Mutagenesis of beta-1,3-Glucanase Genes in Lysobacter enzymogenes Strain C3 Results in Reduced Biological Control Activity Toward Bipolaris Leaf Spot of Tall Fescue and Pythium Damping-Off of Sugar Beet

ABSTRACT Lysobacter enzymogenes produces extracellular lytic enzymes capable of degrading the cell walls of fungi and oomycetes. Many of these enzymes, including beta-1,3-glucanases, are thought to contribute to the biological control activity expressed by several strains of the species. L. enzymogenes strain C3 produces multiple extracellular beta-1,3-glucanases encoded by the gluA, gluB, and gluC genes. Analysis of the genes indicates they are homologous to previously characterized genes in the related strain N4-7, each sharing >95% amino acid sequence identity to their respective counterparts. The gluA and gluC gene products encode enzymes belonging to family 16 glycosyl hydrolases, whereas gluB encodes an enzyme belonging to family 64. Mutational analysis indicated that the three genes accounted for the total beta-1,3-glucanase activity detected in culture. Strain G123, mutated in all three glucanase genes, was reduced in its ability to grow in a minimal medium containing laminarin as a sole carbon source. Although strain G123 was not affected in antimicrobial activity toward Bipolaris sorokiniana or Pythium ultimum var. ultimum using in vitro assays, it was significantly reduced in biological control activity against Bipolaris leaf spot of tall fescue and Pythium damping-off of sugar beet. These results provide direct supportive evidence for the role of beta-1,3-glucanases in biocontrol activity of L. enzymogenes strain C3.     

产酶溶杆菌OH11双精氨酸转运系统关键基因tatC的功能分析

本研究采用同源重组的方法对产酶溶杆菌Lysobacterenzymogenes OH11中双精氨酸转运系统关键基因tatC进行缺失突变,通过对突变体表型及表型相关基因表达分析来研究该基因在菌株OH11中的功能,重点分析该基因与产酶溶杆菌中热稳定抗真菌因子HSAF生物合成的关系。研究结果表明,基因tatC的突变显著提高了HSAF生物合成的产量,以及HSAF生物合成的关键基因pks／nrps转录水平;改变了菌株OHll的细胞形态,使细胞从棍棒状变为椭圆状;并降低了菌株OH11生物膜的形成和滑动能力以及在营养缺陷型（10％TSB）培养基中的生长速率,但不改变其在营养丰富培养基（100％TSB）中的生长速率。此外,与野生型OH11相比,基因tatC突变株不改变其蛋白酶、纤维素酶、β-1,3-葡聚糖酶和几丁质酶的产生水平以及其对瓜果腐霉Pythiumapanidermamm、立枯丝核菌Rhizoctoniasolani和禾谷镰刀菌Fusariumgraminearum的拮抗能力。     

产酶溶杆菌OH11菌株α-水解蛋白酶基因的克隆与表达

α-水解蛋白酶是产酶溶杆菌OH11菌株分泌的一种胞外丝氨酸蛋白酶。利用PCR方法从OH11菌株中克隆到该基因。同源性比较其推测产物与产酶溶杆菌ATCC29478菌株和ATCC29487菌株的α-水解蛋白酶有90%以上的序列一致性。基因经NdeⅠ和XhoⅠ双酶切后连接到含T7启动子的高表达载体pET30a( )上构建重组质粒pαLP,并转化宿主菌BL21(DE3)获得BLαLP表达菌株。经IPTG诱导后发现BLαLP菌株产生一相对分子量约为44kD的蛋白。研究表明,该蛋白能将蛋白质水解,在脱脂奶粉培养基上形成水解圈,并对立枯丝核菌菌丝的生长有抑制作用。     

An antibiotic complex from Lysobacter enzymogenes strain C3: antimicrobial activity and role in plant disease control

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 mug/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 mug/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and DeltaNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.     

