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Two new strains of Streptomyces with metabolic potential for biological control of pear black spot disease

BMC Microbiology volume 24, Article number: 550 (2024) Cite this article

Abstract

Background

Pear black spot is caused by Alternaria tenuissima. It is one of the diseases of concern limiting pear production worldwide. Existing cultivation methods and fungicides are not sufficient to control early blight. Therefore, the aim of this study was to isolate and characterize two strains of Streptomyces and evaluate their potential for biological control of crop diseases caused by Alternaria tenuissima while promoting plant growth. It enriches the resources of biocontrol strains.

Methods

In this study, the genetic background of the strain was elucidated through 16S rRNA gene analysis and multiphase taxonomic identification methods. The metabolic potential of the strain was assessed using a variety of approaches, including antiSMASH, COG, and KEGG databases, RGI tools, as well as the scanning of CAZY and plant-promoting genes. The biocontrol potential of the strain was further substantiated through a combination of plate experiments, gene cluster biopathway resolution and mass spectrometry validation of metabolites. Finally, the biocontrol efficacy of the strain was confirmed through fruit control experiments.

Results

The study identified the potential new species status of the strains. Strain TRM 76130 exhibited a gene size of 5.94 Mbp and a G + C content of 73.65%, while strain TRM 76172 had a gene size of 8.30 Mbp and a G + C content of 71.38%. Both strains contained genes related to amino acid transport and metabolism, along with several CAZY genes and 19 plant growth factors. The resistance genes of strain TRM 76172 were classified as macrolides, and genomic prediction revealed the biosynthetic pathway of the active compound Candidin. Mass spectrometry analysis indicated that strains TRM 76172 and TRM 76130 contained the active compounds amphotericin A and daptomycin, respectively. The pear assays demonstrated that both strains of Streptomyces were capable of reducing the symptoms of pear black spot.

Conclusion

The present study concludes that strains TRM76172 and TRM76130 possess significant potential to control Alternaria tenuissima and promote plant growth, thereby enriching the biocontrol fungal library.

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Background

Pear is a deciduous fruit tree that has been economically cultivated in China for over 3,000 years [1]. According to incomplete statistics, there exist more than 2,000 varieties of pears in the world, with over 1,200 varieties being identified in China. In 2009, the national fruit cultivation area reached 11,139,500 hectares, with an output of 12,246,390 tonnes of fruits. By 2011, the national pear cultivation area grew to to 1,071,300 hectares, yielding 15,057,000 tonnes. This production constituted 10% of the total fruit area of the nation and 63% of the global pear production, thereby contributing 2.2% to China’s overall fruit production [2]. Therefore, pears constitute a significant component of the fruit industry in China.

Alternaria tenuissima is a widely distributed fungus that occurs in natural environments. This pathogen is capable of causing various diseases in pear trees and exists in both saprophytic and pathogenic forms [3,4,5]. The onset of infection is most prevalent from June to August, particularly in conditions of rain, humidity, and high temperatures, with an incidence rate exceeding 30% [6]. When A. tenuissima infects pear trees, it manifests as brown or black spots on the leaves that are either round or irregular in shape. These spots may enlarge, leading to wilting of the leaves. The resulting disease damage can result in significant fruit cracking and early fruit drop, with a rate of affected fruit reaching 75% or more [7].

The control methods for Alternaria. tenuissima primarily include biological control, chemical control, agricultural practices, as well as the selection and breeding of resistant varieties. Despite the efficacy of chemical agrochemicals in inhibiting the occurrence of phytopathogenic fungi, their residues pose significant concerns regarding food safety. However, agricultural measures have several disadvantages, including high labor costs, the periodic selection of disease-resistant varieties, and environmental uncertainties. These measures have had little effect [8]. As a widely utilized management technique in ecological agriculture, biological control possesses the advantages of being environmentally friendly, safe, and effective. In summary, the selection of biological control methods for A. tenuissima is both safe and effective.

Biological control involves the use of microorganisms and their metabolites to defend against and control phytopathogenic fungi. This approach aims to inhibit the growth and reproduction of fungi, thus minimizing their impact on plant health [9, 10]. Streptomyces has garnered significant attention for its broad-spectrum antimicrobial properties [11]. For instance, Streptomyces JCK-8055 possesses the capability to effectively control apple fire blight. Streptomyces violaceusniger niger produces antimicrobial substances that effectively inhibit the growth of Alternaria tenuissima [12]. Additionally, beyond its fungicidal and antimicrobial capabilities, Streptomyces plays a crucial role in various applications, including biopesticides and growth promotion [13].

