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Article

Rhizospheric Bacillus spp. Exhibit Miticidal Efficacy against Oligonychus coffeae (Acari: Tetranychidae) of Tea

by
Popy Bora
1,2,*,
Sukanya Gogoi
2,
Mukund Vinayak Deshpande
3,
Pankaj Garg
4,
Rana P. Bhuyan
5,
Nilofar Altaf
6,
Nikita Saha
1,
Sapna Mayuri Borah
1,
Mousumi Phukon
6,
Nabajit Tanti
5,
Bishal Saikia
1,
Shenaz Sultana Ahmed
7,
Sanjib Ranjan Borah
2,
Ashish Dutta
2 and
Bidyut Kumar Sarmah
1
1
Biocontrol Laboratory, DBT-North East Centre for Agricultural Biotechnology, Jorhat 785013, India
2
AAU-Assam Rice Research Institute, Assam Agricultural University, Jorhat 785013, India
3
National Chemical Laboratory, Pune 411008, India
4
Department of Chemistry, GLA University, Mathura 281406, India
5
Department of Tea Husbandry and Technology, Assam Agricultural University, Jorhat 785013, India
6
Department of Entomology, Assam Agricultural University, Jorhat 785013, India
7
Department of Plant Pathology, Assam Agricultural University, Jorhat 785013, India
*
Author to whom correspondence should be addressed.
Microorganisms 2023, 11(11), 2691; https://doi.org/10.3390/microorganisms11112691
Submission received: 9 June 2023 / Revised: 28 September 2023 / Accepted: 14 October 2023 / Published: 2 November 2023
(This article belongs to the Special Issue Beneficial Microorganisms and Antimicrobials)
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Abstract

:
Oligonychus coffeae (Acari: Tetranychidae), popularly known as red spider mite (RSM) is one of the major pests of commercial tea (Camellia sinensis (L.) O. Kuntze) plantation world over. Many attempts have been made in the past to control this devastating pest using a variety of microbial bioagents, however, area-wise field success is very limited. We carried out an in vitro study to explore the potential of rhizospheric Bacillus spp. (B. amyloliquefaciens BAC1, B. subtilis LB22, and B. velezensis AB22) against O. coffeae through adulticidal and ovicidal activity. The 100% adult and egg mortality was observed with bacterial suspension (1 × 109 CFU/mL) by B. velezensis AB22, showing the lowest LC50 values for both adults and eggs of O. coffeae, i.e., 0.28 × 105 and 0.29 × 105, respectively. The study also throws some insights into the underlying mechanism through electron microscopy study and identification of some putative pesticidal metabolites from all the species. The three Bacillus species were observed to have four commonly secreted putative bioactive secondary metabolites, brevianamide A, heptadecanoic acid, thiolutin, and versimide responsible for their bio-efficacy against O. coffeae. The outcome of our study provides a strong possibility of introducing Bacillus spp. as a biological miticide and developing synthetic metabolites mimicking the mechanistic pathway involved in microbial bioefficacy.

1. Introduction

Tea, Camellia sinensis (L.) O. Kuntze, an evergreen perennial crop of the family Theaceae represents globally one of the oldest organized agricultural industries. Tea made from its tender leaves is the most extensively consumed non-alcoholic beverage throughout the world owing to its multiple health benefits attributable to high antioxidant compounds and polyphenols [1]. The Assam state of India is the single largest contiguous tea growing region in the world, with a production of 0.75 million tons from 0.32 million ha area, thereby, contributing nearly 51% of the global tea basket [2]. The tea plants known as ‘single most forest species’ raised under a monocropping system are subjected to infestation by various pests and diseases, cutting their productive life and deteriorating their quality and quantity, leading to significant damage to the tea industry. Red Spider Mite (RSM), being polyphagous in nature, is one of the major pests of tea [3], causing damage by sucking the sap and lacerating the cells with characteristic reddish-brown marks on the upper surface of mature leaves [4,5], thereby, incurring substantial crop losses ranging between 17% and 46% [4]. Being polyphagous in nature, the pest is reported to cause severe damage to approximately 133 crops in tropical and subtropical regions [6]. Tea growers eventually resort to heavy and frequent use of a number of chemical acaricides. Such intensive, prolonged, and repeated use of synthetic pesticides has many disadvantages, such as non-selective destruction of beneficial microbes, chemical residue-driven soil contamination, and development of pesticide resistance [7,8]. Accumulation of pesticide residues above maximum residue limit in processed tea has resulted in restrictions imposed by tea importers. Such a scenario of tea growing has warranted an immediate necessity to develop an alternative sustainable management option as a green solution without compromising with either green leaf yield or tea quality. Hence, the onus is directed towards exploring microbial pesticides/biopesticides. Different species of Bacillus have been widely exploited for disease management in organic production systems [9]. The entomopathogenic bacteria, Bacillus thuringiensis, has been used as a popular microbial bioagent option over the last 50 years in tea plantations against lepidopteran pests [10,11,12,13], while other species from the genus Bacillus are mostly used as bioagents and growth promoters in an array of crops. Different Bacillus spp. have been reported to be highly effective in reducing the black rot severity caused by Corticium theae [14] and anthracnose in tea caused by Colletotrichum theae-sinensis by 77.30% under glasshouse conditions [15]. A few studies also suggest the entomopathogenic activity of other Bacillus spp. owing to its potential to produce a wide range of secondary metabolites, however, such studies are scarce. Further, studies reporting the role of secondary metabolites synthesized by different Bacillus species against crop pests need a thorough revisit.
In light of these facts, we investigated the biocontrol efficacy of three different Bacillus spp. (B. amyloliquefaciens BAC1, B. subtilis LB22, and B. velezensis AB22) previously isolated from rhizospheric soil of agricultural fields against O. coffeae using their adulticidal and ovicidal properties under controlled laboratory conditions. Furthermore, the identification of effective metabolites responsible for their pesticidal activity was also studied to develop a novel approach against mite pests in the organic tea ecosystem. The results of these studies are likely to put forth some new strategies for organic pest management.

