云斑白条天牛肠道木质素降解菌的分离与鉴定

微生物降解木质素具有环境友好、低成本等优点而受到广泛关注。自云斑白条天牛Batoceralineaolata (Chaevroat)幼虫肠道中筛选高效木质素降解菌,以实现木质素的资源化利用。本研究以愈创木酚为唯一碳源的选择培养基法和苯胺蓝平板法筛选高效降解木质素的菌株,采用形态学观察和16S r DNA基因序列同源性分析方法对菌株进行鉴定。在愈创木酚选择培养基上共筛选出37株细菌菌株,在苯胺蓝平板上,B01和B12菌株的水解圈D/d值最大(1.60和1.58);经鉴定,B01菌株为假单胞菌属(Pseudomonas)的一种,B12菌株为考氏科萨克氏菌属(Kosakonia)的一种。     

解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HAB-7的培养基优化及抑菌活性

研究了解解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HAB-7菌株在不同培养基下生长速率及产生的活性物质对几种植物病原菌的抑菌活性。采用摇瓶法测定了HAB-7耐盐性及生长速率;平板对峙法测定了其抑菌活性;以NA培养基为基础培养基,芒果炭疽菌(Colletotrichum gloeosporioides)为靶标菌,研究了不同碳、氮源组合培养基对HAB-7菌株生长影响,以及对芒果炭疽菌的抑制率;用单因子正交试验,进一步优化碳源、氮源组合培养基。结果表明,HAB-7菌株在0~20%NaCl(g/L)含盐量上均能生长;最佳接种时间应在培养24~28 h;在NA、LB、BPY、AYDA、BPA及牛肉膏蛋白胨培养基上培养的HAB-7,对8种植物病原真菌平均抑制率分别为53.23%、52.03%、55.52%、52.77%、56.58%及53.82%。优化后的最佳碳源为蔗糖(10.0 g/L)和葡萄糖(10.0 g/L)复合碳源;最佳的氮源为酵母粉(20.0 g/L)和胰蛋白胨(20.0 g/L)复合氮源。单因子正交试验结果显示的碳、氮源为蔗糖30.0 g/L、葡萄糖10.0 g/L、酵母粉30.0 g/L、胰蛋白胨40.0 g/L;对芒果炭疽菌抑菌率为78.3%,较BPA培养的HAB-7提高了10.80%,活菌数达到1.97×10~(10)cfu/m L。     

假单胞菌S149对水稻防御酶的诱导及定殖能力

运用分光光度计检测荧光假单胞菌S149对3种水稻防御酶的诱导,利用单抗生素标记法测试该菌株在水稻叶片上的定殖能力。结果表明,各处理喷施S149发酵液(1×108cfu/mL)处理后第1天超氧化物歧化酶(SOD)的酶活性最高,喷施第2天过氧化物酶(POD)和多酚氧化酶(PPO)的活性最高;菌株S149与稻瘟病菌(Magnaporthe oryzae)的混合液诱导的POD和SOD酶活性最高,菌株S149发酵液诱导的PPO酶活性最高。荧光假单胞菌S149可以在含有不同浓度的壮观霉素、利福平、硫酸链霉素的LB平板上生长,能够在低浓度下的四环素、萘啶酮酸、氨苄青霉素和庆大霉素平板上生长,在含有氯霉素、卡那霉素、新霉素和红霉素的LB平板上完全不生长。采用抗生素标记菌株S149发酵液均匀喷雾后,叶片上回收到的菌落数量呈现下降趋势,喷施后第20天回收到7.04×10~4cfu/g的菌落数量。     

拮抗青枯劳尔氏菌的荧光假单胞菌SN15-2分离鉴定及其生防能力分析

为了筛选出对番茄青枯病具有较好防效的生防菌,采用皿内测定法从上海地区的番茄青枯病自然衰退土壤中,分离到一株对番茄青枯病有很强抑制作用的菌株SN15-2,并进行了分子鉴定和对荧光假单胞菌SN15-2产抗生素能力和定殖能力测定。结果表明,菌株SN15-2为荧光假单胞菌(Pseudomonas fluorescens)。其菌株能产生2,4-二乙酰基间苯三酚(2,4-DAPG)、硝吡咯菌素(PRN)、藤黄绿脓菌素(PLT)。同时,可以产生HCN、噬铁素,能够形成生物膜。SN15-2在施入番茄根际后的前20 d定殖数量减少,20 d后基本稳定,60 d时,在干土中的定殖数量可达3.67×105cfu/g。盆栽防效分析表明,荧光假单胞菌SN15-2对番茄青枯病防效达到46.58%。     

