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Article

Bacillus velezensis WZ-37, a New Broad-Spectrum Biocontrol Strain, Promotes the Growth of Tomato Seedlings

by
Xiuling Chen
1,2,
Haifeng Huang
3,
Shumei Zhang
4,
Yao Zhang
3,
Jingbin Jiang
1,
Youwen Qiu
3,
Jiayin Liu
5 and
Aoxue Wang
1,2,3,*
1
College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin 150030, China
2
Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture, Northeast Agricultural University, Harbin 150030, China
3
College of Life Sciences, Northeast Agricultural University, Harbin 150030, China
4
Institute of Microbiology, Heilongjiang Academy of Sciences, Harbin 150001, China
5
College of Sciences, Northeast Agricultural University, Harbin 150030, China
*
Author to whom correspondence should be addressed.
Agriculture 2021, 11(7), 581; https://doi.org/10.3390/agriculture11070581
Submission received: 28 May 2021 / Revised: 17 June 2021 / Accepted: 22 June 2021 / Published: 24 June 2021
(This article belongs to the Special Issue Disease Management of Vegetables)
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Abstract

:
A biological control agent is arguably an ideal alternative to chemical fungicide for the prevention and control of gray mold disease. During this process, a biological control produces low levels of pollution, generates few residues that pose no risk to the environment, and pathogens cannot gain resistance to it easily. A new antifungal strain isolated from plant rhizosphere exhibited high antifungal activity against the phytopathogens Botrytis cinerea, Fusarium oxysporum f. sp. cucumerinum, F. moniliforme, Sclerotinia sclerotiorum, Colletotrichum orbiculare, Alternaria nees, F. equiseti, and F. oxysporum f. sp. lycopersici. It was identified as Bacillus velezensis WZ-37 by morphological and physiological indices and comparisons of 16S rRNA and gyrB genes. WZ-37 can significantly inhibit the mycelia growth of B. cinerea by 96.97%. It can reduce a tomato fruit’s decay rate after 21 days of storage by 33.33% (13.34% less for the control) without significantly affecting its firmness and soluble solids. Plant height, stem diameter, and the fresh and dry weight of tomato seedlings were significantly increased when their seeds were soaked in a WZ-37 suspension (106 cfu/mL) for 3 h and grown for 21 days in soil. WZ-37 has broad-spectrum biocontrol and can prolong a tomato’s storage period and enhance its seedlings’ growth, making it a promising candidate strain for broad-spectrum biocontrol applications in agriculture.

1. Introduction

The tomato (Solanum lycopersicum), a member of the Solanaceae family, is an annual or perennial vegetable native to South America that is currently cultivated worldwide [1]. Tomato gray mold disease, caused by Botrytis cinerea, affects at least 1400 plant species [2], including tomatoes, cucumbers, peppers, strawberries, grapes, and cherries, to name a few [3,4,5]. This disease occurs not only during plant growth but also during the postharvest storage and transportation of fruits and vegetables, causing serious economic losses [6,7,8]. Further, B. cinerea can also systematically colonize host plants without causing symptoms of disease [9,10].
To control gray mold disease, in common practice farmers still mainly rely on chemical fungicides [11], but their efficacy against gray mold has generally decreased over the years because of pathogens evolving resistance to it [12]. Moreover, fungicide residues persist long after applications are made and are not easily degraded, causing serious problems to both the ecological environment and human health [13]. Using a biological control agent offers an ideal alternative to chemical fungicide in the prevention and control of gray mold disease [14,15]. In this respect, biological control has several notable advantages, namely its low pollution levels, few residues with no risk to the environment during the disease prevention and control process, and difficulty for targeted pathogens to acquire resistance [16].
Many microbial strains are now used as biological control agents (BCAs) [17], including yeast, bacteria, fungi, and actinomycetes [18,19,20], and a considerable number of these biological preparations have proven superior to applying chemical fungicides [21]. Trichoderma harzianum and Bacillus subtilis are widely used in gray mold prevention and control strategies [22]. Although the resources for potential effective biocontrol microorganisms are very rich in nature, most researchers have focused on biocontrol mechanisms and fermentation technology [23]. The isolation and characterization of novel broad-spectrum disease-resistant strains with pro-growth effects is a task that still eludes us.
The purpose of this study was to isolate antagonistic bacteria from the rhizosphere soil of plants and screen them against gray mold disease. The antifungal spectrum of candidate biological control strains was further investigated, providing a technological basis for the safe prevention and control of fungus diseases of plants. These findings are of great significance to reducing the use of chemical fungicides to control tomato gray mold disease and to improve the farmland ecological environment, thereby also providing technical support for food safety and sustainable development.

