拮抗细菌B8对烟草黑胫病菌的抑制作用及其菌株鉴定

从烟草根际土壤中分离到对烟草黑胫病菌及其它植物病原菌具有抑制作用的菌株B8,平板对峙培养抑菌带内菌丝畸形、原生质凝集或外渗。其发酵原液和发酵液的无菌滤液均能抑制烟草黑胫病菌和蜡状芽孢杆菌;离体叶片接种法测定其发酵液对烟草黑胫病的防效,结果表明预防作用达100%。室内盆栽试验表明B8菌株发酵液对烟草黑胫病的预防作用可达78.1%,优于治疗作用。该菌菌体杆状,芽孢侧生、椭圆形,经形态学、生理生化性状和16S rDNA序列测定,将其鉴定为侧孢短芽孢杆菌。     

烟草疫霉拮抗细菌B44R-1的筛选及其生防效果

为获得烟草疫霉Phytophthora nicotiana的优良拮抗菌,从烟草根际土壤和根系中分离对烟草疫霉具有较好拮抗作用的菌群,并从中筛选具有较强抑菌效果的拮抗菌株。通过形态学、生理生化试验、16S rDNA基因测序分析鉴定拮抗菌株种属,评价其防病效果及对烟草的促生作用。结果表明,从烟草根际土壤和根系分离得到拮抗菌群39个,并从中筛选得到拮抗效果较好的菌株B44R-1,初步鉴定其为皮特不动杆菌Acinetobater pittii。菌株B44R-1对烟草黑胫病的防效达到55.37%,对烟株根系生长发育影响明显,烟株鲜重、干重、株高、茎围、叶数和最大叶面积相比对照均有提高。本研究筛选获得对烟草黑胫病菌的拮抗菌不动杆菌,对烟草黑胫病防治效果和促生作用显著,具有较好的生防潜力。     

拮抗放线菌TA21对烟草根黑腐病菌的抑制及其控病作用

菌株TA21是一株从重庆烟区土壤中筛选出的对烟草根黑腐病菌Thielaviopsis basicola有较强拮抗作用的放线菌,为了明确其抑菌效果和控病作用及其在烟草根黑腐病生防中的应用潜力,本文进行了菌株TA21抑菌活性检测和温室控病测试;并通过形态学、生理生化和分子分类的方法确定其分类地位。室内测定试验表明,菌株TA21对烟草根黑腐病菌的抑菌带宽达12.5mm;温室控病试验发现其对烟草根黑腐病防治效果达85.3%。无菌滤液试验表明,在试验浓度范围内无菌滤液均能有效抑制烟草根黑腐病菌菌丝生长、减少孢子萌发。根据菌株形态特征、培养性状、生理生化特性、细胞组分及16S rDNA序列分析,确定菌株TA21为链霉菌属吸水链霉菌Streptomyces hygroscopicus。     

烟草黑胫病和根黑腐病生防假单胞杆菌的筛选与鉴定

为获得对烟草黑胫病和根黑腐病具有双重防病效果且能够促进烟草生长的假单胞杆菌,采用稀释涂布法从40份土壤样品中分离出201株细菌,通过平板对峙和含毒介质法,筛选出对烟草黑胫病和烟草根黑腐病病原菌均具有良好拮抗作用的菌株PA2101和PG3402。盆栽促生试验表明,菌株PA2101和PG3402能协调地改善烟草地上部分的生长和烟草根系发育,均在一定程度上增加了烟草的株高、叶面积、株鲜重、根鲜重、叶片数和根长,提高了烟草的根冠比和根活力。盆栽试验结果表明,菌株PA2101对烟草黑胫病和根黑腐病的防效分别为70.11%和62.67%,均高于其对照药剂;菌株PG3402对两种病害的防效分别为60.92%和60.00%,与对照药剂相当。抗性标记菌株的定殖试验结果表明,菌株PA2101和PG3402在接种后第29 d能定殖于烟草根际土壤和根内,在烟草茎和叶内也能长时间存在,表明两菌株能够良好定殖。16Sr DNA序列、菌落形态和生理生化性状分析表明,菌株PA2101为铜绿假单胞杆菌Pseudomonas aeruginosa,菌株PG3402为格拉纳达假单胞杆菌Pseudomonas granadensis。综上所述,菌株PA2101和PG3402对烟草具有良好的促生作用,并对烟草黑胫病和根黑腐病有较好的防病效果,是具有生防潜力的菌株。     

烟草根黑腐病发生规律及综合防控技术

由根串珠霉(Thielaviopsis basicola)引起的烟草根黑腐病是我国烟草产区的主要病害之一。本文总结了烟草根黑腐病的发生与危害、病原学、生物学特性、发病条件及规律等,提出了以农业防治为基础、生物防治为核心,科学合理使用化学药剂的综合防控措施,为烟草根黑腐病的防治提供参考。     

