High Throughput Sequencing-Aided Survey Reveals Widespread Mixed Infections of Whitefly-Transmitted Viruses in Cucurbits in Georgia, USA

Abstract

Viruses transmitted by the sweet potato whitefly (Bemisia tabaci) have been detrimental to the sustainable production of cucurbits in the southeastern USA. Surveys were conducted in the fall of 2019 and 2020 in Georgia, a major cucurbit-producing state of the USA, to identify the viruses infecting cucurbits and their distribution. Symptomatic samples were collected and small RNA libraries were prepared and sequenced from three cantaloupes, four cucumbers, and two yellow squash samples. An analysis of the sequences revealed the presence of the criniviruses cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and the begomovirus cucurbit leaf crumple virus (CuLCrV). CuLCrV was detected in 76%, CCYV in 60%, and CYSDV in 43% of the total samples (n = 820) tested. The level of mixed infections was high in all the cucurbits, with most plants tested being infected with at least two of these viruses. Near-complete genome sequences of two criniviruses, CCYV and CYSDV, were assembled from the small RNA sequences. An analysis of the coding regions showed low genetic variability among isolates from different hosts. In phylogenetic analysis, the CCYV isolates from Georgia clustered with Asian isolates, while CYSDV isolates clustered with European and USA isolates. This work enhances our understanding of the distribution of viruses on cucurbits in South Georgia and will be useful to develop strategies for managing the complex of whitefly-transmitted viruses in the region.

Field Evaluation of Cucurbita Germplasm for Resistance to Whiteflies and Whitefly-transmitted Viruses

“…This could have been because of an increase in inoculum in the whitefly population over time. Kavalappara et al (2021a) detected an increase in the presence of CYSDV in cucurbit crops in Georgia in 2020 compared with 2019. Another explanation is that the detection method used in 2020 was more sensitive than that used in 2019.…”

Wild Radish (Raphanus raphanistrum L.) Is a Potential Reservoir Host of Cucurbit Chlorotic Yellows Virus

“…Finally, symptomatic wild radish samples collected from the field were tested for the presence of CCYV using RT-PCR ( Table 1 ). The recipe and cycling conditions for cDNA preparation and PCR to detect CCYV are detailed in Kavalappara et al [ 22 ].…”

“…Adult whiteflies raised on cotton were provided 24 h of acquisition access period (AAP) on CCYV-infected squash. Viral infection on source plants (squash) was confirmed by RT-PCR before using them in transmissions as previously described [ 22 ]. After AAP, the whiteflies were provided an inoculation access period (IAP) of 24 h on wild radish plants.…”

“…RNA was isolated from leaves of infected samples using Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St Louis, MO, USA) following the manufacturer’s instructions. cDNAs were prepared from 100 ng total RNA following the protocol described in Kavalappara et al [ 22 ]. Five microliters of cDNA was used as template in the qPCR reaction of 25 µL, which also consisted of 12.5 µL of SSOAdvanced Universal SYBR Green Supermix and 1 µL each (10 µM) of forward and reverse primers.…”

“…In the southeastern USA, fall cucurbits are severely affected by CCYV and other WTVs [ 14 , 21 , 22 ]. These viruses are not reported to cause damage to winter vegetables, predominantly the members of Brassicaceae and spring crops that include watermelon.…”