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Development of vermicompost from moso-bamboo, and analysis of its mechanisms of plant-disease suppressiveness

尤 暁東 大阪府立大学 DOI:info:doi/10.24729/00016937

2020.06.24

概要

ミミズによる堆肥化は家畜し尿・敷きわらなど固体廃棄物リサイクルの有効な手段であり、様々な植物病害に抑制効果を示すことがこれまでに報告されている。日本のタケの主要種であるモウソウチクは、かつては食用や建材などに広く利用されていたが、生産者の高齢化によって現在その多くが放置され農地や森林崩壊の原因になっている。モウソウチクを植物病害に抑制効果をもつミミズ堆肥に変えて植物生産現場で利用すれば、植物病害の軽減と放置竹林の有効活用を同時に図ることができる。しかしタケ材をミミズ堆肥化したという報告はこれまでに見られず、その植物病害抑制効果については全く不明である。モウソウチク由来ミミズ堆肥を植物生産で利用するためには、その作出法を開発して植物病害に対する発病抑制効果を確かめるとともに、発病抑制効果に関わる要因を明らかにする必要がある。
 そこで本研究では、モウソウチクから植物病害に抑制効果をもつミミズ堆肥を作出して植物生産に利用することを目的として、堆肥の作出方法の確立、植物病原糸状菌および植物寄生性線虫に対する抑制効果の評価、植物病原糸状菌への抑制に関わる生物的および化学的要因の特定、およびこの堆肥への生物防除微生物添加による病害抑制効果向上の検討を行った。

第1章 モウソウチク由来ミミズ堆肥の作出と植物病原糸状菌に対する抑制効果の評価
 モウソウチクをパウダー状の細粉にした後に水道水に約12時間浸漬することにより、シマミミズがモウソウチクを食べて糞化することがわかった。シマミミズは水道水に浸漬しないモウソウチク細粉中では生存できなかった。モウソウチクからミミズに毒性を示す水溶性物質の存在が確認され、水道水への短時間の浸漬で除去可能であった。また、シマミミズがモウソウチク細粉中で長期間生存するためには、マメ科植物等による有機体窒素の供給も必要であることがわかった。これらの結果に基づき、水道水に一晩浸漬したモウソウチク粉末10kg(湿重)に窒素源としてのクズ乾燥茎葉100gを添加したものにシマミミズ100gを投入し、含水率約80%、温度約28℃に静置した。2週間毎に堆肥の熟度をコマツナ発芽試験で調べた結果、シマミミズ投入の8週間目に完熟することがわかった。
 この方法で作出したモウソウチク由来ミミズ堆肥(vermicomposted bamboo、以下VB)を育苗土として用いた場合のキュウリ苗立枯病に対する抑制効果を調べた。その結果、市販育苗培土commercial nursery medium、以下CNM)に比べてVBでは苗立枯病を起こす糸状菌3種(Pythium aphanidermatum、Globisporangium ultimum var. ultimum および Rhizoctonia solani AG1-IB)による発病が有意に抑制された。VBとCNMを、0、25、50、75および100%(v/v)になるように混合し、上記と同様に接種実験を行った結果、VBの発病抑制効果は濃度依存的に高くなることがわかった。

第2章 植物病原糸状菌に対する抑制効果の要因の解析
 VBをオートクレーブしたもの(aVB)では発病抑制力が完全に消失したことから、VBの発病抑制効果には微生物やその代謝産物が関わっていることが示唆された。そこでVB中の微生物の活性と密度をFDA加水分解活性法と希釈平板法によりaVBやCNMと比較した。その結果、VBの微生物活性と微生物密度はaVBやCNMに比べて有意に高かった。次にMiseqを用いたアンプリコンシーケンス解析により細菌叢を網羅的に調べた結果、VBにのみFlavobacterium属菌が存在し、またBacillus属菌がaVBやCNMに比べて高い割合で存在することがわかった。そこでF. akiainvivens、B. amyloliquefaciens、B. pumilusおよびB. thuringiensisの計4種について、植物病原糸状菌の活性と発病に及ぼす影響を検定した。植物病原糸状菌としてキュウリ苗立枯病菌Rhizoctonia solani AG1-IBとGlobisporangium ultimum var. ultimumを用いた。その結果、いずれの細菌株もR. solaniAG1-IBに対して顕著な抗菌活性と発病抑制力を示した。一方で、G. ultimum var. ultimumに対してはいずれの細菌株も発病抑制力を示さなかった。F. akiainvivensとBacillus属菌3種の由来を明らかにするためにミミズ腸内およびVBの原料であるモウソウチクとクズに含まれる細菌叢を網羅的に調べたところ、F. akiainvivensはミミズ腸内に、Bacillus属菌3種はモウソウチクとクズに由来することがわかった。またこれらのBacillus属菌はミミズの腸内を通ることで約100倍に密度を増加させることがわかった。
 次にVB中に含まれる抗糸状菌物質について調べた。糸状菌として植物病原菌のR. solani AG1-IBを供試した。酢酸エチル等による抽出やペーパーディスク検定を経てVB中の主要な抗糸状菌物質の成分を単離し構造解析を行った結果、VBの酢酸エチルエキスの5つの画分で抗糸状菌活性が確認された。これらの内の1つから主成分を単離して構造解析を行った結果、ergosterol peroxideとそのアナログと同定された。原料であるモウソウチク粉末の酢酸エチルエキスにはこのような活性が見られなかったことから、この物質はモウソウチクが堆肥される過程で産生されたと考えられた。

