Effects of soil treated fungicide fluopimomide on tomato (Solanum lycopersicum L.) disease control and plant growth

Abstract Fluopimomide is a novel acid amide fungicide registered for the control of many plant pathogens. In the present study, the effects of soil-treated fluopimomide on soil micro biomass, disease incidence, plant growth, soil enzyme activity, and marketable yield of tomato (Solanum lycopersicum L.) were investigated via field trial. In addition, the application prospect in China was also evaluated. In the experiment, five treatments with three replications and a randomized complete block design were followed. The treatments were: furrow application of fluopimomide (25% suspension concentrate, SC) at the dosage of 375, 750, and 1,500 g ha−1, which was recommended, double recommended, and quadruple recommended dosages, respectively. Besides, common control fungicide fluopicolide (5% SC) furrow was applied at recommended application dosages of 750 mL ha−1, and a non-treated control was also undertaken. Results indicated that fluopimomide exhibited no effects on the amount of soil bacteria and actinomycetes, and its inhibition effect on fungi amount could be recovered at 60 days after treatment (DAT). With the recommended application dosage, fluopimomide could efficiently reduce the number of plant pathogens in soil by 79.56–85.80%, significantly reduce the disease incidences in tomato plants by 80.00–88.24%, and improve plant height by 13.25–24.05% and marketable yield by 16.88%. Furthermore, soil enzymes exhibited a complex response to fluopimomide, and AOB and nifH gene copy numbers were increased by the double and quadruple recommended dosage of fluopimomide. Based on the above results, fluopimomide could be recommended as an efficient fungicide for the tomato field.


Introduction
Tomato (Solanum lycopersicum L.) is an important vegetable crop [1]. In China, the annual tomato production reached 50 million MT. However, due to long-term continuous cropping and mismanagement, tomato diseases have been increasing over time. The most common diseases are: wilt, blight, and gray mold, which are caused by Fusarium oxysporum, Phytophthora spp., and Botrytis cinerea, respectively [2][3][4]. Yield loss caused by above diseases reached 10-30% in general plots, and 50% in serious plots. Fungicide application is usually one of the main components in tomato production.
Fluopicolide effectively suppressed sporangium formation, zoospore germination, and mycelial growth of Phytophthora pathogen [5], and could significantly reduce the incidence of watermelon fruit rotting in the Carolinas [6]. In China, its preparation named Yinfali Suspension has been widely used for blight control of pepper (Capsicum frutescence L.), potato (Solanum tuberosum L.), tomato (S. lycopersicum), cucumber (Cucumis sativus L.), and other vegetables, its joint application with bio-fungicides could control cucumber diseases more efficiently.
Fluopimomide is a new fluorinated benzamide fungicide developed by Shandong United Pesticide Industry Co. Ltd, China in 2010, it has a similar structure to fluopicolide [7] (Figure 1). Its chemical name is N-(3-chloro-5-trifluoromethyl-pyridine-2-methyl-2,3,5,6-tetrafluoroethane-4-methoxy-benzamide) [8]. In China, fluopimomide has been reported to be efficient for Phytophthora and nematode control [9]. Taking into account the structural similarity of the two compounds, it is recognized that fluopimomide would be effective in preventing oomycetes and other vegetable diseases. However, with the addition of 4 fluorine atoms and a methoxy group in fluopimomide, there might be a great difference between fluopicolide and fluopimomide in sterilization virulence, bactericidal spectrum, mode of action, and mechanism of action, etc.
In the previous study, bactericidal spectrum of the innovative fungicide fluopimomide was measured by in vitro bioassays, and exhibited EC 50 values of 0.97, 2.36, and 3.59 μg mL −1 for B. cinerea, Phytophthora, and F. oxysporum, respectively [10]. However, little information is available about its application effects on the tomato field. The aims of the present study were: (a) to determine the control efficacy of fluopimomide on detrimental pathogens in tomato field, including F. oxysporum, Phytophthora, and B. cinerea, (b) to investigate its micro-ecology effect on tomato planted soil, and (c) to identify the influence of fluopimomide on tomato plant growth and marketable yield.
The soil physicochemical properties of the experimental sites were: sand 29.17%, silt 70.49%, clay 0.34%, organic matter 18.95 g kg −1 soil, and pH 7.1. This site suffered heavily from soil-borne diseases, such as F. oxysporum, Phytophthora, and B. cinerea. In conventional farm operations, compound fertilizer of 15N-15P 2 O 5 -15K 2 O was broadcast applied at the dosage of 750 kg ha −1 as base.
Five treatments with 3 replicates in each were arranged in a random block design, where 25 seedlings were planted per plot (7.20 m × 0.75 m). The treatment programs were: (a) laboratory-made fluopimomide (25% SC, a.i.) furrow-application was done at a dosage of 375 g ha −1 , it is the field recommended application dose; (b) fluopimomide (25% SC, a.i.) furrow-application dose of 750 g ha −1 ; (c) fluopimomide (25% SC, a.i.) furrow-application dose of 1,500 g ha −1 ; (d) laboratory-made fluopicolide (5% SC, a.i.) furrow-application dose of 750 mL ha −1 , it is the field recommended dose; and (e) non-treated control. Evaluation of 2 and 4 folds of recommended application dosage is necessary for novel pesticide crop safety verification.

