Detection of Four Seed-borne Plant Viruses by the Enzyme-Linked Immunosorbent Assay (ELISA)

This study was conducted to evaluate the sensitivity of the ELISA technique in detecting four economically important viruses, namely barley stripe mosaic (BSMV), cucumber green mottle mosaic (CGMMV), bean common mosaic (BCMV), and squash mosaic (BSMV) viruses in single seeds as well as in batches of barley, cucumber, bean and squash seeds, respectively. Results indicated the suitability of the technique in detecting the above viruses in single germinated seeds or embryos. Accordingly, seed transmission rates of BSMV, CGMMV, BCMV and SqMV were found to be 67 %, 17%, 17% and 12%, respectively. In artificially contrived mixtures of infected: healthy seeds or embryos, BSMV, CGMMV, BCMV and SqMV were successfully detected at ratios of 1 : 500, 1 : 25, 1 : 10 and 1 : 10, respectively. Sensitivity of detection was increased in the ease of BSMV by using germinated rather than ground dry BSMV-infected barly seeds; and in the case of SqMV, by using whole germinating emybryos rather than coleoptiles only. Trials on re-using the enzyme-γ-globulin conjugate indicated that CGMMV conjugate used once can be re-used with little loss in reactivity.     

Transmission Efficiency of Cucumber green mottle mosaic virus via Seeds,Soil, Pruning and Irrigation Water

Cucumber green mottle mosaic virus (CGMMV; genus Tobamovirus) infects frequently the grafted watermelon and is widely distributed in China. Investigating the transmission modes and their efficiency is urgently needed to understand the factors contributing to the epidemiology of this viral disease. In the present study, we found that the occurrence of CGMMV in a bottle gourd seed production base reached 100%, while the contamination rate and transmission rate were 100 and 0.92%, respectively. The bottle gourd plants showed obvious mottle symptom on leaves starting 36 days after seed sowing. The long latent period of CGMMV in seedlings implies a potential risk to use contaminated seeds in the production of grafted watermelon. This virus could overwinter in soil with debris of infected plants, and the infection rate of CGMMV from contaminated soils was 10.30%. CGMMV could be transmitted from infected watermelon plants to healthy ones by pruning at least to the ninth plant during the whole growing season. The transmission distance was 1.87 m by drip irrigation and 2.31 m by flow irrigation. This study suggested that contaminated seeds, contaminated soil, pruning and irrigation could transmit CGMMV at different efficiency, and all contribute to the epidemiology of CGMMV.     

Investigation of Ukrainian isolates of cucumber green mottle mosaic virus

Abstract

Isolates of cucumber green mottle mosaic virus (CGMMV) obtained from three greenhouses in Ukraine have been identified. The rabbit antisera have been generated for these isolates. These antisera can be used for diagnostics of CGMMV in plant material by indirect ELISA. Analysis of cDNA sequences of capsid protein of these viruses confirmed that they may be grouped into novel strain of CGMMV.     

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log₁₀. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log₁₀. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log₁₀ lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log₁₀. Conversely, sensitivity was only 0.30 log₁₀ better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.     

Destruction of Cucumber green mottle mosaic virus by heat treatment and rapid detection of virus inactivation by RT-PCR

Heat treatment is commonly used to control viral contamination of seeds. To study virus inactivation, virus was purified from seeds contaminated with Cucumber green mottle mosaic virus (CGMMV) after various heat treatments. CGMMV particles were observed to be physically disrupted by high temperature. Analysis of viral RNA revealed that the 5' and 3' termini of the genome were protected whereas regions between 2-2.5, 3.2-3.7 and 4-4.8 kb from the 5' terminus were not. Heat inactivation of virus on seeds was confirmed by RT-PCR using primers for a heat-sensitive region. The RT-PCR approach developed here may prove preferable to time- and labor-intensive bioassays for assessing virus heat inactivation.     

Viruses infecting winter oilseed rape (Brassica napus ssp. oleifera)

Turnip mosaic virus (TuMV) and cauliflower mosaic virus (CaMV) have been found infecting field crops of winter oilseed rape (Brassica napus ssp. oleifera) in South Warwickshire. Other viruses found include broccoli necrotic yellows virus (BNYV) and a member of the beet western yellows virus group. Systemic leaf symptoms caused by TuMV varied within and between cultivars; the three predominant reaction types were classified as necrotic, mosaic and immune. Some recently introduced cultivars of oilseed rape were more severely affected by TuMV infection than older cultivars. Reactions to CaMV were less varied and immunity was not found. The seed yield from TuMV and CaMV-infected plants was less than that of healthy control plants. This effect was due to infected plants producing either fewer seeds, smaller seeds or both. Germination of seeds from infected plants was unaffected if sown soon after harvest. After storage for one year the germination of seed from a virus infected plant was significantly less than that of seed from a virus-free plant. All commercial cultivars tested were experimentally susceptible to turnip yellow mosaic virus (TYMV) and some American strains of cucumber mosaic virus (CMV).     

