Purification and characterization of two antifungal chitinases extracellularly produced by Bacillus amyloliquefaciens V656 in a shrimp and crab shell powder medium

A Gram-positive bacterium with antagonistic activity was isolated from the soil. It has been identified as Bacillus amyloliquefaciens strain V656 on the basis of 16S ribosomal DNA analysis and standard bacteriological tests. B. amyloliquefaciens V656 produced antifungal enzymes when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine waste. The antifungal enzymes displayed chitinase activities. Two extracellular antifungal chitinases (FI and FII) were purified and characterized, and their molecular weights, isoelectric points, pH and thermal stabilities, and antifungal activities were determined. The characteristics of V656 chitinases are similar to those of the known bacterial chitinases in terms of their isoelectric points, thermal instabilities, and lack of lysozyme activity. In contrast to other known bacterial chitinases, the unique characteristics of V656 chitinases include extremely low molecular weights and nearly neutral optimum pH. Furthermore, this is the first report of the isolation of chitinases from B. amyloliquefaciens that are active against fungi.     

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。     

罗伦隐球酵母对草梅采后灰霉病害的生物防治

测定了罗伦隐球酵母对草莓采后由灰葡萄孢霉引起的灰霉腐烂的抑制效果。在马铃薯葡萄糖琼脂固体培养基（PDA）体外抑菌实验中，把生长有罗伦隐球酵母的营养酵母葡萄糖固体培养基(NYDA)切块放置在涂布有灰霉孢子的马铃薯葡萄糖琼脂固体培养基上一起培养，罗伦隐球酵母并不能抑制霉菌的生长；而在马铃薯葡萄糖液体培养基(PDB)体外抑菌实验中，罗伦隐球酵母活细胞有效地抑制了灰霉孢子的萌发。酵母细胞菌悬液对灰霉的抑菌效果好于酵母培养液，而酵母热杀死液和滤液没有抑菌作用。酵母菌悬液的细胞浓度对其抑菌作用有显著的影响：无论草莓在20℃条件下贮藏3 d或是在2℃条件下贮藏20 d，应用在草莓上的酵母细胞浓度越高，则草莓的霉变率越低。罗伦隐球酵母菌悬液对草莓整果储藏自然条件下霉变的抑制试验也有类似的结果。因此，罗伦隐球酵母可以代替化学杀菌剂作为草莓采后的霉菌抑制剂。     

The invasive plant Solidago canadensis L. suppresses local soil pathogens through allelopathy

Recent studies suggest that invasive plants pose a significant effect on local soil pathogens, which in turn affects on the plant invasion. However, the mechanisms by which invasive plants affect soil pathogens were less well known. We conducted four experiments to test the hypothesis that the invasive plant species Solidago canadensis L. may affect soilborne pathogens through exudation of allelochemicals. Two common soilborne pathogens Pythium ultimum and Rhizoctonia solani were used in the study. Tomato (Lycopersicon esculentum Mill) variety Qianhong No.1 which is sensitive to soil pathogens P. ultimum and R. solani was used to indicate pathogenic activity (in terms of seedling mortality and damping-off). Extracts from root and rhizome of S. canadensis significantly suppressed the growth and pathogenic activity of both pathogens under Petri dish culture and sand culture (experiments 1 and 2), providing direct evidence that S. canadensis exerts allelopathic effects on these pathogens. Subsequently, a pathogen inoculation experiment under sand culture showed that pathogenic activity of both P. ultimum and R. solani was lower under the soil with S. canadensis compared to that under the soil with a common native plant Kummerowia striata (Thunb.) Schindl (experiment 3), implying that invasive S. canadensis had but native K. striata did not have allelopathic effects on soil pathogens through root and rhizome exudation. Finally, results from field soil tests showed that mortality and damping-off rate of tomato seedlings were significantly lower under the soils collected from the fields dominated by S. canadensis than that dominated by native plants at both sampling sites, suggesting that suppression of pathogens also occurs in the field. From the present experimental results we suggest that invasive S. canadensis may acquire spreading advantage in non-native habitat by using “novel weapons” to inhibit not only local plants but also soilborne pathogens.     

