Symbiotic and Antagonistic Functions of the Bacterium Burkholderia cepacia BsNLG8, from the Nilaparvata lugens (Stal)

Abstract

Bacterial symbionts are widespread in insects and other animals. These microbes play crucial roles in many aspects of insect physiology and biology, including immunity, nutrition and confronting plant defenses. In the present study, we isolated and identified the bacterium Burkholderia cepacia BsNLG8 from the brown planthopper (BPHs), Nilaparvata lugens, a devastating pest of rice crops worldwide. Plate confrontation assay indicated that BsNLG8 significantly inhibited the growth of phytopathogenic fungi. In addition, the BsNLG8 strain demonstrated the ability to produce siderophores, which explains its antagonistic mechanism. Lastly, we explored the nicotine degradation ability of BsNLG8 using in vitro and in vivo methods. In vitro, HPLC analysis results showed that BsNLG8 could significantly reduce the concentration of nicotine in the medium at 36 h. Moreover, microinjection of BsNLG8 in axenic BPHs increased the survival rate of the host on nicotine-containing rice seedlings. These findings could serve as the basis of future research in deciphering the interaction between host and symbionts.

1. Introduction

The brown planthopper (BPHs), Nilaparvata lugens (Stal) (Hemiptera: Delphacidae), is one of the most destructive insect pests of rice crops. Notorious for frequent outbreaks, the BPH is a typical migratory monophagous sucking pest that primarily feeds on rice plants and causes severe wilting and drying of plants, a condition known as “hopperburn” [1,2]. Due to its infestation, the estimated annual losses in food production have exceeded one billion US dollars [3]. Currently, chemical control remains the first choice for managing BPHs infestation in Asia. Neonicotinoid insecticides with high efficacy and lasting effects are widely used to control BPHs. However, the widespread use of neonicotinoids has given rise to BPHs resurgence and additional environmental risks [4,5,6,7]. According to previous studies, the resistance of BPHs to nicotinic pesticides is mainly attributed to the enhanced activity of cytochrome P450 detoxification enzymes [8,9,10]; moreover, the symbiotic bacteria of the BPHs can affect the host’s sensitivity to heterologous substances by regulating the expression of host detoxification enzyme genes [2,11,12]. As an analog of neonicotinoids, the research progress on the microbial degradation of nicotine is of great reference value for exploring the mechanisms and diversity of neonicotinoids. Therefore, exploring the role of commensal bacteria in the mechanism of host resistance to nicotine will help us better understand the mechanism of resistance formation in insects and, thus, better control pests.

Microbial symbiosis is a widespread phenomenon in insects, and most insect species harbor symbiotic microorganisms in their body cavity, gut and/or cells [1,2,13,14,15]. Insects have established mutualistic relationships with symbiotic bacteria that are beneficial to both [2]. Symbiotic bacteria are functionally diverse and participate in various important biological activities in their insect host [16]. They help the host degrade toxic substances and improve the host’s insecticide resistance and defense ability. In termites, the intestinal symbiotic bacteria can produce a variety of glucanases to assist in the degradation of cellulose [16,17]. Kikuchi discovered that the intestinal symbiotic bacteria Burkholderia in Riptortus pedestris can improve its tolerance to fenitrothion [18]. Similarly, Arsenophnus in BPHs can affect the resistance of hosts to imidacloprid [11]. The symbiotic bacterium Wolbachia could enhance the expression of the P450 gene NlCYP4CE1 in the host under imidacloprid stress [12].

The long-term selection and evolution of bacterial symbionts can assist the hosts in transforming secondary toxic compounds into non-toxic ones by mineralization and metabolism, thus reducing the damage of plant metabolites to the host [2]. Pseudomonas fulva isolated from the intestinal tract of Hypothenemus hampei can help the host to degrade caffeine in coffee fruit [19]. Acinetobacter in Lymantria dispar can metabolize toxic phenolic glycosides to promote growth [20]. It has been observed that insect symbionts can play different functions under various environmental conditions. Therefore, studying the functions of insect symbionts is of great significance for exploring the host’s insecticide resistance mechanism and coordinating agricultural and chemical control.

