Effect of neutral protease overproduction in Bacillus subtilis 168 via site-directed mutation against Meloidogyne incognita infecting eggplant under greenhouse conditions

doi:10.21203/rs.3.rs-2592246/v1

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Effect of neutral protease overproduction in Bacillus subtilis 168 via site-directed mutation against Meloidogyne incognita infecting eggplant under greenhouse conditions

https://doi.org/10.21203/rs.3.rs-2592246/v1

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Root-knot nematodes (RKN), Meloidogyne incognita, caused significant problems for many important crops. Measuring control with low environmental impact has been required since EU legislation revised pesticide laws for crops. Bacteria-based control methods reduce pollutants and stabilise ecological changes, which makes them promising for controlling plant pathogens. In this study, the derivative of Bacillus subtilis168, termed Bs118, was generated by replacing the native promoter of the extracellular neutral metalloprotease-encoding gene (nprE) with a constitutive promoter of the repU gene responsible for replication of the Staphylococcus aureus plasmid pUB110. As a result, protease production increased to twice that of the wild type. Results revealed that the overproduction of neutral metalloprotease conferred Bs118 high nematocidal activity by inducing 98% mortality in the M. incognita J2 in vitro study. Bs118 stated its priority in affecting root-knot nematode reproduction under greenhouse conditions. The soil drench treatment was more promising than root dipping in controlling M. incognita compared with the untreated control treatment. The same trend happened in the eggplant growth parameters, where Bs118 improved plant health more than Bs168. In conclusion, site-directed mutation via homologous recombination to replace the native promoter with another constitutive one is a promising approach to constructing modified strains with higher protease production that can be employed as an efficient biocontrol agent against root-knot nematodes in addition to the positive impacts on plant growth.

Biological sciences/Biological techniques

Biological sciences/Biotechnology

Biological sciences/Genetics

In Egypt, annual losses in crop production caused by root-knot nematodes, Meloidogyne spp., are considered the major limiting factor in crop production in the tropics ⁽¹⁾. The mode of nematode attack against plant parts and their habitat in the soil make their management more difficult than that of other pests. Meloidogyne incognita is considered one of the most potent plant parasitic nematodes affecting Egyptian ‎vegetables ⁽²⁾. The application and extensive use of chemical nematicide for nematode management caused environmental and health concerns, so alternative strategies have been urged to be used ⁽³⁾. Biological agents have been investigated to control plant-parasitic nematodes. Bacterial and fungal infections of nematodes in the soil create the possibility of employing soil microorganisms to manage them ^{(4, 5)}.

Bacteria are numerically the most prevalent organism in soil; for example, members of the genera Pseudomonas and Bacillus have demonstrated a good potential for nematode biological control^{(6, 7)}. Over the past 20 years, extensive research has been done to evaluate their ability to suppress plant-parasitic nematodes. Several studies have revealed that bacteria are widespread and have a wide range of activity mechanisms. These bacteria influence nematodes in various ways, such as by producing antibiotics, toxins, or enzymes; interfering with plant–nematode recognition; causing systemic plant resistance; and encouraging plant health ^{(8, 9)}. Different Bacillus spp. and Pseudomonas spp., which are the most common prevalent populations in the rhizosphere, have potential efficacy in stimulating induced systemic resistance in plants, which is attributed to a variety of factors, including fortifying cell wall strength through the accumulation of newly produced phenolic compounds⁽¹⁰⁾. In addition, to facilitate the synthesis of defence chemicals against the challenge pathogen, they promote the accumulation of pathogenesis-related proteins like chitinase and peroxidase, as well as the formation of phytoalexin and other secondary metabolites ^{(11, 12)}. The cause of the nematocidal effect in B. subtilis is its ability to create a variety of poisonous metabolites, including benzene acetaldehyde, 2-nonanone, decanal, and 2-undecanone; dimethyl di-sulfide (DDS)^{(13, 14)}, enzyme production, particularly chitinase and protease enzymes^(15–17); antibiotic secretion⁽¹⁸⁾; exotic abilities for promoting growth and stimulating antioxidative potential; protection against various pathogens; and stress ⁽¹⁹⁾. Therefore, it seems to be an ideal agent for relieving nematode infections in plants. Here is why this study manipulates B. subtilis as a bioagent for controlling the root-knot nematode, M. incognita. Different types of structural proteins are present in nematodes, and their proportions alter over the course of their life cycles. In contrast to the mobile stage, which largely includes an exterior cuticle layer made up of glycoproteins and lipids, the egg stage contains a chitin/protein complex⁽²⁰⁾. Proteases, especially those secreted by Bacillus species, are critical nematicides⁽²¹⁾.

