Assessment of the potential of a reduced dose of dimethyl disulfide plus metham sodium on soilborne pests and cucumber growth

Methyl bromide (MB), a dominant ozone-depleting substance, is scheduled to be completely phased out for soil fumigation by December 30th 2018, in China. The combined effects of dimethyl disulfide (DMDS) plus metham sodium (MNa) were assessed in controlling soilborne pests for soil fumigation. A study was designed in laboratory for the evaluation of the efficacy of DMDS + MNa to control major soilborne pests. At the same time, two trials were conducted in cucumber field located in Tongzhou (in 2012) and Shunyi (in 2013), respectively, in order to assess the potential of DMDS + MNa in controlling soilborne pests. Laboratory studies disclosed positive synergistic effects of almost all four used combinations on Meloidogyne spp., Fusarium spp., Phytophthora spp., Abutilon theophrasti and Digitaria sanguinalis. Field trials found that DMDS + MNa (30 + 21 g a. i. m−2), both at a 50% reduced dose, effectively suppressed Meloidogyne spp. with a low root galling index (2.1% and 11.7%), significantly reduced the levels of Phytophthora and Fusarium spp. with a low root disease index (7.5% and 15.8%), gave very high cucumber yields (6.75 kg m−2 and 10.03 kg m−2), and increased income for cucumber growers with the highest economic benefits (20.91 ¥ m−2 and 23.58 ¥ m−2). The combination treatment provided similar results as MB standard dose treatment (40 g a. i. m−2) or DMDS standard dose treatment (60 g a. i. m−2) in pest control and yield, but was more effective than MNa standard dose treatment (42 g a. i. m−2). Usage of all chemical treatments gave better significant results than the untreated group of control. Considering the economic benefits, the DMDS plus MNa combination (30 + 21 g a. i. m−2) could be used for soil fumigation in cucumber production in China.


Results
Laboratory studies. Root-knot nematodes. All four applied rates of DMDS + MNa combination tested treatments showed a positive synergistic effect on root-knot nematodes (Table 1), by decreasing the Meloidogyne spp. numbers by 90.7%. The maximum practiced dose in our study (80 + 40 mg a.i. kg −1 ) decreased the number of Meloidogyne spp. in terms of juveniles by 94.2%.
Soilborne fungi and oomycetes. Positive synergistic efficacy of four rates of DMDS + MNa tested was also observed on both Phytophthora and Fusarium spp. (Table 1). The infestation levels through Fusarium and Phytophthora spp. were decreased by 78.5% and 62.0%, respectively. The maximum applied dose in current study (80 + 40 mg a.i. kg −1 ) decreased Fusarium spp. 93.6% and Phytophthora spp. 74.1%.
Field trials. Root-knot nematodes. The control groups at trial one (in 2012) and trial two (in 2013) were found seriously occupied by Meloidogyne spp. ( Table 2). The significantly higher levels of Meloidogyne spp. were observed in the control group than to all treatments of fumigant (P = 0.05).
The chemical treatments, except for MNa treatment, decreased the nematode infestation levels in trial one and trial two by 86.1% and 88.5%, respectively. The combination treatment of DMDS + MNa suppressed nematode levels by 86.1% and 89.7% in trial one and trial two, respectively; and both results statistically were not found different from MB or DMDS treatment, but higher significantly than MNa treatment at trial one (P = 0.05).
Soilborne fungi and oomycetes. The groups of control were seriously infested by both Phytophthora and Fusarium spp. in both trials ( Table 2). The levels of infestation with Fusarium and Phytophthora spp. were observed maximally significant in the control untreated group than to all fumigant treatments (P = 0.05) ( Table 2).
The lowest Fusarium spp. levels were both provided by MB treatment at both trials, followed by the combination DMDS + MNa treatment, which decreased 93.6% Fusarium spp. and 80.0% at trial one and two. (Table 2).  www.nature.com/scientificreports www.nature.com/scientificreports/ Similarly, MB treatment also had the lowest Phytophthora spp. level, followed by the DMDS + MNa treatment, and reduced 86.2% and 81.1% Phytophthora spp. in trial one and two, respectively (P = 0.05) ( Table 2). While no statistically change was observed between the MNa + DMDS treatments and MB treatment (P = 0.05) ( Table 2).
The first cucumber fruit yield. The 1st production of cucumber fruit was varied with fumigation treatments (Table S1). Cucumbers that were grown in the untreated control plots provided the minimum yield of first fruiting (0.02 and 0.12 kg m −2 , respectively) at both field trials (Table S1). No significant change was shown between the chemical treatments at both trials. And the first fruit yield in all chemical treatments were significantly higher than the untreated group of control except for MNa treatment at trial one, 2012 (P = 0.05) (Table S1).
