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Possible mechanisms of action of Bacillus wiedmannii AzBw1, a biocontrol agent of the root-knot nematode, Meloidogyne arenaria
Egyptian Journal of Biological Pest Control volume 33, Article number: 28 (2023)
With increased environmental concerns and restrictions of chemical control, the importance of other eco-friendly strategies for management of the nematodes is being substantially grown nowadays. One of the most well-known strategies that have attracted the attentions is biological control of these deleterious agents. In our previous study (Moslehi et al. in Egypt J Biol Pest Control 31:1–11, 2021), Bacillus wiedmannii AzBw1 was introduced as a robust antagonistic agent against root-knot nematode Meloidogyne arenaria. Present study addressed the possible mechanisms of action of this strain.
Based on quantitative bioassays it was shown that the strain AzBw1 is able to produce considerable amount of siderophore, protease, and chitinase. In an in vitro assay conducted by bi-plate Petri dishes, it was shown that hatching of the nematode eggs, subjected to bacterial volatile compounds (BVCs) was 34% lower than those of mock-treated control eggs. On the other hand, mortality of BVC-treated juveniles was 33.5% higher than those of mock-treated control juveniles. The secretory proteins from the medium culture of strain AzBw1 were precipitated and fractionated by anion exchange chromatography (AEC). Fractions from AEC were checked for hydrolytic activity and nematicidal effect. It was found that the fractions with the highest protease activity have a strong nematicidal effect. In contrast, significant nematicidal effect in the fraction with Chitinase activity was detected.
The results suggested that protease activity played a key role in strain AzBw1 antagonism against root-knot nematode, M. arenaria. Finally, nonvolatile organic compounds were also extracted from the medium culture after removing secretory proteins and enzymes. Obtained results showed that these metabolites had also a strong anti-nematode effect.
Root-knot nematodes (RKN) (Meloidogyne spp.) are economically important group of highly adapted plant parasites which distributed worldwide (Moens et al. 2009). These nematodes are obligatory endoparasites inhabiting the roots and cause structural, physiological, and biochemical changes in the host plants. They induce the redifferentiation of parenchyma root cells into multinucleate and hypertrophied feeding cells, named giant cells. These giant cells constitute the exclusive source of nutrients for the developing nematode. Hyperplasia of the surrounding cells leads to the formation of the typical root gall, the primary visible symptom of infection (Abad et al. 2009).
Among the well-known species of the Meloidogyne genus, four species, M. incognita, M. javanica, M. arenaria, and M. hapla are the most common and important species (Rusinque et al. 2022). They have vast host ranges, as they all infect many common vegetable crops. M. arenaria is extremely polyphagous, attacking both monocotyledons and dicotyledons and found in most of the warmer regions of the world and frequently encountered in glasshouses in cooler climates (Hunt and Handoo 2009).
With increased environmental concerns and restrictions of chemical control, the importance of other eco-friendly strategies for management of the nematodes is being substantially grown nowadays. The use of resistant or non-host crop plants, fallowing or flooding, application of nematicides and more recently, the use of microbial antagonists and biocontrol agents are the principal methods for management of RKN. Antagonistic plants, fungi and bacteria are among the most important agents for management of the nematodes. One of the most studied examples of natural control of plant parasitic nematodes (PPN) is the bacterial biocontrol agent.
Different mechanisms of action have been reported in different biocontrol agents (Migunova and Sasanelli 2021). Second-stage juveniles can be infected by Pasteuria sp. spores after they enter the roots and start feeding. This bacterium is able to penetrate cuticle by germ tubes that are generated from spores and colonize the body of the developing female (Mankau et al. 1976). The host range of these bacteria is narrow, such that each species of Pasteuria genus is able to infect certain genera of nematodes (de Gives et al. 1999). It seems that this specificity is determined by specific interaction of a carbohydrate ligand on the surface of the spores and a lectin-like receptor on the cuticle of host nematode (Persidis et al. 1991). Apart from Pasteuria species which have distinct parasitic relation with nematodes, many other opportunistic bacteria have been reported that are able to invade PNN in certain circumstances (Tian et al. 2007a). In the most cases these type of microorganisms use hydrolytic enzymes to digest different parts of nematodes (Tian et al. 2006). Moreover, in some of rhizobacteria antinematodal effect was related to antibiotics (Nadeem et al. 2021), toxins (Migunova and Sasanelli 2021) and volatile compounds (Siddiqui et al. 2006).