Biological Control of Bipolaris sorokiniana on Tall Fescue by Stenotrophomonas maltophilia Strain C3

ABSTRACT Stenotrophomonas maltophilia strain C3 was evaluated for control of leaf spot on tall fescue (Festuca arundinacea) caused by Bipolaris sorokiniana. In growth chamber experiments, C3 inhibited conidial germination on leaf surfaces and reduced lesion frequency and percent diseased leaf area compared with nontreated controls. The amount of leaf spot suppression was related to the C3 dose applied. The highest dose tested, 10(9) CFU/ml, prevented nearly all B. sorokiniana conidia from germinating on treated leaf surfaces and provided nearly complete suppression of lesion development. When colloidal chitin was added to C3 cell suspensions of 10(7) or 10(8) CFU/ml, biocontrol efficacy was significantly increased over C3 applied alone, whereas addition of chitin to a C3 cell suspension of 10(9) CFU/ml had no effect. In field experiments, application of C3 to tall fescue turf resulted in significant reductions in infection frequency and disease severity compared with nontreated controls. Strain C3 applied at 10(9) CFU/ml was more effective than C3 applied at 10(7) CFU/ml, and amendment of the lower dose with colloidal chitin enhanced its efficacy. Populations sizes of C3 established on foliage in a growth chamber and in the field were directly related to dose applied. Chitin amendments did not affect C3 population size.     

产酶溶杆菌OH11菌株胞外酶调控相关基因ctp的克隆及功能的初步分析

利用mariner转座子对产酶溶杆菌Lysobacter enzymogenes OH11进行转座诱变,构建菌株OH11的突变体文库.筛选到1株4种胞外酶(蛋白酶、纤维素酶、几丁质酶和β-1,3-葡聚糖酶)产生均减少的突变株D-11.通过亚克隆,鉴定转座子的插入位点,涉及1个羧基末端蛋白酶(carboxy-terminal protease)编码基因ctp.通过同源重组的方法对该基因进行敲除,对缺失突变体Δctp表型分析发现:(1)Δctp与野生型OH11在营养丰富型(2YT)与营养缺陷型(MMX)培养基中生长速率基本一致;(2)Δctp降低了蛋白酶、纤维素酶和β-1,3-葡聚糖酶的产生,但不影响几丁质酶的产生;(3)Δctp生物膜的产生量明显减少.研究还表明,突变体基本不改变其对油菜菌核病菌Sclerotinia sclerotiorum、水稻纹枯病菌Rhizoctonia solani和辣椒疫霉病菌Phytophthora capsici的拮抗能力.互补菌株均恢复了野生型的相关功能.     

Priming as a Mechanism in Induced Systemic Resistance of Plants

Induced systemic resistance is a plant defence state that is associated with an enhanced ability – the so-called priming – to resist pathogen attack by stronger activation of cellular defence responses. So far, however, priming has not been widely appreciated when studying induced plant disease resistance. During the past several years, it has been demonstrated that pre-treatment of cultured parsley cells with inducers of systemic resistance, salicylic acid or a benzothiadiazole, leads to the direct activation of a set of defence-related genes and also primes the cells for stronger elicitation of another set of defence genes including those encoding phenylalanine ammonia-lyase. From these results, it was concluded that the resistance inducers have at least a dual role in plant defence-gene activation. When elucidating whether priming plays a role in induced systemic resistance of Arabidopsis, pre-treating plants with benzothiadiazole was found to augment the subsequent activation of phenylalanine ammonia-lyase genes by Pseudomonas infection, wounding and osmotic stress and also to enhance wound/osmotic stress-induced callose production. The augmentation of phenylalanine ammonia-lyase gene activation or/and callose deposition was not seen in the Arabidopsis non-expresser of pathogenesis-related genes1 mutant which is compromised in induced resistance, while it was present, without benzothiadiazole pre-treatment, in the constitutive expresser of pr genes1 and 5 mutants in which induced resistance is constitutive. Together these studies point to priming as an important cellular mechanism in induced systemic resistance of plants which requires the intact non-expresser of pathogenesis-related genes1 gene.     