2. Material and Methods

Experiments were conducted at the Biological Control Laboratory, Department of Plant Pathology, Assam Agricultural University, Jorhat, Assam (India).

2.1. Bacterial Strains

Three Bacillus spp. isolated from tea rhizosphere with National Centre for Biotechnology Information (NCBI, Bethesda, MD, USA) accessions, B. amyloliquefaciens BAC1 (ON392425), B. subtilis LB22 (ON386193), and Bacillus velezensis AB22 (ON209629) (Figure S1A–C) were collected from author’s Biocontrol Laboratory of Assam Agricultural University, Jorhat, Assam (India). All selected strains were maintained in the freezer at −80 °C in 20% glycerol until further use.
The three strains were further submitted to the national repository of the National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM, Uttar Pradesh, India) with accession numbers, B. amyloliquefaciens BAC1 (NAIMCC-B-03217), B. subtilis LB22 (NAIMCC-B-03226) and B. velezensis (NAIMCC-B-03221). All the isolates were subcultured on Nutrient Agar media (NA, Hi-Media, India) with media composition: Peptone (5 gms), HM Peptone B (1.50 gms), Yeast Extract (1.50 gms), NaCl (5.00 gms), Agar (15.00 gms), pH 7.4 and stored at 4 °C for further experiments.

2.2. Preparation of Bacterial Cell Suspension

To prepare bacterial cell suspensions, the protocol of Zhou et al. (2011) [16] was followed. Briefly, individually purified Bacillus isolates kept in NA-slants were first added to nutrient broth (NB) medium (100 mL) and incubated at 28 °C for 24 h with rotary agitation (150 rpm). Afterward, centrifugation was performed at 5000 rpm for 5 min, and the cells were harvested by discarding the supernatant. The pellets were further washed and resuspended in sterile distilled water to obtain an initial bacterial population density of 1 × 109 colony-forming units (CFU/mL) by adjusting at 640 nm using a spectrophotometer (double beam UV-VIS, Systronics, Gujrat, India). Finally, the suspension was diluted using the serial dilution procedure from 1 × 109 to 1 × 105, 1 × 106, 1 × 107, and 1 × 108 CFU/mL.

2.3. Collection and Rearing of Red Spider Mite

The tested pest, Red Spider Mites (RSM) were collected from the Experimental Organic Tea Garden (26°43′17″ N, 94°11′49″ E) of Assam Agricultural University, Jorhat, Assam (India). A stock culture of RSM population was maintained by following the detached leaf culture technique [17]. The collected mites were immediately transferred onto fresh leaves of susceptible tea cultivar, TV1, keeping the leaves on moistened cotton pads (ca. 1.5 cm thick)placed in plastic rearing trays (42 × 30 × 6.5 cm). Withered leaves were replaced regularly with new ones at 4-day intervals. The rearing trays were kept under controlled conditions in the laboratory at a temperature of 27 ± 2 °C, 75–80% relative humidity, and 16L: 8D photoperiod (Figure S2). Periodically, water was added to rearing trays to keep the cotton moist and prevent further drying of the leaves.

2.4. Screening of Miticidal (Acaricidal and Ovicidal) Potential of Bacillus spp. against O. coffeae

Adulticidal and ovicidal activities of the three Bacillus isolates at three different concentrations (1 × 107, 1 × 108 and 1 × 109 CFU/mL) were evaluated against O. coffeae in order to determine their pesticidal efficiency. The leaf disc method proposed by Ebeling and Pence (1953) [18] was adopted for both the bioassays at a temperature of 27 ± 2 °C, 75–80% relative humidity and 16L: 8D photoperiod [17].

