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Article

Identification of Two Bacillus Strains with Antimicrobial Activity and Preliminary Evaluation of Their Biocontrol Efficiency

by
Erfeng Li
1,†,
Yuxin Li
1,†,
Xinyu Dai
1,
Wanrong Yan
2,* and
Gang Wang
3,*
1
Horticulture and Landscape College, Tianjin Agricultural University, Tianjin 300392, China
2
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
3
Key Laboratory of Agro-Products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture and Rural Affairs, Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2022, 8(8), 744; https://doi.org/10.3390/horticulturae8080744
Submission received: 20 July 2022 / Revised: 12 August 2022 / Accepted: 15 August 2022 / Published: 18 August 2022
(This article belongs to the Special Issue Horticultural Plants Pathology and Advances in Disease Management)
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Versions Notes

Abstract

:
Cabbage Fusarium Wilt (CFW) is a serious disease caused by Fusarium oxysporum f. sp. conglutinans in many parts of the world. The use of chemical fungicides has placed a heavy burden on the environment and is prone to drug resistance in plant pathogens. As a method with great potential, biological control has attracted the attention of many academics both at home and abroad. In this study, we have found that strains B5 and B6 had a strong inhibitory effect on various pathogens and significantly inhibited mycelium growth. They were both identified as Bacillus velezensis by morphological features, biochemical determinations, 16S rRNA gene and gyrA gene sequence analysis. When different concentrations of bacterial suspension were applied to cabbage seeds, hypocotyl and taproot length increased to varying degrees. The in vivo results showed that B5 and B6 decreased the incidence of cabbage seedling wilt disease, with B6 performing significantly better. Furthermore, B. velezensis B6 had the ability to colonize cabbage plants and rhizosphere soil. Thus, strain B6 has great potential for biocontrol development and this research could lead to the development of a promising biological agent for CFW.

1. Introduction

Fusarium wilt is a general term for a class of plant diseases caused by pathogenic fungi that wilt and kill plants. One of the most common fungal pathogens is Fusarium oxysporum. Cabbage Fusarium wilt (CFW) is a soil-borne disease caused by Fusarium oxysporum f. sp. conglutinans, which was first discovered in the United States in 1895 [1]. In the next few decades, CFW was reported in Japan, China, India and other countries around the world. In China, CFW was first found in Yanqing County, Beijing [2]. Since then, the damaged area has been expanding, threatening about one third of the summer and autumn cabbage planting areas in northern China, which has become a major resistance to the production of cruciferous vegetables [3]. At present, the prevention and control of CFW is still dominated by chemical fungicides, but this increases the risk of fungal drug resistance, and has high residual toxicity, which adversely affects both the environment and human health. The main method of disease control in agriculture is the breeding of disease-resistant varieties. This method, however, is slow to take effect and is susceptible to seasonal or geographical constraints. Pathogenic microorganisms may also cause variations and other problems, affecting the ability of plants to control pathogens. As a result, biological control, such as using plant extract and endophytic bacteria against pathogens [4], has received increased attention as a safe and environmentally friendly control method, and the research on antagonistic microorganisms has also entered a new stage [5,6].
In recent years, the microorganisms used to control Fusarium wilt disease mainly include Bacillus [7], Penicillium [8], Pseudomonas [9] and Streptomyces [10], etc., among which Bacillus has advantageous characteristics such as heat tolerance, salt tolerance and UV resistance [11,12,13]. Several Bacillus spp. have been developed as commercial bio-pesticides because of their multi-layer cell wall structure and ability to produce endophytic spores and long-term survival after treatment in the natural environment [14,15]. They are widely used in agricultural production and are of great significance in the biological control of plant diseases. Bacillus spp. are capable of producing two classes of antagonistic factors, lipopeptide antibiotics and antagonist proteins, which have antibiotic effects against plant pathogens [16]. Lipopeptides belong to a class of microbial polypeptides that activate the defense mechanisms of plants [17]. Lipopeptide antibiotics synthesized by the non-ribosomal pathway can be classified into surfactins, iturins and fengycins, which have a good inhibitory effect on bacteria, fungi and viruses [18]. Surfactin can induce resistance in plants, the capacity of Bacillus subtilis S499 to generate surfactin is positively related to its resistance to Botrytis cinerea [19]. Iturin plays an important role in the biological control of plant pathogens due to its hemolytic, surface active and potent antifungal properties. It is an important antimicrobial active substance that causes cytolysis and cytoplasmic efflux of Verticillium dahliae [20]. B. subtilis UTBSP1 has the ability to produce fengycin, which can inhibit the growth of Aspergillus flavus [21]. Antagonistic protein-based antimicrobial substances of Bacillus mainly include cell-wall-degrading enzymes such as chitinases and glucanases, as well as bacteriocins [22]. Fungal cell walls were broken down by cell-wall-degrading enzymes released by B. atrophaeus JZB120050, such as chitinase and glucanase [23]. Pei et al. found that the bacteriocin RC20975 was able to cause extensive cell damage and bacterial lysis against Alicyclobacillus acidoterrestris [24]. Bacteriocins were also found to inhibit plant pathogenic bacteria and nematodes in recent studies [25,26]. Therefore, Bacillus is a biocontrol bacterium with good application prospects.
In this study, we identified two B. velezensis strains B5 and B6, detected their antagonistic activity on various plant pathogenic fungi and researched their effects on disease control against CFW and growth promotion in cabbage.

2. Materials and Methods

2.1. Microorganisms and Growth Condition

The biocontrol strains, used in this study, were isolated from healthy bitter melons (Momordica charantia L.) and deposited in the Plant Disease Laboratory of Tianjin Agricultural University under accession number PATAU191011 and PATAU191012. The pathogens used in this study were provided by the Plant Pathology Laboratory of Tianjin Agricultural University. The fungi cultured in potato glucose agar (PDA) plates at 28 °C and bacteria cultured in LB (Luria-Bertani) plates at 37 °C.

