Virus-free Plants Research Articles

Impact of Sugarcane yellow leaf virus (ScYLV) infection on physiological efficiency and growth parameters of sugarcane under tropical climatic conditions in India

Yellow leaf (YL) of sugarcane caused by Sugarcane yellow leaf virus (ScYLV, a Polerovirus of the Luteoviridae family) is a serious disease affecting the crop production and productivity in India. Although impact of the disease on cane growth is observed, no systematic study has been done so far from the tropical Asian region to establish its impact on various physiological parameters, cane yield and juice quality. We have assessed physiological parameters in symptomatic and asymptomatic plants of ten different cultivars and a genotype. In addition, similar comparisons were made between virus-infected and virus-free plants derived through meristem culture. Our studies established that among several physiological parameters, photosynthetic rate (A), stomatal conductance (g s) and SPAD metre values were significantly reduced in cultivars severely infected with ScYLV. Virus-infected cultivars exhibited significant reduction in growth/yield parameters, viz. stalk height, stalk thickness and number of internodes. Plant growth reductions were found to be 42.9, 42.3 and 38.9 % in susceptible cultivars CoPant 84211, Co 86032 and CoC 671, respectively. In addition to reduction in stalk weight, height and girth, YL disease also reduced juice yield in the affected canes up to 34.15 %. Similarly, comparison of diseased (virus-infected) and virus-free plants derived through meristem culture also revealed a drastic reduction in cane growth/physiological parameters and juice yield due to virus infection. The present study is the first comprehensive report demonstrating that YL disease caused by ScYLV seriously affects cane and juice yield in major sugarcane varieties under tropical climatic conditions (India). Consequently, this situation warrants a massive programme to provide healthy seed material and initiate breeding for YL resistance in sugarcane.

Application of the TOA-MD model to assess adoption potential of improved sweet potato technologies by rural poor farm households under climate change: the case of Kabale district in Uganda

Sweet potato technologies that increase productivity, such as drought resistant varieties and virus free planting material are being promoted in order to reduce the vulnerability of poor farm households to climate change. In this paper, the Trade-off Analysis, Minimum Data Model Approach (TOA-MD) was used to assess the adoption potential of these technologies by resource poor farmers under climate change in Uganda. The model was calibrated and validated using household survey data collected in 2009 from Kabale district. To simulate adoption potential, the base system data was generated from household data and adjusted to reflect impact of climate change on crop yields and prices by 2050. The percentage increase in yields resulting from the use of climate resilient sweet potato technologies were used to estimate yields for alternative systems based on the results from sweet potato trials by the National Agricultural Research Organization (NARO), Uganda. Adoption potential of sweet potato technologies varied across altitudes. Compared with the high and lower altitudes, adoption potential is lowest at moderate altitude despite higher yields and lower costs of production. Paying farmers to adopt new sweet potato technologies is economically rational at the higher and moderate altitudes but not at the lower altitudes. The provision of free planting material (subsidy) for the evaluated technologies resulted in a modest increase of 2 % in adoption potential. Therefore, providing this as a way of increasing adoption of sweet potato technologies to reduce vulnerability of poor farm households to climate change will have a very small impact. Instead, climate change adaptation policy should focus on creating enabling environments for farmers to market their produce so as to raise returns and reduce the opportunity costs of climate change adaptation strategies.

Análise da presença de vírus em alho semente da segunda e quarta gerações, produzidos por termoterapia e cultura de tecido

