Insecticidal characteristics and structural identification of the potential active compounds from Streptomyces sp. KR0006: Strain improvement through mutagenesis.

Pest control by biological means is an effective, eco-friendly, and promising method that typically involves compounds naturally derived from actinomycetes. Thus, the present study aimed to screen, characterize, and identify the structure of insecticidal compounds from Streptomyces sp. KR0006 and increase the activity through mutagenesis. In the examination of the insecticidal activity level of the isolates, Streptomyces sp. KR0006 metabolite showed significant activity against larvae and moths of Plutella xylostella. Taxonomic analyses of the 16S rRNA gene sequences revealed that the isolated KR0006 strain tended to be 99% consistent with Streptomyces cinereoruber strain NBRC 12756. Three active compounds isolated from the culture filtrate of KR0006 were purified by solvent partition, mid-pressure liquid chromatography (MPLC), Sephadex LH20 column chromatography, and high-performance liquid chromatography (HPLC). By performing 1H-NMR, 13C-NMR, and 2D-NMR experiments, and high-resolution electrospray ionization mass spectrometry analysis, the 316-HP2, 316-HP3, and 316-HP5 compounds were inferred as antimycin A3a (MW, 519.; C26H36N2O9), antimycin A8a (MW, 534; C27H38N2O9), and antimycin A1a (MW, 548; C28H40N2O9) respectively. Mutant U67 obtained from exposure to ultraviolet (UV) irradiation (254 nm, height 17 cm) for 70 seconds resulted in a 70% more larval mortality than that of the initial wild culture. The second mutation of the culture broth enhanced insecticidal activity by 80 and 100% compared with the first mutation and initial medium, respectively. Our study found that Streptomyces sp. KR0006 strain produces insecticidal active compounds and could be used for practical pest management.

Introduction

Chemical insecticides are most widely used to protect crop plants from harmful insects since they have high efficiency in pest control. At the same time, there is a common concern associated with the negative impact of insecticides on the environment, humans, and non-target organisms due to chemical pollution, accumulation of toxic residues in soil and water, and development of pesticide resistance in pests [1, 2]. In addition, the use of synthetic pesticides leads to the reproduction of insect pests and the proliferation of secondary pests and ultimately disrupts the ecosystem’s ecological balance by killing natural pest predators [3, 4]. For these reasons, there is a need to develop and implement safe and environmentally friendly biological approaches to conventional chemical practices in pest management.

Bio-insecticidal metabolites as part of biological control in pest management have a great interest and are being broadly studied by researchers as they are eco-friendly, selective, biodegradable, and non-toxic natural products [3, 5–9]. Secondary metabolites of microbes are interpreted as low molecular weight substances [10], and this property is preferred in the development of biocontrol agents. Only in India, about 970 microbial compounds from at least 15 microorganism species were registered for use in pest management [11]. Entomopathogenic fungi such as Beauveria bassiana, Metarhizium anisopliae, Lecanicillium lecanii, Nomuraea rileyi, Hirsutella thompsonii, etc. showed excellent insecticidal activity [12-16]. For instance, Isaria tenuipes against the dengue vector Aedes aegypti (Linn.) and mycotoxins from Beauveria bassiana and Metarhizium anisopliae against Spodoptera litura larvae exhibited target-specific activity by blocking the activity of detoxifying enzymes such as carboxylesterase (α and ß) and superoxide dismutase [17, 18]. Among the biological control agents derived from microorganisms, actinomycetes also represent one of the most important and widely studied microbial resources because they produce various forms of active compounds for use as pesticides [19-21]. Streptomyces spp. is a major group of actinomycetes inhabiting the soil that shows insecticidal activity. An active compound derived from Streptomyces diastatochromogenes isolate showed significant insecticidal activity with LC50 value of 10.26 mg/L against Mythimna separate [22]. Actinomycete isolates inhabiting the soil within the plants of the Asteraceae family in Pakistan showed 100% mortal activity against the first and fourth instar stages of Culex quinquefasciatus larvae [23]. Kim et al. [7] reported that active compounds isolated from Streptomyces exhibited insect growth regulatory and insecticidal activities against Aedes albopictus and Plutella xylostella. Gopalakrishnan et al. [24] also reported a novel metabolite purified from Streptomyces sp. CAI-155 has excellent larvicidal activity against second instar Helicoverpa armigera in both laboratory and greenhouse assays. Prasinons, avermectin doramectin, milbemycin, nanchangmycin, spinosad, and dianemycin have been derived as active compounds from the genus Streptomyces spp., against a variety of pest insects [25-28].

