Biocontrol of citrus bacterial canker caused by Xanthomonas citri subsp. citri by Bacillus velezensis.

Microorganisms with biocontrol capabilities against plant pathogens are considered as one of the most promising approaches for healthy crop management. In this study, ethyl acetate extracts of 25 Bacillus strains were investigated for their antagonistic effect on Xanthomonas citri subsp. citri (Xcc), which causes the citrus bacterial canker (CBC) disease. Among them, 21 strains exerted antibacterial activity against wild-type Xcc strains. Based on the strength of the antibacterial activity, nine Bacillus strains were selected for 16S rRNA analysis. 16S rRNA sequence homology revealed that several strains were closely related to B. velezensis, where strains with no antibacterial activity grouped as the soil-associated community of B. amyloliquefaciens. B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively. Furthermore, B. velezensis Bv-21 strain was tested for biocontrol activity against a streptomycin-resistant XccM4 in detached susceptible citrus leaves. The strain reduced the incidence of CBC by 26.30% and pathogen density of XccM4 by 81.68% over control. The results of the study strongly suggest that B. velezensis can be used as an effective and eco-friendly biocontrol agent either by itself or as an active compound, against both, the wild-type and streptomycin-resistant Xcc.

Introduction

Citrus bacterial canker (CBC) is a severe, economically harmful, and highly contagious disease caused by Xanthomonas citri subsp. citri (Xcc) in most commercial citrus cultivars (Hu et al., 2014). The initial outbreak of the disease reported in Florida, USA in the early 1910s and the disease was caused by imported citrus seedlings from Japan (Li et al., 2007). The symptoms of CBC include elevated hyperplastic lesions on the leaves, stems, and fruits leading to defoliation and premature fruit drop (Gottwald et al., 2002). The most common bactericides used to manage citrus canker are copper-based compounds such as copper hydroxide, cuprous oxide, and copper oxychloride (Marin et al., 2019). However, copper-based compounds posed considerable risks to the environment and long-term usage of these bactericides may result in the development of copper-resistant strains of Xcc (Behlau et al., 2011, Marin et al., 2019). Copper-resistant strains against Xcc and X. alfalfae subsp. citrumelonis were first discovered in Argentina in the mid-1990s in citrus nurseries exposed to copper-based bactericides on a regular basis (Behlau et al., 2011). Furthermore, overuse of these copper-based sprays results in phytotoxicity in the fruit skin and accumulates to toxic levels in the citrus root (Graham et al., 2016). An alternative to copper is zinc, which is generally recognized as safe, and zinc oxide based nano-formulations (Zinkicide) reduced the canker lesions by 38–42% in Pineapple leaves under greenhouse condition. However, the level of protection of zinc-based formulations against Xcc was not satisfactory in the field trials (Graham et al., 2016). Several other potential alternatives are availabe such as antibiotics (e.g., streptomycin). There is also concern of resistant bacteria selection from the overuse of these antibiotics. Moreover, streptomycin is prohibited in Brazil and Europe for agricultural use (Martins et al., 2020). Hence, there is compelling need to develop environmentally acceptable alternative to control the pathogenic strains of Xcc. During last decades, the potential use of Bacillus spp. as a biocontrol agent has been highlighted as a promising alternative to chemical pesticides (Chen et al., 2020, Leal et al., 2021). Bacillus spp. (including more than 300 species) are a wide group of saprophytic, gram-positive, aerobic, spore-forming ubiquitous organisms that can be recovered from almost any niche in the world (Cao et al., 2018).

Bacillus species are considered to be one of the most abundant sources of bioactive secondary metabolites with antipathogenic activity, that largely remain unexplored for plant disease control (Chen et al., 2007, Ongena and Jacques, 2007). Bacillus spp., including B. amyloliquefaciens, B. subtilis, B. cereus, B. licheniformis, B. megaterium, B. mycoides, and B. pumilus are known as a natural factory of biologically active compounds such as cyclic lipopeptides and polyketides. Many of these compounds are potentially useful against deleterious microbes that cause various plant diseases (Huang et al., 2012, Nigris et al., 2014, Wu et al., 2015, Xiong et al., 2015, Zhang and Sun, 2018). In addition to the Bacillus spp., the Pseudomonas spp. has also gained considerable attention as biocontrol agents. Unfortunately, owing to the challenges associated with the preparation of stable and long-lived bio-formulations with the Pseudomonas spp., the commercial application of this biocontrol agent is limited in agriculture (Borriss, 2011). Therefore, Bacillus spp. are commonly recommended in agricultural practices as a result of their ability to form endospores that can withstand exposure to heat and desiccation. In addition, Bacillus spp. based products can be formulated into stable dry powders with longer shelf life (Ongena and Jacques, 2007). Furthermore, Bacillus spp. based natural antagonistic organisms are eco-friendly and can be combined with traditional chemical control systems to reduce the possibility of the development of microbial resistance (Ongena and Jacques, 2007, Wu et al., 2015).