O alho (Allium sativum L.) pode estar naturalmente infectado por um complexo de vírus filamentosos pertencentes aos gêneros Potyvirus, Carlavirus e Allexivirus. O acúmulo destes vírus se dá, principalmente, pela sua propagação vegetativa através dos bulbilhos. Como a planta de alho cultivada não produz semente verdadeira em todo o mundo, a única forma de se obter plantas livres de vírus se dá pela cultura de tecidos dos ápices caulinares e termoterapia. Utilizando estas técnicas, alhos sementes foram produzidos na FCA- UNESP de Botucatu e avaliados via RT-PCR para a presença de potyvirus, carlavirus e allexivirus. Na segunda geração dos microbulbilhos propagados em casa de vegetação, 6,6% de infecção foi verificada por allexivirus. Já na quarta geração foi observada incidência de 60% com allexivirus, 35% com potyvirus e todas foram negativas para carlavirus. A alta taxa de infecção por allexivirus pode estar relacionada à maior dificuldade de remoção de espécies de vírus pertencentes a este gênero, como também já observado por outros autores, pela infecção e transmissão de vírus pelo ácaro, Aceriatulipae, durante o armazenamento dos bulbos de um ano a outro. O alho na quarta geração corresponde a bulbilhos com peso inferior a 1 grama e que não haviam sido selecionados para multiplicação comercial. A seleção para tamanho do bulbilho tem efeito positivo na escolha de bulbilhos com menores taxas de infecção por vírus, já que a técnica de termoterapia e cultura de tecidos não elimina totalmente os vírus. Os resultados também enfatizam a necessidade de se realizar fumigação no alho semente armazenado de um ano a outro a fim de evitar a transmissão de allexivirus durante o armazenamento.

PLANT REGENERATION AND CRYOPRESERVATION OF LILIUM

Lilium is globally one of the most economically important ornamental crops used for flowers, medicine and food. Availability of efficient plant regeneration is necessary for micropropagation, genetic transformation, cryopreservation and production of virus-free plant material of Lilium. In this study, wide-spectrum protocols of plant regeneration via somatic embryogenesis from bulblet transverse thin cell layers (tTCLs) and via organogenesis from leaf segments were successfully established in Lilium. For somatic embryogenesis, 85.0, 75.3, 67.6, 27.8 and 25.0% of embryogenic callus induction frequencies (ECIFs) were obtained in five genotypes including L. longiflorum, L. longiflorum × Oriental 'Triumphator', L. Oriental hybrid 'Siberia', L. Asiatic hybrid 'Elite' and L. davidii var. unicolor, respectively. For organogenesis, similarly high shoot regeneration frequencies (SRFs) at 92-100% and a high number (4.7-7.0) of shoots per segment were obtained in 6 genotypes including L. Oriental hybrid 'Siberia', L. davidii var. unicolor, L. × formolongi, L. Asiatic hybrids 'Elite' and 'Pollyanna' and L. longiflorum × Oriental 'Triumphator'. Shoot tips of adventitious shoots derived from leaf segments of these Lilium genotypes were cryopreserved by droplet-vitrification. Shoot regrowth and somatic embryogenesis could be obtained in cryopreserved shoot tips, depending on post-culture conditions. Assessments of genetic integrity in regenerants from somatic embryogenesis, organogenesis and cryopreserved shoot tips by ISSR revealed that they were genetically stable. Data reported here have potential applications to genetic transformation, cryopreservation and production of virus-free plant material of Lilium by cryotherapy.

Effects of Crop Management Practices on Current-Season Spread of Potato virus Y.

The current-season spread of Potato virus Y (PVY) was monitored in 19 fields under various management practices in New Brunswick, Canada, through the 2011 and 2012 growing seasons. The focus of this study was to evaluate the role of seedborne PVY inoculum, aphid vector abundance, and the numbers, timing, and types of insecticide and mineral oil sprays, and to confirm the reliability and forecasting capacity of midseason PVY testing. In each field, 100 to 110 virus-free plants were identified shortly after emergence and were assessed four times from early July to early September (after top-kill) with enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT-PCR) to track PVY spread. In addition, tubers harvested during development in August and after top-kill were grown-out in the greenhouse for ELISA testing. PVY spread to selected virus-free plants varied widely, ranging from 0 to 76.2% across all studied fields. Of the 19 fields over two seasons, 10 fields were planted with no detectable seedborne PVY, and they showed 0 to 8.7% (mean 2.9%) PVY spread by harvest. The remaining nine study fields with 0.9 to 5.8% seedborne PVY showed 1 to 76.2% (mean 15.2%) PVY spread by harvest. PVY spread was detected in most fields during midseason testing with ELISA and RT-PCR; all tests correlated well with final PVY rates after top-kill, though RT-PCR detection in developing tubers was most sensitive and correlated. Logistic regression modeling was used to identify major factors in PVY spread, including seedborne PVY, early-season aphid abundance, and the numbers of insecticide and mineral oil sprays. The best-fitting model, constructed using these factors as well as a measurement of July PVY incidence (ELISAJuly), strongly explained PVY spread by harvest, with the most significant management factor being the number of mineral oil sprays supplemented with insecticide used during the growing season. A similar model fitted without the ELISAJuly did not adequately predict ultimate PVY spread. The analysis suggests that mineral oil alone was effective at lowering PVY spread, and more effective when combined with insecticide, particularly when used early in the season. No evidence was found for differences in PVY spread across the eight cultivars used or across the range of mineral oil application rates, whereas some evidence was found for differences in the effectiveness of different insecticide types.

