抑制烟草青枯病型生物有机肥的田间防效研究

【目的】烟草青枯病是影响烟叶生产最主要的土传病害之一,生物防控烟草青枯病是近年来的研究热点。为验证抑制烟草青枯病型生物有机肥对生防青枯病的影响,进行2年田间试验研究其防效及其对土壤微生物的影响。【方法】本试验采用从烟草根际原位土壤分离得到的烟草青枯病拮抗菌株L-25和L-9,利用有机肥二次固体发酵技术,制成烟草青枯病拮抗生物有机肥。连续两年在安徽进行田间试验,分别在烟草移栽后50天和105天调查生物防控率,探求生物有机肥对青枯病的田间防效和对烟叶产量的影响;利用平板计数法、Biolog特征性碳源法和梯度变性凝胶电泳法摸索生物有机肥施用后根际土壤微生物数量、群落功能多样性和结构多样性的变化,揭示生物有机肥对青枯病的防控机理。【结果】 1）第一年和第二年施用生物有机肥处理移栽50天后烟草青枯病的生物防控率分别为82.2%和96.2%,105天后烟草青枯病的生物防控率分别达到75.2%和95.4%; 2）生物有机肥处理第一年和第二年烟叶产量分别为2212.5 kg/hm2、1475.5 kg/hm2,是对照的2.4和2.6倍; 3）两年生物有机肥处理的根际土壤可培养细菌、放线菌数量均显著高于对照,真菌数量显著低于对照（P＜0.05）,其中第一年生物有机肥处理50天和105天拮抗菌数量分别为对照的241.8倍和13.4倍,病原菌数量仅为对照的19.7%和56.6%,第二年生物有机肥处理50天和105天拮抗菌数量分别是对照的111.0倍和26.7倍,病原菌数量仅为对照的9.1%和31.4%; 4）两年生物有机肥处理50天和105天根际土壤的微生物群落功能多样性,即Shannon指数、Simpson指数和Mclntosh指数均显著高于对照（P＜0.05）; 5）生物有机肥处理与对照土壤微生物群落结构也不相同,细菌和真菌梯度变性凝胶电泳图谱明显不同,分别属于不同的聚簇,生物有机肥处理的细菌种类较对照有所增加,同时真菌的种类有所减少。【结论】在烟草青枯病发病较为严重的烟田施用生物有机肥,可以显著降低青枯病发病率,增加烟草产量。生物有机肥可以显著提高拮抗菌数量,抑制根际土壤病原菌的数量,提高土壤微生物群落功能多样性,改善微生物种群和组成,丰富土壤微生物群落结构,使土壤保持健康的微生物生态平衡。     

番茄青枯病的土壤微生态防治研究

采用田间试验、微生物培养、BIOLOG GN微平板培养分析，研究了新开地、连作地施用生态有机肥前后土壤微生物多样性与番苗青枯病的关系。结果表明，新开地没有发生青枯病，连作地番茄青枯病发病率为100％，连作地施用生态有机肥后，青枯病发病率降低至49.86%，土壤真菌、放线菌的数量略微增加，细菌的数量变化不大，但平板中真菌、细菌、放线菌的不同形态菌落数明显增多。新开地、施用生态有机肥处理土壤微生物群落在BIOLOG GN微平板温育过程中的AWCD值、Shannon指数、Simpson指数、McIntosh指数、Alatalo均匀度均显著高于连作地土壤，PIE相遇几率则相反，新开地与施用生态有机肥土壤之间无显著差异。由此说明，施生态有机肥能提高土壤微生物多样性，从生态水平上抑制番茄青枯病的发生。     

