*Corresponding author Introduction
Tomato (Solanum lycopersicum L.), a member of the family Solanaceae, is a globally important vegetable crop with a production exceeding 186 million tons in 2020 (Ali et al., 2020; Food and Agriculture Organization of the United Nations Corporate Statistical Database, 2021). Tomato yield and quality are affected by various plant pathogens such as bacteria, fungi, nematodes, and viruses (Arie et al., 2007). Among these, gray mold is considered one of the most devastructive diseases affecting tomato cultivation globally (Lee et al., 2006; Sarven et al., 2020). Gray mold is caused by the ascomycete fungus Botrytis cinerea Persoon: Fries (teleomorph Botryotinia fuckeliana (de Bary) Whetzel). This necrotrophic fungus infects nearly all aerial parts of tomato plant, including leaves, stems, flowers, and fruits (Elad et al., 2016; Ji et al., 2019; Lee et al., 2006; Williamson et al., 2007). Tomato yield losses caused by gray mold typically range from 15% to 20%, but can exceed 50% under severe outbreaks (Ji et al., 2019; Wang and Liu, 2021). Therefore, it is necessary for growers to apply an efficient disease management strategy to control tomato gray mold. Since no tomato varieties exhibit resistance to B. cinerea, gray mold has been mainly managed through chemical fungicides (Leroux, 2007). However, resistance to several fungicides including anilinopyrimidine, benzimidazole, and dicarboximide has emerged in B. cinerea populations (Myresiotis et al., 2007). In western Oregon, 64% of isolates from berries were resistant to two or more fungicides (Stockwell et al., 2018). Similarly, in Korea, 82.5% of 78 isolates from strawberry farms were resistant to benomyl, with resistance to benzimidazole steadily increasing (Kim et al., 2023). In addition to resistance, excessive fungicides use contributes to environmental pollution and ecological imbalance (Abbey et al., 2019; Rosero-Hernández et al., 2019; Sarven et al., 2020; Veloukas et al., 2011). Therefore, alternative strategies such as the use of biological control agents that are environmentally friendly, safe for humans, and effective against B. cinerea are urgently needed. This study aimed to identify potential biological control agents against B. cinerea in tomato. We evaluated four bacterial isolates for their ability to inhibit mycelial growth, production antifungal compounds, and suppress disease symptoms.
Materials and Methods
Isolation and identification of Botrytis cinerea.
B. cinerea 18-042, isolated from tomato leaves showing gray mold symptoms from a farmer's field in Hwaseong, South Korea (location: 37°7’19”N, 126°42’3”E), was used in this study. The rDNA internal transcribed spacer (ITS) and beta-tubulin (TUB2) gene regions of B. cinerea 18-042 were amplified using primers ITS4/ITS5 (White et al., 1990) and Bt2a/Bt2b (Glass and Donaldson, 1995), respectively, and sequenced by BIOFACT (Daejeon, Korea). The ITS and TUB2 sequences were deposited in GenBank (PP106124 and PP208988). A maximum-likelihood phylogenetic tree was constructed based on the TUB2 sequences with 1,000 bootstrap replicates by MEGA 7 (Kumar et al., 2016).
Mycelial growth inhibition assay.
Bacillus isolates were provided from the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science (Hong et al., 2018). To assess their mycelial growth inhibition activity against the gray mold pathogen B. cinerea, a dual culture assay was conducted on potato dextrose agar (PDA) (Becton, Dickinson and Company, Sparks, MD, USA) using four Bacillus isolates (B. amyloliquefaciens PgBE1, PgBE14, PgBE51, and B. subtilis PgBE23). A mycelial agar plug of B. cinerea was inoculated at the center of PDA plates, and each Bacillus isolate was streak-inoculated on both sides of the plug using a sterile loop. Plates were incubated at 25°C for 6 days, the extent of fungal mycelial growth inhibition was measured. Plates inoculated only with the pathogen served as the control.
Enzyme activity assay.
To evaluate protease, cellulase, and phosphate-solubilizing activities, four Bacillus isolates were spot-inoculated onto carboxymethyl cellulose (CMC) agar, Skim milk agar, and Pikovskaya agar, and incubated at 25°C for 3 days. The formation of clear zones (halo) around the colonies was considered a positive indication of enzyme activity. The assay was performed according to the method described by Shin et al. (2023), with minor modifications.
Biocontrol assay.