From the economic point of view, the production and application of the secondary metabolites from wild-type strains do not justify themselves much. Therefore, it is necessary to find ways to improve activity and achieve several times higher efficiency of the isolated secondary metabolites. A number of approaches such as mutagenesis, genetic recombination, selection, or improvement of fermentation processes have been studied and applied in practice to enhance the yield and activity of microbial strains [29-32]. Among them, the UV irradiation method is one of the most convenient, efficient, safe, and widely used in microbial strain improvement [30, 33, 34].

Thus, this study aimed to screen, characterize, and identify the structure of insecticidal compounds from Streptomyces spp. KR0006 and increase the activity through mutagenesis.

Materials and methods

Isolation of actinomycetes and fermentative incubation

A total of 850 Actinomycetes were isolated from the soil collected in the Jeongseon area, Gangwon-do, Korea using HV (humic acid-vitamin) and Bennet’s agar medium. The Actinomycetes strains were fermented in a 500 ml baffled Erlenmeyer flask containing 100 ml M3 culture media (6 g CaCO3, 20 g glucose, 20 g soytone, 40 g soluble starch, and 1 L distilled water, pH of 6.9), which were incubated on a rotary shaker (250 rpm) at 27°C for seven days. Fermentative broth cultures were subjected to centrifugation at 11,300 RCF (Relative Centrifugal Force) for 10 minutes and the supernatant was used for further study of insecticidal activity.

Experimental insect

The diamondback moths (Plutella xylostella) and larvae were reared on cabbage leaves and maintained at 25°C and 65% relative humidity with a 12 h light/12 h dark cycle.

Bio-insecticidal assay

Insecticidal activities of actinobacterial culture media against P. xylostella larvae were evaluated using cabbage leaf disk dipping assay and whole plant assay. Chinese cabbage leaf disks (diameter 3 cm) were dipped for 30 seconds in an undiluted culture filtrate. Distilled water was used as an untreated control. Once the leaf disks dried, they were inoculated with P. xylostella 2nd instars (10 larvae/disk). The number of dead larvae was checked at 24 h intervals and calculated three days after treatment. In the case of the whole plant assay, 10 mL culture filtrate was sprayed on the foliage of Chinese cabbage (2 plants per pot) growing for 10 days under greenhouse conditions (30/25 ± 5°C, light/ dark = 14/10 h). Then, plants were inoculated with P. xylostella 2nd instars (10 larvae/plant). The larvicidal and antifeeding activities were evaluated in the cabbage seedlings 5 days after inoculation (DAI). All experiments were independently repeated three times.

The effect of ovipositional deterrent and controlling the larvae of P. xylostella was investigated using Chinese cabbage (40 seedlings per tray) grown for 10 days in a greenhouse. The culture filtrate 50mLwas first sprayed on the cabbage seedlings and the tray was put in a plastic box (30x25x30cm) and then inoculated with a diamondback moth (50 adults/box). At six DAI, the culture filtrate was secondarily applied to half of the tray. The fecundity and hatching rate and larval mortality were determined by comparing the first and secondary treatments with the untreated control. As a result of preliminary experiments, the activity of biological control agents (BCAs) Bacillus thuringiensis and chemical insecticides (Diflubenzuron) showed 100% insecticidal activity against diamondback moth when treated at the recommended treatment concentration (Table in S1 Table). All experiments were independently repeated three times.

