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Article

Potential of Crude Extract of Streptomyces sp. nov., Strain TRM76147 for Control of A. gaisen

by
Yi-Huang Chen
1,2,
Jia-Xin Zhang
1,2,
Guo Yang
1,2,
Yang Liu
1,2,
Song Ran
1,2,
Jian-Ming Wang
1,2,
Qin Liu
1,2,* and
Xiao-Xia Luo
1,2,*
1
Key Laboratory of Protection and Utilization of Biological Resources, Tarim Basin of Xinjiang Production & Construction Corps, Aral 843300, China
2
China & School of Life Sciences and Technology, Tarim University, Alar 843300, China
*
Authors to whom correspondence should be addressed.
Forests 2024, 15(9), 1605; https://doi.org/10.3390/f15091605
Submission received: 21 August 2024 / Revised: 4 September 2024 / Accepted: 10 September 2024 / Published: 11 September 2024
(This article belongs to the Special Issue Advances in Biological Control of Forest Diseases and Pests)
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Abstract

:
Pear black spot, caused by A. gaisen during fruit growth, is a disease that significantly reduces pear yield. Biological control using antagonistic microorganisms is regarded as a viable alternative to chemical agents. The discovery of TRM76147, a novel species of Streptomyces isolated from the Taklamakan Desert, has demonstrated promising potential in addressing this issue. This study was conducted to determine the potential of crude extract of Streptomyces sp. nov., strain TRM76147, for control of A. gaisen. TRM76147 is closely related to Streptomyces griseoviridis NBRC 12874T, exhibiting an average nucleotide identity (ANI) value of 82.13%. Combined with the polyphasic taxonomic identification, this suggests that TRM76147 is a potentially new species. Through analyses using BigSCAPE and antiSMASH, it was determined that the TRM76147 genome contains 19 gene clusters. The ethyl acetate extract of this strain demonstrates antifungal activity, with the active substance remaining stable at temperatures up to 70 °C, achieving an activity level of 16.23 ± 0.22 mm. Furthermore, the crude extract maintains its antifungal efficacy across a pH range of 2 to 12. Notably, the antifungal diameter was recorded at 16.53 ± 0.12 mm following 80 min of UV irradiation. Under different treatment conditions, TRM76147 fermentation crude extract caused A. gaisen spore crumpling and spore number reduction. In addition, this study also found that the TRM76147 fermentation broth could control the production of pear black spot disease, which initially revealed the inhibition mechanism. The abundant actinomycete resources in this study have good application and development value in the discovery of new species and the study of bioactive substances and biological control.

1. Introduction

Alternaria gaisen is a species known to cause various plant diseases, resulting in the deterioration of agricultural products, including crops, fruits, and vegetables. During storage, A. gaisen not only induces significant rotting in fruit tissues but also diminishes pear quality, shortens the storage period, and reduces the market value [1,2]. These effects ultimately lead to considerable economic losses and pose risks to food safety. By the end of 2021, pear production in Xinjiang alone reached 1,795,900 tons, accounting for about one-ninth of the national production [3], affected by factors such as cultivation and management. The probability of pear black spot disease is also increasing, and the resulting disease causes a large number of cracked fruits and early fruit drop, with the rate of diseased fruits as high as more than 90% [4]. Therefore, it is necessary to prevent and control A. gaisen.
The prevention and management of pear diseases encompass both chemical and biological strategies. Commonly employed control methods are predominantly chemical, such as the application of sodium pentachlorophenate for the control of pear black spot. While this method is effective, it is also associated with recognized harmful effects on the environment [4]. Growing public awareness regarding food safety has led to a widespread acknowledgment of the necessity for safe and effective plant disease control methods [5]. Consequently, exploring more efficient biological control methods would be beneficial. In recent years, biocontrol organisms have shown significant potential in the prevention and management of pear diseases. For instance, the isolation of Aureobasidium pullulans from Portuguese resulted in a 23% reduction in the incidence of blue mold disease in pears and a 36% reduction in lesion diameter [6]. Streptomyces, a genus within the order Actinomycetes [7], can produce antimicrobial agents with antibacterial and bactericidal properties. Notably, approximately 90% of commercial antibiotics, such as the acetomycin [8] and the fungicide ningnanmycin [9,10], are derived from Streptomyces. Furthermore, biocontrol products sourced from various Streptomyces species are already available in the market, and the exploration of novel natural products continues [11,12]. In summary, Streptomyces is one of the good resources of potential strains of biocontrol.
The development and utilization of active substances in Streptomyces are crucial; however, traditional methods for mining active products tend to be ineffective. To address this limitation, genome-guided secondary metabolite mining strategies have emerged [13]. With advancements in genomics technology, researchers have employed genomic analysis to identify secondary metabolites. For instance, Kodani isolated a novel lanolin sulfide active substance from an extract of Actinomadura atramentaria NBRC14695T, which demonstrated antimicrobial activity against S. antibioticus in the spot-on-lawn testing method at a dose of 5 μg (0.5 μL, 10 mg/mL solution) [14]. Additionally, Chen isolated a new wool sulfur antibiotic, mathermycin, which exhibited antimicrobial activity and characterized the key post-translational modifying enzymes associated with mathermycin [15]. In summary, genomic analysis significantly enhances the efficiency of subsequent metabolite mining.
These microorganisms are particularly known for their secondary metabolites, including key active ingredients [16]. Polyoxymycin has demonstrated efficacy in combating fungal diseases such as rice sheath blight [17], while validamycin represents a novel antibiotic that is effective against the same disease [18]. The application of antagonistic strains of Streptomyces has proven successful in both the prevention and management of plant diseases [19,20]. Consequently, certain strains of Streptomyces constitute a valuable biocontrol resource with significant potential for research and development. Generally, Streptomyces are among the largest producers of bioactive compounds. However, the extent of secondary metabolite activity produced by these strains may be influenced by various factors, including pH, temperature, and UV exposure. For instance, investigating the effects of pH and temperature on tropane alkaloids can facilitate the development of safe processing strategies for infant cereals [21]. Therefore, examining the stability of active products is essential for assessing biocontrol potential. Previous studies have primarily concentrated on the role of Streptomyces in managing fungal diseases; however, fewer investigations have been conducted in Xinjiang regarding the utilization of newly acquired Streptomyces for the control of A. gaisen diseases. Moreover, microbial resources available for such studies are limited. In this research, a novel strain of Streptomyces sp., TRM76147, isolated from Tamarisk soil, exhibited significant activity against pear black spot. Genome analysis revealed a biosynthetic gene cluster encoding lanolin peptide with considerable metabolic potential. The active substance demonstrated remarkable stability under varying pH levels, temperatures, and UV treatments. Furthermore, the active crude extract displayed a strong inhibitory effect on both the growth of mycelium and the production of spores of A. gaisen. By expanding the database of antagonistic microbial species in the extreme environments of Xinjiang, this research highlights the potential of the active substance to combat A. gaisen and prevent disease onset in pear fruits. Additionally, this study enhances the understanding of Actinomycetes biodiversity and the exploration of extreme microbial resources.