2. Materials and Methods

2.1. Experimental Strains and Plants

A total of 32 soil samples were collected from the rhizosphere of healthy tomato, cucumber, corn, wheat, and other plants. The established biocontrol bacterium Bacillus subtilis subsp. subtilis strain WXCDD105 served as the reference [24], and the pathogens B. cinerea (WD1), Alternaria nees (AN), Fusarium oxysporum f. sp. lycopersici (FK), F. oxysporum f. sp. cucumerinum (WS3), Colletotrichum orbiculare (CL), F. equiseti (YE), F. moniliforme (YO), F. oxysporium Schelcht (SL), F. oxysporum f. sp. niveum (XK), F. oxysporum f. sp. melonis (TK), F. verticillioides (FV), Sclerotinia sclerotiorum (WS2), and F. raminearum F609 and F1403 were isolated, identified, and preserved in the lab [24]. The fungal pathogens were separately inoculated on the host plants for virulence evaluation. Tomato cv. Dongnong 713, which is susceptible to B. cinerea, was provided by the Tomato Research Institute of the Northeast Agricultural University, China.

2.2. Isolation, Purification, and Screening of Biological Control Strains

From each soil sample, 1 g was added to a sterilized tube containing 9 mL of sterile water and this solution was vigorously vortexed. After letting it stand for 1 h, 1 mL of the ensuing supernatant was transferred to 9 mL of sterile water and gradient-diluted 100-fold [25]; 0.5 mL of this diluted mixture was uniformly spread on LB solid medium of a petri dish and cultured for 48 h at 35 °C. Single colonies were randomly picked according to the distinctive appearance of the colonies and they were preserved for further research.
The potential antipathogen strains were initially screened using a dual-culture method. For this, a 5 mm diameter of B. cinerea was placed on a petri dish, at its center [24]. Then, at 3 cm equidistant to it, three filter paper disks were positioned, to which 10 μL of bacterial suspension was added, and the dish was cultured for 7 days at 28 °C. Each strain had three replicates, and its zone of inhibition was measured.

2.3. Biopotential of Antipathogen Strain

The pathogen inhibition spectrum of WZ-37 was also identified via the dual-culture method, for which 14 plant pathogens were used (Table S1). These pathogens were grown and kept at 28 °C on potato dextrose agar plates. The size of the inhibition zone was measured after 7 days of incubation at 28 °C. This was compared to the WXCDD105 strain.

2.4. Characterization of Biocontrol Strain

2.4.1. Morphological Observations

After incubating at 35 °C on a rotary shaker at 180 rpm for 24 h, a drop of the WZ-37 liquid was streaked and inoculated on a LB agar plate, and this was incubated overnight at 35 °C. The colony shape, size, edge shape, surface and bulge shape, transparency, and medium color were observed. A single colony was taken and spread evenly on a glass slide and then dry-fixed and Gram-stained for 1 min. Next, the morphology, size, and spore characteristics of each strain were observed under a microscope.

2.4.2. Morphological and Biochemical Indices of Strain WZ-37

A glucose oxidation fermentation test was performed, as described by Cappuccino et al. [26]. Both the starch hydrolysis test and contact enzyme test were performed according to Lee et al. [27]. The aerobic or anaerobic test, nitrate reduction test, gelatin liquefaction test, methyl red reaction test, and VP test were all implemented by following Ji et al. [28].

