稻田养鸭抑制水稻纹枯病机制及拮抗细菌A168筛选鉴定研究
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摘要
稻田养鸭不仅能除草、防虫、免耕、减少甲烷排放,提高水稻产量和品质,而且能减轻水稻纹枯病的发生和危害。本研究从两方面就稻田养鸭抑制水稻纹枯病进行探讨。一方面从鸭在稻田中的活动来研究其对水稻及水稻纹枯病菌的影响；另一方面从鸭嘴、鸭毛和鸭粪分泌物中提取粗提物和筛选微生物来研究其对水稻及水稻纹枯病菌的影响。本研究旨在通过系统研究稻田养鸭抑制水稻纹枯病的主要因素及作用机理,探索生物防治水稻纹枯病新途径。主要研究结果如下：
1影响因素及作用机理研究
1.1稻田养鸭对水稻纹枯病菌菌核的影响
在初始菌核相同的情况下,调查放鸭区和未放鸭区纹枯病菌菌核数量。在水稻生长的分蘖末期和齐穗期,放鸭区菌核量为35.7万粒／hm2、41.9万粒／hm2,比未放鸭区菌核72.9万粒／hm2、96.4万粒／hm2,低48.97%、43.46%,表明鸭通过啄食菌核,从而减少纹枯病菌源,减轻病害发生。
1.2封泥对水稻纹枯病的影响
将泥砌封于水稻基部叶鞘研究其对水稻纹枯病的影响。先砌封泥于水稻叶鞘再人工接种纹枯病菌,其发病率为3.08～10.61%、病情指数为5.42～11.82,明显低于井冈霉素处理和对照的发病率3.88～55.36%和3.97～86.22%、病情指数5.66～18.40和19.94～60.72,其控病效果为72.80～81.21%,优于井冈霉素处理和对照。对先砌封泥再接病菌的处理,不同时期测定水稻的过氧化物酶活性均比对照高,但低于井冈霉素处理；同时砌封泥处理使水稻的可溶性糖含量降低。
1.3鸭毛、鸭嘴和鸭粪粗提物对水稻几丁质酶活性的影响
一般认为,水稻抗病性与水稻几丁质酶活性呈正相关的。本研究采用乙酸乙酯和乙醇提取鸭毛、鸭嘴及鸭粪分泌物,并将粗提物施用于水稻,测定水稻几丁质酶活性。其中,鸭粪乙酸乙酯粗提物处理水稻后第8d水稻几丁质酶活性达最大值,为124.7 U·g(-1)·FW(-1)·min(-1),显著高于对照(77.11 U·g(-1)·FW(-1)·min(-1)),且差异显著(P<0.05,P<0.01)；鸭粪乙醇粗提物处理水稻后第8d几丁质酶活性达最大值62.9 U·g(-1)·FW(-1)·min(-1),与对照62.9U·g(-1)·FW(-1)·min(-1)比较无显著差异(P>0.05,P>0.01)；表明鸭粪乙酸乙酯粗提物能提高水稻几丁质酶活性,鸭粪乙醇提取物不能提高水稻几丁质酶活性。而鸭毛和鸭嘴乙酸乙酯、乙醇粗提物处理水稻后,水稻几丁质酶活性与对照比较也无显著变化。
1.4鸭毛、鸭嘴和鸭粪中微生物对水稻几丁质酶活性的影响
采用稀释分离法分别从鸭毛、鸭嘴和鸭粪中共分离出细菌323株,真菌126株。经初步归类,从鸭毛中分离获得1株细菌B106和1株真菌F123,鸭嘴中分离获得1株细菌B75和1株真菌F66,鸭粪中分离获得1株细菌B117和1株真菌F16,并用各菌株悬浮液处理水稻。细菌B117悬浮液处理水稻后,第3d、7d水稻几丁质酶活性达最大值,分别为42.24U·g(-1)·FW(-1)·min(-1)、42.23U·g(-1)·FW(-1)·min(-1),明显高于对照41.74U·g(-1)·FW(-1)·min(-1)、41.69 U·g(-1)·FW(-1)·min(-1),且差异显著(P<0.05,P<0.02),说明鸭粪中存在能提高水稻几丁质酶活性的细菌；其它菌株F16、B106、F123、B75、F66等菌悬液处理水稻后,水稻几丁质酶活性均无显著变化。
1.5鸭毛、鸭嘴和鸭粪粗提物对水稻纹枯病菌的影响
采用平板拮抗法测定鸭毛、鸭嘴和鸭粪乙醇、乙酸乙酯粗提物对水稻纹枯病菌的抑制作用。鸭毛、鸭嘴和鸭粪的乙醇、乙酸乙酯粗提物抑菌效果分别为13%、8%、6%,7%、88.64%、84.11%；鸭粪乙醇粗提物在24h内对水稻纹枯病病菌ECso值为26.11mg／ml；鸭粪乙醇粗提物经萃取分离得到石油醚和乙酸乙酯两种组分,其中石油醚组分抑制效果较弱,乙酸乙酯组分抑制能力较强,对水稻纹枯病抑制率为89.70%,EC(50)值为15.52mg／ml；对乙酸乙酯组分进行硅胶柱层析分离得到5种流份,其中第2流份(L2)有显著抑菌活性,其在24h、48h内对水稻纹枯病抑制率分别为89.58%、80.36%,通过GC-MS分析鉴定,为酚酸类和硫醇类物质。本研究表明：稻田养鸭生态系统中鸭分泌物中存在抑制水稻纹枯病菌的物质。
1.6鸭毛、鸭嘴和鸭粪中微生物对水稻纹枯病菌的影响
采用平板对峙法将菌株B106、B75、B117,F123、F66,F16进行拮抗试验。菌株B106、B75,F123、F66,F16均无拮抗作用,而来自鸭粪中的菌株B117对水稻纹枯病菌具有较强的拮抗作用,进一步分离纯化得菌株A168。稻田养鸭生态系统中鸭分泌物中存在抑制水稻纹枯病菌的拮抗菌。
2鸭粪中拮抗细菌A168筛选、鉴定、生物学特性及拮抗作用研究
2.1鸭粪中拮抗细菌A168筛选、鉴定及生物学特性
采用平板拮抗法,将细菌B117纯化100次,获得拮抗菌株A168,对其进行了鉴定和生物学特性研究。A168菌体杆状,革兰氏染色阳性、芽孢中生、周生鞭毛。A168菌株能利用葡萄糖、阿拉伯糖、木糖和甘露醇产酸,葡萄糖产气试验阴性,淀粉水解阴性,硝酸还原试验阳性,厌氧生长阴性,酪素分解试验阳性,酪氨酸分解试验阴性,接触酶试验阳性,VP反应阳性,苯丙氨酸脱氨酶试验阴性。16S rDNA分析与其亲缘关系较近菌株Bacillus cereus (AJ577288.1)的同源性达99%,初步鉴定A168菌株为蜡状芽孢杆菌(Bacillus cereus),其16SrDNA序列已在GenBank中注册,登录号为GQ118339。A168菌株最优培养时间60h,最适温度28℃、最适pH 7.0。A168菌株能利用的碳源有蔗糖、葡萄糖、甘露醇、α-乳糖、D-木糖、麦芽糖、D-半乳糖、D-果糖；能利用的氮源有胰蛋白胨、蛋白胨、牛肉浸膏和酵母浸膏、干酪素,氯化铵等,但不能利用硝酸钾。