Among the Bacillus spp., B. velezensis has been gaining attention for its eco-friendly nature and versatile mode of action (Kim et al., 2021). B. velezensis is a plant-associated Bacillus that colonizes plant roots, promotes plant growth, and suppresses pathogens (Cao et al., 2018). B. amyloliquefaciens FZB42T (reclassifed as B. velezensis FZB42T) is a gram-positive plant growth promoting rhizobacterium and was first isolated from sugar beet in Brandenburg, Germany (Borriss et al., 2011). This bacterium was originally described in 2005 by Ruiz-Garcia, when it was isolated from the Vélez River in the province of Málaga, Spain (Ruiz-García et al., 2005). Further research has shown that B. velezensis FZB42T is genetically equipped with nine giant gene-clusters that encode secondary metabolites that make up approximately 10% of the total genome (Chen et al., 2007), which have been documented to be useful tools for plant disease control (Ongena and Jacques, 2007). Five of these nine gene clusters were involved in the synthesis of cyclic lipopeptides (i.e., surfactin, fengycin, and bacillomycin D), an unknown peptide, and siderophore producing compound bacillibactin (Chen et al., 2009). Meanwhile, three other polyketide synthase gene clusters were described for the biosynthesis of macrolactin, bacillaene, and difficidin (Chen et al., 2006, 2009). The last gene cluster was found to be responsible for the synthesis and export of the dipeptide antibiotic bacilysin (Chen et al., 2009). Besides, two other ribosomally synthesized bacteriocins (i.e., plantazolicin and amylocyclicin) were identified in B. velezensis and displayed high antibacterial activity against closely related Gram-positive bacteria (Scholz et al., 2014, Scholz et al., 2011).

Besides the direct antagonism of B. velezensis against pathogenic microbes, this bacterium has also been reported to contribute to the plant defense by competing with deleterious microbes for essential nutrients (i.e., iron) through the secretion of siderophore bacillibactin (Xiong et al., 2015) or by expressing the global defense response in the plant by induced systemic resistance (Li et al., 2017). In fact, Bacillus spp. based products account for about half of all commercially available biological control agents (Ongena and Jacques, 2007). To date, commercially available B. velezensis based products include RhizoVital® (B. velezensis FZB42; ABiTEP, GmbH, Berlin, Germany), BioYield™ (B. amyloliquefaciens GB99 + B. subtilis GB122; Bayer Crop Science, USA), Amylo-X® WG (B. amyloliquefaciens subsp. plantarum D747; Certis Europe BV, Netherlands), Taegro® (B. subtilis var. amyloliquefaciens FZB24; Novozymes Biologicals, Virginia, USA etc. (Borriss et al., 2011). The fungicide Serifel® formulated from the B. amyloliquefaciens strain MBI600 has also been shown to have antiviral action against tomato spotted wilt virus (TSWV) and potato virus Y (PVY). A drench application of this biocontrol agent reduced the incidence of tomato spotted wilt virus by 80% and delayed the development of potato virus Y by accumulating the defense-related genes for jasmonic acid and salicylic acid (Beris et al., 2018). Botrybel (Agricaldes, Spain) based on B. velezensis has been found to be effective against Botrytis cinerea, the leading cause of gray mold disease infecting around 200 plant species worldwide (Romanazzi and Feliziani, 2020).

In our previous study, we reported the isolation and identification of B. velezensis EB-39 (an endophytic bacterium) from Dangyuja mandarin citrus and the bacterium was found to have antibacterial activity against the wild-type and streptomycin-resistant mutants of Xcc (Rabbee et al., 2019b). In this study, we perfomred a comparative study of antibacterial activity of various strains of B. velezensis and B. amyloliquefaciens to control both the wild-type and streptomycin-resistant Xcc and found B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc. The biocontrol efficacy of B. velezensis Bv-21 was also examined against streptomycin-resistant XccM4 in detached susceptible citrus leaves. The application of this strain could be benificial for long-term agricultual management and disease control.