2. Materials and Methods

2.1. Strains TRM76147 and A. gaisen Were Obtained

Strain TRM 76147 was isolated from soil samples of Tamarix ramosissima Ledeb collected along the Ahe Highway in Xinjiang, Taklamakan Desert (36.89° E, 82.55° N, 1664.8 m). The strain was isolated on glycerol arginine medium consisting of 2.00 g arginine, 12.00 g glycerol, 0.50 g MgSO4, 1.00 g K2HPO4, and 17.00 g agar, following the method described in a previous study [22]. It was cultured using Gao’s medium at pH 7 [23]. A. gaisen strain CGMCC3.7807 was grown in PDA medium, incubated at 28 °C, and kept at the College of Life Science and Technology, Tarim University, and China Typical Culture Collection Centre.

2.2. 16S rRNA Gene and Multilocus Sequence Analysis (MLSA) Phylogeny

To determine the taxonomic status of strain TRM 76147, this study initiated genome sequencing using the Illumina NovaSeq platform, Parsonage, Nanjing, China. Polymerase chain reaction (PCR) for the 16S rRNA gene was performed on the strains utilizing sequence-specific primers (27F and 1492R). The resulting data were analyzed using the EzBioCloud server (https://www.ezbiocloud.net, accessed on 20 August 2024) [24]. Both TRM76147 and related strains were identified through EzBioCloud, and the genome was retrieved from the National Center for Biotechnology Information (NCBI) [13]. For the construction of a phylogenetic tree based on housekeeping genes, the sequences of strain TRM 76147 were amplified and sequenced in accordance with the Streptomyces multilocus sequence typing protocol (https://kiepczi.github.io/MLST_MicroSoc/, accessed on 20 August 2024). Employing the adjacency linking method in MEGA-X software, the neighbor joining method was utilized to analyze both the 16S rRNA gene and the housekeeping genes of strain TRM 76147, simultaneously establishing a PEPPAN genome tree [25]. Strain TRM 76147 was compared to closely related strains, S. griseoviridis NBRC12874T and S. niveoruber NBRC15428T, through 16S rRNA gene analysis using EZbiocloud, with additional bacterial comparisons conducted via a comprehensive literature review.

2.3. Polyphasic Isolation and Characterization of Strain TRM76147

2.3.1. Culture Characteristics of TRM76147

In accordance with the approach outlined in the rapid identification and systematic classification of Actinomyces study, the mycelium of TRM76147 was identified via culturing on Gao’s medium and examining growth on different ISP media (ISP 1, ISP 2, ISP 3, ISP 4, ISP 5, ISP 6, and ISP 7) for a period of 7 days [26]. Additionally, strain TRM76147’s microscopic morphology was observed using scanning electron microscopy (JSM-6360, jeol Ltd., Singapore) after incubation on Gao’s medium at 30 °C for a week. TRM76147 was also inoculated on Gao’s medium and subjected to various temperatures (4 °C, 12 °C, 20 °C, 25 °C, 32 °C, 37 °C, 40 °C, 45 °C, 50 °C, or 55 °C) and varying NaCl concentrations (0%, 5%, 10%, 15%, 20%, 25%). The pH of the medium was adjusted using different buffers (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) before inoculation of the strain for growth observation at 37 °C. A range of physiological and biochemical characteristics of Streptomyces were identified, as described by Williams [27].

2.3.2. Peptidoglycan, Amino Acids, and Polar Lipids of TRM76147

Standard techniques were utilized to identify the varieties of amino acids present in the cell wall hydrolysates, as well as to quantify the total cellular sugars [28]. The extraction of polar lipids was conducted according to the method described by Minnikin et al. These lipids were subsequently identified using two-dimensional thin-layer chromatography and visualized with phosphomolybdic acid, threonine, and anisaldehyde reagents to facilitate color development [11].

2.4. Genomic Metabolic Potential Analysis of TRM76147

This study investigates the differences in metabolic potential between TRM76147 and closely related strains by obtaining the genomes of 16 strains with the nearest evolutionary distance from the NCBI. These genomes were individually analyzed using antiSMASH [12]. Furthermore, the data were subjected to bigSCAPE analysis [29] (https://blog.csdn.net/Asa12138/article/details/138873339, accessed on 21 August 2024). Through correlation analysis, the study explored the metabolic potential of the strains over time, providing insights for future metabolite exploration.

