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Article

Evaluation of Allyl Isothiocyanate and Ethylicin as Potential Substrate and Space Fumigants in Tomato Greenhouses

by
Guangming Chen
,
Min Zhang
,
Zhaoai Shi
,
Aocheng Cao
,
Qiuxia Wang
,
Dongdong Yan
,
Wensheng Fang
and
Yuan Li
*
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan Road, Beijing 100193, China
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(23), 2502; https://doi.org/10.3390/agriculture15232502 (registering DOI)
Submission received: 3 November 2025 / Revised: 26 November 2025 / Accepted: 30 November 2025 / Published: 1 December 2025
(This article belongs to the Special Issue Diseases Diagnosis, Prevention and Weeds Control in Crops—2nd Edition)
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Abstract

Continuous use of substrate cultivation can easily lead to the accumulation of crop pathogens, leading to widespread crop diseases. It is necessary to screen suitable and efficient substrate and space fumigants to keep the healthy development in substrate and greenhouses. This study systematically evaluated the effects of allyl isothiocyanate (AITC) and ethylicin fumigation on pathogens present on the substrate inside greenhouses. The average populations of Fusarium spp. and Phytophthora spp., bacterial and fungal community structures, tomato growth and yield were investigated and analyzed. The results demonstrated that both AITC and ethylicin exhibited significant inhibitory effects on Fusarium spp. and Phytophthora spp. in the substrate, with control efficiencies of 94.2% and 87.5%. Furthermore, these agents achieved 100% inhibition against Fusarium spp. while exceeding 90% Phytophthora spp. in the greenhouse space. Fumigation treatments significantly reduced pathogenic bacteria and increased beneficial microorganisms like Bacillus, Streptomyces and Brevibacillus in the substrate. Additionally, tomato yields increased significantly by over 45%. This study presents the first report on AITC and ethylicin as potential efficient fumigants easily used for both substrate and greenhouse space fumigation, which demonstrates excellent control effect on crop pathogens, with potential application in commercial tomato production in greenhouses to support sustainable agricultural practices.

Graphical Abstract

1. Introduction

Vegetables crops in greenhouses are less influenced by environmental conditions and temperature fluctuations, enabling stable growth regardless of geographical constraints [1]. As one of the most widely consumed and nutritionally rich vegetables globally, the cultivated area and global production of tomatoes continue to grow each year [2]. However, due to limitations in economic conditions and available land, tomato cultivation often faces challenges associated with continuous cropping. Long-term continuous cropping can lead to a decline in soil fertility, disrupt the physical and chemical properties of the soil and disturb the microbial balance, ultimately resulting in serious crop diseases [2,3]. Therefore, optimizing planting practices and implementing effective strategies to control soil-borne diseases are crucial for achieving a more significant tomato production.
Soilless cultivation is a method of growing plants without relying on soil as a rooting medium [4]. This approach utilizes materials such as peat, coir and wood fiber to anchor the plant roots [5]. The substrate used in this method is diverse in origin and typically offers superior hygiene and ease of sterilization compared to traditional soil. Their excellent drainage and air permeability enhance nutrient absorption, fostering the development of a robust root system [6]. Furthermore, the effective use of the substrate stimulates microbial activity and significantly suppresses the incidence of wilt disease [7]. Nevertheless, continuous use of substrates or poor substrate management still poses significant challenges related to continuous cropping obstacles. For instance, high-nitrogen and high-humidity substrate environments significantly promote the proliferation and growth of pathogenic microorganisms such as Fusarium spp., Phytophthora spp., Pythium spp. and Pectobacterium spp. [8,9]. Currently, substrate disinfestation predominantly employs physical, chemical and biological approaches [10]. However, each method encounters substantial limitations in practical implementation. A primary concern lies in chemical disinfestation, which utilizes various fumigants. While physical and biological disinfestation methods offer more sustainable alternatives, their practical application is hindered by inconsistent efficacy and prolonged treatment durations [11]. Moreover, conventional water-intensive disinfestation practices demand excessive water consumption. The development of safe, efficient, and environmentally friendly chemical disinfestation methods remains a critical research direction for addressing substrate continuous cropping obstacles.
Allyl isothiocyanate (AITC) and ethylicin are two potential alternatives to methyl bromide (MB) [12,13]. The primary component of AITC is isothiocyanate, which is predominantly found in cruciferous vegetables [14]. Ethylicin is a biomimetic chemical pesticide synthesized based on the structure of allicin, which can be extracted from plants or produced through artificial synthesis. These fumigants are characterized by their low toxicity, abundant availability of raw materials, and facile degradability with minimal environmental impact. They can be efficiently administered through multiple application methods, including drip irrigation and spraying [15]. Multiple studies have demonstrated that AITC can effectively control soil-borne diseases, root-knot nematodes and weed seeds [3]. Wu et al. [16] evaluated the biological activity of 15 isothiocyanates (ITCs) against cucumber root-knot nematodes and revealed that AITC was particularly effective when applied in the field conditions at the rate of 1.0 Kg·ha−1. The encapsulation of AITC with modified diatomite granules leads to an extended half-life, providing effective control of soil pathogens for approximately seven months [17]. Drip-irrigated and injected AITC have inhibitory effects on Phytophthora spp. and weed seeds [18]. Ethylicin has shown significant efficacy in the prevention and control of Phytophthora infestans and Xanthomonas oryzae pv. oryzicola (Xoc) [19,20].
Although AITC and ethylicin have demonstrated significant efficacy in controlling soil-borne diseases, their impacts on non-target microorganisms and their effectiveness in greenhouse space disinfestation have not been thoroughly investigated. The research focused on three key aspects: (i) innovative application of allyl isothiocyanate and ethylicin as substrate fumigants in preventing and controlling crop pathogens; (ii) the suppression efficacy against pathogenic microorganisms; (iii) the potential for yield enhancement in tomato cultivation.