Streptomyces has received constant attention due to its comprehensive antimicrobial properties. Streptomyces has been exploited using several approaches, including activity tracking and genome mining. Activity tracking is blind, while genome-guided metabolite mining can avoid this drawback [14]. The extensive sequencing of the Streptomyces genome has demonstrated that Streptomyces possesses an abundance of BGCs that regulate the synthesis of secondary metabolites. Streptomyces has a linear chromosomal genome, the largest known prokaryotic genome. It has a unique metabolic potential due to its complex genome. The conventional DNA sequencing techniques employed for Streptomyces coelicolor A3(2) and Streptomyces avermitilis have undergone a significant transformation due to the advancements in sequencing technology. At present time, more than 1100 Streptomyces genomes have been sequenced, and this number is continuously increasing. Genome-based analysis has the potential to enhance the biological defense potential of bacterial strains [15].

In this study, two strains of Streptomyces ,isolated from the roots of Tamarix tamarisk,were identified as novel species by combining molecular, morphological, and chemical characteristics. The metabolic potentials of the two strains were systematically analyzed, and their biocontrol potentials against Alternaria tenuissima were determined through genome analyses, mass spectrometry assays and biocontrol activity assays.

Results

Identification of the genetic classification status of strains

Morphological, cultural, physiological and biochemical characterization of strain 76172 and strain 76130

Initially, TRM 76172 produced white spores on Gao’s media (Supplementary Fig. 1A a), which subsequently turned gray after 4–5 days of culture (Supplementary Fig. 1A. b). In a similar manner, TRM 76130 also produced white spores on Gao’s media (Supplementary Fig. 1B. a). However, after 4–5 days of culture, it developed purple segments (Supplementary Fig. 1B. b). Both strains showed objective Streptomyces morphology, with flexuous spore chains with hooks observed in TRM76130 and cylindrical spores observed in TRM76172 (Supplementary Fig. 1A. c Fig. 1B. c). Both strains showed antagonistic activity against Alteraria tenuissima in figrue 1 C. The aerial hyphae of TRM76130 appeared folded, whereas those of TRM761720 appeared smooth, twisted, and had straight spare filaments (Supplementary Fig. 1D). The strain TRM76172 grew in the ISP series medium and nutrient agar (NA) medium, Gao’s medium, and Cha’s medium for 7 days at 37℃. The results showed the highest growth on ISP1, limited growth on ISP7, ISP1. It demonstrated the capacity to withstand NaCl concentrations of up to 5.0% and thrived within a pH range of 7.0–11.0, with optimal growth observed at pH 7.0. Strain TRM76172 can utilize cellulose, allowing hydrolysis of starch and the production of urease, oxidase, which reduces nitrates, gelatin liquefaction, which does not produce melanin, and catalase, which peptonizes milk. The strain TRM76130 was cultivated for 7 days at 37℃ in ISP series medium, NA medium, Gao’s medium, and Cha’s medium. The results demonstrated the best growth on Gao’s medium, with the ability to tolerate NaCl concentrations up to 5.0%. It thrived within a pH range of 7.0–10.0, with optimal growth observed at pH 7.0. Strain TRM76130 can utilize cellulose, allow starch hydrolysis, produce urease, reduce nitrates, liquefy gelatin, and cannot produce melanin. It cannot produce catalase, oxidase, or peptonize milk. Supplementary Table 1.

Molecular characterization of TRM 76130 and TRM 76172

The phylogenetic analysis of strains TRM 76172 and TRM 76130 was conducted alongside similar strains Streptomyces fumanus NBRC 13042T, Streptomyces mutabilis NBRC 12800T, and Streptomyces griseoviridis NBRC 12874T. Phylogenetic trees based on the 16 S rRNA gene and multilocus sequences were reconstructed (Fig. 1) [16,17,18]. The comparative average nucleotide identity (ANI) of TRM 76130 and TRM 76172 with phylogenetically related S.mutabilis NRRC 12800T and S. fumanus NBRC 13042T. The ANI of TRM 76172 and S.mutabilis NBRC 12800T was 95.91% identical, and their digital DNA-DNA hybridization (dDDH) was 66.50% alike. This means that they may be a new species. The ANI of TRM 76130 and S. fumanus NBRC 13,042T was 86.30%, and the dDDH between them was 70%, which is significantly lower than the threshold of 70% for the delineation of prokaryotic genomic species. In general, the ANI value between genomes of the same species is greater than 95%. ANI is 86.30% between TRM 76130 and S. fumanus NBRC13042T. TRM 76130 and TRM 76172 as potential new species.

