Seed-borne Virus Research Articles

Molecular detection of Cucumber green mottle mosaic virus variants from cucurbits fields in Iran

Cucumber green mottle mosaic virus (CGMMV) is seed borne virus belonging to the genus Tobamovirus into family Virgaviridae, which transmit mechanically during agricultural practice, seed and through water. In this study, based on virus description cucurbits fields were observed to detect CGMMV. Therefore, biological purification was performed to recognise the type of symptoms incited by each isolates. Accordingly, local lesion host plants were inoculated with virus isolates. Following, the samples having CGMMV symptoms are selected to RNA extraction and molecular tests. Furthermore, to detect different strains of CGMMV, a pair of specific primers corresponding to full coat protein and a part of movement protein gene was applied to amplify a 523 bp fragment in reverse transcription polymerase chain reaction (RT-PCR). Eventually, an expected band with 523 bp length was revealed in nine isolates by RT-PCR. It is a first report of CGMMV from north-west of Iran by molecular methods.

First Report of Olive mild mosaic virus and Sowbane mosaic virus in Spinach in Greece.

Uncommon, viruslike symptoms (yellowing, line patterns, leaf deformation, and necrosis), were observed in spinach fields in the Marathon area, Greece in 2004. Seedlings from the same seed lot, grown in the greenhouse, also developed the same viruslike symptoms, indicating that the causal agent(s) of the disorder is seed-transmissible. Spinach seedlings of the same variety but a different lot and herbaceous indicators (Chenopodium quinoa, C. amaranticolor, Sonchus oleraceus, and Nicotiana benthamiana) were mechanically inoculated with infected material. Spinach developed yellowing or necrotic spots whereas indicators showed variety of symptoms including mosaic, vein banding, and necrotic lesions. Virus purifications, double-stranded RNA extractions, cloning, and sequencing (2,3) followed by a combination of molecular (reverse transcription [RT]-PCR and immunocapture RT-PCR) and serological (ELISA) techniques with antisera provided by Dr. Avgelis were performed as described (4), verifying the presence of two viruses in the diseased seedlings: Sowbane mosaic virus (SoMV), a sobemovirus, was present in spinach and indicators with mottling and leaf deformation, whereas Olive mild mosaic virus (OMMV), a necrovirus, was present in plants with necrotic spots. All RT-PCR products amplified with primers SoMV-F (5'-CAAATGGTCTTGGTCAGCAGTC)/SoMV-R (5'-GCATACGCTCGACGATCTG) and OMMV-F (5'-CAAACCCAGCCTGTGTTCGATG)/OMMV-R (5'-CATCAGTTTGGTAATCCATTGA) were sequenced and found to confirm the other results. The SoMV-spinach isolate polyprotein gene sequence (GenBank Accession No. DQ450973) has 95% sequence identity with the type isolate from C. quinoa (GenBank Accession No. GQ845002), whereas the OMMV-spinach isolate (GenBank Accession No. JQ288895) has 92% sequence identity with the OMMV type isolate from olive (GenBank Accession No. AY616760). SoMV has been found to naturally infect spinach in the Netherlands (1) and, to our knowledge, this is the first report on spinach in Greece. The presence of OMMV in spinach is, to our knowledge, the first report worldwide. Its natural host range is limited to olive, tulip, and now spinach. OMMV might be transmitted by Olpidium spp. and may, according to data of its close relatives, persist in the soil for several decades. Pollen- and seedborne viruses (PSVs) like sobemoviruses and necroviruses are of particular importance for a crop like spinach where crop increase takes place in small, seed production-designated areas. If a PSV spreads in such an area it has the potential to become a major problem for the industry, especially when it remains undetected. Infected seed can be shipped worldwide with PSVs, causing diseases and becoming endemic in areas where they were absent. For this reason and the fact that field losses can exceed 50%, rigorous monitoring for the presence of SoMV and OMMV in seed fields is essential to minimize the possibility of the viruses moving to new areas.

Evaluasi Tiga Metode Preparasi RNA Total untuk Deteksi Turnip mosaic potyvirus dari Benih Brassica rappa dengan Reverse Transcription-Polymerase Chain Reaction