Studies on the diversity of symbiotic bacteria in BPHs have been reported. Yu found that Wolbachia plays a key role in adapting BPHs to Mudgo-resistant rice [21]. Acinetobacter isolated from the intestinal tract of BPHs fed on rice seedlings treated with fungicides demonstrated its role in the resistance of BPHs to fipronil [22]. In addition, it was found that Wolbachia in the fat body of BPHs could produce vitamins B2 and B7, which directly increased the host’s fecundity [23]. These studies prove that the functional diversity of symbiotic bacteria is of great significance for the normal physiological activities of the host.

The Burkholderia cepacia complex (BCC) is a group of closely related bacterial species. Widespread in the environment, BCC can be isolated from soil, water, or plant surfaces [24]. It has been reported that B. cepacia has multiple functions, such as biological control, pesticide degradation and plant growth promotion. Researchers have isolated BCC from rice and found that it can significantly inhibit the growth of Rhizoctonia solani and Fusarium moniliforme in vitro, exhibiting its ability as a potential biocontrol agent [24,25,26]. A strain of B. cepacia Lyc2 from tobacco root soil promoted the growth of cotton seedlings and inhibited the mycelium growth of many plant pathogenic fungi. The efficacy of this bacteria for controlling cotton seedling blight reached 48.8%, stressing its ability to counter fungal diseases in plants [27]. B. cepacia CF-66 isolated from compost showed antifungal activity against plant pathogens such as R. solani and some other fungi [28]. Li indicated that Burkholderia sp. R456 not only suppressed the in vitro mycelial growth of R. solani, but also reduced the incidence and severity of rice sheath blight under greenhouse conditions [29]. Similarly, B. cepacia JBK9 displayed antagonistic activity against phytopathogenic fungi, including Phytophthora capsici, Fusarium oxysporum, and R. solani, and significantly inhibited their growth [30]. However, the function of B. cepacia as an insect symbiont is yet to be explored.

In this study, the B. cepacia BsNLG8 was isolated from the intestines of adult BPHs and subsequently identified and functionally explored. These findings shed light on the various biological functions of BsNLG8 in the host and offer theoretical insight for uncovering the interaction between bacteria and insecticide resistance. At the same time, our results also provide a theoretical basis for the use of BPHs’ symbiotic bacteria to change the development of host resistance.

2. Materials and Methods

2.1. Insects and Microorganisms

The BPHs used in this experiment were collected from the experimental rice farm of South China Agricultural University, Guangzhou, Guangdong, China. The insects were reared in an artificial climate box under controlled conditions: 27 ± 1 °C, relative humidity 75 ± 5%, and photoperiod is 14 L:10 D.

Magnaporthe oryzae, F. oxysporum, Colletotrichum higginsianum Sacc. (C.higginsianum), Botryosphaeria dothidea, Geotrichum candidum and Stagonospora curtisii were provided by the Dr. Shujie Feng, School of Horticulture, South China Agricultural University. These fungal cultures were kept at a constant temperature of 25 ± 1 °C and 75 ± 5% humidity with the PDA (Potato Dextrose Agar) medium.

The intestine tracts of adult BPHs were dissected under a microscope and ground into homogenates, then coated with LB (Luria-Bertani), NA (Nutrient Agar) and EB (Enrichment Broth) selective separation medium, and cultured in a constant temperature incubator at 37 °C. After the single colonies grew in the culture medium, the single colonies were selected and purified continuously on the corresponding culture medium for more than 5 times. After purification, the single colonies were transferred to LB liquid medium. When the bacteria were cultured in the exponential growth phase, they were stored in 25% glycerol aqueous solution and frozen in the refrigerator at −80 °C for later use.

