园林废弃物制造栽培基质过程中微生物的动态变化
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摘要
本文采用传统分离结合荧光定量PCR的方法以及DGGE结合多元统计分析的方法，研究了规模生产过程中不同园林废弃物种类堆腐过程微生物的动态变化，及其与堆腐过程物料理化性质之间的关系。综合考察了不同园林废弃物形成基础物质的微生物生物量、多样性指数及其理化性质。同时，筛选植物病原菌拮抗微生物，以获得具有病原微生物抗性的功能基质，为园林废弃物制造健康栽培基质提供科学和应用基础。
主要研究结果如下：
1、堆腐过程研究及其理化性质分析表明，木质素含量较低的草坪草腐熟速度快，木质素含量较高的枯枝落叶堆腐速度相对较慢；草坪草堆体升温速度快，高温维持时间较枯枝落叶长。两种园林废弃物堆腐升温阶段C/N比均下降迅速而后期较缓；堆体的pH值和EC值均逐渐升高，但草坪草EC值的上升速度显著高于枯枝落叶。同时，两种堆腐材料，腐殖化作用均主要发生在堆体降温和腐熟阶段。堆腐过程中有效磷和有效钾含量变化幅度小，容重呈逐渐下降趋势。
此外，两种园林废弃物堆腐初始C/N比均较一般农业常用固体废弃物如秸秆等为高。
2、可培养法分离微生物计数结果表明，升温快速且堆腐最高温度更高的草坪草堆体中，细菌的生物量并不比枯枝落叶堆体中的生物量低，而堆腐前期更高。两种堆体中可培养微生物生物量整体变化趋势均呈现先升后降，而腐熟阶段略有升高。可培养细菌的生物量高于可培养真菌。高温堆腐对可培养细菌生物量影响小于真菌。荧光定量试验结果表明，堆体中细菌16S rDNA基因的拷贝数远高于真菌18S rDNA基因拷贝数。可培养计数和荧光定量分析均表明，当草坪草堆体温度达到最高温度时，微生物生物量出现明显下降，而枯枝落叶堆体符合一般材料堆腐规律。
3、DGGE研究表明，草坪草和枯枝落叶堆腐过程中真菌的多样性均小于细菌的多样性。草坪草细菌多样性低于枯枝落叶细菌多样性，而两种材料真菌多样性相差不大。草坪草堆腐过程中细菌种群变化较真菌大，枯枝落叶变化趋势亦然但程度较低。对于细菌和真菌两大类群来说，枯枝落叶堆腐过程种群变化均较草坪草缓和。基于冗余分析方法解析微生物群落组成，样点和微生物种群的二维排序图结果表明，草坪草堆腐过程对细菌种群影响较大的理化因子为pH值以及总N含量；对真菌种群影响较大的为容重和有效P。枯枝落叶堆腐过程对细菌种群影响较大为容重和腐植酸，对真菌种群影响较大的为温度和总N含量。
4、三种园林废弃物（草坪草、枯枝落叶和菇渣）堆腐高温期（＞50℃）均超过7d，一般可杀死或抑制堆体中常见病原菌。草坪草和枯枝落叶形成基础物质pH值及EC值较高，而菇渣较低。三种园林废弃物堆腐完成后均含有较高的初始养分。草坪草堆腐后的生物稳定性较其它两种基质高。
同时，初步筛选获得重要园林植物兰花和竹子主要病害兰花黑腐病拮抗菌4株，竹根腐病拮抗菌5株，其中对两种病原菌均具有拮抗作用的菌株2株，16S rDNA测序结果表明，大部分拮抗菌株归属于芽孢杆菌属。
This paper studied the dynamic change of microorganism in different types of gard-enwaste during the composting in the process of mass production process and its relationshipwith the material physicochemical property with the method of traditional separation combinedwith fluorescence quantitative PCR and DGGE combined with multivariate statistical analysis.Comprehensive survey the microbial biomass, diversity index and its physicochemical propertythat are the base of different garden waste. At the same time, screened the antagonistic microbeof phytopathogens to obtain functional matrix that can resist the pathogenic microorganism andprovide science and application foundation for making health cultivation matrix for the gardenwaste.
The main research results are as follows：
1. The composting process and physicochemical property analysis shows that thecomposting rate of grasses with lower lignin content is fast, the composting rate of brancheswith higher lignin content is relatively slow; the tempreture of the pile of grasses heat up fast,its high temperature can maintain longer than branches. These two garden wastes C/N ratioduring temperature rise period drop fast and then slow down in later stage; the pH and ECvalue of the compost body gradually increase, but the grasses EC value rise significantly higherthan that of the branches’. Meanwhile, the humification effect of these two compost materialsmainly occurs in the cooling and rotten stage of the composting. The effective phosphorus andpotassium content are with a small change ranges, the capacity shows a trend of gradualdecline. In addition, the compost initial C/N ratio of these two garden wastes is higher than thatof the general agricultural solid wastes such as straw.
