The effect and mechanism of Dufulin in controlling tomato yellow leaf curl virus on tomato plants

Background: Tomato yellow leaf curl virus (TYLCV) causes critical production loss in tomato cultivation. The control of TYLCV in tomato is done mainly by using pesticide which is dicult and expensive, making it essential to nd an environmentally friendly chemical agent to control TYLCV. Dufulin has been widely used to prevent and control viral diseases in tobacco and rice in recent years. In this study, we investigated the effect and mechanism of Dufulin on TYLCV on tomato plants. Methods: The control effect of Dufulin on TYLCV was evaluated by eld experiments. The expression level of PI II and NPR1 in healthy and TYLCV-infected tomato after treatments were determined by Real-time uorescent quantitative PCR (qRT-PCR). Handheld chlorophyll meter was applied to compare the content of chlorophyll and nitrogen in healthy and TYLCV-infected tomato after treatments. Results: It showed that the relative control effect of 20% Dufulin on TYLCV reached above 68% in 2018 to 2020. Jasmonic acid (JA) level was higher on healthy tomato, but lower on TYLCV-infected tomato plants treated with Dufulin compared to control. Salicylic acid (SA) level was higher on healthy and TYLCV-infected tomato plants treated with Dufulin compared to control. Chlorophyll content on healthy and TYLCV-infected tomato plants was higher after treatment with Dufulin compared to control. Nitrogen content on tomato plants showed no signicant difference after spraying Dufulin compared to control. Conclusions: We found the rst evidence of control effects TYLCV using Dufulin. It induced plant defense and increased plant chlorophyll content to help plants resist infection which is helpful for future control of TYLCV in tomato.

investigated the effect and mechanism of Dufulin on TYLCV on tomato plants.
Methods: The control effect of Dufulin on TYLCV was evaluated by eld experiments. The expression level of PI II and NPR1 in healthy and TYLCV-infected tomato after treatments were determined by Realtime uorescent quantitative PCR (qRT-PCR). Handheld chlorophyll meter was applied to compare the content of chlorophyll and nitrogen in healthy and TYLCV-infected tomato after treatments.
Results: It showed that the relative control effect of 20% Dufulin on TYLCV reached above 68% in 2018 to 2020. Jasmonic acid (JA) level was higher on healthy tomato, but lower on TYLCV-infected tomato plants treated with Dufulin compared to control. Salicylic acid (SA) level was higher on healthy and TYLCVinfected tomato plants treated with Dufulin compared to control. Chlorophyll content on healthy and TYLCV-infected tomato plants was higher after treatment with Dufulin compared to control. Nitrogen content on tomato plants showed no signi cant difference after spraying Dufulin compared to control.
Conclusions: We found the rst evidence of control effects TYLCV using Dufulin. It induced plant defense and increased plant chlorophyll content to help plants resist infection which is helpful for future control of TYLCV in tomato.

Background
Tomato yellow leaf curl virus (TYLCV) belongs to the genus Begomovirus of the family Geminiviridae. It is transmitted by Bemisia tabaci in a persistent, circulative manner and is a plant virus that causes important disease in tomato [1][2][3]. TYLCV-infected tomato plants show various symptoms, for instance, leaf yellowing, curling and stunting, which result in a severe reduction in tomato cultivation production around the world [4,5]. In addition to tomato, other cultivated plants including pepper, common bean, cucurbit and eustoma are also infected by TYLCV [6][7][8][9]. No matter in greenhouse or in open eld production, the TYLCV management is di cult and expensive [10].
There are many different methods to reduce crop losses resulted from TYLCV, such as removing vectors, killing weeds, and changing planting season what have been applied in many crop areas [1]. The development of TYLCV-resistant commercial tomato cultivars is the best approach to control TYLCV so far [11]. However, virus may exist strong genetic variability, in addition, TYLCV-resistant tomato cultivars are easy to vary [12]. Also chemicals or pesticides are used for plant virus diseases and pest control, but they have negative effects on humans, wildlife and environment [13]. Therefore, it is necessary to nd an environmentally friendly chemical agent to control TYLCV.
Dufulin, an amino phosphonate compound with a novel molecular structure ((2-uorophenyl)-(((4methylbenzothiazol-2-yl)-amino) methyl) phosphonic acid diethyl ester, Figure 1) [14][15][16]. It is a new antiviral agent and has highly effect on plant viruses. There are studies have shown that Dufulin inhibit tobacco mosaic virus (TMV) through inducing systemic acquired resistance (SAR) in plants [17]. It is highly effective against TMV, cucumber mosaic virus (CMV), potato virus Y (PVY) and southern rice black-streaked dwarf virus (SRBSDV) [18,19]. Dufulin has been widely used to prevent and control tobacco and rice viral diseases in recent years. It is the rst antiviral agent to meet environmental standards in China [20,21]. In 2007, it was identi ed as a new chemical with high anti-TMV activity by the Ministry of Agriculture of China (LS 20071280 and 20071282). Subsequently, Dufulin was produced on an industrial scale and applied for eld application [22].
The effect of Dufulin in controlling TYLCV is still poorly reported. In this study, we rst investigated the effect of Dufulin in controlling TYLCV, and then we determined the plant defense and plant nutritional properties following treatment with Dufulin in tomato, and uncover the mechanisms of controlling TYLCV by Dufulin in tomato.

