链孢粘帚霉几丁质酶的诱导及其抗真菌活性研究

链孢粘帚霉HL-1-1菌株在不同培养条件下产几丁质酶活性不同,核盘菌菌核对其几丁质酶的诱导作用高于几丁质;首次用菌核配制培养基成功诱导了几丁质酶的产生,其产酶的最适初始pH值为4.5,最适培养时间为6d。几丁质酶对10种病原菌都有不同的抑制作用,对小麦雪腐病、葡萄白腐病、玉米黄斑病及斑点落叶病等病原菌的孢子萌发具有明显的抑制作用;该几丁质酶还能明显抑制核盘菌和立枯丝核菌的菌核萌发。     

粉红螺旋聚孢霉67‐1厚垣孢子生物学特性的研究

粉红螺旋聚孢霉(粉红粘帚霉)是多种植物病原真菌的重寄生菌,具有巨大的生防潜力。以实验室前期分离筛选的高效粉红螺旋聚孢霉67‐1菌株为材料,研究其厚垣孢子和分生孢子的生物学特性。结果表明,以厚垣孢子为接种体,其生长速度和菌丝鲜重与以分生孢子为接种体形成的菌落没有明显差异,但产孢水平提高6倍以上。粉红螺旋聚孢霉对高温和干燥的耐受程度较差,尤其分生孢子更为敏感。当加工温度范围在40–50℃时,厚垣孢子和分生孢子的活性存在显著差异(P<0.05)。菌株67‐1的厚垣孢子耐紫外线能力明显优于分生孢子。在无保护剂条件下,紫外灯照射60s,厚垣孢子活性保持在62.6%,是分生孢子活性1倍以上。土壤抑菌作用对真菌厚垣孢子的影响相对较小,24h孢子萌发率为47.2%,而分生孢子仅为4.6%。除了百菌清,菌株67‐1厚垣孢子对多种化学农药有较强的耐受力,在药剂田间使用浓度下,厚垣孢子萌发率均大于70%,其中对15%恶霉灵、1.8%阿维菌素和3%克百威的耐受力显著高于分生孢子(P<0.05),而对井冈霉素和两种杀虫剂没有显著提高。对菌株67‐1的寄生性测定结果表明,2种孢子对核盘菌菌核的寄生能力没有差异,菌核感染达到4级的占58.9%–60.0%。106/m L浓度厚垣孢子与分生孢子对黄瓜枯萎病的防效均达到65%以上,二者没有显著差异。     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

粘帚霉几丁质酶GcCHI1的结构鉴定及其抑菌作用

【目的】对分离纯化自链孢粘帚霉(Gliocladium catenulatum)HL-1-1菌株的几丁质酶Gc CHI1进行化学结构鉴定,并测定该酶对几种病原真菌的抑菌机制。【方法】几丁质酶Gc CHI1化学结构鉴定采用Nano-ESI-MS/MS技术。该酶对病原真菌菌丝生长、病原菌孢子萌发和病原菌菌核萌发的抑制作用采用牛津杯法等方法。【结果】获得几丁质Gc CHI1胰蛋白酶水解肽段的肽质量指纹谱图,较好的MS/MS图谱,以及3个肽段的氨基酸序列(均15个氨基酸),分别为LYNSNDAIEAFISR、VIGYFTQWGIYGR、LNLGIGYYGR。经Mascot数据库检索认为与来自Stenotrophomonas maltophilials 34S1的几丁质酶A具有高度的相似性。几丁质酶Gc CHI1能明显抑制立枯丝核菌、油菜菌核病菌、番茄灰霉病菌等多种植物病原真菌的菌丝生长、孢子萌发和菌核萌发。【结论】几丁质酶Gc CHI1对多种植物病原菌有抑制作用,因此几丁质酶Gc CHI1是HL-1-1菌株抑菌作用的机制之一。     