2.2. Determination of Antifungal Activity

The pathogeny fungal plugs with a diameter of 5 mm were inoculated on the fresh PDA plates, cultured at 28 °C for 2 days. Bacillus suspension of 2 μL (1 × 108 CFU/mL) and pathogens were inoculated at the same distance on both sides as we reported [27]. As a control, PDA was only inoculated with the pathogen. Then, incubated at 28 °C for 3 days to observe and record the inhibition bandwidth. Each treatment was replicated three times.

2.3. Growth Condition of Pathogenic Fungi on Culture Medium Containing Different Concentrations of Bacillus

Strains B5 and B6 were inoculated on LB plates and incubated at 37 °C for 24 h. Single colonies were picked and inoculated into 100 mL of LB liquid medium and incubated for 20 h (37 °C, 180 rpm). Equivalent volume of the suspensions with specific concentration were transferred to a 50 mL centrifuge tube, which was filled with potato glucose agar (PDA) medium to 20 mL and then mixed well. After that, it was, respectively, put into the Petri dishes to create the medium with the corresponding concentrations of 104, 105, 106, 107 and 108. Finally, we inoculated the pathogen plugs with 5 mm diameter, including Fusarium oxysporum f. sp. conglutinans, Botrytis cinerea, Alternaria solani, F. oxysporum f. sp. zingiberi and Alternaria alternata, in the center of the plates, respectively, and incubated at 28 °C. As a control, PDA was only inoculated with the pathogen. Each treatment was replicated three times. The colony diameters were measured after 7 days using the crossover method, and the inhibition rates were calculated for each treatment. The growth inhibition was calculated using the formula [28]:
Percentage of growth inhibition = (C − T)/C × 100%,
where C is the diameter of fungal colony in control and T is the diameter of fungal colony in the treatment.

2.4. Identification of B5 and B6

B5 and B6 strains were cultured in LB plates at 37 °C for 2 days. The morphological identification was determined by the colony characteristics. Physiological and biochemical identification was based on the manual of systematic identification of general bacteria [29]. The molecular identification was conducted as follows. The DNA was extracted with the bacterial DNA extraction kit. The 16S rRNA and gyrA genes were amplified to identify strain B5 and B6 [30,31]. The sequencing was completed by Shanghai Sangon Biotech Co., Ltd., China. The DNA sequence was analyzed by comparing it with those in NCBI GenBank using the BLASTN program based on the method of Altschul et al. [32]. The DNA multiple sequence homology analysis was performed using MEGA X software to construct a phylogenetic tree using the maximum likelihood method [33].

2.5. The Growth Promotion Ability of B5 and B6 in Cabbage Seedlings

The bacterial cultures were centrifugated at 5000 rpm for 15 min, and the precipitates were diluted by sterile water to obtain suspensions with concentrations of 106, 109 and 1012 CFU/mL. Cabbage seeds were soaked in above three concentrations of suspension for 40 min, sterile water was used as control, then placed in Petri dishes lined with wetted filter paper. Twenty seeds were in each treatment and incubated in a light/dark cycle at 25 °C. During the period, watering 1–2 times a day to keep humidity and provided a suitable culture environment for the seeds. After 4 days, hypocotyl length and taproot length were measured. The seed germination rate was calculated using the formula [34]:
Germination rate = n/N × 100%,
where n is the number of seed germinated and N is the total number of seeds planted.

2.6. Biocontrol Efficacy of B5 and B6 in Potted Cabbage Seedlings Inoculated with F. oxysporum

F. oxysporum was prepared as a suspension of 1 × 107 CFU/mL [7] and the bacterial suspension was prepared as 1 × 108 CFU/mL. Cabbage seedlings with 2–3 true leaves were inoculated by the root-dipping method [35]. Treatment A was the healthy cabbage plants inoculated with sterilized distilled water and these were used as the control group. There was other five treatments in this research. Treatment B was the cabbage plants inoculated with F. oxysporum only; Treatment C was the cabbage seedlings inoculated with F. oxysporum and watered with 30 mL B5 suspension in the concentration of 1 × 108 CFU/mL into soil; Treatment D was the cabbage seedlings inoculated with F. oxysporum and watered with 30 mL B6 suspension in the concentration of 1 × 108 CFU/mL into soil; Treatment E was the cabbage seedlings inoculated with the suspension, including F. oxysporum and B5; and treatment F was the cabbage seedlings inoculated with the suspension, including F. oxysporum and B6. In total, 70 g of soil was weighed and the seedlings were replanted in the seedling pots and maintained in a growth chamber under the condition of 28 °C for 16 h light and 8 h dark. Ten days after inoculation, the disease was recorded and the disease index was calculated [7]. Disease symptoms were assessed based on the disease rating scales [35]. The disease index (DI) and control efficacy were calculated using the formulas [36]:
DI = (a × 0 + b × 1 + c × 2 + d × 3 + e × 4 + f × 5)/[(a + b + c + d + e + f)×5] × 100,
where a, b, c, d, e and f are the number of cabbage plants in the same disease rating scale.
Control efficacy (%) = (C − T)/C × 100%,
where C refers to the disease index of the control group and T denotes the disease index of the treatment group.

2.7. Colonization Characteristics of B6 in Cabbage Seedlings and Soil

To avoid the growth of various bacteria in the cabbage seedlings and soil, the natural antibiotic labeling method was used as described previously [37]. The B6 suspension was spread on LB medium with 2 μg/mL of kanamycin and incubated at 37 °C for 24 h. The colonies with the same morphology as the original strain were selected and transferred to LB medium with 5 μg/mL of kanamycin, then incubated for 24 h. This was repeated to prepare the B6 strain which could grow on LB medium with 10, 15, 20, 25, 50, 75 and 100 μg/mL of kanamycin. The cabbage seedlings were treated by root irrigation with 10 mL of B6 suspension in the concentration of 3 × 108 CFU/mL. The roots, stems and leaves of the cabbage seedlings were, respectively, sampled 1, 4, 7, 11 and 15 days after root irrigation. The six plants were taken as a group, and 2 g of the roots, stems and leaves of each plant was taken, which were ground into a homogenate after washing and diluted to 1 × 102, 1 × 103, 1 × 104 and 1 × 105 times. The 100 μL supernatant was taken, respectively, and spread evenly on antibiotic LB plates and incubated for 48 h to observe the number of colonies. Similarly, the rhizosphere soil of the cabbage seedlings was taken on the 1st, 4th, 7th, 11th and 15th day and placed in a triangular flask containing 10 mL of 0.85% NaCl solution with shaking culture at 160 rpm for 30 min. The supernatant was diluted 1 × 102, 1 × 103, 1 × 104 and 1 × 105 times, respectively, and 100 μL was taken and spread evenly on antibiotic LB plates. The colony number was observed after 48 h incubation.

