Tissue Blot Immunoassay Research Articles

First Report of Cotton Leafroll Dwarf Virus Affecting Chickpea (Cicer arietinum) in Uzbekistan

Chickpea (Cicer arietinum L.; family Fabaceae) plants with symptoms typical of virus infection (chlorosis, stunting, necrosis, yellowing, and reddening) were collected during disease surveys in June 2012 (386 samples from 23 fields) and May to June 2013 (288 samples from 19 fields) from the major chickpea-growing regions (Tashkent, Sirdarya, Jizzah, Samarkand, and Surkhandarya) of Uzbekistan. All samples were tested first by tissue-blot immunoassay (TBIA) (Makkouk and Kumari 1996) with a broad-spectrum legume luteovirus monoclonal antibody (MAb) (5G4; Katul 1992). For comparison of relative reactions, blots from positive legume luteoviruses (bean leafroll virus [BLRV], beet western yellows virus [BWYV], and chickpea chlorotic stunt virus [CpCSV]) and heathy plants were included for each TBIA. To identify the individual legume luteoviruses, the samples with positive reaction to MAb 5G4 were retested further using specific MAbs to BWYV (A5977 from Agdia, U.S.A.), BLRV (4B10; Katul 1992), a mixture of three MAbs (1-1G5, 1-3H4, and 1-4B12) to an Ethiopian isolate of CpCSV, and a mixture of three MAbs (5-2B8, 5-3D5, and 5-5B8) to a Syrian isolate of CpCSV (Abraham et al. 2009). Total RNA was extracted from 35 chickpea samples that showed positive reaction to 5G4 MAb only, or to 5G4 and other MAbs. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using polerovirus group-specific primers AS3 (5′-CACGCGTCIACCTATTTIGGRTTITG-3′) (Abraham et al. 2008) and Pol3870F (5′-ATCACBTTCGGGCCGWSTYTWTCAGA-3′), as described by Sharman et al. (2015), except with the use of MyTaq Red DNA Polymerase (Bioline, U.K.). RT-PCR amplicons of the partial coat protein gene of the expected size (376 bp) were directly sequenced. Four out of 35 tested samples had 96 to 97% nucleotide (nt) sequence identity with the Brazilian (GQ401140 and KP176644) and Argentinean (KF359947) isolates of cotton leafroll dwarf virus (CLRDV, genus Polerovirus, family Luteoviridae). To confirm CLRDV infection in these chickpea samples, they were tested by RT-PCR using CLRDV-specific primer CLRDV3675F (5′-CCACGTAGRCGCAACAGGCGT-3′) and Pol3982R (5′-CGAGGCCTCGGAGATGAACT-3′) (Sharman et al. 2015). Amplicons of the expected size (310 bp) were obtained from all four samples and were sequenced in both directions. These four samples (UzCp137-13, UzCp148-13, UzCp149-13, and UzCp154-13) were collected in 2013 from one field in the Surkhandarya region, and their nucleotide sequences were deposited in GenBank under accession numbers MK461134, MK461135, MK461136, and MK461137, respectively. After removal of primers, the resulting consensus sequences were 268 nucleotides in length and shared greater than 99% nt identity among them, and as such further analysis was done for sample UzCp137-13, for which the closest match by BLAST was 98% nt identity to a CLRDV isolate from Argentina (KF359947). To our knowledge, this is the first report of CLRDV in chickpea from Uzbekistan. Mukherjee et al. (2016) studied the genetic similarity between CLRDV and chickpea stunt disease associated virus (CpSDaV) in India and found the coat protein gene sequences of both viruses to be almost identical (89.4 to 100% depending on the virus isolates), suggesting that CpSDaV is likely a strain of CLRDV. Chickpea stunt is a major disease of chickpeas in India, and thus CLRDV will be of huge concern to both chickpea and cotton production in Uzbekistan.

Lack of transmission of Sugarcane yellow leaf virus in Florida from Columbus grass and sugarcane to sugarcane with aphids or mites.