The preparation method of BsNLG8 seed bacteria solution is as follows: the glycerobacteria stored at −80 °C were inserted into the fresh liquid LB medium at the volume ratio of 1:100, and the seed bacteria solution was obtained by activating it at 220 r/min at 37 °C for 12 h. Later the seed bacteria solution was added to the fresh liquid LB medium at the volume ratio of 1:100, and the bacterial liquid with strong activity was obtained by shaking at 37 °C for 4 h. Finally, the concentration of bacteria solution was adjusted to 1 × 10⁷ cfu/mL [31].

2.2. Identification of BsNLG8

To identify BsNLG8, we employed the Gram staining method described previously [32,33]. The isolated bacteria were stained with Gram-negative and observed under the transmission electron microscope (Thermo Fisher Talos F200S, Waltham, MA, USA) following the method described by Annelies Haegeman [34]. The strains were cultured on nutrient agar medium for two days, and colony morphology was observed. For 16s rDNA sequence analysis, the genomic DNA of the preserved monoclonal strain was extracted by TIANamp Bacteria DNA Kit (Tiangen Biotech Co., Beijing, China), and the extracted DNA was used as the template. The 16s rDNA of bacteria was amplified using universal primers: 27F and 1492R (Table S1). The PCR products were detected, purified and sequenced. The sequence was compared to the GenBank database, and the phylogenetic tree was constructed by Mega 7.0 software to determine its phylogenetic status.

2.3. Mycostatic Ability Test

The mycostatic ability of BsNLG8 against plant pathogenic fungi was determined by the plate confrontation method [35]. The activated plant pathogens were inoculated in the center of PDA culture medium, and BsNLG8 was inoculated at the symmetry of 2.5 cm away from the fungus cake. The plate inoculated with pathogens was used as the control and was placed (inverted) at temperature of 25 ± 1 °C and humidity of 75 ± 5%. According to the growth cycle of pathogens, the colony diameters in the treatment and control groups were measured, and the mycostatic effect of BsNLG8 was evaluated.

The activity of volatile substances was tested by the plate pair buckle method [36]. The pathogenic fungi were inoculated in the center of the PDA plate, BsNLG8 was inoculated in nutrient agar medium simultaneously, and then two dishes were buckled together. The plate inoculated with pathogens was placed above, the plate inoculated with BsNLG8 was placed below, and the plate without BsNLG8 was set as the control. After being cultured at temperature of 25 ± 1 °C and humidity of 75 ± 5%, the growth of pathogens in the control and treatment groups was observed.

The antagonism effect of the sterile filtrate of the bacteria BsNLG8 against the fungi was further determined by following the methods of Wang and Sharifah [37,38] with slight modifications. The bacterial solution of BsNLG8 was centrifuged at 8000 r/min for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane to remove bacteria. The growth rate method was used to prepare the medium according to the ratio of filtrate: medium at 1:5, 1:10 and 1:20, and the plate with the same amount of LB liquid medium instead of the filtrate was used as control. The plant pathogenic bacteria cake with a diameter of 0.5 cm was inoculated on the plate. Each experiment was replicated thrice. The medium was cultured at a temperature of 25 ± 1 °C and humidity of 75 ± 5%. Subsequently, the fungi in the control group attained their maximum growth, and the colony diameters of the treatment and control were measured to compare the mycostatic effect.

2.4. Assessment of Siderophore Production

To assess the siderophore production, we followed the Schwyn and Neilands method [39] which utilizes chrome azurol sulphonate (CAS), hexadecyltrimethylammonium bromide (HDTMA), and FeCl₃·6H₂O from the CAS detection medium. BsNLG8 was inoculated in LB liquid medium and cultured at 180 r/min, 30 °C for 12 h. The 5 μL bacterial liquid was taken into the CAS detection plate, and the plate with only LB liquid medium was used as the control. All bacteria were cultured at a constant temperature of 30 °C for 72 h; the color changes around the colonies were observed, and each experiment was replicated three times.