Materials and methods

Plant materials

A three-year-old Hwanggeum hyang Citrus trees were purchased from the Jeju Hanla Farm (Jeju province, Republic of Korea), planted in pots (18 cm × 20 cm), and grown in a temperature-controlled glasshouse. The leaves of Hwanggeum hyang citrus, which are susceptible to CBC, were used in detached leaf assays for the Xcc infection experiments.

Culture conditions of microorganisms

A total of 25 Bacillus spp. (Table 1) were screened for their biocontrol activity against Xcc. The Korean Agricultural Culture Collection (KACC, RDA, Republic of Korea) kindly supplied 24 strains. Bv-25, an endophytic B. velezensis strain was isolated from the leaves of Hwanggeum hyang citrus in our laboratory. All the Bacillus spp. were cultured on Yeast-Nutrient-Agar (YNA) media (5 g yeast extract, 8 g nutrient broth, 15 g agar in 1L distilled water). Pathogenic Xcc strains included two wild-type Xcc [XccW1 (Xcc 19-18), XccW2 (Xcc 10-5)] and five streptomycin-resistant mutants [XccM4 (Xcc 27-9), XccM5 (Xcc 8-4), XccM6 (Xcc 57-2), XccM7 (Xcc 57-5), XccM8 (Xcc 27-13)], provided by Dr. Hyun at the Citrus Research Station, RDA, Jeju, Korea. These streptomycin resistant Xcc strains were isolated from citrus orchard which was previously sprayed heavily with streptomycin. All Xcc strains were grown in YNA medium.

Table 1

List of B. velezensis and B. amyloliquefaciens used in this study.

Strain no.	KACC*no.	Strain Name	Source of isolation
Bv-1	15814	B. velezensis	Red chili paste (Gochujang)
Ba-2	15815	B. amyloliquefaciens	Soybean paste (Doenjang)
B-3	15816	B. amyloliquefaciens	Soybean paste (Doenjang)
B-4	15817	B. amyloliquefaciens	Soy sauce (Ganjang)
B-5	15818	B. amyloliquefaciens	Soy sauce (Ganjang)
Ba-6	15819	B. amyloliquefaciens	Soy sauce (Ganjang)
B-7	15848	B. amyloliquefaciens	Red chili paste (Gochujang)
B-8	15865	B. amyloliquefaciens	Soybean paste (Doenjang)
B-9	15866	B. amyloliquefaciens	Soybean paste (Doenjang)
B-10	15868	B. amyloliquefaciens	Soybean paste (Doenjang)
Bv-11	15875	B. velezensis	Soybean paste (Doenjang)
B-12	15877	B. amyloliquefaciens	Soybean paste (Doenjang)
B-13	16017	B. amyloliquefaciens	Dried fermented soybeans (Meju)
B-14	16023	B. amyloliquefaciens	Dried fermented soybeans (Meju)
B-15	16024	B. amyloliquefaciens	Dried fermented soybeans (Meju)
B-16	16032	B. amyloliquefaciens	Dried fermented soybeans (Meju)
Bv-17	17029	B. velezensis	Soil
B-18	17030	B. amyloliquefaciens	Soil
Bv-19	17031	B. velezensis	Soil
B-20	17032	B. amyloliquefaciens	Soil
Bv-21	17073	B. velezensis	Onion
Ba-22	18611	B. amyloliquefaciens	Agaricus bisporus media
B-23	18650	B. amyloliquefaciens	Soil
B-24	19163	B. amyloliquefaciens	Pig fecal
Bv-25	Lab strain	B. velezensis EB-39	Endophyte isolated from Dangyuja mandarin citrus

KACC: Korean Agricultural Culture Collection; the strains identified by 16S sequence analysis are highlighted.

Sequencing of the 16s rRNA gene and phylogenetic analysis

To identify the bacterial species, 16S rRNA sequences were compared with the corresponding whole gene sequences available in the GenBank nucleotide database . The phylogenetic analysis was carried out in the MEGA-7 program using the Maximum Likelihood method (Kumar et al., 2016). The phylogenetic tree was constructed using the type strains of Bacillus-related species. To statistically evaluate the nodes in the phylogenetic tree, bootstrap replication (1000) was used.

Morphology of Bacillus strains

Bacillus spp. were grown overnight in YNB or Landy broth (L-glutamic acid, 5.0 g; glucose, 20.0 g; yeast extract, 1.0 g; phenylalanine, 2.0 mg; MgSO4·7H2O, 0.5 g; KCl, 0.5 g; MnSO4, 5.0 mg; CuSO4·5H2O, 0.16 mg; FeSO4·7H2O, 0.15 mg; KH2PO4, 1.0 g; distilled water, 1L; pH was adjusted to 7.0 with 5 N NaOH), then 5 μl cultures were plated on YNA or Landy agar plates. The morphological features were observed after a 24 h and 48 h incubation period at 28 °C.

