# Enhancement of Streptomyces aureoverticillatus HN6 through mutagenesis for improved biocontrol of banana wilt disease: an WGS approach

Xiangnan Yan, Shakil Ahmad, Nazia Manzar, Yunfei Zhang, Coline C Jaworski, Abhijeet Shankar Kashyap, Lanying Wang, Yanping Luo

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## To cite this version:

Xiangnan Yan, Shakil Ahmad, Nazia Manzar, Yunfei Zhang, Coline C Jaworski, et al., Enhancement of Streptomyces aureoverticillatus HN6 through mutagenesis for improved biocontrol of banana wilt disease: an WGS approach. Chemical and Biological Technologies in Agriculture, 2025, 12 (1), pp.12-10.1186/s40538-025-00732-y. http://hal-04961569

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# Enhancement of *Streptomyces aureoverticillatus* HN6 through mutagenesis for improved biocontrol of banana wilt disease: an WGS approach

Xiangnan Yan¹, Shakil Ahmad² ID, Nazia Manzar⁴, Yunfei Zhang¹, Coline C. Jaworski³, Abhijeet Shankar Kashyap⁴, Lanying Wang¹* and Yanping Luo¹*

**Abstract**

*Fusarium oxysporum*-induced Fusarium wilt in bananas is a highly detrimental disease that significantly impedes the progress of the banana industry. In this study, *Streptomyces aureoverticillatus* HN6 was isolated from soil samples of Hainan Botanical Garden and showed a wide spectrum of bacterial inhibition and a substantial effect on the control of banana wilt disease. While the bacterium demonstrates high inhibitory activity, and the use of live strains is effective, there are limitations in the development and application of this particular strain, such as effective secondary metabolites are difficult to extract; unstable metabolites, and low sporulation rates. Consequently, the strain underwent electron beam mutagenesis, ⁶⁰Co-γ ray mutagenesis, two cycles of ultraviolet irradiation mutagenesis, and ethyl methanesulfonate (EMS) mutation to generate multiple mutant strains. The mutant strain Z20-1 exhibited resistance to elevated temperatures and enhanced enzymatic activities, specifically increased levels of both amylase and cellulase. Moreover, the fermentation broth of the mutant strain E95 substantially improved the bacterial inhibitory activity. Comparative genomic analysis revealed the presence of seven single-nucleotide polymorphisms (SNPs), one multiple nucleotide polymorphism (MNP), and three insertion-deletions when comparing the mutant strain E95 with the original strain HN6. Notably, the mutant strain E95 exhibited the highest expression of *rpmC*, a core gene in the niphimycin C synthesis gene cluster. Through mutagenesis of the original strain HN6, several mutant strains with superior characteristics were successfully obtained. These findings serve as a valuable technical reference for the mutation breeding of *Streptomyces*.

**Keywords** Banana wilt disease, Genome-wide assays, Mutagenesis, Niphimycin *Streptomyces aureoverticillatus*

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<footnote>*Correspondence:
Lanying Wang
daivemuwly@126.com
Yanping Luo
yanpluo2012@hainanu.edu.cn
Full list of author information is available at the end of the article</footnote>
## Graphical Abstract

<figure><img src="image_4.png" /></figure>

Quantitative analysis of the niphimycin synthesis gene cluster in the mutant strain E95

# Enhancement of *Streptomyces aureoverticillatus* HN6 through Mutagenesis for Improved Biocontrol of Banana Wilt Disease- An WGS approach

## Introduction

Global crop yields are seriously reduced by plant diseases caused by phytopathogens, including bacterial pathogens and fungi [1]. A variety of synthetic fungicides, such as triazoles and acylalanines, are currently used to control plant fungal diseases [2, 3]. However, the overuse of agrochemicals causes environmental pollution as well as health hazards for humans, and can reduce the susceptibility of pathogenic fungi to agrochemicals [51]. Currently, biocontrol methods that are effective and safe for managing various fungal diseases are increasingly being considered [4-6].

Banana Fusarium wilt is caused by the soil-borne fungus *Fusarium oxysporum*. This disease is highly destructive and has a significant impact on the banana industry [7]. The disease has remained largely unmanaged until recently due to a lack of effective physical and chemical treatments [8]. Biological control agents (BCAs) are promising strategies for limiting the spread of banana Fusarium wilt. Several microbes, such as *Trichoderma*, *Pseudomonas*, and *Bacillus* have been extensively used as BCAs [9-11]. However, there is a need to further enhance disease control effectiveness through microbes. *Streptomyces* of the Actinomycetes bacterial family are potential BCAs against phytopathogenic fungi [9]. While the bacterium demonstrates high inhibitory activity, and the use of live strains is effective, there are limitations in the development and application of this particular strain,

such as effective secondary metabolites are difficult to extract; unstable metabolites, and low sporulation rates.

