Multiple virus infections of lablab [<Emphasis Type="Italic">Lablab purpureus</Emphasis> (L.) Sweet] in Nigeria

Leaf samples of Lablab purpureus collected from two agroecological zones of Nigeria—the northern guinea savanna zone (NGSZ) and the derived savanna zone (DSZ)—were infected with viruses when serologically indexed against available antisera. Approximately 31.1 and 81.1% of the leaf samples collected from the NGSZ and DSZ, respectively, were infected. Seven viruses were found: Bean common mosaic virus (BCMV), Cowpea aphid-borne mosaic virus (CABMV), Cucumber mosaic virus (CMV), Cowpea mottle virus (CPMoV), Cowpea severe mosaic virus (CPSMV), Southern bean mosaic virus (SBMV) and Tobacco mosaic virus (TMV) were detected from samples collected from NGSZ, while CMV, CPMoV, Cowpea mosaic virus (CPMV) and CPSMV were detected from samples from DSZ.     

RNAi‐based enhanced resistance to Cowpea severe mosaic virus and Cowpea aphid‐borne mosaic virus in transgenic cowpea

Cowpea (Vigna unguiculata) is one of the most important legumes cultivated in many parts of the world. The diseases caused by Cowpea severe mosaic virus (CPSMV) and Cowpea aphid‐borne mosaic virus (CABMV) are considered among the most important constraints on yield and quality, especially in Latin America and Africa. Here, the concept of using an RNA interference construct to silence the CPSMV proteinase cofactor gene and the CABMV coat protein gene is explored, in order to generate resistant transgenic cowpea plants. Ten cowpea transgenic lines were produced, presenting a normal phenotype and transferring the transgene to the next generation. Plants were tested for resistance to both CABMV and CPSMV by mechanical co‐inoculation. Seven lines presented milder symptoms when compared to the control and three lines presented enhanced resistance to both viruses. Northern analyses were carried out to detect the transgene‐derived small interfering RNA (siRNA) in leaves and revealed no correlation between siRNA levels and virus resistance. Additionally, in the symptomless resistant lines the resistance was homozygosis‐dependent. Only homozygous plants remained uninfected while hemizygous plants presented milder symptoms.     

Detection and identification of seed-borne viruses from cowpea (Vigna unguiculata (L.) Walp.) germplasm

Seedlings from 182 cowpea ( Vigna unguiculata ) pre-introductions/germplasm accessions from 12 countries were tested under greenhouse conditions for six seed-borne viruses. Twenty-one (13.3%) accessions from eight countries were found to be seed-infected with one of the three following viruses: blackeye cowpea mosaic (BlCMV) and cowpea aphid-borne mosaic (CABMV) potyviruses, and cucumber mosaic cucumovirus (CMV). Natural seed transmission incidence of 0–6.9%, 0–13.3%, and 0–2.0% were determined for BlCMV, CABMV and CMV respectively.   Another set of 2930 cowpea germplasm accessions, mostly from Botswana and Senegal (Africa), were examined under field conditions for detection and identification of seed-borne viruses. Only CABMV was detected in this material. Most of the lines were free from other viruses reported in cowpea seed. Eight isolates of BlCMV and 28 isolates of CABMV were derived from cowpea pre-introductions/germplasm accessions evaluated under greenhouse and field conditions.     

Incidence of viruses in <Emphasis Type="Italic">Alstroemeria</Emphasis> plants cultivated in Japan and characterization of <Emphasis Type="Italic">Broad bean wilt virus-2, Cucumber mosaic virus</Emphasis> and <Emphasis Type="Italic">Youcai mosaic virus</Emphasis>

Alstroemeria plants were surveyed for viruses in Japan from 2002 to 2004. Seventy-two Alstroemeria plants were collected from Aichi, Nagano, and Hokkaido prefectures and 54.2% were infected with some species of virus. The predominant virus was Alstroemeria mosaic virus, followed by Tomato spotted wilt virus, Youcai mosaic virus (YoMV), Cucumber mosaic virus (CMV), Alstroemeria virus X and Broad bean wilt virus-2 (BBWV-2). On the basis of nucleotide sequence of the coat protein genes, all four CMV isolates belong to subgroup IA. CMV isolates induced mosaic and/or necrosis on Alstroemeria. YoMV and BBWV-2 were newly identified by traits such as host range, particle morphology, and nucleotide sequence as viruses infecting Alstroemeria. A BBWV-2 isolate also induced mosaic symptoms on Alstroemeria seedlings.     

