植物内生放线菌活性物质防治猕猴桃溃疡病

以供试内生放线菌发酵液的皿内抑菌活性为指标,得到对猕猴桃细菌性溃疡病菌有抑制活性的菌株76株,其中35株的发酵液对猕猴桃溃疡菌的抑菌圈直径≥20mm,5株对猕猴桃溃疡病菌均具有显著的抑制活性.其中gCLA4菌株发酵液抑菌谱最广,不仅对4种靶标细菌具有抑制活性,还对10种植物病原真菌有较强的抑制活性.通过发酵液制备及大孔吸附树脂D101处理得到gCLA4菌粗提活性物质.田间防治结果表明,gCLA4菌株粗提活性物质100倍稀释液对猕猴桃溃疡病28d的相对防效达64.9%,明显优于化学药剂施那宁.     

枯草芽孢杆菌BS80-6对苹果采后炭疽病的控病效果及作用机制

为提高枯草芽孢杆菌的生防效果,通过离子束诱变筛选出BS80-6菌株.利用生物测定方法研究BS80-6菌株及不同处理方法在不同贮藏温度下对采后苹果炭疽病的控制效果和作用机制.结果表明,BS80-6能显著提高对苹果炭疽菌的抑制作用.枯草芽孢杆菌BS80-6对苹果果实炭疽病的防治效果以活菌液效果最好,菌丝生长抑制率为80.14%,孢子萌发抑制率为98.65%,控病效果为60.34%;灭菌液和过滤液对苹果炭疽菌也有一定的抑制效果.贮藏温度明显影响BS80-6对苹果炭疽病的防治效果;接种方式对控病效果影响显著,先接拮抗菌再接病菌的防效好于先接病菌再接拮抗菌.     

油茶根腐病拮抗内生细菌的筛选及鉴定

为探讨防治油茶根腐病的高效拮抗菌的拮抗机制,采用研磨法从健康油茶根、茎、叶、果等组织中分离内生菌,通过平板对峙试验和抑菌谱检测试验筛选对油茶根腐病菌具有高效拮抗作用的内生菌,并进行形态观察、生理生化指标测定和16S rRNA序列分析.结果显示:从油茶中分离获得内生细菌75株,对油茶根腐病菌有拮抗作用的菌株18株,其中抑菌圈半径在5 mm以上的6株.这6株细菌的发酵液对油茶根腐病菌等6种供试病原真菌均有拮抗作用,其中菌株R6的抑菌效果最好,对各病原菌的抑菌圈半径均在10 mm以上.根据对R6的形态观察和生理生化指标测定,以及16S rRNA序列分析结果,初步鉴定其为枯草芽孢杆菌Bacillus subtilis.     

杨树烂皮病内生拮抗菌的筛选及鉴定

从健康杨树枝条上分离到1株能有效抑制杨树烂皮病菌的内生细菌,编号为NS3。该菌株对供试的13种植物病原菌均有抑制作用,抑菌谱广,其中对杨树烂皮病和杨树溃疡病病原菌抑制作用最强,其活菌的平均抑菌带宽度达到25.6mm以上,发酵液的抑菌圈直径达到48.6mm以上;人工接种防治试验结果表明:发酵液的无菌滤液对杨树烂皮病具有良好的生防作用,保护作用和治疗作用分别达85.4%和69.6%,与生产上用于防治该病的常用药剂防治效果差异不显著;根据其菌株形态、生理生化特征和16SrDNA序列比对,最终将该菌株鉴定为枯草芽胞杆菌(Bacillus subtilis)。     

