【作者】 刘保平；

【导师】 王宏燕；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2009， 博士

【摘要】 本研究利用微生物降解秸秆以修复退化黑土。利用微生物降解秸秆直接还田既能减少秸秆浪费造成的环境污染,又能修复退化黑土,有利于发展绿色农业、促进农业的可持续发展。主要研究内容是寻找既能降解秸秆的微生物,包括分离降解秸秆的真菌、细菌和放线菌;对菌株的降解能力进行定性和半定量分析;试验添加剂对降解效果的影响;试验菌株混合降解效果以及混合后菌株间的竞争性;选取降解秸秆的丝状真菌进行盆栽试验检验其对玉米发芽和苗期生长的影响;试验优良菌株的固体发酵效果。研究得到一批具有降解秸秆和玉米促生的双功能菌,提出秸秆程序性降解新方式。具体研究方法及研究内容如下:建立了一种新的简单的分离降解秸秆的丝状真菌的方法,用此方法从东北寒地黑土中分离出真菌56株,其中丝状真菌46株。这种分离方法简述为:秸秆用土壤或者堆肥浸渍至接近腐烂,用酒精对接近腐烂秸秆进行表面灭菌,用土豆葡萄糖琼脂培养基培养并分离秸秆中微生物,这些分离的微生物大多为产丝真菌。用添加或者不添加硝酸铵、酵母粉和黑土浸液等辅助物的秸秆粉制成不同培养基,这些培养基用来比较其对分离的微生物的生长的影响。结果表明同一微生物在不同培养基中生长区别很小,同一培养基不同微生物生长区别较大,具有多样性。选择了只含秸秆粉而不添加辅助物的培养基作为微生物是否降解秸秆的定性培养基,对分离的56株真菌以水稻、大豆和玉米三种秸秆进行降解定性鉴定,结果显示:该方法筛选的46株丝状真菌都能降解三种供试秸秆。用稻草、大豆和玉米三种干燥秸杆粉碎而成的秸秆粉部分代替LB(一种常用细菌培养基)、高氏培养基中的碳源成分,以此配成液体培养基,用猪粪、牛粪、马粪与黑土混合的土壤作为土样来直接接种,通过延长培养时间来分别富集培养能降解秸杆的细菌、放线菌,用传统的稀释涂平皿挑取单菌落的分离方法,通过菌落形态差异分离出细菌29株,放线菌29株。同时发现大豆秸杆能富集到较多的放线菌,稻草能富集到较多的细菌。根据试验中的平皿中的菌落数计算出菌液中的活菌数浓度。用秸秆粉培养基定性鉴别法对分离的细菌、放线菌进行秸秆的降解的定性鉴别,结果表明:分离的29株细菌中有8株能降解秸秆,分离的29株放线菌中有10株能降解秸秆。选取能降解秸秆的14株菌比较它们在三种秸秆粉培养基上的生长情况,发现供试14株菌都能利用三种秸秆生长,同一菌株在不同秸秆粉培养基上生长的菌落差异较大。测试了硝酸铵、酵母粉和土液等添加物对能降解秸秆的3株细菌和9株放线菌在秸秆粉培养基上的生长影响,结果表明:酵母粉对细菌的生长具有明显促进作用,其它添加物对供试菌的生长影响较小。选取降解秸秆较快且形态差异较大的10株丝状真菌,以玉米秆芯为材料,研究其降解秸秆的协同性。结果表明:供试10株丝状真菌在侵入秸秆内部后都具有排斥其他丝状真菌侵入的倾向。利用平板对峙法研究这10株丝状真菌在培养基PDA上生长的相互影响,结果表明供试10株丝状真菌也普遍具有相互竞争和抑制的作用,没有发现促进生长现象。根据菌落形态、菌丝和孢子形态对10株丝状真菌鉴定到属。在这10株真菌中,有三株(F1、F7、F10)是循环抑制,分类学上分别归属于Mucor,Phytium和Phytophthora。结果表明侵入秸秆内10株丝状真菌阻止外源真菌入侵。盆栽试验研究了接种44株可以降解秸秆的丝状真菌对玉米发芽和苗期生长的影响。结果表明:八株真菌F6、F7、F9、F11、F16、F17、F18和F36可显著(P<0.05,后同)降低玉米发芽率,其中极显著两株为F6和F36,分别降低45.0%和32.5%;真菌F36同时也显著降低单株生物量,降低率为48.0%;两株真菌F5和F45可显著提高玉米苗期单株生物量,分别提高35.0%和48.0%。真菌F5和F45经初步鉴定分别为Fusarium sp.和Trichoderma sp.。结论:降解秸秆丝状真菌Fusarium sp.(F5)和Trichoderma sp(F45)显著促进玉米苗期生长。理论分析表明:可以侵入致密秸秆内部的丝状真菌都能一定程度上降解秸秆,依附于秸秆表面的微生物可能与秸秆的降解有关。依附于秸秆表面的真菌可以通过PDA培养基展示出来,方法是将秸秆片段置于PDA培养基上培养数天,在秸秆表面原位就能见到依附于秸秆的真菌菌落,在秸秆处于潮湿环境下也能展示出表面依附的真菌。根据丝状真菌在PDA和秸秆内的竞争性结果,半腐烂秸秆表面出现块状色斑的实验现象,以及土壤中半腐烂秸秆内只能分离到少量种类真菌的实验结果,分析了土壤中降解秸秆微生物的演替过程。更多还原

