黄龙病菌在柑橘枝条上的分布和多样性分析

 柑橘黄龙病是由“Candidatus Liberibacter asiaticus”(CLas)引起的毁灭性病害,在染病植株的叶片和果实上表现出不同症状。但目前还未见在同一植株或枝条上表现不同症状的叶片或果实中的CLas是否存在多样性的报道。本研究分析来自美国佛罗里达与中国广东两地染病柑橘样品,发现佛罗里达的CLas遗传多样性较低。以3对位于CLas短串联重复位点两端的序列为引物,对广东柑橘黄龙病病株的样品进行PCR扩增和PAGE检测,结果发现:同一植株上的CLas多样性高于同一枝条;同一病枝上显不同症状的叶片和果实中的CLas浓度分布不均匀,但成熟病果中CLas的浓度最高。以同一病枝条为一个组,PCR扩增和PAGE检测53个枝条的样品,发现不同组间CLas多样性不一:3对引物扩增条件下分别有4组(7.55%)、7组(13.21%)和11组(20.75%)样品存在组间多样性,推测同一枝条中含有不同的CLas菌株。以上结果说明了CLas在柑橘枝条上的分布规律,为监测我国CLas的多样性提供了科学依据。     

利用Gateway技术筛选Candidatus Liberibacter asiaticus致病相关基因研究

柑橘黄龙病是危害全球柑橘产业健康发展的最严重病害之一,病原为韧皮部限制性细菌,我国的柑橘黄龙病由亚洲种(Candidatus Liberibacter asiaticus,CaLas)引起。由于该病原菌难以获得离体纯培养,从而限制了其致病分子机制的研究。本试验根据已经报道的植物病原细菌致病相关基因特点,从黄龙病菌亚洲种Psy62菌株全基因组序列(Psy62:NC_012985.1)中选取3个假定致病相关基因,分别标记为PalCa _(Las)、MotCa _(Las)和HlyCa _(Las),利用Gateway技术的同源重组原理,将3个基因通过BP反应和LR反应,分别构建到植物表达载体psk103中,接种本生烟(Nicotiana benthamiana)后,通过瞬时表达筛选可诱导_(N.)benthamiana产生过敏性坏死反应的功能基因。经过PCR克隆和测序验证,PalCa _(Las),、MotCa _(Las)和HlyCa _(Las)基因均成功构建至植物表达载体上。本生烟瞬时表达试验证实:当含有目的基因的根癌农杆菌菌悬液OD600值介于_(0.)6_(~0.)~(8,)测试本生烟苗龄6周左右,PalCa _(Las)基因在接种4 dpi时可引发明显的HR(hypersensitive reaction)反应,12 dpi时HR反应更为强烈。同等条件下MotCa _(Las)和HlyCa _(Las)均不能引发类似反应。由此证实,Gateway技术可成功用于筛选CaLas致病相关基因,PalCa _(Las)基因可能在CaLas致病过程中发挥重要作用。该报道为国内首次采用Gateway技术进行CaLas致病相关基因功能研究,为后续开展CaLas病原寄主互作分子机制研究奠定了基础。     

Detection by PCR of Candidatus Liberibacter asiaticus, the bacterium causing citrus huanglongbing in vector psyllids: application to the study of vector–pathogen relationships

Citrus huanglongbing (HLB), previously called greening, is a serious citrus disease in Asia, eastern and southern Africa. It is caused by Candidatus Liberibacter asiaticus (Las), a phloem-limited, nonculturable bacterium transmitted by the Asian citrus psyllid ( Diaphorina citri ) in Asia. A PCR-based assay was developed for monitoring Las in vector psyllids using a rapid DNA extraction from psyllid bodies and PCR amplification. The entire procedure for Las detection in psyllids can be completed within 5 h. Using this method, Las can be accurately detected in psyllid adults as well as nymphs in different instar stages. The assay is sensitive enough for Las detection in single-psyllid extract from adult, fifth, fourth and third instars. In a transovarial transmission experiment, Las was not detected in eggs or in offspring produced by Las-carrying psyllid females. In a retention test, the Las-carrying psyllids remained Las-positive for 12 weeks after they were moved to common jasmine orange, a Las-immune plant. From these experimental results it was concluded that Las persists in the Asian citrus psyllid vector, but is not transovarially transmitted by the vector. These data help in understanding epidemiological characteristics of Las and psyllids in citrus HLB.     

