土壌菌による有機塩素殺菌剤、ダコニールの吸収と脱クロールについて

Fungi tolerant to an organochlorine fungicide, tetrachloroisophthalonitrile (TCPN), was selected and isolated from various soil samples, and examined for the activity to metabolize TCPN. The population of tolerant fungi was extremely large in the plastic house - soil on which TCPN has been used for six years to control diseases. This is probably due to the change of soil microflora owing to the decline of TCPN-sensitive competitors. Many tolerant fungi took up TCPN very rapidly from culture filtrate. Of ten tolerant isolates tested, two isolates, C-F- I and V-P-5, were found to metabolize TCPN. The metabolite was isolated in pure form by preparative TLC, and identified as trichloroisophthalonitrile by mass spectrometry. C-F-1 was identified as Aspergillus luchuensis and V-P-5 as Penicillium godlewskii. Namely, these soil fungi dechlorinate from tetrachloroisophthalontrile to trichloroisophthalonitrile

エンドウ褐紋病菌の胞子発芽液に含まれるピサチン蓄積誘導物質の分離と性質

An elicitor of pisatin accumulation was isolated and partially characterized from spore germination fluid, mycelial extract, and cell wall preparation of a pea pathogen, Mycosphaerella pinodes. The elicitor was found to be a polysaccharide or glycoprotein and the active component was composed of glucose. The approximate molecular weight was 70,000 daltons. This elicitor elicited pisatin accumulation in pea leaves at a concentration of 10 ug/ml and the maximum activity was ca. 350-400 ug/ml, but did not elicit phytoalexins in soybean, bean, and red clover. High molecular weight preparations from the mycelial extract and mycelial wall of Mycosphaerella melonis and Stemphylium sarcinaeforme, nonpathogens of pea, elicited pisatin accumulation in pea leaves

エンドウ原形質膜におけるATPアーゼとホスファチジルイノシトールリン資質リン酸化酵素の共精製

The plasma membrance ATPase was partially purified by a linear glycerol density gradient centrifugation of the detergent-solubilized plasma membrance poteins and subsequent separation by a size-exclusion column chromatogrphy. A purified ATPase preparation is shown to contain a 97.6kDa protein that was cross-reacted with an antibody raised against mung bean H+-ATPase. The preparation also exhibited the phosphorylation of exogenous phosphatidylionsitol(PI) when supplized with [γ-32P]ATP. These results indicate that one form plasma membrance ATPase is co-purified with PI kinase.エンドウの上胚軸組織から分離した原形質膜画分におけるATPアーゼとホスファチジルイノシトールリン脂質リン酸化酵素との相互作用を解析する目的で、双方の原形質膜画分からの可溶化とそれらの部分精製を試みた。原形質膜のTritonX－100可溶化画分をグリセロール連続密度勾配遠心分画に供し、得られた活性画分をさらに分子ふるいカラムクロマトグラフィーによって分離した。この結果、ＡＴＰアーゼとホスファチジルイイノシトールリン脂質リン酸化酵素は共精製され、非変性条件下では双方の活性を分けることができなかった

共生真菌アクレモニウムエンドファイトの形質転換

Conditions have been developed for transforming protoplasts of the Acremonium endophyte by PEG 4000 and electroporation. Transformation by PEG exhibited a higher number of transformants than by electroporation. lntegration of iaaM gene into the genome was examined by PCR and Southern blot hybridization analysis. PCR product showed that transformants banded at around 1.7 kb corresponding to the size of iaaM gene. Hybridization of the digests of genomic DNA with iaaM gene as DNA probe showed that the number of hybridized band signals was different between transformant and non-transformant. These results might indicate that PEG is an effective method for the transformation of Acremonium endophyte and that there are repeated copies of the iaaM homologous sequences in the genome of Acremonium.アクレモニウムエンドファイトの形質転換の条件を検討した。アクレモニウムエンドファイトからプロトプラストを調製し、PEG4000とエレクトロポレーションを用いて形質転換を試みた。その結果、PEGで形質転換すると、エレクトロポレーションによる場合よりも多くの形質転換体が得られた。PCR解析によってiaaM遺伝子のゲノムへの導入を確認したところ、形質転換体のPCR産物はiaaM遺伝子のサイズに相当する約1.7kbのバンドを持っていた。一方、iaaM遺伝子をプローブとしたサザンブロット解析においては、形質転換体と非形質転換体の間にはハイブリダイズした断片数に違いがあることが明らかとなった。以上の結果は、アクレモニウムエンドファイトの形質転換にはPEG法が有効であること、アクレモニウムエンドファイトのゲノム中にはiaaM遺伝子様配列の反復コピーが存在することを示唆している