The strain AzBw1 was introduced in an attempt to screen rhizobacteria based on their ability to control RKN of tomato plant (Moslehi et al. 2021). This strain outperformed other strains such that in greenhouse conditions, root knots and egg mass development was considerably lower (60–70 percent) in AzBw1-treated plants in comparison to mock-treated plants. Therefore, the aim of this study was to find out possible mechanisms of action of this strain.
Propagation of nematode population
The population of Meloidogyne arenaria was obtained from Phytopathology Laboratory of the Azarbaijan Shahid Madani University, Iran. Nematodes were propagated on the roots of the susceptible tomato cultivar, Super Strain B in greenhouse conditions with 27 ± 2 °C and 16:8 photoperiods.
Bacterial strain and culture conditions
Bacillus wiedmannii strain AzBw1 was obtained from Bacterial Culture Collection, Phytopathology Laboratory of the Azarbaijan Shahid Madani University (AZBC), Iran. Stock cultures were prepared for storage at − 80 °C in 1.5 ml vials by mixing equal volumes of 50% glycerol and 24 h culture broth [from single colony inoculum, 25 ml Luria–Bertani medium, 100 ml flask, 130 rpm]. For preparation of cell suspension and medium culture of strain AzBw1, stock solution of strain AzBw1 was streaked on Nutrient Agar medium and incubated at 28 °C for about 48 h. A single colony was cultured in Luria–Bertani (LB) medium for 72 h at 180 rpm. The grown cells were harvested by centrifugation at 5000 g for 15 min. The supernatant was used for extraction of antinematodal metabolites. The pellet was re-suspended and washed thrice with distilled water. The density of this cells suspension was adjusted to 108 cell/ ml and used for assays.
Nematode mortality and egg hatching tests as affected by bacterial cell suspension
The egg masses were collected from infected roots and dissolved in 0.5% NaOCl to remove the gelatinous matrices (Barker and Hussey 1976). The resulted suspension was used in hatching experiment. For mortality tests, the resulted eggs from the egg masses were transferred into sterile distilled water and incubated in 27 ± 2 °C. Freshly hatched juveniles were collected for 3 days and the resulted suspension was used for experiment. To assess the influence of strain AzBw1 on egg hatch, a volume 0.5 ml of bacterial cell suspension in distilled water (108 CFU ml−1) was transferred into each well of a 24 well-plate to which a volume 0.5 ml egg suspension containing 100 ± 15 surface-sterilized eggs was added. The plate was incubated at 30 °C for four days and the number of hatched eggs in each well was recorded and hatch percentage was calculated by the formula: (number of hatched eggs/total number of eggs) × 100. To assess the influence of AzBw1 strain on M. arenaria juveniles, a volume 0.5 ml of bacterial cell suspension in distilled water (108 CFU ml−1) was transferred into each well of a 24 well-plate to which 0.5 ml of the freshly hatched juvenile suspension containing 50 ± 10 juveniles was added. The plate was incubated at 30 °C for three days and the numbers of dead juveniles were counted and percentage of juvenile mortality was calculated by the formula: (number of dead juveniles/number of total juveniles) × 100 (Siddiqui et al. 2006).
