Comparison of viral RNA populations of pathogenically distinct isolates of Citrus tristeza virus : application to monitoring cross-protection

The population of sequence variants of Citrus tristeza virus (CTV) isolates of different geographic origins and pathogenicity properties was characterized by single-strand conformation polymorphism (SSCP) analysis of cDNA of the genes p18, p13, p20 and p23. The mild isolates analysed here usually yielded a SSCP profile with two DNA bands, suggestive of a predominant sequence variant, whereas the SSCP profile of the most virulent isolates contained more than two DNA bands, indicating that their viral populations are likely to be more complex. The set of SSCP profiles of the four genes allowed identification of individual isolates, but no profile characteristic of a geographic area or a biogroup was found. Sweet orange plants singly inoculated with a mild or with a severe isolate yielded the SSCP profile characteristic of each isolate, whereas the SSCP profile of plants successively inoculated with both isolates was a composite of the two individual profiles. The SSCP profile of plants singly inoculated remained constant, but the profile of doubly inoculated plants varied with time. Plants in which the SSCP profile of the severe isolate became predominant showed stem pitting, and those in which the predominant profile corresponded to the mild isolate remained symptomless. The results indicate that SSCP analysis can be used to study changes in RNA populations of doubly inoculated plants and to monitor cross-protection between mild and severe isolates.     

Systemic spread of Plum pox virus (PPV) in Mariana plum GF 8-1 in relation to shoot growth

Infection of Prunus spp. by Plum pox virus (PPV) is characterized by an uneven distribution of the virus within the tree and branches. In order to gain a better understanding of this distribution, a method for modelling tree growth was used. PPV spread was followed within susceptible Mariana plum clone GF 8-1 shoots for 4 months after inoculation. Shoot growth was unaffected by the presence of the virus. Symptoms appeared on leaves produced in the most actively growing parts of the shoots, i.e. at the beginning of the season. PPV was detected in leaves other than those showing symptoms. The proportion of leaves with detectable virus decreased from the zone showing symptoms, with 100% ELISA-positive responses, to the shoot tip with no detectable virus in leaves produced between 111 and 127 days after inoculation. Furthermore, a higher proportion of positive ELISA results was obtained below the zone showing symptoms (77%) compared with 50% above. PPV was detected in 95% of the most vigorous shoots 71 days after inoculation compared with 37% of slower-growing, later-produced shoots.     

麦二叉蚜传播玉米矮花叶病毒的机制

用胶体金标记法和荧光抗体标记法研究了麦二叉蚜(Schizaphis graminum)传播玉米矮花叶病毒(Maize dwaft mosaic virus,MDMV)的机制。麦二叉蚜传播玉米矮花叶病毒需要辅助成份-蛋白酶(Helper component-proteinase,HC-Pro)的参与。在电镜下观察到HC-Pro可以与MDMV粒子结合。用FITC标记的HC-Pro抗体和MDMV抗体证明,HC-Pro可以直接结合到蚜虫口针上;而MDMV粒子不能直接结合到蚜虫口针,必须在HC-Pro的辅助下才能结合到蚜虫口针上。这为HC-Pro在蚜虫传毒过程中起桥梁作用提供了新的证据。MDMV粒子主要吸附在蚜虫口针的尖端和中间部分。     

晋南冬麦区大麦黄矮病毒流行株系监测及防治策略探讨

连续5年(1996～2000年)采集晋南冬麦区小麦黄矮病标样,采用生物学和血清学(酶联免疫吸附法)相结合的诊断方法对该地区的大麦黄矮病毒流行株系进行了鉴别。结果表明,该小麦黄矮病流行区近五年以GAV株系为主流株系,兼有少量GPV、PAV和混合株系存在。同时对小麦抗黄矮病新品种“临抗1号”进行了GPV和GAV两种株系的抗性测定,明确了该品种兼抗GPV和GAV两种株系。根据小麦黄矮病发生现状,提出了一套以选育推广抗耐病品种为主,以药剂防治为辅的综合防治措施。以期为当地小麦生产服务。     

胶体金免疫层析法快速检测烟草环斑病毒

采用柠檬酸三钠还原法制备胶体金颗粒 ,标记烟草环斑病毒的抗体 ,制成免疫层析检测试纸条。检测粗提纯病毒的灵敏度为 1 0 0 0ng/ml,病汁液稀释 1 0 0 0倍后仍可快速检出。对大豆病种子、烟草冻干病叶等不同材料进行检测也有良好的效果 ,1～ 2min即可出现结果。用 9种不同的病毒进行测试 ,未出现非特异性反应。     

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.     

