In field cultivation, including that of vegetables, the use of production systems based on repeated cultivation of the same crop is common, increasing the likelihood of outbreaks of soil-borne diseases. Methyl bromide soil fumigants have been widely used as a means to eliminate soil-borne pathogens, including fungi and bacteria, as well as disease-causing nematodes. Methyl bromide, however, was designated as an ozone-depleting substance in the 1992 Montreal Protocol¹⁾, and its use in Japan was completely stopped from 2012²⁾. As such, there was a need for a new soil sterilisation technology that could replace soil fumigation using methyl bromide, with the development of reductive soil disinfestation (RSD) as a technology combining soil solarisation and flooding³⁾. RSD is a method that produces a reductive soil environment through mixing easily decomposable organic matter (referred to as RSD materials) into the soil, creating a flooded soil state, covering the soil with plastic, and allowing the soil to heat up through sunlight⁴⁾. In the process of raising the soil temperature and producing reductive soil conditions, the density of soil-borne pathogens decreases. The mechanism for this technology has not been completely explained, but it is thought that soil-borne pathogens are killed through the accumulation of organic acids and metal ions (e.g. Fe2 and Mn2 ), produced as a result of the reductive environment caused by reducing the level of oxygen in the soil⁴⁾. Wheat or rice bran are generally used as RSD materials, making it a sterilisation technology that has a low impact on the health of growers through, for example, pesticide drift, or on the environment due to it being a material and cultural method of pest control that does not make use of chemical agents. Suppression through RSD using wheat bran of various types of plant pathogens - including Fusarium oxysporum, Phomopsis sclerotioides, Phytophthora capsici, and Monosporascus cannonballus - as well as nematodes and weeds has been previously reported²⁾. Disadvantages of the method include the difficulty of removing pathogens that live in deeper soil levels such as bacterial wilt Ralstonia soranacearum, due to the fact that sterilisation cannot be accomplished in deep soil levels for RSD using wheat straw or wheat bran⁵⁾⁶⁾, and the unsuitability of using the method in built-up areas, due to the production of foul odours (`drain` smells) from soil that has been reduced⁴⁾. As measures to deal with pathogens that penetrate into deep soil levels, RSD methods have been developed that use blackstrap molasses or low- concentration ethanol that can penetrate these areas⁷⁾. However, using blackstrap molasses or ethanol comes with issues such as cost, or the need for specialised equipment such as liquid fertiliser blenders for processing⁸⁾⁹⁾.  

We used SMS from enoki and bunashimeji cultivated in Nakano City, Nagano Prefecture, and shiitake SMS purchased from The Japan Kinoko Research Center Foundation Tottori Mycological Institute (Tottori City, Tottori Prefecture). The SMS was kept under 5°C in a cool room up until use. 

2. Production of pathogen-contaminated soil
2-1. Production of tomato bacterial wilt-contaminated soil
The bacterial wilt colony incubated on PSA medium at 30°C was collected using a disposable sample stick (AS ONE DS-2), and suspended in a YPGS liquid medium (1g BactoTM Yeast Extract, 10g peptone, 5g glucose, 5g sucrose, 3g sodium chloride, 1L DW). After static incubation of the suspension for 1 day at 30°C, bacterial density was adjusted to 1.0 x 108 cfu/mL (OD600 = 0.4) using a spectrophotometer (Hitachi, U2900 2J0-0005). A liquid mixture of 2mL of the density-adjusted bacterial suspension with 3mL DW added was inoculated through irrigation into a tomato seedling (variety `Sun Cherry Pure`) grown for 1 week, then further developed for 2 weeks in a plant growth chamber (25°C). After 2 weeks the withered plant material on the surface was removed, and the contaminated soil was used for the experiment. 

 2-2. Production of watermelon Fusarium wilt-contaminated soil
A soil wheat bran growth medium (500mL wheat bran, 200mL vermiculite, 200mL Andosol, 100mL tap water) was placed into a culture bottle (8cm diameter), and autoclaved (121°C, 90 minutes). Into the soil wheat bran medium was inoculated a segment of watermelon Fusarium wilt mycelium which had been incubated for 2 weeks on a PDA plate, which was then further incubated for 1 month at 25°C. After 1 month the soil wheat bran medium colonised with the mycelium was mixed through Andosol such that it constituted 5% of the total soil weight (w/w); it was then left standing at room temperature for 1 week, before being used for the experiment. 

