Next Article in Journal
Vegetative Growth and Productivity of Potted Crocus sativus in Different Growing Media
Next Article in Special Issue
The Complete Genome Sequence, Molecular Detection, and Anatomical Analysis of Hibiscus Chlorotic Ringspot Virus Infecting Hibiscus rosa-sinensis in Peninsular Malaysia
Previous Article in Journal
Transcriptomic Analysis of Salicylic Acid Promoting Seed Germination of Melon under Salt Stress
Previous Article in Special Issue
Comparison of Different Physical Methods and Preservatives for Control of Fusarium proliferatum Rot in Garlic
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 9
Issue 3
10.3390/horticulturae9030376
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Identification, Biocontrol Activity, and Field Application Effect of Bacillus velezensis Yb-1

by
Bao Wang
1,
Erfeng Li
2,
Yang Lin
3,
Tongbin Xiao
1,
Xuncong Ji
1,
Zhixiang Zhao
1,* and
Wanrong Yan
1,*
1
Key Laboratory of Plant Disease and Pest Control of Hainan Province/Institute of Plant Protection, Hainan Academy of Agricultural Sciences (Research Center of Quality Safety and Standards for Agricultural Products of Hainan Academy of Agricultural Sciences), Haikou 571100, China
2
Horticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, China
3
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2023, 9(3), 376; https://doi.org/10.3390/horticulturae9030376
Submission received: 17 February 2023 / Revised: 10 March 2023 / Accepted: 10 March 2023 / Published: 13 March 2023
(This article belongs to the Special Issue Horticultural Plants Pathology and Advances in Disease Management)
Browse Figures
Review Reports Versions Notes

Abstract

:
Colletotrichum capsici is one of the most important pathogens on chili peppers. Unreasonable application of chemical fungicides will lead to threats to human and animal health, environmental damage, and increased fungicide resistance to pathogens. As an alternative strategy, biological control has been paid more and more attention by academics. In this study, the Yb-1 strain was isolated from healthy cucumber leaves, which could significantly restrict the mycelium growth of C. capsici and Botrytis cinerea, with inhibition rates of 93.49% and 74.03%, respectively. Strain Yb-1 was identified as Bacillus velezensis by morphological features and 16S rRNA gene, gyrA gene, and gyrB gene sequence analysis. When pepper seeds were treated with different concentrations of bacterial suspension, we found that a medium-concentration treatment (104 CFU/mL and 105 CFU/mL) could promote seed germination and growth, while high-concentration treatments (108 CFU/mL) limited seed germination and growth. In addition, the root-irrigation method, acupuncture-leaf method, and leaf-cutting method were used to evaluate the safety of strain Yb-1 on peppers. The results indicated that Yb-1 did not affect the normal growth of peppers. The results of further field trials showed that the control effect of strain Yb-1 on pepper anthracnose was 59.45%. Thus, the strain B. velezensis Yb-1 has great biocontrol potential for pepper anthracnose and promotes plant growth.

1. Introduction

Peppers (Capsicum annum L.) are one of the most important vegetable crops in the world for cooking and as a flavoring agent. It originated in the Americas and has spread and grown in countries such as New Zealand, Malaysia, and China [1]. It has become the most widely planted vegetable and the most heavily consumed spicy condiment in China [2]. However, fungal diseases severely limit pepper development, especially anthracnose and gray mold caused by Colletotrichum capsici and Botrytis cinerea. Both of these pathogens can infect the leaves, stalks, flowers, and fruits of peppers, and are serious pre- and postharvest diseases. The current concern regarding the use of chemical fungicides for the prevention and management of fungal diseases not only raises the danger of fungicide resistance, but also presents possible harm to the environment and public health due to the high pesticide residue levels [3,4]. As a result, biological control is an alternate strategy for eradicating pepper diseases because it is cost-effective, environmentally friendly, and sustainable in terms of plant protection.
Many biocontrol agents have been reported for the control of plant diseases, such as bacteria, yeast, and fungi [5,6,7]. Bacillus, belonging to bacteria, has been widely studied for disease control because of its rapid reproduction, salt tolerance, and heat tolerance. The biocontrol mechanisms of Bacillus species mainly involve the production of antagonists, competition for nutrients and space, and induced systemic resistance (ISR) [8,9]. Cui et al. isolated an endophytic Bacillus velezensis strain 8-4 from healthy potatoes, which could limit the growth of a variety of plant fungal pathogens, had a control effect of 51.83% on potato scab in the field, and increased potato yield by 19.91% [10]. Some Bacillus species can promote plant growth through direct or indirect mechanisms to increase crop yield, including improving nutrient availability, altering plant growth hormone homeostasis, and reducing the severity of abiotic stress [11]. Microbiological fertilizers containing B. subtilis could not only increase crop yield but could also increase the nitrogen utilization ratio and microbial population richness in agricultural soil [12]. Phosphorus is an important nutrient for plants but must be mobilized before it can be absorbed. Bacillus can solubilize phosphorus, making it better available to plants [13]. Bacillus spp. may directly increase plant yield through mechanisms of imparting the production of phytohormones or plant growth regulators (PGRs) such as auxins, cytokinins, gibberellins, ethylene, and abscisic acid [14]. Due to the limited ecological niche, some Bacillus species, such as plant-growth-promoting rhizobacteria (PGPR), colonize the surface or interior of plant roots, which may lead to the inviability of some plant-unfriendly microorganisms. Meanwhile, ISR is one of the defense mechanisms in plants, which mainly depends on jasmonate (JA) and/or ethylene (ET) signaling pathways. Elicitation of ISR by B. cereus [15], B. megaterium [16], and B. amyloliquefaciens [17] species has been demonstrated in plants to defend against pathogens. Bacillus is a potential biocontrol microbial agent for the control of pepper anthracnose and gray mold [18,19]. At present, the studies on the biocontrol of Bacillus are mainly about in vitro antagonism and mechanism of action; however, few studies on its field application have been reported.
In this work, we isolated a bacterial strain (Yb-1) from cucumber leaves. It was identified as B. velezensis by morphological observation and molecular identification. Then, the antagonistic activity against C. capsici and B. cinerea and the promotion of pepper-seed germination were studied in vitro, and the control effect of the B. velezensis Yb-1 strain on pepper anthracnose was preliminarily explored through field experiments.