The Role of Chitinase Production by Stenotrophomonas maltophilia Strain C3 in Biological Control of Bipolaris sorokiniana

ABSTRACT The role of chitinase production by Stenotrophomonas maltophilia strain C3 in biological control of leaf spot on tall fescue (Festuca arundinacea), caused by Bipolaris sorokiniana, was investigated in vitro and in vivo. The filtrate of a broth culture of C3, with chitin as the carbon source, was separated into fractions. A high molecular-weight fraction (>8 kDa) was chitinolytic and more inhibitory than a low-molecular-weight, nonchitinolytic fraction to conidial germination and hyphal growth by B. sorokiniana and to leaf spot development. A protein fraction derived by ammonium sulfate precipitation and a chitinase fraction purified by chitin affinity chromatography also were chitinolytic and highly antifungal. The chitinolytic fractions caused swelling and vacuolation of conidia and discoloration, malformation, and degradation of germ tubes. When boiled, the chitinolytic fractions lost chitinase activity along with most of the antifungal properties. Two chitinase-deficient and two chitinase-reduced mutants of C3 were compared with the wild-type strain for inhibition of germination of B. sorokiniana conidia on tall fescue leaves and for suppression of leaf spot development in vivo. The mutants exhibited reduced antifungal activity and biocontrol efficacy, but did not lose all biocontrol activity. An aqueous extract of leaves colonized by wild-type C3 had higher chitinase activity than that of noncolonized leaves and was inhibitory to conidial germination. The addition of chitin to leaves along with the wild-type strain increased both chitinase and antifungal activity. The chitinase activity level of extracts from leaves colonized by a chitinase-deficient mutant of C3, with and without added chitin, was no higher than the background, and the extracts lacked antifungal activity. Chitinolysis appears to be one mechanism of biological control by strain C3, and it functions in concert with other mechanisms.     

枯草芽孢杆菌G3菌剂防治番茄叶霉病田间试验

番茄叶霉病由黄枝孢菌 (Cladosporiumfulvum)引起 ,严重为害番茄叶片和果实。随着温室大棚大面积推广 ,上海郊区番茄叶霉病越发严重 ,成了番茄栽培的重要问题。该病在上海通常于 3月份发病 ,5月达到高峰[1] 。但随着近年来的暖冬效应 ,少数大棚 1月份便开始发病 ,3月份达发病高峰。常规化学杀菌剂 ,如百菌清、多菌灵防效甚微且污染环境 ,急需高效低毒的药剂取而代之。作者曾报道产几丁质酶枯草芽孢杆菌 (Bacillussubtilis)G3菌剂在盆栽试验中对番茄叶霉病有较好的防治效果[2 ] 。本文报道G3菌剂田间防治该病的效果1　材料与方法1 .1　供试…     

枯草芽孢杆菌TR21对香蕉抗病相关基因表达的诱导作用

广谱抗真菌枯草芽孢杆菌Bacillus subtilis菌株TR21在温室和大田对香蕉枯萎病具有较好的防效,其机制已证明与诱导香蕉产生系统抗性有关。本文以巴西蕉（Musa AAA Cavendish subgroup cv.Brazil）为材料,利用半定量RT-PCR法,以香蕉25S rRNA基因为内标,研究灌根接种菌株TR21后对香蕉根系4种抗病相关基因表达的影响。结果表明,PAL、POD、PR-3和PR-1基因在接种后表达水平均表现上调趋势,但PAL和POD基因的表达增幅明显高于PR-3和PR-1基因。PAL和POD基因在接种后12 h表达量最高,与TR21在香蕉根部的定殖规律表现出一致性。系统诱导抗性是枯草芽孢杆菌TR21防治香蕉枯萎病的机制之一。     

青枯病生防菌的筛选、鉴定及其活性物质特性

从发生青枯病的土壤中分离得到4株对植物青枯病病原菌青枯劳尔氏菌具有较强拮抗活性的菌株，其中菌株HY96-2的拮抗活性最强。该菌对其他5种危害严重的植物病原真菌均具很强的拮抗作用。通过对HY96-2的形态、生理生化特征、16S rDNA序列等进行分析，鉴定该菌株为多粘类芽孢杆菌。在此基础上，通过盐析等方法得到HY96-2产生的对青枯病病原菌具有拮抗活性的粗提物，检测了温度和pH对粗提物活性的影响。当温度高于40℃、pH低于4．3或高于8．2时，活性很弱；蛋白酶K作用容易失活；超滤结果表明，该粗提物可能是分子量低于5kD的混合物。     