2.5. Test of Nicotine Degradation by BsNLG8

50 μL BsNLG8 bacteria were added to the fresh medium, and bacteria with strong activity were obtained after activation at 220 r/min at 37 °C for 12 h. The bacteria were collected by centrifugation at 8000 r/min for 5 min, and the degraded seed bacteria were obtained by heavy suspension of sterilized MSM (mineral salt medium). 5% BsNLG8 bacteria were inoculated in 1 g/L nicotine medium and then cultured at 30 °C and 180 r/min. Samples were collected after 6, 12, 24, 36, 48, and 72 h time points. Two samples were collected at 4 °C, 12,000 r/min, and centrifuged for 10 min. The supernatant was removed, and the aseptic phosphate buffer saline (PBS) solution of 2 mL was precipitated and re-suspended. The 200 μL sample was taken to determine the growth of the cell and the absorbance of the culture medium at OD = 595 nm wavelength.

BsNLG8 was inoculated into the nicotine medium of 1 g/L at a proportion of 5%. The nicotine medium without bacteria was used as a blank control and then cultured under similar conditions as mentioned above. The culture medium was absorbed at 6, 12, 24, 36, 48, and 72 h and centrifuged for 10 min at 4 °C and 12,000 r/min. The supernatant was filtered with 0.22 μm membrane, and the bacterial culture medium without inoculation was used as blank control. The content of nicotine in the sample was determined by HPLC. Agilent 1100 liquid chromatograph was used (Agilent, Palo Alto, Santa Clara, CA, USA), the chromatographic column was AgilentTC-C18, the mobile phase was methanol and 0.02 mol disodium hydrogen phosphate buffer (pH = 4.2), the volume ratio was 10:90, the column temperature was 30 °C, the flow rate was 1 mL/min, the injection volume was 10 μL, and the detection wavelength was 259 nm. The changes of nicotine characteristic absorption peak in 259 nm were recorded.

2.6. Effect of BsNLG8 on Nicotine Sensitivity of BPHs

We used K-B method to determine the sensitivity of brown planthopper symbiotic bacteria to mixed antibiotics [40], and then determined the inhibitory effect of antibiotics on BPHs for the subsequent in vivo symbiotic bacteria removal experiment. Subsequent experiments were performed following the protocols of Wari, with some modifications [41]. Rice treated with 300 mL of mixed antibiotic solution (200 mg/L tetracycline and 100 mg/L rifampicin) was fed to 300 BPHs to eliminate commensal bacteria, and rice treated with sterile water was fed to control BPHs. The nymphs of BPH were allowed to feed on treated (mixed antibiotic) and untreated (aseptic water) rice seedlings for 7 days. On the 7th day, we randomly collected the BPHs from the treatment (antibiotic treatment) and the control (aseptic water) to detect whether symbiotic bacteria were removed. The nymphs were used to carry out the follow-up BsNLG8 supplement experiment.

Ten BPHs were randomly selected from the treatment and the control group, and the total RNA was extracted by using Total RNA Kit (Feijie, China). The HiScript III RT Super Mix synthesized the first-strand cDNA for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). Real-Time Quantitative PCR (qRT-PCR) was used to detect the relative content of the symbiotic bacteria. The qRT-PCR was performed on the CFX-96 real-time PCR system (Bio-Rad, Hercules, CA, USA) with the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The qRT-PCR cycling conditions were as follows: initial denaturation at 95 °C for 30 s followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The content of symbiotic bacteria of BPHs was determined by universal quantitative primers 1114F and 1275R of bacterial 16S rDNA (Table S1) [42], and guanine-N(7)-methyltransferase gene (Nl18S) gene was used as an internal reference. The relative symbiotic content of BPHs was calculated by 2^−ΔΔCT method [43].

