In Vitro and In Vivo Plant Growth Promoting Activities and DNA Fingerprinting of Antagonistic Endophytic Actinomycetes Associates with Medicinal Plants.

Endophytic actinomycetes have shown unique plant growth promoting as well as antagonistic activity against fungal phytopathogens. In the present study forty-two endophytic actinomycetes recovered from medicinal plants were evaluated for their antagonistic potential and plant growth-promoting abilities. Twenty-two isolates which showed the inhibitory activity against at least one pathogen were subsequently tested for their plant-growth promoting activities and were compared genotypically using DNA based fingerprinting, including enterobacterial repetitive intergenic consensus (ERIC) and BOX repetitive elements. Genetic relatedness based on both ERIC and BOX-PCR generates specific patterns corresponding to particular genotypes. Exponentially grown antagonistic isolates were used to evaluate phosphate solubilization, siderophores, HCN, ammonia, chitinase, indole-3-acetic acid production, as well as antifungal activities. Out of 22 isolates, the amount of indole-3-acetic acid (IAA) ranging between 10-32 μg/ml was produced by 20 isolates and all isolates were positive for ammonia production ranging between 5.2 to 54 mg/ml. Among 22 isolates tested, the amount of hydroxamate-type siderophores were produced by 16 isolates ranging between 5.2 to 36.4 μg/ml, while catechols-type siderophores produced by 5 isolates ranging from 3.2 to 5.4 μg/ml. Fourteen isolates showed the solubilisation of inorganic phosphorous ranging from 3.2 to 32.6 mg/100ml. Chitinase and HCN production was shown by 19 and 15 different isolates, respectively. In addition, genes of indole acetic acid (iaaM) and chitinase (chiC) were successively amplified from 20 and 19 isolates respectively. The two potential strains Streptomyces sp. (BPSAC34) and Leifsonia xyli (BPSAC24) were tested in vivo and improved a range of growth parameters in chilli (Capsicum annuum L.) under greenhouse conditions. This study is the first published report that actinomycetes can be isolated as endophytes from within these plants and were shown to have antagonistic and plant growth promoting abilities. These results clearly suggest the possibility of using endophytic actinomycetes as bioinoculant for plant growth promotion, nutrient mobilization or as biocontrol agent against fungal phytopathogens for sustainable agriculture.

Introduction

Plants exhibit an intrinsic relationship with a broad range of microbial populations that colonize the rhizosphere (rhizobacteria), the phyllosphere (epiphytic bacteria) and plant tissue (endophytes) [1]. An endophytic microorganism resides within their host plant, for at least a part of its life without causing any apparent disease symptom or infection. They are ubiquitous in nature and are present in all the plant species on earth; however, most of these endophytes and their relationship with plants are not well understood and remain in the initial phase of exploration [2, 3]. The plant endosphere is a complex micro-ecosystem where different niches can be occupied by different types of microorganisms representing rich and genuine sources of novel bioactive metabolites [4, 5].

In spite of ever increasing information about endophytes associated with different host plants, measures to understand their functional diversity is limited especially for ethnomedicinal plants. Parrent et al. [6] suggested that functional diversity could be more effective and powerful than taxonomic measures to understand the mechanistic basis of diversity effects on plant-endophyte relationship. Further, Hardoim et al. [7] suggested the key fitness enhancing traits that are important for the endophytic host relationship, results in the emergence of efficient physiological systems which enable endophytic residence in the host endosphere. The present study focused on understanding the plant growth promoting traits of endophytic actinomycetes, demonstrating the role and importance of endophytic actinomycetes to their host plant.

Endophytes can occupy the cortical tissues of roots and have been effective in the defense against invading pathogens whereas, the cortex evidently confers protection to endophytes from the harsh environment of the rhizosphere [8]. Actinobacteria associated with plants provide a number of benefits to host plants such as the production of phytohormones, nitrogen fixation and secondary metabolites [9]. Although, many species of actinomycetes particularly those belonging to genus Streptomyces inhibit many fungal pathogens and are considered as efficient biocontrol agents, their metabolic products can influence plant growth [5, 10]. The antagonistic activity of Streptomyces against phytopathogens is related to the production of antimicrobial compounds [11, 12] and extracellular hydrolytic enzymes capable of lysing fungal cell walls [13]. They promote plant growth by producing indole-3-acetic acid (IAA) to help root growth [14], siderophores to improve nutrient uptake [15] and a number of antibiotics that are secondary metabolites [16].

Various molecular techniques such as 16S rRNA gene sequencing, amplification of repetitive extragenic palindromic-PCR (REP-PCR) like enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and BOX-PCR are progressively used for identification, comparative analysis and to put Bacteria, Archaea and Eukarya [17, 18]. Here, we compare the robustness of REP-PCR fingerprinting to differentiate among the various potential endophytic actinomycetes isolates obtained from different tissues of plants.

Chilli (Capsicum annuum L) is an essential spice crop that is grown all over the world. It is an economically important and valuable cash crop that is produced and consumed fresh and processed. The production and yield is greatly affected by Fusarium wilt caused by the soil pathogen Fusarium oxysporum [19]. Currently the applications of biological control agents are in practice but they have not provided sufficient disease control [20]. In this study, the potential antagonistic isolates, BPSAC 34 (Streptomyces sp.) and BPSAC 24 (Leifsonia xyli) were evaluated in a greenhouse experiment to determine their potential use for plant growth promotion individually and in consortium by measuring the shoot length, root length and plant weight.