Turnip mosaic virus (TuMV) is an important seedborne virus that infects vegetable crops, especially Brassicae group, in Indonesia. Reliable detection method having high accuracy is required to detect the virus from seeds in order to avoid disease incidence as early as possible. A study was conducted to evaluate three total RNA extraction methods from Brassica seeds for TuMV detection using reverse transcription- polymerase chain reaction. The samples used are Brassica rappa seeds and seedling germinated for 3, 5, and 7 days. Total RNA extraction methods evaluated consisted of Wylie method, Randles method, and commercial kit as comparation. Total RNA extracted using Wylie and Randles methods has high concentration but low level of purity, on the other hand total RNA obtained using commercial kit has low concentration but high level of purity. Detection of TuMV from seed was successfully carried out using Wylie and Randles methods, TuMV from 3 - day old seedlings was detected using all three methodes, TuMV from 5-day old seedlings was only detected using Randles method, whereas TuMV from 7-day old seedlings was not detected using all three methodes. Total RNA extraction method using Wylie and Randles methodes is recommended for detection of TuMV from caisin seed using RT-PCR technique. Key words: Brassica rappa seed, reverse transcription- polymerase chain reaction, Total RNA, Turnip mosaic potyvirus

Virus diseases of annual pasture legumes: incidences, losses, epidemiology, and management

This paper reviews current knowledge concerning the occurrence, losses caused, epidemiology, and management of virus diseases of annual pasture legumes. The viruses commonly present are spread by contact, or aphid vectors either non-persistently or persistently. Whether they are seed-borne and their means of transmission are critical factors determining their incidences within pastures in climatic zones with dry summers or substantial summer rainfall. Large-scale national or state surveys of subterranean clover pastures revealed that some viruses reach high infection incidences. Contamination with seed-borne viruses was widespread in plots belonging to annual pasture legume improvement programs and seed stocks of subterranean clover, annual medics, and alternative annual pasture legumes, and in commercial annual medic seed stocks. Yield loss studies with grazed swards were completed for three common viruses: two in subterranean clover and one in annual medics. These studies demonstrated considerable virus-induced losses in herbage and seed yields, and established that virus infection causes deteriorated pastures with high weed contents even when foliar symptoms are mild. Comprehensive integrated disease management tactics involving phytosanitary, cultural, chemical, or host resistance measures were devised for these three viruses in infected pastures, and for seed-borne viruses in annual pasture legume improvement programs. Several other viruses are potentially important, but, with these, quantification of losses caused in grazed swards is lacking and information on incidences in pastures is currently insufficient. Critical research and development gaps that need addressing are identified.

Application of Multiplex RT-PCR for Detection of Cucurbit-infecting Tobamovirus

Cucumber green mottle mosaic virus (CGMMV) and Kyuri green mottle mosaic virus (KGMMV) are seed borne viruses and they are also transmitted mechanically during agricultural practice and through water. Hence, these viruses have potential diseases widely distributed throughout the world. To detect different strains of CGMMV and KGMMV, several specific primers for each virus were designed for single and multiplex RT-PCR. The results of single and multiplex RT-PCR showed that CGMMV was detected in zucchini isolated in Bali-Indonesia, while KGMMV was detected both in zucchini isolated in Bali-Indonesia and Cucumis metuliferus isolated in Thailand. Furthermore, artificial co-infection of these two viruses was prepared and carried out using two different ways of viral RNAs extraction. Based on the results, it could be reported that viral RNAs for cDNA amplification by multiplex RT-PCR could be extracted from a mixture of infected leaves or separate extraction of each viruses infected leaves. In addition, results presented in this study demonstrated the application of multiplex RT-PCR to simultaneously detect CGMMV and KGMMV from cucurbit leaves using a mixture of four primers and its feasibility as a sensitive and rapid laboratory assay. Since, no multiplex RT-PCR technique has been described for the detection of CGMMV and KGMMV, this technique can be a good option for sensitive and reliable tool for detection of two major cucurbit infecting Tobamoviruses. Keywords : Cucurbit infecting Tobamovirus, multiplex RT-PCR, seed borne viruses