2.5. Setup for Adulticidal Activity

The matured tea leaves (var. TV1) with no past history of pesticidal application for 15 days were collected from the Organic Tea Garden of Assam Agricultural University, Jorhat, Assam (India). Leaf discs (20 mm diameter cut from these mature tea leaves) were placed in Petri dishes lined with water-saturated cotton wool. With a fine camel-hair brush, 10 adult female mites were carefully introduced onto the surface of the discs and counted under a binocular microscope. To evaluate the efficacy of different Bacillus spp. against adult O. coffeae, direct spraying of bacterial isolates was done at three concentrations of 1 × 107, 1 × 108, and 1 × 109 CFU/mL, adding 4 drops of Tween-20 as an emulsifier. Distilled-water-sprayed leaf discs served as control. Each Petri dish containing leaf discs was sprayed with a uniform quantity (1.2 mL/5 s) of Bacillus spp., using a manual glass atomizer (50 mL). The surviving mites were counted at 24 h intervals up to 96 h. Each experiment was under taken with six replications.
The mortality percentage was calculated and corrected using Abbott’s formula [19].
Corrected mortality (%) = Mortality in treatment (%) − Mortality in control (%)/100 − Mortality in control (%) × 100.

2.6. Setup for Ovicidal Activity

As many ten gravid female mites were introduced onto the surface of the discs (24 h before the start of the experiment), allowed to lay eggs, and finally, the number of eggs was adjusted to 30 eggs/leaf disc. The leaf discs containing the eggs were sprayed with different Bacillus isolates at 1 × 107 to 1 × 109 CFU/mL, adding 4 drops of Tween-20, using a glass atomizer (constant pressure 2.5 kg/cm2) and distilled water served as the control. The discs were dried for at least 30 min. After drying, the discs were placed at 27 ± 2 °C and 65 ± 5% relative humidity. All discs were examined on a daily basis for 14 successive days. Hatchability was recorded for both experimental and control batches of eggs. The eggs failing to hatch after this period were considered non-viable. Six replicates were used for each treatment [20].

2.7. Virulence of the Three Bacillus Isolates against Adults and Eggs of O. Coffeae

The virulence of all three strains of Bacillus was evaluated to estimate LC50 following the aforementioned leaf disc method [18]. All three strains showed high pathogenicity in the preliminary screening assay and were further tested against both adults and eggs of RSM at 1 × 105, 1 × 106, 1 × 107, 1 × 108, and 1 × 109 CFU/mL to find out its virulency. For each concentration, six replications were performed. Adult mortality was recorded at every 24 h interval up to 96 h, and in the case of ovicidal activity, data were recorded up to 14 successive days. And subsequently, median lethal concentration (LC50) of all the three treatments to adults and eggs of O. coffeae was calculated.

2.8. Electron Microscopy Study

The virulence of the most effective Bacillus isolate against adults and eggs of O. coffeae was studied with digital bright field microscopy under 40× (Carl Zeiss, Axio Lab 5, Jena, Germany) and was further confirmed by Scanning Electron Microscopic study. Samples of dead mites and unhatched eggs of O. coffeae were prepared for SEM study as per protocol suggested by Orion et al. (1994) [21] with slight modifications. Briefly, the egg samples were fixed at 4 °C in closed tubes containing 1.25% glutaraldehyde and 1.25% paraformaldehyde in 0.05 M cacodylate buffer (pH 7.2) for 4 h. The samples were treated serially for 15 min with 50, 70, 90, and 100% ethanol and butanol mixture. The samples were then vacuum freeze-dried for 24 h. The samples were coated by carbon grid with platinum coating and observed under JEOL (Tokyo, Japan), JSM- 6390LV for morphological changes in insect bodies and egg surface using SAIC Facility at Tezpur University, Tezpur, Assam (India).

2.9. Characterization of Pesticidal Metabolites through LC-MS Profiling

The presence of secondary metabolites with pesticidal properties in the supernatants of three Bacillus isolates was determined by liquid chromatography mass spectrometry (LC-MS) analysis at CSIR-Central Drug Research Institute (CDRI, Uttar Pradesh, India), Lucknow (India). The Bacillus strains were allowed to grow in Nutrient Broth (NB) (Sigma-Aldrich, St. Louis, MO, USA) in conical flasks (500 mL), placed in a rotary shaker (Lark Innovata, Germany) at 120 rpm for 4 days, and incubated at 28 ± 2 °C. The media was then filtered through Whatman No.1 filter paper and kept in another conical flask (500 mL). The filtrates were then mixed with an equal volume of methanol and twice the volume of chloroform (Methanol:Chloroform: 1:2) and kept at 28 ± 1 °C overnight. The solvents were further centrifuged at 12,000 rpm for 10 min and the supernatant obtained was dried in a rotary evaporator system (IKA®, Staufen, Germany). The powdered extract of Bacillus isolates obtained was re-dissolved in methanol. The extract was filtered through 0.22 µ syringe filter and outsourced for performing LC-MS at CSIR-CDRI (India). The LC-MS peaks were analyzed in the MestreNova (Mnova Suite V.11.0.4) software against known insecticidal compounds listed in the literature [22]. The respective retention time and similarity score were recorded for each compound.