2. Materials and Methods

2.1. Microorganisms and Growth Condition

The biocontrol strains used in this study were isolated from healthy cucumbers (Cucumis sativus L.) and deposited at the Institute of Plant Protection, Hainan Academy of Agricultural Sciences, under accession number Yb-1. The pathogens used in this study, including C. capsici ATCC 96157 and B. cinerea bio-52634, were provided by the Biocontrol Research Group of the Institute of Plant Protection, Hainan Academy of Agricultural Sciences. The fungi were cultured in potato dextrose agar (PDA) plates at 28 °C and bacteria were cultured in LB plates at 28 °C. The bacterial suspension was incubated in LB liquid medium at 37 °C and 180 rpm for 24 h.

2.2. Determination of Antifungal Activity

The antifungal activity of Yb-1 against C. capsici ATCC 96157 and B. cinerea bio-52634 was performed according to the method from Zheng et al. [20], with some improvements. The biocontrol bacterial suspension was inoculated at three symmetrical spots on the PDA plate. The 5 µL culture from the Yb-1 strain suspension (1 × 108 CFU/mL) was equidistantly placed on the PDA plate at a specified location. Four millimeter agar discs with fresh PDA cultures of C. capsici ATCC 96157 and B. cinerea bio-52634 were placed, respectively, at the center of the PDA plate for each bacterial isolate and were incubated at 28 °C for 7 days and 3 days, respectively. In addition, the PDA plates inoculated with sterile water were used as blank controls. The same experiment was repeated three times. The antifungal activity against the indicator fungal colony was detected using the agar-disk diffusion assay, measuring the diameter of the fungal colony around the bacterial colony, and the blank control was calculated. The percentage growth inhibition formula refers to that from Khan et al. [21], with some improvements:
Inhibition (%) = [(D − d)/D − 0.4] × 100%,
where d is the diameter of the fungal colony opposite the bacterial colony, and D is the maximum diameter of the fungal colony of the control culture.

2.3. Identification of Yb-1

The Yb-1 strain was cultured in LB plates at 28 °C for 2 days. The morphological identification was determined using the colony characteristics according to the Manual for Systematic Identification of Common Bacteria [22].
The molecular identification was conducted as follows. The DNA was extracted using the bacterial DNA extraction kit (Omega Bio-Tek, Shanghai, China), according to the manufacturer’s recommendations. Molecular identification was performed using the 16S ribosomal RNA gene universal primer pair 27F/1492R [23]. Moreover, the identity of Bacillus spp. was determined using the specific primer pair gyrA-F/gyrA-R [24] and UP1/UP2r [25], which could amplify the previously described gyrase subunit A (gyrA) gene and gyrase subunit B (gyrB) gene. Each 50 µL PCR reaction contained 1 µL of genomic DNA, 0.3 µL of Ex Taq (TaKaRa, Dalian, China), 5 µL of 10× EX PCR Buffer (TaKaRa, Dalian, China), 1 µL of dNTP (10 mM), 1 µL of each primer pair (10 µM), and 40.7 µL of ddH2O. The amplification program included 95 °C for 5 min, 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min (30 cycles), and 72 °C for 10 min, followed by a 4 °C soak. Then, the PCR products were analyzed by electrophoresis in a 1% agarose gel. PCR products were purified using a DNA gel recovery kit (AXYDEN, Silicon Valley, USA). The sequence was completed using Sanger dideoxy sequencing from Hainan Nanshan Biotech Co., Ltd., Kaikou, China. The DNA sequence was analyzed by comparing it with those in the NCBI GenBank using the BLASTN program, based on the method from Altschul et al. [26]. The splicing of DNA sequences was completed using DNAMAN software (Version 6.0, LynnonBiosoft, USA). The DNA multiple-sequence homology analysis was performed using MEGA software (Version 11.0, Richlandtown, PA, USA) to construct a phylogenetic tree using the neighbor-joining method [27]. Experimental sequences of all genes were deposited into the GenBank.

2.4. The Growth-Promotion Ability of Yb-1 in Pepper Seedlings

After 24 h of cultivation (37 °C, 180 rpm), the Yb-1 strain culture was centrifugated at 5000 rpm for 10 min, and the precipitates were diluted with sterile water to obtain suspensions with concentrations of 103, 104, 105, 106, 107, and 108 CFU/mL. The pepper variety was shuaiyou, which is a hybrid F1 seed produced by Anhuikubota Seed Co., Ltd. The pepper seeds were placed in a petri dish lined with wetted filter paper and then the suspension of the above different concentrations was added to the petri dish until the absorbent paper remained moist, with sterile water as the control. This section refers to Li et al. [28], with minor modifications. In each treatment, twenty seeds were used and incubated in a 12 h light/12 h dark cycle at 28 °C. During this period, watering was completed 1 or 2 times a day to keep humidity and provide a suitable culture environment for the seeds. After 7 days, the germination of seeds was investigated, and bud length and root length were measured. The seed germination rate was calculated using the following formula [29]:
Germination rate = n/N × 100%,
where n is the number of seed germinated, and N is the total number of seeds planted.