2. Materials and Methods

2.1. Cultivation Substrate and Greenhouse Space Disinfestation

The experiments were carried out from July 2021 to June 2022 in greenhouses located in Changping, Beijing (GPS: 39°34′ N, 115°46′ E), China, an area with a long history of large-scale tomato production. The substrate used in the study was a composite of peat, coco coir, and vermiculite, mixed in the proportions of 50% peat, 30% coco coir, and 20% vermiculite, with a pH of 7.67, electrical conductivity of 165.00 µS/cm, and organic matter content of 16.56%. The concentrations of nitrogen (N), phosphorus (P), and potassium (K) were measured at 35, 68, and 48.37 mg/100 g of soil, respectively. The methods for determining the soil physicochemical properties were referenced from the study by Shi et al. [21].
The experimental materials utilized in this study are presented as follows: AITC (98%) (Jiangsu Tenglong Biological & Medicinal Co., Ltd., Yancheng, China); Ethylicin (80%) (Hainan Zhengye Zhong nong Hi-tech Co., Ltd., Haikou, China); PE plastic film (Shandong Kang Ye Plastic Products Co., Ltd., Linyi, China); Electrostatic knapsack sprayer Model: SX-LK16 (Máquinas Agrícolas Jacto S.A, Pompéia, Brazil); Electronic bench (Kaifeng Group Co., Ltd., Yongkang, China); Tomato variety Jingfan 309 (Jingyan Yinong Beijing Seed Industry Science and Technology Co., Ltd., Beijing, China).
Five experimental treatments were carried out, each with three replicates: (1) Untreated control (CK); (2–5) Chemical fumigations with AITC and ethylicin (each agent available in two concentrations). The concentrations of AITC and ethylicin were established based on the results of preliminary experiments. Treatments included: High-dose AITC at 14 g/m2 (AH), low-dose AITC at 7 g/m2 (AL), High-dose ethylicin at 8 g/m2 (EH) and low-dose ethylicin at 4 g/m2 (EL). The treatments have been summarized and organized into Table 1.
The substrate beds were constructed as prefabricated furrows with dimensions of 0.3 m in width, 0.3 m in depth, and 15 m in length, and were directly laid on the greenhouse floor. This study was carried out on 14 July to perform substrate fumigation and spatial disinfestation experiments in greenhouse conditions. The row spacing between substrate beds was 0.6 m, and the bottom of each furrow was lined with a root-barrier membrane to prevent downward root penetration. Prior to transplanting, the entire substrate beds underwent fumigation treatment.
The substrate fumigation followed a standardized protocol: initially, the substrate surface was covered with 0.04 mm thick polyethylene (PE) plastic film. Next, trenches were dug around the perimeter of the film using a shovel and backfilled with soil to form a sealed plastic film on all sides, thereby preventing gas leakage from the substrate interior during fumigation and maximizing disinfection efficacy. Subsequently, AITC and ethylicin were applied at previously indicated experimental doses through a drip irrigation system. The fumigants were mixed with water and applied quantitatively using an alternating application method, where fumigant solution and clean water were applied at 30 min intervals to ensure uniform distribution within the substrate. Concurrent spatial disinfestation was performed using an electrostatic knapsack sprayer (Model: SX-LK16), which delivered comprehensive disinfestation treatment to the greenhouse interior, covering films, and ground surfaces, with application rates standardized according to substrate fumigant dosage. After 7 days of fumigation, the plastic film was removed, followed by a 3-day forced ventilation period to ensure complete volatilization of residual fumigants from the substrate.

2.2. Pathogen Analysis

2.2.1. Cultivation Substrate Sampling and Storage Conditions

Substrate samples (500 g each) were randomly collected using a shovel from a depth of 10–20 cm, thoroughly mixed, and replicated in triplicate for each treatment. Two sets of samples were collected: the first set was obtained on the 7th day after fumigants application, and the second set was collected at the time of tomato seedling planting. Immediately after collection, the samples were transferred to the laboratory. The homogenized samples were then divided into two copies: one copy of samples was stored at 4 °C for the detection of Fusarium spp. and Phytophthora spp., while the other one was stored at −80 °C for subsequent DNA extraction and analysis to assess microbial diversity.

2.2.2. Greenhouse Space Sampling and Storage Requirements

The primary objective of space disinfection is to eliminate residual pathogens in the external space of the plastic film following substrate disinfection. To assess the quantity of residual pathogens in the greenhouse space, sterile cotton swabs were primarily used to swab designated space sampling points. These space sampling points mainly encompassed the plastic film surface on the greenhouse roof and the ground. Sampling was performed using a five-point random method, with the cotton swabs immediately transferred into sterile EP tubes and securely sealed thereafter. Two sets of samples were collected: the first set prior to applying the fumigants, and the second set immediately after fumigation and removal of the film. To ensure statistical reliability, three replicate samples were collected from each sampling location. All samples were promptly transferred to the laboratory for subsequent analysis. As previously described, the preservation protocol for these samples was maintained consistent with the methodology employed for substrate sampling.

2.2.3. Pathogen Isolation and Quantification

The average populations of Fusarium spp. and Phytophthora spp., quantified as Colony-Forming Units (CFU) per gram of substrate, were isolated from both greenhouse substrate and space samples using semi-selective medium following the methodologies described by Komada and Masago et al. [22,23].

2.3. Analysis of Microbial Diversity in Cultivation Substrate Samples

2.3.1. DNA Extraction and PCR Amplification

Total genomic DNA was extracted from 0.25 g of substrate samples using the Powersoil® DNA Extraction Kit (Mo Bio, Carlsbad, CA, USA). Two sets of universal primers were employed for the analysis: 338F (5′-ACTCCTACGGGAGCAGCAG-3′) and 806R (5′-GGACTACHGGGTWTCTAAT-3′) for targeting the bacterial 16S rRNA gene [24], as well as ITS1F (5′-CTTGGTCATTTAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCATGATGC-3′) for the fungal ITS (internal transcribed spacer) region [25]. These primers were utilized to study the microbial communities in the substrate samples.

2.3.2. High-Throughput Sequencing of Substrate Samples and Bioinformatic Analysis

To analyze the abundance and community structure of bacteria and fungi in the substrate, operational taxonomic unit (OTU) analysis was performed using the Shengxin Cloud Platform provided by Shanghai Meiji Biotech Company, Shanghai, China.
The raw sequences were processed using Mothur software (V1.48.3) and valid sequences were obtained through processing with FLASH (V1.2.11) and Trimmomatic (V0.39). QIIME software (V1.9.1) was employed to visualize the relationships between different microbial communities via cluster analysis based on the Jaccard distance matrix and principal coordinate analysis (PCoA) utilizing the weighted UniFrac distance matrix. Bar charts representing species composition at the phylum level were generated using R programming language for analysis.

2.4. Investigation of Tomato Growth and Yield

The tomato variety used was Jingfan 309, primarily grown using the substrate furrow cultivation method and directly transplanted into fumigated substrate beds. Each furrow accommodated a single row of plants with a plant spacing of 0.3 m. Following fumigation, the substrate beds were aerated until ventilation standards were met, after which 4–5 leaf stage seedlings were immediately transplanted. Irrigation was delivered via a drip system, with scheduled and quantified applications three times daily (morning, midday, and evening), and total water volume adjusted according to transpiration demands. The experiment was conducted in an automated greenhouse, with environmental parameters monitored and regulated in real-time: daytime temperature of 25–30 °C and nighttime temperature of 15–20 °C (supplemented by shade nets and heaters); relative humidity of 60%; and natural light as the primary source.
In the tomato fruiting period, we randomly selected fruits meeting commercial standards from each treatment group, weighed and recorded them using an electronic bench scale with a maximum capacity of 30 kg after each harvest. This procedure continues until the end of the fruiting cycle. The average weight of marketable tomatoes from each treatment group serves as a crucial indicator for evaluating tomato yield.

2.5. Data Analysis

The efficacy of the fumigants against Fusarium spp. and Phytophthora spp. was calculated according to the following formula:
Y % = X 0 − X 1 X 0 × 100
In the formula, Y represents the control efficacy of fumigation treatment on pathogens, X0 denotes the colony counts of Fusarium spp. and Phytophthora spp. in the untreated control, and X1 indicates the colony counts of Fusarium spp. and Phytophthora spp. after fumigation treatment.
The efficacy of the fumigants on tomato yield was calculated as follows:
Y % = X 1 − X 0 X 0 × 100
In the formula, Y represents the yield increase rate (%) of tomato under fumigation treatment, X0 denotes the tomato yield (kg/666.7 m2) in the untreated control, and X1 indicates the tomato yield (kg/666.7 m2) after fumigation treatment.
All results were expressed as mean ± standard deviation (SD). Analysis of the data for statistical differences was carried out using the SPSS statistical software package (V20.0), which included one−way ANOVA and Duncan’s new multiple range test. The bar charts showing the composition of bacterial and fungal genera were drawn using Origin 2019.

2.6. Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

This manuscript was prepared without the use of AI software.