北方地区小西葫芦黄花叶病毒的酶联检测和西瓜品种抗病性鉴定

应用DAS-ELISA对分别于1998年在河南、陕西和1999年在河南、陕西、河北和山西采集的葫芦科病毒病样本84份、186份进行小西葫芦黄花叶病毒的检测,表明该病毒在这些地区广泛发生,ZYMV阳性检出率分别为79.8%和57.5%熏在北京和河南临颖县ZYMV发生率较低熏≤23.1%,在其余地区即河北石家庄,河南郑州、开封、孟津,山西运城,陕西西安其阳性检出率在40.0%～92.9%之间。除冬瓜样本没有ZYMV外,西瓜、甜瓜、南瓜、丝瓜、小西葫芦、苦瓜、黄瓜和瓠子均受到ZYMV的侵染。不同地区、年份和作物之间ZYMV阳性检出率存在差异。对18份西瓜品种的抗ZYMV温室人工接种鉴定的试验表明,目前推广的一些品种对ZYMV缺乏抗病性。     

Molecular detection of Fusarium solani f. sp. glycines in soybean roots and soil

A polymerase chain reaction (PCR)-based method was developed to detect DNA of Fusarium solani f. sp. glycines , the cause of soybean sudden death syndrome. Two pairs of primers, Fsg1/Fsg2 designed from the mitochondrial small subunit ribosomal RNA gene, and FsgEF1/FsgEF2 designed from the translation elongation factor 1-α gene, produced PCR products of 438 and 237 bp, respectively. Primer specificity was tested with DNA from 82 F. solani f. sp. glycines , 55 F. solani non-SDS isolates, 43 isolates of 17 soybean fungal pathogens and the oomycete Phytophthora sojae , and soybean. The sensitivity of primer Fsg1/Fsg2 was 10 pg while that of FsgEF1/FsgEF2 was 1 ng when using F. solani f. sp. glycines total genomic DNA or down to 10³ macroconidia g⁻¹ soil. Nested PCR increased the sensitivity of the PCR assay 1000-fold to 10 fg using primers Fsg1/Fsg2, and 1 pg using primers FsgEF1/FsgEF2. F. solani f. sp. glycines DNA was detected in field-grown soybean roots and soil by PCR using either single pairs of primers or the combination of two pairs of primers. The occurrence of F. solani f. sp. glycines was determined using nested PCR for 47 soil samples collected from soybean fields in 20 counties of Illinois in 1999. F. solani f. sp. glycines was detected in soil samples from all five Illinois Agricultural Statistic Districts including 100, 89, 50, 92 and 50% of the samples from East, Central, North-east and West Districts, respectively.     

Seasonal distribution of phytoplasmas in Australian grapevines

The distribution and persistence of phytoplasmas were determined in Australian grapevines. Phytoplasmas could be detected using the polymerase chain reaction (PCR) from shoots, cordons, trunks and roots throughout the year, and phytoplasmas appear to persistently infect Australian grapevines from year to year. Phytoplasmas were not always detected in samples from the same sampling area from one sampling period to the next. Phytoplasma detection by PCR was improved by sampling from shoots, cordons and trunks, especially during October (early spring). The diseases expressed by the 20 grapevines used in the distribution and persistence studies were monitored. Australian grapevine yellows disease (AGY) was expressed by 17/20 grapevines at some time during the study, whilst only 4/20 and 15/20 grapevines expressed restricted growth disease (RG) and late season leaf curl disease (LSLC), respectively. All grapevines with RG and LSLC also had AGY. The three diseases were persistently expressed in some grapevines and remission of disease was observed in others. The results of PCR detection in the same grapevines indicated that phytoplasmas were more frequently detected in AGY-affected grapevines that also expressed RG and LSLC compared with grapevines expressing AGY alone. Phytoplasmas were detected in symptomless plant material but less frequently compared with AGY-affected material.     

Purification and some properties of Papaya meleira virus, a novel virus infecting papayas in Brazil

'Meleira', or 'sticky disease', is currently the most damaging papaya disease in the mid-eastern Brazilian growing regions. Consistent disease transmission via latex injection, presence of similar isometric particles in the laticiferous vessels of diseased plants, and detection of double-stranded DNA in naturally and experimentally infected papaya trees suggest that a virus is the causal agent. Conclusive evidence for viral aetiology was previously lacking, mostly because every attempt to purify the putative virus from infected papayas had failed. Following the successful purification and partial characterization of the meleira virus, healthy papaya seedlings injected with purified virus particles later developed typical symptoms of the disease. Negatively stained, isometric, full and 'empty' purified virus particles measured 42 and 38 nm, respectively. The viral genome was a single dsRNA molecule of about 12 kbp. Several capsid proteins, ranging in size from 14·4 to 45 kDa, were consistently revealed by PAGE. Papaya meleira virus (PMeV) appears to represent a novel group of viruses, with no known similar counterpart among known plant-, vertebrate-, invertebrate- or prokaryote-infecting viruses.