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Investigating the plant growth promoting and biocontrol potentiality of endophytic Streptomyces SP. SP5 against early blight in Solanum lycopersicum seedlings

BMC Microbiology volume 22, Article number: 285 (2022) Cite this article

Abstract

Background

Early blight (EB), caused by Alternaria solani, is one of the alarming diseases that restrict tomato production globally. Existing cultural practices and fungicide applications are not enough to control early blight diseases. Therefore, the study aimed to isolate, identify, and characterize an endophytic Streptomyces exhibiting the potential to control early blight in tomato and also promote plant growth.

Results

From a Citrus jambhiri leaf, an endophytic Streptomyces sp. with antagonistic activity against Alternaria solani, Colletotrichum acutatum, Cladosporium herbarum, Alternaria brassicicola, Alternaria sp., Fusarium oxysporum and Fusarium sp. was isolated. It was identified as a Streptomyces sp. through 16S ribosomal DNA sequence analysis and designated as SP5. It also produced indole acetic acid which was confirmed by Salkowski reagent assay, TLC and HPLC analysis. Treatment of pathogen infected plants with Streptomyces sp. SP5 antagonists (culture cells/culture supernatant/solvent extract/ acetone precipitates) decreased the early blight disease incidence and significantly increased the various agronomic traits.

Conclusion

The present study concluded that Streptomyces sp. SP5 possessed antifungal activity against different fungal phytopathogens and had significant potential to control early blight disease and promote plant growth.

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Background

Tomato is the second most widely used vegetable in the world after potato because of its high nutritional value (rich in vitamins A and C) and the presence of lycopene and beta-carotene pigments [1, 2]. Tomato crop occupies an area of 4.85 million hectares in the world with the production of 182.3 million tons and the productivity of 37.6 t/ha [3]. In India, the tomato crop is grown in an area of 0.5 million ha with an annual production of 7.4 million tons [4]. Continuous cropping of tomato plants leads to the prevalence of fungal diseases. One of the most devastating diseases of tomato is early blight caused by the necrotrophic fungus Alternaria solani [5]. Leaf blight, stem blight, and apical fruit rot are the most damaging symptoms of the disease, and in severe cases, this can lead to complete defoliation [6]. Many reports recorded that early blight is responsible for a large proportion of total monetary loss sustained by tomato producers every growing season [7]. Pesticide application is still a valuable and efficient method to controlling plant diseases in modern agriculture. However, the use of agrochemicals is falling into disfavor because of the accretion of toxic compounds potentially perilous to humans and the environment. Also, in the increase of resistance of the pathogens to agrochemicals and effects on a variety of non-target organisms, they are not considered to be a long-term solution. Therefore, recent efforts have been focused on developing eco-friendly safe, long-lasting, and effective agents against plant pathogens for the management of plant diseases. Endophytic microorganisms have become the center of intensive research because they provide numerous advantages over chemical fertilizers [8]. These include environmental protection, safety for humans, reduction of chemical pesticide residues in food, and increased biodiversity in a managed ecosystem. Many recent studies have discussed the possible use of microbe-based biocontrol agents as agrochemical substitutes or supplements [9, 10]. Various bacteria and fungi, such as Streptomyces, Bacillus, Pseudomonas, and Trichoderma as well as nonpathogenic Fusarium, have been successfully used as biocontrol agents [11, 12].

Various beneficial bacteria support their host plants by increasing growth and/or by protecting them against pathogenic diseases [13]. Among all bacterial communities, actinobacteria, especially Streptomyces have been reported to play an important role in the plant rhizosphere by secreting a wide range of antimicrobial products. Streptomyces characterized by a wide range of modes of action like antibiotic production, lysis of fungal cell walls, competition, and hyper parasitism have been proved to be effective biocontrol agents [14]. Besides acting as biocontrol agents, they also possess the potential to stimulate plant growth either directly or indirectly [15]. Streptomyces are also known to develop symbiotic associations with crop plants, colonizing their internal tissues without causing disease symptoms and producing phytohormones such as gibberellic acid and indole-3-acetic acid (IAA) [16, 17]. Indole acetic acid (IAA), one of the most important phytohormones, has a vital role in the stimulation of root growth. The production of IAA has been reported in various Streptomyces species [18, 19]. Commercial formulations from Streptomyces have already been successfully developed to control fungal phytopathogens. Mycostop, a Streptomyces- based (Streptomyces griseoviridis K61) biocontrol product, is registered for use in Canada and 15 other countries against Fusarium, Alternaria and Phytophthora. Another product, Actinovate (Streptomyces lydicus WYEC108) is recommended for foliar and soil-borne fungal diseases of greenhouse and field-grown crops [20].

The natural habitat of most Streptomyces is the soil where they may constitute from 1 to 20% of the culturable population, but Streptomyces are also present inside plant tissues as endophytes [21,22,23]. To discover new biocontrol agents there is a need to screen new Streptomyces strains from untapped sources. Therefore, in the present study an endophytic isolate SP5, exhibiting antifungal and plant growth-promoting activities, was recovered from Citrus jambhiri leaves. The aim of this study was to isolate a potent endophytic Streptomyces strain having potential to control early blight disease in S. lycopersicum plants caused by A. solani.

During this investigation the ability of the strain to promote plant growth in vivo was also assessed by observing its impact on various agronomic traits in the S. lycopersicum plant.

Results

Isolation and screening

A total of 42 different endophytic actinobacteria isolates were recovered from plant samples. Among these, 15 isolates demonstrated antifungal activity against one or more test fungi during primary screening, and 9 isolates were found to have antifungal activity in the culture supernatant, inhibiting various test fungi to varying degrees (Table S1). Isolate SP5 was selected for further studies because it exhibited potent antifungal activity against all the tested phytopathogenic fungi.

Phenotypic and phylogenetic analysis of Streptomyces SP. SP5

Streptomyces sp. SP5 showed good growth on SCNA (Starch casein nitrate agar) and all ISP (International Streptomyces project) media except the ISP2 medium. It exhibited varied cultural characteristics on different media (Table S2). Pigmentation was not observed in any ISP medium. On SCNA medium isolate showed yellowish-white sporulation (Fig. 1a) with strong yellow aerial and brilliant yellow substrate mycelia (Fig. 1b). Under the light microscope (100X), flexuous spore chains with hooks were observed (Fig. 1c), and it was assigned to the Streptomyces retinaculiaperti group. SEM micromorphological analysis showed spore chains, each bearing 20–30 cylindrical spores (1.5–2.0 m length and 1.5 m width) with smooth surface (Fig. 1d) on aerial mycelium. The chemotaxonomic analysis revealed the presence of LL-DAP (LL-diaminopimelic acid) in the cell wall, and no characteristic sugar in whole-cell hydrolysate. In physiological studies, Streptomyces sp. SP5 grew at temperature of 25–45 °C (optimum at 28 °C), pH of 5–10 (optimum at pH 7.0) and showed salt tolerance up to 5%. In biochemical characteristics, isolate SP5 hydrolyzed starch, cellulose, lipid, gelatin and esculin but not able to hydrolyze casein. Isolate SP5 showed positive results for the production of catalase, oxidase, hydrogen sulphide and citrase but gave negative results for both indole production and MRVP (Methyl Red–Voges-Proskauer) tests. The isolate was able to utilize all the tested sugars as the sole carbon source except arabinose (Additional file Table S3). These cultural and chemotaxonomic characteristics showed that SP5 belongs to the genus Streptomyces and was further confirmed by 16S rRNA sequencing. Almost complete 16SrRNA gene sequence (1400 bp) of Streptomyces sp. SP5 was determined and aligned, using the EzTaxon database. The 16S rRNA gene sequence has been deposited in the GenBank database with accession number MW564023. Nucleotide BLAST search analysis showed 100% pairwise similarity with Streptomyces rochei NRRL B- 2410. Streptomyces sp. SP5 formed a clade with Streptomyces rochei NRRL B- 2410 in phylogenetic tree constructed using neighbor-joining method with bootstrap value of 64% (Fig. 2). The culture has been deposited in International Depository Authority (MTCC and Gene Bank, CSIR-IMTECH, Chandigarh (India) with accession number MTCC-13071.