3. Results

3.1. Effect of the Treatments on Fungal Pathogens in the Substrate

Due to the varying doses of fumigants used, it is essential to consider dose effects. In the control of Fusarium spp., both agents demonstrated effective suppression rates compared to the untreated control at different doses. The efficacy of AL reached as high as 87.4%, while EL achieved a control effect of 67%. Effects of fumigation treatments (7 days) on Fusarium spp. and Phytophthora spp. are shown in Table 2.
In contrast, a significant dose-dependent effect was observed in the control of Phytophthora spp. The efficacy of EH reached 87.5%, while EL demonstrated a control effect of 61.7%. The difference in efficacy between AH and AL was not significantly different. Overall, higher doses of fumigants proved to be more effective against pathogens.

3.2. Effects of Fumigation on Fusarium spp. and Phytophthora spp. in Greenhouse Space

The results indicated that using fumigants for disinfecting greenhouse spaces effectively reduced the presence of pathogens. Prior to fumigation, colonies of Fusarium spp. and Phytophthora spp. were observed on plastic greenhouse films and the ground. Effects of fumigation on Fusarium spp. and Phytophthora spp. in greenhouse spaces are shown in Table 3.
Following the application of each fumigant treatment, the number of Fusarium spp. colonies was reduced to zero, achieving 100% efficacy. All treatments, except for the EL treatment, significantly decreased the Phytophthora spp. colonies with reductions exceeding 90% efficacy. Overall, AITC demonstrated greater effectiveness than ethylicin in disinfecting greenhouse spaces, especially at low-dose applications.

3.3. Bacterial Microbial Diversity Analysis in Substrate Samples

3.3.1. Bacterial Diversity Index Analysis

Following high-throughput sequencing of bacterial DNA extracted from the substrate, raw sequence data were processed and optimized, resulting in a total of 1,507,515 high-quality effective reads with an average length of 415 base pairs.
The alpha diversity analysis revealed key insights into community structure: the ACE and Chao1 indices were employed to assess species richness, while the Shannon and Simpson indices were used to evaluate species diversity. Biodiversity exhibited a positive correlation with the Shannon, ACE, and Chao1 indices, indicating that higher values of these indices correspond to greater community diversity and richness. Conversely, a negative correlation was observed with the Simpson index, reflecting its inverse relationship with diversity measures.
During the initial sampling period, all treatment groups demonstrated significant enhancements in both the Shannon and Chao1 indices compared to the control group, with statistically significant variations observed among different treatments (Figure 1a,b). However, this consistent pattern was markedly altered during the subsequent sampling period. Notably, the UAL treatment exhibited a substantial reduction in both diversity indices. Although the remaining treatments maintained an upward trend in microbial diversity relative to the control, the degree of variation among these treatments showed a considerable decrease (Figure 1c,d).

3.3.2. Principal Coordinate Analysis of Bacterial Community in Substrate Samples

Hierarchical clustering analysis employing the weighted UniFrac metric, derived from the β-diversity distance matrix, demonstrated that all triplicate samples within each treatment formed distinct clusters within the bacterial community structure. This clustering pattern indicates high reproducibility across biological replicates. Principal Coordinates Analysis (PCoA) of the initial sampling data revealed significant segregation between fumigated treatments and the control group, with treatments sharing the same fumigant exhibiting closer spatial clustering (Figure 2a). The first two principal coordinates accounted for 64.49% of the total variance, with PC1 and PC2 contributing 38.52% and 25.97%, respectively, to the observed variations in microbial community composition.
PCoA of bacterial community profiles indicated that fumigation treatments induced substantial perturbations to the ecological equilibrium of substrate microbial communities. However, these disturbances showed partial attenuation by the second sampling interval, as evidenced by the spatial convergence of UEH, UEL and UAH treatments with control samples along the horizontal axis in the PCoA ordination, indicative of partial community structure recovery.

3.3.3. Analysis of Differences in Bacterial Community Composition in Substrate Samples

Significant shifts in the composition of dominant bacterial genera were observed between the two sampling periods. During the initial sampling, the bacterial community was predominated by Bacillus, Pseudomonas, Pseudolabrys, Streptomyces and Brevibacillus (Figure 2c). Notably, all treatments exhibited a significant increase in the relative abundance of Bacillus compared to the CK, with the AH treatment showing the most pronounced enhancement. In contrast, the relative abundance of Pseudomonas was significantly reduced across all treatments, particularly in the AH treatment. The abundance of Streptomyces was significantly elevated in the AH, AL and EH treatments, while Brevibacillus showed a significant increase in all treatments relative to CK.
In the subsequent sampling period, the dominant genera shifted to Bacillus, Bauldia, Pseudolabrys, Streptomyces and Devosia (Figure 2d). Consistent with the initial sampling, Bacillus remained significantly enriched in all treatments compared to CK, with the UAL treatment displaying the highest relative abundance. Compared to the control group, the relative abundance of Pseudolabrys exhibited a significant reduction, particularly in the UAL treatment. With the exception of the UAH treatment, the relative abundance of Brevibacillus showed an increase compared to the control, although the magnitude of this change was less pronounced than that observed during the first sampling period.

3.3.4. LEfSe Analysis of Differences in Bacterial Community Composition in Substrate Samples

Linear Discriminant Analysis Effect Size (LEfSe) analysis was performed to identify statistically significant biomarkers (p < 0.05, LDA score > 4) that differentiated treatment groups from the control. The analysis incorporated the nonparametric Kruskal–Wallis (KW) rank-sum test to assess differences in taxonomic abundance, followed by linear discriminant analysis (LDA) to estimate the effect size of each component (species) contributing to the observed variations. Major taxa were screened at the bacterial genus level to identify key biomarkers. In the first sampling period, the number of significant biomarkers identified were as follows: 0 in AH, 1 in AL, 4 in EH, 1 in EL, and 1 in CK (Figure 3a). During the second sampling period, the distribution of biomarkers shifted, with 1 in UAH, 2 in UAL, 0 in UEH, 1 in UEL, and 1 in UCK (Figure 3b). These results suggest that the microbial community in the first sampling was more responsive to fumigation treatments compared to the second sampling.

3.4. Fungal Microbial Diversity Analysis in Substrate Samples

3.4.1. Fungal Diversity Index Analysis

Following high-throughput sequencing of fungal DNA extracted from the substrate, raw sequence data were processed and optimized, yielding a total of 2,869,156 high-quality reads with an average length of 233 base pairs.
Analysis of alpha diversity indices revealed distinct patterns among sampling periods. During the initial sampling, no statistically significant differences were observed in the Shannon index across any of the treatments when compared to the control (Figure 4a). In contrast, the Chao1 index, which reflects species richness, demonstrated significant increases in the AL, EH and EL treatments relative to the control (Figure 4b). In the subsequent sampling period, neither the Shannon nor Chao1 indices exhibited significant variations among the treatment groups, indicating a stabilization of fungal community diversity (Figure 4c,d).

3.4.2. Principal Coordinate Analysis of Fungal Community in Substrate

PCoA of the initial sampling data indicated a marked separation between fumigated treatments and the control group, with treatments using the same fumigant exhibiting closer spatial clustering (Figure 5a). The first two principal coordinates explained 64.89% of the total variance, with PC1 and PC2 contributing 34.99% and 29.9%, respectively, to the observed differences in fungal community composition.
In the second sampling, the PCoA plot revealed a distinct bipartite distribution between the treatment and control groups (Figure 5b). Notably, the UAH treatment group was positioned furthest from the blank control group along the horizontal axis, while the UAL treatment group exhibited substantial separation along the vertical axis. The combined explanatory power of PC1 and PC2 for the differences in community composition was 53.96%, with individual contributions of 31.16% and 22.8%, respectively.
In analyses of fungal communities, PCoA revealed significant differentiation among treatment groups, though fungal assemblages exhibited more rapid recovery kinetics compared to bacterial counterparts, with UEH and UEL treatments demonstrating near-control levels by the second sampling period. The rapid restoration of microbial communities does not exert adverse effects on the subsequent growth phases of crops [26].