2.5. Stability Analysis of Active Crude Extract of Strain TRM76147

This investigation examines the inhibitory effect of strain TRM76147 on A. gaisen using the plate standoff method [30]. We extracted the freeze-dried sample of TRM76147 with a concentration of 0.1 g/mL via ultrasound for thirty minutes, repeating this process three times. Activity testing was conducted by redissolving the sample after rotary evaporation. The study employed a plate confrontation technique to identify the phase segments of TRM76147’s active components and performed multi-stage extractions using methanol, dichloromethane, ethyl acetate, and petroleum ether for activity experiments. The corresponding solvent served as a blank control [31].
In this study, the minimum inhibitory concentration of the crude extract from the TRM76147 fermentation broth was determined using an ethyl acetate extraction method combined with a doubling dilution technique. The samples were extracted with ethyl acetate, freeze-dried, and subsequently diluted with 0.1 mol/L phosphate buffer at concentrations of 6.25%, 12.5%, 25%, 50%, and 100%, respectively [32]. The inhibition of A. gaisen growth using the crude extracts from the TRM76147 fermentation broth was evaluated using the plate stand-off method. The diameter of the inhibition zone was measured after repeating the experiment three times. The inhibitory effect of the strain was assessed by comparing it to the control; if the diameter of the inhibition zone was larger than that of the control, it indicated that the crude extract was active, and conversely, if it was smaller, the extract was deemed inactive.
The activity assay conducted with various reagent extractions indicated that the ethyl acetate extract contains active substances. To determine the inhibition rate of TRM76147 against A. gaisen after treatment at different temperatures, ethyl acetate extracts were subjected to treatments at temperatures of 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C [33]. The ethyl acetate extract served as a blank control. Subsequently, the plate confrontation method was employed to evaluate the inhibitory rate of TRM76147 on A. gaisen without traversing the specified temperature range.
To investigate the effect of ethyl acetate extracts (pH threshold 2–12) on A. gaisen under varying pH conditions, platelet activity experiments were conducted using crude extracts of ethyl acetate at different pH levels. The inhibition zones were measured by repeating the experiments three times, and the data were statistically analyzed using DPS [34].
To investigate the inhibitory effect of the crude extract on A. gaisen under varying UV treatments, the ethyl acetate extract was subjected to UV irradiation for durations of 20, 40, 60, and 80 min. An activity experiment was conducted utilizing the plate confrontation method. The inhibition zones were measured by repeating the experiment three times, and the data were statistically analyzed using DPS [35,36].

2.6. TRM76147 Exploration of the Mechanism of Inhibition of A. gaisen by Crude Extracts and Assessment of Bioprophylactic Potential of Pears

2.6.1. Investigation of the Mechanism of Inhibition of A. gaisen by the Crude Extract of TRM76147

The study aimed to investigate the inhibitory mechanism of TRM76147’s active component on A. gaisen. The impact of fermentation broth on A. gaisen quantity was evaluated by treating A. gaisen spores with phosphate buffer, 2-fold dilution, and 10-fold TRM76147 crude extract. Hyphal and conidial morphology and quantity in A. gaisen were examined using a light microscope [37]. A. gaisen spores treated with strain TRM76147 fermentation broth and normal growth were extracted using sterile gauze and the number of spores was observed under a 10× microscope. Finally, morphological differences in spores and mycelium were compared between treated and untreated groups.

2.6.2. Evaluation for Plant Pathogen Suppression Ability

To investigate the antifungal activity of the strain in vivo, the surface of the fragrant pear was initially disinfected using 2% sodium hypochlorite and 75% ethanol [38]. Three experimental groups were established: one group was inoculated with TRM76147 fermentation broth and A. gaisen, another group was inoculated exclusively with A. gaisen, and a blank control group was also included. The spraying volume was set at 5 mL per pear, with the TRM76147 fermentation broth and the blank treatment consisting of water. The samples were stored in a sterile culture room and incubated at 30 °C. Observations were conducted on days 1, 3, 5, 7, and 9 [39]. The collection site for Pyrus sinkiangensis and the experimental site were both located at Tarim University.

2.7. Statistical Analysis

One-way analysis of variance was used to test the differences in activity of different treatments of TRM76174 fermentation broth on A. gaisen, and Duncan’s new complex polar deviation test was used as a post hoc test to evaluate the statistical significance at 5% probability level. All statistical analyses and calculations were performed in DPS 7.05 Windows version.

3. Results

3.1. Genetic Analysis of Strain TRM76147

A genetic analysis was conducted to determine the taxonomic status of the strain. Utilizing the MEGA-X software, a neighbor-joining time algorithm of the 16S rRNA gene phylogenetic tree was performed (Figure 1). In this analysis, strain TRM76147 clustered closely with Streptomyces griseoviridis NBRC12874T and Streptomyces niveoruber NBRC15428T, yet formed a separate branch, indicating a potential new species. Additionally, a pan-genome phylogenetic tree was constructed using MEGA-X for neighbor-joining-based phylogenetic analysis, as shown in Supplementary Figure S1A. The phylogenetic analysis of the housekeeping gene of strain TRM76147, conducted using the neighbor joining method, is illustrated in Supplementary Figure S1B. Notably, distinct branches were observed for the pan-genome and housekeeping gene of strain TRM76147, suggesting its classification as a new Streptomyces species. Comparison of the 16S rRNA gene sequences between actinomycete TRM76147 and S. griseoviridis NBRC 12874T revealed a sequence identity of 98.96%. Genome sequencing indicated a GC content of 72.38% for strain TRM76147, while the ANI (average nucleotide identity) value compared to S. griseoviridis NBRC 12874T was 82.13%. The ANI value falls below the threshold for prokaryote species classification at 95%, and the dDDH value is 67.22%, significantly lower than the 70% threshold. These findings confirm that strain TRM76147 and strain S. griseoviridis NBRC 12874T represent distinct species. The 16S rRNA gene sequence has been submitted to the NCBI under submission number SUB13726220 and the strain deposited at China Typical Culture Collection Centre: CCTCC AA 2024075.

3.2. Growth and Cultural Characteristics of Strain TRM76147 Compared with Reference Strains

The strain TRM76147 grew in ISP series medium and NA medium, Gao’s medium, and Cha’s medium for 7 days at 30 °C. The results exhibited the best growth on Gao’s medium, limited growth on ISP2, ISP4, Cha’s, and NA media, and no growth on ISP1, ISP3, or ISP6 media.
It demonstrated the ability to tolerate NaCl concentrations up to 5.0% and thrived within a pH range of 7.0–10.0, with optimal growth observed at pH 7.0. In comparison, the strain S. griseoviridis NBRC12874T exhibited higher salt tolerance than TRM76147, and its acid and alkali tolerance surpassed that of TRM76147. The temperature range for optimal growth of strain TRM76147 was determined to be between 28 and 45 °C, with the most favorable temperature being 37 °C. Research results revealed after 7 days of cultivation in Gao’s medium, the colony morphology displayed white spores, as depicted in Figure 2A,B. S. griseoviridis NBRC12874T and S. niveoruber NBRC15428T appear as pale greyish-purple and ivory, respectively. Scanning electron microscopy images in Figure 2C,D revealed straight hyphae for strain TRM76147 and strain S. niveoruber NBRC15428T, while its similar strain S. griseoviridis NBRC12874T, had rectiflexibiles hyphae. Strain TRM76147 did not produce melanin or generate hydrogen sulfide. The reference strains also exhibited growth on all tested media. A comparison of the growth and cultural characteristics of strain TRM76147 with the reference strains is presented in Table 1 [29]. The morphology and color of TRM76147 differed from those of similar strains, and its salt tolerance was lower than that of similar strains; therefore, the strain showed distinctive morphological characteristics and was a potential new species.