3株来源于青稞白酒糟醅的生防菌株鉴定及其生物活性

为寻找高效安全且来源于生物源的除草活性物质,以此为先导化合物开发生物源除草剂。从青稞白酒糟醅中分离到的菌株中筛选到3株高效除草活性菌株,经形态特征和分子生物学鉴定后,确定JZ2?1?2为多粘类芽孢杆菌,JZ2?4?5、JZ2?5?2为枯草芽孢杆菌。3株芽孢杆菌的发酵液对供试禾本科杂草野燕麦的种子萌发具有抑制作用,同时对其幼苗发育影响明显,对野燕麦幼苗根长抑制率分别为92.48%、100.00%、100.00%。枯草芽孢杆菌JZ2?4?5发酵液通过茎叶喷施后,盆栽野燕麦植株7 d后大部分死亡,JZ2?5?2、JZ2?1?2对野燕麦防除效果次之。野燕麦叶片的相对含水量、叶绿素含量均有显著降低,MDA含量和SOD酶活性也变化明显。同时证明了3株芽孢杆菌属菌株对油菜菌核病原菌具有较好的拮抗作用。作物安全性结果表明,3株芽孢杆菌属菌株对小麦、青稞安全,对油菜、蚕豆风险高。综上所述,从青稞白酒糟醅中分离到的多粘类芽孢杆菌JZ2?1?2,枯草芽孢杆菌JZ2?4?5、JZ2?5?2具有作为麦田生物源除草剂开发的潜力。     

水稻稻瘟病拮抗细菌的筛选与防治初探

从水稻作物根际分离筛选得到的 2 0 8个细菌菌株对稻瘟病病菌具有较强的抑制能力 ;抑菌圈直径在 2 0mm以上的有 65株。选用室内拮抗性较稳定的 8株细菌菌株经两年的田间防病试验 ,不同菌株间防效差异显著 ,其中两年防效较好的菌株是Ma 3 2 (蜡状芽孢杆菌 )和Xi 55(枯草芽孢杆菌 )。一些菌株处理种子对秧苗生长有促进作用。     

生防菌株B-3对辣椒枯萎病的防治及其鉴定

为了明确生防菌株B-3对辣椒枯萎病的防效及其分类地位,采用管碟法测定了生防菌株B-3对辣椒枯萎病菌的抑制作用,考察了其对辣椒枯萎病的防效,并采用表型特征培养观察法、生理生化测定法和16SrDNA序列分析法对菌株B-3进行了鉴定。结果表明,生防菌株B-3的发酵液及其滤液对辣椒枯萎病菌均表现出较好的抑制作用,对辣椒枯萎病的盆栽与大田试验的防效都比较好,分别为73.6%和64.8%,显著优于100mg/L多菌灵。菌株B-3的形态与生理生化特性与枯草芽孢杆菌很接近,由16SrDNA序列分析的系统发育树发现,菌株B-3的序列与Bacillussubtilis菌株MO5单独构成一个分支,进化上的距离最近,由此可将其鉴定为枯草芽孢杆菌Bacillussubtilis。     

生防枯草芽孢杆菌L1特性的初步研究

通过对峙培养,测定出枯草芽孢杆菌L1的抑菌谱较宽,特别是对水稻纹枯病菌、大豆菌核病菌、禾谷镰孢菌、辣椒灰霉病菌、玉米小斑病菌抑菌效果明显;枯草芽孢杆菌L1不同发酵时间经湿热灭菌处理后,5 d发酵液中抑菌活性物质含量最高,对水稻纹枯病菌菌丝生长抑制率达83.23%,发酵液随时间延长抑菌效果不再增加;枯草芽孢杆菌L1抑菌活性物质对温度不敏感;枯草芽孢杆菌L1发酵液用硫酸铵梯度沉淀法提取粗蛋白在硫酸铵饱和度达60%～70%(不含60%)沉淀的活性物质抑菌效果最好,对水稻纹枯病菌平均抑菌半径达1.15;枯草芽孢杆菌L1对玉米、大豆、小麦、番茄、菜豆、黄瓜、水稻无致病性,而且还有保鲜和促生作用。     

