番茄灰霉病拮抗内生细菌的筛选、鉴定及其活性

对安徽省淮北市番茄植株根、茎、叶中内生细菌及其数量进行了调查和筛选,并测定了其抑菌活性。结果表明,番茄根、茎和叶中的内生细菌的数量分别为5.69×105、5.16×105和2.83×105CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到267株内生细菌,通过对峙实验,筛选到11株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生细菌总数的4.12%。来自茎组织中的菌株XF136的抑菌效果最佳,抑菌带宽度达32.2mm。根据形态特征、生理生化特性、(G+C)mol%和16SrDNA序列分析,将菌株XF136鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。室内测定菌株XF136发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明,菌株XF136发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为20%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明,10%菌株XF136发酵滤液对番茄灰霉病防治效果与50%多菌灵600倍液相当,20%发酵滤液对番茄灰霉病的防治效果高于50%多菌灵600倍液。本研究表明,菌株XF136是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

番茄内生菌的分离鉴定及菌株FQ-G3抗病促生特性

由灰葡萄孢(Botrytis cinerea)引起的灰霉病是番茄生产中最重要的病害之一,当前使用的杀菌剂因药物残留、病原菌抗药性及食品安全等原因逐渐受到限制。因此,利用拮抗微生物的生物防治逐渐成为灰霉病防控的有效策略。【目的】从番茄植株体内筛选具有抗病促生特性内生菌株并对其生防潜力进行评估,为开发番茄灰霉病生物防治新策略提供理论依据。【方法】采用组织分离法在番茄植株不同部位分离出内生细菌、真菌,结合16SrRNA和ITS序列分析,对候选菌株进行初步鉴定;通过菌株对峙培养、果实离体接种筛选对灰葡萄孢具有拮抗活性的内生菌;进一步测定菌株分泌生长素、嗜铁素的能力及其对拟南芥和番茄幼苗生长的促生特性。【结果】从番茄植株不同部位共分离出72株内生细菌和31株内生真菌,通过平板对峙法筛选出1株对多种病原菌具有较好抑菌活性的内生细菌FQ-G3,分子鉴定为Bacillus velezensis。FQ-G3对灰葡萄孢抑菌率达80.93%,并显著抑制灰葡萄孢在番茄果实上的扩展。该菌株能够分泌生长素、蛋白酶和嗜铁素,且对拟南芥、番茄幼苗具有明显的促生效果。【结论】本研究表明分离自番茄植株的内生菌FQ-G3具...     

番茄灰霉病拮抗内生放线菌的筛选、鉴定及其活性评价

对安徽省淮北市番茄植株根、茎、叶中内生放线菌进行了分离、筛选,并测定了其抑菌活性。结果表明:番茄根、茎和叶中的内生放线菌的数量分别为5.66×104、0.67×104和0.39×104CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到93株内生放线菌,通过对峙实验,筛选到7株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生放线菌总数的7.5%。来自根组织中的菌株HNU-EA27的抑菌效果最佳,抑菌圈直径达28.3mm。根据形态特征、培养特征、生理生化特性、细胞壁组分和16SrDNA序列分析,将菌株HNU-EA27鉴定为毒三素链霉菌(Streptomyces toxytricini)。室内测定菌株HNU-EA27发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明:菌株HNU-EA27发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为30%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明:30%菌株HNU-EA27发酵滤液对番茄灰霉病的预防与治疗效果分别为80.6%和73.8%,均高于50%多菌灵可湿性粉剂600倍液。本研究表明,菌株HNU-EA27是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

抗感青枯病番茄的内生细菌数量动态分析及其对青枯病的生物防治

对番茄内生细菌数量动态及其对青枯病的生物防治研究结果表明:番茄内生细菌可来源于种子内部。番茄不同生育期,内生细菌数量最多在成株期,其中抗病品种根、茎分别为24.3×104CFU/g鲜重和22.9×104CFU/g鲜重,感病品种根、茎分别为9.8×104CFU/g鲜重和13.4×104CFU/g鲜重。抗病品种中具有拮抗青枯菌的内生细菌菌株为17个,感病品种中7个。部分内生细菌具促进番茄种子萌发和防治番茄青枯病的作用,其中5R和3R内生菌株的防病效果分别达91.7%和81.3%。     

红树林内生细菌的分离及拮抗菌筛选

红树林内生细菌及拮抗菌分离筛选结果表明:各品种红树体内均有大量的内生细菌,不同红树品种及部位内生细菌的数量不同,其中以红海榄体内的含量最高,达4．225×104cfu／g(fw),其它依次为木榄、桐花树、秋茄和白骨壤等;不同部位以茎组织体内内生细菌的含量最多,达1．649×104cfu/g(fw),其次为根和叶。获得的内生细菌中约有43．53％的内生细菌菌株对枯萎病菌(Fusarium oxysporum)、炭疽病菌(Colletotrichum sp．)等病原真菌及番茄青枯病菌(Ralstonia solanaceance)具有较强拮抗作用。对番茄生长测定发现,13株菌株中有9株(69．23％)表现为促进生长效果,4株(30．77％)表现为抑制生长作用。经初步鉴定,上述拮抗细菌均为芽孢杆菌(Bacillus sp．)。     