2.4.3. 16S rRNA Sequence Analysis

The total genomic DNA of WZ-37 was extracted via the CTAB method [29]. The PCR amplifications of 16S rRNA and gyrB (all primers are listed in Table S2) were conducted in a 20 μL reaction system consisting of 0.2 μL of TaKaRa LA Taq (5 U/μL), 2.0 μL of 10 × LA Taq Buffer II (Mg2+ plus), 3.0 μL of dNTP mixture (2.5 mM each), 1.0 μL of template DNA (about 2.5 ng), 1.0 μL of forward primer (10 μM), and 1.0 μL of reverse primer (10 μM), topped by adding ddH2O [30]. The PCR cycling conditions were as follows: initial denaturation of 4 min at 95 °C followed by 35 cycles of denaturation (45 s at 94 °C), annealing (45 s at 55 °C), polymerization (90 s at 72 °C), and a final extension of 10 min at 72 °C. The ensuing PCR products were cloned into pCR2.1-TOPO, using the TOPO TA cloning kit (Invitrogen, China) according to the manufacturer’s instructions, and the positive clone was partially sequenced by the Beijing Genomics Institute (BGI) (Beijing, China). Sequence of 16S rRNA and gyrB in WZ-37 were obtained by sequencing. Sequences of other biocontrol strains were obtained using Blastn against the NCBI non-redundant database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 21 June 2021). A neighbor jointing tree was constructed using MEGA 7.05 software with the bootstrapping was 1000.

2.5. Inhibition of Plant Pathogens’ Mycelial Growth by Biocontrol Bacteria

The suspensions of WZ-37 and WXCDD105 were each cultured in flasks containing 50 mL of PDB liquid media, at 35 °C on a rotary shaker (180 rpm) for 24 h. Then, a flask was separately inoculated with a 5 mm diameter portion of B. cinerea, C. orbiculare, F. oxysporum f. sp. cucumerinum, and F. oxysporum f. sp. lycopersici mycelia blocks. The negative control was only inoculated with pathogenic fungi. The treatment and control groups were cultured at 28 °C and 120 rpm for 7 days. The resulting pathogenic mycelium was collected from PDB liquid media and then filtered, dried, and weighed accordingly.

2.6. Effects of the Biocontrol Strain on Tomato Seeds and Seedling Growth

2.6.1. Seed Germination

Different concentrations of WZ-37 and WXCDD105 in suspension (2 × 109 cfu/mL, 2 × 108 cfu/mL, 2 × 107 cfu/mL, 2 × 106 cfu/mL, and 2 × 105 cfu/mL), or just sterile water (control), were used to soak sterilized tomato seeds for 4 h. The seeds were then transferred to a petri dish that contained double-layer filter paper moistened with 3 mL of sterile water; all dishes were incubated at 28 °C for germination. After 4 days, the length of the radicle was measured [31].

2.6.2. Growth of Tomato Seedlings

Sterilized tomato seeds were immersed in a 4 × 106 cfu/mL diluted WZ-37 suspension or sterile water for 4 h. Next, tomato seeds were sown in pots with a sterile mixture of perlite: vermiculite: turfy soil (1: 1: 3) in a greenhouse. Plant height, stem diameter, fresh dry weight, and dry weight of tomato seedlings were all measured after 21 days of growth.

2.7. Effect of Biological Control on Tomato B. cinerea in Greenhouse

The inoculation of tomato plants with B. cinerea and WZ-37 was carried out as described by Kim et al. [32]. The concentration of B. cinerea was 1 × 107 spores per 1 mL, while that of WZ-37 and WXCDD105 was 1 × 107 cfu/mL. Group I: B. cinerea was sprayed on tomato leaves (B). Group II: B. cinerea was inoculated 24 h after the tomato leaves were sprayed with the WZ-37 suspension (WB). Group III: the WZ-37 suspension was sprayed 24 h after the tomato leaves were inoculated with B. cinerea (BW). Group IV: B. cinerea was inoculated 24 h after the tomato leaves were sprayed with the WXCDD105 suspension (XB). Group V: the WXCDD105 suspension was sprayed 24 h after the tomato leaves were inoculated with B. cinerea (BX).
The disease incidence of tomato plants was examined at 20 days post-inoculation. The disease severity index of B. cinerea on the tomato plants was defined as the percentage of leaf area that was in a diseased state, where 0 = no disease symptoms, 1 = 0.1–5%, 2 = 5.1–20%, 3 = 20.1–40%, and 4 = 40.1–100% [33]. The disease severity value per plant was calculated using this formula: Disease severity (%) = {(∑ [number of diseased leaves × disease severity index])/(4 × number of total leaves)} × 100. The disease control value was calculated as follows: Disease control (%) = ([A − B]/A) × 100, where A is the disease severity triggered by pathogen inoculation alone and B is the disease severity after incurring the various treatments.