2. The counting result got from cultivable method to separate microorganism shows thatthe bacterial biomass of grasses compost pile, which temperature rises quickly and composttemperature is higher, are not lower than that of branches, the earlier stage is more higher. Thetwo kinds of compost body culturable microbial biomass in the whole change trend first rises then falls, with maturity stage rising slightly. The cultivable bacterial biomass is higher thanthat of fungal. The effect on culturable bacterial biomass with high temperature composting isless than that of fungal. Fluorescence quantitative test results show that the16S rDNA genecopy number in the bacteria of compost pile is far higher than that of fungi18S rDNA. Thecultivable counting and fluorescence quantitative analysis show that when the grasses composttemperature reaches the highest temperature, the microbial biomass significantly declines,while the branches conform to the rule of general material composting.
3. DGGE research shows that the fungi diversity in both grasses and branches duringcomposting is lower than bacteria. The grasses bacterial diversity is lower than that of thebranches, but their fungal diversity have no big difference. The bacterial community duringcomposting changes a lot compared with fungi, so is the branches but with lower degree. Forthe bacteria and fungi group, the change of community of branches during composting isalleviated than that of grasses. Base on redundancy analysis method to analyse microbialcommunity composition, the sampling point and two-dimensional sequence diagram ofmicrobial population show that the great physicochemical impact factor on bacterialcommunity during grasses composting are pH value and total N content; The density andeffective P have great impact on fungal community. The density and humic acid have greatimpact on the bacterial community of the branches during compost, the temperature and total Ncontent have great impact on fungal community.
4. The compost high temperature period (>50℃) of three kinds of garden waste (grasses,branches, and mushroom residue) are more than7d, which normally can kill or inhibit thecommon pathogenic bacteria in the compost body. The pH and EC value of the base material ofgrasses and branches are higher than that of mushroom residue. These three garden wasteshave higher initial nutrient after composting. The grasses has higher biological stabilitycompared with other two kinds of substrates. At the same time, preliminary screened andobtained4strains that can antagonistic of orchid black rot and5strains that can antagonisticbamboo root rot, which respectively are the main diseases of important landscape plants: orchid and bamboo. There are2bacterial strain have an antagonistic effect on those twopathogenic fungi. According to16S rDNA sequencing, the results showed that mostantagonistic strains belong to Bacillus.
主要研究结果如下：
1、堆腐过程研究及其理化性质分析表明，木质素含量较低的草坪草腐熟速度快，木质素含量较高的枯枝落叶堆腐速度相对较慢；草坪草堆体升温速度快，高温维持时间较枯枝落叶长。两种园林废弃物堆腐升温阶段C/N比均下降迅速而后期较缓；堆体的pH值和EC值均逐渐升高，但草坪草EC值的上升速度显著高于枯枝落叶。同时，两种堆腐材料，腐殖化作用均主要发生在堆体降温和腐熟阶段。堆腐过程中有效磷和有效钾含量变化幅度小，容重呈逐渐下降趋势。
此外，两种园林废弃物堆腐初始C/N比均较一般农业常用固体废弃物如秸秆等为高。
2、可培养法分离微生物计数结果表明，升温快速且堆腐最高温度更高的草坪草堆体中，细菌的生物量并不比枯枝落叶堆体中的生物量低，而堆腐前期更高。两种堆体中可培养微生物生物量整体变化趋势均呈现先升后降，而腐熟阶段略有升高。可培养细菌的生物量高于可培养真菌。高温堆腐对可培养细菌生物量影响小于真菌。荧光定量试验结果表明，堆体中细菌16S rDNA基因的拷贝数远高于真菌18S rDNA基因拷贝数。可培养计数和荧光定量分析均表明，当草坪草堆体温度达到最高温度时，微生物生物量出现明显下降，而枯枝落叶堆体符合一般材料堆腐规律。
3、DGGE研究表明，草坪草和枯枝落叶堆腐过程中真菌的多样性均小于细菌的多样性。草坪草细菌多样性低于枯枝落叶细菌多样性，而两种材料真菌多样性相差不大。草坪草堆腐过程中细菌种群变化较真菌大，枯枝落叶变化趋势亦然但程度较低。对于细菌和真菌两大类群来说，枯枝落叶堆腐过程种群变化均较草坪草缓和。基于冗余分析方法解析微生物群落组成，样点和微生物种群的二维排序图结果表明，草坪草堆腐过程对细菌种群影响较大的理化因子为pH值以及总N含量；对真菌种群影响较大的为容重和有效P。枯枝落叶堆腐过程对细菌种群影响较大为容重和腐植酸，对真菌种群影响较大的为温度和总N含量。
4、三种园林废弃物（草坪草、枯枝落叶和菇渣）堆腐高温期（＞50℃）均超过7d，一般可杀死或抑制堆体中常见病原菌。草坪草和枯枝落叶形成基础物质pH值及EC值较高，而菇渣较低。三种园林废弃物堆腐完成后均含有较高的初始养分。草坪草堆腐后的生物稳定性较其它两种基质高。
同时，初步筛选获得重要园林植物兰花和竹子主要病害兰花黑腐病拮抗菌4株，竹根腐病拮抗菌5株，其中对两种病原菌均具有拮抗作用的菌株2株，16S rDNA测序结果表明，大部分拮抗菌株归属于芽孢杆菌属。
This paper studied the dynamic change of microorganism in different types of gard-enwaste during the composting in the process of mass production process and its relationshipwith the material physicochemical property with the method of traditional separation combinedwith fluorescence quantitative PCR and DGGE combined with multivariate statistical analysis.Comprehensive survey the microbial biomass, diversity index and its physicochemical propertythat are the base of different garden waste. At the same time, screened the antagonistic microbeof phytopathogens to obtain functional matrix that can resist the pathogenic microorganism andprovide science and application foundation for making health cultivation matrix for the gardenwaste.