2.2 A168菌株活性物质的提取条件及抑菌机理研究
采用乙酸乙酯以物料比4：1,在28℃条件下连续超声萃取36h提取A168菌株活性物质效果最好。A168菌株活性物质对水稻纹枯病菌(R.solani)的ECso和EC90值分别为2.15mg／mL和237.86mg／mL；经处理的水稻纹枯病菌菌丝扭曲；当浓度为35mg／mL时,可溶性蛋白质的合成抑制率最大为24.45%,菌核萌发率最低为80.36%；水稻纹枯病菌致病力随活性物质浓度的提高而降低,当浓度为45mg／mL时,纹枯病菌被抑制率为80.29%；A168菌株活性物质对病原菌菌丝体电导率无影响。A168菌株活性物质可通过抑制菌丝生长、蛋白质合成、菌核萌发、降低病菌致病力等途径达到控制纹枯病发生。
2.3 A168菌株最优培养基及发酵条件研究
通过正交试验确定菌株A168的最优发酵培养基配方及发酵条件：豆饼粉2%,葡萄糖7%,CaCO3 0.4%,NaCL 0.35%,蛋白胨6%。摇瓶最优发酵条件：培养时间48h,温度28℃,菌龄24h,接种量6%,转速为200r／min,装液量为210ml(摇瓶500mL),pH值7.0。10L发酵罐最优发酵条件：通气量为180L／h,转速为200r／min,温度为28℃,初始pH为6.5,最优发酵时间为72h。
2.4 A168菌株在水稻上定殖规律及对水稻纹枯病田间防治效果研究
采用回收标记菌株法探索了A168菌株定殖规律及田间防效。以摩擦方式定殖A168菌量最多；在分蘖期菌量最大,水稻齐穗期次之,成熟期最少；水稻基部叶鞘定殖的菌量高于根部和茎部；在接种后32d仍能够回收到活菌；田间防效达78.47%,明显高于井冈霉素。表明A168菌株在水稻上定殖能力强防病效果较好。
2.5 A168菌株促生、抗病及增产效果研究
经A168菌悬液处理水稻幼苗比对照根长、株高、叶长和鲜重分别增长12.35～30.95%、26.52～30.77%、16.37～20.02%和10.26～29.52%；且对"Lemont"“陆两优996”品种的促生效果比“创丰1号”好；经A168菌悬液处理的水稻"Lemont"内吲哚乙酸(IAA)、玉米素核苷(ZR4)、赤霉素(GA3)的含量比对照高,脱落酸(ABA)的含量比对照低；苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活性比对照高,但过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性与对照比较无显著变化；经A168菌悬液处理后的水稻产量比对照高,其中“陆两优996"品种增产最大为13.13%,其次为"Lemont"品种,“创丰1号”品种增产最低。本研究结果表明：A168菌株具有促进水稻生长,提高水稻抗病性及增加水稻产量的作用。
2.6 A168菌株活性物质的分离、纯化及鉴定
采用生化反应、硫酸铵沉淀、DEAE-SepharoseFF、FPLC Phenyl FF疏水色谱柱、HPLC C18液相色谱纯化等方法研究A168菌株活性物质成分并进行了分离、纯化及鉴定。初步鉴定A168过滤液中含有蛋白质和肽,进一步分离、纯化获得拮抗纹枯病菌L-518样品。通过SDS-PAGE电泳可知,L-518样品约有15KD,再经质谱分析其质荷比为15006.899。L-518样品不耐高温,对蛋白酶K稳定,对胰蛋白酶和胃蛋白酶不稳定,这些性质与多肽基本相似,初步鉴定为多肽类物质。
Rice-duck Farming Ecosystem (RDFE) is effective in controlling weeds and pests, reducing methane emission in paddy, and has advantages of zero-tillage, enhancing rice yield and quality. It is reported that RDFE is effectual in controlling Rhizoctonia solani(R.solani). The research was divided into two aspects to explore mechanism of RDFE restricting R.solani. On one hand, the influence of duck movements on rice and R. solani was researched, on the other hand, the effects of extractions and microbes from duck beak, excrement, feather on rice and R.solani were also studied. Both researches were aimed at exploring the factors and mechanisms of RDFE controlling R.solani and pursueing new approaches in R.solani controlling.