십자화과 작물 종자에서 종자전염 세균 및 바이러스 동시 검출을 위한 One-step Multiplex RT-PCR 방법

우리나라에서 주로 재배되는 십자화과 작물(상추, 콜라비, 무, 배추, 양배추)의 종자 전염 병원균 중에서 세균성 병원균 Xanthomonns campestris pv. campestris(Xcc)와 바이러스 병원균 Lettuce Mosaic Virus(LMV)를 종자에서 동시검출하기 위한 One-step multiplex RT-PCR을 개발하였다. 각각의 병원균을 특이적으로 증폭시키는 병원균 검출용 프라이머 2종(Xcc-F/R, LMV-F/R)을 primerblast 프로그램을 이용하여 제작하였고 이들 프라이머 세트는 프라이머간 또는 병원균 cDNA간의 간섭없이 특이적으로 타겟 병원균만을 검출하였다. PCR을 이용한 병원균의 검출 최소 민감도는 1 ng이었다. 십자화과 작물중에서 유통 중인 콜라비 10품종, 상추 50품종, 무 20품종, 배추 20품종 그리고 양배추 20품종에 대한 종자 감염 병원균 검출을 위한 One-step multiplex RT-PCR 수행 결과, LMV는 전체 120품종 중에서 39품종에서 검출되었고, Xcc는 2개 품종에서 검출되었다. 그리고 50품종의 상추 종자 시료 중에 8품종의 시료에서 LMV와 Xcc가 동시 검출되었다. The aim of this research was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant pathogenic bacteria and seed borne virus in commercial Brassicaceae crop seeds, Xanthomonns campestris pv. campestris (Xcc) and Lettuce Mosaic Virus (LMV). Bacterial and virus diseases of Brassicaceae leaves are responsible for heavy losses. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcc and LMV in commercial Brassicaceae crop seeds (lettuce, kohlrabi, radish, chinese cabbage and cabbage), two pairs of specific primer (LMV-F/R, Xcc-F/R) were synthesized by using primer-blast program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The multiplex PCR for the two pathogens in Brassicaceae crop seeds could detect specifically without interference among primers and/or cDNA of other plant pathogens. The pathogen detection limit was determined at 1 ng of RNA extracted from pathogens. In the total PCR results for pathogen detection using commercial kohlrabi (10 varieties), lettuce (50 varieties), radish (20 varieties), chinese cabbage (20 varieties) and cabbage (20 varieties), LMV and Xcc were detected from 39 and 2 varieties, respectively. In the PCR result of lettuce, LMV and Xcc were simultaneously detected in 8 varieties.

Establishing inoculum threshold levels for Bean common mosaic virus strain blackeye cowpea mosaic infection in cowpea seed

Bean common mosaic virus strain blackeye cowpea mosaic (BCMV-BlCM) is an important seed-borne virus infecting cowpea and is transmitted both by seeds and aphids. Infected cowpea seeds can act as primary source of inoculum for disease epidemics. Four field experiments were conducted during 2003 - 2006 to assess the role of different amounts of seed-borne inoculum in the dissemination of BCMVBlCM virus in cowpea under field conditions. The identity of BCMV-BlCM was confirmed by ELISA and IC-RT-PCR. Plants infected at an early growth stage appeared to serve as the primary source for subsequent virus spread by aphids. The mean disease incidence during four field experiments reached 88-93% in plots sown with 10% infected seed. The disease incidence in plots sown with 5% infected seed recorded 46-63% while for plants raised from 3 and 2% BCMV-BlCM seed infection, disease incidence reached 32-49% and 17-23%, respectively. Mean yield losses in terms of seed yield per plant from four field experiments were 74 and 54% for initial seed infection of 10 and 5%, respectively. Seed infection of 2% BCMV-BlCM incidence resulted in an average of 24% mean seed yield loss/plant-1. The infection appeared to decrease the seed yield in terms of number and size. The BCMV incidence in harvested seed ranged from 0.3 - 19% for the different levels of initial seed infection. The field experiments demonstrated that sowing > 1% BCMV-BlCM infected seed can lead to significant losses in grain yield, while the spread of BCMV-BlCM infection resulting from sowing 1% infected seed did not significantly decrease seed yield. The role of establishing damage or inoculum thresholds from BCMVBlCM seed-borne infections is discussed in the present study. Keywords: Cowpea, potyvirus, seed-borne virus, thresholds, yield loss

Elimination of Bean common mosaic virus using an electrotherapy technique

Bean common mosaic virus (BCMV) is a major seed transmitted virus of common bean (Phaseolus vulgaris). The use of virus-free germplasm is a prerequisite for production of certified seeds, which is an important disease control measure. In this study, the efficiency of electrotherapy in eliminating BCMV and producing virus-free plants in two common bean cultivars, Khomein and Capsouli, was evaluated. Nodal cuttings of infected bean plants were exposed to electric currents of 5, 10 and 15 mA for 10 minutes and planted in vitro. One month old regenerated plantlets were tested by a Double Antibody Sandwich (DAS)-ELISA method for BCMV infection. The three intensities of electric current resulted in mean virus elimination percentages of 37.2%, 45.5% and 71.9% and mean plant regeneration of 79.5%, 68.6% and 58.4%, respectively. The Therapy Efficiency Index for the three electrotherapy treatments was estimated as 28.9, 29.2 and 39.1 in cv Khomein and 26.9, 33.3 and 44.8 in cv Capsouli, respectively. Based on the Therapy Efficiency Indices, an electric current of 15 mA for 10 min was the most effective treatment in both cultivars. The technique developed in this study can be a useful procedure for the production of virus-free germplasm and/or nucleus seed stock in crop plants infected with seed-borne viruses.

