JP6253005B2 - Herbicidal enzyme-containing composition and harmful plant control method - Google Patents
Herbicidal enzyme-containing composition and harmful plant control method Download PDFInfo
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- JP6253005B2 JP6253005B2 JP2012288082A JP2012288082A JP6253005B2 JP 6253005 B2 JP6253005 B2 JP 6253005B2 JP 2012288082 A JP2012288082 A JP 2012288082A JP 2012288082 A JP2012288082 A JP 2012288082A JP 6253005 B2 JP6253005 B2 JP 6253005B2
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Description
本発明は酪酸ｐ−ニトロフェニル加水分解活性を有し、植物に対して病原性を有さない微生物由来のクチナーゼ様酵素と、植物病原性微生物とを含む除草用酵素含有組成物、及びこの除草用酵素含有組成物を使用した有害植物の駆除方法に関する。本発明のもう一つの態様は、酪酸ｐ−ニトロフェノール加水分解活性を有するクチナーゼ様酵素を産生し、植物に対して病原性を有さない微生物の培養液を用いる有害植物の駆除方法に関する。 The present invention relates to a herbicidal enzyme-containing composition comprising a cutinase-like enzyme derived from a microorganism having hydrolyzing activity of p-nitrophenyl butyrate and not pathogenic to plants, and the herbicidal composition The present invention relates to a method for controlling harmful plants using an enzyme-containing composition. Another aspect of the present invention relates to a method for controlling harmful plants using a culture solution of a microorganism that produces a cutinase-like enzyme having hydrolyzing activity of p-nitrophenol butyrate and has no pathogenicity to plants.
一般に除草剤と呼称される薬剤のなかには、広い植物種を駆除可能な非選択的な除草剤と、農作物に対する影響は少なく、除草の対象となる有害植物を駆除可能な種間選択的な除草剤が存在する。非選択的な除草剤の典型例である化学除草剤は、農作業従事者を過酷な農作業から解放する上で大きく貢献し、作物生産量の飛躍的な増大をもたらした。その一方で、農業における化学除草剤への依存度が高まるなか、化学除草剤抵抗性の有害植物が出現するなどの新たな問題が生起している。このような状況下にある農業の分野においては、有害植物の駆除方法に関して複数の選択肢を用意し、これらを組合せながら継続して、効率的且つ効果的に有害植物を駆除することが求められている。
非選択的な化学除草剤のみに依存しない除草剤の具体例としては、微生物製剤等の種間選択的な除草剤を挙げることができる。一般に、植物は様々な病原性微生物に感染することで病害を受ける。雑草等の有害植物を含む野外の植物もまた同様に病害を受けている。従って、自然界に存在する病原性微生物の働きによって特定の植物の異常な増殖や蔓延が抑制されている側面がある。このことを利用して、例えば、化学除草剤の使用では困難な外来植物の駆除を行う目的で、人畜や周辺作物等への悪影響がないことを確認した上で、当該外来植物の原産地に生育する病原性微生物を散布する方法が考案されている。
既に、微生物製剤は日本国外で市販されており、日本国内でも芝地に繁殖する雑草であるスズメノカタビラを選択的に駆除可能な微生物製剤が市販された事例がある（例えば、非特許文献１参照）。この微生物製剤に含まれる病原性微生物は、スズメノカタビラの植物体内でのみ増殖可能で、スズメノカタビラが枯死した後の環境からは検出されず、人畜への影響も無いとされている。
Among the drugs generally referred to as herbicides, non-selective herbicides that can control a wide range of plant species, and species-specific herbicides that have little effect on agricultural crops and can control harmful plants targeted for herbicidal use. Exists. Chemical herbicides, typical examples of non-selective herbicides, have greatly contributed to freeing farm workers from harsh farming practices, resulting in dramatic increases in crop production. On the other hand, new problems such as the emergence of chemical herbicide-resistant harmful plants have arisen as the degree of dependence on chemical herbicides in agriculture increases. In the field of agriculture under such circumstances, it is required to prepare multiple options for harmful plant extermination methods and continue to combine them to effectively and effectively eliminate harmful plants. Yes.
Specific examples of herbicides that do not depend only on non-selective chemical herbicides include interspecies selective herbicides such as microbial preparations. In general, plants are affected by infection with various pathogenic microorganisms. Field plants, including harmful plants such as weeds, are similarly affected. Therefore, there is an aspect in which abnormal growth and spread of specific plants are suppressed by the action of pathogenic microorganisms existing in nature. Taking advantage of this, for example, for the purpose of controlling exotic plants that are difficult to use with chemical herbicides, after confirming that there are no adverse effects on human livestock and surrounding crops, etc., grow in the place of origin of the exogenous plants A method of spraying pathogenic microorganisms has been devised.
Microbial preparations have already been marketed outside of Japan, and there are cases in which microbial preparations that can selectively eliminate sparrow caterpillars, which are weeds that grow on turf in Japan, have been marketed (see, for example, Non-Patent Document 1). . It is said that the pathogenic microorganism contained in this microbial preparation can be grown only in the plant of the sparrow leaf but not detected from the environment after the sparrow has died and has no influence on human livestock.
よって、本発明は、効率的且つ効果的な有害植物の駆除に使用される除草用の組成物、及び有害植物の駆除方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a herbicidal composition used for effective and effective harmful plant control, and a method for controlling harmful plants.
一般に、植物の葉面は、貧栄養な環境ではあるが、様々な常在菌が生息している。これらの常在菌の中には、プロテアーゼやリパーゼ、ペクチナーゼなどの加水分解酵素を生産する能力を有するものも多いが、これらの酵素を生産する条件や常在菌の生活におけるこれらの酵素の役割は明らかにされていない。本発明者らは、そのような葉面に生息し、植物に対する病原性を有さない各種微生物の特性について研究する中で、イネの常在菌である葉面酵母Pseudozyma antarcticaの酵素PaE、及びムギの常在菌であり、独立行政法人製品評価技術基盤機構特許微生物寄託センターに、受託番号NITE P-573で寄託された糸状菌株の酵素PCLE（Paraphoma related fungi Cutinase Like Enzyme）が、コハク酸系ポリエステル（例えば、PBS及びPBSA）等の生分解性プラスチックに対する強力な分解性を有することを見出すと共に（例えば、特許４９１５５９３号及び特許５０８２１２５号参照）、これらの酵素の推定アミノ酸配列中にクチナーゼ様酵素に共通するリパーゼボックスと、活性中心を構成する一群のアミノ酸配列が共通して保存されていることを見出した。そして、予想外にも、これら、Pseudozyma antarctica及び受託番号NITE P-573で寄託された糸状菌株等、植物に対して病原性を有さない微生物由来のクチナーゼ様酵素を含む組成物が、植物の乾燥耐性や病原菌耐性を低下させ、各種植物の健全な生育を阻害することを偶然に見出した。よって、本発明は、植物に対して病原性を有さない微生物由来のクチナーゼ様酵素の、斯かる特性を利用した、以下の発明を提供する。 In general, the leaf surface of a plant is in an oligotrophic environment, but various indigenous bacteria live. Many of these resident bacteria have the ability to produce hydrolases such as proteases, lipases, and pectinases, but the conditions for producing these enzymes and the role of these enzymes in the lives of resident bacteria Is not disclosed. In studying the characteristics of various microorganisms that inhabit such leaf surfaces and have no pathogenicity to plants, the present inventors have studied the enzyme PaE of the leaf yeast Pseudozyma antarctica, which is a resident microorganism of rice, and PCLE (Paraphoma related fungi Cutinase Like Enzyme), a filamentous bacterial strain deposited with the NITE P-573, is a succinic acid-based enzyme. It has been found to have strong degradability for biodegradable plastics such as polyester (eg, PBS and PBSA) (see, for example, patents 4915593 and 5082125), and cutinase-like enzymes in the deduced amino acid sequences of these enzymes It was found that a common lipase box and a group of amino acid sequences constituting the active center are conserved in common. Unexpectedly, a composition containing a cutinase-like enzyme derived from a microorganism that does not have pathogenicity to plants, such as the filamentous strain deposited under Pseudozyma antarctica and accession number NITE P-573, It was discovered by chance that the drought tolerance and pathogen resistance were reduced and the healthy growth of various plants was inhibited. Therefore, the present invention provides the following inventions utilizing such characteristics of a cutinase-like enzyme derived from a microorganism that is not pathogenic to plants.
本発明の第一の態様は、有害植物を駆除するための除草用酵素含有組成物であって、酪酸ｐ−ニトロフェニル加水分解活性を有し、植物に対して病原性を有さない微生物由来のクチナーゼ様酵素と、除草用農薬である植物病原性微生物とを含み、除草用酵素含有組成物における前記クチナーゼ様酵素の含有量が、酪酸ｐ−ニトロフェニル加水分解活性に換算して０．０１Ｕ／ｍＬ以上である除草用酵素含有組成物である。
本発明の第二の態様は、植物病原性微生物として、駆除の対象となる有害植物への特異的な病原性を有する微生物を含む本発明の除草用酵素含有組成物を、前記有害植物へ適用する工程を含むことを特徴とする有害植物の駆除方法である。
本発明の第三の態様は、酪酸ｐ−ニトロフェニル加水分解活性を有するクチナーゼ様酵素を産生し、植物に対して病原性を有さない微生物の培養液を、有害植物へ適用する工程を含むことを特徴とする有害植物の駆除方法であって、植物に対して病原性を有さない微生物がPseudozyma属、Cryptococcus属、Acremonium属、Alternaria属、Aspergillus属、Aureobasidium属、Cladosporium属、Epicoccum属、Fusarium属、Leptosphaeria属、Penicillium属、Phoma属、Neoplaconema属、Paraphoma属、Paecilomyces属に属する微生物であることを特徴とする有害植物の駆除方法である。
A first aspect of the present invention is a herbicidal enzyme-containing composition for controlling harmful plants, which has p-nitrophenyl butyrate hydrolyzing activity and has no pathogenicity to plants The cutinase-like enzyme in the herbicidal enzyme-containing composition contains 0.01 U in terms of p-nitrophenyl butyrate hydrolyzing activity. It is a herbicidal enzyme-containing composition that is / mL or more.
The second aspect of the present invention is the application of the herbicidal enzyme-containing composition of the present invention comprising a microorganism having specific pathogenicity to a harmful plant to be controlled as a phytopathogenic microorganism to the harmful plant. It is the extermination method of a harmful plant characterized by including the process to do.
The third aspect of the present invention includes a step of applying a culture solution of a microorganism that produces a cutinase-like enzyme having p-nitrophenyl butyrate hydrolyzing activity and has no pathogenicity to plants to harmful plants. A harmful plant extermination method characterized in that a microorganism that is not pathogenic to the plant is Pseudozyma genus, Cryptococcus genus, Acremonium genus, Alternaria genus, Aspergillus genus, Aureobasidium genus, Cladosporium genus, Epicocccum genus, It is a method for controlling harmful plants, characterized in that it is a microorganism belonging to the genus Fusarium, Leptosphaeria, Penicillium, Phoma, Neoploconema, Paraphoma, Paecilomyces.
