梨火疫病菌噬菌体的初步研究

为找到梨火疫病菌适度专化性的噬菌体，用于该病的检测和鉴定，从英国病山楂枝条上分离到一株噬菌体，测定了其寄主范围、潜育期、繁殖量等，还观察了其形态。分离到的噬菌体为高度专化性噬菌体，不能单位用于梨火疫病菌的检测和鉴定，可与其他梨火疫病菌噬菌体株系配合使用。     

免疫吸附PCR技术提高梨火疫病菌检测灵敏度

本研究采用已经发表的引物和制备的梨火疫病菌抗血清,研制了免疫吸附-PCR技术,使其检测梨火疫病菌纯菌的灵敏度比标准PCR技术提高10倍;检测模拟样品中的梨火疫病菌灵敏度提高了1000倍;从相关混合菌液中能够更加灵敏和准确地检测出梨火疫病菌.该方法简单易行,准确灵敏,具有广阔的应用前景.     

梨火疫病菌拮抗细菌FX1培养基及摇瓶发酵条件优化

贝莱斯芽胞杆菌Bacillus velezensis FX1是从新疆库尔勒香梨生产区果蔬天然酵素发酵液中分离筛选出的对梨火疫病菌Erwinia amylovora具有较强拮抗作用的菌株。为提高其抑菌活性物质的产量和防病效果,本研究以平板抑菌活性和发酵液活菌浓度作为检测指标,采用单因素试验、Plackett-Burman试验和响应曲面法对培养基成分(碳源、氮源、无机盐)及摇瓶发酵条件(温度、转速以及初始pH)进行筛选和优化,确定FX1菌株的最适培养基和发酵条件。结果表明,FX1的最适培养基为：玉米淀粉10.0 g/L,酵母膏25.1 g/L,KH₂PO₄ 0.5 g/L,MnSO₄ 0.001 g/L;最适发酵条件为初始pH 7.0,转速150 r/min,温度30.9℃,装液量100 m L/500 m L,培养时间48 h。优化后FX1菌株活菌数可达1.53×10⁹ CFU/mL,抑菌圈直径可达26.6 mm,相比优化前分别增加了3.10倍和1.32倍。苹果离体花序的防效试验结果显示,喷施FX1菌...     

检测梨火疫病菌的过敏性寄主

梨火疫病（ＦｉｒｅＢｌｉｇｈｔ）是梨、苹果及许多蔷薇科植物上一种毁灭性的细菌病害,也是我国进境植物应检疫的危险性病害之一。植物病原假单胞菌的过敏反应测定通常在烟草和蕃茄上进行,稻白叶枯病菌的过敏性反应测定还可在蚕豆叶上进行［１］,１９７３年Ｌａｉ和Ｈ...     

梨火疫菌的株系和近缘种

多年来 ,学者们一直认为 ,梨火疫菌(Erwiniaamylovora)在遗传上是高度保守的。从世界各地的梨火疫病流行区分离到的梨火疫菌十分相似 ,只有寄生专化性的微小差异。但近些年来 ,随着对梨火疫菌分子生物学研究的深入 ,导致了一些株系和近缘种的发现 ,改变了这一传统认识。按照寄生专化性可将梨火疫菌分为苹果株系、悬钩子株系和亚洲梨株系。按照对抗生素的敏感性可分为抗生素敏感株系和抗性株系。从地理分布和起源又可分为美国株系、韩国株系和日本株系。此外还有基于培养形状 (如菌落大小 )、噬菌体和血清学的株系划分[1] ,但并没有得到广泛…     

亚洲梨火疫病菌入侵中国的风险分析

亚洲梨火疫病(Erwinia pyrifoliae)是一种严重为害梨树的毁灭性病害,本文从其分布状况、寄主、危害及经济重要性、寄主植物经济重要性、传入与定殖可能性和风险管理难度等方面进行了综合分析评估,其综合风险值R为2.52,属于特别危险的有害生物,并据此提出了防止其入侵为害的风险管理措施.     

梨园气溶胶中梨火疫病菌的定量检测

为系统研究梨园气溶胶中梨火疫病菌的含量, 本研究于2019年－2021年在新疆库尔勒市人和农场梨园, 利用病原菌孢子捕捉器在每年春季(4月下旬)、夏季(6月中旬)、秋季(9月中旬)收集梨园气溶胶, 检测梨火疫病菌。结果显示, 健康梨园气溶胶中未检测到梨火疫病菌, 不同发病程度的梨园气溶胶中均能检测到梨火疫病菌, 携菌量均值在102 cfu/(24cm2·h)以上, 其中, 气溶胶中梨火疫病菌含量最高值为2.81×104 cfu/(24cm2·h), 最低值为8.50×102 cfu/(24cm2·h); 重度、中度、轻度发病果园收集的气溶胶中含梨火疫病菌总菌落数均值分别为8.74×103、4.55×103、2.36×103 cfu/(24cm2·h)。此外, 在同一高度收集的气溶胶中, 梨火疫病菌菌落数随收集时间的延长而增加。不同季节气溶胶携菌量检测结果表明, 秋季发病梨园中气溶胶携菌量明显高于夏季和春季, 与梨园梨火疫病发病规律相符。致病性测定结果表明, 气溶胶中分离的梨火疫病菌具有致病性。     