2.5. Safety Evaluation of Yb-1 to Pepper

In order to determine how safe Yb-1 is to peppers, pepper plants were inoculated in three ways, namely, the root-irrigation method, the acupuncture-leaf method, and the leaf-cutting method. The chili pepper plants were planted in a flowerpot for 30 days. Yb-1 was cultured on LB plates at 28 °C for 48 h. A single colony was selected and inoculated in LB liquid medium at 37 °C for shaken culture at 180 rpm for 24 h. The bacteria were collected and diluted to 1 × 104 CFU/mL with sterile water. The sterile water was used as a control, with 3 repeats in each treatment. The state of the pepper plants was observed continuously for one month after treatment, focusing on whether the plant growth was inhibited and infected.

2.6. Field Efficacy Assessment of Strain Yb-1 against Pepper Anthracnose

2.6.1. Field Experiment Design

Chilis were planted and managed according to the local conventional methods. Plots that grow chilis all year round and are infected by C. capsici every year were chosen. The experimental site was located in the experimental base of the Hainan Academy of Agricultural Sciences (110°11′55″ longitude, 19°45′11″ latitude), Yongfa Town, Chengmai County, Hainan Province. The selection of pepper varieties was consistent with that mentioned in Section 2.4. Four different treatments were set up in this experiment (A: Yb-1 suspension of 1 × 104 CFU/mL; B: 10% difenoconazole 70 g/667 m2, produced by Syngenta Nantong Crop Protection Co., Ltd.; C: Yb-1 suspension of 1 × 104 CFU/mL + 10% difenoconazole 70 g/667 m2; D: clean water), each repeated three times. Each cell area was about 30 m2, randomly arranged. The first treatment was in the early stage of pepper anthracnose, and then the second treatment was carried out at an interval of 7 days, on 25 January 2018 and 1 February 2018, respectively. Two surveys were conducted during the experiment, on the day before the first treatment and 10 days after the second treatment.

2.6.2. Methods of Investigation

The disease index was investigated according to the method described in Pesticide—Guidelines for the field efficacy trials (Ⅰ)—Fungicides against pepper anthracnose in standard GB/T 17980.33-2000, issued by the People’s Republic of China [30], and the control efficacy was also calculated as described in standard GB/T 17980.33-2000. The 50 fruits were randomly investigated in each community and were graded according to the percentage of disease spot areas in the whole fruit area:
Level 0: Pepper fruits are healthy and without disease spots.
Level 1: The disease spot area accounts for less than 2% of the fruit area.
Level 3: The disease spot area accounts for 3% to 8% of the fruit area.
Level 5: The disease spot area accounts for 9% to 15% of the fruit area.
Level 7: The disease spot area accounts for 16% to 25% of the fruit area.
Level 9: The disease spot area accounts for more than 25% of the fruit area.
The disease index and control efficiency is calculated as follows:
Disease index = {[∑ (number of diseased fruits at each level × representative value at each level)] / (number of total fruits investigated × highest representative value)} × 100,
Control efficiency = [1 − (CK0 × PT1)/(CK1 × PT0)] × 100%,
where CK0 represents the disease index of the blank control area before treatment, CK1 represents the disease index of the blank control area after treatment, PT0 represents the disease index before treatment in the treatment area, and PT1 represents the disease index after treatment in the treatment area.

2.7. Statistical Analysis

The data were statistically analyzed using an analysis of variance (ANOVA) using data processing system (Version DPS 15.10 advanced edition, China) software, and the means were subjected to Duncan’s multiple-range test at p < 0.05. The phylogenetic tree of different lines was jointly constructed using the neighbor-joining (NJ) method in MEGA software. OriginLab software (Version 2022, Northampton, MA, USA) was used for chart graphing.

3. Results

3.1. Antagonistic Activity of Yb-1

We isolated some strains from cucumber leaves and the cucumber leaves were collected in Wenchang City (110°47′34″ longitude, 19°45′19″ latitude). Except for Yb-1, which showed a strong inhibitory effect on plant pathogenic fungi, other isolates had no antagonistic activity. In vitro inhibition of C. capsici and B. cinerea mycelial growth by Yb-1 was displayed by the formation of a clear zone of inhibition, in which the bacteria and the pathogen did not grow into each other, and the pathogenic colony exhibited reduced growth compared to the control. Isolate Yb-1 was the most effective in the suppressing the mycelial growth of C. capsici, for which the inhibition rate was up to 93.49%. It also restricted the mycelia growth of B. cinerea, with an inhibition rate of 74.03% (Figure 1). Therefore, Yb-1 has great potential for biocontrol development on plant pathogenic fungi.

