A Novel Insecticidal Peptide SLP1 Produced by Streptomyces laindensis H008 against Lipaphis erysimi

Aphids are major insect pests for crops, causing damage by direct feeding and transmission of plant diseases. This paper was completed to discover and characterize a novel insecticidal metabolite against aphids from soil actinobacteria. An insecticidal activity assay was used to screen 180 bacterial strains from soil samples against mustard aphid, Lipaphis erysimi. The bacterial strain H008 showed the strongest activity, and it was identified by the phylogenetic analysis of the 16S rRNA gene and physiological traits as a novel species of genus Streptomyces (named S. laindensis H008). With the bioassay-guided method, the insecticidal extract from S. laindensis H008 was subjected to chromatographic separations. Finally, a novel insecticidal peptide was purified from Streptomyces laindensis H008 against L. erysimi, and it was determined to be S-E-P-A-Q-I-V-I-V-D-G-V-D-Y-W by TOF-MS and amino acid analysis.


Introduction
Actinobacteria are considered as important reservoir of antibiotics and pesticides [1,2].Streptomyces species as actinobacteria are able to produce abundant metabolites with the insecticidal, antimicrobial, anti-tumor, and immunosuppressant activities [3,4].Aphids are exclusive phloem feeders of the great majority of important plants in agriculture, horticulture and forestry, causing damage by direct feeding and transmission of plant diseases [5].Mustard aphid (also known as Turnip aphid), Lipaphis erysimi (Rhopalosiphum pseudobrassicae), is a major insect pest attacking many closely related vegetables, including mustard, broccoli, cabbage, collards and radish [6].Peptides are a valuable alternative to chemical pesticides for control of insect pests in agriculture because of their high kill efficiency, pest-specificity and their safety for the environment [7,8].Recently, the peptides that are from Actinobacteria or other microorganisms exhibited their insecticidal potential [9,10].However, the insecticidal peptide from Actinobacteria against L. erysimi has never been found before as far as we know.This study was to discover a new actinobacteria that can produce a new insecticidal peptide against L. erysimi.Therefore, we isolated many actinobacteria from the soil and investigated their insecticidal activity.Of them, the strain H008 showed a good insecticidal activity against L. erysimi, and S. laindensis H008 was identified as a novel species.Finally, we purified and characterized a novel insecticidal peptide SLP1 (means Streptomyces laindensis H008's peptide No. 1) from S. laindensis H008 against L. erysimi.

Microorganisms
A total of 180 bacterial strains were obtained from the soil samples.The strain H008 exhibited the highest insecticidal activity.The strain H008 was identified by its morphological and physiological traits and molecular biological methods.

Morphological and Physiological Traits of Strain H008
The cultural characteristics of strain H008 are presented in Table 1.The strain H008 belongs to a spiral type, and its sporophore morphology was shown in Figure 1.The physiological and biochemical characteristics of strain H008 was shown in Table 2.The strain H008 utilized D-fructose, D-galactose, D-glucose, D-xylose, D-mannitol and sucrose as carbon sources, indicating its wide pattern of carbon assimilation.It exhibited salt tolerance up to 7% that may be placed in the intermediate group of salt tolerance.The melanin reaction was positive on ISP-6 medium.It was also found positive for biochemical tests like H 2 S, liquefaction of gelatin, starch hydrolysis, nitrate reduction and citrate utilization.Based on its morphological and physiological traits (Tables 1 and 2, Figure 1), the strain H008 was a new species of the genus Streptomyces [11][12][13][14][15].

Morphological and Physiological Traits of Strain H008
The cultural characteristics of strain H008 are presented in Table 1.The strain H008 belongs to a spiral type, and its sporophore morphology was shown in Figure 1.The physiological and biochemical characteristics of strain H008 was shown in Table 2.The strain H008 utilized D-fructose, D-galactose, D-glucose, D-xylose, D-mannitol and sucrose as carbon sources, indicating its wide pattern of carbon assimilation.It exhibited salt tolerance up to 7% that may be placed in the intermediate group of salt tolerance.The melanin reaction was positive on ISP-6 medium.It was also found positive for biochemical tests like H2S, liquefaction of gelatin, starch hydrolysis, nitrate reduction and citrate utilization.Based on its morphological and physiological traits (Tables 1 and 2, Figure 1), the strain H008 was a new species of the genus Streptomyces [11][12][13][14][15].