1 Research on effective factors and mechanisms
1.1 Effects of RDFE on the number of R.solani sclerotium
The number of R.solani sclerotium was investigated in both Duck-raised and non-duck-raised paddies where initial number of R.solani sclerotium were equal. During late tillering stage and full heading stage of rice, the sclerotium number were 357,000 pieces/hm2 and 419,000pieces/hm2 in Duck-raised paddies which were 48.97%and 43.46%lower than non-duck-raised paddies with the number of 729,000 pieces/hm2 and 964,000pieces/hm2. This proved that the reduction number of pathogenic bacteria and the disease controlling were contributed by duck movements.
1.2 Effect of closing mud on R.solani
In order to learn the effects of closing mud on R.solani, the research was carried out by closing mud on the surface of basal leaf sheath of rice. Mud was closed manually on the surface of basal leaf sheath and then R.solani was inoculated, the morbidity was between 3.08～10.61%, disease index was 5.42-11.82 after the treatment, which was significantly lower than Validamycin treatment (3.88～55.36%, 5.66～18.40) and blank control (3.97～86.22%,19.94～60.72). And the control efficiency was 72.80～81.21%. Peroxidase activities were significant higher than blank control but lower than Validamycin treatment in the treatment of mud closing then inoculation, which also lowered soluble sugar content but there was no significant difference among the treatments.
1.3 Effects of extractions from duck beak, excrement, feather on chitinase activity
Previous research has showed that there was positive correlation between disease resistance of rice and its chitinase activity. Compounds that were applied on rice were extracted from duck beak, excrement and feather by ethylacetale and carbinol, and then chitinase activity of rice was tested. Chitinase activity got to the maximum of 124.7 U·g-1·FW-1·min-1 on the 8th day after treated by compound extracted from duck excrement by ethylacetale, significantly higher than blank control(77.11 U·g-1·FW-1·min-1) (p<0.05, p<0.01); Chitinase activity got to the maximum of 62.9 U·g-1·FW-1·min-1 on the 8th day after treated by compound extracted from duck excrement by carbinol, which had no significant difference with 62.9U·g-1·FW-1·min-1 of blank control (p>0.05, p>0.01); This proved that the enhancement of chitinase activity was contributed by the compound extracted from duck excrement by ethylacetale, presumably that the containment of certain somatomedin and compounds could trigger rice disease resistance. While, there were no significance found from the same operations of duck feather and beak.
1.4 Effects of microbes separated from duck feather, beak and excrement on chitinase activity
323 strains of bacteria and 126 strains of fungi were separated from duck feather, beak and excrement by dilution plate method, bacteria B106 and fungi F123 were from duck feather, B75 and fungi F66 were from duck beak, bacteria B117 and fungi F16 were from duck excrement, whose suspensions were treated on rice respectively. Chitinase activity got to maximum of 42.24U·g-1·FW-1·min-1、42.23 U·g-1·FW-1·min-1 at the 3rd and 7th day after B117 treatment, significantly higher than CK 41.74U·g-1·FW-1·min-1、41.69 U·g-1·FW-1·min-1(p<0.05,p<0.01). This proved that microbes from duck excrement could enhance chitinase activity, while others not.
1.5 Effects of extraction from duck feather, beak and excrement on R.solani
Control efficiencies of extractions from duck beak, feather and excrement by ethylacetale and ethanol were tested on R.solani via plate antagonism method, the inhibition effect of which were 13%,8%,6%,7%,88.64%,84.11%respectively; EC50 of excrement-ethanol treatment in 24h was 26.11mg/ml; Crude extraction of duck excrement was separated into petroleum-extraction and ethylacetale-extraction, and the ethylacetale-extraction got relatively higher control efficiency,89.0%, with EC50 of 15.52mg/ml, and the petroleum-extraction got relatively lower control efficiency; Ethylacetale-extraction were filtered and separated by means of silica gel column chromatography and 5 components were acquired, and the L2 fraction had significant control efficiency, with 89.58%and 80.36%in 24h and 48h, which was identified as phenolic acid and mercaptan via GC-MS. This proved that compounds existed in duck secretion could control R.solani in RDFE.
