【作者】 孙超岷；

【导师】 刘开启； 李天飞； 王革；

【作者基本信息】 山东农业大学 ， 植物病理学， 2002， 硕士

【摘要】 绿色木霉菌(Trichoderma viride)Tv-1菌株是玉溪红塔烟草集团王革等从土壤中用诱捕法分离获得的。该菌株生长迅速，在田间对烟草黑胫病、赤星病等重要真菌病害具有良好的防治效果，其固体发酵制剂已经进行到中试阶段。 了解绿色木霉菌Tv-1菌株的部分生态学特性，将有助于其制剂的开发利用。本文通过对绿色木霉菌Tv-1菌株与烟草凝集素的凝集反应、对烟草的吸附性及沿根表的生长繁殖、产纤维素酶、对疫霉菌Ph1菌株的拮抗性等特性进行了研究。埋片法观察结果表明：绿色木霉菌Tv-1菌株在烟草根表面具有很强的繁殖能力，靠近土表处的定殖数量最多；烟草幼苗同绿色木霉菌Tv-1菌分生孢子悬浮液吸附试验表明：Tv-1菌株对烟株根圈具有明显的趋向性，靠近根圈，孢子萌发早且长势好，远离根圈，孢子萌发晚，且长势弱；绿色木霉菌Tv-1菌株的分生孢子悬浮液同烟草凝集素粗提液具有明显的凝集现象，这表明Tv-1菌株具有定殖烟草的能力；通过比色法测定Tv-1菌株液体培养物还原糖的浓度，结果表明：绿色木霉菌Tv-1菌株具有较强的产纤维素酶活性，羧甲基纤维素(CMC)酶、滤纸糖化酶最高酶活力可达149.35μg／ml.min，255.6μg／ml.min，而以往研究结果表明，纤维素酶的含量同菌株定殖能力呈正相关；平板上及生境内绿色木霉菌Tv-1菌株同疫霉菌菌株Ph1的拮抗机制的研究结果表明，两者之间存在位点竞争、营养竞争及重寄生现象。 能否在植株上定殖已成为评价生防菌优劣的一个重要指标。在烟草的根部及叶面撒施或喷施绿色木霉菌Tv-1菌株制剂后，定期取样用扫描电镜观察其定殖状况。结果表明：烟草根部及叶片对绿色木霉菌Tv-1菌株具有显著的吸附作用，吸附是生防菌株定殖植株的最初也是十分重要的步骤：绿色木霉菌Tv-1菌株在烟草外根圈能成功定殖，为其在烟株根表的进一步定殖提供了接种源；绿色木霉菌Tv-1菌株定殖(包括吸附过程)的优先位点主要有：根毛基部、自然孔口处、茎表和叶面腺毛基部以及叶面气孔周围和凹陷处，这些部位分泌物较多，利于Tv-1菌株生长繁殖；结果还表明：绿色木霉菌Tv-1菌株在适宜的侵入位点(如伤口、自然孔口)，可以部分进入植株内部并在一定范围内传导，绿色木霉菌Tv-1菌株在烟株内的传导能力在一定程度上反映了其在烟株体内同病原菌竞争空间和营养的能力。通过定期取样，用木霉半选择性培养基(TSM)培养计数绿色木霉菌Tv-1菌株的定殖数量，跟踪其在烟草根部及叶面的定殖动态。结果表明：绿色木霉菌Tv-1菌株在烟草根部的定殖数量在1个月内变化幅度不大，但均低于原始施入量，主要原因可能是土壤的抑菌作用；在叶面由于相对恶劣的生存环境，定殖数量较根部下降更为明显。 绿色木霉菌Tv-1菌株在植物根部定殖受到多种因子影响。利用平板培养的方法，研究生境的含水量、pH值、营养及烟草品种等对绿色木霉菌Tv-1菌株在烟草根部定殖的影响。用木霉半选择性培养基(TSM)分别在第三天、第六天、第九天取样计数，结果表明：在所测定的0.5％、1.5％、3.0％、5.0％、7.0％、9.0％等6种含水量处理中，偏高的含水量(7.0％、9.0％)及偏低含水量(0.5％)均不利于绿 孙超峨：绿巴木霉菌T。－1菌什在烟草氓部及叶血口b定蚀研穴色木霉菌TV－l菌株的定值；在所测定的4刀、5刀、6．0、7刀、8刀、9刀等6种pH处理中，偏酸性的 pH值（4刀、5刀、6刀）利干绿色木霉菌 TV－l菌株的定值，而偏碱性PH值（8刀、9刀）抑制其定殖：在所测定的葡萄糖琼脂＊叶平板、水琼脂（WA平扳、土壤浸渍液（SA）平板中，添加葡萄糖的GA平板能促进绿色木霉菌TV．l菌 勾株的定殖：不同的烟草品种对绿色木霉菌TV．l 菌株的定殖也有不同程度的影响，所测试的二烟 85、K346、G28、RGll、NC82、NC89等 6个品种中，云烟 85、G28、NC89较适合其定殖。 绿色木霉菌TV－l 菌株定殖烟草后对烟株生长无不良影响，在一定程度上还能促进烟株生长。喷施绿色木霉菌TV－1 菌株制剂后的烟株较未使用制剂的烟珠光合作用速率增强、呼吸作用速率变化不明显，叶绿素含量增加。喷施后的烟株光合作用速率、呼吸作用速率及叶绿素的含量分别为ZI．250mgCO。／din’Al、6厂81mgCO。／din‘．hr、2．647mg／g 鲜重；对照烟株分别为 13．345mgCO。／din’．hr、7石00mgCO，／din’．hr、2．23ling／g鲜重。更多还原

