绿色木霉复配有机肥的筛选及对黄瓜枯萎病的防治作用

单独应用绿色木霉控制蔬菜土传病害效果不理想,探索筛选适合的有机肥与绿色木霉复配,达到提高生防菌防病作用的目的。从供试的7种有机肥中,筛选出菇渣和牛粪适合与木霉菌复配。菇渣和牛粪的提取液对黄瓜枯萎病菌菌丝生长的抑制率分别为74.91%和69.38%,对绿色木霉菌丝生长抑制率分别为-7.90%(促进作用)和4.94%,使黄瓜种子胚根伸长增长率分别为36.75%和28.21%。菇渣、牛粪分别与绿色木霉混合后对黄瓜枯萎病的防效分别为64.98%和63.32%。菇渣和牛粪按1:1混合后再与绿色木霉复配制成菇渣、牛粪木霉有机肥的防效高达85.31%,黄瓜地上部分鲜重和地下部分鲜重分别增加了71.50%和79.55%。菇渣和牛粪与绿色木霉复配可显著提高木霉对黄瓜枯萎病的防病效果和对黄瓜生长的促进作用。     

一株抗药用白菊枯萎病生防菌的分离与生防效应研究

从生长健康的药菊根际土壤分离筛选获得一株对其连作土传枯萎病致病菌（尖孢镰刀菌菊专化型）具有较强拮抗作用的细菌a4,并通过盆钵和田间小区试验研究其生防效果。结果表明,拮抗菌a4对药菊枯萎病病原菌的抑制率达71%。盆栽试验,施用a4二次发酵的生物有机肥（CK+BOF1）,未发生药菊枯萎病,而对照植株则完全发病;对照植株根际土壤尖孢镰刀菌的数量为CK+BOF1处理的6.5倍。田间小区试验,苗床育苗和移栽时都施用BOF并加灌a4发酵液的处理（n+F+a4）,根际土和非根际土中病原菌的数量分别为2.0103和2.8103 cfu/g,而对照则分别为1.4104和1.2104 cfu/g。n+F+a4处理生长120 d的药菊生物量（干重）为对照的4.47倍,生防效果达93.2%。采用苗床育苗和移栽时都施用高效拮抗菌的方法,能有效防止枯萎病发生,促进药菊植株生长,提高产量。     

一株拮抗香蕉枯萎镰刀菌稀有放线菌的分离及鉴定

本研究采用苯酚、干热、SDS和超声波四种不同的预处理方法从五指山原始林区采集的土样中选择性分离得到的702株稀有放线菌,共筛选出4株具有拮抗香蕉枯萎镰刀菌活性的菌株,经过复筛,获得一株高活性的拮抗菌210-1-61。通过对其进行形态鉴定、生理生化特征鉴定和16SrDNA序列分析,结果表明该菌株与M．pattaloongensis JCM12833^T进化关系最近,其16SrDNA序列同源性最高,达98．89％;其形态与生理生化特性也与小单孢菌属M．pattaloongensis JCM12833^T很接近。此外,我们还构建了与该菌株同源性较高的模式菌株16SrDNA序列的聚类分析图,结果发现该菌株与Mpattaloongensis JCM12833^T稳定单独构成一个分支,二者亲缘关系最近,初步鉴定该菌株为Micrombonospora.pattaloongensis。本研究为今后探讨小单孢菌属稀有放线菌对香蕉尖孢镰刀菌具有拈抗活性提供研究实验材料。     

