The role of ornamental plants as hosts of Tomato chlorotic spot virus and its vector thrips affecting tomato production

The tomato industry in South Florida (USA) is seriously affected by recently emerging Tomato chlorotic spot virus (TCSV). Western flower thrips, Frankliniella occidentalis (Pergande), and common blossom thrips, Frankliniella schultzei (Trybom) (Thysanoptera: Thripidae), are the two known vectors of TCSV in Florida. In the present study, the presence of thrips vectors and TCSV in 24 flowering ornamental plant species was observed in South Florida. The two thrips vectors, as well as melon thrips, Thrips palmi Karny (Thysanoptera: Thripidae), were abundant in the area; they were observed with a range from high (83.1 per sample of 10 flowers) in Hibiscus rosa‐sinensis L. to low (3.7 per sample of 10 flowers) in Catharanthus roseus (L.) G. Don in a nursery study. In a subsequent greenhouse study, we selected seven species of ornamentals, among which the species with the highest thrips abundance, and planted them next to tomato, to determine their effects on TCSV incidence and thrips abundance in tomatoes. Tomatoes with Portulaca oleracea L. next to them showed a higher number of TCSV‐infected plants (4.25 plants per plot in 2017, and 3.25 plants per plot in 2018) compared to tomatoes with some of the other ornamentals next to them. We report the presence of TCSV through reverse transcriptase polymerase chain reaction (RT‐PCR) analysis in Lantana camara L., H. rosa‐sinensis, Mandevilla spec., Gazania linearis (Thunb.) Druce, Hemerocallis spec., Agastache spec., and P. oleracea. Identification of alternative hosts of TCSV and thrips vectors can be helpful to evaluate the ongoing management programs and develop future programs in local tomato‐growing areas.

Wild plants, as well as cultivated crops, can be potential hosts of both viruses and virus vectors. Thus, alternative hosts are important to be considered in the epidemiology of plant viruses (Duffus, 1971). Wild host plants are important virus reservoirs from where pathogens can spread to susceptible cultivated crops (Jones, 2014). Because TCSV is an emerging tospovirus established in southern Florida, many new host plants, including ornamental crops, have been recently identified. In the present study, we observed the abundance of thrips in 24 ornamental plants in southern Florida in a nursery study. In a separate greenhouse study, we evaluated the role of selected ornamental plants influencing TCSV incidence and its thrips vectors in the main crop, tomato. We hypothesized that some of these ornamental plants would serve as a host of TCSVvector thrips and thus the tomatoes with those ornamental plants next to them would have more infected plants. The objective of this study was to obtain a thorough view of occurrence, importance, and epidemiology of TCSV in ornamental crops through the identification of its hosts, identification of the thrips vectors involved, and exploring the role of ornamental plants in TCSV virus epidemiology in the tomato agro-ecosystem of South Florida. We also investigated the presence of TCSV in some of these ornamental plants.

Thrips in ornamental plants in the nursery
This study was conducted in a commercial nursery in Homestead,Florida,USA (25°34'49.6"N,80°30'18.8"W,altitude 7 m). Ornamental plant samples were collected between February and April 2017 and March and May 2018. The whole nursery was about 405 000 m 2 (40.5 ha), which consisted of approximately 500 greenhouses, each 30.5 × 12.2 m. Each greenhouse contained one or multiple species of ornamental plants maintained in plastic pots of different sizes (1-4 L). Seeds were planted or seedlings were transplanted into potting soil in pots (Miracle-Gro Potting Soil Mix; Miracle-Gro, Marysville, OH, USA; see Supporting Information for maintenance of the plants). To determine the thrips abundance in ornamental plants, we selected 24 species of commonly grown flowering ornamental plants at their flowering stage for sampling thrips. A profile of these plant species showing their family, habitat, and distribution is presented in Table S1. Samples were collected from four nursery houses (3.1 m long, 91.5 cm wide) placed side by side (1.5 m apart) for each ornamental plant species. There were thus four replications (plots) for each ornamental species.

