Jailani A Abdul Kader, Iriarte Fanny B, Paret Mathews L
University of Florida North Florida Research and Education Center, 316813, Plant Pathology Department, 155, Research Road, Quincy, Florida, United States, 32351;
University of Florida, 3463, North Florida Research and Education Center, Plant Pathology Department, Quincy, Florida, United States;
Plant Dis. 2023 Apr 27. doi: 10.1094/PDIS-01-23-0079-PDN.
Straightneck squash (Cucurbita pepo var. recticollis) is an important cucurbit crop in Florida. In early fall 2022, straightneck squash showing severe virus-like symptoms of yellowing, mild leaf crinkling (Supplementary Figure 1), unusual mosaic patterns and deformation on the surface of the fruit (Supplementary Figure 2), were observed in a ~15-ha straightneck squash field in Northwest FL with a disease incidence of ~ 30%. Based on the distinct symptoms and severity observed, multi-virus infection was hypothesized. Seventeen plants were sampled randomly for testing. Plants tested negative for zucchini yellow mosaic virus, cucumber mosaic virus, and squash mosaic virus, using ImmunoStrips® (Agdia, USA). Total RNA was extracted from 17 squash plants using Quick-RNA Mini Prep (Cat No.11-327, Zymo, USA). A conventional OneTaq® RT-PCR Kit (Cat No. E5310S, NEB, USA) was used to test plants for cucurbit chlorotic yellows virus (CCYV) (Jailani et al., 2021a) and watermelon crinkle leaf-associated virus (WCLaV-1) and WCLaV-2 (Hernandez et al., 2021). Plants were negative for CCYV and 12 out 17 plants were positive for WCLaV-1 and WCLaV-2 (genus Coguvirus, family Phenuiviridae) using specific primers targeting both RNA-dependent RNA polymerase (RdRP) and movement protein (MP) genes of both viruses (Hernandez et al., 2021). In addition, these 12 straightneck squash plants were also positive for watermelon mosaic potyvirus (WMV) based on RT-PCR and sequencing (Jailani et al., 2021b). The partial RdRP sequences for WCLaV-1 (OP389252) and WCLaV-2 (OP389254) shared 99% and 97.6% nt identity with isolates KY781184 and KY781187, respectively from China; the partial MP sequences for WCLaV-1 (OP389253) and WCLaV-2 (OP389255) shared 98.3% and 95.6% nt identity with isolate from Brazil (LC636069) and from China (MW751425), respectively. Additionally, the presence or absence of WCLaV-1 and WCLaV-2 were further confirmed using SYBR® Green-based real-time RT-PCR assay using different specific MP primers for WCLaV-1 (Adeleke et al., 2022), and newly designed specific MP primers for WCLaV-2 (WCLaV-2FP TTTGAACCAACTAAGGCAACATA/WCLaV-2RP-CCAACATCAGACCAGGGATTTA). Both viruses were detected in 12 out of 17 straightneck squash plants validating the conventional RT-PCR results. Co-infection of WCLaV-1 and WCLaV-2 with WMV resulted in more severe symptoms on leaves and fruits. Previously, both viruses were first reported in the USA on watermelon in Texas, (Hernandez et al., 2021), Florida (Hendricks et al., 2021), OK (Gilford and Ali., 2022), GA (Adeleke et al., 2022) and Zucchini in Florida (Iriarte et al., 2023). This is the first report of WCLaV-1 and WCLaV-2 on straightneck squash in the United States. These results indicate that WCLaV-1 and WCLaV-2 either in single or mixed infections are effectively spreading to other cucurbits beyond watermelon in FL. The need to assess mode(s) of transmission of these viruses is becoming more critical to develop best management practices.
直颈南瓜（西葫芦变种）是佛罗里达州一种重要的葫芦科作物。2022年秋初，在佛罗里达州西北部一块约15公顷的直颈南瓜地里，观察到直颈南瓜出现严重的类病毒症状，如叶片发黄、叶片轻度皱缩（补充图1）、异常的花叶图案以及果实表面变形（补充图2），发病率约为30%。基于观察到的明显症状和严重程度，推测存在多种病毒感染。随机采集了17株植株进行检测。使用免疫试纸（美国Agdia公司）检测，植株对小西葫芦黄花叶病毒、黄瓜花叶病毒和南瓜花叶病毒呈阴性。使用Quick-RNA Mini Prep试剂盒（美国Zymo公司，产品编号11 - 327）从17株南瓜植株中提取总RNA。使用常规的OneTaq® RT-PCR试剂盒（美国NEB公司，产品编号E5310S）检测植株是否感染葫芦褪绿黄化病毒（CCYV）（Jailani等人，2021年a）以及西瓜皱叶相关病毒（WCLaV-1和WCLaV-2）（Hernandez等人，2021年）。使用针对两种病毒的RNA依赖性RNA聚合酶（RdRP）和运动蛋白（MP）基因的特异性引物检测，植株对CCYV呈阴性，17株中有12株对WCLaV-1和WCLaV-2呈阳性（Coguvirus属，菲纽病毒科）（Hernandez等人，2021年）。此外，基于RT-PCR和测序结果，这12株直颈南瓜植株对西瓜花叶马铃薯Y病毒（WMV）也呈阳性（Jailani等人，2021年b）。WCLaV-1（OP389252）和WCLaV-2（OP389254）的部分RdRP序列分别与来自中国的分离株KY781184和KY781187具有99%和97.6%的核苷酸同一性；WCLaV-1（OP389253）和WCLaV-2（OP389255）的部分MP序列分别与来自巴西（LC636069）和中国（MW751425）的分离株具有98.3%和95.6%的核苷酸同一性。此外，使用基于SYBR® Green的实时RT-PCR检测方法，分别使用针对WCLaV-1的不同特异性MP引物（Adeleke等人，2022年）和新设计的针对WCLaV-2的特异性MP引物（WCLaV-2FP TTTGAACCAACTAAGGCAACATA/WCLaV-2RP-CCAACATCAGACCAGGGATTTA），进一步确认了WCLaV-1和WCLaV-2的存在与否。在17株直颈南瓜植株中有12株检测到这两种病毒，验证了常规RT-PCR结果。WCLaV-1和WCLaV-2与WMV的共同感染导致叶片和果实出现更严重的症状。此前，这两种病毒首次在美国得克萨斯州的西瓜上被报道（Hernandez等人，2021年），在佛罗里达州（Hendricks等人，2021年）、俄克拉何马州（Gilford和Ali，2022年）、佐治亚州（Adeleke等人，2022年）以及佛罗里达州的西葫芦上也有报道（Iriarte等人，2023年）。这是美国首次关于WCLaV-1和WCLaV-2在直颈南瓜上的报道。这些结果表明，WCLaV-1和WCLaV-2无论是单一感染还是混合感染，都在佛罗里达州有效地传播到了西瓜以外的其他葫芦科作物上。评估这些病毒的传播方式对于制定最佳管理措施变得愈发关键。
