多功能木霉对纤维素和农药的降解特性研究
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摘要
木霉菌(Trichoderma spp.)是一类普遍存在并具有重要经济意义的生防益菌,多年来,木霉菌以其高效、安全、不易引起病原菌抗药性的特点,在以可持续发展为宗旨的生物防治中发挥了重要作用,已作为生防菌剂在美国、印度、瑞典等国家商品化生产,我国近年来也有此类产品问世,其生产方法大多采用固体培养法,以玉米粉、麸皮等农副产品为培养基质,以获得货架期较长的分生孢子。在生产中如何降低生产成本是加速木霉菌实用化进程的关键。
我国是一个农业大国,每年产生的秸秆剩余物数量巨大,而被利用的量却很少。部分农民采取了简单焚烧或随意堆积处理,不但浪费了宝贵的自然资源,还污染了大气环境,对人类健康造成了危害。由于木霉在工业上是一种优良的纤维素酶产生菌,对部分农药亦有一定的降解作用,如果能够利用秸秆为原料生产木霉制剂,且在利用木霉制剂防治病害的同时,使农作物中的农药残留得到降解,这将对农业的可持续发展及人类健康具有重要意义,为此本文展开了以下工作:
1、以本实验室筛选的具有一定生物防治效果的木霉为出发菌株,通过木霉在以纤维素为唯一碳源的CMC培养基、刚果红培养基和滤纸条液体培养基检测生长直径、透明圈和生长程度等指标,筛选出9株能够分解利用纤维素的优势木霉菌株,并研究了其Cx酶活性、滤纸酶活(FPA)和产还原糖能力,筛选出3株降解纤维素最强的木霉菌J15、J16、J18。以农业上常用的高效氯氰菊酯、毒死蜱和吡虫啉3种杀虫剂为唯一碳源制成培养基,考察3种木霉在农药平板培养基上的生长情况,并分别测定其对3种农药72h的降解率。结合各菌株的纤维素降解能力,最终确定J16木霉菌为降解纤维素能力较强,农药降解率最高的目标菌株,经形态学观察和分子生物学分析,将其鉴定为脐胞木霉( Trichoderma brevicompactum)。经检索,目前国内未见有此种的相关报道。
2、对脐胞木霉(Trichoderma brevicompactum)的生物防治特性、利用秸秆生产木霉孢子制剂时不同因素对产孢量的影响进行了研究。结果如下:平板对峙结果表明脐胞木霉(Trichoderma brevicompactum)对萝卜枯萎病菌(Fusarium oxysporum f.sp.)、西瓜枯萎病菌(F. oxysporum f. sp. niveum)、棉花枯萎病菌(F.oxysporum f. sp. vasinfectum)、小麦赤霉病菌(F. graminearum)、黄瓜枯萎病菌(F. oxysporum f. sp. cucumerinum)等病原微生物具有较强的抑制作用,抑制率分别为56.6%;81.2%;65.6%;53.5%;70.6%;用单因素和正交试验法,确定脐胞木霉(Trichoderma brevicompactum)产孢的最优条件为:秸秆:麸皮=2:1;含水量为40%;接种量为12%;温度为28℃;培养时间为9d。
3、脐胞木霉(Trichoderma brevicompactum)在以农药为唯一碳源的液体培养基中培养7d,分别测定培养液中高效氯氰菊酯、毒死蜱和吡虫啉的残余量,可得3种农药的降解率分别为90.43%、86.71%、78.81%。为进一步验证脐孢木霉( Trichoderma brevicompactum)在农业生产中对高效氯氰菊酯和毒死蜱的降解效果,将脐胞木霉(Trichoderma brevicompactum)制成孢子制剂;选择大棚温室中的油菜为供试植物,在喷洒农药12h后喷施木霉孢子制剂,分别在1、5、11、14、17d进行定时取样,用气相色谱检测油菜中的农药残留,结果显示,随着时间的延长,农药降解率逐渐升高,高效氯氰菊酯和毒死蜱的降解率分别提高了45.54%、72.58%。
Trichoderma are well known as biocontrol agents, used worldwide against different plant pathogenic fungi. Trichoderma spp. is free-living fungi that are common in soil and root ecosystems. It has played an important role in controlling plant pathogens for its efficiency, safety, and no-existence pathogens resistance. Some Trichoderma biocontrol agents have been commercialized in the United States, India, Sweden and other countries. It also produced in China in recent years. Corn flour and wheat bran were used as solid culture medium to produce Trichoderma conidia. How to reduce production cost is the key process of accelerating practical utilization of Trichoderma.