黄瓜与西芹间作土壤细菌多样性及其对黄瓜枯萎病发生的影响

本试验以黄瓜与西芹间作种植模式为处理,黄瓜单作和西芹单作种植模式为对照,利用Illumina公司Miseq平台对上述不同处理土壤进行16S rDNA细菌群落多样性高通量测序分析和田间接种黄瓜枯萎病菌,探讨黄瓜与西芹间作模式土壤细菌的多样性及其对田间黄瓜枯萎病发生的影响。16S rDNA测序结果表明,黄瓜与西芹间作土壤的细菌物种总数最多,群落多样性水平最高,与对照相比显著提高了土壤细菌observed species指数、Shannon指数和Chao1指数(P0.05);Beta多样性聚类分析表明,黄瓜与西芹间作土壤的环境群落物种与黄瓜单作和西芹单作有一定差异性。在门分类水平上,共检测到45个菌门,其中变形菌门占明显优势,其次为酸杆菌门和放线菌门等;黄瓜与西芹间作土壤细菌种类所占比例最高,达98.63%。在属水平上,共检测到428类菌属,GP6、GP16、GP4、芽单胞菌属、节细菌属5属的丰度值较大;黄瓜与西芹间作土壤的节细菌属分布比例最高,红游动菌属、鞘氨醇单胞菌属和芽球菌属丰度值较大,为黄瓜与西芹间作土壤细菌明显优势菌属。田间接种黄瓜枯萎病菌试验结果表明,采用上述3种不同种植模式土壤种植黄瓜,在黄瓜苗期接种黄瓜枯萎病菌,黄瓜与西芹间作处理的黄瓜枯萎病的田间发病率较西芹单作和黄瓜单作分别降低57.03%~63.54%和66.95%~72.15%。因此,黄瓜与西芹间作增加了土壤细菌群落多样性,降低了黄瓜枯萎病的发病率,对后茬黄瓜土传病害防控具有一定科学指导意义。     

黄瓜和西芹间作对黄瓜生长及枯萎病发生的影响

为研究黄瓜与西芹间作对黄瓜生长、黄瓜枯萎病菌化感作用及田间黄瓜枯萎病发生的影响,本试验采用室内不同土壤浸提液培养黄瓜枯萎病菌和田间测定黄瓜生长与枯萎病病情指数的方法,探讨与西芹间作土壤浸提液对黄瓜枯萎病菌的化感作用和田间防病效果。结果表明:与西芹间作促进了黄瓜营养生长,降低了第1雌花节位,增加了1~30节内雌花数;与西芹间作土壤浸提液培养的黄瓜枯萎病菌菌落直径最小,分别与西芹单作和黄瓜单作达显著性差异(P0.05);黄瓜与西芹间作土壤的乙醇、丙酮和蒸馏水浸提液均对黄瓜枯萎病菌具有一定化感作用,且表现为抑制作用,化感效果可达38.11%~75.90%;且乙醇浸提液培养的枯萎病菌菌落直径最小,与丙酮和蒸馏水浸提液达极显著差异(P0.01)。采用上述3种土壤田间种植黄瓜,在黄瓜第1片真叶期接种黄瓜枯萎病菌,降低了黄瓜枯萎病的病情指数,对比黄瓜单作和西芹单作的抑病效果可达57.03%~72.15%。因此,黄瓜与西芹间作对黄瓜枯萎病菌具有一定化感抑制效果,能够有效降低田间黄瓜枯萎病的发生,为黄瓜土传病害的化感防控提供科学依据。     

黄瓜连作土壤高温处理对根结线虫和枯萎病的影响

[目的]设施黄瓜连作导致根结线虫和枯萎病发生普遍且严重,利用夏季温室休闲期高温闷棚是解决上述土传病害安全而有效的途径之一。本试验模拟高温闷棚,研究不同温度处理连作土壤根结线虫和枯萎病的变化,旨在为利用太阳光进行温室高温消毒提供理论支撑。[方法]本试验以连作1~19茬‘津优30’黄瓜品种的土壤为试材,首先探明不同连作茬次黄瓜的根结线虫和枯萎病发生程度以及土壤中二者病原物种群数量的动态变化,然后对发病较重的第17茬连作土壤分别进行45℃、50℃、55℃和60℃的高温处理,最后测定比较不同温度处理后的土壤中病原物数量变化,并对高温处理后的土壤进行栽培试验,测量和比较两种病害的发生程度及植株各项生长指标。[结果]随着黄瓜连作茬次的增加,土壤中根结线虫和枯萎病菌的种群数量均增加,两种病害也逐茬加重,至第17茬两种病害的发病程度及其对应病原物的数量均达最高(或次高)水平;对第17茬黄瓜连作土壤进行45℃~60℃的高温处理,随着温度的升高,土壤中的根结线虫和枯萎病菌的数量均减少,当处理温度达55℃时可完全杀灭土壤中根结线虫,达60℃时可同时杀灭枯萎病菌;用高温处理的连作土壤栽培黄瓜秧苗,其根结线虫病和枯萎病的发生程度均随着处理温度的升高而减轻,当处理土壤的温度达到50℃和60℃以上时,根结线虫病和枯萎病分别被完全控制;另外,用60℃处理的连作土壤定植黄瓜,其后期植株生长指标和壮苗指数也显著优于对照。[结论]土传病害随连作茬次增加而加重主要是缘于土壤中病原物积累,定植前对土壤55℃以上的高温处理,可有效减少乃至完全杀灭土壤中根结线虫和枯萎病菌,从而减轻或杜绝两种病害的发生。     

