应用PCR技术快速检测马铃薯环腐病菌

根据马铃薯环腐病菌16SrDNA基因片段核苷酸序列设计引物(引物1∶5-′TGTACTCGGCCAT-GACGTTGG-3′和引物2∶5-′TACTGGGTCATGTTGGT-3)′,进行马铃薯环腐病菌PCR特异性扩增试验。合成的引物能从马铃薯环腐病菌总基因组DNA和细菌纯培养,以及人工接种和自然发病的马铃薯块茎中特异性扩增环腐病菌16SrDNA基因片段1046bp。该试验结果为马铃薯环腐病的鉴定、检测及病害流行学研究提供了新的技术和方法。     

利用体细胞杂交获取马铃薯软腐病的抗性

对马铃薯四倍体栽培种和二倍体野生种Solanumbrevidens的体细胞杂种通过叶片离体培养获得的四倍体植株,以及用四倍体栽培种进行回交获得的五倍体植株的块茎对软腐病的抗性进行了测定。结果表明,由体细胞杂种通过叶片组织离体培养再生植株中,有一个株系SC107对软腐病菌Erwiniacarotovora具有较强的抗性。在用不抗软腐病的马铃薯栽培种对体细胞杂种进行回交获得的杂种后代中,大部分株系对软腐病菌具有高水平的抗性,从而说明Solanumbrevidens对软腐病的抗性基因已转移到马铃薯栽培种。     

嵌套式PCR超灵敏检测马铃薯环腐病菌

马铃薯种薯中存在环腐病菌潜伏侵染,这种潜伏侵染逐代累积、逐渐表现症状,这是马铃薯环腐病无法根除的根本原因。本研究应用国外成功报道的根据存在于pCS1质粒和环腐菌染色体中一个1.3 kb的插入因子IS1121、高度重复的DNA片段设计的环腐病菌亚种特异性引物序列合成出引物对CMSIF1-CMSIR1和引物对CMSIF2-CMSIR2。以环腐标准菌株、黑龙江省环腐菌株以及马铃薯上其它主要的细菌病原菌(青枯病、软腐病、黑胫病)为试验材料进行直接PCR和嵌套式PCR扩增,结果只有环腐标准菌株和黑龙江省环腐菌株出现特异性片段(直接PCR扩增出1046 bp的片段,嵌套式PCR扩增出864 bp的片段)。将环腐菌纯菌种菌悬液稀释成浓度梯度并与马铃薯组织液混合进行直接PCR和嵌套式PCR检测灵敏度比较,结果表明嵌套式PCR检测灵敏度比直接PCR检测灵敏度提高了100￣1000倍。以明显感病症状的块茎、无明显感病症状的块茎和健康块茎为试验材料进行直接PCR和嵌套式PCR,结果除明显感病症状块茎外,所有无明显感病症状的块茎也均被检测出带有环腐病菌。     

不同马铃薯软腐病菌的致病力分析

为了明确马铃薯软腐病菌的致病力,本研究分别使用BL-1、BL-2和KF-1 3个马铃薯软腐病菌接种了‘尤金’和‘夏波蒂’2个马铃薯品种,对出苗率进行了调查,分析、鉴定不同菌种的致病力。结果显示:2个供试品种接种3个马铃薯软腐病菌后的出苗率均有不同程度的下降,平均出苗率为27.5%,与对照(CK)相比,出苗率降低67.5个百分点,其中KF-1是3个病原菌中致病力最强的,平均降低92.5个百分点。     

几种杀菌剂对桃软腐病菌的室内药效试验

桃软腐病是一种真菌性病害,传播快,导致产量大幅度的下降.为了筛选出有效控制桃软腐病的药剂,采用生长速率法测定了4种杀菌剂对桃软腐病菌的抑菌作用.结果表明,20％五氯硝基苯对桃软腐病菌的防治效果最好,抑菌率达到62.2％,对桃软腐病菌有显著的抑制效果.     