连续施用有机肥对连作花生根际微生物种群和 酶活性的影响

花生连作导致土传病害易发,严重制约其产量和品质。为探讨持续施用有机肥缓解连作花生病害发生的防治机理,大田条件下研究了连续6年施用化肥、普通有机肥和生物有机肥(接种内生菌拟茎点霉B3)后对连作花生根腐病、根际土壤典型微生物种群和酶活的影响。结果表明:与单施化肥相比,持续施用普通有机肥和生物有机肥可有效控制连作花生土传病害发生,荚果产量提高23.8%和47.9%。连续施用普通有机肥和生物有机肥均显著提高根际过氧化氢酶、脱氢酶和酚氧化酶活性,荧光定量PCR进一步测定出连续施用有机肥可显著增加连作花生根际细菌和真菌的数量,说明连续施用有机肥后连作花生根际微生物活性显著提升。连续施用普通有机肥和生物有机肥显著增加了连作花生根际有益微生物种群的数量,其中放线菌增加0.9倍和1.6倍,假单胞菌增加10.9倍和13.1倍,伯克氏菌增加2.6倍和1.9倍,芽孢杆菌增加1.1倍和2.1倍;然而连续施用有机肥对连作花生根际主要致病微生物种群(如镰刀菌属和青枯雷尔氏菌)无明显影响。表明通过持续施用有机肥可有效提高连作花生根际有益微生物种群的数量和土壤酶活性,进而改善连作花生根际微生态环境,增强连作土壤抑病能力,以达到控制连作花生土传病害易发的目的。     

烤烟根际微生物群落结构及其动态变化的研究

选择贵州省三种典型的植烟土壤———中性紫色土、黄壤和黄色石灰土为对象,研究了烤烟根际微生物群落结构及其动态变化。结果表明,在烤烟生长过程中,根际细菌的数量在团棵期最低,然后逐渐增加,现蕾期达到峰值,进而又逐渐减少;放线菌和真菌数量从团棵期到成熟期呈增长趋势。土壤类型不同,根际细菌和放线菌的数量不一样,黄壤>黄色石灰土>中性紫色土;真菌数量,中性紫色土>黄壤>黄色石灰土。烤烟根际微生物的种群多样性及其变化在不同土壤中表现也不一样,相对于黄壤和黄色石灰土,在中性紫色土中,根际微生物的优势种群数目较多,根际细菌和放线菌种群更具多样性,而且较稳定,表明其群落结构更为合理,这可能与中性紫色土上的烟草青枯病发生率较低有一定关系。     

生物有机肥防治烟草青枯病及对土壤微生物多样性的影响

为防治烟草青枯病,在邵武地区试验田里施用了一种生物有机肥,考察了该生物肥对烟草青枯病的防治效果,通过对土壤样本的高通量宏基因组测序,研究了该生物肥对烟草根际土壤微生物多样性的影响以及与防治青枯病的初步关系。分析结果表明,施用生物有机肥的试验处理,烟株青枯病病情指数在3次观测中均较对照低,尤其在移栽后95 d时,青枯病病情指数下降了31.43%,而且上等烟比例、最终产量等经济性状较对照高。对土壤样本测序后发现,生物有机肥的施用,提高了烟株根际土壤微生物多样性;不同处理下各样本的土壤细菌和真菌菌群大部分相同,但相对丰度有差别,这与青枯病的发生有着一定的关系。     

有机肥对水稻根际土壤中微生物和酶活性的影响

利用根际箱在红壤上研究了有机肥对水稻根际有效磷、根际微生物和土壤酶活性的影响。试验结果表明,有机肥明显地提高水稻根际和非根际土壤真菌、放线菌和细菌的数量及土壤有效磷的含量。根际土壤各类微生物的数量大于非根际土壤,表现出明显的根际效应。施用有机肥使根际效应增加,其效应为细菌 放线菌 真菌。有机肥还明显地促进水稻根际无机磷溶解菌和有机磷分解数量以及磷酸酶和脲酶的活性,同时对水稻根际土壤磷转化速率有明显的提高,从而加速了土壤养分的转化。     