一株抗真菌海洋放线菌的筛选、分离和多相分类鉴定

为开发利用宁波近海潮间带药用微生物资源,本研究自宁波慈溪海塘采集7份海泥样品,从中筛选、分离抗真菌放线菌,并对目标菌株进行多相分类鉴定,包括16S rRNA、gyrB基因序列分析、形态观察、生理生化鉴定及细胞化学组成分析。通过筛选培养获得了25个菌株,其中2P-4对白色念珠菌、黑曲霉表现出抑制生长的作用。细胞化学组成鉴定分析了2P-4全细胞糖、氨基酸、极性脂质、甲基萘醌和脂肪酸组成,结果表明,2P-4属于链霉菌属。2P-4的16S rRNA、gyrB基因序列分析也表明2P-4属于链霉菌属,并且与毒三链霉菌的关系最密切,其16S rRNA基因序列与Streptomyces toxytricini NBRC 13194^T的相似度为99.56%,gyrB基因序列与Streptomyces toxytricini NRRL B-5426^T的相似度为99.04%,但16S rRNA、gyrB基因序列在系统进化树上均形成独立分枝,支持2P-4与毒三链霉菌分属不同的种;此外,2P-4与其近缘种Streptomyces toxytricini NBRC 13194^T相比,两者在形态和生理生化特征方面存在明显差异。基于以上分析,初步鉴定2P-4为链霉菌属的一个新种。本研究为进一步探究2P-4抗真菌代谢产物提供了理论依据。     

有机肥对黄瓜枯萎病的防治效果及防病机理研究

通过盆栽试验与培养皿培养试验研究了有机肥对黄瓜枯萎病的防治效果,并对其防病机理进行了初步探讨。结果表明,有机肥可有效抑制黄瓜枯萎病的发生,其防病效果受肥料用量、施肥方式、肥料灭菌状况等因素的综合影响。有机肥用量越大,防治效果越好,在出苗50 d后,用量为5%的牛粪与生物有机肥处理较用量为1%的处理发病率降低约34.01%~36.80%;基施较叶面喷施发病率降低66.82%~78.92%,基施和喷施较对照发病率分别降低76.65%~81.17%和17.11%~25.43%;有机肥灭菌后会降低其对黄瓜枯萎病的防治效果,用量为1%和5%的灭菌牛粪处理的黄瓜发病率较未灭菌处理升高18.27%和82.82%;先对土壤进行化学灭菌后再施用有机肥,黄瓜枯萎病的发病率较土壤未化学灭菌处理的发病率降低50%。有机肥对黄瓜枯萎病的防治机理主要在于有机肥可以丰富土壤微生物,并直接抑制黄瓜枯萎病病原菌尖孢镰刀菌的生长,同时有机肥中的微生物及化学物质还能够刺激植物使其增强抗病性。     