In this study, *S. aureoverticillatus* HN6 was isolated from soil samples collected from the Botanical Garden of Hainan Province in 2015 [13]. This strain exhibits significant antagonistic effects against nearly 20 tropical pathogenic fungi and six bacteria, particularly against banana wilt pathogens. The banana wilt pathogen has been used as a target bacterium for tracking secondary metabolites from *S. aureoverticillatus* bacterial strains. These secondary metabolites exert antifungal activity by activating the calcium-dependent ROS signaling pathway of phospholipase C (PLC) [3, 14]. Various studies have investigated the use of live strains. For instance, the colonization of the interroots of banana plants by *S. aureoverticillatus* HN6 can restore the healthy interroots microbiota [15]. Despite studies showing a lack of viable chemical control options, *Streptomyces aureoverticillatus* HN6 exhibits significant inhibitory effects against *F. spinosum*, and could therefore be a promising biocontrol agent against the banana wilt fungus *F. oxysporum* [12].

Mutagenesis serves as a pivotal technique for modifying a target microorganism to attain desired characteristics for biocontrol applications. Acknowledging the developmental potential of *S. aureoverticillatus* HN6 and recognizing its limitations, this study proposes employing electron beam, $^{60}$Co-$\gamma$ rays, and two cycles of ultraviolet irradiation for physical mutagenesis, along with ethyl
methanesulphonate (EMS) for chemical mutagenesis. The evaluation criteria for the mutant strains included cellulase and amylase activity, spore production, potting effectiveness, and defense enzyme activity. A comprehensive analysis, including whole-genome comparisons between the original strain and the optimal mutants, was conducted to identify favorable variants. The outcomes of this research offer a foundation for advancing the development and application of *S. aureoverticillatus* HN6 in biocontrol strategies.

# Materials and methods

## Test media, strains and reagents

Gause's synthetic agar medium was formulated with 20.0 g soluble starch, $KNO_3$ 1.0 g L$^{-1}$, NaCl 0.5 g L$^{-1}$, $MgSO_4$ 0.5 g L$^{-1}$, $KH_2PO_4$ 0.5 g L$^{-1}$, $FeSO_4$ 0.01 g L$^{-1}$, 18.0 g agar, and 1000 mL $H_2O$ (pH 7.2–7.4). Potato dextrose agar consisted of 200.0 g potato, 20.0 g glucose, 18.0 g agar, and 1000 mL $H_2O$. Cellulose Congo red medium was prepared with CMC-Na 2.0 g L$^{-1}$, $(NH_4)_2SO_4$ 2.0 g L$^{-1}$, NaCl 0.5 g L$^{-1}$, $K_2HPO_4$ 1.0 g L$^{-1}$, $MgSO_4 \cdot 7H_2O$ 0.5 g L$^{-1}$, Congo red 0.4 g L$^{-1}$, 12.0 g agar, and 1000 mL $H_2O$ (pH 7.0).

The test strains included *S. aureoverticillatus* HN6 and *Fusarium oxysporum* f.sp. cubense $FOC_4$(ATCC76255), which were provided by the research group of Green Pesticide Creation and Efficient Prevention and Control Technology. Reagents such as $KNO_3$, NaCl, $MgSO_4 \cdot 7H_2O$, $(NH_4)_2SO_4$, $KH_2PO_4$, $FeSO_4$, dextrose, 3,5-dinitrosalicylic acid, and ethyl methanesulphonate (EMS) were obtained from Shanghai Macklin Biochemical Technology Co., Ltd. Soluble starch came from SINOPHARMA Group Chemical Reagent Co. Agar powder, CMC-Na, and sodium thiosulphate were obtained from Beijing Solepol Science and Technology Co. Maltose was supplied by Shanghai Yuan Ye Biotechnology Co. Different strain plates were cultured, the spores were washed with sterile water, and the spore suspension with the required concentration was prepared by the blood cell counting plate.

## Physical mutagenesis

The starting strain was *S. aureoverticillatus* HN6, hereafter referred to as the original strain. A spore suspension of *S. aureoverticillatus* HN6 was placed into Gause's synthetic agar medium and incubated for five days at room temperature. The plate was subjected to mutagenesis using e-beam and $^{60}$Co-γ-ray at Hainan Zheda Irradiation Science and Technology Co., Ltd. Six treatment groups were subjected to electron beam irradiation, ranging from 100 to 600 Gy, and another six groups were subjected to $^{60}$Co-γ-ray irradiation, ranging from 200 to 1200 Gy [16, 17]. Mutant strains

were preliminarily screened according to colony morphology, size and color.