3.4.3. Analysis of Differences in Fungal Community Composition in Substrate

Significant shifts in the composition of dominant fungal genera were observed between the two sampling periods. During the initial sampling, the fungal community was predominantly composed of Aspergillus, Ramophialophora, and Pseudogymnoascus (Figure 5c). Notably, treatments AH, EH, and EL all resulted in a significant increase in the relative abundance of Aspergillus compared to the control (CK), with the AH treatment showing the most pronounced effect. In contrast, the relative abundance of Pseudogymnoascus was significantly reduced across all treatments, while Trichoderma abundance was significantly decreased only in the EH and EL treatments, but not in the AH and AL treatments.
During subsequent sampling periods, a notable shift in fungal community composition was observed, with Ramophialophora, Mortierella, and Tausonia emerging as the dominant genera (Figure 5d), while Aspergillus ranked as the fourth most abundant genus. Notably, the treatment groups consistently exhibited significantly elevated relative abundance of Ramophialophora compared to the control group (p < 0.05). Conversely, a marked reduction in relative abundance was observed for Penicillium, Trichoderma, and Gilmaniella across all treatment conditions relative to the control levels.

3.4.4. LEfSe Analysis of Differences in Fungal Community Composition in Substrate

LEfSe analysis was conducted to identify statistically significant biomarkers that distinguished the treatment groups from the control. In the first sampling period, the number of significant biomarkers identified was as follows: 0 in AH, 1 in AL, 4 in EH, 1 in EL, and 3 in CK (Figure 6a). During the second sampling, the distribution of biomarkers shifted, with 3 in UAH, 2 in UAL, 3 in UEH, 4 in UEL, and 4 in UCK (Figure 6b).
Through LEfSe analysis of the number of significant biomarkers between the treatment and control groups, it was found that the fungal community exhibited more pronounced changes in abundance in response to fumigation treatments compared to the bacterial community. This result is consistent with the microbial community composition analysis across different treatments, indicating that the fungal community undergoes more significant alterations.

3.5. Significant Improvement of Tomato Yield

Both fumigant treatments significantly increased tomato yield compared to the untreated control group. The AL and EL treatments consistently resulted in yield increases of up to 20%. Effects of fumigation on tomato yield are shown in Table 4. The AH and EH treatments exhibited varying yield increases of 32.68% and 45.16%, respectively. The EH treatment is recommended for field application. The observed yield increase is likely positively associated with the enhanced inhibition of pathogenic fungi following fumigation treatments. Specifically, the inhibition rates of Fusarium spp. and Phytophthora spp. were higher under the AH treatment compared with the AL treatment, while the EH treatment exhibited a greater inhibitory effect on Phytophthora spp. than the EL treatment. These results suggest that higher dosages of fumigants are accompanied by stronger suppression of soil-borne pathogens, which in turn contributes to greater yield improvements. Cheng et al. [27] demonstrated that fumigant application significantly reduces soil pathogen populations, thereby enhancing tomato growth parameters and yield indices. These findings are in strong agreement with our field yield data, further validating the beneficial impact of fumigation on crop productivity.

4. Discussion

The scientific novelty of this study was to evaluate allyl isothiocyanate and ethylicin as potential substrate and space fumigants in tomato greenhouses and develop an eco-compatible substrate regeneration protocol through the optimized application of fumigants in order of sustainable intensive agriculture with appropriate management of agricultural pests.

4.1. Investigation and Monitoring of Fusarium spp. and Phytophthora spp. in Substrate

Numerous studies have been carried out to evaluate the efficacy of fumigants against soil-borne pathogens. The efficacy of AITC in substrate aligns with the findings reported by Ren et al. [28]. In particular, AITC exhibits a superior suppression rate against Fusarium spp. compared to Phytophthora spp. However, its efficacy against Phytophthora spp. remains limited. It is important to consider the effects of soil properties, the amount of agent applied, as well as soil temperature and humidity on the performance of AITC [29]. Furthermore, the combination of AITC with standard fumigants such as chloropicrin, 1,3-dichloronitromethane, and methyl isothiocyanate significantly enhances overall effectiveness [30].

4.2. Detection of Pathogens in the Space of Tomato Greenhouse

Disinfectants such as sodium hypochlorite (NaOCl), hydrogen peroxide (H2O2), and chlorine dioxide (ClO2) are widely employed in greenhouses for disinfecting machinery and preserving food [31]. For instance, Bhakta et al. [32] sprayed weakly acidic electrolytic water with a chlorine concentration of approximately 30 mg/L that significantly reduces the viability of airborne microorganisms without adversely affecting the growth of greenhouse crops. However, these disinfectants are highly effective against bacterial and viruses but less so against fungi [33,34].
Applying AITC and ethylicin as fumigants has traditionally been limited to soil treatment. This study innovatively applied fumigants for greenhouse space disinfestation, achieving comprehensive three-dimensional disinfestation of the treatment environment. This finding confirms the effectiveness of AITC and ethylicin as space fumigants, with their effective control against pathogens hidden in space and substrate. It is noteworthy that the efficacy of ethylicin was relatively lower, suggesting that further optimization of its control effect could be achieved by increasing the applied dosage.

4.3. Analysis of Microbial Diversity of Substrate

4.3.1. Analysis of Taxonomic Diversity

Maintaining an optimal rhizosphere soil microenvironment is crucial for ensuring crop yield and enhancing crop quality [35]. Fumigation represents a critical factor influencing microbial community dynamics within the substrate. Extensive prior research has established that soil fumigants exert significant effects on soil microbial communities. For example, chloropicrin fumigation has been shown to not only substantially reduce bacterial diversity but also induce profound alterations in the structural composition of both bacterial and fungal communities within the soil matrix [36,37]. Supporting this observation, Fang et al. [38] documented that dazomet fumigant application resulted in a notable decrease in soil bacterial taxonomic diversity. In contrast to these findings, our investigation revealed that soil bacterial α-diversity indices exhibited an increase following fumigation with AITC and ethylicin, compared to control conditions. Notably, bacterial microbial diversity demonstrated heightened sensitivity to fumigant exposure [39]. These collective findings suggest that fumigant application for substrate disinfection does not induce substantial perturbations to the soil microenvironment, with transient inhibitory effects being rapidly ameliorated during crop establishment phases.
Regarding bacterial community dynamics, our analysis revealed a general upward trajectory in microbial diversity indices. The suppressive effects of fumigants on substrate microbial diversity were transient in nature, with diversity metrics progressively returning to levels analogous to the control group during the seedling establishment period. This observation is consistent with previous investigations into the effects of AITC on soil microbial populations and further substantiates the capacity of AITC and ethylicin to facilitate rapid reconstitution of substrate microbial communities [40]. Furthermore, our study demonstrated that fumigant dosage significantly influences substrate microbial diversity profiles. Specifically, increasing concentrations of AITC and ethylicin were associated with a progressive decline in bacterial diversity, potentially attributable to fumigation-mediated modifications of specific soil environmental parameters, resulting in subsequent alterations in bacterial community architecture and substrate microecological conditions. This interpretation is supported by complementary findings from Romanowicz et al. [41].