Sugarcane yellow leaf virus (SCYLV), the causal agent of yellow leaf disease, naturally infects at least three plant species in Florida: sugarcane (Saccharum spp.), the weed Columbus grass (Sorghum almum) and cultivated sorghum (S. bicolor). All three hosts are also colonized by the sugarcane aphid (Melanaphis sacchari), the main vector of SCYLV worldwide. To understand the high incidence of SCYLV observed in sugarcane commercial fields and in germplasm collections, we investigated the transmission efficiency of SCYLV from sugarcane and Columbus grass to sugarcane using the sugarcane aphid and a spider mite (Oligonychus grypus) that also tested positive for SCYLV in Florida. Healthy and SCYLV-infected leaf pieces of sugarcane and Columbus grass carrying viruliferous aphids or spider mites were transferred to virus-free plants of the yellow leaf susceptible sugarcane cultivar CP96-1252. Three- and 6-months post inoculation, the 108 aphid-inoculated plants of Columbus grass and the 90 mite-inoculated plants of sugarcane tested negative for SCYLV by tissue blot immunoassay (TBIA) or reverse transcription polymerase chain reaction (RT-PCR). Similar results were obtained for 162 aphid-inoculated plants of sugarcane, except for two plants that tested positive for SCYLV by TBIA and RT-PCR. In two field experiments planted with SCYLV-free and virus-infected sugarcane (cultivar CP96-1252), only 18–28% of healthy plants became infected during a 24- to 28-month period. SCYLV prevalence in these field experiments did not differ between aphicide treated and untreated plots. Incidence of M. sacchari haplotypes in the Everglades agricultural area also indicated that the predominant haplotype that is currently colonizing sugarcane was not a vector of SCYLV in Florida. Lack of virus transmission by the spider mite suggested that this arthropod only acquired the virus when feeding on infected plants but was unable to transmit SCYLV. The current vector of SCYLV in Florida remains to be identified.

Application of biological and single-strand conformation polymorphism assays for characterizing potential mild isolates of Citrus tristeza virus for cross protection

Citrus tristeza virus (CTV) by killing millions of citrus cultivars grown on sour orange rootstock worldwide has become one of the most dangerous viral pathogen. Characterization of 12 CTV isolates was analyzed by biological indexing. Infected samples of citrus were collected from citrus growing areas of Pakistan and CTV was detected by symptoms on indicator plants and confirmed by direct tissue blot immunoassay (DTBIA). CTV positive samples were graft inoculated on six biological indicator hosts in the study. A standardized protocol was deployed to study biological characteristics of these isolates. All biological indicators induced mild and from mild to moderate reactions against all of the CTV isolates tested. About two isolates produced stem-pitting symptoms from moderate to severe on Mexican lime. CTV strains were further characterized and confirmed by the analysis of p25 gene of CTV isolates using single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed that most isolates confined only one predominant sequence variant. SSCP profiles of PCR amplified products from CTV isolates showed bands patterns corresponding to mild and sever strain. Three isolates (4MF, 8KBS and 10GS) from different regions and cultivars were identified as potential source of mild strains for cross protection. These results are the best base for mild strain cross protection (MSCP) in the country.

Identification of New QTL Contributing to Barley Yellow Dwarf Virus-PAV (BYDV-PAV) Resistance in Wheat.

Barley yellow dwarf (BYD) is a major virus disease which dramatically reduces wheat yield. Introducing BYD resistance genes into commercial varieties has been proven to be effective in reducing damage caused by barley yellow dwarf virus (BYDV). However, only one major resistance gene is readily deployable for breeding; Bdv2 derived from Thinopyrum intermedium is deployed as a chromosomal translocation. In this study, a double haploid (DH) population was developed from a cross between XuBYDV (introduced from China showing very good resistance to BYD) and H-120 (a BYD-sensitive Chinese accession), and was used to identify QTL for BYD resistance. The population was genotyped using an Infinium iSelect bead chip array targeting 90K gene-based SNPs. The disease resistance of DH lines inoculated with BYDV was assessed at the heading stage. The infections were assessed by tissue blot immunoassay (TBIA). Three new QTL were identified on chromosomes 5A, 6A, and 7A for both symptom and TBIA, with all three resistance alleles being inherited from XuBYDV. Some DH lines with the resistance alleles from all three QTL showed high level resistance to BYD. These new QTL will be useful in breeding programs for pyramiding BYD resistance genes.