几种杀菌剂对梨黑斑病菌抑制作用研究

经室内研究表明,噻菌灵、咪鲜胺、克菌丹、抑霉唑4种杀菌剂对:梨黑斑病菌(Alternaria alternata)均有很好的抑菌效果,其中有效浓度为5×10-8的抑霉唑和6×10-8的咪鲜胺浸泡处理10min能完全杀灭梨黑斑病菌,是极具潜力的浸果处理杀菌药剂。     

免疫捕获PCR检测进境苹果果实中梨火疫病菌

利用梨火疫病菌抗体和PCR技术建立了梨火疫病菌免疫捕获PCR方法,检测苹果模拟样品的灵敏度达到了1.5×102cfu/reaction。与直接PCR进行比较,免疫捕获PCR方法检测苹果模拟样品的灵敏度提高了10倍以上,而且省去了DNA提取等步骤。利用该方法成功从进境苹果样品中检测到梨火疫病菌,产物测序结果表明,产物序列和梨火疫病菌的相应序列高度一致。试验结果表明,免疫捕获PCR法能除去样品中的大部分PCR反应抑制物质,可以有效检测进境苹果中梨火疫病菌,在口岸水果检疫中具有一定的应用潜力和推广价值。     

应用聚合酶链反应技术检测梨火疫菌的研究

应用梨火疫菌ＰＣＲ技术可以准确，快速地检测出梨火疫菌潜伏侵染的样品，检出样品中梨火疫菌的最低带菌量为５０个细菌，未发现它与其它植病细菌有交叉反应，从ＰＣＲ扩增、电泳分离到获得最后结果仅需８小时。此法适宜对进口苗木、接穗和水果上的梨火疫菌快速检疫和对引种苗圃和果园里梨火疫病发生进行监测。     

进境苹果果实中梨火疫病菌的套式PCR检测

 针对进境商用苹果果实携带梨火疫病菌Erwinia amylovora数量有限的特点,选取源于病菌pEA29质粒的2对引物P29A/P29B和PEANT1/PEANT2配对组合成套式PCR,其检测灵敏度可达0.15 pg菌体DNA,检测灵敏度高于EPPO推荐的单管套式PCR方法和常规PCR方法。分别利用这3种PCR检测方法对美国、新西兰、日本和智利等国进境的166批苹果样品进行检测,3种检测方法的样品阳性率分别为53.6%、38.0%和8.4%,试验结果表明此套式PCR检测方法可用于进境商用苹果的梨火疫病菌快速检测。进境样品的检测结果证实了进境商用苹果果实中存在梨火疫病菌的可能性。     

吩嗪类化合物对梨火疫病菌的抗菌活性研究

由解淀粉欧文氏菌Erwinia amylovora引起的梨火疫病是危害梨、苹果等蔷薇科果树的一种重要细菌病害,在世界范围内造成严重损失,是果树上重要的防控对象。药剂防治是控制梨火疫病传播蔓延的一种重要手段。吩嗪类化合物是绿针假单胞菌Pseudomonas chlororaphis和抗生素溶杆菌Lysobacter antibioticus等生防细菌产生的抗菌活性物质,在农药、医药等领域有广阔的应用前景。为明确吩嗪类化合物对梨火疫病菌的抗菌活性,本研究通过测定含不同浓度药剂的菌悬液OD₆₀₀的方法,评价了本课题组分离的5个吩嗪类化合物以及常见的抗细菌药剂对梨火疫病菌的室内抗菌活性。结果表明,堆囊粘菌素(myxin)和吩嗪-1-羧酸(PCA)活性较好,EC₅₀分别为7.20μg/mL和46.41μg/mL。中生菌素、噻霉酮、春雷霉素等药剂EC₅₀为7.21～50.74μg/mL。进一步对活性较好的药剂进行了复配筛选,结果显示,吩嗪-1-羧酸∶中生菌素=9∶1复配组合的EC₅₀为27.15μg/mL,增效...     

梨火疫细菌实时荧光PCR和诱捕PCR-ELISA检测方法的建立

根据梨火疫细菌中独特而保守存在的质粒pEA29，设计了1对引物和3条探针，建立了实时荧光PCR检测方法和诱捕PCR-ELISA检测方法。实时荧光PCR采用带荧光标记的核酸杂交探针，边扩增边检测，步骤简单，不需PCR后处理，可避免假阳性和交叉污染；诱捕PCR-ELISA检测方法只需简单处理的样品就能检测，减少了核酸不纯出现的漏检，由于增加了核酸杂交探针，可不需凝胶电泳EB染色检测，不会出现假阳性问题。     

Identification by PCR analysis on plasmid pEA29 of isolates of Erwinia amylovora responsible of an outbreak in Central Europe

A collection of 127 strains of Erwinia amylovora, the causative agent of fire blight, was tested by PCR amplification of a fragment of the plasmid pEA29. A variability in the length of the DNA fragment obtained was observed after digestion by MspI and Sau3A restriction enzymes. Strains were distributed into three groups according to the length of the DNA product. Most of the strains analysed were placed into two groups. Thirteen strains were clustered into a third group which was linked with the geographical origin of strains: they were all isolates from recently reported outbreaks of fire blight in Austria and in southern Bavaria in Germany. The variation in the length of the amplified fragment is probably due to an insertion into this fragment.     