Sampling
20, 40, and 60 DAT, a soil column cylinder with a diameter of 5 cm was used to sample soil nearby tomato plants at a depth of 0-10 cm, and random 20 points were included for each plot. Each collected soil sample (0.5-1.0 kg) was sieved (1 mm mesh) and separated into two parts. Part one was stored at 4°C for the microbiological and enzyme activities test, and the test was completed within less than a week. Part two was treated with liquid nitrogen and stored at −80°C for DNA extraction.

Disease assessment in plants
After the transplant, incidences of wilt, blight, and gray mold of ten random plants in each plot were recorded at intervals of 20, 40, and 60 days. Levels of disease severity were assessed by visually estimating the percentage of diseased surface, and graded as: 0 = 0%, 1 = 1-9%, 2 = 10-24%, 3 = 25-49%, 4 = >50% of surface affected. Wilt was calculated by spot on stem base, while blight and gray mold were weighted by round and angle spots on leaves, respectively. The disease index was calculated using the following formula:

Disease index
The relative score Plant numbers in the score Total plant numbers 4 100,

Assays of soil enzymatic activities
Analysis of soil dehydrogenase and urease activity was done according to the methods of Lebrun et al. [16]. Soil phosphatase activity was tested using the method of Wang et al. [17]. The invertase activity was measured following the modified method of Ohshima et al. [18].

Quantitative PCR analysis of soil nitrogen-related genes
The qPCR analysis of AOA, AOB, nifH, and nirS was performed with ABI 7500 Real-Time PCR System and 7500 System Software-SDS 2.2 by absolute quantification method, and the primer information is shown in Table 1.
The procedure of qPCR was performed at 95°C for 15 min and followed by 40 cycles of 95°C for 10 s, annealing (53°C for AOA, 56°C for AOB, 58°C for nifH and nirS) for 20 s, and 72°C for 1 min.  2.8 Influence of fluopimomide on tomato plants 20,40, and 60 DAT, the plant height of randomly chosen ten plants in each plot was measured. Fruits were harvested twice (85 and 120 DAT) when they were mature, and the marketable yield was calculated.

Statistical analysis
As there were no significant differences between experiments over the 2 years, data from the two experiments were combined for analysis and interpretation. The data were analyzed statistically by the Duncan test with a significance level of p < 0.05.

Results and discussion
3.1 Effect of fluopimomide on the abundance of fungi, bacteria, and actinomycetes Soil microbes are basic components of soil ecology and are highly sensitive to environmental changes [19]. The diversity and abundance of soil microbial communities are the important indices for gaining knowledge of modern soil microbiology. Still, it is the main indicator of risk assessment for pesticide application in agricultural fields [20]. So, in the present study, the application of novel fungicide fluopimomide required tests to determine the effect on the soil environment. As shown in Table 2

Effects of fungicide fluopimomide on the amount of soil-borne pathogens
As a greenhouse vegetable, disease management is one of the most essential components of tomato production. The primary soil-borne diseases, wilt (F. oxysporum), blight (P. infestans), and grey mold (B. cinerea), have caused severe yield loss throughout the world [21], and current systematic fungicides are efficient for these disease control [22,23]. Manikandan et al. reported that the high Fusarium gene level in tomato planted soil suffered from wilt [24]. In our assay, the soil application of fungicide fluopimomide has reduced the amounts of three typical soil-borne pathogens, especially B. cinerea and F. oxysporum (the inhibition ratios >80%). As for Phytophthora, fluopimomide exhibited similar efficiency (79.56-89.21%) to fluopicolide (84.64-87.59%). As time passed, the inhibition efficiency of fluopimomide remained constant until 60 DAT. Combined with the results of Table 2, we could conclude that the recovery of fungi amount in fluopimomide treatment at 60 DAT has nothing to do with the target pathogens. Moreover, one-off soil treatment of fluopimomide could effectively control soilborne pathogens ( Table 3).