Molecular Biological Identification of Strain H008
The 16S rDNA sequence of Streptomyces sp.H008 was amplified by the general primers for the Streptomyces species.The PCR yielded 1.053 kb 16S rDNA of Streptomyces sp.H008.The GenBank Accession number of Streptomyces sp.H008 was KX002028.The 16S rDNA sequence of Streptomyces sp.H008 was investigated by a similarity analysis (BLAST: https://blast.ncbi.nlm.nih.gov/Blast.cgi) and a phylogenetic analysis (Figure 2).The Hasegawa-Kishino-Yano model was selected to construct the Maximum Likelihood (ML) tree by finding the best model for ML tree after the alignment.All of these results indicated that strain H008 was a new species of genus Streptomyces that was named S. laindensis H008.

Molecular Biological Identification of Strain H008
The 16S rDNA sequence of Streptomyces sp.H008 was amplified by the general primers for the Streptomyces species.The PCR yielded 1.053 kb 16S rDNA of Streptomyces sp.H008.The GenBank Accession number of Streptomyces sp.H008 was KX002028.The 16S rDNA sequence of Streptomyces sp.H008 was investigated by a similarity analysis (BLAST: https://blast.ncbi.nlm.nih.gov/Blast.cgi) and a phylogenetic analysis (Figure 2).The Hasegawa-Kishino-Yano model was selected to construct the Maximum Likelihood (ML) tree by finding the best model for ML tree after the alignment.All of these results indicated that strain H008 was a new species of genus Streptomyces that was named S. laindensis H008.

Insecticidal Activity
To investigate the stability of the insecticidal activity against L. erysimi, the culture filtrates of the S. laindensis H008 were stored at different pH values (from 2.0 to 11.0) for 24 h, or they were treated with different temperatures (20 to 80 • C) for 1 h.After storage at pH 2.0 to 11.0 for 24 h, the insecticidal activity of the culture filtrate was slightly (but significantly) changed and the mortality was 81.73% (the lowest one at pH 11.0) to 88.81% (the highest one at pH 6.5) by using one-way ANOVA with the Fisher least significant difference (LSD) post hoc test (p < 0.05).In addition, the mortalities of the culture filtrates against L. erysimi at 20, 30, 40, 50, 60, 70 and 80 • C were 88.82%, 85.65%, 82.57%, 78.74%, 75.86%, 73.32% and 70.11%, respectively.During the purification, each fraction was investigated its insecticidal activity and the result was shown in Table 3.In two steps of purification, the fraction No. 1B 12 -1C 1 in the first step and the fraction No. 2D 1 -2D 2 in the second step showed the highest activity, respectively.

Purification
To obtain the insecticidal active extract, 20 L fermentation medium of S. laindensis H008 was subjected to filtration, precipitation, centrifugation, concentration and gel filtration.With the bioassay-guided method, the insecticidal extract was purified by preparative HPLC in two steps.First, the extract was purified by HPLC as shown in Figure 3a.In the first step, eleven fractions were obtained and each fraction was investigated its insecticidal activity as shown in Table 3.The tenth fraction (No. 1B 12 -1C 1 ) exhibited the highest insecticidal activity (95% mortality).Second, the fraction No. 1B 12 -1C 1 was purified by HPLC as shown in Figure 3b.In the second step, seven fractions were obtained and each fraction's insecticidal activity was also investigated as shown in Table 3.The fifth fraction (No. 2D 1 -2D 2 ) exhibited the highest insecticidal activity (100% mortality).Then, the purified insecticidal peptide's (fraction No. 2D 1 -2D 2 ) purity (more than 97.12%) and concentration (0.7 mg/mL) was investigated.Finally, a total of 258.2 mg of the purified insecticidal peptide was ultimately obtained from 20 L of fermentation medium.

