Genetic mapping and utilization of quantitative trichome-mediated insect resistance in potato

Introgression of trichome-mediated insect resistance from the wild speciesSolanum berthaultii has become a major focus of the potato improvement program at Cornell University during the past twelve years. Several quantitative characters are involved in this resistance which is effective against a wide range of pest types. Correlative biochemical assays have been developed to assay specific components of the resistance, and the effects of the resistance on the target pests have been studied. Quantitative laboratory assays and specific measurements of insect behavior and biology have increased the precision of selection and enable the investigation of the genetic control of the resistance.We are currently using restriction fragment length polymorphisms (RFLPs) for genetic mapping of factors controlling the trichome traits fromS. berthaultii. Backcrosses to both the wild and the cultivated species parents have been evaluated for phenotypes contributing to the resistance mechanism, including trichome density, sucrose ester and polyphenol oxidase production by the trichomes, and the enzymatic browning reaction responsible for insect entrapment. Genetic maps are being developed for these progenies, using RFLP markers previously mapped in potato. Field and greenhouse trials under insect infestations are also being conducted with the mapping progeny. Our goal is to locate genes responsible for quantitative insect resistance by correlating RFLP variation at mapped loci with the trichome phenotypes and insect resistance. Genetic markers for these traits will be useful in transfer of the effective wild chromosomal segments into and among tetraploid potatoes, and for a better understanding of the resistance mechanism.     

安徽省鸡免疫抑制性疾病的流行病学调查

采集了安徽省6个主要养鸡地区65群鸡的185只病鸡共986份组织样品，对可引起免疫抑制性疾病的5种最常见病毒进行了PCR检测。结果，传染性腔上囊病病毒（IBDV）、鸡传染性贫血病毒（CIAV）、马立克氏病病毒（MDV）、禽白血病病毒（ALV）和网状内皮增生症病毒（REV）的群阳性率和个体阳性率分别为73．08％和40．91％、46．15％和25．95％、41．54％和17．84％、18．46％和7．57％、13．85％和4．86％，其中被检鸡之二重或多重混合感染的总阳性率为24．33％。调查结果证实，免疫抑制性疾病在安徽省的商业鸡群中普遍存在，并与鸡群疾病多而复杂、损失大相关。     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

陕甘宁老区脆弱生态环境定量评价——以榆林、延安两市为例

位于黄土高原中部的陕甘宁老区生态环境极为脆弱 ,近年来由于气候、人类开发资源等自然和人为原因 ,使生态环境的脆弱程度升高。选定年降水量、年均温、蒸发量等 8个指标 ,定量评价各县 1 970 - 2 0 0 0年的脆弱度状况 ,结果表明榆林、延安两市生态环境整体脆弱 ,脆弱度存在空间差异但差异不明显 ,时间段上的波动幅度不大。陕甘宁老区脆弱生态环境具有不稳定性 ,对外界干扰较敏感。     

应用Nested PCR技术检测柑桔木虱及其寄主九里香的柑桔黄龙病带菌率

应用PCR及Nested PCR技术检测柑桔木虱及其寄主九里香的结果表明:PCR只可检测最低2头带菌木虱,Nested PCR可检测到单个带菌木虱。100头带菌木虱中,单虫检出率为96％。检测田间重、中等、轻病的柑桔园内的木虱,其带菌率依次为87％、53％和21％。在病芦柑上饲菌不同天数的木虱均能检测出带菌,其饲菌时间最短为1d。城市九里香叶片及在其叶上取食的木虱单虫,均能用Nested PCR检测出病原。饲菌木虱接种九里香及芦柑健苗,在植株尚未表现症状时,常规PCR难检测出病原,但用Nested PCR则能检测到病原,说明九里香不仅是木虱的寄主,而且是黄龙病病原的隐症寄主。     

番木瓜环斑病毒株系的分子生物学方法鉴定

 以PRSV株系特异性引物对PRSV的PRSV126(PRSV日本分离物)、Ys、Vb和Sm等株系进行RT-PCR方法鉴定,引物PR21/PR22能把Ys从Vb和Sm中鉴定出来,PR300/PR301则能把Vb从Ys、Sm和PRSV126中鉴定出来;用限制性内切酶Hae Ⅱ、Sau3A I和Hinf I对PRSV的PRSV126、Ys、Vb和Sm等株系进行单酶切RT-PCR-RFLP分析,Hinf I能把PRSV126与Ys、Vb和Sm鉴别开来,Sau3A I能把Ys与Vb和Sm鉴别开来,Hae Ⅱ则能把Ys与PRSV126、Vb和Sm鉴别开来;以P1/P2为引物,对Vb、Ys和Sm株系进行RT-PCR-RFLP-SSCP分析,结果能一次把三者较好地区别开来。     

