Nuances of Whitefly Vector–Crinivirus Interactions Revealed in the Foregut Retention and Transmission of Lettuce Chlorosis Virus by Two Bemisia tabaci Cryptic Species

Lettuce infectious yellows virus is the first crinivirus for which the retention of purified virions ingested into the whitefly (Bemisia tabaci New World (NW)) vector’s foregut, has been demonstrated to be a requisite for successful virus transmission. This key finding supports the hypothesis that the determinant of foregut retention and transmission is present on the virion itself. However, whether this is also true for other criniviruses has not been established. Here, we provide evidence that lettuce chlorosis virus (LCV) acquired from plants is retained in the foreguts of both the B. tabaci NW and Middle East–Asia Minor 1 (MEAM1) vector species and transmitted upon inoculation feeding. An association between foregut retention and transmission by NW vectors is also observed following the acquisition and inoculation feeding of LCV virions purified using a standard procedure involving 2% or 4% (v/v) Triton™ X-100 (TX-100). However, while virions purified with 2% or 4% TX-100 are also retained in the foreguts of MEAM1 vectors, transmission is observed with the 4% TX-100-purified virions or when more vectors are used for acquisition and inoculation feeding. These results suggest that an intrinsic difference exists between NW and MEAM1 vectors in their interactions with, and transmission of, LCV virions.

Bemisia tabaci Vesicle-Associated Membrane Protein 2 Interacts with Begomoviruses and Plays a Role in Virus Acquisition

Begomoviruses cause substantial losses to agricultural production, especially in tropical and subtropical regions, and are exclusively transmitted by members of the whitefly Bemisia tabaci species complex. However, the molecular mechanisms underlying the transmission of begomoviruses by their whitefly vector are not clear. In this study, we found that B. tabaci vesicle-associated membrane protein 2 (BtVAMP2) interacts with the coat protein (CP) of tomato yellow leaf curl virus (TYLCV), an emergent begomovirus that seriously impacts tomato production globally. After infection with TYLCV, the transcription of BtVAMP2 was increased. When the BtVAMP2 protein was blocked by feeding with a specific BtVAMP2 antibody, the quantity of TYLCV in B. tabaci whole body was significantly reduced. BtVAMP2 was found to be conserved among the B. tabaci species complex and also interacts with the CP of Sri Lankan cassava mosaic virus (SLCMV). When feeding with BtVAMP2 antibody, the acquisition quantity of SLCMV in whitefly whole body was also decreased significantly. Overall, our results demonstrate that BtVAMP2 interacts with the CP of begomoviruses and promotes their acquisition by whitefly.

First report of cucurbit yellow stunting disorder virus and cucurbit chlorotic yellows virus in cucurbit crops in Alabama