Collecting and processing of samples in the nursery study
All ornamental plant samples were collected at their full bloom flowering stage, between 10:00 and 12:00 hours EST, as this is a peak activity time for thrips (RA Khan, unpubl.). Ten flowers (one flower per plant) were randomly collected from each selected plot of an ornamental species. Each sample of 10 flowers from each ornamental species was placed in a 0.47-L plastic cup (Deli containers; Uline, Pleasant Prairie, WI, USA) marked with ornamental plant species and plot number. Thus, we had four plastic cups (one cup per plot for a sample of 10 flowers) for each species. Immediately after collection, the cups were closed with well-fitted lids to prevent the thrips escaping and were brought back to the Integrated Pest Management (IPM) Laboratory at the Tropical Research and Education Center (TREC), UF-IFAS, Homestead, FL, USA, for further processing. Flower samples were processed using 70% ethanol following the protocol of Seal & Baranowski (1992). Finally, the thrips species in ethanol were counted under a Leica Wild M3Z stereo microscope (Micro Optics of Florida, St. Petersburg, FL, USA) at 10-30× magnification. Thrips were slide mounted and identified under a VHX-6000 digital microscope (Keyence, Itasca, IL, USA) at 50-200× magnification. The thrips were identified to species by observing taxonomic characters including antennal segments, the position of post ocellar setae in the ocellar triangle, and the microtrichial comb on the eighth abdominal tergite (Nakahara, 1997). The presence of virus was not assessed in these plants.

Greenhouse study to assess Tomato chlorotic spot virus and its thrips vectors
The study was performed in a greenhouse at the TREC, UF/ IFAS to assess the effect of selected ornamental plants on TCSV prevalence and its thrips vectors on tomatoes. The temperature of the greenhouse ranged from 28 to 34 °C with an average of 31 °C, and the r.h. ranged from 51 to 75% with an average of 69%. This study was conducted in April-June 2017 and repeated in May-July 2018. Data were collected from the study over the 2 years.

Treatments and experimental design of greenhouse study
The greenhouse experiment was conducted using tomato (S. lycopersicum cv. Sanibel) as the main crop. Planting, irrigation, and crop management followed standard practices (Freeman et al., 2016). Tomato transplants (Mobley Plant World, Labelle, FL, USA) were planted in 3.8-L plastic pots (Black Thermoformed Nursery Pot, black/matte; Grower's solution, Cookeville, TN, USA) containing garden soil (PRO-MIX; Premier tech home and garden, Quakertown, PA, USA). The selected ornamental plants were also grown in 3.8-L plastic pots like the tomato crop. Plants in the greenhouse were irrigated twice a day (at 08:00 and 16:00 hours EST), delivering 0.6 cm of water each time using a sprinkler system set at 120 cm above the plants. Overgrowing parts of ornamental plants were clipped off once every 3 weeks to ensure normal growth and development of the main crop (tomato).
Seven ornamental species including P. oleracea, Hibiscus rosa-sinensis L. (Malvaceae), Lantana camara L. (Verbenaceae), Mandevilla spec. (Apocynaceae), Gazania linearis (Thunb.) Druce (Asteraceae), Hemerocallis spec. (Asphodelaceae), and Agastache spec. (Lamiaceae), were selected to plant in association with tomatoes in the same research plot and were referred to as treatments. The ornamental plants were selected based on the abundance of thrips in them as found in our earlier study (nursery study). There was an untreated plot (control) of tomatoes with no ornamental plants. All plants were placed on a bench, considered a research plot (2.44 × 1.83 m, 0.91 m from the ground). There were 3.05-m-long non-planted spaces between the treatments. Thus, there were eight such benches for seven treatments and an untreated plot, which constituted a block ( Figure 1). The treatments were arranged in a randomized complete block design replicated 4× using 32 benches in four blocks in the same greenhouse. We placed the tomato plants as a center row on each bench with three potted ornamental plants, 0.6 m apart from each other in a parallel row spaced 0.30 m from the center on each side. In total, we had five potted tomato plants in each plot and six ornamental plants of one species.