长枝木霉对禾谷胞囊线虫的寄生和致死作用

【目的】明确长枝木霉(Trichoderma longibrachiatum)孢子悬浮液对小麦禾谷胞囊线虫(Heterodera avenae)的防治潜力和可能的作用机理。【方法】通过室内显微观察和测定不同时期长枝木霉(T.longibrachiatum)孢子悬浮液对小麦禾谷胞囊线虫(H.avenae)胞囊的寄生和致死作用及其可能的机理。【结果】显微观察结果表明,侵染初期长枝木霉孢子寄生于胞囊的表面,并且萌发产生大量的菌丝,侵染后期整个胞囊被致密的菌丝包围,胞囊内卵的胚胎发育停止和内容物凝集,甚至有的胞囊表面突起形成深褐色的小液泡,或者胞囊破裂和溶解。室内测定结果表明,不同浓度长枝木霉孢子悬浮液对胞囊具有明显的寄生和致死作用,并且其可能的寄生和致死作用机理主要是通过胞囊诱导和提高长枝木霉菌几丁质酶、葡聚糖酶和酪蛋白酶的活性,进而使胞囊体壁溶解和内容物外渗。处理后第18天浓度为1.5×108CFU/mL的长枝木霉孢子悬浮液对胞囊的寄生率为93.3%,第10天对胞囊孵化的相对抑制率为93.6%;经胞囊诱导后第4天浓度为1.5×108CFU/mL的长枝木霉孢子悬浮液几丁质酶和葡聚糖酶活性最高为0.78和0.96 U/(min·mL),第6天浓度为1.5×108CFU/mL的长枝木霉孢子悬浮液酪蛋白酶活性最高为4.03 U/(min·mL),并且诱导后其几丁质酶、葡聚糖酶和酪蛋白酶活性随着长枝木霉孢子悬浮液浓度的增加而增加。【结论】长枝木霉对小麦禾谷胞囊线虫胞囊具有较强的寄生和致死作用,且可能的寄生和致死作用机理主要通过胞囊诱导和提高长枝木霉菌几丁质酶、葡聚糖酶和酪蛋白酶的活性。     

捕食线虫丝孢菌的菌寄生性测定

研究了节丛孢Arthrobotrys、单顶孢Monacrosporium和隔指孢Dactylella三个捕食线虫丝孢菌属16个菌株,对水稻立枯丝核菌RhizoctoniasolaniAG1、大豆核盘菌Sclerotiniasclerotiorum、茄科镰刀菌Fusariumsolani和恶疫霉Phytophthoracactorum四种常见土壤植物病原真菌的菌寄生性。结果表明供试菌可以通过弹簧式菌丝圈缠绕、类附着胞结构吸附、简单的菌丝缠绕或者贴附寄主菌丝生长四种方式寄生病原菌。其中,绝大多数菌株对立枯丝核病菌有寄生作用,一些供试真菌对其它三种病原真菌有寄生现象。利用孢子液浸泡法测定了其中5种捕食线虫真菌对核盘菌菌核的寄生能力,显示有较高寄生率。     

盾壳霉在油菜菌核病菌生物防治中的应用

油菜核病菌(Sclerotiniasclerotiorum)是一种世界性病原菌,其分布广、危害大、难根治。盾壳霉(Coniothyriumminitans)是该病原菌的破坏性寄生真菌,可以有效、专一地降低病原菌菌核的形成与萌发,在该病原菌的生物防治方面具有较大的应用潜力。从油菜核盘菌的致病过程与特点、盾壳霉的生长特性、盾壳霉和油菜核盘菌间相互作用的规律及途径等几个方面阐述了盾壳霉对油菜核盘菌的生防特性,讨论了盾壳霉在生产实践中的应用潜力及存在问题,并提出了一些解决问题的可能途径及需要进一步研究的内容与方向 。     