A generic RT‐PCR assay for the detection of Luteoviridae

This study, using RT‐PCR, is the first comprehensive assessment since 1991 of a generic detection method for the Luteoviridae. Thirteen Luteoviridae species were detected using three separate sets of low‐degeneracy generic primers with RT‐PCR to amplify 68‐, 75‐ and 129/156‐bp regions of the Luteoviridae coat‐protein gene. Species detected include all members of the genus Luteovirus [Barley yellow dwarf virus (BYDV)‐PAV, BYDV‐PAS, BYDV‐MAV (129 and/or 156 bp amplicons), Soybean dwarf virus, Bean leafroll virus (68 bp amplicon)] and eight of nine species from the genus Polerovirus [Beet western yellows virus, Beet chlorosis virus, Beet mild yellowing virus, Turnip yellows virus, Potato leafroll virus, Cucurbit aphid‐borne yellows virus, Cereal yellow dwarf virus‐RPV (68‐bp amplicon) and Sugarcane yellow leaf virus (75‐bp amplicon)]. These primers were not able to detect Carrot red leaf virus, Sweet potato leaf speckling virus (both belong to unassigned Luteoviridae) and Pea enation mosaic virus‐1 (genus Enamovirus). A synthetic positive control containing all primer sequence priming sites was designed to facilitate this method as a generic tool for use with a variety of host plants. The Luteoviridae primers described in this study present a simple infection‐detection tool of benefit to biosecurity authorities in nursery‐stock surveillance, disease management or outbreak prevention, and may also be useful in detection of as‐yet undiscovered species within the Luteovirus and Polerovirus genera.     

Leonotis nepetaefolia: An important plant virus reservoir in central Mexico

The presence of viruses in the weedLeonotis nepetaefolia in central México is reported from two field surveys.L. nepetaefolia, with viral-like symptoms such as mosaic, leaf deformation and calico, was observed growing next to cultivated fields in the Valley of Atlixco, Puebla, an important agricultural region in Mexico. The viruses harbored by this plant were characterized biologically, serologically and by molecular methods. The viruses detected wereAlfalfa mosaic virus (AMV),Cucumber mosaic virus (CMV), a satellite RNA of CMV (CMV satRNA) andTobacco mosaic virus (TMV). This last one was detected only during the first survey. CMV was the predominant virus found in both surveys, and was associated mostly to mosaic symptom. Phylogenetic analysis based on the coat protein gene sequence of CMV indicated that this isolate belongs to subgroup IA and confirmed that it is a mosaic-inducing isolate, whereas AMV belongs to subgroup II. Finally, CMV satRNA was found to be a non-necrogenic ameliorative variant, both by symptomatology and by phylogenetic analysis. Our results suggest thatL. nepetaefolia is a reservoir for several viruses in central Mexico, and given its wide distribution in several parts of the world, its role as a virus reservoir could be more general. http://www.phytoparasitica.org posting Aug. 31, 2005.     

Viruses affecting tomato crops in Serbia

In a two-year survey (2011–2012), 3220 samples were collected and analyzed in order to determine the presence and distribution of viruses in tomato crops at 56 localities of 18 districts in Serbia. Out of 12 viruses tested, Cucumber mosaic virus (CMV), Potato virus Y (PVY), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) were detected in 42.1, 40, 11, 8.6, 2.3 and 1.3% of the total tested samples, respectively. The results revealed that CMV was prevalent in 2011 and PVY in 2012. CMV and PVY, apart from being predominant, were also the most widespread viruses. In general, single infections were the most frequent type of infection. Additionally, the most common mixed infections were double infections and the most prevalent combination was CMV and PVY. In 2011, the incidence of diseases and the percentage of all infection types were significantly higher than in 2012. Furthermore, in 2011, regardless of total single infections being prevalent compared to mixed infections, two prevailing viruses were commonly detected in mixed infections. The additional molecular testing of ELISA-negative samples using virus specific primers did not reveal the presence of Pepino mosaic virus (PepMV), Tomato yellow leaf curl virus (TYLC), Tomato infections chlorosis virus (TICV) and Tomato chlorosis virus (ToCV).     

