氏   名
アラム コシェド
ALAM S.M. Khorshed
本籍(国籍)
バングラデシュ
学位の種類
博士(農学)
学位記番号
甲 第126号
学位授与年月日
平成11年3月24日
学位授与の要件
学位規則第4条第1項該当
専  攻
生物資源科学専攻
学位論文題目
Ecological Studies on the Soft Rot Disease of Chinese Cabbage
(ハクサイ軟腐病の発生生態に関する研究)

論文の内容の要旨

 The soft rot of Chinese cabbage (Brassica campestris L.,pekinensis group)has been known as soil-borne disease caused by pathogenic bacterium (Erwinia carotovora subsp.carotovora) inhabiting in soil. The incipient symptoms of the disease appear on the pe-tioles at first then it develops systemically from the wrapping stage following destru-ctive damage. It is listed up as one of the destructive diseases in Japan. Though there re many reports on the disease cycle consisting of saprophytic phase in soil and para-sitic phase on host plants up to date,it has not come to a satisfactory solution.
  The present study was carried out to clarify the ecological aspects of the soft rot disease with reference to the establishment of source of inoculum for the disease in Yamagata University Farm. The results obtained are as follows.

1. Growth of the organisms in sterilized and non-sterilized soil
  The soil was collected from Yamagata University Farm and sterilized by autoclaving at 121℃ for 2 hrs. The soft rot bacteria inoculated to the sterilized soil at 101cfu/gof dry soil grow rapidly and population level of 106-107 cfu/g of dry soil was obtainedat 3 days after incubation at 25℃. On the contrary, even the organisms of 106 cfu/g ofdry soil did not grow in non-sterilized soil and could not even be detected by the phage technique at 7 days after incubation showing to be the level below 101cfu/g of dry soil. The results may indicate that most of the organisms inoculated externally to non-sterilized soil may die out within a short period.

2. Growth of the organisms in field and occurrence of the soft rot
  The organisms have been reported to inhabit widely in soil. In ordinary circum stan-ces, the population of the organism is below 103cfu/g of dry soil which is undetectablelevel by routine procedures. However, when Chinese cabbage (cv. Matsushima Kohai W-1116,unless otherwise stated) is grown, the organism can exclusively grow in the rhizospheresoils in a wrapping stage at 40-50 days after seeding in both the spring and summer-seeded crops. The population level was 105-107cfu/g of dry soil and was continued untilharvest. The organism was also isolated from leaf surface of the plant from a wrapping stage. The soft rot occurred at about 50 days after seeding corresponding with the latewrapping stage. Susceptibility index of the disease in the spring-seeded crops was hi-gher than that of the summer-seeded crops.

3. The factors concerning with the growth of the organisms in the rhizosphere soils at a wrapping stage
  The high population level of the organisms in the rhizosphere soils at a wrapping stage is decreased by cutting the leaves of the plants and can not be detected at 7days after cutting. The results may show that the exclusive growth of the organisms in the rhizosphere soils depends upon the nutrient substances secreted by roots of the plant in which the leaves conduct photosynthesis. Root exudates were sampled at early, middle and wrapping stages, respectively and 10ml of them were inoculated with level of 101cfu/ml of the organisms. The number of the organisms was 107-108cfu/ml after incuba-tion at 25℃ for 24 hrs regardless of sampling time. There was an evident that the rootexudates collected at a wrapping stage had a significant effect on the growth of the organisms. Application of kinetin to the leaves (0.3 ppm) had some effect on the exclu-sive growth of the organisms in the rhizosphere soils.
  All the 540 bacterial isolates were obtained from the rhizosphere soils at early,middle and wrapping stages by the dilution plating method with PDA using 10-4 diluent of the soil in the spring and the summer-seeded crops. The 249 isolates on NA plates and the 271 isolates on PDA plates out of the 540 isolates were found to be antagonis-tic to five indicator strains of the soft rot bacterium. Though the antagonistic bacte-ria were isolated from the rhizosphere soils in spite of different growth stages, the number of the bacteria showed a trend to decrease in a wrapping stage. The decrease seems to be one of the factors inducing an exclusive growth of the organisms in rhizo-sphere soils.