The relationship and relativity between three isolates of Potato virus Y Potyvirus infecting potato (Solanum tuberosum L.) at Alexandria and El-Beheira governorates, northern Egypt

Potato virus Y (PVY) is a highly pathogenic virus, causing enormous economic losses in potato (S. tuberosum) crop. Three isolates of PVY were obtained from naturally infected potato plants showing mosaic; yellowing and vein necrosis symptoms, during 2017-2018 growing seasons at certain locations of El-Beheira and Alexandria governorates, Egypt. PVY could be easily transmitted mechanically by aphids. Detection of the PVY-3 in different organs of infected Nicotiana glutinosa plants by Indirect-ELISA; Dot blot immunoassay (DBIA) and Tissue blot immunoassay (TBIA), indicated the possibility of using these methods for viral detection. Egyptian PVY (MK376452) isolate was of close homology to PVY isolated from South Africa. The other Egyptian isolates were found to be close to a French PVY (KJ741115) isolate. There were variations on comparing nucleotide and amino acid sequences; however, nucleotide sequencing could be more reliable. Application of sequence inspection allowed us to identify the PVY isolates by phylogenetic analysis. Phylogenetic analysis of the genomic coat protein (CP) from 24 PVY isolates registered in GenBank indicated the presence of relationships between each other's. This reflected the high degree of genetic variability among our local Egyptian isolates. The aims of the current work were to; isolate and detect PVY from naturally infected potato plants in northern Egypt, characterize the PVY isolates using different assays, detect PVY in different organs of infected potato plants, study the CP gene of the PVY isolates using Reverse transcription-Polymerase Chain Reaction (RT-PCR), and register these isolates in GenBank.

First Report of Barley virus G in Australia

Wheat (Triticum aestivum), barley (Hordeum vulgare), and oats (Avena sativa) are some of the most economically important cereals in Australia and worldwide. Several cereal viruses are present in southeastern Australia with varied incidence and prevalence depending on year, location, and climatic conditions. Barley and cereal yellow dwarf viruses (family Luteoviridae) are increasingly prevalent in southeastern Australia (Nancarrow et al. 2018). They are transmitted by aphid vectors, mainly the bird cherry-oat aphid (Rhopalosiphum padi) and the corn leaf aphid (Rhopalosiphum maidis). High Plains wheat mosaic virus (HPWMoV) (family Fimoviridae) has been reported in Western Australia (Coutts et al. 2014), and there have been unconfirmed reports of its presence in Victoria; wheat streak mosaic virus (WSMV) (family Potyviridae) was reported in Victoria and other Australian states in 2003 (Ellis et al. 2003). Both HPWMoV and WSMV are transmitted by the wheat curl mite (Aceria tosichella) (Coutts et al. 2014). In late April 2018, a high number of R. maidis was observed on volunteer cereal plants consisting of wheat, barley, and wild oats (Avena fatua) in fields near Horsham, Victoria, Australia, together with yellowing and streaking discoloration on some plants. No evidence of wheat curl mite infestation was observed. Initially, 148 samples were collected from symptomatic and nonsymptomatic plants and tested for WSMV (DSMZ-AS0544), luteoviruses (DSMZ, AS-0227/1), and the yellow dwarf viruses BYDV-PAV, BYDV-MAV, and CYDV-RPV by tissue blot immunoassay (TBIA) (Nancarrow et al. 2018); 16 samples were positive for WSMV, 34 were positive for luteoviruses, and of the 34 luteovirus-positive samples, six were positive for at least one of the yellow dwarf viruses typically found in the Horsham region. The remaining 28 luteovirus-positive samples required further identification. In early May, a further 68 samples, prioritizing plants with streaking discoloration, were collected from the same area and tested for WSMV, luteoviruses, and HPWMoV (Agdia, SRA17200) by TBIA; 26 samples were positive for WSMV, three were positive for luteoviruses, and two were positive for HPWMoV. Two of the 216 samples tested by TBIA had mixed infections. Virus was detected in symptomatic and nonsymptomatic samples, and a number of symptomatic plants tested negative. A subset of 23 samples requiring either further identification or confirmation was additionally tested by reverse transcription polymerase chain reaction (RT-PCR) for WSMV (French and Robertson 1994), luteoviruses (Robertson et al. 1991), and HPWMoV (Lebas et al. 2005). Amplicons of the expected sizes were obtained with 11 samples positive for WSMV, 12 positive for luteoviruses, and two positive for HPWMoV. Amplicons were directly sequenced. Nucleotide sequences of the WSMV (GenBank accession MK103385) and HPWMoV (accession MK103386) amplicons were identical to accessions AY327865 (WSMV) and KT988872 (HPWMoV), respectively, confirming the presence of both viruses and hence the presence of HPWMoV in Victoria. The nucleotide sequence of the luteovirus amplicon showed 99% identity to barley virus G (BVG) (accession NC_029906). The 12 samples that tested positive for luteoviruses by TBIA and RT-PCR were further tested using two sets of BVG-specific primers (Jo et al. 2018; Park et al. 2017), each targeting a distinct region of the BVG genome; all samples were positive. An amplicon from each BVG-specific RT-PCR test was directly sequenced (accessions MK411425 and MK103388), with each showing 96% nucleotide identity to BVG (accession NC_029906), confirming the presence of BVG in these samples. Some BVG-positive samples showed yellow discoloration of the leaf tips. This is the first reported detection of BVG in Australia. BVG was reported for the first time in South Korea in 2016 (Zhao et al. 2016). Epidemiology and yield losses associated with BVG are not known in Australia or elsewhere.

