Diagnosis and molecular analysis of Potato leafroll virus isolates in Tunisia

Potato leafroll virus (PLRV) is a major constraint to potato production in North Africa. Serological (sandwich and cocktail ELISA) and molecular (RT-PCR) tests were used to detect PLRV in 131 potato samples collected in different areas of Tunisia. RT-PCR proved to be usable as a routine diagnostic test for epidemiological purposes, being more sensitive and reliable, and less time-consuming, than serological tests. One RT-PCR-amplified portion of ORF3 (336 nt) was cloned and sequenced, and used for molecular characterization of Tunisian PLRV isolates. These showed high sequence identity with PLRV retrieved from GenBank.     

中国不同地区亚洲韧皮杆菌遗传多样性分析

 柑桔黄龙病是最严重的柑桔病害之一,现已威胁世界柑桔产业。已被证实在中国南部该病害通过嫁接和木虱传播。因此黄龙病的流行病学研究就显得尤为重要。本研究中,基于外膜蛋白(omp)基因的PCR-RFLP的方法被用来研究中国南部各柑桔主产区的亚洲韧皮部杆菌的遗传多样性。研究采用不同的限制性内切酶消化各地区的亚洲韧皮部杆菌的omp基因产生了不同的RFLP指纹图谱,并对各地亚洲韧皮部杆菌的omp基因进行克隆测序然后构建系统发育树。结果显示:中国不同地理区域的亚洲韧皮部杆菌具有一定的遗传多样性,即便在某一特定的地区也有一定的差异。序列分析显示中国地区的亚洲韧皮部杆菌有很高的同源性。这些结果对揭示中国柑桔黄龙病的流行病学及制定科学有效的防治策略具有一定的指导意义。     

中国不同地理来源稻曲病菌rDNA-IGS的初步分析

 The rDNA-IGS of 11 Ustilaginoidea virens isolates from different geographical regions in China were amplified by PCR and sequenced. The results showed that the rDNA-IGS fragments consisted of one cen-tral variable region and two lateral conservative regions. The two conservative sequences among the11 isolates shared the sequence similarity of 99.44%. The variable region consisted of 2, 4, or 6 of 77 bp-length-repeats in the isolates and the sequences of the repeat units were high conservative. Primers UIGS Ⅲ and UIGS IV were designed for amplification of the variable region in the rDNA-IGS.     

Aggressive and mild Potato virus Y isolates trigger different specific responses in susceptible potato plants

Differences in the early responses of two potato cultivars, Igor and Nadine, to two isolates of Potato virus Y (PVY), the aggressive PVY^NTN and the mild PVY^N, were monitored. Microarray and quantitative real‐time PCR analyses were carried out to identify differentially expressed genes after inoculation with each virus isolate. Additionally, symptom severity and development was observed and the amount of virus isolate accumulated in systemically infected leaves was evaluated, where a significantly higher amount of PVY^NTN was detected. Microarray analysis revealed 572, 1288 and 1706 differentially expressed genes at 0·5, 12 and 48 h post‐inoculation, respectively in cv. Igor, with a similar pattern observed in cv. Nadine. Microarray and quantitative real‐time PCR results implied an earlier accumulation of sugars and lower photosynthesis in leaves inoculated with the aggressive isolate than in leaves inoculated with the mild isolate. The PVY^NTN isolate did not activate early differential expression of the Fe‐superoxide dismutase and pectin methylesterase inhibitor (PMEI) genes, indicating a delay in plant response relative to that following PVY^N inoculation. Differences in the expression of the β‐glucanase‐I gene were also observed in early plant responses to inoculation with each virus isolate.     

Transmission of Tomato chlorotic dwarf viroid by Myzus persicae assisted by Potato leafroll virus

Journal of Plant Diseases and Protection - In this study, we have shown Myzus persicae transmissibility of Tomato chlorotic dwarf viroid (TCDVd) by exposing Potato leafroll virus (PLRV) plus TCDVd...     

Some characteristics of the transmission of grapevine leafroll associated virus 3 by Planococcus citri Risso

Some characteristics of the acquisition and transmission of GLRaV-3 by Planococcus citri were determined by ELISA testing and transmission experiments. Groups of five insects were used, i.e. the advisable minimum group size suggested by the results of ELISA of insect groups of various sizes. The virus was transmitted to only 1/10 test plants each of which had been exposed to a group of insects fed on GLRaV-3 infected plants for at least three days, eventhough more than 80% of the insect groups were expected to contain viruliferous individuals under these conditions. Viruliferous mealybugs transferred to potato plants could retain the virus for up to 24 h, but lost the capacity for effective transmission to vines within 1 h after transfer. In newly infected vines, the virus remained latent or undetectable by ELISA for at least 13 months.     