4. Role of the organisms growing in rhizosphere soils and inhabiting on leaf surface of the plants as a source of inoculum for the disease
  As the time of occurrence of the soft rot coincides with that of growth of the orga-nisms in rhizosphere soils, the organisms growing in the rhizosphere soils and inhabit-ing on leaf surface of the plants were supposed to be an effective source of inoculum for the soft rot. The organism marked with rifampicin-resistant and phage sensitivity was inoculated to sterilized soils, cut straws of rice and rice husks in 300ml conical flask, respectively, then the soil and plant materials were inoculated with the organi-sm. After incubation at 25℃ for 7 days, they were buried at a depth of about 10 cm in the field, respectively. The slices of carrot root were dipped for 4 hrs in the water suspension (108cfu/ml) of the organism and were similarly buried. Chinese cabbage were preliminary grown for 14 days in paper pots filled up with sterilized soils. The plantswere immediately replanted in the buried sites. When the soft rot occurred and develop-ed about 50-80 of susceptibility index, the rifampicin-resistant organism buried in thefield with sterilized soil or plant materials were frequently reisolated from the soft rotten lesions and rhizosphere soils of the diseased plants. The sprayed organisms on leaves of the plants remained there until harvest time and were frequently reisolated from the soft rotten lesions and rhizosphere soils in spite of different spraying time and concentrations of the organism. It was found that the organism in soil and on leav-es played a role as an effective inoculum source for the disease. In other word, the source of inoculum for the disease can not only originate from soil but can also origi-nate from leaves of the plants.

5. An attempt to control of the disease biologically by the antagonistic bacteria
  The efficacy of biological control by using the two strains out of the antagonistic bacteria as mentioned above was examined by three different methods such as spraying ofthe organisms on the leaves of the plants with water suspension (106 cfu/ml), dipping of seeds in the water suspension before seeded in the field (bacterization of seeds by the organisms) and transplantation of the plants grown in the organisms-grown sterilized soil (107 cfu/g of soil). It was found that there is a high possibility of controlling the soft rot disease biologically by antagonistic bacteria.

和文抄録

 ハクサイBrassica campestris L., pekinensis group) 軟腐病は土壌生息性の病原細菌(Erwinia carotobovora subsp.carotovora) によってひきおこされる土壌伝染病である。本病は地域や年次によってはハクサイ栽培に壊滅的な被害を与え、ウイルス病、根こぶ病と共に難防除病害の一つであり、その効果的防除法の確立が要請されている。本研究は山形大学農学部附属農場において毎年4月(春播)と8月(夏播)に播種し、露地栽培したハクサイを用いて本病の発生生態、とくにほ場における感染源の成立を中心に検討したもので結果は次のように要約される。

 1.殺菌および非殺菌土壌における軟腐病菌の増殖
 本学部附属農場のハクサイ連作ほ場から採取した土壌を高圧滅菌(121℃、2時間)後、2系統の軟腐病菌を各々乾土1gあたり101 cfu(以下同じ)のレベルで接種し、25℃で培養した。この結果、本菌は急速に増殖し3日後には106−107 のレベルに達した。しかし、非殺菌土壌では106−107 のレベルで接種しても増殖することはなく、7日後にはファージ法でも検出不能となり、101以下のレベルに低下していることが明らかとなった。これらの結果より、非殺菌土壌に外部から導入された軟腐病菌の大部分は定着することなく死滅するものと推察された。