多功能放线菌Act12 对土传病原真菌的拮抗性及其鉴定

采用琼脂块法及抑菌试验, 研究了多功能放线菌Act12 对6 种常见土传病原真菌的拮抗作用; 采用形态特征、生理生化特性及16 S rDNA 序列分析, 确定了Act12 的分类地位。结果表明, Act12 对6 种供试土传病原真菌具有不同程度的拮抗作用, 其中, 对木贼镰刀菌(Fusarium equiseti)和尖孢镰刀菌西瓜专化型(F. oxysporum f. sp. niveum)的拮抗圈直径分别达到20.5 mm 和18.4 mm; 培养48 h, Act12 无菌发酵滤液对木贼镰刀菌的抑菌率达到83.2 %。菌株显微形态、培养特征、生理生化特性及16 S rDNA 序列分析结果表明, 多功能放线菌Act12 为链霉菌属的密旋链霉菌(Streptomyces pactum)。     

固体生物肥防治黄瓜枯萎病、青椒疫病的田间应用效果评价

利用田间小区试验进行固体生物肥料防治黄瓜枯萎病和青椒疫病的效果研究，对田间应用效果进行评价。结果表明，固体生物肥料对黄瓜枯萎病的防治效果为45．12％、对青椒疫病防治效果达51．79％，达到了差异显著的效果，而且分别增产11．06％和37．19％，其防病与增产效果显著。     

拮抗菌防治西瓜枯萎病的试验

从西瓜、黄瓜、柑桔等作物根际中分离到的B11等芽孢杆菌的哈茨木霉T31、粘帚霉G1等拮抗真菌，对防治西瓜枯萎病菌有一定抑制作用。经田间防治试验，T31、G1和B11单个菌的田间防效在35％－50％左右；施用T31、G1、B11的混合拮抗菌和采取的一些措施（有机肥作基肥，拮抗菌与杀菌霸混施）对西瓜枯萎病有更好的防治作用，防效达60％－85％。     

Control of Fusarium wilt disease of cucumber plants with the application of a bioorganic fertilizer

Two field experiments were conducted to evaluate the effect of organic fertilizer application either with or without antagonistic bacteria (Bacillus subtilis SQR-5 and Paenibacillus polymyxa SQR-21) on the control of Fusarium oxysporum f. sp. Cucumerinum J. H. Owen wilt disease in cucumber. The incidence of Fusarium wilt disease was 5.3–13.5% for cucumber plants treated with bioorganic fertilizer, while it was 30.3–51% in controls (only with organic fertilizer). Higher yields and lower disease incidences were observed in the dry season when compared with the wet season for both types of organic fertilizer treatments. Biolog analysis showed a significant change in soil bacterial composition and activity after bioorganic fertilizer application. The numbers of colony-forming units of F. oxysporum f. sp. Cucumerinum J. H. Owen for bioorganic-fertilizer-treated soils were significantly decreased compared with control. Scanning electron micrographs of cucumber basal stems showed a presence of mycelia-like mini strands accompanied by an amorphous substance within the xylem vessels. This amorphous substance and mini strands were richer in calcium and phosphorus but had low carbon and oxygen than the living mycelia. Reverse-phase high-pressure liquid chromatography and mass spectroscopic analysis showed that the antagonistic bacteria produced the antifungal compounds fusaricidin A, B, C, and D with molecular weights of 883.5, 897.5, 947.5, and 961.5 Da, respectively. The application of bioorganic fertilizer has a great potential for the control of F. oxysporum wilt disease in cucumber plants.     

拮抗菌发酵的生物有机肥对茄子生长和青枯病的防治效果

采用盆栽试验研究了拮抗菌发酵的生物有机肥(BIO-36和BIO-23)对茄子植株的生长以及青枯病防治效果的影响。结果表明:①苗期茄子植株生长30天时,施用BIO-36和BIO-23的处理鲜重显著增加,尤其BIO-36处理的茄子根系的总长、总表面积和总体积提高8.58倍、2.74倍和16.57倍;②茄子苗移栽35天时,施用BIO-36和BIO-23的处理株高、地上部和地下部生物量都有显著提高,青枯病的发病率低,分别为4.1%和8.3%;③施用BIO-36和BIO-23的处理的茄子根际的真菌数量比对照CK增加了4.1~4.6倍和3.89~4.64倍,放线菌数量增加了3.38~3.78倍和3.08~3.47倍,根际茄科劳尔氏菌的数量分别为1.5×105cfu/g、2.1×105cfu/g,都控制在106cfu/g数量级以下,而对照处理数量级达到了1.23×106cfu/g。施用拮抗菌发酵的生物有机肥能显著促进茄子植株的生长,有效地降低茄子青枯病的发病率。     