本発明の除草用酵素含有組成物は、酪酸ｐ−ニトロフェニル加水分解活性に換算して００．１Ｕ／ｍＬ以上の含有量で、植物に対して病原性を有さない微生物に由来するクチナーゼ様酵素を含有することにより、予想外にも、有害植物の植物病原性微生物に対する耐性を低下させ、除草用酵素含有組成物が含有する植物病原性微生物の補助的な作用により、有害植物を駆除することができる。また、本発明の除草用酵素含有組成物に含まれて、補助的に作用する植物病原性微生物は、有害植物に対して種間選択的に作用するので、化学除草剤と異なり、農作物や人畜に対して悪影響を及ぼすことなく、有害植物のみを駆除することができる。
本発明の第二の態様の有害植物の駆除方法は、本発明の除草用酵素含有組成物を使用しているので、上記除草用酵素含有組成物の効果と同等の効果が得られることに加え、除草用酵素含有組成物が駆除の対象となる有害植物への特異的な病原性を有する微生物を含むので、人畜や作物等には実質的な影響を及ぼすことなく、駆除の対象となる有害植物のみを特異的に除草することができる。
本発明の第三の態様の有害植物の駆除方法は、酪酸ｐ−ニトロフェニル加水分解活性を有するクチナーゼ様酵素を産生し、植物に対して病原性を有さない微生物の培養液を、有害植物へ適用するので、クチナーゼ様酵素の作用により有害植物の乾燥耐性や病原菌耐性を低下させ、環境中の植物病原性微生物による有害植物への感染を誘導し、有害植物を効果的且つ効率的に駆除することができる。
The herbicidal enzyme-containing composition of the present invention is cutinase-like derived from a microorganism that has a content of 00.1 U / mL or more in terms of p-nitrophenyl butyrate hydrolyzing activity and is not pathogenic to plants. Unexpectedly, by containing an enzyme, the tolerance of harmful plants to phytopathogenic microorganisms is reduced, and harmful plants are controlled by the auxiliary action of phytopathogenic microorganisms contained in the herbicidal enzyme-containing composition. be able to. In addition, phytopathogenic microorganisms contained in the herbicidal enzyme-containing composition of the present invention and acting in an auxiliary manner act interspecificly against harmful plants. It is possible to control only harmful plants without adversely affecting the plant.
The harmful plant extermination method of the second aspect of the present invention uses the herbicidal enzyme-containing composition of the present invention, so that the same effect as that of the herbicidal enzyme-containing composition is obtained. Since the herbicidal enzyme-containing composition contains microorganisms that have specific pathogenicity to harmful plants to be controlled, harmful effects to be controlled without substantially affecting human livestock and crops. Only plants can be specifically weeded.
The method for controlling harmful plants according to the third aspect of the present invention is a method for producing a cutinase-like enzyme having hydrolyzing activity of p-nitrophenyl butyrate and having a culture solution of microorganisms that are not pathogenic to plants. Therefore, the action of cutinase-like enzymes reduces the drought tolerance and pathogen resistance of harmful plants, induces infection of harmful plants by phytopathogenic microorganisms in the environment, and effectively and efficiently eliminates harmful plants. can do.
以下、本発明について詳細に説明する。
＜除草用酵素含有組成物＞
本発明の除草用酵素含有組成物は、有害植物を駆除するためのものであって、酪酸ｐ−ニトロフェニル加水分解活性を有し、植物に対して病原性を有さない微生物由来のクチナーゼ様酵素を含む。本発明の除草用酵素含有組成物は、植物に対して病原性を有さない微生物由来のクチナーゼ様酵素のみを含むものであってもよいし、更に、除草用農薬としての植物病原性微生物を含むものであってもよいが、除草用農薬としての植物病原性微生物も含むことが好ましい。本発明の除草用酵素含有組成物が植物に対して病原性を有さない微生物由来のクチナーゼ様酵素のみを含む場合、除草用農薬としての植物病原性微生物を、当該除草用酵素含有組成物を植物に適用した後に適用してもよい。
［クチナーゼ様酵素］
クチナーゼと病原性微生物の病原性との関係については、病原菌として知られるFusarium solaniやイネイモチ病菌において、一部のクチナーゼ欠損株で、植物への感染率が低下することから、クチナーゼが病原性微生物の病原性に関与していることが示唆されていたものの（Rogers et. al., "Cutinase Gene Disruption in Fusarium solani f sp pisi Decreases Its Virulence on Pea", The Plant Cell, Vol. 6, 935-945, 1994; Skamnioti and Gurr, "Cutinase and hydrophobin interplay", Plant Signaling & Behavior 3:4, 248-250, April 2008）、病原機構におけるクチナーゼの役割は未だ明らかにはなっていなかった。また、クチナーゼの作用のみにより病原菌が植物に侵入できるとは考えられてはおらず、また、クチナーゼを外部から供給することにより、病原性微生物における植物への病原性が高まるといった知見は全く得られていなかった。
逆に、病原性微生物の産生するクチナーゼや、クチナーゼによるクチンの分解産物で植物を処理することにより、植物が病原性微生物の存在を察知し、植物による抵抗性反応が誘導されて病害への耐性が向上するといった報告すらなされている（Chassot et al., "Cuticular defects lead to full immunity to a major plant pathogen", The Plant Journal (2007) 49, 972-980; Curvers et al., "Abscisic Acid Deficiency Causes Changes in Cuticle Permeability and Pectin Composition That Influence Tomato Resistance to Botrytis cinerea", Plant Physiology, October 2010, Vol. 154, p. 847-860; L'Haridon et al., "A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity", PLos Pathogens, July 2011, Vol. 7, Issue 7, e1002148）。
更に、Pseudozyma antarcticaが産生するクチナーゼ様酵素PaEや、受託番号NITE P-573で寄託された糸状菌のクチナーゼ様酵素PCLEにおいては、クチナーゼ様酵素において一次構造が共通するリパーゼボックスは１００％保存されてはいるものの、植物病原性微生物として知られているColletotrichum gloesporioidesクチナーゼとの、これらの部分アミノ酸配列における相同性は、８５％であって相同性が低い。また、クチナーゼ活性の指標の一つとされている脂肪酸ｐ−ニトロフェニル加水分解活性についても、Pseudozyma antarcticaが産生するクチナーゼ様酵素PaEや、受託番号NITE P-573で寄託された糸状菌のクチナーゼ様酵素PCLEにおいては、長鎖脂肪酸エステルも効率よく分解するなど、基質特異性が非常に広いのに対し、病原性微生物のFusarium solaniのクチナーゼでは、長鎖脂肪酸エステルの分解活性が非常に低い。このように、酵素反応の特異性の観点からもPseudozyma antarcticaが産生するクチナーゼ様酵素PaEや、受託番号NITE P-573で寄託された糸状菌のクチナーゼ様酵素PCLEと、病原性微生物のクチナーゼは異なるものである（Kodama et al., "Crystal structure and enhanced activity of a cutinase-like enzyme from Cryptococcus sp. Strain S-2", Proteins p. 709-717, 2009; Shinozaki Y, Morita T, Cao XH, Yoshida S, Koitabashi M, Watanabe T, Suzuki K, Sameshima-Yamashita Y, Nakajima-Kambe T, Fujii T, Kitamoto HK Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization. Appl Microbiol Biotechnol (in press) DOI: 10.1007/s00253-012-4188-8）。
このため、病原性微生物において、クチナーゼが病原性発現の一翼を担っていたとしても、病原性微生物のクチナーゼを外部から植物に適用した際に病原性微生物の病原性が増強されるということや、植物に対して病原性を有さない微生物由来のクチナーゼ様酵素を外部から植物に適用した際に共存する病原性微生物の病原性が増強されるといったことは、当業者が容易に想到しえないものであった。
Hereinafter, the present invention will be described in detail.
<Enzyme-containing composition for herbicidal>
The herbicidal enzyme-containing composition of the present invention is for controlling harmful plants, has a p-nitrophenyl butyrate hydrolyzing activity, and is a cutinase-like derived from a microorganism that does not have pathogenicity to plants. Contains enzymes. The herbicidal enzyme-containing composition of the present invention may contain only a cutinase-like enzyme derived from a microorganism that is not pathogenic to plants, and further contains a phytopathogenic microorganism as a herbicidal pesticide. Although it may contain, it is preferable that the phytopathogenic microbe as a herbicide for weeding is also included. When the herbicidal enzyme-containing composition of the present invention contains only a cutinase-like enzyme derived from a microorganism that is not pathogenic to plants, the phytopathogenic microorganism as a herbicidal pesticide is used as the herbicidal enzyme-containing composition. You may apply after applying to a plant.
[Cutinase-like enzyme]
Regarding the relationship between cutinase and the pathogenicity of pathogenic microorganisms, Futinium solani and rice blast fungus, which are known as pathogenic microorganisms, have reduced cutaneous infection rates in some cutinase-deficient strains. Although suggested to be involved in virulence (Rogers et. Al., "Cutinase Gene Disruption in Fusarium solani f sp pisi Decreases Its Virulence on Pea", The Plant Cell, Vol. 6, 935-945, 1994; Skamnioti and Gurr, "Cutinase and hydrophobin interplay", Plant Signaling & Behavior 3: 4, 248-250, April 2008), the role of cutinase in the pathogenic mechanism has not yet been clarified. In addition, it is not considered that pathogenic bacteria can invade plants by the action of cutinase alone, and the knowledge that pathogenic microorganisms have increased pathogenicity by supplying cutinase from the outside has not been obtained at all. There wasn't.
Conversely, by treating plants with cutinase produced by pathogenic microorganisms or by the degradation products of cutin by cutinase, plants detect the presence of pathogenic microorganisms and induce resistance reactions by the plants, thereby resisting disease. (Chassot et al., "Cuticular defects lead to full immunity to a major plant pathogen", The Plant Journal (2007) 49, 972-980; Curvers et al., "Abscisic Acid Deficiency Causes Changes in Cuticle Permeability and Pectin Composition That Influence Tomato Resistance to Botrytis cinerea ", Plant Physiology, October 2010, Vol. 154, p. 847-860; L'Haridon et al.," A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity ", PLos Pathogens, July 2011, Vol. 7, Issue 7, e1002148).
Furthermore, in the cutinase-like enzyme PaE produced by Pseudozyma antarctica and the cutinase-like enzyme PCLE of the filamentous fungus deposited under the accession number NITE P-573, 100% of the lipase box having the same primary structure in the cutinase-like enzyme is preserved. Nevertheless, the homology in these partial amino acid sequences with Colletotrichum gloesporioides cutinase, known as a phytopathogenic microorganism, is 85% and low. In addition, the fatty acid p-nitrophenyl hydrolyzing activity, which is one of the indicators of cutinase activity, is also related to the cutinase-like enzyme PaE produced by Pseudozyma antarctica and the fungal cutinase-like enzyme deposited under accession number NITE P-573. In PCLE, the long-chain fatty acid ester also decomposes efficiently, and the substrate specificity is very wide. On the other hand, the pathogenic microorganism Fusarium solani cutinase has a very low degradation activity of the long-chain fatty acid ester. Thus, from the viewpoint of the specificity of the enzyme reaction, the cutinase-like enzyme PaE produced by Pseudozyma antarctica and the cutinase-like enzyme PCLE of the filamentous fungus deposited under the deposit number NITE P-573 are different from those of pathogenic microorganisms. Kodama et al., "Crystal structure and enhanced activity of a cutinase-like enzyme from Cryptococcus sp. Strain S-2", Proteins p. 709-717, 2009; Shinozaki Y, Morita T, Cao XH, Yoshida S, Koitabashi M, Watanabe T, Suzuki K, Sameshima-Yamashita Y, Nakajima-Kambe T, Fujii T, Kitamoto HK Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization.Appl Microbiol Biotechnol (in press) DOI : 10.1007 / s00253-012-4188-8).