Collecting and processing of samples in the greenhouse study
Five fully expanded young leaves from fifth/sixth nodes of a tomato plant and five fully expanded yellow flowers, one of each plant part from each tomato plant, were randomly collected from each research plot and placed separately in the collection cups (0.47 L). Five flowers and leaves of ornamental plants from each plot were also randomly collected following the same method and placed separately in the collection cups. The samples were collected every week starting from 4 weeks after planting up to 10 weeks. The collection and processing of all these samples followed the procedure outlined in the previous section 'Collecting and processing of samples in the nursery study'.

Tomato chlorotic spot virus infected tomato plants and marketable fruit yield
Tomato chlorotic spot virus infected tomato plants were confirmed by visual symptoms including necrosis on leaves, chlorotic and necrotic ring spots followed by dwarfing and wilting of a portion or the entire plant, and necrotic ring spots on fruits (Polston et al., 2013). The number of TCSV-symptomatic plants was counted in F I G U R E 1 Experimental design of greenhouse study with tomatoes with ornamental plants next to them. Tomatoes (main crop) were placed in the middle row of the plot and ornamental plants were placed on each side of those tomatoes 0.3 m apart. B = unplanted buffer. This figure shows one block; the placement of treatments (ornamental plants) differed in the four blocks. each plot at the end of the study. All marketable fruits were harvested from all tomato plants of each plot 12 weeks after transplanting. Marketable fruits (green stage) were weighed, using a scale of 31.8 kg capacity (CCI Scale, Ventura, CA, USA) and graded, representing USA no. 1, 2, and 3 comprising small (5.08 cm diameter), medium (5.72 cm diameter), and large (6.35 cm diameter) sizes following market standards (USDA, 2022).

Collection and processing of samples for detection of Tomato chlorotic spot virus
Five leaves and five flowers (one of each per plant) from each ornamental species planted next to tomatoes across all replicates in 2017 and 2018 were collected 8 weeks after transplanting the tomatoes and were placed in a Ziploc bag (SC Johnson & Son, Racine, WI, USA) and stored in a freezer (VIP series −86 °C; Sanyo North America, San Diego, CA, USA) at −80 °C in 2018. Five tomato leaves were also collected across all replicates in both years. The samples were tested for TCSV using reverse transcriptase polymerase chain reaction (RT-PCR) and sent for DNA sequencing, following the protocol mentioned by Poudel et al. (2018). Results are showing the positive results for the ornamental plant samples.

Statistical analysis
The mean number of thrips from each treatment was compared separately for each year. The number of thrips per ornamental species (for nursery studies) and the number of thrips per treatment (for greenhouse studies) were averaged to a single measure per block and treatment combination. Multiple sampling dates were averaged to remove the effects of repeated measures and the large number of zero counts. The resulting average was then subjected to square root transformation before statistical analysis to meet the assumption of normality. Marketable yield and TCSV (for greenhouse studies) were only measured once per year and therefore were not averaged but were still square root transformed. Non-transformed means are reported in the tables. All responses were analyzed using a linear mixed model (randomized complete block design) with the fixed effect treatment, and the random effect block (Proc GLIMMIX model). Degrees of freedom were estimated using the Kenward-Roger's method. If the F-value for the overall treatment effect was significant, differences of means among treatments (least square means) were separated using Tukey's multiple comparisons procedure. Pearson's correlation coefficient analysis was conducted to explore the correlation between the thrips population and the TCSV infected tomatoes for the greenhouse study (Benesty et al., 2009). The Wilcoxon twosample test was applied to measure the t-approximation of the number of thrips. All analyses (α = 0.05) were done using SAS v.9.3 (SAS Institute, Cary, NC, USA).

