Virus-free Planting Material Research Articles

Efficient immunodiagnosis of Citrus yellow mosaic virus using polyclonal antibodies with an expressed recombinant virion-associated protein.

Citrus yellow mosaic virus (CYMV) is a member of genus Badnavirus of the family Caulimoviridae. It is the causal agent of citrus yellow mosaic disease in citrus and causes reduction in yield. As the virus is vegetative propagated by grafting, development of high-throughput diagnosis methods based on serological techniques is a prerequisite for production of healthy virus-free planting material. The current study describes the development of polyclonal antibodies raised in rabbits against purified recombinant virion-associated protein (rVAP) encoded by ORF-II of CYMV. The specificity of developed antiserum was evaluated in immunosorbent electron microscopy (ISEM), antigen-coated plate-enzyme linked immunosorbent assay (ACP-ELISA) and immunocapture PCR (IC-PCR). The antiserum specifically reacted up to a dilution of 1:2000 in ACP-ELISA for detection of CYMV-infected plants. The antiserum was validated by screening CYMV-infected plants maintained in the glass house through ACP-ELISA. To the best for our knowledge, this is the first report on production of polyclonal antiserum using recombinant virion-associated protein as fusion protein, which could be used for screening CYMV-infected plants by ELISA and IC-PCR. These immunodiagnostic methods can be effectively employed in routine indexing of citrus and in quarantine process.

Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus

Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India.

English

Cassava production in eastern and Central Africa is severely threatened by the current epidemic of cassava brown streak disease (CBSD). The disease is caused by cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) of the genus Ipomovirus, family Potyviridae. Using virus-free planting material is one effective way to reduce CBSD yield losses in farmer fields. The effects of chemical and heat treatment on tissue cultured plants was investigated to eliminate CBSV and UCBSV infections in three cassava varieties with differing levels of resistance to CBSD. Cassava node buds taken from young shoots of the varieties Kaleso (CBSD resistant), Kiroba (tolerant) and Albert (susceptible) infected with CBSV and UCBSV were grown in vitro in standard Murashige and Skoog tissue culture media, which alone eliminated viruses from up to 30% of the plants. To increase the number of virus-free plants, two-week old virus-infected in vitro plants were exposed to constant temperature regimes of 30, 35, 40 or 45°C for three weeks. Heat treatment at 40°C had the optimum effect as up to 50% virus-free plants were obtained for Kiroba; however, incubation at 45°C was lethal to all three varieties while the lower temperatures produced fewer virus-free plants. Chemical treatment of in vitro plants was performed using the antiviral chemical ribavirin at various concentrations. Ribavirin at 0.10 mM generated up to 40% virus-free plants in all three varieties but concentrations of 0.21 mM were lethal. These findings indicate important methods to produce virus-free planting material; their use by breeders and in the field, will help develop effective control strategies for CBSD management. Key words: Africa, cassava, chemical and heat treatment, viruses, tissue culture, disease resistance.

Hormonal Effect on Mandarin Orange (Citrus reticulata Blanco) Micro-propagation

Tissue culture is the best option to produce disease free seedling of the fruit crop rapidly. Micro-propagation and use of the in-vitro grafting (micro-grafting) is very helpful for production of virus free planting materials in mandarin. Different levels of the in-vitro hormone affect the success of callusing, shooting and plant regeneration in mandarin. Shoot bud, flower bud and in-vitro seedling epicotyl was used as explants to study the hormonal effect on mandarin micro-propagation. Similarly, 10 levels of BAP and IAA combination on MS media for mandarin tissue culture were used. Observation was done for 100 test tubes per treatment combination after 4, 8 and 12 weeks of culture. Data was arc sine transformed for analysis. Shooting from explants was significantly higher (71.72%) on medium level of the BAP (0.5 mg/L) and IAA (0.2 mg/L) using in-vitro seedling stem as explant, however, it was 27.91% for stem bud as explant. Stem bud showed higher level of callusing (6.15%, p<0.001) in mandarin orange. However, flower bud didn’t develop shoot in mandarin tissue culture. Increment of the in-vitro regeneration of the shooting and callusing was observed by the increment of the in-vitro incubation duration in mandarin orange tissue culture.