Endophytic actinomycetes from seven selected ethnomedicinal plants in Mizoram, North-East, India, were used to represent medicinal plants from a biodiversity hotspot [21, 22]. This study described the functional and genetic characterization of endophytic actinomycetes from seven selected medicinal plants from two protected forest areas of Mizoram, India. The results underscore the importance of endophytic actinomycetes community for host plants and can be used for the development of microbial formulations to enhance growth of Capsicum annuum L.

Materials and Methods

Plant collection and description

Seven healthy medicinal plants, viz. Eupatorium odoratum, Musa superba, Mirabilis jalapa, Curcuma longa, Clerodendrum colebrookianum, Alstonia scholaris and Centella asiatica were collected from Phawngpuii National Park [Phawngpuii NP] (22°40′N; 93°03′E) and Dampa Tiger Reserve Forest [Dampa TRF] (23°25′N; 92°20′E) in Mizoram, India during the month of November, 2012. Permission for the collection of medicinal plants was obtained from the Chief wildlife warden, Environment and forest department, Government of Mizoram, India issued by Mr. Liankima Lailung, Conservator of forest (WL), Mizoram, India. The selection of plant species was based on their ethnomedicinal history and abundance (Table 1). The tissues from root, stem, leaf and flower were carefully collected. The tissue samples were placed in sterile polythene bags and brought into the laboratory in an icebox and processed within 24 h of collection.

Table 1

Summary of plant sample collection, taxonomic status, uses Sharma et al. [21] and endophytic actinomycetes isolated from different tissues.

Location	Local Name (Voucher No.)	Scientific Name	Traditional medicinal value	Tissue of origin	Isolates obtained	Family
Dampa Tiger Reserve Forest	Tlangsam (MZU/BT/001)	Eupatorium odoratum	Used as an antiseptic and to remove pinworm from the anus	Root	5	Asteraceae
				Stem	2
	Changel (MZU/BT/002)	Musa superb Roxb.	Treatment of chilling sensation, convulsion, cough, snake bites and bee stings	Flower	2	Musaceae
	Artukhuan (MZU/BT/003)	Mirabilis jalapa	Treatment of sexually transmitted diseases, kidney and urinary infection. Reduce swelling due to bone fracture or twisting	Root	6	Nyctaginaceae
				Stem	3
				Leaf	1
				Petiole	2
	Lambak (MZU/BT/004)	Centella asiatica	Popularly used as memory stimulator. Treatment of asthma and eye problems and also used in hypertension	Root	3	Apiaceae
				Flower	1
Phawngpuii National Park	Aieng (MZU/BT/005)	Curcuma longa	Treatment of cancer, heart diseases and stomach colic	Root	2	Zingiberaceae
				Leaf	3
	Phuihnam (MZU/BT/006)	Clerodendrum colebrookianum	Treatment of hypertension, diabetes and colics in infants	Root	4	Verbenaceae
				Stem	3
				Leaf	2
	Thuamriat (MZU/BT/007)	Alstonia scholaris	Treatment of malaria, diarrhea, heart diseases and hypertension	Root	2	Apocynaceae
				Stem	1

Isolation of endophytic actinomycetes

All the collected samples were washed with tap water to remove debris. Tissues were cut into small pieces and subjected to surface sterilization [23]. The tissue samples were placed on five different isolation media, supplemented with filter sterilized nystatin and cycloheximide to suppress fungal growth and nalidixic acid to inhibit bacterial growth. Isolation media used were; (1) Starch Casein Nitrate Agar (SCNA); (2) Actinomycetes Isolation Agar (AIA); (3) Tap Water Yeast Extract Agar (TWYE); (4) Yeast Malt Extract Agar (ISP2) and (5) Glycerol Asparagine Agar (ISP5). The surface sterilization process was confirmed by spreading aliquots of the sterile distilled water from the final rinse on ISP2 medium, followed by incubation at 28°C, and observation of microbial growth. If there was no visible growth of microorganism on the surface of agar plates, the sterilization was assumed completed [24]. Pure isolates of endophytic actinomycetes were obtained by repeated streaking, and the pure isolates were stored at -20°C in 20% glycerol.

Identification and antagonistic activity of endophytic actinomycetes isolates

The phenotypic characterization of the endophytic actinomycetes were based on cultural and morphological characteristics including colony colour and characteristics, aerial and substrate colour, spore mass colour, production of diffusible pigment and spore chain morphology according to Bergey`s manual of determinative bacteriology [25]. Field emission gun-Scanning electron microscopy (FEG-SEM) was used for observation of the spore chain morphology for 10 d grown culture on ISP4 media [26]. The identification of the isolates was further confirmed by 16S rRNA gene amplifications sequencing.

All isolates were evaluated for their antagonistic activity against six major plant pathogenic fungi, Rhizoctonia solani (MTCC-9666), Fusarium oxysporum f. sp. ciceri (MTCC-2791), Fusarium proliferatum (MTCC-286), Fusarium oxysporum (MTCC-284), F. graminearum (MTCC-1893) and Colletotrichum capsici (MTCC-8473) using a dual culture in vitro assay [27]. The pathogens were obtained from Microbial Type Culture Collection, Institute of Microbial Technology (IMTECH), Chandigarh, India. Plates with only pathogen culture served as control. All plates were incubated at 28°C for 14 d and the percent inhibition was calculated using the formula C-T/C x 100, where, C is the colony growth of fungal pathogen in control, and T is the colony growth in dual culture. All experiments were carried out in triplicate.