Siratro latent polerovirus (SLPV): a new polerovirus from Australia with a non-functional open reading frame 0

Macroptilium atropurpureum (siratro) samples from Queensland, Australia were collected as part of virus surveys and screened using tissue blot immunoassays for the presence of luteoviridae viruses. Sequencing of a reverse transcription polymerase chain reaction (RT-PCR) product, generated with degenerate primers for poleroviruses, revealed the non-symptomatic virus in siratro was a novel polerovirus, given the proposed name siratro latent polerovirus (SLPV). SLPV-specific RT-PCR showed that SLPV was widely distributed in siratro throughout Queensland. SLPV was transmitted via Aphis craccivora (cowpea aphids) to siratro and Pisum sativum (field pea). The complete genome of one isolate of SLPV was found to be 6090 nucleotides in length and shared only 69% nucleotide identity with its closest match, phasey bean mild yellows virus. Poleroviruses contain six major open reading frames (ORFs) labelled ORF 0–5. The SLPV genome has a similar arrangement. However, the region that should encode for ORF 0 is missing an AUG start codon. Sequencing of this region, from eight SLPV isolates, from different geographic regions, showed all lacked the ORF 0 AUG site and some isolates contained point mutations and deletions in this region, thus inhibiting translation of ORF 0. These results suggest that SLPV does not have a functional ORF 0 which may affect viral pathogenicity and restrict host range. This is the first report, to date, of a polerovirus with an apparently non-functional ORF 0 region.

Detection of Cucumber mosaic cucumovirus in infected cowpea plants (Vigna unguiculata L.) from northern Egypt

Cucumber mosaic cucumovirus (CMV) was isolated from naturally infected Cowpea plants (Vigna unguiculata L.) showing different symptoms of mosaic; mottle, dwarfing, and vein clearing, grown at certain locations of Alexandria governorate, Egypt, during the growing seasons from 2011- 2012. CMV isolate was transmitted by Aphid nerii, A. gossypii, and Myzus persicae in non-persistent manner. The virus was partially purified using polyethylene glycol (PEG) 6000, and differential centrifugation. The ratio of A 260/280 was 1.622 and A 280/260 was 0.617. Whereas, the ratio of A max/ min was 1.915. Concentration of the virus in the preparation was estimated using an extinction coefficient of E 2600.1% = 5. Yield of purified virus was about 6.88 mg/ 100g fresh weight of leaves of Nicotiana glauca. Antiserum titer was determined by Indirect enzyme linked immunosorbent assay (ELISA). Positive ELISA values were obtained up to dilutions of 1: 25600. The virus was detected by indirect ELISA in infected sap at 8, 16 and 24 days after inoculation; and by Tissue blot immunoassay (TBIA) on nitrocellulose membrane after the same period. The unused face of the processed nitrocellulose membrane already printed with plant tissues was tested. Results revealed that both faces of nitrocellulose membrane and Canson paper could be used as solid carriers in TBIA, for detection of CMV in infected leaves. According to Reverse transcription polymerase chain reaction (RT-PCR); the size of amplification of the obtained product was approximately 870 bp for CMV isolate; and was assigned accession number of LN606587. The Phylogenetic tree was generated using partial sequence of CMV isolate, with those of other CMV isolates obtained from GenBank. The aims of the current work were; to produce specific polyclonal antiserum against the purified CMV isolated from cowpea plants, and to register this isolate in GenBank.