2.8. Statistical Analysis

The experimental data were subjected to variance analysis (ANOVA) using the SPSS software version 19.0 (SPSS Inc., Armonk, NY, USA). Different treatment mean values were separated using Duncan’s multiple range test at p < 0.05.

3. Results

3.1. Determining the Antimicrobial Spectrum of Strains B5 and B6

The inhibitory effects of the strains B5 and B6 on the seven pathogenic fungi were shown in Table 1. B5 and B6 had different antagonistic abilities against the pathogens, but they all showed good inhibition effects. Strains B5 and B6 had a significant antagonistic effect on Fusarium wilt disease on watermelon, cotton and cucumber, and demonstrated fungal inhibitory bandwidths more than 10 mm. Apart from that, B5 also exhibited obvious inhibitory effect against F. oxysporum f. sp. conglutinans, Botrytis cinerea and Alternaria alternata, while B6 had an obvious inhibitory effect on F. oxysporum f. sp. conglutinans. Therefore, B5 and B6 both have great potential for biocontrol development on plant Fusarium wilt.

3.2. Inhibition Effect of B5 and B6 Strains at Different Concentrations on Plant Pathogens

The strains B5 and B6 exhibited antagonistic activities against the five-tested fungal pathogens on PDA plates. In general, the diameter of pathogen colonies was negatively correlated with the concentration of bacterial suspension contained in the plates, namely, the higher the concentration, the smaller the diameter of the pathogen colonies compared to the control group (Figure 1). This indicated that B5 and B6 both had significant inhibitory effects on each pathogen.
With the increasing concentration of B5 and B6 suspension in the cultural medium, the inhibition rate was generally on the rise. F. oxysporum f. sp. conglutinans race 1 reached the maximum inhibition rate of 73.75% at the concentration of 107, but there was no significant difference in the inhibition rate with 108. The rest of the pathogens were inhibited most significantly at 108, and the inhibition rates were all above 75%, indicating that the higher the concentration, the greater the inhibition effect.

3.3. Identification of B5 and B6

The colonies of strains B5 and B6 on LB plates both showed a creamy yellow color, with an opaque, moist and smooth surface and untidy edges (Figure 2a). The single colony is nearly circular (Figure 2b). They are Gram-positive bacteria and capable of producing spores. They can grow well in a temperature of 28–37 °C. They were positive for methylred staining, the Voges-Proskauer test, hydrolysis of starch, citrate utilization and sucrose fermentation. They also had NaCl resistance, with growing occuring with 0–8% NaCl. Furthermore, they were negative for carboxymethyl cellulose and phenylalanine deaminase production.
The strains B5 and B6 were tested with genomic DNA as a template, and the PCR products were detected by agarose gel electrophoresis. The 16S ribosomal RNA gene and gyrA gene fragments were obtained by sequencing, respectively, which were homology matched by BLASTN in NCBI, and phylogenetic evolutionary trees were constructed. The phylogenetic trees demonstrated that B5 and B6 strains formed a cluster closely related to B. velezensis pattern strains CAU B946 and FZB42, indicating that B5 and B6 strains belong to the species B. velezensis (Figure 2c,d). Combining the morphological characteristics and molecular identification results, B5 and B6 were identified as B. velezensis. The 16S ribosomal RNA gene sequence and partial gyrA of B5 and B6 were deposited in the GenBank database with the accession numbers ON564870, ON564871, ON584562 and ON584563, respectively.

3.4. Growth Promoting Ability of B5 and B6 in Cabbage Seedlings

To investigate the effect of B5 and B6 on seed germination and growth, cabbage seeds were treated with different concentrations of B5 and B6 suspension, sterile water was used as a control. They significantly improved the germination rate and seed viability to different degrees (Table 2). The hypocotyl length and taproot length under the treatment of B6 suspension with 1012 CFU/mL were the longest, with 1.25 cm and 2.55 cm, respectively (Figure 3). The seed germination rate increased the concentration of suspension increased. The germination rate of B5 or B6 suspension in concentrations 109 and 1012 CFU/mL was 100%. To summarize, B5 and B6 suspension could promote cabbage germination and growth significantly, and B6 performed better.

3.5. Biocontrol Efficacy of B5 and B6 in Cabbage Seedlings Inoculated with F. oxysporum

The disease assessment was conducted 10 days after inoculation. The disease index of each treatment group was calculated separately according to the disease rating scales. There was no disease symptoms for treatment A (Figure 4a). Compared with treatment B, the disease index of Fusarium wilt in cabbage was significantly reduced under the effects of B5 and B6 suspension (Figure 4). There was no significant difference in disease indexes and control effects among treatments C, D and E. The control effects were all less than 35%. Treatment F, the roots dipped with B6 suspension, had a good control effect that reached 55.18%. The disease index also decreased significantly, which was 27.08 (Table 3). Therefore, we chose B6 for further research on its colonization characteristics in cabbage and soil.