Determination of plant growth promoting activities

Phosphate solubilization

Qualitative phosphate solubilization activity of potential antagonistic endophytic actinomycetes isolates were analyzed on PKV agar media. Those forming clear halo zone around their colonies after day 5 incubation were considered to have P-solubilization and were selected for quantitative estimation of P- solubilization using a standard method [28]. Endophytic actinomycetes suspension (1ml, 10−3 cfu/ml) was added to 50 ml of NBRIP broth which contained the following ingredients: 10 g of tricalcium phosphate (TCP), 10 g of MgCl2.6H2O, 5 g of MgSO4.7H2O, 0.25 g of KCl and 0.2 g of (NH4)2SO4. The culture was incubated at 30°C with shaking at 125 rpm for 10 d, centrifuged at 10,000 rpm for 10 min and the amount of phosphate in the supernatant was estimated by the ascorbic acid method [16]. The absorbance was measured at 880 nm using a spectrophotometer and compared with the standard curve of KH2PO4. The amount of KH2PO4 was expressed in mg/100ml.

Indole acetic acid (IAA) production

The production of IAA by endophytic actinomycetes isolates were estimated according to Gordon and Weber (1951) [29]. The actinomycetes isolates were grown on ISP2 medium at 30°C for 7 d. Four-millimeter-diameter agar discs were cut using sterile cork borer and inoculated into 100 ml of ISP-2 broth containing 0.2% L-tryptophan. The culture was incubated at 30°C with continuous shaking at 125 rpm for 14 d. After incubation the suspension was centrifuged at 11,000 rpm for 15 min and the supernatant (1 ml) was mixed with 2 ml of Salkowski’s reagent and further incubated for 25 min in dark at 30°C. IAA production was observed as the development of a pink-red color and the absorbance was measured at 530 nm using a Thermo scientific (Multiskan GO) spectrophotometer and compared with the standard curve of IAA and the amount of IAA was expressed in μg/ml.

Ammonia production

The endophytic actinomycetes isolates were tested for the production of ammonia using the method described by Cappucino and Sherman [30]. In this method 20 μl of seed culture was propagated in 10 ml of peptone water and incubated at 30°C with shaking at 120 rpm for 15 d. Subsequently, 0.5 ml of Nesseler’s reagent was added to the culture and the development of brown to yellow color indicated a positive test for ammonia production. The absorbance was measured at 530 nm using a Thermo scientific (Multiskan GO) spectrophotometer, compared with the standard curve of (NH4)2SO4 and expressed in mg/ml.

Siderophore production

Siderophore production of the endophytic actinomycetes was determined by the method of Schwyn and Neilands [31]. A loop full of culture was inoculated on Chrome azurol S (CAS) agar medium and incubated at 28± 2°C for 5 d. The colony with a halo zone of yellow-orange color was considered positive for siderophore production. The positive isolates were cultured on modified Gaus No. 1 [32] and incubated at 30°C with shaking at 120 rpm for 10–15 d. Catechol-type siderophores were estimated by Arnow’s method [33] and hydroxamate siderophores were determined using the Csaky test [34].

Chitinase activity of the isolates

Chitinase enzyme activity of the selected isolates was tested on the colloidal chitin agar medium. The colloidal chitin medium contained:- colloidal chitin—15 g, yeast extract—0.5 g, (NH4)2 SO4−1 g, MgSO4 · 6H2O—0.3 g, KH2PO4−1.36 g, agar– 15 g and distilled water—1000 ml. The clear halo zone measured on colloidal chitin agar medium showed positive chitinase activity [35].

Hydrogen cyanide production

Hydrogen cyanide production was evaluated according to the methods of Lorck, [36]. The isolates were grown in Bennett agar amended with 4.4 g/l glycine. A Whatman filter paper No. 1 flooded with 0.5% picric acid in 2% sodium carbonate for a minute and stuck underneath the Petri dish lids. The plates were sealed with parafilm and incubated at 28 ± 2°C. After 7 d, an orange to red color on the filter paper was indicative of positive HCN production.

In vivo plant growth promotion assay

The two most potent endophytic actinomycetes isolates Streptomyces sp. 34 (BPSAC34) and Leifsonia xyli 24 (BPSAC24) were grown on ISP1 broth at 30°C for seven d with continuous shaking at 150 rpm. The cells were centrifuged at 10,000 rpm for 15 min and the pellets diluted with distilled water to yield a final concentration of 106 CFU/ml. The endophytic actinomycetes suspension was used to treat targeted plants under green house conditions.

The chilli seeds were surface sterilized according to the protocol previously described [37]. Healthy chilli seeds were immersed in distilled water for 24 h, surface sterilized with 70% ethanol for 5 min followed by 1% NaOCl treatment for 5 min and five washes with sterile distilled water before planting in sterile soil. The seeds were transferred onto wet sterile tissue paper at 28°C. The seeds were supplied with 2 ml distilled water for 2–3 d to promote seed germination under dark conditions. After 3 weeks, the germinated chilli seedlings were planted separately in plastic pots filled with greenhouse soil. Four greenhouse treatments included T1: untreated chilli seedlings as control. T2: Streptomyces sp. 34 (BPSAC 34) inoculum treated chilli seedlings. T3: Leifsonia xyli 24 (BPSAC 24) inoculum treated chilli seedlings T4: the mixture of two endophytic actinomycetes inoculum (BPSAC 24 and BPSAC 34) applied to the seedlings. All treatments were carried out in seven d interval repeated four times with plants were watered twice daily. Root length, shoot length and fresh plant weight were measured between inoculated and uninoculated plants. Data were statistically analysed using one way ANOVA and turkey tests and P = 0.05 was considered significant. All experiments were performed in triplicates.