烤烟根际土壤微生物对根系酚酸类物质的响应

【目的】烟草连作导致化感物质累积,探索化感物质中主要的酚酸类物质对根际土壤微生物的影响,可为克服烟草连作障碍提供理论依据。【方法】采用盆栽试验方法,将前期分离、鉴定出的烟草根系分泌物中主要酚酸类物质苯甲酸和3-苯丙酸接种到土壤中,模拟烟草多年连作土壤。试验设4个处理:对照(T0),向土壤中加入等量灭菌去离子水;添加苯甲酸3μg/kg土(T1);3-苯丙酸8μg/kg土(T2);同时添加苯甲酸3μg/kg土和3-苯丙酸8μg/kg土(T3),每处理5次重复。以MiSeq测序平台对根际土壤微生物进行高通量测序,探索其对根际土壤微生物的影响,同时采用荧光定量PCR法检测土壤中的茄科劳尔氏菌、短短芽孢杆菌、固氮菌、无机磷细菌、硅酸盐细菌等功能微生物及细菌和真菌的数量变化。【结果】T 1、 T 2处理土壤细菌OTUs(Operational Taxonomic Units)数目分别比对照T0降低了21.5%和17.0%,T3处理OTU数量低于T1和T2处理;T2处理土壤中优势微生物种群增多,结构平衡性降低,门上分类构成和微生物群落构成显著不同于对照。主成分分析与聚类分析显示,T2或T3处理土壤微生物聚类关系较近,都与T0处理较远;T3处理土壤中病原菌数量显著提高,拮抗菌、固氮菌、无机磷细菌、硅酸盐细菌、细菌和真菌数量显著减少,且减少幅度大于T1、T2处理。【结论】根系分泌物中主要酚酸类物质苯甲酸和3-苯丙酸均能明显改变根际土壤微生物区系,降低土壤微生群落多样性,显著增加有害微生物数量的同时大大降低有益微生物数量。两种酚类同时存在的危害效果远大于单一酚类。     

重迎茬对大豆根际土壤微生物数量的影响

通过对不同茬口、迎茬条件下施用不同有机肥的大豆根际土壤微生物数量进行测定,结果表明,重迎茬根际土壤中细菌数量明显低于正茬,真菌数量高于正茬,而放线菌数量在不同时期有很大变化。施入不同有机肥后,能明显增加土壤中细菌和放线菌的数量,真菌中青霉菌数量增多,致病菌数量减少。     

生物有机肥对樱桃生长及根际土壤生物学特征的影响

以一年生‘吉塞拉,樱桃苗为试材,通过盆栽试验,利用传统的平板计数法和现代的T-RFLP技术,研究有机肥(OF)和生物有机肥(BF)对樱桃根际土壤微生物数量、微生物量碳和氮、细菌群落结构多样性、土壤酶活性、樱桃生长及养分吸收的影响.结果表明:施用OF和BF后可不同程度地提高根际土壤中的微生物数量及微生物量碳、氮;同OF处理比,BF处理中的放线菌、真菌、微生物总量、微生物量碳和微生物量氮分别提高了115.65％,115.96％,48.3％,58.68％和23.72％,细菌数量差异未达显著水平.同对照相比,OF和BF的施用可提高根际土壤细菌群落结构的多样性及丰富度.同对照相比,OF提高了土壤脲酶、蔗糖酶活性.同OF相比,BF处理的脲酶、磷酸酶、蔗糖酶和多酚氧化酶活性显著提高.OF的施用可促进樱桃根系的生长,提高N和P的养分吸收量.同OF处理相比,BF处理根、茎和叶的生物量分别增加9.55％,9.98％和11.16％,N、P和K养分吸收量分别提高15.35％,21.06％和32.09％.由此可见,盆栽条件下,施用有机肥和生物有机肥均可有效改善樱桃根际土壤生态环境,促进樱桃生长和养分的吸收,但生物有机肥的施用效果明显优于有机肥.     

重迎茬对大豆根际土壤微生物数量的影响

通过对不同茬口、迎茬条件下施用不同有机肥的大豆根际土壤微生物数量进行测定，结果表明，重迎茬根际土壤中细菌数量明显低于正茬，真菌数量高于正茬，而放线菌数量在不同时期有很大变化。施入不同有机肥后，能明显增加土壤中细菌和放线菌的数量，真菌中青霉菌数量增多，致病菌数量减少。     