Many molecular genetic techniques have been reported to improve the nematocidal effect of bacterial species. Microbial virulence determinants, for example, are bacterial proteases whose corresponding genes were cloned and expressed in another organism to maximise product ⁽²²⁾. Other genetic approaches are directed mutagenesis, in which a gene encoding a putative virulence determinant responsible for a certain pathogenic trait is replaced or disrupted for the construction of a mutant strain, or random mutagenesis that has been demonstrated by various current research projects; for example, Tn5 transposon-mediated mutagenesis was utilised by Wechter et al. ^{(23, 24)} to construct a library of mutants and generate five BG33R mutants that have no wild-type ovicide activity of Pseudomonas sp. BG33R. Another approach is protoplast fusion, this method was manipulated by Mohamed et al ⁽²⁾. They successed in formation of ten stable bacterial fusants that gave higher nematocidal effect against M. incognita compared with parental strains B. cereus and B. thuringiensis subsp tenebrionis. Here, in this study, the aim is to overproduce protease enzyme from B. subtilis subsp. subtilis strain 168 (Bs168) by site directed mutagenesis, as well as to study and compare the effect of modified strain and wild-typeagainst M. incognita in laboratory and greenhouse trials.

Bacterial strains, plasmids, and cloning

The bacterial strains and plasmids used in this research are E. coli DH5α, used as a host for cloning and plasmid preparation, and Bacillus subtilis subsp. subtilis strain 168, accession no. AL009126.3 (Bs168), used as a source for genes isolation and an expression host. Genetic characteristics of bacterial strains, in terms of purchased and constructed plasmids containing genetic fragments, are listed in Table 1. The structures of plasmids are shown in fig. 1.

Media, growth conditions, and preparation of culture supernatants

Bacterial strains were grown in Luria-Bertani (LB) medium⁽²⁵⁾ for 18 hours at 37 °C and 150 rpm in a shaking incubator (Thermoscientific, UK) supplemented with ampicillin and neomycin (50µg/ml and 20µg/ml, respectively; Sigma, St. Louis, MO). Protease production was checked on agar plates containing 1% (v/v) skim milk. Centrifugation for 20 minutes at 10,000 rpm at 4°C was employed to obtain culture supernatant, which was then used immediately to determine proteolytic activity. Also, preparation of cell-free culture supernatants by microfiltration (membrane filter, cellulose acetate/nitrate, pore size, 0.22 m) was used to measure the proteolytic activity simultaneously to ensure the proteolytic activity was due to cell-free, i.e., secreted proteases ⁽²⁶⁾.

Assay of protease activity

Proteolytic activity on casein was determined using a slightly modified method described by Han and Damodaran ⁽²⁷⁾. The reaction mixture was composed of 1 mL of supernatant as a source of enzyme, and 1 mL of 1% casein solution in a 0.1 M Tris–HCl buffer (pH 8.9). Proteolysis was carried out at 37°C for 30 min and stopped by the addition of 2.0 ml of 15% w/v trichloroacetic acid (TCA). Also, TCA was used to inhibit the enzyme before incubation with casein solution and was considered a blank reaction. The sample was incubated on ice for 10 minutes and then centrifuged at 12000 rpm for 15 minutes. According to Lowry et al ⁽²⁸⁾, the reaction mixture was centrifuged for 10 minutes at 10,000 rpm at 4°C, and the protein released in the clear supernatant was measured. A unit of protease activity has been defined as the amount of enzyme necessary to release 1 μg of tyrosine per minute under test conditions.

Protease overproduction using the site-direct mutation protocol

Primer design

During the construction of pGEM-derived pMG vectors dedicated to inducing the genetic exchange by homologous recombination in Bs168, a specific cassette was designated to replace the metallo-neural protease promoter (PnprE) for Bs168. The cassette is made up of ylaA partial sequences, tag="BSU_14710," and nprE partial sequences, encoding extracellular neutral metalloprotease locus_tag="BSU_14700."These two partial genes are upstream and downstream from the promoter that regulates the expression of the protease-encoding gene. Two primer pairs were designed for each partial gene sequence with artificial restriction sites. The sense primer (ylaA fwd) and anti-sense primer (ylaA rev) containing the two artificial restriction sites, SalI (GTCGAC) and SacI (GAGCTC), were designed to amplify partial fragment from ylaA, while amplification of the nprE gene was achieved by nprE fwd and nprE rev primers containing the two artificial restriction sites, NcoI (CCATGG) and SphI (GCATGC), respectively. The sequence of primers is shown in Table 2.