Characterization of the Chemical Components of Bacterial Cell Walls

In order to distinguish the independence of the bacterial strains, it is necessary to analyze the chemical components in the strains, such as amino acids, and to identify the relevant chemical markers. The results are shown in Figure 3, the cell wall fractions analyzed via ninhydrin staining were found to contain PIDM, PIM, PIM, PI, PC, LPG, PG, DPG, PME, and PE; the cell wall fractions analyzed using polylipids staining were found to contain PC, LPG, PG, OH-PE, and NPG; the cell wall fractions analyzed via molybdophosphoric acid staining were found to contain PC, PG, LPG, DPG, DPG, and NPG. The cell wall fraction was found to contain PC, PG, LPG, DPG, PME, and PE, indicating that the whole-cell hydrolyzed sugar fraction of this strain contained mannose and glucose and that the diaminoheptanoic acid type was LL-DAP (LL-2,6-diaminoheptanoic acid). The chemical classification of strain TRM76147 is consistent with members of the genus Streptomyces.

3.3. Analysis of Secondary Metabolites in TRM76147 Strain Using antiSMASH and bigSCAPE Software

In this study, the 16S rRNA gene based on TRM76147 was compared in EZbiocloud. The 16 most closely related strains were selected for genomic analysis based on Mega (10.0) analysis. The antiSMASH statistical method was used to analyze 16 strains, with the gene cluster analysis results provided in Figure 4. Among these 16 strains, the most common gene clusters identified included terpenes and siderophores. Figure 4 illustrates the gene clusters present in all 16 strains as well as those specific to the TRM76147 strain. Notably, S. swartbergensis HMC 13 had the highest metabolic potential with 47 gene clusters, while similar strain S. griseoviridis NBRC 12874T and S. niveoruber NBRC15428T contained 33 and 32 gene clusters, respectively. The TRM76147 strain exhibits a variety of metabolites such as terpenes and peptides. Of particular interest is the presence of gene clusters related to wool sulfur peptides in the genome of the TRM76147 strain, which display antifungal properties. This study used antiSMASH analysis to predict a type of compound called geosmin, which is composed of one gene, the geosmin synthase Cyc2 gene. The corresponding metabolites in the MIBIG database could not be found in any of the other 18 types of gene clusters, suggesting that the strain has the potential to produce new metabolites.
As illustrated in Figure 5, bigSCAPE software was used to analyze 305 types gene clusters of 16 bacteria. A total of 427 gene clusters were identified, as depicted in Figure 5. The 16 bacteria were analyzed for their terpene compound clusters, which revealed that S. swartbergensis HMC 13T had 47 gene clusters, S. minuticleroticus NBRC 13000T had 39 gene clusters, S. brasiliensis NBRC 101283T had 37 gene clusters, TRM76147 had 20 gene clusters, and S. chromofuscus NBRC 12851T had 19 gene clusters.

3.4. Strain 76147 Antimicrobial Activity Analysis

3.4.1. Investigation of Fungistatic Effects of Streptomyces TRM76147 on A. gaisen Using Stepwise Extraction Method

The study employed a stepwise extraction method in conjunction with a plate confrontation technique to evaluate the inhibitory effects of the crude extract derived from the fermentation of TRM76147 on A. gaisen. The fermentation broth of the Streptomyces TRM76147 strain demonstrated significant activity against A. gaisen, exhibiting an antifungal zone size of 23 ± 1.23 mm. Furthermore, the TRM76147 plate also showed activity against A. gaisen, with a restricted zone size of 13 ± 1.16 mm. These results suggest that the active substance from the strain is present extracellularly and is soluble in the fermentation broth. Notably, the active component of TRM76147 did not display antagonistic activity in the dichloromethane, water, or methanol phases. Conversely, activity was observed in the ethyl acetate phase, where a fungistatic circle with a diameter of 20 ± 1.43 mm was recorded (Figure 6). Different blank solvents are showing activity. To determine the minimum concentration of the active substance, a minimum inhibitory concentration (MIC) experiment was conducted. The fungistatic effect of TRM76147 was evident at a concentration of 6.25%, resulting in a fungistatic circle measuring approximately 16 ± 1.23 mm. The minimal inhibitory concentration was established at 6.25%, as illustrated in Figure 6.

3.4.2. Impact of Temperature on Antifungal Efficacy of TRM76147 Fermentation Crude Extract

In this study, different temperatures were utilized to explore the stability of the active substance. Research findings demonstrate a decrease in the antifungal efficacy of TRM76147 fermented crude extracts as the temperature increases. Figure 7 visually represents this correlation, showing inhibition zone sizes of 19.71 ± 0.04 mm, 16.23 ± 0.22 mm, and 15.72 ± 0.23 mm at 40 °C, 50 °C, and 60 °C, respectively. This study found that untreated crude ethyl acetate extract exhibited significant activity in all treatment groups, while the activity of samples treated at different temperatures decreased with increasing temperature, as the activity of the crude extract remained largely unchanged. It is worth noting that the antifungal activity significantly decreases at 70 °C and stops above this temperature threshold, highlighting the impact of temperature on the overall antifungal effect (Figure 7). In summary, the surface exhibits resistance to temperatures ranging from 40 °C to 60 °C.