2.10. Statistical Analysis

Data on mortality and ovicidal activities of Bacillus cell suspension against O. coffeae were generated, arcsine transformed, and subjected to analysis of variance (Tukey’s test of significance at 5% level) using SPSS version 20.0 [23].

3. Results

3.1. In Vitro Adulticidal Activity of Bacillus spp. against O. Coffeae

All the evaluated Bacillus spp., B. amyloliquefaciens BAC1, B. subtilis LB22, and Bacillus velezensis AB22, expressed both adulticidal (Figure 1) and ovicidal activity to varying proportions at the three different concentrations tested (1 × 107, 1 × 108, and 1 × 109 CFU/mL).
At 1 × 109 CFU/mL, the isolate B. velezensis AB22 showed 100% adult mortality earliest at 72 h after treatment (HAT), making it the most efficient among the other treatments. Another isolate, B. amyloliquefaciens BAC1 at the same concentration, also showed 100% mortality of adult O. coffeae at 96 HAT, which was found to be statistically at par with B. velezensis AB22, showing 96.66% mortality at 1 × 108 CFU/mL, followed by B. subtilis LB22 with 93.33% mortality at 1 × 109 CFU/mL (Table S1).

3.2. In Vitro Ovicidal Activity of Bacillus spp. against O. coffeae

As shown in Figure 2 and Figure 3C,D, the three isolated Bacillus spp. showed varying degrees of egg mortality after 14 days after spray (DAS) at all three concentrations tested. The isolate B. velezensis AB22 showed 100% ovicidal activity of O. coffeae at 1 × 109 CFU/mL followed by B. amyloliquefaciens BAC1 (92.22%) and B. subtilis LB22 (86.67%). The results further indicated that higher concentrations of Bacillus isolates were associated with proportionately higher pesticidal efficacy against the insect.
The bio-efficacy of B. velezensis AB22 showed higher adulticidal and ovicidal activity against O. coffeae. The Bacillus spp., B. velezensis registered a net 6.00% higher ovicidal activity over the other two Bacillus spp. (B. amyloliquefaciens and B. subtilis). However, the ovicidal activity of all three Bacillus spp. was observed to be significantly higher by comparing the response at 1 × 107 versus 1 × 109 CFU/mL (Table S2).

3.3. Virulence of the Three Bacillus Isolates against Adults and Eggs of O. Coffeae

The median lethal concentration (LC50) of all three Bacillus spp. was measured for their efficacy against the mortality of adults and eggs of O. coffeae. The LC50 values for adult mortality by Bacillus velezensis AB22, B. amyloliquefaciens BAC1 and B. subtilis LB22 were 0.28 × 105, 1.06 × 105 and 5.12 × 107 CFU/mL, respectively (Table 1). In case of egg mortality, LC50 values were found to be in the same trend i.e., 0.29 × 105, 0.41 × 105 and 0.43 × 107 CFU/mL for Bacillus velezensis AB22, B. amyloliquefaciens BAC1 and B. subtilis LB22 (Table 2). Thus, Bacillus velezensis AB22, showing the lowest LC50 values for both adults and eggs of O. coffeae, i.e., 0.28 × 105 and 0.29 × 105,respectively, was considered the most virulent isolate amongst the tested Bacillus spp.

3.4. Morphological Changes in O. coffeae in Response to Bacillus Treatment

SEM study on morphological changes in O. coffeae adults and eggs in response to the most effective Bacillus isolate, B. velezensis AB22 treatment showed complete paralysis of adult mites with distorted abdomen and folded legs at 96 h after treatment (HAT). The inner body content of the mite dried completely and remained as a mass of cast skin (Figure 4A). The eggs of O. coffeae after treatment (14 DAS) were also disfigured, flattened on one side, and remained unhatched (Figure 4B).

3.5. Profiling and Identification of Pesticidal Metabolites of Bacillus spp.

Bacillus species are known to secrete various metabolites inhibitory to many phytopathogens. Our study identified an array of insect-pests attacking diversified host plants. The LC-MS chromatogram of extracts from three Bacillus isolates depicted several peaks corresponding to bioactive compounds recognized by their peak retention time, peak area (%), height (%), and mass spectral fragmentation patterns of known compounds described by the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) library. The presence of different compounds was observed to have insecticidal as well acaricidal activities. These compounds comprised brevianamide A, heptadecanoic acid, thiolutin, and versimide. In addition to these four metabolites, B. velezensis was observed to produce citromycin, emodin, peramine, and zwittermicin A; while B. subtilis was characterized by some unique compounds, namely, anhydrofusarubin, emodin, nikkomycins, and sterigmatocystin. On the contrary, B. amyloliquefaciens produced tenuazonic acid and milbemycins D as additional metabolites (Table 3, Table 4 and Table 5 and Figure 5, Figure 6 and Figure 7).