Location of Soybean Mosaic Virus in Seed-Parts of Individual Dormant and Germinating Mottled and Non-mottled Soybean Seeds

Soybean mosaic virus (SMV) was detected in individual embryos of both dormant mottled (50%) and non-mottled (50%) seeds of certified soybean cvs Davis. Essex. Hutchinson, Ransom, Stonewall, and Young by using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). SMV was not detected in individual strained seed coats of mottled or non-stained seed coats of non-mottled dormant seeds and endosperms of either type of seed. SMV was not detected m individual stained and nonstained seed coats, epicotyls, and endosperms of mottled and non-mottled seeds at 72 h post-germination. However, the virus was detected in individual hypocotyls at an average level of 31% in post-germinated seeds (mottled and non-mottled) of all cultivars. Symptomless glasshouse-grown 6–8-week-old seedlings from mottled and non-mottled seeds of certified soybean cultivars occurred twice as often as those showing early symptoms. However, almost half of these symptomless plants were found to be SMV-infected by DASELISA. Virus-free soybean plants grown to maturity from non-mottled seeds also produced mottled seeds.     

Seed-transmission in the ecology of nematode-borne viruses

Virus-free populations of vector nematodes can acquire tomato black ring (TBRV), raspberry ringspot (RRV) and arabis mosaic (AMV) viruses from weed seedlings grown from virus-carrying seed. When soils from fields where nematode-borne viruses occurred naturally were air-dried to kill vector nematodes and then moistened, TBRV and RRV occurred commonly in the weed seedlings that grew, but AMV occurred only rarely. Similar tests did not detect tobacco ringspot, grapevine fanleaf or tobacco rattle viruses in weed seeds in the single soil studied in each instance, although these three viruses are also seed-borne in some of their hosts. Many weed species, when infected experimentally, readily transmit TBRV and RRV to their seed, but the viruses were much commoner in naturally occurring seed of some of these species than of others. These discrepancies between the frequency of seed-transmission of viruses from experimentally infected plants and the extent of natural occurrence of infected seed seem largely to reflect the host preferences of the vectors. Infective Longidorus elongatus kept in fallow soil retained TBRV and RRV only up to 9 weeks. When weed seeds in the soil were then allowed to germinate, the nematodes reacquired virus from the infected seedlings. Some weed species were better than others as sources of virus. Persistence of these viruses in fields through periods of fallow or fasting of the vector therefore depends on a continuing supply of infected seedlings produced by virus-containing weed seeds. This is probably less true of viruses like AMV and grapevine fanleaf, which persist for 8 months or more in their vectors (Xiphinema spp.). A few seeds containing TBRV and RRV were found in soils free of vector nematodes, suggesting that the viruses are disseminated in weed seed. This probably explains how TBRV and RRV have reached a large proportion of L. elongatus populations in eastern Scotland.     

Occurrence and distribution of lettuce mosaic disease in Tehran province from Iran

Lettuce mosaic virus (LMV) Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) were identified in fields of Tehran province. In this study 452 leaf samples were collected from the fields throughout the Tehran province during 2002 and 2003. Distribution of Lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV)and Arabis mosaic virus (ArMV) was determined with DAS-ELISA. Percentage of single Infection to LMV. CMV or TSWV was 20.58, 15.93 and 9.96% respectively. Also 15.28% of samples were co- infected with LMV+CMV, 8.19% with LMV+TSMV and 7.74% with CMV+TSWV. 4.65% of samples were Infected to all of these three viruses. LMV was found in 48.69%, CMV in 43.59% and TSWV in 30.54% of samples totally. Therefore LMV is major dominant agent of lettuce mosaic disease in Tehran province. This is the first report of occurrence of TSWV on lettuce in Iran and first report of CMV and LMV in Tehran province.     

Increase in Cucurbit Aphid–Borne Yellows Virus Concentration by Co–infection with Sap–transmissible Viruses does not Increase its Aphid Transmissibility

The cucurbit aphid–borne yellows virus (CABYV) is a new tentative member of the luteovirus group which is transmitted persistently by Myzus persicae and Aphis gossypii.
In muskmelon plants, mixed infection with CABYV and zucchini yellow mosaic virus (ZYMV) induced an increase in CABYV concentration estimated by double antibody sandwich–enzyme linked immunosorbent assay (DAS–ELISA), which was maximum after 3 weeks, of co–infection.
Assays, conducted with other cucurbit hosts and sap–transmissible viruses showed that a similar increase occurred with most of the potyviruses tested. However, cucumber mosaic virus (CMV) and squash mosaic virus (SqMV) were inefficient for less efficient than potyviruses) in increasing CABYV concentration.
Aphid transmission experiments were conducted to check whether increased virus multiplication could either enhance transmission rates or modify the mode of CABYV acquisition by aphids. However, when A. gossypii was used, no increases in CABYV, transmissibility nor in its acquisition mode were detected.     