農業用殺菌剤、植物病原菌の有害代謝産物によるファイトアレキシンの生産

The endocarp of the fresh pea pod incubated with solution or suspension of agricultural fungicides, phytotoxic metabolites of plant pathogenic fungi formed the pea phytoalexin, pisatin. Among the compounds tested, cycloheximide, triazine, dichlone, phenyimercury acetate, UV degradation product of phenylmercury acetate, triphenyltin fungicides, and 3-hydroxy-5-methylisoxazole induced pisatin. Ophiobolin, a toxin from Cochliobolus mtyabeanus, and ascochitine, a toxic metabolite from Ascochyta fabae, also induced pisatin. The possibilities of the development of harmless plant disease control agents was discussed in relation- to the induced synthesis of phytoalexins

Chitin Component in Haustorial Wall of Powdery Mildew Fungus of Barley

純寄生性病害の感染成立に中心的役割を演ずると考えられている吸器の性質を明らかにするために,オオムギうどんこ病菌の吸器形成過程における吸器壁のキチン質の生成に関して組織化学的に検討した. その結果,吸器壁はキトーサン反応陽性で,菌糸壁と同様キチン質がその骨格をなしているものと考えられる. さらに,経時的な検討の結果,感染初期に形成される吸器原基において,すでにキチン反応が陽性であり,キチン合成系の活性化は,吸器機能発現のための一つの重要な過程であると推定される

A Volatile Substance, β-Caryophyllene, from Talaromyces wortmannii Promotes Growth and Tolerance to Diseases on Several Plants

A plant growth-promoting fungus, Talaromyces wortmannii strain FS2 was isolated from an agricultural field at Okayama Pref. FS2 enhanced seed germination, root elongation and leaf growth of Brassica rapa var perviridis (Komatsuna). Such plant growth-promoting effect was observed in the same sealed chamber where FS2 was cultured on PDA medium separated from seedlings, suggesting effective volatile compound(s). GC‒MS analysis showed that FS2 emitted at least seven terpenoids, of which a volatile was identified as β‒caryophyllene. β‒caryophyllene alone promoted the growth of cucumber, Nicotiana benthamiana and barley. Furthermore β‒caryophyllene increased the yield of cucumber fruits. Interestingly, we found that β‒caryophyllene conditioned these plants to be resistant to respective diseases caused by Colletotrichum orbiculare, Botrytis cinerea or Blumeria graminis f. sp hordei. The findings indicate that β‒caryophyllene has desirable dual features and therefore, it is available to cultivation of many crops.岡山県総社市の圃場から分離した植物生育促進菌Talaromyces wortmannii FS2が生産するβ-caryophylleneは，コマツナ（アブラナ科）のみならず，キュウリ（ウリ科），タバコ（ナス科）およびオオムギ（イネ科）など広汎な植物に対して，生育促進作用および耐病性増進作用を示したことから，有用な農業資材として利用可能であるものと考察した

病原菌シグナルによるエンドウ原形質膜におけるホスファチジルイノシトールリン脂質のリン酸化とリゾリン脂質生成の制御

Effects of elicitor and suppressor from a pea pathogen, Mycosphaerella pinodes, on Pl etabolism in pea plasma membrane were examined in vitro. The elicitor induced rapid phosphorylation of phosphatidylinositols as well as production of lysophospholipid in plasma membranes, but these responses were severely inhibited by the suppressor. These results indicate that a membrane-associated phospholipase A is regulated coordinately by fungal signals, together with Pl metabolism, and that it may participate in signal transduction pathways leading to defense responses. To evaluate a possible rote of phospholipase A activation in induction of a pea defense response, the effect of free fatty acid on induction of a phytoalexin accumulation was also examined. When pea leaves were treated with linoleic- or linolenic acid, most commonly released in plant cells by phospholipase A, the accumulation of pisatin was induced even in the absence of the elicitor. It is, therefore, conceivable that free fatty acid(s) released from plasma membrane is also implicated in the early stage of elicitor-signal transduction in pea.エンドウの上胚軸組織により分離した原形質膜画分を褐紋病菌の生産するエリシターで処理すると、ホスファチジルイノシトールリン脂質の急速なリン酸化とリゾリン脂質の生成が誘導されたが、同菌より調製したサプレッサーの共存下では双方とも著しく阻害された。本結果は、ポリホスホイノシチド代謝系と同調的に作動するホリパーゼA活性化が存在すること、さらに、原形質膜における病原菌シグナルの受容・応答には複数の資質代謝系が介在する可能性を示唆している。一方、ホスホリパーゼAの活性化の役割を調べる目的で、本酵素によって原形質膜から生成されると考えられる脂肪酸(リノール酸ならびにリノレン酸)をエンドウ葉に処理したところ、エリシターの非存在下においてもファイトアレキシンであるピサチンの生成が誘導されることが示された。以上から、ポリホスホイノシチド代謝系と同調的に働くホスホリパーゼAがエリシターシグナルの初期伝達に深く関連しているものと考えられた