Molecular epidemiology of cassava mosaic disease in Madagascar

Cassava is the staple food for hundreds of millions of people in Africa but its cultivation is seriously constrained by cassava mosaic disease (CMD) in Madagascar, and in Africa in general. This study identified the cassava mosaic geminiviruses (CMGs) involved in CMD in Madagascar and their associated epidemiological characteristics from countrywide surveys. Molecular characterization of CMGs in Madagascar revealed an unprecedented diversity and co‐occurrence of six viruses: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Kenya virus (EACMKV), East African cassava mosaic virus (EACMV), South African cassava mosaic virus (SACMV) and the recently described Cassava mosaic Madagascar virus (CMMGV). Distinct geographical distributions were observed for the six viruses. While ACMV was more prevalent in the central highlands, EACMV and EACMKV were prevalent in lowlands and coastal regions. Both EACMCV and SACMV occurred in almost all the localities visited. PCR diagnosis revealed that mixed infection (up to four co‐infected viruses) occurred in 21% of the samples and were associated with higher symptom severity scores. Pairwise comparisons of virus associations showed that EACMCV was found in mixed infections more often than expected while ACMV and SACMV were mostly found in single infections. A greater abundance of whiteflies was observed in lowland and coastal areas. Nevertheless, infected cuttings remain the primary source of CMD propagation (95%) in Madagascar.     

Inter‐laboratory validation of PCR‐based protocol for detection of olive viruses

Sanitary selection and certification of olive cultivars require sensitive diagnostic methods and effective sanitation protocols. Although much attention has been paid in the past few years to the development of diagnostic tools for reliable virus identification, the need to define a common and standardized diagnostic protocol led to the implementation of a ring test among nine Italian diagnostic laboratories. A one‐step RT‐PCR protocol and different primer sets, targeting the most common olive viruses covered by phytosanitary rules, were tested in each laboratory, using the same batch of positive and healthy controls as well as the same amplification conditions and reaction components. The one‐step RT‐PCR, performed using several specific primer sets, was able efficiently to detect the target viruses in all laboratories. Furthermore, a one‐step RT‐PCR protocol was used successfully for the first time for detection of Tobacco necrosis virus (TNV) and Olive mild mosaic virus (OMMV). Results showed that all target viruses were not uniformly distributed in the canopy, and that at least two subsets of samples must be collected from each plant. This standardized protocol is now being used to produce nuclear stocks for 70 different Italian olive cultivars, in the framework of the national project OLVIVA, which involves 25 national research institutions.     

Biodiversity of viruses infecting tomato in Italy: methods for diagnosis and diversification*

An account is given of strain‐specific diagnostic methods currently adopted in Italy to detect alfalfa mosaic alfamovirus (AMV), cucumber mosaic cucumovirus (CMV) and tomato spotted wilt tospovirus (TSWV). These viruses are highly detrimental to tomato crops as the introduction of new virus strains has deeply changed the population structure of resident viruses. RT‐PCR coupled with restriction analysis of amplicons proved suitable for differentiating CMV and AMV strains whereas single‐stranded conformational polymorphism (SSCP) was used for the identification of TSWV isolates. Application of strain‐specific diagnostic methods to phytosanitary inspection of plant propagules is suggested as a powerful tool for timely identification of new virus strains     

Occurrence of viruses in field-grown pepper crops and some of their reservoir weed hosts in Samsun, Turkey

Pepper production is affected by several viral diseases in Samsun, Turkey. To determine the identity of these viruses, a total of 313 samples from field-grown peppers were collected during surveys in 1998 and 1999, and tested by enzyme linked immunosorbent assay (ELISA). Six viruses,Alfalfa mosaic virus (AMV),Cucumber mosaic virus (CMV),Potato virus Y (PVY),Tomato mosaic virus (ToMV),Tobacco mosaic virus (TMV) andTomato spotted wilt virus (TSWV) were detected in the samples. Of 313 plants tested, 42 were doubly infected, and TMV+PVY (15.4%) was the most common double infection. This is also the first report of AMV in pepper fields in Turkey. The effect of some weed species that may act as reservoir of these viruses was also investigated in the region and of 24 weed species belonging to 15 families tested, 16 were found to be infected with at least one virus.Amaranthus retroflexus (Redroot pigweed) appeared to be a common host of CMV, PVY, ToMV, TMV and TSWV, whereasHibiscus trionum (Venice mallow) was recorded as a new weed host of PVY and TSWV. The majority of weed species found to be virus infected were very common in the pepper growing areas of the region. This indicates that pepper fields contaminated with these weeds are under risk of viral infections. http://www.phytoparasitica.org posting July 21, 2005.     