1.6 Effect of microbes from duck feather, beak and excrement on R.solani
Antagonisms of B106, B75, B117, F123, F66 and F16 against R.solani were tested via plate-confront method. No antagonism were found in strains of B106, B75, F123, F66 and F16, while B117 isolated from duck excrement showed high antagonism against R.solani, and A168 strain was acquired via further screening of B117. This proved that antagonistic bacteria could be isolated from duck secretion in RDFE.
2. Research on screening, identification, biological property and antagonism of A168
2.1 Research on screening, identification and biological property of A168
By purifying B117 for 100 times via plate-antagonistic method, A168 strain was selected, identified and biologically characterized. A168 was rhabditiform, Gram-positive, central spore, peritrichate. It can utilize Glucose, Arabinose, Xylose and mannitol to produce acid, and is negative in Glucose gas producing test, Amylohydrolysis test, anaerobic growth test, Tyr degradation test, Phenylalanine deaminase test, while positive in Hydrogen nitrate reduction test, Casein degradation test, Catelase test and VP reaction test. It had homology of 99% with its consanguinity Bacillus according to 16S rDNA analysis.16S rDNA sequence was registered in GenBank with the accession number of GQ118339. Population of A168 had maximum in 60h cultivation, its optimum temperature was 28℃, and it lived well at pH7.0. Sucrose, Glucose, Mannitol, a-lactose, D-xylose, maltose, D-galactose and D-fructose could be utilized as carbon source, and tryptone, peptone, beef extract, yeast extract, casein and salmiac could be utilized as nitrogen source by A168.
2.2 Research on process of active materials extraction and fungistatic mechanism of A168
Optimum extraction process were worked out:ratio of ethylacetate and zymotic fluid was 4:1, temperature 28℃,36h constantly ultrasound extraction. EC50 and EC90 of extracted active material against R.solani were 2.15mg/mL and 237.86mg/mL; mycelium twisted after active material treatment; Inhibition ratio of soluble protein had maximum of 24.45% and germination ratio of sclerotium had minimum of 80.36% at treatment concentration of 35mg/mL; Pathogenicity of R.solani decreased along with increasing concentration of active material, and inhibition ratio reached to 80.29% at concentration of 45mg/ml; Effect of active material on electrical conductivity of mycelium was not found yet. This proved that active material of A168 could control R.solani by inhibiting mycelium growth, protein synthesis and sclerotium germination, as well as reducing pathogenicity.
2.3 Research on fermentation condition and optimum substrate for A168
Optimum fermentation and cultural condition were researched via orthogonal test:bean-cake powder 2%, glucose 7%, CaCO3 0.4%, NaCl 0.35%, peptone 6%. Optimum in vibrating flask:48h,28℃, cell age 24h, inoculation volume 6%, rotate speed 200r/min, liquid capacity 210ml (500ml in vibrating flask), pH7.0. Optimum in lOL-fermentor:VC 180L/h, rotate speed 200r/min,28℃, nascent pH6.5, duration 72h.
2.4 Research on colonizing regulation and control efficiency of A168 on rice
Colonizing regulation and field control efficiency of A168 were explored via strain recycling marked method. Friction was optimum for colonizing; A168 had largest population at tillering stage in rice after inoculation, then full headin stage and mature period had smallest; Basal sheath had denser population than root and stem; Live strain could be obtained after 32 days of inoculation; Field control efficiency reached to 78.47%, significant higher than Validamycin treatment.This proved that A168 had a higher colonizing capacity and control efficiency on rice.
2.5 Research on growth promotion, disease resistance and yield improvement effects of A168
After treated by A168 suspensions, root length, plant height, leaf length and fresh weight of rice seedling were respectively increased by 12.35～30.95%、26.52～30.77%、16.37～20.02%and 10.26～29.52%, compared to blank control; A168 had better growth promotion effects in rice variety "Lemont" and "Luliangyou996" than in "ChuangfengNo.1"; Compared to blank control, "Lemont" had higher content of IAA, ZR4, GA3 and lower content of ABA, and had higher activity of PAL, POD but no significantly different activity of CAT and APX after treatment of A68 suspensions; A168 suspensions increased rice yield, with increasing rate of 13.13% on "Luliangyou 996", and then "Lemont" and "Chuangfeng No.1". These proved that A168 had effect of promoting growth, improving disease resistance and yield on rice.