松材线虫病综合治理模式及其成效分析

从2002~2004年连续3年对松材线虫病治理区进行全面、及时、彻底清除松材线虫病枯死木及健康松树上的枯死侧枝;清除治理区范围内松墨天牛的一切孳生材料;在架放松墨天牛诱捕器的同时,结合设置诱木诱杀天牛成虫和诱集成虫产卵,并在天牛成虫羽化出木前清除诱木。通过2002年的除治,治理区内当年松材线虫病枯死木发生率下降到万分之三以下;通过2003年的除治,当年松材线虫病枯死木发生率下降到万分之一以下;通过2004年的除治,当年松材线虫病枯死木发生率下降到十万分之一以下。首次对治理措施中清除衰弱木和健康松树上的枯死侧枝以及在林间设置天牛产卵诱木的必要性进行了分析,并提出了验收松材线虫病治理项目的新指标。     

The rhizosphere soil of diseased tomato plants as a source for novel microorganisms to control bacterial wilt

Plant growth promoting rhizobacteria (PGPRs) are used for biocontrol of bacterial wilt caused by Ralstonia solanacearum. They are commonly isolated from the rhizosphere of healthy plants and are scarce in the rhizosphere of diseased plants. We hypothesized that a pathogen-prevalent environment, such as the rhizosphere of infected plants, would be a good or better source for isolating PGPRs than the rhizosphere of healthy plants. In order for these PGPRs to survive successfully in a pathogen-prevalent environment, they must have particularly well-developed survival strategies under the stresses exerted by pathogen activities, which would be of value for their use as biocontrol agents. To test this hypothesis, R. solanacearum-antagonistic bacteria were screened from the rhizospheres of diseased and healthy tomato plants. In total, 110 rhizobacteria were isolated, 18 of which showed antagonism to R. solanacearum in vitro. Among the 18 antagonistic strains, 11 (out of 60) were from the rhizosphere of diseased plants, with inhibition diameter zones ranging from 11.2 to 15.2 mm, whereas 7 (out of 50) were from the rhizosphere of healthy plants, with inhibition diameter zones ranging from 11.5 to 30.5 mm. Strains WR4, WR21, and WR42 from diseased plants rhizosphere, and HR61, HR62, and HR92 from healthy plants rhizosphere, were chosen to investigate their biocontrol efficacies (BCEs) in greenhouse condition. Results showed that WR-isolates performed better in reducing disease incidence (DI) than those HR-isolates. Population densities of R. solanacearum in the rhizosphere soil and crown section of tomato plants were lower in WR-isolate treatments than those in HR-isolate treatments. The best biocontrol effect was achieved by inoculating the strain WR21, followed by WR4, WR42, HR92, HR62, and HR61. Root colonization test showed WR21 had the highest root-colonizing capacity compared with 5 other antagonists. BCEs were positively (r = 0.747) correlated with root-colonizing capacities, but were negatively (r = −0.797) correlated with inhibition zones. In conclusion, the rhizosphere of diseased tomato plants is a good reservoir of biocontrol bacteria.     