菌核寄生菌Talaromyces flavus的生物学特性及寄生菌核规律初探

本文对核盘菌菌核寄生菌Ｔａｌａｒｏｍｙｃｅｓｆｌａｖｕｓ的基本生物学特性及影响其寄生菌核的生态因子进行了初步研究。结果表明：Ｔ。ｆｌａｖｕｓ在我国南北方菌核病发生区土壤中普遍存在。其菌丝在３－３５℃及ｐＨ值２－１０范围内均能生长及产孢,最适生Ｋ温度为３０℃,最适生长ｐＨ值为４。对峙培养结果表明Ｔ,Ｆｌａｖｕｓ对核盘菌的抗生作用微弱,对菌丝和菌核均有很强的寄生致腐作用。除核盘菌外它还能寄生三叶草核盘菌、小核盘菌及离菌上的一种待鉴定的产菌核真菌。Ｔ,ｆｌａｖｕｓ寄生菌核的最适温度为２５－３０℃,土壤类型也影响到其寄生致腐菌核的效果。     

黄绿木霉菌代谢产物对大豆菌核病核盘菌的抑菌能力研究

利用室内实验的方法,研究了黄绿木霉对大豆菌核病菌的抑菌能力。黄绿木霉在与核盘菌（Sclerotinia sderotiorum）对峙培养过程中,5-7d即可将菌核病菌完全覆盖,在形成的菌核上会有黄绿木霉孢子出现,扫描电镜中观察到核盘菌的菌丝体发生变形;其代谢产物在经121℃处理25min后,抑菌作用仍可高达100％;在对黄绿木霉发酵过程中,利用PD培养基即可达到有效的控制大豆菌核病菌菌丝生长的目的,而且利用PD培养基发酵3-4d的黄绿木霉代谢产物抑菌效果最好,抑菌中浓度最低,发酵时间过长与过短,均不利于对病原菌的抑制。经黄绿木霉代谢产物处理的菌核菌电导率与呼吸强度均发生变化,在光学显微镜下观察到菌核菌菌体发生变化,且处理后的菌核菌致病力明显降低。     

菌核寄生菌Talaromyces flavus的生物学特性及寄生菌核规律初探

本文对核盘菌菌核寄生菌Ｔａｌａｒｏｍｙｃｅｓｆｌａｖｕｓ的基本生物学特性及影响其寄生菌核的生态因子进行了初步研究。结果表明：Ｔ.Ｆｌａｖｕｓ在我国南北方菌核病发生区土壤中普遍存在。其菌丝在３－３５℃及ｐＨ值２－１０范围内均能生长及产孢,最适生Ｋ温度为３０℃,最适生长ｐＨ值为４。对峙培养结果表明Ｔ,fｌａｖｕｓ对核盘菌的抗生作用微弱,对菌丝和菌核均有很强的寄生致腐作用。除核盘菌外它还能寄生三叶草核盘菌、小核盘菌及离菌上的一种待鉴定的产菌核真菌。Ｔ,ｆｌａｖｕｓ     

Mycoparasites effect on reproductive ability of Sclerotinia sclerotiorum sclerotia.

The ability to parasitise Sclerotinia sclerotiorum and the effect on apothecia production was evaluated for the following antagonists: Trichoderma harzianum; Trichoderma koningii; Gliocladium roseum and Chaetomium globosum. Plastic trays were filled with of steam-sterilized soil. Each one of them was infested with sclerotia of S. sclerotiorum and the culture of the antagonists. The trays were kept in a greenhouse and after 30, 60 and 90 days, evaluations were made. The rates of carpogenic germination, myceliogenic germination, mycoparasitism and destruction were evaluated. To assess carpogenic germination, the sclerotia were put in a growth chamber over moistened filter paper at 20 -/+ 2 degrees C and 12 light hours. The rates of myceliogenic germination and mycoparasitism were evaluated on Petri dishes with 2% APD. Antagonists effect on carpogenic germination was observed one month after the start of the assay. In the evaluation made at 60 and 90 days, T. harzianum; T. koningii and G. roseum kept inhibitory properties. Such inhibition was not observed in the trays containing C. globosum. In the evaluations made at 30 days, mycoparasitism rate was high in the trays with T. harzianum; T. koningii and G. roseum. G. roseum and T. harzianum destroy S. sclerotiorum sclerotia.     