Identification of ALS inhibitor‐resistant Amaranthus biotypes using polymerase chain reaction amplification of specific alleles

Summary Polymerase chain reaction (PCR) amplification of specific alleles (PASA) was adapted as a molecular marker‐based method for the rapid detection of point mutations in Amaranthus retroflexus and Amaranthus rudis leading to ALS inhibitor resistance. Two pairs of primers were designed for the specific amplification of alleles of the ALS gene of susceptible and resistant biotypes. The allele‐specific primer matched the desired allele, but mismatched the different allele at its 3′ end. Differentiation was carried out by comparison of the amplified DNA fragments in gel electrophoresis after PASA‐PCR. In A. rudis, differentiation was possible with one PCR and genomic DNA as probe. A ‘nested’ PCR was necessary for the differentiation of sensitive and resistant A. retroflexus. PASA is useful for the identification of resistant weed biotypes and also as a monitoring tool to map resistance occurrence and distribution. Advantages include the fast and clear separation of those plants with and without mutations at an early stage of development, its easy and consistent performance and quick results compared with existing resistance detection tests. These advantages, when combined with management strategies, enable further activities to reduce herbicide resistance.     

Detection of Colletotrichum coccodes and Helminthosporium solani in soils by bioassay

The sensitivity of a bioassay in detecting soil inoculum of Colletotrichum coccodes and Helminthosporium solani was examined using potato minitubers and microplants. Tests were conducted on soils which were collected from fields in which the interval after a previous potato crop differed, and which were also artificially infested with conidia or microsclerotia. For C. coccodes , determining plant infection based on the occurrence of infected roots after 9–12 weeks was a sensitive method for detecting and quantifying the amount of inoculum in soil. Infestations of less than 0·4 microsclerotia per g soil were detected in artificially infested soils. A semiselective medium, developed for isolating C. gloeosporioides from pepper, detected soil infestations by C. coccodes as low as nine conidia or one microsclerotium per g soil in artificially infested soil. For H. solani , infection on minitubers was a sensitive measure, with soil inoculum of fewer than 10 conidia per g soil being detected. Soil infestation could be quantified by assessing the percentage surface area of minitubers covered by sporulating lesions, which was strongly related to the amount of soil infestation. The results of these bioassay tests were compared with published results for real-time quantitative PCR assays on the same soils. The two methods were in good agreement in artificially infested soils, but the bioassay appeared to be more sensitive with naturally infested soils.     

Further Contributions to the Development of a Differential Set of Pea Cultivars (Pisum sativum) to Investigate the Virulence of Isolates of Aphanomyces euteiches

Common root rot (Aphanomyces euteiches Drechs.) has become a very destructive disease in the French pea crops since 1993. For an accurate investigation of the virulence variability among French A. euteiches populations and between French and foreign populations, a new set of differential pea genotypes was developed. Thirty-three American and European pea lines, displaying different levels of resistance, were screened in a growth chamber against two French isolates. Symptoms (disease severity from 0 to 5, evaluating symptom surface on roots and epicotyl) and percentage of top fresh weight (inoculated/uninoculated top fresh weight ratio) were measured. From this screening 12 relatively resistant lines, from various genetic backgrounds, were identified along with a highly susceptible control. This set of 13 genotypes was inoculated under controlled conditions with 14 isolates from France, Sweden, USA, Canada and New Zealand, to investigate genotype–isolate interactions. Root symptoms were rated (disease severity), and a susceptibility/resistance threshold was established at disease severity = 1. Significant quantitative interactions were observed, and five 'resistance patterns' were identified, leading to a set of six pea genotypes: Baccara (susceptible), Capella, MN313, 902131, 552 and PI180693. Fields trials of this set in 1999 and 2000 gave the same resistance rankings than in growth chamber conditions. This set will allow more accurate assessments of the variability in virulence/aggressiveness of A. euteiches isolates from France and foreign countries, and further investigations of the epidemiological and genetic basis of pea–A. euteiches interactions.     

Association of a Geminivirus with a Leaf Curl Disease of Sunn Hemp (Crotalaria juncea) in India

Natural occurrence of a geminivirus causing severe leaf curl disease on sunn hemp (Crotalaria juncea) was recorded in India. The association of a geminivirus with the disease was demonstrated by whitefly transmission tests and polymerase chain reaction (PCR) amplification of DNA fragments of expected sizes with three pairs of degenerate geminivirus primers. The PCR-amplified viral DNA fragments were further characterized by Southern hybridization with a geminivirus probe consisting of the cloned coat protein (CP) gene of Indian tomato leaf curl virus (ITLCV). Restriction fragment length polymorphism analysis of a PCR-amplified CP fragment revealed that the geminivirus from sunn hemp was different than ITLCV.