2-3. Production of spinach Fusarium wilt-contaminated soil

Spinach seeds (variety `Okame`) were sown onto a petri dish lined with moistened filter paper, and left to stand for 1 week at 15°C. The germinated seeds were planted into a seedling box (319 x 241 x 72mm) filled with 1500g of Andosol, before being grown for 2 weeks at 25°C. A segment of spinach Fusarium wilt mycelium incubated for 1 week on a PDA plate was inoculated into a conical flask filled with 300mL of PDB medium (24g DifcoTM Potato Dextrose Broth, 1L DW), and then cultured while shaking for 1 week. The culture liquid was filtered through a double layer of KimWipes, to extract the mycelium. Centrifugal separation was conducted at 1500 x g for 5 minutes (KUBOTA, Model 2410), with the supernate removed. After the spores had been sufficiently washed through resuspension of precipitated spores in DW and centrifugal separation, a Thoma cell counting chamber was used to adjust the spore density to 1.0 x 106/mL, producing a spore suspension. This was inoculated into the seedling box in which the spinach plants were growing at a ratio of 1mL of spore suspension to 200g of soil. Once the plant material had been confirmed to have died, it was tilled into the soil which was then used for the experiment. 

3. Evaluation of soil reduction effect with SMS treatment
In order to examine the appropriate mixture quantities of SMS, soil reduction tests were carried out using a method adapted from Ōtani⁹⁾ and Ōsaki et al.¹⁶⁾. Into 800g of Andosol (fresh weight) was mixed each type of SMS dried for 24 hours at 100°C using a constant-temperature drying oven (SANYO, MOV-212), at 1, 2, and 5% (w/w) of dry soil weight. This was then filled into 1L wide-mouthed glass bottles. As the control group we used Andosol mixed in the same way with wheat bran. For the water treatment group, we used Andosol only. Following this we added tap water as 61% of the soil dry weight to the bottles. The bottle openings were sealed using agricultural polyethylene film (0.05mm) and a cable tie (301mm). One EP-201 type platinum electrode (Fujiwara Factory) was inserted into a point 2cm left and right of the centre of the agricultural polyethylene film for each bottle, and bonding resin (Bath Bond Q, KONISHI) was applied to the edges of the film to prevent air from entering (Figure 1). The bottles were incubated at 30°C, and the oxidation reduction potential (Eh value) was measured using an Eh meter (PRN-41, Fujiwara Factory) every 2-3 days. 

The reduction effect was also evaluated in the same way for dried and un-dried enoki SMS applied to each contaminated soil sample produced as described above.

4. Odour evaluation of reduction-treated soil 
In order to control the effect of wind, measurements were taken within a plastic case (19.5 x 45 x 24cm). First, a portable odour sensor SP-329m (NEW COSMOS ELECTRIC) was placed into the plastic case, with the sensor value calibrated to 0. Next, measurement was taken by placing the air inlet of the odour sensor at the opening of the reduction-treated soil bottle. As the odour sensor shows relative odour strength, indicated values are evaluated not as units, but through comparison with the control group. 

6. Examination of pathogen density within reduction-treated soil   
The density for each pathogen present within each soil treatment group before and after reduction, was examined using the soil dilution plate method. Into a 200mL conical flask was placed a 10g soil sample and 100mL DW, and a soil extraction liquid was produced by shaking for 30 minutes at 140rpm. The soil extraction liquid was sequentially diluted at 10-1, 10-2, and 10-3, with the 100 μL diluted soil extraction liquid applied to each selected medium. For the bacterial wilt selected medium, Hara-Ono selected medium was used (300g potato, 0.5g calcium nitrate, 2g disodium hydrogen phosphate, 5g peptone, 20g sucrose, 15g agar, 1L DW, 5mL crystal violet [1mg/mL], 5mL cycloheximide [10mg/mL], 1mL polymyxin B sulphate [50mg/mL], 1mL chloramphenicol [10mg/mL], tetrazolium chloride [10mg/mL]). For the Fusarium selected medium, FO-G1 medium was used (1g potassium phosphate, 0.5g potassium chloride, 0.5g magnesium sulphate heptahydrate, 2g sodium nitrate, 0.5g boric acid, 5mg econazole nitrate, 0.2mL micronutrient solution [5g citric acid, 5g ferrous nitrate, 1g zinc sulphate heptahydrate, 0.5g copper sulphate, 0.05g manganese sulphate pentahydrate, 0.05g sodium molybdate, 95mL DW, mixed and autoclaved], 0.25g chloramphenicol, 20g agar, 1L DW, autoclaved and then post-sterilisation adjusted with the addition of 20g l-sorbose, 0.05mL iminoctadine triacetate 25% solution, and 1mg tolclofos methyl 50% wettable powder). Hara-Ono medium was incubated at 30°C and FO-G1 at 25°C for 1 week. The number of colonies that formed on top of the medium was measured, and the pathogen density (cfu/1g soil) was calculated for 1g soil. For the untreated group, pathogen density was similarly measured for contaminated soil that had been left to stand for 1 month. Through comparison with the untreated group, evaluation of the disinfestation effectiveness was carried out. 