Cucumber plant height, root galling index and root disease index. The height of cucumber fruit plant at 4 WAT varied with fumigation treatments at both field trials (Table 3). Throughout the chemical treatments at both trials, significantly higher plant height was observed compared to the untreated group of control. And no significant change was shown between the chemical treatments apart from the DMDS treatment at trial one, 2012 (P = 0.05) ( Table 3).
By the end of all trials, the nematode infestation was calculated through index of root galling. Root galling index was significantly affected after fumigant treatments (P = 0.05). The maximum root galling indexes were both noticed in the untreated plots (61.4% and 78.3% at trials one and two, respectively). At trial one, the lowest index of root galling was provided by MB treatment, which was not different statistically from DMDS + MNa or DMDS alone, but observed lower significantly than MNa alone. At trial two, the minimum root galling index was also gained in MB treated plots, which did not change significantly from DMDS + MNa treatment but was lower significantly than DMDS alone or MNa alone (P = 0.05) ( Table 3).  Table 3. Effect of fumigation programs on cucumber plant height, root galling index, and root disease index. While comparing with the untreated control groups, chemical treatments affected root disease index significantly (P = 0.05). The maximum index for root disease (50.0% and 67.5%) were provided by the untreated control plots at two trials. At trial one, the lowest root disease index (3.3%) was provided by MB treatment, which was not different statistically from DMDS + MNa (7.5%), but was lower significant than DMDS or MNa alone. At trial two, the lowest root disease index (4.2%) was observed in treated MB plots, which was not significantly different from DMDS + MNa treatment or DMDS alone, but was observed lower than MNa alone, significantly (P = 0.05) ( Table 3).
Similarly, the gross income of grower through the production of cucumber also varied with chemical treatments (Table 4). Minimum gross income from cucumber cultivation without treatment was obtained (9.67 and 12.50 ¥ m −2 , respectively). The plots treated with MB provided the highest gross incomes (22.47 and 26.24 ¥ m −2 , with 132.0% and 109.9% increase, respectively, at trials one and two), which was not different significantly from the DMDS + MNa treatment (22.33 and 25.00 ¥ m −2 , with 129.9% and 100.0% increase, respectively, at trials one and two) or DMDS alone (19.14 and 23.31 ¥ m −2 , with 96.9% and 86.5% increase, respectively, at trials one and two), but significantly higher than MNa alone (16.93 and 20.05 ¥ m −2 , with 74.2% and 60.4% increase, respectively, at trials one and two) (P = 0.05) ( Table 4).
In addition, the economic benefits (also called net income) of different soil fumigation treatments were assessed. Fumigation cost of different soil treatments were calculated by the sum of fumigants cost and film cost (Table S2). The DMDS + MNa treatment had a relatively low fumigation cost (1.42 ¥ m −2 ), which was lower than MB treatment (4.07 ¥ m −2 ) or MNa alone (1.60 ¥ m −2 ) ( Table 4). Net income of growers was finally calculated by the difference of the gross income and fumigation cost (Table 4). In trial one and two, the treated plots with combination DMDS + MNa both had the highest net income (116.2% and 88.6% increase, respectively), followed by MB treated plots (90.3% and 77.4% increase, respectively), DMDS alone treated plots (85.0% and 76.5% increase, respectively), MNa alone treated plots (58.5% and 47.6% increase, respectively) and the untreated plots (P = 0.05) ( Table 4).

Discussion
To control root-knot nematode, DMDS is proven equally effective to MB not only in China but elsewhere 16,17,29 , which was confirmed in our both field trials. Anyhow, the overall formulations of DMDS exhibit poor efficacy against soilborne fungi and oomycetes, and moderate activity against weeds. So, additional herbicide and fungicide would be needed to improve soilborne fungi, oomycetes and weed control. MNa, a MITC producer, can control soilborne fungi, oomycetes and weeds efficiently. In the current study, it was observed that the MNa was comparatively less effective ton soilborne fungi and oomycetes than weeds. Thus, the combined use of DMDS and MNa as a promising soil fumigant is at least possible theoretically. All of the combinations of DMDS and MNa in laboratory studies exhibited positive synergy effects on root-knot nematodes, 2 major weed seeds and key soilborne fungi and oomycetes.  Table S2. e Cucumber net income was calculated by the difference of cucumber gross income and fumigation cost. f In each column, data are means of three repeats. Means followed by the same letter are not different (P = 0.05) according to the LSD test.