Ultrastructural and cytochemical aspects of the interaction between the ectomycorrhizal fungus Laccaria laccata and the saprotrophic fungi, Trichoderma harzianum and T. virens

Different interactions between soil fungi competing in the rhizosphere with each other are necessary to understand their influence on plant growth and health. The interactions between the ectomycorrhizal (ECM) fungus Laccaria laccata and soil saprotrophic fungi (T. harzianum, T. virens) were studied by transmission electron microscopy, and by gold cytochemistry to assess the potential role of cell wall lytic enzymes in mycoparasitism. Anti-β-1,3-glucan antibody, WGA/ovomucoid-gold complex and PATAg test were used to localize β-1,3-glucan, chitin and polysaccharides. Cytoplasm disorganisation of the saprotrophic fungi occurred concurrently with dissolution of β-1,3-glucan in walls of hyphae and conidia of the saprotrophic fungi. Then digestion of polysaccharides and chitin of colonised fungal structures occurred. The studies suggest sequential contribution of cell wall lytic enzymes and importance of disturbing the host's cell integrity during mycoparasitism. We conclude that the ECM fungus can parasitise on the saprotrophic fungi not only in dual culture on artificial medium but also in the rhizosphere of Scots pine.     

Hydrolytic enzymes and ability of arbuscular mycorrhizal fungi to colonize roots

The production of hydrolytic enzymes from external mycelia associated with roots and colonized soybean roots (Glycine max L.) inoculated with different arbuscular-mycorrhizal (AM) fungi of the genus GLOMUS:, and the possible relationship between these activities and the capacity of the AM fungi to colonize plant roots was studied. There were differences in root colonization and plant growth between the GLOMUS: strains, and also between two isolates of G. mosseae. Hydrolytic activities in the root and external mycelia associated with roots differed in the AM fungi tested. Correlations were only found between the endoxyloglucanase activity of the external mycelia associated with roots of the AM fungi tested and the percentage root colonization or plant growth. However, hydrolytic activities of roots colonized by the different endophytes correlated with those of external mycelia. The hydrolytic activities were not qualitatively different because the endoxyloglucanase from AM colonized roots and the external mycelia did not show a high degree of polymorphism in the different species of fungus tested. The possible role of the hydrolytic activity of external hyphae of AM fungi was discussed as a factor affecting fungal ability to colonize the root and influence plant growth.     

Mycoparasitism of Endophytic Fungi Isolated From Reed on Soilborne Phytopathogenic Fungi and Production of Cell Wall-Degrading Enzymes In Vitro

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of β-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of β-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.     

Evaluation of Gliocladium roseum Against Wood-Degrading Fungi in vitro and on Major Canadian Wood Species

The protection of wood from fungal stain using biological agents has considerable potential for reducing discoloration of freshly sawn logs and lumber, while decreasing fungicide use. A number of biocontrol candidates have been reported worldwide, and Gliocladium roseum is one of such microorganisms. In this study, the bio-activity of G. roseum was investigated against different wood-degrading fungi on agar plates and wafers of 12 major Canadian wood species. Of the four sap-staining fungi tested on agar plates, Ophiostoma piceae and Alternaria alternata showed greater sensitivity than Aureobasidium pullulans or Cladosporium sphaerospermum to G. roseum . On wood wafers, a spore suspension of G. roseum (1 10 6 spores/ ml) provided satisfactory protection of wood from stain on western hemlock ( Tsuga heterophylla ), white spruce ( Picea glauca ), amabilis fir ( Abies amabilis ), balsam fir ( Abies balsamea ) and jack pine ( Pinus banksiana ). The antagonist also restricted the development of moulds and stain on black spruce ( Picea mariana ), lodgepole pine ( Pinus contorta ) and white pine ( Pinus strobus ), but did not protect Douglas fir ( Pseudotsuga menziesii ), red pine ( Pinus resinosa ), white birch ( Betula papyrifera ) and trembling aspen ( Populus tremuloides ). Logs of black spruce and jack pine treated with G. roseum were much less stained than untreated ones after a 4-month period of summer storage in the field. In an anti-decay test, no significant difference was found for weight loss between wood blocks treated with G. roseum and untreated samples. Application of G. roseum with low levels of an anti-sap stain chemical (NP-1) to wood wafers simultaneously did not produce a noticeable improvement in wood protection against stain compared with the chemical treatment alone.     