Identification
The purified insecticidal peptide (fraction No. 2D 1 -2D 2 ) was analyzed by molecular mass determination and amino acid sequence analysis.The TOF-MS analysis showed that the molecular was 1691.377Da (Figure 4).The analysis of amino acid sequence revealed that it was a 15 amino acid peptide (named SLP1).The amino acid sequence of SLP1 was determined to be S-E-P-A-Q-I-V-I-

Discussion
According to the Kuster's key for identification of Streptomyces spp.[14], S. laindensis H008 is only closed to S. actuosus by comparing 274 named taxa of Streptomyces spp.However, S. actuosus can utilize L-arabinose, L-rhamnose and raffinose [15], and S. laindensis H008 cannot utilize anyone of them.In addition, 16SrDNA of S. laindensis H008 is closed to S. sampsonii and S. albus.However, S. laindensis H008 belongs to the group of gray aerial mycelium and no distinctive reverse pigment, and both S. sampsonii and S. albus are not in this group [14].Therefore, S. laindensis H008 is a novel species of genus Streptomyces based on its morphological and physiological traits and 16SrDNA analysis.Our result indicated that S. laindensis H008, a new species, produced a new insecticidal peptide SLP1 against L. erysimi.Therefore, Streptomyces spp.still deserve investigation of their new insecticidal peptide, although the Streptomyces species from soil have been widely researched [9,[16][17][18].
The insecticidal peptide SLP1 exhibited similar insecticidal activity against L. erysimi (100% mortality at 700 mg/L; molecular weight: 1691.38) with the insecticidal compounds of low molecular weight such as the ethylene glycol diesters (LC 50 : 1320 to 11,500 mg/L) [19], the two derivatives of Harmine (LC 90 : 240.10 mg/L and 418.63 mg/L) [20] and three insecticidal alkaloids from Cynanchum mongolicum (LC 50 : 292.48 mg/L, 367.21 mg/L and 487.791 mg/L) [21].Although the peptide SLP1 showed lower activity against L. erysimi than the protein of higher molecular weight from plants such as defensins (BjD and RiD) (LC 50 : 9.01 and 45.31 mg/L) [22] and Allium sativum leaf agglutinin (LC50: 20 mg/L) [23], the peptide SLP1 showed the good stability of pH and heat, and it is easier to be produced by a cost-effective and time-saving microbial fermentation method than cultivating plants [1,4].It is because the species of Streptomyces has a shorter life cycle and a smaller genome than plants.It is easier to get a high-producing mutant of Streptomyces spp.than plants.The production of Streptomyces spp.metabolites is more easily improved by fermentation process optimization than optimizing plant cultivation.
Taken together, the insecticidal peptide SLP1 from S. laindensis H008 had the promising potential to be developed as a new biopesticide according to its stability of pH and heat, insecticidal activity and molecular character.The biosynthesis, active mechanism, structure-activity relationship and fermentation optimization of the insecticidal peptide SLP1 deserve to be further researched in future.

Isolation
The soil samples were taken with an auger (diameter 2.5 cm) to a depth of 20 cm.Ten auger holdings were randomly collected from an agricultural field at the Institute of All-Russian Plant Conservation Biology, Russia.The soil dilution technique on glycerol-asparagine-salts agar medium.The strain was maintained on yeast extract-malt extract-dextrose (YMD) agar medium at 4 • C for further study [24].

Morphological and Physiological Traits
Cultural characters of strain H008 were studied on International Streptomyces Project (ISP) and non-ISP media [25].The micromorphology of the strain cultured on ISP medium 2 at 37 • C for 2 days was examined under scanning electron microscopy (model JOEL-JSM 5600, JOEL Limited, Akishima, Tokyo, Japan) at various magnifications [24,26].Utilization of carbon sources was investigated [27].

Molecular Biological Identification
Extraction of genomic DNA of the strain was performed according to the method described by Rainey et al. [28].The 16SrRNA gene was amplified with primers 8-27f (5 -AGAGTTTGATCCT GGCTCAG-3 ) and 1115-1100r (5 -AGGGTTGCGCTCGTTG-3 ) [29][30][31][32].The amplified DNA fragment was separated on 1% agarose gel, eluted and purified using the Qiaquick gel extraction kit (Qiagen, Hilden, Germany).The purified PCR product was sequenced using the Big-Dye terminator kit ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).The phylogenetic position of the isolated strain (H008) was assessed by performing a nucleotide sequence database search using the BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).The sequence data of related species were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/genbank/).The alignment and the phylogenetic tree were carried out by using Mega software (version 7.0 for windows) [33].The number of Bootstrap Replications was set as 1000, and the 16S ribosomal RNA gene of Kitasatospora setae strain KM-6054 was used as an outgroup sequence.