Fig. 1
figure 1

Streptomyces sp. SP5 morphological characteristics: a Aerial mycelium, b Substratum mycelium, c Aerial hyphae spore chains with open hooks under Light Microscope (100X), d Electron microscopic image of spores with smooth surface (Magnification = 5.00 KX

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Fig. 2
figure 2

Phylogenetic tree obtained by neighbour joining analysis of 16S rRNA gene sequences showing the relationship between SP5 and related species belonging to genus Streptomyces obtained from EzTaxon database. Numbers on branch nodes are bootstrap values (expressed as percentage of 1000 replications)

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Antifungal potential

Streptomyces sp. SP5 showed antifungal activity against all the tested fungal phytopathogens. The production of active metabolites in SCN culture broth against fungal pathogens was detected after 2 days of incubation. The maximum activity reached after 5 days, and then began to decrease as the incubation period was extended further. Additionally, during the 10-day activity profile, there was a positive correlation between antifungal activity and biomass (Fig. 3, Table S4). The maximum biomass was obtained after fifth day of incubation, which coincided with the maximum activity obtained on the same day. In vitro bioassay demonstrated broad-spectrum antifungal activity of Streptomyces sp. SP5 against test fungi (Fig. 4). Among the tested fungi, higher activity was observed against Fusarium oxysporum, Cladosporium herbarum, Fusarium sp. (25-28 mm) as compared to Alternaria brassicicola, Alternaria sp., Colletotrichum acutatum, Alternaria solani, and Fusarium solani (20-25 mm) (Table S5).

Fig. 3
figure 3

Growth and antifungal activity of Streptomyces sp. SP5 against test organisms viz. Fusarium oxysporum, Colletotrichum herbarum, Fusarium sp. Alternaria solani and Alternaria sp

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Fig. 4
figure 4

Antifungal activity of Streptomyces sp. SP5 cultural supernatant against fungal phytopathogens: a) Fusarium sp.; b) Cladosporium herbarum; c) Colletotrichum acutatum; d) Alternaria sp.; e) Fusarium oxysporum; f) Fusarium solani; g) Alternaria solani; h) Alternaria alternata (Production carried out in SCN broth, pH 7.0 at 28 °C using 2.5% inoculum)

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Antifungal metabolite extraction

The active metabolites from culture supernatant were extracted in diethyl ether. After the concentration of the organic phase using rotavapor, a light-yellow colored crude extract was obtained, which was redissolved in chloroform. Diethyl ether solvent extract showed pronounced activity against A. brassicicola, A. solani, F. oxysporum and F. solani (15-20 mm). However, the activity was also observed in the aqueous phase which indicated that antifungal compounds were not fully recovered by solvent extraction. Therefore, another technique i.e., acetone precipitation was employed to recover the bioactive metabolites from the culture supernatant. The acetone precipitates showed antifungal activity against A. brassicicola, A. solani, C. acutatum, F. oxysporum, F. solani and C. herbarum (20-30 mm).

Stability of antifungal metabolites in the culture supernatant

The stability of antifungal metabolites present in the culture supernatant was investigated under various physical stresses. Active metabolites remained completely stable at 37 °C after 1 h exposure. However, a loss of 20, 48, and 68% in the activity was observed at 70 °C, 100 °C, and autoclaving (121 °C for 20 min), respectively. The metabolites remained active at extreme pH values (from 2 to 14) with 92 and 88% residual activity, respectively. The metabolites were photostable in both UV and sunlight (Table 1).

Table 1 Effect of various parameters on antifungal activity of culture supernatant against A. solani
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IAA production from Streptomyces SP. SP5

Streptomyces sp. SP5 produced 40 μg/ml indole acetic acid in an initial screening for indole acetic acid production. TLC and HPLC analyses further demonstrated the ability of Streptomyces sp. SP5 to produce IAA. The ethyl acetate crude extract displayed a clear pink-colored spot on the TLC plate after being treated with Salkowski regent at Rf value corresponding to standard IAA (0.9 retention factor). After the detection of IAA in TLC, partial purification of crude extract containing IAA was done using silica gel chromatography. HPLC analysis was used to precisely classify and quantify the IAA production. The partially purified extract and corresponding reference authentic standard both displayed peaks at the same retention time (18.6 min) (Fig. S1). For the highest IAA production by Streptomyces sp. SP5, experiments were carried out with different values of the incubation period, temperature, pH, and L-tryptophan concentration. As shown in Fig. 5a, IAA production began within 24 hours of incubation, increased steadily over the time, reached a maximum of 58.43 μg/ml after 6 days, then began to decline with additional incubation, reaching 5.45 μg/ml after 10 days. The impact of pH on IAA production was investigated within the pH range of 5 to 10. Streptomyces sp. SP5 produced the maximum IAA of 58.63 μg/ml at an initial pH of 7.0. Acidity and high alkalinity of the medium led to a marked decrease in growth and IAA production (Fig. 5b). Incubation temperature had a significant impact on IAA production. The maximum production of 58.63 μg/ml IAA was achieved at 28 °C (Fig. 5c). In the absence of L- tryptophan i.e. IAA precursor, Streptomyces did not produce IAA. With the increase in concentration of L-tryptophan from 0.5 to 6 mg/ml, there was an increase in the production, and the maximum biosynthesis of 99.65 μg/ml IAA was achieved at 5 mg/ml L-tryptophan (Fig. 5d). IAA production decreased slightly at higher L-tryptophan concentrations. Additionally, a positive correlation between IAA production and biomass was observed.