棉花内生细菌数量动态及其对棉花黄、枯萎病菌的拮抗作用

摘要：【目的】了解棉花内生细菌数量动态,从棉花中获得拮抗黄、枯萎病菌的内生细菌资源【方法】对棉花根、茎、叶表面灭菌,采用稀释平板法分离棉花内生细菌;通过对峙培养法体外鉴定分离的棉花内生细菌对棉花黄、枯萎病菌的拮抗作用,并对拮抗棉花黄、枯萎病菌的内生细菌16S rDNA序列进行了分析【结果】棉花根中内生细菌的数量显著高于茎、叶;根的苗期总体内生细菌数量低于开花期、吐絮期,茎、叶中的内生细菌数量在不同生育期呈现一定的波动性,但趋势性不明显;6个棉花品种根中内生细菌平均数量差异并不显著,但茎、叶中内生细菌数量不同品种间呈现一定程度差异。平板对峙鉴定显示:棉花根中具有较高比例的拮抗棉花黄、枯萎病菌的内生细菌,拮抗强致病落叶型黄萎病菌（V107）的内生细菌比例不仅低于枯萎病菌（F108）,而且低于非落叶型黄萎病菌（V396）。同时拮抗棉花黄、枯萎病菌的内生细菌有44株,16S rDNA分子序列分析表明：这些拮抗内生细菌的类群包括了两个门(变形杆菌门、拟杆菌门)8个属,其中10个菌株与已报道菌株相似性<97%,可能是新的种（属）,优势种群为肠杆菌属（18株）、泛菌属（15株）。【结论】棉花的品种、生育期与器官影响棉花内生细菌数量;棉花拮抗黄、枯萎病菌的内生细菌具有优势种群,且具多样性。     

一株拮抗赤霉病的小麦内生细菌的筛选和抑菌活性

对安徽省淮北市小麦植株根、茎、叶中内生细菌的数量进行了调查和筛选,并测定了其抑菌活性.小麦根、茎和叶中的内生细菌的数量分别为7.01×105、4.26×105和0.94×105CFU·g-1鲜重.从健康小麦植株体内分离到131株内生细菌,通过对峙实验,筛选到6株对禾谷镰刀菌有拮抗作用的菌株,占所分离内生细菌总数的4.58%.来自根系组织中的菌株HB022的抑菌效果最佳,抑菌半径为17.0mm.根据形态特征、生理生化特性和16SrDNA序列分析,将菌株HB022初步鉴定为多粘类芽孢杆菌(Paenibacillus polymyxa).抑菌试验结果表明,100倍稀释的菌株HB022无菌发酵滤液对禾谷镰刀菌菌丝生长具有明显抑制作用,5倍稀释的无菌发酵滤液可完全抑制禾谷镰刀菌分生孢子萌发.可见,内生细菌HB022具有潜在生防应用前景.     

黄瓜青枯病内生拮抗菌株的分离及ARDRA分析

在黄瓜生长的不同阶段从根系分离内生细菌共469株。通过青枯菌平板拮抗试验,从中筛选到具明显拮抗作用的菌株59株。将内生拮抗菌纯培养物扩增近全长的16SrDNA并用限制性内切酶AluⅠ对PCR产物进行ARDRA(amplifiedrDNArestrictionanalysis)多态性分析,共得到5种不同的操作分类单元(OperationalTaxonomicUnit,OTU)。其中属于OTU1共有39株分离物,占内生拮抗菌总数的66%,为优势种群。进一步通过ERIC-PCR指纹图的方法在菌株水平上分析OTU1类群。结果表明,OTU1可分为12种不同的菌株,其中菌株HE-1和HE-2在黄瓜生长的5个不同阶段均可分离到。通过标记天然不具有利福平抗性的HE-1和HE-2菌株,获得抗利福平突变体菌株,回收检测结果表明,在栽培的不同时期,黄瓜植株根内均有HE-1和HE-2菌株的定殖。经防病效果的盆栽试验,发现HE-1和HE-2的浸种处理能有效降低黄瓜青枯病的病发率,与对照比较差异显著。因此确定HE-1和HE-2为黄瓜青枯病生物防治的优良菌株。     