For this reason, even if cutinase plays a role in pathogenic expression in pathogenic microorganisms, the pathogenicity of pathogenic microorganisms is enhanced when the cutinase of pathogenic microorganisms is externally applied to plants, Those skilled in the art cannot easily imagine that the pathogenicity of pathogenic microorganisms coexisting when a cutinase-like enzyme derived from a microorganism that does not have pathogenicity to plants is externally applied to the plant is enhanced. It was a thing.
本発明の除草用酵素含有組成物が含有するクチナーゼ様酵素は、植物に対して病原性を有さない微生物から得られたものであり、好ましくは、Pseudozyma属、Cryptococcus属、Acremonium属、Alternaria属、Aspergillus属、Aureobasidium属、Cladosporium属、Epicoccum属、Fusarium属、Leptosphaeria属、Penicillium属、Phoma属、Neoplaconema属、Paraphoma属、Paecilomyces属に属する微生物から選択される少なくとも１種である、植物に対して病原性を有さない微生物から得られたものであり、より好ましくは、これら属に属する植物常在菌から得られたクチナーゼ様酵素から得られたものである。より具体的には、Pseudozyma antarctica、Pseudozyma ruglosa、Pseudozyma tsukubaensis、Pseudozyma aphidis、Aspergillus oryzae、Cryptococcus flavus、Cryptococcus laurentii、Cryptococcus rajasthanensis、Cryptococcus magnus、Alternaria alternata、Cladosporium cladosporioides、Cladosporium oxysporum、及びPenicillium pinophilumから得られたクチナーゼ様酵素、並びに受託番号NITE P-573で寄託された糸状菌株から得られたクチナーゼ様酵素PCLEを挙げることができる。
これらのクチナーゼ様酵素は、葉面酵母Pseudozyma antarctica由来のクチナーゼ様酵素のPaEの推定アミノ酸配列のうち、第１０６アミノ酸残基から第１１３アミノ酸残基のアミノ酸配列に相当するGYSQGAAA（リパーゼボックス）と９０％以上の相同性を有する部分アミノ酸配列を有し、かつ第１８２アミノ酸残基から第２０４アミノ酸残基のアミノ酸配列に相当するCIYGDGVCDVSSGFGITPQHLTY（活性中心を構成するアミノ酸配列）と３０％以上の相同性を有する部分アミノ酸配列を有していることが好ましい。また、上記クチナーゼ様酵素は、Pseudozyma antarctica由来のクチナーゼ様酵素のPaEのリパーゼボックスと、より好ましくは９５％以上、更に好ましくは９７％以上、最も好ましくは１００％の相同性を有する部分アミノ酸配列を有する。同様に、上記クチナーゼ様酵素は、Pseudozyma antarctica由来のクチナーゼ様酵素PaEの活性中心を構成するアミノ酸配列と、より好ましくは３４％以上、更に好ましくは６０％以上、最も好ましくは７８％以上の相同性を有する部分アミノ酸配列を有する。
以上のクチナーゼ様酵素の中でも、本発明の除草用酵素含有組成物は、Pseudozyma antarcticaのクチナーゼ様酵素PaE、受託番号NITE P-573で寄託された糸状菌株から得られたクチナーゼ様酵素PCLE、Cryptococcus sp. S-2株のクチナーゼ様酵素CLE、Aspergillus oryzaeのCutL1、及びCryptococcus magnusru類縁菌BPD1A株から得られたクチナーゼ様酵素CmCut1を含有することがより好ましく、Pseudozyma antarcticaのクチナーゼ様酵素PaE、及び受託番号NITE P-573で寄託された糸状菌株から得られたクチナーゼ様酵素PCLEを含有することが最も好ましい（Suzuki K, Shinozaki Y, Sakamoto H, Tabata J, Watanabe T, Mochizuki A, Koitabashi M, Fujii T, Tsushima S, and Kitamoto HK Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis Appl. Microbiol. Biotechnol. (in press)も参照）。
The cutinase-like enzyme contained in the herbicidal enzyme-containing composition of the present invention is obtained from a microorganism that is not pathogenic to plants, and preferably, the genus Pseudozyma, Cryptococcus, Acremonium, Alternaria A plant that is at least one selected from microorganisms belonging to the genus Aspergillus, Aureobasidium, Cladosporium, Epicocumcum, Fusarium, Leptosphaeria, Penicillium, Phoma, Neoplocanone, Paraphoma, Paecilomyces Those obtained from microorganisms having no pathogenicity, and more preferably those obtained from cutinase-like enzymes obtained from plant resident bacteria belonging to these genera. More specifically, Pseudozyma antarctica, Pseudozyma ruglosa, Pseudozyma tsukubaensis, Pseudozyma aphidis, Aspergillus oryzae, Cryptococcus flavus, Cryptococcus laurentii, Cryptococcus rajasthanensis, Cryptococternaria magnus And the cutinase-like enzyme PCLE obtained from the filamentous strain deposited under the deposit number NITE P-573.
These cutinase-like enzymes include GYSQGAAA (lipase box) and 90 corresponding to the amino acid sequence from the 106th amino acid residue to the 113th amino acid residue in the deduced amino acid sequence of PaE of the cutinase-like enzyme derived from the foliar yeast Pseudozyma antarctica. 30% or more homology with CIYGDGVCDVSSGFGITPQHLTY (amino acid sequence constituting the active center) having a partial amino acid sequence having a homology of at least% and corresponding to the amino acid sequence from amino acid residues 182 to 204 It preferably has a partial amino acid sequence. The cutinase-like enzyme has a partial amino acid sequence having a homology of more than 95%, more preferably more than 97%, most preferably 100% with the PaE lipase box of the cutinase-like enzyme derived from Pseudozyma antarctica. Have. Similarly, the cutinase-like enzyme has a homology of 34% or more, more preferably 60% or more, most preferably 78% or more with the amino acid sequence constituting the active center of the cutinase-like enzyme PaE derived from Pseudozyma antarctica. Having a partial amino acid sequence.
Among the above cutinase-like enzymes, the herbicidal enzyme-containing composition of the present invention includes a cutinase-like enzyme PCE, Cryptococcus sp. Obtained from the cutinase-like enzyme PaE of Pseudozyma antarctica, the filamentous strain deposited under accession number NITE P-573. More preferably it contains cutinase-like enzyme CLE of S-2, CutL1 of Aspergillus oryzae, and cutinase-like enzyme CmCut1 obtained from Cryptococcus magnusru related strain BPD1A, cutinase-like enzyme PaE of Pseudozyma antarctica, and accession number Most preferably it contains the cutinase-like enzyme PCLE obtained from the filamentous strain deposited at NITE P-573 (Suzuki K, Shinozaki Y, Sakamoto H, Tabata J, Watanabe T, Mochizuki A, Koitabashi M, Fujii T, Tsushima S, and Kitamoto HK Affinity purification and characterization of a biodegradable plastic-degrading enzyme from a yeast isolated from the larval midgut of a stag beetle, Aegus laevicollis Appl.Microbiol. Biotechnol. See also ess).
本発明の除草用酵素含有組成物におけるクチナーゼ様酵素の含有量は、酪酸ｐ−ニトロフェニル加水分解活性に換算して、０．０１Ｕ／ｍＬ以上の酵素活性を有する量であり、０．１Ｕ／ｍＬ以上であることが好ましく、０．２Ｕ／ｍＬ以上であることがより好ましく、０．５Ｕ／ｍＬ以上であることが最も好ましい。なお、本発明の除草用酵素含有組成物に含まれるクチナーゼ酵素の酵素活性は、当該酵素の至適ｐＨ近傍のｐＨ、好ましくは至適ｐＨで測定した場合に、上記の酵素活性以上の酵素活性を有することが好ましい。
ここで、本発明の除草用酵素含有組成物において、個別のクチナーゼ様酵素の酪酸ｐ−ニトロフェニル加水分解活性は、例えば、Pseudozyma antarcticaのPaEの場合であれば、ｐＨ８．０の５０ｍＭ Ｔｒｉｓ塩酸緩衝液中、３０℃で５分間反応させることにより測定した酪酸ｐ−ニトロフェニルの加水分解活性に換算して算出したものであり、１分間に１μｍｏｌの酪酸ｐ−ニトロフェニルを加水分解可能な酵素活性を１Ｕとした。また、基質として、酪酸ｐ−ニトロフェニルに代えてポリブチレンサクシネートアジペート（ＰＢＳＡ）を用いてもよいが、ｐＨ８．８の２０ｍＭ Ｔｒｉｓ塩酸緩衝液中、３０℃で測定したPaEのＰＢＳＡ加水分解活性１Ｕ／ｍＬは、上記条件下で測定した酪酸ｐ−ニトロフェニル加水分解活性１．９２Ｕ／ｍＬに換算される。また、クチナーゼ様酵素の各種基質の加水分解活性は、所定濃度以上のカルシウムイオンを含む緩衝液中で、より高まる場合があり、クチナーゼ様酵素の活性を定めるにあたって必要に応じて緩衝液にカルシウムイオンを添加することが好ましいが、例えば、２ｍＭ塩化カルシウムを含むｐＨ６．８の２０ｍＭ Ｔｒｉｓ塩酸緩衝液中で測定したPCLEのＰＢＳＡ加水分解活性１Ｕ／ｍＬは、上記条件下で測定した酪酸ｐ−ニトロフェニル加水分解活性１．１１Ｕ／ｍＬに換算される。
なお、PaEのＰＢＳＡ加水分解活性における至適ｐＨは９〜１０であり、PCLEのＰＢＳＡ加水分解活性における至適ｐＨは７〜８である。
The content of the cutinase-like enzyme in the herbicidal enzyme-containing composition of the present invention is an amount having an enzyme activity of 0.01 U / mL or more in terms of p-nitrophenyl butyrate hydrolyzing activity, and 0.1 U / It is preferably at least mL, more preferably at least 0.2 U / mL, and most preferably at least 0.5 U / mL. Note that the enzyme activity of the cutinase enzyme contained in the herbicidal enzyme-containing composition of the present invention is higher than the above enzyme activity when measured at a pH near the optimum pH of the enzyme, preferably at the optimum pH. It is preferable to have.
Here, in the herbicidal enzyme-containing composition of the present invention, the p-nitrophenyl butyrate hydrolyzing activity of individual cutinase-like enzymes is, for example, 50 mM Tris hydrochloric acid buffer at pH 8.0 in the case of Pseudozyma antarctica PaE. It was calculated in terms of the hydrolysis activity of p-nitrophenyl butyrate measured by reacting in liquid at 30 ° C. for 5 minutes, and the enzyme activity capable of hydrolyzing 1 μmol of p-nitrophenyl butyrate per minute. Was 1U. In addition, polybutylene succinate adipate (PBSA) may be used as a substrate instead of p-nitrophenyl butyrate, but the PBSA hydrolysis activity of PaE measured at 30 ° C. in 20 mM Tris hydrochloric acid buffer at pH 8.8. 1 U / mL is converted into p-nitrophenyl butyrate hydrolysis activity of 1.92 U / mL measured under the above conditions. In addition, the hydrolytic activity of various substrates of cutinase-like enzyme may be further increased in a buffer solution containing calcium ions at a predetermined concentration or higher, and calcium ions may be added to the buffer solution as necessary to determine the activity of cutinase-like enzyme. For example, 1U / mL of PCLE in PBSA hydrolyzing activity measured in a 20 mM Tris-HCl buffer solution containing 2 mM calcium chloride at pH 6.8 is p-nitrophenyl butyrate measured under the above conditions. Hydrolysis activity is converted to 1.11 U / mL.
