# Microbial Flora Associated with the Halophyte–*Salsola imbricate* and Its Biotechnical Potential

Fehmida Bibi 1 \*, Gary A. Strobel 2 , Muhammad I. Naseer 3 , Muhammad Yasir 1 , Ahmed A. Khalaf Al-Ghamdi 4 and Esam I. Azhar 1,4

*1 Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia, 2 Department of plant sciences, Montana State University, Bozeman, MT, United States, 3 Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia, 4 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia*

#### *Edited by:*

*Iftikhar Ahmed, Institute of Microbial Culture Collection of Pakistan, National Agricultural Research Centre, Pakistan*

#### *Reviewed by:*

*Samina Mehnaz, Forman Christian College, Pakistan Yuki Chan, University of Hong Kong, Hong Kong Kauser Abdulla Malik, Forman Christian College, Pakistan*

> *\*Correspondence: Fehmida Bibi<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology*

*Received: 16 November 2017 Accepted: 11 January 2018 Published: 31 January 2018*

#### *Citation:*

*Bibi F, Strobel GA, Naseer MI, Yasir M, Khalaf Al-Ghamdi AA and Azhar EI (2018) Microbial Flora Associated with the Halophyte–Salsola imbricate and Its Biotechnical Potential. Front. Microbiol. 9:65. doi: 10.3389/fmicb.2018.00065* Halophytes are associated with the intertidal forest ecosystem of Saudi Arabia and seemingly have an immense potential for yielding useful and important natural products. In this study we have aimed to isolate and characterize the endophytic and rhizospheric bacterial communities from the halophyte, *Salsola imbricata,* In addition these bacterial strains were identified and selected strains were further studied for bioactive secondary metabolites. At least 168 rhizspheric and endophytic bacteria were isolated and of these 22 were active antagonists against the oomycetous fungal plant pathogens, *Phytophthora capsici* and *Pythium ultimum*. Active cultures were mainly identified with molecular techniques (16S r DNA) and this revealed 95.7–100% sequence similarities with relevant type strains. These microorgansims were grouped into four major classes: *Actinobacteria, Firmicutes*, β*-Proteobacteria,* and γ *-Proteobacteria*. Production of fungal cell wall lytic enzymes was detected mostly in members of *Actinobacteria* and *Firmicutes*. PCR screening for type I polyketide synthases (PKS-I), type II polyketide synthases (PKS-II) and nonribosomal peptide synthetases (NRPS) revealed 13 of the 22 strains (59%) were positive for at least one of these important biosynthetic genes that are known to be involved in the synthesis of important antibiotics. Four bacterial strains of *Actinobacteria* with potential antagonistic activity including two rhizobacteria, EA52 (*Nocardiopsis* sp.), EA58 (*Pseudonocardia* sp.) and *two* endophytic bacteria *Streptomyces sp.* (EA65) and *Streptomyces* sp. (EA67) were selected for secondary metabolite analyses using LC-MS. As a result, the presence of different bioactive compounds in the culture extracts was detected some of which are already reported for their diverse biological activities including antibiotics such as Sulfamethoxypyridazine, Sulfamerazine, and Dimetridazole. In conclusion, this study provides an insight into antagonistic bacterial population especially the *Actinobacteria* from *S. imbricata*, producing antifungal metabolites of medical significance and characterized taxonomically in future.

Keywords: halophyte, antifungal activity, enzyme production, 16S rRNA gene sequence, LC/MS analyses

# INTRODUCTION

Coastal dune plants consist of shrubs that are associated with tropical and subtropical coastal areas. This ecosystem is considered as a low nutrient environment due to the sparseness of nitrogen and phosphorus (Frosini et al., 2012). Coastal dune areas of the world usually represent a unique flora as a result of variations in salinity, anaerobicity, and temperature fluctuations during different seasons. All of these conditions make this habitat unusual and thus may have been selective for the presence of truly unique microorganisms having a rich biodiversity. Under these conditions microorganisms associated with these costal dune plants play a role in their survival and perform diverse biological functions in plants (Del Vecchio et al., 2015).

Bacteria associated with Coastal dune plants are responsible for various activities including resistance against different pathogens by producing different bioactive metabolites and plant growth promotion compounds. Bacteria are associated with these plants either as rhizospheric or endophytic forms and use nutrients from plants for their growth (de Melo Pereira et al., 2012). Endophytic bacteria get access to internal parts of plants by producing enzymes to hydrolyze cell wall of plants (Cho et al., 2007). These hydrolytic enzymes are also involved in defense of plants against various bacterial and fungal pathogens (Luo et al., 2013).

Endophytes produce metabolites that possess antimicrobial activities (Strobel, 2003). Due to their beneficial effects and broad range, endophytes have been isolated from plants widely ranging from the poles of the earth to the steaming tropics. Plants provide these microorganisms, particularly endophytic bacteria and fungi, a nutrient-rich environment for their growth and development (Strobel et al., 2004; de Melo Pereira et al., 2012). Endophytic Actinobacteria associated with plant roots or leaves are a source of novel potential bioactive substances. Actinobacteria produce various secondary metabolites including antibiotics, antitumor, immunosuppressive, and enzymes widely used in industrial, agriculture, and pharmaceutical industry (Manivasagan et al., 2014). Recently, various antimicrobial compounds and enzymes have been isolated from marine Actinobacteria (Hong et al., 2009; He et al., 2012). Secondary metabolite salinosporamide A, isolated from a marine Actinobacteria is currently in clinical trials as a cancer treatment (Feling et al., 2003). Several other bioactive compounds have been isolated from different marine Actinobacteria (Fu et al., 2012).

The Genus Salsola is comprised of different halophyte species including Salsola imbricata that inhabit the coastal area of the Thuwal region in Jeddah, Saudi Arabia. Recent studies on S. imbricata reported the presence of triterpenesaponins and a new isorhamnetin from the methanolic extract of this plant (Osman et al., 2016). In a recent study from Saudi Arabia, phytochemical analysis of S. imbricata showed presence of bioactive biphenylpropanoids i.e., biphenylsalsonoid A and B. These two bioactive compounds exhibited antioxidant and antibacterial activity against human pathogenic bacteria (Oueslati et al., 2017). Although the coastal dune environment is an unexplored source of natural compounds with diverse activities there has been little or no work on the bacterial flora of this plant. Based on previous studies, it would appear that certain microbial flora components may be present in this plant that has biotechnical potential. Thus, this study was undertaken to explore potential antifungal microflora of halophyte that may produce products of medicinal, agricultural, and industrial significance.

# MATERIALS AND METHODS

#### Collection of Plant Samples and Isolation of Rhizobacteria and Endophytic Bacteria

The halophyte, S. imbricata was collected from the coastal area of Jeddah, Saudi Arabia (22◦ 15′ 54′′ North, 39◦ 6 ′ 44′′ East). We used soil, roots and leaves of plant sample for the isolation of both rhizopheric and endophytic bacteria and the sample plant were placed in sterile plastic bag at the time of collection. For isolation of bacteria from soil associated with roots they were dipped in sterile distilled water and serial dilutions were made (10−3 , 10−4 , and 10−5 ) in filtered autoclaved sea water (FAS) and spread in triplicate on four different media used for culturing of bacteria. We have used half strength R2A (½ R2A) [0.25 g yeast extract, 0.25 g proteose peptone No. 3 (Difco), 0.25 g casamino acid, 0.25 g dextrose, 0.25 g soluble starch, 0.15 g sodium pyruvate, 0.15 g K2HPO4, 0.03 g MgSO4], half Tryptic soy agar (½ TSA) [Pancreatic Digest of Casein, 7.5g Papain Digest of Soybean, 2.5 g Sodium Chloride, 2.5 g Agar, 15.0 g], marine agar (MA) [Peptone, 5.0 g Yeast Extract, 1.0 g Ferric Citrate, 0.1 g Sodium Chloride, 19.45 g Magnesium Chloride, 8.8 g Sodium Sulfate, 3.24 g Calcium Chloride, 1.8 g Potassium Chloride, 0.55 g Sodium Bicarbonate, 0.16 g Potassium Bromide, 0.08 g Strontium Chloride, 34.0 mg Boric Acid, 22.0 mg Sodium Silicate, 4.0 mg Sodium Fluoride, 2.4 mg Ammonium Nitrate, 1.6 mg Disodium Phosphate, 8.0 mg Agar,15.0 g] and half nutrient agar (½ NA) [Beef Extract, 1.5 g Peptone, 2.5 g Agar, 15.0 g] (Difco Laboratories, Detroit, MI) in 1000 ml filtered Sea water for bacterial culturing. For isolation of endophytic bacteria, roots, and leaves were surface sterilized. Both roots and leaves were washed several times with tap water to remove adhering soil particles and then sterilized by washing with disinfectants as described previously (Bibi et al., 2012). After sterilization, small pieces of sterilized root and leaf segments were ground in FAS using a sterile mortar with pestle. Aliquots were further serially diluted and plated in triplicate on four different media mentioned above. To suppress fungal growth, 50µg/ml cycloheximide was mixed to the medium before pouring. The plates were incubated at 28◦C for 2 weeks for bacterial growth.

#### Isolation of Rhizopheric and Endophytic Actinobacteria

For the isolation of rhizosphere and endophytic Actinobacteria, the same dilutions from soil, roots, and leaves were inoculated onto starch-casein agar (Himedia) in sea water supplemented with cycloheximide and nystatin (50µg/ml). Further, plates were incubated at 28◦C for up to 10-12 days. By checking morphological characteristics, Actinobacteria were selected and subcultured using the same isolation medium. Individual colonies were streaked to check purity of the strains and stored in 15% (v/v) glycerol. All bacterial strains were labeled according to acquisition numbers of the King Abdulaziz University special infectious agents unit and stored at −70 C.

# Screening of Bacteria for Antifungal Activity

All isolated bacteria were screened for their antifungal activity. We used five different fungal pathogens i.e., Phytophthora capsici (P. capsici), Pythium ultimum (Py. ultimum), (obtained in this laboratory from Capsicum annuum and Cucumis sativus respectively), Magnaporthe grisea (KACC 40415), Altenaria malli (KCTC 6972) and Fusarium moniliforme (KCTC 6149) were obtained from Korean Agricultural Culture Collection (KACC) and Korean type culture collection center (KCTC). Antifungal activity was determined by using the cross streak method (Bibi et al., 2012). All isolates were streaked on PDA medium supplemented with ½ R2A in sea water. Each 6 mm mycelial disc of freshly grown fungal pathogen was placed in the center of plate perpendicular to streak of isolates at 4 cm distance from edges of plate and incubated for 4–6 days at 28◦C. Antagonistic strains were checked in duplicate for antagonistic activity evaluated by measuring the inhibition zone of fungal pathogens around each bacterial streak.

### Bacterial DNA Extraction and 16S rRNA Gene Sequencing

Genomic DNA was extracted from the selected antagonistic bacterial isolates using a DNA extraction kit (Thermo Scientific, Waltham, USA). To identify antagonistic bacteria, 16S rRNA gene sequencing was performed. Approximately 1500 bp fragment of the 16S rRNA gene was amplified using bacterial universal primers 27F (5′ -AGAGTTTGATCCTGGCTCAG-3′ ) and 1492R (5′ - GGTTACCTTGTTACGACTT-3′ ), the 16S rRNA gene fragment was amplified under the following PCR conditions: one cycle of 95◦C for 5 min followed by 28 cycles of 95◦C for 1 min, and annealing at 58◦C for 50 s with extension at 72◦C for 50s, and a final extension step at 72◦C for 10 min (Bibi et al., 2012). PCR products were purified using PCR purification kit (Thermo Scientific, Waltham, USA), and sequenced commercially (Macrogen, South Korea). Antagonistic bacteria were identified by blast searches of their 16S rRNA gene sequences obtained using the EzTaxon server (https:// www.ezbiocloud.net) (Kim et al., 2012) to identify antagonistic bacteria. Phylogenetic positions of the antagonistic bacteria were confirmed using CLUSTALX (Thompson et al., 1997) multiple alignments of the bacterial sequences and BioEdit software (Hall, 1999) was used to edit the gaps. The neighbor-joining method in the MEGA6 Programme with bootstrap values based on 1,000 replications was used for construction of the phylogenetic tree based on the 16S rRNA gene sequences (Tamura et al., 2013).

# Evaluation of Hydrolytic Enzymatic Activity

Antagonistic bacterial isolates were further evaluated for their lytic enzyme production. Protease activity was checked using skim milk ½ R2A agar plates. Bacteria exhibiting protease production made clear zone on skim milk agar plates. Amylase production was checked on starch medium. Starch hydrolysis was seen as a clear zone on agar plates by positive strains (Kumar et al., 2012). For lipase activity, tributyrin ½ R2A agar medium was used. After 48 h of incubation at 28◦C a clear zone was detected around bacterial colonies after hydrolysis of tributyrin. To check cellulase activity, CMC agar (carboxy methyl cellulose agar) medium was used. Bacteria were streaked on plates with the respective enzyme medium and incubated at 28◦C for 2 days. Congo red (0.1%) solution was added to plates and put on orbital shaker for 30 min and then rinsed with 1MNaCl (Hendricks et al., 1995). Cellulase activity was observed as a clear zone on CMC agar plates around bacterial colonies.

# Detection of Antimicrobial Genes

It has been well established that many antimicrobial products contain polyketides. Thus, to determine if the antagonistic bacteria might contain such enzymes, a search of the bacterial genome was done to check for the presence of genes coding for these enzymes. To detect biosynthetic genes, polyketide synthetase I (PKS-I) and nonribosomal peptide synthetase (NRPS) genes, the following primer pairs K1F/M6R (5′ - TSAAGTCSAACATCGGBCA-3′ ; 5′ -CGCAGGTTSCSGTAC CAGTA-3′ ) and A3F/A7R (5′GCSTACSYSATSTACACSTCS GG-3′ ; 5′ -SASGTCVCCSGTSCGGTAS-3′ ), respectively were used (Ayuso-Sacido and Genilloud, 2005). Amplification of polyketide synthetase II (PKS-II) gene was performed using primer pair KSα/Kβ (5′ -TSGCSTGCTTGGAYGCSATC-3′ ; 5 ′ -TGGAANCCGCCGAABCCTCT-3′ ) (Metsa-Ketela et al., 1999). PCR reaction mixture (25 µl) contained 12.5 µl of PCR mastermix (promega), 1 µl of each primer, 1 µl of template DNA, and 9.5 µl of Nuclease-Free Water. Amplifications were performed under following PCR conditions: one cycle of 95◦C for 5 min followed by 28 cycles of 95◦C for 1 min, and annealing at 58◦C (K1F/M6R), 56◦C (A3F/A7R) and 62◦C (KSα/Kβ) for 50 s with extension at 72◦C for 50 s, and a final extension step at 72◦C for 10 min. PCR amplification products were then checked by using 0.8% agarose through gel electrophoresis, and bands of 1.2–1.4 kb, 600 bp, and 700–800 bp were detected as products of PKS-I, PKS-II, and NRPS genes, respectively.