Molecular characterization and phylogenetic analysis of antagonistic endophytic actinomycetes

DNA extraction, amplification of 16S rRNA gene and sequencing

Genomic DNA extraction was carried using the Pure link genomic DNA extraction kit (In-vitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Genomic DNA was quantified by using Cary 60 UV-VIS (Agilent Technologies). The 16S rRNA gene was amplified using the universal primers set PA (5′-AGAGTTTGATCCTGGCTCA-3′) and PH (5′-ACGGCTACCTTGTTACGACT-3′) [38]. The DNA sequencing was carried out commercially (Sci-Genome Labs Private Ltd., Chennai, India) and the nucleotides obtained were subjected to BLAST analysis using the NCBI database and deposited in NCBI GenBank.

ERIC-PCR fingerprinting

The PCR reactions were carried out as described by Versalovic et al. [39] using a set of primer sequences ERIC-1R (5′-CACTTAGGGGTCCTCGAATGTA- 3′) and ERIC-2F (5′-AAGTAAGTGACTGGGGTGAGCG- 3′) to amplify the regions in the bacterial genome positioned between the ERIC sequences. The PCR amplification was performed on Veriti thermal cycler (Applied Biosystem, Singapore) in a total reaction mixture volume of 25 μl. The reaction mixture consist of 1 μl of DNA template (50 ng), 2.5 μl of 10 x reaction buffer, 0.5 μl of dNTP mix (10 mM), 10 pmol of each primer (ERIC 1R and ERIC 2F), 1.5 μl of MgCl2 (25 mM), and 1.5 U of DreamTaq DNA polymerase (In-vitrogen, USA). PCR was performed under following conditions; initial denaturation at 95°C for 7 min and then subjected to 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min and extension at 68°C for 8 min with a final extension step at 65°C for 16 min. A negative control reaction mixture without DNA template of actinomycetes was also included with each set of PCR reactions. The amplified products were separated by electrophoresis on a 1.5% agarose gel using 1x TAE buffer solution. The PCR bands were analyzed under UV light and documented using a BioRad Gel Doc XR+ system (Hercules, CA, USA).

BOX PCR fingerprinting

BOX primer sequences were used in PCR to detect differences in the number and distribution of these bacterial repetitive sequence elements dispersed throughout the bacterial genome. BOXA1R PCR fingerprinting was carried out using primer sequences BOXA1R (5′-CTACGGCAAGGCGACGCTGACG-3′) as previously described by Rademaker et al. [40]. The PCR amplification was carried out in 25 μl total volume reaction mixture, containing 50 ng of genomic DNA, 2.5 μl of 10 x Taq Buffer, 1.5 μl of 25mM MgCl2, 2.0 μl of 2.5mM dNTPs, 1 μl of 10 pmol BOXA1R primer, 1 μl of DMSO (100%), 0.5 μl of BSA (10 mg/ml) and 1μl of 2U Taq DNA polymerase. The DNA was amplified under the following conditions: initial denaturation at 95°C for 7 min followed by 30 cycles at 94°C for 30 sec, at 57°C for 1 min, and at 65°C for 8 min with a final extension step at 65°C for 16 min. The amplified fragments were separated on 1.2% agarose gel using 1x TAE buffer at 60V for 3h and the restriction patterns were examined using the gel documentation system as described earlier.

Phylogenetic analysis

16S rRNA sequences based phylogeny and identification was reported earlier (Passari et al. 2015) [41]. In the present study the antagonistic isolates were phylogenetically compared using ERIC and BOX-PCR. Polymorphic DNA fingerprints were scored in the binary form i.e. 1 for presence of a band and 0 when there is absence band, to generate a binary matrix [42] for ERIC and BOX markers. The binary matrix was used to calculate the Simple Matching (SM) coefficient, and a phylogenetic tree was constructed using the Unweighted Pair Group with Arithmetic Mean (UPGMA) methods [43] supported by Numerical Taxonomy SYStem (NTSYS version 2.2).

Detection of iaaM gene from indole acetic acid in endophytic actinomycetes

The indole acetic acid biosynthetic gene (iaaM) was amplified by using forward primer iaaM F(5’-ATGACGTCCACCGTGCCCAACGCG-3’) and iaaM R (5’-CTAGTCCTCGGGGAGTTCCACGGG-3’) as described by Lin and Xu, 2013 [44]. The PCR amplifications were performed in Veriti thermal cycler (Applied Bio-system, Singapore) under the following conditions: initial denaturation at 94°C for 5 min followed by 30 cycles at 94°C for 40 s, at 59°C for 30 s and at 72°C for 2 min and a final extension step at 72°C for 10 min. The amplified fragments were separated on 1.2% agarose gel using 1 x TAE buffer at 80V for 3h and the restriction patterns were examined using the gel documentation system as described earlier.

Detection of chitinase gene (chiC gene) from potential chitinase producing endophytic actinomycetes

The forward (5’- AAGCTCGCSGCSTTCCTSGC—3’) and reverse (5’- GCACTCGAGSGCGCCGTTGAT—3’) primers were used for amplification of the chitinase gene under the following conditions: initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation for 30 s at 98°C, annealing for 30 s at 50°C, extension for 1.0 min at 72°C were used and 10 min at 72°C for the final extension [45].