In addition, the optimal pH in PBSA hydrolysis activity of PaE is 9-10, and the optimal pH in PBSA hydrolysis activity of PCLE is 7-8.
以上に説明したクチナーゼ様酵素については、上述した植物に対して病原性を有さない微生物の培養ろ液に由来するクチナーゼ様酵素であることが好ましい。
上記微生物の培養ろ液を得る方法については、上記微生物について従来行われている方法を採用すればよく、特に限定されるものではないが、例えば、Pseudozyma antarcticaの場合、以下の培養方法を採用することができる。
まず、培地としては、Pseudozyma antarcticaの培養に通常使用される、通常の合成培地、半合成培地、及び天然培地に、必要に応じて炭素源としてグルコース及び／又はキシロースを加え、２５℃以上３２℃以下の培養条件で、２４時間の前培養を行った後、同様の温度で７２時間以上培養する。前培養の培地としては、YM培地（Yeast Extract Malt extract medium）を使用することが好ましく、本培養の培地としては、FMM培地（Fungal Minimum Medium）、又は各成分の濃度を高濃度に調整したFMM培地、好ましくは炭素源としてキシロースのみを添加し、各成分の濃度を通常使用される濃度の３倍に調整したFMM培地を使用することが好ましい。
また、受託番号NITE P-573で寄託された糸状菌の場合は、一般に糸状菌の培養に用いられる合成培地であるCzapek-Dox液体培地に、唯一の炭素源としてＰＢＳＡエマルジョン（商品名：ＥＭ−３０１（昭和電工社製）等）を加えて培養することにより培養ろ液中にクチナーゼ様酵素PCLEを生産させることができる。
斯くして培養した培地を遠心処理した後、孔径０．４５μｍ以下のろ過膜でろ過し、Pseudozyma antarcticaや受託番号NITE P-573で寄託された糸状菌の菌体を取り除くことにより、それぞれの菌が産生したクチナーゼ様酵素を含有する培養ろ液を得ることができる。
更に、本発明の除草用酵素含有組成物に用いられるクチナーゼ様酵素は、培養ろ液から精製したものであってもよい。たとえば、Pseudozyma antarcticaのクチナーゼ様酵素PaEの粗酵素液を得る場合は、培養ろ液に、硫酸アンモニウムを最終濃度５０％になるように加え、４℃で一夜静置後、４０，０００ｇで遠心分離して得られるPaEを含む沈殿物を緩衝液で可溶化し、透析による脱塩を行って得ることができる。なお、本発明の除草用酵素含有組成物に用いられるクチナーゼ様酵素は、粗酵素液をさらに精製したクチナーゼ様酵素であって、酪酸ｐ−ニトロフェニル加水分解活性やＰＢＳＡ加水分解活性を有するものや、遺伝子組換え技術を使用して他の生物で生産したクチナーゼ様酵素の各種溶液や精製物を用いてもよい。
The cutinase-like enzyme described above is preferably a cutinase-like enzyme derived from a culture filtrate of a microorganism that is not pathogenic to the above-described plant.
The method for obtaining the culture filtrate of the microorganism is not particularly limited as long as a conventional method for the microorganism is employed. For example, in the case of Pseudozyma antarctica, the following culture method is employed. be able to.
First, as a medium, glucose and / or xylose is added as a carbon source to a normal synthetic medium, a semi-synthetic medium, and a natural medium that are usually used for culturing Pseudozyma antarctica. After pre-culture for 24 hours under the following culture conditions, the cells are cultured at the same temperature for 72 hours or more. It is preferable to use a YM medium (Yeast Extract Malt extract medium) as a pre-culture medium. As a main culture medium, an FMM medium (Fungal Minimum Medium) or an FMM in which the concentration of each component is adjusted to a high concentration is used. It is preferable to use a medium, preferably an FMM medium in which only xylose is added as a carbon source and the concentration of each component is adjusted to 3 times the concentration normally used.
In addition, in the case of the filamentous fungi deposited under the deposit number NITE P-573, the Czapek-Dox liquid medium, which is a synthetic medium generally used for the cultivation of filamentous fungi, is added with PBSA emulsion (trade name: EM- 301 (manufactured by Showa Denko Co., Ltd.) and the like is added to the culture and the cutinase-like enzyme PCLE can be produced in the culture filtrate.
After culturing the thus cultured medium, the medium is filtered through a filtration membrane having a pore size of 0.45 μm or less, and the fungus bodies deposited under Pseudozyma antarctica and accession number NITE P-573 are removed, whereby each bacterium is removed. A culture filtrate containing the cutinase-like enzyme produced by can be obtained.
Furthermore, the cutinase-like enzyme used in the herbicidal enzyme-containing composition of the present invention may be purified from a culture filtrate. For example, when obtaining a crude enzyme solution of the cutinase-like enzyme PaE of Pseudozyma antarctica, ammonium sulfate is added to the culture filtrate to a final concentration of 50%, and the mixture is allowed to stand overnight at 4 ° C. and then centrifuged at 40,000 g. The precipitate containing PaE obtained in this manner can be solubilized with a buffer solution and desalted by dialysis. The cutinase-like enzyme used in the herbicidal enzyme-containing composition of the present invention is a cutinase-like enzyme obtained by further purifying the crude enzyme solution, which has p-nitrophenyl butyrate hydrolyzing activity or PBSA hydrolyzing activity, Various solutions and purified products of cutinase-like enzymes produced in other organisms using genetic recombination techniques may be used.
［植物病原性微生物］
本発明の除草用酵素含有組成物は、除草用農薬である植物病原性微生物を含有することが好ましい。植物病原性微生物は、従来一般に使用されているものを使用すればよく、その種類については特に限定されるものではないが、例えば、灰色かび病菌（例えば、Botrytis属に属する病原菌）、さび病菌（例えば、Puccinia属に属する病原菌）を、特に好ましく使用することができる。また、それら以外の主要な植物病原菌としては、いもち病（Pyricularia oryzae）、苗腐病菌（Pythium graminicola）、紋枯病菌（Rhizoctonia solani）、ばか苗病菌（Gibberella fujikuroi）、ごま葉枯病菌（Cochliobolus miyabeanus）、苗立枯病菌（Rhizopus oryzae）、赤かび病菌（Fusarium graminearum）、紫斑病菌（Cercospora kikuchii）、うどんこ病菌（一例としてSphaerotheca fuliginea）、菌核病菌（一例としてSclerotinia sclerotiorum）、炭疽病菌（一例としてColletotrichum orbiculare）、疫病菌（一例としてPhytophthora melonis）、各種立枯性病害を引き起こすフザリウム病菌（一例としてFusarium oxysporum）、葉かび病菌（Passalora fulva）、輪紋病菌（Alternaria solani）、褐色輪紋病菌（Corynespora cassiicola）、斑点病菌（Stemphylium lycopersici）、褐紋病菌（Phomopsis vexans）、すすかび病菌（Mycovellosiella nattrassii）、褐色円星病菌（Paracercospora egenula）、べと病菌（Pseudoperonospora cubensis）、褐斑病菌（Corynespora cassiicola）、つる枯病菌（Didymella bryoniae）、斑点病菌（Cercospora capsici）、萎凋病菌（Fusarium oxysporum f.sp.spinaciae）、斑点落葉病菌（Alternaria mali）、黒星病菌（Venturia inaequalis）、黒斑病菌（Alternaria kikuchiana）、黒星病菌（Venturia nashicola）、赤星病菌（Gymnosporangium asiaticum）、黒点病菌（Diaporthe citri）、青かび病菌（Penicillium italicum）、晩腐病菌（Glomerella cingulata）、べと病菌（Plasmopara viticola）等を挙げることができる。これらの植物病原性微生物は、駆除の対象となる有害植物の種類に応じて適宜、選択して使用すればよく、駆除の対象となる有害植物への特異的な病原性を有する微生物を使用することが好ましい。
[Plant pathogenic microorganisms]
The herbicidal enzyme-containing composition of the present invention preferably contains a phytopathogenic microorganism that is a herbicidal agricultural chemical. The plant pathogenic microorganisms may be those conventionally used, and the type thereof is not particularly limited. For example, gray mold fungi (for example, pathogens belonging to the genus Botrytis), rust fungi ( For example, pathogenic bacteria belonging to the genus Puccinia can be particularly preferably used. Other major plant pathogens include Pyricularia oryzae, Pythium graminicola, Rhizoctonia solani, Baka seedling fungus (Gibberella fujikuroi), Sesame leaf blight fungus (Cochliobolus) miyabeanus), Rhizopus oryzae, Fusarium graminearum, Purcobacterium (Cercospora kikuchii), powdery mildew (for example, Sphaerotheca fuliginea), mycorrhizal fungus (for example, Sclerotinia sclerotiorum), anthrax Colletotrichum orbiculare), plagues (Phytophthora melonis as an example), Fusarium fungus causing various diseases (Fusarium oxysporum as an example), fungi (Passalora fulva), ring rot (Alternaria solani), brown ring Fungus (Corynespora cassiicola), Spot fungus (Stemphylium lycopersici), Brown rot fungus (Phomopsis vexans), Fungus fungus (Mycovellosiella nattrassii) Brown crustacean (Paracercospora egenula), downy mildew (Pseudoperonospora cubensis), brown blight (Corynespora cassiicola), vine blight (Didymella bryoniae), bacilli (Cercospora capsici), wilt (Fusariumspoxyfporum.oxysporum. ), Spotted deciduous fungus (Alternaria mali), black spot fungus (Venturia inaequalis), black spot fungus (Alternaria kikuchiana), black spot fungus (Venturia nashicola), red stink fungus (Gymnosporangium asiaticum), black spot fungus (Diaporthe citri), blue mold fungus (Pili) italicum), late rot fungus (Glomerella cingulata), downy mildew (Plasmopara viticola) and the like. These phytopathogenic microorganisms may be appropriately selected and used according to the type of harmful plant to be controlled, and microorganisms having specific pathogenicity to the harmful plant to be controlled are used. It is preferable.