DNA manipulation and PCR amplification

Genomic DNA isolation was performed using the Wizard® Genomic DNA Purification Kit from Promega (Madison, WI, U.S.A.). The PCR amplification steps were performed as follows: denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 seconds, and hybridization at 50 °C for 30 seconds for both genes, ylaA and nprE. An elongation step was at 72 °C for 45 sec, and final elongation steps were at 72 °C for 10 min.

Preparation of competent cells

Competent cells from E. coli DH5 were generated by following the method modified by Yang et al ⁽²⁹⁾. While competent B. subtilis 168 cells were briefly prepared, a ten-fold dilution of an overnight LB culture was performed by adding new LB medium. When the cell density reached 0.7 at 600 nm, arabinose was added at a final concentration of 0.6% w/v, and the culture was agitated for 1 hour. The culture was then ready to be altered. A total of 5 µL of the generated vector was mixed with 100 µL of competent cells and incubated at 37 oC, 180 rpm shaking conditions ⁽³⁰⁾.

Bacterial strains	Description	Source
Bacillus subtilis subsp. subtilis strain 168	trpC², sfp⁰	Lab stock
E. coli DH5α	F^–endA1glnV44thi-1recA1relA1gyrA96deoRnupGpurB20 φ80dlacZΔM15 Δ(lacZYA-argF) U169, hsdR17(r_K^–m_K⁺), λ^–	Lab stock
Plasmids	Description	Source
pGEM-T Easy	Cloning vector, Ap^r	Promega
pBG106	εpbp, P_repU-neo, εfenF	(Leclère et al. 2005)
pMG112	1340 bp SalI and NotI p_repU-neofragment from pBG106 cloned into pGEM-T Easy	Lab stock
pMG115	576 bp ylaA gene fragment of B. subtilis 168 cloned into pGEM-T Easy	This study
pMG116	658 bp nprE gene fragment of B. subtilis 168 cloned into pGEM-T Easy	This study
pMG117	ylaA gene fragment SalI and SacI double digested and inserted into pMG112	This study
pMG118	nprE partial gene fragment NcoI and SphI double digested and inserted into pMG117	This study

Table 1. Bacterial strains and plasmids used in this study.

Name	Primer sequence (5'-3')	Product size (bp)
ylaA fwd	GTCGAC TTATACGTTCGACCTTGCTG	576
ylaA rev	GAGCTC TAAAGTGTTTCATCCGTAGG	576
nprE fwd	CCATGGTATCAATCAGCCTGCCAGGT	658
nprE rev	GCATGCAACAGTTGCGCCCTTTAGC	658

Table 2. Primers sequences for isolation of partial fragments from ylaA, and nprE

Construction of the modular cassette

This protocol was performed to replace the native PnprE by a constitutive promoter p_repU in several steps by designing plasmid construction to include the two partial upstream and downstream genes, ylaA and nprE, respectively, of P_nprE in Bs168 and the new promoter, p_repU and marker gene of neomycin, neo. The expected PCR products for the ylaA and nprE gene fragments were separated on a 1.5% agarose gel, then purified using a gel purification kit (Thermo Scientific, USA), then cloned separately in pGEM-T Easy vector, and the ligation protocol was performed as described in the pGEM®-T Easy Vector Systems Technical Manual from Promega Corp. (Madison, WI, USA). The vector pBG106 (fig. 1b), which contained cassettes pbp, P_repU-_neo, fenF31, and pGEM, was double digested by SalI and NotI to obtain the P_repU-_neo fragment, which was then inserted into pGEM-T Easy (fig. 1a), yielding pMG112 (fig. 2). Double digestion of ylaA and pGEM by SalI and SacI, followed by insertion, was implemented to obtain pMG115 (fig. 3a). Along the same lines, the pMG116 vector was generated by the insertion of the nprE gene fragment into pGEM-T after double digestion by NcoI and SphI (Fig. 3b). Then, vectors pMG115 and pMG112 were double digested via SalI and SacI and ligated, and a fragment of ylaA was inserted in the corresponding sites to form pMG117 (Fig. 4). Finally, pMG118 was constructed by double digestion of both pMG116 and pMG117 plasmids with Nco1 and Sph1 enzymes and inserting the released nprE fragment into pMG117 (Fig. 5). The recombinant vectors were transformed into competent cells of E. coli DH5α. Following Sambrook and Russell ⁽³²⁾ standard procedures for restriction endonuclease digestions and agarose gel electrophoresis, Plasmids from transformant colonies were purified using the Mini Plasmid Kit (Thermo Scientific, USA). The vectors, pMG118, were then transformed into comptent Bs168 cells. Transformant cells were plated on LB agar supplemented with neomycin and incubated at 37°C to select the recombinants.

Trials of nematode biocontrol

Nematode inoculum.

Root-knot nematodes, initially isolated from the infested fields in Giza governorate and maintained on tomato roots, were used as inoculum in the experiment. The experiment was implemented in the greenhouse belonged to the Plant Pathology Department, National Research Centre, Egypt. By light microscopic investigations of a perennial pattern, separation, and identification of females as Meloidogyne incognita were implemented ⁽³³⁾. The infected roots were cut and incubated in tap water. After three days, M. incognita juveniles (J2s) were extracted according to method reported by Hussey and Barker ⁽³⁴⁾.

Preparation of bacterial suspension.

Single colonies of bacterial strains were allowed to grow on nutrient agar plates for Bs168 and on nutrient agar supplemented with neomycin in the case of the modified strain at 37 ^oC for 18 h. Vegetative cells of each stain were suspended in 10 ml of sterile distilled water in a 50 ml sterile falcon tube to get a concentration of approximately 1x10⁶ CFU/ml ⁽³⁵⁾.

Bioassay test

The nematocidal efficacy of both the wild-type (Bs168) and the modified strain (Bs118) was assessed by mixing 1 mL of nematode suspension (approximately 100 ± 3 of M. incognita J2 with 2 mL of bacterial suspension containing 1x10⁶ CFU/mL in test tubes separately. Test tubes containing only 2 mL of distilled water and 1 mL of nematode suspension were assigned as control. All treatments were replicated five times and incubated at 30 ^oC. Dead juveniles were counted after 24 and 48 h of bacterial treatment. At the end of incubation, the nematode suspensions in all treatments were washed and resuspended in 2 mL distilled water for another 24 h, then the average percentage of nematode recoveries was determined. The percentage of mortality was calculated according to the equation: mortality% = [C1-C2/C1] x 100, where C1 is the number of live nematode larvae in the control treatment and C2 is the number of live nematode larvae in the other treatments. Net mortality was calculated according to % mortality after 48 h of bacterial treatment minus nematode recovery in distilled water ^(1,16).

The greenhouse experiments

A pot experiment was conducted in the experimental greenhouse of the Plant Pathology Department, National Research Centre, Giza, Egypt. The plastic pots (20 cm in diameter) were filled with 2 kg of sterilised sandy and clay soil (1:1 w/w). Instantly, three weeks old eggplant seedlings of Solanum melongena (cv. Alabaster) were transplanted, two seedlings per pot. One week later, seedlings were thinned to one seedling per pot. Each pot was inoculated with 2000 M. incognita J2, followed by five mL of the bacterial suspension (1x10⁶ CFU/mL) of each strain, besides the control treatment. Simultaneously, all treatments were replicated five times. The pots were then watered and arranged in a completely random design on the bench in the greenhouse at a temperature of 27–32 ºC ^(1,16).

Recorded data

Estimation of nematodes parameters

Sixty days after nematode inoculation, eggplants in the five replicates were gently uprooted, and the roots were washed and cleaned from the adhering soil particles. J2 was extracted from 200 g of soil using the sieving and decanting technique⁽³⁶⁾ and examined under a light microscope with a Hawksley counting slide. The number of galls and egg masses was determined for the whole root system. For each parameter, the percentage of nematode reduction was calculated and compared with the control.

Measurement of eggplant growth parameters

Plant parameters such as length of shoots (cm), fresh weights of shoots and roots, and dry weight of shoots (g) of eggplant were measured. The percentage of plant growth increase for each criterion was calculated and compared to the untreated control ⁽²⁾.

Measurement of Biochemical parameters in eggplants

Two months after eggplant treatment by bacterial cultures, one gram of collected leaves from each treatment was used to demonstrate biochemical parameters represented by the assessment of polyphenol oxidase, PPO (units. g-1 fresh weight of leaves) following the method of Vamos-Vigyazo and Nadudvari-Marlcus³⁷, β-1,3-glucanase (GLU) (units. g^-1 fresh weight of leaves) using the method of Gupta et al ⁽³⁸⁾, chitinase ⁽³⁹⁾ (units. g-1 fresh weight of leaves), and total phenolic compounds (U/mg) following the assay of Saikia et al ⁽⁴⁰⁾. Enzymes extraction was accomplished according to McCord and Fridovich ⁽⁴¹⁾. Protein content was determined according to Lowry et al ⁽²⁸⁾. Bovine serum albumin was used as a standard.

Ethical approval

The eggplant seedlings identified by the Agricultural Research Center, were supplied from Egyptian nurseries and agriculture. All the methods and handling of the cultivated eggplants included in experimental research were performed in accordance with relevant guidelines and regulations.

Protease overproduction using site directed mutation

PCR amplification of ylaA and nprE partial sequences

The sense primer (ylaA fwd) and anti-sense primer (ylaA rev) succeeded in generating ylaA with a size of 576 bp. Furthermore, the nprE gene was amplified at 658 bp using nprE forward and nprE reverse primers (Fig. 6). Each of them was cloned separately in pGEM-T Easy to construct pMG115 and pMG116, respectively. The constructed vector pMG112, which included the P_repU-neocassette fragment, received the cloned fragment of ylaA in a vector named pMG116, which was then inserted with the fragment of P_repU-neocassette in a new construct pMG118 (Fig. 5). Taking this into consideration, the direction (5' 3') of P_repU-neocassette and nprE is linear, while that of the ylaA gene fragment is in the opposite direction. After transformation of competent cells of BS168 via pMG118, the positive transformants successfully grew on LB agar media supplemented with neomycin. Homologous recombination between the constructed vector pMG118 and the Bs168 chromosome is represented in Fig. 7. The crossingover between homologous fragments leads to the replacement of pnprE with constitutive P_repU-neo that will consecutively enhance and increase protease yield. The modified strain was referred to as Bs118.

Estimation of protease activity for wild-typeand modified strain

Genetic modification is believed to be an efficient tool to achieve potent modified strains of industrial enzymes, such as protease, for different applications. In this study, site-directed mutations employed the PrepU promoter to replace the native extracellular neutral protease promoter to construct the plasmid pMG118 used to transform B. subtilis 168 for protease overproduction. The enzyme activity of wild-type was measured at 43.33 u/ml, while the enzyme activity of the modified strain, Bs118, was 90 u/ml, which was two times more than wild-type (Fig. 8).

Nematocidal effect of Bs118 and Bs168 bacterial strain suspensions on M. incognita J2 in vitro test

The modified bacterial strain and the wild-typewere examined for their nematocidal properties via the effects of their different biochemical components in vitro. Table 3 shows that Bs168 and the modified strain Bs118 have a significant lethal effect on M. incognita J2 after 24, and 48 hours of exposure compared to the control.

Generally, the mortality gradually increased with time, and it was noticed that the highest J2 mortality (98%) after 48 h was obtained by Bs118 treatment, whereas Bs168 recorded an 88.67% mortality as compared with the control. M. incognita J2 did not recover after being washed with distilled water and left for 24 hours, indicating that the death was real.

Treatments	Nematode mortality After different exposure time*		% Net mortality^#
Treatments	24h	48 h	% Net mortality^#
Control	0.00^c**	0.00^c	0.00^c
Bs118	81.67^b	88.67^b	88.67^b
Bs168	93.33^a	98.00^a	98.00^a

Table 3. The effect of Bs118 and Bs168 on M. incognita juveniles mortality under laboratory conditions.

*Values are average of five replicates **Means followed by the same letter(s) are not significantly (P≤ 0.05) different according to Duncan’s Multiple Range Test. # % Net mortality = mortality after 48 h minus nematode recovery in distilled water which =zero, Bs118 = modified B. subtilis, Bs168 = wild-typeof B. subtilis

Greenhouse experiment

In a greenhouse experiment, the effect of the wild-typeBs168 and the modified strain Bs118 on root-knot nematodes M. incognita in infected eggplant was evaluated using soil drench and/or root dipping application methods.The data in Table 4 revealed that the tested Bs168 and Bs118 significantly (P ≤0.05) reduced nematode multiplication, and the modified strain was more effective in decreasing the number of M. incognita J2 in soil, root galls, and egg masses/root systems and reducing nematode infection than the wild-type as compared with the control.The soil drench treatment was more effective than the root dipping treatment in reducing nematode counts in soil and eggplants. In soil drench application, data showed that the modified strain Bs118 resulted in 86.05%, 68.80%, and 79.94% reductions in the number of J2s in soil, galls, and egg-masses numbers, respectively, while Bs168 recorded 40.04%, 42.23%, and 63.41% reductions in the number of J2s, galls, and egg-masses numbers, respectively, compared with the control. Also, the same trend was noticed in root dipping, where the Bs118 achieved a reduction of 61.41%, 62.83%, and 72.35% in the aforementioned parameters, respectively. While the BS168 reduced the same parameters by 21.38 percent, 28.50%, and 61.95 percent, respectively.

Treatments	No. J₂ in soil	R%.**	No. galls / root system	R%.	No. egg-masses / root system	R%.
Control	276.00^a	---	528.00^a	----	240.50a	---
Soil drenches application
Bs118	38.50^e	86.05	164.75^e	68.80	48.25^d	79.94
Bs168	165.50^c	40.04	305.00^c	52.23	88.00^b	63.41
Root dipping application
Bs118	106.50^d	61.41	196.25^d	62.83	66.50^c	72.35
Bs168	217.00^b	21.38	377.50^b	28.51	91.50^b	61.95

Table 4. The nematocidal effects of Bs168 and Bs118 strains against M. incognita infecting eggplant

#Values are average of five replicates. Different letters within the same column indicate significant differences among treatments according to least significant difference test (P ≤ 0.05) ** R. % = % Reduction, Bs118 = modified B. subtilis, Bs168 = wild-type of B. subtilis

Effect of Bs168 and Bs118 on the eggplant growth parameters

Table 5 indicated that the two evaluated strains significantly (P≤0.05) increased plant growth parameters compared to the control, and Bs118 was more promising than Bs 168. In soil drench applications, the percentage increases in shoot length, fresh weight, and flower numbers due to Bs118 were 81.94%, 175.22%, and 300%, respectively, compared with the control. While Bs168 recorded a 44.56%, 35.32%, and 280% increase in the same parameters, respectively, compared with the control. In root dipping application, Bs118 exhibited the same trend by increasing the previously mentioned plant parameters by 18.69%, 35.41%, and 120%, respectively, more than Bs168, which recorded 10.28%, 23.32%, and 60% in the same parameters, respectively, compared with the control. In general, soil drenching led to greater increases in plant parameters under investigation than root dipping, and the modified strain Bs118 was more effective rather than the wild-type Bs168.

Treatments	Shoot System				Length (cm)	% Inc.	No. of Flowers	% Inc.
Treatments	Length (cm)	*% Inc.	fresh weight (g)	% Inc.	Length (cm)	% Inc.	No. of Flowers	% Inc.
Control	26.75^d*	---	10.25^b	----	18.00^c	---	1.25^c
Soil drench
Bs118	48.67^a	81.94	28.21^a	175.22	32.67^a	76.59	5.00^a	410
Bs168	38.67^b	44.56	13.87^ab	35.32	28.92^b	56.32	4.75^ab	280
Root dipping
Bs118	31. 67^c	18.69	13.88^b	35.41	20.33^c	9.46	2.75^b	120
Bs168	29.67^c	10.28	12.97^b	23.32	20.00^c	8.11	2.00^c	60

Table 5. The effect of Bs168 and Bs118 on eggplant growth parameters under greenhouse conditions

#Values are average of five replicates. *Within a column, means followed by the same letter(s) are not significantly different according to Duncan’s Multiple Range Test. ** R. % = % Reduction %Inc.- % Increase, Bs118 = modified B. subtilis, Bs168 = wild-type of B. subtilis

Estimation of Biochemical analysis in eggplant leaves

The potential to induce plant resistance against nematode infection was assessed by assessing the biochemical compounds such as protein, total phenols, and enzyme activity (glucanases (GLU), polyphenol oxidase (PPO), and chitinase (CHI) activities). The data in Fig. 9 demonstrated that the preceding treatments greatly increased PPO, GLU, and CHI activities. The plant analysis revealed that the two treatments had a high level of PPO activity when compared to the control. Generally, plants treated with Bs118 as a soil drench application produced the most PPO, GLU, and CHI, which recorded at 3.90, 6.95, and 0.39 U/mg, according to the findings of this study. While Bs168 had the lowest recorded increase, with 3.05, 0.95, and 0.29 U/mg compared to the control, which had 0.14 U/mg.However, chitinase showed a non-significant value in eggplant leaves between the Bs118 and Bs168 in both applications compared to the control. Notably, soil drench application stimulated activity in eggplant leaf companies when combined with root dipping application. The same trend was observed with the obtained data, demonstrating that Bs118 was more effective than Bs168 in activating PPO, GLU, and CHI in eggplant leaves after two applications. Noteworthy, Bs118 showed the best activity with the wild-typeand untreated control at two applications. Generally, the soil drenches are definitely more applicable than the root dipping for stimulating the antioxidant enzymes in plants.

Effect of the bacterial strains, Bs168 and Bs118, on the total phenolic compounds (TPC) in eggplant leaves

The effect of Bs168 and Bs118 on TPC in eggplant leaves infected with M. incognita was studied in a greenhouse setting. The results in Fig. 10 showed that Bs118 treatments were quite helpful in enhancing TPC in leaves (7.68 U/mg) compared to Bs168 (0.46 U/mg) and the control (0.46 U/mg) in the case of the soil drench. The same trend was observed when root dipping was applied: Bs118 achieved a higher level of TPC in leaves (7.68 U/mg) compared to Bs118 (7.06 U/mg).

Due to climate change, a rising human population, and a declining supply of farmland, there is a growing demand for food production. These issues have so far been resolved via molecular methods and chemical applications (using fertilisers and pesticides). A sustainable alternative to replace chemical fertilizers and pesticides is the use of bio-derived materials. Various microorganisms, such as Fungi ⁽⁴²⁾, bacteria ⁽¹²⁾ and actinomycetes ⁽⁴³⁾, all have a history of producing compounds with anti-RKN efficiency. More research is required to identify and develop novel nematocidal metabolites ⁽¹⁷⁾ specifically fungal and bacterial metabolites that exhibit nematocidal action, which seems to be limited.

More research is needed to discover and create new nematocidal metabolites ⁽¹⁷⁾ since, in contrast to fungal and bacterial metabolites, those with nematocidal activity are uncommon. The nematocidal activity of microbial metabolites has also been linked to their enzymatic capacity, particularly chitinase and protease enzymes ^(13,14). Bacillus species are considered the main producers of extracellular proteases, which have a crucial role as nematocidal nematocidal ⁽¹⁶⁾. In this study, enhancement of protease production originating from Bs168 was achieved by site-directed mutation, a powerful approach to increase nematocidal activity. The native promoter of the Bs168 extracellular neutral protease was replaced by a constitutive promoter controlling the gene repU responsible for replication of the Staphylococcus aureus plasmid pUB110. This resulted in the discovery of Bs118, a modified strain that produced twice as much protease as the wild type. It is obvious that P_repU-regulated metalloprotease overproduction is implicated in the nematocidal effect evolved in vivo by the modified strain, Bs168 that has been conferred extra nematode biocontrol potential. The P_repU promoter previously proved its efficiency in previous publications, as reported by Lecle`re et al⁽³¹⁾. They replaced the native promoter of the mycosubtilin operon with P_repU, and that increased mycosubtilin up to 15-fold more than B. subtilis ATCC 6633 as a wild type. Hussein and Fahim ⁽⁴⁴⁾ created a mutant strain, BMG06, that overexpressed plipastatin and fengycin by about 35 and 4 folds, respectively, more than wild-type B. subtilis 168, using constitutive promotor PrepU to replace Ppps (the plipastatin promoter) and Pfen (the fengycin promoter).Different genetic approaches were employed to improve the protease production from microbial sources for different applications; for example, UV-laser random mutagenesis was employed to treat B. subtilis to select high protease producers for increasing the peptide level of fermented soybean meal, which helps to improve its nutritional value ⁽⁴⁵⁾. Suberu etal ⁽⁴⁶⁾established the heterologous expression of the serine alkaline protease gene from Bacillus subtilis RD7 using the pET15b vector in E. coli BL21 cells, and the purified recombinant protease enzyme was employed to eliminate egg yolk stains at 40 °C and pH 10. The study conducted by Degering et al ⁽⁴⁷⁾ presented a novel approach to increasing the extracellular production of a protease enzyme, subtilisin BPN from B. amyloliquefaciens, using various Bacillus host strains and the fusion of 393 N-terminal signal peptides (SPs), with 173 SPs originating from B. subtilis (termed homologous SPs) and 220 SPs originating from B. licheniformis DSM (termed heterologous SPs). The fusion constructs were then cloned and produced in B. subtilis and B. licheniformis, resulting in improved protein export not only in the original screening host but also in two distinct B. licheniformis strains. Darwesh et al ⁽⁴⁸⁾ investigated other applications of thermostable alkaline protease secreted by Saccharomonospora viridi to control harmful nematodes.

Several studies have also shown that the biochemical changes caused by the interaction of several factors within the biological control strategy and influencing the plant's immune system are an effective way to reduce nematode damage to plants. PGPB also play an important role in improving plant health and inducing resistance mechanisms by overproducing plant defence hormones and enzymes like catalase, chitinase, peroxidase, phenylalanine ammonia-lyase, and polyphenol oxidase ^{(49, 50)}. Pooled data collected after the experiment of root dipping with either the wild-type strain Bs168 or the overproducer derivative Bs118 proved that they have plant resistance-inducing activity. Moreover, plantlets inoculated with bacterial cultures induce growth in the absence of nematode infections, providing evidence that Bs168 and Bs118 lack any phytotoxic effects per se.

Under greenhouse conditions, Bs118 achieved significantly higher levels of oxidative enzymes than Bs168. These results are in agreement with Khanna et al ⁽⁵¹⁾, since they reported that applied organic fertilisers and bio-agents increased the activities of defense-related enzymes peroxidase (POX), PPO, and phenylalanine ammonia oxidase, phenylalanine ammonia. PPO's effect as a defense-related enzyme is caused by the oxidation of TPC to quinones, which are more toxic than the original phenols and frequently induce plant pathogen resistance ⁽⁵²⁾. Also, improvement of nematocidal effect is attributed to the increase in GLU's which is due to its ability to degrade pathogenic agent cell walls and hydrolyze the corresponding substrates ⁽⁵³⁾. In contrast, this result disagrees with Abd-Elgawad et al ⁽⁵⁴⁾, who found nematode infection had no effect on GLU activity but increased PPO activity in inoculated roots compared to uninoculated roots. By comparing the efficiency of soil drench and root dipping experiments as an approach to applying biocontrol, soil drench is preferable, and that can be explained by the higher effective performance of optimised bacterial cells than in the case of root dipping. These results agree with Khan and Tarannum ⁽⁵⁵⁾; Ramadan and Soliman ⁽⁵⁶⁾ proved the higher efficacy of soil drench than root dipping treatment in enhancing plant growth and nematode infection reduction. A recent study by Mohammad et al ⁽⁵⁷⁾ concluded that when tomato plants were exposed to sweet annie and garden cress aqueous extracts, they produced a response that included the accumulation of phenolic compounds and an increase in the activity of defensive enzymes.

The use of biological agents as an alternative to chemical pesticides offers considerable potential for controlling a variety of plant infections, including root-knot nematodes (Meloidogyne spp.). Enhancement of microbial protease by means of genetic engineering techniques, such as replacement of the native promotor with a constitutive promotor, results in increased suppression of Meloidogyne incognita and improved plant parameters. This method is thought to be effective for releasing potently modified strains as bioagents in a promising manner. And that qualifies them for additional research to increase their effectiveness in the field.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Author contribution

All authors participated in the development and implementation of the reviewing plan and subsequently written it. They discussed the different parts of the article, finalize the experimentation, and write the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

Financial support was partially made by National Research Centre, Egypt (by supplement chemicals and instruments) to develop and analyze the data

Acknowledgement

This study was supported in part by the NRC via providing chemicals and instruments. The facilities offered by The National Research Centre are appreciated.

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No competing interests reported.

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Effect of neutral protease overproduction in Bacillus subtilis 168 via site-directed mutation against Meloidogyne incognita infecting eggplant under greenhouse conditions

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