4.3.2. Difference Analysis of Community Composition

In the bacterial community, the results showed that fumigation treatment significantly increased the relative abundance of beneficial bacteria and significantly decreased the relative abundance of pathogenic bacteria. We found that the relative abundance of Bacillus spp. was significantly increased after fumigation. It was reported that Bacillus is an effective biological control agent and can reduce the incidence rate [42]. Huang et al. [43] reported that Bacillus can effectively control Fusarium wilt. The results showed that the relative abundance of Streptomyces spp. increased significantly in AH, AL, and EH treatments, and the relative abundance of Brevibacillus spp. increased significantly in all treatments. Streptomyces in soil has antibacterial, fungal, viral and anticancer activities, and is an effective resource for screening biocontrol agents [44]. Brevibacillus is a natural strain widely used in microbial fertilizers. It has strong vitality and rapid reproduction. It can promote plant growth through its own metabolism to produce organic acids, enzymes, physiologically active substances, etc., and can also reduce toxic substances such as pesticides by secreting polysaccharides. It has the dual effects of improving soil quality and increasing crop production and income [45,46]. The increase of these bacteria may make a significant contribution to the ability of AITC and ethylicin to control crop pathogens.
Van De Veerdonk et al. [47] have shown that Aspergillus is a saprophytic fungus that exists and grows in soil or decaying vegetation. Aspergillus also plays an important role in carbon and nitrogen cycling. The biological nitrification inhibition ability of Mortierella can effectively inhibit the ammonia oxidation in soil [48]. In conclusion, after AITC and ethylicin fumigation, the fungal community changed significantly, which also changed the composition of the substrate, which was more conducive to tomato colonization.

5. Conclusions

This study demonstrated that fumigation with AITC and ethylicin effectively suppressed key soil-borne pathogens in greenhouse substrate cultivation systems. Specifically, AITC and ethylicin achieved control efficacies of 94.2% and 87.5%, respectively, against Fusarium spp. and Phytophthora spp. in the substrate, while exhibiting even higher inhibition rates of 100% and >90% against these pathogens in the greenhouse space. Microbial diversity analyses revealed that these treatments significantly reduced pathogenic bacterial populations and enriched beneficial taxa, including Bacillus, Streptomyces, and Brevibacillus, thereby reshaping the bacterial and fungal community structures toward greater microbial diversity and functionality. Consequently, tomato growth was enhanced, culminating in a substantial yield increase exceeding 45%.
In short, soilless cultivation as a critical complement to traditional soil-based agriculture, has become an indispensable component of modern agricultural systems. However, the persistent challenges of pests and diseases associated with continuous cropping necessitate the implementation of scientifically validated disinfection strategies [49,50]. Environmentally friendly disinfectants with low toxicity, such as ethylicin and AITC, have emerged as efficient and sustainable solutions for substrate sterilization. These agents not only ensure a pathogen-free environment in both greenhouse spaces and cultivation substrate but also markedly prolong the functional lifespan of the substrate, thereby fostering optimal crop growth conditions and significantly improving the yield and quality. The scientifically informed and judicious application of these green disinfectants has great potential to promote the sustainable development of soil-free cropping systems, providing a substantial technical support for the transition to a modern agriculture for greater ecological sustainability, operational efficiency, and long-term resilience.

Author Contributions

G.C.: Writing—review and editing, Writing—original draft, Investigation. M.Z.: Writing—review and editing, Writing—original draft, Validation. Z.S.: Formal analysis, Data curation. A.C.: Resources, Conceptualization. Q.W.: Supervision, Resources. D.Y.: Software, Project administration. W.F.: Visualization, Funding acquisition. Y.L.: Supervision, Methodology. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key R & D Program of China (Grant No. 2024YFD2001100).

Data Availability Statement

No new data were created or analyzed in this study.

Acknowledgments

We gratefully acknowledge the State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, for providing technical support. Declaration of generative AI and AI-assisted technologies in the writing process: After the preparation of this work, AI tools were only used to check the grammar and spelling errors in order to improve language accuracy. After using this tool, the authors reviewed the whole manuscript and take full responsibility for the content of the publication.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

Methyl Bromide (MB), Allyl isothiocyanate (AITC), Principal Coordinates Analysis (PCoA), Linear Discriminant Analysis Effect Size (LEfSe).