Healthy tomato leaves were collected from 5-week-old tomato plants, disinfected in a 1% NaClO solution for 2 min, and thoroughly washed in sterile distilled water (SDW). Each Bacillus isolate was grown in LB broth for 16 hr at 25°C with shaking at 150 rpm, and adjusted to optical density at 600 nm (OD600) of 1.0 in SDW. The bacterial suspension was diluted 10 times in SDW and sprayed onto the tomato leaves. SDW was used as the control. After drying the tomato leaves for 2 hr naturally, a mycelial agar plug of B. cinerea grown on PDA was placed onto the middle of tomato leaves. Inoculated tomato leaves were incubated at 25°C in a moistened plastic box. After 3 days of incubation, the percent diseased area was determined using ImageJ v1.53c (National Institutes of Health, Bethesda, MD, USA) (Kim et al., 2020). The experiment was repeated three times with three replications.
Six-week-old tomato plants were sprayed with the bacterial suspension (10-fold dilution from bacterial suspension at OD600 of 1.0 in SDW), SDW (mock), or the registered fungicide Mirabit (pyriflumetofen 18.35% suspension concentrate), air-dried for 2 hr naturally, and sprayed with conidial suspension of B. cinerea (1×105 conidia/ml) or SDW (mock). Control plants were treated in the same way but only sprayed with conidial suspension. All the plants were incubated in a humid chamber at 25°C. After 5 days of incubation, disease index and biocontrol efficiency were evaluated. Disease severity was defined according to the following scale (0-4): 0, no infection; 1, 1-25%; 2, 26-50%; 3, 51-75%; and 4, 76-100% infected leaf area. Disease index was calculated according to the following formula: disease index = [(0n0 + 1n1 + 2n2 + 3n3 + 4n4) / 4N] × 100, where n0, n1, n2, n3, and n4 are the number of leaves with score 0, 1, 2, 3, and 4, respectively, and N the total plant leaves investigated (Ganphung et al., 2019; Taheri and Tarighi, 2010). Control efficiency was calculated as follows: control efficiency (%) = (1 - T / C) × 100, where T and C are the disease index in the treatment and control, respectively. The experiment was repeated three times with three replications.
Results and Discussion
Identification of Botrytis cinerea.
To identify the tomato gray mold 18-042 isolate used in this study, ITS and TUB2 gene sequences were analyzed to determine its taxonomic position. National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) analysis showed that the ITS and TUB2 sequences of the isolate 18-042 were 100% identical to the sequence of B. cinerea in the GenBank database (GenBank accession nos. KU992694 and MF426036). The isolate 18-042 was grouped in the same clade as B. cinerea, but clearly distinct from other Botrytis species (Fig. 1).
Mycelial growth inhibition assay.
Four Bacillus isolates were co-cultured with B. cinerea 18-042, the causal agent of gray mold, to assess their antagonistic effects. We found that the four isolates showed strong inhibitory effects on mycelial growth of B. cinerea, ranging from 67% to 73% inhibition (Table 1). In addition, the four isolates exhibited strong mycelial growth inhibition of the pepper anthracnose fungus Colletotrichum scovillei and lettuce sclerotinia rot fungus Sclerotinia sclerotiorum, indicating a broad-spectrum inhibitory effect against other plant pathogenic fungi (Table 1).
Table 1.
Inhibition of mycelial growth of by antagonistic bacteri
| Pathogens | Treatment | ||||
|---|---|---|---|---|---|
| PgBE1 | PgBE14 | PgBE23 | PgBE51 | Control (untreated) | |
| Botrytis cinerea | ++++a | ++++ | ++++ | ++++ | + |
| Colletotrichum scovillei | +++++ | +++++ | ++++ | +++++ | + |
| Sclerotinia sclerotiorum | +++++ | ++++ | +++++ | +++++ | + |
Enzyme activity assay.
Several extracellular enzymes secreted by bacteria, such as cellulase, chitinase, glucanase, and protease are known to play a role in suppression of fungal or fungal-like phytopathogens through cell wall hydrolysis (Amaria et al., 2024; Fatima et al, 2023; Shin et al., 2023; Viswanathan et al., 2003; Woo et al., 2006; Xu et al., 2016). Meanwhile, phosphate-solubilizing ability promotes plant growth by aiding the phosphate availability to plants (Lee et al., 2012; Rodríguez and Fraga, 1999). To assess these functional traits, the four Bacillus isolates were tested for their production of pathogen inhibiting degradative enzymes and phosphate solubilizing activity using CMC agar, skim milk agar, and Pikovskaya's agar. All isolates exhibited cellulase, protease, and phosphate solubilizing activities, as evidenced by halo zone formation on the respective media, although PgBE1 produced a slightly smaller halo on CMC agar (Fig. 2). These findings suggest that the antifungal enzymes produced by the four Bacillus isolates may contribute to their inhibitory effect on mycelial growth of B. cinerea.