一种适宜从小麦种子上分离细菌性黑颖病原菌的半选择性琼脂培养基

一种半选择性琼脂培养基(XTS 琼脂)被研制出。经试验,它适宜从小麦种子上分离细菌性黑颖病原菌 Xanthomonas campe-stris PV.translucens。这种培养基的成分如下:Difco 营养琼脂 23g葡萄糖 5g放线菌酮 200mg     

黄瓜土传病害拮抗细菌的筛选、鉴定及生防特性研究

为了有效防治黄瓜的土传病害,本文从黄瓜根际土壤中分离得到256株细菌,利用平板对峙试验筛选到一株拮抗菌ZM-1。通过形态、生理生化和分子生物学方法对菌株进行鉴定,测定菌株产生的抑菌物质。通过正交实验法与单因素法对菌株进行发酵优化,并通过盆栽试验测定菌株防病能力。结果表明,菌株ZM-1为铜绿假单胞菌Pseudomonas aeruginosa,该菌对黄瓜枯萎病菌Fusarium oxysporum f.sp.cucumebrium和黄瓜疫病菌Phytophthora drechsleri的抑制率分别为62.80%和65.58%。该菌株可以产生纤维素酶、铁载体等多种抑菌物质。经过最佳发酵优化,菌株发酵液菌含量可达到1×10¹⁰ cfu/mL以上。最适发酵培养基配方为蔗糖5 g/L、胰蛋白胨10 g/L、K₂HPO₄ 10 g/L,发酵条件温度25℃、pH 7.5、时间48 h、接种量2%。盆栽试验表明,菌株ZM-1对黄瓜枯萎病和黄瓜疫病的防效分别为53.97%和51.11%。本研究表明拮抗菌ZM-1在防治黄瓜土传病害方面具有...     

绿色荧光蛋白标记荧光假单胞菌P303及其生存能力检测

构建了含有荧光假单胞菌自身启动子PP303的中间质粒载体pGFP.然后利用mini-Tn5转座子,通过接合转移,将两个带有不同启动子的绿色荧光蛋白基因分别整合到荧光假单胞菌P303染色体上,获得了在488nm波长下发光稳定的P303m1和P303m3菌株.PCR鉴定和Southern印迹结果均证明绿色荧光蛋白已随机插入P303染色体.SDS-PAGE结果表明,含有PA1/04/03启动子的P303m3菌株GFP表达量低于含有PP303启动子的P303m1菌株,染色体标记的GFP表达量低于质粒标记.室内平板抑菌试验结果表明,P303m1与出发菌株P303抑菌活性相当,对九种植物病原真菌有较强的拮抗作用.定殖、生存竞争能力研究表明,荧光假单胞菌在自然土壤中和大白菜根际都具有较强的定殖能力.P303和P303m1在自然土壤中第60天的菌量分别为1.63×104和3.3×102cfu/g土(湿重),大白菜根际第50天的菌量分别为3.29×106和4.1×104cfu/g根(湿重).     

适合 Pilidium lythri 菌丝生长和产孢的培养基

由Pilidium lythri引起的草莓褐色叶斑病是在草莓Fragaria×ananassa上发现的一种新病害。病原菌在PDA(马铃薯葡萄糖琼脂)培养基上生长速度慢,产孢量低。为了探讨不同培养基对P.lythri菌丝生长和产孢的影响,筛选适合该病原菌菌丝生长和大量产孢的培养基,本文比较了10种培养基对P.lythri生长和产孢的影响,结果表明,SPDA(添加1.2%草莓果汁的马铃薯葡萄糖琼脂)培养基可以促进P.lythri菌丝生长;CA(胡萝卜琼脂)培养基、V8培养基和TPDA(胰蛋白胨马铃薯葡萄糖琼脂)培养基则可以促进P.lythri大量产生分生孢子。     