Development of multiplex polymerase chain reaction assay for simultaneous detection of clostero-, badna- and mandari-viruses along with huanglongbing bacterium in citrus trees

Citrus trees harbor a large number of viral and bacterial pathogens. Citrus yellow vein clearing virus (CYVCV), Indian citrus ringspot virus (ICRSV), Citrus yellow mosaic virus (CYMV), Citrus tristeza virus (CTV) and a bacterium, Candidatus Liberibacter asiaticus (CLa) associated with huanglongbing (HLB) disease, the most prevalent pathogens in citrus orchards of different regions in India and are responsible for debilitating citriculture. For detection of these viral and bacterial pathogens a quick, sensitive and cost effective detection method is required. With this objective a multiplex polymerase chain reaction (mPCR) assay was developed for simultaneous detection of four viruses and a bacterium in citrus. Several sets of primers were designed for each virus based on the retrieved reference sequences from the GenBank. A primer pair published previously was used for greening bacterium. Each pair of primers was evaluated for their sensitivity and differentiation by simplex and mPCR. The constant amplified products were identified on the basis of molecular size in mPCR and were compared with standard PCR. The amplicons were cloned and results were confirmed with sequencing analysis. The mPCR assay was validated using naturally infected field samples for one or more citrus viruses and the huanglongbing bacterium. The mPCR assay developed here will aid in the production of virus free planting materials and rapid indexing for certification of citrus budwood programme.

English

In Zimbabwe, the average sweet potato yield (6 t/ha) is relatively low when compared to Asian counterparts (17 t/ha). These low crop yields have been blamed on weevil infestations and viral infections which account for 60-90% of sweet potato yield losses in Africa. Meristem tip culture, a Centre for Potato Improvement (CIP) initiated tissue culture technique, has been widely used to eradicate viruses from clonally propagated crops and has been noted to be one of the instrumental techniques that helped China to increase sweet potato yields. In an effort to adopt the meristem tip culture technique for the production of virus-free planting material of a local sweet potato (cv Brondal), a study was conducted to evaluate the effect of Benzylamino purine (BAP), 1-Naphthaleneacetic acid (NAA) and Gibberellic acid (GA3) (either alone or in combination) on cultured Brondal meristems. The different hormonal treatments were assessed on the following parameters: plantlet regenerative capacity, multiple plantlet production, shoot height, average leaf number per shoot and average node number per shoot, ten weeks after meristem culture. All treatments containing a combination of BAP (1 mg - L) and GA3 (at either 5 mg - L, 10 mg - L, or 20 mg - L) had a significantly (p<0.01) higher plantlet regenerative capacity of 33-66% when compared to other treatment combinations. Only treatments, 10 mg - L GA3 + 1 mg - L BAP and 20 mg - L GA3 + 1 mg - L BAP were capable of inducing multiple plantlet formation, producing an average of three plantlets/meristem and two plantlets/meristem respectively. Overall, treatment 10 mg - L GA3 + 1 mg - L BAP gave rise to significantly (p<0.01) taller shoots (20 mm) compared to the rest of the treatments used. For average leaf number per shoot, all GA3 treatments (5 mg - L, 10 mg - L, or 20 mg - L) supplemented with 1 mg - L BAP gave significantly (p<0.01) higher numbers of leaves (six leaves/shoot) than the rest of the treatments. Treatments 10 mg - L GA3 + 1 mg - L BAP and 20 mg - L GA3 + 1 mg - L BAP gave rise to the highest number of nodes per shoot, producing an average of three nodes per shoot. In sharp contrast to treatments containing a combination of BAP and GA3, all treatments containing a combination of BAP and NAA performed poorly in all parameters tested for plant regeneration of Brondal sweet potato variety. In conclusion, the best hormonal treatment for culturing Brondal meristems proved to be 10 mg - L GA3 + 1 mg - L BAP. Keywords: Ipomoea batatas , meristem, regeneration, Benzylamino purine, Gibberellic acid, 1-Naphthaleneacetic acid

Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification

Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam production globally. Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential component of disease control. Current serological and PCR-based diagnostic methods for YMV are time consuming involving a succession of target detection steps. In this study, a novel assay for specific YMV detection is described that is based on isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown to be reproducible and able to detect as little as 14pg/μl of purified RNA obtained from an YMV-infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages over the RT-PCR; positive samples can be detected in less than 30min, and amplification only requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay a promising candidate for adapting into a field test format to be used by yam breeding programmes or certification laboratories.

Sharka epidemiology and worldwide management strategies: learning lessons to optimize disease control in perennial plants.