2.8. Effect of Biocontrol Bacteria on Physiological Indices of Tomato Fruit

Cherry tomatoes (S. lycopersium var. cerasiforme TS-54) were harvested from a greenhouse at the firm-ripe stage. For each treatment, 30 cherry tomatoes were weighed and soaked in 1 × 107 cfu/mL of WZ-37 or WXCDD105, or sterilized water, for 4 min. Then all the tomato fruits were air dried and placed in a sterile box and stored at room temperature; their fruit rot and weight loss rates were calculated every 3 days. After 21 days of storage, the samples were removed to determine their fruit hardness and content of soluble solids [34]. For the analyses, the treatment using the WXCDD105 suspension group served as a reference comparison [35]. Fruit decay rate (%) = rotten number of fruits/total number of fruits × 100; Fruit weight loss (%) = (initial fruit weight − survey fruit weight)/initial fruit weight × 100. Fruit firmness was measured with a GY-4 fruit hardness tester, while the soluble solid content was measured with a KEM refractometer [36].

2.9. Statistical Analysis

Unless otherwise stated, the statistical program GraphPad Prism 7.0 (GraphPad Software Institute, San Diego, CA, USA) was used for all data analyses. One-way ANOVA analysis of variance or t-tests were used to compare among and between means, for which differences were considered significant at p ≤ 0.05. Every assay was performed in a completely randomized design with three replications. Data from repeated experiments were tested for homogeneity of variance prior to pooling. The percentage inhibition or disease reduction relative to the corresponding control was calculated as 100 × (C − T)/C, where C is the value for the water control group and T is that per treatment.

3. Results

3.1. Isolation, Purification, and Screening of Biocontrol Bacteria Strains

In total, 209 strains were isolated from 32 soil samples (File S1), of which six strains (No. 37, 85, 89, 101, 116, and 169) demonstrated more inhibition efficiency to B. cinerea (Table 1 and Figure 1). Among these six strains, No. 37 had the strongest inhibitory effect against the pathogens B. cinerea, F. oxysporum f. sp. cucumerinum, and F. moniliforme. Because No. 37 was adept at inhibiting the growth of both B. cinerea and F. moniliforme, we designated No. 37 as ‘WZ-37’ for further study.
Antagonistic assays between WZ-37 and a variety of plant pathogens were conducted (Table S3). Evidently, WZ-37 can produce the antifungal zone against 14 phytopathogens, for which its inhibitory effect was stronger than WXCDD105. In the dual-culture with the tested pathogens, WZ-37 produced pronounced inhibition zones against F. moniliforme, S. sclerotiorum, B. cinerea, C. orbiculare, A. Nees, F. equiseti, F. oxysporum f. sp. cucumerinum, and F. oxysporum f. sp. lycopersici in the process, generating a significantly better inhibitory effect than WXCDD105.

3.2. Morphological, Physiological, Biochemical Characteristics, and Identification of Biocontrol Strain

3.2.1. Morphological Observations

Colonies of WZ-37 were grayish white and round with surface moisture, appearing wrinkled, opaque, and with neat edges (Figure 2A). The bacterial cells were rod-shaped (0.5–0.7 μm × 1.6–5.0 μm), single or in pairs, and the gemmae were cylindrical, mesogenic, or proximal. The cytocysts were not expansion and were Gram positive (Figure 2B).

3.2.2. Molecular Identification of the WZ-37 Strain

WZ-37 is an aerobic, fermentative acid-producing bacterium that is Gram positive, according to the detection results of Bergey’s Manual of Determinative Bacteriology (Table S4). The methyl red reaction and VP test for WZ-37 were negative, but it tested positive for nitrate reduction reaction, amylohydrolysis, contact enzyme reaction, gelatin liquefaction, and citrate utilization.
Phylogenetic trees of the 16S rRNA and gyrB genes (Figure 3) for WZ-37 were constructed using MEGA 7.05. The higher homology of WZ-37 to the Bacillus sp. gyr B gene sequence was analyzed to more accurately identify the species. Bacillus velezensis BCRC 17467(DQ903176), positioned within the same branch as WZ-37, had the highest similarity (99.1%), whereas the similarity of WZ-37 to the other model strains was <95%. In combination with the compact automatic bacterial identification system (Table S5), WZ-37 was thus reliably identified as Bacillus velezensis.