3.4.3. Stability and Activity of TRM76147 Extract under UV and pH Conditions

UV irradiation and pH are significant factors influencing the activity of active substances. The plate confrontation method revealed that UV radiation exhibited antifungal activity between 20 and 80 min, as illustrated in Figure 8A. The differences in A. gaisen activity across the various treatments were statistically significant (p < 0.05). Calculations of the antifungal rate indicate a decrease in activity with prolonged UV irradiation times, with a notable reduction observed after 60 min. At 20, 40, 60, and 80 min, the inhibition diameters were 18.34 ± 0.07 mm, 17.66 ± 0.47 mm, 17.19 ± 0.13 mm, and 16.53 ± 0.12 mm, respectively, for the different durations of UV exposure. Despite this decrease, TRM76147 maintained its antifungal activity throughout all four time periods of UV irradiation, indicating its resistance to UV exposure for 20–80 min. Furthermore, despite extreme pH levels of 2 and 12, the TRM76147 ethyl acetate extract still exhibited some level of activity. The optimal pH for antifungal activity was found to be 2, as illustrated in Figure 8B. It was observed that antifungal activity diminishes with increasing pH levels, with the smallest antifungal zone measuring 15.23 ± 0.16 mm, recorded at a pH value of 12. This trend is corroborated by the data in Figure 8B, which indicate that TRM76147 activity decreases at higher pH levels.

3.5. Effect of Crude Extract of Strain 76147 on the Activity of A. gaisen

3.5.1. Impact of TRM76147 Compounds on A. gaisen Mycelium and Conidium Integrity

The spores were diluted in a sterile phosphate buffer solution to achieve a concentration of approximately 150 to 200 spores per field of view at 10× magnification. A subsequent 2-fold dilution with ethyl acetate extract resulted in a drastic reduction of spores, bringing their number down to 40 to 60 at 10× magnification. Following a 10-fold dilution with the ethyl acetate extract, the observed count of spores at 10× magnification was further reduced to approximately 60 to 100 spores, as illustrated in Figure 9. In conclusion, the fermented crude extract of strain TRM76147 effectively inhibited A. gaisen.
The mycelium of A. gaisen appeared plump and smooth, as illustrated in Figure 10A (1). In contrast, the mycelium treated with the TRM76147 crude extract exhibited rough and irregular characteristics, displaying visible damage, as shown in Figure 10A (2). Similarly, the conidia of A. gaisen presented round and plump features with intact cell integrity in Figure 10B (1). However, the conidia treated with the TRM76147 crude extract revealed shriveled cells and deformed sporophytes, as depicted in Figure 10B (2).

3.5.2. Impact of TRM76147 Fermentation Broth on A. gaisen Growth

After 9 days of treatment using various methods, this study observed differences in fungal growth under identical treatments over different days, with a progressive increase in the area of pear black spot over time. Notably, the area of pear black spot was reduced in fruits treated with the TRM76147 fermentation broth after 9 days of incubation, compared to those subjected solely to inoculation with A. gaisen and the blank control (Figure 11). In conclusion, the fermentation broth of TRM76147 demonstrates potential for the biological control of A. gaisen.

