as Rhizobium luzonensis which is closely related to Rhizobium undicola LMG 11875T. Strain 56b isolated from Desmodium scorpiurus is also a close neighbor of Rhizobium undicola LMG 11875T and proposed as Rhizobium mindanawensis. Strains M30a and P-Ab isolated from Aeschynomene indica and Desmodium triflorum, respectively. Strain M30a was proposed as Rhizobium mindoronensis. Whereas, strain P-Ab as Rhizobium isabelanensis.

With regards to the presence of the symbiotic genes, the nitrogen fixing (nifH) and nodulating (nodD) genes were not detected in all the aforementioned novel species by PCR amplification using several primer sets. Moreover, all strains failed to infect the roots of the promiscuous Macroptilium atropurpureum (siratro) as host plant in the nodulation/infection assay. This was also observed in the recognized species of Mesorhizobium thiogangeticum and Bradyrhizobium betae isolated from the roots of Clitoria ternatea and Beta vulgaris, respectively, both devoid of symbiotic genes.

In conclusion, our study successfully isolated and classified 7 novel species of rhizobia belonging to family Rhizobiaceae and Hyphomicrobiaceae. The use of 16S rRNA sequences in conjunction with DNA-DNA hybridization and physico-chemical tests are important in delineating closely related species. The newly described strains will further add to the ever growing members of the Order Rhizobiales. Moreover, the rhizobia group is biological diverse not only in terms of their heterogeneous characteristics and taxonomy but also with their interaction/association with leguminous plants (host range).

フィリピンは豊富な微生物多様性を産出する農業国である。農業上最も重要な微生物の一つは窒素固定細菌Rhizobiaであり、それは植物生産のために土壌へ肥沃さを供給するという利点がある。しかし、フィリピン国内の根粒菌の分類学的研究はまだ十分とは言えない。マメ科植物と細菌の種多様性の豊富さ、環境の違いから、根粒菌の新種が見出される可能性は十分あるものと考えられる。このように、フィリピン根粒からの分離株の分類学的研究を行うことは、それらの分離株について分類学的に正しい位置付けを行うために、ひいては新規の細菌を見出すためにも重要である。本研究の主な目的は新しいRhizobiales目細菌を分離し、多相分類学の手法を用い引て新規分離株の分類学的位置を決めることにある。今日までRhizobiales目には12科(Rhizobiaceae, 'Aurantimonadaceae',Bartonellaceae,Brucellaceae,Phyllobacteriaceae,Methylocystaceae,Beijerinkiaceae,Bradyrhizobiaceae,Hyphmicrobiaceae,Methylobacteriaceae,Rhodobiaceae,Xanthobacteraceaeが知られている。本研究は主にこれらの分類群に含まれる新属、新種の細菌を見出すことにある。

第1章では研究の背景と目的について述べ、第2章では実験材料と実験方法についてまとめた。

第3章ではフィリピンのマメ科植物の根粒から分離を試みた根粒菌の分離結果について述べた。48種のマメ科植物を採集し、それらの根粒から計411株の細菌分離株を得た。これらの「分離株について16SrRNA遺伝子の部分塩基配列を調べ、同定を行った。分離源であるマメ科章物のうち、Leucaenaleucocephala,Desmodium scorpiurusでは菌種が一定でない傾向を示した。これらの植物での根粒では分離地の異なった試料の根粒からBradyrhizobium, Rhizobium, Mesorhizobium, Ensiferの菌種が分離された。一方、Sesbania sesban, Mimosa sp.のようなものはRhizobium属の種が主であった。Rhizobium, Bradyrhizobium は複数の植物種の根粒から同時に分離されており、このことは両属の菌種の宿主域の広さを物語ろている。他のグループの細菌種は混入雑菌として分離されたものと考えられる。というのは、本研究で用いた培地は根粒菌の分離に特異的な選択培地ではないためである、また、non-rhizobiaの菌種としてAmorphomonas, Crabtreella, Devosia Labrys Herbaspirillumなどが分離されている。

これらの菌株の中で新規性の可能性の高い8株(strains M9cRIT,T25aT,56bT,P5bT,M30aT,P-AbT,ELS-4T,Yak96BT)については16SrRNA遺伝子の全配列を調べ、系統樹を作成した。その結果、これらの株はfamily Rhizobiaceaeまたはfamily Hypomicrobiaceaeに含まれ、新種の可能性が高い株であることがわかった。これらの菌株について分類学的検討を行い、その新種記載を以下の章に記す。

第4章では屋久島海岸のクズの根粒から分離された菌株の同定について述べた。屋久島海岸に生育のクズ(Pueraria lobata)から分離したYak96BT株は16SrRNA遺伝子塩基配列、化学分類、生理・生化学的性状に基づき、1)evosia属の新種Devosia yakushimanensis sp.nov.と同定した。Devosis属はHyphomicrobiaceae科に属する。

第5章ではPterocarpus indicus根粒から分離したRhizobiaceae科の新種Rhizobium philippinensisについて述べた。フィリピンの国木Pterocarpus indicusから分離されたP5bT株はDNA-DNA相同性実験、生理・生化学試験、化学分類のデータに基づき、Rhizobium属の新種Shinella philippinensis sp.nov.と同定した。

第6章ではDesmzodium属マメ科植物から分離された3新菌種Rhizobium mindanawensis,Rhizobium isabelanensis Rhizobium orientalisについて述べた。16SrRNA塩基配列、DNA-DNA相同性実験、生理・生化学試験、化学分類のデータに基づいてそれぞれの分類学的位置を決定した。すなわち、Desmodium scorpiurusからの分離株56bTはRhizobium mindanawensis sp.nov.と同定、Desmodium triflorumからの分離株P-AbTはRhizobium isabelanensis sp.nov.と同定、Desmodium styracifoliumからの分離株ELS-4TはRhizobium orientalis sp.nov.とそれぞれ同定した。

第7章ではマメ科植物Aeschynomene indicaの根粒から分離した新種Rhizboium mindorensisについて述べた。16SrRNA塩基配列、DNA-DNA相同性実験、生理・生化学試験、化学分類のデータに基づいてM30aTはRhizboium mindorensis sp.nov.と同定した.

第8章ではマメ科植物Cajanus cajan,Vigna radiataから分離された新種Rhizobium luzonensisにっいて述べた。Vigna radiataから分離されたT25aT株およびCajanus cajanから分離されたM9cRIT株は同一の菌種に含まれ、DNA-DNA相同性実験、生理・生化学試験、化学分類のデータに基づき、Rhizobium属の新種、Rhizobium luzonensis sp.nov.と同定した。

第9章は結論と考察である。

以上、本論文は新たに分離されたRhizobiales目細菌の系統分類学的位置を明らかにしたもので、学術上、応用上、貢献するところが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。