四个品种烟草内生微生物的分布特征

对4个常规优质烟草品种(K326、G-80、红花大金元、净叶黄)的种子、根、茎、叶进行了内生菌的分离计数、初步分类与拮抗筛选研究.结果表明:烟草中有大量的内生菌分布,以细菌最多,放线菌次之,真菌最少,与土壤中的分布规律一致.烟草不同器官中内生菌的分布表现为根和种子中数量相当,茎次之,叶最少的规律.不同烟草品种间的分布为:种子中是K326和红花大金元内生菌分布较多,净叶黄次之,G-80最少;苗期植株中4个品种的内生菌数没有明显差别;大田期是净叶黄烟草的内生菌数量最少.烟草中内生细菌以革兰氏阴性菌为主,而种子中内生细菌的芽胞菌数量要明显多于植株中的.通过对大肠杆菌和金黄色葡萄球菌的抑菌实验,从红花大金元品种中筛选到有抑菌活性的菌株5株,从净叶黄和K326中分别筛选到1株,体现出了4个品种的抗病性与拮抗菌之间的一定相关性.     

拮抗辣椒疫病菌的红树内生细菌筛选及RS261菌株鉴定

红树内生细菌分离及拮抗辣椒疫霉(Phytophthora capsici)筛选结果表明：各红树体内均有大量的内生细菌, 不同红树种类及部位内生细菌的数量均不同, 被测定的红树内生细菌中约有27.97%的可培养菌株对辣椒疫霉具有拮抗作用; 其中18株拮抗作用较强的细菌在辣椒果上对辣椒疫病菌均有一定的抑制效果, 以来自红海榄叶片内的RS261菌株效果最好; 经形态、生理生化特征和分子生物学等测定分析, 将RS261菌株初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。     

香蕉植株内生细菌群落多态性研究

采用平板法对香蕉(Musa nana)植株的内生细菌进行分离纯化,并采用细菌脂肪酸法进行鉴定。结果表明,从香蕉的健康植株和感病植株中共分离得到内生细菌21属24种。从健株分离得到9种内生细菌,其中根、茎和叶分别分离到6种、2种和8种内生细菌。从病株分离得到15属17种内生细菌,其中根、茎和叶分别分离到3种、11种和6种。香蕉健株根部的内生细菌含量最高,达5.195×106cfu g-1,下部叶片内生细菌的含量最低,仅为30 cfu g-1;香蕉病株茎部内生细菌的数量显著高于其他部位,达1.05×107cfu g-1。这说明香蕉在不同生长状态下,其内生细菌的种类和数量存在多样性。     

大豆内生细菌的分离及根腐病拮抗菌的筛选鉴定

内生细菌存在于健康植物体内,一些内生细菌具有促生长、抗病和固氮等生物学功能.本项研究采用化学药剂表面灭菌方法从黑龙江省大豆品种合丰25的根、茎、叶和种子中分离到大量内生细菌,其种群数量在根部最多,为3.4×103CFU/g,在叶部次之,为2.8×103CFU/g,在茎部和种子中最少,为2.9×102 CFU/g和1.4×102CFU/g.从121株内生细菌中筛选到31株对大豆根腐病菌Fusarium oxysporum f. sp.soybean具有较强抑制作用的拮抗内生细菌,其中菌株TF28抑菌谱广,抑菌率高,对不同植物的病原菌F. oxysporum的抑菌率为80.2%～96.7%.经形态、生理生化和16S rRNA鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).     

香樟产芽孢内生细菌的系统发育多样性

为了解香樟产芽孢内生细菌的多样性,采用改良的NA培养基分离、去除冗余及芽孢染色,得到40株产芽孢内生细菌,占分离所得内生细菌总数的29.9%,其中根、茎、叶中分别分离到25、5和10株。16SrRNA序列系统发育分析结果表明,这40株菌分属于Bacillus、Lysinibacillus、Paenibacillus和Brevibacillus属的12个种;7株菌的16SrRNA部分序列与数据库中模式菌株对应序列相似性小于97%,代表着潜在新类群的存在。同时,3个部位分离出的产芽孢内生细菌既呈现出一定程度的细菌区系相似性,又表现出细菌区系的器官特异性。     

Specific and Functional Diversity of Endophytic Bacteria from Pine Wood Nematode Bursaphelenchus Xylophilus with Different Virulence