3.2. Identification of Yb-1

The colony of strain Yb-1 on the LB plates was round or oval, light yellow, and transparent, with a smooth surface and irregular edges after culture for 48 h (Figure 2A,B). To further identify strain Yb-1, the 16S RNA gene, the gyrA gene, and the gyrB gene were analyzed via PCR amplification and sequencing. After 1% agarose gel electrophoresis, it was found that the bands of the 16S RNA gene, the gyrA gene, and the gyrB gene were 1500 bp, 1000 bp, and 1200 bp, respectively, which was in line with our expectations. According to the obtained gene sequence information, and through multiple comparative alignment analyses in the NCBI database, we found that the similarity between Yb-1 and the two strains was the highest. The degrees of similarity between Yb-1 and B. velezensis FZB42 and Yb-1 and B. velezensis JS25R were 98.38% and 98.45%, respectively. The phylogenetic tree was constructed through the concatenation of the three genes. The phylogenetic tree demonstrated that strain Yb-1 formed a cluster closely related to the B. velezensis strains FZB42, JS25R, SQR9, and CBMB205, indicating that Yb-1 belongs to the species B. velezensis (Figure 2C). Combined with the results of the morphological and molecular biology identification, Yb-1 was identified as B. velezensis. The 16S ribosomal RNA gene, the gyrA gene, and the gyrB gene partial sequences of Yb-1 were deposited into the GenBank database with the accession numbers OP776915, OP797799, and OP797798, respectively.

3.3. Effect of Yb-1 on the Germination and Growth of Pepper Seeds

In order to study the effect of Yb-1 on the germination and growth of pepper seeds, the seeds were treated with different concentrations of Yb-1 suspension, with sterile water as the control. Yb-1 increased the germination rate of seeds to different degrees within a certain concentration range (Table 1). With the continuous increase in the treatment concentration, the seed germination rate at first increased and then decreased. At the same time, the bud length and root length were the longest under the treatment of 104 CFU/mL, with 15.66 mm and 29.34 mm, respectively (Figure 3A,B). However, it was worth noting that when the concentration increased to 108 CFU/mL, the germination and growth of the pepper seeds were completely inhibited. In short, the treatment with an intermediate concentration of Yb-1 could promote the germination and growth of pepper seeds, while the treatment with a high concentration limited the germination and growth.

3.4. Safety Evaluation of Yb-1 to Peppers

Safety is critical for plants when biocontrol bacteria are inoculated back into plants. In this study, we used three different methods to inoculate potted pepper plants, namely, the root-irrigation method, the injection method, and the leaf-cutting method. During the 30 days after treatment, we continued to observe the growth of the pepper plants, especially observing whether the plants were deciduous or infected. The results showed that the roots, stems, leaves, flowers, and fruits of the peppers could grow normally, and there was no difference when compared with the control (Figure 4). In other words, pepper plants inoculated with Yb-1 with the concentration of 104 CFU/mL are safe for growth and development.

3.5. Evaluation of the Biocontrol of Pepper Anthracnose with Strain Yb-1 in the Field

Many biocontrol agents show good biocontrol activity in laboratory research, but the practical application effect in the field is very poor, so the field evaluation of biocontrol agents is very important. Therefore, we evaluated the effect of Yb-1 on pepper anthracnose in the field (Figure 5). The disease index of peppers was investigated before the first treatment, and the results showed that the disease index was 0.89~1.04. The difference in the variance analysis was not significant, indicating that the infection of C. capsici was relatively uniform before treatment. The disease index of the peppers was investigated again 10 days after the second treatment. The results showed that the disease index of treatment D was 13.34, while the disease indexes of treatment A, treatment B, and treatment C were 5.41, 5.11, and 3.31, respectively. The control effects of treatment A, treatment B, and treatment C on C. capsici were 59.45%, 64.63%, and 71.98%, respectively (Table 2), although the control effect of the Yb-1 suspension alone was low when compared with the commercial 10% difenoconazole. However, the combined use of the two can control pepper anthracnose by more than 70%. As a result, the Yb-1 strain has the potential to be developed into a commercial biological control agent for the control of C. capsici.