Alfalfa mosaic virus infects the tropical legume Desmanthus virgatus in Australia and the potential role of the cowpea aphid (Aphis craccivora) as the virus vector

Severe yellowing and stunting of plant growth was observed in experimental plots of Desmanthus virgatus (desmanthus) at the Tamworth Agricultural Institute during the 2015/16 summer season. Both symptomatic and non-symptomatic plants were tested for the presence of a range of viruses by Tissue blot immunoassay and symptomatic plants consistently reacted positive to Alfalfa mosaic virus (AMV), while non-symptomatic plants were negative to all viruses tested. AMV was not detected in seedlings grown from seed collected from AMV-positive desmanthus plants. AMV was readily transmitted from AMV-positive desmanthus plants by mechanical inoculation to faba bean, but attempts to transmit the virus from AMV-positive Medicago sativa (lucerne) plants to desmanthus by mechanical inoculation were unsuccessful. However, AMV was successfully transmitted from lucerne to desmanthus by Aphis craccivora (cowpea aphid). Aphid feeding studies showed that the cowpea aphid, but not Acyrthosiphon pisum (pea aphid), could colonise and multiply on desmanthus. AMV could become a limiting factor for the adoption of desmanthus as a pasture legume in NSW, particularly as AMV has been reported to be seed transmitted in desmanthus.

Barley yellow dwarf virus infection affects physiology, morphology, grain yield and flour pasting properties of wheat

Barley yellow dwarf virus (BYDV) is a phloem-limited virus that is persistently transmitted by aphids and causes significant yield losses in wheat (Triticum aestivum L.). The present study was conducted to investigate the effects of BYDV in wheat on physiological and morphological traits, yield attributes and pasting properties of flour, and to determine any differences for these traits between susceptible and resistant genotypes under BYDV infection. Significant impact on physiological and morphological traits and yield was observed in plants inoculated at the 2-leaf stage (Zadoks scale, Z12), with a greater impact in the three susceptible genotypes than in the resistant genotype. Yield reduction with inoculation at Z12 was 18–49%, and yield reduction with inoculation mid tillering (Z25) was 6–31%. There was a significant reduction in effective tiller number with both inoculation times, but 1000-kernel weight was affected only with early inoculation. Pasting properties were little affected by BYDV infection, with genotype having a larger effect than infection. Grain yield showed negative correlation with tissue-blot immunoassay and visual symptom score, and positive correlation with all gas-exchange parameters, chlorophyll fluorescence, leaf area and biomass weight. The results suggest that stomatal conductance, transpiration rate and chlorophyll fluorescence measurements are suitable for assessment of BYDV infection and for screening BYDV of susceptible and resistant wheat genotypes.

Prevalence and Incidence of Yellow Dwarf Viruses Across a Climatic Gradient: A Four-Year Field Study in Southeastern Australia.

Yellow dwarf viruses (YDVs) form a complex of economically important pathogens that affect cereal production worldwide, reducing yield and quality. The prevalence and incidence of YDVs including barley yellow dwarf viruses (BYDV-PAV and BYDV-MAV) and cereal yellow dwarf virus (CYDV-RPV) in cereal fields in Victoria, Australia were measured. As temperature decreases and rainfall increases from north to south in Victoria, fields in three geographical regions were evaluated to determine potential differences in virus prevalence and incidence across the weather gradient. Cereal samples randomly collected from each field during spring for four consecutive years (2014-2017) were tested for BYDV-PAV, BYDV-MAV, and CYDV-RPV using tissue blot immunoassay. BYDV-PAV was the most prevalent YDV species overall and had the highest overall mean incidence. Higher temperature and lower rainfall were associated with reduced prevalence and incidence of YDVs as the northern region, which is hotter and drier, had a 17-fold decrease in virus incidence compared with the cooler and wetter regions. Considerable year-to-year variation in virus prevalence and incidence was observed. This study improves our understanding of virus epidemiology, which will aid the development of more targeted control measures and predictive models. It also highlights the need to monitor for YDVs and their vectors over multiple years to assess the level of risk and to make more informed and appropriate disease management decisions.

Development of Infectious cDNA Clones of Citrus Yellow Vein Clearing Virus Using a Novel and Rapid Strategy.