Fig. 5
figure 5

Streptomyces sp. SP5 produces IAA in response to a) incubation period, b) pH, c) temperature, and d) tryptophan concentration. Columns and bars represent the mean ± SE

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In vivo biocontrol of early blight disease by Streptomyces sp. SP5 and its impact on Solanum lycopersicum plant growth

Figure 6 shows the in vivo biocontrol potential of Streptomyces sp. SP5 against A. solani in S. lycopersicum plants. Plants treated with Streptomyces sp. SP5 antagonists (culture cells/culture supernatant/solvent extract/ acetone precipitates) showed no symptoms of early blight disease throughout the 3 months of experiments whereas leaf blight symptoms were observed on the negative control plants ((plants treated with pathogen only) after 1 month of experiments (Fig. 7) and at the end of experiment plants were defoliated. In addition, treatment of pathogen infected plants with Streptomyces sp. SP5 antagonists increased the various agronomic traits viz. shoot length (120–202.35%), root length (432–516.12%), fresh weight of shoot (135.48–270.09%), fresh weight of root (243.05–428.30%), dry weight of shoot (644.98–1346.99%) and dry weight of root (1332.4–2467.5%) significantly over the fungal pathogen infected plants (Additional file Table S6, Fig. 7a). Among the four treatments, acetone precipitates and culture cells were found to be more effective as compared to the culture supernatant and solvent extract. In the absence of pathogen stress also, Streptomyces sp. SP5 antagonists enhanced shoot length (71.875 to 108.54%), root length (76.82 to 131.70%), fresh weight of shoot (42.85 to 115.63%), fresh weight of root (74.15 to 264.23%), dry weight of shoot (100.98 to 264.23%) and dry weight of root (265.90 to 538.63%) over the control plants (Additional file Table S6, Fig. 7b). During the period of experimental study, early flowering and fruiting were also observed in plants treated with antagonists (culture cells/ acetone precipitates) both in the presence and absence of pathogen stress whereas no such stages were observed in control and pathogen infected plants. The Streptomyces sp. SP5 was re-isolated from roots, stems, and leaves of treated S. lycopersicum plants (Fig. S2). It showed 58.3, 66.6, and 91.6% of colonization in leaves, stems, and roots, respectively (Table S7). This data indicated the biocontrol and plant growth-promoting potential of Streptomyces sp. SP5.

Fig. 6
figure 6

Effect of Streptomyces sp. SP5 and its metabolites as root treatments on S. lycopersicum (tomato) plants to control Alternaria solani causing early blight disease: a) shoot lengths of plants, b) root lengths of plants, c) fresh weights of shoots, d) fresh weights of roots, e) dry weights of shoots, f) dry weights of roots. C, control (Water only); P, pathogen only; CC + P, Streptomyces sp. SP5 cells + pathogen; CS + P, culture supernatant + pathogen; SE + P, Streptomyces sp. SP5 solvent extract + pathogen; AP + P, acetone precipitates + pathogen; CC, SP5 cells only; CS, culture supernatant only; SE, solvent extract only; AP, acetone precipitates only. Values were expressed in means ± standard deviation and diffrent alphabets on the graphs indicate that the average mean values of treatments are significantly different according to Tukey’s multiple comparison test (P ≤ 0.01)

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Fig. 7
figure 7

a): Effect of Streptomyces sp. SP5 and its metabolites on growth of S. lycopersicum (tomato) plants infected with A. solani; b): Plant growth promoting potential of Streptomyces sp. SP5 and its metabolites on S. lycopersicum (tomato) plants

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Discussion

In this research study, a Streptomyces sp., designated as Streptomyces sp. SP5, was isolated from leaves of Citrus jambhiri, and characterized through a polyphasic approach. The formation of flexuous spore chains by Streptomyces sp. SP5 as well as chemotaxonomical markers (LL-DAP in cell wall and no characteristic sugar in whole cell hydrolysate), indicated that it belonged to the Streptomyces genus. In phylogenetic characterization, Streptomyces sp. SP5 displayed 100% sequence similarity with Streptomyces rochei NRRL B- 2410.

Streptomyces sp. SP5 inhibited varying phytopathogenic fungi (F. moniliforme, A. brassicicola, C. herbarum, A. solani, F. solani, C. acutatum) to varying degrees. The test pathogen may play a role in the variation in antagonistic behaviour. The genus Streptomyces has been studied broadly in the past decades and reported for the production of antifungal compounds [24,25,26,27]. Since the biocontrol agent's shelf-life is dependent on environmental conditions such as soil temperature, soil pH, nutrient availability and water status, one of the major issues in the commercialization of biocontrol agents is their loss of viability over time [28]. However, antifungal metabolites produced by Streptomyces sp. SP5 were found to be photo-thermostable and pH stable. These characteristics of metabolites make them suitable for the development of effective biocontrol agents for varied climate conditions throughout the globe.

The Streptomyces sp. SP5 also exhibited production of plant growth-promoting hormone, indole acetic acid (IAA). It is one of the most physiologically active auxins which stimulates and facilitates plant growth [18, 29, 30]. Biosynthesis of IAA in soil-dwelling Streptomyces spp. and endophytic Streptomyces has been documented in various studies [17, 18, 31,32,33]. Streptomyces sp. SP5 has been found to produce a significant amount (40 μg ml− 1) of phytohormone IAA when grown in YMB broth supplemented with tryptophan. HPLC is a reliable method for the identification of auxins, thus characterization of IAA was performed by using HPLC. Results of the classical optimization experiment showed that the incubation time, temperature, and pH played an important role in IAA production by Streptomyces sp. SP5 which is in agreement with other reports [34, 35]. Plant-associated microbes synthesize IAA via tryptophan (trp)-dependent and -independent pathways [36]. IAA production from Streptomyces sp. SP5 increased two-folds in the presence of L- tryptophan which strongly indicated that Streptomyces sp. SP5 synthesized IAA via the tryptophan-dependent pathway. It was also observed that beyond optimized concentrations of tryptophan, IAA production was adversely affected in Streptomyces sp. SP5. Myo et al. [18] also reported that high tryptophan concentrations affect the capability of strain to produce IAA. Manulis et al. [37] have reported that various Streptomyces spp. including S. violaceus, S. griseus, S. scabies, S. coelicolor S. exfoliatus, and S. lividans, produced indole- 3-acetic acid (IAA) when grown in the presence of tryptophan.