#### Optimization of Bacterial Culture Conditions for Production Bioactive Compounds

To optimize culture conditions of selected bacterial strains four different media i.e., ½ R2A broth, ½ TSB, ½NB in sea water and Marine broth in distilled were used for culturing. Bacterial cultures were grown for 72 h and after every 24 h optical density (OD) was checked and antifungal activity was assessed against P. capsici, Py. ultimum using the disc diffusion method. The effect of temperature was optimized within the range of temperatures (20–40◦C) in ½ R2A broth. For pH optimization, different ranges of pH values (5–12) were used for the growth and antifungal compound production in ½ R2A broth.

#### LC-MS Analysis of Bacterial Culture

In order to assess the actual presence of antimicrobial products in bacterial cultures that had been previously screened and shown to be biologically active we performed LC-MS analyses of the bacterial supernatants. A Bacterial culture (5 ml) was placed on −80◦C for 5 min, and then transfer to 37◦C water bath for 5 min and this procedure was repeated 5 times. It was centrifuged at 15,000 g for 10 min and transfer 3 ml supernatant fluid was transferred to a tube. To it was added 12 ml of acetonitrile and vortexed for 30 s. It was centrifuged again at 15,000 g for 10 min and 300 µl supernatant was taken for LC-MS metabolomic analysis. The injection volume was 3 µl and samples were analyzed on an Agilent 6540B TOF/Q-TOF Mass Spectrometer coupled with an Agilent 1290 UPLC and Dual AJS ESI ion source. An ACQUITY UPLC HSS T3 (100 × 2.1mm, 1.8 um) column and pre-column (Phenomenex Security GuardTM ) was used to separate the sample. The column temperature was set to 45◦C and flow rate was 0.5 ml/min. The acquisition range was from 50 m/z to 1500 m/z and the scan rate was 1.00 spec/sec. MS conditions were set as follow: capillary voltage 3500 V, nebulizer pressure 35 psi, drying gas 10 L/min, gas temperature 325◦C, vaporizer 200 V, voltage charge 1000 V; negative-ion mode capillary voltage 3500 V, corona negative 15.0 V, fragmentor 175 V, skimmer1 65.0 V, octopole RF Peak 750 V; positive ion mode capillary voltage 3500 V, corona positive 4.0 V, fragmentor 175 V, skimmer1 65.0 V, and octopole RF Peak 750 V. Raw data was imported to the Agilent MassHunter Qualitative Analysis B.06.00 software. Metabolites were identified by using National Institute of Standards and Technology database (NIST) database. Further bioactive metabolites from other secondary metabolites in culture extract were identified using different public databases such as PubChem (http://pubchem.ncbi.nlm.nih.gov/), ChemSpider (http://www.chemspider.com/), SciFinder (http:// www.cas.org/products/scifinder), and ChEMBL (https://www. ebi.ac.uk/chembl/).

#### Nucleotide Sequence Numbers

Nucleotide sequences of the antagonistic bacteria isolated from the halophyte have been deposited in the GenBank database under accession numbers KY436424–KY436445.

# RESULTS

#### Isolation of Rhizospheric and Endophytic Bacteria

In this study, rhizospheric and endophytic bacteria were isolated from the halophyte, S. imbricata growing in a selected site on the Saudi Arabian peninsula. A total of 168 rhizospheric and endophytic bacteria were isolated from soil, roots, and leaves samples. Various methods were used to acquire these bacteria including the use of four different culturing media. It turns out that both high and low nutrient medium content favored the growth of different groups of bacteria. It has been noted that the ½ R2A in SW is a favorable medium for the isolation of bacteria from the marine environment. Thus, all isolated bacteria culture on different media were sub-cultured on ½ R2A in SW. In total 168 bacteria were isolated from different parts of halophyte and soil as well (**Table 1**). From soil, 85 rhizobacteria were isolated by culturing on different media listed above for isolation. Roots samples used for isolation of endophytic bacteria yielded 48 endophytic bacteria. While from leaves 35 bacteria were recovered. Details of bacteria number their antagonistic activity and dominance of different bacterial phyla is mentioned in **Table 1**.

### Screening of Bacteria against Pathogenic Fungi

All isolated bacteria were tested for their antagonism against plant pathogenic oomycetes. Of the 168 bacteria isolated, 22 (13%) displayed inhibition to both oomycete plant pathogens that was the established bioassay test. The percentage of antagonistic bacteria varied in different parts of halophyte, being highest in roots (n = 9; 5.3%), followed by soil (n = 8; 4.7%), and leaves (n = 5; 2.9%). These antagonistic bacterial isolates were tested further against three other fungal pathogens. Different groups of antagonistic bacteria were identified from S. imbricata, and Actinobacteria was the dominant phylum (**Table 1**. Both rhizospheric and endophytic antagonistic Actinobacteria i.e., Nocardiopsis dassonvillei (EA52), Pseudonocardia sp. (EA58), Arthrobacter sp. (EA59), and Streptomyces sp. (EA64) showed strong inhibition against the oomycetes (**Table 2**). Some of the antagonistic isolates (EA56, EA62, EA68-EA70) showed inhibition against oomycetes but did not show inhibition against other pathogenic fungi used (**Table 2**).

#### Identification and Phylogenetic Analysis of Rhizopheric and Endophytic Antagonistic Bacteria

In total, 22 antagonistic bacteria were identified by using 16S rRNA gene sequence analysis. Result of sequencing showed that eight different genera of bacteria were identified belonging to four major classes: Actinobacteria (n = 15; 68%), Firmicutes (n = 5; 23%), γ -Proteobacteria (n = 1; 4.5%), and β-Proteobacteria (n = 1; 4.5%). Sequence identities of antagonistic bacteria were recorded from 95.7−100% (**Table 1**). Sequencing results indicated Actinobacteria as the dominant phylum and 26% of

TABLE 1 | Distribution of rhizopheric and endophytic antagonistic bacteria isolated from halophyte.

*a Isolation source of rhizopheric and endophytic bacteria.*

*bTotal number of rhizopheric and endophytic bacteria isolated from soil, roots and leaves of halophyte.*

*cTotal number of antagonistic rhizopheric and endophytic bacteria.*

*dPercentage of antagonistic rhizopheric and endophytic bacteria.*

*eDominant phylum in all rhizopheric and endophytic antagonistic bacteria.*

+ + +*, between 7 and 9 mm;* + + ++*, between 10 and 12.*

the antagonistic isolates belonged to the genus Streptomyces (**Table 1**). Using 16S rRNA gene data, a neighbor Joining (NJ) phylogenetic tree was constructed (**Figure 1**). High bootstrap values were recovered in phylogenetic tree. A separate cluster has been generated for isolates of class Actinobacteria. Antagonistic strains in this cluster were identified with high bootstraps values (51–100%). Representatives of this class belong to four different genera i.e., Nocardioides, Nocardiopsis, Arthrobacter, and Streptomyces. One strain of β-Proteobacteria was identified with separate cluster also showing high bootstrap value (100%). One strain of γ -Proteobacteria made a distinct cluster with closely related type strain Pseudomonas sp. (EA54) with high a bootstrap value of 100 %. Antagonistic bacterial strains of class Firmicutes were placed in separate cluster belonging to the genus Bacillus. In this study, two antagonistic endophytic bacterial strains were also identified showing low 16S rRNA gene sequence similarity (<98%). One strain of Actinobacteria i.e., Streptomyces sp. (EA64) and one strain belonging to Firmicutes i.e., Bacillus sp. (EA68) were identified with similarities of <98% to the closest type strains (**Table 3**). Four antagonistic Actinobacteria were selected for secondary metabolites identification depending on their antagonistic activity against oomycetes.

# Enzymatic Activities of Antagonistic Bacteria

Antagonistic bacteria were evaluated for their ability to produce fungal cell wall lytic enzymes. Protease, amylase, lipase, and cellulase activities of these bacteria were examined (**Table 2**). The number of bacteria exhibiting protease activity (n = 9; 41%) was high as compared to other enzymatic activities. Most of protease producing bacteria were endophytes (n = 6) and recovered from leaves and roots of halophyte. Amylase activity was observed for 7 (32%) antagonistic bacteria. Mostly strains of Actinobacteria showed amylase activity. Endophytic strains of Actinobacteria exhibited high production of amylase. Lipase activity was observed in 7 (32%) antagonistic bacteria. Strains of Actinobacteria and Firmicutes showed high production of protease. Cellulase activity was observed in only 1 (4.5%) bacterial strain. A strain of N. dassonvillei (EA52) exhibited cellulase activity belonged to Actinobacteria.

### Detection of PKS and NRPS Genes

Bacteria possess NRPS/PKS gene clusters especially common in the phyla Proteobacteria, Actinobacteria and Firmicutes. Antagonistic bacterial isolates from this study showed that of

FIGURE 1 | Phylogenetic distribution of antagonistic bacteria isolated from *S.imbricata* on the basis of 16S rRNA gene sequences obtained from bacteria and closely related sequences of the type strains of other species. The phylogenetic relationships were inferred from the 16S rRNA gene sequences (1 kb) by using the neighbor-joining method from distances computed with the Jukes-Cantor algorithm. Bootstrap values (1,000 replicates) are shown next to the branches. GenBank accession numbers for each sequence are shown in parentheses. Bar, 0.01 accumulated changes per nucleotide. Isolates selected for bioactive metabolites identification are highlighted.

TABLE 3 | Tentative novel taxa based on partial 16S rRNA gene sequence analyses from this study.

22 isolates, PCR amplification revealed that 13 strains (59%) were positive for at least one of the biosynthetic genes checked (**Table 2**). For the NRPS gene, 8 (36%) strains were positive. NRPS genes were detected in both classes of Actinobacteria and Firmicutes. The PKS-I gene was detected in only 4 (18%) antagonistic bacterial strains. While PKS-II gene was detected in 7 (32%) strains. All three genes PKS-I and PKS-II and NRPS were detected in strain Bacillus sp. (EA72). Three isolates belong to Actinobacteria, Streptomyces enissocaesilis sp. (EA52), Streptomyces sp. (EA63), and Streptomyces sp. (EA64) were positive for the presence of both NRPS and PKS-II.

#### Identification of Strain EA52, EA58 (Rhizobacteria), and EA65, EA67 (Endobacteria) Metabolites by LC-MS

On the basis of high inhibition activity against fungal pathogens we have selected four antagonistic bacterial strains from our study to identify secondary metabolites. These four antagonistic isolates belong to class Actinobacteria i.e., Nocardiopsis sp. (EA52) and Pseudonocardia sp. (EA58) were rhizobacteria while Streptomyces sp. (EA65) and Streptomyces sp. (EA67) are endophytes. Production of antifungal compounds was influenced by culturing in R2A as culture medium using optimum culture conditions. The maximum antifungal activity was observed after 48 h of growth at 28◦C with pH 7.5. Metabolites in cultures extract of selected bacteria were analyzed using the LC-MS. LC-MS technique confirms presence of various bioactive compounds although not novel but already known for their bioactivity. Identification of metabolites was determined by LC-MS analysis and comparing results from NIST database. LC-MS analysis of culture extract of these selected strains identified different bioactive metabolites (**Table 4**). Strain of Nocardiopsis sp. (EA52) produced 7 identified peaks of different bioactive metabolites in both the positive- and negative-ion mode (**Figures 2A,B**). These compounds include Thiabendazole, Sulfamethoxypyridazine, N-Nitrosodiethylamine, Ibuprofen, Laberalol, Isoxsuprine, and Oxibendazole. For Pseudonocardia carboxydivorans (EA58), 2 bioactive compounds have been detected among hundreds of peaks known for other different metabolites. These three compounds include Sulfamethoxypyridazine and Dimetridazole (**Figures 2C,D**). Strain EA65 genetically closely associated as a Streptomyces diastatochromogenes showed presence of nine different bioactive compounds in the culture extract i.e., Sulfamethoxypyridazine, Dimetridazole, Salbutero, Moxidectin, Atenolol, Timolol, Benzydamine, Acebutolol, and Bambuterol (**Figures 2E,F**). These bioactive secondary metabolites are already known for their biological activities. LC-MS analysis of strain EA67, Streptomyces europaeiscabiei showed the presence of six bioactive compounds among several compounds detected. These bioactive compounds include, Sulfamethoxypyridazine, Benzamide, Sulfamerazine, Dimetridazole, Metronidazole-oh, and Carazolol (**Figures 2G,H**). These compounds are already known for antimicrobial, antiphytopathogenic and biocontrol properties.

#### DISCUSSION

There is a need for the discovery of new drugs due to the emergence of resistant bacteria to different antibiotics and cancer cells to anticancer drugs. Currently, marine habitats have been identified as a source of discovering many important novel therapeutic agents (Malve, 2016). S.imbricata, is a halophyte containing various chemical compounds of medicinal use and it is widely spread in the sea shore areas in Saudi Arabia. Generally halophytes with traditional health benefits in folk medicine are a source of attraction for modern comprehensive microbial and chemical investigations. In our previous study (Bibi et al., 2017) we have isolated two rhizobacteria from Salsola exhibiting cellulase and amylase activity on culturing media. One strain EA151 with cellulase activity belonged to Firmicutes while other strain EA152 exhibited amylase activity closely related to type strain of Actinobacteria.

In this study, rhizopheric and endophytic bacterial populations with potent antagonistic activity against phytopathogens were studied. One hundred and sixty eight rhizopheric and endophytic bacteria were isolated and only 22 were found to be potent antagonists of oomycetes. Different cultivation media were used to culture bacteria to isolate novel isolates with unique bioactivities. A study has reported that high nutrient medium favors growth of diverse groups of bacteria, while low nutrient media increase number of bacteria (Vishnivetskaya et al., 2000). To our knowledge, this work is the first study on the isolation, screening, characterization and metabolites identification of both rhizospheric and endophytic bacteria from S. imbricata. In this study, culture-dependent approach was used to study the diversity of antagonistic bacteria

from halophyte against oomycetes plant pathogens. Screening of 168 rhizospheric and endophytic bacteria and their 16S rRNA gene sequencing resulted in 22 different bacteria belong to four major classes. Bioactive compounds production by rhizo and endophytic bacteria is one of the phenomenon used by bacteria to defend the host against different pathogens.