Selection of antagonistic Bacillus strains against Xcc

Ethyl acetate extracts of 25 Bacillus isolates were prepared according to Goodson et al. (2017) and tested for their antibacterial activity against XccW1 by the disk diffusion method. Briefly, the XccW1 strain grown overnight was mixed with 5 mL of 0.7% YNA soft agar and poured directly onto the YNA plates (1.5% agar). After air-drying, the ehtyl acetate extracts were dissolved in HPLC grade methanol to a concentration of 1.0 mg mL−1 for the antibiotic assay. Ethyl acetate was selected in the metabolite extraction process due to its low boiling point and polarity. Consequently, 30 µl of extracts were placed on a sterile filter paper disk. The antibacterial activity of the ethyl acetate extract was compared to those of the two positive controls composed of streptomycin S1 (streptomycin 1.0 mg mL−1) and S2 (streptomycin 0.1 mg mL−1) and a negative control where the filter paper discs were impregnated with 30 µl of methanol. After 24 h incubation of the plates at 28 °C, diameter of inhibition zone was measured around the paper disk (8 mm in size). Based on the activity against XccW1, the metabolite extracts of nine Bacillus isolates (Bv-1, Ba-2, Ba-6, Bv-11, Bv-17, Bv-19, Bv-21, Ba-22, and Bv-25) were further selected for evaluating their in vitro antagonistic effect against the two wild-type (XccW1 and XccW2) and the two streptomycin-resistant Xcc strains (XccM4 and XccM6). The experiment was performed twice with three replicates. The error bars in a two-tailed t-test represented the standard deviation, and the result was found to be statistically significant at p < 0.05.

Determination of the MIC and MBC of Bv-17 and Bv-21

To perform MIC and MBC experiments, we selected the most effective strains of Bv-17 and Bv-21. The MIC and MBC of ethyl acetate extracts of Bv-17 and Bv-21 and streptomycin were determined by the broth microdilution method (Adimpong et al., 2012). For the MIC analysis, various concentration of ethyl acetate extracts of Bv-17 and Bv-25 were prepared ranging from 15.625 to 1000 μg mL−1 as two-fold serial dilution in 96-well microtiter plates. Each well contained 100 µl of the ethyl acetate extract from Bv-17 or Bv-21, 90 µl of YNB broth, and 10 µl of Xcc strains at around 2.2 × 104 colony-forming units/ mL. The positive control was composed of YNB broth inoculated with Xcc whereas the negative control was only the YNB broth. After incubation at 28 °C for 24 h, MIC was recorded as the lowest metabolite extract concentration that prevented the visible growth of the indicator bacterial strain. However, MBC was determined by plating 10 µl of each well's cultures onto YNA plates and incubating the plates at 28 °C for 24 h. MBC was defined as the lowest concentration of metabolite extract that, after an incubation period of 24 h at 28 °C, did not show any bacterial growth in the YNA plates. MIC and MBC were both expressed in µg mL−1.

In vivo CBC disease suppression by B. velezensis Bv-21

In vivo biological control tests were performed on detached Hwanggeum hyang citrus leaves (Randhawa, 1985). For the assay, fully expanded immature (four-week-old) leaves were excised from Hwanggeum hyang (highly susceptible) cultivar, surface-sterilized with 70% ethanol for 1 min, 2% NaOCl (sodium hypochlorite) solution for 3 min, and 100% ethanol for 30 s. The leaves were washed three times with sterile distilled water (SDW) and then dried with sterilized filter paper. XccW4, a streptomycin-resistant mutant, and B. velezensis Bv-21 were used for inoculation. Both the strains were grown on liquid YNB media at 28 °C in a rotary shaker incubator at 180 rpm for two days and harvested by centrifugation at 4 °C at 6000 rpm for 15 min. The pellet was then re-suspended in SDW and adjusted to an approximate density of OD600 = 0.4 [approximately 108 colony-forming units (cfu) mL−1]. For each treatment of the freshly detached leaves of Hwanggeum hyang, approximately 0.1 mL of bacterial suspension, were infiltrated into the intercellular spaces of the leaves by applying steady and gentle pressure and holding the open end of the syringe to the leaf. For each concentration, three separate leaves were infiltrated and around 6–8 mm area of the leaves were water-soaked. The bacterial suspensions were prepared by mixing 0.8 OD600nm XccW4 with 0.8 OD600nm Bv-21. The freshly detached leaves of Hwanggeum hyang were infiltrated with SDW (negative control), XccM4 at 0.4 OD600nm (positive control), and a mixture of XccM4:Bv-21 at 0.4 OD600nm and Bv-21 at 0.4 OD600nm.