进口大豆中麦角鉴定方法新研究

我国进口大豆逐年迅猛增多,针对进口大豆中麦角和菌核难以鉴定的现状进行分析研究。根据麦角中毒性成分主要是麦角胺、麦角生碱和麦角新碱等成分,比较了食品领域中麦角生物碱不同检测方法的优缺点,选择快速有效的检测方法。根据现状提出3种研究方法:第1种是传统感官生物碱显色法,参考GB 2715-2016《食品安全国家标准粮食》中的检测方法;第2种是液相色谱-质谱/质谱法,结合食品领域中麦角生物碱的检测方法,采用提取、分离、纯化、成分分析等步骤;第3种是分子生物学方法。对麦角菌核酸的制备提取,然后采用普通PCR仪或者荧光定量PCR进行分析检测。研究为病原菌的检验鉴定提供了一种新的思路,采用液相色谱-质谱/质谱法对病原菌产生的致病物质进行分析,丰富了植物病原菌的检验鉴定理论。首次提出对麦角菌利用分子生物学原理进行鉴定,为鉴别进口大豆中的菌核与麦角提供了一个新的标准,为一线人员提供鉴定基础。     

海南槟榔黄化病病原物的分子鉴定

槟榔黄化病是槟榔上的一种重要病害,如何快速检测该病原菌是防治该病的重要基础。利用植原体16SrDNA通用引物对海南感染黄化病的槟榔花苞总DNA进行巢式PCR扩增,获得约1.2kb的特异片段,并对扩增产物进行核苷酸序列测定。通过BLAST程序比较、系统进化树构建及iPhyClassifier分析表明,引起海南槟榔黄化病病原植原体属于翠菊黄化植原体组(16SrⅠ组),且为该组中一个新的亚组,即G亚组,现将其暂命名槟榔黄化植原体(Arecanut yellow leaf phytoplasma,AYL)。     

马铃薯黑胫病菌的分离、纯化及PCR检测

由Pectobacterium atroseptica引起的马铃薯黑胫病是影响马铃薯生产的重要种薯传播细菌病害,从种薯发芽到生长后期均可发病,严重影响马铃薯的产量和品质。为构建马铃薯黑胫病菌的分离、鉴定技术体系,从广东省惠州市惠东县大水坑马铃薯基地采集了疑似感染马铃薯黑胫病植株,对其进行病原菌分离和纯化,得到7株病菌,利用引物Eca 1 g/Eca 2 g对其进行PCR扩增、将PCR产物测序并将所测定序列递交Gen Bank数据库,使用Blast软件进行比对确定目标菌株。结果表明,其中一株菌株能扩增出长度大约688 bp的特异性条带,其DNA序列符合Eca 3762序列,鉴定为马铃薯黑胫病菌。此方法可以成功地从发病植株中分离出黑胫病菌,成本较低廉,为广东省建立马铃薯种薯质量检测和控制体系提供技术支撑。     

香草兰细菌性软腐病病原菌鉴定

1988年,在海南岛兴隆地区首次发现香草兰细菌性软腐病。发病叶片呈水渍状软腐,腐烂病痕的边缘有褐色线纹。在潮湿情况下病痕扩展迅速,以致全叶软腐塌萎。根据对病原菌的细菌学性状、致病性和血清学反应的测定,确认本病的病原菌属于胡萝卜欧氏软腐菌胡萝卜病原型[Erwina carotovora pv.carotovora(Jones)Bergey]。     

黑龙江省东部地区马铃薯块茎病害调查及快速通关检疫

2002年对黑龙江省东部地区11个县(市),13个点进行调查,初步掌握了东部地区马铃薯块茎病害的发生种类、危害程度、流行趋势,并提出了防治马铃薯块茎病害几点建议,为今后更好地指导东部地区马铃薯生产奠定基础。针对本地区发生比较重的马铃薯块茎病害,进行快速出关检疫方法的研究,对本地区尚未发生的马铃薯块茎检疫性病害,进行快速入关检疫识别。     

Resistance to soft rot (Erwinia carotovora subsp.atroseptica) in tetraploid potato families obtained from 4x-2x crosses