Thrips in ornamental plants in the nursery
Frankliniella occidentalis, F. schultzei, melon thrips (Thrips palmi Karny), chilli thrips (Scirtothrips dorsalis Hood), and onion thrips (Thrips tabaci Lindeman; all Thysanoptera: Thripidae) were observed in the flowers collected from 24 ornamental flowering plants. The numbers of S. dorsalis and T. tabaci were very low and they were only observed in a few plant species in an inconsistent pattern. Only F. occidentalis, F. schultzei, and T. palmi were considered for result interpretation.
Frankliniella occidentalis (ca. 75% of the total adult thrips population) was the dominant species on the 24 ornamental plants in the nursery during 2 years of sampling (Tables 1 and 2). In 2017, the number of F. occidentalis in flowers differed among the ornamental plants (F 23,72 = 59.74, P < 0.001). The highest number was recorded in H. rosa-sinensis (mean ± SE = 59.08 ± 13.22 per sample of 10 flowers), followed by G. linearis (47.16 ± 4.18) ( Table 1). The abundance of F. schultzei differed among the ornamental flowers (F 23,72 = 54.25, P < 0.001). The numbers of F. schultzei were highest in Helianthus annuus L. and the numbers of T. palmi were highest in Hibiscus spec. More thrips larvae were recorded in Agastache spec. than in other ornamental flowers (Table 1). Similar thrips population trends were found in both years for F. occidentalis, F. schultzei, T. palmi, total adult thrips, and the thrips larvae (Table 2).

Thrips in ornamental plants in the greenhouse
A higher number of F. occidentalis was recorded in H. rosasinensis (mean ± SE = 27.75 ± 6.05 per sample of five flowers) than in other ornamental flowers in 2017 (Table 3). The number of F. schultzei was higher in H. rosa-sinensis (1.00 ± 0.45 per five flowers) than in Hemerocallis spec. and Agastache spec.; the number of T. palmi was higher in H. rosa-sinensis (1.75 ± 0.66 per five flowers) than in P. oleracea and Hemerocallis spec. (Table 3). More thrips adults were recorded in H. rosa-sinensis (31.14 ± 6.21) than in other ornamental flowers. Also more thrips larvae were found in H. rosa-sinensis (52.14 ± 12.90) than in other ornamental plants, except Agastache spec. (Table 3).
In 2018, numbers of F. occidentalis were higher than those of F. schultzei and T. palmi. Higher numbers of F. occidentalis (35.75 ± 8.45 per five flowers) and total adult thrips (38.85 ± 8.74) were recorded in H. rosa-sinensis than in other ornamental flowers (Table 3). More F. schultzei were recorded in G. linearis flowers (1.03 ± 0.35 per five flowers) than in L. camara, Hemerocallis spec., and Agastache spec. flowers; more T. palmi were found in H. rosa-sinensis (3.21 ± 1.20 per five flowers) than in Hemerocallis spec. (Table 3). The number of thrips larvae was highest in H. rosa-sinensis, followed by Agastache spec. and G. linearis (Table 3). Overall, very few ornamental leaf samples had thrips (data not shown).

Thrips in tomatoes in the greenhouse
Thrips numbers were low in tomato flowers (mean ± SE = 0.50 ± 0.21 to 2.16 ± 1.01 adult thrips per sample of five flowers; Table 4) as well as on tomato leaves (<1.0 per five leaves; Table 5) in both 2017 and 2018. The numbers of F. schultzei in tomato flowers were almost zero. In both years, there were no significant differences between tomatoes without (control) vs. with ornamental plants next to them in the abundance of F. occidentalis, F. schultzei, T. palmi, total adult thrips, and thrips larvae, both in tomato flowers (Table 4) and on tomato leaves (Table 5).