3.3. Inhibitory Effects of Strain WZ-37 upon Mycelial Growth

WZ-37 can significantly inhibit the growth of B. cinerea, C. orbiculare, F. oxysporum f. sp. cucumerinum, and F. oxysporum f. sp. Lycopersici (Table 2). The WZ-37-treated group had almost no mycelial growth when compared to the pathogen-only control. The corresponding inhibitory rates of WZ-37 against the four pathogenic fungi were 96.97%, 93.82%, 95.59%, and 96.38%, respectively.

3.4. Effects of Strain WZ-37 Suspension on the Growth of Tomato Seeds and Seedlings

3.4.1. Tomato Seeds

The effect of a WZ-37 suspension on tomato seeds was tested under different concentrations (Figure 4). We found that the concentration of 106~107 cfu/mL was the optimal concentration for promoting tomato seed germination. Then the suspension was diluted to 1 × 107 cfu/mL, 4 × 106 cfu/mL, and 2.5 × 106 cfu/mL. The radicle length was 1.54 times higher in the WZ-37-treated group than the WXCDD105-treated group when 4 × 106 cfu/mL was applied. This was deemed the optimal concentration for promoting seed germination, given the 3.15 cm long radicle it yielded. The positive impact of WZ-37 for enhancing seed germination surpassed that of WXCDD105 (Figure S1).

3.4.2. Growth of Tomato Seedlings

The WZ-37 suspension had a better promoting effect on tomato seedlings when diluted to 4 × 106 cfu/mL (Figure 5). These results showed that all size traits—plant height, stem diameter, fresh weight, and dry weight—were significantly increased vis-à-vis the control (Table 3). Compared with the water control, the plant height, stem diameter, fresh weight, and dry weight of tomato seedlings treated with WZ-37 suspension increased, respectively, by 23.60%, 17.97%, 38.89%, and 33.33%.

3.5. Biocontrol Effect of WZ-37 against Tomato Gray Mold in the Greenhouse

On the fifth day post-inoculation with B. cinerea, individual leaves began to display disease symptoms, and the disease index reached 52.59% on the 20th day (Table 4). The disease index was significantly lower for the WB group (20.95%) than the control group. The disease index of the BW treatment group exceeded that of the WB disease prevention group. The results showed that WZ-37’s control effect reached as high as 60.16%, being significantly better than that of WXCDD105 (55.64%).

3.6. Effect of Strain WZ-37 on the Physiological Indices of Tomato Fruits

The fruit decay rate of a tomato in the WZ-37 treatment group was significantly lower than that in the control group after 21 days of storage. The decay rates of WZ-37, WXCDD105, and the negative control were 33.33%, 36.67%, and 46.67%, respectively (Figure 6A).
The fruit weight loss rate of tomato fruit generally increased with prolonged storage time. By the third day, there was little change in the weight loss rate of the treatments, but, from the sixth day onward, the weight loss rate of each group, especially that of the control group, increased significantly. However, the weight loss rate of tomato fruit was significantly lower in the WZ-37 treatment group than in the control group (Figure 6B).
After the 21 day storage, the firmness of the tomato fruits treated with WZ-37, WXCDD105, and the control decreased by 3.59%, 3.8%, and 4.13%, respectively (Figure 6C).