3.6. Colonization Characteristics of B6 in Cabbage Seedlings and Soil

The colonization of biocontrol bacteria is essential for its biocontrol efficacy. Figure 5 showed the colonization of B6 in cabbage seedlings and soil. It could colonize in roots, stems and leaves, especially in roots. In total, 7 days after irrigation, the number of colonies isolated from the roots, stems and leaves reached the maximum, which was 12 × 105 CFU/g, 1.9 × 105 CFU/g and 0.5 × 105 CFU/g, respectively. After that, colonies started to gradually decline and disappeared on the 15th day. In addition, the number of colonies isolated from the seedlings was “root > stem > leaf” in the days after irrigation. Overall, 3 × 105 CFU/g was monitored in the roots as early as the first day after root irrigation. At this time, B6 had not been detected in the stems and leaves. Figure 5d showed that B6 also colonized in rhizosphere soil. In general, the colonization amount of B6 in rhizosphere soil was the highest for the whole experiment. The number of colonies decreased gradually with time after root irrigation, with the greatest decrease of 25% on the 7th day until it decreased to 0 after 15 days. As a result, B6 could colonize effectively in the cabbage host and rhizosphere soil for more than 11 days.

4. Discussion

In recent years, biological control technology has become an important initiative for plant disease control in modern agricultural production. Due to the ability of Bacillus to secrete a variety of antifungal substances, it has become one of the most popular strains in the field of biocontrol. Studies showed that under the artificial control condition, the application of biocontrol bacteria can often achieve good control effects [38]. It has been an effective strategy to screen biocontrol strains from plants. We have isolated microbes from various plants, among which B. velezensis strains B5 and B6 demonstrated excellent effects. In this study, the two strains had high antifungal activities, which could act against a variety of pathogens on different hosts, not only on cabbage. These findings suggested that they were potential biocontrol agents in agriculture.
Furthermore, B5 and B6 both had significant roles in promoting growth. The germination rate of seeds treated with B5 and B6 in concentrations over 109 CFU/mL was 100%. For cabbage roots, the growth effect become better along with the increase in B6 concentration, while the situation of B5 is different. The results showed that 109 CFU/mL of B5 was the most suitable for the growth of cabbage seeds, and there was no significant difference between 109 CFU/mL and 1012 CFU/mL from the germination rate. Liu et al. (2020) found that B. methylotrophicus DD-1 may create a range of plant growth inducers, including Indole-3-acetic acid (IAA), Gibberellic acid (GA) and Siderophore, which can improve the dry weight, root length and shoot length of rice seedlings [39]. This study showed the increase in the bacterial concentration promoted the growth of plants. That may be due to the production of antifungal substances or growth regulators. Microbial mechanisms for promoting plant growth in practical application including nitrogen fixation [40], phosphorus solubilization [41] and secretion of plant hormones [39], etc. Additionally, several systems frequently cooperate. B. cereus YN917 was reported to promote seed germination and seedling plant growth by producing siderophores, ACC deaminases, indole-3-acetic acid (IAA), phosphate solubilizing and enzymes such as β-1,3-glucanase, protease, cellulase and amylase [42]. Overall, the mechanism is complex and diverse, which should be explored further.
In addition, our results also showed that B. velezensis B5 and B6 decreased the incidence of cabbage seedlings’ wilt disease. When the roots were dipped with B6 suspension, the control efficacy reached 55.18%. The disease index also decreased significantly, which was 27.08. Several previously described potential biocontrol agents have similar or higher efficacy. For example, the biocontrol efficacy of Paenibacillus polymyxa HX-140 against Fusarium wilt of cucumber seedlings was reported to be 55.6 % in 2021 [43].
B. velezensis B6 was able to colonize cabbage seedlings efficiently and rapidly. During this process, bacteria can exploit a number of strategies to enhance colonization, such as surfactin production [44], cell-wall-degrading enzyme production [45] and biofilm formation [46,47]. The production of multiple lipopeptides enhances and broadens the antifungal and antibacterial spectrum of Bacillus and facilitates the colonization of ecological niches [48]. In this study, the maximum amount of colonization in the root, stem and leaf was 12 × 105 CFU/g, 1.9 × 105 CFU/g and 0.5 × 105 CFU/g, respectively, and even up to 100 × 105 CFU/g in the rhizosphere soil. This suggested that the B6 strain could mainly colonize in the root and rhizosphere soil during the process of colonization, which was consistent with the conclusions of Coy’s study [49]. Biocontrol bacteria could compete with pathogens for sites and play roles in disease control [50]. The amount of colonization in the rhizosphere soil showed a continuous decreasing trend, but increased in the roots, stems and leaves, indicating that B6 may transferred from the rhizosphere soil. Then, after 7 days, the colonies isolated from the roots, stems and leaves started to gradually decline and disappeared on the 15th day. This phenomenon indicated that the activity of the strain B6 was decreasing after 7 days, which led to weaker effects on cabbage seedlings. Since F. oxysporum is often infected from the root [51] and B6 could colonize the root, root irrigation treatment during cabbage planting could form an effective biological barrier to prevent the invasion of F. oxysporum. In this study, B. velezensis B6 was also found in the stems and leaves of cabbage, which proved that the B6 strain could be transferred from the roots to the stems and leaves, thus preventing the infection of pathogens more effectively. The colonization of Bacillus FZB42 on Zea mays, Arabidopsis thaliana and Lemna minor also showed similar results [52]. This strain was previously designated to B. amyloliquefaciens and reclassified to B. velezensis in 2018 [53]. The same was true for B. velezensis CAU B946, which was previously known as B. amyloliquefaciens subsp. plantarum [53,54]. Another study found that B. subtilis EA-CB0575 not only effectively colonized banana and tomato rhizosphere, but also showed a certain role in promoting growth [55]. Endophytes colonize host plants and form a tight mutualistic symbiotic connection. It is critical to investigate the colonization and growth-promoting mechanisms of endophytes, as well as the molecular recognition mechanism between endophytes and plants, in order to fully exploit the entire function of the endophyte-plant microecosystem and ensure the long-term development of green agriculture.
In conclusion, we reported the biological control effects of two B. velezensis strains on CFW for the first time. This study has laid a theoretical foundation for the development and further application of B. velezensis; however, the above experiments were conducted in an indoor environment and the antagonistic bio-protection effect under natural conditions is easily affected by many factors such as soil, indigenous microorganisms and climate, etc. As a result, numerous experiments must be carried out to determine whether it can be successfully used as a biocontrol bacterium in actual production.