Field control effect of Dufulin on TYLCV
The eld experiment was conducted in Chunhua vegetable-growing areas, Changsha City, Hunan Province, where TYLCV diseases occur every year. Tomato (Solanum lycopersicum, "Zuanhongmeina", Hunan Academy of Agricultural Sciences) was planted on April from 2018 to 2020. After planting for 3 days, the dead tomato plants were replaced. In the experiments, there were 2 treatments (Dufulin and water control). There were 3 replicates for each treatment, with 6 plots in total. Plots were arranged in random groups and 60 tomato plants were planted in each plot. Protective rows were set between plots.
The reagent was 20% Dufulin suspension (Guangxi Garden Biochemical Joint Stock Company, Guangxi, China), water was used as control. Prior to Dufulin and water treatment, no other antiviral reagents were applied at the experimental eld.
The rst reagent application was made in accordance with the manufacturer's instructions on April from 2018 to 2020. The Singapore AGROLEX SPRAYER JACTO HD400 16-litre knapsack sprayer (Linon Private Limited Company, Singapore) was used to spray the leaf surface, with a spray pressure of 1-2 kgf/cm 2 and a ow rate of 255-950 g/min. The relatively steady rate of spraying was maintained and the weather was sunny. The reagent of 5 liters was sprayed on each plot, and was applied 3 times in a plot. Each interval is 7 days. Any phytotoxicity in tomato was observed after each application of 2 days.
Healthy and TYLCV-infected tomato plants were used. TYLCV-infected tomato plants were obtained according to Ning et al. [24].
TYLCV-infected tomatoes and nonviruliferous tomatoes were sprayed with Dufulin puri cation after transplanting 35 days, water was used as control. Each tomato plant was sprayed with about 15 mL of the reagent. There were nine tomato plants for each treatment. About 0.1 g tomato leaf was collected into RNA free sampling tubes after spraying of 1, 2, 3, 4, 5 and 6 days, respectively. They were stored at -80°C for further detection. All tomato leaves RNA were extracted with TRIzol (TransGen Biotech, Beijing, China). The RNA concentration and purity were measured by NanoDrop 2000 (Thermo Fisher Scienti c, Beijing, China). qRT-PCR (real-time uorescent quantitative reverse transcriptase polymerase chain reaction) was used to measure the expression level of JA and SA associated genes in tomato after treatments. The rst strand of cDNA for qRT-PCR was synthesized by using cDNA synthesis kit (TransGen Biotech, Beijing, China) in accordance with the manufacturer's instructions. qRT-PCR was carried out on qTOWER3G qPCR system (Analytik Jena, Jena, Germany) by using qPCR kit (TransGen Biotech, Beijing, China) in accordance with the manufacturer's instructions. We took PI II and NPR1 as target gene. PI II, a downstream gene associated with JA pathway in tomato [25] and NPR1, a downstream gene associated with SA pathway in tomato [26]. Corresponding qPCR primers were listed in Table 1. Normalized gene expression was calculated using the 2 −ΔΔCT method [27] with ACT and UBI as reference gene [28].

Effects of Dufulin on chlorophyll and nitrogen content in tomato
Handheld chlorophyll meter (Okechi Instrument Company, Henan, China) was used to measure the chlorophyll and nitrogen content in tomato leaves. The treatments were the same as above. The same leaves were measured after spraying of 1, 2, 3, 4, 5 and 6 days, respectively. The third leaf from the top of the plant was marked and measured in accordance with the manufacturer's instructions.

Data analysis
Data were analyzed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Independent sample t-test was used to compare the eld control effect of Dufulin. The general linear model (GLM) repeatedmeasure was used to compare the effects of Dufulin on plant defense and nutrition in tomato. Differences between groups were considered statistically signi cant when the p value was less than 0.05.

Results
Field control effect of Dufulin on TYLCV Three days after the application of 20% Dufulin, eld observation showed that the tomato grew normally.

Effects of Dufulin on JA and SA in tomato
After spraying Dufulin, the relative expression level of PI II gene showed no signi cant difference on healthy tomato compared with control on the whole (F = 3.305, p 0.05) (Fig. 4A). The relative expression level of PI II on healthy tomato was increased from 1 to 6 days. But the relative expression level of PI II gene showed signi cant lower on TYLCV-infected tomato compared with control (F = 37.813, p = 0.001) (Fig. 4B). The relative expression level of PI II gene on TYLCV-infected tomato was increased rstly, then declined from 2 to 6 days. The relative gene expression was lower after spraying Dufulin than control from 1 to 6 days.
The relative expression level of NPR1 gene showed signi cant higher on healthy tomato and TYLCVinfected tomato compared with control (Healthy plant: F = 434.469; p 0.001; TYLCV-infected plant: F = 179.074; p 0.001). The relative expression level of NPR1 gene on healthy tomato plants increased from 1 to 6 days (Fig. 5A). But the relative expression level of NPR1 on TYLCV-infected tomato plants rst declined and then increased from 2 to 6 days (Fig. 5B). And the relative gene expression was higher on healthy and TYLCV-infected tomato plants after spraying Dufulin compared to control.