吡咯伯克霍尔德氏菌JK-SH007的发酵条件及其对杨树溃疡病的防治效果

本文研究了拮抗细菌吡咯伯克霍尔德氏菌Burkholderia pyrrocinia菌株JK-SH007产抗菌物质的最佳发酵条件及其对杨树溃疡病的野外防治效果。结果表明,牛肉膏、蛋白胨是发酵培养基中最佳营养物质,有利于菌株JK-SH007抗菌物质的产生;培养基初始pH、培养时间、温度、培养体积、不同牛肉膏、蛋白胨组合等对菌株生长及其抗菌物质的产生有明显的影响,初始pH7、牛肉膏2g、蛋白胨20g、以1/2装液量装液、30℃振荡培养36h可获得较高产量的胞外分泌型抗菌物质;菌株JK-SH007的发酵液对杨树溃疡病的野外防效可达40.54%。     

小麦全蚀病生防细菌的筛选和鉴定

为获得对小麦全蚀病菌有良好拮抗效果的生防菌株,分别从河南省商丘市及驻马店市小麦全蚀病发生田块中采集小麦根际土样,采用稀释平板涂布法共分离到1051株细菌,通过与全蚀病菌G1037菌株进行平板对峙筛选,最终获得9株具有明显拮抗效果且生长状况良好的菌株。16S rDNA序列比对及生理生化性状分析结果表明：菌株P155、P154、P16及P147为荧光假单胞菌Pseudomonas fluorescens,菌株P188和P97为恶臭假单胞菌Pseudomonas putida,菌株LY3为产酶溶杆菌Lysobacter enzymogenes,菌株S38为嗜根寡养单胞菌Stenotrophomonas rhizophila,菌株B20为洋葱伯克霍尔德Burkholderia cepacia。产抗生素相关基因的检测结果发现,菌株P147含吩嗪和硝吡咯菌素合成基因,菌株B20含硝吡咯菌素合成基因。除B20、LY3、S38和P147外,其余菌株均可产生嗜铁素。9株细菌都产蛋白酶。除P97、B20和S38外,其余菌株均可产脂肽类物质。盆栽试验结果表明,9株生防菌对小麦全蚀病都具有较好的防治效果,菌株P155和P154的防治效果最好,相对防效分别为67.11%和63.82%,略高于3%的苯醚甲环唑种衣剂的防治效果。研究结果表明这些细菌具有作为小麦全蚀病生防菌的潜力。     

A new medium for the detection of fluorescent pigment production by pseudomonads

Two media (King’s B [KB] and CSGA) commonly used for the detection of fluorescent pigment by Pseudomonas spp. were compared to a new medium proposed in this study, PGS agar. Thirty‐nine strains of 10 different species of Pseudomonas from several geographic regions were screened. The efficacies of these media were examined under several conditions, including the addition of iron‐binding substances or supplementation with extra iron. The medium developed, which included an iron‐binding agent, was the most permissive for production of fluorescent pigments when compared to KB and CSGA. Thirty‐seven of the 39 pseudomonad strains screened were highly fluorescent on this new medium compared to 15 and 16 strains, respectively, on KB or CSGA. The optimal composition of the medium per litre was Bacto peptone 10 g, gelatin 20 g, sucrose 20 g, agar 15 g, dipotassium hydrogen phosphate 1 g, magnesium sulphate heptahydrate 1 g and conalbumin 2 g. Protocol validation tests performed through an intra‐laboratory study in comparison to KB demonstrated the effectiveness of the new PGS medium.     