7. Disease suppression effects for watermelon and spinach seedlings in reduction-processed soil
Contaminated soil that had undergone reduction processing using enoki SMS for 1 month was mixed with Andosol at a 1:1 ratio and place into pots (8cm diameter), before watermelon seedlings (grafted scion, `Tsukuna no Kaori`; root stock, `Kachidoki No. 2) and spinach seedlings (variety, `Okame`) were planted, and grown for 3 weeks in a growth chamber (14 hours of light/day, 30°C). 

Watermelon stems were cut horizontally, and the level of browning was observed. Evaluation of the degree of disease progression, and calculation of disease rate was carried out according to the following indices/formula. The experiment was conducted twice. 
• Degree of disease progression (indices). 0: no browning; 1: less than 1/3 browning; 2: less that 2/3 browning; 3: more than 2/3 browning. 
• Disease rate (%) = {Σ(index x no. plants of each degree of disease progression)/(3 x no. plants surveyed)} x 100

RSD experiments were conducted using enoki SMS on contaminated polytunnels plots at the Tottori Horticultural Research Center between 2021 to 2023. This was conducted in 2021 and 2022 on tomato bacterial wilt-infected fields, and in 2023 on spinach Fusarium wilt-contaminated fields.  

Into a 15m2 field (3m x 5m) contaminated with tomato bacterial wilt was ploughed, at a depth of 20~30cm using a rotavator, 15kg of enoki SMS processed by drying for more than 48 hours at 80°C, broken up lightly by hand, and then sieved to a 2mm mesh size. As a control group, wheat bran was processed in the same way. Agricultural polyethylene film (0.05mm) was used as a cover, and more than 100L/m2 of tap water was applied to the area using 5 irrigation tubes. The polytunnel was left for 27~29 days in a sealed state. A water-treated group, where only irrigation was carried out, and an untreated group were also produced in the same way. Temperature of the soil at 20cm and 60cm, as well as within the polytunnel, was measured. The Eh value of the soil in each treatment group was measured at a depth of 20cm. Furthermore, a dipyridyl solution (1g dipyridyl reagent dissolved into 500mL 10% acetic acid solution) was sprayed on the soil of each treatment group, with soil reduction evaluated through observation of the colour reaction. From each treatment group 6 soil samples (20cm and 60cm depth) were taken (3 samples x 2 locations), for both pre- and post-reduction treatment, with disinfestattion effectiveness evaluated through measurement of pathogen density using soil dilution plate method with Hara-Ono medium, as described above. 

Once soil reduction treatment was complete the film was removed and the polytunnel was opened up. The soil was then given shallow cultivation and left for a week. Grafted tomato plants (grafted scion `Sun Cherry Pure`; bacterial wilt-resistant root stock `Caddy No.1 [Tokita]) and non-grafted (variety `Sun Cherry Pure`) cherry tomato plants were planted out with 20 plants to each treated plot, spaced in rows 80cm apart (2 rows per plot), and 40cm between plants. Fertiliser was not applied before or during cultivation, and standard management of cultivation was carried out. Evaluation of the degree of bacterial wilt disease progression, and calculation of disease rate was carried out according to the following indices/formula.

• Disease rate (%) = {Σ(index x no. plants of each degree of disease progression)/(4 x no. plants surveyed)} x 100

Into a 20m2 field (8m x 2.5m) contaminated with spinach Fusarium wilt was ploughed, at a depth of 20~30cm using a rotavator, 20kg of dried, or 44kg of undried, enoki SMS. A control plot was prepared in the same way using wheat bran. As with (1), plastic film was laid down and the plot was irrigated with tap water before the polytunnel was left in a sealed state for 33 days. The temperature of the soil at a depth of 20cm, as well as inside the polytunnel, was measured, as was the Eh value of the soil for each treated plot. Soil samples were taken for each control plot (depth 20cm) before and after reduction treatment, with disinfestation effectiveness evaluated through measurement of pathogen density using soil dilution plate method with FO-G1 medium.