Variation in virus populations and growth characteristics of two sugarcane cultivars naturally infected by Sugarcane yellow leaf virus in different geographical locations

Two sugarcane cultivars (R570 and SP71-6163) naturally infected by Sugarcane yellow leaf virus (SCYLV) were each imported from several geographical locations into a sugarcane yellow leaf-free environment (Montpellier, France). Plants were grown as plant cane for 5–6 months and the experiment was repeated for three consecutive years (2003–2005) in a greenhouse. Several sugarcane-growth and disease characteristics were monitored to identify variation in pathogenicity of SCYLV. Depending on their geographical origin, sugarcane cvs R570 and SP71-6163 were infected by SCYLV genotypes BRA-PER or REU, or a mixture of the two. Severity of symptoms did not vary between plants of cv. R570, but variation in disease severity between plants of cv. SP71-6163 from different geographical locations suggested the occurrence of pathogenic variants of SCYLV. For each sugarcane cultivar, differences in stalk length, number of stalk internodes, virus titre in the top visible dewlap leaf, and percentage of infection of leaf and stalk phloem vessels were also found between plants from different geographical origins. However, these differences were not always reproducible from one year to another, suggesting occurrence of different plant responses to SCYLV isolates under varying environmental conditions.     

Potato leafroll virus: antiserum preparation and detection in potato leaves and sprouts with the enzyme-linked immunosorbent assay (ELISA)

Potato leafroll virus (PLRV) was purified fromPhysalis floridana, applying freezing, low-speed centrifuging, ammonium sulphate precipitation, clarification with chloroform and butanol, ultracentrifuging and sucrose-gradient centrifuging. Three antisera with titers from 64 to 256 were prepared, one of them being sufficiently specific to be used in the enzyme-linked immunosorbent assay (ELISA).With this test PLRV could be detected reliably in the foliage of secondarily infected, glasshouse-grown potato plants of six cultivars tested, and in sprouts of four or five of them. The results indicate that ELISA may be used successfully for routine testing of foliage of glasshouse-grown potato plants.Samenvatting Aardappelbladrol, veroorzaakt door het aardappelbladrolvirus (PLRV) is reeds meer dan een eeuw in Europa bekend en is in veel landen vermoedelijk nog steeds de ernstigste virusziekte van de aardappel. De bestrijding wordt ernstig bemoeilijkt door het ontbreken van een betrouwbare toetsmethode. De callosetoets heeft als routinetoets voor het aantonen van PLRV in knollen op beperkte schaal ingang gevonden, maar is niet 100% betrouwbaar.Sinds kort is voor virussen een zeer gevoelige serologische methode beschikbaar gekonen, de enzyme-linked immunosorbent assay (ELISA). Hierbij wordt het virus in plantemateriaal aangetoond door middel van een enzymreactie, waarvan de kleuromslag met het blote oog of (bij voorkeur) met een fotometer wordt afgelezen. De uitslag van de fotometer (extinctie) is een aanduiding voor de aanwezigheid van het virus. De methode lijkt bruikbaar voor routinematige toepassing. Om de bruikbaarheid voor het aardappelbladrolvirus te onderzoeken werd dit virus gezuiverd, werden antisera bereid en werden loof en spruiten van bladrolvirusvrije en-zieke aardappels onderzocht.Bij de viruszuivering uitPhysalis floridana, werd gebruik gemaakt van bevriezing, precipitatie door middel van ammoniumsulfaat, klaring met chloroform en butanol, centrifugering bij laag en hoog toerental en suikergradiëntcentrifugering. Met de viruspreparaten werden drie konijnen geïnjiceerd. De verkregen antisera bereikten titers van 64 tot 256 in de micro-precipitatietoets (Tabel 2). Eén hierven (B) was voldoende specifiek om in ELISA gebruikt te worden.Toetsingen werden uitgevoerd met blad van in een kas opgekweekte bladrolvrije en secundair geïnfecteerde aardappelplanten en met in het donker gekweekte spruiten. Tabel 1 geeft een overzicht van de getoetste rassen, de aantallen knollen, die ter kieming waren weggelegd en waarvan telkens één oog in de kas werd uitgeplant, en van de virussen waarmee ze geïnfecteerd waren. De resultaten van de toetsingen, weergegeven in de Tabellen 3 en 4, laten zien, dat het bladrolvirus betrouwbaar kan worden aangetoond in het blad van alle zes cultivars, maar in de spruiten van slechts vier of vijf, nl. Bintje, Element, Ostara, Resy en mogelijk Désirée. ELISA kan derhalve goed worden gebruikt voor het aantonen van het bladrolvirus in blad van secundair geïnfecteerde kasplanten. Of dit ook geldt voor veldplanten moet nog worden onderzocht.ELISA zal voor de Nederlandse pootgoedteelt het meest waardevol zijn wanneer met deze toets het virus in vers gerooide, slapende knollen kan worden aangetoond (nacontrole). Helaas waren zulke knollen niet beschikbaar toen we onze proeven met blad en spruiten uitvoerden.     