本発明の除草用酵素含有組成物に用いられる植物病原性微生物は、従来公知の方法により培養することにより得ることができる。そのような培養方法としては、例えば、植物病原性微生物を、これが増殖可能な培地で培養し、刷毛でかき取ったり、遠心分離を行ったりする等の手段を用いて、菌体や胞子を回収する方法を挙げることができる。人工培養ができない植物病原性微生物（絶対寄生菌）の場合は、植物体の上で増殖させた試料から上記と同様の方法で菌体や胞子を回収することが一般的である。
本発明の除草用酵素含有組成物に用いられる植物病原性微生物は、微生物の生菌であるが、当該微生物の生菌は、除草用酵素含有組成物の保存安定性の観点から、胞子の形態であることが好ましい。このため、上記植物病原性微生物の培養方法においては、培養の終期において微生物の生菌を胞子の形態にするために、培地等の種類や組成、培地等のｐＨ、培養温度、培養湿度、培養する際の酸素濃度などの培養条件を、その胞子の形成条件に適合させるように培養することが好ましい。なお、人工培養の不可能な絶対寄生菌を培養する場合においては、当該病原菌が通常寄生する植物を適宜選択し、当該病原菌が通常生育する条件下で培養し、胞子等を収集することが好ましい。
また、本発明の除草用酵素含有組成物を流通させる際に、クチナーゼ様酵素を含有する水溶液（好ましくはクチナーゼ様酵素を産生し、植物に対して病原性を有さない微生物の培養ろ液）と、植物病原性微生物を別容器に収容して販売して、除草用酵素含有組成物の使用直前に両者を混合する形態としてもよく、クチナーゼ様酵素のみを含む除草用酵素含有組成物と、植物病原性微生物を別々に有害植物に適用してもよい。
本発明の除草用酵素含有組成物に含まれる植物病原性微生物の含有量は、本発明の効果を損なわない限りにおいて特に限定されないが、懸濁液中に含まれる胞子の数が、１０spores/mL以上１０8spores/mL以下であることが好ましく、１０4spores/mL以上１０7spores/mL以下であることが更に好ましい。
The phytopathogenic microorganism used in the herbicidal enzyme-containing composition of the present invention can be obtained by culturing by a conventionally known method. As such a culture method, for example, phytopathogenic microorganisms are cultured in a medium in which the phytopathogenic microorganisms can grow and scraped with a brush or centrifuged, and then the cells and spores are collected. The method of doing can be mentioned. In the case of phytopathogenic microorganisms (absolute parasitic fungi) that cannot be artificially cultured, it is common to collect bacterial cells and spores from a sample grown on a plant by the same method as described above.
The phytopathogenic microorganism used in the herbicidal enzyme-containing composition of the present invention is a living microorganism, but the living microorganism is a spore form from the viewpoint of storage stability of the herbicidal enzyme-containing composition. It is preferable that For this reason, in the above cultivating method of phytopathogenic microorganisms, in order to make viable microorganisms into spore form at the end of the culture, the type and composition of the medium, the pH of the medium, the culture temperature, the culture humidity, the culture It is preferable that the culture conditions such as the oxygen concentration at the time of culturing are adapted to match the spore formation conditions. In the case of culturing an absolute parasitic bacterium that cannot be artificially cultured, it is preferable to appropriately select a plant in which the pathogenic bacterium normally parasitizes, culture under conditions where the pathogenic bacterium normally grows, and collect spores and the like. .
In addition, when distributing the herbicidal enzyme-containing composition of the present invention, an aqueous solution containing a cutinase-like enzyme (preferably a culture filtrate of a microorganism that produces a cutinase-like enzyme and has no pathogenicity to plants) And the plant pathogenic microorganism may be stored in a separate container and sold, and both may be mixed immediately before use of the herbicidal enzyme-containing composition, the herbicidal enzyme-containing composition containing only a cutinase-like enzyme, and The phytopathogenic microorganisms may be applied separately to harmful plants.
The content of the phytopathogenic microorganism contained in the herbicidal enzyme-containing composition of the present invention is not particularly limited as long as the effects of the present invention are not impaired, but the number of spores contained in the suspension is 10 spores / mL. It is preferably 10 8 spores / mL or less, more preferably 10 4 spores / mL or more and 10 7 spores / mL or less.
本発明の除草用酵素含有組成物は、更に、一般に農薬等の散布の際に用いられる展着剤等、クチナーゼ様酵素や植物病原性微生物の植物体表面への付着・伸展性を高めるための成分を含んでいてもよい。 The herbicidal enzyme-containing composition of the present invention further enhances adherence / extensibility of cutinase-like enzymes and phytopathogenic microorganisms to the plant surface, such as spreading agents generally used when spraying agricultural chemicals, etc. Ingredients may be included.
＜有害植物の駆除方法＞
本発明は、植物病原性微生物として、駆除の対象となる有害植物への特異的な病原性を有する微生物を含む除草用酵素含有組成物を、有害植物へ適用する工程を含むことを特徴とする有害植物の駆除方法にも関する。除草用酵素含有組成物を、有害植物へ適用するにあたっては、従来公知の手法を使用して、除草用酵素含有組成物を有害植物へ散布又は噴霧することにより、除草用酵素含有組成物を有害植物の植物体の表面と接触させればよい。
なお、有害植物への特異的な病原性を有する植物病原性微生物を使用する場合、除草用酵素含有組成物の一成分として当該組成物中に混合して散布してもよいし、除草用酵素含有組成物の散布時期とは時期を変えて散布してもよい。植物病原性微生物を、除草用酵素含有組成物の散布時期と時期を変えて散布する場合には、除草用酵素含有組成物の散布後の１日後から５日後に植物病原性微生物を散布することが好ましく、除草用酵素含有組成物の散布後の３日後に植物病原性微生物を散布することが更に好ましい。
このようにして、除草用酵素含有組成物を有害植物の植物体に接触させることにより、除草用酵素含有組成物に含まれるクチナーゼ様酵素が、有害植物の病害菌耐性を低下させ、除草用酵素含有組成物中に含まれる病原性微生物又は除草用酵素含有組成物とは別途散布される病原性微生物が、有害植物に病害を引き起こすことにより、有害植物を駆除することができる。
なお、本発明の除草用酵素含有組成物は、好ましくは、植物に対して病原性を有さない微生物の培養ろ液として提供されるため、植物病原性微生物を含む液状の組成物を調製して散布又は噴霧されることが好ましいが、クチナーゼ様酵素を含有する組成物を粉体組成物として提供可能な場合は、これを植物病原性微生物の胞子等と混合することにより、粉体の組成物として除草用酵素含有組成物を調製し、当該粉体の除草用酵素含有組成物を有害植物に適用することが好ましい。
<How to eliminate harmful plants>
The present invention includes a step of applying, as a plant pathogenic microorganism, a herbicidal enzyme-containing composition containing a microorganism having specific pathogenicity to a harmful plant to be controlled to a harmful plant. It also relates to methods for controlling harmful plants. In applying the herbicidal enzyme-containing composition to harmful plants, the herbicidal enzyme-containing composition is harmful by spraying or spraying the herbicidal enzyme-containing composition onto the harmful plants using a conventionally known method. What is necessary is just to make it contact with the surface of the plant body of a plant.
In addition, when using a phytopathogenic microorganism having specific pathogenicity to harmful plants, it may be mixed and sprayed as one component of the herbicidal enzyme-containing composition, or the herbicidal enzyme You may spread by changing a time with the dispersion | spreading time of a containing composition. When phytopathogenic microorganisms are to be sprayed at different spraying periods of herbicidal enzyme-containing composition, spray phytopathogenic microorganisms 1 to 5 days after spraying herbicidal enzyme-containing composition. It is more preferable to spray the phytopathogenic microorganism 3 days after spraying the herbicidal enzyme-containing composition.
In this way, by bringing the herbicidal enzyme-containing composition into contact with the plants of harmful plants, the cutinase-like enzyme contained in the herbicidal enzyme-containing composition reduces the resistance of harmful plants to harmful bacteria, Pathogenic microorganisms sprayed separately from the pathogenic microorganisms contained in the containing composition or the herbicidal enzyme-containing composition cause diseases to harmful plants, so that harmful plants can be controlled.
In addition, since the herbicidal enzyme-containing composition of the present invention is preferably provided as a culture filtrate of microorganisms that are not pathogenic to plants, a liquid composition containing phytopathogenic microorganisms is prepared. However, when a composition containing a cutinase-like enzyme can be provided as a powder composition, it is mixed with spores of phytopathogenic microorganisms to obtain a composition of the powder. It is preferable to prepare a herbicidal enzyme-containing composition as a product and apply the powdered herbicidal enzyme-containing composition to harmful plants.
また、本発明は、酪酸ｐ−ニトロフェニル加水分解活性を有するクチナーゼ様酵素を産生し、植物に対して病原性を有さない微生物の培養液を、有害植物へ適用する工程を含むことを特徴とする有害植物の駆除方法であって、植物に対して病原性を有さない微生物がPseudozyma属、Cryptococcus属、Acremonium属、Alternaria属、Aspergillus属、Aureobasidium属、Cladosporium属、Epicoccum属、Fusarium属、Leptosphaeria属、Penicillium属、Phoma属、Neoplaconema属、Paraphoma属、Paecilomyces属に属する微生物であることを特徴とする有害植物の駆除方法にも関する。
クチナーゼ様酵素を含有する微生物の培養液としては、当該微生物の培養ろ液を用いることが好ましく、その製造方法や使用条件は、上記＜除草用酵素含有組成物＞の［クチナーゼ様酵素］の項で説明した製造方法や使用条件と同様の方法及び条件を使用することができる。また、クチナーゼ様酵素を含有する溶液としては、クチナーゼ様酵素の粗精製溶液や精製液を用いてもよい。
クチナーゼ様酵素を含有する微生物の培養液や培養ろ液、粗精製溶液や精製液を有害植物に適用することにより、有害植物の乾燥耐性や病原菌耐性を低下させ、有害植物の植物組織が水分を失ったり、環境中の病原性微生物に感染したりすることにより、有害植物を駆除することができる。
Further, the present invention includes a step of producing a cutinase-like enzyme having p-nitrophenyl butyrate hydrolyzing activity and applying a culture solution of a microorganism having no pathogenicity to plants to harmful plants. A method for controlling harmful plants, wherein microorganisms that are not pathogenic to plants are Pseudozyma, Cryptococcus, Acremonium, Alternaria, Aspergillus, Aureobasidium, Cladosporium, Epicocccum, Fusarium, The present invention also relates to a method for controlling harmful plants characterized by being a microorganism belonging to the genus Leptosphaeria, Penicillium, Phoma, Neoplocanone, Paraphoma, and Paecilomyces.
As a culture solution of a microorganism containing a cutinase-like enzyme, it is preferable to use a culture filtrate of the microorganism, and the production method and use conditions thereof are described in the section [Cutinase-like enzyme] of the above-mentioned <herbicidal enzyme-containing composition>. The method and conditions similar to the manufacturing method and use conditions described in the above can be used. Moreover, as a solution containing a cutinase-like enzyme, a crude purified solution or purified solution of a cutinase-like enzyme may be used.
By applying the culture solution or culture filtrate of microorganisms containing cutinase-like enzymes, crude purification solutions or purification solutions to harmful plants, the drought tolerance and pathogen resistance of harmful plants are reduced, and the plant tissue of harmful plants loses moisture. Harmful plants can be controlled by losing them or infecting them with pathogenic microorganisms in the environment.
以下、本発明について実施例を挙げて詳細に説明する。なお、本発明は、以下に示す実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples. In addition, this invention is not limited to the Example shown below at all.