Yellow vein clearing disease (YVCD) causes significant economic losses in lemon and other species of citrus. Usually, citrus yellow vein clearing virus (CYVCV) is considered to be the causal agent of YVCD. However, mixed infection of CYVCV and Indian citrus ringspot virus (ICRSV) or other pathogens is often detected in citrus plants with YVCD. In this study, we re-examined the causal agent of YVCD to fulfill Koch's postulates. First, the full-length genome of CYVCV isolate AY (CYVCV-AY) was amplified by long-distance RT-PCR from a Eureka lemon (Citrus limon) tree with typical YVCD symptoms. The genomic cDNAs were then cloned into a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector, pCY, using transformation-associated recombination (TAR) strategy, and 15 full-length cDNA clones of CYVCV-AY were obtained. Subsequently, four of these clones were selected randomly and inoculated on Jincheng (C. sinensis) seedlings through Agrobacterium-mediated vacuum-infiltration, and it was found that 80 to 100% of inoculated plants were infected with CYVCV by RT-PCR at 20 to 40 days postinoculation (dpi) and by direct tissue blot immunoassay at 60 dpi. The progeny of CYVCV-AY from cDNA clones caused typical symptoms of YVCD such as yellow vein clearing, leaf distortion, and chlorosis, which were the same as that elicited by wild-type virus. Finally, the regeneration of CYVCV-AY genome was confirmed by long-distance RT-PCR in lemon trees inoculated with the infectious cDNA clone. These results proved that CYVCV was the primary causal agent of YVCD. This is the first report on the development of infectious cDNA clones of CYVCV, which lays the foundation for further studies on viral gene functions and virus-host interactions.

First Report of Cucurbit aphid-borne yellows virus Affecting Chickpea (Cicer arietinum L.) in Sudan.

A total of 204 chickpea samples with symptoms typical of virus infection including stunting, yellowing, necrosis, reddening, and tip wilting were collected during February 2013 and March 2015 from 14 chickpea fields located in Gizera, middle Gizera, Barbar, Shendi and Hudiba, Sudan. All samples were tested using tissue-blot immunoassay (TBIA) (Makkouk and Kumari, 1996) against a battery of legume virus antibodies including a broad-spectrum legume luteovirus monoclonal antibody (McAb) (5G4; Katul, 1992). To identify the luteoviruses affecting chickpea in Sudan, 41 chickpea samples that gave positive reaction to 5G4 McAb were tested using generic reverse transcription polymerase chain reaction (RT-PCR) Luteovirus primer pairs (Asaad et al. 2009; Abraham et al. 2009). Direct sequencing of these products indicated that 12 samples were related to Cucurbit aphid-borne yellows virus (CABYV). To confirm this data, all samples were tested again by RT-PCR using CABYV-specific primer CABYV3635F (5’-GAAACCGCCGACGCCCTAAT-3’; this study) and primer AS3 (Abraham et al. 2009). Results showed that 12 of the 41 samples that gave positive reactions to 5G4 McAb generated RT-PCR amplicons of the partial coat protein gene of the expected size (481 bp) and were considered positive for CABYV. Amplicons from two chickpea samples (isolates SuCp106-13 and SuCp122-13) were directly sequenced in both directions. After removal of primers, the resulting sequences were 435 nucleotides in length (GenBank accessions numbers MG933685 and MG933686). The two isolates shared 100% nt identity with each other and 96% nt identity with the type reference sequence for CABYV, NC_003688. Moreover, we confirmed the presence of CABYV in RT-PCR positive samples serologically with TBIA using polyclonal antisera (provided by Lecoq, INRA, France). The observed symptoms in CABYV-infected chickpea plants were stunting, accompanied by leaf yellowing and necrosis and is likely to reduce yield similar to other Luteoviruses. Of the 204 virus-symptomatic plants we tested in this study, 12 were positive for CABYV (6% of the samples tested). Chickpea is an economically important crop in many countries of the world and knowing the exact identity of a virus affecting this crop in a region is essential for breeding for resistance and crop management purposes. CABYV and its aphid vector (Aphis gossypii Glover) were reported on cucurbit crops in many countries of the world with high yield loss. Further disease surveillance will be needed to determine if CABYV continues to impact chickpea production in Sudan and if it is also present in other chickpea production regions of the world. CABYV was reported recently on broad bean (Vicia faba L.) in Turkey (Buzkan et al. 2017), but based on our knowledge this is the first report of CABYV on chickpea in Sudan and worldwide.