Assessment of antinematodal properties of strain AzBw1
The in vitro antinematodal ability of washed cell of strain AzBw1 was assessed as described previously (Moslehi et al. 2021). To assess antinematodal effect of medium culture of strain AzBw1, the medium culture was prepared as described above and the supernatant was filtered by 0.22 µM syringe filter. One-half ml of this solution was mixed with either same volume of eggs or juvenile suspensions (containing 100 of each) and hatching of eggs and juvenile’s mortality were assessed for 6 days. Sterile non-inoculated LB medium was used as negative control. Qualitative determination of proteolytic activity was determined by using skimmed-milk agar plates (Kumar et al. 2005). Qualitative determination of chitinase activity was evaluated by chitin agar medium (Hsu and Lockwood 1975). The siderophore production was determined by performing chrome azurol S (CAS) assay (Alexander and Zuberer 1991). Production of hydrogen cyanide (HCN) was assessed by the alkaline picrate test (Lorck 1948). The effects of bacterial volatile compounds (BVC) on egg hatching and juvenile’s viability was assessed using the bi-plate Petri dish method. In one side of the plate, strain AzBw1 was streaked on NA medium and incubated in 30 °C. After 5 h, either eggs suspension (200 eggs) or juveniles were spread on water agar medium by paintbrush in other side of the dish. Petri dishes were sealed by Parafilm and incubated in 30 °C for 6 days. Hatching of the eggs and juvenile viability was assessed on third and sixth days.
Extraction and fractionation of secretory proteins of strain AzBw1
Secretory proteins of strain AzBw1 were precipitated by ammonium sulfate method. Fine salt was gradually added to medium culture of strain AzBw1, while stirring and kept at 4 °C overnight. Then, the solution was centrifuged to separate the protein precipitates. The precipitates were floated in 20 mM tris–HCl buffer with pH 8.0 and dialyzed against a similar buffer to achieve desalting. The desalting leads to dissolving the protein precipitates and then, proteins gain their native structures and activities (Abdollahzadeh et al. 2020). Fractionation of prepared protein solution was performed by anion exchange chromatography (AEC). Briefly, a Q-sepharose column (Fast Flow, cytiva, UK) was equilibrated with 20 mM Tris–HCl at pH 8, loaded with protein preparation, washed with 20 mM Tris–HCl and eluted with different concentrations of NaCl solution (50, 150, 250, 350, 450 and 1000 mM). The flow-through and eluted proteins were collected as one ml fractions. Concentration of protein in these fractions was estimated by measuring the optical density at 280 nm and Bradford assay (Bradford 1976) and then they preserved at − 20 °C until analysis.
Assays for determination of hydrolytic activities
The quantitative determination of protease activity was performed by a modified colorimetric method (Souissi et al. 2008). Briefly, a 50 µl aliquot of the samples (fractions from AEC) was mixed with 500 µl of 20 mM Tris–HCl (pH 8.0) containing 0.5% casein, and incubated for 15 min at 37 °C. The reaction was stopped by addition of 250 µl 20% trichloroacetic acid. The mixture was allowed to stand at room temperature for 15 min and then centrifuged at 13,000 rpm for 15 min to remove the precipitate. The absorbance of the supernatant was measured at 280 nm. A standard curve was generated using solutions of 0–50 mg l−1 tyrosine. One unit of protease activity was defined as the amount of enzyme required to liberate 1 mg of tyrosine in 1 min under the experimental conditions used. The quantitative determination of chitinase activity was determined by colorimetric method in which colloidal chitin was used as substrate and the concentration of released reducing sugars was measured by dinitrosalicylic acid (DNS) method (Miller 1959). Colloidal chitin was prepared by a method as described by (Hsu and Lockwood 1975). For determination of enzyme unit, serial dilutions of N-acetylglosamine (from 0 to 50 mM) were prepared. One unit (U) of the chitinase activity was defined as amount of enzyme required to release 3.8 µmol ml−1 of N-acetyl d-glucosamine in 1 h (Shirazi et al. 2007).