Biological and molecular diagnosis of seedborne viruses in cowpea germplasm of geographically diverse sub‐Saharan origins

A total of 983 cowpea accessions obtained from the University of California, Riverside (UCR) Cowpea Repository were analysed for seedborne viruses. A majority of the accessions originated from 11 countries representing different agroclimatic zones in sub‐Saharan Africa, and included landraces, local cultivars and breeding lines. Following the initial grow‐out tests, 69 cowpea accessions, mostly with symptoms of virus infection, were selected for further evaluation using a combination of host range, reverse transcription‐polymerase chain reaction (RT‐PCR) and sequence analyses. The analyses revealed that samples from 46 (67%) accessions harboured one or more known seedborne viruses of cowpea. These included seed samples of accessions originating from Botswana (13 accessions), Ghana (6), Nigeria (6), Mali (1), Kenya (5), Cameroon (7), Niger (4), Côte d’lvoire (1), Benin (1), India (1) and China (1). Viruses were identified by RT‐PCR analysis of total RNAs extracted from suspected virus‐infected samples using virus species‐specific primers, as well as the cloning and sequencing of RT‐PCR products amplified using virus genus‐ and family‐specific degenerate oligonucleotide primers. The viruses identified included Cowpea aphid‐borne mosaic virus (CABMV), Cucumber mosaic virus (CMV) and Southern bean mosaic virus (SBMV). Phylogenetic analysis of the deduced coat protein (CP) amino acid sequences of selected CMV isolates recovered from five agroclimatically distinct locations confirmed their affiliations as new members of CMV subgroup IB. This is the first time that seedborne viruses of cowpea accessions in a major collection (UCR) have been identified using RT‐PCR and sequencing approaches.

Serological detection of seed borne viruses in cowpea regenerated germplasm using protein a sandwich enzyme linked immunorsorbent assay

Cowpea (Vigna unguiculata (L.) Walp) is susceptible to a number of fungal, bacterial and viral diseases. The effects of viruses can be devastating and are a major constraint to the production of cowpea. A study was carried out to detect the presence of seed borne viruses in fourteen cowpea accessions screened from 300 regeneratedcowpea germplasm from the genebank collection of the International Institute of Tropical Agriculture (IITA) in Nigeria. Protein A Sandwich Enzyme linked Immunosorbent Assay (PAS ELISA) was used to detect seed borne viruses in the accessions. Out of 36 accessions of TVu 033 indexed, 31 were infected with Cowpea aphid-borne mosaic virus genus Potyvirus, which is of great economic importance in germplasm conservation and exchange between countries as it causes great loss. Southern bean mosaic virus was not detected in any of the accessionsevaluated. Seeds were harvested from the infected mother plants at maturity and the embryos excised in vitro onto hormone free medium composed of 4.43 g MS basal salt, 30 g sucrose and 8 g of agar. The seedlings obtained were serologically indexed to detect any seed-borne viruses after acclimatisation to screen house conditions. Theseedlings obtained from excised embryos got from harvested seeds gave a negative ELISA result.

Effectiveness of Chemo- and Thermotherapeutic Treatments on Pepino mosaic virus in Tomato Seed.

Pepino mosaic virus (PepMV) is a seedborne virus of importance in greenhouse tomato. The ease of mechanical transmission of PepMV from contaminated tomato seed to seedlings makes commercial tomato seed a potential source of initial virus inoculum. The objective of this study was to evaluate the effectiveness of chemo- and thermotherapeutic treatments on PepMV using seed from a commercial hybrid tomato seed lot. The effect of various seed treatments was evaluated through bioassay with mechanical inoculation of the treated seed extract on indicator plants (Nicotiana benthamiana). Three commonly used seed-treatment chemicals and two thermo-treatment methods were evaluated. The most effective chemical was the commercial bleach solution (0.5 and 1.0% sodium hypochlorite), followed by trisodium phosphate. On the other hand, hydrochloric acid treatments were not effective. Under the stated chemotherapeutic treatment conditions, the rate of seed germination was not significantly affected. With thermotherapeutic treatments, although hot water soaking for 2 h at 55°C deactivated virus infectivity, it also resulted in a detrimental effect on seed germination. However, treatment with dry-heat baking (72 or 80°C for 48 to 72 h) was effective in reducing PepMV infection with minimum impact on seed germination.