The main research results are as follows：
1. The composting process and physicochemical property analysis shows that thecomposting rate of grasses with lower lignin content is fast, the composting rate of brancheswith higher lignin content is relatively slow; the tempreture of the pile of grasses heat up fast,its high temperature can maintain longer than branches. These two garden wastes C/N ratioduring temperature rise period drop fast and then slow down in later stage; the pH and ECvalue of the compost body gradually increase, but the grasses EC value rise significantly higherthan that of the branches’. Meanwhile, the humification effect of these two compost materialsmainly occurs in the cooling and rotten stage of the composting. The effective phosphorus andpotassium content are with a small change ranges, the capacity shows a trend of gradualdecline. In addition, the compost initial C/N ratio of these two garden wastes is higher than thatof the general agricultural solid wastes such as straw.
2. The counting result got from cultivable method to separate microorganism shows thatthe bacterial biomass of grasses compost pile, which temperature rises quickly and composttemperature is higher, are not lower than that of branches, the earlier stage is more higher. Thetwo kinds of compost body culturable microbial biomass in the whole change trend first rises then falls, with maturity stage rising slightly. The cultivable bacterial biomass is higher thanthat of fungal. The effect on culturable bacterial biomass with high temperature composting isless than that of fungal. Fluorescence quantitative test results show that the16S rDNA genecopy number in the bacteria of compost pile is far higher than that of fungi18S rDNA. Thecultivable counting and fluorescence quantitative analysis show that when the grasses composttemperature reaches the highest temperature, the microbial biomass significantly declines,while the branches conform to the rule of general material composting.
3. DGGE research shows that the fungi diversity in both grasses and branches duringcomposting is lower than bacteria. The grasses bacterial diversity is lower than that of thebranches, but their fungal diversity have no big difference. The bacterial community duringcomposting changes a lot compared with fungi, so is the branches but with lower degree. Forthe bacteria and fungi group, the change of community of branches during composting isalleviated than that of grasses. Base on redundancy analysis method to analyse microbialcommunity composition, the sampling point and two-dimensional sequence diagram ofmicrobial population show that the great physicochemical impact factor on bacterialcommunity during grasses composting are pH value and total N content; The density andeffective P have great impact on fungal community. The density and humic acid have greatimpact on the bacterial community of the branches during compost, the temperature and total Ncontent have great impact on fungal community.
4. The compost high temperature period (>50℃) of three kinds of garden waste (grasses,branches, and mushroom residue) are more than7d, which normally can kill or inhibit thecommon pathogenic bacteria in the compost body. The pH and EC value of the base material ofgrasses and branches are higher than that of mushroom residue. These three garden wasteshave higher initial nutrient after composting. The grasses has higher biological stabilitycompared with other two kinds of substrates. At the same time, preliminary screened andobtained4strains that can antagonistic of orchid black rot and5strains that can antagonisticbamboo root rot, which respectively are the main diseases of important landscape plants: orchid and bamboo. There are2bacterial strain have an antagonistic effect on those twopathogenic fungi. According to16S rDNA sequencing, the results showed that mostantagonistic strains belong to Bacillus.
引文
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