Extraction of nonvolatile organic compounds
After precipitation of peptides and proteins from medium culture, the remaining medium was used for extraction of nonvolatile organic compounds. Liquid culture was acidified to pH 2 with 200 ml of 5 N HCl and extracted with equal volume of ethyl acetate for 30 min with vigorous shaking. Phase separation was accelerated by 10 min of centrifugation at 6000 rpm. The organic phase was transferred to a round-bottom glass flask and flash-evaporated, and the residue was dissolved in 1 ml of HPLC-grade methanol and chloroform solution (2:1 v/v) (Shirzad et al. 2012).
Data were processed by analysis of variance, followed by the Fisher’s multiple range test (< 0.05), with Minitab 19 software (Minitab LLC, Pennsylvania, USA).
Assessment of antinematodal properties of strain AzBw1
The results from in vitro assays showed that both washed cells and filtered sterile medium of this strain have robust anti-nematode activity such that hatching of eggs treated with cell suspension and filtered medium were14.75 and 29.28 percent, respectively, which is 4.27 and 2 times lower than that of mock-treated control eggs suspension (Table 1). In addition, mortality of juveniles treated with bacterial cells suspension and filtered medium were 82.5 and 58.58 percent, respectively, which is 3.5 and 3.7 times higher than that of mock-treated control juveniles. To find clues about the possible mechanism of action, this strain was assessed for its ability to produce antinematodal metabolites. Obtained results showed that strain AzBw1 was able to form clear transparent halo zone on skimmed-milk agar medium (average of 1.2 cm in diameter), which indicated its ability to produce proteases. To assess its ability to produce siderophores, CAS ager medium was spot inoculated by strain AzBw1. Almost one day after inoculation, an orange halo (average of 2 cm in diameter) was appeared around colonies. Chitin agar medium was used to assess the ability of strain AzBw1 to produce chitinase. After three days of incubation, small colonies were appeared on this medium and after four days, transparent halos were appeared around colonies. Alkaline picrate test was used to detect HCN production by this strain. It was found that strain AzBw1 was a weak producer of HCN as it could turn the yellow color of picrate-impregnate filter papers to light brown. The effect of bacterial volatile compounds (BVCs) produced by strain AzBw1 on nematode was also investigated. Hatching of the eggs that were subjected to BVCs was 34% lower than those of control eggs. On the other hand, mortality of BVC-treated juveniles was 33.5% higher than those of mock-treated control juveniles (Fig. 1).
Fractionation of secretory proteins from strain AzBw1
Secretory proteins from the medium culture of strain AzBw1 were precipitated and fractionated by AEC. By increasing NaCl concentration, molecules started to elute, reaching to the highest concentration in fraction 7 (Fig. 2). However, nematicidal activity was only detected in fractions 8 and 9. The mortality of juveniles treated with only 50 µl of fraction 8 was 70% (Fig. 2). There was slight increase in mortality of juveniles treated with fractions F10-F14. The same pattern was observed as the effect of these fractions on eggs hatching was assessed (data not shown). To evaluate the possible role of hydrolytic enzymes in mortality of juveniles and reduction of egg hatching, chitinase and protease activity in all bound eluted and unbound, flow-through fractions was estimated. The highest protease activity was detected in fractions 8 and 9 but no chitinase activity was detected in any bound eluted fractions (Fig. 2, Table 2). On the other hand, considerable amount of chitinase activity was detected in flow-through fractions (8.4 ± 1.86 U mL −1) but no protease activity detected in these fractions (Table 2).