两种丛枝菌根真菌对黄瓜苗期枯萎病的防效及根系抗病相关酶活性的影响

由尖孢镰刀菌引起的黄瓜枯萎病是设施黄瓜生产的主要障碍之一,丛枝菌根真菌(AM真菌)可以和包括黄瓜在内约80%的维管植物的根系形成菌根共生体,适宜的共生体组合对于寄主的生长与抗病性的提高十分有益。为明确Glomus versiforme与Glomus intraradices两种AM真菌对"津绿3号"黄瓜苗期枯萎病的防治效果。试验采用盆钵培养的方法,研究了两种AM真菌对幼苗生长及其根系3种抗病相关酶活性的影响。结果表明:两种AM真菌均可促进黄瓜幼苗的生长,并能减轻病害,但以G.versiforme的促生及生防作用更显著,接种G.versiforme处理的黄瓜幼苗株高、茎粗、叶面积及干重均显著大于对照,该处理的幼苗病情指数较对照降低26.6%。菌根化黄瓜幼苗抗病性的提高一方面与接种病原菌Fusarium oxysporum f.sp.cucumerinum前幼苗生长健壮有关;另一方面与根系抗病相关酶活性的提前诱导有关。接种F.oxysporum f.sp.cucumerinum前,G.versiforme处理的黄瓜幼苗壮苗指数显著高于G.intraradices处理与对照,分别为G.intraradices处理与对照的1.19倍与1.22倍;G.versiforme处理的黄瓜幼苗根系几丁质酶、β-1,3-葡聚糖酶与PAL酶分别比对照提前2 d、7 d、7 d被诱导,且酶活性分别为对照的1.44倍、2.16倍和92.00倍。     

三七设施栽培根际微生物菌群变化及其与三七根腐病的相关性研究

本研究通过对三七免耕种植的定位研究,探讨三七根腐病发病过程中的菌群变化及其与三七根腐病的相关性。结果表明:设施栽培条件下三七根际土壤中各类微生物数量及种群结构均随着季节气温而变化,其消涨变化规律与三七根腐病高发期吻合,是诱发三七根腐病发生的根本原因。进一步的研究表明三七根腐病的发生,与土壤中厌氧细菌、真菌及放线菌数量的增加及其种群数量比例变化有密切的关联性,这三类菌群的数量及其比例变化是诱导三七根腐病害发生的主要原因;土壤温度与根腐发病率没有直接的关联作用,但是温度可改变土壤微生物种群变化并导致三七根腐病的发生发展;根际土壤中好氧细菌数与厌氧细菌数比例及真菌与细菌总数比值可作为三七根腐发病及病害防治的预期参考指标。     

Dynamic relationships between soil properties and foliar disease as affected by annual additions of organic amendment to a sandy-soil vegetable production system

Additions of organic amendments to agricultural soils can lead to improved soil quality and reduced severity of crop diseases. However, the relationship between disease severity and soil properties as affected by repeated additions of these amendments is poorly understood. The primary objectives of this study were to (i) resolve multivariate relationships between soil properties and foliar disease severity and (ii) identify soil properties that contribute to disease severity in an intensive irrigated vegetable production system receiving annual additions of fresh and composted paper mill residuals (PMR). Foliar diseases caused by Pseudomonas syringae pv. syringae on snap bean (bacterial brown spot) and P. s. pv. lachrymans on cucumber (angular leaf spot) are the focus of this report. The experiment consisted of a 3-year crop rotation of potato (1998 and 2001), snap bean (1999 and 2002), and cucumber (2000). Treatments included a non-amended fertilizer control and two rates of fresh PMR, PMR composted alone (PMRC), and PMR composted with bark (PMRB). Soil measures included total soil carbon (TC) and nitrogen (TN), particulate organic matter carbon (POMC) and nitrogen (POMN), volumetric soil moisture (VM) and in situ NO₃-N. Multiple regression (MR) and principal component analyses (PCA) were conducted to identify key soil properties that influenced the amount of disease. On average, the amount of TC in plots amended with PMR composts increased 77-178% from 1999 to 2002 compared to the non-amended soils. In 1999, a year in which compost additions reduced the amount of bacterial brown spot of bean, TC explained 42% of the total variation in disease severity in the best MR model. Midseason TN alone was inversely related to angular leaf spot incidence in 2000, while POMN explained 51% of the variation in the best MR model for that year. In 2002, a year in which PMRC-amended soils exacerbated brown spot symptoms, midseason quantities of TN explained 80% of the variation in disease severity. Unique to 2002, NO₃-N alone positively correlated with disease severity. Overall, the influence of soil carbon on disease severity was displaced by the increasing importance of TN and NO₃-N, indicating a transition from a C-dependent to an N-dependent system.