Diploid potato clones, interspecific hybrids ofSolanum species, having in their originS. tuberosum,S. chacoense, S. yungasense, S. phureja, S. gourlayi, andS. demissum, with resistance to soft rot, were crossed to tetraploid potato clones in 4x-2x crosses. The 24 tetraploid families obtained in a North Carolina II design were examined for tuber resistance to soft rot in a laboratory test and for basic agronomic traits in field trials conducted for two consecutive years. In addition, one family originating from a 4x-2x cross of two susceptible parents was tested. Statistical analysis of the data revealed significant general (GCA) and specific combining ability (SCA) effects, year, GCA (female) × year, GCA (male) × year, and SCA × year upon the inheritance of resistance to soft rot. About 35% of the progeny was selected as resistant to tuber soft rot, and of these 11% showed high resistance combined with good tuber yield, tuber weight, and tuber appearance. The relationships between resistance to soft rot and chosen agronomic traits were not noted or were weakly significant and sporadic. The resistance to tuber soft rot found in diploid potato hybrids can be transferred to the cultivated tetraploid pool through 4x-2x crosses, and a high frequency of offspring posses resistance.     

马铃薯环腐病菌Real-time Taqman-PCR检测体系的建立

本研究根据环腐病菌16S rRNA保守序列设计特异性引物及探针,建立了马铃薯环腐病菌实时定量荧光PCR检测体系。研究表明,该体系可以准确、稳定、定量的检测出样品中最低浓度为1fg·μL-1(即3.21×103copies·μL-)1的目的基因,检测极限可以达到几个拷贝。该检测体系可对马铃薯环腐病菌进行微量检测,对于保证马铃薯各级种薯、商品薯及其相关产品的质量,提高市场竞争力具有重要意义。     

Introgression and stabilization oferwinia tuber soft rot resistance into potato after somatic hybridization ofsolanum tuberosum ands. brevidens

Resistance to potato tuber soft rot caused byErwinia carotovora was transferred fromSolanum brevidens to the cultivated potato over the course of four backcross generations originating from a somatic hybrid. Soft rot reactions were determined via a tuber plug inoculation method developed during the course of these experiments. Soft rot resistance was highest in the somatic hybrid (only ca. 20% of tubers and plugs showed evidence of severe rotting) and lowest among progeny of control potato x potato crosses (ca. 80% of tuber plugs showed severe rot). Backcross generations involving somatic hybrids were intermediate in their reaction, and resistance stabilized to about 60% of tuber plugs showing severe rot in the BC₂ through the BC₄. Reciprocal crosses showed no difference in the inheritance of soft rot resistance, indicating that neitherS. brevidens norS. tuberosum donor cytoplasm had a significant effect on the expression of resistance. Crosses between BC₃ siblings where noS. brevidens genetic markers were detected but resistance was segregating demonstrated a dosage effect for soft rot resistance. We conclude that introgression of soft rot resistance has occurred and that at least one locus responsible for resistance inS. brevidens now resides in theS. tuberosum genome.     

Potato powdery scab, caused bySpongospora subterranea f. sp.subterranea, in Costa Rica

Summary Seven samples of potato powdery scab like-lesions were used to determine and confirm the occurrence ofSpongospora subterranea f.sp.subterranea in Costa Rica. Light microscope observations of spore balls and zoosporangia in tomato bait plants identified the organism asS. subterranea. Positive DAS-ELISA test results from all seven samples and PCR specific amplification products from five samples confirmed its identity. The geographical location of the samples source plantations suggests wide distribution of the plant pathogen across Costa Rica main potato growing areas. Different lesion types may indicate host-pathogen or environment-pathogen interactions. The symptoms differences and PCR amplification results among specific primer pairs suggest that some degree of genetic variation among theS. subterranea samples may exist; however, further testing is required.     