Fig. 1
figure 1

Phylogenetic analysis of TRM 76172 and TRM 76130 based on the 16S rRNA gene

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Characterization of the chemical components of TRM76172 and TRM76130

In order to distinguish the independence of bacterial strains, it is imperative to analyze the chemical constituents present in the strains, such as amino acids, and to identify the pertinent chemical indicators. The cell wall fractions analyzed by nhydrin, polylipids, molybdophosphoric acid, and strain TRM76172 were found to contain PC and PE, while strain TRM76130 contained PIDM, PIM, PG, PE and PLS. Strain TRM76130 and strain TRM76172 contained the diaminoheptanoic acid type was LL-DAP (LL-2,6-diaminoheptanoic acid) (Fig. 2). Whole-cell hydrolyzed sugars of strain TRM76130 were predominantly Ara, while those of strain TRM76172 were predominantly Gal. The chemical classification of strain TRM76172 and strains TRM76130 is in accordance with the members of the genus Streptomyces. Supplementary picture 2.

Fig. 2
figure 2

Type of cell wall polar lipid of strain TRM76172 and TRM76130 (A: TRM76172, B: TRM76130)

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Genome analysis of strain TRM76172 and strain TRM76130

Comparative genomics of TRM 76172 and TRM 76130 with strain S. mutabilis NBRC 12800T and S. fumanus NBRC 13042T

TRM 76130 has nine synthetic gene clusters (Fig. 3A), whereas TRM 76172 has thirty-three (Fig. 3B). The genome sizes of TRM 76130 and S. fumanus NBRC 13042 were 5.94 Mbp and 8.15 Mbp, respectively (Fig. 3C). The content of the G + C was 73.65% and 73.00%. The total genome size of TRM 76172 and S. mutabilis NBRC12800T and 8.30 Mbp and 7.79 Mbp, respectively. (Fig. 3D). It was found that the G + C content was 71.38% and 71.15%. In the TRM 76130 and TRM 76172 genomes, respectively, 10,661 and 241 scaffolds were found. The predicted protein encoding genes (peg) were 8030 and 7063 in TRM 76130 and S. fumanus NBRC 13042T. The predicted protein encoding genes (PEGs) were 7450 and 7254 in TRM 76172 and S. mutabilis NBRC 12800T, respectively. Furthermore, 60 and 74 noncoding genes were observed in TRM 76130 and S. fumanus NBRC 13042T, respectively, 72 and 101 noncoding genes were also observed in TRM 76172 and S. mutabilis NBRC 12800T, respectively. The comparison of the genomes of TRM 76130 and TRM 76172 with their closest phylogenetic relationship to S. mutabilis NBRC 12800T was presented in Supplementary Table 2, (see Fig. 4).

Fig. 3
figure 3

Unveiling metabolite biosynthesis gene clusters in Streptomyces strains TRM 76172 and TRM 76130. Note: A synthetic gene clusters of TRM 76130 B synthetic gene clusters of TRM 76172.C Whole genome circle map of TRM 76172.D Whole genome circle map of TRM 76130

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Fig. 4
figure 4

Genomic metabolic potential analysis based on 16 S rDNA phylogenetic analysis of strain TRM76172 and strain TRM76130 and similar strains

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KEGG of TRM 76172 and TRM 76130

The results of the KEGG metabolic pathway analysis for TRM 76172 genome are shown in Supplementary Fig. 3. These genes are cateporized into 22 metabolic pathways that correspond to the expression products of diverse gene sequences. The annotated outcomes of TRM 76172 and TRM 76130 database primarily pertain to the processing of genetic information, signal and cellular processes, carbohydrate metabolism, and amino acid metabolism. The functionally encoded genes were generally of TRM 76172 were generally superior to TRM 76130.

COG of TRM 76172 and TRM 76130

According to the COG function prediction, TRM 76172 contains 825 genes with unknown functions, followed by genes closely related to transcription and amino acid transport and metabolism. TRM 76130 is also enriched in 725 genes closely associated with transcription and 571 genes closely associated with amino acid transport metabolism. While TRM 76172 and TRM 76130 have been classified into 26 cluster of orthologous groups of proteins (COG). The functional protein annotations of TRM 76130 are lower than those of 76172, both of which have good activity against Alternaria tenuissima. The COG function circle diagram for TRM 76172 and TRM 76130 (Supplementary Fig. 4).

RGI (Resistance Gene Identifier)

The genome of TRM 76172 strain comprises a gene that encodes a macrolide glycotransferase (Mgt) and also encodes for the production of macrolide antibiotics. Macrocyclic lactones are a class of compounds that possess potent antifungal properties. It is anticipated that the resistance gene of TRM 76130 is associated with tetracycline (Supplementary Table 3).