四种替代药剂对梨火疫病菌的抑制活性及其田间防效

为筛选农用链霉素替代药剂,以其为对照药剂,测定春雷霉素、中生菌素、噻霉酮和噻菌铜这4种常用细菌病害防治药剂对梨火疫病菌Erwinia amylovora的抑制活性、对梨火疫病的田间防效及不同药剂处理后梨中的农药残留,并对这4种药剂的作物安全性进行评价。结果表明,中生菌素、噻霉酮和春雷霉素对梨火疫病菌有抑制作用,抑制中浓度EC50分别为1.60、6.64和60.57 mg/L,中生菌素的抑制效果高于链霉素。在连续2年田间试验中,中生菌素1 000倍液和春雷霉素400倍液对梨火疫病的保护效果和治疗效果均达90.33%以上,噻霉酮500倍液和噻菌铜200倍液对梨火疫病的保护效果均达100.00%,治疗效果均超过86.97%,与对照药剂农用链霉素2 000倍液的效果相当。所有药剂处理后均未检出中生菌素、春雷霉素、高效氯氰菊酯和农用链霉素的农药残留,而噻虫嗪、阿维菌素和矮壮素虽有检出,但其残留量均低于国家限量标准。花期前后、幼果期和果实膨大期施药,中生菌素、春雷霉素、噻霉酮和噻菌铜对梨花、叶片、枝梢及果实生长均无不良影响。综合来看,中生菌素、春雷霉素、噻霉酮和噻菌铜均可作为农用链霉素的替代药剂,...     

Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 10³ cells mL^−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.     

梨火疫病菌的RPA快速检测技术的研发

梨火疫病菌Erwinia amylovora是我国进境植物检疫性有害生物, 主要侵染梨、苹果、山楂、海棠等多种蔷薇科植物引起梨火疫病。该病自1780年在美国被发现以来, 已经传播到世界多个国家, 对我国的果树生产也构成一定的威胁。严格植物检疫是全球防控梨火疫病菌扩散危害的重要方法之一, 目前我国植物检疫部门主要从实验室分离鉴定、分子检测和田间症状识别及快速检测方面开展工作。本研究基于重组酶聚合酶扩增(RPA)技术和E.amylovora 16S-23S ITS区间的保守序列设计的引物和探针, 建立了实时荧光RPA和侧流层析试纸条RPA两种检测方法。经测试, 在39℃条件下, 这两种检测方法都具有良好的特异性, 分别能在20 min和10 min内完成扩增。实时荧光RPA对E. amylovora菌株DNA的检测阈值为1.38×10-2 ng/μL;侧流层析试纸条RPA对E. amylovora菌悬液和DNA的检测阈值分别为104 cfu/mL和1.38×10-3 ng/μL。侧流层析试纸条RPA体系因反应快速、读取结果方便、不需要复杂仪器操作可以满足现场快速检测的需求。     

Baseline sensitivity of natural populations and resistance of mutants of Xanthomonas oryzae pv. oryzae to a novel bactericide,zinc thiazole

During 2009–2010, a total of 323 isolates of Xanthomonas oryzae pv. oryzae were obtained from rice with symptoms of bacterial leaf blight (BLB) in four provinces (Zhejiang, Jiangsu, Anhui and Hubei) in China. These isolates were tested for baseline sensitivity to zinc thiazole, a novel bactericide with strong antibacterial activity against Xanthomonas. The sampled pathogenic population had similar sensitivity to zinc thiazole (0·1–16·8 mg L^?1) in all four regions and over the whole two‐year study period. The baseline sensitivity was distributed as a unimodal curve with a mean EC₅₀ value of 6·79 ± 1·61 mg L^?1. The risk of mutation to resistance of zinc thiazole in X. oryzae pv. oryzae was further evaluated in vitro and in vivo. Twelve zinc thiazole‐resistant mutants were obtained through ultraviolet (UV) irradiation, culturing on zinc thiazole‐amended nutrient agar (NA) plates, and culturing on zinc thiazole‐treated rice plants. These zinc thiazole‐resistant mutants had resistance factors (RF = EC₅₀ value of a mutant / EC₅₀ value of the wildtype parent of this mutant) of 12·4 to 186·1 with a mean RF value of 44·1. Mutants obtained via UV irradiation, culturing on NA plates and culturing on rice plants had mean RF values of 51·8, 24·5 and 14·4, respectively. All mutants showed decreases in resistance to zinc thiazole after 20 successive transfers on bactericide‐free media or 10 successive inoculation–reisolations on bactericide‐free rice plants. No significant difference was found in bacterial growth and sensitivity to bismerthiazol between zinc thiazole‐resistant mutants and their parents. However, a significant decrease was observed in the pathogenicity of zinc thiazole‐resistant mutants compared with their parents, especially for mutants obtained via UV irradiation.