DIFFERENCES OF IN VITRO THERMOTOLERANCE TO HIGH TEMPERATURES IN AUTOCHTHONOUS SWEET CHERRY VARIETIES FROM THE JERTE VALLEY (SPAIN)

Different clones of the autochthonous sweet cherry varieties ‘Ambrunes’, ‘Pico Negro’ and ‘Pico Colorado’ from the ‘Clonal Selection Program’ were subjected to different temperature regimes to optimize in vitro heat therapy to obtain virus-free plants. These varieties were especially sensitive to high temperature treatments: ‘Ambrunes and ‘Pico Colorado’ had similar thermotolerance, and ‘Pico Negro’ was less tolerant. However, one of the ‘Pico Negro’ clones had similar thermotolerance to ‘Ambrunes’ and ‘Pico Colorado’. It is necessary to establish a period of adaptation to high temperatures for increasing both the plant and the isolated apex survival.

Establishment of an Efficient and Practical Virus-free Seedling Supply System by Means of Culture of Shoot Apexes, RT-PCR and Clonal Propagation in Sweet Potato (Ipomoea batatas)

Aims: Sweet potato (Ipomoea batatas) cv. “Miyazakibeni” was used as material for shoot apex culture, reverse transcription-polymerase chain reaction (RT-PCR) and clonal propagation to establish an efficient and practical virus-free seedling supply system in production of vegetatively reproductive plants. Study Design: At first, efficient plant regeneration was achieved from shoot apex culture of sweet potato. Secondly, RT-PCR method was used to detect the sweet potato feathery Original Research Article British Biotechnology Journal, 4(1): 51-63, 2014 52 mottle virus (SPFMV) viral infection of tuber surface of edible sweet potato using the RNAs from the plants obtained from shoot apex culture. Finally, the virus-free plants verified by RT-PCR were propagated clonally by culture of suckers cut from stems of the virus-free plants. Place and Duration of Study: Faculty of Environmental and Horticultural Science, Minami Kyushu University, between June 2008 and December 2012. Methodology: The best efficiency for material sterilization was tested using different concentrations (0.1% 1.5%) of sodium hypochlorite solution (SHS) and the treated times (5 min – 20 min). Theshoot apexes less than 0.3mm in size were cultured on Komamine and Nomura (1998) (KN) medium and Murashige and Skoog (1962) (MS) medium. The regenerated plants were used for RNA extraction and then, used for RT-PCR for detection of SPFMV. Based on the result of RT-PCR, the suckers cut from stems of virus-free plants were cultured and propagated clonally and routinely within a short period. Results: The combination of 0.3% of SHS and 10 and/or 20 min gave the best result (100%) of surviving rate for material sterilization. The culture of shoot apexes less than 0.3 mm in size gave plant regenerating rates of 82% and 65% on KN and MS medium, respectively. The results of RT-PCR of RNAs from plants obtained from shoot apex culture and plants of SPFMV infection showed that SPFMV virus was clearly removed by shoot apex culture conducted in this study. For clonal propagation, 80-100% of suckers cut from the stems of the virus-free plants detected grew into complete plants after 6 weeks of culture, indicating that the virus-free plants could be routinely propagated 5 times in number each time and repeatable by the short circle. The sweet potato produced in field showed no symptom called as russet crack-like symptom (RC-LS) even after cultivation two seasons. Conclusion: Overall, an efficient and practical virus-free seedling supply system was established in sweet potato by the three steps of 1) virus-free plant regeneration from shoot apex culture, 2) quick detection of SPFMV using RNA of the regenerated plants by RT-PCR, and 3) the verified virus-free plants were propagated clonally and routinely within a short period using culture of suckers cut from the stems of virus-free plants.