葡萄霜霉病拮抗细菌N22的筛选鉴定及其防效初探

采用叶盘法筛选到1株防治葡萄霜霉病效果较好的芽孢杆菌N22菌株,并对该菌株进行了鉴定。结果表明,用N22菌株的16S r DNA序列与其相关种属进行比对,其序列与解淀粉芽孢杆菌(Bacillus amyloliquefaciens)及枯草芽孢杆菌(Bacillus subtilis)都具有100%的相似性。系统发育分析也表明,N22菌株和枯草芽孢杆菌及解淀粉芽孢杆菌聚在一个枝上。经进一步克隆了该菌株的gyr A基因序列,其比对结果中相似度高的菌株中只出现了解淀粉芽孢杆菌,其Query cover达到91%。因此,该菌株最终鉴定为解淀粉芽孢杆菌。以解淀粉芽孢杆菌N22菌株的田间防效显示,当发酵液菌体孢子含量为10~7个/m L的防治效果最好,防效可达76.94%。     

马铃薯黑胫病生防菌株的筛选与鉴定

利用本实验室前期分离保存的58株芽孢杆菌进行马铃薯黑胫病生防菌株的筛选与鉴定,旨在为内蒙古地区马铃薯黑胫病的防治探索新方法。经平板初筛,共有17个菌株对马铃薯黑胫病菌(Pectobacterium atrosepticum)具有拮抗活性,占供试菌株的29.31%;进一步复筛获得拮抗效果较好的5个菌株,分别为Chi8-7、Genyun4-4、Gen7-4、Jing6-7、S-7。其中,菌株Genyun4-4的抑菌效果最为明显,抑菌圈宽度达到20.7 mm,显著高于其他供试菌株处理,且生物活性测定结果也表明该菌株的抑菌效果较好。通过形态学观察、分子鉴定和生理生化特征测定,菌株Chi8-7鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、Genyun4-4鉴定为枯草芽孢杆菌(B. subtilis)、菌株Gen7-4、Jing6-7和S-7均鉴定为甲基营养型芽孢杆菌(B. methylotrophicus)。     

The Effect of Irrigation Practices on the Spatio-temporal Increase of Asiatic Citrus Canker in Simulated Nursery Plots in Reunion Island

Asiatic citrus canker is a potentially severe disease of several citrus species and cultivars in many tropical and subtropical areas. In such areas, infected nursery plants constitute an important source of primary inoculum for newly established citrus groves. The influence of overhead, drip, and mist irrigation systems on the development of Asiatic citrus canker was studied in simulated, Mexican-lime nurseries in Reunion Island. Overhead irrigation exacerbated the increase of disease incidence and severity caused by a streptomycin-resistant strain of Xanthomonas axonopodis pv. citri. The temporal development of Asiatic citrus canker for overhead irrigated nursery plots was best described by an exponential model, because disease incidence in these plots did not come close to an asymptote during the experimental period. This can be explained by the continuous production of new growth, susceptible to infection by Xanthomonas axonopodis pv. citri, and splash dispersal of Xanthomonas axonopodis pv. citri associated with overhead irrigation. Based on spatial correlation and spatio-temporal analyses, aggregated disease patterns were found irrespective of the irrigation system. In overhead-irrigated plots, the spread of Xanthomonas axonopodis pv. citri lacked directionnality. Rainstorms of short duration and high intensity were apparently associated with disease increase in drip-irrigated plots. There is a need to improve cultivation practices in Reunion Island citrus nurseries to minimize Asiatic citrus canker incidence in nurseries and to minimize the introduction of Xanthomonas axonopodis pv. citri to new groves.     