Spore suspensions were prepared from the original HN6 strain and the F1 generation mutant strain after exposure to e-beam and $^{60}$Co-γ-ray mutagenesis. The spores were coated in Gause's synthetic agar medium and subjected to UV irradiation at a wavelength of 254 nm and a power of 20 W. UV irradiation was applied six times, ranging from 30 to 150 s. The plates were exposed to UV radiation and incubated at room temperature [18, 19]. The growth of a single colony was monitored and recorded. According to the growth size, shape and color of the strain, the preliminary screening was carried out, and the plate culture was carried out to obtain the second round of UV mutagenesis F1 strain.

## Chemical mutagenesis

Chemical mutagenesis was performed by EMS. A spore suspension of the original strain (200 µL of $1 \times 10^6$ cfu/mL) was aspirated and centrifuged. The supernatant was removed, and 200 µL of the EMS mutagens were added to the pellet at concentrations of 0.05, 0.1, 0.5 and 1 mol/L. The treatment was carried out for 60 min. After centrifugation, the supernatant was removed, and 1 mL of 5% sodium thiosulfate solution was added to terminate the reaction. The supernatant was again removed, and 1 mL of sterile water was added to obtain a bacterial suspension, which was spread onto Gause's synthetic agar plate and incubated at room temperature [20]. The growth of the plant was carefully observed and recorded. After the spores were grown on the mutagenic plate, the colony morphology, size and color were observed, and the mutant strain was obtained by preliminary screening.

## Determination of in-dish bacterial inhibitory activity of mutant strains

The plate confrontation method was used to determine the antifungal activity in the mutant strain dish. A 0.5-cm layer of the banana *Fusarium* wilt pathogen was inoculated in the center of the PDA plate, and the same size of the original strain or the mutant strain was inoculated 2 cm from the center, four in each dish, with no biocontrol bacteria as the control. After the control plate was grown to 2/3 of the plate size, the diameter of the pathogenic fungus was determined by the cross-crossing method [21]. Strains with improved antagonistic effects were screened to determine the activity of the fermentation broth.

## Determination of the antifungal activity and stability of the mutant fermentation broth

### Determination of antifungal activity

The growth rate was used to determine the antifungal activity of the fermentation broth. A spore suspension
of the mutant strain (1×10⁷ cfu/mL) was prepared and inoculated into 100 mL of liquid Gause No. 1 medium for 5 days of shaking culture. The sample was centrifuged at 5000 r/min and 4 °C for 25 min, after which the supernatant was collected. A sterile bacterial filter with a 0.22-µm pore size was used to remove bacteria. The filtrate was mixed with 15% PDA in a plate, and 0.5 cm of banana fusarium wilt fungus cake was inoculated in the center. A plate without fermentation broth was used as a control. The inoculation was repeated three times, and inverted culture was conducted at room temperature. After the strains in the control group grew to 2/3 plate density, the colony diameter was measured by the cross method. The average value of the two measurements was taken as the colony diameter, and the bacteriostatic rate was calculated by the formula. Mutant strains with significantly greater antibacterial effects than the original strain were subjected to gradient dilutions (1%, 3%, 5%, 7%, and 10%) of the fermentation filtrate to prepare a gradient plate for culture of the target bacteria. The virulence regression equation of the fermentation broth was determined, and the correlation coefficient and IC₅₀ value were calculated [22]:

$$I = \frac{(D_1 - 0.5) - (D_2 - 0.5)}{D_1 - 0.5} \times 100\%$$

where I—inhibitory rates; D₁—the colony diameter formed in the control plate (cm); and D₂—the colony diameter formed in the test plate (cm).

### Determination of stability

The thermal stability of strains with outstanding fermentation broth (E8, E87 and E95) was determined. The highly active mutant strains were fermented and shaken. After 30 min of culture in a water bath at 4 °C, 27 °C, 37 °C, 55 °C, 75 °C or 100 °C, the strains were supplemented with PDA. The growth rate method was used to detect the antifungal activity of the fermentation filtrate after treatment against banana *Fusarium* wilt at different temperatures.

### Screening of functional enzyme activity of mutant strains

Qualitative screening of functional enzymes was carried out using a plate test. The obtained mutant strains were subjected to quantitative sequencing of functional enzyme activities. For qualitative screening of cellulase, a Congo red cellulose solid plate with CMC-Na as the main carbon source was screened. The original and mutant strains were inoculated on plates and cultured at room temperature for 5 days. The diameter of the transparent ring (H, mm) and the colony diameter (C, mm) were measured. The diameter ratio (H/C) was calculated to screen the strong lysing cellulose mutant

strain [23]. For qualitative screening of amylase, the original and mutant strains were inoculated on solid plates with starch as the main carbon source and cultured at room temperature for 5 days. Lugol's iodine solution was added to the culture, and the diameter of the transparent ring (H, mm) and the colony diameter (C, mm) were measured. The diameter ratio (H/C) was calculated to screen for mutant strains with strong starch solubilization ability [24, 25]. Cellulase and amylase were quantified using the 3,5-dinitrosalicylic acid method following [26, 27]. The standard curves of glucose and maltose were drawn following [28, 29].

