3. Research and Findings
The whitefly
B. tabaci transmits more than 100 species of viruses to plants. The mode of the transmission is related to the taxonomical status of the viruses and is characterized as semipersistent (criniviruses, ipomoviruses, and torradoviruses) and persistent (begomoviruses) [
17]. These viruses generally differ in the values of the transmission parameters, such as the probability of viruliferous insects to infect a plant, the time it takes a vector to ingest virus and to inoculated the virus into plants by feeding, and in the time the whiteflies remain infective following acquisition of the virus. In the present paper, we have compared the parameters of transmission of the bipartite begomovirus ToLCNDV-ES by the vector,
B. tabaci Med-Q1, in two different crop species, tomato and zucchini.
A single insect is able to acquire monopartite begomovirus TYLCV and transmit it to tomato plants. The reported minimum AAP and IAP of TYLCV by
B. tabaci biotype B (MEAM1) varies from 15 to 60 min and from 15 to 30 min, respectively [
22,
23,
24]. Similar values were reported for other monopartite geminiviruses infecting tomato such as TYLCV Sardinia virus (TYLCSV) from Italy [
25]. The mean infectivity of infected
B. tabaci adults was retained during 7 to 14 days. In general, begomoviruses are retained in the vector for almost its entire life. Adults of MEAM1
B. tabaci that acquired tomato severe rugose virus (ToSRV) during an AAP of 24 h on infected tomato remained viruliferous for 25 days, the maximum period that the insects survived when kept on cabbage plants that are immune to the virus [
26]. Tomato leaf curl Sinaloa virus (TOLSCI) was detected in adults of
B. tabaci for up to nine days [
27]. However, the squash leaf curl virus (SLCV) was retained in the insect for 26 days [
28].
Thus, the maximum retention of 14 days has been measured after a six-hour acquisition period [
29]. The retention values of 20 days have also been reported for Chino del tomato virus (CdTV) and tomato yellow vein streak virus (ToYVSV) [
30,
31], and life-long retention in the vector was reported for tomato yellow leaf curl Thailand virus (TYLCTHV) [
32]. Comparisons of the transmission of ToLCNDV-ES and other begomoviruses should be done with caution. Despite differences in vector species, plant host species, and in the experimental circumstances, the values for IAP, AAP, and the persistence of ToLCNDV-ES were similar to those of monopartite and other bipartite begomoviruses. Transmission efficiencies, however, were different, and in other begomovirus pathosystems, this type of difference has been shown to be associated with feeding habits and preferences on the plant hosts that were used for acquisition and transmission [
33,
34,
35,
36]. The efficiency is further complicated by differences in the amount and distribution of begomoviruses in the different plant hosts being studied [
37]. Also, the presence of selected endosymbionts (ex.
Hamiltonella spp.) has been shown to affect the transmission efficiency of begomoviruses [
38,
39]. Finally, ToLCNDV-ES in Mediterranean countries occurs in cucurbitaceous crops that are potentially co-infected with
B. tabaci-transmitted cucurbit yellow stunting disorder crinivirus (CYSDV) and cucumber vein yellowing ipomovirus (CVYV) [
40]. These semipersistant transmitted viruses have no latent period after ingestion and the retention in the vector lasts from hours to days, depending on the species [
41]. There is no evidence of interference between CYSDV and CVYV in the transmission by
B. tabaci [
42,
43], but the effect of coinfecting plant viruses on vector-transmission has been suggested in other combinations of mixed infections, i.e., of CYSDV and aphid-transmitted watermelon mosaic virus (WMV) [
44]. Therefore, the effect of criniviruses and ipomoviruses on the transmission of ToLCNDV-ES requires further investigation.
The efficiency of ToLCNDV-ES transmission by single insects was low in tomato and very high in zucchini. These differences in the inoculation efficiency were used to explain the predominance of different begomoviruses that are acquired at similar rates by the same vector species in the same crop species, such as tomato severe rugose virus (ToSRV) versus tomato golden vein virus (ToSRV) in Brazilian tomato fields [
19,
45]. But transmission efficiencies may also reflect host plant resistance and the ability of a virus to replicate inside the host plant [
46]. The higher the host plant's resistance, the lower the transmission rate, and the lower is the amount of virus that is detected in the host plant. This has been shown for TYLCV where viral DNA accumulation was shown to be lower in the resistant source plants compared with the susceptible plants [
47].