4. Discussions

Streptomycetes are abundant in soil, but after decades of screening, it has become increasingly difficult to obtain Streptomycetes capable of producing new active substances from common habitats [40,41]. In recent years, we have gradually increased the exploration of microbial resources in extreme environments to provide raw materials for microbial science research and application development [42]. A total of 129 new strains of Streptomycetes were isolated and reported from 35 desert environments around the globe during the period 2000–2021 [43]. Desert environments, including extremely saline regions, alpine areas, and the inter-root zones of plants have garnered significant attention from researchers due to their capacity to produce Actinomycetes with a higher utilization of active substances. Studies indicate that inter-root microorganisms associated with various plant types, such as agricultural crops, cash crops, and forest plants, exhibit greater richness and diversity compared to those found in non-inter-root environments [44]. Consequently, the isolation of Actinomycetes from plant roots holds considerable ecological significance and economic value. Actinomycetes extracted from the soil of medicinal plants belong to seven suborders, ten families, and fourteen genera. Notably, all Streptomyces species demonstrate bacteriostatic activity, with approximately 70% of the strains of Streptomyces sp. showing effectiveness against maize small blotch disease (SBD), a major contributor to this condition. These strains also exhibit strong antibacterial properties against Exserohilum turcicum [45]. Tou et al. successfully isolated 154 strains of actinobacteria from 24 inter-root soil samples collected from 2 types of sandy plants in the Lop Nor region, distributed across 12 genera, among which CA15-2 may represent a potential new species [46]. The isolation of Streptomyces sp. TRM76147 from the Tamarix rhizosphere was accomplished using the plate method. Through multiphase classification identification, TRM76147 was differentiated from a similar strain, S. griseoviridis NBRC12874T. Comparisons with S. niveoruber NBRC15428T revealed unique physiological characteristics and salt tolerance in strain TRM76147, classifying it into a distinct branch. Strain TRM76147 exhibits a straight spore chain morphology, whereas S. griseoviridis NBRC12874T displays rectiflexibile characteristics. Additionally, strain TRM76147, along with similar strains, does not produce melanin; however, both strains can liquefy gelatin, with strain TRM76147 also capable of producing hydrogen sulfide. Analysis of the 16S rRNA gene and genome indicated that TRM76147 is a potential new species of Streptomyces, with findings aligning with previous research by Zheng et al. [47]. Discovery of a new species of Streptomyces TRM76147 enriches the diversity of biological resources.
The rational exploitation of biological resources must be underpinned by the judicious application of modern biotechnology. Current methods for natural product mining primarily encompass bioactivity-oriented isolation, genome mining, metabolomics analysis, and co-culture technology. However, activity-oriented mining and co-culture techniques often lack precision and can be repetitive. Genomic approaches are essential for the development of bioactivity and the discovery of metabolites [48]. This study employed bigSCAPE and antiSMASH tools for an initial assessment of the metabolic capabilities of the strains. Iftime et al. successfully isolated the active compound lanthipeptide through heterologous expression following a genomic analysis of Streptomyces collinus Tü 365 [49]. In the present study, a similar gene cluster was identified in strain TRM76147 via genomic analysis. The findings from the analysis of strain TRM76147 and its gene clusters indicate a robust metabolic capacity. Notably, the genome of strain TRM76147 contains gene clusters associated with wool sulfur peptides that exhibit antifungal properties. The biosynthetic gene clusters encoding lanthipeptide class III are integral to the antifungal activity produced by Streptomyces sp. TRM76147. These gene clusters are structurally diverse and hold significant biological relevance. The analysis of genomic potential greatly facilitates the identification of active strains.
Plant diseases caused by pathogenic fungi result in significant agricultural losses and pose a considerable threat to global food security. The management of these diseases primarily involves chemical and biological control methods. Chemical pesticides are essential in agricultural production; farmers have utilized these substances to combat plant diseases, and it has been reported that approximately one-third of agricultural output relies on pesticide application. Without pesticides, 78% of fruit production, 54% of vegetable production, and 32% of cereal production would be lost [50]. However, the use of chemical pesticides is toxic to non-target organisms and contributes to environmental pollution, affecting air, water, and soil quality, which in turn poses risks to human health [51]. Biotechnological approaches are crucial in addressing these fungal diseases in plants, with Streptomyces playing a significant role [52]. Active bacterial strains and their metabolites are vital components of biotechnology for the prevention and management of plant diseases. Specific secondary metabolites produced by Streptomyces exhibit broad-spectrum antifungal properties, making them a promising alternative for disease management [52]. Meghashyama has reported that the Streptomyces sp. strain YC69 can effectively antagonize fungal pathogens affecting chili plants [53]. Research by Yuan has underscored the potential of Streptomyces setonii WY228, along with its volatile compound 2-ethyl-5-methylpyrazine, in effectively managing black spot disease in sweet potatoes [54]. Additionally, Meghashyama et al. identified antifungal activity against chili pathogens in Streptomyces strain KF15 [55]. In summary, the application of Streptomyces is a crucial component of biocontrol strategies for the effective suppression of plant diseases; thus, the isolation and identification of antagonistic microorganisms are essential for developing biocontrol agents [56,57]. Furthermore, Streptomyces sp. TRM76147 has demonstrated promising potential for the biological control of A. gaisen. This study found that the crude extract could inhibit the growth of A. gaisen spores. Pear black spot typically occurs in the spring when conidia are dispersed by wind and rain, landing on pear leaves, where, under optimal temperature and humidity conditions, they germinate. This disease generally begins to develop in late April, with a higher incidence observed in June and July. A critical factor in controlling this disease is stability. The study investigated the effects of temperature, UV irradiation, and pH on the compounds to evaluate their utility. The results revealed that the substances in the strains exhibited peak activity between 40 °C and 60 °C. Notably, the antifungal activity of strain TRM76147 diminished progressively following UV treatment, with a significant reduction noted after 60 min of irradiation. Moreover, the crude extract showed considerable acid stability within a pH range of 2–12, a resilience likely attributable to the harsh environmental conditions in Xinjiang, characterized by high heat and UV radiation. Microorganisms in such extreme environments tend to produce substances that can withstand these conditions. For instance, Rahul et al. isolated a cryophilic strain of Pseudomonas aeruginosa from a mountainous region capable of producing phenazine-1-carboxylic acid at temperatures ranging from 14 °C to 25 °C [58]. This compound has demonstrated biocontrol and plant growth promoting abilities, supporting the conclusion that environmental factors influence the microbial secondary metabolic capacity. Additionally, Jain et al. identified acidophilic and alkalophilic strains in extreme environments, suggesting that extreme microorganisms possess the capacity to adapt to environmental changes [59]. Studies by Yao et al. and Santos underscore the significant impact of UV radiation and pH on the stability and activity of certain compounds [53,60,61]. Overall, the findings highlight the crucial role of pH, temperature, and UV radiation in modulating the activity of active substances, primarily through alterations in microbial metabolites due to environmental influences. This also shows that TRM76147 is tolerant to temperature, UV, etc. and has good potential for application as a biocontrol strain.
This study contributes to the expansion of the database of antagonistic microbial species in extreme environments in Xinjiang, providing valuable insights into Actinomycetes biodiversity and facilitating the exploration of extreme microbial resources.

5. Conclusions

In the present study, a strain of Streptomyces sp., TRM76147, isolated from tamarisk, was identified by combining genotypic characterization, physiological and biochemical features, and other characteristics, which showed good antagonistic activity. Through genomic analysis, the metabolic capacity of the strain was predicted, and the stability of TRM76147 active substance against UV, pH, and temperature was investigated. We found that strain TRM76147 has an impact on the cell wall and growth rate of A. gaisen. The biocontrol potential of the strain was validated through fruit experiments. These findings provide a theoretical basis for developing pathogen prevention and control strategies. These findings not only contribute to the understanding of controlling pear black spot, but also underscore the importance of preserving and utilizing microbial resources for sustainable development in the world.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f15091605/s1, Figure S1: Analysis of the pan-genome and housekeeping genes of strain TRM76147.

Author Contributions

All the authors contributed to the study conception and design. Y.-H.C., J.-X.Z., G.Y. and S.R. analyzed samples and edited the manuscript. Y.L. and J.-M.W. managed the resources. Q.L. and X.-X.L. designed the experiment and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Tarim University President gene project “Preparation and application of phosphonic acid producing biocontrol agents” (TDZKZD202202), supported by the study on the biosynthesis mechanism and combination biosynthesis of indole oxazole antibiotics in actinomycetes (32360009) and diversity of typical soil actinomycetes in different habitats of the Tarim River and screening of phosphonic acid producing strains (2022-XBQNXZ-019) and Tarim University Doctoral Entrepreneurship Project (TDBSCX202302).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. If you need to obtain the original data, you can contact the first author, Chen Yihuang, email: [email protected], or the corresponding authors, Qin Liu and Luo Xiaoxia, email: [email protected].