FFC Ceramic Water(TM) Enhances Plant Apyrase Activity

The FFC ceramics(TM) from FFC Japan Co., Ltd. are now widely used in the fields of agriculture, fishery and food industry in Japan. Recently the FFC ceramic beads-based technology has been also applied to meet several environmental problems including pollution in sea, lakes and rivers. In this study the FFC ceramic water was tested for effect on plant enzyme, potato apyrase (EC 3.6.1.5; ATP-diphosphohydrolase), which hydrolyses nucleoside triphosphate (NTP) and -diphosphate (NDP) to produce corresponding nucleoside monophosphate (NMP) and inorganic phosphate (Pi). Addition of the FFC ceramic water to the enzyme reaction mixture markedly enhanced ATP-hydrolyzing activity, when used as ATP as substrate. However, the concomitant presence of Ca(2+) chelator, EGTA (O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid) with the FFC ceramic water, completely abolished the enzyme activation. In fact, exogenous calcium ion such as CaSO4 mimicked the FFC ceramic water. These results indicate that apyrase activation by the FFC ceramic water largely depends on calcium ions. On the other hand, when the FFC ceramic water prepared from "used" ceramics was tested for the apyrase activity, the enhanced effect on apyrase was decreased compared to the FFC ceramic water from "new" ones. This result, consistent with our present data covering concentration of calcium ions and conductivity, indicates that long and/or successive usage of the ceramic beads results in decrease of contents of released minerals, especially calcium ions. The apyrase-based enzyme assay presented here is probably applicable to estimate and quantify the effect of FFC ceramic water.本報は，FFC セラミックス(TM)（㈱エフエフシー・ジャパン）で調製した FFC セラミック水（FFC 水）の植物アピラーゼ（EC 3.6.1.5）の活性に及ぼす直接的な作用について調べたものである．FFC 水はアピラーゼがもつ ATP加水分解活性を促進し，その作用は反応液への添加量に依存した．先の無機元素分析結果から，FFC 水に含まれる主要な塩類はCa(2＋)であることが判明している．そこで，Ca(2＋)キレート剤EGTAを反応液へ加え，その影響について調べたところ，FFC 水による活性化作用は消失することが明らかとなった．また，FFC 水と類似の作用は，硫酸カルシウム，塩化カルシウムまたは硝酸カルシウムの添加で認められ，陰イオンの種類によって明確な違いはなかった．これらの結果から，FFC 水が植物アピラーゼに及ぼす活性化作用の一因は，セラミックスから遊離する Ca(2＋)に依存しているものと推察された．一方，アピラーゼ活性を指標として，使用済のセラミックスから調製した FFC 水の効果について検討したところ，未使用からの水と比べて，カルシウム濃度ならびに活性化作用の顕著な低下が認められた．このことは，継続的な使用によってセラミックスから遊離する塩類，特にカルシウムの溶出量が大きく変わることを意味し，アピラーゼを用いた本検定が，FFC 水の効果を定量的に確かめる方法の一つとして利用できると考えられた．以上，これらの結果を総合して，FFC水の植物酵素への直接的作用，ならびに植物への施用によって効果が現れる耐病性獲得作用との関連について考察した

殺菌剤、ベノミル、チウラムの土壌微生物相と2～3の土壌生息性糸状菌に及ぼす影響

Effects of Benomyl and Thiram on the soil microflora and some soil inhabitant fungi were studied using the_ soil (sandy loam) of the experimental field, Okayama University. Under field conditions, heavy application of Benomyl did not affect significantly the soil microflora. Thiram, however, reduced the fungal population in soil to 1/6 at the next day of treatment, but recovered to the normal level after 6 days. Under laboratory conditions, both fungicides did not affect soil microflora. Population of Benomyl-to lerant fungi was about 1/10 of the total fungi and increased slightly in the field soil by treatment with Benomyl at the later stage of experiment during June to October. Neither Thiram-tolerant fungus nor bacterium was found in both Thiram-treated and non-treated soils. A fungus highly tolerant to Benomyl was isolated and identified as Aspergillus versicolor, and found to not have the metabolic activity to degrade BCM. The absorption of BCM by the mycelia of this tolerant fungus, A. versicolor, was less than half of the BCM-sensitive one, such as Cladosporium harbarum