Marketable yield and Tomato chlorotic spot virus infected tomato plants
In both years the yield differed among treatments (2017: F 7,24 = 8.14; 2018: F 7,24 = 7.90, both P < 0.001) -the yield in tomatoes with P. oleracea next to them was lower than in tomatoes with any of the other ornamentals next to them or than in tomatoes without plants nearby (control) (Figure 2). Also TCSV prevalence differed among treatments (2017: F 7,24 = 3.24, P = 0.015; 2018: F 7,24 = 4.46, P = 0.0035) -on tomato plants with P. oleracea next to them TCSV prevalence was significantly higher than on tomatoes with L. camara or Hemerocallis spec. next to them (2017) or than on tomatoes with Hemerocallis spec. next to them (2018) (Figure 3). In both years, we observed TCSV symptoms in tomatoes without (control) and with ornamental plants next to them in the study area.  T A B L E 2 Mean (± SE) number of thrips per sample of 10 flowers of ornamental plants in a nursery study in 2018. T A B L E 4 Mean (± SE) number of thrips per sample of five flowers of tomatoes with selected ornamental plants next to them, in the greenhouse study.  The numbers of adult and larval thrips in tomato leaves were positively correlated with the prevalence of TCSV disease (i.e., the number of plants with visible symptoms) in both years (2017, adults: Pearson's r = 0.09, larvae: r = 0.12; 2018, adults: r = 0.03, larvae: r = 0.11). Also, the numbers of adult and larval thrips in tomato flowers were positively correlated with the prevalence of TCSV disease (2017, for adults: r = 0.10, larvae: r = 0.15, 2018, adults: r = 0.14, larvae: r = 0.15). F I G U R E 2 Mean (± SE) marketable yield per plot (kg) in tomatoes with selected ornamental plants next to them in 2017 and 2018. The ornamental plants, and therefore the tomatoes, varied in thrips densities (see Tables 3-5). Control: no plant next to the tomato plants. Means within a year capped with the same letter are not significantly different (Tukey's test: P > 0.05).

Ornamental plants as reservoir of Tomato chlorotic spot virus in the greenhouse
The RT-PCR analysis showed that both leaf and flower samples of six out of the seven tested ornamental species were positive for TCSV in both 2017 and 2018: P. oleracea, H. rosa-sinensis, L. camara, G. linearis, Hemerocallis spec., and Agastache spec. For Mandevilla spec., only the flowers were positive, but not the leaves (Figure 4). The results were not separated for symptomatic and nonsymptomatic plants.