2.6 Research on screening, purification and identification of active materials in A168
Chemical reaction, Ammonium sulfate precipitation, DEAE-SepharoseFF and FPLC Phenyl FF lyophobic chromatographic column, HPLC C18-purification were used in order to study, isolate, purificate and identificate the active materials in A168. Protein and peptide were isolated from filtrate of A168, and such active material was named L-158, and then contributed to further separation and purification. Via SDS-PAGE electrophoresis analysis,15KD of L-518 was found; Via mass spectrum analysis, L-518 was identified to have charge-to-mass ration of 15006.899. Sample L-518 was not well resistant to high temperatures, and stable when exposed to protease K while instable to trypsase and pepsase. Considered these characters were similar to polypeptide, L-518 was presumably identified to be polypeptide.
1影响因素及作用机理研究
1.1稻田养鸭对水稻纹枯病菌菌核的影响
在初始菌核相同的情况下,调查放鸭区和未放鸭区纹枯病菌菌核数量。在水稻生长的分蘖末期和齐穗期,放鸭区菌核量为35.7万粒／hm2、41.9万粒／hm2,比未放鸭区菌核72.9万粒／hm2、96.4万粒／hm2,低48.97%、43.46%,表明鸭通过啄食菌核,从而减少纹枯病菌源,减轻病害发生。
1.2封泥对水稻纹枯病的影响
将泥砌封于水稻基部叶鞘研究其对水稻纹枯病的影响。先砌封泥于水稻叶鞘再人工接种纹枯病菌,其发病率为3.08～10.61%、病情指数为5.42～11.82,明显低于井冈霉素处理和对照的发病率3.88～55.36%和3.97～86.22%、病情指数5.66～18.40和19.94～60.72,其控病效果为72.80～81.21%,优于井冈霉素处理和对照。对先砌封泥再接病菌的处理,不同时期测定水稻的过氧化物酶活性均比对照高,但低于井冈霉素处理；同时砌封泥处理使水稻的可溶性糖含量降低。
1.3鸭毛、鸭嘴和鸭粪粗提物对水稻几丁质酶活性的影响
一般认为,水稻抗病性与水稻几丁质酶活性呈正相关的。本研究采用乙酸乙酯和乙醇提取鸭毛、鸭嘴及鸭粪分泌物,并将粗提物施用于水稻,测定水稻几丁质酶活性。其中,鸭粪乙酸乙酯粗提物处理水稻后第8d水稻几丁质酶活性达最大值,为124.7 U·g(-1)·FW(-1)·min(-1),显著高于对照(77.11 U·g(-1)·FW(-1)·min(-1)),且差异显著(P<0.05,P<0.01)；鸭粪乙醇粗提物处理水稻后第8d几丁质酶活性达最大值62.9 U·g(-1)·FW(-1)·min(-1),与对照62.9U·g(-1)·FW(-1)·min(-1)比较无显著差异(P>0.05,P>0.01)；表明鸭粪乙酸乙酯粗提物能提高水稻几丁质酶活性,鸭粪乙醇提取物不能提高水稻几丁质酶活性。而鸭毛和鸭嘴乙酸乙酯、乙醇粗提物处理水稻后,水稻几丁质酶活性与对照比较也无显著变化。
1.4鸭毛、鸭嘴和鸭粪中微生物对水稻几丁质酶活性的影响
采用稀释分离法分别从鸭毛、鸭嘴和鸭粪中共分离出细菌323株,真菌126株。经初步归类,从鸭毛中分离获得1株细菌B106和1株真菌F123,鸭嘴中分离获得1株细菌B75和1株真菌F66,鸭粪中分离获得1株细菌B117和1株真菌F16,并用各菌株悬浮液处理水稻。细菌B117悬浮液处理水稻后,第3d、7d水稻几丁质酶活性达最大值,分别为42.24U·g(-1)·FW(-1)·min(-1)、42.23U·g(-1)·FW(-1)·min(-1),明显高于对照41.74U·g(-1)·FW(-1)·min(-1)、41.69 U·g(-1)·FW(-1)·min(-1),且差异显著(P<0.05,P<0.02),说明鸭粪中存在能提高水稻几丁质酶活性的细菌；其它菌株F16、B106、F123、B75、F66等菌悬液处理水稻后,水稻几丁质酶活性均无显著变化。
1.5鸭毛、鸭嘴和鸭粪粗提物对水稻纹枯病菌的影响
采用平板拮抗法测定鸭毛、鸭嘴和鸭粪乙醇、乙酸乙酯粗提物对水稻纹枯病菌的抑制作用。鸭毛、鸭嘴和鸭粪的乙醇、乙酸乙酯粗提物抑菌效果分别为13%、8%、6%,7%、88.64%、84.11%；鸭粪乙醇粗提物在24h内对水稻纹枯病病菌ECso值为26.11mg／ml；鸭粪乙醇粗提物经萃取分离得到石油醚和乙酸乙酯两种组分,其中石油醚组分抑制效果较弱,乙酸乙酯组分抑制能力较强,对水稻纹枯病抑制率为89.70%,EC(50)值为15.52mg／ml；对乙酸乙酯组分进行硅胶柱层析分离得到5种流份,其中第2流份(L2)有显著抑菌活性,其在24h、48h内对水稻纹枯病抑制率分别为89.58%、80.36%,通过GC-MS分析鉴定,为酚酸类和硫醇类物质。本研究表明：稻田养鸭生态系统中鸭分泌物中存在抑制水稻纹枯病菌的物质。
1.6鸭毛、鸭嘴和鸭粪中微生物对水稻纹枯病菌的影响
采用平板对峙法将菌株B106、B75、B117,F123、F66,F16进行拮抗试验。菌株B106、B75,F123、F66,F16均无拮抗作用,而来自鸭粪中的菌株B117对水稻纹枯病菌具有较强的拮抗作用,进一步分离纯化得菌株A168。稻田养鸭生态系统中鸭分泌物中存在抑制水稻纹枯病菌的拮抗菌。