First Report of Freesia sneak virus in Commercial Freesia hybrida Cultivars in Korea

In order to investigate the virus infection rate of commercial freesia cultivars in early February 2013, 19 freesia cultivars showing necrotic purple speckles or streaks on leaves, purple streaks parallel to the midrib, and necrotic speckles on leaves were collected from three different regions (Suwon and Icheon in Gyeonggi Province and Jeonju in North Jeonla Province) and used for virus detection. Nucleic acid extracts were analyzed for detection of major freesia-infecting viruses including Freesia sneak virus (FreSV) by reverse transcription (RT)-PCR with specific primer pairs. The FreSV CP gene was amplified using primer pair FreSV-F (5'-TTAGATAGTGAATCCATAAGCTGC-3') and FreSV-R (5'-ATGTCTGGAAAATACTCCGTCCAA-3'). The approximately 1.3-kb fragment of the FreSV amplified product was cloned and sequenced (GenBank Accession No. KC771891 to 98). The nucleotide sequences of CP gene of FreSV korean isolates showed 99.2 to 99.8% similarity to other FreSV isolates DQ885455, FJ807730, and GU071089, which are registered in GenBank. FreSV was detected from 71.7% of 138 plants tested while the infection rate of Freesia mosaic virus (FreMV) was 34.8%. Neither Bean yellow mosaic virus (BYMV) nor Tobacco rattle virus (TRV) were detected from any plants tested in this study. In certain cultivars, such as 'Bluebau' (II) and 'Pretty women,' most plants planted in the field showed purple streak symptoms on the leaves. In conclusion, FreSV was detected from some symptomatic freesia cultivars showing purple streak or speckles on leaves with or without necrotic spots and necrotic speckles on leaves. FreSV is currently widespread in Korea and some freesia plants were mixed infected with FreMV. FreSV has been occurring in the Netherlands for over 40 years (2). It is a plant virus in the family Ophioviridae and Ophiovirus genus. Once it occurs in freesia plantation fields, eradication is almost impossible because FreSV is transmitted by zoospores of Olpidium brassicae, which is a soilborne root-infecting fungus (3). Resting spores of O. brassicae can remain dormant in the soil and can be infective for 20 years (1). To produce virus-free freesia plants, growers should consider whether or not their fields are contaminated with O. brassicae carrying FreSV. To our knowledge, this is the first report of FreSV in freesia plants in Korea.

IMPROVEMENT OF SOME NUTRITIONAL VALUES OF TOMATOES VIA SALINITY

The antioxidant activity status is important to determine the nutritional value of fruits and vegetables. Increased antioxidant levels not only have high benefits in preventing widespread human diseases, including cancer and cardiovascular pathogens but also it enhances shelf life as well. This study was carried out to test the effects of 100 mM NaCl on some antioxidants (vitamins C and E) and some monosaccharides (glucose and fructose) of TYLCV virus-free tomato plants (Solanum lycopersicon) cultivar Castle Rock. The plants were treated at 30 days old with 100 mM NaCl for the next 45 days. At 75 days old, mature leaves were harvested and tested for TYLCV infection, as this virus affects the amount of antioxidants in tomato plants. Plants were kept in cages to avoid infection with TYLCV. The plants gave negative results, confirming that they are virus-free. Levels of vitamin C and vitamin E were determined by a reverse-phase HPLC technique while levels of glucose and fructose were determined using UPLC-MS in 75 days treated tomato leaves. Plants showed an increase in vitamin C, α-tochopherol, glucose and a slight increase in fructose contents versus the control plants. These results are of great importance from the nutritional and health points of view where salt stress improved the plant quality by increasing the concentrations of important antioxidants (vitamin C and vitamin E). In conclusion, the use of controlled salinity level can be an effective method to produce tomatoes of good nutritional quality and with higher market price.