Author Contributions

Conceptualization, G.W., E.L. and W.Y.; methodology, E.L., Y.L. and X.D.; validation, E.L., Y.L. and X.D.; formal analysis, E.L. and Y.L.; data curation, E.L. and Y.L.; writing—original draft preparation, E.L. and Y.L.; writing—review and editing, G.W. and W.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Natural Sciences Foundation of China (grant number “31801682”), Scientific Research Project of Tianjin Municipal Education Commission (grant number 2017KJ179) and National College Students’ Innovation and Entrepreneurship Training Program (grant number “202210061012”).

Institutional Review Board Statement

Not applicable.

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. Smith, E.F. The fungus infestation of agricultural soils in the United States. Sci. Am. Suppl. 1899, 48, 1981–1982. [Google Scholar]
  2. Li, M.Y.; Zhang, T.T.; Li, X.H.; Yan, H. Wilt disease of cruciferous vegetables and its pathogen identification. Plant Prot. 2003, 29, 44–45. (In Chinese) [Google Scholar]
  3. Liu, X.; Ling, J.; Xiao, Z.L.; Xie, B.Y.; Fang, Z.Y.; Yang, L.M.; Zhang, Y.Y.; Lv, H.H.; Yang, Y.H. Characterization of emerging populations of Fusarium oxysporum f. sp. conglutinans causing cabbage wilt in China. J. Phytopathol. 2017, 165, 813–821. [Google Scholar]
  4. Gava, C.A.T.; Pinto, J.M. Biocontrol of melon wilt caused by Fusarium oxysporum Schlect f. sp. melonis using seed treatment with Trichoderma spp. and liquid compost. Biol. Control. 2016, 97, 13–20. [Google Scholar]
  5. Phong, N.H.; Pongnak, W.; Soytong, K. Antifungal activities of Chaetomium spp. against Fusarium wilt of tea. Plant Protect. Sci. 2016, 52, 10–17. [Google Scholar] [CrossRef]
  6. Fernando, W.G.D.; Ramarathnam, R.; Krishnamoorthy, A.S.; Savchuk, S.C. Identification and use of potential bacterial organic antifungal volatiles in biocontrol. Soil Biol. Biochem. 2005, 37, 955–964. [Google Scholar] [CrossRef]
  7. Zhang, J.X.; Gu, Y.B.; Chi, F.M.; Ji, Z.R.; Wu, J.Y.; Dong, Q.L.; Zhou, Z.S. Bacillus amyloliquefaciens GB1 can effectively control apple valsa canker. Biol. Control 2015, 88, 1–7. [Google Scholar] [CrossRef]
  8. Alam, S.S.; Sakamoto, K.; Inubushi, K. Biocontrol efficiency of Fusarium wilt diseases by a root-colonizing fungus Penicillium sp. Soil Sci. Plant Nutr. 2011, 57, 204–212. [Google Scholar] [CrossRef]
  9. Rania, A.B.A.; Jabnoun-Khiareddine, H.; Nefzi, A.; Mokni-Tlili, S.; Daami-Remadi, M. Endophytic bacteria from Datura metel for plant growth promotion and bioprotection against Fusarium wilt in tomato. Biocontrol Sci. Technol. 2016, 26, 1139–1165. [Google Scholar] [CrossRef]
  10. Wang, J.; Cai, B.Y.; Li, K.; Zhao, Y.K.; Li, C.Y.; Liu, S.W.; Xiang, D.D.; Zhang, L.; Xie, J.H.; Wang, W. Biological control of Fusarium oxysporum f. sp. cubense tropical race 4 in banana plantlets using newly isolated Streptomyces sp. WHL7 from marine soft coral. Plant Dis. 2022, 106, 254–259. [Google Scholar]
  11. Obagwu, J.; Korsten, L. Integrated control of citrus green and blue molds using Bacillus subtilis in combination with sodium bicarbonate or hot water. Postharvest Biol. Technol. 2003, 28, 187–194. [Google Scholar] [CrossRef]
  12. Li, H.Q.; Yue, H.W.; Li, L.; Liu, Y.; Zhang, H.Y.; Wang, J.H.; Jiang, X.W. Seed biostimulant Bacillus sp. MGW9 improves the salt tolerance of maize during seed germination. AMB Expr. 2021, 11, 74. [Google Scholar] [CrossRef] [PubMed]
  13. Nicholson, W.L.; Galeano, B. UV Resistance of Bacillus anthracis spores revisited: Validation of Bacillus subtilis spores as UV surrogates for spores of B. anthracis sterne. Appl. Environ. Microb. 2003, 69, 1327–1330. [Google Scholar] [CrossRef] [PubMed]
  14. Hu, X.J.; Roberts, D.P.; Maul, J.E.; Emche, S.E.; Liao, X.; Guo, X.L.; Liu, Y.Y.; McKenna, L.F.; Buyer, J.S.; Liu, S.Y. Formulations of the endophytic bacterium Bacillus subtilis Tu-100 suppress Sclerotinia sclerotiorum on oilseed rape and improve plant vigor in field trials conducted at separate locations. Can. J. Microbiol. 2011, 57, 539–546. [Google Scholar] [CrossRef]
  15. Santoyo, G.; Orozco-Mosqueda, M.C.; Govindappa, M. Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas: A review. Biocontrol Sci. Technol. 2012, 22, 855–872. [Google Scholar] [CrossRef]
  16. Yamamoto, S.; Shiraishi, S.; Suzuki, S. Are cyclic lipopeptides produced by Bacillus amyloliquefaciens S13-3 responsible for the plant defence response in strawberry against Colletotrichum gloeosporioides? Lett. Appl. Microbiol. 2015, 60, 379–386. [Google Scholar] [CrossRef]
  17. Shafi, J.; Tian, H.; Ji, M.S. Bacillus species as versatile weapons for plant pathogens: A review. Biotechnol. Biotechnol. Equip. 2017, 31, 446–459. [Google Scholar] [CrossRef]
  18. Schofield, B.J.; Skarshewski, A.; Lachner, N.; Ouwerkerk, D.; Klieve, A.V.; Dart, P.; Hugenholtz, P. Near complete genome sequence of the animal feed probiotic, Bacillus amyloliquefaciens H57. Stand. Genom. Sci. 2016, 11, 60. [Google Scholar] [CrossRef]