Many plant epidemics that cause major economic losses cannot be controlled with pesticides. Among them, sharka epidemics severely affect prunus trees worldwide. Its causal agent, Plum pox virus (PPV; genus Potyvirus), has been classified as a quarantine pathogen in numerous countries. As a result, various management strategies have been implemented in different regions of the world, depending on the epidemiological context and on the objective (i.e., eradication, suppression, containment, or resilience). These strategies have exploited virus-free planting material, varietal improvement, surveillance and removal of trees in orchards, and statistical models. Variations on these management options lead to contrasted outcomes, from successful eradication to widespread presence of PPV in orchards. Here, we present management strategies in the light of sharka epidemiology to gain insights from this worldwide experience. Although focused on sharka, this review highlights more general levers and promising approaches to optimize disease control in perennial plants.

Sweet potato cultivar degeneration rate under high and low sweet potato virus disease pressure zones in Uganda

Sweet potato is a vegetatively propagated crop where vine cuttings from previous crops or volunteer plants are used as planting material. This practice can lead to the accumulation of systemic pathogens, especially viruses. However, the contribution of this practice to degeneration of sweet potato cultivars in Uganda has been only speculative, hence the need to document the rate of cultivar degeneration in high and low sweet potato virus disease pressure zones. Four cultivars of sweet potato – ‘Beauregard’, ‘Dimbuka’, ‘Ejumula’ and ‘NASPOT 1’ – were planted in a series of field trials in central (Kabanyolo) and Eastern (Serere) Uganda over five generations (G1, G2, G3, G4 and G5). The trials started with virus-free planting material and each succeeding trial retained planting material from the previous one, as well as receiving fresh clean material. Data were recorded on virus incidence and severity monthly for 4 months, root yield and vine weight at harvest after 6 months. Virus symptoms were observed 1 month after planting in all the plant generations, with Sweet potato feathery virus and Sweet potato chlorotic stunt virus being the most prevalent viruses detected. The cultivars ‘Beauregard’ and ‘Ejumula’ had highest disease incidence and severity, with the latter collapsing after a single season in both locations. Storage root yields and numbers were greatest in G1 but remained similar although less in all subsequent generations (G2, G3, G4 and G5) for each cultivar. Since it is impractical to provide fresh planting material each year for farmers, the focus should be on breeding more resistant varieties of sweet potato.

Production of virus-free garlic (Allium sativum L.) through meristem tip culture after solar or hot air treatment of cloves

SummaryGarlic (Allium sativum L.) is a clonally propagated crop that can be infected by many virus species which tend to accumulate year-after-year, leading to yield reductions and varietal degeneration. Therefore, the production and use of virus-free planting material is vital to sustain productivity. In this study, Onion yellow dwarf virus (OYDV; Potyvirus), Garlic common latent virus (GarCLV; Carlavirus), Shallot latent virus (SLV; Carlavirus), and Garlic virus X (GarV-X; Allexivirus) were detected in cloves of the commercial garlic cultivar, ‘G-1’ (‘Jamuna Safed’) by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). DAC-ELISA was performed using specific antisera against each virus species, and confirmed through reverse transcription-polymerase chain reactions (RT-PCR) using virus or genus-specific oligonucleotide primers. To obtain virus-free plants of ‘G-1’, cloves were subjected to solar heat treatment for 5, 10, or 15 d, or to hot air treatment (at 37°C, 40°C, or 42°C for 7, 14, or 21 d) combined with meristem isolation and in vitro culture, then indexed for all viruses using DAC-ELISA and RT-PCR. Meristems from solar heat-treated or hot air-treated cloves regenerated after 90 d and 45 d in culture, respectively. Solar heat treatment for 15 d resulted in 17 - 18% regeneration, and all were free from GarCLV, SLV, and OYDV. However, when solar heat-treated for 10 d, only 66% of the 17 - 18% regenerated plants were free from all viruses, including GarV-X. Hot air treatment at 37°C or 40°C eliminated GarCLV, SLV, and OYDV in 20 - 38% and 42 - 62% of regenerated plants, respectively, for the different durations of exposure. However, GarV-X could not be eliminated from any of the regenerated plants by hot air treatment, irrespective of the duration of exposure. Meristems from cloves that had been hot air-treated at 42°C resulted in 50 - 66% regeneration, of which 53 - 57% were free from GarCLV, SLV, and OYDV when treated for 7 d or 14 d, and 29% were free from all viruses when treated for 21 d. For most treatments, an increase in the duration of exposure to heat resulted in reduced regeneration, but increased the rate of recovery of virus-free plants. To our knowledge, this is the first study to report the use of solar treatment for virus elimination in garlic and the development of an efficient tissue culture protocol for the production of virus-free garlic plants using different thermotherapy treatments coupled with meristem tip culture.