Taxonomic characteristics of the strain

To conduct taxonomic characteristics of the selected strain codded as KR0006, 16S rRNA gene nucleotide sequencing, and PCR amplification analyses were performed as reported by Bo et al. [35]. PCR products were commissioned to Macrogen Co., Ltd. (Daejeon, Korea) to perform gene sequencing. The sequences of the secured 16S rRNA gene obtained were analyzed through the BLASTn program of NCBI (http://www.ncbi.nlm.nih.gov) and were aligned with similar sequences using the CLUSTAL W program (MEGA 6.0). The phylogenetic tree of the strain was created using the Neighbor-joining method in the MEGA 6 program [36, 37]. The bootstrap analyses were implemented with 1000 replications to evaluate the stability of the constructed phylogenetic tree.

Purification and structural identification of the active compounds

To separate the active substance produced by the strain KR0006, the culture filtrate(6L) was portioned twice with an equal volume of the Ethyl acetate (EtOAc) as reported by Kim et al. [38] and concentrated in vacuo. EtOAc extract(1.85g) was purified by Mid-pressure liquid chromatography (MPLC) system eluted with a gradient of increasing methanol concentration (70% to 100%) in water, followed by Sephadex LH-20 (Phamarcia, Uppsala, Sweden) column chromatography eluted with 100% methanol.

The insecticidal activity of each obtained fraction was evaluated by conducting the leaf disk-feeding assay (see bio-insecticidal assay). The active fraction was finally purified by preparative HPLC on an ODS C18 reverse-phased silica gel column (Atlantis T3, 5μm, 10x250mm; WATERS) using 75% acetonitrile containing 0.02% trifluoroacetic acid over 60 minutes at a flow rate of 3ml minute-1.

NMR spectra were recorded on a Bruker AVANCE HD 800 NMR spectrometer (800 MHz for 1H and 200 MHz for 13C) and Bruker AVANCE HD 700 NMR spectrometer (700 MHz for 1H and 175 MHz for 13C) at Korea Basic Science Institute (KBSI) in Ochang, Korea. NMR spectra were recorded in DMSO-d6 and chemical shifts were referenced to the residual solvent signal (δC 39.5, δH 2.50). High-resolution electrospray ionization mass spectrometry (HR-ESIMS) data were acquired on a Q-TOF mass spectrometer (SYNAPT G2, Waters) at KBSI, Ochang, Korea.

Strain activity improvement through Mutagenesis–UV mutation

The strain KR0006 spores was exposed to UV rays at a distance of 17 cm from the UV lamp (DESAGA, Sarstedt-Gruppe, MinUVIS UV Lamp) (λ = 254nm) for 30, 45, 60, and 90 seconds. All these exposures were performed in a dark room to avoid any photoreaction in the production of mutants. The spores were then spread in plates containing Bennet’s agar. The plates were incubated at 27°C for 72 hours. The method employed for UV mutagenesis was described earlier by Khattab & Mohamed [39] and Wang et al. [40].

The survival and lethality rates were assessed after UV exposure and survived mutant colonies were cultured on an M3 liquid medium as described earlier (see fermentative incubation). Once mutant culture supernatants were harvested, the activity of each mutant was screened on the larvae of P. xylostella by conducting the leaf disk-feeding assay as described earlier (see bio-insecticidal assay).

The mutant strain that exhibited the highest insecticidal activity was selected and subjected to second-time mutagenesis aimed to increase the activity of the mutant obtained from the first mutation. The procedures of mutation and screening taken in the first mutation were carried out exactly the same way in the second mutation.

Statistical analysis

One-Way ANOVA statistical analysis was performed using OriginPro 8.1 [41]. The Fisher’s LSD test was used to compare the mean value, and the means values < 0.05 were considered statistically significant.

Results

Insecticidal activities of Streptomyces isolate

As a result of screening 850 actinomycetes for their insecticidal activity on the larvae of P. xylostella using the leaf disc feeding assay, high insecticidal activity (more than 80% mortality) was found in six actinomycete strains (KRA18-68, KRA18-104, KRA18-290, KR0512, KR0006 and KR0603) (Fig 1). Among these six strains, the KR0006 strain showed high insecticidal activities with mortalities of 100% after three days of treatment and was selected for further studies.

Fig 1

Insecticidal activity of the actinobacteria strains against Plutella xylostella.