References

  1. Chen, Z.; Wu, Z.; Gao, J. A convolutional neural network for large-scale greenhouse extraction from satellite images considering spatial features. Remote Sens. 2022, 14, 4908. [Google Scholar] [CrossRef]
  2. Castello, I.; Baglieri, A.; Montoneri, E.; Vitale, A. Utilization of municipal biowaste-derived compounds to reduce soilborne fungal diseases of tomato: A further step toward circular bioeconomy. GCB Bioenergy 2025, 17, 70027. [Google Scholar] [CrossRef]
  3. Castello, I.; D’Emilio, A.; Danesh, Y.; Vitale, A. Enhancing the effects of solarization-based approaches to suppress Verticillium dahliae inocula affecting tomato in greenhouse. J. Agric. Food Res. 2024, 18, 101355. [Google Scholar] [CrossRef]
  4. Barrett, G.E.; Alexander, P.D.; Robinson, J.S. Achieving environmentally sustainable growing media for soilless plant cultivation systems—A review. Sci. Hortic. 2016, 212, 220–234. [Google Scholar] [CrossRef]
  5. Wang, Y.; Zhang, X.; Du, X. Effects of different straw breeding substrates on the growth of tomato seedlings and transcriptome analysis. Sci. Rep. 2024, 14, 22181. [Google Scholar] [CrossRef] [PubMed]
  6. Hodge, A.; Berta, G.; Doussan, C. Plant root growth, architecture and function. Plant Soil 2009, 321, 153–187. [Google Scholar] [CrossRef]
  7. Montagne, V.; Capiaux, H.; Cannavo, P. Protective effect of organic substrates against soil-borne pathogens in soilless cucumber crops. Sci. Hortic. 2016, 206, 62–70. [Google Scholar] [CrossRef]
  8. Liu, W.; Zhang, Z.; Zhang, B. Role of bacterial pathogens in microbial ecological networks in hydroponic plants. Front. Plant Sci. 2024, 15, 1403226. [Google Scholar] [CrossRef]
  9. Sela Saldinger, S.; Rodov, V.; Kenigsbuch, D. Hydroponic agriculture and microbial safety of vegetables: Promises, challenges, and solutions. Horticulturae 2023, 9, 51. [Google Scholar] [CrossRef]
  10. Liu, Y.; Wu, J. Research status and prospects of organic cultivation substrates. North. Hortic. 2011, 172–176. [Google Scholar]
  11. Dong, Y.; Guo, S.; Li, X. Effects of disinfection, companion planting, and microbial agents on the properties of continuous cropping substrates and the growth and development of cucumbers. Northwest J. Plants 2023, 43, 1136–1145. [Google Scholar]
  12. Li, W.; Ren, L.; Li, Q. Evaluation of ethylicin as a potential soil fumigant in commercial tomato production in China. Sci. Total Environ. 2023, 854, 158520. [Google Scholar] [CrossRef] [PubMed]
  13. Dahlin, P.; Hallmann, J. New Insights on the Role of Allyl Isothiocyanate in Controlling the Root Knot Nematode Meloidogyne hapla. Plants 2020, 9, 603. [Google Scholar] [CrossRef] [PubMed]
  14. Shin, J.; Kathuria, A.; Lee, Y.S. Effect of hydrophilic and hydrophobic cyclodextrins on the release of encapsulated allyl isothiocyanate (AITC) and their potential application for plastic film extrusion. J. Appl. Polym. Sci. 2019, 136, 48137. [Google Scholar] [CrossRef]
  15. Wang, X.; Wang, Q.; Zhang, D. Fumigation alters the manganese-oxidizing microbial communities to enhance soil manganese availability and increase tomato yield. Sci. Total Environ. 2024, 919, 170882. [Google Scholar] [CrossRef]
  16. Wu, H.; Wang, C.; Bian, X. Nematicidal efficacy of isothiocyanates against root-knot nematode Meloidogyne javanica in cucumber. Crop Prot. 2011, 30, 33–37. [Google Scholar] [CrossRef]
  17. Zhang, Y.; Fang, W.; Yan, D. Encapsulated allyl isothiocyanate improves soil distribution, efficacy against soil-borne pathogens and tomato yield. Pest Manag. Sci. 2024, 80, 4583–4593. [Google Scholar] [CrossRef]
  18. Zhang, Y.; Fang, W.; Yan, D. Comparison of drip-irrigated or injected allyl isothiocyanate against key soil-borne pathogens and weeds. Pest Manag. Sci. 2023, 79, 3860–3870. [Google Scholar] [CrossRef]
  19. Huang, Y.; Li, H.; Zhao, G. Ethylicin inhibition of Xanthomonas oryzae pv. oryzicola in vitro and in vivo. J. Agric. Food Chem. 2023, 71, 1405–1416. [Google Scholar] [CrossRef]
  20. Zhang, S.; Zhang, M.; Khalid, A.R. Ethylicin prevents potato late blight by disrupting protein biosynthesis of Phytophthora infestans. Pathogens 2020, 9, 299. [Google Scholar] [CrossRef]
  21. Shi, Z.; Zhang, M.; Chen, G. Evaluation of newly combination of Trichoderma with dimethyl disulfide fumigant to control Fusarium oxysporum, optimize soil microbial diversity and improve tomato yield. Ecotoxicol. Environ. Saf. 2025, 292, 117903. [Google Scholar] [CrossRef]
  22. Komada, H. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev. Plant Prot. Res. 1975, 8, 114–124. [Google Scholar]
  23. Masago, H.; Yoshikawa, M.; Fukada, M. Selective inhibition of Pythium spp. on a medium for direct isolation of Phytophthora spp. from soils and plants. Phytopathology 1977, 67, 425–428. [Google Scholar] [CrossRef]
  24. Xu, N.; Tan, G.; Wang, H. Effect of biochar additions to soil on nitrogen leaching, microbial biomass and bacterial community structure. Eur. J. Soil Biol. 2016, 74, 1–8. [Google Scholar] [CrossRef]
  25. Adams, R.; Miletto, M.; Taylor, J. Dispersal in microbes: Fungi in indoor air are dominated by outdoor air and show dispersal limitation at short distances. ISME J. 2013, 7, 1262–1273. [Google Scholar] [CrossRef]
  26. Jiang, W.; Chen, R.; Zhao, L. Chemical fumigants control apple replant disease: Microbial community structure-mediated inhibition of Fusarium and degradation of phenolic acids. J. Hazard. Mater. 2022, 440, 129786. [Google Scholar] [CrossRef]
  27. Cheng, H.; Zhang, D.; Ren, L. Bio-activation of soil with beneficial microbes after soil fumigation reduces soil-borne pathogens and increases tomato yield. Environ. Pollut. 2021, 283, 117160. [Google Scholar] [CrossRef] [PubMed]
  28. Ren, Z.; Li, Y.; Fang, W. Evaluation of allyl isothiocyanate as a soil fumigant against soil-borne diseases in commercial tomato (Lycopersicon esculentum Mill.) production in China. Pest Manag. Sci. 2018, 74, 2146–2155. [Google Scholar] [CrossRef]
  29. Liu, J.; Wang, X.; Fang, W. Soil properties, presence of microorganisms, application dose, soil moisture and temperature influence the degradation rate of allyl isothiocyanate in soil. Chemosphere 2020, 244, 125540. [Google Scholar] [CrossRef]
  30. Hoffmann, M.; Ajwa, H.A.; Westerdahl, B.B. Multitactic preplant soil fumigation with allyl isothiocyanate in cut flowers and strawberry. HortTechnology 2020, 30, 251–258. [Google Scholar] [CrossRef]
  31. Guan, J.; Lacombe, A.; Rane, B. Scaling up gaseous chlorine dioxide (ClO2) treatment in the cold storage of fresh apples. Postharvest Biol. Technol. 2024, 217, 113118. [Google Scholar] [CrossRef]
  32. Bhakta, B.; Yamane, S.; Bhakta, J.N. Microbial control in greenhouses by spraying slightly acidic electrolyzed water. Horticulturae 2023, 9, 81. [Google Scholar] [CrossRef]
  33. Jefri, U.H.N.M.; Khan, A.; Lim, Y.C. A systematic review on chlorine dioxide as a disinfectant. J. Med. Life 2022, 15, 313–318. [Google Scholar] [CrossRef] [PubMed]
  34. Koide, S.; Shitanda, D.; Note, M. Effects of mildly heated, slightly acidic electrolyzed water on the disinfection and physicochemical properties of sliced carrot. Food Control 2011, 22, 452–456. [Google Scholar] [CrossRef]
  35. Li, S.M.; Li, L.; Zhang, F.S. Acid phosphatase role in chickpea/maize intercropping. Ann. Bot. 2004, 94, 297–303. [Google Scholar] [CrossRef]
  36. Fang, W.; Yan, D.; Wang, X. Evidences of N2O emissions in chloropicrin-fumigated soil. J. Agric. Food Chem. 2018, 66, 11580–11591. [Google Scholar] [CrossRef]
  37. Li, J.; Huang, B.; Wang, Q. Effect of fumigation with chloropicrin on soil bacterial communities and genes encoding key enzymes involved in nitrogen cycling. Environ. Pollut. 2017, 227, 534–542. [Google Scholar] [CrossRef]
  38. Fang, W.; Yan, D.; Wang, X. Responses of nitrogen-cycling microorganisms to dazomet fumigation. Front. Microbiol. 2018, 9, 2529. [Google Scholar] [CrossRef]
  39. Zhang, S.; Liu, X.; Jiang, Q. Legacy effects of continuous chloropicrin-fumigation for 3-years on soil microbial community composition and metabolic activity. AMB Express 2017, 7, 178. [Google Scholar] [CrossRef] [PubMed]
  40. Zhu, J.; Ren, Z.; Huang, B. Effects of fumigation with allyl isothiocyanate on soil microbial diversity and community structure of tomato. J. Agric. Food Chem. 2020, 68, 1226–1236. [Google Scholar] [CrossRef] [PubMed]
  41. Romanowicz, K.J.; Freedman, Z.B.; Upchurch, R.A. Active microorganisms in forest soils differ from the total community yet are shaped by the same environmental factors: The influence of pH and soil moisture. FEMS Microbiol. Ecol. 2016, 92, fiw149. [Google Scholar] [CrossRef]
  42. Hollister, E.B.; Hu, P.; Wang, A.S. Differential impacts of brassicaceous and nonbrassicaceous oilseed meals on soil bacterial and fungal communities. FEMS Microbiol. Ecol. 2013, 83, 632–641. [Google Scholar] [CrossRef]
  43. Huang, Y.; Xiao, X.; Huang, H. Contrasting beneficial and pathogenic microbial communities across consecutive cropping fields of greenhouse strawberry. Appl. Microbiol. Biotechnol. 2018, 102, 5717–5729. [Google Scholar] [CrossRef]
  44. Kawicha, P.; Laopha, A.; Chamnansing, W. Biocontrol and plant growth-promoting properties of Streptomyces isolated from vermicompost soil. Indian Phytopathol. 2020, 73, 655–666. [Google Scholar] [CrossRef]
  45. Gangola, S.; Bhandari, G.; Joshi, S. Esterase and ALDH dehydrogenase-based pesticide degradation by Bacillus brevis 1B from a contaminated environment. Environ. Res. 2023, 232, 116332. [Google Scholar] [CrossRef]
  46. Li, Z.; Wang, J.; Deng, R. Mechanisms of manganese-tolerant Bacillus brevis MM2 mediated oxytetracycline biodegradation process. Environ. Pollut. 2024, 363, 125060. [Google Scholar] [CrossRef] [PubMed]
  47. Van De Veerdonk, F.L.; Gresnigt, M.S.; Romani, L. Aspergillus fumigatus morphology and dynamic host interactions. Nat. Rev. Microbiol. 2017, 15, 661–674. [Google Scholar] [CrossRef] [PubMed]
  48. Sadhukhan, R.; Jatav, H.S.; Sen, S. Biological nitrification inhibition for sustainable crop production. In Plant Perspectives to Global Climate Changes; Aftab, T., Roychoudhury, A., Eds.; Academic Press: Cambridge, MA, USA, 2022; pp. 135–150. ISBN 978-0-323-85665-2. [Google Scholar]
  49. Negawo, W.J.; Beyene, D.N. The role of coffee based agroforestry system in tree diversity conservation in Eastern Uganda. J. Landsc. Ecol. 2017, 10, 1–18. [Google Scholar] [CrossRef]
  50. Suman, S.; Verma, U. State space modelling and forecasting of sugarcane yield in Haryana, India. J. Appl. Nat. Sci. 2017, 9, 2036–2042. [Google Scholar] [CrossRef]
Agriculture 15 02502 g001
Figure 1. Changes in alpha diversity of substrate bacteria at the OUT level after fumigation (a,b) and at the time of seedling planting (c,d). Note: Student’s t-test was used to determine differences in alpha diversity between soil fumigation conditions. Statistical differences between the two groups are marked with 1–3 asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001). AH = 14 g/m2 AITC after 7 days of fumigation; AL = 7 g/m2 AITC after 7 days of fumigation; EH = 8 g/m2 ethylicin after 7 days of fumigation; EL = 4 g/m2 ethylicin after 7 days of fumigation; CK = untreated control after 7 days of fumigation; UAH = 14 g/m2 at the time of seedling planting; UAL = 7 g/m2 AITC at the time of seedling planting. UEH = 8 g/m2 ethylicin at the time of seedling planting; UEL = 4 g/m2 ethylicin at the time of seedling planting; UCK = untreated control at the time of seedling planting.
Figure 1. Changes in alpha diversity of substrate bacteria at the OUT level after fumigation (a,b) and at the time of seedling planting (c,d). Note: Student’s t-test was used to determine differences in alpha diversity between soil fumigation conditions. Statistical differences between the two groups are marked with 1–3 asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001). AH = 14 g/m2 AITC after 7 days of fumigation; AL = 7 g/m2 AITC after 7 days of fumigation; EH = 8 g/m2 ethylicin after 7 days of fumigation; EL = 4 g/m2 ethylicin after 7 days of fumigation; CK = untreated control after 7 days of fumigation; UAH = 14 g/m2 at the time of seedling planting; UAL = 7 g/m2 AITC at the time of seedling planting. UEH = 8 g/m2 ethylicin at the time of seedling planting; UEL = 4 g/m2 ethylicin at the time of seedling planting; UCK = untreated control at the time of seedling planting.
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Figure 2. PCoA of classified bacteria at OTU level (a,b). Differences in bacterial genera composition in the presence of different doses of AITC and ethylicin (c,d). Note: The number of asterisks indicates significant differences among treatments according to one−way ANOVA (p < 0.05): * 0.01 < p ≤ 0.05, ** 0.001 < p ≤ 0.01, and *** p ≤ 0.001. In the subsequent sampling analysis, the PCoA plot displayed a clear bipartite distribution pattern among treatment groups (b). Notably, the UAL treatment exhibited substantial spatial separation along the horizontal axis, distinct from other treatment clusters. The cumulative explanatory power of PC1 and PC2 for community composition variations was 52.43%, with individual contributions of 30.38% and 22.05%, respectively.
Figure 2. PCoA of classified bacteria at OTU level (a,b). Differences in bacterial genera composition in the presence of different doses of AITC and ethylicin (c,d). Note: The number of asterisks indicates significant differences among treatments according to one−way ANOVA (p < 0.05): * 0.01 < p ≤ 0.05, ** 0.001 < p ≤ 0.01, and *** p ≤ 0.001. In the subsequent sampling analysis, the PCoA plot displayed a clear bipartite distribution pattern among treatment groups (b). Notably, the UAL treatment exhibited substantial spatial separation along the horizontal axis, distinct from other treatment clusters. The cumulative explanatory power of PC1 and PC2 for community composition variations was 52.43%, with individual contributions of 30.38% and 22.05%, respectively.
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Figure 3. LEfSe cladogram analysis of the differentially abundant species in substrate bacterial (LDA = 4, p = 0.05): (a) after 7 days of fumigation and (b) at the time of seedling planting. Note: The five rings in the LEfSe cladogram represent the phylum, class, order, family and genus, respectively, when read from the inside. The different color nodes (except yellow) on the ring represent significant changes in taxonomic composition due to the treatments.
Figure 3. LEfSe cladogram analysis of the differentially abundant species in substrate bacterial (LDA = 4, p = 0.05): (a) after 7 days of fumigation and (b) at the time of seedling planting. Note: The five rings in the LEfSe cladogram represent the phylum, class, order, family and genus, respectively, when read from the inside. The different color nodes (except yellow) on the ring represent significant changes in taxonomic composition due to the treatments.