To avoid splashing of inoculum, the leaves were lightly blotted with sterile tissue to remove excess inoculum. Following inoculation, the leaves were placed in a humid box, upward (abaxial side), on two layers of sterile tissue paper moistened with SDW and later sealed with a parafilm to maintain high humidity. The boxes containing the inoculated leaves were kept at 24 °C in a plant growth chamber for the development of disease symptoms. The lesion area was measured using a Vernier digital electronic caliper and photographed at 7 days post-infiltration (dpi). All experiments were carried out three times and yielded the same results each time. Data were analyzed by one way ANOVA (analysis of variance) test and result was found significant (p < 0.0001) among the groups.

Quantification of Xcc populations in leaf tissues

Following the onset of CBC symptoms on the leaves of Hwanggeum-hyang citrus citrus at 7 dpi, the populations of XccW4 in the inoculated sites were counted on the YNA plates. In brief, the leaves were rinsed five times with SDW and dried under a laminar-flow hood to remove the moisture. The lesions (10 mm in diameter) were removed individually with a sterilized cork borer. The leaf disks were homogenized using a grinder in 1.0 mL of SDW and the resulting suspension was serially diluted from 10−2 to 10−8 in a microtiter plate. By plating10 µl of each suspension onto YNA supplemented with streptomycin, the number of viable cells was calculated. The YNA plates were incubated for 3 days at 28 °C for colony formation and counted to calculate the cfu mL−1. Data were analyzed by one way ANOVA test and result was found significant (p < 0.0001) among the groups.

Results

Identification of bacteria based on 16S rRNA gene sequencing

A total of 25 strains of B. velezensis or B. amyloliquefaciens (Table 1) were used in this experiment. After 16S rRNA sequence homology analysis with corresponding gene sequences for which complete genomes are available, we found that the 24 B. amyloliquefaciens strains provided by KACC were actually two groups of Bacillus spp., namely B. velezensis and B. amyloliquefaciens. Furthermore, the phylogenetic tree constructed from the 16S rRNA gene sequences revealed that all the isolates (except Bv-11) that suppressed the growth of Xcc belong to B. velezensis clade (Fig. 1). Both the species of B. velezensis and B. amyloliquefaciens differed in morphological features obtained on two different growth media namely the Landy and YNA media after an incubation period of 2 days at 28 °C (Fig. S1).

Fig. 1

Phylogenetic analysis of 16S rRNA gene sequences that highlight the position of selected Bacillus species closely related to the representatives of the Bacillus genus, such as, B. amyloliquefaciens, B. subtilis, B. licheniformis, B. tequilensis, B. cereus, and B. pumilus. Phylogenetic tree was constructed using Maximum Likelihood method. Parentheses indicate the accession numbers obtained from the National Center for Biotechnology Information (NCBI) database. The bootstrap values (%) are mentioned at the nodes and are obtained by repeating the analysis 1000 times. The scale bar represents a nucleotide substitution rate of 0.005 per nucleotide position.

Antagonistic activity of ethyl acetate extracts of B. velezensis against Xcc

In the disc diffusion assay, ethyl acetate extracts of 25 Bacillus strains were initially screened for potential antibacterial activity against the wild-type XccW1 (Fig. 2). Among these, Bv-21 and Bv-17 showed the strongest activity against XccW1 with an inhibition zone of 22.16 ± 1.58 and 18.29 ± 0.57 mm, respectively. Four isolates (Ba-2, Ba-6, Bv-11, and Ba-22) had no antibacterial effect against XccW1.

Fig. 2

In vitro antagonistic effect of the ethyl acetate extract of Bacillus spp. (1–25) and streptomycin on the virulence of X. citri subsp. citri (Xcc). (a) Bactericidal activity was conducted against the wild-type XccW1. 1–25: Ethyl acetate extract of 25 Bacillus strains (1.0 mg mL−1); C: Control (methanol); S1: Streptomycin (1.0 mg mL−1); S2: Streptomycin (0.1 mg mL−1). (b) Measurement of inhibition zones (mm) of ethyl acetate extracts of Bacillus spp. against XccW1. The error bars in a two-tailed t-test represented the standard deviation, and the result was found to be statistically significant at p < 0.05.