山茶花灰斑病菌生物学特性研究

 山茶花(Camellia japonica L.)灰斑病菌[Pestalotiopsis guepini (Desm.) Stey.]^[1,2,3,4]菌丝生长适温20-25℃,适宜pH值是5-7。在不同培养基里以PDA和燕麦培养基最佳。对碳源的利用以葡萄糖,蔗糖最佳,木糖最差,氮源以门冬酰胺、蛋白胨、酵母液最佳,尿素最差。
分生孢子萌发适温20-25℃,适宜pH值是4-5,在不同营养液里以酵母汁,茶花叶汁萌发最好。在不同光照条件下,自然光和连续黑光灯利于产孢,完全黑暗不产孢。不同培养基里以燕麦和PDA培养基最适宜病菌产孢,在山茶花叶汁与水洋菜培养基里不产孢。     

Development of a semiselective medium for isolating Xanthomonas campestris pv. musacearum from insect vectors, infected plant material and soil

A semiselective medium was developed for isolating Xanthomonas campestris pv. musacearum ( Xcm ) from infected banana plants, soil and insect vectors. The new medium was named cellobiose-cephalexin agar (CCA) and it contained (L⁻¹): 1 g yeast extract, 1 g glucose, 1 g peptone, 1 g NH₄Cl, 1 g MgSO₄·7H₂O, 3 g K₂HPO₄, 1 g beef extract, 10 g cellobiose, 14 g agar, 40 mg cephalexin, 10 mg 5-fluorouracil and 120 mg cycloheximide. The medium was evaluated for selectivity using 21 bacterial isolates and for plating efficiency using Xcm . The bacterial isolates included a soilborne Xanthomonas species and three pathogenic Xanthomonas strains that infect cassava, cabbage and beans. Although the plating efficiency of Xcm on CCA was lower (59%) than on non-selective yeast extract peptone glucose agar (YPGA), its selectivity was significantly higher, averaging 60 and 82%, when isolating from banana fruits and soil, respectively. CCA was also superior when isolating Xcm from insect vectors, with selectivity of 48–75%, compared with 8–17% on YPGA. Xanthomonas campestris pv. phaseoli did not grow on CCA, while X. campestris pv. campestris and X. axonopodis pv. manihotis grew, but their colonies were smaller than those of Xcm . Twenty-nine out of 33 suspected Xcm strains isolated from plants, soil and insects using CCA were pathogenic when inoculated onto banana plants, indicating that CCA can be a reliable tool in isolating Xcm populations. The medium should prove useful in studies on ecology, epidemiology and management of the banana bacterial wilt pathogen that is currently ravaging bananas in East and Central Africa.     

棉籽带枯萎病菌检验方法的研究

棉籽带菌是远距离传病的主要途径。病棉株种子带菌率0.01—46.8%。带菌部位以种壳为主,但子叶、胚均带菌。棉籽上除带枯萎病菌外,还带有半棵、串珠、茄病、木贼等几种镰刀菌及其他杂菌,干扰检验。文中介绍一种选择性培养基,其成分为:甲基纤维素1克、KH_2PO_4克、蛋白胨5克、MgSO_4·7H_2O0.5克、K_2S_20.2克、KCl 0.6克,NH_4NO_30.3克、五氯硝基苯0.1克、蔗糖20克、链霉素0.1克、琼脂20克、蒸馏水1000毫升。应用其进行分离培养棉籽,可根据菌落形态特征及颜色反应,较容易地检验出棉花枯萎病菌。     

Cultural characteristics of Sporisorium sorghi and detection of the pathogen in plant tissue by microscopy and polymerase chain reaction

Despite the economic importance of covered kernel smut of sorghum (Sporisorium sorghi) in many African states and other parts of the world, only limited information is available on laboratory cultivation methods for this fungus and techniques for its diagnosis in plant tissue. The current paper describes laboratory and greenhouse experiments performed with field material of S. sorghi. When intact sori were kept at 5°C, 80% of the spores germinated even after 24?months of storage. Spore germination on agar medium and production of mycelial dry weight in still culture were highest between 20° and 35°C, with a peak at 30°C. Both showed a steady increase from pH 4.5 to pH 7.5, followed by a decline at pH 8.5 and 9.5. In shake culture in different broth media the addition of 0.3% peptone from soybean caused an increase in fungal growth compared with the media alone. Of the media tested, mycelial production was highest in malt dextrose broth supplemented with peptone. When cultivated on different agar media, the morphology of single spore isolates differed both among isolates and depending on the agar medium. In greenhouse experiments, five short, early maturing sorghum breeding accessions proved to be partially or fully resistant to covered kernel smut. Among the plant materials tested, cv. ??Dorado?? appeared to be the one best suited for greenhouse experiments with covered kernel smut. By microscopy of hand-cut sections stained with trypan-blue, hyphae of S. sorghi were seen in apical buds and in nodes of young sorghum plants. Diagnostic PCR amplified a 903?bp element comprising the internal region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding gene and enabled the detection of S. sorghi in both nodes and apical buds of infected sorghum seedlings. Both techniques, i.e., microscopy and diagnostic PCR, have the potential to be used in studies for the identification of effective sorghum seed treatments already at the seedling stage.     