Insecticidal Activity
The aphids L. erysimi were used as a targeted aphid and they came from a laboratory colony originating from the Heilongjiang Province Academy of Sciences, Harbin, China.The wingless adults of the aphid L. erysimi were selected for the insecticidal test.The insecticidal activity of the culture filtrates, the fractions (in the purified process) and peptide SLP1 were tested by the leaf dipping method [20,21].The leaf of cabbage with the L. erysimi was cut into the leaf discs.Then, the leaf disc with 20 (for the bioassay-guided purification) or 30 (for the stability) wingless adults of the L. erysimi was dipped into the culture filtrate or other solution (the fraction or peptide SLP1 solution) for 10 s, and the excess solution was immediately absorbed with a filter paper.Water was used as a control.The leaf disc with the L. erysimi in the petri dish were held at 25 ± 0.5 • C, light period of a 14/10-h light/dark cycle (LD), and 75%-85% relative humidity (RH).Each treatment was repeated three times.The mortality and the adjust mortality (with reference to the control) were calculated after 48 h.

Purification and Identification of Insecticidal Peptide
To the culture filtrate (20 L) of strain H008 was added acetone (50:50, v/v).The extract of peptide was obtained after the centrifugation, vacuum concentration and gel filtration (Sephadex G-15 column, 1.5 cm × 50 cm) [34].With the bioassay-guided method, the extract of peptide was purified by preparative HPLC (Zorbax 300SB-C18 reverse-phase column, 4.6 mm × 100 mm, Agilent Technologies, Wilmington, DE, USA) (Solution A: 80% acetonitrile containing 0.05% TFA; Solution B: 0.065% TFA plus 2% acetonitrile) in two steps: First, the extract was purified by HPLC (0% Solution A for 6 min, followed by 0%-60% Solution A over 25 min, 1.5 mL/min, at 214 nm).Second, the fraction with the highest activity was also subjected to the second purification by HPLC (0% Solution A for 6 min, followed by 0%-50% Solution A over 20 min, 1.5 mL/min, at 214 nm) [35].After purification of HPLC in two steps, the purity of the purified peptide with the insecticidal activity was investigated by HPLC.The concentration of the peptide was determined by the different concentrations of BSA solution.
The insecticidal peptide was analyzed using the Triple TOF 5600 TOF MS Analyzer (Applied 130 Biosystems, Concord, ON, Canada), and the data were acquired in the MS scanning mode with a scan range of 250-2000 (m/z) [34,36].The insecticidal peptide was also submitted to an amino acid sequence analysis.The N-terminal amino acid sequences of the peptides were determined by Edman degradation carried out in a Shimadzu PSQ-23A protein sequencer (Shimadzu, Kyoto, Japan).

Figure 2 .
Figure 2. The phylogenetic analysis of strain H008.The sequences used in the analysis were obtained from the GenBank Database.The numbers at the branch nodes are the percentage bootstrap supports based on 1000 resample data sets.The scale bar corresponds to 0.01 substitutions per nucleotide position.

Figure 2 .
Figure 2. The phylogenetic analysis of strain H008.The sequences used in the analysis were obtained from the GenBank Database.The numbers at the branch nodes are the percentage bootstrap supports based on 1000 resample data sets.The scale bar corresponds to 0.01 substitutions per nucleotide position.

Figure 3 .
Figure 3. Purification by preparative HPLC in two steps: (a) eleven fractions in the first step; and (b) seven fractions in the second step.

Figure 3 .
Figure 3. Purification by preparative HPLC in two steps: (a) eleven fractions in the first step; and (b) seven fractions in the second step.

Table 2 .
Physiological and biochemical characteristics of strain H008.
Note: + means positive results; − means negative results.

Table 2 .
Physiological and biochemical characteristics of strain H008.

Table 3 .
Insecticidal activity of each fraction purified by HPLC.