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. The phylogenetic analysis of the 16S rRNA gene of strain TRM76147. Note: The phylogenetic analysis of strain TRM76147’s 16S rRNA was carried out based on neighbor joining. Kitasatospora setae NBRC14216T was used as an external reference strain.
Figure 1. The phylogenetic analysis of the 16S rRNA gene of strain TRM76147. Note: The phylogenetic analysis of strain TRM76147’s 16S rRNA was carried out based on neighbor joining. Kitasatospora setae NBRC14216T was used as an external reference strain.
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Figure 2. Morphological characteristics of TRM76147 colony. (A) Colony morphology after 7 days of cultivation in Gao’s medium. (B) TRM76147 single colony. (C) Scanning electron microscopy images of the mycelium of strain TRM76147. (D) Scanning electron microscopy images of the spore chain of strain TRM76147.
Figure 2. Morphological characteristics of TRM76147 colony. (A) Colony morphology after 7 days of cultivation in Gao’s medium. (B) TRM76147 single colony. (C) Scanning electron microscopy images of the mycelium of strain TRM76147. (D) Scanning electron microscopy images of the spore chain of strain TRM76147.
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Figure 3. Chemical index identification of strain TRM76147. (A) Type of cell wall polar lipid of strain TRM76147 (1: ninhydrin staining, 2: molybdenum phosphoric acid staining, 3: 4-anisaldehyde staining). (B) Strain TRM76147 cell wall whole cell hydrolytic sugar type. (C) Strain TRM76147 fine cell wall acetaminophen acid type. DPG: diphosphatidyl glycerol. PME: phosphatidyl methyl ethanolamine. PE: phosphatidyl ethanolamine. PG: phosphatidyl glycerol. LPG: lyso-phospholipids glycerol. NPG (Gliu Nu): phospholipids of unknown structure containing glucosamine. PIM: phosphatidylinositol mannosides. PI: phosphatidyl inositol. PIDM: phosphatidylinositol dimannoside. PC: phosphatidyl choline. OH-PE: hydroxyl-phosphatidylethal phosphatidylinositol. Man: mannose. Glu: glucose. Rha: Rhamnose. Xly: xylopyranose. Gal: galactose. Rib: Ribosome. Ara: Arabinose. LL-DAP: LL-2,6-Diaminopimelic acid. Meso-DAP: Meso-Diaminopimelic. D,D-DAP: DD-Diaminopimelic.
Figure 3. Chemical index identification of strain TRM76147. (A) Type of cell wall polar lipid of strain TRM76147 (1: ninhydrin staining, 2: molybdenum phosphoric acid staining, 3: 4-anisaldehyde staining). (B) Strain TRM76147 cell wall whole cell hydrolytic sugar type. (C) Strain TRM76147 fine cell wall acetaminophen acid type. DPG: diphosphatidyl glycerol. PME: phosphatidyl methyl ethanolamine. PE: phosphatidyl ethanolamine. PG: phosphatidyl glycerol. LPG: lyso-phospholipids glycerol. NPG (Gliu Nu): phospholipids of unknown structure containing glucosamine. PIM: phosphatidylinositol mannosides. PI: phosphatidyl inositol. PIDM: phosphatidylinositol dimannoside. PC: phosphatidyl choline. OH-PE: hydroxyl-phosphatidylethal phosphatidylinositol. Man: mannose. Glu: glucose. Rha: Rhamnose. Xly: xylopyranose. Gal: galactose. Rib: Ribosome. Ara: Arabinose. LL-DAP: LL-2,6-Diaminopimelic acid. Meso-DAP: Meso-Diaminopimelic. D,D-DAP: DD-Diaminopimelic.
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Figure 4. Analysis of metabolic potential of strain TRM76147 and similar strains.
Figure 4. Analysis of metabolic potential of strain TRM76147 and similar strains.
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Figure 5. Statistical analysis of gene cluster of TRM76147 and similar strains. (A) BigSCAPE software analyses of the total number of genes clusters. (B) The total number of gene clusters class analyzed via BigSCAPE software.
Figure 5. Statistical analysis of gene cluster of TRM76147 and similar strains. (A) BigSCAPE software analyses of the total number of genes clusters. (B) The total number of gene clusters class analyzed via BigSCAPE software.
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Figure 6. Inhibitory effect of strain TRM76147 on A. gaisen. Different letters indicate a significant difference (p < 0.05) among treatments via Duncan’s new complex polar deviation test. Having the same letter means that the difference between the samples is not significant and not having the same letter means that the difference between the samples is significant. Note: (A) TRM76147 plate and fermentation broth inhibition of A. gaisen, (1: TRM76147 plate, 2: TRM76147 fermentation solution). (B) Inhibition of A. gaisen via different solvent extracts of fermentation broths (3: dichloromethane phase, 4: aqueous phase, 5: methanol phase, 6: ethyl acetate phase). (C): Inhibition of A. gaisen via different concentrations of crude extracts (a: 6.5%, b:12.5%, c: 25%, d: 50%, e: 100%, CK: Tris buffer solution).
Figure 6. Inhibitory effect of strain TRM76147 on A. gaisen. Different letters indicate a significant difference (p < 0.05) among treatments via Duncan’s new complex polar deviation test. Having the same letter means that the difference between the samples is not significant and not having the same letter means that the difference between the samples is significant. Note: (A) TRM76147 plate and fermentation broth inhibition of A. gaisen, (1: TRM76147 plate, 2: TRM76147 fermentation solution). (B) Inhibition of A. gaisen via different solvent extracts of fermentation broths (3: dichloromethane phase, 4: aqueous phase, 5: methanol phase, 6: ethyl acetate phase). (C): Inhibition of A. gaisen via different concentrations of crude extracts (a: 6.5%, b:12.5%, c: 25%, d: 50%, e: 100%, CK: Tris buffer solution).
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Figure 7. Inhibition effect of ethyl acetate extract on A. gaisen under different temperature treatments. (A) Ethyl acetate extract treated at 40 °C. (B) Ethyl acetate extract treated at 50 °C. (C) Ethyl acetate extract treated at 60 °C. (D) Ethyl acetate extract treated at 70 °C. (E) Ethyl acetate extract treated at 80 °C. CK: ethyl acetate extract without temperature treatment.