It is common knowledge that a strain's ability to suppress disease in vitro does not imply that it can be used in the fields as biocontrol agent since the strain might not be able to demonstrate its potential in natural conditions [38]. Therefore, in this study, in vivo pot experiments were conducted against the early blight of S. lycopersicum caused by A. solani to explore the possibility of using Streptomyces sp. SP5 as a biocontrol agent under natural conditions. Several Streptomyces spp. and their bioactive compounds have been reported as biocontrol agents, effective against numerous plant pathogens [9, 39, 40]. The results of the present study revealed a remarkable effect of Streptomyces sp. SP5 in mitigating early blight stress in S. lycopersicum plants. The treatment of soil, infected by A. solani, with Streptomyces sp. SP5 cells/supernatant/solvent extract and acetone precipitates exhibited a significant increase in various growth traits of S. lycopersicum plants and disease suppression in comparison to the plants infected with the pathogen only. To the best of our knowledge, only a few reports are available in the literature pertaining to in vivo biocontrol of early blight [11, 41,42,43]. The data obtained from the in vivo experimental study suggested the superiority of Streptomyces sp. SP5 over the earlier reported studies to control early blight of S. lycopersicum. Khan et al.[43] demonstrated the potential of Paenibacillus lentimorbus B-30488r to control early blight and reported that treatment of pathogen-infected plants with P. lentimorbus B-30488r exhibited 16% defoliation and increased the shoot length, fresh plant weight, dry plant weight and no effect on root length. On the other hand, in our study Streptomyces sp. SP5 completely suppressed the early blight with 0% defoliation, and no disease symptoms appeared in treated plants while complete defoliation was observed in plants exposed to the pathogen. Additionally, Streptomyces sp. SP5 significantly enhanced shoot length (120–202.35%), root length (432.25–516.12%), fresh weight of shoot (135.48–270.09%), fresh weight of root (243.05–428.30%), dry weight of shoot (644.98–1346.99%), and dry weight of root (1332.4–2467.5%). Similarly, Cuppels et al. [11] evaluated efficacy of strains S. griseoviridis K61 and S. lydicus WYEC108 isolated from the formulated commercial products Mycostop and Actinovate, respectively to control early blight, anthracnose and bacterial spot of field S. lycopersicum. Both BCAs (biocontrol agents) strongly suppressed the growth of A. solani in dual culture agar diffusion bioassay. S. griseoviridis successfully controlled anthracnose and early blight diseases whether applied before pathogen or simultaneously with the pathogen but S. lydicus provided protection only when applied 48 h before the exposer of pathogen.

Moreover, during the pot trial Streptomyces sp. SP5 also significantly increased various growth parameters of S. lycopersicum plants such as shoot and root lengths in the absence of pathogen stress. These findings support previous research which found that Streptomyces spp. not only protected plants from pathogens, but also enhanced plant growth and physiology [9, 44,45,46]. Dias et al. [45] observed a significant increase in shoot length and root length of S. lycopersicum plants treated with different Streptomyces isolates. However, Streptomyces sp. SP5 reported in the present study showed highly pronounced effect on all the observed plant growth traits in S. lycopersicum plants. Additionally, during this in vivo study, enhancement in some plant growth traits (root length, root fresh weight, root dry weight, shoot dry weight) was more under the pathogen stress than in the absence of pathogen stress. The reason behind these results might be the induced systemic resistance (ISR) in plants caused by pathogen stress [47, 48]. Plants possess a range of active defense apparatuses that are actively expressed in response to biotic stresses (like pathogen stress). Similarly, in our previous study we reported additional enhancement of plant growth traits (root fresh weight, shoot length) in S. lycopersicum pathogen-infected plants treated with Streptomyces sp. MR14 as compared to the plants treated with Streptomyces sp. MR14 alone [9].

Conclusion

The results of in vitro and in vivo studies are highly significant and data reveals that Streptomyces sp. SP5 is superior to the previously reported Streptomyces spp. and might be used as a potential biological agent to control fungal phytopathogens and as an effective biofertilizer to promote plant growth.

Material and methods

Sample collection

Leaves and roots of healthy plants (Cinnamon basil, Ricinus communis, Epipremunum aureum, Citrus jambhiri, Hibiscus rosasinensis) were collected from “Botanical Garden” of Guru Nanak Dev University, Amritsar, Punjab, India (31°37′45″N and 74°49′36″N). The plants were identified and deposited in university herbarium vide accession numbers 5839, 2409, 4312, 5871 and 6462, respectively. Plant samples were wrapped aseptically in plastic bags and taken to the laboratory where they were processed within 4 hours.

Test fungi

Different test phytopathogenic fungi viz. Alternaria brassicicola (MTCC2102), Alternaria solani (MTCC2101), Colletotrichum acutatum (MTCC1037), Fusarium oxysporum (MTCC284), Cladosporium herbarum (MTCC351) were procured from Microbial Type Culture Collection (MTCC), CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. Whereas, Fusarium solani (NFCCI 91) was obtained from the National Fungal Culture Collection of India NFCCI, Pune. Fusarium sp. and Alternaria sp. (accession number GU004283) were isolated in the lab [49]. All the fungal cultures were maintained at 4 °C on Potato dextrose agar (PDA) slants.

Isolation of endophytic strains from plant samples

The collected plant samples were washed under running tap water for 1–2 minutes to remove soil particles. The samples were then surface sterilized by 70% ethanol for 10 min followed by treatment with 1% sodium hypochlorite solution for 15 min [50]. Plant samples were then repeatedly washed with sterilized water, air-dried in a laminar airflow hood, and cut into small pieces using a sterile razor blade. The small pieces of sterile samples were placed on SCNA medium (starch casein nitrate agar). To inhibit the growth of fungi and Gram negative bacteria, cycloheximide (50 g/ml) and nalidixic acid (50 g/ml) were added to the medium. The plates were then incubated at 28 °C for 7–21 days. Isolated actinobacteria colonies were subcultured and purified on SCNA plates. Isolate spores were reserved as a stock in 20% glycerol at − 20 °C for potential use.

Screening and selection of the isolate

Primary screening determines the ability of the microorganisms to produce an antifungal metabolite without providing a significant idea about the production potential of the organism. Modified Kirby Bauer antibiotic susceptibility test was used for primary screening [51]. The actinobacterial isolates were cultured for 7 days on the SCNA medium at 28 °C. Six mm agar discs from well-grown cultures of actinobacterial isolates were placed on PDA plates which were already seeded with the test phytopathogenic fungi (100 μl of 106spores ml− 1). The plates were then kept at 4 °C for 30 min for the diffusion and then incubated at 28 °C. The zones of inhibition were determined after 48 h.

Following primary screening, the active isolates were subjected to secondary screening [52]. Three discs of actinobacterial culture with a diameter of 6 mm were transferred to 250 ml Erlenmeyer flasks containing 50 ml of starch casein nitrate broth and incubated for 7 days at 28 °C in a rotary shaker at 180 rpm. Mycelia were removed by centrifuging at 10,000 x g for 20 minutes at 4 °C and supernatant was used for bioassay. The PDA plates inoculated with test fungi (100 μl of 106spores ml− 1) were punctured with sterile cork borer to create wells (6 mm in diameter) and 200 μl of culture supernatant was transferred to each well under aseptic conditions. The plates were incubated at 28 °C for 3 days and observed for antifungal activity of the isolates as clear zones of inhibition around wells. Out of 9 isolates from secondary screening, isolate SP5 was selected for further studies based on its broad-spectrum antifungal activity.

Morphological, physiological, and biochemical characterization of Streptomyces SP. SP5

Cultural characteristics were determined as per methods described by Shirling and Gottlieb [53] in the International Streptomyces Project (ISP). ISCC-NBS standard color chart was used to determine the colors of the substrate and aerial mycelia [54]. Brightfield light microscopy and electron microscopy were used to examine the isolate morphological characteristics. Physiological and biochemical characterization such as growth at different temperatures (20–50 °C), pH (5.0–12.0), salt concentration (0–20% w/v), and capacity to generate different hydrolytic enzymes were carried out according to standard protocols [55]. The assimilation of sugars as carbon source was investigated as per Shirling and Gottlieb [53]. The major diagnostic features of Streptomyces, sugar pattern in whole-cell hydrolysate and isomer of diaminopimelic acid (DAP) in the cell wall, were determined using Lechevalier and Lechevalier [56].