4. Discussion

In recent years, there have been many reports on the biological control function of Bacillus as an antagonistic bacterium. A member of this genus is B. velezensis, a species considered valuable because of its potent bioactive molecules [37]. It was previously found that B. velezensis can prevent and control various kinds of plant-disease-causing pathogens, such as Verticillium dahliae Kleb, Alternaria brassicae, Pyricularia grisea (Cooke) Sacc, Lettuce Root Rot (Pythium), Pythium, Rhizoctonia, Alternaria, Fusarium, Phytophthora, and Thielaviopsis [38,39,40,41]. In the present study, we isolated one strain of B. velezensis whose ability to control tomato gray mold disease was impressive. Surprisingly, few prior studies have reported on using B. velezensis to prevent and control tomato gray mold disease. Crucially, WZ-37 is more effective than other strains previously tested in the literature, for which the lowest disease index reported was 22.67% when inoculated for 10 days, whereas it was even lower for WZ-37 at just 20.95% [42].
When compared with the antagonistic results of other experimental studies, WZ-37 is also noteworthy because of its broader host spectrum and greater efficacy in combating fungi diseases that afflict crop plants. It can significantly inhibit the growth of B. cinerea, C. orbiculare, F. oxysporum f. sp. cucumerinum, and F. oxysporum f. sp. lycopersici by 96.97%, 93.82%, 95.59%, and 96.38%, respectively. However, the similar B. velezensis ZSY-1 strain can only limit infection by B. cinerea, F. oxysporum f. sp. capsicum, and F. oxysporum f. sp. niveum at an inhibition rate of 93.8%, 76.7%, and 57.0%, respectively [39].
More kinds of bioactive metabolites are produced by B. velezensis than by other bacterial strains [37], and B. velezensis is noted for its broad-spectrum resistance to pathogens, aphids, and nematodes [43,44,45]. Therefore, B. velezensis has great potential for improving food production and quality and biopesticide development and application. We know that B. velezensis can produce amylocyclicin, bacilysin, bacillomycin-D, bacillibactin, bacillaene, difficidin, fengycin, and macrolactin [46]. The congener B. subtilis can produce bacillibactin, bacillaene, bacilysin, difficidin, bacitracin, fengycin, locillomycin, subtilosin, and surfactin [47,48], while B. amyloliquefaciens can produce bacillibactin, bacillaene, bacillomycin-D, bacilysin, fengycin, and surfactin [47,49]. There are few studies on their active properties and mechanisms for biocontrol [50]. The volatiles released by B. velezensis that are capable of inhibiting fungi have rarely been reported, but the antibacterial and antifungal activity of volatile substances has been demonstrated in other Bacillus strains. Antifungal and antibacterial active substances can be further utilized as a kind of new biocontrol agent that can be easily produced and preserved. Moreover, many functional genes in B. velezensis that antagonize pathogens have been investigated [51]. For example, a strain of B. velezensis isolated from rice rhizosphere can induce resistance to disease in Arabidopsis thaliana and an overproduction of the PAD4 gene in plants, thereby triggering hydrogen peroxide, cell death, and sputum precipitation. When applied to plants, B. velezensis can stimulate increases in SOD, POD, and CAT activity levels in their leaves, suggesting that strains of this bacterial species can induce host plant resistance [52].
Bacillusvelezensis occurs in various ecological environments, such as the plant rhizosphere, various soils, plants’ interior, and even rivers. Most of the B. velezensis isolated from the rhizosphere can co-colonize plant roots and play an important role in inhibiting pathogens [53]. Existing studies have shown that many biocontrol bacteria can curtail the growth of pathogenic microorganisms while promoting the growth of host plants. Some Bacillus can even enhance plant growth by producing IAA, NH3, and ACC deaminase [54,55]. Furthermore, it has been confirmed that B. velezensis can promote seed germination and seedling growth of beet, carrot, cucumber, pepper, potato, radish, squash, tomato, and turnip plants [56]. Consistent with those findings, our study also showed that B. velezensis is capable of significantly improving the germination of tomato seeds and the early growth of tomato seedlings. Chemical preservatives have a significant impact on the quality of fruits and vegetables after their harvest but pose a threat to human health and cause environmental pollution. As a safe, effective, environmentally friendly, and promising way to preserve fresh fruits and vegetables, microbial preservatives have gradually emerged as robust candidates for such practical applications. Their use meets the public’s rising demand for safe and natural fresh produce, and we anticipate they can replace chemical preservatives in the future. Immersing the tomato fruits in the WZ-37 fermentation liquid led to their fruit weight being diminished but not their fruit quality. However, the mechanisms by which WZ-37 fosters plant growth and contributes to fruit preservation await further study.

5. Conclusions

We isolated a novel antifungal strain from the rhizosphere of plants, one that is capable of string antifungal activity against the crop pathogens B. cinerea, Fusarium oxysporum f. sp. cucumerinum, F. moniliforme, S. sclerotiorum, B. cinerea, C. orbiculare, A. nees, F. equiseti, and F. oxysporum f. sp. lycopersicie. Using morphological and physiological indices and comparisons of 16S rRNA and gyrB genes, we identified the strain as Bacillus velezensis WZ-37. Importantly for tomato crops, WZ-37 can significantly inhibit the mycelia growth of B. cinerea by 96.97%, and it can extend the storage period of harvested tomato and also promote the growth of tomato seedlings. Taken together, this suggests that the WZ-37 strain is perhaps the best candidate for use as a broad-spectrum biocontrol bacterium strain in agricultural settings and applications.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/agriculture11070581/s1: Figure S1—The effects of different concentrations of suspension on the growth of tomato seeds’ radicle; Table S1—Pathogens used for the determination of the inhibition spectrum of WZ-37; Table S2—Primers used for the amplification of 16S rRNA and gyrB; Table S3—Determination of the inhibition spectrum of WZ-37; Table S4—Physiological and biochemical characteristics of WZ-37; Table S5—The results of the bacterial automatic identification system of WZ-37; Excel S1—Screening results for the biocontrol strains against WD1.