产脂肽菌株发酵生物有机肥的生物防治与促生作用研究

选取对番茄青枯菌有明显抑制作用的产脂肽菌株XZ-173,与有机肥混合发酵制成生物有机肥。通过温室盆栽试验,评价了该生物有机肥对番茄青枯病的防治效果及对番茄的促生效用。结果表明,生物有机肥能够显著降低青枯病发病率,相对防效达56.8%。与施用化肥和未添加XZ-173的有机肥处理相比,施用该生物有机肥能显著提高番茄植株叶绿素含量、番茄地上部和地下部生物量、根际土壤细菌和放线菌数量,显著降低根际土壤中真菌数量。生物有机肥的有益效果使其在作物种植中具有广阔的应用前景。     

Effects of bio-organic fertilizer plus soil amendment on the control of tobacco bacterial wilt and composition of soil bacterial communities

Tobacco bacterial wilt (TBW) is caused by Ralstonia solanacearum (R. solanacearum), a severe pathogenic agent with a wide host range. In this study, lime?+?ammonium bicarbonate (L?+?AB), organic fertilizer (OF), bio-organic fertilizer (BOF), and integrated treatment (L?+?AB?+?BOF) were assessed for the ability to control TBW and to influence the composition of native soil bacterial communities. The results showed that disease incidence of L?+?AB?+?BOF for two growth seasons in pot experiment was the lowest, with only 15.56 and 11.11 % at seasons 1 and 2, respectively. The integrated treatment could also significantly suppress TBW in the field, with a disease incidence of only 14.27 % compared with 35.41, 50.03, and 31.32 % in L?+?AB, OF, and BOF treatments, respectively. With application of the integrated treatment in pot and field experiments, the abundances of R. solanacearum were both significantly lower than those with other treatments. Denaturing gradient gel electrophoresis (DGGE) patterns showed that application of BOF significantly affected composition of bacterial communities of rhizosphere. The analysis of 454 sequencing data showed that application of integrated treatment recruited more beneficial bacteria than other treatments, such as Bacillus, Paenibacillus, Arthrobacter, and Streptomyces, while the abundance of Ralstonia with the integrated treatment was decreased. Overall, these results suggested that application of integrated agricultural management could effectively suppress bacterial wilt by affecting the composition of bacterial community and reducing the population of R. solanacearum.     

Evaluation of Bacillus-fortified organic fertilizer for controlling tobacco bacterial wilt in greenhouse and field experiments

Bacterial wilt caused by Ralstonia solanacearum is one of the most serious tobacco diseases worldwide, and no effective control measures are available to date. Three Bacillus isolates (Bacillus amyloliquefaciens SQR-7 and SQR-101 and Bacillus methylotrophicus SQR-29) were obtained from the rhizosphere soil of tobacco. These bacilli exhibited strong inhibition against R. solanacearum and produced indole acetic acid and siderophores. The three antagonistic strains were used to fortify organic fertilizers to produce bioorganic fertilizers (BOFs named for each isolate) for the control of tobacco bacterial wilt. The application of BOFs delayed wilt development and effectively decreased the disease incidence under both greenhouse and field conditions. The tobacco bacterial wilt control efficacy was 44.3%, 70.5%, and 85.1% using BOF101, BOF29, and BOF7 in the greenhouse. Although the control efficacies in the field were lower, the application of BOF7 still achieved 58.0% and 56.2% control efficacies in two years field experiments. The application of bioorganic fertilizer significantly (p < 0.001) repressed the pathogen R. solanacearum in soil in both pot and field experiments, though the abundance of R. solanacearum increased as during the growth period of the tobacco plants. In general, the populations of the antagonistic bacterial strains declined after soil application and as the tobacco plants grew; however, the density of SQR-7 and SQR-29 in the rhizosphere soil remained at a high level (≥10⁶ cfu/g) in the later growth stages. Additionally, the application of bioorganic fertilizers promoted tobacco growth and increased the leaf yield.     