### Screening of mutant strains for high-temperature resistance

The most promising mutant strains for enzymatic activity (E8, E87, E95, Z20-1 and Z21) were tested for high-temperature resistance. The strains were inoculated into Gause's synthetic agar solid medium plate by point inoculation and cultured in constant-temperature incubators at 27 °C, 38 °C, 55 °C and 75 °C for 5 days, after which the growth of the strains was measured. Each treatment was repeated three times.

The most promising mutant strains for high-yield spore production were prepared as 1×10⁶ cfu/mL spore suspensions, inoculated into liquid Gause's synthetic medium and cultured at 180 r/min under room temperature oscillation for 5 days. Plate coating was used to determine the sporulation of the strain.

### Determination of the disease control effect and induced resistance of mutant strains to bananas

The spore suspensions of the original and mutant strains were prepared with 1×10⁹ cfu/mL spores and a spore suspension of 1×10⁶ cfu/mL *F. oxysporum* f. cubense for later use. Individually potted healthy banana plants were purchased from Two Houses Seed Industry, Danzhou City, Hainan Province, and used for stage 3–4 leaves. The roots of the banana plants were washed clean and transplanted into flower pots. After 15 days, the spore suspension of the mutant strain was inoculated by root irrigation. Sterile water and the original strain were used as controls, with five strains per treatment, and the process was repeated three times. Five days after the application of the mutant strain, the spore suspension of *F. oxysporum* f.sp. cubense plants were inoculated via the root-wounding method [14]. Related defense enzymes were quantified at 0, 7, 14 and 21 days after inoculation [30, 31]. The disease control effect of each strain was determined 21 days after inoculation [32, 33].
## Whole-genome sequencing of the mutant strain E95

The strain with the most favorable mutation effect, E95, was selected for whole-genome determination. Genomic DNA sequencing was performed using the Illumina HiSeq×10 sequencing platform combined with the Pacbio third-generation platform. The candidate gene clusters of secondary metabolite biosynthesis were detected and analyzed by antiSMASH7.0. The mechanism for the higher activity of the mutant strain E95 relative to the other strains was determined, and the composition of possible antibacterial substances was predicted. The strain was inoculated into liquid Gause's synthetic medium at 180 r/min at room temperature for three days and centrifuged at 8000 rpm for 5 min. The supernatant was removed, and the mycelium was washed three times with sterile water, placed in liquid nitrogen and sent to Meiji Biological Company for whole-genome sequencing.

## Quantitative determination of key differential gene clusters of secondary metabolites in the mutant strain E95

The mutant strain E95 was cultured in a shaker at room temperature, and bacteria were collected by centrifugation. Total RNA was extracted from the bacteria according to the instructions of the TIANGEN® RNAprep Pure Bacterial Total RNA Extraction Kit. The purity of the extracted RNA was determined using the agarose gel method, and the concentration was measured by a nucleic acid concentration detector. Reverse transcription was carried out according to the instructions of the TOLOBIO™ reverse transcription kit to obtain cDNA.

According to the instructions of TOLOBIO™ qPCR, 2×10 µL of Q3 SYBR qPCR Master Mix (Universal), 0.4 µL of primer 1 (10 µM), 0.4 µL of primer 2 (10 µM) and 1 µL of template DNA/cDNA were added to the PCR tube. Finally, ddH₂O was added to the 20 µL volume for fluorescence quantitative qRT-PCR verification. The primers used are shown in **Table S1**.

qRT-PCR was performed on a real-time fluorescent quantitative PCR instrument (Applied Biosystems). The internal reference gene 16S rRNA was selected for fluorescence quantification using cDNA as a template [12]. The reaction conditions were set according to the instructions of the qPCR kit, and qRT-PCR was completed at 95 °C for 30 s, 95 °C for 10 s, 60 °C for 30 s for 40 cycles, 95 °C for 15 s, 60 °C for 60 s and 95 °C for 15 s. The differences in the expression of the core genes of the secondary metabolite synthesis gene clusters were analyzed by relative quantitative methods [34].