Evaluation of chloropicrin gelatin capsule formulation as a soil fumigant for greenhouse strawberry in China

Gelatin capsules containing chloropicrin (Pic gel cap) were developed as a new formulation to reduce the potential human exposure risks associated with injection application methods. The objective of this study was to test the efficacy of a Pic gel cap formulation on soilborne pathogens and to determine the effects on strawberry plant growth and fruit yield. Three field experiments were conducted in strawberry greenhouses located in Mancheng County, China, in 2008-2010. The results demonstrated that effects of Pic gel cap on soilborne pathogens were similar to Pic injection; Pic gel cap effectively reduced key soilborne pathogens population, was partially effective against weeds, improved strawberry plant growth, and increased fruit yield significantly compared to the untreated control. Pic gel cap applied to preformed beds uses less fumigant than broadcast applications of Pic gel cap and can provide an equivalent level of disease control. The present study confirms that the Pic gel cap is a promising new formulation which provides field efficacy and marketable yields similar to Pic injection or methyl bromide in strawberry cultivation in China.     

棉花黄萎病生防菌脂肽类抑菌物质分离和鉴定

棉花黄萎病(Cotton Verticillium Wilt)是一种由大丽轮枝菌(Verticillium dahliae Kleb)引起的严重危害棉花生产的土传维管束病害[1]。因大丽轮枝菌菌丝体产生的大量微菌核在土壤中存活时间长以及病原菌变异快等特点,造成培育抗病品种、使用化学药剂和利用新的栽培技术等方法防病效果均不理想。近年来,以生物防治为主的综合防治措施日益受到人们重视,并取得了一定效果[2-4];该措施     

棉花内生解淀粉芽孢杆菌489-2-2对棉花黄萎病的防效研究

为更好地防治棉花黄萎病,在冀棉11根系中分离得到一株对大丽轮枝菌抗性明显的内生细菌,经分子鉴定为解淀粉芽孢杆菌489-2-2.本试验以489-2-2为材料,研究该菌株对棉花黄萎病的防效和机理.结果表明,解淀粉芽孢杆菌489-2-2能够抑制黄萎病菌Vd080的生长,可导致Vd080菌丝形态异常.用该菌株发酵液浸泡棉花种子...     

蓝莓根部内生木霉分离鉴定及其拮抗作用

为探究贵州省蓝莓根部内生木霉多样性,筛选具有拮抗作用的内生木霉,采用组织分离法、对峙培养法及无菌发酵液法,分析蓝莓根部内生木霉多样性及内生檑对7种植物病原真菌的拮抗作用。结果表明,通过组织分离、形态观察、rDNA-ITS和EF-1α基因序列鉴定得到16株内生木霉,属于Trichoderma virens、T. koningiopsis、T. harzianum和T. pubescens 4种,相似度为98%~100%。蓝莓根部内生木霉分离率(IR)为8%,分离频率(IF)为15.53%,Shannon-Weiner多样性指数为0.450 8,Simpson多样性指数为0.988 7、Margalef丰富度指数为2.408 2,Pielou均匀度指数为0.345 0。16株蓝莓根部内生木霉对交链格孢菌、禾谷镰孢菌、Diaporthe longicolla、小新壳梭孢、尖镰孢菌、胶孢炭疽菌、尖孢炭疽菌7种植物病原真菌的抑制率分别为35.01%~76.38%、58%~67.68%、55.10%~73.53%、52.24%~70.00%、25.80%~52.99%、37.18%~66.76%、37.37%~70.53%。选取T. harzianum GL11、T. harzianum GL15、T. harzianum GL19、T. harzianum GL39、T. virens GL42共5株木霉菌进行无菌发酵液试验,结果显示5株木霉菌发酵液对除小新壳梭孢外的其他6种植物病原菌具有一定的抑制作用,对Diaporthe longicolla的抑制效果最好,抑制率为46.71%。本研究结果可为蓝莓病害生物防治提供理论依据。     

Activity of meadowfoam (Limnanthes alba) seed meal glucolimnanthin degradation products against soilborne pathogens

Meadowfoam (Limnanthes alba L.) is a herbaceous winter-spring annual grown as a commercial oilseed crop. The meal remaining after oil extraction from the seed contains up to 4% of the glucosinolate glucolimnanthin. Degradation of glucolimnanthin yields toxic breakdown products, and therefore the meal may have potential in the management of soilborne pathogens. To maximize the pest-suppressive potential of meadowfoam seed meal, it would be beneficial to know the toxicity of individual glucolimnanthin degradation products against specific soilborne pathogens. Meloidogyne hapla second-stage juveniles (J2) and Pythium irregulare and Verticillium dahliae mycelial cultures were exposed to glucolimnanthin as well as its degradation products. Glucolimnanthin and its degradation product, 2-(3-methoxyphenyl)acetamide, were not toxic to any of the soilborne pathogens at concentrations up to 1.0 mg/mL. Two other degradation products, 2-(3-methoxymethyl)ethanethioamide and 3-methoxyphenylacetonitrile, were toxic to M. hapla and P. irregulare but not V. dahliae. The predominant enzyme degradation product, 3-methoxybenzyl isothiocyanate, was the most toxic compound against all of the soilborne pathogens, with M. hapla being the most sensitive with EC(50) values (0.0025 ± 0.0001 to 0.0027 ± 0.0001 mg/mL) 20-40 times lower than estimated EC(50) mortality values generated for P. irregulare and V. dahliae (0.05 and 0.1 mg/mL, respectively). The potential exists to manipulate meadowfoam seed meal to promote the production of specific degradation products. The conversion of glucolimnanthin into its corresponding isothiocyanate should optimize the biopesticidal properties of meadowfoam seed meal against M. hapla, P. irregulare, and V. dahliae.     