4. Discussion

The target pest, Oligonychus coffeae, is one of the major mite pests causing a serious threat to tea crops, deteriorating both production as well as quality [5]. Microorganisms, such as antagonistic fungi and bacteria, provide a much safer, more sustainable and environmentally friendly alternative to the commercially available synthetic acaricides [24,25,26]. Numerous investigations revealed that certain Bacillus species have pesticidal effects against various diseases and insect pests in a wide range of crops [14,27,28,29,30]. In this investigation, we employed three rhizospheric Bacillus spp. (B. amyloliquiefaciens BAC1, B. subtilis LB22 and B. velezensis AB22) to explore their miticidal potential, if any. Our study demonstrated the adulticidal and ovicidal efficacy of all three Bacillus spp. Against O. coffeae under laboratory conditions at three different concentrations:1 × 107, 1 × 108, and 1 × 109 CFU/mL. However, the highest efficacy on adults and eggs of mites was recorded in B. velezensis AB22 (1 × 109 CFU/mL), followed by B. amyloliquefaciens BAC1 (1 × 109 CFU/mL), and B. subtilis LB22 (1 × 109 CFU/mL) (Tables S1 and S2). B. velezensis AB22, with a lower LC50 value of 0.28 × 105, was found superior to the other two Bacillus isolates, B. amyloliquefaciens BAC1 and B. subtilis LB22, recording LC50 values of 1.06 × 105 and 5.12 × 107, respectively, after 96 HAT. Further, histopathological studies of the adults and eggs of O. coffeae treated with the most effective strain, i.e., B. velezensis, showed complete paralysis of adult mites with distorted abdomen and folded legs at 96 h after treatment when observed under compound and Scanning Electron Microscope. Additionally, the treated eggs of O. coffeae showed disfiguration, flattened on one side, and remained unhatched at 14 days after treatment. This may be attributed to the pesticidal compounds, namely, microbial lipopeptides and extracellular cuticle-degrading enzymes, including chitinases and proteases documented in Bacillus species [31]. The cuticle-degrading hydrolytic enzymes can effectively degrade the chitin and protein components in the cuticle, which impairs the exoskeleton’s essential function in the life of pests, leading to lethal effects and significantly decreasing the ecological fitness of the surviving insects [31].
The pesticidal efficacy of the genus Bacillus has been documented in some previous studies, showing bioefficacy against various insect pests. Choi et al. (2023) [27] tested the crude enzymes and bacterial broth culture of Bacillus velezensis CE 100 against gall midge larvae and found that both treatments caused mortality rates of up to 85.5% and 96.7%, respectively. B. amyloliquefaciens strains QST2808, FZB25, D747, and QST 2808 were found to exhibit high mortality of adults and eggs of spider mites in pepper [29] and aphicidal effect against bean aphids via treatment with a suspension of bacterial cells (CS), cell-free supernatant (CFS), and isolated lipopeptide fraction (LF) of strains CBMDDrag3, PGPBacCA2, and CBMDLO3 on artificial diet [30] were reported. Al-Azzazy et al. (2020) [32] also tested the effectiveness of Bacillus subtilis (2.470 × 108 CFU/mL) and Bacillus qassimus (3.320 × 108 CFU/mL) against Tetranychus urticae, infesting eggplant, under laboratory conditions and found that seven days post-treatment with B. subtilis and B. qassimus caused 72.22 and 70.74% reductions in mite populations, respectively. Similarly, at 96 h after exposure, a Bacillus thuringiensis strain was reported to have median lethal doses (LC50) against larvae of Helicoverpa armigera ranging from 1.7 to 1.8 × 105 CFU/mL [33].
Besides the well studied cuticle degrading enzymes and proteins, some more pesticidal secondary metabolites may also contribute in the acaricidal efficacy of the Bacillus spp. Our study through LC-MS profiling of putative pesticidal compounds of the three Bacillus isolates identified a total number of nine (9) metabolites in Bacillus velezensis AB22, six (6) in Bacillus amyloliquifaciens BAC1 and eight (8) in Bacillus subtilis LB22. Four proven insecticidal metabolites, Brevianamide A [34], Heptadecanoic acid [31], Thiolutin [35], and Versimide [36], were also observed in all three studied Bacillus spp. Two identified secondary metabolites, sterigmatocystin and milbemycins D reported from B. subtilis and B. amyloliquefaciens, respectively, have proven acaricidal properties. Mishima (1983) [37] reported high potency of milbemycins D against a variety of mites supporting our finding, while sterigmatocystin is reported to be highly toxic to mold mites, Tyrophagus putrescentiae [38]. From all the experiments carried out in our study, the isolate Bacillus velezensis AB22 was found to be superior to the other two Bacillus species. This may be due to the presence of identified pesticidal compounds compared to the other two isolates or due to the secretion of Zwittermicin, which has been widely reported antibacterial and antifungal compound [39] and is also known for its insecticidal action as well-known entomopathogenic bacteria. Zwittermicin A enhancing the activity of endotoxin from B. thuringiensis [40,41] is reported to increase the mortality of third instar gypsy moth, Lymantria dispar (L) treatment with B. thuringiensis [42]. Hence, the present investigation established the miticidal (adulticidal and ovicidal) property of all three Bacillus isolates with varying efficacy, reporting such findings for the first time against O. coffeae of tea to the best of our knowledge.
The present bioassay, coupled with the characterization of key pesticidal compounds, clearly demonstrated the untapped acaricidal and entomopathogenic potential of Bacillus spp. However, it further calls for a much greater depth of study on decoding molecular and biochemical principles of tripartite interaction involving bioagent tea as host plant mite pest followed by isolation of key functional metabolites for robust field management of O. coffeae. These studies also put forth an additional possibility of biocloning secondary metabolites as synthetic microbes.