Molecular characterization of genetic basis of Sugarcane Yellow Leaf Virus (SCYLV) resistance in Saccharum spp. hybrid

AbstractSugarcane (Saccharum Spp.) produces 80% of the world's sugar along with other by‐products. The production of sugarcane is vulnerable to infestation of sugarcane yellow leaf virus (SCYLV) worldwide. A study was conducted using an F1 segregating population derived from CP95‐1039 × CP88‐1762 to identify the genetic factors underlying SCYLV resistance. The disease infection data were measured using tissue blot immunoassay after 6 years of exposure to the virus under natural field conditions. Genetic maps were created using genotyping by sequencing‐based markers for each parent separately following a pseudo‐testcross approach. Two quantitative trait loci (QTL) were detected for SCYLV resistance accounting for 28% of the phenotypic variation. A major QTL qSCYLR79 located on linkage group 79 and linked with marker 3PAV3154 appears to be unique for SCYLV resistance in sugarcane. Progeny having a combination of two major alleles had 31% less SCYLV incidence than progeny with a combination of major and minor alleles in the genomic region of qSCYLR79. Thus, selection against the minor allele may decrease the SCYLV incidence in sugarcane.

A screening method to detect BYDV‐PAV resistance in cereals under glasshouse conditions

A reliable method was developed to screen cereal crops for BYDV‐PAV resistance in glasshouse experiments. This also entailed the evaluation of traits associated with Barley yellow dwarf virus (BYDV) infection such as leaf discolouration, reduction in growth, biomass and yield traits, and percentage of virus‐infected plants, using enzyme‐linked immunosorbent assay (ELISA) and tissue blot immunoassay (TBIA). Four glasshouse experiments were conducted with eight wheat, barley and oat varieties inoculated with BYDV‐PAV at the 2‐leaf stage, using different numbers of viruliferous aphids and different inoculation periods and temperatures. Inoculation with 5–10 viruliferous aphids per plant for 4 days led to a high percentage of infection in susceptible varieties, indicating that this is an effective BYDV screening method when selecting for resistance in cereal crops. For barley and oat, visual evaluation of symptoms is considered adequate for assessing BYDV resistance. However, for wheat it is necessary to evaluate BYDV resistance by ELISA/TBIA tests and plant biomass (at early stage) and grain number and yield (at late stage) measurements.

Non‐cultivated grass hosts of yellow dwarf viruses in Ethiopia and their epidemiological consequences on cultivated cereals

AbstractThe yellow dwarf (YD) disease complex epidemics in cultivated cereals grown in a specific period of the year mainly depend on the presence of potential reservoir alternative hosts harbouring both the viruses and the vectors over the off‐season and serve as a source of inoculum in subsequent cropping season, further spread being supported by efficient aphid vectors. As such, an extensive and intensive exploration to generate base line information on the identity and prevalence of YD viruses [barley yellow dwarf virus (BYDV)‐PAV, BYDV‐MAV and BYDV‐SGV; cereal yellow dwarf virus (CYDV)‐RPV; and maize yellow dwarf virus (MYDV)‐RMV] on wild annual and perennial grasses and forage cereals alternative hosts was conducted consecutively during 2013–2015 main‐ and short‐rainy seasons in cereals growing belts of Ethiopia. Random sampling was employed to collect the samples that were tested by the tissue blot immunoassay (TBIA) to identify the YDVs associated with the hosts using a battery of virus‐specific polyclonal antibodies. Of 13,604 samples analysed, YDVs were detected in 392 (2.9%) samples, which consisted of various wild grasses, forage cereals and three cultivated crops. YDVs were identified from at least 26 grass species and forage cereals, some of them are new records, and some are previously documented hosts. To our knowledge, this is the first report of YDV infection of Andropogon abyssinicus (FresenR.Br. ex Fresen.) (BYDV‐PAV), Avena abyssinica Hochst (BYDV‐PAV), Bromus pectinatus Thunb. (BYDV‐PAV and BYDV‐MAV), Eragrostis tef (Zuccagni) Trotter (BYDV‐PAV), Eragrostis sp. (BYDV‐PAV), Hyparrhenia anthistrioides Stapf. (BYDV‐PAV), Panicum coloratum L. (BYDV‐PAV), Polypogon monspeliensis (L.) Desf. (BYDV‐PAV), Setaria pumila (Poir.) Roem & Schult (BYDV‐PAV, BYDV‐SGV and MYDV‐RMV), Setaria australiensis (Scribn. & Merrill) Vickery (BYDV‐PAV, BYDV‐MAV and CYDV‐RPV) and Snowdenia polystachya (Fresen.) Pilg (BYDV‐PAV, BYDV‐MAV, BYDV‐SGV, CYDV‐RPV and MYDV‐RMV).