Genomic Distribution of CarbohydrateActive Enzymes (CAZymes) in TRM 76130 and TRM 76172

The 60 and 48 CAZY enzyme genes were identified in TRM 76172 and TRM 76130 using eCAMI. In TRM 76172 and TRM76130, 21 and 59 CAZY enzyme genes were identified using DIAMON. 162 and 253 CAZY enzyme genes were identified using the HMMER, respectively. In these three databases, two and one CAZY enzymes were predicted for TRM 76130 and TRM 76172 (Supplementary Fig. 5). CAZY predominantly belongs to glycosyl hydrolases (GH) families.

Plant growthpromotion gene factors

Supplementary Fig. 6 shows the presence and absence of gene factors encoding enzymes and gene cluster in plant growth-promotion pathways in the the Streptomyces family. Red square indicates the presence and absence of the enzymes and cluster, blue square indicates the absences of the enzymes and cluster. It was discovered that all of the strains utilized possessed related enzymes involved in IAA synthesis, and futher research revealed that related enzymes invoved in trehalose synthesis were founded to be more prevalent in all strains. ACC deaminase was absent in all strains, and enzymes involved in acetoin and 2,3-butanediol synthesis were also less abundant. There was major focus on desferoxamine in all strains regarding siderophore. It is noteworthy that strain TRM 76172 is involved in other growth promoting factors for plants besides ACC deaminase, whereas strain TRM 76130 is solely involved in the synthesis of polyamines and IAAs.

Bioproofing potential analysis and validation

Genome mining in gene cluster analysis TRM 76172 and TRM 76130

These strains possessed a greater number of T1PKS, terpene, and siderphore, NRPS clusters. Furthermore, some of the smBGCs were strain-specific (Fig. 4). For example. TRM 76172 contains 33 gene clusters in which candidin is predicted to be the reported active metabolite; TRM 76130 contains 9 gene clusters in which cyphomycin is predicted to be the reported active metabolite; Streptomyces sp. mutabilis NBRC 12,800T contains 29 gene clusters in which actinomycin D is predicted to be the reported active metabolite; Streptomyces fumanus NBRC 13,402T contains 36 gene clusters in which planosporicin is predicted to be the reported active metabolite. The most common clusters in this family were involved in the biosynthesis of T1PKS, T2PKS, T3PKS, Melanin, RiPP-like and terpene. The smBGCs frequently found in Streptomyces species are those for ectoine and deferoxamine. The number of TRM 76130 gene clusters is minimal, but it has excellent resistance to Alternaria tenuissima. Cyphomycin is an active gene cluster of TRM 76130. By analyzing the similar strain Streptomyces swartergensis HMC 13T, and Streptomyces griseovirdis NBRC 12,874T. The phosphonic acid synthesis gene cluster was found in two strains.

Through genome analysis, TRM 76172 was found to have a candidin biosynthesis gene cluster, which has 90% similarity to BGC0000034 of Streptomyces sp. FR-008 [19, 20], as shown in Table 1. The candidate gene cluster of TRM 76172 comprises 16 genes, whereas the gene cluster of FR-008 comprises 20 genes. TRM 76172 lacks four genes, such as fscC, fscB, fscE and fscF with FR-008 (Fig. 5A). These four genes produce polyketase. TRM 76172 possesses a gene that is comparatively complete. These four genes encode 17 domains, and the candidate protein biosynthesis gene clusters ctg54_2 and ctg54_5 of TRM 76172 encode two modules (Fig. 5B). These two modules comprise KS, MAT, KR and ACP domains, which consist of acyl carrier protein domain (ACP), keto thioester synthase (KS), keto reductase (KR) and dehydratase (DH). It is a complete gene cluster, and similar compounds or new secondary metabolites may be formed upon comparisn. This study utilized thin layer chromatography to determine the fermentation crude extract of TRM 76172. A spot appeared using methanol 30:1 as the developing agent and using UV coloration (Fig. 5C). It has a good activity of inhibiting Candida albicans (Fig. 5D). It appeared to be a singular ensemble. It is deemed to posses a robust conjugate system. Through literature investigation and analysis, we found that only the cyclofosfomy-cingene cluster showed good antifungal activity in TRM 76130 genome. It is possible that the activity of TRM 76130 itself against A. tenuissima could be triggered by this antibiotic. Coelichelin, it is an organochlorine insecticide. Concanamycin A, a novel antibiotc discovered in Brazilian fungus breeding ant, has been identified. An enzyme inhibitor. A54145 is an antimicrobial compound lipopeptide. They were all detected genome-wide in TRM 76172. Cyphomycin, a novel antibiotic discovered in a Brazilian fungus breeding ant. It was also discovered within the genome of TRM 76130.