Development of Strain-specific Primers for a Strain of Gliocladium catenulatum Used in Biological Control

The randomly amplified polymorphic DNA (RAPD) technique was used to develop strain-specific primers for Gliocladium catenulatum strain J1446, which is promising in biological control. One of the primer pairs developed proved to be strain-specific; strain J1446 was differentiated from 16 G. catenulatum strains and six other strains of two Gliocladium species, as well as from Trichoderma virens, and isolates of Nectria spp. and Fusarium spp. Specific primers were also tested with DNA isolated from cucumber leaves, treated or untreated with a solution made from Gliocladium powder. The expected amplification product was produced only from treated leaves. DNA isolated from Gliocladium-treated potato tubers and fungi grown in peat was also used in amplification reactions. Strain-specific primers detected strain J1446 when the amount of DNA was 5pg or more. Some variation between the Gliocladium strains was found by the random amplified microsatellites method (RAMS) and the universally primed polymerase chain reaction method (UP-PCR), but no clear fragments specific to strain J1446 were produced. Cross-blot hybridisation of UP-PCR products differentiated strain J1446 from T. virens, but not from the Gliocladium isolates. The 28S rDNA sequences and -tubulin sequences were identical or very similar in all Gliocladium strains. Thus, it is possible that the Gliocladium strains of the present study are conspecific, which means that a revision in the taxonomy of Gliocladium species may be necessary.     

寡雄腐霉生防机理及应用研究进展

寡雄腐霉是一种重要的微生物农药,因其对病原菌具有广谱、高效的防治特性及对作物具有促生长和增产等特点被广泛关注。本文从寡雄腐霉对植物病原菌抗性、诱导植物产生诱导系统抗性、促进植物生长等方面对其生防的分子机理和信号转导的研究进展进行了综述,总结了寡雄腐霉在防治植物病害方面的应用,并对研究和应用过程中存在的问题及解决途径提出展望。     

堆肥提取液诱导草莓对黄萎病抗性及抑菌机理研究

为了明确菌糠堆肥提取液的生物防治效果,进一步探讨其抑菌防病机理,以便于有针对性地利用堆肥提取液达到防病、治病的效果。采用体内和体外2种试验方法,以草莓黄萎病菌为靶标病原菌,研究了堆肥提取液原液、高温灭菌液和过滤除菌液对大丽轮枝菌菌丝生长和分生孢子萌发的抑制作用,通过温室盆栽试验验证其对草莓黄萎病的防病促生效果,并测定根施堆肥提取液对草莓叶片内防御酶系的影响。研究表明,堆肥提取液对病原菌菌丝生长和分生孢子萌发均有显著的抑制作用,抑制率分别为92.43%和82.94%,经高温灭菌和过滤除菌后抑制效果显著降低。盆栽试验结果表明,预先用堆肥提取液灌根处理对草莓黄萎病的防治效果高达42.23%,且能诱导草莓叶片内防御酶活性显著增强,接病原菌后第6 d该处理下的POD和PPO活性分别比施清水对照提高了45.45%和39.47%,而MDA含量则下降了14.83%。说明微生物对病原菌的直接抑制作用以及诱导系统抗性是菌糠堆肥提取液发挥抑菌防病功效的主要因素。     

壳寡糖诱导植物抗病性研究进展

壳寡糖是由2-10个氨基葡糖通过β-1,4-糖苷键连接而成的低聚糖,具有多种生物学活性。本文对壳寡糖诱导的植物抗病性及其诱导植物抗性机理进行了评述。     

真核表达产物Y3诱导烟草对烟草花叶病毒的抗性研究

从毛头鬼伞Coprinus comatus中提取的碱性糖蛋白Y3可以降低烟草花叶病毒(TMV)的侵染.克隆获得Y3蛋白cDNA后与真核表达载体pPIC-9k连接,重组载体pPIC-9k-Y3成功电转化入毕赤酵母Pichia pastoris后,转化子在28℃、250 r/min培养条件下,使用1.0％甲醇诱导表达6d,...     