Results

Isolation and identification of endophytic actinomycetes

A total of 42 endophytic actinomycetes were isolated from the seven healthy medicinal plants, and identified based on their morphological characteristics. Frequency of the endophyte isolation demonstrated that nutrient poor media such as ISP2, ISP5, TWYE, AIA and SCNA was most effective for the isolation of endophytic actinomycetes from different tissues of the medicinal plants and that Streptomyces was the most dominant genus. The SEM images showed aerial mycelia with spiral spore chains (Fig 1).

Fig 1

Field emission gun-scanning electron microscopy (FEG-SEM) micrographs of (A) Streptomyces sp. (BPSAC34) and (B) Leifsonia xyli (BPSAC24) showing spore chain morphology.

All the antagonistic endophytic actinomycetes isolates were amplified for the 16S rRNA gene and an amplified fragment of about 1.5 Kb was obtained. All sequences were submitted to the Genbank, NCBI and accession numbers assigned to KF255557, KF255560, KF255576 and KJ584866 to KJ584884. In the present study the isolates were screened for plant growth promoting activities and were compared genotypically using ERIC and BOX—PCR fingerprinting.

In vitro antagonistic screening against phytopathogens

To select the potential antagonistic isolates, the antimicrobial activity of all the isolates was tested against six fungal phytopathogens. Among the 42 actinomycetes isolates, 22 (52.3%) isolates showed inhibitory activity against at least one tested pathogen and were selected for further studies. Out of 22 isolates, 14 isolates (63.6%) showed significant inhibitory activity against three important plant-pathogens, i.e. Rhizoctonia solani (MTCC-9666), Fusarium graminearum (MTCC-1893) and Fusarium oxysporum (MTCC-284). The three isolates i.e. Microbacterium sp. 21 (BPSAC21), Leifsonia xyli 24 (BPSAC24) and Streptomyces sp. 34 (BPSAC34) demonstrated significant antagonistic activity ranging from 35% to 92%, against all the tested pathogens (Table 2).

Table 2

Top 10 best endophytic actinomycetes isolates and their antagonistic activity, antifungal mechanisms in addition to their plant growth promoting traits and general assessment and ranking for their ability to function as PGPR.

Code	Isolates Organism	Antagonistic Activities						Antifungal Mechanisms			Plant growth promoting traits				Total Ass. (29) n	Rank
		GI percentage (%)
		Rs a	Fg b	Fo c	Fp d	Foc e	Cc f	Chi g	Sid h	HCN i	PS j	IAA k	Am l	Sid h
BPSAC34	Streptomyces sp.	2	3	3	3	2	2	1	1	1	3	3	1	1	26	1st
BPSAC24	Leifsonia xyli	3	3	1	3	2	2	1	1	1	3	3	1	1	25	2nd
BPSAC21	Microbacterium sp.	2	3	2	3	3	1	1	1	1	2	2	1	1	23	3rd
BPSAC27	Microbacterium sp.	1	0	2	2	2	0	1	1	1	2	2	1	1	16	4th
BPSAC37	Actinomycete	1	2	0	1	3	2	1	1	0	1	2	1	1	16	4th
BPSAC42	Streptomyces mutabilis	3	1	2	3	2	1	1	0	0	1	1	1	0	16	4th
BPSAC28	Microbacterium sp.	2	0	2	2	1	0	1	1	1	2	1	1	1	15	5th
BPSAC35	Brevibacterium sp.	1	1	1	1	1	0	1	1	1	0	1	1	1	11	6th
BPSAC32	Streptomyces sp.	0	1	3	2	2	0	1	0	0	0	0	1	0	10	7th
BPSAC2	Streptomyces sp.	1	1	1	1	1	0	1	0	0	1	1	1	0	9	8th

GI (%): Growth inhibition percentage (1 = 30–54.5%; 2 = 55–74.5%; 3 = 75–95%).

aRs: Rhizoctonia solani;

bFg: Fusarium graminearum;

cFo: Fusarium oxysporum;

dFp: Fusarium prolifratum;

eFoc: Fusarium oxysporum ciceri;

fCc: Colletotrichum capsici.

gChi: Chitinase production;

hSid: Siderophores production;

iHCN: Hydrogen cyanide.

jPS: Phosphate solubilization (1 = 1.0–11.9; 2 = 12–22.9; 3 = 23–33);

kIAA: Indole acetic acid production (1 = 1.0–11.9; 2 = 12–23.9; 3 = 24–35.9).

lAm: Ammonia production.

nTotal assessment points.

Plant growth promoting traits of antagonistic endophytic actinomycetes

Phosphate solubilization and IAA production

Among the 22 endophytic actinomycetes isolates, 14 (63.6%) were able to solubilize inorganic phosphate and were identified as potential phosphate solubilizing isolates based on a clear halo zone around the colony on Pikovskaya`s medium. The phosphate solubilization efficiency varied from 35 to 73% among the isolates, and the highest phosphate solubilization were detected in Streptomyces sp. 34 (73%) followed by Leifsonia xyli 24 (64%) and Microbacterium sp. 21 (59%). Quantitative estimation of phosphate solubilization by the endophytic actinomycetes ranged from 3.2 to 32.6 mg/100ml (Table 2), with the highest by Streptomyces sp. 34 (32.6 mg/ 100 ml) followed by Leifsonia xyli 24 (31.5 mg/ 100 ml).