Fig. 5
figure 5

Genetic analysis of candidin biosynthesis. Note A TRM 76172 and FR-008 synthetic gene cluster. B TRM 76172 gene cluster module. C FR-008 gene cluster module. D TLC spot plate and antimicrobia activity picture in TRM 76172

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Table 1 Prediction of biosynthetic gene clusters using antiSMASH in TRM 76172 and TRM 76130
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Analysis of metabolites of strain TRM76130 and strain TRM76172

The crude extracts of strain TRM76172 and strain TRM 76130 showed that strain TRM 76172 detected amphotericin A, which belongs to the same class of diene macrolides, as the biosynthesis gene cluster containing candidin in the strain predicted in the prestage. A congener of candidin was also obtained based on molecular weight prediction with the molecular formula C21H36O5Na. The strain TRM 76130 detected daptomycin, which belongs to the T1PKS type of compounds obtained from genome prediction. and all of the above compounds have antagonistic activities (Fig. 6).

Fig. 6
figure 6

Mass Spectrometric analysis of crude material from strain TRM76172 and TRM76130. Note: A C TRM76172 B TRM76130

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Activity verification

To investigate the bioprophylactic potential of the strains, we examined the effects of plate confrontation between strain TRM 76172 and strain TRM 76130 on the activity of Alternaria tenuissima and Candida using the plate confrontation method. The size of the activity circle was measured to determine the antagonistic activity. Additionally, we conducted experiments to evaluate the effects of the fermentation broths of TRM 76172 and TRM 76130 on pear infected with Alternaria tenuissima. The pear were initially soaked in 75% ethanol for 30 s, followed by sterilization through immersion in 1% sodium hypochlorite for 5 min. After this, they were rinsed three times with sterilized distilled water and air-dried on an ultra-clean bench in preparation for subsequent experiments. The pear inoculated with Alternaria tenuissima using the spot inoculation method were cultured in an aseptic chamber at 28 ℃ for 7 days to observe the symptoms of black spot disease (see Fig. 7).

Fig. 7
figure 7

Comparative study of TRM76172 and TRM76130 fermentation broth effects. A effect of TRM76172 fermentation broth on pear. B C Pears inoculated with Alternaria tenuissima only D effect of TRM76130 fermentation broth on pear

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Discussion

In this research study, TRM76172 grew best on ISP1 medium, with limited growth on ISP7. It tolerated up to 5.0% NaCl concentration, and grew vigorously at pH 7.0–11.0, and grew best at pH 7.0. Strain TRM76172 utilizes cellulose, hydrolyzes starch, produces urease, oxidase, and gelatin liquefaction. However, it does not produce melanin or catalase. TRM76130 grows best on Gao’s medium, tolerates up to 5.0% NaCl, and grows vigorously in the pH range of 7.0–10.0, with optimal growth at pH 7.0. Strain TRM76130 was capable of utilizing cellulose, hydrolyzing starch, producing urease, reducing nitrate, and liquefing gelatin. However, it was unable to produce melanin, catalase, oxidase, and milk peptones.TRM 76172 was found to contain PC and PE, whereas strain TRM 76130 contained PIDM, PIM, PG, PE, PLS. Whole-cell hydrolyzed sugars of strain TRM76130 were predominantly Ara, whereas whole-cell hydrolyzed sugars of strain TRM76172 were predominantly Gal. Compared to similar strains, the strain had an optimal pH of 4.3 and tolerated 8% salt concentration, S. mutabilis NBRC 12,800T utilized L-rhamnose, D-glucose, L-arabinose, D-mannitol, D-ribose production, nitrate reduction and lipase reaction were positive, and S. mutabilis NBRC 12,800T utilized L-rhamnose, L-arabinose, D-mannitol, D-ribose production, nitrate reduction and lipase reaction were positive. The reduction and lipase reaction were found to be positive, while urease, gelatin, oxidase and catalase reaction were found to be negative, The whole cell hydrolyed sugars, including glucose, ribose and other phospholipids, were found to be DPG, PE, PI, PC, PG, PIM. In conclusion, it can be inferred that strains TRM 76130 and TRM 76172 have the potential to be novel species [16,17,18]. The alteration of metabolic pathways by lignans influences insulin production, and the strength of metabolic capacity may affect the extent of active capacity [21]. Genomic analysis reveals that strain TRM 76172 possesses superior metabolic potential compared to TRM 76130, as evidenced by a greater number of genes involved in amino acid metabolic pathways. This finding aligns with the metabolic capacity predicted by KEGG and COG [22, 23]. Furthermore, plate activity assays demonstrate that strain TRM 76172 exhibits both a higher and broader spectrum of activity than strain TRM 76130. Glycoside hydrolases play a crucial role in metabolic pathways; the literature suggests that elevated levels of glycoside hydrolases (GHs) and glycosyltransferases (GTs) in the microbiome during plant infection by phytopathogenic bacteria are associated with disease suppression, which correlates with the strain’s effectiveness in mitigating plant diseases [24,25,26]. Both strains TRM 76172 and TRM 76130 contain a significant number of members from this enzyme family. Notably, the highest percentage of glycosyl hydrolases was observed in strain TRM 76172, further indicating the metabolic potential of these two new species.