The results of tests on odours produced during the process of reduction disinfestation using enoki SMS were as follows: for the wheat bran control treatment, the odour value was between 100~250, whereas for enoki SMS the value was low, between 50~160 (Figure 3). While no significant difference was observed through t testing between the two treatment groups, for the 1% and 2% mixes, throughout the treatment period a tendency for progression at low odour values was observed for the SMS treatment group in comparison to the wheat bran treatment group. Offensive odours caused by RSD are thought to be caused by the breakdown of proteins that exist within organic material such as wheat bran¹⁹⁾, suggesting suppression of offensive odours through difference in protein content between SMS and wheat bran.

2. Disinfestation effect using enoki SMS treatment on pathogen-contaminated soil
The results of reduction treatment using enoki SMS on each type of contaminated soil are as follows: for all the contaminated soils, where SMS was used an Eh value of -100 was reached within 1 week, subsequently maintaining a stable reductive state, and with a similar level of reduction observed in the wheat bran control group (Figure 4). The Fusarium contaminated soil mixed with 2% undried enoki SMS showed a similar level of reduction to that treated with dried enoki SMS. 

The elution-tested concentration of metal ions Fe² and Mn² was measured for the supernatant of each type of contaminated soil for which reduction disinfestation was carried out for 1 month using enoki SMS (Table 1). The results showed the same Mn2 concentration value as for the wheat bran control group, with Fe2 concentration the same value or slightly lower than the wheat bran control group. Soil reduction for the SMS treatment group was confirmed through the higher concentration of metal ions in comparison to the water treatment group. Furthermore, the fact that elution of both metal ions was confirmed during the water treatment of Fusarium-infected soil can be thought to be the impact of reduction caused by wheat bran included within the base soil substrate inoculated during the process of producing contaminated soil.  

The results of investigation into the density of each pathogen contained within the soil of each treatment group before and after soil disinfestation (Table 1) showed an increase, for bacteria-wilt contaminated soil, in the bacterial density of the untreated and water-treated group in comparison to before reduction disinfestation. In contrast, the bacterial density decreased by, respectively, 87% and 93% for the SMS treatment group and the wheat bran treatment group. In watermelon Fusarium-wilt contaminated soil, fungal density was 0 for the SMS and wheat bran treatment groups in comparison to before treatment, demonstrating a striking level of suppression. For spinach Fusarium wilt contaminated soil as well, fungal density reduced by more than 99% for both the undried and dried enoki SMS treatment groups, and the wheat bran treatment group. A substantial reduction in pathogen density was observed in all the contaminated soils through RSD using SMS, demonstrating a disinfestation effect. 

Next, watermelon and spinach seedlings were planted into contaminated soil that had undergone reduction disinfestation, to examine the onset of disease. For watermelon seedlings the results were as follows: the untreated group had a disease rate of 55.0±2.9%; in contrast, the SMS treatment group and the wheat bran treatment group both had a disease rate of 7.5±0.5%, and a suppression rate of 86.4% (Figure 5). A statistically significant level of suppression of disease onset was thus shown through reduction treatment using SMS. For spinach seedlings the results were as follows: the untreated group had a disease rate of 86.7±10.9%; in contrast, the undried and dried enoki SMS treatment groups both had a disease rate of 13.3±10.9%, and a suppression rate of 84%, showing statistically significant suppression of disease onset for SMS treatment. The wheat bran treatment group had a suppression rate of 88%, meaning that a similar disease suppression effect was observed in the SMS treatment group as with the wheat bran treatment group. 

Soil samples were collected following reduction disinfestation in 2021 and 2022 for each treatment group at depths of 10cm, 20cm, and 60cm. Examining the reduction state of the soil through dipyridyl reaction showed a reddish-brown colour for soil at depth of 10cm and 20cm for the SMS and wheat bran treatment groups, but soil at a depth of 60cm showed either a light red colour, or no colour at all. This suggests that reduction occurred up to a depth of 20cm, but that at a depth of 60cm adequate reduction did not occur, and there is low disinfestation effect for pathogens existing at lower soil depths. 

Measuring the EC value of soil samples taken before and after reduction gave 0.10 mS/cm for soil at a depth of 20cm for the SMS treatment group, and 0.16 mS/cm at 60cm, lower than that prior to processing (0.24mS/cm at 20cm, 0.22mS/cm at 60cm). The pH value of the soil was measured at pH6.78 at 20cm and pH5.64 at 60cm, with no striking difference observed with that prior to treatment (pH6.89 at 20cm, pH6.32 at 60cm).