4. Discussion

Pepper anthracnose seriously restricts the growth of pepper plants and causes huge losses to the yield. At present, chemical fungicides are the most commonly used strategy for the control of fungal diseases. However, high concentrations of pesticide residues are unfriendly to people, animals, and the environment, and some of their use has been explicitly banned in many countries [31,32,33]. Therefore, many scientists have been looking for new, low-residue, human and animal-friendly, and environmentally friendly alternatives. Biological control is an optional strategy because it does not cause pesticide residues and is environmentally friendly. Bacillus spp. has been widely studied as a biological control agent to control plant diseases because of its rapid reproduction and strong tolerance to the environment [34].
In this study, strain Yb-1 was identified as a strain of B. velezensis based on the morphological characteristics and sequence analysis of the 16S rRNA gene, the gyrA gene, and the gyrB gene. Polygene molecular identification can improve the accuracy of identification, which has been fully affirmed in modern molecular biology [35,36,37]. B. velezensis has been reported to have a variety of biocontrol functions, such as inhibiting pathogen growth in vitro [38], promoting plant growth [39], and controlling plant diseases [40]. A potential biocontrol strain can usually inhibit the growth of a variety of pathogens [41,42]. In vitro, the inhibition rate of strain Yb-1 on B. cinerea was 74.03%, and the inhibition rate on C. capsici was 93.49%. The results showed that pepper seeds treated with Yb-1 suspensions with the concentrations of 104 CFU/mL and 105 CFU/mL could significantly improve the germination rate of seeds and promote the growth of buds. The results of this part are consistent with those of Li et al. [28] and Jiang et al. [43]. We speculate that Yb-1 may produce some plant hormones that promote seed germination and growth. An interesting phenomenon was that when the concentration of Yb-1 was 108 CFU/mL, the germination and growth of the pepper seeds were completely restricted. At the same time, the Yb-1 concentration of 108 CFU/mL showed excellent antagonistic activity in vitro, which seemed to be contradictory. The ability to inhibit the growth of pathogens indicates that Yb-1 can produce antifungal substances, such as lipopeptides. It can promote seed germination and growth, indicating that Yb-1 may produce some plant growth hormones. These also proved, once again, that Yb-1 is a potential biocontrol strain. Why does Yb-1 at the concentration of 108CFU/mL restrict seed germination and growth? In this study, we suspect that the possible reason was that the seeds were treated differently. Different from the study by Li et al. [28], during the first moisturizing, we directly added the soaked suspension to the absorbent paper. We believe that this was equivalent to soaking the seed in a high concentration of Yb-1 suspension for a long time, although the subsequent addition of sterile water may dilute it. This may be because Yb-1 produces some plant hormone that inhibits seed germination because the concentration is too high.
In addition, studies have shown that the results of in vitro bacteriostatic tests of many biocontrol agents are quite different from those of practical applications in the field. This is because, in the field, they will be affected by many aspects, such as soil, climate, inoculation mode, and so on [44]. To further verify the actual effects of strain Yb-1 on the control of pepper anthracnose in the field, we selected the 104 CFU/mL concentration for the field application. Moreover, before the field experiment, we used three different methods to inoculate potted pepper plants and observed the plants for 30 days. The results showed that strain Yb-1 could not infect the peppers, cause pepper leaves to fall, or affect the normal growth of the peppers. In the field experiment, the control effect of treatment A on pepper anthracnose was 59.45%, which was consistent with the control effect found by Cui et al. [10] on potato scabs by using B. velezensis strain 8-4. Treatment C had the best control effect on pepper anthracnose, which was up to 71.98%, indicating that the mixed use of strain Yb-1 and commercial 10% difenoconazole had a synergistic effect. The results of this part were similar to the control of potato late blight by B. velezensis strain SDTB038 and fluopimotide in the field [45]. Strain Yb-1 had a control effect on anthracnose of peppers in the field experiment. At the same time, we can also consider it as being an additive in the application of difenoconazole. Usually, the study of biocontrol endophytic bacteria with biocontrol potential should also include their colonization and action mechanism, which are also the focus of our next work.
As far as we know, the use of B. velezensis isolated from healthy cucumber leaves to control pepper anthracnose is being reported for the first time. In conclusion, this study proved that Yb-1 is a great potential biocontrol strain. B. velezensis Yb-1 had strong inhibitory effects on C. capsici and B. cinerea, which could promote the germination and growth of pepper seeds. Further results also indicated that Yb-1 had a control effect on pepper anthracnose in the field. The application of Yb-1 as a bacterial agent can provide a reference for the biological control of anthracnose in pepper plantations.

Author Contributions

Conceptualization, W.Y., B.W., Z.Z., T.X. and X.J.; methodology, B.W., Y.L., W.Y. and E.L.; software, B.W.; validation, B.W., T.X. and Z.Z.; formal analysis, B.W.; investigation, W.Y. and Z.Z.; resources, T.X. and X.J.; data curation, B.W. and W.Y.; writing—original draft preparation, B.W.; writing—review and editing, W.Y., Z.Z., E.L. and Y.L.; visualization, B.W. and Z.Z.; supervision, W.Y. and T.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Project of Young Scientists Fund of the Hainan Natural Science Fund (grant number “321QN0958”), the Innovative team Project of Safety Prevention and Control of Major Tropical Fruit and Vegetable Pests of the Hainan Academy of Agricultural Sciences (grant number “HAAS2023TDYD12”), and the Finance Science and Technology Project of Hainan Province (grant number “SQKY2022-004”).