5. Conclusions

In our work, we unraveled the miticidal potential of rhizospheric Bacillus spp. (B. amyloliquiefaciens, B. subtilis, and B. velezensis) against O. coffeae of tea, with the highest efficacy of B. velezensis, and the putative metabolites employed by all the bacterial strains against the pest. Light microscopy coupled with SEM study further revealed the morphological deformities due to the most efficient strain B. velezensis BAC1 in adults and eggs of O. coffeae. The study, with the initial clue on the pesticidal efficacy of Bacillus spp., can open up a new frontier in harnessing these PGP bacterial species (unpublished data) also as a biocontrol option against mite pests. However, further investigation is required for in planta acaridical efficacy of the strains under field conditions in O. coffeae and in other pests as well. Furthermore, purification and screening of individual pesticidal compounds can be a new-generation technology alternative to chemical acaricides in tea.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11112691/s1.

Author Contributions

Conceptualization, P.B.; Methodology, S.G. and N.S.; Software, A.D.; Formal analysis, S.G., S.M.B. and S.S.A.; Investigation, M.V.D., M.P. and N.T.; Resources, S.G. and P.B.; Data curation, N.T., S.R.B., N.A. and P.G.; Writing—original draft, P.B.; Writing—review & editing, P.B. and B.K.S.; Visualization, R.P.B., B.S. and S.R.B.; Supervision, P.B.; Funding acquisition, R.P.B., and B.K.S. All authors have read and agreed to the published version of the manuscript.

Funding

The work was funded by Department of Biotechnology, Government of India, Grant number: BT/PR45283/NER/95/1919/2022 (Research Project “Development of sustainable agriculture practices for biotic and abiotic stress management in conventional and organic tea plantations”).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare that there is no conflict of interest.

Abbreviations

RSM: Red Spider Mite; SEM: Scanning Electron Microscopy; CFU: Colony Forming Units; LC-MS: Liquid Chromatography-Mass Spectrometry; ECI/LC-MS: Electrospray Ionization/Liquid Chromatography-Mass Spectrometry; NB: Nutrient Broth.