Testing Methods For Seed‐transmitted Viruses: Principles and Protocols ‐ S. E. Albrechtsen

Testing Methods For Seed-transmitted Viruses: Principles and Protocols. S. E. Albrechtsen. 288 pp. Wallingford, UK: CABI Publishing (http://www.cabi-publishing.org), 2005. £55. ISBN 0 85 199016 9 (spiralbound softback). I was recently asked to recommend some key plant virology texts for a new diagnostic laboratory. Good reference material is easy: buy the latest edition of Matthews’ Plant Virology (Hull, 2002) and then go online for VIDE (http://image.fs.uidaho.edu/vide/refs.htm) and DPVweb (http://www.dpvweb.net). There are also some excellent crop-specific texts available, e.g. Viruses and Virus Diseases of Poaceae (Lapierre & Signoret, eds, 2004). But what about practical guides? Well, then it gets more tricky: Methods in Plant Virology (Hill, 1984) is long out of press. There is Diagnosis of Plant Virus Diseases (Matthews, 1993), which is excellent on the principles but is somewhat dated, e.g. PCR only gets one page. The more recent and soon to be revised Plant Virology Protocols (Foster & Taylor, 1998) is much better for molecular methods, but is aimed more at research and development rather than diagnostic requirements. Beyond that there is little else; that is, I believe until now, with the publication of this new book. As far as I am aware, this is the first book to be dedicated to seed virus-testing, but it goes well beyond that and actually provides one of the best single-source, practical guides to plant virus diagnostic methods available. Split into seven chapters, the main bulk of the book is dedicated to the three methods chapters: biological assays, serological tests and nucleic acid-based tests. All provide excellent coverage of their respective fields, with good, established protocols. I particularly like the fact that there are plenty of options given, e.g. a table of different recipes for one-step RT-PCR. I also appreciated that support techniques such as isolate storage are fully covered. As anyone who has run a routine ELISA laboratory will know, maintaining a supply of positive control material is a major problem. All laboratories need good advice and protocols on this, and this book provides it. And good advice on troubleshooting is another strong point, as is the fact that the latest techniques, such as real-time PCR methods, are covered. I guess one disappointment is that the author did not go as far as including any real-time protocols, in contrast to the comprehensive coverage given for conventional PCR. While real-time PCR is still a relatively new technique, it has become a routine technique in some laboratories and is rapidly establishing itself. Similarly electron microscopy does not really feature. However in fairness, EM is not a great tool for seed testing, which has very much become a niche technique used routinely by very few. The other good thing about this book is that it really does do what it says in the title; covering both principles and protocols. It is often easy for books to become either laboratory manuals with precious little background or reference texts without practical use. However, this is a book that is equally at home in the laboratory and the library. It has excellent review chapters covering the background to seedborne viruses, providing information on a range of subjects including the mechanism of seed transmission, control and economic importance – all good stuff. It also has an extremely useful section (co-written by Steve Roberts) on sampling and testing strategies, arguably the most important step in any seed test. The quick reference list of seedborne viruses is another helpful addition. Overall, this is an excellent guide to seed testing and virological diagnostic methods in general. It is a very welcome addition to the literature and a book that should have a place on the shelf of every diagnostic plant virology laboratory, whether or not they are involved in seed testing.

Immunolocalization of Pepper mild mottle virus in Capsicum annuum seeds

To clarify the mechanism of seed transmission of Pepper mild mottle virus (PMMoV), the virus was immunolocalized in Capsicum annuum seeds using fluorescence microscopy. Two distinct patterns were observed: In the first, PMMoV was present in the epidermis and parenchyma but not in the endosperm or embryo; in the second, the virus was restricted to the surface of the epidermis and parenchyma. These findings shed light on the fundamental mechanisms of seed transmission of tobamoviruses and may aid in the design of new methods to prevent the spread of seedborne virus diseases.

Occurrence and Relative Incidence of Viruses Infecting Soybeans in Iran.