【Abstract】 Triclioderma viride Persoon strain Tv-1 was isolated from soil by Dr. Wang Ge who worked in Hongta Group.This strain grew fast and controlled fungous diseases of tobacco effectively.The solid fermention preparation of T.viride strain Tv-1 has been in production.Understanding the ecology characterristics of T.viride strain Tv-1 clearly especially relative to colonization.is important to know the colonization mechanisms and promote its colonization efficiency.The experiment by the method of immersed piece results showed that T.viride strain Tv-1 could grow and reproduct fast along the root of tobacco.The colonization number of T.viride strain Tv-1 near to soil surface was more than that on other section of tobacco.The adhesion experiment results indicated that T.viride strain Tv-1 showed obvious topotaxis to tobacco seedling.The ones near to tobacco grew much better than those far from tobacco.The crude conidia suspension of T.viride strain Tv-1 was able to agglutinate with crude lectin of tobacco,vvhich suggested the colonization capability of T.viride strain Tv-I. T.viride strain Tv-I could product cellulase and the highest peak of carboxymethyl cellulase(CMCasc) and filter paper cellulase was 149.35 n g/ml.min and 255.60 u g/ml.min.A lot of reserches reveaed that the cellulase activity was positively correlated to colonization capability of biocontrol agents.The antagonistic experiment of by T.viride strain Tv-1 and Phytophthora strain Phi showed that the action mode of antagonisms included niches and nutrition competion and hyperparasitism.Whether or not the biocontrol agents are able to colonize on plant is an important evaluation index.The experiment samples were taken at regular intervals and were observed by Scanning Electronic Microscope.The results illustrated that T.viride strain Tv-1 was able to adhere to tobacco sifnificantly,which was the first and important step for colonization.The colonization of external rhizosphere act as the an inocula for second colonization.The major suitable colonization niches of Tv-1 were the base of root hairs.natural openings,wound.the base of glandular hairs of stem and phylloplane,around stoma and recessed of phylloplane,where there were much more metabolins.And T.viride strain Tv-1 was able to penetrate into root and stem in some suitable niches and conducted in a short distance.The consduction capability showed the potential of competing space and nutrition with pathogy within tobacco of T.viride strain Tv-1.The colonization number of T.viride strain Tv-1 on the root and phylloplane of tobacco was measured by Trichoderma Semiselective Medium(TSM).The results showed that the colonization number of T.viride strain Tv-1 was stable,but the number were always bellow the original one.The phenomena perhaps showed the soil mycostasis and population competetion and bad adverse circumstances.The colonization of T.viride strain Tv-1 was influenced by many facors.The factorsaffecting colonization of T.viride strain Tv-1 on the tobacco root were studied by testing on the agar plate. The isolation and counting of strain Tv-1 were performed on 7hc/ifera-Semiselective Medium(TSM).The results demonstrated that the following factors,including moisture,nutrition,pH and varieties of tobacco influenced the colonization ability of strain Tv-1 on root. 97.0% moisture on the agar plate ,the pH ranging from 4.0 to 6.0 and glucose as the carbon sources were optimum conditions for the colonization of strain Tv-1,and Yunyan85, G28 and NC89 were most favourable tobacco varieties for the colonization of strain Tv-1 on the root of tobacco.The colonization of T.viride strain Tv-1 did not hamper the growth of tobacco.Inversely, T.viride strain Tv-1 was able to promote the growth of tobacco to some extent.The results showed that the photosynthetic rate chlorophyll content of tobacco with T.viride strain Tv-1 were higher than that of the control.And the respiration rate kept constant.The photosynthetic rate and respiration rate and chlorophyll content were 21.250mgCO2/dnr.hr,6.781更多还原