Genomic characterization

The genomic DNA of the isolate was extracted using Marmur method [57]. The 16S rRNA gene sequence was amplified by polymerase chain reaction (PCR) using primers 27f (5′-AGAGTTTGATCC TGGCTCAG-3′) and 1492r (5′-AGAAAGGAGGTGATC CAGGC-3′). Amplification was done for 40 cycles in a PCR machine (Eppendorf-gradient). Each cycle included a denaturation stage of 1 minute at 94 °C, an annealing step of 1 minute at 50 °C, an extension step of 2 minutes at 72 °C and a final extension of 10 minutes at 72 °C. The QIA quick gel extraction kit (Qiagen, Germany) was used to purify the obtained PCR product. The Institute of Microbial Technology (IMTECH), Chandigarh, India, sequenced the 16S rRNA gene of Streptomyces sp. SP5. For the detection of phylogenetic neighbors and calculation of pairwise 16S rRNA gene sequence similarities, the EzTaxon server (http://www.ezbiocloud.net) was used [58]. The Clustal W program was used to align the nearly complete sequence (1400 bp). Phylogenetic tree based on bootstrap values (1000 replications with MEGAX software) was constructed using neighbor-joining method [59, 60].

Time course experiment related to antifungal metabolite production and growth

For antifungal profiling, seed culture was prepared in Erlenmeyer flask (250 ml) containing 50 ml of starch casein nitrate broth, inoculated with three discs (6 mm diameter) of 7 days old culture grown on SCNA and incubated at 28 °C for 3 days at 180 rpm. Production was carried out by inoculating the production medium (SCN broth) with 2% seed culture (106 colony forming units /ml)) and incubating at 28 °C for 10 days of agitation at 180 rpm. After every 24 h, the flasks were harvested and culture broth was centrifuged at 10,000×g for 10 min. The biomass was separated from the culture supernatant and dried at 60 °C for 2 days. The activity against test fungal phytopathogens was determined using the remaining cell-free culture supernatant. The test fungi (100 μl of 106spores ml− 1) were seeded on PDA plates, which were punctured with sterile cork borer to create 6 mm wells. The plates were held in the refrigerator for 1 hour after adding 200 μl of culture supernatant to allow active metabolites to diffuse. In the control wells, culture supernatant was replaced with water/uninoculated broth. After diffusion, the plates were incubated at 28 °C for 2–3 days. The antifungal activity of the isolate was measured in millimetres as zones of inhibition around wells.

Recovery of antifungal metabolites

For the production of antifungal active metabolites, fermentation was carried out by inoculating the culture (at a concentration of 2.5% from seed culture) in production medium and incubating at 28 °C under shaking (180 rpm). Fermentation was terminated when the maximum antifungal activity was observed (5th day) and culture broth was centrifuged at 10,000×g at 4 °C for 10 min to separate the mycelium.

The active metabolites from culture supernatant were recovered by solvent extraction. Different organic solvents (hexane, diethyl ether, chloroform, ethyl acetate, butanol) having a wide range of polarity were screened to work out the best extractant. The supernatant of Streptomyces sp. SP5 was extracted twice in the ratio of 1:1 (supernatant: solvent). Rotavapor (BUCHI Rotavapor R-210) was used to concentrate the obtained organic phase to dryness. The obtained crude extracts were redissolved in respective solvents (1 ml) and tested for antifungal activity against test fungi. Acetone precipitation was also employed to extract out the antifungal metabolites from the culture supernatant. For acetone precipitation, two volumes of 100% chilled acetone were added to the culture supernatant and kept for 1 hour at -20 °C. The precipitates were recovered by centrifugation at 10,000×g for 10 minutes at 4 °C, and were redissolved in sterile distilled water. The antifungal activity of precipitates was determined against different test phytopathogenic fungi using the agar well diffusion method.

Stability of active metabolites in the culture supernatant

The supernatant was heated at various temperatures (37 °C, 50 °C, 70 °C, 100 °C and autoclaving at 121 °C) for 1 hr, and was also exposed to a lower temperature (− 20 °C) for 1 hour to determine thermostability. The effect of pH on activity was evaluated by adjusting the pH at 2.0 and 14.0, incubating for 1 hr at 28 °C, and then reverting the pH of the supernatant to 7.0 to check antifungal activity. Photostability was tested by exposing the supernatant separately to UV (200 – 600 nm) and sunlight (1 hr). The antifungal activity of all the treated samples was then tested using well diffusion assay and values were represented as their mean ± SD of three independent experiments.

Plant growth promoting potential

Indole acetic acid (IAA) production

For Indole acetic acid (IAA) production, 50 ml of yeast malt broth (YMB (Himedia); g/l: yeast extract 4.0, malt extract 10.0, dextrose 4.0; pH 6.8 ± 0.2) containing 0.2% L-tryptophan was inoculated with three plugs (6 mm) of seven-days-old Streptomyces sp. SP5 culture grown on SCNA and then incubated at 28 °C for 7 days under shaking (180 rpm). After incubation, the broth was centrifuged at 10,000x g for 15 minutes to remove mycelia. To detect IAA production, 2 ml Salkowski reagent was added into tubes containing 1 ml of cell-free supernatant and incubated for 25 min at room temperature. The intensity of developed pink color was measured by taking absorbance at 530 nm using Shimadzu UV-visible spectrophotometer (UV-1601, Japan), which indicates indole acetic acid production. The concentration of IAA produced was estimated against the standard curve of IAA using a concentration range of 10–100 μg/ml.

Extraction, purification, and identification of indole acetic acid

The culture supernatant was acidified to pH 2.5 with HCl and extracted twice with ethyl acetate using solvent extraction. The separated organic phase was concentrated using a rotary evaporator and redissolved in ethyl acetate. For IAA analysis, the ethyl acetate extract was separated using thin-layer chromatography (TLC) with propanol: water (8:2, v/v) as solvent system, and the chromatogram was observed after spraying the plates with Salkowski regent. IAA was partially purified from the crude solvent extract by using silica gel column chromatography and fractions were collected with the solvent system of ethyl acetate and hexane (20:80 v/v). Each fraction was checked for the presence of IAA by TLC using Salkowski regent. The indole containing fraction was further quantified using reversed-phase high performance liquid chromatography (RP-HPLC): Shimadzu Micros orb MV, 100 mm × 10 mm ID, 10 μm, using C-18 reversed-phase column with methanol: 1% acetic acid in water (40:60 v/v as mobile phase) as an eluent at 1 ml min− 1 flow rate. For the analysis, 20 μl of partially purified IAA extract was spiked into the column and pure IAA compound was used as a standard. The identical retention time of the respective standard was used to assess the presence of IAA in the purified fraction.