A survey was conducted to determine the incidence of Alfalfa mosaic virus (AlMV), Bean common mosaic virus (BCMV), Bean yellow mosaic virus (BYMV), Blackeye cowpea mosaic virus (BlCMV), Cucumber mosaic virus (CMV), Pea enation mosaic virus (PEMV), Peanut mottle virus (PeMoV), Soybean mosaic virus (SMV), Tobacco mosaic virus (TMV), Tobacco ringspot virus (TRSV), Tobacco streak virus (TSV), Tomato ringspot virus (ToRSV), and Tomato spotted wilt virus (TSWV) on soybean (Glycine max) in Iran. Totals of 3,110 random and 1,225 symptomatic leaf samples were collected during the summers of 1999 and 2000 in five provinces of Iran, where commercial soybean is grown, and tested by enzyme-linked immunosorbent assay (ELISA) using specific polyclonal antibodies. Serological diagnoses were confirmed by electron microscopy and host range studies. The highest virus incidence among the surveyed provinces was recorded in Mazandaran (18.6%), followed by Golestan (15.7%), Khuzestan (14.2%), Ardabil (13.9%), and Lorestan (13.5%). Incidence of viruses in decreasing order was SMV (13.3%), TSWV (5.4%), TRSV (4.2%), TSV (4.1%), PEMV (2.9%), BYMV (2.2%), ToRSV (2.1%), AlMV (1.3%), BCMV (0.8%), and CMV (0.6%). Additionally, 1.5% of collected leaf samples had positive reactions in ELISA with antiserum to TMV, indicating the possible infection of soybeans in Iran with a Tobamovirus that is related serologically to TMV. Of 195 leaves from plants showing soybean pod set failure syndrome (PSF) in Mazandaran and Lorestan, only 14 (7.2%) samples had viral infection. No correlation was observed between PSF and presence of the 13 viruses tested, suggesting the involvement of other viruses or factors in this syndrome. To investigate the presence of seed-borne viruses, including SMV, TRSV, ToRSV, and TSV, 7,830 soybean seeds were collected randomly at harvesting time from the major sites of soybean seed production located in Mazandaran and Golestan provinces. According to ELISA analyses of germinated seedlings, 7.1 and 8.9% of the seed samples from Golestan and Mazandaran provinces, respectively, transmitted either SMV, TRSV, ToRSV, or TSV through seed. We also showed that SMV and other seed transmissible viruses, as well as TSWV, usually are the most prevalent viruses in soybean fields in Iran. In this survey, natural occurrence of AlMV, BCMV, BlCMV, BYMV, CMV, PEMV, PeMoV, and TSWV was reported for the first time on soybeans in Iran.

Moisture Contents in Bottle Gourd Seeds during Dry Heat Treatment and Subsequent Germination

Dry heat treatment (DHT), a powerful and agrochemical-free means of inactivating seed-borne virus and other pathogens, has been extensively used for value-added vegetable seeds in Korea, Japan, and some other countries. Since seeds are treated with extremely high heat (75 °C or higher) for a long time (72 h or longer), heat-induced phytotoxicity symptoms are frequently observed. Even though various internal and external factors, such as seed maturity and vigor, maximum temperature and duration of DHT, are known to influence the severity of phytotoxicity, precise control of seed moisture contents during DHT is regarded as one of the most important factors for successful DHT. In an ideal condition using a specifically designed DTH machine, seed moisture content of bottle gourd, initially around 6.20% to 0.64% when stored in a storage room with 50% RH, decreased by 1% after 24 h at 35 °C (5.20% to 0.23%), and further decreased below 4% after 24 h pretreatment at 50 °C (3.64% to 0.37%). The seed moisture content was further reduced down to about 2% after 72 h DHT at 75 °C (2.16% to 0.28%). During the post-treatment conditioning at 50 °C and 70% RH for 24 h, the moisture contents were raised to about 6%(5.94% to 0.45%), thus approaching the initial moisture content of 6% to 7%. During the germination period, treated seeds showed slower absorption of water as compared to the intact seeds, thus suggesting that this slow absorption of initial moisture absorption may be responsible for the slow initial germination frequently observed in treated seeds. Final germination and seedling vigor were not affected by DHT.

Using epidemiological information to develop effective integrated virus disease management strategies

Virus diseases cause serious losses in yield and quality of cultivated plants worldwide. These losses and the resulting financial damage can be limited by controlling epidemics using measures that minimise virus infection sources or suppress virus spread. For each combination of virus, cultivated plant and production system, there is an ‘economic threshold’ above which the financial damage is sufficient to justify using such measures. However, individual measures used alone may bring only small benefits and they may become ineffective, especially over the long term. When diverse control measures that act in different ways are combined and used together, their effects are complementary resulting in far more effective overall control. Such experiences have led to the development of integrated management concepts for virus diseases that combine available host resistance, cultural, chemical and biological control measures. Selecting the ideal mix of measures for each pathosystem and production situation requires detailed knowledge of the epidemiology of the causal virus and the mode of action of each individual control measure so that diverse responses can be devised to meet the unique features of each of the different scenarios considered. The strategies developed must be robust and necessitate minimal extra expense, labour demands and disruption to standard practices. Examples of how epidemiological information can be used to develop effective integrated disease management (IDM) strategies for diverse situations are described. They involve circumstances where virus transmission from plant-to-plant occurs in four different ways: by contact, non-persistently or persistently by insect vectors, and by root-infecting fungi. The examples are: Subterranean clover mottle virus (SCMoV) (contact-transmitted) and Bean yellow mosaic virus (BYMV) (non-persistently aphid-transmitted) in annually self-regenerating clover pasture; three seed-borne viruses (all non-persistently aphid-transmitted) plots of pasture legume improvement programmes; Tomato spotted wilt virus (TSWV) (persistently thrips-transmitted) in vegetables in seedling nurseries, protected cropping or field systems; and lettuce big-vein disease (fungus-transmitted) in lettuce in seedling nursery, hydroponic, infested field or uninfested field situations. By describing the kinds of approaches required, this article is intended to help future research and extension programmes devise integrated disease management strategies that not only function effectively to diminish the losses caused by economically important plant virus diseases but also fulfill the requirement of being environmentally and socially responsible.