Use of phages for identifying the citrus canker bacterium Xanthomonas campestris pv. citri in Taiwan

Phages CP115 and CP122, which were isolated from canker lesions on grapefruit and Liucheng sweet orange, respectively, showed a high degree of specificity with respect to lysis of test bacterial strains. When used jointly, they lysed 135 (97·8%) out of 138 Xanthomonas campestris pv. citri strains isolated from the canker lesions on leaves, twigs, and fruits of various citrus species, cultivars, and hybrids grown throughout Taiwan, but they did not lyse other X. campestris pathovars and other phytopathogenic bacteria, nor other bacteria isolated from soil, clinical or environmental samples. Of 252 CP115/CP122-sensitive and 78 CP115/CP122-resistant bacterial strains with colony characteristics typical of or similar to those of X. campestris pv. citri , isolated from canker lesions of various citrus plants in diverse growing regions in Taiwan, 250 (99·2%) and 76 (97·4%) strains were pathogenic and non-pathogenic, respectively, when inoculated into Liucheng sweet orange or Mexican lime. Thus, phages CP115 and CP122, when used jointly, appear to be applicable for identifying X. campestris pv. citri in Taiwan.     

Bacterial antagonists and their cell-free cultures efficiently suppress canker disease in citrus lime

Journal of Plant Diseases and Protection - Proliferation of citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) has increasingly become a serious threat and has resulted in a...     

Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.     

柑橘溃疡病检疫与防治

细菌性溃疡病是严重危害世界柑橘产业的重大检疫性病害之一,柑橘溃疡病引起落叶、枯枝和落果,溃疡病斑导致果品质量降低,影响外贸出口。世界各国长期以来对病害采取严格苗木检疫、疫区病树铲除、零星病害药剂防控的综合治理措施;新近美国农业部推出"柑橘健康种植行动计划";2007年7月中国农业部正式启动"柑橘非疫区建设和维护"项目,总体目标在于防控柑橘溃疡病的发生和传播,确保柑橘产业的安全。     

柑桔溃疡病菌PCR快速检验检疫技术研究

 柑桔溃疡病是严重影响全世界柑桔生产的重大检疫性病害,根据柑桔溃疡病菌(Xanthomonas axonopodis pv. citri)新近公布的全基因组中独有的保守蛋白基因序列,设计筛选出一对种特异性引物(JYF5/JYR5),能专一地扩增检出柑桔组织表面所带溃疡病菌的DNA靶带(413 bp)。而柑桔叶面附生的非致病性黄单胞菌、野油菜黄单胞菌近缘种以及健康柑桔样品都不能扩增;靶细菌DNA检测下限1.56 pg/μL,靶细菌悬浮液检测下限10 cfu/μL;在不同PCR仪及各种控温方式下都能稳定地扩增出特征性靶带。这一特异、准确的柑桔溃疡病菌PCR检验技术和研制的预包被固相化PCR检测试剂盒已开始用于我国非疫生产区建设中柑桔苗木、果实的病害检疫检验。     

柑橘溃疡病菌抗铜相关基因copA和 copB的克隆及原核表达

 以柑橘溃疡病菌DNA为模板,对抗铜相关基因copA和copB进行了PCR扩增和克隆,获得了大小分别为1 782 bp 和1 095 bp的目标片段。构建了这2个基因的原核表达载体 (pET-copA和pET-copB),并在大肠杆菌 [Escherichia coli BL21(DE3)] 中成功诱导表达。利用原核表达的融合蛋白免疫大耳白兔,制备了抗copA和copB原核表达蛋白的多克隆抗体。用间接ELISA法测定了所制备的多克隆抗体的效价均为1∶6 400。用所制备的抗体分别对copA和copB的原核表达产物进行Western blot分析的结果显示,在相应位置产生了较强的免疫反应条带,表明所制备抗体能特异性地与相应的抗原发生免疫反应。     

Effect of temperature and leaf wetness duration on infection of sweet oranges by Asiatic citrus canker