Antagonistic bacteria belong to four major classes, Firmicutes, γ -Proteobacteria, and β-Proteobacteria. Our results show that Actinobacteria is recognized as a dominant phylum among these antagonitistic bacteria isolated from halophyte (**Table 1**). Actinobacteria are distributed in various ecological habitats especially in terrestrial and aquatic ecosystems where they play a potential role in decomposition of plants and animals remains. They are biotechnologically important due to their ability to produce secondary metabolites (Berdy, 2005). Therefore, we have selected four different strains of Actinobacteria for further study of bioactive metabolites produced by these antagonistic rhizopheric and endophytic bacteria. Results of 16S rRNA sequence analyses showed a diversity of different genera of Actinobacteria and the genus Streptomyces remain which are dominant in our study. Results of this study are also compatible with previous studies where many novel Actinobacteria have been recovered from different unexplored marine sources (Fiedler et al., 2005; Bull and Stach, 2007). Halophytes growing near the sea shore in tropical and subtropical areas have become valuable sources for the isolation of microbial flora especially in the group Actinobacteria because of the chemodiversity of environmental factors prevailing there. The presence of major numbers of Streptomyces strains amongst antagonists from the halophyte in this study are in accordance with the results reported

previously (Eccleston et al., 2008; Ravikumar et al., 2012). In Actinibacteria, the most active bacterial strains in addition to Streptomyces are, Nocardiopsis, Pseudonocardia, and Arthrobacter which have been reported for their inhibitory activities and production of antimicrobial metabolites (Anandan et al., 2016).

The metabolic diversity of Streptomycetes is remarkable and novel strains with new bioactive compounds including antibiotics have been isolated from extreme habitats (Fiedler et al., 2005). Until now, several studies have reported diverse groups of antimicrobial compounds by bacteria isolated from halophytic endophytes, such as steroids, peptides, alkaloids, terpenoids, quinines, flavonoids, and phenols (Newman and Cragg, 2007). Our results showed that most of the endophytes in this study belonging to Streptomyces and were recovered from root samples. This finding is supported by previous results where maximum number of endophytes were recovered from roots specimens (Taechowisan et al., 2003; Passari et al., 2015) This may be due to the reason that the rhizosphere has a high concentration of nutrients and various bacteria enter different root tissues of the host plant through the rhizosphere and become endophytic in their mode of life (Rosenblueth and Martinez-Romero, 2006).

The second dominant class of bacteria in this study was Firmicutes. The group of bacteria belonging to the class Firmicutes are commonly found as they are easily culturable. Also, from marine sources, species of Bacillus are dominant for the production of antibacterial, antifungal antibiotics, surfactants, and lytic enzymes (Mondol et al., 2013). Endophytic bacterial strains of Bacillus from halophytes are already reported to show inhibition against bacterial and fungal plant pathogens (Menpara and Chanda, 2013). In this study, only two antagonistic rhizopheric and endophytic bacteria were related to the class Proteobacteria belonging to two different classes γ -Proteobacteria and β-Proteobacteria. Members of class γ - Protobacteria from marine source produce the highest number of secondary metabolites (Long and Azam, 2001).

Hydrolytic enzymes from marine bacteria are produced and used by them in catalyzing different biochemical processes in the marine environment (Thatoi et al., 2013). In addition to production of useful secondary metabolites and antibiotics Actinobacteria from marine sources are also known for the production of lytic enzymes of industrial use (Anandan et al., 2016). In this study, mostly strains of Actinobacteria and Firmicutes were active candidates for production of different enzymes. Protease, lipase and amylase activity was mostly observed (**Table 2**). Only one strain EA59 exhibited protease, amylase and lipase activity. This strain is endophytic and belongs to Actinobacteria. It seems as if the productions of these hydrolytic enzymes is necessary for antagonism as well as for intracellular colonization of bacteria in the host plant. Only four strain of rhizobacteria belong to Actinobacteria were able to produce at least one type of enzyme tested in this study. While most of the endophytic bacteria were able to show enzymatic activities belong to Actinobacteria. Marine Actinobacteria have the potential to produce extracellular enzymes of industrials use. These extracellular enzymes are usually used by endophytic bacteria for their antagonism against different pathogens invading host plants (Zhao et al., 2016).

(H) positive mode LC/MS analysis.

mode LC/MS analysis of *Streptomyces* sp. (EA65) (F) positive mode LC/MS analysis (G) negative mode LC/MS analysis of *Streptomyces* sp. (EA67)

Polyketide synthetase (PKS) and nonribosomal polyketide synthetase (NRPS) genes are considered as hallmarks for the production of secondary metabolites. These biosynthetic genes of bacterial secondary metabolites are involved in the biosynthesis of many bioactive products. The presence of the biosynthetic genes PKS and NRPS in rhizospheric and endophytic bacteria associated with this halophyte suggests that these beneficial bacteria are dominant producers of bioactive natural products. Amplification of these three biosynthetic gene clusters domains is an indirect approach to test the knowledge that bioactive products are being produced by the organism. Of 22 isolates screened for detection of PKS-I, PKS-II, and NRPS gene, only 13 strains were positive for at least one of the biosynthetic genes tested. Several species of Streptomyces possess two types of PKSs. One is responsible for antibiotic synthesis, while the other is involved in spore pigments production in various Streptomyces species (Bergh and Uhlén, 1992). In this study, PKS-II and NRPS were detected in most of the Streptomyces. Among them, strains EA52, EA61, EA63, and EA64 were positive for the presence of NRPS and PKS-II, and also exhibited antifungal activity against tested pathogens (**Table 2**). While strains EA53, EA56, EA59, EA60, EA62, EA68-EA71 were negative for the presence of PKS-I, PKS-II, and NRPS genes and positive for antifungal activity. Therefore, there is no correlation between presence of PKS and NRPS gene and antimicrobial properties (Qin et al., 2009). It is evident that NRPS PKS-I and PKS-II gene screening using PCR in different bacterial strain help to find out potential strain of bacteria producing important secondary metabolites.

Strains of Streptomycetes are active producers of antimicrobial compounds and more than 70% of antibiotics in the world are produced by them. Endophytic Streptomycetes occupy a biologically important niche in tissues of the host plant, taking nutrition from host and in turn providing protection to the plant. These endophytes produce important metabolites that are not toxic to their host therefore, can be considered as important bioactive metabolites in drug discovery (Castillo et al., 2007). Selection of Actinobacteria for identification of active secondary metabolites resulted in a number of known synthetic bioactive compounds not of microbial origin (**Table 4**). Based upon antifungal activity and low 16S rRNA gene similarity four out of twenty-two rhizopheric and endophytic strains were ultimately selected for LC-MS analyses. Production of antifungal compounds was influenced by culturing the organism in R2A as a culture medium using optimum culture conditions. The maximum antifungal activity was observed after 48 h of growth with pH 7.5 at 28◦C. Metabolites in cultures extracts of selected bacteria were analyzed using LC-MS. Two rhizopheric strains namely EA52 closely related to Nocardiopsis sp. and EA58 showed similarity with Pseudonocardia sp. in producing diverse metabolites including Thiabendazole, Sulfamethoxypyridazine, N-Nitrosodiethylamine, Ibuprofen, Laberalol, Isoxsuprine, Oxibendazole, and Dimetridazole. Strain EA52 showed close similarity to Nocardiopsis sp. This marine non-Streptomycete genus of Actinobacteria, contained a variety of bioactive compounds and showed a wide range of activities including cytotoxicity, antibacterial, antifungal, and anti-angiogenesis. Nocardiopsis sp. contained α-pyrone (nocapyrone S1) and (4-aminophenyl) acetic acid, N-(2- hydroxyphenyl)-acetamide, cyclo-(L-Pro-L-Val), cyclo- (L-Pro-L-Leu), and cyclo-(L-Pro-L-Ile) when evaluated for secondary metabolite production (Zou et al., 2017) but different secondary metabolites have been detected in closely related strain EA58 in the present study (**Figures 2A,B**). Antimicrobial activity of strains of Pseudonocardia is already evident from previous studies but no such antifungal activity and metabolites detected in our study were produced from the closely related strain EA58 (**Figures 2C,D**; Tanvir et al., 2016).

Two endophytic Streptomycetes EA65 and EA67 were analyzed by LC-MS and they exhibited spectra for 15 bioactive compounds including antibacterial, antifungal, antiprotozoal and anthelmintic compounds. Strain EA65 showed close similarity at the 97% level to a rhizobacterium S. diastatochromogenes, a promising source of antifungal metabolites and novel natural antibiotics and used as biocontrol agents (Shentu et al., 2016). S. diastatochromogenes reportedly produces polyketomycin, momofulvenoneA, azdimycin, toyocamycin, concanamycin, and oligomycins. Strain EA65 exhibited the presence of bioactive compounds not reported in S. diastatochromogenes including Sulfamethoxypyridazine, Dimetridazole, Salbutero, Moxidectin, Atenolol, Timolol, Benzydamine, Acebutolol, and Bambuterol (**Figures 2E,F**). Another endophytic strain EA67 related to S. europaeiscabiei showed the presence of bioactive compounds such as Sulfamethoxypyridazine, Benzamide, Sulfamerazine, Dimetridazole, Metronidazole-oh, and Carazolol (**Figures 2G,H**). These compounds are already known for antimicrobial, antiphytopathogenic and biocontrol properties. Sulfamethoxypyridazine, Nitroimidazole including Dimetridazole and Metronidazole-oh and Moxidectin are major antimicrobial compounds detected in all four strains in this study. Sulfamethoxypyridazine was detected in all four strains in our study. These antibacterial sulfonamides are synthetic antimicrobial agents used as antibiotics in different inflammatory diseases (Vicente and Pérez-Trallero, 2010). This is first study showing the isolation and screening of antagonistic bacteria from S. imbricata and the identification of active metabolites from crude extracts. Compounds produced by these strains although not new, have been not reported in literature from other organisms and most of them are synthetic so this is the first reported of these Actinobacteria as a natural source for production of these antimicrobial compounds.

In conclusions, present study of antagonistic bacteria from S. imbricata exhibited wide range antifungal and enzymatic activities indicating their industrial, biotechnological and agricultural potential. Further analyses of bioactive metabolites from selected Actinobacteria exhibited production of known antibiotics and bioactive compounds of synthetic nature not reported from bacteria before. These results suggest that halophytes are potential source of bioactive metabolites via their associated bacterial microflora. This study points to an enormous potential of discovering novel bioactive compounds using the methodology described. Currently we are working on some selected novel strains for deep bioactive analysis and genome sequencing that will be presented in our future work.

#### AUTHOR CONTRIBUTIONS

FB and GS: designed the study and write manuscript; MN and EA: identified the plant samples and help in collection of sample and experimental work; MY and AK collect and analyzed the data. All authors read and approved the final manuscript.

#### REFERENCES

#### ACKNOWLEDGMENTS

This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH)– King Abdulaziz City for Science and Technology-the Kingdom of Saudi Arabia-award number (12-BIO2724-03). The authors also, acknowledge with thanks Science and Technology Unit, King Abdulaziz University for technical support.

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their bioactivity and metabolites evaluation. Microbiol. Res. 185, 22–35. doi: 10.1016/j.micres.2016.01.003

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

Copyright © 2018 Bibi, Strobel, Naseer, Yasir, Khalaf Al-Ghamdi and Azhar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Isolation, Diversity, and Antimicrobial and Immunomodulatory Activities of Endophytic Actinobacteria From Tea Cultivars Zijuan and Yunkang-10 (Camellia sinensis var. assamica)

Wei Wei1,2, Yu Zhou2 , Fanjie Chen1,2, Xiaomei Yan2 , Yongmin Lai1 , Chaoling Wei2 , Xiaoyun Chen1 , Junfeng Xu1 \* and Xu Wang2 \*

#### Edited by:

Sheng Qin, Jiangsu Normal University, China

#### Reviewed by:

Bao-Zhu Fang, Sun Yat-sen University, China Bhim Pratap Singh, Mizoram University, India Samina Mehnaz, Forman Christian College, Pakistan

#### \*Correspondence:

Junfeng Xu<EMAIL_ADDRESS>Xu Wang<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology

Received: 27 February 2018 Accepted: 29 May 2018 Published: 18 June 2018

#### Citation:

Wei W, Zhou Y, Chen F, Yan X, Lai Y, Wei C, Chen X, Xu J and Wang X (2018) Isolation, Diversity, and Antimicrobial and Immunomodulatory Activities of Endophytic Actinobacteria From Tea Cultivars Zijuan and Yunkang-10 (Camellia sinensis var. assamica). Front. Microbiol. 9:1304. doi: 10.3389/fmicb.2018.01304 1 State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Quality and Standard for Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou, China, 2 State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, China

Endophytic actinobacteria exist widely in plant tissues and are considered as a potential bioresource library of natural products. Tea plants play important roles in human health and in the lifestyles of Asians, especially the Chinese. However, little is known about the endophytic actinobacteria of tea plants. In this study, 16 actinobacteria of 7 different genera and 28 actinobacteria of 8 genera were isolated and analyzed by 16S rRNA gene sequencing from tea cultivars of Zijuan and Yunkang-10 (Camellia sinensis var. assamica), respectively. The diversity of actinobacteria species from Zijuan were higher in July than December (6 vs. 3 genera), but the diversity of species from Yunkang-10 were higher in December than July (7 vs. 3 genera). No actinobacteria isolates were obtained from any tea cultivar in September. Ten isolates from Yunkang-10 exhibited antimicrobial activity against at least one human pathogenic microorganism (Staphylococcus epidermidis, Shigella flexneri, and Escherichia coli), but none of the isolates from Zijuan exhibited antimicrobial activities. Fourteen strains were further exammined the genes of polyketide synthetase (PKS)-I and PKS-II and non-ribosomal peptide synthetase (NRPS). Brevibacterium sp. YXT131 from Yunkang-10 showed strong inhibitory activity against S. epidermidis, Sh. flexneri, and E. coli, and PKS-I and PKS-II and NRPS genes were obtained from the strain. In in vitro assays, extracts from 14 actinobacteria that were tested for antibiotic biosynthetic genes showed no inhibition of concanavalin A (ConA)-induced murine splenocyte proliferation. In in vivo assays, the crude extract of YXT131 modulated the immune response by decreasing the proinflammatory cytokines interleukin (IL)-12/IL-23 p40 and tumor necrosis factor (TNF)-α in the serum of mice. These results confirm that endophytic actinobacteria from tea plants might be an undeveloped bioresource library for active compounds.