  19. Cawoy, H.; Mariutto, M.; Henry, G.; Fisher, C.; Vasilyeva, N.; Thonart, P.; Dommes, J.; Ongena, M. Plant defense stimulation by natural isolates of Bacillus depends on efficient surfactin production. Mol. Plant Microbe Interact. 2014, 27, 87–100. [Google Scholar] [CrossRef]
  20. Han, Q.; Wu, F.L.; Wang, X.N.; Qi, H.; Shi, L.; Ren, A.; Liu, Q.H.; Zhao, M.W.; Tang, C.M. The bacterial lipopeptide iturins induce Verticillium dahliae cell death by affecting fungal signalling pathways and mediate plant defence responses involved in pathogen-associated molecular pattern-triggered immunity. Environ. Microbiol. 2015, 17, 1166–1188. [Google Scholar] [CrossRef]
  21. Farzaneh, M.; Shi, Z.Q.; Ahmadzadeh, M.; Hu, L.B.; Ghassempour, A. Inhibition of the Aspergillus flavus growth and Aflatoxin B1 contamination on pistachio nut by fengycin and surfactin-producing Bacillus subtilis UTBSP1. Plant Pathol. J. 2016, 32, 209–215. [Google Scholar] [CrossRef] [PubMed]
  22. Leclère, V.; Béchet, M.; Adam, A.; Guez, J.S.; Wathelet, B.; Ongena, M.; Thonart, P.; Gancel, F.; Chollet-Imbert, M.; Jacques, P. Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism’s antagonistic and biocontrol activities. Appl. Environ. Microb. 2005, 71, 4577–4584. [Google Scholar] [CrossRef] [PubMed]
  23. Ni, M.; Wu, Q.; Wang, J.L.; Liu, W.C.; Ren, J.H.; Zhang, D.P.; Zhao, J.; Liu, D.W.; Rao, Y.H.; Lu, C.G. Identification and comprehensive evaluation of a novel biocontrol agent Bacillus atrophaeus JZB120050. J. Environ. Sci. Health B 2018, 53, 777–785. [Google Scholar] [CrossRef]
  24. Pei, J.J.; Yue, T.L.; Jin, W.G. Application of bacteriocin RC20975 in apple juice. Food Sci. Technol. Int. 2016, 23, 166–173. [Google Scholar] [CrossRef]
  25. Chowdhury, S.P.; Hartmann, A.; Gao, X.W.; Borriss, R. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42-a review. Front. Microbiol. 2015, 6, 780. [Google Scholar] [CrossRef] [PubMed]
  26. Muñoz, C.Y.; Zhou, L.; Yi, Y.H.; Kuipers, O.P. Biocontrol properties from phyllospheric bacteria isolated from Solanum lycopersicum and Lactuca sativa and genome mining of antimicrobial gene clusters. BMC Genom. 2022, 23, 152. [Google Scholar] [CrossRef]
  27. Li, L.; Yang, B.L.; Humza, M.; Geng, H.R.; Wang, G.; Zhang, C.X.; Gao, S.; Xing, F.G.; Liu, Y. A novel strain Lactobacillus brevis 8-2B inhibiting Aspergillus carbonarius growth and ochratoxin a production. LWT 2021, 136, 110308. [Google Scholar] [CrossRef]
  28. Khan, M.S.; Gao, J.L.; Zhang, M.F.; Xue, J.; Zhang, X.H. Pseudomonas aeruginosa Ld-08 isolated from Lilium davidii exhibits antifungal and growth-promoting properties. PLoS ONE 2022, 17, e0269640. [Google Scholar] [CrossRef]
  29. Dong, X.Z.; Cai, M.Y.B. Systematic Determinative Manual of General Bacteria; Science Press: Beijing, China, 2001; pp. 353–370. [Google Scholar]
  30. Wang, B.B.; Yuan, J.; Zhang, J.; Shen, Z.Z.; Zhang, M.X.; Li, R.; Ruan, Y.Z.; Shen, Q.R. Effects of novel bioorganic fertilizer produced by Bacillus amyloliquefaciens W19 on antagonism of Fusarium wilt of banana. Biol. Fertil. Soils 2013, 49, 435–446. [Google Scholar] [CrossRef]
  31. Chun, J.; Bae, K.S. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. Antonie Van Leeuwenhoek 2000, 78, 123–127. [Google Scholar] [CrossRef]
  32. 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]
  33. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef] [PubMed]
  34. Wang, L.M.; Xi, N.; Lang, D.Y.; Zhou, L.; Zhang, Y.J.; Zhang, X.H. Potential biocontrol and plant growth promotion of an endophytic bacteria isolated from Glycyrrhiza uralensis seeds. Egypt. J. Biol. Pest Control 2022, 32, 55. [Google Scholar] [CrossRef]
  35. Li, E.F.; Wang, G.; Xiao, J.L.; Ling, J.; Yang, Y.H.; Xie, B.Y. A SIX1 homolog in Fusarium oxysporum f. sp. conglutinans is required for full virulence on cabbage. PLoS ONE 2016, 11, 0152273. [Google Scholar]
  36. Jing, C.L.; Zhao, J.; Han, X.B.; Huang, R.H.; Cai, D.S.; Zhang, C.S. Essential oil of Syringa oblata Lindl. As a potential biocontrol agent against tobacco brown spot caused by Alternaria alternata. Crop Prot. 2018, 104, 41–46. [Google Scholar] [CrossRef]
  37. Ding, T.; Su, B.; Chen, X.J.; Xie, S.S.; Gu, S.Y.; Wang, Q.; Huang, D.Y.; Jiang, H.Y. An endophytic bacterial strain isolated from Eucommia ulmoides inhibits Southern corn leaf blight. Front. Microbiol. 2017, 8, 903. [Google Scholar] [CrossRef] [PubMed]
  38. Finkel, O.M.; Castrillo, G.; Paredes, S.H.; González, I.S.; Dangl, J.L. Understanding and exploiting plant beneficial microbes. Curr. Opin. Plant Biol. 2017, 38, 155–163. [Google Scholar] [CrossRef]
  39. Liu, Z.P.; Wang, H.X.; Xu, W.H.; Wang, Z.G. Isolation and evaluation of the plant growth promoting rhizobacterium Bacillus methylotrophicus (DD-1) for growth enhancement of rice seedling. Arch. Microbiol. 2020, 202, 2169–2179. [Google Scholar] [CrossRef]
  40. Liu, X.M.; Li, Q.; Li, Y.B.; Guan, G.H.; Chen, S.F. Paenibacillus strains with nitrogen fixation and multiple beneficial properties for promoting plant growth. PeerJ 2019, 7, e7445. [Google Scholar] [CrossRef]