Effect of the ring rot pathogen and latent potato viruses on ring rot symptoms and yield of potatoes

The ring rot bacterium,Corynebacterium sepedonicum (Spieck. and Kotth.) Skapt. and Burkh., and latent potato viruses (potato virus S and potato virus X) were investigated for their effect on ring rot symptom development on potato plants in the greenhouse and on symptom development and yield of potatoes in the field. Both virus-free (VF) and virus-infected (VI) Red Pontiac stem cuttings root-inoculated with ring rot bacteria in the greenhouse developed typical (T) ring rot symptoms, and symptom severity did not differ between VF and VI plants. In a field study, both VF and VI Russet Burbank seed pieces knife-inoculated with ring rot bacteria produced plants with atypical (A) and T ring rot symptoms as well as a combination of both types. The data suggest that more A than T symptoms develop on VI plants and more T than A symptoms develop on VF plants. Combined infection with the ring rot pathogen and the latent potato viruses resulted in greater yield losses of total and marketable Russet Burbank tubers than infection with the bacterial or viral pathogens alone.     

The inheritance of resistance to soft rot (Erwinia carotovora subsp.atroseptica) in diploid potato families

Soft rot of potato tubers, caused byErwinia (Pectobacterium), is a serious disease affecting potato crops during storage. Studies on the inheritance of resistance to tuber soft rot were undertaken on diploid potato hybrids. A total of 480 clones, derived from 12 families, were examined for resistance to soft rot in laboratory tests over 3 years and for basic agronomic traits in field experiments over 2 years. General (GCA) and specific (SCA) combining abilities were significant in the inheritance of resistance to soft rot; however, GCA for female parents were significant in 1 or 2 years during 3 years of evaluation and SCA was significant for eight, two, and six families out of 10 tested in 1994, 1995, and 1996, respectively. There were also significant differences between years. Moreover, marked interactions for GCA_females × year and SCA × year in the variation of soft rot resistance were found. Broad-sense and narrowsense heritability of resistance, measured as diameter of rotten tissue, was estimated as 0.92 and 0.89, respectively. The maternal effect, evaluated in two sets of reciprocal crosses, was not significant for the genetic determination of resistance to soft rot. No significant relationships were found between resistance to soft rot and the main agronomic traits in the tested diploid families. These results suggest that diploid potato clones with resistance to soft rot can be selected after being evaluated over a few seasons. It is also possible to select clones combining good resistance to soft rot with high yield, superior tuber characteristics, and acceptable starch content. These resistant diploids can be used as male parents in 4x-2x crosses in breeding tetraploid potato resistant to soft rot.     

Application of a PCR-based test for detection of potato late blight and pink rot in tubers

A polymerase chain reaction (PCR)-based test for potato late blight (Phytophthora infestans) and pink rot (P.erythroseptica, P. nicotianae) diseases has been developed for use with potato tuber tissue. Primers based on sequence analysis of the ITS2 region of ribosomal DNA of late blight and pink rot pathogens were utilized in PCR assays of inoculated tubers and tubers harvested from plots known to have late blight and/or pink rot. Assays of artificially inoculated Kennebec and Russet Burbank tubers revealed thatP. infestans was detected by PCR as early as 72 h after inoculation and in the absence of visible symptoms. Much higher detection frequencies were obtained by PCR compared with plating on selective medium or placement of tissue in moist chambers. Tubers from plots known to have late blight and/or pink rot were tested using the PCR assay. Assay of late blight lesions showed ca. 80% recovery for late blight-infected tubers from the field. Results indicate that the PCR assay provides a rapid and accurate test for diagnosis of late blight and pink rot in potato tubers.     

一株对红棕象甲幼虫和卵有致病力的病原菌的分离鉴定

从自然死亡的红棕象甲卵和幼虫尸中分离得到一株产红色色素的昆虫病原菌HN-1菌株,经形态学鉴定、生理生化测定和分子鉴定,确定为:为粘质沙雷氏菌亚种(Serratia marcescens subsp)；16S rDNA通用引物扩增该菌得到了预期的1 408 bp条带,并与粘质沙雷氏菌亚种Serratia marcesce...