Optimization of IAA production

To optimize IAA production, various factors such as incubation period, pH, temperature, and tryptophan concentration were studied using one variable at a time approach. In order to determine the effect of incubation time, 50 ml of YMB was added in each of 250 ml Erlenmeyer flasks which were then inoculated with 2% v/v inoculum (106 cfu/ml, developed as mentioned above in the time course experiment) and incubated at 28 °C at 180 rpm for 10 days. The effect of pH on IAA production was determined by changing the initial pH of the medium from 2 to 10 using 1 N HCl and 1 N NaOH. Similarly, the effect temperature of incubation on production was investigated by incubating Streptomyces sp. SP5 into the same medium at 20, 25, 28, 30, 35, and 40 °C for 6 days (at which the maximum production was achieved). The effect of L-tryptophan concentration was determined by varying L-tryptophan over a range of 1–5 mg/ml in the medium (pH 7.0). All experiments were performed in triplicates and average values were observed.

In vivo biocontrol of A. solani by Streptomyces SP. SP5 and its effect on S. lycopersicum plant growth

Efficacy of Streptomyces sp. SP5 against early blight disease was tested by conducting three independent pot trials at Guru Nanak Dev University (Amritsar, Punjab, India) using soil drenching method. The aim was to observe whether Streptomyces sp. SP5 culture cells, culture supernatant, solvent extract as well as acetone precipitates could regulate early blight caused by A. solani and promote various plant growth traits. S. lycopersicum seeds susceptible to A. solani (S. lycopersicum Mill. “Pusa Ruby”) were sown in sterile soil for 3 weeks at 28 ± 2.0 °C and after true leaf stage, a single plant was transplanted into a pot of 8 cm diameter containing 100 g sterile soil for each treatment. The plants were subjected to a variety of treatments with Streptomyces sp. SP5 antagonists viz. Streptomyces sp. SP5 cells, supernatant, solvent extract and acetone precipitates, with and without pathogen (Table 2). Three replications were maintained for each treatment and the pots were kept under natural conditions (temperature 22 ± 2.0 °C, photoperiod L16: D8). Plants were watered daily and observed for disease symptoms, flowering, and fruiting stages. After disease appearance in the control plants, all plants were uprooted carefully, and washed with running tap water to remove the adhered soil. The shoot & root lengths and fresh & dry weights of seedlings were recorded, and data obtained were subjected to statistical analysis. Values were represented as mean ± SD of three independent experiments, and to compare the mean difference, one-way analysis of variance (ANOVA) with Tukey’s post hoc test was carried out using SPSS statistical analysis software (Version 20.0, IBM SPSS). To determine the endophytic relationship, Streptomyces sp. SP5 was re-isolated from different parts (roots, stems, and leaves) of treated S. lycopersicum plants. Culture obtained from plant parts was identified by morphological, physiological, and biochemical characterization. The recovery of colony growth of Streptomyces sp. SP5 on the explant pieces was evaluated and calculated for colonization percentage using the following formula:

$$\textrm{Colonization}\left(\%\right)=\frac{\textrm{Number}\kern0.17em \textrm{of}\kern0.17em \textrm{plant pieces}\kern0.17em \textrm{with}\kern0.17em \textrm{colony}\kern0.17em \textrm{growth}}{\textrm{Total}\kern0.17em \textrm{number}\kern0.17em \textrm{of}\kern0.17em \textrm{plant}\kern0.17em \textrm{pieces}}\times 100$$
(1)
Table 2 Treatment of S. lycopersicum plants with Streptomyces sp. SP5 antagonists by soil drenching method
Full size table

Availability of data and materials

All data generated or analysed during this study are included in this article.

Abbreviations

SCNA:

Starch Casein Nitrate Agar

ISP:

International Streptomyces Project

TLC:

Thin Layer Chromatography

PDA:

Potato Dextrose Agar

DAP:

Diaminopimelic Acid

MTCC:

Microbial Type Culture Collection

IAA:

Indole Acetic Acid

References

  1. Niu K, Guo H, Kakizaki M, Cui Y, Ohmori-Matsuda K, Guan L, et al. A tomato-rich diet is related to depressive symptoms among an elderly population aged 70 years and over: a population-based, cross-sectional analysis. J Affect Disord. 2013. https://doi.org/10.1016/j.jad.2012.04.040.

  2. Rais M, Sheoran. A scope of supply chain managementin fruits and vegetables in India. J Food ProcessTechnol. 2015. https://doi.org/10.4172/2157-7110.1000427.

  3. Food and Agriculture Organization. World tomato production. Food and agriculture Organization of the United Nations, FAO Rome, 2018.

  4. Shanmugasundaram S. Vegetables. In: Ram N, editor. The Hindu survey of Indian Agriculture. Chennai: Kasturi and Sons Ltd; 2004. p. 126–8.

  5. Ellis MB, Gibson IAS. Alternaria solani no. 45 set 48. Kew: Commonwealth mycological institute; 1975.

    Google Scholar 

  6. Peralta IE, Knapp S, Spooner DM. New species of wild tomatoes (Solanum section Lycopersicon: Solanaceae) from northern Peru. Syst Bot. 2005. https://doi.org/10.1600/0363644054223657.

  7. Adulrahman O. Diagnosis and in vitro control of tomato leaf spot caused by Alternaria alternata in sulaimanig overnorate. J Zankoy Sulaimani-For Pure and App Science. 2018. https://doi.org/10.17656/jzs.10710.

  8. Rabiey M, Hailey LE, Roy SR, Grenz K, Al-Zadjali MA, Barrett GA, et al. Endophytes vs tree pathogens and pests: can they be used as biological control agents to improve tree health. Eur J Plant Pathol. 2019. https://doi.org/10.1007/s10658-019-01814-y.

  9. Kaur T, Rani R, Manhas RK. Biocontrol and plant growth promoting potential of phylogenetically new Streptomyces sp. MR14 of rhizospheric origin. AMB Express. 2019. https://doi.org/10.1186/s13568-019-0849-7.

  10. Li Y, Guo Q, He F, Li Y, Xue Q, Lai H. Biocontrol of root diseases and growth promotion of the tuberous plant Aconitum carmichaelii induced by actinomycetes are related to shifts in the rhizosphere microbiota. Micrb Eco. 2019. https://doi.org/10.1007/s00248-019-01388-6.

  11. Cuppels DA, Higham J, Traquair JA. Efficacy of selected streptomycetes and a streptomycete+ pseudomonad combination in the management of selected bacterial and fungal diseases of field tomatoes. Biol Control. 2013. https://doi.org/10.1016/j.biocontrol.2013.09.005.

  12. Heydaria A, Pessarakli M. A review on biological control of fungal plant pathogens using microbial antagonists. J Biol Sci. 2010. https://doi.org/10.3923/jbs.2010.273.290.

  13. Goudjal Y, Zamoum M, Sabaou N, Mathieu F, Zitouni A. Potential of endophytic Streptomyces spp. for biocontrol of Fusarium root rot disease and growth promotion of tomato seedlings. Biocontrol sci techn. 2006. https://doi.org/10.1080/09583157.2016.1234584.