2鸭粪中拮抗细菌A168筛选、鉴定、生物学特性及拮抗作用研究
2.1鸭粪中拮抗细菌A168筛选、鉴定及生物学特性
采用平板拮抗法,将细菌B117纯化100次,获得拮抗菌株A168,对其进行了鉴定和生物学特性研究。A168菌体杆状,革兰氏染色阳性、芽孢中生、周生鞭毛。A168菌株能利用葡萄糖、阿拉伯糖、木糖和甘露醇产酸,葡萄糖产气试验阴性,淀粉水解阴性,硝酸还原试验阳性,厌氧生长阴性,酪素分解试验阳性,酪氨酸分解试验阴性,接触酶试验阳性,VP反应阳性,苯丙氨酸脱氨酶试验阴性。16S rDNA分析与其亲缘关系较近菌株Bacillus cereus (AJ577288.1)的同源性达99%,初步鉴定A168菌株为蜡状芽孢杆菌(Bacillus cereus),其16SrDNA序列已在GenBank中注册,登录号为GQ118339。A168菌株最优培养时间60h,最适温度28℃、最适pH 7.0。A168菌株能利用的碳源有蔗糖、葡萄糖、甘露醇、α-乳糖、D-木糖、麦芽糖、D-半乳糖、D-果糖；能利用的氮源有胰蛋白胨、蛋白胨、牛肉浸膏和酵母浸膏、干酪素,氯化铵等,但不能利用硝酸钾。
2.2 A168菌株活性物质的提取条件及抑菌机理研究
采用乙酸乙酯以物料比4：1,在28℃条件下连续超声萃取36h提取A168菌株活性物质效果最好。A168菌株活性物质对水稻纹枯病菌(R.solani)的ECso和EC90值分别为2.15mg／mL和237.86mg／mL；经处理的水稻纹枯病菌菌丝扭曲；当浓度为35mg／mL时,可溶性蛋白质的合成抑制率最大为24.45%,菌核萌发率最低为80.36%；水稻纹枯病菌致病力随活性物质浓度的提高而降低,当浓度为45mg／mL时,纹枯病菌被抑制率为80.29%；A168菌株活性物质对病原菌菌丝体电导率无影响。A168菌株活性物质可通过抑制菌丝生长、蛋白质合成、菌核萌发、降低病菌致病力等途径达到控制纹枯病发生。
2.3 A168菌株最优培养基及发酵条件研究
通过正交试验确定菌株A168的最优发酵培养基配方及发酵条件：豆饼粉2%,葡萄糖7%,CaCO3 0.4%,NaCL 0.35%,蛋白胨6%。摇瓶最优发酵条件：培养时间48h,温度28℃,菌龄24h,接种量6%,转速为200r／min,装液量为210ml(摇瓶500mL),pH值7.0。10L发酵罐最优发酵条件：通气量为180L／h,转速为200r／min,温度为28℃,初始pH为6.5,最优发酵时间为72h。
2.4 A168菌株在水稻上定殖规律及对水稻纹枯病田间防治效果研究
采用回收标记菌株法探索了A168菌株定殖规律及田间防效。以摩擦方式定殖A168菌量最多；在分蘖期菌量最大,水稻齐穗期次之,成熟期最少；水稻基部叶鞘定殖的菌量高于根部和茎部；在接种后32d仍能够回收到活菌；田间防效达78.47%,明显高于井冈霉素。表明A168菌株在水稻上定殖能力强防病效果较好。
2.5 A168菌株促生、抗病及增产效果研究
经A168菌悬液处理水稻幼苗比对照根长、株高、叶长和鲜重分别增长12.35～30.95%、26.52～30.77%、16.37～20.02%和10.26～29.52%；且对"Lemont"“陆两优996”品种的促生效果比“创丰1号”好；经A168菌悬液处理的水稻"Lemont"内吲哚乙酸(IAA)、玉米素核苷(ZR4)、赤霉素(GA3)的含量比对照高,脱落酸(ABA)的含量比对照低；苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活性比对照高,但过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性与对照比较无显著变化；经A168菌悬液处理后的水稻产量比对照高,其中“陆两优996"品种增产最大为13.13%,其次为"Lemont"品种,“创丰1号”品种增产最低。本研究结果表明：A168菌株具有促进水稻生长,提高水稻抗病性及增加水稻产量的作用。
2.6 A168菌株活性物质的分离、纯化及鉴定
采用生化反应、硫酸铵沉淀、DEAE-SepharoseFF、FPLC Phenyl FF疏水色谱柱、HPLC C18液相色谱纯化等方法研究A168菌株活性物质成分并进行了分离、纯化及鉴定。初步鉴定A168过滤液中含有蛋白质和肽,进一步分离、纯化获得拮抗纹枯病菌L-518样品。通过SDS-PAGE电泳可知,L-518样品约有15KD,再经质谱分析其质荷比为15006.899。L-518样品不耐高温,对蛋白酶K稳定,对胰蛋白酶和胃蛋白酶不稳定,这些性质与多肽基本相似,初步鉴定为多肽类物质。
Rice-duck Farming Ecosystem (RDFE) is effective in controlling weeds and pests, reducing methane emission in paddy, and has advantages of zero-tillage, enhancing rice yield and quality. It is reported that RDFE is effectual in controlling Rhizoctonia solani(R.solani). The research was divided into two aspects to explore mechanism of RDFE restricting R.solani. On one hand, the influence of duck movements on rice and R. solani was researched, on the other hand, the effects of extractions and microbes from duck beak, excrement, feather on rice and R.solani were also studied. Both researches were aimed at exploring the factors and mechanisms of RDFE controlling R.solani and pursueing new approaches in R.solani controlling.