Genomes offer significant opportunities for exploring the biological functions of Streptomyces. Analyzing strain genomes provides essential theoretical guidance for their exploitation. For instance, the genomic analysis of the biosynthetic gene cluster in the genome of TRM 76172, along with the confirmation of amphotericin A’s presence via mass spectrometry, has established a theoretical foundation for subsequent genomic studies. This research predicted the existence of a complete polyene macrolide synthesis gene cluster in strain TRM 76172. According to the literature, amphotericin A exhibits antifungal activity, which aligns with the antagonistic properties of strain TRM 76172 against pear blackspot. Additionally, mass spectrometry analysis of sample TRM 76130 revealed the presence of daptomycin, a compound known for its antagonistic effects against pathogenic fungi [27, 28]. Both strain TRM 76172 and strain TRM 76130 exhibit various plant growth-promoting factors, including indole-3-acetic acid (IAA), which plays a crucial role in plant growth. Notably, these strains were isolated from the soil of tamarisk, and those obtained from the roots of the plant displayed a range of growth-promoting factors. This observation underscores the potential of microorganisms to enhance plant growth [29]. It is predicted that the plant growth factors encoded in the genomes of these strains may provide valuable insights for their application in promoting plant growth.

In vitro experiments of strains are more responsive to the biocontrol potential of strains, and the ability of a strain to inhibit disease in vitro does not necessarily indicate its efficacy as an in situ biocontrol as it may not be able to show its potential under natural conditions, among which, Chen et al. found that strain XDS1-5 strain showed the strongest inhibition of brown rot, with 80% inhibition in vitro and 66% inhibition in vivo [30]. In this study, in vitro experiments were conducted to evaluate the efficacy of the strain against pear black spot disease caused by A. tenuissima. The results showed that strains TRM76172 and TRM76130 were significantly effective in reducing the incidence of pear black spot.

Conclusion

In this study, we determined the genetic taxonomic status of two new Streptomyces species, TRM76172 and strain TRM76130. The metabolic potential of these strains was analyzed from a genomic perspective using various software tools. Additionally, we explored the biocontrol potential of the strains by examining the genes involved in the biosynthetic pathways of potential metabolites, which was validated through mass spectrometry detection. Finally, we conducted fruit control experiments to assess their efficacy. This research investigated the ability of these two new strains to control Alternaria tenuissima using multiple methodologies, while also enriching the diversity of strain resources to provide a foundation for subsequent metabolite mining.

Materials and methods

Classification status of TRM76172 and TRM76130

Analysis of morphological, cultural, physiological, and biochemical characteristics

TRM 76172 and TRM 76130 were isolated from Tamarix rhinosphere soil in salwuzeke township, Xinjiang The oil samples with continuous dilution of 10− 4 times were placed on glycerol arginine medium for Actinomycetes isolation. Incubated at 28℃ for 3–5 days and observed the growth every day. The Gao’s culture medium was cultivated and stored at 4℃ on Actinomycetes isolation agar. The cultures were stored in 20% glycerol at − 80℃. A .tenuissima was purchased from China General Microbiological Culture Collection Center, strain NO. CGMCC 3.3546, in the College of Life Science and Technology, Tarim University.

The bacterial cultures were cultured in ISP4 medium at 28℃ at 150 rpm in orbital shaker for 2 to 3 days. The cultures were centrifuged at 6000 rpm at 4℃ for 5 min and resuspended in 100 mM of phosphate buffer containing 2.5% glutaraldehyde at pH 7.2. After incubation for 4 h, the cell pellets were washed 3 times at 4℃ with 100 mM phosphate buffer pH 7.2 for 5 min. The samples were dehydrated with a gradient of 30–100% ethanol for 15 min at a time. Finally, the samples were dried directly from 100% ethanol to a critical point dryer. When the sample is placed, 100% ethanol should be continuously added to the sample until all samples are discharged and dried immediately. After critical point drying, the samples were sputtered and coated with an ion sputtering instrument and observed by SEM (JSM-6360, jeol), The morphological characteristics of TRM 76130 and TRM 76172 were analyzed using SEM [31].