During the summer and fall of 2020, foliar yellowing symptoms, including leaf mottle and interveinal yellowing with green veins were observed on several melon, squash, and cucumber plants in commercial fields in Alabama, USA. These foliar symptoms were similar to those caused by the whitefly-transmitted yellowing viruses, cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) (both genus Crinivirus, Closteroviridae). A total of 231 leaf samples showing yellowing, interveinal chlorosis, and mottling (e-Xtra 1, 2) were collected from individual plants from 25 commercial fields in Alabama (70 watermelon, 52 melon, 34 pumpkin, 50 squash, and 25 cucumber) during two sampling periods, June (spring/summer season) and October (fall season) 2020. Total RNA, extracted as described in Tamang et al. (2021), was used in reverse transcription polymerase chain reaction (RT-PCR) with primer sets designed to amplify portions of the CCYV and CYSDV RNA-dependent RNA polymerase (RdRp) genes encoded on RNA1 of these viruses (Mondal et al. 2021, submitted; Kavalappara et al., 2021). Single infections of either CYSDV or CCYV were found in 53 of 57 infected cucurbit samples (of 231 total plants), whereas both viruses were detected in four samples, all squash. In June 2020 near the end of the spring season, CYSDV was identified from 20 of 114 total cucurbit plants tested (17.5%), but CCYV was not identified from any plants. During the fall season, 37 of 117 plants (32%) tested positive for the presence of one or both criniviruses. Of the 37 virus-positive samples from the fall season, 26 were singly infected with CCYV (70%), seven were singly infected with CYSDV (19%), and four were infected with both CYSDV and CCYV (11%). The RdRp amplicon was sequenced from three CCYV-infected plants (2 squash; GenBank Accession No. MZ073347, MZ073348; 1 cucumber, MZ073349) and one CYSDV-infected plant (melon, MZ073350); the 857 nt sequenced portion of the CCYV RdRp gene was found to share 99% identity with the same sequence of CCYV RNA1 isolates from Israel (MH477611.1) and California (MW680157), whereas the 494 nt CYSDV amplicon shared 100% sequence identity with the comparable sequence from RNA1 of a CYSDV isolate from Arizona (EF547827.1). In addition, all of the CYSDV and CCYV infections were confirmed using a second set of primers that amplified 394 and 372 nt sections of the coat protein gene of each virus, respectively (Wintermantel et al., 2009; 2019), encoded on RNA2 of each viral genome. Furthermore, a recently developed multiplex RT-qPCR method (Mondal et al. 2021, submitted) was used to confirm four representative CYSDV and CCYV infections each. This is the first report of CYSDV and CCYV in cucurbit crops from Alabama. Surprisingly, CYSDV was only found in melon plants (20 of 52, 38%), whereas CCYV was only found in squash, pumpkin, and cucumber (26 of 109, 24%); no watermelon plants were infected with either virus, even though watermelon is a known host of both viruses. The identification of CCYV and CYSDV in Alabama, along with a recent report of both criniviruses from nearby Georgia (Kavalappara et al., 2021) illustrates the need for a more thorough sampling of cucurbit crops, further monitoring of the whitefly vector, Bemisia tabaci, and the identification of alternate hosts of these viruses to better understand the epidemiology of these viruses in Alabama and throughout the Gulf Coast region.

Tomato leaf curl New Delhi virus (tomato New Delhi virus).

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite, single-stranded DNA virus transmitted by the whitefly, Bemisia tabaci. The virus was first identified in India in 1995 affecting solanaceous crops (Padidam et al., 1995) and thereafter, causing major damage to cucurbit crops on the Indian subcontinent (Zaidi et al., 2017). ToLCNDV was first detected in Europe in 2012, affecting zucchini squash crops in Spain (Juárez et al., 2014), with subsequent detections in Tunisia (Mnari-Hattab et al., 2015), Italy (Panno et al., 2016) and Morocco (Sifres et al., 2018). ToLCNDV is responsible for severe outbreaks of disease in cucurbit crops in the Mediterranean basin (Juárez et al., 2019; Panno et al., 2019) and represents a serious threat to economically imp1ortant solanaceous crops in the region (Moriones et al., 2017). ToLCNDV appears to be spreading rapidly and is listed as a quarantine pest by EPPO (EPPO, 2019a). There are quarantine measures to control its whitefly vector (Bertin et al., 2018).

Tomato leaf curl New Delhi virus (Tomato New Delhi virus).

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite, single-stranded DNA virus transmitted by the whitefly, Bemisia tabaci. The virus was first identified in India in 1995 affecting solanaceous crops (Padidam et al., 1995) and thereafter, causing major damage to cucurbit crops on the Indian subcontinent (Zaidi et al., 2017). ToLCNDV was first detected in Europe in 2012, affecting zucchini squash crops in Spain (Juárez et al., 2014), with subsequent detections in Tunisia (Mnari-Hattab et al., 2015), Italy (Panno et al., 2016) and Morocco (Sifres et al., 2018). ToLCNDV is responsible for severe outbreaks of disease in cucurbit crops in the Mediterranean basin (Juárez et al., 2019; Panno et al., 2019) and represents a serious threat to economically important solanaceous crops in the region (Moriones et al., 2017). ToLCNDV appears to be spreading rapidly and is listed as a quarantine pest by EPPO (EPPO, 2019). There are quarantine measures to control its whitefly vector (Bertin et al., 2018).