Data Availability Statement

The data sets supporting the results of this article will be freely available upon request to the first author.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Ali, A.; Bordoh, P.K.; Singh, A.; Siddiqui, Y.; Droby, S. Post-harvest development of anthracnose in pepper (Capsicum spp.): Etiology and management strategies. Crop Prot. 2016, 90, 132–141. [Google Scholar] [CrossRef]
  2. Zou, Z.; Zou, X. Geographical and Ecological Differences in Pepper Cultivation and Consumption in China. Front. Nutr. 2021, 8, 718517. [Google Scholar] [CrossRef] [PubMed]
  3. Droby, S.; Wisniewski, M.; Teixidó, N.; Spadaro, D.; Jijakli, M.H. The science, development, and commercialization of postharvest biocontrol products. Postharvest Biol. Technol. 2016, 122, 22–29. [Google Scholar] [CrossRef]
  4. Shafi, J.; Tian, H.; Ji, M. Bacillus species as versatile weapons for plant pathogens: A review. Biotechnol. Biotec. Equip. 2017, 31, 446–459. [Google Scholar] [CrossRef]
  5. Carmona-Hernandez, S.; Reyes-Pérez, J.; Chiquito-Contreras, R.; Rincon-Enriquez, G.; Cerdan-Cabrera, C.; Hernandez-Montiel, L. Biocontrol of Postharvest Fruit Fungal Diseases by Bacterial Antagonists: A Review. Agronomy 2019, 9, 121. [Google Scholar] [CrossRef]
  6. Freimoser, F.M.; Rueda-Mejia, M.P.; Tilocca, B.; Migheli, Q. Biocontrol yeasts: Mechanisms and applications. World J. Microbiol. Biotechnol. 2019, 35, 154. [Google Scholar] [CrossRef]
  7. De Lamo, F.J.; Takken, F.L.W. Biocontrol by Fusarium oxysporum Using Endophyte-Mediated Resistance. Front. Plant Sci. 2020, 11, 37. [Google Scholar] [CrossRef]
  8. Fira, D.; Dimkic, I.; Beric, T.; Lozo, J.; Stankovic, S. Biological control of plant pathogens by Bacillus species. J. Biotechnol. 2018, 285, 44–55. [Google Scholar] [CrossRef]
  9. Chen, K.; Tian, Z.; He, H.; Long, C.-a.; Jiang, F. Bacillus species as potential biocontrol agents against citrus diseases. Biol. Control 2020, 151, 104419. [Google Scholar] [CrossRef]
  10. Cui, L.; Yang, C.; Wei, L.; Li, T.; Chen, X. Isolation and identification of an endophytic bacteria Bacillus velezensis 8-4 exhibiting biocontrol activity against potato scab. Biol. Control 2020, 141, 104156. [Google Scholar] [CrossRef]
  11. Blake, C.; Christensen, M.N.; Kovacs, A.T. Molecular Aspects of Plant Growth Promotion and Protection by Bacillus subtilis. Mol. Plant Microbe Interact. 2021, 34, 15–25. [Google Scholar] [CrossRef]
  12. Sun, B.; Gu, L.; Bao, L.; Zhang, S.; Wei, Y.; Bai, Z.; Zhuang, G.; Zhuang, X. Application of biofertilizer containing Bacillus subtilis reduced the nitrogen loss in agricultural soil. Soil Biol. Biochem. 2020, 148, 107911. [Google Scholar] [CrossRef]
  13. Prakash, J.; Arora, N.K. Phosphate-solubilizing Bacillus sp. enhances growth, phosphorus uptake and oil yield of Mentha arvensis L. 3 Biotech 2019, 9, 126. [Google Scholar] [CrossRef]
  14. Miljakovic, D.; Marinkovic, J.; Balesevic-Tubic, S. The Significance of Bacillus spp. in Disease Suppression and Growth Promotion of Field and Vegetable Crops. Microorganisms 2020, 8, 1037. [Google Scholar] [CrossRef]
  15. Nie, P.; Li, X.; Wang, S.; Guo, J.; Zhao, H.; Niu, D. Induced Systemic Resistance against Botrytis cinerea by Bacillus cereus AR156 through a JA/ET- and NPR1-Dependent Signaling Pathway and Activates PAMP-Triggered Immunity in Arabidopsis. Front. Plant Sci. 2017, 8, 238. [Google Scholar] [CrossRef]
  16. Kildea, S.; Ransbotyn, V.; Khan, M.R.; Fagan, B.; Leonard, G.; Mullins, E.; Doohan, F.M. Bacillus megaterium shows potential for the biocontrol of septoria tritici blotch of wheat. Biol. Control 2008, 47, 37–45. [Google Scholar] [CrossRef]
  17. Gond, S.K.; Bergen, M.S.; Torres, M.S.; White, J.F., Jr. Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol. Res. 2015, 172, 79–87. [Google Scholar] [CrossRef]
  18. Jayapala, N.; Mallikarjunaiah, N.H.; Puttaswamy, H.; Gavirangappa, H.; Ramachandrappa, N.S. Rhizobacteria Bacillus spp. induce resistance against anthracnose disease in chili (Capsicum annuum L.) through activating host defense response. Egypt. J. Biol. Pest Control 2019, 29, 45. [Google Scholar] [CrossRef]
  19. Marquez, R.; Blanco, E.L.; Aranguren, Y. Bacillus strain selection with plant growth-promoting mechanisms as potential elicitors of systemic resistance to gray mold in pepper plants. Saudi J. Biol. Sci. 2020, 27, 1913–1922. [Google Scholar] [CrossRef]
  20. Zheng, T.-w.; Liu, L.; Nie, Q.-w.; Hsiang, T.; Sun, Z.-x.; Zhou, Y. Isolation, identification and biocontrol mechanisms of endophytic bacterium D61-A from Fraxinus hupehensis against Rhizoctonia solani. Biol. Control 2021, 158, 104621. [Google Scholar] [CrossRef]
  21. Khan, M.S.; Gao, J.; Chen, X.; Zhang, M.; Yang, F.; Du, Y.; Moe, T.S.; Munir, I.; Xue, J.; Zhang, X. Isolation and Characterization of Plant Growth-Promoting Endophytic Bacteria Paenibacillus polymyxa SK1 from Lilium lancifolium. BioMed Res. Int. 2020, 2020, 8650957. [Google Scholar] [CrossRef] [PubMed]