To determine the optimal growth medium, Strains TRM 76172 and TRM 76130 were grown on a variety of International Streptomyces Project (ISP) media types, including ISP1, ISP2, ISP3, ISP4, ISP5, ISP6 and ISP7. Cultures were also grown on Gause’s medium, potato dextrose agar and nutrient agar. The pH of all these media was adjusted to 7.0. The strain was maintained on ISP4 synthetic medium for growth and maintenance. The basal medium recommended by Pridham [32] was used for carbon source utilization tests. The growth capacity of strain TRM 76172 and TRM 76130 was evaluated at temperatures between 4 and 50℃ and pH values between 4 and 11. The tolerance of the system to different concentrations of NaCl ranging from 0 to 25% (w/v) was also evaluated. Additional tests examined the production of peroxidase, urease, esterase and catalase according to the methods described by Gerhardt et al. [33]. Other tests included the use of a sole carbon source (0.5%; w/v), cellulose degradation, starch hydrolysis, gelatine liquefaction, milk peptization and solidification, nitrate reduction and H2S production.

Molecular Characterization of TRM 76172 and TRM 76130

The genomic DNA of TRM 76172 and TRM 76130 was isolated according to the protocol and analyzed with 1% agarose gel. The 16 S rRNA universal primer pair 27 F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 1492R (5ʹ-TACGGTTACCTTGTTACGACT-3ʹ) were used to identify [34]. PCR reaction volume was 20 µL. PCR rea-gent 20 µL, template (total genomic DNA) 0.5 µL (~ 100 ng/µL), primer (10 pmol/µL) 1 µL each. T100 thermal cycler (Biorad) was used for PCR amplification. Initial denaturation was performed at 94℃ for 5 min, followed by 30 cycles of 94 ℃ for 30 s, 52℃ for 30 s, and 72 ℃ for 1.5 min and 72℃ for 10 min PCR products were electrophoretized on 1% agarose gel and 1 µL Gold VIEW (10 mg/mL of stock solution) was prepared in 1×TAE buffer. The gel was visualized using the Molecular imager ChemiDos XRS Gel Recording (BIO-RAD USA) system. Information based on 16S rRNA sequence was used to identify the closest strainsin the type strains retrieved at https://www.ezbiocloud.net/taxon omy database. The 16 S rRNA-based information was used to identify the nearest neighbor. Amplification and sequencing of the 16 S rRNA gene were performed. Multiple alignments with sequences from the most closely related members of the genus Streptomyces and calculations of sequence similarity were carried out using the EzTaxon- server (www.ezbiocloud.net/ identify). The housekeeping gene sequences of strain.TRM 76172 and TRM 76130 were amplified and sequenced according to the Streptomyces multilocus sequence typing protocol. (http://199.133.98.43/Streptomyces/documents/Streptomyces_MLST_Protocols 2013. pdf). Sequences of related strains were obtained from the ARS Microbial Genome Sequence Database server (http://199.133.98.43). Each locus for each strain was concatenated head-to-tail inframe as follows: atpD, gyrB, recA, ropB and trpB. Phylogenetic trees were constructed by neighbor-joining (NJ) method with 1000 bootstrap values of MEGA X 10.0 [35, 36].

Chemotaxonomic characteristics

Such as amino acid types in cell wall hydrolysates and whole cell sugar content were identified [37]. Polar lipids were determined by two-dimensional thin layer chromatography using 10% ethanolic molybdophosphoric acid as described by Minnikin et al. (1984) [38].

Genomics of TRM 76172 and TRM 76130

Metabolic potential analysis of TRM 76172 and TRM 76130

The genome of TRM 76172 and TRM 76130 was sequenced using a custom protocol recommended by Illumina (personalbio, Nanjing, China) to prepare a paired-end DNA sequencing library. The DNA was then se-quenced using 400 bp paired end blocks. Standard Illumina TruSeq nanoDNA LT li-braries were created using Pico Green Quantitative Libraries with stringent RAST, SEED Viewer, and RASTtk parameters [39,40,41,42]. TRM 76172 and TRM 76130 were sequenced using the Illumina Hiseq2000 sequencing platform and spliced with ABYSS. KEGG analysis [40] and COG analysis were performed on TRM 76172 and TRM 76130. Protein analysis of TRM 76172 and TRM 76130 was conducted as well [43]. Antibiotic-based synthetic resistance was evaluated by pre-dicting drug resistance genes using the RGI software database (https://card.mcmaster.ca/analyze/rgi). CAZY (Carbohydrate active enzymes) analysis [44] was used to predict CAZY for the two Streptomyces strains. AntiSMASH (6.0) [45] was employed to analyze secondary metabolite biosynthetic gene clusters (smBGCs) and predict antibiotics. Additionally, a phylogenetic tree was constructed using 16S rRNA gene data for secondary data analysis. Since all the strains were derived from soil, each strain was examined for plant growth-promoting genetic factors [46]. These factors include the synthesis of IAA, trehalose, ACC deaminase, polyamines, acetyl and 2,3-butanediol, and siderophores.