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Figure 4. Changes in alpha diversity of substrate fungi at the OUT level after 7 days of fumigation (a,b) and at the time of seedling planting (c,d). Note: Student’s t-test was used to determine differences in alpha diversity among soil fumigation conditions. Statistical differences between the two groups are marked with 1–3 asterisks (* 0.01 < p ≤ 0.05; ** 0.001 < p ≤ 0.01; *** p ≤ 0.001).
Figure 4. Changes in alpha diversity of substrate fungi at the OUT level after 7 days of fumigation (a,b) and at the time of seedling planting (c,d). Note: Student’s t-test was used to determine differences in alpha diversity among soil fumigation conditions. Statistical differences between the two groups are marked with 1–3 asterisks (* 0.01 < p ≤ 0.05; ** 0.001 < p ≤ 0.01; *** p ≤ 0.001).
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Figure 5. PCoA of classified fungi at OTU level (a,b). Differences in fungi genera composition in the presence of different doses of AITC and ethylicin (c,d). Note: The number of asterisks indicates significant differences between treatments according to one−way ANOVA: * 0.01 < p ≤ 0.05; ** 0.001 < p ≤ 0.01; *** p ≤ 0.001.
Figure 5. PCoA of classified fungi at OTU level (a,b). Differences in fungi genera composition in the presence of different doses of AITC and ethylicin (c,d). Note: The number of asterisks indicates significant differences between treatments according to one−way ANOVA: * 0.01 < p ≤ 0.05; ** 0.001 < p ≤ 0.01; *** p ≤ 0.001.
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Figure 6. LEfSe cladogram analysis of the differentially abundant species in substrate fungi (LDA = 4, p = 0.05): (a) after 7 days of fumigation and (b) at the time of seedling planting. Note: The five rings in the LEfSe cladogram represent the phylum, class, order, family and genus, respectively, when read from the inside. The different color nodes (except yellow) on the ring represent significant changes in taxonomic composition due to the treatments.
Figure 6. LEfSe cladogram analysis of the differentially abundant species in substrate fungi (LDA = 4, p = 0.05): (a) after 7 days of fumigation and (b) at the time of seedling planting. Note: The five rings in the LEfSe cladogram represent the phylum, class, order, family and genus, respectively, when read from the inside. The different color nodes (except yellow) on the ring represent significant changes in taxonomic composition due to the treatments.
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Table 1. Summary of All Treatments.
Table 1. Summary of All Treatments.
TreatmentDosage (g/m2) Label 1Post-Treatment TimeLabel 2Post-Treatment Time
Untreated control CK UCK
AITC14AHafter 7 days of fumigationUAHat the time of seedling planting
AITC7ALUAL
ethylicin8EHUEH
ethylicin4ELUEL
Note: CK-EL represents the sampling after 7 days of fumigation, while UCK-UEL represents the sampling at the time of tomato seeding planting. This design was used to investigate changes in bacterial and fungal communities over time. The time interval between these two sampling dates was 15 days.
Table 2. Relative control efficacy of fumigation against Fusarium spp. and Phytophthora spp. in the substrate.
Table 2. Relative control efficacy of fumigation against Fusarium spp. and Phytophthora spp. in the substrate.
TreatmentDose (g/m2)Fusarium spp. (%)Phytophthora spp. (%)
AH1494.2 ± 4.60 a73.9 ± 3.48 b
AL 787.4 ± 7.99 a69.2 ± 4.06 b
EH 868.9 ± 4.63 b87.5 ± 2.35 a
EL467.0 ± 1.99 b61.7 ± 4.89 c
CK --
Note: Each value is the mean of three replications ± standard deviation (SD); AH = 14 g/m2 AITC after 7 days of fumigation; AL = 7 g/m2 AITC after 7 days of fumigation; EH = 8 g/m2 ethylicin after 7 days of fumigation; EL = 4 g/m2 ethylicin after 7 days of fumigation. AITC = Allyl isothiocyanate. Data followed in each column by the same letter are not statistically different according to Duncan’s new multiple-range test (p = 0.05).
Table 3. Effects of fumigation on Colony Forming Units of Fusarium spp. and Phytophthora spp. in greenhouse space.
Table 3. Effects of fumigation on Colony Forming Units of Fusarium spp. and Phytophthora spp. in greenhouse space.
TreatmentDose
(g/m2)		Spatial LocationFusarium spp.Phytophthora spp.
Before FumigationAfter FumigationInhibition Rate (%)Before FumigationAfter FumigationInhibition Rate (%)
AH 14film13.2 ± 2.71 c0100.0133.5 ± 34.32 d5.23 ± 3.33 e96.2 ± 1.22 a
ground65.34 ± 12.87 a 0100.0435.16 ± 61.12 b30 ± 3.45 c93.1 ± 0.26 b
AL7film30.23 ± 8.14 b0100.0195.34 ± 24.77 c18 ± 2.59 d90.7 ± 0.74 c
ground45.22 ± 11.37 a0100.0124.85 ± 18.72 d11 ± 4.28 e91.1 ± 1.53 bc
EH8film16.43 ± 3.77 c0100.0953.67 ± 134.38 a29 ± 2.76 c96.9 ± 0.42 a
ground42 ± 8.27 ab0100.0780.21 ± 84.21 a73 ± 8.13 b90.6 ± 0.66 c
EL4film13 ± 2.54 c0100.0153.35 ± 27.89 cd31 ± 4.75 c79.7 ± 1.84 d
ground56 ± 7.65 a0100.0865.77 ± 92.17 a97 ± 9.24 a88.7 ± 0.24 c
Note: Each value is the mean of three replications ± standard deviation (SD); AH = 14 g/m2 AITC after 7 days of fumigation; AL = 7 g/m2 AITC after 7 days of fumigation; EH = 8 g/m2 ethylicin after 7 days of fumigation; EL = 4 g/m2 ethylicin after 7 days of fumigation. AITC = Allyl isothiocyanate. Data followed in each column by the same letter are not statistically different according to Duncan’s new multiple-range test (p = 0.05).
Table 4. Effects of fumigation on tomato yield.
Table 4. Effects of fumigation on tomato yield.
TreatmentDose
(g/m2)		Yield (kg/666.7 m2)Yield Increase (%)
AH14575.6 ± 12.29 b32.68 ± 1.42 b
AL7521.59 ± 22.62 c20.23 ± 1.81 c
EH8629.73 ± 12.20 a45.16 ± 2.74 a
EL4516.97 ± 11.44 c19.17 ± 2.73 c
CK 433.81 ± 13.44 d-
Note: Each value is the mean of three replications ± standard deviation (SD); AH = 14 g/m2 AITC after 7 days of fumigation; AL = 7 g/m2 AITC after 7 days of fumigation; EH = 8 g/m2 ethylicin after 7 days of fumigation; EL = 4 g/m2 ethylicin after 7 days of fumigation. AITC = Allyl isothiocyanate. Data followed in each column by the same letter are not statistically different according to Duncan’s new multiple-range test (p = 0.05). The yield unit “kg/666.7 m2” refers to the standardized mu yield (1 mu ≈ 666.7 m2, a common unit in Chinese agriculture, facilitating comparison with field cultivation). The actual experimental yields were calculated based on the fresh fruit weight from each plot and then proportionally converted to mu yields.
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Chen, G.; Zhang, M.; Shi, Z.; Cao, A.; Wang, Q.; Yan, D.; Fang, W.; Li, Y. Evaluation of Allyl Isothiocyanate and Ethylicin as Potential Substrate and Space Fumigants in Tomato Greenhouses. Agriculture 2025, 15, 2502. https://doi.org/10.3390/agriculture15232502

AMA Style

Chen G, Zhang M, Shi Z, Cao A, Wang Q, Yan D, Fang W, Li Y. Evaluation of Allyl Isothiocyanate and Ethylicin as Potential Substrate and Space Fumigants in Tomato Greenhouses. Agriculture. 2025; 15(23):2502. https://doi.org/10.3390/agriculture15232502

Chicago/Turabian Style

Chen, Guangming, Min Zhang, Zhaoai Shi, Aocheng Cao, Qiuxia Wang, Dongdong Yan, Wensheng Fang, and Yuan Li. 2025. "Evaluation of Allyl Isothiocyanate and Ethylicin as Potential Substrate and Space Fumigants in Tomato Greenhouses" Agriculture 15, no. 23: 2502. https://doi.org/10.3390/agriculture15232502

APA Style

Chen, G., Zhang, M., Shi, Z., Cao, A., Wang, Q., Yan, D., Fang, W., & Li, Y. (2025). Evaluation of Allyl Isothiocyanate and Ethylicin as Potential Substrate and Space Fumigants in Tomato Greenhouses. Agriculture, 15(23), 2502. https://doi.org/10.3390/agriculture15232502

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