China is a large agricultural country, the quantity of straw residues is huge, but its utilization is little. Burning or abandoning them not only a waste of valuable natural resources, but also polluted the atmosphere and is harmful to human health. It is known that Trichoderma spp. can produce cellulase and degrade some pesticides. It will be of great significance to agricultural if we could use straw materials as solide to produce Trichoderma biological control agents and use it to degrade pesticide. The main conclusions of this paper are as follows:
1. A multi-functional Trichoderma stain J16 was selected which having a certain ability of biological control, degrading cellulose and degrading pesticide .It indentified as Trichoderma brevicompactum by morphological and rDNA-ITS sequence analysis. It is the first report of this genus in China.
2. Trichoderma brevicompactum had a strong antagonist on pathogenic microorganisms such as Fusarium oxysporum f.sp, F. oxysporum f. sp. niveum, F.oxysporum f. sp. vasinfectum, F. graminearum, F. oxysporum f. sp. cucumerinum, which ihibitory rates were 56.6%; 81.2%; 65.6%; 53.5%; 70.6% respectively. Different factors on sporulation when using straw as the nutrientsubstance was further studied. The optimal conditions of spore production were determined by single-factor method and orthogonal experimentation. straw: wheat = 2:1, 40% of water content, 12% of inoculum, 28℃, culture time is 9 days.
3. Under laboratory conditions, when Trichoderma brevicompactum were cultured with pesticides (beta-cypermethrin, chlorpyrifos and imidacloprid) as the single source of carbon for 7 days. The three kinds of pesticide degradation rates were 90.43%, 86.71% and 78.81% respectively.To research the degradation effects of Trichoderma brevicompactum on beta-cypermethrin and chlorpyrifos in practical applications, the biocontrol agent of the Trichoderma brevicompactum was applied in the rapes (Brassica campestris L.) in the greenhouse, the degradation rates of Trichoderma brevicompactum against insecticides (beta-cypermethrin in synthetic pyrethroids and chlorpyrifos in organophosphate pesticides) were determined by GC-FID. The degradation rates were 45.54% and 72.58% at the 17 days respectively.
我国是一个农业大国,每年产生的秸秆剩余物数量巨大,而被利用的量却很少。部分农民采取了简单焚烧或随意堆积处理,不但浪费了宝贵的自然资源,还污染了大气环境,对人类健康造成了危害。由于木霉在工业上是一种优良的纤维素酶产生菌,对部分农药亦有一定的降解作用,如果能够利用秸秆为原料生产木霉制剂,且在利用木霉制剂防治病害的同时,使农作物中的农药残留得到降解,这将对农业的可持续发展及人类健康具有重要意义,为此本文展开了以下工作:
1、以本实验室筛选的具有一定生物防治效果的木霉为出发菌株,通过木霉在以纤维素为唯一碳源的CMC培养基、刚果红培养基和滤纸条液体培养基检测生长直径、透明圈和生长程度等指标,筛选出9株能够分解利用纤维素的优势木霉菌株,并研究了其Cx酶活性、滤纸酶活(FPA)和产还原糖能力,筛选出3株降解纤维素最强的木霉菌J15、J16、J18。以农业上常用的高效氯氰菊酯、毒死蜱和吡虫啉3种杀虫剂为唯一碳源制成培养基,考察3种木霉在农药平板培养基上的生长情况,并分别测定其对3种农药72h的降解率。结合各菌株的纤维素降解能力,最终确定J16木霉菌为降解纤维素能力较强,农药降解率最高的目标菌株,经形态学观察和分子生物学分析,将其鉴定为脐胞木霉( Trichoderma brevicompactum)。经检索,目前国内未见有此种的相关报道。
2、对脐胞木霉(Trichoderma brevicompactum)的生物防治特性、利用秸秆生产木霉孢子制剂时不同因素对产孢量的影响进行了研究。结果如下:平板对峙结果表明脐胞木霉(Trichoderma brevicompactum)对萝卜枯萎病菌(Fusarium oxysporum f.sp.)、西瓜枯萎病菌(F. oxysporum f. sp. niveum)、棉花枯萎病菌(F.oxysporum f. sp. vasinfectum)、小麦赤霉病菌(F. graminearum)、黄瓜枯萎病菌(F. oxysporum f. sp. cucumerinum)等病原微生物具有较强的抑制作用,抑制率分别为56.6%;81.2%;65.6%;53.5%;70.6%;用单因素和正交试验法,确定脐胞木霉(Trichoderma brevicompactum)产孢的最优条件为:秸秆:麸皮=2:1;含水量为40%;接种量为12%;温度为28℃;培养时间为9d。