We utilized KEGG [47] to predict plant growth-promoting gene factors. Enzyme sequences were queried using NCBI and compared with HMMER (http://hmmer.janelia.org). The gene family HMM model was obtained by accessing the Pfam database. HMM search software was employed to search for protein gene families, and the protein genomes of 19 strains were scanned and compared. Core proteins were scanned based on the HMM model, and Orthofinder was used to identify homologous protein families. Species tree files were utilized for tree construction. The software was built using ITOL (https://itol.embl.de/itol.cgi). The genetic data were visualized using imageGP (https://www.bic.ac.cn/ImageGP/index.php/Home/Index/index.html).

Mass spectrometry of TRM 76172 and TRM 76130

Strain TRM76172 and strain TRM76130 were inoculated in ISP4 liquid medium and incubated at 37℃ for 7 days, and the samples were freeze-dried and sent to Xi’an Research Dog for testing on the Bruker solanX 70 FT-MS; Agilent 6540TOF, and the data obtained from the testing were compared with the molecular weights of the metabolites predicted by the genome, which were used to initially determine the active metabolites of the strain.

Evaluation for plant pathogen suppression ability reelaborated

The antibacterial effect of strain TRM76172 and strain TRM76130 on the agar plate was tested using the plate confrontation method [48]. To evaluate the biological control effect of the fermentation broth of these strains on A. tenuissima, the invasive inoculation method was employed. Xiangli was first sterilized with 5% sodium hypochlorite and 75% ethanol. The experiment consisted of two treatments: fermentation broth treatment (7 days) and inoculation with pathogenic bacteria only. Firstly, after 24 h of inoculation with pathogenic bacteria, fermentation solution were sprayed on the fruits, then blown dry and incubated in sterile bags for 7 days. Observations were made during this period, determination of bacteriostatic effect by comparing onset diameters.

Nucleotide sequence accession numbers

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain TRM 76172T and TRM 76130T is JAMYEN000000000 and JAMYEO000000000. The genome of TRM 76172 and TRM 76130 were submitted to NCBI with accession numbers PRJNA850657 and PRJNA850660, respectively (Supporting material 1).

Data availability

Availability of data and materials: https://www.ncbi.nlm.nih.gov/bioproject/ JAMYEN000000000. https://www.ncbi.nlm.nih.gov/bioproject/ JAMYEO000000000.

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Funding

This research was funded by The National Natural Science Foundation project focuses on the biosynthesis mechanism and combination biosynthesis of indole oxazole antibiotics in actinomycetes (32360009)” and “The APC was funded by X X L”; “Tarim University Bochum Program (TDBSCX202302)” and “The APC was funded by Y H C”. and Preparation and application of biocontrol agents producing phosphonic acid components, grant number (TDZKZD202202)” and “The APC was funded by X X L”.

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  1. State Key Laboratory Incubation Base for Conservation and Utilization of Bio-Resource in Tarim Basin, Alar, Xinjiang Uygur Autonomous Region, 843300, China

    Yi-Huang Chen, Jia-Xing Zhang, Yan Min, Yang Liu, Jian-Ming Wang & Xiao-Xia Luo

  2. School of Life Science and Technology, Tarim University, Alar, Xinjiang Uygur Autonomous Region, 843300, China

    Yi-Huang Chen, Jia-Xing Zhang, Yan Min, Yang Liu, Jian-Ming Wang & Xiao-Xia Luo

  3. State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, China

    Lin-Quan Bai

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C Y H carried out the methodology of the research article, thesis writing, Y M for validation, J X Z for investigation, Y L for data organization, W J M B L Q for verification, and X.X L for project management funding acquisition. All authors have read and agreed to the published version of the manuscript.

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Chen, YH., Zhang, JX., Min, Y. et al. Two new strains of Streptomyces with metabolic potential for biological control of pear black spot disease. BMC Microbiol 24, 550 (2024). https://doi.org/10.1186/s12866-024-03609-6

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