 2.ハクサイの生育にともなう軟腐病の増殖と軟腐病の発生
 普通、土壌中の軟腐病菌の密度は103 以下のレベルで希釈平板法では検出できない。しかし、本菌は軟腐病抵抗性の強弱に関係なくいずれの品種のハクサイでも播種後40−50日の根圏で105−107のレベルに増殖し、収穫期まで保持された。同時期でも本菌は根圏以外の土壌からは検出されなかったことから、結球後期のハクサイ根がほ場における本菌の増殖に重要な役割を担っていることが明らかとなった。本菌は同時期からハクサイ葉面にも組織1gあたり105−106のレベルで生存していた。軟腐病は春、夏播とも播種後約50日の結球後期から発生し、発病指数(被害)は春播の方で高い傾向が見られた。

 3.ハクサイ根圏における軟腐病菌の増殖に関与する要因
 結球期に達したハクサイの全茎葉を切断したところ、根圏土壌の軟腐病菌は急速に減少し7日後には検出不能となった。この事実から茎葉の切断処理により根からの分泌物が量的、質的に変化し、本菌の減少をひきおこしたものと推察された。次に生育別にハクサイ根の水浸出液を調整し、 101/mlの菌数で本菌を接種して25℃、24時間培養した結果、いずれの生育期でも107−108/mlのレベルに増殖したが、結球後期の浸出液で増殖が促進された。カイネチンを茎葉部切断処理したハクサイに散布した結果、根圏における本菌の減少が緩やかになる傾向があった。また、生育別にハクサイ根圏土壌からPDAを用いた希釈平板法により540菌株の細菌を分離した。それらの中でNA平板上で271菌株、PDA平板上で271菌株が5系統の指示菌(軟腐病菌)に拮抗性を示した。これらの拮抗菌は全生育期にわたり分離されたが、その密度は生育中期に高く、軟腐病菌が増殖する結球期には低下する傾向があることから、根圏における本菌の増殖は主に根からの分泌物と拮抗菌の密度に依存しているものと推察された。

 4.ハクサイ根圏および葉面上の軟腐病菌の感染源としての役割
 ハクサイ軟腐病は病原菌が根圏や葉面上で増殖する結球後期から発生することから、根圏や葉面上の病原菌が感染源の役割を担うことが想定されたので、リファンピシン耐性菌を用いて検討した。土壌、イネモミガラ、切断したイナワラ組織片を各々300ml容コルベンに入れ、高圧滅菌後供試菌を接種した。25℃、7日間培養し充分定着させた後、ほ場の深さ約10cmの部位に各々埋没し直ちに殺菌土壌を充填したペーパーポットで14日間育苗したハクサイをその部位に移植した。また、供試菌の殺菌水懸濁液(108/ml) に4時間浸漬したニンジン根部組織の円盤も供試した。軟腐病は無処理区と同様に結球後期から発生したが、その病斑および根圏土壌からリファンピシン耐性菌が高い頻度で分離された。また、ハクサイの生育期および菌濃度を変えて葉面に散布した場合にも同様に高い頻度で分離された。これらの分離菌株のファージ感受性を検定したところ、供試のリファンピシン耐性菌と同一であることが確認された。以上の結果より、土壌で低密度で生存している軟腐病菌は根圏で特異的に増殖して感染源の役割を担うこと、および葉面上の軟腐病菌も同様の役割を担うことが判明した。また、ハクサイ軟腐病の発生は感染源の成立に加えてハクサイの生育期に強く依存していることが示唆された。

 5.拮抗菌によるハクサイ軟腐病の生物的防除法の検討
 ハクサイ根圏土壌から分離した拮抗菌の中から広い抗菌スペクトルを持つ2菌株を供試し、それらの殺菌水懸濁液(106/ml)の葉面散布、同懸濁液への種子の浸漬および拮抗菌が増殖した殺菌土壌(107/g)で育苗後本畑へ移植する方法により、ハクサイ軟腐病に対する拮抗菌の防除効果を調査した。この結果、拮抗菌による種子および根面のバクテリゼーションによりハクサイ軟腐病を効果的に防除できる可能性が示唆された。