Surveys were conducted on plant growth and disease rate after planting out both grafted and non-grafted tomato plants (in 2022, grafted tomatoes only) in each of the control plots following reduction disinfestation. For both years no impact was observed on reduction-treated tomato growth. In the 2021 survey into disease level, non-grafted tomatoes planted into the wheat bran treated plot and the untreated plot showed a disease rate at 21 days after planting of, respectively, 30.0±10.6% and 26.3±2.7%; no disease was observed at the same point in the SMS treated plot (Figure 8A). At 25 days after planting, the disease rate for the SMS treated plot was 55.0±10.6%, but had the slowest onset of disease. For the grafted tomatoes, while a disease rate of 3.8±0.9% was confirmed in the untreated plot at 25 days after planting, for the wheat bran and SMS treatment plots the disease rates 32 days after planting were 10.0±0.0% and 2.5±1.8% respectively, showing statistically significant disease suppression in comparison to the untreated plot (Figure 8A). At 57 days after planting the disease rate was 95.0±3.5% for the untreated plot, 67.5±5.3% for the wheat bran treatment plot, and 60.0±14.1% for the SMS treatment plot, with the SMS treatment plot progressing at the slowest rate (Figure 8A). For the 2022 disease rate survey, onset of disease in the untreated plot was seen on the 25th day after planting, whereas for the SMS treatment plot this occurred on day 32 after planting, having the slowest disease onset among the treatment plots (Figure 8B). The disease rate at day 86 after planting was 48.8±4.4% for the SMS treatment plot, 72.5±0.01% for the wheat bran treatment plot, and 75.0±12.4% for the untreated plot, with the SMS treatment plot progressing at the slowest rate (Figure 8B). 

3-2. Disinfestation effect in fields contaminated with spinach Fusarium wilt
Tests were conducted on spinach Fusarium wilt-contaminated fields from 29th July to 1st September 2023 (34 days). For the dried and undried SMS treatment plots an Eh value of -100mV was reached on the 4th day after treatment, with -100mV subsequently maintained (Figure 6C). For the untreated plot, no drop in potential was observed. Soil samples following reduction disinfestation were taken at a depth of 20cm for every treatment plot, with the results of tests on the reduction state through dipyridyl reaction showing a red colour for both the undried and dried SMS treatment plots and the wheat bran treatment plot, confirming reduction. The soil prior to reduction treatment was at pH6.7 which, following treatment, was at pH 7.3 for the undried and dried SMS treatment groups, with generally no change. In contrast, the EC value dropped, from 0.46 mS/cm prior to treatment, to 0.25 mS/cm for the undried and dried SMS treatment plots. 

Next, soil samples taken both before and after reduction disinfestation were measured for the density of Fusarium wilt. While the density prior to treatment was 104~105 cfu/1g of soil, following treatment for the undried and dried SMS treatment plots and the wheat bran treatment plots no colonies were detected, with a significant decrease in fungal density (Table 2). The density for the untreated plot was 103 cfu/1g of soil. Results from tests on the growth and disease rate of spinach plants planted into each treatment plot following reduction disinfestation (Table 2) showed a statistically significant increase in overall plant length and above-ground weight for the undried/dried SMS treatment plots and wheat bran treatment plot in comparison to the untreated plot. The rate of Fusarium wilt disease also reduced, with the number of infected plants at 1.0~1.5% for the undried/dried SMS treatment plots and the wheat bran treatment plot, in comparison to 39.0% for the untreated plot (Table 2). Previous research²⁰⁾ has suggested that application of used shiitake substrate in soil used for growing sweetcorn improves harvest weight in comparison to untreated soil, having a similar fertilisation effect to that of artificial fertiliser. Through the use of RSD using SMS it can be thought that components within the SMS contribute to the stimulation of plant growth. It can be inferred from the research shown here that the main factor in the promotion of spinach growth for the undried SMS treatment plot in comparison to the dried SMS treatment plot is the decomposition or denaturing of fertilising compounds within SMS through drying at high temperatures. Further study is necessary on the fertilisation effects of RSD using SMS, for each agricultural crop. 

As shown above, similar disinfestation effects have been observed for RSD tests using undried or dried SMS in spinach Fusarium wilt-contaminated fields to reduction tests conducted inside the laboratory. As Fusarium wilt does not live deep below the soil surface, but rather at a soil depth of 20cm at which adequate reduction has been shown, we can consider there to be a high disease suppression effect.  

Part of the results of this research received support from the JST Adaptable and Seamless Technology Transfer Program through Target Driven R&D (A-STEP) Try-out JPMJTM20FT. 

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