短短芽胞杆菌产抗菌物质——羟苯乙酯发酵培养基的优化

羟苯乙酯是短短芽胞杆菌FJAT-0809-GLX的主要抑菌活性物质,为提高该菌株发酵液中羟苯乙酯的产量,本研究采用响应面法对其发酵培养基成分进行优化。首先通过单因素试验,对发酵培养基中的碳源、氮源和无机盐进行了优化,进一步通过Plackett-Burman设计对培养基的影响因素进行筛选,最后采用最陡爬坡路径逼近最大响应区域,通过Box-Behnken设计,结合响应面分析,获得短短芽胞杆菌FJAT-0809-GLX发酵产生羟苯乙酯的最佳培养基配方。结果表明,培养基最佳碳源、氮源和无机盐分别为DL-苹果酸、蛋白胨和NaCl。影响显著的3个因素分别为DL-苹果酸、豆饼粉和NaCl。最佳培养基配方为可溶性淀粉8 g/L、DL-苹果酸29.68 g/L、豆饼粉25.18 g/L、蛋白胨2 g/L、NaCl 13.18 g/L,pH 7.0~7.2。采用此培养基配方进行短短芽胞杆菌FJAT-0809-GLX发酵,羟苯乙酯平均产量为8.15μg/mL,较基础发酵培养基提高了286.26%。     

Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry

Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml.Plating efficiency of Cms in yeast glucose mineral medium (YGM) was 104% compared with yeast peptone glucose medium. Nalidixic acid, polymyxin B sulphate and the experimental disinfectant S-0208 inhibited colony growth of cattle slurry bacteria as compared with Cms in YGM. The optimal concentration of these inhibitors in combination was determined by modified agar diffusion tests and by pour plating in 24-well tissue culture plates. The semi-selective medium YGMI consisted of YGM supplemented with nalidixic acid (2 mg/l), polymyxin B sulphate (30 mg/l) and S-0208 (125 mg/l). Plating efficiency varied for Cms between 50.9 and 69.6%, for cattle slurry bacteria between 1.8 and 2.5% and for saprophytes from potato heel end extracts between 11.5 and 27.4%.Differentiation of Cms colonies from other colonies was based on their small and bluish colony morphology in pour plates and on immunofluorescence colony-staining (IFC). IFC of a pure culture of micro colonies of Cms in YGM was possible after one day incubation (colonies c. 5 cells). Green background fluorescence in the agar gels was prevented by addition of Tween 20 (0.1%) to the washing buffer and the use of 1% agar gels. IFC of macro colonies of Cms in YGMI, visible with 4x objective magnification, was possible after 4 days. The detection level of the target organism in artificially inoculated cattle slurry in YGMI based on colony morphology varied between 1.4×10³ and 2.3×10⁴ cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (=16 mm), proved suitable for detection, but was c. 30 times les sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R ²=0.74).     