Second instar larvae of P. xylostella were treated with undiluted culture filtrate of each actinobacterium strain using the cabbage leaf dipping method. The mortality was evaluated three days after inoculation. The vertical bars indicate the standard errors of the means and different letters above the error bars indicate a significant difference in the Fisher’s LSD test (P < 0.05).

In an experiment using whole plants, it was confirmed that the larvae control effect was 90% in 5-fold dilution at 5 DAI, but the mortality rate was significantly reduced when treated with 10-fold dilution. It was confirmed that the insecticidal activity of the KR0006 culture filtrate was concentration-dependent and the insecticidal effect increased with time after treatment (Fig 2). In addition, cabbage seedlings treated with even high-concentration of KR0006 culture filtrate, some feeding marks appeared on the cabbage leaves due to larvae activity at the initial stage of treatment, but the larvae were confirmed to be completely dead (Fig 3).

Fig 2

Concentration-dependent insecticidal activities of KR0006 against Plutella xylostella.

The 10mL diluted solutions of the KR0006 were sprayed on the foliage of Chinese cabbage (2 plants) growing in the greenhouse. Plants were inoculated with P. xylostella 2nd instars (10 larvae/plant) and the mortality was calculated five days after inoculation. The vertical bars indicate the standard errors of the means.

Fig 3

Insecticidal activities of the broth filtrate of KR0006 against Plutella xylostella 2nd instars in greenhouse conditions.

Representative pictures were taken five days after inoculation. (A); 2-fold dilution of broth filtrate, (B); untreated control.

The oviposition deterrent activity evolution experiments revealed that the occurrence of the larvae was considerably less than that of the untreated control due to the inhibition of fecundity and the hatching of diamondback moths by the broth filtrate of KR0006. Also, the feeding marks caused by larvae after the secondary treatment of the culture filtrate were similar to those of the primary treatment, which is because the feeding ability of the larvae was inhibited by the secondary treatment of culture filtrate (Fig 4). Thereby, we confirmed that the culture filtrate of the KR0006 strain showed insecticidal activity by inhibiting the oviposition of moths and the growth and development of larvae.

Fig 4

The ovipositional repellency and larvicidal activity of Streptomyces sp. KR0006 against diamondback moth.

The culture filtrate was first sprayed on the cabbage seedlings grown in the tray, and then 50 diamondback moths were inoculated. Six days after inoculation (DAI), the culture filtrate was secondarily applied to half of the tray. (A); 6 DAI control, (B); 1st treatment of broth filtrate, (C); 10 DAI control, (D); 2nd treatment of broth filtrate.

Taxonomic classification of the strain KR0006

The PCR technique using universal primers was performed to obtain the nucleotide sequence of the 16S rRNA gene of the KR0006 strain. An obtained nucleotide sequence was compared with the nucleotide sequence of other Streptomyces strains through the BLASTn program of NCBI (http://www.ncbi.nlm.nih.gov). Results of comparative analysis with similar bacterial strains through 16S rRNA sequencing analysis showed that this strain tended to be 99% consistent with the Streptomyces cinereoruber NBRC 12756 strain (Fig 5).

Fig 5

Neighbor-joining tree of strain KR0006 based on its 16S rRNA gene sequence showing the phylogenetic relationship with related Streptomyces species.

Numbers at each branch node indicate the bootstrap percentage of 1000 replications.

Purification and structural determination of the active compounds

The fermentation broth (10 liters) of strain KR0006 was partitioned between the EtOAc and H2O. The EtOAc soluble portion was further separated by HPLC analysis using 75% acetonitrile containing 0.02% trifluoroacetic acid to yield five compounds 316-HP1–5 (Fig 6). Among the five compounds, the chemical structure of 316-HP2, 3, and 5 compounds that showed insecticidal activity against P. xylostella were determined by spectroscopic methods.

Fig 6

High-performance liquid chromatography chromatogram of compounds 1~5 produced by Streptomyces sp. KR0006 and UV spectrum.

Column, waters Atlantis T3 column (i.d. 4.6x250 mm); mobile phase, 70% acetonitrile/0.02% TFA; flow rate, 0.8 ml/min; detection, 230nm.

