Report Of Cucurbit Aphid-borne Yellows Virus Research Articles

First report of Cucurbit aphid-borne yellows virus causing yellowing disease on pumpkin (Cucurbita pepo) and cucumber (Cucumis sativus) in Bulgaria.

Cucurbit aphid-borne yellows virus (CABYV) was first reported in France in 1992 but has since been observed worldwide (Lecoq et al. 1992; Choi and Choi 2016; Buzkan et al. 2017; Zindovic et al. 2017; Vidal et al. 2018; Khanal and Ali, 2018). This virus has caused severe losses to different crops especially to the members of Cucurbitaceae and yield losses can reach up to 40-50% if infection occurs at early stages (Lecoq et al. 1992). In July 2017, leaf samples showing virus-like symptoms were collected from five pumpkin (Cucurbita pepo L. var. Clypeata Alefield) and two cucumber (Cucumis sativus L. cv. Azuma matsunari) plants, growing in а field near Sadovo, Bulgaria. Nearly all plants in the field were affected and displayed green or yellow mosaic, interveinal yellowing, blisters, and leaf deformation (Fig. 1). The collected samples were all symptomatic and were subjected to double antibody sandwich (DAS) or triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA) to determine the viral agent(s). Specific monoclonal antibodies (Leibniz institute DSMZ, Germany) raised against Cucumber leaf spot virus, Cucurbit chlorotic yellows virus, Cucurbit yellow stunting disorder virus, Cucumber mosaic virus (CMV), Melon necrotic spot virus, Beet western yellows virus (BWYV), CABYV, Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Cucumber green mottle mosaic virus, were used. The total number of tested samples was seven (n=5 from pumpkin and n=2 from cucumber). All of them displayed positive signals for CABYV and BWYV, both belonging to genus Polerovirus, family Luteoviridae. In addition, ZYMV and/or WMV were detected in pumpkins while CMV and/or WMV were detected in cucumber samples, respectively. To confirm the presence of CABYV and/or BWYV, total RNA was isolated from all seven samples by TRI Reagent® (Sigma, St. Louis, USA) and converted to cDNA with First Strand cDNA Synthesis Kit, Thermo Scientific™. Reverse transcription (RT)-polyemerase chain reaction (PCR) was performed using two pairs of primers (CABYV1FW: 5'-TTATCAGGGGACTATGTTTA-3' and CABYV14REV: 5'-GAGGGGATTTTAACTGACTG-3', and BWYV1FW: 5'-AGTAAGTCCTCCCCAACTGA-3' and BWYV2REV: 5'-CTACCCACGACCGTATTCAT-3'), specifically designed to detect CABYV and BWYV, respectively. Amplicons with expected sizes of 1,930 bp were obtained only with CABYV primers for all samples while no fragments were amplified with BWYV primers. The obtained products from two samples (pumpkin and cucumber) were purified and sent to Macrogen Inc., South Korea, for direct sequencing in both directions. High quality nucleotide sequences were submitted to GenBank We have evaluated the quality of the sequencing and trimmed those parts that did not comply the needed quality. The obtained smaller fragments Nucleotide sequences were submitted to GenBank with accession numbers MK671010 (656 bp) and MK671014 (712bp). These sequences contained ORFs encoding CABYV P1-P2 fusion proteins as determined by Blastp analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). A phylogenetic tree constructed by the Neighbor-joining method using 18 CABYV accessions and Potato leafroll virus as an outlier (Fig. 2) showed that the closest accessions to MK671010 and MK671014 were NC003688 (France) and EU636992 (China) with respective nucleotide identity of 98% and 99%. In 2019, another outbreak was observed in the same field near Sadovo and in a field near Plovdiv planted with pumpkins. Nearly 30% of the plants showed leaf yellowing typical for Polerovirus infection. Screening of collected samples (n=17) by RT-PCR confirmed CABYV presence in 15 samples. Based on available reports and according to our knowledge this is the first report of CABYV in Bulgaria.

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.