Biology, etiology, and control of virus diseases of banana and plantain.

Banana and plantain (Musa spp.), produced in 10.3 million ha in the tropics, are among the world's top 10 food crops. They are vegetatively propagated using suckers or tissue culture plants and grown almost as perennial plantations. These are prone to the accumulation of pests and pathogens, especially viruses which contribute to yield reduction and are also barriers to the international exchange of germplasm. The most economically important viruses of banana and plantain are Banana bunchy top virus (BBTV), a complex of banana streak viruses (BSVs) and Banana bract mosaic virus (BBrMV). BBTV is known to cause the most serious economic losses in the "Old World," contributing to a yield reduction of up to 100% and responsible for a dramatic reduction in cropping area. The BSVs exist as episomal and endogenous forms are known to be worldwide in distribution. In India and the Philippines, BBrMV is known to be economically important but recently the virus was discovered in Colombia and Costa Rica, thus signaling its spread into the "New World." Banana and plantain are also known to be susceptible to five other viruses of minor significance, such as Abaca mosaic virus, Abaca bunchy top virus, Banana mild mosaic virus, Banana virus X, and Cucumber mosaic virus. Studies over the past 100 years have contributed to important knowledge on disease biology, distribution, and spread. Research during the last 25 years have led to a better understanding of the virus-vector-host interactions, virus diversity, disease etiology, and epidemiology. In addition, new diagnostic tools were developed which were used for surveillance and the certification of planting material. Due to a lack of durable host resistance in the Musa spp., phytosanitary measures and the use of virus-free planting material are the major methods of virus control. The state of knowledge on BBTV, BBrMV, and BSVs, and other minor viruses, disease spread, and control are summarized in this review.

Comparing the Effects of Benzyladenine and &lt;i&gt;meta&lt;/i&gt;-Topolin on Sweet Basil (&lt;i&gt;Ocimum basilicum&lt;/i&gt;) Micropropagation

&#x0D; Micropropagation of aromatic plants reveals an effective way of obtaining high volume, virus-free plant material of uniform quality. The application of meta-Topolin (mT) (N6-(2-hydroxybenzyl) adenine-9-riboside) and aromatic cytokinin as Benzyladenine (BAP) in the micro propagation of sweet basil (Ocimum basilicum L.) was tested for the first time and plant growth parameters assessed to determine the optimum level of these cytokinins. Additionally, the rate of root-growth inhibition due to these two cytokinins was also assessed. Our results show that 1 mg/l (4.43 mM) BAP and 0.5 mg/l (2.07 mM)mT produced the most favourable effects on new shoot developments. Meta-Topolin was shown to increase the quality of the plants and in comparison with BAP fewer distortions were observed. No significant differences in root-growth inhibition between the mT and BAP were detected. &#x0D;

A degenerate pair of primers for simultaneous detection of four alpha- and betanecroviruses

The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5′ and parCoat3′ was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found.The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced.

Occurrence of Telosma mosaic virus causing passion fruit severe mosaic disease in Thailand and immunostrip test for rapid virus detection

The cause of leaf and fruit severe mosaic disease in purple passion fruit (Passiflora edulis) in Chiang Mai province, Thailand, was identified and virus diagnostic kit was developed. Symptomatic plants collected from diseased mother plants in the nursery at the Royal Pangda research station (RPRS) was used for virus isolation through single lesion transfer in Chenopodium amaranticolor, followed by virus propagation for purification in Nicotiana benthamiana, and back inoculation to purple passion fruit. Electron microscopy of leaves with typical symptoms revealed potyvirus-like flexuous rod particles, ca. 750 nm long and pinwheel inclusion bodies in the cytoplasm of infected cells. The deduced amino acid sequence of the complete coat protein revealed that the isolated virus was a strain of Telosma mosaic virus with which it shared 84% identity. A rabbit polyclonal antiserum was produced against purified virions, and used to develop a gold-labeled immunostrip for rapid virus diagnosis. The test strip could detect the virus in diseased passion fruit sap up to a dilution of 1: 640, and positive test line could be read within 3–5 min. Application of strip test for virus assay in RPRS nursery's mother plants help screen clean stocks. This strip test kit supports RPRS program of producing virus-free planting materials for farmers.

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.

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.

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.

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.