The bacteria were centrifuged at 8000 r/min for 3 min, the supernatant was removed, and the same volume of aseptic PBS was added. An ultra-micro ultraviolet spectrophotometer determined the OD value of bacteria, and the concentration of bacteria was adjusted to 1 × 10⁶ cfu/mL. We used a microinjection technique to inject BsNLG8 into the BPHs [44]. Nymphs were placed on an agarose plate for injecting 0.05 μL of bacteria. Injections were made between the coxa of the second and third pair of feet of each test worm using a microinjection apparatus [45]. A similar volume of PBS was injected into the control. After injection, BPHs were moved to the rice seedling and later shifted in the artificial climate box. The survival of BPHs was observed 24 h after injection. After 24 h of injection of BsNLG8 solution, 10 surviving BPHs were collected, and the total RNA was extracted. The content of BsNLG8 in BPHs was analyzed by qRT-PCR. The primers used in the experiment are listed in Table S1. The surviving BPHs were transferred to rice seedlings soaked in nicotine solution with a concentration of 10 mg/L, and the mortality was recorded every 24 h, and the experiment was replicated three times.

2.7. Statistical Analysis

ClustalW software for multi-sequence alignment was used, and Mega7.0 software was used to construct the phylogenetic tree by the neighbor-joining method, adjust the bootstrap value and test the reliability of the evolutionary tree.

Graphpad Prism 8.0 software was used to analyze the data. One-way analysis of variance (ANOVA) was employed to test the significance of the difference in our experiment. The data with p values equal to or lower than 0.05 were considered statistically significant. “*” and “**” showed significant differences at p < 0.05 and p < 0.01, and “ns” represents non-significant differences.

3. Results

3.1. Identification of Bacteria

The BsNLG8 is a gram-negative rod-shaped bacterium. The colony is round, smooth, neatly edged and yellow in color (Figure S1). Observation of BsNLG8 by transmission electron microscope showed that the internal structure of the bacterium was clearly visible, and it was a rod-shaped bacterium with flagella (Figure 1). The identification results of the physiological and biochemical functions of BsNLG8 are shown in

Table 1. Physiological and biochemical characteristics of BsNLG8.

No.	Reactant	Result	No.	Reactant	Result
1	catalase	+	11	Urease test	-
2	oxidase	+	12	Qiyeling	+
3	Anaerobic growth	-	13	Growth at 37 °C	+
4	Nitrate reduction	-	14	Growth at 42 °C	+
5	Starch hydrolysis	-	15	Lysine decarboxylase	+
6	D-glucose fermentation	+	16	Ornithine decarboxylase	-
7	maltose fermentation	+	17	arginine dihydrolase	-
8	lactose fermentation	+	18	β- Galactosidase test	+
9	D-xylose fermentation	-	19	Gelatin liquefaction	+
10	Sucrose fermentation	-

“+” denotes positive, and “-” denotes negative.

. BsNLG8 can grow at 37 °C and 42 °C. It can react with catalase and oxidase. BsNLG8 was positive in D-glucose, maltose and lactose fermentation, but it could not decompose sucrose and urease. BsNLG8 could liquefy gelatin but was unable to degrade ornithine decarboxylase and arginine dihydrolase.

The 16S rDNA nucleotide sequence of this bacteria was compared with the rRNA/ITS database in NCBI (https://www.ncbi.nlm.nih.gov accessed on 10 September 2022). B. cepacia BsNLG8 was the most similar to Burkholderia contaminans strain J2956 (99.72%) (NR_104978.1) and Burkholderia puraquae strain CAMPA 1040 (99.79%) (NR_159299.1). (Figure S2). Based on a comprehensive analysis of biological, physiological and biochemical identification, we concluded that the bacteria belong to B. cepacia, and the specific taxonomic status is Burkholderia puraquae. We named it BPHs B. cepacia BsNLG8 (MZ749716).