Keywords: Camellia sinensis, endophytic actinobacteria, diversity, antimicrobial activity, immunomodulatory activities

# INTRODUCTION

fmicb-09-01304 June 14, 2018 Time: 17:48 # 2

Actinobacteria are aerobic, gram-positive bacteria and are wellknown producers of a vast array of secondary metabolites, including antibiotics, immunosuppressive agents, antitumor agents, and enzymes, many of which are of great importance to the pharmaceutical and agricultural industries (Saini et al., 2015; Landwehr et al., 2016; Salcedo et al., 2016; Yang et al., 2016). Endophytes are microorganisms that ubiquitously colonize the internal tissues of plants without causing any negative effects, and some endophytes are able to control plant pathogens and promote the growth of plants (Santoyo et al., 2016; Kandel et al., 2017). Although numerous species of actinobacteria occur in the soil, other microbial habitats, such as leaf litter and plants, are potential sources of actinobacteria for the isolation of biologically active compounds (Sardi et al., 1992; Takahashi and Omura, 2003; Gos et al., 2017). In recent years, endophytic actinobacteria have been isolated from many crop plants (such as wheat, rice, and potatoes) (Coombs and Franco, 2003; Sessitsch et al., 2004; Tian et al., 2007) and medicinal plants (Qin et al., 2009; Gos et al., 2017). Endophytic actinobacteria are a potential source for the production of secondary metabolites that are used in the direct antagonism of pests and diseases (Cao et al., 2005) as well as various natural products with antimicrobial, antitumor, and antiinfection activities (Qin et al., 2011; Gos et al., 2017). Endophytic actinobacteria can also confer salt tolerance to host plants and promote host-plant growth (Qin et al., 2017).

As a popular non-alcoholic beverage, tea and tea drinks play important roles in human health and lifestyle, such as by reducing cardiovascular mortality and treating digestive disorders (Yang C.S. et al., 2009; Persson et al., 2010; Begas et al., 2017). As the same plant species, the tea cultivars of Zijuan and Yunkang-10 belong to the taxonomic species of Camellia sinensis var. assamica, which originated from the Yunnan province of China (Wang et al., 2017; Zhu et al., 2017). The two closely related cultivars of Zijuan and Yunkang-10 are the major raw materials for Pu'er tea (a kind of dark tea) produced in Yunnan. Catechins are the major secondary metabolic products in tea leaves (especially in green tea), and polyphenols are known to confer health benefits (Khan and Mukhtar, 2007). Anthocyanin is another functional flavonoid with health benefits and is widely distributed in higher plants (e.g., vegetables, flowers, fruits, cereals, and tea plants) (Joshi et al., 2015; Fabroni et al., 2016; Chen et al., 2017). The total anthocyanin content in Zijuan has been reported to be almost three times that found in other Chinese tea cultivars (including Yunkang-10), and thus Zijuan is considered to be an anthocyanin-rich cultivar (Yang X.R. et al., 2009). However, only a few studies have observed on tea plant endophytes, and those studies have mainly been performed on endophytic fungi and for plant disease protection (Rabha et al., 2014). For example, the endophytic fungus Colletotrichum gloeosporioides from healthy tea plant leaves shows strong inhibitory activity against tea pathogens of Pestalotiopsis theae and Colletotrichum camelliae; the inhibitory factors may be the highly efficient fungal chitinase and protease (Rabha et al., 2014). Compared to endophytic fungi, endophytic actinobacteria have received almost no attention in tea plants, and the interactions between endophytic actinobacteria and tea plants have not been investigated. Based on recent research progress in other plants, the study of endophytic actinobacteria culture and bioactivity is of great significance for developing bioresource libraries. In this study, the endophytic actinobacteria from tea plants of Zijuan and Yunkang-10 were isolated at different seasons, and the bioactivities of antimicrobial and immunomodulatory were screened and further evaluated for the isolates.

#### MATERIALS AND METHODS

#### Tea Sample Collection

Three-year-old tea plants of Zijuan and Yunkang-10 (Camellia sinensis var. assamica) were obtained from Dechang tea plantation (Shucheng China, 31◦ 11' N, 116◦ 47' E). The leaf samples of Zijuan and Yunkang-10 were collected on July 5, September 10, and December 5 of 2015. At the each time of sampling, 15 branches with the same growth and no pest damage were selected for each tea cultivar (considered as 1 sample). The healthy branches were placed in fully soaked floral foam and transported to the laboratory within 2 h for actinobacteria isolation; the isolation procedures were performed within 96 h.

#### Endophytic Actinobacteria Isolation

The leaf samples were pretreated following the method described by Qin et al. (2009) with minor modifications. The samples were air-dried for 48 h at room temperature and then washed with ultrasonic cleaning (160 W, 15 min). After drying, the samples were sterilized in the following order: a 6-min wash in 5% NaOCl, followed by a 10-min wash in 2.5% Na2S2O3, a 5-min wash in 70% ethanol, a 5-min wash in sterile water, and a final rinse in 10% NaHCO3 for 10 min. The sterilized tissues were imprinted on nutrient agar (NA, Difco) and tryptic soy agar (TSA, Difco), and incubated at 28◦C for 2 weeks to ensure the sterilization effectiveness. After surface sterilization and thorough drying under aseptic conditions, the samples were cut up in a sterile mortar and ground to a homogenate, followed by dilution to 10−1 to 10−3 with sterile water. Aliquots of 200 µL of the dilutions were spread-plated onto a series of isolation media as indicated in **Table 1** and incubated at 28◦C for 2–3 weeks for actinobacteria cultivation. The pH of the selected media was adjusted to 7.2. Each isolation medium was amended with nalidixic acid (50 mg/L) and nystatin (100 mg/L) to prevent the growth of gram-negative bacteria and fungi. As colonies appeared on the plates, candidate colonies were observed and selected carefully according to phenotypic characteristics.

#### DNA Extraction, 16S rRNA Gene Sequencing, and Phylogenetic Analysis

The obtained isolates were subjected to 16S rRNA gene sequence analysis for genus and species identification. The genomic DNA was extracted using the method of Li et al. (2007). The 16S rRNA gene of each isolate was amplified using primer pairs 27F and 1492R (**Table 2**), and polymerase chain reaction (PCR) amplification was carried out as described by Li et al. (2007).

TABLE 1 | Culture medium composition for endophytic actinobacteria isolation.

TABLE 2 | Polymerase chain reaction (PCR) primers used in this study.

The reagents for PCR reaction were purchased from TaKaRa (Dalian, China). The PCR -products were separated by agarose gel electrophoresis, purified using QIA quick gel extraction kits (Qiagen, Hilden, Germany), then ligated into a pMD-19T vector (TaKaRa). Positive clones were screened further, and insert DNA sequencing was performed by Invitrogen (Shanghai, China) on an Applied Biosystems PRISM 3730 DNA sequencer.

The 16S rRNA gene analysis was performed by BLAST searches in the National Center for Biotechnology Information database1 and EzBioCloud2 . Multiple sequence alignment of selected 16S rDNA sequence was carried out using CLUSTAL\_X (version 2.0) (Thompson et al., 1997), and a phylogenetic tree was constructed using MEGA v6.0 (Tamura et al., 2013). Distances (distance options according to the Kimura two-parameter model) (Kimura, 1980) and clustering were based on the neighborjoining (Saitou and Nei, 1987) method. Bootstrap analysis based on 1000 resamplings was used to evaluate the topology of the neighbor-joining tree (Felsenstein, 1985). The 16S rRNA gene sequences of the 44 isolates have been deposited in GenBank under the accession numbers (MH298662–MH298705).

#### Detection of PKS-I, PKS-II, and NRPS Genes

Three sets of degenerate primers for amplification of the genes encoding polyketide synthases I and II (PKS-I and PKS-II) and non-ribosomal peptide synthetase (NRPS) were selected (**Table 2**), and amplification was carried out as recommended by Metsa-Ketela et al. (1999) and Ayuso-Sacido and Genilloud (2005). The reaction mixture contained 2.5 U of Taq DNA polymerase, 1 mM MgCl2, 0.4 mM deoxynucleoside triphosphates, 2 µM each primer, and 5% dimethyl sulfoxide in a 50-µL reaction volume. A reaction mixture with no actinobacterial DNA template was used as a negative control. Thermocycling conditions consisted of one denaturation step of 94◦C for 5 min, 30 amplification cycles of 94◦C for 1 min, 57◦C (for K1F–M6R and A3F–A7R) or 58◦C (for KSα–KSβ) for 1 min, and 72◦C for 2 min; and a final extension at 72◦C for 5 min.

#### Active Compound Extraction and Bioactivity Evaluation

The endophytic isolates were cultured in GAUZE's liquid medium at 28◦C and 180 r/min (Rehacek, 1959). After 7– 12 days of cultivation, the 100 mL culture broth was collected by centrifugation at 12,000 × g for 10 min and extracted by 100 mL ethyl acetate for three times. The organic phase was evaporated under reduced pressure to yield a dry extract. The dry extract was resuspended by 5 mL sterile water and used for antimicrobial screening. The antimicrobial susceptibility was examined by placing antimicrobial testing disks (7 mm diameter) containing 25 µL test extract suspension onto LB plates (Mearns-Spragg et al., 1998). The tested plates were incubated at 37◦C, and the diameters of the inhibition zones were measured after 24 h. A 25-µL volume of sterile water was used as a negative control. The pathogenic bacteria Staphylococcus epidermidis, Shigella flexneri, Escherichia coli, and Bacillus cereus were used as the indicator microorganisms for antimicrobial determination. The pathogenic microorganisms were obtained from the Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences.

1https://blast.ncbi.nlm.nih.gov/Blast.cgi

2https://www.ezbiocloud.net/

# Animal Experiments and Physiological Tests

Female BALB/c mice at 8–9 weeks of age were purchased from the Zhejiang Laboratory Animal Center (Hangzhou, China). The mice were maintained in pathogen-free conditions with standard laboratory chow and water ad libitum. Animal experiments were approved and performed in accordance with the guidelines of the Animal Care Committee of Zhejiang province, China. Dried extracts from the test endophytic actinobacteria were resuspended by saline with concentration of 2 mg/mL and injected i.p. 100 µL per mouse once daily for 2 weeks. The same volume (100 µL per mouse) of saline was given as a vehicle control. Three independent experiments were repeated, and in each experiment five mice were used. Clinical signs of poisoning were assessed and the weights of mice were recorded daily. Blood samples (∼500 µL per mouse) were taken from the retro-orbital venous plexus at the end of the experiment and incubated at 4◦C for 30 min. Serum was collected by centrifugation at 4500 × g for 10 min and stored at −20◦C until analysis. Cytokines [interleukin-2 (IL-2), interleukin-6 (IL-6), the shared p40 subunit of IL-12 and IL-23 (IL-12/IL-23 p40), and tumor necrosis factoralpha (TNF-α)] in the serum were determined with sandwich enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (Dakewe Biotech, Shenzhen, China).

#### Splenocyte Proliferation

Freshly isolated splenocytes were obtained from BALB/c mice and incubated in 200 µL RPMI 1640 with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 5 µg/mL of concanavalin A (ConA) in a humidified, 37◦C, 5% CO2 containing incubator for 48 h in the presence or absence of extracts. Cyclosporin A (CsA) (500 ng/mL) was used as a positive control. Three independent experiments were repeated, and in each experiment five wells of splenocytes were used. RPMI 1640 medium was purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, United States). ConA and CsA were purchased from Sigma (St. Louis, MO, United States). The cell number was determined by a Millipore Guava easyCyte 8HT Flow Cytometer (Millipore, Billerica, MA, United States).

#### Statistical Analysis

Data are presented as means ± standard deviation. Statistical analyzes were performed using Student's t test. P < 0.05 was considered statistically significant.

#### Ethics Statement

This study was carried out in accordance with the guidelines of the Animal Care Committee of Zhejiang province, China (Government Decree No. 263). The protocol was approved by the Committee on the Ethics of Animal Experiments of Zhejiang Academy of Agricultural Science.

# RESULTS

### Evaluation of Surface Sterilization

Surface sterilization is critical for the study of plant endophytic actinobacteria. In this study, the surface-sterilized leaves were examined by NA and TSA, and no microbial colony was observed after 2 weeks of incubation at 28◦C. This indicated that the surface-sterilization protocol modified from Qin et al. (2009) was effective in removing phyllospheric microorganisms of tea plants.

# Selective Isolation of Culturable Endophytic Actinobacteria From Zijuan and Yunkang-10

To obtain as many endophytic actinobacteria as possible, five selective isolation media were used simultaneously in this study (**Table 1**). Endophytic actinobacteria were isolated on all of five media. In total, 44 actinobacterial strains (28 from Yunkang-10 and 16 from Zijuan) were isolated from 3 samples of Zijuan and 3 samples of Yunkang-10 (**Table 3**).

### Diversity of Endophytic Actinobacteria Analyzed by 16S rRNA Gene Sequencing

The endophytic actinobacteria obtained from Zijuan were distributed among 7 genera [i.e., Brachybacterium sp. (3 isolates), Brevibacterium sp. (1 isolate), Kocuria sp. (4 isolates), Leucobacter sp. (1 isolate), Micrococcus sp. (4 isolates), Microbacterium sp. (1 isolate), and Streptomyces sp. (2 isolates)] within the class actinobacteria (**Figure 1**). Among them, Brachybacterium sp. and Kocuria sp. were two mutual groups both isolated in July and December, while the others were only isolated in July or December, and no endophytic actinobacteria were obtained in September. The diversity of endophytic actinobacteria from the Zijuan cultivar was in the order of July (6 genera) > December (3 genera) > September (0 genera). The 28 endophytic actinobacteria isolated from Yunkan-10 were distributed among 8 genera [i.e., Brevibacterium sp. (10 isolates), Micrococcus sp. (3 isolates), Mycobacterium sp. (3 isolates), Pseudarthrobacter sp. (1 isolate), Brachybacterium sp. (1 isolate), Kocuria sp. (4 isolates), Microbacterium sp. (5 isolates), and Saccharomonospora sp. (1 isolate)] within the class actinobacteria (**Figure 1**). Isolates of Brevibacterium sp. and Micrococcus sp. were obtained from both July and December specimens, and isolates of Mycobacterium sp. were found only in July. The isolates from other genera were only obtained in December, while no endophytic actinobacteria were obtained in September. The diversity of endophytic actinobacteria from Yunkang-10 was in the order of December (7 genera) > July (3 genera) > September (0 genera). In comparing the two cultivars, isolates of Leucobacter sp. and Streptomyces sp. were endemic actinobacterial groups to the Zijuan cultivar, while Mycobacterium sp., Pseudarthrobacter sp., and Saccharomonospora sp. were endemic to Yunkang-10.