Antibiosis Contributes to Biological Control of Fire Blight by Pantoea agglomerans Strain Eh252 in Orchards

ABSTRACT Fire blight, caused by Erwinia amylovora, is the most serious bacterial disease of pear and apple trees. Biological control with strains of Pantoea agglomerans (syn. Erwinia herbicola) may provide an effective disease management strategy for fire blight. Most strains of P. agglomerans evaluated for suppression of fire blight produce compounds that inhibit the growth of E. amylovora in culture. The role of these inhibitory compounds in fire blight suppression in orchard environments has not been studied. In seven field trials in Oregon, we compared the population dynamics and disease suppression with P. agglomerans Eh252, a strain that produces a single antibiotic, with its near-isogenic antibiotic-deficient derivative, strain 10:12. Water or suspensions of Eh252 or 10:12 (1 x 10(8) CFU/ml) were applied at 30 and 70% bloom to pear or apple trees. Aqueous suspensions of freeze-dried cells of E. amylovora (3 x 10(5) CFU/ml) were applied at full bloom. Additional trees were treated with streptomycin or oxytetracycline at 30 and 70% bloom and in some experiments, 1 day after application of the pathogen. Population sizes of Eh252 or 10:12 on pear blossoms were estimated by spreading dilutions of blossom washes on culture media. Average population sizes of Eh252 and 10:12 on blossoms ranged from 10(5) to 10(7) CFU, and in five of six trials, the relative area under the population curve of Eh252 was not significantly different than that of its derivative 10:12. Both Eh252 and 10:12 reduced the growth of the pathogen on blossoms compared with inoculated water-treated controls. Eh252 significantly decreased the incidence of fire blight in six of seven field trials compared with the incidence on water-treated trees, and 10:12 similarly reduced the incidence of fire blight in four of seven trials. In three of seven field trials, trees treated with Eh252 had a significantly lower incidence of fire blight compared with trees treated 3 with 10:12. Overall,3 Eh252 reduced the incidence of fire blight by 55 +/- 8%, 10:12 by 30 +/- 6%, streptomycin by 75 +/- 4%, and oxytetracycline by 16 +/- 14%. The effectiveness of strain 10:12 compared with water treatment indicates that other mechanisms (e.g., competitive exclusion or habitat modification) also contribute to disease suppression by P. agglomerans. The increased suppression of fire blight by the parental strain Eh252 compared with the antibiotic-deficient mutant 10:12 indicates that antibiosis is an important mechanism of biological control of fire blight.     

土壤杆菌K1026对果树根癌病的生物防治

由致病性根癌土壤杆菌Agrobacterium spp.侵染引起的根癌病对山东及我国其他省份的樱桃、桃及其他果树生产造成严重的影响.土壤杆菌Agrobacterium rhizogenes K1026在澳大利亚已制成生防菌剂并在全球多个国家实现商品化,但在我国果树育苗和生产行业中还未得到广泛应用.本文从山东樱桃和桃树根癌组织及感病土壤中分别分离到100余株致病性土壤杆菌,经鉴定均属于生物Ⅱ型土壤杆菌;利用双层平板抑菌试验,检测了37株病原菌对菌株K1026的敏感性,结果显示,所有菌株均对K1026产生的抗生物质敏感,可以在Stonier,s培养基上产生5种以上不同类型的抑菌圈;胡萝卜切片试验显示,菌株K1026可完全抑制37株病原菌对胡萝卜的致瘤活性.上述结果为土壤杆菌K1026用于山东省樱桃及桃树根癌病的生物防治奠定了基础.     

β-氨基丁酸诱导植物抗病作用及其机理

β-氨基丁酸(BABA)作为一种结构简单的非蛋白质氨基酸,不仅具有广谱的诱导抗病活性,而且与多种化合物具有协同增效作用,是一种极具潜力的植物化学诱抗剂。综述了BABA诱导植物抗病性的特点、作用机理、使用方法及其代谢方式,并对其应用前景进行了展望。     

Resistance as a Means of Control of the Potato Root Eelworm

A survey of the resistance of wild potatoes to the potato root eelworm and the use made of this property in potato-breeding is presented. Several biotypes of this parasite have been found to exist. The use of resistant varieties in the control of the potato root eelworm, in connection with other methods of control (crop rotation and soil disinfestation), is discussed.