Figure 7. Inhibition effect of ethyl acetate extract on A. gaisen under different temperature treatments. (A) Ethyl acetate extract treated at 40 °C. (B) Ethyl acetate extract treated at 50 °C. (C) Ethyl acetate extract treated at 60 °C. (D) Ethyl acetate extract treated at 70 °C. (E) Ethyl acetate extract treated at 80 °C. CK: ethyl acetate extract without temperature treatment.
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Figure 8. Effect of UV and pH on the active crude extract of TRM76147. Different letters indicate a significant difference (p < 0.05) among treatments via Duncan’s new complex polar deviation test. Having the same letter means that the difference between the samples is not significant and not having the same letter means that the difference between the samples is significant. Note: (A). Effect of UV on active crude extracts (1: ethyl acetate extract treated with UV for 20 min; 2: ethyl acetate extract treated with ultraviolet radiation for 40 min; 3: purple ethyl acetate extract treated with external line for 60 min; 4: ethyl acetate extract treated with ultraviolet light for 80 min). (B) Effect of pH on active crude extracts (5: ethyl acetate extract with pH = 2; 6: ethyl acetate extract with pH = 4; 7: ethyl acetate extract with pH = 6; 8: ethyl acetate extract with pH = 8; 9: ethyl acetate extract with pH = 10; 10: ethyl acetate extract with pH = 12).
Figure 8. Effect of UV and pH on the active crude extract of TRM76147. Different letters indicate a significant difference (p < 0.05) among treatments via Duncan’s new complex polar deviation test. Having the same letter means that the difference between the samples is not significant and not having the same letter means that the difference between the samples is significant. Note: (A). Effect of UV on active crude extracts (1: ethyl acetate extract treated with UV for 20 min; 2: ethyl acetate extract treated with ultraviolet radiation for 40 min; 3: purple ethyl acetate extract treated with external line for 60 min; 4: ethyl acetate extract treated with ultraviolet light for 80 min). (B) Effect of pH on active crude extracts (5: ethyl acetate extract with pH = 2; 6: ethyl acetate extract with pH = 4; 7: ethyl acetate extract with pH = 6; 8: ethyl acetate extract with pH = 8; 9: ethyl acetate extract with pH = 10; 10: ethyl acetate extract with pH = 12).
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Figure 9. Changes in spore count before and after treatment with ethyl acetate extract. (A) Changes in spores before and after different solvents (1: number of spores treated sterile phosphate buffer solution, 2: number of spores treated twice with the ethyl acetate extract). (B) Changes in spore germination before and after different solvents (1: number of spores treated sterile phosphate buffer solution, 2: number of spores treated ten-fold with ethyl acetate extract).
Figure 9. Changes in spore count before and after treatment with ethyl acetate extract. (A) Changes in spores before and after different solvents (1: number of spores treated sterile phosphate buffer solution, 2: number of spores treated twice with the ethyl acetate extract). (B) Changes in spore germination before and after different solvents (1: number of spores treated sterile phosphate buffer solution, 2: number of spores treated ten-fold with ethyl acetate extract).
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Figure 10. Morphological changes of hyphae and conidium before and after treatment with ethyl acetate extract. (A) Mycelia before and after treatment with ethyl acetate extract (1: A. gaisen mycelium, 2: hyphae treated with crude extract). (B) Spore bodies before and after treatment with ethyl acetate extract (1: A. gaisen spores, 2: spore bodies treated with crude extract).
Figure 10. Morphological changes of hyphae and conidium before and after treatment with ethyl acetate extract. (A) Mycelia before and after treatment with ethyl acetate extract (1: A. gaisen mycelium, 2: hyphae treated with crude extract). (B) Spore bodies before and after treatment with ethyl acetate extract (1: A. gaisen spores, 2: spore bodies treated with crude extract).
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Figure 11. Fermentation broth treatment, pathogenic treatment, blank processing, morphological changes of fragrant pear.
Figure 11. Fermentation broth treatment, pathogenic treatment, blank processing, morphological changes of fragrant pear.
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Table 1. Physiological indicators of TRM76147 and similar strains.
Table 1. Physiological indicators of TRM76147 and similar strains.
FeatureTRM76147Streptomyces griseoviridis NBRC12874TStreptomyces niveoruber NBRC15428T
Color of aerial hyphaeWhitePale greyish-purpleIvory
Matrix hyphal colorYellowPurplish-redPale yellow
Spore chain morphologyStraightRectiflexibilesStraight
Pigment productionYellowNoneBrown
Temperature range (°C)20–45≤45<45
pH range7–105–126–11
NaCl range (%)≤5≤7<7
Sucrose+NDND
Maltose+++
L-arabinose+(+)+
Galactose+NDND
LactoseND+(+)
Inositol++ND
Dextrin++ND
Melanin production−−−
Hydrogen sulfide production+−−
Gelatin liquefaction+++
Protease production++−
Amylase production+NDND
Nitrate reduction+NDND
Oxidase production+NDND
Production of catalase+NDND
Urease production−NDND
Tween 20+++
Cellulose decomposition+NDND
Notes: + indicates positive; − indicates negative; (+); weakly positive; ND, not deterministic.
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MDPI and ACS Style

Chen, Y.-H.; Zhang, J.-X.; Yang, G.; Liu, Y.; Ran, S.; Wang, J.-M.; Liu, Q.; Luo, X.-X. Potential of Crude Extract of Streptomyces sp. nov., Strain TRM76147 for Control of A. gaisen. Forests 2024, 15, 1605. https://doi.org/10.3390/f15091605

AMA Style

Chen Y-H, Zhang J-X, Yang G, Liu Y, Ran S, Wang J-M, Liu Q, Luo X-X. Potential of Crude Extract of Streptomyces sp. nov., Strain TRM76147 for Control of A. gaisen. Forests. 2024; 15(9):1605. https://doi.org/10.3390/f15091605

Chicago/Turabian Style

Chen, Yi-Huang, Jia-Xin Zhang, Guo Yang, Yang Liu, Song Ran, Jian-Ming Wang, Qin Liu, and Xiao-Xia Luo. 2024. "Potential of Crude Extract of Streptomyces sp. nov., Strain TRM76147 for Control of A. gaisen" Forests 15, no. 9: 1605. https://doi.org/10.3390/f15091605

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