Nine Bacillus spp. (Bv-1, Ba-2, Ba-6, Bv-11, Bv-17, Bv-19, Bv-21, Ba-22, and Bv-25) were further selected from the initial screening data to check the antibacterial activity against two wild-type (XccW1 and XccW2) and two streptomycin-resistant strains of Xcc (XccM4 and XccM6). The ethyl acetate extract of Bv-1, Bv-17, Bv-19, Bv-21, and Bv-25 inhibited the growth of both wild-type and streptomycin-resistant Xcc strains (Fig. 3a, 3c). However, streptomycin S1 and S2 exerted no antibacterial effect on the streptomycin-resistant Xcc strains (Fig. 3c, 3d). S1 and S2 had antagonistic effects on the wild-type Xcc strains, with inhibition zones ranging from 28.17 ± 0.69 to 28.88 ± 0.61 mm and 15.10 ± 0.81 to 15.54 ± 0.58 mm, respectively. Bv-17 showed antibacterial activity with inhibition zones ranging from 20.0 ± 1.21 to 21.58 ± 0.81 and 19.70 ± 0.81 to 19.81 ± 1.49 for both wild-type and mutant Xcc strains, respectively. However, Bv-21 exhibited the highest antibacterial activity against XccW2 and XccM4 with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively (Fig. 3b, 3d).

Fig. 3

In vitro antagonistic effect of the ethyl acetate extract of selected Bacillus spp. and streptomycin on wild-type and streptomycin-resistant X. citri subsp. citri (Xcc) strains. (a) Bactericidal activity was conducted using the pathogenic wild-type XccW1 and XccW2. (b) Measurement of inhibition zones (mm) of ethyl acetate extracts of Bacillus spp. against XccW1 and XccW2. (c) Bactericidal activity was conducted using the pathogenic strains XccM4 and XccM6 (streptomycin-resistant). (d) Measurement of inhibition zones (mm) of ethyl acetate extracts of Bacillus spp. against the streptomycin-resistant XccW4 and XccW6. C: control (methanol); 1, 2, 6, 11, 17, 19, 21, 22, and 25: Ethyl acetate extract (1.0 mg mL−1) of selected Bacillus spp.; S1: streptomycin (1.0 mg mL−1); S2: streptomycin (0.1 mg mL−1). The error bars in a two-tailed t-test represented the standard deviation, and the result was found to be statistically significant at p < 0.05.

Determination of MIC and MBC of the ethyl acetate extracts against Xcc

The MIC and MBC of crude ethyl acetate extracts of strain Bv-17 and Bv-21 were determined against all Xcc strains by using the broth micro dilution method. Among the antagonistic strains of B. velezensis, Bv-17 and Bv-21 showed the highest antibacterial activity during the in vitro study against Xcc. Therefore, the metabolites of these two isolates were further selected for the determination of MIC and MBC against the wild-type Xcc strain (XccW1) and the streptomycin-resistant mutant Xcc strains (XccM4, XccM5, XccM6, XccM7, and XccM8). For the Bv-17 isolate, the MIC and MBC values of the ethyl acetate extract ranged from 62.5 to 125.0 µg mL−1 and for B. velezensis Bv-21 the MIC and MBC ranged from 31.25 to 125.0 µg mL−1. However, against XccM5, the MIC and MBC of ethyl acetate extract of Bv-21 were determined to be 31.25 µg mL−1 and 62.5 µg mL−1, respectively. The MIC of streptomycin against wild-type XccW1 strain ranged from 78 to 156 µg mL−1. However, the MBC of streptomycin against the mutant Xcc strains was relatively high and ranged from 375 to 1500 µg mL−1 (Fig. S2; Table 2).

Table 2

MIC and MBC of the ethyl acetate extract of Bv-17 and Bv-21, and streptomycin against Xcc strains.

Xcc strains	Bv-17 ethyl acetate extract		Bv-21 ethyl acetate extract		Streptomycin
	MIC (µg mL−1)	MBC (µg mL−1)	MIC (µg mL−1)	MBC (µg mL−1)	MIC (µg mL−1)	MBC (µg mL−1)
XccW1	62.5	125.0	62.5	125.0	78.0	156
XccM4	62.5	125.0	62.5	125.0	375.0	750.0
XccM5	62.5	125.0	31.25	62.5	375.0	750.0
XccM6	62.5	125.0	62.5	125.0	375.0	750.0
XccM7	62.5	125.0	62.5	125.0	750.0	1500.0
XccM8	62.5	125.0	62.5	125.0	375.0	750.0