Out of 22 isolates 20 endophytic actinomycetes (90.9%) were positive for IAA production, among them the 14 isolates belongs to Streptomyces sp. Quantitative range of IAA production was found from 10–32 μg/ml (Table 2). Leifsonia xyli 24 and Streptomyces sp. 34 produce the most IAA, with 30.5 and 32 μg/ml, respectively. The results of PCR amplification of IAA gene demonstrated the presence of 150 bp fragment in all the 20 positive isolates (Fig 2A).

Fig 2

PCR amplification of (A) iaaM gene and (B) chiC gene for endophytic actinomycetes isolates. M: low range (100bp -3 kb) molecular marker; N: negative control; numerical numbers represents different isolates.

Ammonia and siderophore production by endophytic actinomycetes

All 22 endophytic actinomycetes isolates were positive for the production of ammonia at levels ranging from 5.2 to 54 mg/ml. Isolate Streptomyces sp. 34 produced the maximum amount of ammonia (54 mg/ml). Siderophore production was detected in 16 (72%) isolates on CAS agar media, forming clear orange halo zone around the colonies. The five isolates produced catachol type siderophore (at levels ranging from 3.2–5.4 μg /ml), whereas, 16 isolates produced hydroxamate- type siderophore (range from 5.2–36.4 μg /ml). Isolates Leifsonia xyli 24 and Streptomyces sp. 34 produced mostly catechol type siderophores (5.4 μg/ ml), while the isolate Streptomyces sp. 34 produced the greatest amount of hydroxamate type siderophores (36.4 μg /ml) on modified Gaus No.1 broth. All the Microbacterium sp. (BPSAC 21, 27, 28 and 29) produced hydroxamate type siderophores at levels ranging from 15.4–34.5 μg /ml (Table 2).

Chitinase production

Nineteen isolates were positive for chitinase production and formed clear halo zone around the colonies. Chitin degrading activity was found to be high in Leifsonia xyli 24 and Microbacterium sp. 21 which exhibited a colloidal chitin degradation zone of 15 and 17mm, respectively (Table 2). All the 19 positive isolates were subjected to the amplification of the chitinase gene and an amplified fragment that was approximately 400 bp was obtained from these isolates (Fig 2B).

HCN production

Among the 22 isolates, 15 isolates were positive for HCN production. Most of the HCN production positive isolates belongs to Streptomyces sp. and Microbacterium sp. (Table 2). Isolate Streptomyces sp. 34 exhibited the highest amount of HCN production as indicated by a very deep red color on the filter paper.

In vivo plant growth activity of chilli seedlings

The most potent isolates Streptomyces sp. 34 and Leifsonia xyli 24 identified by 16S rRNA gene sequencing as strains of Streptomyces sp. and Leifsonia xyli were used for in vivo greenhouse experiments on chilli seedlings. Inoculation of seedling with Leifsonia xyli 24 and Streptomyces sp. 34, showed a significant (p<0.05) increase in root and shoot height in comparison to control (Fig 3). The mixture of two actinomycetes isolates (Leifsonia xyli 24 and Streptomyces sp. 34) demonstrated the maximum increase in shoot and root length of the chilli plant when compared with the control after 30 and 45 d of sprouting (Table 3).

Fig 3

Effect of Streptomyces sp. (BPSAC34), Leifsonia xyli (BPSAC24) and combined inoculation of BPSAC 34 and BPSAC 24 on shoot length (A1), root length (A2) and plant total weight (A3) on chilli seedlings in greenhouse conditions.

Table 3

Effect on different growth parameters of chilli seedlings treated with Streptomyces sp. 34 (BPSAC34) and Leifsonia xyli 24 (BPSAC24) in greenhouse pot trials within 30 and 45 d.

Treatments	Shoot length (cm)	Root length (cm)	Plant weight (grams)
After 30 d
Control	9.7 ± 0.05	2.6 ± 0.12	1.8 ± 0.02
Inoculation with BPSAC 24	11.5 ± 0.20	3.7 ± 0.15	2.9 ± 0.03
Inoculation with BPSAC 34	12.5 ± 0.13	5.6 ± 0.11	3.1 ± 0.04
Mix inoculation with BPSAC 24 & 34	18.6 ± 0.08	8.4 ± 0.17	8.6 ± 0.02
After 45 d
Control	11.4 ± 0.115	3.0 ± 0.05	2.1 ± 0.02
Inoculation with BPSAC 24	14.5 ± 0.173	4.1 ± 0.12	3.6 ± 0.11
Inoculation with BPSAC 34	15.2 ± 0.088	6.2 ± 0.17	4.1 ± 0.12
Mix inoculation with BPSAC 24 & 34	22.4 ± 0.202	9.4 ± 0.23	9.2 ± 0.08

Data presented are mean ± SE from three replicates: Each replica consisted of three plants per jar. Means are significantly different from control at P = 0.05 (Tukey test).

Concluding assessment of the in vitro PGPR traits

An attempt was made to select the best isolates among the screened endophytic actinomycetes with high plant growth promoting potential; a bonitur scale was generated and used to assess the PGPR traits [46, 47]. In this scale, the maximum bonitur score is 29 points. The assessment showed that out of 22 isolates screened, 10 isolates obtained three points each against the fungal pathogen for antagonistic activity (totaling 18 points). Production of the chitinase enzyme, siderophore and HCN was evaluated with one point each (totaling 3 points). For plant growth promoting traits, it is possible to obtain three points each for phosphate solubilization and IAA and one point each for ammonia and siderophores (totaling 8 points). Streptomyces sp. 34 was the most effective isolate showing the highest ∑ assessment value of 26 points (Table 2).