Secretory proteins of strain AzBw1 were precipitated by ammonium sulfate method. Fine salt was gradually added to medium culture of strain AzBw1 while stirring and kept at 4 °C overnight. Then, the solution was centrifuged to separate the protein precipitates. The precipitates were floated in 20 mM tris–HCl buffer with pH 8.0 and dialyzed against a similar buffer to achieve desalting. The desalting led to dissolving the protein precipitates and then, proteins gain their native structures and activities (Abdollahzadeh et al. 2020). Fractionation of prepared protein solution was performed by anion exchange chromatography (AEC). Briefly, a Q-sepharose column (Fast Flow, cytiva, UK) was equilibrated with 20 mM Tris–HCl at pH 8, loaded with protein preparation, washed with 20 mM Tris–HCl and eluted with different concentrations of NaCl solution (50, 150, 250, 350, 450 and 1000 mM). The flow-through and eluted proteins were collected as one ml fractions. Concentration of protein in these fractions was estimated by measuring the optical density at 280 nm and Bradford assay (Bradford 1976) and then they preserved at − 20 °C until analysis.
Role of organic compounds in anti-nematode effect of strain AzBw1
After removing proteins and peptides, organic compounds were extracted from medium culture of strain AzBw1. It was found that this preparation still contained antinematodal compounds such that the mortality of juveniles treated with 5 µg of extracted organic compounds was about 57% higher than those of mock-treated juveniles (Fig. 3). Hatching of eggs treated with these compounds was also significantly reduced in comparison to mock-treated control eggs (Fig. 3).
Several species of the genus, Bacillus has been introduced as efficient agents for biological control of RKN (Migunova and Sasanelli 2021). B. wiedmannii is a facultative anaerobic, spore-forming Bacillus strain which initially was isolated from dairy foods (Miller et al. 2016). To our knowledge, there have not been any reports investigating biocontrol capability of B. wiedmannii. However, in the recent work, a novel isolate from this species (nominated as AzBw1) was introduced as a robust anti-nematode bacterium (Moslehi et al. 2021). Considering the method used for isolation of rhizobacteria, this strain was supposed to be an endophytic bacterium. Therefore, it seems that it had a close relation with tomato plants.
To find out possible mechanisms of action of strain AzBw1, common bioassays were performed to assess antagonistic attributes of this strain. Standard CAS test was used to detect siderophore production. In this test, siderophores removed the iron from the blue color CAS-ferric complex, turning its color to orange. The strain AzBw1 was considered as a strong producer of siderophore, as it was able to create large orange hallo in a short time. Siderophores could suppress PPN via different mechanisms. It was reported that chelation of iron by bacterial siderophores could limit iron access of nematode and this could lead to nematode death (Proença et al. 2019). They also could indirectly suppressed PPN by induction of plant systemic resistance (Romera et al. 2019) and plant growth promotion (Pahari et al. 2017).
Bacterial volatile compounds (BVCs) have been reported as key player in performance of plant probiotic bacteria (Sharifi and Ryu 2016, 2018). Some of them can stimulate plant growth and some others can suppress pathogenic microbes, nematodes and even insects (Popova et al. 2014; De Vrieze et al. 2015; Xu et al. 2015; Cheng et al. 2017; Sharifi and Ryu 2018). Emission of variety of nematocidal BVC such as benzeneacetaldehyde, 2-nonanone, decanal, 2-undecanone and dimethyl disulfide (DMDS), phenyl ethanone, nonane, phenol, 3,5-dimethoxy-toluene, 2,3-dimethyl-butanedinitrile, 1-ethenyl-4-methoxy- benzene and methyl isovalerate (MIV) was reported from different species of Bacillus (Huang et al. 2010; Ayaz et al. 2021; Diyapoglu et al. 2022). Among all of the mentioned compounds above, DMDS was the most reported anti-nematode compound in variety of microorganisms, therefore it was registered by Arkema as a pesticide with the trade name of Paladin (Diyapoglu et al. 2022). Bi-plate Petri dish method was used to find out possible role of BVCs in anti-nematode activity of the strain AzBw1. Obtained results showed that the BVCs emitted from this strain had nematocidal effect on M. arenaria. Ability of this strain to produce HCN was also investigated. However, this strain AzBw1 was rated as a weak producer of HCN, therefore it might have minor role in AzBw1’s anti-nematode activity.