## Statistical analysis

Data analysis was conducted using SPSS (SPSS Inc., Chicago, IL, USA) with one-way analysis of variance

(ANOVA). Duncan's multiple comparative analysis test was performed to determine significant differences (*P* < 0.05). The results are expressed as three independent experiments, and the data are presented as the mean values ± standard errors.

## Results

### Physical mutagenesis

Twelve mutagenized strains were obtained by electron beam irradiation and ⁶⁰Co-γ-ray irradiation. A total of 74 mutants were obtained by two rounds of ultraviolet irradiation of the original strain, six mutants were obtained by electron beam mutagenesis, and six mutants were obtained by ⁶⁰Co-γ ray mutagenesis.

### Chemical mutagenesis

The original strain was chemically mutagenized by EMS. The growth of the strain was not affected by treatment with 0.05 or 0.1 mol/L EMS, and the mortality rate of the strain was greater under both EMS treatments. After treatment with 0.5 mol/L EMS, 69 mutant strains were obtained, and the colony growth rate was 27.3/mL. After treatment with 1 mol/L EMS, 48 mutant strains were obtained, and the colony growth rate was 15.3/mL. After the original strain of the 117 mutant was subjected to two rounds of ultraviolet irradiation, six mutants were obtained by electron beam mutagenesis, and six mutants were obtained by ⁶⁰Co-γ-ray mutagenesis.

## Antifungal activity and stability in mutant strain fermentation broth

The antagonistic activity of eight mutant strains, namely, E3, E4, E6, E8, E10, E87, E95 and E106, on the banana *Fusarium* wilt pathogen was significantly greater than that of the original strains. Among them, E3 had the highest antifungal activity of 72% in the initial screening (Fig. 1A). The antifungal activity of the fermentation broth of the highly antagonistic strains in the dish was then measured. The fermentation broth of the mutant strains E8, E87 and E95 completely inhibited the growth of the target bacteria at the tested concentrations (Fig. 1C). The antifungal activity increased by more than 70% compared with that of the original strain (Fig. 1B). The fermentation filtrates of E8, E87 and E95 were diluted by a series of concentration gradients to determine their virulence equations (Table S2). The R² values of E87 and E95 equations were all greater than 0.9, indicating high reliability, the R² values of E8 equation was greater than 0.85, shows reliability. The IC₅₀ values of the mutant strains E8, E87 and E95 were 1.75, 1.28 and 1.34 mg/mL, respectively (Table 1). E87 and E95 had the strongest virulence, and their difference was not significant (Fig. 1D-F).
<figure><img src="image_5.png" /><figcaption>**Fig. 1** (A) Initial screening of mutant strains for antagonistic effects against *Fusarium oxysporum* strain (only strains with positive mutagenesis are shown here). (B, C) Evaluation of the antagonistic effects of fermentation broth derived from mutagenic strains on Fusarium banana wilt. (D) Measure of virulence for highly active mutagenic strains, specifically, E8(D), E87(E), and E95(F), at different concentrations used to calculate virulence equations. In (A, B), different letters above the bars indicate significant differences between the tested groups according to Duncan's multiple range test (*P* < 0.05)</figcaption></figure>

<table><caption>**Table 1** Mutagenic strain virulence equation</caption><thead><tr><th>Strain</th><th>Virulence regression equation</th><th>R<sup>2</sup></th><th>IC50(mg/mL)</th></tr></thead><tbody><tr><td>E8</td><td>y = 6.58 + 24.76x</td><td>0.86</td><td>1.75 Aa</td></tr><tr><td>E87</td><td>y = 0.46 + 38.73x</td><td>0.94</td><td>1.28 Bb</td></tr><tr><td>E95</td><td>y = 0.75 + 36.68x</td><td>0.96</td><td>1.34 Bb</td></tr></tbody></table>

"a" represents a comparison at the 0.05 level, where the difference is significant;

"A" represents a comparison at the 0.01 level, where the difference is highly significant

## Determination of thermal stability

The fermentation filtrates of the highly active mutant strains E8, E87 and E95 were treated at different temperatures. The fermentation broth of the mutant strain E95 still had strong antifungal activity after 30 min at 75 °C (Fig. 2). Compared with the two other mutant

strains, E95 had a significantly better thermal stability at high temperatures (> 37°C).