A Survey of Viruses Affecting Faba Bean (Vicia faba) in Tunisia Includes First Record of Soybean dwarf virus.

A survey was conducted in April 2003 to identify viruses infecting faba bean (Vicia faba L.) in six regions (Beja, Bizerte, Cap-bon, Le Kef, Siliana, and Zaghouan) in Tunisia. A total of 292 faba bean samples with symptoms of viral infection (leaf rolling, yellowing, and mosaic) were collected. The samples were tested at the virology laboratory of the International Center for Agricultural Research in the Dry Areas (ICARDA), Syria, for 11 viruses using the tissue-blot immunoassay procedure (3). Specific rabbit polyclonal antisera were used to test for Chickpea chlorotic dwarf virus (CpCDV) (provided by H. J. Vetten, BBA, Braunschweig, Germany), Alfalfa mosaic virus (AMV), Bean yellow mosaic virus (BYMV), Broad bean mottle virus (BBMV), Broad bean stain virus (BBSV), Cucumber mosaic virus (CMV), and Pea seedborne mosaic virus (PSbMV) (ICARDA, Aleppo, Syria). In addition, four specific monoclonal antibodies were used to detect Bean leaf roll virus (BLRV) (4B10) (2), Beet western yellows virus (BWYV) (ATCC PVAS-647; American Type Culture Collection, Manassas, VA), Faba bean necrotic yellows virus (FBNYV) (3-2E9) (1), and Soybean dwarf virus (SbDV) (ATCC PVAS-650). Serological tests showed that BBMV, a beetle-transmitted and seedborne virus identified in 23.3% (68 samples) of the samples tested, was the most common. BLRV, FBNYV, BWYV, BYMV, SbDV, and PSbMV were detected in 56, 33, 31, 10, 5, and 1 sample(s) of 292 samples tested, respectively. AMV, BBSV, CMV, and CpCDV were not detected in any samples tested. In Tunisia, BLRV, BWYV, BYMV, FBNYV, and PSbMV have previously been reported in faba bean (4), but to our knowledge, this is the first record of SbDV affecting faba bean in Tunisia, where it was detected in two fields in the Cap-bon Region. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by western blots, extracts from SbDV-infected plants were observed to contain 23-kDa structural proteins, which reacted strongly with SbDV monoclonal antibodies. Transmission tests showed that the samples, which reacted with SbDV monoclonal antibodies, were transmitted to faba bean plants by the pea aphid (Acyrthosiphon pisum Harris) in a persistent manner. To our knowledge, this is the first report of SbDV naturally infecting faba bean in Tunisia and it could cause a serious problem to other leguminous crops grown in Tunisia, such as French bean and peas, which are hosts for the virus.

First record of Chickpea chlorotic dwarf virus (CpCDV) affecting faba bean (Vicia faba) crops in Egypt

A field visit was conducted in March 2002 to identify viruses infecting the faba bean ( Vicia faba ) crop in four governorates (Fayoum, Beni Suef, El-Nobareia and Bihera) in Egypt. A total of 71 faba bean samples with symptoms of viral infection (leaf rolling, yellowing and mosaic) were collected. These were tested at the Virology Laboratory of ICARDA, Syria, for seven viruses using the tissue-blot immunoassay procedure (Makkouk & Comeau, 1994). Specific rabbit polyclonal antisera were used to test for Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus , family Geminiviridae ; provided by H. J. Vetten, BBA, Braunschweig, Germany); for Broad bean stain virus (BBSV, genus Comovirus , family Comoviridae ); for Bean yellow mosaic virus (BYMV, genus Potyvirus , family Potyviridae ) and for Pea seed borne mosaic virus (PSbMV, genus Potyvirus , family Potyviridae ). In addition, three specific monoclonal antibodies were used to detect Faba bean necrotic yellows virus (FBNYV, genus Nanovirus , family Nanoviridae ) (Franz et al ., 1996); Bean leaf roll virus (BLRV, family Luteoviridae ) (4B10; Katul, 1992) and Beet western yellows virus (BWYV, genus Polerovirus , family Luteoviridae ) (ATCC PVAS-647; American Type Culture Collection, Manassas, VA, USA). Serological tests showed that BYMV, an aphid-transmitted and seedborne virus that was identified in 89% of samples tested, was the most common virus. In most of the fields surveyed, BYMV symptoms were noted to occur at high levels (80–100% infection). PSbMV was detected in nine samples (one from Fayoum and eight from Beni Suef); FBNYV was detected in 11 samples (all from Beni Suef). CpCDV, a leafhopper-transmitted virus, was, by contrast, detected in only two samples collected from the El-Nobareia governorate showing leaf rolling, yellowing and stunting. BWYV, BLRV and BBSV were not detected in any of the samples tested. In Egypt, FBNYV, BYMV and PSbMV have previously been reported in faba bean (Makkouk et al ., 1994), but this is the first record of CpCDV affecting faba bean in Egypt. CpCDV is the only member of the Geminivirus that is reported to naturally infect faba bean.