ToLCNDV-ES has been found as natural and experimental infections in cucurbit and solanaceous species [
4,
8]. However, relative incidences of the virus are very different among crops in the same agronomic region of the southeast of Spain. Among commercial crops, the percentages of plants that are infected with ToLCNDV varied from 95% in zucchini, 80% in melon, 50% in cucumber, 0% in watermelon, and 15% in tomato [
8]. Viral loads in tomato that were experimentally infected with ToLCNDV-ES were also reduced when compared with zucchini (
Figure 1). This conforms to previously published comparisons of the viral loads of ToLCNDV-ES in zucchini and in tomato [
10]. As such, both the natural incidences of ToLCNDV-ES in zucchini and tomato, the viral loads in this paper, and those that were published before suggest that these are in line with the significantly different efficiencies of transmission in both crop species. Since the transmission efficiency in zucchini is almost 100% for single insects, and because the disease can be fatal in this crop species, control of ToLCNDV in zucchini is a big challenge and requires efficient physical vector exclusion using greenhouses and careful planning and application of biological and integrated pest management [
48,
49]. Moreover, the success of these control strategies could change whenever evidence of differences in the ToLCNDV-ES transmission by co-infecting criniviruses or ipomoviruses is obtained, following the future research as suggested above.
Natural incidences of ToLCNDV-ES are lower in commercial tomato crops [
8], which is consistent with the reduced transmission efficiency in this host plant. However, crop protection against
B. tabaci and ToLCNDV should also be applied with care in tomato because these plants may often be co-infected with other begomoviruses, such as monopartite TYLCV species [
10], which may compromise the interpretation of the observed symptoms.
The present paper established the retention of ToLCNDV in adult vectors to be around between 7 and 14 days, but here we also determined a different aspect of virus retention: we showed that 20% of adults that emerged from pupae on drying zucchini plant leaves were actually infective. This ratio is comparable to TYLCV in tomato where 28% of emerging adults were found to be infective [
30]. We also established that the amounts of the virus in emerging adults (ranging between approx. 10
2 and 10
7), were generally lower than the amounts in the plants (between approx. 10
9 and 10
11). Since the 20% higher virus levels in adults from the dried zucchini leaves were approx. 10
5 or more, this value may well represent the minimum viral load in vectors that is necessary to achieve a successful infection. In comparison, all single adults that were collected from infected zucchini plants had values above 7 × 10
4, and 96% of single adults that successfully infected zucchini. In contrast, none of the adults emerging from pupae on the infected dried tomato leaves were able to infect zucchini plants, and 95% of these
B. tabaci had viral loads below 10
5.
The efficiency of transmission and the viral loads in adults that were emerging from pupae may explain why biological control using predators of
B. tabaci eggs and immature stages can significantly reduce the short-distance spread of the virus in greenhouses [
48,
49]. However, it may be also significant in the long-distance control of ToLCNDV-ES, i.e., in the trade and transport of infected plant material, when no
B. tabaci adults are spotted, these commodities may contain small-sized immature stages that could well be a source of infection [
50]. On the other hand, the infectivity of emerging adults from infected dried plant materials provides evidence that plants that are removed from the crop either during the growing and harvesting stage, or after the crops have finished, should be carefully covered, in sealed boxes, containers, etc., and carefully treated or destroyed, but not exposed or transported as such as that would permit the spread of emerging viruliferous vectors and, consequently, the spread of the virus. So, ToLCNDV-ES management of crops not only should involve hygiene, vector exclusion, and control during the nursery, production, and harvesting stages but also once the production and harvesting have finished and the plants are removed from the field or the greenhouse because even as dried materials, they can contain immature vector stages and produce viruliferous emerging insects.