【Abstract】 The work was microbial degradation of the stalks for restoring the degenerated black soil. Return of the stalks degraded by microorganisms to the field benefit not only to the environments but also to the restoration of the degenerated black soil, it benefit to the development of green agriculture and continuation development of agriculture. The main content of the work was screening microorganisms that could both degrade stalks and promote crop growth. Following work included: isolation of fungi, bacteria and actinomycetes that could degrade stalks; measurement of the capability and rough extent of the degradation; evaluation of the effects of the addings to the degradation; tests of the effects of the degradation inoculated with mixed microorganisms and the competition between the microorganisms; tests of the effects of inoculation with selected filamentous fungi that could degrade stalks on the growth and germination of maize seedling in the potted trials; evaluation of solid fermentation of the stalks using chosed microorgansms. Some strains of microorganisms that could both degrade stalks and promote maize growth were got, programmed degradation of the stalks using microorganisms was put forward. The detailed methods and contents of the research work could be seen as following:A new simple method to isolate filamentous fungi that could degrade stalks was built. Fifty-six strains of fungi include 46 strains of filamentous fungi were isolated from the black soil in the northeast in China by the method. The isolation method could be summarized as: Stalks were nearly rotten after being covered with soil or compost suspension for a few days, ethanol were used to disinfect the surface of the stalks, the culture medium (Potato Dextrose Agar,PDA) were used to isolate the microorganisms in the stalks, most of the microorganisms isolated by the method are filamentous fungi. Three dry stalks include straw, soybean stalk and corn stalk are collected to be pulverized to made stalk powder. Stalk powder added with different matter (NH4NO3, yeast abstract, black soil solution) or not were recruited to made solid culture media to compare the growth of the microorganisms isolated. Results showed that there are little differences between the growths of the same strain on different media but varified differences between the growth of the different strains on the same medium. The simple medium made by stalk powders without any of the adding matters were chosen to determine whether the three stalks were degraded by the 56 strains of fungi. Results showed that all of the 46 strains of filamentous fungi isolated by the method could degrade the three stalks.Twenty-nine strains of bateria and 29 strains of actinomycetes, judging by the colonies differences on the plates, were isolated from the soil (mixture of swine manure,cattle manure,horse manure and black soil) by conventional dilution-plating method, Gaose`s medium and LB medium in which carbon component partly replaced by the three stalk powder(straw,soybean and maize stalks) were used and the cultural time were prolonged to enrich the content of the bacteria and actinomycetes that could degrade stalks. More actinomycetes were discovered on the plates of the soybean stalk enrichment and more bacteria on that of the straw enrichment. The densities of the bacteria and actinomycetes in the suspention of the microorganisms were calculated from the number of the colonies on the plates. Eight strains in the 29 bacteria and 10 strains in the 29 actinomycetes could degrade stalks tested by the determination method from the growth of the microorganisms on the medium made of the mixture powder of the three stalks. Fourteen strains of microorganisms that could degrade the mixture of the three stalks were used to make trials of the growth on the three media (each made of one of the three stalks), results showed that each of the 14 microorganisms could grow on each of the three media, and the colonies` formation of each strains on the three media were different. Stalk powder added with different matter (NH4NO3, yeast abstract, black soil solution) or not were recruited to made solid culture media to compare the growth of the 3 bacteria and 9 actinomycetes that could degrade stalks, results showed that yeast abstract promoted the growth of the 3 bacteria on the stalk powder and the other addings have little effect on them.Cooperativity in the degradation procedure in the core of the maize stalks between each 2 strains among 10 strains of filamentous fungi that could most effectively degrade stalks and colony morphologically different were studied.Results showed that each of the 10 filamentous fungi have the tendency to prevent other fungi penetrating into the space it already occupied. The method of two fungi face to face on the plates was recruited in the study of the effects between each two strains among the 10 strains of fungi on the PDA medium. Results showed that competition and antagonism were seen between most of the 10 fungi, no growth acceleration were showed.The 10 strains of fungi were identified to genus according the morphology of the colonies, the mycelium and the spores. Three (F1、F7、F10) of the 10 strains of fungi form an inhibition cycle,the three fungi taxonomically belong to Mucor,Phytium and Phytophthora. Results showed that the 10 filamentous fungi in the stalks prevent other fungi penetrating.Pot experiments effects of inoculation with each of the 44 filamentous fungi that could degrade stalks on the germination and seedling growth of maize were investigated. The results indicated that germination rate of the maize significantly (P< 0.05) reduced by inoculation of the 8 strains of fungi (F6,F7,F9,F11,F16,F17,F18 and F36) , included two most significantly treatments inoculated with F6 and F36,which 45.0% and 32.5% decreased ; The seedling biomass were also reduced 48.0% by inoculation of the fungus F36; the seedling biomass of maize prominently increased by inoculation of the two strains F5 and F45,which 35.0% and 48.0% increased .The fungi F5 and F45 were identified as Fusarium sp. and Trichoderma sp.. In conclusion, growth of the seedling of maize were significantly promoted by inoculation of the two fungi that could degrade stalks (Fusarium sp.(F5) and Trichoderma sp.(F45)). Theoretically analysis showed that filamentous fungi that could penetrate into the compact stalk all could degrade the stalk to some extent, microorganisms attached on the surface of the stalk may involved in the degradation procedure.Fungi attached on the surface of the stalk could be displayed on the PDA medium, the method is to put the stalk segments on the PDA medium and cultivate for some days,the fungi colonies could be seen on the original surface of the stalks,the fungi could also displayed in the humidity environment. A succession procedure of the microorganisms degrading stalks in soil was analysed based on the following facts and phenomena: competition results of the filamentous fungi in stalks and on PDA, the experimental phenomenon that the appearance of the colorful massive slump on the surface of half decomposed stalks, and the experimental results that only few strains of fungi could isolated from the half decomposed stalk in the soil.更多还原