Comparison of the potential rate of population increase of brown and green color morphs of Sitobion avenae (Homoptera: Aphididae) on barley infected and uninfected with Barley yellow dwarf virus

Life tables of brown and green color morphs of the English grain aphid, Sitobion avenae (Fabricius) reared on barley under laboratory conditions at 20 ± 1°C, 65% ± 5% relative humidity and a photoperiod of 16 : 8 h (L : D) were compared. The plants were either: (i) infected with the Barley yellow dwarf virus (BYDV); (ii) not infected with virus but previously infested with aphids; or (iii) healthy barley plants, which were not previously infested with aphids. Generally, both color morphs of S. avenae performed significantly better when fed on BYDV-infected plants than on plants that were virus free but had either not been or had been previously infested with aphids. Furthermore, when fed on BYDV-infected plants, green S. avenae developed significantly faster and had a significantly shorter reproductive period than the brown color morph. There were no significant differences in this respect between the two color morphs of S. avenae when they were reared on virus-free plants that either had been or not been previously infested with aphids. These results indicate that barley infected with BYDV is a more favorable host plant than uninfected barley for both the color morphs of S. avenae tested, particularly the green color morph.

CRYOTHERAPY: A NEW TOOL FOR THE ELIMINATION OF ARTICHOKE VIRUSES

Globe artichoke (Cynara scolymus L.) is a highly valuable vegetable crop characterized by a broad diversity of ecotypes that need preservation to avoid genetic erosion. This species is affected by many viruses which cause significant economic losses, thus virus-free planting material is essential for its sustainable production. Cryogenic techniques are one option for the long-term storage of germplasm (cryopreservation) and virus eradication (cryotherapy). In order to achieve these two objectives, vitrification was applied to artichoke ecotypes selected from several Italian regions. A protocol was established to obtain high regeneration rates of early ecotypes after liquid nitrogen treatment. Cryogenic treatment successfully eliminated Artichoke latent virus (ALV) from infected shoot tips with high frequency. Sanitized shoot tips regenerated plantlets that maintained their virus-free status over time and were micropropagated for successful transfer to the field.

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.

Transmitting Plant Viruses Using Whiteflies

Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year. Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus. There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.

Rapid detection of Grapevine leafroll-associated virus type 3 using a reverse transcription loop-mediated amplification method

Grapevine leafroll disease (GLD) is the most important disease of Grapevines in South Africa. Grapevine leafroll-associated virus type 3 (GLRaV-3) has a close association with the disease and is prevalent in South African vineyards. GLD can be controlled using a combination of virus-free planting material, systemic insecticides to control vector populations and removal of infected vines by roguing. Infected vines are identified each autumn using either symptom display (in red cultivars) or ELISA (in white cultivars). While ELISA is a simple, reliable means of testing for GLRaV-3, it is time consuming, laborious and insensitive and a quicker, more sensitive method of detecting GLRaV-3 in the field is needed. A single-tube one-step reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay combined with a simple RNA extraction protocol was developed for the rapid and easy detection of GLRaV-3. Hydroxy napthol blue was included as an indicator and under isothermal conditions at 60°C the target viral gene could be amplified in under 2h and positive results could be easily seen by examining the colour change from violet to sky blue. Using this method, 50 samples could be also pooled together with a single positive sample still being detected. A direct comparison of ELISA, nested PCR and RT-LAMP showed that RT-LAMP is as sensitive as nested PCR and could be performed in a much shorter time with less equipment. This assay is may be a possible alternative to ELISA for the detection of GLRaV-3 in the field.

Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein

Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constrain in large cardamom production in India. Purification of LCCV from large cardamom tissues is difficult and therefore immunodiagnostic reagents are not available. In the present study, we have successfully expressed coat protein (CP) gene of LCCV in Escherichia coli. The purification of expressed protein by Ni-NTA affinity chromatography was inefficient due to precipitation of protein during renaturation. We have optimized a simple, inexpensive and efficient method for purification of the expressed CP through gel extraction with 5% SDS followed by renaturation in Milli-Q water, which resulted in high yield (4.7mg/ml) and good quality of the protein. A higher titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced, which strongly recognized LCCV in crude leaf extract and showed minimal background reaction with the healthy leaf extract in enzyme linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The sensitivities of the ELISA and DIBA were 5 and 0.1ng of expressed protein, respectively. Both the ELISA and DIBA were validated with 100% accuracy in detecting LCCV in field samples. The PAb differentiated Cardamom mosaic virus, another close relative of LCCV. Our study is first to report highly efficient immunodiagnosis with PAb to E. coli expressed recombinant CP of a virus under the genus Macluravirus. The antigen expression construct and PAb developed in the present study will be useful in production of virus free planting materials of large cardamom.

Determination of scanned virus-free potato planting materials by positive selection and screening of tubers from seed stores in the western highlands of Cameroon

Positive selection for the identification of virus–free potato planting material was evaluated in four locations in Cameroon. Leaves from asymptomatic plants were randomly collected, the plants marked and tubers collected four weeks later, and screened with DAS-ELISA for PLRV, PVY, PVA, PVX, PVS and PVM presence. Five viruses were prevalent in leaves while four prevalent in tubers. Potato virus M was highly prevalent in leaves and tubers while PVY was high only in leaves. Potato virus A was absent in both leaves and tubers while PVX was free only in tubers. A positive correlation was observed between virus prevalence in leaves and tubers (r=0.806). The prevalence of the six viruses in potato seed tubers from four seed stores was tested. Potato virus M was the most prevalent, while PLRV was the least. Small, medium and large tuber sizes were tested for the viruses, and infection rates decreased significantly the bigger the tuber size. Positive selection though not highly efficient can be recommended for resource-poor farmers, to control the economically important potato viruses. Tuber size can serve as a guide to identify healthy tubers, but must be combined with laboratory tests for effective use in selecting seeds for planting. Keywords : Potato viruses, virus-free tubers, positive selection.

Occurrence of Stone Fruit Viruses in Plum Orchards in Latvia

To evaluate the occurrence of nine viruses infecting Prunus a large-scale survey and sampling in Latvian plum orchards was carried out. Occurrence of Apple mosaic virus (ApMV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), Apple chlorotic leaf spot virus (ACLSV), and Plum pox virus (PPV) was investigated by RT-PCR and DAS ELISA detection methods. The detection rates of both methods were compared. Screening of occurrence of Strawberry latent ringspot virus (SLRSV), Arabis mosaic virus (ArMV), Tomato ringspot virus (ToRSV) and Petunia asteroid mosaic virus (PeAMV) was performed by DAS-ELISA. In total, 38% of the tested trees by RT-PCR were infected at least with one of the analysed viruses. Among those 30.7% were infected with PNRSV and 16.4% with PDV, while ApMV, ACLSV and PPV were detected in few samples. The most widespread mixed infection was the combination of PDV+PNRSV. Observed symptoms characteristic for PPV were confirmed with RT-PCR and D strain was detected. Comparative analyses showed that detection rates by RT-PCR and DAS ELISA in plums depended on the particular virus tested. The results obtained in this study revealed that commonly grown plum cultivars in Latvia are infected with economically important stone fruit viruses and highlight the need to implement a programme to produce and propagate virus-free planting material.

Combination of stem-disc dome culture and thermotherapy to eliminate Allexiviruses and Onion yellow dwarf virus from garlic (Allium sativum cv. Hamedan)

Onion yellow dwarf virus (OYDV) and Garlic viruses (GarVs) A–D belonging to the genera Potyvirus and Allexivirus, respectively, cause serious disease problems and yield losses in garlic (Allium sativum L.). Possibility of obtaining virus-free garlic using stem-disc dome (SDD) culture and its combination with thermotherapy (36 °C for 4 and 5 weeks) were studied. Duplex reverse transcriptase-polymerase chain reaction was used for detecting these viruses. Results showed that the efficiency of viral elimination is depended on viral type, applied methods and duration of thermotherapy. For SDD culture, an overall average efficiency of virus-free plant production was 61 and 77% for GarVs and OYDV, respectively. Thermotherapy combined with SDD culture had a deleterious effect on OYDV elimination. For GarVs, it was more effective than SDD culture by itself and a high GarVs elimination of 75% was achieved by thermotherapy for four weeks of treatment. The efficiency of GarVs elimination (90%) was improved as treatment duration prolonged for five weeks.