施用石灰氮对烟株根际土壤微生物区系的影响

为研究施用石灰氮对烟株根际土壤微生物区系变化及其对烟草青枯病发病率的影响,分别在烟株不同生长期采集根部土壤进行16S/18S rDNA基因测序,以分析烟株生长过程中微生物数量、多样性以及群落结构的变化。结果表明,施用石灰氮后,在烟株生长的团棵期硝化螺旋菌门(Nitrospirae)、担子菌门(Basidiomycota)相对丰度相比对照分别增加了55.0%和105.6%,子囊菌门(Ascomycota)降低了20.2%,旺长期细菌的Sobs指数、Shannon指数和Chao指数分别增加了6.3%、3.1%和3.4%。PCA分析显示,施用石灰氮的处理与对照的根际土壤微生物区系结构从团棵期开始表现出差别,且随烟株生长差异逐渐增大。物种差异分析显示施用石灰氮的处理中鞘脂单胞菌属(Sphingomonas)、硝化螺旋菌属(Nitrospira)、黄杆菌属(Flavisolibacte)等与土壤氮循环有关的功能菌丰度显著增多。成熟期对青枯病发病率和烟叶产量等统计表明,施用石灰氮处理对烟草青枯病的防控率达到了49.9%,烟叶产量提高了11.7%。     

连续施用生物有机肥对烟草青枯病的防治效果

分离获得一株对烟草青枯病病原菌茄科劳尔氏菌(Ralstonia solanacearum,简称RS)具有较强拮抗能力的拮抗菌(SQR11)并制成生物有机肥,研究了连续施用该生物有机肥对烟草青枯病的防治效果。结合生理生化和16SrDNA技术鉴定,菌株SQR11被鉴定为解淀粉芽孢杆菌。施用该生物有机肥后第一季烟草青枯病的生物防治率达到47%以上,第二季为69%以上,第三季达到89%以上。第三批盆栽实验表明,当根际土中病原菌数量达到2×105cfu/g干土时,植株出现发病症状,随着病原菌数量的增加,发病症状加重。当根际土中拮抗菌活菌数量达到2×107cfu/g干土时,病原菌繁殖得到有效抑制,可有效阻止植株染病;若低于107cfu/g干土,则不能有效抑制病原菌增殖,植株表现发病症状。植株各组织内拮抗菌数量检测发现,未发病植株茎部拮抗细菌数量为4×104cfu/g(组织鲜重,下同)左右,而同处理中发病症状的植株茎部拮抗细菌数量仅为6×103cfu/g;相对应的病原菌数量分别为1.5×102cfu/g(健康植株)和3×103cfu/g(发病植株)。SQR11菌株制成的生物有机肥还具有较好的促生作用。总之,利用拮抗菌SQR11菌株制成的生物有机肥对烟草青枯病具有显著的生物防治作用,在根部进行大量定殖后可有效防止病原菌的侵入,能够获得显著的生防效果。     

Tobacco bacterial wilt can be biologically controlled by the application of antagonistic strains in combination with organic fertilizer

Bacterial wilt caused by Ralstonia solanacearum is one of the most serious tobacco diseases worldwide. Brevibacillus brevis (L-25) and Streptomyces rochei (L-9) with strong inhibitory effects on R. solanacearum in vitro were isolated from the rhizosphere of a healthy tobacco plant in a severely wilt-diseased field. Pot and field experiments were conducted to evaluate the biocontrol effect of the isolated antagonists alone and in combination with organic fertilizer. In pot experiment, the control efficacy was 92.3–100 % in the treatments applied with L-25 and L-9 alone or together with organic fertilizers. When bioorganic fertilizer containing L-9 and L-25 was applied to the soil in field condition, the control efficacies were 95.4 and 30.0 in the Anhui and Guizhou field plots, respectively. The counts of bacteria and actinomycetes in rhizosphere soil were significantly increased (p?≤?0.05) under all antagonist applications compared with CK (P_R). In contrast, fungal and R. solanacearum densities in the rhizosphere soil applied with antagonists were much lower than the CK (P_R) rhizosphere. Combined application of the two antagonists had better effect than single antagonist treatments. The antagonists were more effective when they were combined with organic fertilizer as compared with the antagonistic strains only. These results allow us to conclude that a combination of the biocontrol agents, L-25 and L-9, together with organic fertilizers can effectively control bacterial wilt by affecting soil microbial structure.     