## Comprehensive screening of enzyme activities and traits in mutant strains

Z20-1 had the strongest cellulose-decomposing ability, and the measured enzyme activity reached 41.07 U/mL, which was significantly different from that of the original strain (Fig. 3A, B). Z21 had the strongest ability to dissolve starch, and the measured enzyme activity reached 46.52 U/mL, which was significantly greater than that of the original strain (Fig. 3C, D). The most promising mutant strains, E8, E87, E95, Z20-1 and Z21, were screened for high-temperature resistance and sporulation. Only the Z20-1 mutant survived above 38 °C, and the other strains, including the original strain, did not survive above 38 °C (Fig. 3E). Only E8 had a significantly greater (3 times greater) sporulation level than the original strain (Fig. 3F).
<figure><img src="image_6.png" /><figcaption>**Fig. 2** Determination of thermal stability in the fermentation broth of the most promising mutant strains. A different '*' above the bars indicates a significant difference between the tested groups according to Duncan's multiple range test (*P* < 0.05)</figcaption></figure>

## Disease control effect of mutant strains and determination of their ability to induce resistance in banana plants

The mutant strain E95 had the most prominent disease control effect on banana Fusarium wilt on potted banana plants; the final control effect reached 84%, which was significantly greater than that of the other strains. No significant difference was found between the other strains and the original strain (Table 2, Fig. S1).

## Determination of the induced resistance ability of mutant banana seedlings

The induced activities of defense enzymes in potted banana plants were further determined. The activities of POD, PAL and PPO in all treated banana plants increased over time. Beginning on the seventh day, the PPO activity in the banana plants treated with the original strain and the mutant strain was greater than that in the plants in the blank control treatment. The PPO activity of the mutant strain E95 increased most significantly, and the difference from that of the other strains was significant (Fig. 4C). On the 14th day, the PAL and POD activities of the banana plants treated with the original and mutant strains were greater than those of the plants in the blank control treatment group. The PAL and POD activities of the mutant strain E95 increased most significantly and were significantly different from those of the other treatments. The other mutants showed significant differences from the blank control after 14 days (Fig. 4A, B).

## Whole-genome sequencing of the original strain and mutant strain E95

In-depth genomic analysis through whole-genome sequencing was conducted on both the original strain and the mutant strain E95, revealing notable distinctions in their basic genome structures. Comparative

examination revealed a 0.45% reduction in GC content, a 1.7% decrease in coding genes, a one-unit increase in transfer RNA content, and unaltered ribosomal RNA numbers in the mutant strain E95 compared to the original strain (Table S3). Functional annotations revealed key roles for the original strain's 6983 genes in amino acid, carbohydrate, and lipid metabolism; energy production; genome expression and repair; inorganic ion transport; cell wall and membrane synthesis; signal transduction; and secondary metabolite synthesis (Table S4). While the mutant strain E95 retained similar functional categories, it exhibited a sevenfold increase in genes of unknown function. Analysis of secondary metabolite synthesis gene clusters revealed shared and unique elements between the strains, pinpointing specific differential gene clusters such as aurantimycin A, funisamine, and niphimycin (Table S5). Comparative single-nucleotide polymorphism (SNP) analysis highlighted seven SNPs, one MNP, two insertions, and one deletion in the mutant strain E95, which are predominantly associated with coenzyme transport, carbohydrate metabolism, transcription, secondary metabolite synthesis, amino acid transport, and energy production.

## Quantitative determination of gene cluster differences in key secondary metabolites in the mutant strain E95

The genome size of the original strain HN6 is 9738430bp. The GC content was 71.94%. The total number of coding genes was 8248, containing 64 tRNA and 18 rRNA (Fig. 5A). The genome size of mutant E95 was 9632675bp, the GC content was 71.49%, and the total number of coding genes was 8107, containing 65 tRNA and 18 rRNA (Fig. 5B).

AntiSMASH7.0 was used to predict the secondary metabolite synthesis gene cluster in the genome data of
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 3** Screening of mutant strains for cellulase activity via (A) strain growth plate screening and (B) quantitative determination. The amylase activity of the mutant strains was screened via (C) strain growth plate screening and (D) quantitative determination. (E) Assessment of heat tolerance in Z20-1. (F) Evaluation of sporulation ability in mutant strains. Different ‘*’ above the bars indicate significant differences; \*p < 0.05; \*\*p < 0.01; \*\*\*p ≤ 0.001</figcaption></figure>

the original strain and the mutant strain E95. The ada-
mantine a gene cluster and doxorubicin in the original
strain were not detected in the mutant strain E95, while
the nifamycin gene cluster in the mutant strain was not
detected in the original strain. The expression of the core
gene cluster in the niphimycin synthesis gene cluster of
the mutant strain E95 was determined by fluorescence
quantitative qRT-PCR. *npmA*, *npmB*, *npmC*, *npmE*,
*npmH*, *npmD* and *npmI* were expressed in the mutant
strain E95, and the expression of *npmC* was significantly

greater than that of the other genes in the niphimycin
gene cluster (Fig. 5).