# Endophyte Antimicrobial Activity and Sequencing of PKS and NRPS Genes

In antimicrobial screening test, several culture media indicated in **Table 1** were preliminary evaluated, and GAUZE's medium

TABLE 3 | The isolated endophytic actinobacteria of Zijuan and Yunkang-10.

was the best one for the majority of actinobacteria isolates from Zijuan and Yunkang-10. The extracts from GAUZE's medium showed no obvious antimicrobial difference from other media, but all the tested strains could be cultured by the GAUZE's medium. The 44 isolates were screened for antimicrobial activities against the pathogenic bacteria S. epidermidis, Sh. flexneri, E. coli, and B. cereus. Ten of the 28 isolates (35.7%) from Yunkang-10 exhibited activity against at least one of the tested pathogenic microorganisms. Surprising, none of the 16 isolates from Zijuan showed obvious antimicrobial activity.

16S rRNA gene sequences.

TABLE 4 | Endophytic actinobacteria isolated from Zijuan and Yunkang-10, and similarity values for 16S rRNA gene sequences.

A total of 14 isolates selected (10 antimicrobial positive strains and four negative strains) were selected for the determination of antibiotic biosynthetic gene sequences of PKS-I, PKS-II, and NRPS by PCR amplification using specific primer sets K1F– M6R, KSα–KSβ, and A3F–A7R, respectively (**Table 4**). As shown by primary screening, the inhibitory effect on Sh. flexneri was the most frequent detected antimicrobial activity in this study. Eight isolates were active against S. epidermidis, and 7 isolates were found to inhibit two or more pathogenic microorganisms. However, none of the isolates of this study exhibited activity

8-YKFG1122, 9-YKFT1130, 10-YKFH1122, 11-YKFG1112, 12-ZFY142, 13-ZFG130, 14-ZJFT1121.

against B. cereus. Two isolates, YXT131 and YKFG1221, which belong to the genus Brevibacterium, appeared to have a broad spectrum of antimicrobial activity (three pathogenic microorganisms). Brevibacterium sp. YXT131 exhibited high inhibitory effects against S. epidermidis, Sh. flexneri, and E. coli (**Figure 2** and **Table 5**).

The PKS-I sequence was detected in 9 isolates (64.3%), while the PKS-II and NRPS sequences were detected in 6 and 4 of the 14 strains (**Table 5** and **Supplementary Figure S1**), respectively. The isolates YXT131 and YKFG1221, which have broad spectrum antimicrobial activity, gave positive amplification products with PKS-I, PKS-II, and NRPS primers. The isolates from Yunkang-10 that exhibited antimicrobial activity against pathogenic microorganisms also gave positive amplification products for at least one of the PKS-I, PKS-II, and NRPS genes. The isolate YKFG1122 neither exhibited antimicrobial activity to the four tested pathogenic microorganisms nor provided any positive amplification products for the three biosynthetic genes. The three antimicrobial negative isolates from Zijuan still provided positive amplification products PKS-I or PKS-II.

#### Immunomodulatory Activity in Selected Actinobacteria

Chattopadhyay et al. (2012) showed that black tea has potential anti-inflammatory and immunomodulatory effects in animal models and in human peripheral mononuclear cells, and actinobacteria can produce secondary metabolites with immunosuppressive activity by suppressing cytokine expression and T cell proliferation (Yang et al., 2016). In in vitro splenocyte proliferation assays, extracts from 14 actinobacteria that were tested for antibiotic biosynthetic genes showed no inhibition of ConA-induced murine splenocyte proliferation (**Figure 3**), indicating that extracts from actinobacteria might not directly affect splenocyte proliferation. In the animal model, six isolates, YFT113, YXN111, YXN112, YXT131, YKFG1221, and YKFT1130, were examined for their potential immunomodulatory activity. Compared with the control group, BALB/c mice treated by fermentation extracts of the six isolates showed no obvious clinical signs of poisoning or other atypical signs throughout the trial. No significant differences in body weight were observed between control and fermentation extract-treated mice during the 2 weeks of testing (data not shown). CD4+ T cells are the key components of the adaptive immune system, and naïve CD4+ T cells can differentiate into effector T helper cell subsets (e.g., Th1, Th2, or Th17) by the coordinated functioning of distinct cytokines, including IL-6, IL-12, IL-23, and IL-2 (Murphy and Stockinger, 2010). TNF-α is a multifunctional cytokine that coordinates tissue homeostasis by regulating cytokine production, cell survival, and cell death (Annibaldi and Meier, 2018). In this study, no significant differences in IL-2 and IL-6 concentrations in serum were observed between control and fermentation extracttreated mice (**Figures 4A,B**). IL-12 and IL-23 are heterodimeric cytokines that share a common p40 subunit. To our surprise, the serum levels of IL-12/IL-23 p40 and TNF-α in Brevibacterium sp. YXT131 fermentation extract-treated mice were significantly lower than in the control group, but other fermentation extracttreated groups showed no significant differences (**Figures 4C,D**). These results indicated that the isolate YXT131 appeared to have immunosuppressive activity.

### DISCUSSION

As described by previous studies, the natural characteristics and planting environment of Zijuan and Yunkang-10 are similar, and the two cultivars both originate from the Yunnan province of China (Wang et al., 2017; Zhu et al., 2017).

TABLE 5 | Antimicrobial activities and PKS/NRPS genes of culturable actinobacteria from Zijuan and Yunkang-10.

aEstimated by measuring the diameter of the clear zone of growth inhibition. Symbols: −, no activity; +, ++, and +++, weak activity, moderate activity, and strong activity, respectively. b+, present; −, absent.

The high anthocyanin content of Zijuan is one of the most important differences between the two cultivars (Yang X.R. et al., 2009). Previous studies have indicated that plants secondary metabolites such as alkaloids, phenolics, and terpenoids can interfere with cancer cells, bacteria, and fungi (Wink et al., 2012), and that anthocyanins act as antimicrobial agents of natural plant origin (Cisowska et al., 2011). The different anthocyanin content of Zijuan and Yunkang-10 may influence the microbial community, diversity and bioactivity of endophytic actinobacteria. In this study, the endophytic actinobacteria from Zijuan and Yunkang-10 were isolated and compared for their diversity and antimicrobial and immunomodulatory activities.

In our study, 44 isolates were isolated in July and December, but none of the endophytic actinobacteria was obtained in September. This result might be an incidental, alternatively, endophytic actinobacteria community in Zijuan and Yunkang-10 changed in September and is unculturable on these media. Many studies indicated that the endophytes of plant tissues [such as maple tree sap (Filteau et al., 2010), the buds of Scots pine trees (Pirttilä et al., 2005) and the grape endosphere (Baldan et al., 2014; Bulgari et al., 2014)] were shown to be sensitive to seasonal changes. Other than culture-dependent method, a variety culture independent methods including T-RFLP, PCR fingerprinting and 16S rRNA specific probes were used to investigate the seasonal community changes, and these results were consistent to the present culture-dependent investigation from tea plant (Shen and Fulthorpe, 2015).

In recent years, the antibiotics abuse has been resulted serious bacterial resistance, and become a heavy threat to public health. For instance, methicillin-resistant staphylococcal infections are an important cause of catheter-associated disease, and 75–90% among hospital isolates are S. epidermidis (Otto, 2009). Antibiotic resistance by Shigella species is also a global issue now (Lampl, 2015). E. coli and B. cereus are abundant in nature, and many factors make them a potential threat for the food industry (Gundogan and Avci, 2014). Endophytic actinobacteria are wellknown producers of a vast array of secondary metabolites, including antibiotics. In our study, all of 44 isolates were screened for antimicrobial activities against S. epidermidis, Sh. flexneri, E. coli, and B. cereus. Ten isolates from Yunkang-10 exhibited antimicrobial activity against at least one of the tested pathogenic microorganisms, but none of the isolates from Zijuan showed obvious activity. The high inhibitory activity and broad antimicrobial spectrum of these tested strains suggested

that the endophytic actinobacteria from tea cultivar Yunkang-10 are potential candidates for novel antimicrobial agents. For PKS-I, PKS-II, and NRPS screening, the antimicrobial activity results and the biosynthetic genes seemed to be positively correlated in isolates from Yunkang-10, but 3 isolates from Zijuan that showed no antimicrobial activity still provided positive amplification products for PKS-I or PKS-II. The 3 isolates from Zijuan that showed negative antimicrobial results in this study might produce the antimicrobial agents to other pathogenic microorganisms. The 6 isolates with a broad spectrum of antimicrobial activity and high inhibitory effects were then selected for potential immunomodulatory activities in in vivo test. The isolate Brevibacterium sp. YXT131, which have broad spectrum antimicrobial activity, gave positive amplification products with PKS-I, PKS-II, and NRPS primers, also exhibited high inhibitory effects of the serum levels of IL-12/IL-23 p40 and TNF-α. These results indicated that endophytic actinobacteria from Yunkang-10 might be an undeveloped bioresource library for active compounds.

#### CONCLUSION

In this study, we found that the endophytic actinobacterial communities in the tea cultivars Zijuan and Yunkang-10 were quite different. The isolates of Leucobacter sp. and Streptomyces sp. were endemic actinobacterial groups for the Zijuan cultivar, while Mycobacterium sp., Pseudarthrobacter sp., and Saccharomonospora sp. were endemic actinobacterial groups for Yunkang-10. Ten of the 28 isolates (35.7%) from Yunkang-10 exhibited activity against at least one of the tested pathogenic microorganisms, but none of the 16 isolates from Zijuan showed obvious antimicrobial activity. Brevibacterium sp. YXT131 and Brevibacterium sp. YKFG1221 from Yunkang-10 appeared to have broad-spectrum antimicrobial activity (against S. epidermidis, Sh. flexneri, and E. coli) and gave positive amplification products for the PKS-I, PKS-II, and NRPS genes. The crude extract from Brevibacterium sp. YXT131 showed no inhibition of ConA-induced splenocyte proliferation but decreased IL-12/IL-23 p40 and TNF-α levels in the serum of

#### REFERENCES

a mouse model, indicating that Brevibacterium sp. YXT131 had immunosuppressive activity. Endophytic actinobacteria from Yunkang-10 might be an undeveloped bioresource library for active compounds.

### AUTHOR CONTRIBUTIONS

JX, XW, and YZ conceived of and designed the experiments. WW, XW, FC, XY, and YL performed the experiments. WW drafted the manuscript. WW, YZ, JX, CW, and XC analyzed the data. JX and XW revised the manuscript. All authors read and approved the final manuscript.

#### FUNDING

This work was supported by grants from the Natural Science Foundation of Anhui Province (1608085QC57), the Natural Science Foundation of Zhejiang Province (LQ17C180001), and the Open Fund of State Key Laboratory of Tea Plant Biology and Utilization (SKLTOF20150102).

#### ACKNOWLEDGMENTS

We thank LetPub for its linguistic assistance during the preparation of this manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.01304/full#supplementary-material

FIGURE S1 | DNA gel electrophoresis of PKS-I, PKS-II, and NRPS. The PCR amplification products were resolved using electrophoresis in 1.5% agarose gels. M-Marker, 1-YFT113, 2-YXN120, 3-YXN111, 4-YXN112, 5-YXT131, 6-YKFG1221, 7-YKFG1121, 8-YKFG1122, 9-YKFT1130, 10-YKFH1122, 11-YKFG1112, 12-ZFY142, 13-ZFG130, 14-ZJFT1121.

oxide in healthy volunteers. Public Health Nutr. 13, 730–737. doi: 10.1017/ S1368980010000170

fmicb-09-01304 June 14, 2018 Time: 17:48 # 10

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

Copyright © 2018 Wei, Zhou, Chen, Yan, Lai, Wei, Chen, Xu and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Actinobacteria Associated With Arbuscular Mycorrhizal Funneliformis mosseae Spores, Taxonomic Characterization and Their Beneficial Traits to Plants: Evidence Obtained From Mung Bean (Vigna radiata) and Thai Jasmine Rice (Oryza sativa)

#### Edited by:

Wen-Jun Li, Sun Yat-sen University, China

#### Reviewed by:

Sheng Qin, Jiangsu Normal University, China Wensheng Xiang, Northeast Agricultural University, China Khan Inam Ullah, Yunnan Institute of Microbiology, China

\*Correspondence:

Wasu Pathom-aree<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology

Received: 29 December 2017 Accepted: 23 May 2018 Published: 11 June 2018

#### Citation:

Lasudee K, Tokuyama S, Lumyong S and Pathom-aree W (2018) Actinobacteria Associated With Arbuscular Mycorrhizal Funneliformis mosseae Spores, Taxonomic Characterization and Their Beneficial Traits to Plants: Evidence Obtained From Mung Bean (Vigna radiata) and Thai Jasmine Rice (Oryza sativa). Front. Microbiol. 9:1247. doi: 10.3389/fmicb.2018.01247 Krisana Lasudee1 , Shinji Tokuyama2 , Saisamorn Lumyong1 and Wasu Pathom-aree1,3 \*

1 Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand, 2 Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan, 3 Center of Excellence in Bioresources for Agriculture, Industry and Medicine, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand

In this study, we report on the isolation of actinobacteria obtained from spores of Funneliformis mosseae and provide evidence for their potential in agricultural uses as plant growth promoters in vitro and in vivo. Actinobacteria were isolated from spores of F. mosseae using the dilution plate technique and media designed for the selective isolation of members of specific actinobacterial taxa. Six strains namely 48, S1, S3, S4, S4-1 and SP, were isolated and identified based on16S rRNA gene sequences. Phylogenetic analysis showed that isolate SP belonged to the genus Pseudonocardia with P. nantongensis KLBMP 1282T as its closest neighbor. The remaining isolates belonged to the genus Streptomyces. Two isolates, 48 and S3 were most closely related to S. thermocarboxydus DSM 44293T . Isolates S4 and S4-1 shared the highest 16S RNA gene similarity with S. pilosus NBRC 127772T . Isolate S1 showed its closest relationship with the type strain of S. spinoverrucosus NBRC14228T . The ability of these isolates to produce indole-3-acetic acid (IAA), siderophores and the ability to solubilize phosphate in vitro were examined. All isolates produced siderophores, four isolates produced IAA and two isolates solubilized inorganic phosphate at varying levels. S. thermocarboxydus isolate S3 showed the highest IAA production with high activities of phosphate solubilization and siderophore production. The inoculation of mung beans (Vigna radiata) with this strain resulted in a significant increase in fresh weight, root length and total length as an effect of IAA production. In an experiment with rice (Oryza sativa), S. thermocarboxydus isolate S3 promoted the growth of rice plants grown in low nutritional soil under induced drought stress. This report supports the view that the inoculation of rice with plant growth promoting actinobacteria mitigates some adverse effects of low nutrient and drought stress on rice.

