Field control effect of Dufulin on TYLCV
The field 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 field.
The first 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/cm2 and a flow 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. The morbidity was investigated by 5 point diagonal sampling method following the last application of 14 days, and 6 plants were investigated at each point. A total of 60 plants were investigated. The total number of investigated plants and the number of diseased plants at all levels were recorded. The disease index and control effect were calculated by the following formulate:
Disease index = Σ (Number of diseased plants at every levels × Level value) / (Total survey plants × the highest value);
Control effect = [(Disease index of control group - Disease index of treatment group) / Disease index of control group] × 100%.
Each plant was assigned a disease level according to the following scale [23]:
0 = Asymptomatic;
1 = Slight symptoms (apical leaves slightly yellowing and margin slightly curling);
2 = Moderate symptoms (apical leaves moderate yellowing, marginal moderate curling and wrinkling)
3 = Severe symptoms (severe marginal / interveinal yellowing, wrinkling and curling);
4 = Very severe symptoms (foliar symptoms similar to 3 accompanied by reduction in leaf size, branching, and severe stunting).
Effects of Dufulin on jasmonic acid (JA) and salicylic acid (SA) in tomato
This experiment was conducted in the laboratory. Tomato (Solanum lycopersicum, “Zuanhongmeina”) plants were grown in pots (d = 12.5 cm, h = 15 cm) kept in whitefly-proof screen-cages (60 × 40 × 60 cm) in a greenhouse under photoperiod L16: D8, temperature 25 ± 1°C, and relative humidity 60 ± 10%. 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 purification 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 Scientific, Beijing, China). qRT-PCR (real-time fluorescent quantitative reverse transcriptase polymerase chain reaction) was used to measure the expression level of JA and SA associated genes in tomato after treatments. The first 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 field control effect of Dufulin. The general linear model (GLM) repeated-measure was used to compare the effects of Dufulin on plant defense and nutrition in tomato. Differences between groups were considered statistically significant when the p value was less than 0.05.