Author Contributions

Conceptualization, X.C. and A.W.; methodology, H.H. and S.Z.; software, X.C.; validation, J.J., S.Z. and A.W.; formal analysis, X.C. and Q.Y; investigation, X.C., H.H. and J.J.; resources, Y.Z.; data curation, S.Z. and Y.Q.; writing—original draft preparation, X.C. and H.H.; writing—review and editing, A.W.; visualization, J.L.; supervision, A.W.; project administration, A.W.; funding acquisition, X.C. and A.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the National Key Research and Development Program of China (grant number 2017YFE0105000), the National Natural Science Foundation of Heilongjiang Province (grant number LH2020C011), the Postdoctoral Scientific Research Developmental Fund of Heilongjiang Province (grant number LBH-Q18023), and the National Natural Science Foundation of China (grant number 31872120).

Institutional Review Board Statement

This research did not involve any human participants and/or animals.

Informed Consent Statement

This research did not involve any human participants and/or animals.

Data Availability Statement

This study did not report any data.

Conflicts of Interest

The authors declare no conflict of interest.

Compliance with Ethical Standards

We certify that this paper is new. Neither the entire paper nor any part of its content has been published or has been accepted elsewhere. It is not being submitted to any other journal. We confirm that we have the authority to publish the material presented. All authors have read the manuscript and approved the final manuscript. This research did not involve any human participants and/or animals.