Keywords: actinobacteria, arbuscular mycorrhizal spore, Funneliformis mosseae, plant growth-promoting activity, indole-3-acetic acid (IAA), rice (Oryza sativa), drought stress, low nutritional soil

# INTRODUCTION

fmicb-09-01247 June 11, 2018 Time: 14:41 # 2

Actinobacteria are Gram-positive mostly filamentous bacteria with high %G+C content in their genomes. They are prolific producers of useful bioactive metabolites with a wide spectrum of uses in particular antibiotics (Bérdy, 2005; Kurtboke, 2012; Tiwari and Gupta, 2012). It is also well accepted that some actinobacteria are considered potential plant growth promoting bacteria (Doumbou et al., 2001; Hameda and Mohammadipanah, 2015; Qin et al., 2017).  The growth promotion of several plants by actinobacteria was reported in peas (Tokala et al., 2002), beans (Nassar et al., 2003), alfalfa (Le et al., 2016), wheat (Sadeghi et al., 2012; Anwar et al., 2016), and rice (Rungin et al., 2012; Gopalakrishnan et al., 2014).

Arbuscular mycorrhizal fungi (AMF) are well-known plant growth promoters and have been used in the agricultural industry worldwide (Smith and Smith, 2011). AMF can only be grown with plants as obligate symbionts (Owen et al., 2015). This symbiosis improves the plant's nutrient uptake and provides AM fungi with carbon sources. Funneliformis is a member of AMF in the family Glomeraceae, which form symbiotic relationships with plant roots. It was formerly known as Glomus mosseae until the genus Funneliformis was established in 2010 with F. mosseae as a type species (Schubler and Walker, 2010). F. mosseae is widely used in horticulture as a bioinoculant (Kruger et al., 2012). This symbiosis improves the plant's nutrient uptake and provides AM fungi with carbon sources. Recently, microorganisms associated with AM fungal spores have been reported, including several bacterial species that are both Gram-negative (Xavier and Germida, 2003; Bharadwaj et al., 2008a,b; Battini et al., 2016; Lasudee et al., 2017) and Gram-positive bacteria including actinobacteria of the genus Streptomyces (Schrey et al., 2012; Mohandas et al., 2013; Poovarasan et al., 2013; Battini et al., 2016). These mycorrhizal associated bacteria have shown interesting properties such as those associated with biocontrol and plant growth promoting activities (Mohandas et al., 2013; Poovarasan et al., 2013; Battini et al., 2016). The beneficial properties of these bacteria are considered attractive for sustainable agricultural practices. However, few research studies have been published on actinobacteria that are associated with mycorrhizal spores. This has led to our interest in mycorrhizal-associated actinobacteria, which may be a good source of novel taxa for the purposes of agricultural bioprospecting. Hence, it is the aim of this study to isolate actinobacteria from F. mosseae spores and to screen for their plant growth promoting properties both in vitro and in planta using mung beans (Vigna radiata) and Thai jasmine rice (Oryza sativa). We have given special attention to testing the ability of these actinobacteria to promote rice growth under drought conditions in soil possessing low nutritional content. Their taxonomic positions were also identified.

# MATERIALS AND METHODS

#### Selective Isolation of Actinobacteria From Funneliformis mosseae Spores

Funneliformis mosseae CMU-RYA08 was used for the isolation of actinobacteria in this study. It was originally isolated from the soil of a Aquilaria crassna plantation in Rayong Province, Thailand (Chaiyasen et al., 2014) and was kindly supplied to us by Dr. Amornrat Chaiyasen, Department of Biology, Faculty of Science, Chiang Mai University. Briefly, spores of F. mosseae were surface sterilized by vortex mixing them sequentially in 500 µl of 2% (v/v) sodium hypochlorite solution for 1 min, followed by 500 µl of 70% (v/v) ethanol for 1 min. After that, spores were washed with 500 µl of distilled water for 1 min (three times) and used for isolation of actinobacteria. The efficiency ofsurface sterilization method was determined by spreading the final washing water onto the nutrient agar as well as the International Streptomyces Project (ISP) medium 2 agar (Shirling and Gottlieb, 1966), and the plates were incubated at 30◦C for 48 h. Twenty surface sterilized spores of F. mosseae were used for each isolation. Aseptically, 20 surface sterilized spores were added to microcentrifuge tubes containing 200 µl of soil extract broth and were ground by sterile micropestle. Soil extract was prepared according to the method of Taylor and Lochhead (1938). One kilogram of garden soil was sterilized in 1 l of tap water for 20 min at 121◦C. The suspension was allowed to settle and the supernatant was filtered through Whatman's No. 41 filter paper. One gram of glucose was added to the filtrate and the pH value was adjusted to 6.8–7.0 prior to being autoclaved. The resultant solution was used as a soil extract broth for enrichment. The resulting spore suspension was enriched by shaking at 120 rpm for 1 h at room temperature. Two hundred microliters of the sample were spread on starch casein agar (Küster and Williams, 1964) and humic acid-vitamin agar (Hayakawa and Nonomura, 1987) supplemented with 25 µl/ml nalidixic acid and 100 µg/ml ketoconazole. Plates were then incubated at 30◦C for up to 4 weeks. All isolates were routinely cultivated on International Streptomyces Project (ISP) medium 2 agar (Shirling and Gottlieb, 1966) at 30◦C and spore suspension was maintained in 20% (w/v) glycerol at −20◦C for long term preservation.

# Molecular Identification by 16S rRNA Gene Sequencing

Biomass for molecular study was prepared by growing the isolates on ISP medium 2 agar for 7 days. Genomic DNA was extracted using FavorPrep Tissue Genomic DNA Extraction Mini Kit (Favogen) according to the manufacturer's protocol. All isolates were subjected to amplification of 16S rRNA gene using universal primer 27F (50 -AGAGTTTGATCMTGGCTCAG-30 ) and 1525R (50 -AAGGAGGTGWTCCARCC-30 ) (Lane, 1991). PCR was carried out in a GeneAmp PCR System 9700 with the following reactions. The initial denaturation step was done at 94◦C for 2 min, followed by 30 cycles of 94◦C for 30 s, 55◦C for 30 s and 72◦C for 30 s and final extensions at 72◦C for 7 min. PCR products of expected size were confirmed by 1% (w/v) agarose gel electrophoresis. The purified PCR products were sequenced

commercially at 1st BASE DNA Sequencing Division, Malaysia. The obtained sequences were compared with related sequences in the EzBioCloud database1 using BLAST program. Phylogenetic analysis based on a neighbor-joining method (Saitou and Nei, 1987) was carried out using MEGA version 5.2 program (Tamura et al., 2011). The resultant tree topology was evaluated by bootstrap analysis (Felsenstein, 1985) of the neighbor-joining data based on 1000 re-sampled datasets

### In Vitro Plant Growth Promoting Potential

#### Indole-3-Acetic Acid (IAA) Production

The production of IAA of all isolates was determined by the colorimetric method as has been described previously (Khamna et al., 2010). Agar plug (5 mm diameter) of actinobacteria grown on ISP medium 2 agar was inoculated into 5 ml of ISP medium 2 broth containing 2 mg ml−1 of L-tryptophan (Gordon and Weber, 1951). Tubes were incubated at 28 ± 0.2◦C with shaking at 110 rpm for 7 days. The supernatant was collected by centrifugation at 11,000 rpm for 5 min. IAA production was detected by mixing one ml of the supernatant with 2 ml of Salkowski's reagent (Glickmann and Dessaux, 1994). The appearance of a pink color after incubation at room temperature for 30 min in the dark was an indicator of IAA production. The optical density was measured at 530 nm using a spectrophotometer (GENESYS TM 20 Visible Spectrophometer, Thermo Fishcer Scientific). The IAA concentration in the culture broth was estimated based on a calibration curve of pure IAA standard. The confirmation of IAA production was also confirmed by TLC analysis with 10 mg/ml IAA standard (Mohite, 2013). In addition, the iaaM, the key IAA biosynthetic gene of the highest IAA producing isolate, was examined by PCR amplification (Lin and Xu, 2013). The amplified gene (1,698 bp) was cloned and sequenced at 1st BASE DNA Sequencing Division, Malaysia. The obtained sequences were compared with related sequences in the GenBank database using BLAST program.

#### Phosphate Solubilization

Phosphate solubilizing activity of all isolates was determined on Pikovskaya (PVK) agar (Pikovskaya, 1948) containing 0.5% (w/v) tricalcium phosphate. The agar plug (5 mm diameter) of actinobacterial growth on ISP medium 2 agar was transferred to PVK agar plates and incubated at 28 ± 0.2◦C for 7 days. The occurrence of a clear zone around the agar plug was considered a positive indication of phosphate solubilization (Nautiyal, 1999). Quantitative analysis of tricalcium phosphate solubilization in liquid medium was also carried out using the Fiske and Subbarow (1925). Briefly, 5 agar plugs (5 mm diameter) of fully grown actinobacteria were inoculated into 50 ml of Pikovskaya's broth and were incubated with shaking at 120 rpm for 7 days at 28 ± 0.2◦C. The supernantant was collected by centrifugation at 12,000 rpm for 15 min. The resultant supernatant (500 µl) was mixed with 500 µl of 10% (w/v) trichloroacetic acid in a test tube. Four milliliters of the color reagent (1:1:1:2 ratio of 3M H2SO4, 2.5% ammonium molybdate (w/v), 10% ascorbic acid (w/v) and sterile distilled water) was added and the mixture was incubated at room temperature for 15 min. The pH value of the culture broth was also measured on day 0 and day 7. Sterile PVK medium served as a control. The optical density was read at 820 nm using a spectrophotometer (GENESYS TM 20 Visible Spectrophometer, Thermo Fishcer Scientific). Soluble phosphorus was estimated using a standard curve.

#### Siderophore Production

Siderophore production in all isolates was determined by chrome azurol S (CAS) assay (Schwyn and Neilands, 1987). Actinobacteria were grown on ISP medium 2 agar for 5 days at 28 ± 0.2◦C. Agar plugs (5 mm diameter) of actinobacterial growth that were grown on ISP medium 2 agar were transferred to CAS agar and incubated for 7 days at 28 ± 0.2◦C in a dark room. A yellow to orange zone around the actinobacterial disks indicated siderophore production. Siderophore quantity and types were determined and quantified by ferric perchlorate assay (Atkin et al., 1970) for hydroxamate type and Arnow assay (Arnow, 1937) for catecholate type.

#### In Vitro Assay for Drought Tolerance in Actinobacteria

All actinobacteria were grown on ISP medium 22 agar for 7 days. Agar plugs (5 mm diameter) of fully grown actinobacteria were transferred to 10% Tryptic soy agar (TSA) that was supplemented with 0 gL−1 ; 85 gL−1 ; 285 gL−1 ; 405 gL−1 ; 520 gL−1 , and 660 gL−1 of sorbitol to adjust the aw value of the media in order to simulate water stress at 40◦C for 7 days. The final aw value of the media were 0.998, 0.986, 0.957, 0.919, 0.897, and 0.844, respectively (Hallsworth et al., 1998). Growth recorded at aw of 0.919 was considered a drought-tolerant property.

#### Growth Promotion of Vigna radiata by Selected Actinobacteria

Actinobacteria isolate S3 was selected based on its in vitro plant growth promoting properties. Mung beans (Vigna radiata) were used as a representative of dicotyledon plants. Mung bean seeds (Raithip Brand, Thai Cereal World Company, Thailand) were surface sterilized by being immersed sequentially in 2% (v/v) sodium hypochlorite for 1 min, 95% (v/v) ethanol for 1 min, 70% (v/v) ethanol for 1 min and then washed with sterile distilled water for 1 min (five times). Five decontaminated seeds were randomly selected to check for surface sterility on nutrient agar. Cell suspension was prepared from 7-day-old culture of isolate S3 grown in ISP medium 2 broth. Surface sterile seeds were mixed with 108 CFU ml−1 cell suspension in 25 ml sterile water on a shaker at 120 rpm for 3 h before being sown in a sand pot. The following treatments, each with three hundred replicates of mung bean seeds, were conducted: (1) control (without bacterial inoculation), (2) cell suspension of isolate S3 (108 CFU ml−1 ), and (3) standard IAA solution adjusting the concentration to 11.12 µg ml−1 . The experiment was carried out in 2.5 cm × 2.5 cm pots and kept in a greenhouse at 30◦C for 7 days. Pots were arranged in a completely randomized

1https://www.ezbiocloud.net/taxonomy

arrangement. After 7 days, the following growth parameters were recorded: fresh weight (g), root length (cm), seed germination (%) and total length (cm). Seed germination was calculated as a percentage of the number of germinated seeds from a total of 100 sown seeds per pot.

### Growth Promotion of Rice Under Drought Conditions in Low Nutritional Soil by Selected Actinobacteria

In this experiment, Thai jasmine rice (Oryza sativa) KDML105 was used as a representative monocotyledon plant. The soil used in the experiment was of a sandy loam type and had poor nutritional value. The soil was bought from Lan Sai Company, Chiang Mai, Thailand. Soil properties were determined by Central Laboratory, Faculty of Agriculture, Chiang Mai University. The soil was sterilized by being autoclaved twice for 20 min over two consecutive days. The sterility of the soil was determined by spreading the autoclaved soil suspension on ISP medium 2 and nutrient agar.

Rice seeds were kindly supplied by Chiang Mai Rice Seed Center, Rice Seed Division, Rice Department, Ministry of Agriculture and Cooperatives, Thailand. Seeds were surface sterilized by being immersed sequentially in 2% (v/v) sodium hypochlorite for 1 min, 95% (v/v) ethanol for 1 min and 70% (v/v) ethanol for 1 min, and then being washed with sterile distilled water for 1 min (three times). Five decontaminated seeds were randomly selected to check for surface sterility on ISP medium 2 and nutrient agar. Surface sterile seeds were mixed with 108 CFU ml−1 of isolate S3 and incubated on a rotary shaker at 120 rpm at 30◦C for 16–18 h before being sown. Seedlings were prepared by sowing 100 surface sterile seeds in a tray (25 cm × 35 cm × 9 cm) containing 1 kg of sterile soil. Seedlings were watered once a day with sterile distilled water for 7 days at room temperature. Seed germination was calculated as a percentage of the number of germinated seeds from a total of 100 sown seeds per tray. Seven-day-old seedlings having two leaves were transferred to a bigger pot for further growth promoting experimentation.