East African cassava mosaic virus.

Like other CMGs, cassava is the primary host of East African cassava mosaic virus (EACMV) and related viruses, although the virus has been detected in other plant species (Ogbe et al., 2006; Alabi et al. 2015). Analysis of the genomes of different isolates of EACMV-type viruses show considerable genetic variability and genome plasticity relative to ACMV isolates. The primary means of virus spread is via movement of contaminated vegetative cassava cuttings and secondary spread occurs via the whitefly vector, Bemisia tabaci. Perhaps the most notable documentation of invasiveness of EACMV-type viruses is the regional pandemic of a severe CMD in East Africa caused by EACMV-UG which began in Uganda in the early to mid-1990s (Gibson et al., 1996; Otim-Nape et al., 1997) on popular and widely cultivated cassava varieties and soon spread to other countries in East Africa, including Kenya and Tanzania (Otim-Nape et al., 1997; Legg, 1999). The pandemic resulted in famine-related deaths (Otim-Nape et al., 1998) due to complete devastation of affected cassava farms in the region. EACMV is not on the IUCN or ISSG alert list.

Cotton leaf curl Gezira virus (African cotton leaf curl Begomovirus).

Cotton leaf curl Gezira virus (CLCuGV) is endemic to the African Sahel region (Idris et al., 2000). It is an economically important cotton-infecting begomovirus, and poses a serious threat to cotton production. It causes yield loss in all affected cotton-growing areas in Africa. Losses are difficult to assess, but estimates range up to 20% when infection occurs early in the growing season and/or with highly susceptible cultivars. Natural spread is mainly by the whitefly vector, Bemisia tabaci, which transmits the virus in a persistent, circulative manner. Viruliferous whiteflies on infested/infected plants harbouring CLCuGV imported to other countries are of concern for preventing introduction.

African cassava mosaic virus (African cassava mosaic).

Cassava is vegetatively propagated therefore ACMV and other CMGs are primarily transmitted via movement of contaminated cuttings. Consequently, introductions of specific CMGs into new localities mirror patterns of cassava cuttings exchange among farmers. Once infected cuttings are planted, the virus establishes easily and can be transmitted within and between fields through the feeding behaviour of the whitefly vector, Bemisia tabaci. ACMV is particularly invasive in that it is the most widespread of all known CMGs, occurring across all cassava-producing countries of Africa in cassava and several alternative host plants (Thottappilly et al., 2003; Alabi et al. 2015). ACMV has also been reported infecting non-cultivated exotic cotton species in Pakistan (Nawaz-Ul-Rehman et al., 2012) further underscoring its invasive nature. Yield loss due to CMD can range from 12 to 82%, depending on the cassava variety and infection type (Owor et al., 2004). ACMV is not on the IUCN or ISSG alert list.

Tomato yellow leaf curl virus (leaf curl).

The wide global distribution of tomato crops and the dramatic outbreaks of the populations of the TYLCV vector, the whitefly Bemisia tabaci, led to a pandemic of this devastating disease. The virus probably arose in the Middle East between the 1930s and 1950s. Its global invasion began in the 1980s after the emergence of two strains: TYLCV-IL and TYLCV-Mld. The long-distance transportation of viruliferous whiteflies contaminating commercial shipments of tomato seedlings and ornamentals is probably the major reason for the virus pandemic (Caciagli, 2007). Sequence analyses allowed Lefeuvre et al. (2010) to trace the history of TYLCV spread. For instance, TYLCV-IL has invaded the Americas at least twice, once from the Mediterranean basin in 1992-1994 and once from Asia (a descendant of imported Middle Eastern TYLCV) in 1999-2003. As a result the estimated losses caused by TYLCV reached about 20% of tomato production in the USA, and 30-100% in the Caribbean Islands, Mexico, Central America and Venezuela. Therefore several countries (Australia, EU) have established severe quarantine measures to control the whitefly vector.