By performing 1H-NMR, 13C-NMR, and 2D-NMR experiments, and high-resolution electrospray ionization mass spectrometry analysis, the 316-HP2, 316-HP3, and 316-HP5 compounds were inferred as antimycin A3a, antimycin A8a, and antimycin A1a respectively (Fig 7). The structural features of antimycin A having methyl substituents at 2- and 6-positions, n-hexyl substituents at 8-positions, acyloxy substituents at 7-positions and an aroylamido substituent at 3-positions in the three active compounds are confirmed. Antimycin A is an optically active, nitrogenous phenol with the molecular formula C28H40 N2O9 isolated from a culture broth of Streptomyces strain in 1949 [42]. The isolation of antimycins A1 to A16 have been reported so far, and each of antimycins A1~A8 is a mixture of two isomers containing a closely related alkylacyl group [43].

Fig 7

The structures of the antimycins purified from Streptomyces sp. KR0006.

For antimycin A8a (316-HP3), high-resolution mass spectrometry ([M+H]+, m/z 535.2667; [M+Na]+, m/z 557,2486) gave a molecular formula of C27H38N2O9. This requires that the alkyl side chain has a formula of C5H11. COSY connectivities from a methine at 2.53ppm (7-H) to methylene hydrogens at 1.65 and 1.36 ppm (Hα), from Ha to methylene at 1.06ppm (Hβ), from Hα to methane at 1.45 ppm (Hγ), and from Hγ to a six hydrogen methyl doublet at 0.83 ppm (Hα and Hβ), indicated that the alkyl side chain is isopentyl (3-methyl butyl). HMBC connectivities from Hδ and Hε to Cγ and Cβ support this side-chain structure [44]. The structure determination of antimycin A8 was first reported in 1997 by Barrow et al. [45] and was determined similarly to that of A7, and differed from antimycins A1 and A3 only at the 7-alkyl side chain.

Active compound 316-HP2 was inferred as antimycin A3a with the formula of C26H36N2O9 ([M+H]+, m/z 520.2327). It was confirmed that this compound is significantly similar to the compound 316-HP3. However, there was no branched CH peak, as it appeared to be a linear alkyl in the side chain. The compound 316-HP5 with the formula of C28H40N2O9 ([M+H]+, m/z 549.2819; [M+Na]+, m/z 571.2637) was identified as antimycin A1a, a tautomer of the 316-HP3. Spectroscopic data for antimycins A1~A4 were in agreement with literature data [46-48].

Strain activity improvement through mutagenesis–UV mutation

The number of mutant colonies decreased with the increases in UV exposure time. Among the tested UV mutants, the highest activity was witnessed in mutant U67. Larval mortality at five-fold dilution broth filtrate of mutant U67 was 70% after 24 hours of treatment, and 100% after 48 hours of treatment, confirming that the insecticidal effect was higher compared to other mutants and wild type (Table 1).

Table 1

Insecticidal activity of the mutants against Plutella xylostella larvae.

Treatments	Larval mortality (%)
	After 24h	48h	72h
Mutant—U21	0±0e	60±5c	95±4a
U22	0±0e	70±5b	70±5b
U23	30±2c	75±5b	100±0a
U24	0±0e	50±4c	80±10b
U25	0±0e	15±3d	92±2a
U26	0±0e	0±0e	100±0a
….	. . .	…	…
U67	70±5a	100±0a	100±0a
U73	50±5b	80±10b	100±0a
….	. . .	…	….
Wild type	5±2d	20±5d	45±5c

To estimate the larvicidal activity, Chinese cabbage leaf disks (diameter 3 cm) were dipped for 30 seconds in five-fold diluted (x1/5) each mutant and wild broth filtrate solutions. Once the leaf disks surface-dried they were inoculated with P. xylostella 2nd instars (10 larvae/disk). Values are the mean of three replication and different letters in each column indicate significant differences in the Fisher’s LSD test (P < 0.05).