DISCUSSION
In the nursery study, various species such as F. occidentalis, F. schultzei, T. palmi, S. dorsalis, T. tabaci, and thrips larvae were recorded from 24 ornamental plants. The highest numbers of both adult thrips and larvae were recorded in H. rosa-sinensis among all tested ornamentals. These thrips species are commonly found in ornamental plants in Florida and are known to transmit different species of tospoviruses (Rotenberg, 2015;Cluever & Smith, 2017). The current study on thrips abundance in ornamental plants was conducted during late spring to early summer when the vegetable growing season was nearly over. A high number of both adult and larval thrips, as we determined from the present study, indicated that the thrips migrated to flowers of ornamental plants from the nearby vegetable growing area (Ramachandran et al., 2001). Frankliniella occidentalis was the dominant thrips species in the nursery study; this is a vector of tospoviruses including TSWV and TCSV (Riley et al., 2011;Wijkamp et al., 1995). The second dominant thrips species in ornamental plants was T. palmi, which was reported to be the vector of some tospoviruses (Riley et al., 2011). Besides their role in transmitting tospoviruses, both F. occidentalis and T. palmi are known to feed and reproduce on the terminal growth of some ornamental plants (Funderburk et al., 2007). The numbers of F. schultzei, another confirmed vector of TCSV in Florida, were comparatively low in ornamental flowers. However, a small number of viruliferous thrips can play a significant role in transmitting tospoviruses (Boonham et al., 2002).
Seven ornamental plant species were selected in a greenhouse study to observe their effect on the incidence of TCSV and thrips vectors in tomatoes. We found a high number of thrips in the beginning of the study period. Thrips numbers in the ornamental flowers decreased as the study progressed. The experiment was started in late spring when thrips are usually found in higher numbers and numbers decrease with the approach of the summer, with heavy rain (RA Khan, unpubl.). Among several thrips species, F. occidentalis were found in higher numbers than other species in ornamental flowers in the present study. Thrips species recorded in this study are considered as pests of ornamental plants in Florida, where they inflict injury to these plants by feeding on flowers and terminal shoots. These thrips species were also found to aggregate on flowers (Funderburk et al., 2007). Whereas some plant species are only feeding hosts for thrips, the presence of both adults and larvae on ornamental flowers indicated that these plants are reproductive hosts for thrips. From these plants, thrips can migrate to the neighboring crop fields (Northfield et al., 2008).
The number of thrips in tomatoes, the main crop in the greenhouse study, was very low. Frankliniella occidentalis were found in comparatively higher numbers than F. schultzei and T. palmi in tomatoes, and this coincided with the higher abundance of F. occidentalis in the selected ornamental plants used in combination treatments with tomatoes. The presence of higher numbers of adult thrips and larvae in tomato flowers than in the leaves show their greater attraction to the flowers compared to the leaves. The lower number of adult thrips and larvae in tomatoes compared to the ornamental plants indicated that tomato is not a favored host for thrips. Hirano et al. (1994) identified the presence of α-tomatine, a steroidal glycoalkaloid in tomatoes, that acts as a strong antifeedant for thrips.
Although there was no significant difference between the numbers of thrips in tomatoes without (control) and with ornamental plants next to them, planting ornamentals had a significant effect on the disease incidence of TCSV (i.e., number of plants with visible symptoms) and marketable yield in tomatoes. Tomatoes with P. oleracea next to them showed a higher incidence of TCSV compared to other treatments. The RT-PCR analysis of leaf samples from symptomatic ornamental plants revealed that all samples were positive for TCSV. Leaf samples from TCSV infected tomato plants were also positive for TCSV (results not shown). Portulaca oleracea was already identified as a reservoir of TCSV (Raid et al., 2017), and this was confirmed in the present study. In a greenhouse study, Costa & Carvalho (1960) observed that the tissue of P. oleracea can maintain a high titer of TSWV for a long time. There is a possibility of P. oleracea tissues maintaining a high TCSV titer as well, which could lead to thrips infection with the virus. Our study showed a relatively high number of TCSV infected tomatoes with P. oleracea next to them, possibly indicating high virus titer in P. oleracea.
Plant hosts that support both vectors and viruses are important in disease epidemiology. In this study, we found that ornamental plants support the growth and development of thrips populations. The same ornamental plants were also observed to be reservoirs of TCSV. Although thrips numbers in tomatoes were low, tomatoes without (control) and with ornamental plants next to them were all infected with TCSV. These results indicated that thrips recorded on the tomatoes migrated from nearby ornamental plants, which can be considered as viruliferous. Coutts et al. (2004) revealed that tospovirus spread in crops was monocyclic, where the viruliferous thrips vectors were coming from outside sources and infecting the crops. Khan et al. (2020) observed the presence of thrips and the edge effect of TCSV spread in infected tomato fields, which concurred with our present study. The geographical distribution of TCSV occurrence is still restricted in southern Florida. More potential hosts of TCSV can be expected from weeds, ornamental plants, or other cultivated crops in southern Florida. Considerable disease prevalence can be expected because TCSV is present in the region of southern Florida where growers produce vegetable crops, with ornamental nurseries, tropical fruits, and transplants in proximity (Zhang et al., 2019).
Results from the current study revealed the ornamental plants P. oleracea, H. rosa-sinensis, L. camara, Mandevilla spec., G. linearis, Hemerocallis spec., and Agastache spec. as TCSV reservoirs in southern Florida where the disease is most prevalent. Perennial ornamental crops can be natural reservoirs for plant viruses and help in virus circulation and transmission to other economically important crops (Mitrofanova et al., 2018). Ornamental flowering plants are mostly grown during late spring and summer, whereas most of the vegetable crops are grown in fall and spring in southern Florida. Various vegetable crops, ornamental nursery crops (both foliage and flowering plants), and fruits are grown side by side, as close as 9 m apart (Seal & RA Khan, unpubl). Thrips species with a polyphagous nature can migrate from the ornamentals to the vegetable plants. Tomato chlorotic spot virus detected in ornamental plants as found in this study revealed the fact that more alternative hosts of this virus may exist in the vicinity. In the future, other ornamental plants, weeds, and vegetable plants need to be examined for TCSV. Thrips abundance and seasonal cycles also need to be evaluated in those plants.