＜実施例１＞
硫酸アンモニウムを最終濃度５０％になるように加え、４℃で一夜静置後、４０，０００ｇで遠心分離して得られるPaEを含む沈殿物を緩衝液で可溶化し、透析による脱塩を行って粗精製し、２０ｍＭトリス塩酸緩衝液（ｐＨ６．８）で透析した葉面酵母Pseudoyzma antarctica由来のクチナーゼ様粗精製酵素（PaE；植物に対して病原性を有さない微生物由来のクチナーゼ様酵素）溶液（酪酸ｐ−ニトロフェニル加水分解活性は１４．４Ｕ／ｍＬ）を上記緩衝液で５倍に希釈し、２．８８Ｕ／ｍＬとした。矮性のトマト（MicroTom）および、シロイヌナズナの切葉に対してこれを噴霧処理した後、密閉容器の中で保湿しつつ保温した。その結果、粗精製したPaEが含まれる緩衝液を噴霧したトマト及びシロイヌナズナの葉面は、黄化または液化し、カビなどによる病徴が確認できた。また、同濃度の粗酵素溶液の噴霧７２時間後に、それぞれの植物に感染する病原性糸状菌（灰色カビ病菌）の胞子（５×１０4spores/mL；植物病原性微生物）を噴霧すると、感染効率が促進され病徴の発現が助長された（図１参照）。
なお、図１中、左図はトマトの切葉を粗酵素液希釈用バッファーで処理したもの、中央図は２．８８Ｕ／ｍＬの酪酸ｐ−ニトロフェニル加水分解活性を有する粗酵素液で処理したもの、右図は２．８８Ｕ／ｍＬの酪酸ｐ−ニトロフェニル加水分解活性を有する粗酵素液と、灰色カビ病菌の胞子とで処理したものを示す。
<Example 1>
Ammonium sulfate was added to a final concentration of 50%, and after standing overnight at 4 ° C., the precipitate containing PaE obtained by centrifugation at 40,000 g was solubilized with a buffer solution and desalted by dialysis. A crudely purified cutinase-like crude enzyme (PaE; a cutinase-like enzyme derived from a microorganism that is not pathogenic to plants) derived from the foliar yeast Pseudoyzma antarctica, dialyzed with 20 mM Tris-HCl buffer (pH 6.8) (The p-nitrophenyl butyrate hydrolyzing activity was 14.4 U / mL) was diluted 5-fold with the above buffer to give 2.88 U / mL. This was sprayed on dwarf tomato (MicroTom) and cut leaves of Arabidopsis thaliana, and then kept warm in a sealed container while being moisturized. As a result, the leaf surfaces of tomato and Arabidopsis sprayed with a buffer solution containing roughly purified PaE were yellowed or liquefied, and symptom due to mold or the like could be confirmed. In addition, after spraying the crude enzyme solution of the same concentration 72 hours after spraying spores (5 × 10 4 spores / mL; phytopathogenic microorganisms) of pathogenic filamentous fungi (gray mold fungus) that infect each plant, infection Efficiency was promoted and expression of disease symptoms was promoted (see FIG. 1).
In addition, in FIG. 1, the left figure processed tomato cut leaves with the crude enzyme solution dilution buffer, and the middle figure treated with 2.88 U / mL crude enzyme solution having p-nitrophenyl butyrate hydrolyzing activity. The right figure shows what was treated with 2.88 U / mL crude enzyme solution having p-nitrophenyl butyrate hydrolyzing activity and spores of gray mold.
＜実施例２＞
PaE（植物に対して病原性を有さない微生物由来のクチナーゼ様酵素）を含有するPseudozyma antarcticaの培養液から菌体を除去した後（培養ろ液）は、PaEの酪酸ｐ−ニトロフェニル加水分解活性は１５．３６Ｕ／ｍＬであった。
なお、Pseudozyma antarcticaの培養に当たっては、５Ｌ容のジャーファーメンターに、表１に示した組成の培地３Ｌを加え、Pseudozyma antarcticaをYM培地で培養した前培養液３０ｍＬを接種して、３０℃、撹拌速度５００ｒｐｍ、通気量８ＬＰＭで培養を行った。高泡形成による培養液の流出を防止するため、消泡剤（商品名「信越シリコーンKM-72F」、信越化学工業株式会社製）を、消泡センサーを利用して断続的に自動滴下し、培養開始後７２時間までにおおよそ６０ｍＬ添加した。
培地中のアンモニウムイオンの消費によるｐＨの低下をセンサーで感知し、窒素源の追加とｐＨの調整のため、培地にアンモニア水をアルカリ調整溶液として自動滴下してｐＨを６．０に調整した。培養開始２４時間後から、表２に示した組成の流加培地を０．５Ｌ／ｄの速度で流加した。
表１：ジャーファーメンター用培地
表２：PaE生産誘導用流加培地
※ディフコ社製
<Example 2>
After removing cells from the culture solution of Pseudozyma antarctica containing PaE (a cutinase-like enzyme derived from a microorganism that is not pathogenic to plants) (culture filtrate), p-nitrophenyl butyrate hydrolysis of PaE The activity was 15.36 U / mL.
When cultivating Pseudozyma antarctica, add 3 L of medium having the composition shown in Table 1 to a 5 L jar fermenter, inoculate 30 mL of a preculture solution in which Pseudozyma antarctica was cultured in YM medium, and stir at 30 ° C. Culturing was performed at a speed of 500 rpm and an aeration rate of 8 LPM. In order to prevent the culture fluid from flowing out due to the formation of high bubbles, an antifoaming agent (trade name “Shin-Etsu Silicone KM-72F”, manufactured by Shin-Etsu Chemical Co., Ltd.) is automatically and intermittently dropped using an anti-foam sensor, Approximately 60 mL was added by 72 hours after the start of culture.
A decrease in pH due to consumption of ammonium ions in the medium was detected by a sensor, and in order to add a nitrogen source and adjust the pH, aqueous ammonia was automatically added to the medium as an alkaline adjustment solution to adjust the pH to 6.0. From 24 hours after the start of the culture, a fed-batch medium having the composition shown in Table 2 was fed at a rate of 0.5 L / d.
Table 1: Jar fermenter medium
Table 2: PaE production induction fed-batch medium
* Difco
上記培養ろ液をポット栽培の矮性のトマト（MicroTom）の植物体に噴霧処理し、人工気象室内で２７℃で栽培した。対照区には、培養ろ液をオートクレーブで１２１℃、１５分間処理し、酪酸ｐ−ニトロフェニル加水分解活性が失われたことを確認した溶液（失活酵素液）を散布した。その結果、培養ろ液散布区では、７日後には、すでに展開している成熟葉の淵および先端が褐変化し、カーリングした。また、若芽の未熟葉では、全体的な褐変化とカーリングが観察された。未熟花芽では先端が褐変化した（図２Ａ参照）。また、培養ろ液を噴霧処理したトマトを屋外に設置し、給水を減らしたところ、７日後には落葉、黄化・褐変葉の割合が対照区に対して顕著に増大した（表３、図２Ｂ参照）。
表３
The culture filtrate was spray-treated on pot-cultivated dwarf tomato (MicroTom) plants and cultivated at 27 ° C. in a climate chamber. In the control group, the culture filtrate was treated with an autoclave at 121 ° C. for 15 minutes, and a solution (inactivated enzyme solution) in which p-nitrophenyl butyrate hydrolyzing activity was confirmed to be lost was sprayed. As a result, in the culture filtrate spraying group, after 7 days, the wrinkles and tips of the mature leaves that had already developed turned brown and curled. Moreover, overall browning and curling were observed in immature leaves of young shoots. The tip of the immature flower bud turned brown (see FIG. 2A). In addition, when the tomato treated with sprayed culture filtrate was installed outdoors and the water supply was reduced, the ratio of leaf fall, yellowing and browning leaves increased markedly with respect to the control group after 7 days (Table 3, Fig. 3). 2B).
Table 3
同様の培養ろ液をシロイヌナズナに対して噴霧処理した後、人工気象室内で栽培したところ、７日後には、矮性のトマトと同様にロゼッタ葉の淵が褐変化し、張りを失ってカーリングした。また、葉が重なり合った部位では、葉が溶け合って融合した。（図３参照）
なお、図２Ａにおいて、左図は失活酵素液を適用した若芽部位、中央図及び右図は通常の培養ろ液を適用した若芽部位の様子を示し、図２Ｂにおいて、左図は失活酵素液を適用した対照区、右図は通常の培養ろ液を適用した処理区の植物体を示し、図３において、左図は失活酵素液を適用したロゼッタ葉、右図は通常の培養ろ液を適用したロゼッタ葉を示す。
After the same culture filtrate was sprayed on Arabidopsis thaliana, it was cultivated in an artificial weather chamber. After 7 days, the rosette leaf buds turned brown, curling with no tension, like dwarf tomatoes. Also, at the site where the leaves overlap, the leaves melt and fuse. (See Figure 3)
In FIG. 2A, the left figure shows the young bud site to which the inactivated enzyme solution is applied, the center figure and the right figure show the young bud site to which the normal culture filtrate is applied, and in FIG. 2B, the left figure shows the inactivated enzyme. The control panel to which the solution was applied, the right figure shows the plant body of the treatment group to which the normal culture filtrate was applied. In FIG. 3, the left figure is the rosetta leaf to which the inactivated enzyme solution was applied, and the right figure is the normal culture filter. The rosetta leaf which applied the liquid is shown.
＜実施例３＞
実施例２で用いたPaE（植物に対して病原性を有さない微生物由来のクチナーゼ様酵素）を含むPseudozyma antarcticaの培養ろ液と同じ培養ろ液（酪酸ｐ−ニトロフェニル加水分解活性が１５．３６Ｕ／ｍＬ）を、野外雑草に散布処理した。対照区ではPaEの失活酵素液を散布した。なお、野外雑草として生育していた植物種は、主としてイネ科雑草のメヒシバとエノコログサであった。培養ろ液は、１ｍ2当たり約１Ｌを散布した。その結果、培養ろ液を散布した植物で黄化と病害の発病率、発病度が上昇した。ここで、発病率、発病度を、日本植物防疫協会で記載されている発病度の調査法（農薬実用化試験の基準）に則って評価したところ、培養ろ液処理を行った領域では発病率が８８．７％、発病度が５６．３だったのに対し、対照区では、発病率が７０％、発病度が２６．２であった。
即ち、対象区に対して培養ろ液処理区においては、発病率は２５．８％上昇し、発病度には統計的な有意差が認められた。これは、自然状態でも病原菌の感染が起こっているが、培養ろ液で処理を行うことにより、その病徴を示す発病度が著しく上昇していることを示している。特に、噴霧した溶液の水滴が付着した部位から発病していたことから、培養ろ液中に含まれるPaEによる植物のクチンの分解効果により、病害が誘導されたと認められる（図４及び表４参照）。
なお、図４のＡにおいて、左図は散布１日後の対照区を、右図は散布１日後の処理区を示し、図４のＢにおいて、左の葉は散布７日後の対照区を、右の葉は散布７日後の処理区の葉を示す。更に、図４のＣは、散布７日後の処理区を示す。図４のＡより、対象区及び処理区ともに、培養ろ液が付着した箇所が付着痕を形成しているが、処理区ではカーリングを起こす等、病害受けていると見られる徴候を示している。また、図４のＣより、培養ろ液が付着した箇所に病害が発生していることが確認できる。
表４
※発病度は、各評価基準に割り当てられた得点を当該評価基準に該当する葉数で積算し、全評価基準を通して合算した数値を合計調査葉数に４を乗じた値で除算し、更に１００を乗じた値を示す。
<Example 3>
The same culture filtrate (p-nitrophenyl butyrate hydrolyzing activity as 15) of Pseudozyma antarctica containing PaE (a cutinase-like enzyme derived from a microorganism having no pathogenicity to plants) used in Example 2 was used. 36 U / mL) was sprayed on field weeds. In the control, PaE inactivated enzyme solution was sprayed. The plant species that had grown as field weeds were mainly grasses weeds and enokorogusa. About 1 L of the culture filtrate was sprayed per 1 m 2 . As a result, the incidence of yellowing and disease and the degree of disease increased in plants sprayed with culture filtrate. Here, when the disease incidence and disease severity were evaluated according to the disease severity survey method described in the Japan Plant Protection Association (the standard for practical application of agricultural chemicals), the disease incidence was observed in the area where culture filtrate treatment was performed. Was 88.7% and the disease severity was 56.3, while in the control plot, the disease incidence was 70% and the disease severity was 26.2.