The mechanism of specific synergistic activity of DMDS and MNa was still not clearly understood but the laboratory outcomes presented above, preliminarily indicated the possibility of their synergistic usage and affirmed the synergy activity of DMDS and MNa as previously reported by Gerik and Hanson 28 . DMDS is found to inhibit complex IV of mitochondrial electron transport chain (ETC), decrease the intracellular ATP concentration subsequently, which thereby activated neuronal KATP channels mediating membrane hyperpolarization and reduction of neuronal activity 30 . MITC as the active biocidal product of MNa is believed to react with amines and thiols in biological molecules 31 . It will be very difficult to assess a single mechanism of action that accounts for the synergism observed for the combination of DMDS plus MNa. However, two possible mechanisms may offer insight into the synergistic response of the fumigant combination 32 . One mechanism suggested is that sub-lethal exposure of soilborne pests to one fumigant may alter cell processes to such an extent that exposure to a second fumigant is more deleterious than would normally be expected 33 . Another suggested mechanism is that one fumigant causes changes in cell-wall permeability that increases the uptake or decreases the efflux of a second fumigant from the cell 34 . Further understanding of the activity of DMDS and MNa is necessary before the specific synergism mechanism can be deduced.
Our field trials showed that the combined use of DMDS and MNa at 30 + 21 g a.i. m −2 rate successfully inhibited Meloidogyne spp. root galling, decreased the colony-forming units (cfu) of Fusarium spp. sharply and Phytophthora spp. on media, sustained maximum production of cucumbers at the same time, and was not different significantly from 40 g m −2 MB dose or DMDS alone at 60 g m −2 dose, but improved than MNa alone at 42 g m −2 dose (P = 0.05). The results of field trials above confirmed that the combinations of DMDS and MNa are good choices than alone MB application. Furthermore, we compared the combination of DMDS plus MNa with the used soil fumigant combinations, such as 1,3-D/DZ, 1,3-D/Pic, DMDS/DZ, 1,3-D/DMDS and 1,3-D/MNa (Table S3). Indeed, the combination of DMDS plus MNa is not the best alternative of MB, if only considering the control efficacy. However, considering the application methods and forbidden use issue at the same time, the combination of DMDS plus MNa is more promising than other combinations to replace MB.
Commercial preparations of MNa typically consist of 42% MNa in an aqueous solution at a self-buffered pH > 7. True MNa and halogenated chemicals mixtures (e.g. 1,3-D and Pic) are not available currently due to chemical incompatibility arising when combining them 35,36 . However, the different combinations of DMDS and MNa did not show any degradation according to the synergy effect in our laboratory in the current tests, which may be attributed to the relatively high rates of application 4 .
MNa requires relatively rigorous prerequisites just like DZ for the effective application. The aqueous solution of MNa is diluted by water and applied to the moist soil, where the compound rapidly decomposes to produce MITC. In the vapor and liquid phase of the soil, MITC is toxic to various soilborne pests. However, the physical and chemical properties of MITC (low water solubility, low vapor pressure, and low Henry's constant) limit its movement and distribution in the soil following MNa application 37,38 . DMDS is highly volatile and a small molecule, so it diffuses and moves in the soil more quickly than MITC 30 . In this point, DMDS could make up the disadvantage of MITC and improve the control efficacy of the combination. Anyhow, uniform distribution in soil is needed by effectual mechanical injection or chemigation system. For better distribution of fumigant in soil, MNa is suitable to be applied by broadcast application or shank injection rather than chisel injection. If there was a good chemigation system available in the field, applying the fumigants by chemigation couldn't be better.
In concluding remarks, the combination of DMDS and MNa fumigant applied by chemigation was almost equally to MB in terms of controlling knot-nematodes along with soilborne fungus while sustaining high cucumber yields and income for growers. However, much of the detailed work on its application type including its formulations and suitable composites with biological agents (eg. Trichoderma asperellum, Bacillus subtilis or others) are desirable before the DMDS and MNa combination can be suggested as an efficient substitute to MB for the yield of cucumber in China. The side effects of the combinations on soil beneficial organisms (eg. earthworms, actinomycetes, etc.) should also be considered in further studies.

Materials and Methods
Laboratory studies. The combined and single efficacy of DMDS and MNa, was assessed under laboratory study. The collection of soil samples was taken from the 20 cm top in a cucumber field at Shunyi, Beijing, where nematodes and soilborne pests' presence was abundant. The composition of soil sample was 20.34% sand, 76.25% silt, and 3.41% clay, with pH 6.40 and organic matter content 35.64 g kg −1 soil. A soil sieve of 2-mm mesh was used and then mixing was done thoroughly. The moisture of soil was recorded 23.80% (w/w). Analysis for particle size were estimated through pipette method 39 . pH was determined in a 1:2.5 soil to water extract. Organic carbon contents were determined by wet oxidation followed the method of Walkley and Black 40 . Soil moisture contents were performed by remaining the soil at 105 ± 5 °C for 6 hours until constancy of mass was reached 41 .