FIRST REPORT OF CUCURBIT APHID-BORNE YELLOWS VIRUS AFFECTING SUMMER SQUASH AND MELON IN MONTENEGRO

The phytosanitary status of cucurbit crops in the central and coastal regions of Montenegro was evaluated with a survey in 2015. Leaf samples of summer squash, zucchini squash (Cucurbita pepo L.) and melon (Cucumis melo L.) showing mosaic, interveinal mottling or yellowing, bright yellowing, blisters and distortions were collected from 101 plants. All samples were screened by DAS-ELISA (Bioreba, Switzerland) for Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Moroccan watermelon mosaic virus (MWMV), Zucchini yellow mosaic virus (ZYMV), Zucchini yellow fleck virus (ZYFV) and Papaya ringspot virus (PRSV). Serological analysis showed that 91.1% of the samples were infected with at least one of three viruses, the most prevalent of which was CMV (62.4%), followed by WMV (50.5%) and ZYMV (12.9%). Samples from 20 plants showing symptoms resembling those caused by poleroviruses were analyzed by RT-PCR using the coat protein (CP) gene-specific primers CE9/CE10 of Cucurbit aphid-borne yellows virus (CABYV) (Juarez et al., 2004) An amplicon of the expected size (600 bp) was generated from six samples of summer squash and one of zucchini squash. Single infections by CABYV were found in two of seven positive samples, while other five were co-infected with CMV, WMV and/or ZYMV. Nucleotide sequences of the complete CP gene from four Montenegrin isolates (accession Nos. KX398662-KX398665) were identical to one another, sharing the highest nucleotide identity (99.1%) with an isolate from France (X76931). A previous survey in the former Yugoslavia did not reveal the presence of CABYV (Dukic et al., 2002). Afterwards, the virus has been reported in several neighboring countries (Tomassoli and Meneghini, 2007; Vucurovic et al., 2011), but to our knowledge, this is the first report of CABYV infecting C. pepo in Montenegro.

First report of Cucurbit aphid‐borne yellows virus in Vicia faba

There are 17 formally recognised virus species in the genus Polerovirus (family Luteoviridae) according to the ICTV classification released in 2014. Three polerovirus species have been described that affect production in economically important cucurbit crops: Cucurbit aphid-borne yellows virus (CABYV), Melon aphid-borne yellows virus (MABYV) and Suakwa aphid-borne yellows virus (SABYV). Two other poleroviruses, Beet western yellows virus (BWYV) and Pepper vein yellows virus (PVYV) have been detected recently in field-grown pepper plants in Turkey (Buzkan et al., 1). During surveys in March 2016, symptoms including chlorosis of young leaves and yellowing of older leaves, suggestive of polerovirus infection, were observed in broad bean (Vicia faba) plants (Fig. 1) in Tarsus, a province in the Mediterranean region of Turkey. A total of 36 diseased plants were tested for the presence of poleroviruses using DAS-ELISA (Dombrovsky et al., 2) and 26 tested positive. A 1.1 kb portion of the polerovirus genome was amplified from all 26 ELISA-positive samples using the general polerovirus primer pair Pol-G-F and Pol-G-R in RT-PCR (Knierim et al., 3). PCR amplicons were subsequently sequenced (GenBank Accession No. KY112798) and subject to a BLASTn search to identify the polerovirus species. According to an alignment of 473 nucleotides of the RdRp, the broad bean isolate showed 98% nucleotide identity with a CABYV isolate from cucumber from Iran (KF425567). To the best of our knowledge this is the first report of CABYV in V. faba in Turkey and globally. CABYV has been reported previously in a range of cucurbit crops in Turkey using DAS-ELISA (Yardımcı & Özgönen, 6). However, the virus species was not confirmed since the CABYV polyclonal antibody used cross-reacts with other poleroviruses (Dombrovsky et al., 2). Since CABYV was first identified in 1992, the virus has been detected worldwide in temperate and subtropical areas (Lecoq et al., 4). Apart from cucurbit crops, other species of agronomic importance that are CABYV hosts include Beta vulgaris and Lactuca sativa, as well as common weed species such as Capsella bursa-pastoris, Papaver rhoeas and Senecio vulgaris which are thought to be virus reservoirs (Knierim et al., 3; Mnari-Hattab et al., 2009). CABYV is a phloem-limited virus that is transmitted in a persistent, non-propagative mode by aphids, including Aphis gossypii and Myzus persicae (Lecoq et al., 4). Mixed polerovirus infections increase the risk of the emergence of recombinant viruses. Poleroviruses must be considered as important emerging pathogens in open-field and greenhouse vegetable cultivation. This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 113 O 423).