  22. Dong, X.Z.; Cai, M.Y. Manual of Identification of Common Bacterial Systems; Science Press: Beijing, China, 2001. [Google Scholar]
  23. Dees, P.M.; Ghiorse, W.C. Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA. FEMS Microbiol. Ecol. 2001, 35, 207–216. [Google Scholar] [CrossRef] [PubMed]
  24. Chun, J.; Bae, K.S. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. Antonie Van Leeuwenhoek J. Microbiol. Serol. 2000, 2, 78. [Google Scholar]
  25. Kenichi, K.; Masao, F.; Ryuji, K. Detection and Identification of Escherichia coli, Shigella, and Salmonella by Microarrays Using the gyrB Gene. Biotechnol. Bioeng. 2003, 83, 721–728. [Google Scholar]
  26. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
  27. Tamura, K.; Nei, M.; Kumar, S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc. Natl. Acad. Sci. USA 2004, 101, 30. [Google Scholar] [CrossRef]
  28. Li, E.; Li, Y.; Dai, X.; Yan, W.; Wang, G. Identification of Two Bacillus Strains with Antimicrobial Activity and Preliminary Evaluation of Their Biocontrol Efficiency. Horticulturae 2022, 8, 744. [Google Scholar] [CrossRef]
  29. Wang, L.; Xi, N.; Lang, D.; Zhou, L.; Zhang, Y.; Zhang, X. Potential biocontrol and plant growth promotion of an endophytic bacteria isolated from Glycyrrhiza uralensis seeds. Egypt. J. Biol. Pest Control 2022, 3, 55. [Google Scholar] [CrossRef]
  30. GB/T 17980.33-2000; National Standards Pesticide-Guidelines for the Field Efficacy Trials (Ⅰ) Fungicides against Pepper Anthracnose. Ministry of Agriculture and Rural Affairs: Beijing, China, 2000.
  31. Panuwet, P.; Siriwong, W.; Prapamontol, T.; Ryan, P.B.; Fiedler, N.; Robson, M.G.; Barr, D.B. Agricultural Pesticide Management in Thailand: Situation and Population Health Risk. Environ. Sci. Policy 2012, 17, 72–81. [Google Scholar] [CrossRef]
  32. Donley, N. The USA lags behind other agricultural nations in banning harmful pesticides. Environ Health 2019, 18, 44. [Google Scholar] [CrossRef]
  33. Kristoffersen, P.; Rask, A.M.; Grundy, A.C.; Franzen, I.; Kempenaar, C.; Raisio, J.; Schroeder, H.; Spijker, J.; Verschwele, A.; Zarina, L. A review of pesticide policies and regulations for urban amenity areas in seven European countries. Weed Res. 2008, 48, 201–214. [Google Scholar] [CrossRef]
  34. Ibarra-Villarreal, A.L.; Gándara-Ledezma, A.; Godoy-Flores, A.D.; Herrera-Sepúlveda, A.; Díaz-Rodríguez, A.M.; Parra-Cota, F.I.; de los Santos-Villalobos, S. Salt-tolerant Bacillus species as a promising strategy to mitigate the salinity stress in wheat (Triticum turgidum subsp. durum). J. Arid Environ. 2021, 186, 104399. [Google Scholar] [CrossRef]
  35. Franco-Duarte, R.; Cernakova, L.; Kadam, S.; Kaushik, K.S.; Salehi, B.; Bevilacqua, A.; Corbo, M.R.; Antolak, H.; Dybka-Stepien, K.; Leszczewicz, M.; et al. Advances in Chemical and Biological Methods to Identify Microorganisms-From Past to Present. Microorganisms 2019, 7, 130. [Google Scholar] [CrossRef]
  36. Cabral, A.; Rego, C.; Nascimento, T.; Oliveira, H.; Groenewald, J.Z.; Crous, P.W. Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines. Fungal Biol. 2012, 116, 62–80. [Google Scholar] [CrossRef]
  37. Bakhshi, M.; Arzanlou, M.; Babai-Ahari, A.; Groenewald, J.Z.; Crous, P.W. Multi-gene analysis of Pseudocercospora spp. from Iran. Phytotaxa 2014, 184, 245. [Google Scholar] [CrossRef]
  38. Liu, Y.; Teng, K.; Wang, T.; Dong, E.; Zhang, M.; Tao, Y.; Zhong, J. Antimicrobial Bacillus velezensis HC6: Production of three kinds of lipopeptides and biocontrol potential in maize. J. Appl. Microbiol. 2020, 128, 242–254. [Google Scholar] [CrossRef]
  39. Meng, Q.; Jiang, H.; Hao, J.J. Effects of Bacillus velezensis strain BAC03 in promoting plant growth. Biol. Control 2016, 98, 18–26. [Google Scholar] [CrossRef]
  40. Jiang, C.H.; Yao, X.F.; Mi, D.D.; Li, Z.J.; Yang, B.Y.; Zheng, Y.; Qi, Y.J.; Guo, J.H. Comparative Transcriptome Analysis Reveals the Biocontrol Mechanism of Bacillus velezensis F21 Against Fusarium Wilt on Watermelon. Front. Microbiol. 2019, 10, 652. [Google Scholar] [CrossRef]
  41. Myo, E.M.; Liu, B.; Ma, J.; Shi, L.; Jiang, M.; Zhang, K.; Ge, B. Evaluation of Bacillus velezensis NKG-2 for bio-control activities against fungal diseases and potential plant growth promotion. Biol. Control 2019, 134, 23–31. [Google Scholar] [CrossRef]
  42. Zhang, X.; Zhou, Y.; Li, Y.; Fu, X.; Wang, Q. Screening and characterization of endophytic Bacillus for biocontrol of grapevine downy mildew. Crop Prot. 2017, 96, 173–179. [Google Scholar] [CrossRef]
  43. Jiang, C.-H.; Liao, M.-J.; Wang, H.-K.; Zheng, M.-Z.; Xu, J.-J.; Guo, J.-H. Bacillus velezensis, a potential and efficient biocontrol agent in control of pepper gray mold caused by Botrytis cinerea. Biol. Control 2018, 126, 147–157. [Google Scholar] [CrossRef]
  44. Ferraz, L.P.; Cunha, T.D.; da Silva, A.C.; Kupper, K.C. Biocontrol ability and putative mode of action of yeasts against Geotrichum citri-aurantii in citrus fruit. Microbiol. Res. 2016, 188–189, 72–79. [Google Scholar] [CrossRef] [PubMed]