The experiment was carried out in pots (20 cm diameter × 15 cm height) containing 3 kg of sterile soil and kept in a greenhouse at ambient light and temperature for 45 days with five replicates per treatment and five seedlings per pot (n = 25). Pots were arranged in a completely randomized arrangement in a greenhouse. Tap water was supplied once a day to full container capacity and kept at 1 cm above soil level. Fertilizer was not added in order to maintain the low nutritional value of the soil. The following treatments were investigated: (1) control (without bacterial inoculation) and (2) drought-induced treatment [seeds mixed with cell suspension of isolate S3 (108 CFU ml−1 )]. In addition, a well-watered treatment [seeds mixed with cell suspension of isolate S3 (108 CFU ml−1 )] with its control (without bacterial inoculation) was also carried out for comparison purposes. Drought stress was induced by completely withholding water starting on day 36 for 10 days. At the end of the experiment (day 46 after being sown), the following growth parameters were recorded: fresh weight (g), dry weight (g), root length (cm), total length (cm), height (cm), stem diameter (mm), number of leaves, relative water content (RWC), chlorophyll content (mg g−1 ) and proline content (µmol g−1 ). Proline content was determined by rapid colorimetric method (Bates et al., 1973). Chlorophyll content was determined spectrophotometrically based on the standard method of Arnon (1949). RWC of rice leaves (n = 5) was calculated from the formula RWC (%) = [(FM-DM)/(TM-DM)] × 100 according to Oukarroum et al. (2007).

Colonization of rice root by isolate S3 was confirmed by dilution spread-plate and microscope examination. Briefly, the root was ground in TE buffer and shaken on a rotary shaker for 3 h at 180 rpm. The root suspension (1 ml) was serially diluted and spread on ISP medium 2 agar that was supplemented with 25 µl/ml nalidixic acid and 100 µg/ml ketoconazole. The suspension was then incubated at 30◦C for 7 days. 16S rRNA gene sequencing was also performed on the obtained isolate.

Isolate S3 was also screened for 1-aminocyclopropane-1-carboxylate (ACC) deaminase production as described by Palaniyandi et al. (2014). Inoculation was done on DF minimal salts agar (Dworkin and Foster, 1958) with no nitrogen source and DF minimal salts agar that had been supplemented with either 5 mmol l−1 of ammonium sulfate or ACC. Plates were then incubated at 30◦C for 10 days. The level of growth of the isolates on three different media was compared. Strains that were able to utilize ACC as a nitrogen source (ACC deaminase positive) exhibited a level of growth equal to the growth on (NH4)2SO4 containing medium, whereas strains that could not utilize ACC showed no growth, which was compatible to that of the medium without a nitrogen source.

#### Nucleotide Sequence Accession Numbers

The 16S rRNA gene sequences of all actinobacterial isolates were deposited in the DDBJ, EMBL and GenBank nucleotide sequence databases under accession numbers LC062606-LC062608, LC065388-LC065389 and LC207997.

#### Statistical Analysis

The data were analyzed using one-way analysis of variance (ANOVA) and Tukey's multiple range tests (TMRT). The detection for all treatments in this study was calculated using SPSS (version 16.0) at p = 0.05.

# RESULTS

#### Selective Isolation of Actinobacteria From Funneliformis mosseae Spores

In this section, we tried to isolate cultivable actinobacteria that are associated with the spores of arbuscular mycorrhiza, Funneliformis mosseae. The first attempt at isolation without

employing an enrichment step in the soil extract broth resulted in no actinobacterial growth on both selective media. Six presumptive actinobacteria were successfully isolated from the spores of F. mosseae using an enrichment step. Isolates 48, S1 and S3 were recovered from starch casein agar, whereas the remaining isolates were grown on humic acid vitamin agar (**Table 1**). The total actinobacterial count was 5 × 102 CFU/20 spores on the starch casein agar. No microorganisms were grown on the nutrient agar and the ISP medium 2 agar obtained from the final washing water.

#### Identification of Actinobacteria Based on 16S rRNA Gene Analysis

The 16S rRNA genes obtained from all isolates were amplified with primers 27F and 1525R and sequenced. The resultant sequences ranged from 1200 to 1422 bp were identified using the BLAST program in the EzBioCloud database. Their closest phylogenetic neighbors are shown in **Table 1**. Five isolates were identified as members of the genus Streptomyces. Isolates 48 (99.86% similarity) and S3 (99.93% similarity) were closely related to S. thermocarboxydus DSM 44293T , whereas isolates S4 and S4-1 shared 99.75% and 99.79% similarity with S. pilosus NBRC 127772T . Isolate S1 was closely related to S. spinoverrucosus NBRC14228T (99.28% similarity). The remaining isolate, isolate SP was identified as Pseudonocardia and closely related to P. nantongensis KLBMP 1282T (98.56% sequence similarity). Phylogenetic analysis based on an almost complete sequence of the Streptomyces isolates is showed in **Figure 1A**. Isolates 48 and S3 were in the S. thermocarboxydus clade and supported by 88% bootstrap value, whereas isolates S4 and S4-1 fell into the S. pilosus clade with 68% bootstrap support. Isolate S1 was clustered with S. spinoverrucosus. The Pseudonocardia isolate SP shared a clade with P. nantongensis KLBMP 1282T , a relationship supported by a 100% bootstrap value (**Figure 1B**). The sequences from all isolates were deposited in the DDBJ database under the accession numbers shown in **Table 1**.

# In Vitro Plant Growth Promoting Potential

#### IAA Production

All 5 isolates of Streptomyces (48, S1, S3, S4, S4-1) were positive for IAA production in a range of 0.74 – 11.12 µg ml−1 (**Table 2**). Only the Pseudonocardia isolate SP showed a negative result. Isolate S3 produced the highest IAA value of 11.12 µg ml−1 . This isolate also produced 1.12 µg ml−1 without L-tryptophan supplement. In addition, during periods of reduced water activity, isolate S3 still produced IAA at up to aw = 0.919 (data not shown). TLC analysis of the IAA sample showed a pink color spot at similar Rf when compared with the chemical IAA standard (Supplementary Figure S1). PCR amplification of the IAA gene in isolate S3, the highest IAA producer, showed a product of approximately 1600 bp and was similar to that of the positive control, S. coelicolor A3(2). Sequence analysis of this PCR fragment using the BLAST program (blastx) revealed a similarity to the amino oxidase gene in Streptomyces coelicolor A3(2) (NP\_625735).

#### Phosphate Solubilization

Half of the isolates (isolates 48, S1, S3) were found to produce clear zones around their colonies on Pikovskaya's agar (**Table 2**). The largest clear zone diameter of 7.00 ± 0.05 mm was observed (Supplementary Figure S2a). Quantitative estimation of phosphate solubilization in the culture broth was in a range of 215.6 – 224.27 mg L−1 , with the highest value obtained from isolate S3 (224.27 ± 0.06 mg L−1 ). A decline in the pH value of the culture filtrate was observed in all these positive isolates. The lowest pH value was recorded at pH 4.46 ± 0.25 for isolate S3.

TABLE 1 | Identification of actinobacteria associated with F. mosseae's spores based on 16S rRNA gene sequence analysis.

SC, starch casein agar; HV, humic acid-vitamin agar.

representative of their related taxa. Bootstrap values are based on 1,000 re-samplings. Only values of >50% are shown at the nodes.

#### Siderophore Production

fmicb-09-01247 June 11, 2018 Time: 14:41 # 7

All isolates could produce siderophore on CAS agar as an orange halo zone was observed around the agar plugs (**Table 3**). Isolate 48 produced the largest zone of siderophore production (17.83 ± 0.58 mm) followed by isolate S3 (15.83 ± 0.58 mm) (Supplementary Figure S2b). All isolates could produce both catecholate and hydroxamate type siderophores at various levels. Isolate S3 produced the highest catecholate type siderophore (39.17 ± 0.002 µmol L−1 ), whereas isolate S1 produced the highest hydroxamate type siderophore (97.50 ± 3.63 µmol L−1 ) (**Table 2**).

# In Vitro Assay for Drought Tolerance in Actinobacteria

Two Streptomyces isolates, S1 and S3, showed a high tolerance to drought in vitro, as they were able to grow in medium with reduced water availability (aw = 0.919). The remaining isolates could grow up to aw of 0.957 (**Table 3**). Though both isolates could grow under water stressed conditions, isolate S3 was selected as a candidate for rice growth promotion under drought conditions due to its better performance with regard to in vitro plant growth promoting properties.

# Growth Promotion of Vigna radiata by Selected Actinobacteria

The effects of S. thermocarboxydus isolate S3 inoculation on mung bean seeds were observed. The percentage of seed germination in all treatments involving isolate S3 was in a range of 95–98%, which was statistically higher than the control. The highest value of enhancement of all growth parameters was obtained from Treatments 2 and 3 (**Figure 2A**). Treatments with isolate S3 showed higher fresh weight, root length and total length when compared to the control. The increases in fresh weight, root length and total length were not significantly different in Treatments 2 and 3. In addition, though root intensity was not directly observed, it is evident from **Figure 2B** that Treatments 2 and 3 displayed more intense roots than the control.

#### Growth Promotion of Rice Under Drought Conditions in Soil With Low Nutritional Value by Selected Actinobacteria

The soil used in this study was of poor quality as can be seen in **Table 4**. The organic matter content was only 0.75%, which determined it to be a low nutritional type of soil for rice cultivation as recommended by the Rice Department, Ministry of Agriculture and Cooperatives, Thailand. In addition, the nitrogen, phosphate and potassium levels were also low and this was especially true for nitrogen. The effects of S. thermocarboxydus isolate S3 inoculation on Thai jasmine rice KDML105 is presented in **Figures 3**–**5**. Seed germination was recorded between 98 and 99% for all treatments, which was higher than the control (96%). In the well-watered treatment, the positive effects of S. thermocarboxydus isolate S3 was seen in the rice as indicated by the following observed growth parameters, namely fresh weight, dry weight, root length and total length, which were all significantly higher than in the control (**Figure 3A**). However, the chlorophyll and proline contents were similar between the two treatments (**Figure 3B**). The RWC value of the rice leaves obtained from the treatment using isolate S3 was 96.1% compared to 81.0% in the control.

For the drought-induced treatment, a similar level of growth enhancement was observed in all tested parameters. The root, stem and total length measurements of the rice in S. thermocarboxydus isolate S3 treatment were higher than in the control (**Figure 4A**). The proline and chlorophyll contents, as well as the fresh and dry weight, were also higher than in the control (**Figure 4B**). At the end of the experiment, all rice plants in the control treatment withered and died, whereas in the treatment with S. thermocarboxydus isolate S3, some plants were still green (**Figures 5A,B**). The RWC value of the rice leaves in the inoculated rice with isolate S3 was 48.3% as compared to 43.3% in the control under drought stress conditions. S. thermocarboxydus isolate S3 was re-isolated from the rice root after harvesting. A count of 2 × 103 cfu/g fresh weight was obtained. The reisolated strain showed 100% 16S rRNA gene sequence, which was identical to that of the S. thermocarboxydus isolate S3. In addition, filamentous cells of S. thermocarboxydus isolate S3 were observed under a light microscope as is shown in the Supplementary Figure S3. ACC deaminase activity was detected in isolate S3 as it could grow on DF salt minimal agar that was supplemented with 5 mmol l−1 of ACC as the nitrogen source.

# DISCUSSION

# Isolation and Identification of Actinobacteria From Spores of Funneliformis mosseae

Cultivable actinobacteria associated with arbuscular mycorrhizal spores were reported to be an interesting source of both biocontrol and plant growth-promoting activities (Schrey et al., 2012; Mohandas et al., 2013; Poovarasan et al., 2013; Battini et al., 2016). These findings encouraged us to explore actinobacteria that was obtained from the spores of Funneliformis mosseae. In the present study, we successfully isolated 6 actinobacteria from the spores of Funneliformis mosseae. The predominant isolates were members of the genus Streptomyces, a finding consistent with previous reports (Schrey et al., 2012; Mohandas et al., 2013; Poovarasan et al., 2013; Battini et al., 2016). The fact that we could isolate actinobacteria from surface sterilized spores suggested that they should be obtained from inside the spores. No bacterial growth was observed on nutrient agar and ISP2 agar obtained from the final wash water. These findings suggested that these actinobacteria are likely to be endophyte.

The interaction between actinobacteria and their mycorrhizal symbiosis have not been well-documented. Most research studies were conducted on Gram-negative bacteria such as Pseudomonas. However, several researchers have reported on the enhanced spore germination or mycorrhiza formation of various AM fungi in the presence of actinobacteria (Frey-Klett et al., 2007).

TABLE 2 | Indole-3-acetic acid (IAA) production, phosphate solubilization and siderophore production of actinobacteria associated with F. mosseae's spores.

PVK, Pikovskaya's agar; CAS, Chrome Azurol Sulphonate agar. Data represent mean values of three replicates ± SD. Different letters indicate the existence of significant differences according to Tukey test (P = 0.05).

TABLE 3 | Growth of actinobacteria associated with F. mosseae's spores under reduced water activity.

+, growth; −, no growth.

Tylka et al. (1991) found that volatiles obtained from S. orientalis stimulated the germination of both Gigaspora margarita and G. mosseae spores on water agar. Similarly, the spore germination of G. margarita was found to be stimulated by volatiles of soil-isolated actinobacteria, especially those with straight spore chains and 2-methylisoborneol (MIB) producers (Carpenter-Boggs et al., 1995). The same authors speculated that the stimulation of AM spore germination enhanced the probability of the formation of mycorrhizal associated with nearby plants. Inoculation of S. coelicolor 2389 with G. intraradices increased the intensity of mycorrhizal root colonization and arbuscular formation in sorghum (Abdel-Fattah and Mohamedin, 2000). Streptomyces was also found to enhance mycorrhiza formation in the ectomycorrhizal fungus of Amanita muscaria as a result of the promotion of fungal growth (Schrey et al., 2005). In addition, mycorrhiza-associated bacteria together with AM fungi contributed to protection against root pathogens (Frey-Klett et al., 2007). For example, mycorrhizal associated streptomycetes obtained from Norway spruce trees produced secondary metabolites displaying antifungal activity against the growth of several plant pathogenic fungi including Fusarium oxysporum (Schrey et al., 2012). Streptomyces obtained from G. mossaea spores also showed strong antifungal activity against F. oxysporum and Alternaria solani (Mohandas et al., 2013). Considering the plant growth promoting ability of the isolated actinobacteria, it is possible that these actinobacteria are "mycorrhiza helper bacteria" that can contribute to nutrient mobilization from the soil and impact root structure through the production of growth promoters, as has been suggested by Frey-Klett et al. (2007). However, the possibility of the isolated actinobacteria being involved in the process of promoting mycorrhization could not be ruled out and requires further study.