Hairy nightshade as a potential Potato leafroll virus (Luteoviridae: Polerovirus) inoculum source in Pacific Northwest potato ecosystems

Hairy nightshade, Solanum sarrachoides, is a solanaceous weed found abundantly in Pacific Northwest potato ecosystems. It serves as a reservoir for one of the important potato viruses, Potato leafroll virus (PLRV) (Luteoviridae: Polerovirus), and its most important vector, the green peach aphid, Myzus persicae (Homoptera: Aphididae). Laboratory research indicated an increased green peach aphid settling and performance on S. sarrachoides than on potato. It also revealed that green peach aphids transmitted PLRV more efficiently from S. sarrachoides to potato than from potato to potato. To test the efficiency of S. sarrachoides as an inoculum source in the field, a two season (2004 and 2005) trial was conducted at Kimberly, Idaho. Two inoculum sources, PLRV-infected potato and PLRV-infected S. sarrachoides, were compared in this trial. Green peach aphid density and temporal and spatial PLRV spread were monitored at weekly intervals. Higher densities of green peach aphids were observed on plots with S. sarrachoides and inoculum sources (PLRV-infected S. sarrachoides and potato) than on plots without S. sarrachoides and inoculum sources. PLRV infection in plots with PLRV-infected S. sarrachoides was similar to or slightly higher than in plots with PLRV-infected potato as an inoculum source. Temporal and spatial PLRV spread was similar in plots with either inoculum source. Thus, S. sarrachoides is as efficient as or a better PLRV inoculum source than potato.     

The effects of co‐infection by different Potato virus Y (PVY) isolates on virus concentration in solanaceous hosts and efficiency of transmission

The influence of co‐infection on concentration and accumulation of genetically different isolates of Potato virus Y (PVY) in potato and tobacco plants and the efficiency of transmission by Myzus persicae of PVY isolates from doubly versus singly infected plants were evaluated. The vector ability to simultaneously transmit two virus isolates was examined. Eight PVY isolates represented three strain groups: PVY^O (pathotype and serotype O), PVY^NW (pathotype N and serotype O), and PVY^NTN (pathotype and serotype N). Different diagnostic methods, including DAS‐ELISA, multiplex RT‐PCR, aphid transmission tests and bioassays, were applied to detect the presence of PVY isolates in source and assay plants. Significant reductions in concentrations of certain PVY isolates during co‐infection with other isolates were found both in potato and tobacco plants. The observed effects were both isolate‐ and host‐dependent in form. The highest rates of virus transmission by single aphids were recorded with PVY^NTN isolates, and the lowest ones with PVY^O isolates. Individual aphids of M. persicae were able to simultaneously transmit two PVY isolates. The frequency of transmission was generally low, but it reached as high as 20% for one of the isolate combinations. The findings presented in the work provide proof for antagonistic within‐plant interactions between isolates of PVY, with some implications of these interactions for virus transmission by aphid vectors. Consequently, this research contributes to a better understanding of the epidemiology of the disease caused by PVY.     

中国不同地理来源谷瘟病菌rDNA-IGS序列分析

由谷瘟病菌引起的谷瘟病是我国谷子的主要病害之一,谷瘟病发生可造成谷子大量减产。为了研究谷瘟病菌群体遗传结构和进化关系,本研究对64个谷瘟病菌的r DNA-IGS序列进行扩增检测和测序分析,结果表明,谷瘟病菌之间IGS序列的长度存在着明显的多态性。进一步序列分析发现,IGS序列中重复单元(GGGGGTGCAGGGTA和CATTTTT)的重复次数的不同是造成序列长度差异的主要原因,并且发现IGS基因序列具有寄主特异性。采用最大似然法对所获得的IGS序列进行系统发育树分析,将谷瘟病菌划分为3个谱系。谷瘟病菌遗传分化明显,具有丰富的多样性,分析表明影响谷瘟病菌IGS基因遗传分化的环境因素主要为寄主因素。研究结果为今后深入研究谷瘟病菌群体遗传结构提供理论支持。     

Molecular and biological characterization of Potato mop‐top virus (PMTV,Pomovirus) isolates from the potato‐growing regions of Colombia

Potato mop‐top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil samples were taken from the main potato‐producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons, three types of RNA‐CP (RNA2) and RNA‐TGB (RNA3) were found. The isolates from the centre of the country (CO3 and CO4) were similar to isolates from Europe. The genomes of CO1, CO2 and CO5 differ from other PMTV isolates, placing them in a separate clade in phylogenetic trees. The three Colombian isolates (CO1, CO2 and CO5) only induced slightly different symptoms in the indicator plants. However, the isolate from the northwest of the country (CO1) induced stronger symptoms in N. benthamiana including severe stunting. A correlation between the genotype of the isolates and the symptoms they induced on indicator plants was not found.     