In the secondary UV mutagenesis using mutant U67, the mutant U67-46 exhibited the highest larval mortality and was selected. The larvicidal activity of mutant U67-46 in the leaf disk feeding assay was 98% and 65% when treated with 20-fold and 30-fold dilutions, respectively (Table 2). In the case of mutant U67, the larval mortality was 55% when treated with 10-fold diluted culture filtrate indicating that the insecticidal activity of the mutant U67-46 was increased 3 times compared to the U67, and more than 5 times compared to the wild type (Table 2).

Table 2

Insecticidal activity of KR0006 (WT) and mutants (first and second) against Plutella xylostella.

Dilution fold	Larval mortality (%)
	KR0006 (WT)	U67	U67-46
1	100±0a	100±0a	100±0a
2	100±0a	100±0a	100±0a
5	90±3a	100±0a	100±0a
10	15±4b	55±3b	100±0a
20	0±0c	25±3c	98±2a
30	0±0c	0±0d	65±10b

Values are the mean of three replication and different letters in each column indicate significant differences in the Fisher’s LSD test (P < 0.05).

Discussion

To overcome the unwanted effects of synthetic pesticides such as chemical pollution, accumulation of toxic materials, and development of pesticide resistance, studies for eco-friendly compounds as alternatives have been extensively conducted and continuously utilized [49]. More specifically, insecticidal, insect growth regulatory, and antifeedant activities of actinomycetes have been studied and confirmed that actinomycetes produce a range of chemical structures [4, 6, 50]. In this study, larvacidal activity against P. xylostella was investigated in 850 actinomycetes targeting the discovery of active compounds with insecticidal activity. The larval mortality was over 80% in the initially selected six strains, and among them, strain KR0006 showed the highest activity.

Antimycins are known as secondary metabolites produced by Streptomyces and other types of soil bacteria and provide competitive advantages to kill neighbouring organisms such as insects, nematodes, fungi [51] and virus [52]. Several studies reported that the antimycin family of compounds has exhibited a broad spectrum of insecticidal and antifungal activities. Antimycin A9 isolated from fermentative broth of Streptomyces sp. K01-0031 exhibited insecticidal activity against Artemia salina and nematocidal activity against Caenorhabtitis elegans [53] and antimycins A10-16 isolated from fermentative broth of Streptomyces spp. SPA-10191 and SPA-8893 showed antifungal activity against Candida utilis [43]. Yan et al. [54] found the moderate fungicidal activity of antimycin A18 produced by Streptomyces albidoflavus. In our study, three active compounds identified as antimycin A3a, antimycin A8a, and antimycin A1a were purified from the culture broth of Streptomyces sp. KR0006. Our results coincided with recent results by Kim et al. [7] who had isolated five antimycins as active compounds from Streptomyces celluloflavus displayed significant insecticidal activity against P. xylostella, A. albopictus, T. urticae, and F. occidentalis. In addition to insecticidal and larvicidal activity, our results revealed that the culture broth of Streptomyces sp. KR0006 showed a considerable oviposition suppressing activity against the Diamondback moth. Although the antifeedant, larvicidal, and growth inhibitory activity of the secondary metabolites isolated from Streptomyces spp. has been reported [4, 7, 54, 55], the inhibition of oviposition has not been adequately studied yet. Taking into account high-level insecticidal, larvicidal, and spawn suppressing the activities of the strain KR0006, interest has arisen in conducting additional experiments of increasing the activity of this strain.

Strain improvement can be successfully achieved through conventional selection, UV mutation, or genetic recombination [30]. The activity or productivity of several microbial strains had been enhanced by UV treatment [56-59]. It became clear from our experiments that exposure to ultraviolet (UV) irradiation (254 nm, height 17 cm) for 70 seconds to KR0006 strain improved the larvicidal activity by 70%. There are very rare studies available on UV mutation for increasing the insecticidal or herbicidal activity of metabolites produced by Streptomyces. However, our results were closely in agreement with research finding where two times enhancement was observed in endoglucanase activity of UV treated Aspergillus niger strain [60] and up to 57.4% increase in Streptomyces griseoaurantiacus strain [58]. To achieve an even better activity, we subjected the selected mutant strain to the secondary UV irradiation which resulted in an improvement in insecticidal activity by 80 and 100% compared with the first mutant and initial wild strains respectively. There was no back mutation observed even after the second mutation which is common in most microbe mutation studies [56]. Our mutagenizes experiments confirmed that proper UV mutation can be forwarded as a tool to increase the activity of secondary metabolites produced by Streptomyces.