Asiatic citrus canker, caused by Xanthomonas smithii ssp. citri , formerly X. axonopodis pv. citri , is one of the most serious phytosanitary problems in Brazilian citrus crops. Experiments were conducted under controlled conditions to assess the influence of temperature and leaf wetness duration on infection and subsequent symptom development of citrus canker in sweet orange cvs Hamlin, Natal, Pera and Valencia. The quantified variables were incubation period, disease incidence, disease severity, mean lesion density and mean lesion size at temperatures of 12, 15, 20, 25, 30, 35, 40 and 42°C, and leaf wetness durations of 0, 4, 8, 12, 16, 20 and 24 h. Symptoms did not develop at 42°C. A generalized beta function showed a good fit to the temperature data, severity being highest in the range 30–35°C. The relationship between citrus canker severity and leaf wetness duration was explained by a monomolecular model, with the greatest severity occurring at 24 h of leaf wetness, with 4 h of wetness being the minimum duration sufficient to cause 100% incidence at optimal temperatures of 25–35°C. Mean lesion density behaved similarly to disease severity in relation to temperature variation and leaf wetness duration. A combined monomolecular-beta generalized model fitted disease severity, mean lesion density or lesion size as a function of both temperature and duration of leaf wetness. The estimated minimum and maximum temperatures for the occurrence of disease were 12°C and 40°C, respectively.     

Quantitative real-time polymerase chain reaction for bacterial enumeration and allelic discrimination to differentiate xanthomonas strains on citrus

ABSTRACT Quantitative real-time polymerase chain reaction (QRT-PCR) was developed for identification and enumeration of bacteria in citrus plant samples infected with Xanthomonas axonopodis pvs. citri and citrumelo, the cause of citrus bacterial canker (CBC) and citrus bacterial spot (CBS), respectively. Three sets of primers based on the pathogenicity gene (pth) in X. axonopodis pv. citri, a ribosomal gene in X. axonopodis pv. citrumelo, and the leucine-responsive regulatory protein (lrp) in both pathovars were combined with TaqMan probes and applied for specific strain detection and quantification. Calibration curves for bacterial abundance in plant samples obtained with the three primer-probe combinations were congruent with colony counts on plates of semiselective medium in most of the cases. However, apparent overestimation of bacterial cells by QRT-PCR indicated the presence of nonculturable or nonviable cells in some samples. In addition to quantification, the lrp primers and probes permitted differentiation by allelic discrimination of Xanthomonas strains infecting citrus tissues. This technique is based on the utilization of two probes that detect a single nucleotide difference in the target sequence between different strains and was validated with a collection of cultured Xanthomonas strains as well as tissue with CBC and CBS lesions. Allelic discrimination is demonstrated to be a more specific and sensitive protocol than previously developed PCR-based methods for strain identification and quantification.     

N‐acetylcysteine interferes with the biofilm formation,motility and epiphytic behaviour of Xanthomonas citri subsp. citri

Citrus canker is caused by Xanthomonas citri subsp. citri. Bacterial biofilm formation is important in the development of this disease because it is a factor in epiphytic bacterial survival on leaves and in infection. N‐acetylcysteine (NAC), in addition to having antibacterial properties, reduces biofilm formation by a variety of bacteria and was therefore tested for impairing biofilm formation by X. citri. Copper is currently the antimicrobial compound most commonly applied in agriculture to control citrus canker. Therefore, this study also evaluated a possible synergistic effect between NAC and copper to improve the strategy for controlling this phytopathogen. NAC was found to decrease biofilm formation, the production of extracellular polysaccharides and bacterial stickiness. Motility was also affected in the presence of NAC. The best combination of NAC and copper for controlling X. citri was application of NAC followed by copper 48 h later. The concentrations of 6 mg mL^?1 of NAC and 3·5 μg mL^?1 of copper were able to kill X. citri. NAC inhibited the epiphytic behaviour of X. citri on leaves, altering cell growth and the bacterial ability to form biofilms. The addition of copper to cells previously treated with NAC enhanced its bactericidal activity. In conclusion, NAC has antibacterial properties against X. citri, interfering with bacterial growth, motility and biofilm formation. Under epiphytic conditions, NAC made the cells more susceptible to copper by affecting X. citri biofilm formation. This study opens new possibilities for the use of NAC in combination with copper, possibly resulting in more sustainable management of citrus canker.