Control efficiency of fungicide
fluopimomide on tomato seedling diseases The fluorine atom has four effects: analog, electronic, hindering, and penetration, C-F bond with much higher energy than the C-H, and significantly increases the stability and physiological activity of organic fluorine compounds. In this study, the added four fluorine atoms and a methoxy group in fluopimomide have expanded its fungicidal range. This is consistent with the report of Ji et al., in which fluopimomide was revealed to have excellent efficiency on tomato gray mold [25]. Mulugeta et al. have reported that phosphite could protect tomato against blight but were not effectively under higher disease pressure [26]. In this study, with recommended application dosage, fungicide fluopimomide could significantly reduce the seedling disease incidences of tomato, with inhibition ratios of 80.00, 88.24, and 84.63% for wilt, blight, and gray mold, respectively. Still, when the concentration of fluopimomide doubles or quadruples, the infection of above soil-borne pathogens can be definitely inhibited. In contrast with the novel broad-spectrum fungicide, control fungicide fluopicolide can only inhibit the disease incidence of blight, with the inhibition ratio of 94.12% at the recommended application dosage. The results confirmed the previous indoor toxicity tests and showed that fluopimomide could be recommended as an excellent fungicide for tomato disease management ( Table 4).

Effect of fluopimomide on soil enzyme activities 3.4.1 Effect of fluopimomide on soil dehydrogenase activities
Dehydrogenase, representative of soil organism metabolism [27], can transfer hydride groups from a substrate to an acceptor such as NAD + . It plays an important role in the organic decomposition process, particularly for bacteria, which are the main ultimate consumers and metabolizers of aromatic compounds, such as pesticides [28,29]. As shown in Table 5, at 20 DAT, recommended dosage of fluopimomide significantly increased soil dehydrogenase activities (p < 0.05), while quadruple recommended dosage exhibited a significant inhibition effect. The results showed as mean value ± std. error, different letters in each column showed the significant difference at 0.05 level, and the same as follows.
At 40 days after treatment, the dehydrogenase activities of soil treated with a quadruple recommended dosage of fluopimomide increased drastically. This may be because of the soil ecosystem alteration with the increasing fungicide concentration, and microorganisms can increase their metabolic activity in response to xenobiotics in the soil [30]. This is in agreement with Monkiedje et al. [31], who had reported a significant inhibition effect of fungicides mefenoxam and metalaxyl on soil dehydrogenase activity. However, Tejada et al. [32] had found a non-significant increase in dehydrogenase activity in pesticide polluted soils. According to Bending et al. [33], the variant responses of soil dehydrogenase activity to fungicides input are determined by soil type and other factors, such as microbial community structure and types of fungicide. At the end of the incubation (60 DAT), the dehydrogenase activities of treated soil recovered to be similar to control, indicating high safety of tested fungicide on soil dehydrogenase.

Effects of fluopimomide on soil phosphatase activities
As shown in Table 6, fluopimomide exhibited a significant activation effect on soil phosphatase activities with recommended dosage at 20 DAT. However, when treated with a quadruple dosage, the soil phosphatase activity decreased significantly compared with the control. Still, the inhibition effect of higher fungicide concentrations The results showed as mean value ± std. error, different letters in each column showed the significant difference at 0.05 level, and the same as follows. The results showed as mean value ± std. error, different letters in each column showed the significant difference at 0.05 level, and the same as follows.
continued until 40 DAT. On 60 DAT, the influences of fluopimomide and fluopicolide in various dosages were reduced to be non-significant. Our result is in accordance with that of Jastrzębska et al. [34], which reported an increase in soil phosphatase activity after being treated with cyprodinil, dimoxystrobin, and epoxiconazole, and indicated the direct relationship between the increase in soil phosphatase activity and dosage of the applied fungicide. Monkiedje and Spiteller [35] also found the stimulating effects of metalaxyl and prochloraz on soil phosphatase activity and speculated that both fungicides were used as sources of energy by soil microorganisms. Nevertheless, Chen et al. [36] reported the inhibitory effect of benomyl, captan, and chlorothalonil on soil phosphatase activity. In this assay, fluopimomide has exhibited a gentle and restorable effect on soil phosphatase activities.