Using skimmed-milk agar and chitin agar mediums, it was found that strain AzBw1 had strong protease and chitinase activities. In our initial bioassays, it was also observed that even washed cells of this strain were inhibitory against juvenile and egg suspensions in deionized water. Regarding the fact that bacteria become inactive in nutrient-free environment (deionized water), it was concluded that they digested certain components of nematodes which resulted in reduction of egg hatching and increasing juvenile mortality. To prove this hypothesis, the secretory proteins from the medium culture of strain AzBw1 were precipitated and fractionated by AEC. Fractions from AEC were checked for their hydrolytic activity and nematocidal effect. It was found that the fractions with the highest protease activity had a strong nematocidal effect. In contrast, significant nematocidal effect in the fraction with chitinase activity could not detect. These results suggested that protease activity play a key role in strain AzBw1 antagonism against nematode. Slight increase in the mortality of juveniles treated with fractions F10–F14 was detected (Fig. 2), which could due to the high concentration of NaCl in these fractions. The dominant role of bacterial proteases had been shown in the invasion process of several bacteria (Huang et al. 2005; Qiuhong et al. 2006; Tian et al. 2006, 2007b; Geng et al. 2016; Hu et al. 2020). Histopathological observations and molecular biological analyses demonstrated that an extracellular alkaline serine protease (BLG4) from Brevibacillus laterosporus G4 could be the main virulence factor in this bacterium (Huang et al. 2005; Tian et al. 2006). The BLG4-deficient strain BLG4-6 was only 43%, as effective as the wild-type strain at killing nematodes, and showed only 22% as much cuticle degrading activity (Tian et al. 2006, 2007a). In a whole genome sequencing analysis of Bacillus firmus DS-1 (an excellent biocontrol agent of PNN) multiple potential virulence factors were detected among which, a peptidase S8 superfamily protein called Sep1 was reported as an important virulence factor in this biocontrol agent (Geng et al. 2016). This serine protease degraded multiple intestinal and cuticle associated proteins and destroyed host physical barriers. On the other hand, in another study which conducted on Bacillus cereus BCM2, it was shown that secreted proteases do not damage the internal organization of the nematode, but instead, they severely damage nematode cuticle, which leads to content leakage (Hu et al. 2020).
Nematicidal effect of medium culture of strain AzBw1 was not completely removed after extraction of enzymes and proteins by acetone precipitation. Obtained results showed that ethyl acetate extracted nonvolatile metabolites from this medium culture have also a strong anti-nematode effect. This effect could be attributed to one or several secondary metabolites. Sphingosine was reported as the main anti-nematode metabolite of B. cereus strain S2 which was a successful biocontrol agent of M. incognita (Gao et al. 2016). In sphingosine-treated nematodes, ROS was induced in intestinal tract and genital area was disappeared.
Taken together, biocontrol ability of strain AzBw1 could be related to several mechanisms. Present results showed that protease activity and production of volatile and nonvolatile compounds play an important role in antagonistic ability of this strain. However, considering the fact that it had strong inhibitory effect, even in pure water against juveniles and eggs, it seemed that protease activity is the main mechanism of action of this strain.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Bacterial volatile compounds
Anion exchange chromatography
Plant parasitic nematodes
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The present study was supported financially by Azarbaijan Shahid Madani University, Tabiz, Iran.
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Fallahzadeh-Mamaghani, V., Shahbazi-Ezmareh, R., Shirzad, A. et al. Possible mechanisms of action of Bacillus wiedmannii AzBw1, a biocontrol agent of the root-knot nematode, Meloidogyne arenaria. Egypt J Biol Pest Control 33, 28 (2023). https://doi.org/10.1186/s41938-023-00668-1
- Meloidogyne arenaria
- Bacillus wiedmannii
- Secretory proteins