3、脐胞木霉(Trichoderma brevicompactum)在以农药为唯一碳源的液体培养基中培养7d,分别测定培养液中高效氯氰菊酯、毒死蜱和吡虫啉的残余量,可得3种农药的降解率分别为90.43%、86.71%、78.81%。为进一步验证脐孢木霉( Trichoderma brevicompactum)在农业生产中对高效氯氰菊酯和毒死蜱的降解效果,将脐胞木霉(Trichoderma brevicompactum)制成孢子制剂;选择大棚温室中的油菜为供试植物,在喷洒农药12h后喷施木霉孢子制剂,分别在1、5、11、14、17d进行定时取样,用气相色谱检测油菜中的农药残留,结果显示,随着时间的延长,农药降解率逐渐升高,高效氯氰菊酯和毒死蜱的降解率分别提高了45.54%、72.58%。
Trichoderma are well known as biocontrol agents, used worldwide against different plant pathogenic fungi. Trichoderma spp. is free-living fungi that are common in soil and root ecosystems. It has played an important role in controlling plant pathogens for its efficiency, safety, and no-existence pathogens resistance. Some Trichoderma biocontrol agents have been commercialized in the United States, India, Sweden and other countries. It also produced in China in recent years. Corn flour and wheat bran were used as solid culture medium to produce Trichoderma conidia. How to reduce production cost is the key process of accelerating practical utilization of Trichoderma.
China is a large agricultural country, the quantity of straw residues is huge, but its utilization is little. Burning or abandoning them not only a waste of valuable natural resources, but also polluted the atmosphere and is harmful to human health. It is known that Trichoderma spp. can produce cellulase and degrade some pesticides. It will be of great significance to agricultural if we could use straw materials as solide to produce Trichoderma biological control agents and use it to degrade pesticide. The main conclusions of this paper are as follows:
1. A multi-functional Trichoderma stain J16 was selected which having a certain ability of biological control, degrading cellulose and degrading pesticide .It indentified as Trichoderma brevicompactum by morphological and rDNA-ITS sequence analysis. It is the first report of this genus in China.
2. Trichoderma brevicompactum had a strong antagonist on pathogenic microorganisms such as Fusarium oxysporum f.sp, F. oxysporum f. sp. niveum, F.oxysporum f. sp. vasinfectum, F. graminearum, F. oxysporum f. sp. cucumerinum, which ihibitory rates were 56.6%; 81.2%; 65.6%; 53.5%; 70.6% respectively. Different factors on sporulation when using straw as the nutrientsubstance was further studied. The optimal conditions of spore production were determined by single-factor method and orthogonal experimentation. straw: wheat = 2:1, 40% of water content, 12% of inoculum, 28℃, culture time is 9 days.
3. Under laboratory conditions, when Trichoderma brevicompactum were cultured with pesticides (beta-cypermethrin, chlorpyrifos and imidacloprid) as the single source of carbon for 7 days. The three kinds of pesticide degradation rates were 90.43%, 86.71% and 78.81% respectively.To research the degradation effects of Trichoderma brevicompactum on beta-cypermethrin and chlorpyrifos in practical applications, the biocontrol agent of the Trichoderma brevicompactum was applied in the rapes (Brassica campestris L.) in the greenhouse, the degradation rates of Trichoderma brevicompactum against insecticides (beta-cypermethrin in synthetic pyrethroids and chlorpyrifos in organophosphate pesticides) were determined by GC-FID. The degradation rates were 45.54% and 72.58% at the 17 days respectively.
引文
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