烟草疫霉拮抗放线菌的生防潜力评价

由寄生疫霉烟草致病变种Phytophthora parasitica var. nicotianae引起的烟草黑胫病是连作烟田的重要土传病害之一,重发年份常给烟农造成巨大的经济损失。本研究通过稀释分离法从贵州省遵义市烟草黑胫病重病区的健康烟草根际土壤中共分离获得179株放线菌。通过平板对峙培养法筛选得到15株对烟草黑胫病菌菌丝生长有明显抑制作用的菌株。测定了这15个菌株代谢液对烟草疫霉菌丝生长的抑制作用,其中H-3菌株代谢液对烟草疫霉菌丝生长抑制率最高,达到85.68%。研究了H-3代谢液对烟草疫霉游动孢子萌发的抑制作用。结果显示培养6、12和24h对游动孢子萌发的抑制率分别为72.75%、65.20%和63.45%。结合形态特征及16S rDNA序列分析,初步鉴定H-3菌株属于链霉菌属Streptomyces sp.。盆栽防效和田间防效试验结果表明,施用H-3对烟草黑胫病具有一定的防效,当添加H-3固体发酵物22.5 kg/hm2时田间防效最好,防效可达70.42%。通过荧光定量PCR技术检测了烟草根际土壤中烟草疫霉的量,结果发现,随着H-3用量的增加,根际土壤中烟草疫霉的拷贝数呈下...     

烟草疫霉拮抗菌株P 72 10的鉴定及其拮抗代谢 产物 初步分析

 为探讨烟草根际生防细菌的生防机制,从重庆地区连作烟田健康烟株根际土壤中分离筛选到1株对烟草疫霉具有较强拮抗作用和对黑胫病具有良好防效的细菌菌株P-72-10。根据培养性状、形态特征、生理生化特性、基因组DNA的(G+C)mol%含量测定以及16S rDNA基因序列分析确定该菌株的分类地位。该菌株菌落乳白色,能产生水溶性荧光色素,革兰氏染色反应阴性,菌体杆状、大小(8.1~16.2)μm×(1.8~4.8)μm,单端生鞭毛,不形成芽孢。The BIOLOG GN2 结果显示该菌株属于假单胞菌属Pseudomonas。该菌株基因组DNA的(G+C)mol%含量为 60.72 mol%。16S rDNA基因序列分析显示该菌株与假单胞菌属荧光假单胞杆菌多个菌株的序列同源性达到99%,GenBank上的登录号为：HQ888871。结合其形态特征和生理生化特性,将菌株P-72-10鉴定为荧光假单胞杆菌P. fluorescens。平板检测拮抗代谢产物结果表明：该菌株具有分解纤维素、蛋白质和结合Fe ³⁺的能力,但不能分解几丁质。     

烟草赤星病菌拮抗菌解淀粉芽胞杆菌YW-2-6鉴定及生防效果

为获得有效拮抗烟草赤星病菌的生防资源,采用稀释分离法和平板对峙法从烟草根际土壤中分离筛选烟草赤星病菌拮抗菌株。通过形态学观察、生理生化特征和16S rDNA序列分析进行鉴定,并测定其对烟株的促生作用,验证菌株的产IAA能力,评价生防菌发酵液对烟草赤星病的田间防效。结果表明,从分离的65株细菌菌株中筛选到5株烟草赤星病拮抗细菌,其中菌株YW-2-6对烟草赤星病菌拮抗效果最好,抑制率为71.32%,初步鉴定菌株YW-2-6为解淀粉芽胞杆菌Bacillus amyloliquefaciens。菌株YW-2-6对小麦赤霉病菌、辣椒疫霉病菌、烟草靶斑病菌等病原菌具有显著的拮抗作用。YW-2-6发酵液对烟草赤星病防效为56.93%,且菌株YW-2-6对烟株有明显的促生作用。菌株YW-2-6具有良好的生防潜力和开发前景。     

烟草根黑腐病综合防治技术

烟草根黑腐病Thielaviopsis basicola (Brek．et Br．) Ferr．是世界性的烟草病害之一,在我国主要分布于河南、山东、安徽、云南、重庆等地[1]。从幼苗到成株期均可发病,主要为害烟株根系,使根出现黑色腐烂。在有的烟区,烟草根黑腐病已经上升为主要病害,常与黑胫病混合发生,造成烟草损失。     