A study of viral coat protein accumulation in lily chloroplasts from mixed virus infections of Lily mottle virus and Cucumber mosaic virus

Two viruses that frequently occur in many Lilium species are Lily mottle virus (LMoV) and Cucumber mosaic virus (CMV), which usually co-infect lilies causing severe disease symptoms. Recent reports have revealed that the viral coat protein (CP) affects chloroplast ultrastructure and symptom development. This study used western blot analysis to confirm that in leaves infected by mixed virus infections of LMoV and CMV, CPs of both viruses were accumulated in lily chloroplasts. Immunogold labelling further demonstrated that both the LMoV CP and CMV CP were localized in the stroma and the thylakoid membranes of the chloroplasts. In addition, it was found that CPs of both viruses were rapidly transported into isolated, intact chloroplasts (in vitro), and their transport efficiencies were positively related to CP concentrations. The lowest transmembrane concentration of CMV CP decreased from 38 μg mL⁻¹ recorded in the single CMV CP import system to 10 μg mL⁻¹ in the mixed import system of LMoV CP and CMV CP. CPs of both viruses exhibited species selection in their transmembrane transport into chloroplasts. This is the first report that the CPs from two viruses (LMoV and CMV) are simultaneously present in lily chloroplasts. Accumulation of high levels of LMoV CP and CMV CP inside the chloroplast appears to contribute to a synergistic interaction inducing the development of mosaic symptoms.     

侵染西瓜的5种病毒ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR检测体系的建立与检测应用

 根据5种病毒小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、烟草花叶病毒(Tobacco mosaic virus,TMV)、南瓜花叶病毒(Squash mosaic virus,SqMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)的核苷酸保守区序列,设计特异性引物对,从影响多重RT-PCR (mRT-PCR)扩增的引物浓度、Mg²⁺浓度、Taq DNA聚合酶浓度、dNTPs浓度、退火温度等方面进行反应体系的优化,建立了一种能够同时检测ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR技术体系,并进行了实际应用。在一个体系中对上述5种病毒复合侵染的西瓜材料进行多重RT-PCR扩增,得到与试验设计相符的5条特异性条带,依次是542、485、410、354和293bp。该体系实现了对侵染西瓜的5种病毒的同时检测,极大地提高了检测效率,降低了检测成本,体现了多重RT-PCR的优越性。     

Rhizobacteria‐mediated resistance against the blackeye cowpea mosaic strain of bean common mosaic virus in cowpea (Vigna unguiculata)

BACKGROUND: The present study investigated the effect of seven Bacillus‐species plant‐growth‐promoting rhizobacteria (PGPR) seed treatments on the induction of disease resistance in cowpea against mosaic disease caused by the blackeye cowpea mosaic strain of bean common mosaic virus (BCMV). RESULTS: Initially, although all PGPR strains recorded significant enhancement of seed germination and seedling vigour, GBO3 and T4 strains were very promising. In general, all strains gave reduced BCMV incidence compared with the non‐bacterised control, both under screen‐house and under field conditions. Cowpea seeds treated with Bacillus pumilus (T4) and Bacillus subtilis (GBO3) strains offered protection of 42 and 41% against BCMV under screen‐house conditions. Under field conditions, strain GBO3 offered 34% protection against BCMV. The protection offered by PGPR strains against BCMV was evaluated by indirect enzyme‐linked immunosorbent assay (ELISA), with lowest immunoreactive values recorded in cowpea seeds treated with strains GBO3 and T4 in comparison with the non‐bacterised control. In addition, it was observed that strain combination worked better in inducing resistance than individual strains. Cowpea seeds treated with a combination of strains GBO3 + T4 registered the highest protection against BCMV. CONCLUSION: PGPR strains were effective in protecting cowpea plants against BCMV under both screen‐house and field conditions by inducing resistance against the virus. Thus, it is proposed that PGPR strains, particularly GBO3, could be potential inducers against BCMV and growth enhancers in cowpea. Copyright © 2009 Society of Chemical Industry