3.2. Inhibitory Effect of BsNLG8 on Fungi

3.2.1. Inhibitory Effect of Bacteria on Fungal Growth

The results of antagonistic culture between BsNLG8 and six pathogenic fungi are shown in Figure 2 and

Table 2. Inhibition rate of BsNLG8 against phytopathogenic fungi.

Pathogenic Fungi	Cultivation Time (d)	Colony Diameter (mm)		p Value	Inhibition Rate %
		Control	Treatment
M. oryzae	7	853.33 ± 8.66	135.56 ± 8.82	p < 0.001	84.11
F. oxysporum	6	854.44 ± 7.26	167.78 ± 9.72	p < 0.001	80.36
C. higginsianum	7	627.78 ± 9.72	110.00 ± 6.67	p < 0.001	82.48
S. curtisii	7	722.22 ± 6.67	144.44 ± 8.82	p < 0.001	80.00
G. candidum	7	834.44 ± 11.30	127.78 ± 8.33	p < 0.001	84.69
B. dothidea	10	858.89 ± 3.33	171.11 ± 6.01	p < 0.001	80.08

Inhibition rate % $=(\frac{\mathrm{Diameter} \mathrm{of} \mathrm{pathogen} \mathrm{colonies} \mathrm{in} \mathrm{the} \mathrm{control}-\mathrm{Diameter} \mathrm{of} \mathrm{pathogen} \mathrm{colonies} \mathrm{in} \mathrm{the} \mathrm{treatment}}{\mathrm{Diameter} \mathrm{of} \mathrm{pathogen} \mathrm{colonies} \mathrm{in} \mathrm{the} \mathrm{treatment}}$) × 100%.

. BsNLG8 strain significantly inhibited M. oryzae, F. oxysporum, C. higginsianum, B. dothidea, G. candidum and S. curtisii (Figure 2). Furthermore, the inhibition rate of BsNLG8 strain was tested on each fungus (Table 2). The inhibition rate of BsNLG8 on pathogenic fungi reached more than 80%, which indicated that this bacterium had an excellent inhibitory effect on plant pathogenic fungi.

3.2.2. Mycostatic Effect of BsNLG8 Volatiles

The plates inoculated with pathogenic fungi were cultured in pairs with the plates inoculated with BsNLG8. After 7 days of inoculation, the results showed that the volatile compounds of BsNLG8 could significantly inhibit the growth of M. oryzae, C. higginsianum, B. dothidea, G. candidum and S. curtisii, in comparison to control (Figure 3). Additionally, the colony diameter was measured for comparative analysis. The results showed that the colony diameter of pathogenic fungi in the treatment was significantly smaller than that in control (Figure 4). These findings indicated that the volatile compounds from BsNLG8 could significantly inhibit the normal growth of plant pathogenic fungi.

3.2.3. Mycostatic Effect of BsNLG8 Sterile Solution

The pathogenic fungi were inoculated into the medium containing BsNLG8 aseptic filtrate in different proportions. The culture results showed that all of them could grow on the medium of BsNLG8 aseptic filtrate. The colony diameters of the F. oxysporume, C. higginsianum, B. dothidea, G. candidum and S. curtisii in the treatment group were significantly smaller than that of the control (Figure 5a–e). For example, the diameter of colonies of C. higginsianum grown in 1:20 sterile filtrate of BsNLG8 was only 434.44 ± 8.82 mm, compared with 607.78 ± 9.72 mm in control. There was no significant difference between the treatment and the control group of M. oryzae. These results suggested that the production of mycostatic substances during the growth of BsNLG8 can effectively inhibit the growth of most pathogenic fungi.

3.3. BsNLG8 Can Secrete Siderophore

To explore the mycostatic mechanism of BsNLG8, we used the CAS plate test to investigate whether it can produce siderophores. After 72 h, a clear yellow halo on the center of the CAS detection plate was observed (Figure S3). This phenomenon indicates that BsNLG8 can produce siderophores with a high affinity for iron ions [39], which takes the iron ions away, resulting in a yellow halo. Our experimental results further showed that one of the inhibitory effects of BsNLG8 on plant pathogenic fungi is due to its ability to produce siderophores, presenting its mycostatic effect on pathogenic fungi.

