豆叶病毒(BLRV;豆叶病毒),Luteovirus属的单链RNA病毒,韧皮部受限,主要由蚜虫在非繁殖中传播,坚持不懈的方式(Rashed等人。,2018;基德纳马里亚姆和亚伯拉罕,2023年)。BLRV感染各种豆类,并已在全球主要的豆类生长地区报道(Agindotan等人。,2019年),但不是在加拿大大草原。其对作物产量的影响因植物和病毒的基因型以及感染时间而异。一些豌豆田的发病率高达80%(Clement等人。,2020年;汉普顿,1983).在整个2022年的生长季节(6月和7月),来自萨斯喀彻温省各地的脉冲场被随机选择和调查,收集并在处理前在-80°C下储存显示叶片黄化和萎黄病的有症状植物。观察到的症状包括坏死点,萎黄病,叶子斑驳,叶子在豌豆中滚动,严重的明亮泛黄,鹰嘴豆的叶边缘坏死。使用酶联免疫吸附测定(ELISA)和逆转录聚合酶链反应(RT-PCR)对收集的样品的35片叶片进行BLRV检测。ELISA测试遵循制造商的方案,使用商业试剂盒(NanoDiagnostics,圣何塞,CA,美国)。使用TRIzol(Invitrogen,卡尔斯巴德,CA,美国)。为了检测不同的BLRV分离株,对各种分离株的序列进行比对,并在内部专门设计引物,针对GP3和3UTR上病毒的高度保守区域(见补充材料)。还设计了针对先前用于BLRV检测的外壳蛋白(CP)编码区的其他引物(Agindotan等人。2019年;Larsen&Webster1999)。对35个有症状的样本进行PCR检测,包括12个豌豆植物和23个鹰嘴豆植物,在两个有症状的样本中确定了BLRV的存在,一个来自豌豆(PisumsativumL.var。CDC印加)和desi型鹰嘴豆(CicerarietinumL.var。CDC负责人)。在萨斯卡通发现了受感染的豌豆和鹰嘴豆样本,SK(坐标:52°9\'27\'\'N,106°34\'14\“W),和领导区,萨斯喀彻温省西南部,SK(坐标:50°52\'14\"N,109°23\'11\“W),分别。纯化PCR扩增子并送去进行Sanger测序。组装读段以从豌豆和鹰嘴豆产生1666和323个核苷酸,分别,最低覆盖2倍。从豌豆(PsSK1)和鹰嘴豆(CaSK1)获得的BLRV分离株的部分核苷酸序列(GenBank登录号:PP240429,PP266588)显示(1521/1574bp)96.63%和(316/323bp)97.83%的相似性与BLRV参考分离株序列(NC_003369)和来自阿根廷的分离株(KR261610)的15323/9相应地。通过ELISA证实两种感染的样品是BLRV感染的,并且在450nm测量的30分钟后与阳性对照(0.292)相比表现出高的相互作用比(PsSK1:0.319和CsSK1:0.245)。这是BLRV在加拿大大草原脉搏生长地区的第一份报告。在萨斯喀彻温省,尽管有大量易感作物种植,但没有BLRV的历史。因此,本研究参与的调查项目并非旨在评估BLRV的严重程度,而是旨在确定是否存在可能被忽视的病毒.因此,样本是随机抽取的,专注于田地数量和地理覆盖范围,而不是专注于每个田地的多个植物。此外,字段不是根据症状选择的,而是随机选择的。虽然,选择田地内的植物是因为它们表现出症状。通常,疾病记录包括严重程度和潜在风险的估计;然而,这对这项研究来说是不可能的。相反,它被检测到的事实表明风险比以前想象的要大,因为假设BLRV不存在。这些发现凸显了对病毒现状进行进一步研究的必要性,它对作物生产的影响,以及萨斯喀彻温省种植的脉冲品种的抗性。
Bean leafroll virus (BLRV; Bean leafroll virus), a single-stranded RNA virus in the genus Luteovirus, is phloem-limited and primarily transmitted by aphids in a non-propagative, persistent manner (Rashed et al., 2018; Kidanemariam and Abraham, 2023). BLRV infects various legumes and has been reported from major pulse-growing regions worldwide (Agindotan et al., 2019) but not in the Canadian Prairies. Its impact on crop yield varies with plant and virus genotypes and the timing of infection. Some
pea fields have experienced disease rates of up to 80% (Clement et al., 2020; Hampton, 1983). Throughout the 2022 growing season (June and July), pulse fields from across Saskatchewan were randomly selected and surveyed, and symptomatic plants demonstrating leaf yellowing and chlorosis were collected and stored at -80°C before processing. Observed symptoms included necrotic spots, chlorosis, leaf mottling, leaf rolling in peas, severe bright yellowing, and leaf marginal necrosis in chickpeas. BLRV detection was performed on 35 leaves of the collected samples using both Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse transcription polymerase chain reaction (RT-PCR). ELISA testing followed the manufacturer\'s protocol using a commercial kit (Nano Diagnostics, San Jose, CA, USA). Total RNAs were extracted from the frozen samples using TRIzol (Invitrogen, Carlsbad, CA, USA). For