  41. Chen, Y.P.; Rekha, P.D.; Arun, A.B.; Shen, F.T.; Lai, W.A.; Young, C.C. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl. Soil Ecol. 2006, 34, 33–41. [Google Scholar] [CrossRef]
  42. Zhou, H.; Ren, Z.H.; Zu, X.; Yu, X.Y.; Zhu, H.J.; Li, X.J.; Zhong, J.; Liu, E.M. Efficacy of plant growth-promoting bacteria Bacillus cereus YN917 for biocontrol of rice blast. Front. Microbiol. 2021, 12, 684888. [Google Scholar] [CrossRef] [PubMed]
  43. Zhai, Y.; Zhu, J.X.; Tan, T.M.; Xu, J.P.; Shen, A.R.; Yang, X.B.; Li, J.L.; Zeng, L.B.; Wei, L. Isolation and characterization of antagonistic Paenibacillus polymyxa HX-140 and its biocontrol potential against Fusarium wilt of cucumber seedlings. BMC Microbiol. 2021, 21, 75. [Google Scholar] [CrossRef] [PubMed]
  44. Chen, X.H.; Koumoutsi, A.; Scholz, R.; Schneider, K.; Vater, J.; Süssmuth, R.; Piel, J.; Borriss, R. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J. Biotechnol. 2009, 140, 27–37. [Google Scholar] [CrossRef] [PubMed]
  45. Selim, H.M.M.; Gomaa, N.M.; Essa, A.M.M. Application of endophytic bacteria for the biocontrol of Rhizoctonia solani (Cantharellales: Ceratobasidiaceae) damping-off disease in cotton seedlings. Biocontrol Sci. Technol. 2017, 27, 81–95. [Google Scholar] [CrossRef]
  46. Beauregard, P.B. Chapter One—Not just sweet talkers: How roots stimulate their colonization by beneficial bacteria. Adv. Bot. Res. 2015, 75, 1–20. [Google Scholar]
  47. Raaijmakers, J.M.; Bruijn, I.D.; Nybroe, O.; Ongena, M. Natural functions of lipopeptides from Bacillus and Pseudomonas: More than surfactants and antibiotics. FEMS Microbiol. Rev. 2010, 346, 1037–1062. [Google Scholar] [CrossRef]
  48. Falardeau, J.; Wise, C.; Novitsky, L.; Avis, T.J. Ecological and mechanistic insights into the direct and indirect antimicrobial properties of Bacillus subtilis lipopeptides on plant pathogens. J. Chem. Ecol. 2013, 39, 869–878. [Google Scholar] [CrossRef]
  49. Coy, R.M.; Held, D.W.; Kloepper, J.W. Rhizobacterial colonization of bermudagrass by Bacillus spp. in a Marvyn loamy sand soil. Appl. Soil Ecol. 2019, 141, 10–17. [Google Scholar] [CrossRef]
  50. Walker, T.S.; Bais, H.P.; Grotewold, E.; Vivanco, J.M. Root exudation and rhizosphere biology. Plant Physiol. 2003, 132, 44–51. [Google Scholar] [CrossRef]
  51. Steinkellner, S.; Mammerler, R.; Vierheilig, H. Germination of Fusarium oxysporum in root exudates from tomato plants challenged with different Fusarium oxysporum strains. Eur. J. Plant Pathol. 2008, 122, 398–401. [Google Scholar] [CrossRef]
  52. Fan, B.; Borriss, R.; Bleiss, W.; Wu, X.Q. Gram-positive rhizobacterium Bacillus amyloliquefaciens FZB42 colonizes three types of plants in different patterns. J. Microbiol. 2012, 50, 38–44. [Google Scholar] [CrossRef] [PubMed]
  53. Pandic, C.; Coq, D.L.; Deschamps, J.; Védie, R.; Rousseau, T.; Aymerich, S.; Briandet, R. Complete genome sequence of Bacillus velezensis QST713: A biocontrol agent that protects Agaricus bisporus crops against the green mould disease. J. Biotechnol. 2018, 278, 10–19. [Google Scholar] [CrossRef] [PubMed]
  54. Blom, J.; Rueckert, C.; Niu, B.; Wang, Q.; Borriss, R. The complete genome of Bacillus amyloliquefaciens subsp. plantarum CAU B946 contains a gene cluster for nonribosomal synthesis of Iturin A. J. Bacteriol. 2012, 194, 1845–1846. [Google Scholar] [PubMed]
  55. Posada, L.F.; Álvarez, J.C.; Romero-Tabarez, M.; de-Bashan, L.; Villegas-Escobar, V. Enhanced molecular visualization of root colonization and growth promotion by Bacillus subtilis EA-CB0575 in different growth systems. Microbiol. Res. 2018, 217, 69–80. [Google Scholar] [CrossRef]
Horticulturae 08 00744 g001 550
Figure 1. Inhibition effects of strain B5 (white rectangle) and B6 (grey rectangle) against F. oxysporum f. sp. conglutinans race 1 (a), F. oxysporum f. sp. conglutinans race 2 (b), Botrytis cinerea (c), Alternaria solani (d), F. oxysporum f. sp. zingiberi (e) and A. alternata (f). Values were means of three replicates. Error bars represented standard error of the mean. For each pathogen, different lowercases above the columns indicated statistical difference at p < 0.05.
Figure 1. Inhibition effects of strain B5 (white rectangle) and B6 (grey rectangle) against F. oxysporum f. sp. conglutinans race 1 (a), F. oxysporum f. sp. conglutinans race 2 (b), Botrytis cinerea (c), Alternaria solani (d), F. oxysporum f. sp. zingiberi (e) and A. alternata (f). Values were means of three replicates. Error bars represented standard error of the mean. For each pathogen, different lowercases above the columns indicated statistical difference at p < 0.05.
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Figure 2. The morphological and molecular identification of strains B5 and B6. Morphological and microscopic observation of strains B5 (a) and B6 (b). Phylogenetic tree based on the sequences of 16S rRNA gene (c) and gyrA gene (d).