Identification of Alfalfa Mosaic Virus in Greek Alfalfa Crops

Alfalfa mosaic virus (AMV) was found to be the prevalent virus disease in alfalfa crops in Central and Northern Greece. The virus was identified on the basis of the host symptomatology, aphid and seed transmission, particle morphology and serological tests. AMV was largely detected in commercial seeds of local grown alfalfa cvs by ELISA. Infected seed lots appear to be the main source responsible for the widespread distribution of the virus in Greek alfalfa fields.     

Occurrence, distribution and relative incidence of mosaic viruses infecting field--grown squash in Tehran province, Iran

Squash (Cucurbita pepo) belongs to Cucurbitaceae family. Every year Cucurbitaceae are planted world wide. They are one of the most important economic crops. Cucurbitaceae are threatened by viruses. Many viruses damage the plants of this family. Since nine viruses have been reported on squash from Iran. In this survey, during 2002--2003, to determine the distribution of Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV), 466 samples were collected from squash field in Tehran province. Infected plants showing symptoms such as: mosaic, yellowing, deformation, shoestring of leaves and fruit deformation and yield reduction. Distribution of CMV, ZYMV and WMV were determined by DAS-ELISA. Thepercentage of ZYMV, WMV and CMV were 35.6, 26.1 and 25.1% respectively. Triple infection (CMV+ZYMV+WMV) were found in 6.4% of samples. ZYMV were found the most frequently the viruses. This is the first report of WMV on squash in Tehran province.     

The appearance and spread of mosaic infection in the tomato crop and the relation to seed transmission of the virus

Appearance and spread of infection with mosaic-inducing viruses were studied for three seasons in tomato crops under glass. Comparison was made between the reactions of plants raised from virus-free seed and those of plants raised from virus-infected seed, on plots distributed at random in a house in which no precautions against entry and spread of virus were taken. Freedom from mosaic infection was maintained longest in plants raised from virus-free seed. An experiment was carried out after steam sterilization of the soil and under exceptionally favourable weather conditions. Appearance of mosaic symptoms occurred later in the life of the plants in this season and plants raised from virus-free seed did not react differently from other plants.
The location of plants first showing mosaic symptoms was related to the depth and texture of soil beneath those plants.
Tests were made of the apparent virus content of infected tomato seed during germination and differences were found in the persistence of virus during germination in seeds of differing origin.
Apparent, 'delayed' seed transmission of mosaic-inducing viruses occurs in the tomato crop, but as yet, this condition can only be interpreted in terms of differences in the resistance of plants raised from seed of differing origin to the multiplication and systemic spread of those viruses. The use of virus-free seed taken from well-nourished vigorous plants is essential to the production of a virus-free tomato crop under commercial conditions.     

Serological and conductimetric assays for the detection of Pseudomonas syringae pathovar pisi in pea seeds

Test protocols for detecting Pseudomonas syringae pv. pisi , the causal agent of bacterial blight, in pea seeds are generally based on dilution-plating assays. These assays are usually very specific and reliable, but are time-consuming and laborious. Tests suited for large scale screening of seed lots are therefore needed. Conductimetric assays, immunofluorescence microscopy (IF) and an enzyme-linked immunosorbent assay (ELISA) for detecting Ps. syr. pv. pisi in pea seed extracts were compared with dilution-plating by two extraction methods, viz. 6 h soaking of seeds and 2 h soaking of flour of ground pea seeds in water. In general, the detection of Ps. syr. pv. pisi with conductimetric, IF and dilution-plating assays in the suspension water of the ground and 2 h-soaked pea samples was less sensitive than detection in suspension water of the 6 h-soaked pea seeds. The detection threshold of these assays varied per seed lot between 0 and 4.08 log cfu ml^-1 for the 6 h soaking procedure. The detection threshold of ELISA varied for both extraction methods generally between 4.08 and 6.08 log cfu ml^-1. Detection times recorded in conductimetric assays correlated well (— 0.89 < r < —0.98) with the log colony-forming units of Ps. syr. pv. pisi added to seed extracts at 27 as well as 17°. However, confirmation of results by isolation on semi-selective media after conductimetry was more successful at 17° than at 27°, because of the relatively lower activity of saprophytic Pseudomonas spp. at this temperature.     

利用异种动物抗血清双抗夹心法检测小麦黄花叶病毒

小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV),属于马铃薯Y病毒科(Potyviridae),大麦黄花叶病毒属(Bymovirus),传播介体为禾谷多粘菌(Polymyxa graminis),与发生在欧美的小麦梭条花叶病毒(WSSMV)为同一属内的两种病毒。该病毒在我国分布广泛,在长江流域各省份以及济南、陕西等都有分布,对小麦生长.发育构成严重危害。一般可引起小麦减产10％～30％,严重时达70％,甚至绝产。以往对该病害的诊断主要是根据田间的症状表现,有时很难与由其他病原或环境因子引致症状相区分,目前,关于WYMV的问接酶