Effects of fluopimomide on soil urease activities
As reported, urease is externalized due to parent cell death and lysis. This enzyme plays an important role in the nitrogen cycle in soils. Its substrate, urea, is incorporated into the soil from fertilizer, animal excreta, or nucleic acids [17]. In this study, with recommended dosages of fluopimomide and fluopicolide, a non-significant increase has been detected. Still, large dosages of fluopimomide could inhibit urease activity to a certain extent (<10%). With passage of time, the effects of fungicides on soil urease activities became lighter and lighter. In previous studies, similar results have been reported. Monkiedje et al. [35] observed a slight inhibitory effect of metalaxyl and prochloraz on urease in the short term. It was speculated that soil microorganisms took fungicides as an energy source. Uyanőz et al. [37] also reported the activation effect of captan, quintozene, and propamocarb hydrochloride on soil urease activity ( Table 7).

Effects of fluopimomide on soil invertase activities
Soil invertase is of particular importance in carbon cycles [38]. A previous study had documented that bioorganic fertilizer application could always improve invertase activity [39]. Our assay results showed a significant increase in invertase activities when the soil was treated with a recommended dosage of fluopimomide and fluopicolide at 20 DAT, which were probably used as carbon sources by microbes. However, the activation effects were converted to inhibition at 40 DAT, and recovered to be similar to control at 60 DAT. The complicated influence of fluopimomide may be due to the complex response of microorganisms to fungicide The results showed as mean value ± std. error, different letters in each column showed the significant difference at 0.05 level, and the same as follows. The results showed as mean value ± std. error, different letters in each column showed the significant difference at 0.05 level, and the same as follows.
dosages, including the ecology change in the microorganism community.
Overall, the effects of fluopimomide on the four soil enzymes are temporary and reversible, indicating relatively high safety to the soil environment ( Table 8).

Quantitative PCR analysis of soil nitrogen-related genes
Soil microbes are essential in soil nutrient mineralization and accumulation [40]. Soil N cycling is participated by varieties of microorganisms, of which nifH encodes for N 2 fixation, AOA and AOB for ammonia oxidation, and nirS for denitrification [41]. Jiang et al. has reported that five fluoroalkylether compounds could reduce amoA gene abundance in soil and had different effects on nirS [42].
In this study, 20, 40, and 60 DAT, the copy numbers of AOA and nirS fluctuated greatly, but the differences among treatments were not obvious, indicating that the effects of chemical treatment on AOA and denitrifying bacteria in soil were not regular. At 40 and 60 DAT, the AOB and nifH gene copy numbers were higher in the double and quadruple dosages of fluopimomide treatments than in the control, indicating that fluopimomide could promote the proliferation of soil AOB, nitrogen- The results showed as mean value ± std. error, different letters in each column showed the significant difference at 0.05 level, and the same as follows. fixing bacteria, and other bacteria (Figure 2), which might be helpful for tomato plant growth [43].

Effect of fluopimomide on plant height and marketable yield
Soil management for disease suppressive could help to improve plant growth [44]. Jakl et al. has reported the increased effect of soil-treated triazole fungicides on tomato fruit yield [45]. As shown in Table 9, 20 DAT, fluopimomide had increased tomato plant height nonsignificantly by 13.25, 25.65, and 20.03% with 1-, 2-, and 4-folds of recommended dosages. Still, the stimulated efficiencies continued with time-lapse until 60 DAT. A similar tendency could be found in fluopicolide as well. Meanwhile, the two fungicides significantly improved marketable tomato yield by reducing disease incidences. Among which fluopimomide had improved the total marketable yield by 16.88, 18.16, and 17.47%, with the application dosage of 375, 750, and 1,500 g ha −1 , respectively. Furthermore, fluopicolide exhibited lower stimulation efficiency by 9.87%. Thus, the conclusion could be drawn out that fungicide fluopimomide could improve tomato yield, possibly via the inhibition of soil-borne pathogens.

Conclusion
Via soil treatment, the new fluorinated benzamide fungicide fluopimomide could significantly reduce the amounts of soil-borne pathogens in soil, reduce the disease incidences in tomato plants, and eventually increase the marketable yield of tomatoes. During the inoculation period, the soil enzymes had been influenced differently, and AOB and nifH gene copy numbers were increased by the double and quadruple dosages of fluopimomide treatment. Compared with the control fungicide fluopicolide, fluopimomide exhibited more efficiency in tomato plant height and marketable yield. Therefore, as a broadspectrum fungicide, fluopimomide could be popularized to manage tomato diseases. Still, the broader application scope remains to be investigated.
Funding information: The research was supported by The National Science Foundation of Shandong Province (ZR2019PC013), and the National Natural Science Foundation of China (32102259).