Conclusion

In conclusion, Streptomyces sp. KR0006 containing antimycin A8a and its tautomers antimycin A3a, and antimycin A1a as active substances showed high insecticidal activity against Plutella xylostella. The culture filtrate of the KR0006 strain showed an insecticidal and larvicidal effect by inhibiting the oviposition of moths and the growth and development of larvae. The insecticidal activity of the culture filtrate was concentration-dependent. The yield of effective insecticidal substances produced by KR0006 was increased through mutagenesis by UV radiation. The insecticidal activity of 2nd mutant U67-46 increased 3 times compared to 1st mutant U67, and more than 5 times compared to the wild type.

In our study, Streptomyces sp. KR0006 was found to be a valuable biological control tool against diamondback moth (P. xylostella) and can be used to form an environmentally safe natural product in the ecofriendly pest management. However, further research is needed to make clear the mechanisms of insecticide action and evaluate the effectiveness in the open field.

Insecticidal activity of the biological control agent (Bacillus thuringiensis) and chemical insecticide (Diflubenzuron) against Plutella xylostella larvae.

To estimate the larvicidal activity, Chinese cabbage leaf disks (diameter 3 cm) were dipped for 30 seconds in the recommended concentration of insecticide solutions. Once the leaf disks were surface-dried they were inoculated with P. xylostella 2nd instars (10 larvae/disk). Distilled water was used as untreated control. Letters in each column indicate significant differences in the Fisher’s LSD test (P < 0.05).

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Reviewer #1: This manuscript presents “Insecticidal characteristics and structural identification of the potential active compounds from Streptomyces sp. KR0006: Strain improvement through mutagenesis.”. This manuscript formulated in clear and concise manner with well-supported statements. The problems arisen were well-addressed.

The regulatory mechanisms of these naturally derived compounds from actinomycetes were exhibited well enough, while these findings provide new insights how to control pests, which depends on biological approaches rather than chemicals.

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Please refer to the observations/suggestions available as notes in the enclosed file.

The authors should also check the following references since are related to the same topic:

1. Cao, Haijing, et al. "5′-Epi-SPA-6952A, a new insecticidal 24-membered macrolide produced by S treptomyces diastatochromogenes SSPRC-11339." Natural product research 33.5 (2019): 659-664.

2. Gopalakrishnan, Subramaniam, et al. "Insecticidal activity of a novel fatty acid amide derivative from Streptomyces species against Helicoverpa armigera." Natural Product Research 30.24 (2016): 2760-2769.

3. Tanvir, Rabia, Imran Sajid, and Shahida Hasnain. "Larvicidal potential of Asteraceae family endophytic actinomycetes against Culex quinquefasciatus mosquito larvae." Natural Product Research 28.22 (2014): 2048-2052.

**********

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Reviewer #1: Yes: Botir Khaitov

Reviewer #2: No

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Submitted filename: PONE-D-22-11701.pdf

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6 Jul 2022

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

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• After carefully reading the formatting style, the authors followed wholly to meet the manuscript PLOS ONE’s style requirements.

2. Financial disclosure:

• This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agricultural Machinery/Equipment Localization Technology Development Program, funded by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) (321056-05). However, the funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. This manuscript should be published in OPEN ACCESS. I will state the role of the Funder statement in the cover letter too and please change the online submission form on our behalf.

Additional Editor Comments:

3. The manuscript needs substation revision and the reference section should be updated and discuss more bio-pesticides including microbial pesticides in the introduction as well in the discussion least the author should refer to the following documents and rewrite the advantages of biocontrol potential of bio-pesticides. Please refer to several articles published recently in Plos One.

I suggest the author go through all the points suggested by the reviewer and provide a point-by-point explanation about your revision. In not please provide a suitable rebuttal to avoid the next round of revision.