Figure 2. The morphological and molecular identification of strains B5 and B6. Morphological and microscopic observation of strains B5 (a) and B6 (b). Phylogenetic tree based on the sequences of 16S rRNA gene (c) and gyrA gene (d).
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Figure 3. The hypocotyl length (white rectangle) and taproot length (grey rectangle) under different treatments. Different lowercases indicated statistical difference at p < 0.05.
Figure 3. The hypocotyl length (white rectangle) and taproot length (grey rectangle) under different treatments. Different lowercases indicated statistical difference at p < 0.05.
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Figure 4. The disease symptoms of cabbage seedlings under different treatments. (a) Treatment A, which refers to cabbage seedlings inoculated with sterilized distilled water as a control group; (b) Treatment B, which refers to cabbage seedlings inoculated with F. oxysporum only; (c) Treatment C, which refers to cabbage seedlings inoculated with F. oxysporum and watered with B5 suspension into soil; (d) Treatment D, which refers to cabbage seedlings inoculated with F. oxysporum and watered with B6 suspension into soil; (e) Treatment E, which refers to cabbage seedlings inoculated with the suspension, including F. oxysporum and B5; (f) Treatment F, which refers to cabbage seedlings inoculated with the suspension, including F. oxysporum and B6.
Figure 4. The disease symptoms of cabbage seedlings under different treatments. (a) Treatment A, which refers to cabbage seedlings inoculated with sterilized distilled water as a control group; (b) Treatment B, which refers to cabbage seedlings inoculated with F. oxysporum only; (c) Treatment C, which refers to cabbage seedlings inoculated with F. oxysporum and watered with B5 suspension into soil; (d) Treatment D, which refers to cabbage seedlings inoculated with F. oxysporum and watered with B6 suspension into soil; (e) Treatment E, which refers to cabbage seedlings inoculated with the suspension, including F. oxysporum and B5; (f) Treatment F, which refers to cabbage seedlings inoculated with the suspension, including F. oxysporum and B6.
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Figure 5. Colonization of B6 in the roots (a), stems (b), leaves (c) and rhizosphere soil (d) of cabbage seedlings. Different lowercases indicated statistical difference at p < 0.05.
Figure 5. Colonization of B6 in the roots (a), stems (b), leaves (c) and rhizosphere soil (d) of cabbage seedlings. Different lowercases indicated statistical difference at p < 0.05.
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Table
Table 1. Antifungal activity of B5 and B6 against different pathogens in vitro.
Table 1. Antifungal activity of B5 and B6 against different pathogens in vitro.
StrainsInhibition Effects 1
F. oxysporum
f. sp.
niveum		F. oxysporum
f. sp.
vasinfectum		F. oxysporum
f. sp.
cucumerinum		F. oxysporum
f. sp.
conglutinans Race 1		F. oxysporum
f. sp.
conglutinans Race 2		Botrytis
cinerea		Altenaria
alternata
B5+++++++++++++++++
B6+++++++++++++++
1 Mean values of inhibitory zone diameters were obtained from three replicates for each treatment. The inhibitory zone diameters of <5 mm, 5−10 mm and >10 mm were shown as +, ++ and +++, respectively.
Table
Table 2. Effect of B5 and B6 suspension at different concentrations on cabbage seed germination.
Table 2. Effect of B5 and B6 suspension at different concentrations on cabbage seed germination.
Treatment (CFU/mL)
CKB5B6
10610910121061091012
Germination
rate		85% a90% a100% b100% b90% a100% b100% b
Different lowercases indicated statistical difference at p < 0.05.
Table
Table 3. Control effects of B5 and B6 on cabbage Fusarium wilt.
Table 3. Control effects of B5 and B6 on cabbage Fusarium wilt.
Treatments 1Disease IndexControl Effect/%
A0−
B60.42 a−
C39.58 b34.49 a
D47.92 b20.69 a
E43.75 b27.59 a
F27.08 c55.18 b
1 Treatment A refers to plants inoculated with sterilized distilled water as a control. Treatment B refers to plants inoculated with F. oxysporum only. Treatment C/D refers to plants inoculated with F. oxysporum and watered with B5/B6 suspension into soil. Treatment E/F refers to plants inoculated with the suspension, including F. oxysporum and B5/B6. Different lowercases indicated statistical difference at p < 0.05.
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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. https://doi.org/10.3390/horticulturae8080744

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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(8):744. https://doi.org/10.3390/horticulturae8080744

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Li, Erfeng, Yuxin Li, Xinyu Dai, Wanrong Yan, and Gang Wang. 2022. "Identification of Two Bacillus Strains with Antimicrobial Activity and Preliminary Evaluation of Their Biocontrol Efficiency" Horticulturae 8, no. 8: 744. https://doi.org/10.3390/horticulturae8080744

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