  14. Kanini GS, Katsifas EA, Savvides AL, Karagouni AD. Streptomyces rochei ACTA1551, an indigenous Greek isolate studied as a potential biocontrol agent against Fusarium oxysporum f. Sp lycopersici Biomed Res Int. 2013. https://doi.org/10.1155/2013/387230.

  15. Karnwal A. Production of indole acetic acid by fluorescent Pseudomonas in the presence of L-tryptophan and rice root exudates. J Plant Pathol. 2009. https://doi.org/10.4454/jpp.v91i1.624.

  16. Goudjal X, Toumatia O, Sabaou N, Barakate M, Mathieu F, Zitouni A. Endophytic actinomycetes from spontaneous plants of Algerian Sahara: indole-3-acetic acid production and tomato plants growth promoting activity. World J Microbiol Biotechnol. 2013. https://doi.org/10.1007/s11274-013-1344-y.

  17. Lin L, Xu X. Indole-3-acetic acid production by endophytic Streptomyces sp. En-1 isolated from medicinal plants. Curr Microbiol. 2013. https://doi.org/10.1007/s00284-013-0348-z.

  18. Myo EM, Ge B, Ma J, Cui H, Liu B, Shi L, et al. Indole-3-acetic acid production by Streptomyces fradiae NKZ-259 and its formulation to enhance plant growth. BMC Microbiol. 2019. https://doi.org/10.1186/s12866-019-1528-1.

  19. Suarez M, Vinchira-Villarraga DM, Vergara-Morales DI, Castellanos L, Ramos FA, Guarnaccia C, et al. Plant-growth promotion and biocontrol properties of three Streptomyces spp. isolates to control bacterial rice pathogens. Front Microbiol. 2019. https://doi.org/10.3389/fmicb.2019.00290.

  20. Cuppels DA, Higham J, Traquair JA. Efficacy of selected streptomycetes and a streptomycete+ pseudomonas combination in the management of selected bacterial and fungal diseases of field tomatoes. Biol Control. 2013. https://doi.org/10.1016/j.biocontrol.2013.09.005.

  21. Ngalimat MS, Mohd Hata E, Zulperi D, Ismail SI, Ismail MR, Mohd Zainudin NAI, et al. Characterization of Streptomyces spp. from rice fields as a potential biocontrol agent against Burkholderia glumae and Rice Plant growth promoter. J Agron. 2021. https://doi.org/10.1111/1751-7915.13909.

  22. Hong-Thao PT, Mai-Linh NV, Hong-Lien NT, Van Hieu N. Biological characteristics and antimicrobial activity of endophytic Streptomyces sp. TQR12-4 isolated from elite Citrus nobilis cultivar ham yen of Vietnam. Int J Microbiol. 2016. https://doi.org/10.1155/2016/7207818.

  23. Gonzaga de Oliveira L, Sigrist R, Sachetto Paulo B, Samborskyy M. Whole-genome sequence of the endophytic Streptomyces sp. strain CBMAI 2042, isolated from Citrus sinensis. Microbiol Resour Announc. 2019. https://doi.org/10.1128/MRA.01426-18.

  24. Chen X, Hu LF, Huang XS, Zhao LX, Miao CP, Chen YW, et al. Isolation and characterization of new phenazine metabolites with antifungal activity against root-rot pathogens of Panax notoginseng from Streptomyces. J Agric Food Chem. 2019. https://doi.org/10.1021/acs.jafc.9b04191.

  25. Lyu A, Liu H, Che H, Yang L, Zhang J, Wu M, et al. Reveromycins a and B from Streptomyces sp. 3–10: antifungal activity against plant pathogenic fungi in vitro and in a strawberry food model system. Front Microbiol. 2017. https://doi.org/10.3389/fmicb.2017.00550.

  26. Zhang K, Gu L, Zhang Y, Liu Z, Li X. Dinactin from a new producer, Streptomyces badius gz-8, and its antifungal activity against the rubber anthracnose fungus Colletotrichum gloeosporioides. Microbiol Res. 2020. https://doi.org/10.1016/j.micres.2020.126548.

  27. Sharma M, Manhas RK. Purification and characterization of salvianolic acid B from Streptomyces sp. M4 possessing antifungal activity against fungal phytopathogens. Microbiol Res. 2020. https://doi.org/10.1016/j.micres.2020.126478.

  28. Dhanasekaran D, Sivamani P, Panneerselvam A, Thajuddin N, Rajakumar G, Selvamani S. Biological control of tomato seedling damping off with Streptomyces sp. Plant Pathol. 2005. https://doi.org/10.3923/ppj.2005.91.95.

  29. Liu WH, Chen F, Wang CE, Fu HH, Fang XQ, Ye JR, et al. Indole-3-acetic acid in Burkholderia pyrrocinia JK-SH007: enzymatic identification of the indole-3-acetamide synthesis pathway. Front Microbiol. 2019. https://doi.org/10.3389/fmicb.2019.02559.

  30. Sadeghi A, Karimi E, Dahaji PA, Javid MG, Dalvand Y, Askari H. Plant growth promoting activity of an auxin and siderophore producing isolate of Streptomyces under saline soil conditions. World J Microbiol Biotechnol. 2012. https://doi.org/10.1007/s11274-011-0952-7.

  31. Shrivastava P, Kumar R, Yandigeri MS. In vitro biocontrol activity of halotolerant Streptomyces aureofaciens K20: a potent antagonist against Macrophomina phaseolina (Tassi) Goid. Saudi J Biol Sci. 2017. https://doi.org/10.1016/j.sjbs.2015.12.004.

  32. Tamreihao K, Ningthoujam DS, Nimaichand S, Singh ES, Reena P, Singh SH, et al. Biocontrol and plant growth promoting activities of a Streptomyces corchorusii strain UCR3-16 and preparation of powder formulation for application as biofertilizer agents for rice plant. Microbiol Res. 2016. https://doi.org/10.1016/j.micres.2016.08.005.

  33. Vurukonda SSKP, Giovanardi D, Stefani E. Plant growth promoting and biocontrol activity of Streptomyces spp. as endophytes. Int J Mol Sci. 2018. https://doi.org/10.3390/ijms19040952.

  34. Khamna S, Yokota A, Peberdy JF, Lumyong S. Indole-3-acetic acid production by Streptomyces sp. isolated from some Thai medicinal plant rhizosphere soils. Eur Asian. J Biosci. 2010. https://doi.org/10.5053/ejobios.2010.4.0.4.

  35. Goudjal X, Toumatia O, Sabaou N, Barakate M, Mathieu F, Zitouni A. Endophytic actinomycetes from spontaneous plants of Algerian Sahara: indole-3-acetic acid production and tomato plants growth promoting activity. World J Microbiol. 2013. https://doi.org/10.1007/s11274-013-1344-y.