1 Research on effective factors and mechanisms
1.1 Effects of RDFE on the number of R.solani sclerotium
The number of R.solani sclerotium was investigated in both Duck-raised and non-duck-raised paddies where initial number of R.solani sclerotium were equal. During late tillering stage and full heading stage of rice, the sclerotium number were 357,000 pieces/hm2 and 419,000pieces/hm2 in Duck-raised paddies which were 48.97%and 43.46%lower than non-duck-raised paddies with the number of 729,000 pieces/hm2 and 964,000pieces/hm2. This proved that the reduction number of pathogenic bacteria and the disease controlling were contributed by duck movements.
1.2 Effect of closing mud on R.solani
In order to learn the effects of closing mud on R.solani, the research was carried out by closing mud on the surface of basal leaf sheath of rice. Mud was closed manually on the surface of basal leaf sheath and then R.solani was inoculated, the morbidity was between 3.08～10.61%, disease index was 5.42-11.82 after the treatment, which was significantly lower than Validamycin treatment (3.88～55.36%, 5.66～18.40) and blank control (3.97～86.22%,19.94～60.72). And the control efficiency was 72.80～81.21%. Peroxidase activities were significant higher than blank control but lower than Validamycin treatment in the treatment of mud closing then inoculation, which also lowered soluble sugar content but there was no significant difference among the treatments.
1.3 Effects of extractions from duck beak, excrement, feather on chitinase activity
Previous research has showed that there was positive correlation between disease resistance of rice and its chitinase activity. Compounds that were applied on rice were extracted from duck beak, excrement and feather by ethylacetale and carbinol, and then chitinase activity of rice was tested. Chitinase activity got to the maximum of 124.7 U·g-1·FW-1·min-1 on the 8th day after treated by compound extracted from duck excrement by ethylacetale, significantly higher than blank control(77.11 U·g-1·FW-1·min-1) (p<0.05, p<0.01); Chitinase activity got to the maximum of 62.9 U·g-1·FW-1·min-1 on the 8th day after treated by compound extracted from duck excrement by carbinol, which had no significant difference with 62.9U·g-1·FW-1·min-1 of blank control (p>0.05, p>0.01); This proved that the enhancement of chitinase activity was contributed by the compound extracted from duck excrement by ethylacetale, presumably that the containment of certain somatomedin and compounds could trigger rice disease resistance. While, there were no significance found from the same operations of duck feather and beak.
1.4 Effects of microbes separated from duck feather, beak and excrement on chitinase activity
323 strains of bacteria and 126 strains of fungi were separated from duck feather, beak and excrement by dilution plate method, bacteria B106 and fungi F123 were from duck feather, B75 and fungi F66 were from duck beak, bacteria B117 and fungi F16 were from duck excrement, whose suspensions were treated on rice respectively. Chitinase activity got to maximum of 42.24U·g-1·FW-1·min-1、42.23 U·g-1·FW-1·min-1 at the 3rd and 7th day after B117 treatment, significantly higher than CK 41.74U·g-1·FW-1·min-1、41.69 U·g-1·FW-1·min-1(p<0.05,p<0.01). This proved that microbes from duck excrement could enhance chitinase activity, while others not.
1.5 Effects of extraction from duck feather, beak and excrement on R.solani
Control efficiencies of extractions from duck beak, feather and excrement by ethylacetale and ethanol were tested on R.solani via plate antagonism method, the inhibition effect of which were 13%,8%,6%,7%,88.64%,84.11%respectively; EC50 of excrement-ethanol treatment in 24h was 26.11mg/ml; Crude extraction of duck excrement was separated into petroleum-extraction and ethylacetale-extraction, and the ethylacetale-extraction got relatively higher control efficiency,89.0%, with EC50 of 15.52mg/ml, and the petroleum-extraction got relatively lower control efficiency; Ethylacetale-extraction were filtered and separated by means of silica gel column chromatography and 5 components were acquired, and the L2 fraction had significant control efficiency, with 89.58%and 80.36%in 24h and 48h, which was identified as phenolic acid and mercaptan via GC-MS. This proved that compounds existed in duck secretion could control R.solani in RDFE.