First Report of Raspberry bushy dwarf virus in Rubus multibracteatus from China.

Raspberry bushy dwarf virus (RBDV), genus Idaeovirus, has been reported in commercial Rubus spp. from North and South America, Europe, Australia, New Zealand, and South Africa. Infection can cause reduced vigor and drupelet abortion leading to crumbly fruit and reduced yields (3,4). In recent years, Rubus germplasm in the form of seed, was obtained on several collection trips to The People's Republic of China to increase the diversity of Rubus spp. in the USDA-ARS National Clonal Germplasm Repository, (Corvallis, OR). Before planting in the field, seedlings were tested for the presence of RBDV, Tomato ringspot virus, and Tobacco streak virus using triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) (antiserum produced by R. R. Martin). One symptomless plant of R. multibracteatus H. Lev. & Vaniot (PI 618457 in USDA-ARS GRIN database), from Guizhou province in China, tested positive for RBDV (RBDV-China). After mechanical transmission on Chenopodium quinoa Willd., this isolate produced typical symptoms of RBDV (3). To determine if RBDV-China was a contaminant during the handling of the plants, or if the source was a seedborne virus, the coat protein gene was sequenced and compared to published sequences of RBDV. RNA was extracted from leaves of R. multibracteatus and subjected to reverse transcription-polymerase chain reaction (RT-PCR) using primers that flank the coat protein gene. Products from four separate PCR reactions were sequenced directly or were cloned into the plasmid vector pCR 2.1 (Invitrogen, Carlsbad, CA) and then sequenced. The coding sequence of the coat protein gene of RBDV-China was 87.5% (722/825) identical to that isolated from black raspberry (Genbank Accession No. s55890). The predicted amino acid sequences were 91.6% (251/274) identical. Previously, a maximum of five amino acid differences had been observed in the coat proteins of different RBDV strains (1). The 23 differences observed between RBDV-China and the isolate from black raspberry (s55890) confirm that the RBDV in R. multibracteatus is not a greenhouse contaminant but is indeed a unique strain of RBDV. In addition, monoclonal antibodies (MAbs) to RBDV (2) were tested against RBDV-China. In these tests, MAb D1 did not detect RBDV-China, whereas MAb R2 and R5 were able to detect the strain. This is the first strain of RBDV that has been clearly differentiated by MAbs using standard TAS-ELISA tests. Although RBDV is common in commercial Rubus spp. worldwide, to our knowledge, this is the first report of RBDV in R. multibracteatus, and the first report of RBDV from China. The effects of this new strain of RBDV could be more or less severe, or have a different host range than previously studied strains. It is more divergent from the type isolate than any other strain that has been studied to date. Phylogenetic analysis of coat protein genes of RBDV may be useful in understanding the evolution and spread of this virus.

Identification of Pepper mild mottle virus in Commercial Bell Pepper in Florida.

Pepper mild mottle virus (PMMoV) has been identified on pepper in Florida several times since 1997. Initial observations were on ornamental peppers (Capsicum chinense, C. frutescens, and C. annuum). However, in the winter growing seasons of 1999 and 2000, PMMoV was detected in several bell pepper (C. annuum) cultivars in commercial production fields in south Florida. Symptoms in bell pepper plants were observed to vary with plant age and cultivar, and included stunting of young plants and puckering and yellow mottling of leaves. Fruit was deformed (lumpy and mottled) and slightly reduced in size. Older fruit exhibited brown streaks and, in some cultivars, an undesirable color at maturity. Incidence in commercial bell pepper fields varied from <1 to 30%. Symptoms on mechanically inoculated indicator hosts, analysis of viral-associated double-stranded RNA, and inclusion body morphology suggested the presence of a tobamovirus. PMMoV was specifically identified by serological testing using ELISA (Agdia, Elkhart, IN). To our knowledge, this is the first definitive identification of PMMoV (2) in Florida, although a prior isolation of the same or a similar virus was made in 1964 and reported as the Samsun latent strain of Tobacco mosaic virus (1). The occurrence of this seedborne virus in an area of intensive commercial pepper production requires careful monitoring to avoid future significant losses.