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most devastating diseases of Pinus spp. The PWN was therefore listed as one of the most dangerous forest pests in China meriting quarantine. Virulence of the PWN is closely linked with the spread of PWD. However, main factors responsible for the virulence of PWNs are still unclear. Recently epiphytic bacteria carried by PWNs have drawn much attention. But little is known about the relationship between endophytic bacteria and virulence of B. xylophilus. In this research, virulence of ten strains of B. xylophilus from different geographical areas in six provinces of China and four pine species were tested with 2-year-old seedlings of Pinus thunbergii. Endophytic bacteria were isolated from PWNs with different virulence to investigate the relationship between the bacteria and PWN virulence. Meanwhile, the carbon metabolism of endophytic bacteria from highly and low virulent B. xylophilus was analyzed using Biolog plates (ECO). The results indicated that ten strains of PWNs showed a wide range of virulence. Simultaneously, endophytic bacteria were isolated from 90% of the B. xylophilus strains. The dominant endophytic bacteria in the nematodes were identified as species of Stenotrophomonas, Achromobacter, Ewingella, Leifsonia, Rhizobium, and Pseudomonas using molecular and biochemical methods. Moreover, S. maltophilia, and A. xylosoxidans subsp. xylosoxidans were the predominant strains. Most of the strains (80%) from P. massoniana contained either S. maltophilia, A. xylosoxidans, or both species. There was a difference between the abilities of the endophytic bacteria to utilize carbon sources. Endophytic bacteria from highly virulent B. xylophilus had a relatively high utilization rate of carbohydrate and carboxylic acids, while bacteria from low virulent B. xylophilus made better use of amino acids. In conclusion, endophytic bacteria widely exist in B. xylophilus from different pines and areas; and B. xylophilus strains with different virulence possessed various endophytic bacteria and diverse carbon metabolism which suggested that the endophytic bacteria species and carbon metabolism might be related with the B. xylophilus virulence.     

盐地碱蓬内生中度嗜盐菌的分离与系统发育多样性分析

为了了解东营滨海盐地碱蓬植株内生中度嗜盐菌的多样性,采用传统分离鉴定技术和基于16S rRNA序列分析对样品中可培养细菌的多样性进行研究。根据其生理生化特征、16S rRNA序列测定和系统发育分析,分离获得的15株内生菌可分为4个类群,涉及Halomonadaceae科的Chromohalobacter属、Kushneria属、Halomonas属以及Bacillaceae科的Bacillus属。类群I中4菌株的16S rRNA序列与Chromohalobacter israelensis的最高相似性为95%。类群II共7株菌,归属于Kushneria属,是碱蓬内生中度嗜盐菌中的优势类群。类群III菌株的16S rRNA序列与一株尚无明确分类地位的Gammaproteobacteria亚门耐盐固氮细菌Haererehalobacter sp.JG11的相似性为99%。类群IV中的芽孢杆菌的16S rRNA序列与已知细菌的相似性为96%,很可能代表了Bacillus属的新种。各种水解酶类的分析表明,在分离的15株菌中有3株菌产蛋白酶,14株产酯酶,8株产DNA酶,11株产半乳糖苷酶,14株产脲酶。研究结果揭示,盐地碱蓬中存在较为丰富的中度嗜盐菌多样性和系统发育多样性,并且潜藏着较多的新的微生物类群。     

Ability of Moderately Halophilic Bacteria to Control Grey Mould Disease on Tomato Fruits

Tomato is one of the leading crops in Tunisia in terms of weight consumed (20 kg/per person/year). Preserving the quality of the fruit from field to consumer is essential to successful marketing. Grey mould rot induced by Botrytis cinerea is an important cause of postharvest loss depending on season and handling practices. We describe here the ability of halotolerant to moderately halophilic bacteria isolated from different Tunisian Sebkhas (hypersaline soils) to protect fresh‐market tomato fruits from B. cinerea. The tomatoes tested were at two different stages of ripening, (i) mature‐green and (ii) red. Six strains significantly reduced growth of the pathogens from 67% to 87%. The effectiveness of these antagonists was also confirmed on green tomatoes; in which the fruit rot protection rate ranged from 74% to 100%. The antagonists were characterized by morphological, biochemical and physiological tests as well as 16S rDNA sequencing. The halotolerant effective isolates were identified as belonging to one of the species Bacillus subtilis (M1‐20, J9) or B. licheniformis (J24). One effective moderately halophilic isolate (M2‐26) was identified as Planococcus rifietoensis. These strains are a source of hydrolytic enzymes such as chitinases, proteases, laminarinases, amylases, lipases and cellulases. For comparison, 12 halotolerant or moderately halophilic strains obtained from DSM culture collection were also evaluated for their antifungal activity against B. cinerea on tomato fruits. The most effective strains were Halomonas subglaciescola, Halobacillus litoralis, Marinococcus halophilus, Salinococcus roseus, Halovibrio variabilis and Halobacillus halophilus with a percentage of grey mould rot reduction ranging from 71% to 97%. Inoculation of mature‐green tomatoes by the bacterial antagonist of Halobacillus trueperi resulted in no disease development. Our results indicate that the use of halotolerant to halophilic micro‐organisms should be helpful in reducing grey mould disease of stored tomatoes.