第3章 植物寄生性線虫に対する抑制効果の評価
 植物寄生性線虫のネコブセンチュウとニセフクロセンチュウに対するVBの防除効果を、ササゲを用いた温室および圃場レベルで調べた。この実験では、圃場で施用しやすい水抽出液にしてVBを供試した。比較として、野菜残渣ミミズ堆肥(vermicomposted vegetable waste、以下 VV)の水抽出液と水道水を用いた。評価項目は、1)二期幼虫への殺効果と卵の孵化抑制、2)線虫の根への侵入の抑制、および3)ササゲの生長・収量の影響およびネコブ形成の抑制とした。その結果、VBとVVで殺線虫効果や卵の孵化抑制および線虫の根への侵入の抑制が見られた。VBとVVとの比較では、ニセフクロセンチュウの卵の孵化でVBの方が高い抑制効果が見られたが、ネコブセンチュウの孵化では有意な差が見られなかった。またVBは両線虫の根侵入に対してVVより高い抑制効果を示したが、ネコブセンチュウの二期幼虫に対する殺効果はVVよりも低かった。さらにVBとVVのいずれも、ササゲの生長・収量への影響が見られなかったが、VBではササゲの線虫によるネコブ形成において、時期が異なる2つの試験で有意な抑制が見られた。このようにVBとVVの両方で線虫に対する抑制効果が見られたが、VBの方が、試験の時期に関わらず安定した抑制を示した。これはモウソウチク由来ミミズ堆肥では材料が時期によらず均質であるのに対し、野菜残渣由来ミミズ堆肥では時期によって材料の種類や質が異なることが原因と考えられた。

第4章 生物防除微生物Pythium oligandrumの添加によるVBの発病抑制効果の向上
 上述のようにVBは植物病原糸状菌や植物寄生性線虫に対する抑制効果を示すことが明らかになったが、その効果は殺菌剤と比較すると低い。そこでVBの発病抑制効果の向上を目的として、苗立枯病を抑制する生物防除微生物として既に良く知られているP. oligandrumの添加の影響を調べた。VBを25および50%(v/v)の比率になるようにCNMに混合し、P. oligandrumの培養物を1%(v/v)になるように添加した。これらを用いてキュウリおよびダイズを育成し、各植物の苗立枯病菌を接種して発病抑制効果を評価した。比較として標準的な殺菌剤を供試した。その結果、VBにP. oligandrumを添加した試験区では、VBやP. oligandrumの単独で処理した場合よりもキュウリ苗立枯病に対する発病抑制効果が高く見られ、さらに殺菌剤と同等のレベルに抑制効果が向上することがわかった。またダイズ苗立枯病に対してもP. oligandrumの添加による発病抑制効果の向上が確認された。育苗土中でのP. oligandrumの菌密度を調べたところ、VBが一定割合で培土に存在する場合に有意な菌密度の増加が見られた。これらの結果から、VBは植物病原糸状菌や植物寄生性線虫を抑制するが、生物防除微生物のP. oligandrumに対してはその活性を促進することがわかった。

結論
 本研究では、まずモウソウチク由来ミミズ堆肥の作出法を確立し、その植物病原糸状菌に対する抑制効果を確認した。そして、この抑制効果をもたらす主要な要因が、VB中に多様かつ高密度の微生物が存在することによる栄養や生育場所をめぐる競合、F. akiainvivensやB. amyloliquefaciens等の拮抗作用、およびergosterol peroxideなどの抗菌活性物質の存在であることを明らかにした。また温室および圃場レベルでVBが2種の植物寄生性線虫に対して抑制効果を示すことも確認した。さらに生物防除微生物Pythium oligandrumをVBに添加することによってキュウリ等の苗立枯病に対して、標準的な殺菌剤と同等のレベルにまで発病抑制効果が向上することを明らかにした。今後、VBの植物病害抑制力をさらなる圃場試験で評価することにより、有機栽培や減農薬栽培などを行う作物生産現場でVBを活用することが期待される。

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