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Microorganisms 11 02691 g001
Figure 1. Effect of different Bacillus spp. at 1 × 109 CFU/mL on O. coffeae adults at 24-hourly interval for 96 h of treatment. T1: Bacillus velezensis AB22; T2: B. amyloliquefaciens BAC1; T3: B. subtilis LB22; T4: Control. Different lower-case letters (a, b, and c) show significant differences (p = 0.05) between treatments.
Figure 1. Effect of different Bacillus spp. at 1 × 109 CFU/mL on O. coffeae adults at 24-hourly interval for 96 h of treatment. T1: Bacillus velezensis AB22; T2: B. amyloliquefaciens BAC1; T3: B. subtilis LB22; T4: Control. Different lower-case letters (a, b, and c) show significant differences (p = 0.05) between treatments.
Microorganisms 11 02691 g001
Microorganisms 11 02691 g002
Figure 2. Ovicidal effect of Bacillus spp. at three different population doses (1 × 107–1 × 109 CFU/mL) against O. coffeae during 14 days of treatments. T1: Bacillus velezensis AB22; T2: B. amyloliquefaciens Bac1; T3: B. subtilis LB22; T4: Control. Different lower-case letters (a, b, and c) show significant differences (p = 0.05) between treatments.
Figure 2. Ovicidal effect of Bacillus spp. at three different population doses (1 × 107–1 × 109 CFU/mL) against O. coffeae during 14 days of treatments. T1: Bacillus velezensis AB22; T2: B. amyloliquefaciens Bac1; T3: B. subtilis LB22; T4: Control. Different lower-case letters (a, b, and c) show significant differences (p = 0.05) between treatments.
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Microorganisms 11 02691 g003
Figure 3. Photographs showing adulticidal and ovicidal activity of Bacillus velezensis AB22 against O. coffeae under 40×. (A) Adult mite in control (96 HAT); (B) Distorted adult mite (96 HAT); (C) Eggs of mite in control (14 DAS); (D) Damaged mite eggs (14 DAS).
Figure 3. Photographs showing adulticidal and ovicidal activity of Bacillus velezensis AB22 against O. coffeae under 40×. (A) Adult mite in control (96 HAT); (B) Distorted adult mite (96 HAT); (C) Eggs of mite in control (14 DAS); (D) Damaged mite eggs (14 DAS).
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Microorganisms 11 02691 g004
Figure 4. Morphological changes in O. coffeae in response to treatment with Bacillus velezensis AB22 under Scanning Electron Microscope. (A) Morphological distortion in treated adults; (B) Morphological distortion in treated eggs.
Figure 4. Morphological changes in O. coffeae in response to treatment with Bacillus velezensis AB22 under Scanning Electron Microscope. (A) Morphological distortion in treated adults; (B) Morphological distortion in treated eggs.
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Microorganisms 11 02691 g005
Figure 5. Detection of insecticidal metabolites by LC-MS from methanol crude extract of Bacillus velezensis AB22 (ON209629).
Figure 5. Detection of insecticidal metabolites by LC-MS from methanol crude extract of Bacillus velezensis AB22 (ON209629).
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Microorganisms 11 02691 g006
Figure 6. Detection of insecticidal metabolites by LC-MS from methanol crude extract of Bacillus amyloliquifaciens BAC1 (ON392425).
Figure 6. Detection of insecticidal metabolites by LC-MS from methanol crude extract of Bacillus amyloliquifaciens BAC1 (ON392425).
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Microorganisms 11 02691 g007
Figure 7. Detection of insecticidal metabolites by LC-MS from methanol crude extract of Bacillus subtilis LB22 (ON386193).
Figure 7. Detection of insecticidal metabolites by LC-MS from methanol crude extract of Bacillus subtilis LB22 (ON386193).
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Table 1. LC50 of Bacillus isolates against adults of O. coffeae using different concentrations (1 × 105 to 1 × 109 CFU/mL) after 96 h of treatment.
Table 1. LC50 of Bacillus isolates against adults of O. coffeae using different concentrations (1 × 105 to 1 × 109 CFU/mL) after 96 h of treatment.
TreatmentConc.
(CFU/mL)		Adult Mortality (%)
after 96 h		Log of Conc.Probit MortalityRegression Statistics
(a = Slope, b = Intercept)		Regression Equation
Y = aX + b		LC50 (Y = 5)
LC50 = antilogX		LC50 Value
T1: Bacillus velezensis AB221 × 10570.00 (56.79)55.18a = 0.262;
b =3.832 		Y = 0.262x + 3.83228,708.797370.28 × 105
1 × 10681.67 (64.60)65.39
1 × 10793.33 (75.00)75.67
1 × 10896.67 (79.37)85.81
1 × 109100.00 (90.00)96.28
T2: B. amyloliquefaciens BAC11 × 10563.33 (52.71)55.08a = 0.273;
b = 3.643 		Y = 0.273x + 3.643106,151.44251.06 × 105
1 × 10676.67 (61.07)65.28
1 × 10788.33 (70.00)75.52
1 × 10891.67 (73.15)85.61
1 × 109100.00 (90.00)96.28