It is interesting to note that a member of the genus Pseudonocardia was isolated from mycorrhiza spores for the first time in this study. It was previously only detected in the spores of Gigaspora margarita and Gigaspora rosea by DGGE (Long et al., 2008). Currently, DGGE analysis of F. mosseae spores has revealed the presence of actinobacteria in the genera Amycolatopsis, Arthrobacter, and Propionibacterium (Agnolucci et al., 2015). Glomus mosseae spores were reported to harbor cultivable actinobacteria of the genus Arthrobacter, Curtobacterium, Liefsonia, Microbacterium, Micrococcus, and Streptomyces (Bharadwaj et al., 2008b; Mohandas et al., 2013; Poovarasan et al., 2013; Selvakumar et al., 2016).

It is also worth mentioning that we could not isolate any actinobacteria without an enrichment step in the soil extract solution. Enrichment is a common practice for effectively increasing the population of target microorganisms (Kamagata, 2015). Soil extract was successfully used to isolate novel actinomycetes from the soil (Hamaki et al., 2005). Conventional cultures and isolation media that are generally nutritionally rich usually result in a failure to cultivate most bacteria in nature. Our

results suggest that the addition of the soil extract could support the growth of actinobacteria, which represents a small proportion of the bacteria that is associated with mycorrhizal spores and can increase the numbers to a detectable level on the isolation plate.

It is evident from the 16S rRNA gene sequences and the phylogenetic analysis that 5 isolates were members of the genus Streptomyces. These isolates were closely related to S. thermocarboxydus, S. pilosus, and S. spinoverrucosus (**Table 1** and **Figure 1A**). Based on this data, we identified isolates 48 and S3 as S. thermocarboxydus, isolates S4 and S4-1 as S. pilosus and isolate S1 as S. spinoverrucosus. These three Streptomyces species have never been isolated from mycorrhizal spores. Isolate SP was identified as Pseudonocardia and is closely related to P. nantongensis KLBMP 1282T (**Table 1**). It shared a well support

branch with P. nantongensis KLBMP 1282T (**Figure 1B**). To the best of our knowledge, this is the first Pseudonocardia strain isolated from mycorrhizal spores. This strain shared only 98.56% similarity with P. nantongensis KLBMP 1282T , a value below the proposed 98.7% (Stackebrandt and Ebers, 2006) or 98.65% (Kim et al., 2014) cut-off values for the delineation of novel species. It is likely that isolate SP will represent a novel species within the genus Pseudonocardia though a further characterization using the polyphasic approach is needed. However, the detailed formal description of this strain as a novel species will be the subject of another publication.

#### In Vitro Plant Growth Promoting Activities

Indole-3-acetic acid (IAA) is a phytohormone of the auxin type, which helps promote plant growth and development especially in the root system. The production of IAA is common in actinobacteria. Several Streptomyces species are known to produce IAA including S. coelicolor, S. griseus, or S. scabies (Manulis et al., 1994). A high proportion (70%) of IAA producing actinobacteria that are associated with the mycorrhizal spores of R. intraradices was also reported (Battini et al., 2016). In the present study, all Streptomyces isolates were able to produce IAA in the presence of L-tryptophan (0.74 – 11.12 µg ml−1 , **Table 2**). Streptomyces and Leifsonia poae isolated from G. mosseae spores were reported to produce IAA at similar levels to our isolates (5.0–10.1 µg ml−1 ) (Mohandas et al., 2013). A higher level of IAA production of 43.8 µg ml−1 compared to S. thermocarboxydus isolate S3 (11.12 µg ml−1 ) was recorded in S. thermocarboxydus DBT219 using endophytic actinobacteria associated with tomatos (Solanum lycopersicum) (Passari et al., 2016). However, those authors did not indicate the amount of supplemented L-tryptophan in the experiment. Streptomyces isolates obtained from Thai medicinal plants produced IAA in a range of 11-144 µg ml−1 , while S. viridis CMUH-009 could produce IAA at a level as high as 300 µg ml−1 under optimized conditions (Khamna et al., 2010). The expected PCR product of approximately 1,600 bp from S. thermocarboxydus isolate S3, as well as S. coelicolor A3(2), indicated the presence of the IAA encoding gene. However, BLAST identification of this PCR

product as an amino oxidase gene in isolate S3 suggested that this Streptomyces may use the tryptamine (TAM) pathway in IAA synthesis instead of the indole-3-acetamide (IAM) pathway, as was the case with several Streptomyces species including S. coelicolor A3(2) and S. scabies (Manulis et al., 1994).

Several actinobacteria associated with mycorrhizal spores were reported to have phosphate solubilization properties. Three of our Streptomyces isolates, namely isolates 48, S3 and S1, produced a clear zone of tricalcium phosphate solubilization on Pikovskaya's agar. Streptomyces and Leifsonia isolated from G. mosseae spores were also found to produce clear zones of tricalcium phosphate solubilizing activity (Mohandas et al., 2013). Similarly, Arthrobacter ilicis obtained from the spores of G. intraradices could solubilize phosphate on Pikovskaya's

agar (Bharadwaj et al., 2008a). However, the quantitative determination of phosphate solubilization in the culture broth was not reported in these previous research studies. High phosphate solubilization in the range of 215.6 – 224.27 mg L −1 as was recorded in this study was higher than that of the Streptomyces isolates obtained from the sediment of Chorao Island, India (89.3 – 112.1 µg mL−1 ) (Dastager and Damare, 2013). Nevertheless, a higher degree of phosphate solubilizing activity of 72.13 mg/100 ml was reported in Streptomyces obtained from the wheat rhizosphere soil in Pakistan (Anwar et al., 2016). The ability to solubilize phosphate in actinobacteria obviously varies from strain to strain and species to species, as exemplified by our results and the previous research reports.

The acidification of the culture filtrate was observed in all isolates that could release soluble phosphate in the culture broth. This observation suggested that the solubilization process was involved with the production of organic acids. Microbial solubilization of mineral phosphates is proposed to either be due to the production of organic acids (Surapat et al., 2013) or the production of chelating substances such as siderophores (Hamdali et al., 2008). A preliminary investigation on the quality and quantity of organic acids in the culture broth by HPLC analysis confirmed the presence of several organic acids including gluconic acid, malonic acid, oxalic acid and propionic acid (data not shown). Lactic and 2-ketogluconic acids were the predominant acids found in phosphate-solubilizing Streptomyces (Banik and Dey, 1982). Additionally, phosphate-solubilizing actinobacteria, Micromonospora endolithica, was reported to be capable of producing organic acids namely citric, oxalic, gluconic, malic, succinic, acetic, and lactic acids (El-Tarabily et al., 2008).

All actinobacteria that were isolated from the spores of F. mosseae were able to produce siderophores in both hydroxamate and catecholate types at varying concentrations. Siderophores are small molecules of iron chelating compounds produced by several microorganisms including actinobacteria under iron starvation (Wang et al., 2014). Siderophores are known to promote plant growth and inhibit plant pathogens (Verma et al., 2011; Sadeghi et al., 2012). Several Streptomyces species have been found to be able to produce desferrioxamine (hydroxamte type siderophores) including S. pilosus (Wang et al., 2014). Catecholate types are not common among siderophoreproducing actinobacteria. However, Streptomyces were found to be able to secrete enterobactin, a catecholate type siderophore of Enterobacteriaceae (Lee et al., 2012). All actinobacteria obtained from G. mosseae spores including Streptomyces were also able to produce siderophores on CAS agar (Mohandas et al., 2013). Similarly, 79% of the cultivable actinobacteria obtained from R. intraradices spores could produce siderophores (Battini et al., 2016). The siderophore-producing endophytic Streptomyces was clearly shown to be able to promote the growth of Thai jasmine rice by significantly increasing root and shoot biomass, as well as root length, in comparison with the siderophore-deficient mutant (Rungin et al., 2012).

It is worth mentioning that three of our Streptomyces isolates (48, S1, S3) displayed all of the plant growth-promoting activities we investigated. The observation that the actinobacteria associated with the spores of F. mosseae are equipped with plant growth promoting properties in vitro suggests that these actinobacteria are potential candidates for in planta testing. This strategy has been used successfully to identify potential actinobacteria that can promote the growth of several plant species, e.g., guava (Mohandas et al., 2013), rice (Rungin et al., 2012; Gopalakrishnan et al., 2014) tomato (Passari et al., 2016) and wheat (Anwar et al., 2016).

# In Vitro Assay for Drought Tolerance in Actinobacteria

Bacteria in general prefer a high aw value for growth. A minimum aw for bacterial growth was defined at 0.900aw for a majority of bacteria (Grant, 2004). In the present study, Streptomyces isolates S1 and S3 grew up to 0.919 aw which could indicate xerotolerance. Drought is a water deficit condition that does

not allow for the growth of most plants. To use plant growth promoting bacteria to alleviate drought stress for plants, one basic requirement is the growth of plant growth-promoting bacteria under such condition. The ability to grow during periods of low water availability is an important criterion for selecting potential actinobacterial isolates when testing the growth promotion of the plant under the conditions of drought. This screening approach was successfully used to obtain rhizosphere bacteria from cacti for the growth promotion of Zea mays L. under drought conditions (Kavamura et al., 2013). The ability of actinobacteria to grow at low aw is not surprising as several species of actinobacteria, including several Streptomyces species, have been isolated from even the driest places on earth like the Atacama desert (Okoro et al., 2009). This report and our results strongly support the conclusion that these xerotolerant actinobacteria could survive under extreme water deficit environments and we recommended them as promising materials for plant growth-promoting bacteria under conditions of drought stress.

#### Plant Growth Promotion Growth Promotion of Vigna radiata by Selected Actinobacteria

This experiment was designed to test the ability of the selected S. thermocarboxydus isolate S3 to promote plant growth in vivo. Mung beans (V. radiata) have been widely cultivated throughout Southeast Asia. This plant species has been used as a test plant due to its short life cycle. The ability of isolate S3 to promote the growth of mung beans was evident (**Figure 2**). Similar results from treatment 2 (cell suspension and supernatant) and treatment 3 (addition of 11.12 µg ml−1 IAA) indicated that the observed level of growth enhancement in the root and total length might be a result of the contribution of IAA that was produced by S. thermocarboxydus isolate S3 as this isolate was found to produce 11.12 µg ml−1 IAA in vitro (**Table 2**). More developed root systems were also observed in the treatment with isolate S3. This observation suggested the effect of IAA production by isolate S3. IAA is known for its effect on the production of lateral roots and the promotion of root-length (Etesami et al., 2015). Similar incidences of the promotion of plant growth that occurred due to the production of plant phytohormones by G. mosseae spores associated with actinobacteria in pomegranate seedlings have been reported (Poovarasan et al., 2013). S. canus which produced 10.10 µg ml−1 of IAA in vitro was found to increase shoot and root growth and plant dry matter in 6 month-old pomegranate seedlings. The production of bacterial phytohormones such as IAA is considered to be a major mechanism for drought endurance and resilience in microbial strains (Kaushal and Wani, 2016; Forni et al., 2017). This is the reason that S. thermocarboxydus isolate S3 was selected for further experimentation on rice growth promotion under induced drought conditions in the greenhouse.

#### Growth Promotion of Rice Under Drought Conditions in Soil With Low Nutritional Content by Selected Actinobacteria

It is evident that in soil having low nutritional content and under non-stress conditions, inoculation with isolate S3 improved the growth of rice seedlings as was observed in terms of root length, stem length, and fresh and dry weight (**Figure 3**). Our results provide evidence that the most prominent beneficial effects of inoculation with a potential PGPR is to be expected in poor soil when the development of the indigenous microbial community is inhibited (Ramos Solana et al., 2006). Similar results have been reported under other unfavorable conditions such as in rice (Yuwono et al., 2005) or wheat (Yandigeri et al., 2012) under conditions of drought and in tomatos (Palaniyandi et al., 2014) under conditions of salinity.

Rice is more susceptible to drought stress than most crop plants. According to the International Rice Research Institute (IRRI), when rice plants experience drought, they have a reduced ability to extract nutrients from the soil (Lafitte and Bell, 2017). Thai jasmine rice KDML105 is a dominant rice variety that has been widely cultivated in Thailand and is sensitive to drought (Cha-um et al., 2010). Therefore, it has been selected as a test plant in the investigation of the ability of plant growth promoting S. thermocarboxydus isolate S3 to protect KDML105 under conditions of induced drought stress in soil of low nutritional value. In this study, we induced conditions of drought stress in rice during the early vegetative stage, which requires large amounts of water for the development of a complete panicle formation2 . This drought stress induces a reduction in rice growth and development as was observed in many growth parameters in this study. With the use of soil having low nutritional content in this study, the adverse effects on rice growth was expected to be more severe as was seen in reductions of fresh and dry weights (**Figures 3B**, **4B**).

The growth and development of rice was observed to decline due to the fact that drought stress induces damage to biochemical and physiological mechanisms (Pandey and Shukla, 2015). This stress could be alleviated through the application of a microbial inoculant that induces biochemical and physiological changes in plants to sustain their growth (Yang et al., 2009; Yandigeri et al., 2012; Vurukonda et al., 2016; Rubin et al., 2017). This study provides evidence that induced drought stress conditions could be relieved by inoculation with plant growth promoting S. thermocarboxydus isolate S3.

Fresh weight is another common morphological growth parameter that is severely affected by drought (Jaleel et al., 2009). Reduced biomass was reported in rice under conditions of water stress (Manickavelu et al., 2006; Farooq et al., 2009, 2010). Many studies have reported a significant decrease in the fresh and dry weights of the shoots (Centritto et al., 2009; Mostajeran and Rahimi-Eichi, 2009) and roots (Ji et al., 2012) under conditions of drought. Our results revealed a reduction in both the fresh (3.18 g) and dry (0.52 g) weights of droughtinduced rice compared to well-watered rice in treatments without S. thermocarboxydus isolate S3 (5.6 g, 0.92 g). However, the fresh and dry weight measurements in drought-induced rice that has been inoculated with S. thermocarboxydus isolate S3 were higher than in the control. This observation implies the ability of plant growth promoting S. thermocarboxydus isolate S3 to reduce the damaging effects caused by drought stress.