  36. Dimkpa CO, Zeng J, McLean JE, Britt DW, Zhan J, Anderson AJ. Production of indole-3-acetic acid via the indole-3-acetamide pathway in the plant-beneficial bacterium Pseudomonas chlororaphis O6 is inhibited by ZnO nanoparticles but enhanced by CuO nanoparticles. Appl Environ Microbiol. 2012. https://doi.org/10.1128/AEM.07424-11.

  37. Manulis S, Shafrir H, Epstein E, Lichter A, Barash I. Biosynthesis of indole-3-acetic acid via the indole-3-acetamide pathway in Streptomyces spp. Microbiology. 1994. https://doi.org/10.1099/13500872-140-5-1045.

  38. Li Q, Jiang Y, Ning P, Zheng L, Huang J, Li G, et al. Suppression of Magnaporthe oryzae by culture filtrates of Streptomyces globisporus JK-1. Biol Control. 2011. https://doi.org/10.1016/j.biocontrol.2011.04.013.

  39. Shakeel Q, Lyu A, Zhang J, Wu M, Li G, Hsiang T, et al. Biocontrol of aspergillus flavus on peanut kernels using Streptomyces yanglinensis 3-10. Front Microbiol. 2018. https://doi.org/10.3389/fmicb.2018.01049.

  40. Wei Y, Zhao Y, Zhou D, Qi DF, Li K, Tang W, et al. A newly isolated Streptomyces sp. YYS-7 with a road-spectrum antifungal activity improves the banana plant resistance to Fusarium oxysporum f. sp. cubense tropical race 4. Front Microbiol. 2020. https://doi.org/10.3389/fmicb.2020.01712.

  41. Awan ZA, Shoaib. Combating early blight infection by employing Bacillus subtilis in combination with plant fertilizers. Curr Microbiol. 2019. https://doi.org/10.1016/j.cpb.2019.100125.

  42. Attia MS, El-Sayyad GS, Abd Elkodous M, El-Batal AI. The effective antagonistic potential of plant growth-promoting rhizobacteria against Alternaria solani-causing early blight disease in tomato plant. Sci Hortic. 2020. https://doi.org/10.1016/j.scienta.2020.109289.

  43. Khan N, Mishra A, Nautiyal CS. Paenibacillus lentimorbus B-30488r controls early blight disease in tomato by inducing host resistance associated gene expression and inhibiting Alternaria solani. Biol Control. 2012. https://doi.org/10.1016/j.biocontrol.2012.03.010.

  44. Ambrosini A, Beneduzi A, Stefanski T, Pinheiro FG, Vargas LK, Passaglia LMP. Screening of plant growth promoting rhizobacteria isolated from sun-flower (Helianthus annuus). Plant Soil. 2012. https://doi.org/10.1007/s11104-011-1079-1.

  45. Dias MP, Bastos MS, Xavier VB, Cassel E, Astarita LV, Santarém ER. Plant growth and resistance promoted by Streptomyces spp. in tomato. Plant Physiol Biochem. 2017. https://doi.org/10.1016/j.plaphy.2017.07.017.

  46. Shirling EB, Gottlieb D. Methods for characterization of Streptomyces species. International journal of systemic and evolutionary. Microbiology. 1966;16(3):313–40.

    Google Scholar 

  47. Heil M, Bostock RM. Induced systemic resistance (ISR) against pathogens in the context of induced plant defences. Ann Bot. 2002. https://doi.org/10.1093/aob/mcf076.

  48. Kuć J. Concepts and direction of induced systemic resistance in plants and its application. Eur J Plant Pathol. 2002. https://doi.org/10.1023/A:1008718824105.

  49. Kaur T, Manhas RK. Antifungal, insecticidal, and plant growth promoting potential of Streptomyces hydrogenans DH16. J Basic Microbiol. 2014. https://doi.org/10.1002/jobm.201300086.

  50. Cao L, Qiu Z, Dai X, Tan H, Lin Y, Zhou S. Isolation of endophytic actinomycetes from roots and leaves of banana (Musa acuminata) plants and their activities against Fusarium oxysporum f. Sp cubense World J MicrobBiot. 2004. https://doi.org/10.1023/B:WIBI.0000040406.30495.48.

  51. Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotics susceptibility testing by a standardized single disc method. Am J Clin Pathol. 1966;45:493.

    Article  CAS  PubMed  Google Scholar 

  52. Manhas SM, RK. Purification and characterization of actinomycins from Streptomyces strain M7 active against methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus. BMC Microbiol. 2019. https://doi.org/10.1186/s12866-019-1405-y.

  53. Shirling EB, Gotllieb D. Methods for characterization of streptomycetes sp. Int J Syst Bacteriol. 1966. https://doi.org/10.1099/00207713-16-3-313.

  54. Kelly KL. Inter-society color council—national bureau of standards color-name charts illustrated with centroid colors. Washington: US government printing office; 1964.

    Google Scholar 

  55. Cowan ST, Steel KJ. Manual for the identification of medical Bacteria. London: Cambridge University Press; 1965.

    Google Scholar 

  56. Lechevalier MPA, Lechevalier HA. The chemotaxonomy of actinomycetes. In: Dietz A, Thayer DW, editors. Actinomycete taxonomy: Society for Industrial Microbiology; 1980. p. 22–291.

    Google Scholar 

  57. Marmur J. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol. 1961. https://doi.org/10.1016/S0022-2836(61)80047-8.

  58. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, et al. EzTaxon: a web-based tool for the identifcation of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol. 2007. https://doi.org/10.1099/ijs.0.64915-0.

  59. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol bio evol. 1987. https://doi.org/10.1093/oxfordjournals.molbev.a040454.

  60. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013. https://doi.org/10.1093/molbev/mst197.

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Acknowledgements

We duly acknowledge Department of science & technology for the financial support to accomplish this research work. One of the authors, Sapna Devi acknowledges the grant of fellowship under university with potential for Excellence scheme of DST- Promotion of University Research and Scientific Excellence.

The study complies with relevant institutional, national, and international guidelines and legislation.

Funding

This work was financially supported by DST- Promotion of University Research and Scientific Excellence scheme and RUSA 2.0 Rashtriya Ucchatar Shiksha Abhiyan, MHRD, India.

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  1. Department of Microbiology, Guru Nanak Dev University, Amritsar, Punjab, India

    Sapna Devi, Manish Sharma & Rajesh Kumari Manhas

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RM as research supervisor of SD and MS was involved in the planning of research work; investigation and interpretation of data; drafting and critical editing of the manuscript in terms of intellectual content. MS conducted and interpreted the data related to isolation of the strain and SD was involved in the planning and accomplishment of the research work; analysis and interpretation of data; manuscript writing following the suggestions of the research supervisor. All of the authors agreed to be responsible for all aspects of the writing, including ensuring that any concerns about the work’s quality or credibility are thoroughly investigated and resolved. The manuscript has been read and accepted by all contributors.

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Devi, S., Sharma, M. & Manhas, R.K. Investigating the plant growth promoting and biocontrol potentiality of endophytic Streptomyces SP. SP5 against early blight in Solanum lycopersicum seedlings. BMC Microbiol 22, 285 (2022). https://doi.org/10.1186/s12866-022-02695-8

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