That is, in the culture filtrate treatment group, the disease incidence increased by 25.8%, and a statistically significant difference was observed in the disease severity. This indicates that although infection with pathogenic bacteria has occurred even in the natural state, the disease severity indicating the disease symptoms has been remarkably increased by treatment with the culture filtrate. In particular, since the disease was caused from the site where the water droplets of the sprayed solution adhered, it is recognized that the disease was induced by the effect of decomposition of plant cutin by PaE contained in the culture filtrate (see FIG. 4 and Table 4). ).
In FIG. 4A, the left figure shows the control plot one day after spraying, the right chart shows the treatment plot one day after spraying, and the left leaf in FIG. 4B shows the control plot seven days after spraying. The leaves indicate the leaves of the treatment area 7 days after spraying. Furthermore, C of FIG. 4 shows the treatment area 7 days after spraying. From A in FIG. 4, in both the target area and the treatment area, the place where the culture filtrate adhered forms an adhesion trace, but the treatment area shows signs that it is suffering from disease such as curling. . Moreover, it can confirm that the disease has generate | occur | produced in the location where the culture filtrate adhered from C of FIG.
Table 4
* Disease severity is calculated by adding the scores assigned to each evaluation criterion by the number of leaves corresponding to the evaluation criterion, dividing the total number obtained through all the evaluation criteria by the value obtained by multiplying the total number of surveyed leaves by 4, and then adding 100. Indicates the value multiplied by.
＜実施例４＞
実施例１で用いた、硫安沈殿により粗精製したPaE（植物に対して病原性を有さない微生物由来のクチナーゼ様酵素）溶液を、２０ｍＭトリス塩酸緩衝液（ｐＨ６．８またはｐＨ８．８）で希釈し、０．７２Ｕ／ｍＬ（２０倍希釈液）、２．８８Ｕ／ｍＬ（５倍希釈液）の粗酵素液を調製した。対照区として２０ｍＭトリス塩酸緩衝液（ｐＨ６．８またはｐＨ８．８）を用いた。これら溶液とトマトに感染する病原性糸状菌（灰色カビ病菌）の胞子（５×１０4spores/mL；植物病原性微生物）を矮性のトマト（MicroTom）切葉に対して同時に噴霧処理した後、密閉容器の中で保湿しつつ人工気象室内で、２７℃で保温した。噴霧処理７日後に各切葉の様子を無変化、黄化、病徴発現の３段階で評価し、その結果を表５に示した。表５より明らかなように、２．８８Ｕ／ｍＬの酪酸ｐ−ニトロフェニル加水分解活性を有する粗精製したPaEとカビ胞子を同時に噴霧したトマトの葉面は希釈に用いた緩衝液のｐＨに関わらず、２５％で黄化し、７５％でカビによる病徴が確認できた。０．７２Ｕ／ｍＬの粗酵素処理区ではｐＨ８．８条件で処理した方がｐＨ６．８条件で処理したものより病徴の発現が助長された。
<Example 4>
The PaE (cutinase-like enzyme derived from a microorganism having no pathogenicity to plants) solution roughly purified by ammonium sulfate precipitation used in Example 1 was diluted with 20 mM Tris-HCl buffer (pH 6.8 or pH 8.8). Dilution was performed to prepare a crude enzyme solution of 0.72 U / mL (20-fold diluted solution) and 2.88 U / mL (5-fold diluted solution). As a control, 20 mM Tris-HCl buffer (pH 6.8 or pH 8.8) was used. After spraying these solutions and spores (5 × 10 4 spores / mL; phytopathogenic microorganisms) of pathogenic filamentous fungi (grey mold fungus) that infect tomatoes simultaneously on dwarf tomato (MicroTom) cut leaves, The temperature was kept at 27 ° C. in an artificial weather chamber while keeping the moisture in a sealed container. Seven days after the spray treatment, the state of each cut leaf was evaluated in three stages: unchanged, yellowing, and symptom expression. The results are shown in Table 5. As is apparent from Table 5, the leaf surface of tomato sprayed with 2.88 U / mL of crudely purified PaE having a hydrolysis activity of p-nitrophenyl butyrate and mold spores at the same time depends on the pH of the buffer used for dilution. First, yellowing occurred at 25%, and symptom due to mold was confirmed at 75%. In the 0.72 U / mL crude enzyme-treated section, the treatment with the pH 8.8 condition promoted the development of disease symptoms than the treatment with the pH 6.8 condition.
表５
Table 5
＜実施例５＞
培養ろ液希釈液（PaE；植物に対して病原性を有さない微生物由来のクチナーゼ様酵素１５．３６Ｕ／ｍＬ）を２０ｍＭトリス塩酸緩衝液（ｐＨ８．８）で希釈し、０．１９Ｕ／ｍＬ、０．９６Ｕ／ｍＬ、１．９２Ｕ／ｍＬ、１５．３６Ｕ／ｍＬの酵素溶液を調製した。これらを用い、実施例１と同様の手法で矮性のトマト（MicroTom）切葉に対して噴霧処理した後、密閉容器の中で保湿しつつ保温した。噴霧処理直後、処理後３日、処理後１０日の各切葉の様子を無変化、黄化、病徴発現の３段階で評価し、その結果を表６に示した。表６に示すように、酵素溶液中のPaE濃度に応じて、黄化や病徴発現の割合が向上し、特に１５．３６Ｕ／ｍＬの酵素溶液を適用した切葉では１０日経過後に全ての葉が病徴を示した。
<Example 5>
Diluted culture filtrate (PaE; 15.36 U / mL of a cutinase-like enzyme derived from a non-pathogenic microorganism) with 20 mM Tris-HCl buffer (pH 8.8), 0.19 U / mL 0.96 U / mL, 1.92 U / mL, and 15.36 U / mL enzyme solutions were prepared. Using these, after spraying fertile tomato (MicroTom) cut leaves in the same manner as in Example 1, it was kept warm while being kept moist in an airtight container. Immediately after the spraying treatment, 3 days after the treatment, and 10 days after the treatment, the state of each cut leaf was evaluated in three stages: no change, yellowing, and symptom expression. The results are shown in Table 6. As shown in Table 6, the rate of yellowing and symptom expression increases according to the PaE concentration in the enzyme solution. In particular, in the cut leaves to which the 15.36 U / mL enzyme solution was applied, The leaves showed symptoms.
表６
Table 6
＜実施例６＞
近年、日本産のタデ科植物であるイタドリFallopia japonicaが欧米諸国で大繁殖し、その被害拡大が深刻な状況にある。高コストの化学除草剤による化学的防除法ではイタドリの蔓延を防ぐことが困難なため、原産地の病原菌を活用する生物防除法の開発が急務となっている。本実施例では、イタドリに対する病原性微生物として有望なさび病菌Puccinia polygoni-amphibii var. tovariae（植物病原性微生物）を用い、除草用酵素含有組成物がさび病菌の病徴発現に及ぼす影響を調べた。イタドリの葉の両面に酵素液（PaE；実施例２で用いたPaE（植物に対して病原性を有さない微生物由来のクチナーゼ様酵素）を含むPseudozyma antarcticaの培養ろ液と同じ培養ろ液；酪酸ｐ−ニトロフェニル加水分解活性は１５．３６Ｕ／ｍＬ）を噴霧処理し、処理後の植物体を温室で維持した（処理区）。一方、対照区では滅菌水を噴霧した。処理区及び対照区において、噴霧７２時間後、さび病菌Puccinia polygoni-amphibii var. tovariaeの夏胞子（植物病原性微生物）とタルカムパウダーを１：１０（ｗ／ｗ）に調製してイタドリの葉の両面に塗抹接種した。処理後の植物体を湿度１００％の条件下で４８時間静置後、温室（２５℃）にて維持した。処理区及び対照区において、発病度や落葉率を経日調査した。発病度は以下の評価基準で評価した。
［発病度］
０：健全
１：斑点形成（夏胞子堆形成はなし）
２：夏胞子堆形成（葉面の５０％未満）
３：夏胞子堆形成（葉面の５０％以上）
４：葉全体の黄化
５：落葉枯死
<Example 6>
In recent years, the Japanese knotweed Fallopia japonica, which has grown in Japan and Europe, has been proliferating in Europe and the United States, and its damage is seriously growing. Chemical control methods using high-cost chemical herbicides make it difficult to prevent the spread of Japanese knotweed, so the development of a biocontrol method that uses pathogenic bacteria in the country of origin is urgently needed. In this example, Puccinia polygoni-amphibii var. Tovariae (phytopathogenic microorganism), a promising rust pathogenic microorganism for Japanese knotweed, was used to examine the effects of herbicidal enzyme-containing compositions on the symptom expression of rust pathogens. . The same culture filtrate as the culture filtrate of Pseudozyma antarctica containing an enzyme solution (PaE; PaE (a cutinase-like enzyme derived from a microorganism having no pathogenicity to plants) used in Example 2) on both sides of the itadori leaves; The p-nitrophenyl butyrate hydrolyzing activity was 15.36 U / mL), and the treated plants were maintained in a greenhouse (treated section). On the other hand, sterilized water was sprayed in the control group. In the treated area and the control area, after 72 hours of spraying, the summer spores (phytopathogenic microorganisms) and talcum powder of the rust fungus Puccinia polygoni-amphibii var. Tovariae were prepared at 1:10 (w / w) and Smoke was inoculated on both sides. The treated plant body was allowed to stand for 48 hours under the condition of 100% humidity and then maintained in a greenhouse (25 ° C.). In the treatment group and the control group, the disease severity and leaf fall rate were investigated daily. The severity was evaluated according to the following evaluation criteria.
[Severity]
0: Healthy 1: Spot formation (no formation of summer spores)
2: Summer spore formation (less than 50% of leaf surface)
3: Summer spore formation (more than 50% of leaf surface)
4: Yellowing of the entire leaf 5: Fallen leaf death
その結果、（１）培養ろ液を噴霧しただけで４８時間以内に新葉数枚が枯死し（図５）、さらに（２）さび病菌の接種後、処理区の発病度と落葉率が対照区より高く推移し、培養ろ液の病原菌感染の助長効果が認められた（図６）。 As a result, (1) just by spraying the culture filtrate, several new leaves died within 48 hours (Fig. 5), and (2) after inoculation with rust fungus, the incidence and defoliation rate of the treated area were controlled. It was higher than the ward, and the culture filtrate promoted the pathogen infection effect (FIG. 6).