中国胭脂鱼的细胞遗传学分析

采用植物血凝素、秋水仙素活体注射和肾细胞直接制片法分析了中国胭脂鱼(Myxiocyprinus asiaticus)的染色体核型和Ag-NORs显带;并以中国胭脂鱼血细胞为样本,鸡血细胞DNA含量为标准(2.50 pg),用流式细胞仪测定了中国胭脂鱼的 DNA 含量.结果表明:(1)中国胭脂鱼染色体数目2n=100,核型公式为10 m+4 sm+12 st+74 t, NF=114,未观察到次缢痕及随体,亦未发现有性染色体.(2)中国胭脂鱼染色体具有1对Ag-NORs,位于第1对染色体短臂上,间期核中Ag-NORs的数目为1~2个,未见Ag-NORs的联合现象.(3)PI和DAPI作为荧光染料,得出中国胭脂鱼DNA含量分别为5.50±0.24 pg和6.16±0.03 pg.(4)中国胭脂鱼为四倍体,在特定的分类阶元中属于较原始的鱼类.     

三种PCR方法检测柑橘黄龙病菌的效果比较

为了比较常规PCR、巢式PCR和实时荧光定量PCR方法在大田检测中对柑橘黄龙病(Huanglongbing, HLB)的检测效果, 首先比较了3种检测方法对柑橘黄龙病菌检测的灵敏度, 结果发现：3种检测方法的灵敏度依次为常规PCR<巢式PCR<实时荧光定量PCR。运用3种检测方法对广东5个柑橘品种上的189个黄龙病疑似病样进行检测, 结果发现：黄龙病检出率依次为常规PCR<巢式PCR<实时荧光定量PCR。研究表明：常规PCR适合以较低成本大规模检测黄龙病; 实时荧光定量PCR具有最大的检测灵敏度; 巢式PCR检测技术同时具有前两者的一些优点, 但操作较复杂, 适合技术熟练的研究者使用。     

转食和联合投喂对胭脂鱼仔稚鱼生长和消化能力的影响

为考察转食及联合投喂对胭脂鱼仔稚鱼生长和消化能力的影响,在水温(24±0.5)°C下,用丰年虫、水蚯蚓、微颗粒饲料和螺旋藻作为开口饵料,按照不同配比共设置8个处理组来投喂出膜后10 d(10 dph)的胭脂鱼开口仔鱼,40 dph后有2组(水蚯蚓组和丰年虫组)继续投喂先前的饵料,其余处理组全部进行转食投喂微颗粒饲料。统计养殖各阶段的成活率,并在30,40,45,55和65 dph取材测量全长、体质量及相关酶的活性。结果如下:1在转食前,各个处理组间的存活率无明显差异,均大于90%,40 dph后进行转食的处理组存活率逐渐下降,在实验结束时未进行转食的丰年虫组、水蚯蚓组成活率明显高于相应的转食组(丰年虫/饲料组、水蚯蚓/饲料组)和其他转食组(P0.05);2转食对胭脂鱼仔稚鱼的淀粉酶活性没有太大影响,对丰年虫/饲料组仔稚鱼胰蛋白酶活性影响也不大,但能显著影响水蚯蚓/饲料组仔稚鱼的胰蛋白酶活性(P0.05),对于脂肪酶活性,丰年虫组和水蚯蚓组在40 dph后存在差异,但转食后它们之间的变化趋势接近;3进行联合投喂的2个处理组(丰年虫+饲料+螺旋藻/饲料组、水蚯蚓+饲料+螺旋藻/饲料组)相对未进行联合投喂的处理组(丰年虫+螺旋藻/饲料组、水蚯蚓+螺旋藻/饲料组),在转食前后联合投喂组的体质量和全长均要小于相应的处理组;42个联合投喂组与相应的处理组在转食后,淀粉酶活性都呈现上升趋势,其中2个联合投喂组的淀粉酶活性在转食后均高于相应的处理组(P0.05);2个联合投喂组在转食前后胰蛋白酶和碱性磷酸酶活性整体波动趋势不大,但在转食后5 d脂肪酶活性均呈现先下降后上升的趋势。结果表明:丰年虫、饲料和螺旋藻进行联合投喂胭脂鱼仔鱼,并在40 dph时转食饲料是可行的投喂方案,但饲料投喂比例及添加时间还需进一步研究。     