禾谷镰刀菌拮抗菌株的筛选及鉴定

禾谷镰刀菌引起的小麦赤霉病能够导致小麦产量的大幅降低和品质的严重损失。为探索赤霉病生物防治方法,本试验从扬花期被禾谷镰刀菌侵染的麦穗上分离得到28株芽孢杆菌,利用平板对峙法对其进行筛选,其中20株菌对禾谷镰刀菌表现出明显的拮抗作用。根据生理生化特性和16S rDNA序列分析,这20株拮抗菌分别被鉴定为甲基营养型芽孢杆菌(11株)、解淀粉芽孢杆菌(2株)、枯草芽孢杆菌(2株)、暹罗芽孢杆菌(3株)和特基拉芽孢杆菌(2株)。这些菌株能够有效降低小麦赤霉病的发病率、发病程度和病情指数,其中菌株JS62N、JS15E和JS29I能够将赤霉病发病率降低80%以上;菌株JS29I、JS62N、JS39C和JS39D能够将赤霉病发病程度降低80%以上。16株菌能将病情指数降低80%以上;所有菌株均能极显著增加小麦的百粒重,JS12Q效果最好(+101.4%)。本研究结果为小麦赤霉病的防治提供了生物防治材料。     

温室及田间条件下解淀粉芽孢杆菌菌株W19对香蕉生长、产量的促进和对香蕉镰刀菌萎蔫病的抑制研究

Plant growth-promoting rhizobacteria (PGPR) are considered to be the most promising agents for cash crop production via increasing crop yields and decreasing disease occurrence. The Bacillus amyloliquefaciens strain W19 can produce secondary metabolites (iturin and bacillomycin D) effectively against Fusarium oxysporum f. sp. cubense (FOC). In this study, the ability of a bio-organic fertilizer (BIO) containing W19 strain to promote plant growth and suppress the Fusarium wilt of banana was evaluated in both pot and field experiments. The results showed that application of BIO significantly promoted the growth and fruit yield of banana while suppressing the banana Fusarium wilt disease. To further determine the beneficial mechanisms of the strain, the colonization of green fluorescent protein-tagged strain W19 on banana roots was observed using confocal laser scanning microscopy and scanning electron microscopy. The effect of banana root exudates on the formation of biofilm of strain W19 indicated that the banana root exudates may enhance colonization. In addition, the strain W19 was able to produce indole-3-acetic acid (IAA), a plant growth-promoting hormone. The results of these experiments revealed that the application of strain W19-enriched BIO improved the banana root colonization of strain W19 and growth of banana and suppressed the Fusarium wilt. The PGPR strain W19 can be a useful biocontrol agent for the production of banana under field conditions.     

Effects of novel bioorganic fertilizer produced by Bacillus amyloliquefaciens W19 on antagonism of Fusarium wilt of banana

Banana production has been severely hindered by the long-term practice of monoculture agriculture. Fusarium wilt, caused by the Fusarium oxysporum f. sp. cubense (FOC), is one of the most destructive diseases that can afflict banana plants. It is both necessary and urgent to find an efficient method for protecting banana production worldwide. In this study, 57 antagonistic bacterial strains were isolated from the rhizospheres of healthy banana plants grown in a heavily wilt-diseased field; of the 57 strains, six strains with the best survival abilities were chosen for further study. Compared with the control and the other strains in the greenhouse experiment, W19 strain was found to observably decrease the incidence of Fusarium wilt and promote the growth of banana plants when combined with the organic fertilizer (OF). This strain was identified as Bacillus amyloliquefaciens based on its morphological, physiological, and biochemical properties, as well as 16S rRNA analysis. Two kinds of antifungal lipopeptides (iturin and bacillomycin D) produced by W19 strain were detected and identified using HPLC–ESI-MS. Another lipopeptide, called surfactin, was also produced by the thick biological film forming W19 strain. In addition to lipopeptides, 18 volatile antifungal compounds with significant antagonistic effect against F. oxysporum were detected and identified using gas chromatography–mass spectrometer (GC–MS). The work described herein not only highlights how the bioorganic fertilizer with B. amyloliquefaciens can be used to control Fusarium wilt of banana but also examines some of the potential mechanisms involved in the biocontrol of Fusarium wilt.     

Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter

Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence. A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors. The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively. Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site. These results indicate that the proteases are natural variants of subtilisin and bacillolysin.     

解淀粉芽孢杆菌YN-1抑菌蛋白TasA基因的克隆及原核表达

本文以解淀粉芽孢杆菌YN-1菌株为研究对象,利用PCR方法从基因组DNA中扩增出编码TasA抑菌基因的全长DNA,并构建pGEX-4T2/TasA原核表达载体,获得TasA-GST融合表达的抑菌蛋白。测序结果表明,解淀粉芽孢杆菌YN-1TasA基因核苷酸序列全长为786bp(GenBank登录号:EU131674),编码261个氨基酸残基;同源性分析表明,它与解淀粉芽孢杆菌B.amyloliquefaciens FZB42(YP_001421886)序列的同源性最高,达98%,预测蛋白分子量为31kD。SDS-PAGE分析表明,TasA基因能在大肠杆菌BL21中表达,Western印迹分析pGEX-4T2/TasA原核表达载体,检测到约57kD的TasA-GST融合外源蛋白,与预测的融合蛋白分子量大小相符。表达产物细胞裂解液上清经Ni柱层析,表明浓度为500mmol/L咪唑洗脱缓冲液时获得较高浓度和纯度的纯化蛋白。进一步抑菌活性分析表明表达产物对黄瓜灰霉病病原菌检测平板上显示出较好的抑菌活性。本研究结果将为今后深入研究TasA抑菌蛋白基因以及抗病转基因工程提供了参考。     

Hydrogen cyanide production by soil bacteria: Biological control of pests and promotion of plant growth in sustainable agriculture

Currently, plant diseases and insect infestations are mainly controlled by the extraneous application of pesticides. Unfortunately, the indiscriminate use of such agrochemicals can cause ecological and environmental problems, as well as human health hazards. To obviate the potential pollution arising from the application of agrochemicals, biological control of soilborne pathogens or insect pests using antagonistic microorganisms may be employed. Certain soil bacteria, algae, fungi, plants and insects possess the unique ability to produce hydrogen cyanide(HCN), which plays an important role in the biotic interactions of those organisms. In particular, cyanogenic bacteria have been found to inhibit the growth of various pathogenic fungi, weeds, insects, termites and nematodes. Thus, the use of HCN-producing bacteria as biopesticides offers an ecofriendly approach for sustainable agriculture. The enzyme, HCN synthase,involved in the synthesis of HCN, is encoded by the hcnABC gene cluster. The biosynthetic regulation of HCN, antibiotics and fluorescent insecticidal toxins through the conserved global regulatory GacS/GacA system is elaborated in this review, including approaches that may optimize cyanogenesis for enhanced pest control. In addition, the effects of bacterially synthesized HCN on the production of indole acetic acid, antibiotics and fluorescent insecticidal toxins, 1-aminocyclopropane-1-carboxylate deaminase utilization and phosphate solubilization may result in the stimulation of plant growth. A more detailed understanding of HCN biosynthesis and regulation may help to elaborate the precise role of this compound in biotic interactions and sustainable agriculture.     

Influence of thaxtomins in different combinations and concentrations on growth of micropropagated potato shoot cultures

Plant-pathogenic Streptomyces species produce a variety of different phytotoxic 4-nitroindol-3-yl-containing 2,5-dioxopiperazines (thaxtomins) that induce scab symptoms on potato tubers (Solanum tuberosum). The possible mutual synergistic or antagonistic effects of thaxtomins are unknown. Modified methodology using column chromatography allowed the purification of thaxtomin A in large quantities (27 mg, HPLC purity of 97%). Thaxtomin A ortho isomer, thaxtomin B, and C-14 deoxythaxtomin B (thaxtomin D) were also purified. All four compounds induced similar symptoms of reduced root and shoot growth, root swelling (10-200 ppb), or necrosis (200-1000 ppb) on micropropagated in vitro cultures of potato. The scab-resistant potato cvs. Sabina and Nicola were more tolerant to thaxtomins than was the scab-susceptible cv. Matilda. Thaxtomins applied in combinations showed additive effects but no synergism, whereas thaxtomins A and B displayed antagonism with thaxtomin A ortho isomer.     