ERIC-PCR fingerprinting

All the antagonistic endophytic actinomycetes generated a specific pattern with ERIC-PCR and genetic diversity of isolates was not significant from either location. The fingerprinting pattern yielded discriminatory patterns with 3 to 12 fragments ranging in size from approx. <100 bp to 3.0 kb which demonstrate the usefulness of this technique in differentiate the isolates. Dendrogram generated by ERIC-PCR divided the isolates into four clusters (A-D). Cluster A consist of 10 isolates, belonging to the genus Streptomyces. Isolate Streptomyces 5 (BPSAC5) and Streptomyces 33 (BPSAC33) showed 100% similarity and both identified as Streptomyces sp. based on 16S rRNA gene sequencing. Cluster B consist of four isolates all belonging to the genus Microbacterium. Cluster C consist of five isolates comprising different genera belonging to Leifsonia, Brevibacterium and Streptomyces and the cluster D consists of two isolates both belonging to the genus Streptomyces (Fig 4).

Fig 4

Dendrogram generated from ERIC PCR genomic fingerprints of endophytic actinomycetes isolates using Ntsys 2.0.

BOX-PCR fingerprinting

The BOX-PCR fingerprinting of all the antagonistic endophytic actinomycetes were developed and size recognizable bands were between <100bp to 2kb. Less visible fragments above 2kb were also observed in some isolates. Dendrogram analysis divides the isolates into two major clusters (A & B). Cluster A consist of 16 isolates whereas cluster B consists of six isolates. Most of the isolates showed different BOX fingerprinting patterns, which confirms a significant diversity exist in between the different endophytic actinomycetes isolated in this study (Fig 5).

Fig 5

Dendrogram generated from BOX PCR genomic fingerprints of endophytic actinomycetes isolates using Ntsys 2.0.

Discussion

The environment pollution problems resulting either directly or indirectly from the use of chemical fertilizers, pesticides, herbicides etc. are a major concern in crop production. Therefore, forcing researchers to seek an alternative path based on natural sources for the sustainable plant growth in agriculture and horticulture [48, 49]. Endophytic actinomycetes are of special interest as they have many properties which enhance the growth of the plants [50]. These findings encouraged us to explore ethnomedicinal plants used by local tribes of Mizoram state, India to better understand the endophytic actinomycetes community and their plant growth promoting potential.

Our results demonstrated that endophytic actinomycetes colonizing medicinal plants promote plant growth through production of plant growth regulators (IAA, siderophore, chitinase), phosphate solubilization, siderophore production and promote antagonistic activity against pathogens such as Rhizoctonia solani, F. oxysporum f. sp. ciceri, F. graminearum, Fusarium oxysporum, F. prolifratum and Colletotrichum capsici. Here we identified 22 endophytic actinomycetes with potential antagonistic and PGPR activity. Among them the fifteen isolates belongs to genus Streptomyces, four isolates belongs to Microbacterium and one each belongs to Actinomycete sp., Leifsonia and Brevibacterium. The most frequently isolates endophytic actinomycetes from various plants in our study and in previous studies belongs to genus Streptomyces [5, 51]. This suggests that the strains of Streptomyces are able to reside in the variety of plant tissues. On the other hand, the genera Leifsonia and Brevibacterium were among the rare genera reported from endosphere of plants [41].

All isolates were screened for their antagonistic activity against six fungal phytopathogens. Fourteen isolates (63.6%) showed significant antimicrobial activity against three pathogens, i.e. Rhizoctonia solani (MTCC-9666), Fusarium graminearum (MTCC-1893) and Fusarium oxysporum (MTCC-284). Similar, antimicrobial activity against fungi was reported by Varma et al. [5]. Most of the antagonistic activity positive isolates belongs to Streptomyces sp. (n = 11, 50%), Leifsonia xyli, Brevibacterium sp. and Microbacterium sp. However, the isolates Leifsonia xyli 24 and Streptomyces sp. 34 demonstrated a significant antagonistic activity against all the tested phyto-pathogens. Similarly, Debananda et al. [52] also reported that S. vinaceusdrappus having greater antagonistic potency against rice fungal pathogens F. oxysporum with plant growth promoting properties. Metabolites produced by microbe plays an active role in resistance development by functioning as signals to mediate cross-talk between the endophytes and their host [53]. Since the endophytic actinomycetes isolated from medicinal plants produce a wide variety of antifungal and plant growth regulatory bioactive metabolites [54, 55], they can be explored as novel sources of natural products as well as novel biocontrol agents.

The maximum phosphate solubilization activity was detected in the isolate BPSAC34, identified as Streptomyces sp. (32.6 mg/100 ml). These results are in agreements with Hamdali et al. [56] who reported 83.3, 58.9 and 39 mg/100 ml phosphate solubilization by Streptomyces cavourensis, Streptomyces griseus and Micromonospora aurantiaca, respectively. It was reported that the phosphate solubilizing strains plays an important role in acidification of the medium, and the pH and the soluble phosphate concentration were inversely proportional [57]. This result is consistent with our findings in which 63.6% (14 out of 22) isolates were identified as phosphate solubilizers. This may be either due to the acidification of external medium by production of low molecular weight organic acids like gluconic acid [58]. Hence, endophytic actinomycetes with phosphate solubilization efficiency play an important role in the improvement of plant growth.