Introduction:

Please refer my first point, it needs more information about biopesticides through microbial culture with recent updates. # refer doi:10.3390/jof6040196, 10.1016/j.jip.2018.10.008, 10.1016/j.ecoenv.2019.109474, 0.1007/978-81-322-2056-5_3,

The result, discussion, and Conclusion section need to develop according to your result and appropriate citations must be provided to justify your claim.

• Thank you for your valuable comments. The manuscript was revised and references and all the citations were also updated. In the introduction and discussion sections, the authors tried to discuss more detail bio-pesticides, especially microbial pesticides. Authors read all the literature suggested by the editor and reviewers and cited where needed. A number of articles published in PLOS ONE were also cited in the manuscript. Almost all the sections of the manuscript were rewritten and the manuscript was developed (refer to the manuscript file).

Review’s Comments to the Author

Reviewer #2: There are several aspects that need clarifications/implementation.

Please refer to the observations/suggestions available as notes in the enclosed file.

The authors should also check the following references since are related to the same topic:

Cao, Haijing, et al. "5′-Epi-SPA-6952A, a new insecticidal 24-membered macrolide produced by Streptomyces diastatochromogenes SSPRC-11339." Natural product research 33.5 (2019): 659-664.

Gopalakrishnan, Subramaniam, et al. "Insecticidal activity of a novel fatty acid amide derivative from Streptomyces species against Helicoverpa armigera." Natural Product Research 30.24 (2016): 2760-2769.

Tanvir, Rabia, Imran Sajid, and Shahida Hasnain. "Larvicidal potential of Asteraceae family endophytic actinomycetes against Culex quinquefasciatus mosquito larvae." Natural Product Research 28.22 (2014): 2048-2052.

• Thank you for your valuable comments. The authors read carefully the suggested papers by the reviewers and cited them where needed. The authors took into consideration the suggestions and comments in the enclosed file given by the reviewers and the manuscript was revised according to those.

Submitted filename: Response to Reviewers.docx

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1 Aug 2022

Submitted filename: PONE-D-22-11701_rev.pdf

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10 Aug 2022

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

• After carefully reading the formatting style, the authors followed wholly to meet the manuscript PLOS ONE’s style requirements.

2. Financial disclosure:

• The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

3. The guidelines for resubmitting your figure files

• Thank you for showing us the guideline for adjustments of figure files. Now, all figure files are adjusted to the journal requirements and resubmitted.

Review’s Comments to the Author

1 80- it should be better to report the G value instead rpm

2 115- did you compare the results also with those available on Genebank?

3 205- you should compare the experimental data with those reported in literature for these compounds since are all already described. The relevant references should be provided.

4 209- if you are referring to the molecular formula it should be considered that the species [M+H]+ is the protonated form of and its empirical formula is different. It is formally one ion positively charged. It should be more clear.

5 Fig7- HMBC correlations cannot be checked without the experimental spectra (mono and bidimensional NMR)

• Thank you for your valuable comments.

1 Rpm value of centrifuge was changed into g value in other word RCF (Relative Centrifugal Force)

2 Yes, obtained sequences of the secured 16S rRNA gene were analyzed through the BLASTn program of NCBI (http://www.ncbi.nlm.nih.gov).

3 Yes, we compared the experimental data with those reported in the literature for these compounds, and relevant references were cited where they are needed. Please refer to the manuscript file.

4 Thank you for your valuable comment. The structural determination part of the manuscript was deeply reviewed and revised. Please refer to the tracked manuscript or manuscript file to see corrections.

5 Figure seven is now provided with the experimental spectra (mono and bidimensional NMR).

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

4 Sep 2022

Insecticidal characteristics and structural identification of the potential active compounds from Streptomyces sp. KR0006: Strain improvement through mutagenesis

PONE-D-22-11701R2

Dear Dr. Umurzokov,

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Additional Editor Comments (optional):

The manuscript can be accepted now

Reviewers' comments:

8 Sep 2022

PONE-D-22-11701R2

Insecticidal characteristics and structural identification of the potential active compounds from Streptomyces sp. KR0006: Strain improvement through mutagenesis

Dear Dr. Choi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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