植物土传病原菌拮抗细菌的筛选与鉴定

从作物根际土壤中分离到1056株细菌，筛选出7个具有较强拮抗活性的菌株。室内测定对水稻纹枯病菌、辣椒疫病菌、瓜果腐霉、油菜菌核病菌、棉花枯萎病菌、茄根腐病菌、棉花黄萎病菌、番茄青枯病菌、甘蓝黑腐病菌等重要土传病原菌有较强拮抗作用；温室盆栽试验对番茄青枯病表现出较好防效，其中以BOH2和OH11效果较为明显，防效分别为90．9％和86．4％。通过形态观察、生理生化试验和16SrDNA序列分析，确定OH11为产酶溶杆菌。BOH3为荧光假单胞菌，其余5个菌株为不同芽孢杆菌。     

Interactions between the biocontrol agent Pseudomonas fluorescens CHA0 and Thielaviopsis basicola in tobacco roots observed by immunofluorescence microscopy

Pseudomonas fluorescens CHA0 protects plants from damage caused by several soilborne fungi. In this work, immunofluorescence microscopy was used to investigate the colonization of tobacco roots by CHA0 and its physical relationship with the black root rot fungus Thielaviopsis basicola . The pseudomonad colonized the rhizoplane shortly after planting of tobacco seedlings in sterile soil microcosms, in which it had been introduced as soil inoculant. CHA0 was found between and inside cells in the epidermis and the cortex, as well as in the xylem vessels, within 4–7 days after planting of seedlings. The presence of CHA0 delayed the colonization of the interior of tobacco roots by T. basicola compared with the treatment in which only the fungus had been inoculated. Likewise, the pseudomonad reduced the extent of black root rot from 82% to 28%. However, CHA0 was seldom found in contact with the mycelium of T. basicola or in its vicinity, indicating that direct colonization of the mycelium of T. basicola by CHA0 was not required for protection of tobacco against black root rot. Overall, the results suggest that the interior of the root is a key site for implementation of the strain's biocontrol activity against soilborne plant-pathogenic fungi.     

西葫芦根腐和茎基腐病新病原-露湿拟漆斑菌Paramyrothecium roridum

 2013年12月,甘肃省白银市水川镇日光温室中的西葫芦发生了严重的根腐和茎基腐,部分棚室病株率达50%,从病根和病茎上分离得到拟漆斑菌属真菌3株,病株分出率27.3%。采用胚根和茎基部接种法测定了菌株FG-62对西葫芦的致病性:茎基部接种后27 d,植株开始出现凋萎;接种后40 d,两种接种法的西葫芦苗均呈现严重根腐和茎基腐症状,茎基部接种的西葫芦凋萎株率达30%;从病根和病茎上均可再分离出原接种菌。菌株FG-62在PDA平板上25℃培养14 d,产生大量墨绿色至黑色分生孢子座,分生孢子无色至淡榄黑色,单胞,杆状或腰鼓状,两端钝圆,大小为(7.04~9.15)μm ×(1.97~2.46)μm,聚集的分生孢子呈黑色。BLASTn分析结果显示,菌株FG-62(GenBank 登录号 MK252098)的rDNA-ITS序列与露湿拟漆斑菌Paramyrothecium roridum分离物E-469 (GenBank 登录号KY582183.1)和CBS 357.89(源自模式材料,GenBank 登录号NR_145077.1)的序列相似性分别达99.65%和96.83%。依据病原菌形态学和rDNA-ITS序列,将其鉴定为露湿拟漆斑菌P. roridum (Basionym:Myrothecium roridum)。这是露湿拟漆斑菌引起西葫芦根腐和茎基腐的首次报道。     

烟草根黑腐病菌致病力分化及品种抗性差异研究

 烟草根黑腐病由根串珠霉（Thielaviopsis basicola (Berk.et Br.)Ferr.）引起,主要为害烟株的根部,可使根部组织呈特异性黑色坏死而导致烟苗死亡或地上部分生长不良。该病是世界性的烟草病害,在各产烟国家如美国、日本、加拿大等国普遍发生,在我国主要分布于河南、山东、安徽、云南等地     

Soil properties related to suppression of Rhizoctonia solani on tobacco fields from northwest Argentina

Biotic and abiotic factors from soils have been implicated in the disease suppression of Rhizoctonia solani. This study included a Eucalyptus twig baiting assay, disease index and qPCR quantification of R. solani, and physicochemical analysis of 10 tobacco soils from five different locations (V: Vaqueros, C: Cerrillos, R: Rosario de Lerma, SA: San Agustín, CH: Chicoana) in the northwest of Argentina. Levels of Rhizoctonia soil inoculum quantified by baiting assay and qPCR were positively correlated. However, there was no correlation with root rot disease index in tobacco fields. Soils from V1, SA2 and CH2 fields, which reduced root rot disease on tobacco plants, were suppressive to R. solani infection. High clay, pH, organic matter content and physical stability in tobacco soils were the main physicochemical properties that limited Rhizoctonia development. Interestingly, growth of R. solani subgroups AG4-HGI and AG4-HGIII was highly suppressed in V1 and CH2 fields, and in SA2 fields, respectively. Undisturbed soil from a local forested mountain also resulted in reduction of growth of AG4-HGIII and AG4-HGI, while AG2-1 was less affected, suggesting that high soil organic matter contributed to suppression of R. solani. Soils highly suppressive of R. solani had significantly different populations of culturable bacteria, Pseudomonas and fungi, but populations of actinobacteria and Trichoderma spp. did not differ. These different populations may be involved in the inhibition of fungal growth. The results demonstrated that physicochemical and biological properties of soil suppressive to R. solani could act as an alternative for controlling Rhizoctonia diseases on tobacco.     