**Discussion**

Environmentally friendly methods for crop plant disease
control are needed, and the biocontrol strain *Streptomyces*
has attracted increasing amounts of attention due to
its potential [35–37]. Wild *Streptomyces* species often
face limitations such as environmental sensitivity, unsta-
ble metabolites, and low sporulation rates, hindering
<table><caption>**Table 2** Control effect of mutagenic strains on banana *Fusarium* wilt</caption><thead><tr><th rowspan="2">Control</th><th colspan="3">Disease index</th><th rowspan="2">Efficacy (%)</th></tr><tr><th>7d</th><th>14d</th><th>21d</th></tr></thead><tbody><tr><td>Blank check</td><td>22.22 ± 2.3</td><td>61.11 ± 1.8</td><td>84.44 ± 3.2</td><td>—</td></tr><tr><td>HN6</td><td>0</td><td>8.89 ± 1.2</td><td>24.48 ± 2.0</td><td>71.01 ± 2.88b</td></tr><tr><td>E95</td><td>0</td><td>4.56 ± 0.9</td><td>13.54 ± 1.1</td><td>83.96 ± 3.2 Aa</td></tr><tr><td>E87</td><td>0</td><td>5.56 ± 1.1</td><td>22.54 ± 2.3</td><td>73.31 ± 4.3Bb</td></tr><tr><td>E8</td><td>0</td><td>7.89 ± 0.8</td><td>24.39 ± 2.1</td><td>71.11 ± 3.7Bb</td></tr><tr><td>Z20-1</td><td>0</td><td>6.56 ± 2.4</td><td>23.45 ± 1.9</td><td>72.23 ± 3.5 Bb</td></tr><tr><td>Z21</td><td>0</td><td>7.22 ± 1.5</td><td>26.02 ± 3.1</td><td>69.19 ± 4.2Bb</td></tr></tbody></table>

"a" represents a comparison at the 0.05 level, where the difference is significant; "A" represents a comparison at the 0.01 level, where the difference is highly significant

Determination of thermal stability

their production and application [38–40]. To address this, mutagenesis was performed on the original strain *S. aureoverticillatus* HN6 using various methods, such as electron beam, ⁶⁰Co-γ ray, electron beam-ultraviolet irradiation, ⁶⁰Co-γ-ultraviolet irradiation, and EMS mutagenesis. Among the 203 mutant strains obtained and screened, strains Z20-1 and Z21, generated through electron beam-ultraviolet irradiation, exhibited significantly improved cellulase and amylase production

activities. Moreover, Z20-1 displayed enhanced heat resistance. Eight mutant strains demonstrated improved activity, with E8, E87, and E95 fermentation broths showing over 70% increased inhibition rates after live bacteria removal. Notably, the E95 fermentation broth maintained strong antifungal activity even after 30 min of incubation at 75 °C, facilitating the subsequent extraction of active compounds from the strain.

Physical mutagenesis is widely used because of its simple operation and diverse mutation sites. The earliest application of physical mutagenesis was ultraviolet mutagenesis. With the development of science and technology, ion beam mutagenesis, γ-ray mutagenesis and other technologies have been gradually developed and widely used [41, 42]. In the present study, 12 mutagenized strains were obtained by electron beam and ⁶⁰Co-γ-ray mutagenesis, and the number of strains obtained was small. The mutation efficiency of a single mutagenesis method is relatively low, and the selection of two or more mutagenesis methods for compound mutagenesis can improve the strain acquisition rate [43–45]. Therefore, two rounds of ultraviolet irradiation mutagenesis were carried out using an electron beam and ⁶⁰Co-γ. This process led to an increase in the number of mutant strains (n=74). Chemical mutagenesis has the advantages of easy dose control, a high mutation ratio and strong specificity. EMS acts on DNA guanine, which can alkylate the

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>**Fig. 4** Effects of mutant strains on the activity of defense enzymes in banana plants over time. The different letters on the right indicate significant differences between the activity levels of the mutant strains at 21 days according to Duncan's multiple range test (*P* < 0.05). **A** PPO activity, **B** PAL activity and **C** indicates POD activity</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Fig. 5** *Streptomyces aureoverticillatus* Circos map and Quantitative analysis of the niphimycin synthesis gene cluster in the mutant strain E95. (A) *Streptomyces aureoverticillatus* HN6 Circos map; (B) *Streptomyces aureoverticillatus* E95 Circos map; C differential gene expression in mutant strains</figcaption></figure>

bases and eventually lead to base substitution to produce
mutants for mutation breeding [46, 47]. In the present
work, EMS was selected for chemical mutagenesis to
mutate *S. aureoverticillatus* HN6. A total of 117 mutants
were obtained. Our study showed that using single physi-
cal mutagenesis to obtain fewer strains can significantly
improve the acquisition rate of strains, but chemical
strain mutagenesis had a greater overall acquisition rate
than physical mutagenesis.