Biocontrol efficacy of antagonistic bacteria against Botrytis cinerea in tomato.
The effect of four Bacillus isolates on the lesion formation in detached tomato leaves was evaluated. Diseased area of the SDW-treated control leaves was about 30.6%, whereas PgBE14, PgBE23, and PgBE51 treatment significantly reduced the lesion area of B. cinerea, showing 2.3%, 2.6%, and 2.5% of diseased area, respectively (Fig. 3). The treatment of registered fungicide Mirabit resulted in 2.4% of diseased area, which was comparable to the PgBE14, PgBE23, or PgBE51 treatment. The PgBE1 isolate also reduced the diseased area of B. cinerea compared to the control, but it was less effective than other isolates.
Fig. 3.
Control of gray mold on detached tomato leaves by antagonistic bacteria. Bacterial suspension (10-fold dilution from bacterial suspension at OD600 of 1.0 in sterile distilled water) was sprayed onto the tomato leaves. After air drying, a mycelial agar plug of Botrytis cinerea was inoculated onto the middle of tomato leaves. Photographs were taken after 3 days of incubation in a moistened box (A) and the percent diseased area was determined using the ImageJ software (National Institutes of Health, Bethesda, MD, USA) (B). A registered fungicide Mirabit (pydiflumetofen 18.35% suspension concentrate) was used to compare the effectiveness of bacterial isolates. Different letters on the bars indicate significant differences according to Duncan's multiple range at P<0.05.
Next, we evaluated the effect of the isolates PgBE14, PgBE23, and PgBE51 in whole tomato plants. The disease index of plants inoculated with B. cinerea without bacterial suspension was 75.0 (Fig. 4, Table 2). However, when treated with PgBE14, PgBE23, and PgBE51, the disease index decreased to 21.9, 12.7, and 21.0, respectively (Fig. 4, Table 2). A registered fungicide Mirabit was used to compare the effectiveness of the bacterial isolates. The control efficiency of Mirabit was 81.4%, which was not significantly different from the PgBE23 treatment (83.1%), demonstrating the effectiveness of the bacterial isolate. Other isolates, PgBE14 and PgBE51, also exhibited considerable control efficiencies; 70.8% and 71.9% of control efficiencies, respectively.
Fig. 4.
Control of gray mold on 6-week-old tomato plants. Tomato plants were sprayed with the bacterial suspension and conidial suspension of Botrytis cinerea. Mock plants were sprayed with sterile distilled water without bacteria or conidia, and control plants were sprayed with only conidial suspension of B. cinerea. A fungicide treatment using Mirabit (pydiflumetofen 18.35% suspension concentrate) was applied at the manufacturer-recommended rate for comparison. Photographs were taken 5 days of incubation in a humid chamber at 25°C.
Table 2.
Control efficiency of bacterial isolates against Botrytis cinerea on tomato plants
| Treatments | Disease indexa | Control efficiency (%)c |
|---|---|---|
| PgBE14 | 21.9±1.9 bb | 70.8±2.5 b |
| PgBE23 | 12.7±1.0 c | 83.1±1.3 a |
| PgBE51 | 21.0±2.5 b | 71.9±3.4 b |
| Fungicide | 14.0±1.6 c | 81.4±2.1 a |
| SDW (control) | 75.0±3.1 a | - |
In recent decades, the use of biological control agents against B. cinerea has gained great attention and many studies have evaluated the efficacy of a variety of bacteria possessing antagonistic properties against B. cinerea (Compant et al., 2013; Elmer and Reglinski, 2006; Haidar et al., 2016; Sarven et al., 2020). There are several commercialized products for the biological control of B. cinerea, including Double Nickel 55WDG/LCTM, which is an B. amyloliquefaciens strain D747 based product (Haidar et al., 2016). In this study, we found B. amyloliquefaciens PgBE14 and PgBE51 and B. subtilis PgBE23 exhibited strong inhibitory effect against gray mold caused by B. cinerea on tomato. The B. amyloliquefaciens PgBE1 isolate also exhibited inhibitory effect of mycelial growth of B. cinerea, but was less effective than other isolates. Thus, we expect that B. amyloliquefaciens PgBE14 and PgBE51 and B. subtilis PgBE23 can be used as biological control agents to suppress tomato gray mold, which requiring further validation through field trials and formation development of the isolates.
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