Root‐knot nematodes do not affect black root rot disease severity on watermelon and bottle gourd

The effects of root‐knot nematodes on black root rot of watermelon and bottle gourd were studied using field surveys and co‐inoculation tests with Meloidogyne incognita (southern root‐knot nematode) and Diaporthe sclerotioides. The results of the field survey suggested that root‐knot nematodes had little effect on either the severity of black root rot or infection with D. sclerotioides. None of the three fields showed a significant positive correlation between disease severity and nematode gall index, with low correlation coefficients. Co‐inoculation experiments under controlled conditions found no significant effect of root‐knot nematodes on black root rot of watermelon and bottle gourd based on area under disease progress curves (AUDPC). These results were supported by the quantities of DNA of the two agents in root tissues because no significant difference was found between dual‐ and single‐inoculation treatments with M. incognita and/or D. sclerotioides. These findings suggest that root‐knot nematodes probably do not affect the infection of watermelon or bottle gourd roots by D. sclerotioides or the incidence of black root rot in these crops caused by this fungus.     

The first report of tomato foot rot caused by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-3 PT and AG-2-Nt and its host range and molecular characterization

Foot rot of mature tomato plants was found in four cities of Hokkaido, Japan, from 2004 to 2007. Six of eight isolates obtained from damaged tissues were identified as Rhizoctonia solani anastomosis group (AG)-3, and the remaining two isolates belonged to AG-2-1. We compared these isolates with nine reference isolates including the different subgroups in AG-3 (PT, TB and TM) and AG-2-Nt (pathogen of tobacco leaf spot) within AG-2-1 in terms of pathogenicity to tomato, tobacco and potato. All eight isolates caused foot rot on tomato. The six AG-3 isolates caused stem rot on young potato plants. While, all reference isolates of AG-3 PT causing stem rot of young potato plants incited foot rot on tomato. The two AG-2-1 isolates and an AG-2-Nt reference isolate caused severe leaf spot on tobacco leaves. The sequences of rDNA- ITS region and rDNA-IGS1 region of the AG-3 isolates showed high similarity to that of AG-3 PT isolates. Phylogenetic tree based on ITS and IGS1 regions of rDNA indicated that the AG-2-1 isolates from tomato formed a single clade with AG-2-Nt isolates and that they were separate from Japanese AG-2-1 isolates (culture type II). Pathogenicity tests and DNA sequence evaluation of the causal fungi revealed that the present isolates of AG-3 and AG-2-1 belonged to AG-3 PT and AG-2-Nt, respectively. This is the first report of tomato foot rot caused by R. solani in Japan.     

烟草尖孢镰刀菌拮抗真菌的筛选鉴定及促生作用研究

为获得烟草镰刀菌根腐病拮抗真菌菌株,试验以河南省豫中烟区烟草根际土壤为试材,以主要致病菌尖孢镰刀菌Fusarium oxysporum为主要靶标,采用稀释涂布平板法分离获得土壤真菌371株,平板对峙培养法测定获得1株具有明显抑制效果的拮抗菌株,利用形态学和分子生物学的方法将其鉴定为棘孢木霉Trichoderma asperellum Tr-0111。抑制效果测定表明,该菌株对尖孢镰刀菌的抑制率最高可达93.13%,对根串珠霉菌、拟茎点霉、链格孢菌等其他8种烟草病原菌均具有较强的抑制效果。促生作用测定结果表明,菌株发酵液使烟草种子萌发率提高5.34%,烟苗根长增长率为62.39%,对盆栽烟草的最大叶面积和地上部鲜重也具有显著的促生效果。为进一步相关机理的研究和生防菌剂的研制奠定了基础。     

Salicylic Acid Biosynthetic Genes Expressed in Pseudomonas fluorescens Strain P3 Improve the Induction of Systemic Resistance in Tobacco Against Tobacco Necrosis Virus

ABSTRACT Application of salicylic acid induces systemic acquired resistance in tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in tobacco against tobacco necrosis virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of tobacco, but did not improve the ability of CHA0 to induce systemic resistance in tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobacco in a gnotobiotic system.