In this study, 90.9% of the isolates were positive for IAA production, among them 63.6% of isolates belonged to genus Streptomyces. The IAA production ranges between 10–32 μg/ml, which is in accordance with Verma et al. [50] and Khamna et al. [15]. Nimnoi and Pongslip, [59] reported that IAA synthetic bacteria enhanced root and shoot development of Raphanus sativus and Brassica oleracea more than five-fold when compared with control. The endophytic actinomycetes present inside root tissues produce IAA that may play an important role in host plant development and growth.

Here we detected most of the endophytic actinomycetes produced ammonia. Marques et al. [60] suggested that bacteria can produce ammonia and supply nitrogen to the host plant. The ammonia produced by endophytes is beneficial for the root and shoot elongation, consequently increasing plant biomass. Moreover, it is very useful for the over production of ammonia which can serve as a triggering factor for the virulence of opportunistic plant pathogens [61].

Siderophore production is another feature that promotes plant growth by binding to the available iron form (Fe3+) in the rhizosphere making iron unavailable to the phytopathogens [62]. Siderophores display considerable structural variability and affinity for iron that determines the growth of a microbe under competitive conditions when iron availability itself is a limiting factor [10]. Streptomyces species are well known for the production of hydroxamate-type siderophores, which can inhibit phytopathogen growth by competing for iron in rhizosphere soils [15]. Tan et al. [63] suggested that the production of siderophore is an important factor for phytopathogen antagonism and developing growth of the plant. In our study, we detected the siderophore production in 72% isolates. The maximum amount of catechol type and hydroxamate type siderophores were produced by isolate Streptomyces sp. 34 (5.4 and 36.4 μg/ml) these are in agreement with Nimnoi et al. [64] who demonstrated that Pseudonocardia halophobica isolated from the roots of Aquilaria crassna possess high capacity for hydroxymate type siderophore production (39.30 μg/ ml). Similarly, Khamna et al. [15] has showed that Streptomyces CMU-SK 126 isolated from Curcuma mangga rhizospheric soil exhibited high amount of siderophore, catechol type and hydroxamate types production.

Chitinase from microorganisms is crucial for the degradation and recycling of carbon and nitrogen trapped in insoluble chitin [65] and is widely used for the preparation of biopesticides and mosquito control [66]. Our results showed that 19 (86.3%) endophytic actinomycetes isolates were positive for extracellular chitinase production, and demonstrated the presence of chitinase gene. These results were consistent with Taechowisan et al. [11] who reported 4.56% isolates had chitinase gene.

HCN plays an important role in disease suppression [67]. Here we detected fifteen isolates (68.1%) were positive for HCN production, and most of which belongs to Streptomyces sp. Similarly, Hastuti et al. (2012) [51] reported Streptomyces sp. LSW05 strain as a potent HCN producer.

Streptomyces sp. 34 and Leifsonia xyli 24 demonstrated a significant PGPR activity under green house experiment. And a mixture of two actinomycetes isolates (BPSAC24 and BPSAC34) displayed the maximum increase in shoots and root length of the chilli plant when compared with the control after 30 and 45 d of sprouting. Many reports have shown that actinobacteria can increase root and shoot length in different plants [68], and such an increase may confer advantages to the host plant with respect to health and overall growth. Taken together, these results show that Streptomyces sp. 34 and Leifsonia xyli 24 are well suited as an efficient biocontrol and plant growth promoting inoculam for sustainable agriculture.

All the antagonistic endophytic actinomycetes isolates were characterized by PCR amplification of the 16S rRNA gene. The DNA sequence of most isolates showed of 97–100% identity with BlastN sequences and phylogenetic analysis based on 16S rRNA gene amplification showed that Streptomyces formed a major group consistent with previous studies [41, 69].

The dendrogram generated by ERIC-PCR divided the isolates into four groups (A, B, C and D) and the fingerprinting pattern clearly differentiate among the Streptomyces sp. whereas Microbacterium sp. (BPSAC29) falls in group A and other genera like Leifsonia, Brevibacterium and Microbacterium were clearly found in a separate group B which was in agreement with the findings of De-Bruijn et al. [70]. ERIC-PCR fingerprinting could be a reliable tool for the detection of similarities and differences in the relationships among different isolates in the same bacterial genus and species [71]. They are genetically diverse enough to allow the construction of a phylogenetic tree showing the relative relatedness of the different strains. BOX-PCR fingerprinting has proved a very useful tool to discriminate highly related strains and has been applied to study the genetic diversity at the species level among the endophytic actinomycetes isolates [72, 73]. In this study, however, we observed that genetic variation was too high among the 22 isolates, when analyzed by BOX-PCR fingerprinting.

Conclusions

From this study, we conclude that endophytic actinomycetes have great potential as a potential antagonistic agent against major fungal phytopathogens. These species enhance the growth of plants by producing phytohormones (IAA), solubilizing inorganic phosphorous, producing siderophores and ammonia, as well as by providing protection to plant from phytopathogens. Since, in vitro studies are a prelude to any green house or field studies, these findings provides compelling evidence that the endophytic actinomycetes residing in the healthy tissues of plants posses the ability to supply sustainable options for agriculture. The identification and characterization of these endophytic actinomycetes having in vitro PGPR traits from medicinal plants help us understand the behavior of actinomycetes in the endosphere of plants while identifying potential strains to improve the growth of agricultural plants affected by fungal phytopathogens.