Utilization of trehalose, benzoate, valerate, and seed and root exudates by genotypes of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens

Isolates of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are effective biocontrol agents against soilborne pathogens. A previous study showed that the superior (“premier”) root colonizer P. fluorescens Q8r1-96 (genotype D) utilized trehalose, benzoate and valerate as sole carbon sources but average colonizers Q2-87 (genotype B) and 1M1-96 (genotype L) did not. We tested the utilization of these three carbon sources by a collection of 55 2,4-DAPG-producing P. fluorescens strains from 17 genotypes and found no correlation between a strain's ability to utilize these carbon sources and superior rhizosphere competence on wheat and pea. Of the strains tested, 73%, 48% and 69% were able to utilize trehalose, benzoate and valerate as sole carbon sources, respectively. With some exceptions, we found a correlation between the utilization of these compounds and previous groupings of these strains by BOX-PCR; genotype D strains utilized all three compounds. Twenty-three strains grew efficiently on root and seed exudates from wheat and pea, with doubling times between 0.9 and 1.6 h generation⁻¹ and lag phases between 5 and 8 h, comparable to growth on glucose as a sole carbon source. Only 10 strains, including those with “premier” (Q8r1-96) and “average” (Q2-87) rhizosphere competence, showed slower growth in wheat root exudates, with lag phases between 16 and 22 h. Results were the same when soil was added to the culture medium. Growth of four strains in media containing glucose or wheat or pea seed exudates as a sole carbon source was not influenced by whether the bacterial cells used as inoculum were harvested from wheat seeds or broth culture. We conclude that the superior ability of some strains to colonize the roots of certain crops cannot be explained by the utilization of the carbon sources tested in our study.     

连作土壤微生物区系分析、调控及高通量研究方法

土壤连作后导致土壤微生物生态失衡、病原微生物富集、有益微生物减少,土壤微生物从细菌主导型向真菌主导型转化,使病原菌更容易侵染植物而引发植物的各种土传病害。施用微生物有机肥可以将失衡的微生物区系恢复到健康的状态,从而可以起到防治土传病害的作用。本文围绕连作引起的土壤中各大类微生物数量和种类的变化以及连作土壤施用微生物有机肥修复后其微生物数量和种类的变化进行了综述,并通过与传统技术比较,介绍了454高通量测序研究方法的优越性,指出了其在土壤微生物区系研究中的重要性。     

Bacillus amyloliquefaciens Pb-4对穴盘育苗番茄生长及生理特性的影响

【目的】 以穴盘育苗番茄为试材,研究分析了Bacillus amyloliquefaciens Pb-4对植物的促生作用及应用潜力,为该菌在番茄穴盘育苗上的应用提供理论依据。 【方法】 采用穴盘育苗的方法,以Bacillus amyloliquefaciens Pb-4在PYJ培养基中培养48 h的发酵液为接种剂,设置不接种 (CK) 和接种20 (T20)、60 (T60)、100 (T100) 和200 (T200) mL/L 基质5个处理,番茄幼苗四叶一心时取样分析其生理指标。 【结果】 1) Pb-4显著促进了番茄幼苗地上部的生长,其中T100处理的促生作用最强,株高、茎粗、叶面积以及地上部干重较CK处理显著增加了28.5%、23.9%、57.4%和42.4%,而T200处理的促生作用下降;Pb-4促进了番茄幼苗根系的生长,显著增加了根系直径大于0.5 mm的根长占总根长的比重,T20处理的根表面积和根体积最大,较CK处理显著增加了16.9%和34.2%,但与T100处理间差异不显著。2) Pb-4显著增加了番茄幼苗光合色素含量,其中T100处理的总叶绿素含量最高,CK处理最低,T20、T60、T100和T200处理分别较CK处理增加5.8%、9.4%、12.6%和7.6%;Pb-4提高了番茄幼苗茎、叶中IAA和GA₃的含量,而对根中IAA和GA₃的含量没有影响。3) 相关性分析表明,番茄株高、茎粗、叶面积、地上部干重与叶片光合色素含量、茎中IAA、GA₃以及叶片中GA₃的含量显著相关,而根系干重与光合色素含量、茎中IAA含量显著相关,根表面积与叶片IAA以及根系中GA₃的含量显著相关,根体积与叶片中IAA含量显著相关。 【结论】 Bacillus amyloliquefaciens Pb-4可促进番茄幼苗地上部生长,改变根系形态特征,提高番茄叶片光合色素以及不同器官中IAA和GA₃含量,其对番茄幼苗的促生作用在施用量为100 mL/L基质时最佳,超过该施用量促生作用降低。