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Figure 1. Antifungal activity of the WZ-37 strain against B. cinerea (A) and F. moniliforme (B).
Figure 1. Antifungal activity of the WZ-37 strain against B. cinerea (A) and F. moniliforme (B).
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Figure 2. Morphological characteristics of WZ-37 on LB medium plate culture (A), and observed under an optical microscope (B).
Figure 2. Morphological characteristics of WZ-37 on LB medium plate culture (A), and observed under an optical microscope (B).
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Figure 3. Phylogenetic tree of the WZ-37 strain based on 16S rRNA (A) and gyrB (B).
Figure 3. Phylogenetic tree of the WZ-37 strain based on 16S rRNA (A) and gyrB (B).
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Figure 4. Effects of different concentrations of suspension on the growth of tomato seeds’ radicle. Note: Bars are means of three replications and their standard errors. Different letters above the bars indicate significant differences at p ≤ 0.05 (according to Duncan’s Multiple Range Test).
Figure 4. Effects of different concentrations of suspension on the growth of tomato seeds’ radicle. Note: Bars are means of three replications and their standard errors. Different letters above the bars indicate significant differences at p ≤ 0.05 (according to Duncan’s Multiple Range Test).
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Figure 5. Bioaccumulation of the WZ-37 strain on tomato seedlings.
Figure 5. Bioaccumulation of the WZ-37 strain on tomato seedlings.
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Figure 6. Effects of the WZ-37 strain on the physiological indices of tomato fruits: (A) decay rate; (B) weight loss; (C) fruit quality. Bars are means of three replications and their standard errors. Different letters above the bars indicate significant differences at p ≤ 0.05 (according to Duncan’s Multiple Range Test).
Figure 6. Effects of the WZ-37 strain on the physiological indices of tomato fruits: (A) decay rate; (B) weight loss; (C) fruit quality. Bars are means of three replications and their standard errors. Different letters above the bars indicate significant differences at p ≤ 0.05 (according to Duncan’s Multiple Range Test).
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Table
Table 1. Screening results for the biocontrol strains.
Table 1. Screening results for the biocontrol strains.
Strain NumberInhibition Zone/mm
WD1WS3FO
378.50 ± 0.57 a8.00 ± 0.51 a9.00 ± 0.68 a
858.00 ± 0.49 a3.00 ± 0.48 b2.50 ± 0.37 b
1016.00 ± 0.39 b1.50 ± 0.29 c1.50 ± 0.31 bc
893.50 ± 0.41 c1.00 ± 0.29 c1.00 ± 0.27 c
1693.00 ± 0.40 c0.50 ± 0.23 c1.00 ± 0.29 c
1163.00 ± 0.11 c0.50 ± 0.11 c0.50 ± 0.11 c
WD1: Botrytis cinerea; FO: Fusarium moniliforme; WS3: Fusarium oxysporum f. sp. cucumerinum. Different letters indicate significant differences at p ≤ 0.05 (according to Duncan’s Multiple Range Test).
Table
Table 2. Inhibition of mycelium growth of plant pathogenic fungi by the WZ-37 strain.
Table 2. Inhibition of mycelium growth of plant pathogenic fungi by the WZ-37 strain.
Strain WD1FKCLWS3
WZ-37Mycelium Dry Weight (mg)14.00 ± 1.78 a23.00 ± 2.04 a22.00 ± 1.78 a16.00 ± 2.12 a
Inhibition Rate (%)96.9793.8295.5996.38
WXCDD105Mycelium Dry Weight (mg)15.00 ± 1.87 a65.00 ± 2.04 b95.00 ± 1.78 b117.00 ± 2.86 b
Inhibition Rate (%)96.7582.5380.9673.53
CKMycelium Dry Weight (mg)462.00 ± 24.91 b372.00 ± 15.12 c499.00 ± 28.08 c442.00 ± 23.72 c
Inhibition Rate (%)----
Different letters indicate significant differences at p ≤ 0.05 (according to Duncan’s Multiple Range Test).
Table
Table 3. Effects of the WZ-37 strain on promoting the growth of tomato seedlings.
Table 3. Effects of the WZ-37 strain on promoting the growth of tomato seedlings.
TreatmentHeight/mmStem Diameter/mmFresh Weight/gDry Weight/g
WZ-3769.65 ± 0.60 a1.51 ± 0.03 a0.25 ± 0.02 a0.016 ± 0.02 a
CK56.35 ± 0.46 b1.28 ± 0.03 b0.18 ± 0.03 b0.012 ± 0.01 b
Different letters indicate significant differences at p ≤ 0.05 (according to Duncan’s Multiple Range Test).
Table
Table 4. Biocontrol effect of WZ-37 against B. cinerea.
Table 4. Biocontrol effect of WZ-37 against B. cinerea.
TreatmentDisease Index %Control Efficiency %
B52.59 ± 0.71 a-
WB20.95 ± 0.57 c60.16%
BW26.67 ± 0.74 c49.29%
XB23.33 ± 0.72 b55.64%
BX28.15 ± 0.59 b46.47%
Treatment B denotes B. cinerea being sprayed onto tomato leaves; treatment WB denotes B. cinerea being inoculated 24 h after the tomato leaves were sprayed with the WZ-37 suspension; treatment BW denotes the WZ-37 suspension being sprayed at 24 h after the tomato leaves were inoculated with B. cinerea; treatment XB denotes B. cinerea being inoculated 24 h after the tomato leaves were sprayed with the WXCDD105 suspension; treatment BX denotes the WXCDD105 suspension being sprayed 24 h after the tomato leaves were inoculated with B. cinerea. Different letters indicate significant differences at p ≤ 0.05 (according to Duncan’s Multiple Range Test).
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Chen, X.; Huang, H.; Zhang, S.; Zhang, Y.; Jiang, J.; Qiu, Y.; Liu, J.; Wang, A. Bacillus velezensis WZ-37, a New Broad-Spectrum Biocontrol Strain, Promotes the Growth of Tomato Seedlings. Agriculture 2021, 11, 581. https://doi.org/10.3390/agriculture11070581

AMA Style

Chen X, Huang H, Zhang S, Zhang Y, Jiang J, Qiu Y, Liu J, Wang A. Bacillus velezensis WZ-37, a New Broad-Spectrum Biocontrol Strain, Promotes the Growth of Tomato Seedlings. Agriculture. 2021; 11(7):581. https://doi.org/10.3390/agriculture11070581

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Chen, Xiuling, Haifeng Huang, Shumei Zhang, Yao Zhang, Jingbin Jiang, Youwen Qiu, Jiayin Liu, and Aoxue Wang. 2021. "Bacillus velezensis WZ-37, a New Broad-Spectrum Biocontrol Strain, Promotes the Growth of Tomato Seedlings" Agriculture 11, no. 7: 581. https://doi.org/10.3390/agriculture11070581

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