  45. Yan, H.; Qiu, Y.; Yang, S.; Wang, Y.; Wang, K.; Jiang, L.; Wang, H. Antagonistic Activity of Bacillus velezensis SDTB038 against Phytophthora infestans in Potato. Plant Dis. 2021, 105, 1738–1747. [Google Scholar] [CrossRef] [PubMed]
Horticulturae 09 00376 g001 550
Figure 1. Inhibition effects of strain Yb-1 against C. capsici (B) and B. cinerea (D). (A,C) are the control, and the lower-right corner is the scale.
Figure 1. Inhibition effects of strain Yb-1 against C. capsici (B) and B. cinerea (D). (A,C) are the control, and the lower-right corner is the scale.
Horticulturae 09 00376 g001
Horticulturae 09 00376 g002 550
Figure 2. The morphological and molecular identification of strain Yb-1. Morphological and microscopic observation of strain Yb-1 (A,B). Phylogenetic tree is based on the sequences of the 16S rRNA gene, the gyrA gene, and the gyrB gene (C). The phylogenetic tree was constructed with the neighbor-joining method. The number at each branch is the percentage of times the group of strains in that branch occurred, based on 1000 cycles, via bootstrap analysis.
Figure 2. The morphological and molecular identification of strain Yb-1. Morphological and microscopic observation of strain Yb-1 (A,B). Phylogenetic tree is based on the sequences of the 16S rRNA gene, the gyrA gene, and the gyrB gene (C). The phylogenetic tree was constructed with the neighbor-joining method. The number at each branch is the percentage of times the group of strains in that branch occurred, based on 1000 cycles, via bootstrap analysis.
Horticulturae 09 00376 g002
Horticulturae 09 00376 g003 550
Figure 3. Effect of Yb-1 suspension at different concentrations on pepper seeds. (A) Effect of strain Yb-1 on the bud length and root length of peppers. Different letters indicate that mean values are significantly different between treatments (p < 0.05). (B) Comparison between the control and the optimal concentration treatment.
Figure 3. Effect of Yb-1 suspension at different concentrations on pepper seeds. (A) Effect of strain Yb-1 on the bud length and root length of peppers. Different letters indicate that mean values are significantly different between treatments (p < 0.05). (B) Comparison between the control and the optimal concentration treatment.
Horticulturae 09 00376 g003
Horticulturae 09 00376 g004 550
Figure 4. Safety evaluation of peppers inoculated with Yb-1 by three different methods.
Figure 4. Safety evaluation of peppers inoculated with Yb-1 by three different methods.
Horticulturae 09 00376 g004
Horticulturae 09 00376 g005 550
Figure 5. Field control experiment of pepper anthracnose. (A) Experimental plot division. (B) Field spray treatment.
Figure 5. Field control experiment of pepper anthracnose. (A) Experimental plot division. (B) Field spray treatment.
Horticulturae 09 00376 g005
Table
Table 1. Effect of Yb-1 suspension at different concentrations on pepper seed germination.
Table 1. Effect of Yb-1 suspension at different concentrations on pepper seed germination.
Yb-1 Suspension Treatment (CUF/mL)
CK103104105106107108
Germination rate/%86.67 b85.00 b98.33 a96.66 a86.67 b83.33 b0.00 c
Different letters indicate that mean values are significantly different between treatments (p < 0.05).
Table
Table 2. Effects of different treatments on pepper anthracnose in field.
Table 2. Effects of different treatments on pepper anthracnose in field.
TreatmentDisease Index Before TreatmentDisease Index After TreatmentControl Effect/%
A0.96 a5.41 b59.45 c
B1.04 a5.11 b64.63 b
C0.89 a3.41 c71.98 a
D1.04 a13.34 a/
Different capital letters indicate different treatments. A: Yb-1; B: 10% difenoconazole; C: A + B; D: clean water. Different small letters indicate that mean values are significantly different between treatments (p < 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wang, B.; Li, E.; Lin, Y.; Xiao, T.; Ji, X.; Zhao, Z.; Yan, W. Identification, Biocontrol Activity, and Field Application Effect of Bacillus velezensis Yb-1. Horticulturae 2023, 9, 376. https://doi.org/10.3390/horticulturae9030376

AMA Style

Wang B, Li E, Lin Y, Xiao T, Ji X, Zhao Z, Yan W. Identification, Biocontrol Activity, and Field Application Effect of Bacillus velezensis Yb-1. Horticulturae. 2023; 9(3):376. https://doi.org/10.3390/horticulturae9030376

Chicago/Turabian Style

Wang, Bao, Erfeng Li, Yang Lin, Tongbin Xiao, Xuncong Ji, Zhixiang Zhao, and Wanrong Yan. 2023. "Identification, Biocontrol Activity, and Field Application Effect of Bacillus velezensis Yb-1" Horticulturae 9, no. 3: 376. https://doi.org/10.3390/horticulturae9030376

APA Style

Wang, B., Li, E., Lin, Y., Xiao, T., Ji, X., Zhao, Z., & Yan, W. (2023). Identification, Biocontrol Activity, and Field Application Effect of Bacillus velezensis Yb-1. Horticulturae, 9(3), 376. https://doi.org/10.3390/horticulturae9030376

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop