Protein Elicitor PeBL1 of Brevibacillus laterosporus Enhances Resistance Against Myzus persicae in Tomato

Myzus persicae, a destructive aphid of tomato usually managed by chemical pesticides, is responsible for huge annual losses in agriculture. In the current work, a protein elicitor, PeBL1, was investigated for its capacity to induce a defense response against M. persicae in tomato. Population growth rates of M. persicae (second and third generation) decreased with PeBL1 treatments as compared with controls. In a host selection assay, M. persicae showed preference for colonizing control plants as compared to tomato seedlings treated with PeBL1. Tomato leaves treated with PeBL1 gave rise to a hazardous surface environment for M. persicae due to formation of trichomes and wax. Jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) showed significant accumulation in tomato seedlings treated by PeBL1. The following results showed that PeBL1 significantly modified the tomato leaf surface structure to reduce reproduction and deter colonization by M. persicae. Defense processes also included activation of JA, SA, and ET pathways. The study provides evidence for use of PeBL1 in the protection of tomato from M. persicae.


Introduction
During the course of evolution, a complex relationship has formed between plants and herbivores. Plants damaged by herbivores show accumulation of toxic or volatile organic compounds with modification of their physical structures. These compounds and structures affect colonization by herbivores and their development, feeding, survival, and oviposition, which in turn attract natural enemies and induce defense [1]. Two defense mechanisms, mainly constitutive, have been developed by plants to deal effectively with this damage [2]. Physical barriers, including cuticle trichomes, callose, cell walls, and suberin, prevent plants from being colonized, whereas allelochemicals with antibiotic effects affect pest development, growth, fecundity, and durability of insects, or induce repellent effects [3].
Aphids are phloem-feeding insects, which transmit plant viruses by consuming plant sap, thus resulting in severe agricultural losses [4,5]. Defense responses induced by aphids have been analyzed in various aphid-plant systems. Green peach aphid showed decreasing fecundity in infested leaves of Arabidopsis thaliana [6]. In chilli plants, feeding caused confronting effects and increased emission of volatile organic compound, with a repulsive outcome versus infesting Bemisia tabaci [7]. Reduction in survival rate and population growth parameters of immature Plutella xylostella were found in Brassica napus due to resistance to Brevicoryne brassicae [8].
guard SUBSTRAT). Three weeks-old tomato seedlings were sprayed with the concentrations of PeBL1 solution after 7 days, followed after 24 h by inoculation with 8-10 adults of M. persicae per plant. The number of aphids after inoculation was then recorded every 5 days. Positive and negative controls were tested with water and 88.72 µg mL −1 buffer (50 mM Tris-HCl, pH 8.0). Transparent air-permeable cages were used to separate seedlings of each pot from others. The experiment was performed twice with four replications.

Increase in Intrinsic Rate of M. persicae Population
Tomato seeds were soaked for 24 h in PeBL1 purified protein solution of 88.72 µg mL −1 and then were moved to petri dishes for germination for 2-3 days in distilled water. Seedlings were sprayed with PeBL1 purified protein solution of 88.72 µg mL −1 after 24 h. Inoculation with a newly born nymph of M. persicae was then performed for each seedling. A cotton-gauze-covered glass tube was used to separate every seedling from each other. The newly born aphid was observed twice a day to record the total time and number of offspring it produced that were then taken out each day. An identical test on seeds and seedlings were performed after 5 days. The experiment was individually repeated two times using 30 replicates per treatment. The increase of intrinsic rate of each aphid was measured by the formula: where Md represents the number of new born nymphs in a development time equal to T d which is the period from the aphid birth till its first reproduction.

Feeding Preference
Seeds and seedlings of tomato were tested as listed above. Three-weeks-old seedlings of tomato were treated with PeBL1 solution. The positive and negative controls were treated with water and buffer of 88.72 µg mL −1 (50 mM Tris-HCl, pH 8.0), respectively. Transparent and breathable cage (60 × 60 × 60 cm) with leaves cross touching and a white cardboard bridge (12 × 4 cm) connecting the basal part of stems were used to place treated PeBL1 and control seedlings. Thirty adults of wingless M. persicae were released in the middle of the bridge. The experiment was repeated 15 times counting the numbers of aphids on each seedling after 24 h.

Aphids Bioassay
PeBL1 elicitor purified protein with different concentrations (i.e., 88.72, 53.23, 26.61, and 22.17 µg mL −1 ) as treatment, positive control (water only), and negative controls (Buffer 88.72 µg mL −1 ) were bio-assayed against M. persicae on tomato plants. Bradford assay was used to determine different protein concentrations. At three-leaf stage of tomato plant, around 2-3 mL of PeBL1 solution was applied with a precise spray bottle until the solution drained off from plants. Water and buffer (50 mM Tris-HCl, pH 8.0) were applied to positive and negative controls. Plants were allowed to dry overnight, and, on the next day, 3 to 5 numbers of freshly moulted (0-6 h), old adult winged aphids per leaf were allowed to feed on these plants. Nymphal development time was recorded, by consecutive observations at 3 h intervals until the completion of the bioassays for each instar, as the total number of offsprings produced by all aphid instars, whereas the longevity was considered as the number of days the aphids used to live on. Bioassays at three non-identical temperature regimes (16,22, and 27 • C) were repeated three times individually, using ten replicates per each treatment.

Surface Structure Observation of Leaves
Seven-day-old seedlings and seeds of tomato plant were treated similarly as above. Seeds were soaked for 8 days, one day after the spraying of seedlings. The central part was obtained and tested for first leaves, and 3.5% glutaraldehyde diluted in 0. 1

Determining Plant Hormone with HPLC/MS
Seven-day-old seedlings and seeds were treated as mentioned above. The aerial part of seedlings, weighing about 0.5 g, was used to extract JA, SA, and ET, as described earlier [25]. Around 20 µL of extract was inoculated into a high-performance liquid chromatography mass spectrometer (HPLC/MS; Shimazu Scientific Instruments, ODS-C 18 , 3 µm, 2.1 × 150 mm, Kyoto, Japan). HPLC analysis was carried out at a flow rate of 0.2 mL min −1 , 60% methanol as a mobile state, with 4 • C sample temperature and 40 • C column temperature, a desolvation temperature of 250 • C set with a selected ion monitoring (SIM) in negative ion mode (SA, m/z: 137.00; JA, m/z: 209.05), 200 • C of heat block, 10 L min −1 drying gas flow rate, 1.5 L min −1 nebulizing gas flow, 1.30 kV voltage of detector, and interface voltage of −3.5 kV.

Data Analysis
Data collected for each treatment pair were statistically compared with the independent Levene's test and two-tailed t-test. Data obtained from three or more treatments were statistically compared by least significant difference (LSD) and one-way analysis of variance (ANOVA). For statistical data analyses, Statistix version 8.1 (Analytical Software, Tallahassee, FL, USA) was used. Data on fecundity of aphids were square-root transformed prior to analysis. In order to take out differences, one-way factorial analysis of variance was performed among treatment factors such as the concentrations of PeBL1 elicitor and different temperature regimes, followed by least significant different test, at a probability of 95%. The expressions of genes (RT-qPCR) were obtained by the comparative CT (2 −∆∆CT ) method. Student's t-test (α = 0.05) was used for comparing fold changes in the plant samples treated with elicitor and buffer.

Evaluation of Recombinant PeBL1
The PET30-TEV/LIC recombinant expression vector was transformed into E. coli BL21 (DE3) cells. After successful transformation, the expressed His6-PeBL1 was soluble in E. coli. PeBL1 was purified using a His-Trap HP column (GE Healthcare, Waukesha, WI, USA) ( Figure 1a) and desalted in a HiTrap Pathogens 2020, 9, 57 5 of 20 desalting column (GE Healthcare) as described by Wang et al. [20]. At 12 kDa on Tricine SDS-PAGE, a single band showed the characteristics of the pure recombinant protein.
Pathogens 2020, 9, 57 5 of 21 The PET30-TEV/LIC recombinant expression vector was transformed into E. coli BL21 (DE3) cells. After successful transformation, the expressed His6-PeBL1 was soluble in E. coli. PeBL1 was purified using a His-Trap HP column (GE Healthcare, Waukesha, WI, USA) ( Figure 1a) and desalted in a HiTrap desalting column (GE Healthcare) as described by Wang et al. [20]. At 12 kDa on Tricine SDS-PAGE, a single band showed the characteristics of the pure recombinant protein.

Indoor Performance of M. persicae
PeBL1 induced resistance to the tomato aphid M. persicae in two ways. Firstly, PeBL1-treated tomato seedlings showed a population decrease; Table 1 shows the percentage decrease in population in the PeBL1 treatment compared with the buffer and control treatments. M. persicae preferred feeding on control tomato plants in the host selection test. One day after aphid inoculation and two days after plant spraying, the number of M. persicae colonizing PeBL1-treated plants (7.13 ± 0.34) was significantly lower than the control (13.65 ± 0.18) and "Elsewhere" is colonization of aphid at places other than the buffer-and PeBL1-treated areas. Some aphids, based on their feeding behavior, showed colonization in areas opposite to that treated with buffer and PeBL1 ( Figure 2). Secondly, in case of PeBL1 treatment, tomato aphid developmental time was more extended as compared to control, while daily reproductive abilities of M. persicae that fed on PeBL1treated seedlings were decreased (second and third nymphal instars). Furthermore, second and third generations showed lower growth rates ( Table 2).

Indoor Performance of M. persicae
PeBL1 induced resistance to the tomato aphid M. persicae in two ways. Firstly, PeBL1-treated tomato seedlings showed a population decrease; Table 1 shows the percentage decrease in population in the PeBL1 treatment compared with the buffer and control treatments. M. persicae preferred feeding on control tomato plants in the host selection test. One day after aphid inoculation and two days after plant spraying, the number of M. persicae colonizing PeBL1-treated plants (7.13 ± 0.34) was significantly lower than the control (13.65 ± 0.18) and "Elsewhere" is colonization of aphid at places other than the buffer-and PeBL1-treated areas. Some aphids, based on their feeding behavior, showed colonization in areas opposite to that treated with buffer and PeBL1 ( Figure 2). Secondly, in case of PeBL1 treatment, tomato aphid developmental time was more extended as compared to control, while daily reproductive abilities of M. persicae that fed on PeBL1-treated seedlings were decreased (second and third nymphal instars). Furthermore, second and third generations showed lower growth rates ( Table 2). Data compared by least significant difference (LSD), one-way analysis of variance, ANOVA, and Levene's test with SPSS 18.0. Letters in rows show significant differences at different treatments; same day after aphid inoculation (P = 0.05).

Figure 2.
Colonization of M. persicae on PeBL1-treated and control tomato seedlings after infestation (mean ± SD); data compared by Latin square design (LSD), one-way ANOVA and Levene's test with SPSS 18.0. Different lower style alphabets letters indicate significant differences among treatments (P = 0.05).

Influence of PeBL1 on Nymphal Development Time
Factorial analysis showed an impact of different concentrations of PeBL1 (F 5,468 = 77.84; p < 0.0000) at three different temperature regimes (F 2,468 = 158.43; p < 0.001) and of their interaction (F 10,468 = 1.61; p < 0.0079) on the overall developmental time of M. persicae as shown in Table A2 of Appendix A. A differential trend was found for the effect of the protein elicitor on nymphal development time, at different temperature regimes. The developmental time of each nymphal instar was prolonged with increasing concentrations of PeBL1. Maximum nymphal developmental time was 3 days for 1st instar and 3.7 days for 4th instar nymphs for high concentration (88.72 µg mL −1 ) at low temperature (16 • C). Minimum nymphal development time 1.6 days was recorded for 1st instar for low elicitor concentration (22.17 µg ml −1 ) at high temperature regime (27 • C). In buffer-treated (control) plants, the nymphal development time varied from maximum 2 days for 4th instar, at 16 • C, to minimum 1.1 day for 1st instar, at 27 • C. In water-treated (control) plants, there was no significant increase in the nymphal development with 1.8 days for 4th instar at 16 • C, and 1 day for 1st instar, at 27 • C.

Effect of PeBL1 on Aphid Fecundity
Data showed that the fecundity of M. persicae adults was significantly influenced by the concentrations of PeBL1 F , = 33.59; < 0.0001 and temperature regimes F , = 9.12; <

Effect of PeBL1 on Aphid Fecundity
Data showed that the fecundity of M. persicae adults was significantly influenced by the concentrations of PeBL1 (F 5,162 = 33.59; p < 0.0001) and temperature regimes (F 2,162 = 9.12; p < 0.0006) as shown in Table A3 of Appendix A. Less offspring were produced by M. persicae individuals that fed on PeBL1-treated plants, as compared to those that fed on the positive (water) and negative controls. Moreover, the maximum fecundity was noted at minimum temperature (16 • C) while minimum fecundity was noted at maximum temperature (27 • C) (Figure 4).
Pathogens 2020, 9, 57 9 of 21 0.0006 as shown in Table A3 of Appendix A. Less offspring were produced by M. persicae individuals that fed on PeBL1-treated plants, as compared to those that fed on the positive (water) and negative controls. Moreover, the maximum fecundity was noted at minimum temperature (16 °C) while minimum fecundity was noted at maximum temperature (27 °C) (Figure 4).

Effect of PeBL1 on tomato leaves
The surface of tomato leaves was significantly modified by the PeBL1 protein. Seedlings treated with PeBL1 showed more trichomes than controls (PeBL1 treatment, 90.71 ± 1.20 mm −2 ; control, 48.49 ± 0.36 mm −2 ; P= 0.05). A more refined wax structure was formed that gave rise to a much more refined surface environment, which is a trait known for being unfavorable for the aphid colonization and other behaviors [28].
All three signaling pathways were shown to participate in aphid-induced resistance in tomato [29]. Furthermore, JA, SA, and ET in PeBL1-treated seedlings accumulated, suggesting that the defense response in tomato plants was at least partially induced by the protein elicitor. JA, SA, and ET inductions are known to be affected by numbers, infestation time, and aphid species [30,31].

Effect of PeBL1 on Tomato Leaves
The surface of tomato leaves was significantly modified by the PeBL1 protein. Seedlings treated with PeBL1 showed more trichomes than controls (PeBL1 treatment, 90.71 ± 1.20 mm −2 ; control, 48.49 ± 0.36 mm −2 ; P= 0.05). A more refined wax structure was formed that gave rise to a much more refined surface environment, which is a trait known for being unfavorable for the aphid colonization and other behaviors [28].

Figure 5.
Contents in tomato seedlings (mean ± SD). Treatment with PeBL1 was carried out one-day after spraying. In both treatments, the aphids were inoculated one-day after seedlings were sprayed, and the samples were collected one-day after inoculation. (Data were compared by LSD, one-way ANOVA, and Levene's test using SPSS 18.0. Lower letters show significant differences among various treatments performed in jasmonic acid (JA), salicyclic acid (SA), and ethylene (ET), P = 0.05.). Figure 5. Contents in tomato seedlings (mean ± SD). Treatment with PeBL1 was carried out one-day after spraying. In both treatments, the aphids were inoculated one-day after seedlings were sprayed, and the samples were collected one-day after inoculation. (Data were compared by LSD, one-way ANOVA, and Levene's test using SPSS 18.0. Lower letters show significant differences among various treatments performed in jasmonic acid (JA), salicyclic acid (SA), and ethylene (ET), P = 0.05.). All three signaling pathways were shown to participate in aphid-induced resistance in tomato [29]. Furthermore, JA, SA, and ET in PeBL1-treated seedlings accumulated, suggesting that the defense response in tomato plants was at least partially induced by the protein elicitor. JA, SA, and ET inductions are known to be affected by numbers, infestation time, and aphid species [30,31].

Relative Fold Change of Defense-Related Expression
PeBL1 increased the defense response in tomato seedlings. All marker genes were up regulated by PeBL1 treatment, showing transcripts statistically more expressed than in the control. It was considered that induced resistance was caused by the aphid infestation and enhanced by PeBL1. Although genes involved in JA pathway were moderately expressed, all JA-associated genes were up regulated after 12, 18, 24, and 48 h of aphid infestation ( Figure 6). Similar trends were observed for all SA-and ET-associated genes that were significantly up regulated and significantly different from control samples for all observation times (Figures 7 and 8). Heat map of the expression levels of all 24 genes (Figure 9) suggest that resistance against aphid was due to increased transcription of the JA, SA, and ET genes.
Pathogens 2020, 9, 57 11 of 21 PeBL1 increased the defense response in tomato seedlings. All marker genes were up regulated by PeBL1 treatment, showing transcripts statistically more expressed than in the control. It was considered that induced resistance was caused by the aphid infestation and enhanced by PeBL1. Although genes involved in JA pathway were moderately expressed, all JA-associated genes were up regulated after 12, 18, 24, and 48 h of aphid infestation ( Figure 6). Similar trends were observed for all SA-and ET-associated genes that were significantly up regulated and significantly different from control samples for all observation times (Figures 7 and 8). Heat map of the expression levels of all 24 genes (Figure 9) suggest that resistance against aphid was due to increased transcription of the JA, SA, and ET genes.

Discussion
Use of elicitors represents a novel biological pest management technique, as they play a vital role in defense and signaling mechanisms of plants under attack of sap-feeding insects [32]. Various strains of B. laterosporus have shown different, broad-spectrum, antimicrobial activities against microbes such as bacteria and fungi, acting as antimicrobial peptides. They can enter the cell and be relocated in the cytoplasm and nucleus where they can interrupt the synthesis of proteins by intermingling to DNA and RNA [33]. Pathogenic bacteria and fungi, either necrotrophic or biotrophic, constitute an important source of elicitors such as PAMPs or MAMPs [34]. This study showed potential activity of PeBL1 derived from B. laterosporus A60 strain for the control of M. persicae. Previously, other studies showed that the application of chemical elicitors such as methyl jasmonate, benzothiadiazole, and other plant defense proteins such as proteinase inhibitors significantly reduced the activity of herbivore pests in tomato crop [35]. Data from this study validate previous findings showing that the soybean aphid Aphis glycines was reduced up to 40% with the use of methyl salicylate elicitor [34]. Bioassays data demonstrated that the population development was significantly slower on PeBL1-treated plants as compared to the buffer-treated control. Previous studies indicated a negative impact of exogenous applications of different elicitors, including methyl jasmonate (MJ), JA, and benzothiadiazole (BTH), on the population growth and fitness of different aphid species, an effect confirmed by the present findings [35]. Likewise, a biocontrol potential of this entomopathogenic bacterium has been found versus several Diptera, Coleoptera, and Lepidoptera, as well as versus mollusks and nematodes [23,24].
The present study revealed the potential of PeBL1 for the suppression of sap-feeding herbivores, affecting growth parameters and population performance. Trichomes are first lines of physical resistance against herbivores and pathogenic microorganisms. These hair-like appendages of plant epidermal cells [36] affect herbivore behavior, morphology, and density. Solanum spp. tests have shown a role of trichomes in defense, i.e., seven types of trichomes with two important effects in defense [37]. First, a plant surface constitutes a physical barrier because its dense hairs mat confers resistance, limiting the possibility of feeding and reducing the access of insects to the surface. Excessively hairy plants, such as S. hirsutum, are avoided by M. persicae. Trichomes are also associated with fundamental defense in tomato plant, as unicellular or multicellular hairs appendages arising from epidermal cell cover the surface conferring resistance to several pests due to the plant "pubescence." Leaf beetle (Coleoptera: Chrysomelidae) colonization with dense trichomes was reduced in soybean pods as compared with the trichome-removed pods, which attracted more beetles [38].
PeBL1-treated seedlings and leaves possessed more trichomes as compared to controls. PeBL1-treated tomato seedlings and leaves with increased trichomes numbers were supposedly disadvantageous for aphid reproduction and colonization. Non-glandular, high-density, tomato trichomes negatively affected the feeding behavior of the Colorado potato beetle Leptinotarsa decemlineata [39,40]. The cell wall is another vital part of physical barrier lignin, underpins plant resistance, and is an indicator of the cell-wall enhancement [41,42]. Aphid tolerance in chrysanthemum was improved by an increased lignin content [43]. Physical defenses in plants include trichomes and wax production, in response to biotic or abiotic stresses. Their formation can be induced by direct damage, i.e., as induced by leaf-cut, methoxyfenozide, and manganese [44,45]. Cuticular wax deposition and trichome density may be also affected by application of exogenous phyto-hormones, MJ or JA, as shown in Arobidopsis and tomato, respectively [46]. Wax deposition in B. napus was induced with SA application [36]. Therefore, it can be speculated that accumulations of SA and JA in PeBL1-treated tomato seedlings were related to the increased density of trichomes and deposition of cuticular wax.
However, various proteinase inhibitors were produced by chemical elicitors previously described in tomato plants [47]. Furthermore, negative effects were exerted by the application of PeBL1 elicitor on the aphid fecundity. PeBL1-treated plants produced much lower number of aphids as compared to buffer-treated and control seedlings. Results are in line with previous studies evidencing that exogenous application of SA and MJ induced a lower mean lifetime fecundity in aphids [34,48].
Likewise, optimum temperature, i.e., 22 • C, showed maximum fecundity of aphids while higher temperatures, i.e., 27 • C showed minimum fecundity, reduced metabolic rate at high temperature being responsible for the effect [48,49]. Similarly, analysis of variance showed that nymphal developmental time was prolonged in plants treated with PeBL1 as compared to control. Also at lower temperature (16 • C), maximum nymphal development time was observed, confirming that an increase of one degree in temperature affects the insect's life cycle to become shorter [50]. Further studies are required to understand the underlying mechanism induced by PeBL1 in tomato plants, in particular, concerning its effect on fecundity and nymphal development time.
Additionally, JA, SA, and ET increased transcription of marker genes, which suggest they play a role in aphid resistance in tomato. Aphid infestation in Arabidopsis significantly increased the transcripts of SA-related genes (PR5, PR1, and BGL2) and JA marker genes (PDF1.2 and LOX2, LOX12). Actin is a structural component in the plant cell wall [51], which undergo depolymerization through regulation in cell and cross-linking [52]. Actin depolymerization negatively correlated with aphid fecundity and population [53]. JA, SA, and ET molecules confer a certain degree of resistance against insect herbivory and pathogenic attack, enhancing plants' defense responses [35,[54][55][56]. All key genes in this study associated with JA, SA, and ET showed significant and strong up regulation, in particular, the AOS, AOC, OPR, 4CL, ACX, AIM1, KAT2, and LOX12 marker genes for the JA pathway. Increased transcription of AOS (coding for allene oxide synthase) improved aphid resistance as shown in tomato by Thompson and Moran [54]. AOC, coding an allene oxide cyclase reduced the feeding activity and survival rate by increased crop resistance [57]. PAL and 4CL, coding for a phenylalanine ammonia-lyase and a 4-coumarate-CoA ligase family protein, respectively, are involved in the construction of the cell wall, as shown in Arabidopsis [58]. ACX codes for an acyl-coenzyme A oxidase necessary for anti-insect defensive process and reproduction. AIMI and LOX code for fatty acid beta-oxidation multifunctional proteins. Lipoxygenase up regulation in JA occurs upon Pseudomonas inoculation in tomato plants [59]. KAT2 codes A 3-ketoacyl-CoA thiolase, carrying out wound-activated responses, as shown in the biosynthesis of JA in wounded Arabidopsis plants [60]. PAD4 codes for a phytoalexin-deficient 4-1 protein that mainly works in the balance of resistance (R) genes [61,62]. NPR1 is a pathogenesis-related protein involved in systemic acquired resistance [63]. SAMT codes for S-adenosyl-L-methionine-a carboxyl methyl transferase that functions in systemic and local defense responses [64]. SlERF3/LeERF3b ethylene response factors are involved in stress response and significantly increase broad spectrum resistance in tomato [65]. TAR 2 codes for tryptophan amino transferase in ethylene-mediated signaling functions in plant defense against bacteria [66]. Findings from this study confirm the activation by M. persicae of JA, SA, and ET pathways-associated genes [56,67].

Conclusions
Data showed aphid resistance in tomato with increased developmental time of each nymphal instar, associated to a lower fecundity of M. persicae. Aphid colonization was also affected by increased concentrations of PeBL1. The resistance factors were confirmed by increasing number of trichomes and quantity of wax, which mostly are involved in mechanical defenses. Physical defense response induced by PeBL1, JA, SA, and ET participated in a global plant physical response. However, some problems need to be resolved in future, such as "whether composition of wax influences or not the aphid behavior"; "how JA, SA, and ET function in induction of resistance"; and "whether or not other plant hormones are involved in this process." However, the current study provided evidence that PeBL1 isolated from B. laterosporus strain A60 could be applied to tomato seeds and seedlings to protect plants from M. persicae.
Author Contributions: K.J. designed and performed the whole experiment, data analysis, and paper writing. D.Q. provided help in managing the whole experiment and revising the article. All authors have read and agreed to the published version of the manuscript. Acknowledgments: Humayun Javed provided help in data analysis and revising the article. Kashan Khan helped with extensive language editing and data presentation of article. The experiment was carried out in the key Laboratory of Bio pesticides and Engineering, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, P.R. China.

Conflicts of Interest:
The authors declare no conflicts of interest.