基于微卫星标记对长江中上游胭脂鱼增殖放流效果的评估

利用已经发表的11对胭脂鱼多态性微卫星标记, 对长江中上游胭脂鱼(Myxocyprinus asiaticus)增殖放流效果进行评估。结果表明, 5个胭脂鱼养殖场内所有的149尾繁殖亲本和长江中上游采集的65尾胭脂鱼中共观测到140个等位基因, 其观测杂合度和期望杂合度的平均值(范围)分别为0.771(0.519~0.906)和0.759(0.469~0.894), 多态信息含量(范围)为0.726(0.392~0.882)。通过软件Cervus统计得到, 11个微卫星座位累计排除率为99.998 3%, 并且在长江中上游采集的65个样本中, 11个样本与养殖场内繁殖亲本确定存在亲子关系, 据此确定这11尾为增殖放流的胭脂鱼, 并由此推算增殖放流的胭脂鱼对长江中上游野生群体的贡献量为16.92%。研究结果表明, 增殖放流是实现胭脂鱼野生种群资源恢复的有效途径。

     

Quantification of viable <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17–31% and 16–28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management. The authors P. Trivedi and U. S. Sagaram contributed equally to this work.     

Effects of dietary protein to energy ratios on growth and body composition of juvenile Chinese sucker,Myxocyprinus asiaticus

A growth experiment was conducted to investigate effect of dietary protein to energy ratios on growth and body composition of juvenile Myxocyprinus asiaticus (initial mean weight: 10.04 ± 0.53 g, mean ± SD). Nine practical diets were formulated to contain three protein levels (340, 390 and 440 g kg^?1), each with three lipid levels (60, 100 and 140 g kg^?1), in order to produce a range of P/E ratios (from 22.4 to 32.8 mg protein kJ^?1). Each diet was randomly assigned to triplicate groups of 20 fish in 400‐L indoors flow‐through circular fibre glass tanks provided with sand‐filtered aerated freshwater. The results showed that the growth was significantly affected by dietary P/E ratio (P < 0.05). Fish fed the diets with 440 g kg^?1 protein (100 and 140 g kg^?1 lipid, P/E ratio of 31.43 and 29.22 mg protein kJ^?1) had the highest specific growth rates (SGR) (2.16 and 2.27% day^?1, respectively). However, fish fed the diet with 390 g kg^?1 protein and 140 g kg^?1 lipid showed comparable growth (2.01% day^?1), and had higher protein efficiency ratio (PER), protein productive value (PPV) and energy retention (ER) than other groups (P < 0.05). No significant differences in survival were found among dietary treatments. Carcass lipid content was positively correlated with dietary lipid level, but irrespective of protein level and inversely correlated with carcass moisture content. Carcass protein contents increased with increasing dietary lipid at each protein level. The white muscle and liver composition showed that lipid increased with increasing dietary lipid level (P < 0.05). Dietary protein concentrations had significant effect on condition factor (CF), hepatosomatic index (HSI) and viscerosomatic index (VSI) (P < 0.05). However, dietary lipid concentrations had no significant effect on CF, HSI (P > 0.05). Based on these observations, 440 g kg^?1 protein with lipid from 100 to 140 g kg^?1 (P/E ratio of 29.22 to 31.43 mg protein kJ^?1) seemed to meet minimum requirement for optimal growth and feed utilization, and lipid could cause protein‐sparing effect in diets for juvenile Chinese sucker.     

长江胭脂鱼人工放流子一代遗传多样性初步研究

运用微卫星分子标记对80尾胭脂鱼人工放流子一代个体的遗传多样性和种群遗传结构进行了分析。用磁珠富集法构建了胭脂鱼(AAAG)n富集文库,从中分离并鉴定得到的22个微卫星位点最终设计合成18对引物。对长江中、上游的80尾人工增殖放流的胭脂鱼子一代进行了遗传多样性的初步研究,其中5对引物呈现多态性,等位基因数目为 2～8个;多态信息含量0.2957～0.8038;Shannon多样性指数0.5466～1.8840;观测杂合度0.3056～0.7222;期望杂合度0.3658～0.8381;Hard-Weinberg遗传偏离指数（D）-0.1818～0.4287。结果初步表明,胭脂鱼人工放流子一代处于较高的遗传多样性水平,但很大程度上仍受到人类活动的影响。