1.6 Effect of microbes from duck feather, beak and excrement on R.solani
Antagonisms of B106, B75, B117, F123, F66 and F16 against R.solani were tested via plate-confront method. No antagonism were found in strains of B106, B75, F123, F66 and F16, while B117 isolated from duck excrement showed high antagonism against R.solani, and A168 strain was acquired via further screening of B117. This proved that antagonistic bacteria could be isolated from duck secretion in RDFE.
2. Research on screening, identification, biological property and antagonism of A168
2.1 Research on screening, identification and biological property of A168
By purifying B117 for 100 times via plate-antagonistic method, A168 strain was selected, identified and biologically characterized. A168 was rhabditiform, Gram-positive, central spore, peritrichate. It can utilize Glucose, Arabinose, Xylose and mannitol to produce acid, and is negative in Glucose gas producing test, Amylohydrolysis test, anaerobic growth test, Tyr degradation test, Phenylalanine deaminase test, while positive in Hydrogen nitrate reduction test, Casein degradation test, Catelase test and VP reaction test. It had homology of 99% with its consanguinity Bacillus according to 16S rDNA analysis.16S rDNA sequence was registered in GenBank with the accession number of GQ118339. Population of A168 had maximum in 60h cultivation, its optimum temperature was 28℃, and it lived well at pH7.0. Sucrose, Glucose, Mannitol, a-lactose, D-xylose, maltose, D-galactose and D-fructose could be utilized as carbon source, and tryptone, peptone, beef extract, yeast extract, casein and salmiac could be utilized as nitrogen source by A168.
2.2 Research on process of active materials extraction and fungistatic mechanism of A168
Optimum extraction process were worked out:ratio of ethylacetate and zymotic fluid was 4:1, temperature 28℃,36h constantly ultrasound extraction. EC50 and EC90 of extracted active material against R.solani were 2.15mg/mL and 237.86mg/mL; mycelium twisted after active material treatment; Inhibition ratio of soluble protein had maximum of 24.45% and germination ratio of sclerotium had minimum of 80.36% at treatment concentration of 35mg/mL; Pathogenicity of R.solani decreased along with increasing concentration of active material, and inhibition ratio reached to 80.29% at concentration of 45mg/ml; Effect of active material on electrical conductivity of mycelium was not found yet. This proved that active material of A168 could control R.solani by inhibiting mycelium growth, protein synthesis and sclerotium germination, as well as reducing pathogenicity.
2.3 Research on fermentation condition and optimum substrate for A168
Optimum fermentation and cultural condition were researched via orthogonal test:bean-cake powder 2%, glucose 7%, CaCO3 0.4%, NaCl 0.35%, peptone 6%. Optimum in vibrating flask:48h,28℃, cell age 24h, inoculation volume 6%, rotate speed 200r/min, liquid capacity 210ml (500ml in vibrating flask), pH7.0. Optimum in lOL-fermentor:VC 180L/h, rotate speed 200r/min,28℃, nascent pH6.5, duration 72h.
2.4 Research on colonizing regulation and control efficiency of A168 on rice
Colonizing regulation and field control efficiency of A168 were explored via strain recycling marked method. Friction was optimum for colonizing; A168 had largest population at tillering stage in rice after inoculation, then full headin stage and mature period had smallest; Basal sheath had denser population than root and stem; Live strain could be obtained after 32 days of inoculation; Field control efficiency reached to 78.47%, significant higher than Validamycin treatment.This proved that A168 had a higher colonizing capacity and control efficiency on rice.
2.5 Research on growth promotion, disease resistance and yield improvement effects of A168
After treated by A168 suspensions, root length, plant height, leaf length and fresh weight of rice seedling were respectively increased by 12.35～30.95%、26.52～30.77%、16.37～20.02%and 10.26～29.52%, compared to blank control; A168 had better growth promotion effects in rice variety "Lemont" and "Luliangyou996" than in "ChuangfengNo.1"; Compared to blank control, "Lemont" had higher content of IAA, ZR4, GA3 and lower content of ABA, and had higher activity of PAL, POD but no significantly different activity of CAT and APX after treatment of A68 suspensions; A168 suspensions increased rice yield, with increasing rate of 13.13% on "Luliangyou 996", and then "Lemont" and "Chuangfeng No.1". These proved that A168 had effect of promoting growth, improving disease resistance and yield on rice.
2.6 Research on screening, purification and identification of active materials in A168
Chemical reaction, Ammonium sulfate precipitation, DEAE-SepharoseFF and FPLC Phenyl FF lyophobic chromatographic column, HPLC C18-purification were used in order to study, isolate, purificate and identificate the active materials in A168. Protein and peptide were isolated from filtrate of A168, and such active material was named L-158, and then contributed to further separation and purification. Via SDS-PAGE electrophoresis analysis,15KD of L-518 was found; Via mass spectrum analysis, L-518 was identified to have charge-to-mass ration of 15006.899. Sample L-518 was not well resistant to high temperatures, and stable when exposed to protease K while instable to trypsase and pepsase. Considered these characters were similar to polypeptide, L-518 was presumably identified to be polypeptide.
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