the detection of the diverse BLRV isolates, sequences of various isolates were aligned and primers were specifically designed in-house, targeting the virus\'s highly conserved regions on the GP3 and 3\' UTR (see Supplementary material). Additional primers were also designed targeting coat protein (CP) coding regions which were previously used for BLRV detection (Agindotan et al. 2019; Larsen & Webster 1999). PCR testing of 35 symptomatic samples including 12
pea plants and 23 chickpea plants, identified the presence of BLRV in two symptomatic samples, one each from a field
pea (Pisum sativum L. var. CDC Inca) and a desi-type chickpea (Cicer arietinum L. var. CDC Leader). The infected
pea and chickpea samples were found in Saskatoon, SK (Coordinates: 52°9\'27\'\'N,106°34\'14\"W), and the Leader area, southwest of Saskatchewan, SK (Coordinates: 50°52\'14\"N,109°23\'11\"W), respectively. PCR amplicons were purified and sent for Sanger sequencing. The reads were assembled to generate 1666 and 323 nucleotides from
pea and chickpea, respectively, with a minimum of 2X coverage. Partial nucleotide sequences of the BLRV isolates obtained from pea (PsSK1) and chickpea (CaSK1) (GenBank accession numbers: PP240429, PP266588) showed (1521/1574 bp) 96.63% and (316/323 bp) 97.83% similarity with a BLRV reference isolate sequence (NC_003369) and to an isolate from Argentina (KR261610) which was reported on Medicago sativa L. with (1555/1574 bp) 98.79% and (319/323 bp) 98.76% similarity, correspondingly. Both infected samples were confirmed to be BLRV-infected through the ELISA and exhibited a high interaction ratio (PsSK1: 0.319 and CsSK1: 0.245) compared to a positive control (0.292) after 30 minutes as measured at 450 nm. This is the first report of BLRV in the pulse-growing region of the Canadian Prairies. In Saskatchewan, there is no history of BLRV despite the large amount of area growing susceptible crops. Therefore, the survey project that this study was part of was not intended to evaluate the severity of BLRV but rather to determine if there is any virus present that might have been overlooked. The samples were therefore taken randomly, with a focus on the number of fields and geographic coverage rather than focusing on multiple plants per field. Moreover, fields were not chosen based on symptoms but rather at random. Although, plants within fields were chosen because they displayed symptoms. Typically, a disease note includes estimates of severity and potential risk; however, that is not possible for this study. Rather, the fact that it was detected indicates a greater risk than previously perceived, since it was assumed that BLRV was not present. These findings highlight the need for further research on the virus\'s current status, its impact on crop production, and the resistance of pulse varieties grown in Saskatchewan.