In vivo biocontrol efficacy of B. velezensis Bv-21

Among the metabolites of Bacillus spp. tested, B. velezensis Bv-17 and B. velezensis Bv-21 held the highest antibiotic activity. Furthermore, B. velezensis Bv-21 was tested for biocontrol efficacy against a streptomycin-resistant mutant XccM4. In the detached leaf assay, all the citrus leaves were infiltrated with 0.1 mL of SDW, XccM4, and a mixture of XccM4:Bv-21. After a period of 7 dpi, both XccM4 and the mixture of XccM4:Bv-21 generated lesions of CBC (Fig. 4a). Lesions developed on XccM4-infiltrated leaves were visible on both faces and appeared in the form of water-soaked margins that were surrounded by yellow rings. However, the leaves infiltrated with Bv-21 exhibited no visible toxic effects or disease symptoms on the leaves of Hwanggeum hyang citrus (Fig. 4a). The leaves infiltrated with the mixture of XccM4:Bv-21 developed smaller necrotic lesions than those infiltrated by XccM4 alone (15.67 ± 1.6 mm vs. 11.54 ± 3.7 mm; Fig. 4b). The artificial inoculation of XccM4:Bv-21 suspension reduced the Xcc populations (XccM4) in citrus leaves by approximately 81.68% compared to those in the XccM4 alone (Fig. 4c). On the other hand, leaves infiltrated with SDW and Bv-21 showed no colonies on the YNA plates containing streptomycin.

Fig. 4

Development of disease symptoms in the leaves of Hwanggeum hyang citrus at 7 dpi through the inoculation of Xanthomonas citri subsp. citri (XccM4) and suspension of XccM4:Bv-21 (a) The leaves were infiltrated with 0.1 mL aliquots of OD600nm 0.4 XccM4 mixed with SDW (XccM4); OD600nm 0.4 XccM4 mixed with OD600nm0.4 Bv-21 (XccM4:Bv-21) and Bv-21. As a control, leaves were inoculated with SDW (b) Diameter of disease lesions measured at 7 dpi. (c) Quantification of XccM4 from the leaves treated with XccM4 and suspension of XccM4:Bv-21. Three independent replicates were tested for each treatment. One way ANOVA (analysis of variance) test was performed and result was found significant (p < 0.0001) among the groups.

Discussion

The role of biological agents in the control of crop diseases is gradually gaining the attention of microbiologists and plant pathologists. Unlike conventional pesticides, biological agents are considered as sustainable, safer to the environment and enhance the soil bacterial diversity (Jaffuel et al., 2019, You et al., 2016). In this study, we have investigated 25 Bacillus strains that were initially identified and stored as B. amyloliquefaciens (except Bv-25) in KACC (Table 1). However, the homology analysis of 16S rRNA gene sequences indicated that several KACC strains were indeed B. velezensis. According to previous studies, B. amyloliquefaciens is closely related to B. velezensis and is difficult to distinguish using traditional phenotypic approaches (Rabbee et al., 2019a). Furthermore, B. amyloliquefaciens strains are genetically related to B. subtilis species complex, which includes B. subtilis, B. velezensis, B. pumilus, and B. licheniformis (Fan et al., 2017). In 2017, Fan et al. analyzed the nucleotide sequence of 66 B. velezensis and related bacterial species based on its core genomes and rpoB (RNA polymerase beta-subunit) gene. Dendrograms based on sequence analysis revealed the presence of three tightly linked branches of (1) B. siamensis (2) soil-borne B. amyloliquefaciens, and (3) a conspecific group that included B. methylotrophicus, B. amyloliquefaciens subsp. plantarum and B. velezensis (Fan et al., 2017). Phylogenetic analysis and extended genome sequence data revealed that B. amyloliquefaciens subsp. plantarum and B. methylotrophicus is actually classified as B. velezensis (Dunlap et al., 2016, Rabbee et al., 2019a). B. amyloliquefaciens strains were well known for producing industrial enzymes such as amylase, protease, and glucanase (Fan et al., 2018) and demonstrated reduced potentiality in the synthesis of non-ribosomal secondary metabolites that act as antimicrobial compounds (Rückert et al., 2011). On the other hand, B. velezensis produces abundant secondary metabolites with antimicrobial activities against pathogenic microorganisms (Chen et al., 2018, Niazi et al., 2014).