T3: B. subtilis LB221 × 10561.67 (51.71)55.05a = 0.161;
b = 4.229		Y = 0.161x + 4.22951,286,138.45.12 × 107
1 × 10668.33 (55.73)65.15
1 × 10781.67 (64.60)75.39
1 × 10888.33 (70.00)85.52
1 × 10993.33 (75.00)95.67
Y = Probit kill; X = Log concentration.
Table 2. LC50 of the Bacillus spp. most effective against eggs of O. coffeae using different concentrations (1 × 105 to 1 × 109 CFU/mL) after 14 days of treatment.
Table 2. LC50 of the Bacillus spp. most effective against eggs of O. coffeae using different concentrations (1 × 105 to 1 × 109 CFU/mL) after 14 days of treatment.
TreatmentConc.
(CFU/mL)		Ovicidal Activity (%)
after 14 Days		Log of Conc.Probit MortalityRegression Statistics
(a = Slope,
b = Intercept)		Regression Equation
Y = aX + b		LC50 (Y = 5)
LC50 = antilogX		LC50 Value
T1: Bacillus velezensis AB221 × 10573.33 (58.89)55.61a = 0.238;
b =3.936		Y= 0.238x + 3.93629,552.090.29 × 105
1 × 10681.11 (64.23)65.88
1 × 10787.22 (69.06)76.13
1 × 10892.78 (74.41)86.48
1 × 109100.00 (90.00)98.95
T2: B. amyloliquefaciens BAC11 × 10563.89 (53.01)55.36a = 0.130;
b = 4.400		Y = 0.130x + 4.4041,246.260.41 × 105
1 × 10676.67 (61.07)65.74
1 × 10784.44 (66.77)75.99
1 × 10887.22 (69.06)86.13
1 × 10992.22 (73.81)96.41
T3: B. subtilis LB221 × 10561.11 (51.41)55.28a = 0.110;
b = 4.490		Y = 0.110x + 4.4943,287.610.43 × 105
1 × 10671.67 (57.80)65.58
1 × 10777.78 (61.87)75.77
1 × 10881.67 (64.65)85.92
1 × 10986.67 (68.58)96.13
Y = Probit kill; X = Log concentration.
Table 3. Details of secondary metabolites (based on ECI/LC-MS) produced by Bacillus velezensis AB22 (ON209629).
Table 3. Details of secondary metabolites (based on ECI/LC-MS) produced by Bacillus velezensis AB22 (ON209629).
Sl. No.Compound NameFormulaMatch ScoreRetention Time (RT)Predicted
m/z		Matched m/z
1.Brevianamide AC21H23N3O30.96127.27404.1371403.8991
2.CitromycinC13H10O50.90910.18285.0160284.8307
3.EmodinC15H10O50.91228.51303.0863302.8130
4.Heptadecanoic acidC17H34O20.95328.36303.2894302.9980
5.PaxillinesC27H33NO40.87212.46458.2302458.0682
6.PeramineC12H17N5O0.90510.18285.1202284.8307
7.ThiolutinC8H8N2O2S20.9279.71246.0365245.7579
8.VersimideC9H11NO40.92618.91220.0580219.8944
9.Zwittermicin AC13H28N6O80.85631.86414.2307413.9633
Table 4. Details of secondary metabolites (based on ECI/LC-MS) produced by Bacillus amyloliquifaciens BAC1 (ON392425).
Table 4. Details of secondary metabolites (based on ECI/LC-MS) produced by Bacillus amyloliquifaciens BAC1 (ON392425).
Sl. No.Compound NameFormulaMatch ScoreRetention Time (RT)Predicted
m/z		Matched m/z
1.Brevianamides AC21H23N3O30.93327.24404.1371404.0471
2.Heptadecanoic acidC17H34O20.90428.23303.2894302.9980
3.Milbemycins DC33H48O70.88825.11574.3738574.1025
4.Tenuazonic acidC8H16O20.95627.51485.9953486.2257
5.ThiolutinC8H8N2O2S20.9709.01246.0365245.7579
6.VersimideC9H11NO40.92318.98220.0580219.7834
Table 5. Details of secondary metabolites (based on ECI/LC-MS) produced by Bacillus subtilis LB22 (ON386193).
Table 5. Details of secondary metabolites (based on ECI/LC-MS) produced by Bacillus subtilis LB22 (ON386193).
Sl. No.Compound NameFormulaMatch ScoreRetention Time (RT)Predicted
m/z		Matched m/z
1.AnhydrofusarubinC15H12O60.95527.14306.0972305.9951
2.Brevianamide AC21H23N3O30.96127.27404.1371403.8991
3.EmodinC15H10O50.91228.51303.0863302.8130
4.Heptadecanoic acidC17H34O20.90628.33303.2894303.0721
5.NikkomycinsC20H25N5O100.92523.99518.1494518.3794
6.SterigmatocystinC18H12O60.87829.35342.0972341.8858
7.ThiolutinC8H8N2O2S20.9829.73246.0365245.7949
8.VersimideC9H11NO40.92618.91220.0580219.8944
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Bora, P.; Gogoi, S.; Deshpande, M.V.; Garg, P.; Bhuyan, R.P.; Altaf, N.; Saha, N.; Borah, S.M.; Phukon, M.; Tanti, N.; et al. Rhizospheric Bacillus spp. Exhibit Miticidal Efficacy against Oligonychus coffeae (Acari: Tetranychidae) of Tea. Microorganisms 2023, 11, 2691. https://doi.org/10.3390/microorganisms11112691

AMA Style

Bora P, Gogoi S, Deshpande MV, Garg P, Bhuyan RP, Altaf N, Saha N, Borah SM, Phukon M, Tanti N, et al. Rhizospheric Bacillus spp. Exhibit Miticidal Efficacy against Oligonychus coffeae (Acari: Tetranychidae) of Tea. Microorganisms. 2023; 11(11):2691. https://doi.org/10.3390/microorganisms11112691

Chicago/Turabian Style

Bora, Popy, Sukanya Gogoi, Mukund Vinayak Deshpande, Pankaj Garg, Rana P. Bhuyan, Nilofar Altaf, Nikita Saha, Sapna Mayuri Borah, Mousumi Phukon, Nabajit Tanti, and et al. 2023. "Rhizospheric Bacillus spp. Exhibit Miticidal Efficacy against Oligonychus coffeae (Acari: Tetranychidae) of Tea" Microorganisms 11, no. 11: 2691. https://doi.org/10.3390/microorganisms11112691

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