3.4. Role of BsNLG8 in Degrading Nicotine

According to Li’s method [46], a nicotine curve was made, and the absorbance value of different concentrations of nicotine was measured at 259 nm. Nicotine concentration determined by liquid chromatography had a good linear relationship in the range of 0.005–0.1 g/L (y = 36.498x − 0.1839, R2 = 0.994).

The changes in bacteria and nicotine after BsNLG8 inoculation into nicotine medium were detected. BsNLG8 proliferated rapidly in the first 6 h, followed by a gradual decrease in concentration until 24 h. The results showed a slight increase in bacterial concentration between 24 and 48 h, followed by a significant decrease at 72 h. In parallel, we detected nicotine concentration in the presence of BsNLG8 and observed a negative correlation. While nicotine concentration was effectively reduced at 24 h, the nicotine content in the medium was either too low to be detected by liquid chromatography or degraded entirely at 36 to 72 h (Figure 6). These results showed that under laboratory conditions, the BsNLG8 could effectively degrade nicotine in 36 h, advocating its excellent degrading ability against nicotine.

3.5. The Sensitivity of BPHs to Nicotine Is Affected by Bacterial BsNLG8

We tested the inhibition effect of mixed antibiotics on BPHs symbiotic bacteria, and the results showed that BPHs symbiotic bacteria have strong sensitivity to mixed antibiotics (Figure S4). In order to determine whether BsNLG8 can also degrade nicotine in vivo and affect the sensitivity of the host to nicotine, the BPH nymphs were fed on rice containing mixed antibiotics for 7 days, and the bacterial load was calculated. The bacterial content decreased dramatically after mixed antibiotics treatment (Figure S4 and Figure 7a), and the axenic population of BPHs were collected for further experiments.

We used a microinjection technique to inject BsNLG8 (treatment) and PBS (control) into the BPHs. The bacterial abundance was calculated 24 h post-injection. The results showed that the bacterial contents were significantly higher in the treatment than in the control group (Figure 7b). Subsequently, the treated and control populations were transferred to rice seedlings containing nicotine for survival analysis. The survival rate of BPHs fed on nicotine seedlings was lower compared to those as lower than those fed on nicotine-free rice plants. However, it is worth mentioning that the survival rate of BPHs injected with BsNLG8 was higher than that of BPHs injected with PBS (Figure 7c). Our results indicated that BsNLG8 had a positive effect on the survival of BPHs after nicotine treatment, and it may still play a critical role in nicotine degradation, thus helping the host to resist the threat of nicotine.

4. Discussion

Insect symbiotic bacteria participate in a variety of physiological activities of the host. The symbiotic bacteria in the BPHs are functionally diverse and maintain the microecological balance in the host [15,16,47]. Currently, the in vitro plate culture is the primary method for the culture and identification of intestinal microorganisms [22]. In our study, B. cepacia BsNLG8 was isolated and identified from BPHs by in vitro culture. We used the plate confrontation method and co-cultured BsNLG8 with several plant pathogenic fungi. The results showed that BsNLG8 could significantly inhibit fungal growth. In parallel, it was also found that the volatiles and metabolites of BsNLG8 had an inhibitory effect on the growth of pathogenic fungi. Similar to our findings, among the different genotypes of B. cepacia isolated by Zhang from the roots of maize and rice, 46 strains of B. cepacia showed strong mycostatic activity against pathogenic fungi, and their studies supported our results [48]. The current discovery provides a valuable experimental case for studying the function of B. cepacia. At the same time, exploring the mycostatic function of microorganisms is also conducive to a deeper understanding of the relationship between microorganisms.