Virus incidence in wheat increases under elevated CO2: A 4-year study of yellow dwarf viruses from a free air carbon dioxide facility

The complexities behind the mechanisms associated with virus-host-vector interactions of vector-transmitted viruses, and their consequences for disease development need to be understood to reduce virus spread and disease severity. Climate has a substantial effect on viruses, vectors, host plants and their interactions. Increased atmospheric carbon dioxide (CO2) is predicted to impact the interactions between them. This study, conducted under ambient and elevated CO2 (550μmolmol−1), in the Australian Grains Free Air Carbon Enrichment facility reports on natural yellow dwarf virus incidence on wheat (including Barley/Cereal yellow dwarf viruses (B/CYDV)). A range of wheat cultivars was tested using tissue blot immunoassay to determine the incidence of four yellow dwarf virus species from 2013 to 2016. In 2013, 2014 and 2016, virus incidence was high, reaching upwards of 50%, while in 2015 it was relatively low, with a maximum incidence of 3%. Across all years and most cultivars, BYDV-PAV was the most prevalent virus species. In the years with high virus incidence, a majority plots with the elevated levels of CO2 (eCO2) were associated with increased levels of virus relative to the plots with ambient CO2. In 2013, 2014 and 2016 the recorded mean percent virus incidence was higher under elevated CO2 when compared to ambient CO2 by 33%, 14% and 34%, respectively. The mechanism behind increased yellow dwarf virus incidence under elevated CO2 is not well understood. Potential factors involved in the higher virus incidence under elevated CO2 conditions are discussed.

Serological and molecular detection of Bean leaf roll and Chickpea chlorotic stunt luteoviruses in chickpea from Iran

AbstractChickpea (Cicer arietinumL.) is an important legume crop and widely cultivated in northwestern provinces of Iran. During a survey in the 2015 growing season a total of 170 selected chickpea plants with general yellowing symptoms including stunting and leaf bronzing were collected. Serological Elisa and tissue blot immunoassay (TIBA) tests revealed the presence ofBean leaf roll virus(BLRV) andChickpea chlorotic stunt virus(CpCSV) as the predominant viruses in the region. Some serologically positive samples of BLRV and CpCSV were selected and rechecked by RT-PCR. The results of amplified PCR products using a specific pair of primers towards theCpgene region of the viruses were approximately 413 bp for CpCSV and 391 bp for BLRV. Results obtained from sequence comparison of BLRV (IR-F-Lor-5) isolate form two subgroups with eight other BLRV isolates from GeneBank indicating a high homology of 96% with isolates from Argentina, Germany, Tunisia, USA, Spain, and Colombia. An isolate from Norabad (Iran) (IR-Nor) had 98% homology with HQ840727 Libyan isolate. CpCSV sequence comparison with six other GeneBank isolates indicated 98% homology with isolates from Tunisia and Azerbaijan. The overall results of this research revealed the CpCSV and BLRV (luteoviruses) associated with the yellowing disease syndrome of chickpea crops in the surveyed region.

Distribution of Tomato spotted wilt virus in dahlia plants.

In this study, the distribution of Tomato spotted wilt virus (TSWV) in various parts of dahlia plants was investigated for the first time. The distribution of TSWV was uneven in compound leaves, leaflets, stems, and bulbs. The middle parts of the stem or the petiole and leaf veins should be sampled to detect TSWV when selecting healthy plants.

The Comparison of Antibodies Raised Against PLRV with Two Different Approaches - Viral Particles Purification and Recombinant Production of CP

Serology is one of the most important techniques which extensively used in different fields especially the agriculture one. Serological methods usually which are extensively used because of their specificity in disease diagnosis and relative ease of completion. The methods are widely used in plant virology include enzyme-linked immunosorbent assay, dot-blot immunoassay, immunospecific electron microscopy, and tissue-blot immunoassay. The main stone of the serology test is antiserum which is either mono or poly. Due to the high cost of the monoantisera production and its low sensitivity of the ones immunized by the purified virus particles, the recombinant protein could be good substitution. In this study, we aimed to produce polysera using the viral particles and with the recombinant coat protein of the Potato Leaf Roll Virus as well and used both in rabbit immunization separately. The sensitivity, specificity, and reactivity of the two produced sera were tested in comparing with the real-time polymerase chain reaction. Results revealed that polysera produced by recombinant coat protein are more specific and sensitive than the sera produced by the purified particles. Moreover, the results obtained by dot and tissue blot confirmed the results obtained by enzyme-linked immunosorbent assay techniques. The real time-polymerase chain reaction results were similar to that obtained by serological methods except the time and the substrate. In conclusion, production of the polysera using recombinant protein is test easy, cheap, and sensitive test for on line vial detection.