＜実施例７＞
［実験方法］
培養ろ液（PaE；実施例２で用いたPaE（植物に対して病原性を有さない微生物由来のクチナーゼ様酵素）の溶液）に、夏胞子の濃度が約１．５×１０5spores/mLとなるようにさび病菌Puccinia polygoni-amphibii var. tovariaeの夏胞子を混合した除草用酵素含有組成物を、イタドリの葉の両面に噴霧接種した（以下、酵素処理区とも称する）。また、対照として、失活酵素液（実施例２と同様に培養ろ液をオートクレーブ処理したもの）に、夏胞子の濃度が約１．５×１０5spores/mLとなるようにさび病菌の夏胞子を混合したものを、イタドリの葉の両面に噴霧接種した（以下、対照区とも称する）。酵素処理区及び対照区の植物体は２０℃、湿度１００％条件下で４８時間静置後、温室（２５℃）にて維持した。１試験区でそれぞれ７個体を処理し、各試験を２回、反復して行った。
［結果］
図７に示すように、酵素処理区及び対照区の平均落葉率は、PaEを含む培養ろ液とさび病菌の夏胞子を含む除草用酵素含有組成物で処理した処理区では、失活酵素液を使用した対照区と比較して、さび病菌のより激しい病徴が確認されたとともに、病勢進展が有意に早いことが明らかとなった。平均落葉率においては、酵素処理区では３日後に１．０２％、６日後に２６．２％、９日後に３４．１％、１２日後に３８．７％、対照区では３日後に０％、６日後に１６．２％、９日後に３２．４％、１２日後に３９．３％であり、PaEを含む培養ろ液を用いた場合においてさび病菌の同時接種から６日後までに有意に差のある落葉率を示した。
<Example 7>
[experimental method]
In the culture filtrate (PaE; PaE used in Example 2), the concentration of summer spores is about 1.5 × 10 5 spores / A herbicidal enzyme-containing composition in which summer spores of the rust fungus Puccinia polygoni-amphibii var. tovariae were mixed so as to be mL was spray-inoculated on both sides of the Japanese knotweed leaf (hereinafter also referred to as an enzyme-treated section). In addition, as a control, the inactivated enzyme solution (the one obtained by autoclaving the culture filtrate as in Example 2) was added to the summer rust fungus so that the concentration of summer spores was about 1.5 × 10 5 spores / mL. A mixture of spores was spray-inoculated on both sides of the Japanese knotweed leaf (hereinafter also referred to as a control group). Plants in the enzyme-treated group and the control group were allowed to stand for 48 hours at 20 ° C. and 100% humidity, and then maintained in a greenhouse (25 ° C.). Seven individuals were treated in one test group, and each test was repeated twice.
[result]
As shown in FIG. 7, the average defoliation rate in the enzyme-treated group and the control group is as follows. In the treated group treated with the herbicidal enzyme-containing composition containing PaE-containing culture filtrate and rust fungus summer spores, the inactivated enzyme solution Compared with the control group using, a more intense symptom of rust fungus was confirmed, and it was revealed that the disease progression was significantly faster. The average leaf fall rate was 1.02% after 3 days in the enzyme treatment group, 26.2% after 6 days, 34.1% after 9 days, 38.7% after 12 days, and 0% after 3 days in the control group. , After 6 days, 16.2%, after 9 days, 32.4%, after 12 days, 39.3%, and in the case of using culture filtrate containing PaE, it was significantly less than 6 days after the simultaneous inoculation of rust fungus. The leaf fall rate was different.
＜実施例８＞
［実験方法］
Czapek-Dox液体培地に、唯一の炭素源としてＰＢＳＡエマルジョン（ＥＭ−３０１（昭和電工社製））を加え、受託番号NITE P-573の糸状菌株を液体振とう培養し、培養液から糸状菌の菌体を除去し培養ろ液（PCLE：植物に対して病原性を有さない微生物由来のクチナーゼ様酵素を含む溶液）を調整した。培養ろ液のＰＢＳＡ加水分解活性は、２ｍＭ塩化カルシウムを含むｐＨ６．８の２０ｍＭ Ｔｒｉｓ塩酸緩衝液で測定したところ、１０Ｕ／ｍＬであった。この培養ろ液を２ｍＭ塩化カルシウムを含むｐＨ６．８の２０ｍＭ Ｔｒｉｓ塩酸緩衝液、又は２ｍＭ塩化カルシウムを含まないｐＨ６．８の２０ｍＭ Ｔｒｉｓ塩酸緩衝液で希釈し、ＰＢＳＡ加水分解活性が１Ｕ／ｍＬのクチナーゼ様酵素PCLEの溶液を調製した。なお、このクチナーゼ様酵素PCLEの溶液のＰＢＳＡ加水分解活性は、ｐＨ８．０の５０ｍＭ Ｔｒｉｓ塩酸緩衝液中の酪酸ｐ−ニトロフェニル加水分解活性１．１１Ｕ／ｍＬに換算される。
実施例１と同様に、矮性のトマト（MicroTom）の切葉各１１枚に対してクチナーゼ様酵素PCLEの溶液を噴霧処理した後、密閉容器の中で保湿しつつ保温した。酵素溶液の噴霧後、７２時間が経過した後に、矮性のトマトに感染する病原性糸状菌（灰色カビ病菌）の胞子（５×１０4spores/ｍL；植物病原性微生物）を噴霧して、保湿しつつ保温した。７日後に切葉の様子を観察したところ、対照区（２ｍＭ塩化カルシウムを含むｐＨ６．８の２０ｍＭ Ｔｒｉｓ塩酸緩衝液を散布した矮性トマトの切葉）に比べてクチナーゼ様酵素PCLEの溶液を散布した処理区では、酵素溶液中の塩化カルシウム有無に関わらず、黄化する葉の出現率が高くなった。更に、病徴を示す葉の割合は、対照区では１８．２％（２葉）であったのに対し、処理区では病徴を示す葉の割合が高くなり、塩化カルシウムを加えていないクチナーゼ様酵素PCLEの溶液を散布した処理区では４５．５％（５葉）、塩化カルシウムを加えたクチナーゼ様酵素PCLEの溶液を散布した処理区では６３．６％（７葉）と感染の効率が向上し病徴が助長された（表７参照）。
表７
<Example 8>
[experimental method]
PBSA emulsion (EM-301 (manufactured by Showa Denko KK)) is added to Czapek-Dox liquid medium as the sole carbon source, and the filamentous strain with the accession number NITE P-573 is subjected to liquid shaking culture. The microbial cells were removed and a culture filtrate (PCLE: a solution containing a cutinase-like enzyme derived from a microorganism having no pathogenicity to plants) was prepared. The PBSA hydrolysis activity of the culture filtrate was 10 U / mL as measured with a 20 mM Tris hydrochloric acid buffer solution having a pH of 6.8 containing 2 mM calcium chloride. This culture filtrate is diluted with 20 mM Tris hydrochloric acid buffer with pH 6.8 containing 2 mM calcium chloride or 20 mM Tris hydrochloric acid buffer with pH 6.8 not containing 2 mM calcium chloride, and a cutinase having a PBSA hydrolysis activity of 1 U / mL. A solution of the enzyme PCLE was prepared. The PBSA hydrolysis activity of this cutinase-like enzyme PCLE solution is converted to p-nitrophenyl butyrate hydrolysis activity of 1.11 U / mL in 50 mM Tris hydrochloric acid buffer at pH 8.0.
In the same manner as in Example 1, 11 cut leaves of dwarf tomato (MicroTom) were each sprayed with a solution of cutinase-like enzyme PCLE, and then kept warm in a sealed container while moisturizing. 72 hours after spraying the enzyme solution, spraying spores (5 × 10 4 spores / mL; phytopathogenic microorganisms) of pathogenic filamentous fungi (gray mold fungus) that infect fertile tomatoes, moisturizing While keeping warm. After 7 days, the state of the cut leaves was observed, and the cutinase-like enzyme PCLE solution was sprayed compared to the control group (cut leaves of fertile tomato sprayed with 20 mM Tris hydrochloric acid buffer solution containing 2 mM calcium chloride, pH 6.8). In the treated group, the appearance rate of yellowing leaves increased regardless of the presence or absence of calcium chloride in the enzyme solution. Furthermore, the proportion of leaves showing symptoms was 18.2% (two leaves) in the control group, whereas the proportion of leaves showing symptoms was high in the treated group, and cutinase without added calcium chloride. In the treatment group sprayed with the solution of PCLE-like enzyme PCLE, the efficiency of infection was 45.5% (5 leaves), and in the treatment group sprayed with the solution of cutinase-like enzyme PCLE added with calcium chloride, 63.6% (7 leaves). The symptoms improved and the symptoms were promoted (see Table 7).
Table 7
Claims (6)
酪酸ｐ−ニトロフェニル加水分解活性を有し、植物に対して病原性を有さない微生物由来のクチナーゼ様酵素を含み、
除草用酵素含有組成物における前記クチナーゼ様酵素の含有量が、酪酸ｐ−ニトロフェニル加水分解活性に換算して０．０１Ｕ／ｍＬ以上であり、
前記植物に対して病原性を有さない微生物が、Pseudozyma属、Cryptococcus属又はParaphoma属に属する微生物であることを特徴とする、除草用酵素含有組成物。 A herbicidal enzyme-containing composition for controlling harmful plants,
Has butyrate p- nitrophenyl hydrolytic activity includes cutinase-like enzyme derived from microorganisms having no pathogenic to plants,
The content of the cutinase-like enzyme in herbicidal enzyme containing composition state, and are 0.01 U / mL or more in terms of acid p- nitrophenyl hydrolytic activity,
A herbicidal enzyme-containing composition, wherein the microorganism having no pathogenicity to the plant is a microorganism belonging to the genus Pseudozyma, Cryptococcus or Paraphoma .
前記植物に対して病原性を有さない微生物が、Pseudozyma属、Cryptococcus属、又はParaphoma属に属する微生物であることを特徴とする、有害植物の駆除方法。 Produce cutinase-like enzyme having acid p- nitrophenyl hydrolytic activity, a culture of a microorganism having no pathogenic to plants, a method of combating harmful plants comprising applying to the harmful plants ,
Microorganisms having no pathogenic to the plants, Pseudozyma spp., Cryptococcus spp, or characterized in that it is a microorganism belonging to Paraphoma genus, method of combating harmful plants.
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| JP3184967B2 (en) * | 1999-04-01 | 2001-07-09 | 農林水産省蚕糸・昆虫農業技術研究所長 | Microbial herbicide and herbicidal method |
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| JP4915593B2 (en) * | 2007-02-28 | 2012-04-11 | 独立行政法人農業環境技術研究所 | Biodegradable plastic-degrading bacteria and method for producing the degrading enzyme |
| JP5448135B2 (en) * | 2008-05-27 | 2014-03-19 | クミアイ化学工業株式会社 | Pest control composition |
| JP5082125B2 (en) * | 2008-09-29 | 2012-11-28 | 独立行政法人農業環境技術研究所 | Novel microorganisms and biodegradable plastic degrading enzymes |
| CN103229785B (en) * | 2012-12-28 | 2014-08-27 | 沈阳农业大学 | Method for controlling ambrosia trifida by using rust fungus |
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