Microorganisms can produce a variety of unique bioactive substances, including the secretion of siderophore, one of the primary mechanisms for microbial control of fungal diseases [49]. Siderophores play an important role in agricultural production, pest control, and other aspects of biocontrol. Studies have found that Pseudomonas secreted siderophores can effectively and persistently inhibit the occurrence of Fusarium wilt of carnation [50]. Similarly, Burkholderia pyrrocinia, JK-SH007, displayed an evident siderophore production ability [51]. In this experiment, we used the CAS plate detection technique and found that BsNLG8 can produce siderophores. The siderophores can reduce the concentration of iron in the environment and inhibit the growth of pathogenic fungi. The results of this study revealed the biocontrol mechanism of this strain against plant pathogenic fungi to some extent.

Nicotine is the major alkaloid in the tobacco plant. With large quantities of tobacco products being produced and consumed, tobacco waste, such as nicotine, is entering the environment [52]. The United States Environmental Protection Agency (EPA) listed nicotine in the list of environmentally restricted release compounds in 1994, and the European Union also has relevant laws and regulations for nicotine waste [53,54]. If these wastes are not treated, they will threaten the life and health of humans and animals [55]. Microbial degradation of environmental pollutants is not only efficient and economical but also causes little to no secondary pollution. Many studies have proved that microorganisms can absorb and degrade nicotine [55]. Neurospora crassa, an endophytic fungus isolated from flue-cured tobacco leaves, could effectively reduce (33.9%) the nicotine content [56]. Likewise, an endophytic bacterial strain Microbacterium barkeri 11L140 significantly (97.76%) degraded the nicotine when cultured with 1% nicotine medium for 54 h [57]. Our results demonstrated the BsNLG8 had an excellent nicotine degradation ability. It not only flourished in the nicotine medium continuously but also reduced the nicotine content in the culture medium. The nicotine concentration decreased significantly after 36 h, and BsNLG8 could completely degrade the nicotine. These findings confirmed that Burkholderia spp. BsNLG8 has nicotine tolerance and nicotine biodegradability capacity.

To analyze and verify the function of symbionts in the host, antibiotics or high-temperature treatments are often used. The identified symbiotic bacteria are then re-inoculated in the host to explore their functions. In Neoseiulus californicus, antibiotic and temperature treatments proved to be effective for removing bacterial microorganisms [2,58]. Similarly, a combination of three antibiotics (oxytetracycline hydrochloride, penicillin G potassium and rifampicin) was administered to Cicadella viridis, effectively reducing the bacterial load [59]. To further elucidate the function of BsNLG8 in the host, we used antibiotics to remove the bacterial load and determine the sensitivity of BPHs to nicotine. Once the axenic population of the BPHs was acquired, we microinjected the BsNLG8 into the host insects, subsequently allowing the injected BPHs to feed on rice seedlings containing nicotine. Results showed that BsNLG8 could significantly reduce the sensitivity of BPHs to nicotine, thus improving their survival ability. We theorize that symbiotic bacteria BsNLG8 could play a crucial role in reducing the susceptibility of BPHs to nicotine by degrading nicotine or participating in the process of nicotine detoxification in the host.

Symbiotic bacteria play a pivotal role in the physiological activities of the BPHs, which is crucial for the maintenance of the micro-ecological balance in the host. Studying the diversity of symbiotic bacteria and exploring their species composition is of great significance in understanding the relationship between symbiotic bacteria and BPHs. Our findings not only provide a valuable case for the further study of the function of B. cepacia BsNLG8 but also lay a theoretical foundation for the use of biotechnology to degrade nicotine waste and reduce nicotine content in tobacco leaves [46]. However, additional research is required to investigate the mechanisms driving nicotine degradation by BsNLG8 and its role in the host’s detoxification process.

5. Patents

Xu Xiaoxia, Wang Xuemei, Jin Fengliang, Yang Rongrong.A Burkholderia BsNLG8 and its application. China, CN 114437962 A.