Effect of Carbon, Nitrogen, and C:N Ratio on Growth, Sporulation, and Biocontrol Efficacy of Talaromyces flavus

ABSTRACT Engelkes, C. A., Nuclo, R. L., and Fravel, D. R. 1997. Effect of carbon, nitrogen, and C:N ratio on growth, sporulation, and biocontrol efficacy of Talaromyces flavus. Phytopathology 87:500-505.Five-day biomass production by the biocontrol fungus Talaromyces flavus was measured in a liquid basal medium, pH 5.5, containing each of 37 carbon (C) sources with a single nitrogen (N) source, and each of 42 N sources with a single C source. In general, production of biomass was greatest on complex sugars such as polysaccharides (32 g/liter of medium) and beta-glucosides (2.4 g/liter of medium), and was least on monosaccharides (1.3 g/liter of medium). Ascospore production at 6 weeks on solid basal medium with the same amount of these same 37 degrees C sources was greatest on oligosaccharides (2.9 x 10(8) spores per 5.5-cm-diameter petri dish), and least on polysaccharides and monosaccharides (1.6 and 1.4 x 10(8) spores per 5.5-cm-diameter petri dish, respectively). For C sources, there was no correlation between production of ascospores and hyphal dry weight. The various N sources yielded 0 to 10(9) ascospores per 5.5-cm-diameter petri dish and 10(-4) to 10(-5) g of hyphal dry weight per milliliter. In general, N sources that resulted in the greatest number of ascospores also yielded the greatest hyphal dry weights. For the two C and two N sources tested, the number of ascospores increased as the ratio of C to N increased from 5:1 to 30:1. This effect was most obvious as the C:N ratio increased from 5:1 to 15:1. At low C:N ratios (<15:1), treatments with hypoxanthine as a N source resulted in significantly greater production of biomass than treatments with ammonium tartrate; no difference was observed at C:N ratios >/=15:1. Incidence of Verticillium wilt was 50% lower for eggplants drenched with ascospores grown on potato dextrose agar (PDA) compared with eggplants either nondrenched or drenched with ascospores grown on media with hypoxanthine plus lactose or maltose. Thus, C and N sources that slightly increased ascospore production of T. flavus reduced efficacy of biocontrol of Verticillium wilt compared with ascospores produced on PDA.     

The effect of different carbon sources on phenotypic expression by Fusarium graminearum strains

Two Fusarium graminearum strains were cultured in glucose yeast extract peptone broth or minimal medium broth to measure the production of mycelial biomass, pH, mycotoxins, and aurofusarin pigment, when limited to single carbon sources (at 1%), including xylan, cellulose, starch, or glucose. A random complete block design with factorial arrangement and analysis of variance at a significance level of 0.01 were employed to test for treatment differences. Overall, the F. graminearum strains produced significantly more biomass, deoxynivalenol, and aurofusarin with xylan than with cellulose. No significant differences were found in terms of 15–acetyldeoxynivalenol production from the four carbon sources. The presence of significant interactions between the strains, carbon sources, and media led to the following specific differences. In yeast extract peptone broth, R-9828 strain yielded significantly more deoxynivalenol production with xylan than cellulose and R-9832 produced significantly more mycelium (biomass) with xylan than cellulose. R-9828 strain yielded significantly more deoxynivalenol production than the R-9832 strain. Also in yeast extract peptone broth, cellulose led to significantly higher pH values than other carbons, which might be due to the limited ability of the Fusarium strains to utilize cellulose as an energy source. Aurofusarin was the only expressed analyte to show a significant difference in minimal medium broth, and R-9832 produced significantly more aurofusarin with xylan than with cellulose in the broth. These results suggest that xylan may induce Fusarium growth and deoxynivalenol production to assist the infection process and may support the theory that F. graminearum invades through xylan in the cell walls of cereals.     

New Selective Medium for Isolation of Burkholderia glumae from Rice Seeds

A new selective medium for Burkholderia glumae was developed, which has a simpler composition and greater selectivity compared to Tsushima's S-PG medium currently used. This selective medium, designated CCNT, contains 2 g of yeast extract, 1 g of polypepton, 4 g of inositol, 10 mg of cetrimide, 10 mg of chloramphenicol, 1 mg of novobiocin, 100 mg of chlorotharonil and 18 g of agar in 1000 ml of distilled water, and is adjusted to pH 4.8. B. glumae produced a yellowish white colony with a diffusible yellow pigment on CCNT medium, which was distinguishable from other bacterial species when incubated at 41°C for 2 to 4 days. On CCNT medium, B. glumae was detected at a rate similar to that on S-PG medium in rice seeds, while other microorganisms were detected at a much lower rate. Received 16 September 1999/ Accepted in revised form 4 January 2000