To test the antibacterial efficacy of ethyl acetate extracts from all Bacillus spp. against Xcc, a disk diffusion assay was performed. The result showed that the ethyl acetate extracts of 21 Bacillus isolates exhibited different degrees of inhibition against XccW1. After initial screening against XccW1, we evaluated nine Bacillus strains for checking the antagonistic effect against two wild-type and two streptomycin-resistant Xcc strains. We found that ethyl acetate extracts of B. velezensis have antibacterial activity against Xcc pathogens. Moreover, the solvent extract of B. velezensis Bv-21 displayed the strongest antibacterial activity against both wild-type and streptomycin-resistant Xcc strains. Previously, the methanol extracts of B. velezensis displayed antimicrobial activity against Ralstonia solanacearum (causative agent of tomato wilt) and Fusarium oxysporum (causative agent of banana Fusarium wilt) under both laboratory and greenhouse conditions (Cao et al., 2018). In a another study, it was shown that ethyl acetate extract of B. velezensis Lle-9 exhibited antifungal activity against F. oxysporum with the growth inhibition percentage of 68.56 ± 2.35% (Khan et al., 2020). Inoculation of B. velezensis into the root of different plant seedlings also reported to increase the plant height as well as root and shoot biomass (Balderas-Ruíz et al., 2020).

Host plant colonization is a critical process for the survival of any plant pathogens. In our experiment, detached citrus leaves were infiltrated with a bacterial mixture of XccM4: Bv-21 and reduced the disease symptoms as compared to those infiltrated with XccM4 alone. Hwanggeum hyang leaves infiltrated with Bv-21, on the other hand, showed no signs of disease, indicating that Bv-21 is not pathogenic to citrus leaves. Furthermore, the number of phytopathogenic bacteria was reduced in citrus leaves that had been artificially inoculated with XccM4:Bv-21. More specifically, the suspension of XccM4: Bv-21 reduced Xcc populations in citrus leaves by approximately 81.68% relative to XccM4 alone. In our previous work, co-infiltration of endophytic bacteria B. thuringiensis TbL-22 and Xcc reduced the lesions of CBC by 64.05% relative to positive controls (Islam et al., 2019). Biocontrol property of B. velezensis AP-3 was reported to control the severity of Fusarium wilt (caused by F. oxysporum f. sp. lycopersici) in tomato by 50% when compared with control (Medeiros and Bettiol, 2021).

Bacillus spp. are well known as biocontrol agents against pathogenic microbes due to their capability to produce a wide variety of antimicrobial substances, such as lipopeptides, polyketides and some other volatile organic compounds (Chen et al., 2020). B. velezensis and B. amyloliquefaciens are morphologically similar, however; genome analysis revealed that some of the B. amyloliquefaciens strains with biocontrol potential were B. velezensis (Magno- erez-Bryan et al., 2015). B. velezensis exhibits biocontrol activity against the rice blight and leaf streak pathogens X. oryzae pv. oryzae and X. oryzae pv. oryzicola, by producing the antibacterial compounds difficidin and bacilysin (Wu et al., 2015). Fengycins produced by B. amyloliquefaciens MEP218 exhibited antibacterial potentiality against X. axonopodis pv. vesicatoria, a plant pathogen causing bacterial spot disease (Medeot et al., 2020). In a similar study, the B. methylotrophicus strain NKG-1 isolated from China exerted biocontrol activity against B. cinerea and thirteen phytopathogenic fungi. Strain NKG-1 reduced gray tomato mold infection by 60% in a detached leaf assay and enhanced tomato plant growth in both greenhouse and field trial experiments (Ge et al., 2016). In another research, it was shown that bacilysin synthesize by B. velezensis contributed biocontrol potentiality against Soybean root rot disease pathogen Phytophthora sojae (Han et al., 2021).

Conclusions

The ability of B. velezensis to synthesize antibacterial compounds has opened a new possibility in order to control plant pathogenic microbes. Our findings strongly suggest that metabolic extracts of B. velezensis can control the spread of Xcc under both in vitro and in vivo conditions. Therefore, identification of the antibacterial compounds of B. velezensis, and investigation of their biosynthetic pathways and regulatory genes under different culture conditions would be the additional steps in order to control Xcc. Furthermore, B. velezensis could be developed as a bio-fertilizer for more sustainable agriculture practices. All these findings point to B. velezensis can be the basis of biocontrol agent to combat Xcc in an efficient and eco-friendly manner.

Author contributions

M.F.R., Md. N. I., and K.-H.B. collected the data and wrote the manuscript. All authors have read and approved the final manuscript.

Funding

This research was funded by NRF-2019R1F1A1052625.

Declaration of Competing Interest

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