The activity of microbial enzymes and their tolerance
to the environment can be improved by mutagenesis.
A recent study used heavy ion mutagenesis to improve
the high-temperature resistance of *Bacillus subtilis* [48]. Another study improved the tolerance of the
strain to isobutanol by mutagenesis of yeast with EMS
[49]. The phytase activity of *Streptococcus thermophilus*
ST20 was improved by EMS mutagenesis [50]. In this
study, the original strain, *S. aureoverticillatus* HN6, was
mutagenized by electron beam mutagenesis, ⁶⁰Co-γ-
ray mutagenesis, electron beam-ultraviolet irradiation
and ⁶⁰Co-γ-ultraviolet irradiation. A total of 86 mutants
were obtained. After screening, strains Z20-1 and Z21
obtained by electron beam-ultraviolet irradiation had
significantly improved cellulase and amylase production
activities, respectively, and the heat resistance of Z20-1
was also enhanced. Similarly, the cellulose and amylase
activity of the strain was not sufficient for composting
the original strain; as such, exogenous bacteria should be
added to achieve the ideal composting effect. The effects
of strains Z20-1 and Z21 have improved [14]. In follow-
up studies, these strains can be directly applied to com-
posting fermentation to reduce the types of exogenous
bacteria and increase composting efficiency.

Sequence alignment analysis of the mutant strain E95
and the original strain revealed that the niphimycin
gene cluster sequence also existed in the original strain,
but the gene cluster appeared in a linkage with other

gene clusters, making it impossible to express domi-
nantly in the original strain. The mechanism of EMS
mutagenesis is that it can alkylate guanine, resulting in
base mismatch, which can act on multiple sites contain-
ing guanine. This study speculates that when the origi-
nal strain is mutagenized, the drug mutation site is just
at the linkage between the niphimycin gene and other
genes, so that the niphimycin gene can be dominantly
expressed. It is also possible that a point mutation just
becomes the promoter of the transcription of the nipi-
mycin synthesis gene cluster, so that niphimycin can be
dominantly expressed in the mutant strain.

**Conclusion**

In summary, this study explored the impact of different mutagenesis techniques on *S. aureoverticillatus* HN6, leading to the identification of the biocontrol strain E95, which has significant antifungal activity. E95 exhibited a 70% increase in bacteriostatic activity compared to the original strain HN6 and demonstrated variations in heat resistance, cellulose, and starch-decomposing abilities. The E95 fermentation broth still exhibited strong antifungal activity after 30 min in a water bath at 75 °C, which provides convenient conditions for the subsequent separation and extraction of active compounds from the strain and guarantees the acquisition of active compounds. Experiments on banana plants revealed greater resistance to E95 than to HN6. Whole-genome sequencing revealed seven single-nucleotide polymorphisms (SNPs), one polynucleotide polymorphism (MNP), and three insertion-deletions (indels) in E95 compared to the original strain, primarily affecting genes related to coenzyme transport, metabolism, carbohydrate transport, transcription, secondary metabolite biosynthesis, amino acid transport, and energy production.
**Supplementary Information**

The online version contains supplementary material available at https://doi.org/10.1186/s40538-025-00732-y.

Additional file 1

**Acknowledgements**

Not applicable.

**Author contributions**

XY, SA contributed to conceptualization, investigation, validation and performed experiments. SA, NM, YZ, CJ, and ASK contributed to writing, editing and formal analysis. LW, and YL involved in data curation, funding acquisition and supervision. All authors reviewed the manuscript.

**Funding**

This work was supported by the Natural Science Foundation of China (32360683), the Special fund of Science and Technology Hainan Province (ZDYF2022XDNY274).

**Data availability**

The datasets generated during the current study are available in the [NCBI] repository and in the supplementary files of this submission.

No datasets were generated or analysed during the current study. Chromosome Accession ID: CP176002.

Plasmid Accession ID: CP176003.

The SRA records will be accessible with the following link: https://www.ncbi.nlm.nih.gov/sra/PRJNA1109548

The BioProject accession number: PRJNA1109548.

**Declarations**

**Ethics approval and consent to participate.**

Not applicable.

**Consent for publication**

Not applicable.

**Competing interests**

The authors declare no competing interests.

**Author details**

¹School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China. ²College of Life Sciences, Hainan Normal University, Haikou 571158, China. ³UMR ISA, INRAE, Université Côte d'Azur, 06000 Nice, France. ⁴Plant Pathology Lab, ICAR-National Bureau of Agriculturally Important Microorganisms, Mau 275103, UP, India.

Received: 24 August 2024 Accepted: 16 January 2025

Published online: 28 January 2025

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