'자랑' 포도 생장점 배양으로부터 무병주 식물 대량번식에 미치는 배지 구성물질과 생장 조절제의 영향

본 연구는 바이러스 무병묘의 대량 증식을 위한 기초 자료를 얻고자 포도 '자랑' 품종의 신초와 뿌리의 형성 및 생장에 미치는 배지조성물질과 생장조절제의 영향을 구명하고자 수행되었다. 배지의 무기염 농도가 낮을수록 신초의 형성은 많아지는 경향으로, 특히 1/2MS배지에서 신초의 형성 및 생장이 가장 양호하였다. 부정근의 형성은 무기염의 농도가 높아질수록 양호하여 2MS배지에서 부정근의 수가 2.1개로 가장 많았다. Sucrose의 경우, 신초의 형성은 1%, 부정근의 형성은 3%, 생장은 1% 농도구에서 가장 좋았다. 활성탄의 0.05% 첨가에 의해 신초의 생장은 양호했으며, 특히, 부정근의 형성 및 생장에 효과적이었다. '자랑'의 대량 증식을 위한 배지의 pH는 6.8이 가장 적합하여 신초의 마디수와 길이가 각각 3.9개와 1.3 cm로 가장 좋았다. 기내 대량증식에 미치는 생장조절제의 영향으로 BA 1.0 mg/L에서 신초의 생장이 가장 왕성하였으며, NAA 1.0 mg/L에서 절편체당 16.9개의 신초가 형성되어 양호한 결과를 얻었다. This study was performed to clarify the effect of medium compositions and plant growth regulators on the shoot, root formation and growth of 'Jarang' grape for mass propagation of virus-free plant. The formation and growth of shoot were considerably favorable in half-concentration of MS medium. However, the formation of adventitious root per explants (avg. 2.1) was effective in higher concentration (two times) of MS medium. For sucrose concentration, 1% for the shoot formation, 3% for the adventitious root formation and 1% for the growth were observed as yield significant results. With the addition of 0.05% of activated carbon, the shoot growth was improved, and it was effective for the adventitious root formation and growth as well. A pH of 6.8 in the medium was the most suitable for mass propagation; the results showed significant enhancement in the number of nodes and the length of the shoot, 3.9 and 1.3 cm, respectively. The shoot growth was the most vigorous in BA 1.0 mg/L due to the impact of the growth regulator on the mass propagation in it. Consequently, 16.9 shoots per explant were formed in NAA 1.0 mg/L so good results were obtained.

Modification of Non-Vector Aphid Feeding Behavior on Virus-Infected Host Plant

Virus-infected host plants can have positive, neutral or negative effects on vector aphids. Even though the proportion of non-vector aphids associated with a plant far exceeds that of vector species, little is known about the effect of virus-infected plants on non-vector aphids. In the present study, the English grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphididae), a non-vector of Wheat dwarf virus (WDV) and Cereal yellow dwarf virus-RPV (CYDV-RPV), was monitored on, virus-infected, virus-free and leafhopper/aphid-infested, and virus- and insect-free (control) barley, Hordeum vulgare L. (Poales: Poaceae), plants. Electrical penetration graph recordings were performed. Compared with the control plants, S. avenae on infected plants exhibited reduced non-probing and pathway phase, and increased phloem sap ingestion phase, and more aphids reached sustained phloem ingestion. However, the electrical penetration graph parameters described above showed no significant differences in aphid feeding behavior on virus-free and vector pre-infested plants and the control barley plants during S. avenae feeding. The results suggest that WDV/CYDV-RPV-infected host plants positively affected the feeding behavior of the non-vector aphid S. avenae. Based on these results, the reasons and trends among the virus-infected host plants' effects on the feeding behavior of non-vector aphids are discussed.