Soil samples with six hundred grams weight were placed into each of 27 (2.5 L) desiccators. In each desiccator, 10 Abutilon theophrasti seeds and fifteen Digitaria sanguinalis seeds were buried at 2 cm depth, respectively. The following fumigant treatments were performed with three replicates: DMDS alone (40, 80 mg a.i. kg −1 soil), MNa alone (20, 40 mg a.i. kg −1 soil), DMDS + MNa (40 + 20, 40 + 40, 80 + 20, 80 + 40 mg a.i. kg −1 soil) and untreated group of control. DMDS or MNa was (Eppendorf, Germany) injected into the soil by pipette, and then the desiccator was sealed immediately. The laboratory study photo (see Fig. 1a.) was taken by the authors. The desiccators were placed at 28°C for five days, and then covers were remained open for one day to release the fumigant residues. The height of weed was then determined. Fusarium and Phytophthora spp. were both separated from the fumigated soil quantitatively based on the methods of Komada 42 and Masago et al. 43  Randomized blocks design of chemical treatments was performed with three replicates (Table 5). In field trial one, the size of each plot was 17.92 m 2 (3.2 m wide by 5.6 m long). A treatment as a reference of MB, DMDS alone, MNa alone, DMDS combination with MNa and untreated group of control were established. The application of DMDS was done through chemigation at 60 g a.i. m −2 rate; in order to increase the solubility of DMDS in the water, approximate 4% addition of Tween 80 is necessary 13 . MNa was practiced by chemigation at 42 g a.i. m −2 rate. Combined treatment of DMDS and MNa was practiced as follows: apply MNa by chemigation at 21 g a.i. m −2 rate, and then apply DMDS by chemigation 30 g a.i. m −2 rate. To cover the above treatments, PE film was placed. Application of MB between PE sheet and soil was through the hot gas method at 40 g m −2 rate. The fumigants were applied on 27 July 2012, and the tarps were removed on 17 August 2012. The cucumbers (cultivar "No.16, Zhongnong") were transplanted on 28 August 2012, and the final root disease investigation was conducted on 3 December 2012. In field trial two, the size of each plot was 20.16 m 2 (3.6 m wide by 5.6 m long). All of the treatments in field trial two were the same as those in field trial one ( Table 5). The fumigation period was from 10 September 2013 to 26 September 2013. The field trial photo (see Fig. 1b.) was also taken by the authors. The cucumbers (cultivar "No.12, Jinyou") were transplanted on 5 November 2013, and the final root disease evaluation was conducted on 28 May 2014.
Populations of soil fungi [colony-forming units (cfu) g −1 soil] were evaluated after fumigation treatment from soil samples at 0-20 cm depth from the surface of soil. Soil samples were collected from 3 spots in each plot along the diagonal lines. Root-knot nematode densities in the soil were estimated after soil fumigation from 0-20 cm   Statistical analyses. Laboratory studies. Mortality of nematode was calculated by the equation following.
where X is % nematode mortality, N 1 is the number of dead nematodes, and N 2 is the number of the live nematodes. Corrected mortality of nematode was considered by the equation following.
where Y is the % corrected nematode mortality, while X 1 is the % nematode mortality of fumigated treatments, and X 2 is the % nematode mortality of untreated control. Controlling fungi or weed seeds efficacy was measured according to the equation following.
where Y is the fungi or weed seeds control efficacy, X 1 is the fungal populations or weed height in untreated control group, X 2 is the fungal populations or weed height in treated fumigated plots. The DMDS plus MNa control efficacy was calculated by the equation following, using methods described by Limpel et al. 46 . where E 0 is the expected control efficacy of DMDS plus MNa, X 1 is the actual measured control efficacy of DMDS, X 2 is the actual measured control efficacy of MNa. E is the actual measured control efficacy of DMDS plus MNa. If E > E 0 , the combination of DMDS plus MNa was synergistic; if E < E 0 , the combination of DMDS plus MNa was antagonistic.
Field trials. The effectiveness of controlling root-knot nematode or soilborne fungi or oomycetes was measured according to the Eq. (3). Root galls scores or disease scores recorded for each plot were respectively converted into root galling index (% RGI) or root disease indices (% RDI) using the formula described by McKinney 45 : where ƒ = number of plants in each class, v = class value, N = number of observed plants, and X = highest value of the evaluation scale. Statistical software SAS (SAS, version 8.0 for Windows) was run to conduct analysis of variance (ANOVA) for data analysis. Data for populations of soilborne fungus and knot-nematode were transformed as necessary (log10 for large numbers [>100] and square root transformations for small numbers [<100] for statistical analyses), however, all of the data reported here as non-transformed values. Fisher's LSD test at P = 0.05 was used for significant differences determination among means.