The influence of detergents and active components of detergent on bioproduction of organic matters and enzymatic activity of some species of fungi.

Detergent (Merix, "Merima " Krusevac) applied in concentration of 1% vol. showed specific influence on the bioproduction of some 15 different amino acids and on the enzyme activity of the species of fungi A. niger, A. alternata and T. roseum. Detergent has significantly stimulated the production of 15 analyzed amino acids of the fungi species A. niger. The same applied concentration of detergent has decreased or considerably decreased the production of some 14 of totally 15 analyzed amino acids of investigated fungi species A. alternata and T. roseum. The enzyme activity of the fungi A. niger was more intensive in relation to the species A. alternata and T. roseum during the experimental period or in some phases of the experimental period. The detergent component, ethoxyled oleyl-cetyl alcohol, in concentration of 0.01%, 0.1% and 1% showed an inhibitory effect, or significant inhibitory effect on the enzyme activity of the examined species of fungi (A. niger, A. alternata and T. roseum).     

木霉菌防治植物真菌病害研究进展

木霉菌是一种重要的植物病害生防因子,尤其在防治植物病原真菌病害中一直受到极大的关注。木霉菌依靠其菌株在包括趋向生长、识别、接触、缠绕与穿透等步骤的真菌寄生过程中分泌产生的几丁质酶、葡聚糖酶、纤维素酶、蛋白酶等一系列细胞壁降解酶,进行重寄生作用,拮抗其他植物病原菌,行使其生防功能。我们简要概述了木霉菌的种类、拮抗对象、抑菌机制、诱导抗性、促生作用、基于分子生物学的转基因工程研究,以及木霉菌在植物病原真菌生物防治中的应用。     

Characterization of Chitinolytic and Antifungal Activities in Marine-Derived Trichoderma bissettii Strains

Trichoderma fungi have been intensively studied for mycoparasitism, and the latter is closely related to their cell-wall degrading enzymes including chitinase. Here, we studied marine-derived Trichoderma spp., isolated from distinct sources and locations, for chitinolytic and antifungal activity. Based on morphological and phylogenetic analyses, two strains designated GJ-Sp1 and TOP-Co8 (isolated from a marine sponge and a marine alga, respectively) were identified as Trichoderma bissettii. This species has recently been identified as a closely related species to Trichoderma longibrachiatum. The extracellular crude enzymes of GJ-Sp1 and TOP-Co8 showed activities of chitobiosidase and β-N-acetylglucosaminidase (exochitinase) and chitotriosidase (endochitinase). The optimum chitinolytic activity of the crude enzymes was observed at 50 °C, pH 5.0, 0–0.5% NaCl concentrations, and the activities were stable at temperatures ranging from 10 to 40 °C for 2 h. Moreover, the crude enzymes showed inhibitory activity against hyphal growth of two filamentous fungi Aspergillus flavus and Aspergillus niger. To the best of our knowledge, this is the first report of the chitinolytic and antifungal activity of T. bissettii.     

Role of zearalenone lactonase in protection of Gliocladium roseum from fungitoxic effects of the mycotoxin zearalenone

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of < or =10 microg/ml. The fungitoxic effect declined in the order zearalenone > alpha-zearalenol > beta-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.