Effects of Dufulin on chlorophyll and nitrogen content in tomato
After spraying Dufulin, the content of chlorophyll was signi cant higher on healthy tomato and TYLCVinfected tomato compared with control (Healthy plant: F = 184.902; p 0.001; TYLCV-infected plant: F = 104.757; p = 0.001). The chlorophyll content on healthy tomato plants was increased from 1 to 6 days (Fig. 6A). But the chlorophyll content on TYLCV-infected tomato plants was decreased from 1 to 4 days, then rosed (Fig. 6B). The content of chlorophyll on healthy and TYLCV-infected tomato plants was higher than control from 1 to 6 days.
The content of nitrogen on healthy and TYLCV-infected tomato were not signi cant difference compared with control (Healthy plant: F = 1.174; p 0.05; TYLCV-infected plant: F = 0.023; p 0.05;) The nitrogen content on healthy tomato plants decreased from 1 to 6 days (Fig. 7A). However the nitrogen content on TYLCV-infected tomato plants was rst decreased, then rose from 3 to 6 days after spraying with Dufulin ( Fig. 7B). The nitrogen content of nitrogen remained relatively stable on healthy and TYLCV-infected tomato plants in these two treatments.

Discussion
Dufulin has been widely used to prevent and control many tobacco, rice and other viral diseases. As a kind of highly effect on resist plant viral diseases, its effectiveness, active mechanism, environmental biotoxicity, and safety evaluation have been widely researched [29,30]. Currently, TYLCV is a disease that seriously damages tomato production. In this study, Dufulin was applied to control TYLCV for the rst time. There was a signi cant reduction in the incidence of TYLCV on tomato, and the average relative control effect reached more than 68% from the year of 2018 to 2020. It indicates that Dufulin has potential application value in the prevention and control of TYLCV.
Plants cannot move to elude environmental threats. As a result, plants have evolved complex mechanisms for sensing aggression, such as viruses, fungi, bacteria, insects and so on, and then translate that perception into an adaptive response [31]. Plant defense plays an important role in its virus resistance. JA plays important roles in plant defense against insects. White y nymphs are in uenced by JA treatments, the development become slower and have a lower survival rate [32]. In this study, PI II relative expression level increased after spraying Dufulin on healthy tomato. However, PI II relative expression level rose rst and then fell after spraying Dufulin on TYLCV-infected tomato. This phenomenon may be caused by TYLCV infection. Previous research showed that TYLCV infection of host plants are bene cial to Bemisia tabaci vectors by inhibiting JA signaling pathway [33]. Our results also illustrate this hypothesis. SA mediates plant defense against pathogens through accumulating in both infected and distal leaves, which can response to pathogen attack [34][35][36][37][38]. It was reported that Dufulin is an activator of systemic acquired resistance (SAR) by regulating the SA signaling pathway in tobacco, then the plant cells are given antiviral activity [29]. In our study, NPR1 relative expression level was higher on healthy and TYLCV-infected tomato plant after spraying Dufulin compared to control. It is consistent with previous studies, suggesting Dufulin can resist TYLCV by regulating the SA signaling pathway in tomato. Treatment with Dufulin can control TYLCV by strengthening plant defenses, such as JA and SA. However, what speci c regulatory mechanisms in the process need further veri cation.
Chlorophyll is the main pigment in photosynthesis and plays a central role in light absorption. Nitrogen is an essential macronutrient which is needed for plant growth and development. Chlorophyll and nitrogen concentration in leaves were also measured because they can be affected by the plant nutritional status [39]. There are studies showed that plant chlorophyll content was positively correlated with nitrogen content [40]. Therefore, the value of chlorophyll content in leaves can help understand the nutritional status of plants, guide fertilization management scienti cally, and ensure the quality and yield of crops [41,42]. Chlorophyll content on healthy and TYLCV-infected tomato plants was higher after treatment with Dufulin compared to control. This indicated that the effect of Dufulin in controlling TYLCV was related to the increase of chlorophyll content on tomato. Nevertheless, nitrogen content on healthy and TYLCV-infected tomato plants were not signi cant difference compared with control. This may indicate that Dufulin does not directly affect the nitrogen content of tomato plants. Further research is still needed to con rm the mechanism of the chlorophyll increase with Dufulin treatment.

Conclusions
In conclusion, we found the effects of Dufulin on controlling TYLCV for the rst time, by inducing plant defense and plant nutrition. Our study uncovered the mechanisms of TYLCV controlling by Dufulin in tomato. Dufulin will be used more widely on other viral diseases in plants.      The content of chlorophyll (SPAD) on healthy and TYLCV-infected tomato after spraying Dufulin and water for 1, 2, 3, 4, 5, 6 days. (A) Healthy tomato. (B) TYLCV-infected tomato. Data are given as mean ± SD; Error bar: mean ± SEM; *p ≤ 0.05.