不同施肥结构对酸性黄棕壤修复效果研究

利用3年田间定位试验,研究了不同施肥结构对酸性黄棕壤酸性、交换性能以及微生物区系的影响。结果表明:连年施用有机肥以及配施石灰和白云石粉均能显著降低土壤中交换性酸和交换性Al~(3+)的含量,提高土壤pH,且不同施肥结构对修复黄棕壤酸性的效果为有机肥配施石灰有机肥配施白云石粉有机肥;连年施用有机肥以及配施石灰主要提高了土壤中交换性Ca~(2+)含量,而配施白云石粉则主要提高了土壤中交换性Mg~(2+)含量;有机肥配施石灰和白云石粉均降低了土壤中真菌的数量,但提高了土壤中细菌/真菌比、放线菌/真菌比以及细菌和放线菌数量,改善了土壤微生物群落结构,增加了烟叶的产量和产值。     

Application of Bio-organic Fertilizer to Control Tomato Fusarium Wilting by Manipulating Soil Microbial Communities and Development

A pot culture experiment was conducted in soils with a 3-, 5-, or 10-year history of continuous cropping. Results showed that with increasing years of continuous cropping, biomass, soil and plant analyzer development (SPAD) readings, root activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content gradually decreased. After bio-organic fertilizer (BOF) application, biomass, SPAD readings and root activity, SOD activity, and MDA content gradually increased compared to controls. Similarly, catalase (CAT) and guaiacol peroxidase (POD) activity decreased with increasing years of continuous cropping, and increased substantially in BOF-treated soil compared to the control soil. The diversity and richness of soil bacterial community declined, while those of fungal community increased as continuous cropping increased. BOF application led to substantial increases in quantities of soil rhizosphere bacteria and actinomycetes and significant decreases in quantities of soil fungi. The soil bacterial community showed the greatest changes, whereas the fungal community showed the fewest changes.     

生物有机肥育苗防控烟草青枯病

【目的】烟草青枯病是影响贵州烟叶生产的主要土传病害之一。生物防控方法作为防控烟草青枯病体系的有效措施之一,寻求经济可行的途径势在必行。【方法】利用实验室自主筛选到的烟草青枯菌拮抗菌NJL-14,通过二次固体发酵研制成烟草专用拮抗青枯病型生物有机肥 (BIO),选取K326和红花大金元两个烟草品种分别进行育苗和盆栽试验。育苗床基质分别添加BIO 1%、2%、3%、4%和5%,以确定适宜的BIO育苗添加量。盆栽试验两个品种分别设定五个处理:常规基质育苗 + 植烟健康土壤(CKn+CKp）、常规基质育苗?+?青枯病病土（CKn+Rs）、常规基质育苗?+?青枯病病土?+?有机肥(CKn+OFp)、常规基质育苗?+?青枯病病土?+?BIO(CKn+BIOp)、育苗和盆栽病土中都施入BIO(BIOn+BIOp)。分别采用荧光定量PCR和Biolog-ECO研究育苗基质和盆栽土壤中细菌和真菌数量变化,以及微生物功能多样性,特别是烟草青枯菌及其拮抗菌的消长关系。【结果】当BIO浓度在2%以内时,烟草的发芽率与对照无显著差异;与普通漂浮育苗相比,托盘育苗不但能稳定和延长拮抗菌NJL-14在根际的定殖,而且显著提高烟苗生物量,K326经BIO育苗处理的烟苗地上部和地下部的干重分别比常规基质育苗的处理高17.2%和30.8%,而红花大金元分别高14.9%和20.0%;采用育苗与移栽土壤双重使用BIO模式对烟草青枯病的防控效果明显,防控效果可以达到100%,明显优于只在盆栽中使用BIO模式,并且能够显著抑制土壤中病原菌数量在107 cfu/g土以下,同时有效提高土壤微生物生态多样性。【结论】采用育苗与移栽土壤双重使用烟草专用拮抗青枯病型生物有机肥模式可有效防控烟草青枯病,控制土壤中病原菌数量,改善土壤微生物功能多样性,为有效地生物防控烟草青枯病奠定基础。