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

2.1 Chemicals and reagents

Fluopimomide (purity = 98%) and fluopicolide (purity = 96%) were both provided by Shandong United Pesticide Industry Co. Ltd, China.

2.2 Field experiment design

Field experiments were arranged in the autumn cropping seasons of 2018–2019 and 2019–2020 in a commercial greenhouse near Fang county, Tai’an, China (N35°58′13″, E117°12′13″). Six-week-old “Jinpeng” tomato seedlings were transplanted on August 23, 2018 and August 18, 2019.

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–15P₂O₅–15K₂O 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.

2.3 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.

2.4 Enumeration of microbial population

The amounts of fungi, bacteria, and actinomycetes were evaluated using the serial ten-fold dilution (10-2–10-7) method [11], where 45 mL of sterile water mixed with 5 g soil was regarded as 10⁻¹ dilution. Fungi, including yeast, were counted on Martin’s medium, with pH 6 and containing 150 mg L⁻¹ streptomycin. Bacteria were counted on a selective medium prepared with glucose 1 g L⁻¹, proteose peptone 3 g L⁻¹, yeast extract 1 g L⁻¹, K₂PO₄ 1 g L⁻¹, agar 15 g L⁻¹, and cycloheximide 100 mg L⁻¹. Actinomycetes were counted on improved GAO No.1 medium with pH 7.4–7.6 [12]. The number of F. oxysporum in soil was counted on the PCNB selective medium containing KH₂PO₄ 1.0 g L⁻¹, MgSO₄·7H₂O 0.5 g L⁻¹, peptone 5.0 g L⁻¹, agar 20.0 g L⁻¹, with streptomycin 0.30 g L⁻¹ and 75% quintozene wettable powder 1.0 g L⁻¹ added before usage [13]. The amount of Phytophthora was measured in a PDA medium with 3-hydroxy-5-methylisoxazole, benomyl, nyastatin, pentachloronitrobenzen, rifampicin, and ampicillin added at the concentration of 100, 10, 25, 25, 10, and 50 mg L⁻¹, respectively [14]. The B. cinerea selective medium included basic components (NaNO₃ 1.0 g L⁻¹, KCl 0.15 g L⁻¹, K₂HPO₄ 1.2 g L⁻¹, MgSO₄·7H₂O 0.5 g L⁻¹, glucose 20.0 g L⁻¹, agar 25.0 g L⁻¹) as well as fungicide components (pentachloronitrobenzen 0.012 g L⁻¹, penicillin 0.05 g L⁻¹, chloramphenicol 0.05 g L⁻¹, sulphate streptomycin 0.05 g L⁻¹, CuSO₄ 2.2 g L⁻¹, and Rubigan 0.01 mL L⁻¹) added after sterilization [15]. Dilution ratios were chosen properly according to their present amounts in the soil. From the diluted solution, 100 μL was smeared on the various medium plates, each with five replications. After cultivation for 5 days at 25°C, the plate with colony amounts between 10 and 100 was used for the eventual calculation of microbe numbers and population densities. The data were reported as cfu g⁻¹.

2.5 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:

2.6 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].

2.7 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 reaction system consisted of 2 × SuperReal PreMix 10 μL, primers 0.3 μmol L⁻¹, and cDNA template 2.5 μL. 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.

2.9 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.

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, under the concentration of 1, 2, and 4 folds of field recommended dosage, fungicide fluopimomide reduced the amounts of fungi significantly (p < 0.05), while it showed no influence on bacteria and actinomycetes. This result falls following Ji et al., who had reported the excellent compatibility of fluopimomide with Bacillus. 20, 40, and 60 DAT, the amounts of fungi treated by fluopimomide increased gradually with the time-lapse and recovered to the control level at 60 DAT under the recommended dosages. However, the treatments with 2- and 4-fold still inhibited the amount of fungal population. The control fungicide fluopicolide exhibited similar effects with fluopimomide on the amounts of fungi, bacteria, and actinomycetes. Overall, fluopimomide had no significant negative effects on soil bacteria and actinomycetes, and the effects on fungal populations could be recovered within 60 days, indicating a less negative effect on soil microbe.

3.2 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 soil-borne pathogens (Table 3).

3.3 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).

3.4 Effect of fluopimomide on soil enzyme activities

3.5 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 N₂ 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-fixing bacteria, and other bacteria (Figure 2), which might be helpful for tomato plant growth [43].

Figure 2

Effects of fluopimomide on soil nitrogen-related genes.

3.6 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 non-significantly 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.