Biological characterisation of various geographical isolates of potato virus Y inducing superficial necrosis on potato tubers

A range of selected PVY isolates that induce superficial necrosis on potato tubers, originating from several countries, were compared with standard strains of PVA, PVV and PVY. Biological properties (e.g., host range, aphid transmissibility and relationships based on cross-protection between virus isolates) were studied. PVY^NN isolates differ from the normal PVY^C, PVY^N and PV^O strains by their ability to infect Capsicum annuum but not Chenopodium amaranticolor and C. quinoa. All PVY^NN isolates are transmissible by Myzus persicae , without any significant differences from one standard strain. These additional data confirm that these tuber-necrosing isolates belong to PVY. However, they could be ranged in a homogeneous and distinct group inside the PVY^N group, based on the differences revealed in the host range, in addition to the specific ability naturally to induce necrosis on tubers.     

Localization of potato leafroll virus in leaves of secondarily-infected potato plants

Potato leafroll virus (PLRV) antigen was localized by immunogold labelling in semi-thin leaf sections of secondarily-infected potato plants cv. Bintje. Viral antigen was present in all cell types of the phloem tissue. but occurred most abundantly in the companion cells. Detectable amounts of PLRV antigen were found only in the sieve elements in veins with a large number of infected companion cells. Occasionally, parenchyma cells were also found to be infected. PLRV was not exclusively limited to the phloem tissue in the infected potato plants, but was also found in mesophyll cells neighbouring minor phloem vessles. Spread of virus from cell to cell in the mesophyll was not observed. The distribution of PLRV in the potato leaf tissue has implication on its availability, for acquisition by aphids.     

水稻条纹病毒中国分离物和日本分离物RNA1片段序列比较

 首次测定了我国水稻条纹叶枯病常年流行区的云南楚雄(CX)及病害暴发区的江苏洪泽(HZ)的RSV 2个分离物RNA1片段的全长序列,这2个分离物RNA1片段的全长序列均为8970 nts。同源性分析结果表明,HZ与日本T分离物的亲缘关系较CX与T分离物的亲缘关系更为接近。通过对纤细病毒属病毒RNA1编码的依赖于RNA的RNA聚合酶(RdRp)氨基酸序列分析的结果表明,该蛋白除了具有RNA聚合酶特征的基元序列结构外,还存在mRNA的转录过程中所采取的加帽起始机制的保守性结构域位点,这表明,纤细病毒属病毒和布尼安病毒科病毒及甲型流感病毒一样,都是采取加帽起始机制进行转录的。     

The effect of potato leafroll virus on the biology of Myzus persicae

Since potato leafroll virus multiplies in the green peach aphid,Myzus persicae, the effect of the virus on the biology of its vector was investigated. Observations were made regarding the longevity and the reproduction rate of viruliferous and non-viruliferous aphids on leafroll-diseased and healthy plants ofPhysalis floridana. The same matters were investigated for both viruliferous and non-viruliferous aphids on seedlings of Chinese cabbage (Brassica pekinensis). It was shown that on leafroll-diseased plants ofP. floridana the aphids produced more progeny than on healthy ones, although the average number of progeny produced per day in both cases was almost the same. On healthy Chinese cabbage seedlings there was no difference in average length of the larval and adult stages, number of progeny per aphid, and number of progeny per day, between viruliferous and non-viruliferous aphids. Evidence was obtained that the virus does not influence the development of its vector. Measurements of oxygen consumption of both viruliferous and non-viruliferous aphids point in the same direction.     

不同来源的柑橘衰退病毒分离物基因组5'端A、F变异区序列克隆及分析

 柑橘衰退病毒(Citrus tristeza virus,CTV)组群自然条件下存在株系分化现象。本研究利用RT-PCR技术扩增、克隆了来自我国不同地区的21个柑橘衰退病毒分离物的5'端A、F变异区。通过分析发现,不同来源的各分离物在5'端A、F区存在较大的变异。21个分离物A区序列相似性最低为85.8%,最高可达99.8%,平均为95.9%;与GenBank中9个代表性株系的平均相似性为84.2%。F区序列相似性较A区高,为98.0%;相似性最低为94.3%,最高达99.1%。结果显示不同来源的CTV分离物5'端序列A、F区变异较大。