Plant growth-promoting of olive and walnut actinobacteria: isolation, screening PGP traits, antifungal activities, identification, and hydroponic production of wheat

ABSTRACT Bacteria are among the most frequently studied microorganisms in plant growth-promoting traits and fighting against different pathogens. Actinobacteria are prokaryotic organisms that produce mycelium, spores, and can form various secondary metabolites, especially antibiotics. In our study, free-living and endophytic Actinobacteria were isolated from rhizosphere soils and plant parts of olive (Olea europaea L.) and walnut (Juglans regia L.) trees, which are commercially essential agricultural products for Turkey. The study aimed to explore the plant growth-promoting activities of these Actinobacteria as biological fertilizers. Some of the properties of purified Actinobacteria that regulate the plant growth-promoting (PGP) traits; phosphate solubilization, indole-3-acetic acid, siderophore, ammonia, protease, beta-galactosidase, chitinase production, and antagonistic activities against various plant-pathogen fungi were investigated. As a result, 422 Actinobacteria were isolated, and 88 Actinobacteria determined to be possible plant bio-fertilizers were identified as Streptomyces spp. (70), Amycolatopsis spp. (12), Micromonospora spp. (four) and Nocardiopsis spp. (two) after the sequencing of their 16S rRNA genes. Moreover, four Streptomyces spp. and two Amycolatopsis spp. were used in hydroponic germination of wheat seeds (Triticum aestivum L.), and plant growth in terms of root and stem parts increased.


Introduction
The rhizosphere is defined as the root zone surrounding the plant soil and all organisms around it. After defining the rhizosphere (Hiltner 1904), interactive mechanisms related to the plant root system and the microbiome in this region were examined and discovered (Berg 2009;Dede 2013;Schlaeppi and Bulgarelli 2015;Dede and Guven 2018). The mucilage (Knee et al. 2001) and other root exudates (Shi et al. 2011) secreted by the plant form nutrients for microorganisms. The different organisms are positively drawn toward this area and interact with the relevant plants. Moreover, the saprophytic interactions with the dead biomass of plants and animals (Lowenfels and Lewis 2010) also attracted different microbiomes.
Microorganisms that interact around the rhizosphere can be beneficial, pathogenic or commensal (Berendsen et al. 2012). Beneficial microorganisms directly or indirectly interact with the plants for the interest of the plant (Lugtenberg and Kamilova 2009). The beneficial microorganisms

Actinobacteria isolation, purification and preservation
The soil samples were collected from the regions where plants' surface soil and first encountered plant roots soil. The soil samples were collected from different depths of the rhizosphere of olive and walnut trees in Balıkesir, Turkey. Stem, leaves and root parts of olive and walnut trees were collected from different trees in Balıkesir, Turkey (geographical sampling zones were given in Supplementary file 1. All Actinobacteria were incubated on ISP3 (Himedia, India) and ISP4 (Himedia) agar to distinguish their morphological form at 28°C for 15-30 days. Spores and mycelia of pure Actinobacteria were stocked in 20% glycerol (Sigma, Germany) at −86°C.

Isolation of endophytic actinobacteria
Isolation of endophytic Actinobacteria was performed by modification of the method (Chanway et al. 2000). 'Healthy' plant parts were washed with running water and the soil, dirt, etc., parts were removed. Leaves, shoots and fruit pieces were cut to have an area of approximately 1 cm 2 and root pieces were cut to approximately 1 cm. Plant parts were kept in 70% ethanol for 5 min. Washed in sterile distilled water 1-3 times. Plant parts were kept in sodium hypochlorite (Sigma) solution (0.9%) for 20 min. Washed in sterile distilled water 1-3 times. Kept in 2% sodium bicarbonate (Sigma) for 15 min. Wash 1-3 times in sterile distilled water.
The surface-sterilized parts were incubated at 28°C for 15-30 days by inoculating the medium created by adding cycloheximide to the modified glycerol yeast extract agar and Actinobacteria isolation agar (AIA) (Himedia) at 50 µg/ml. Semi-solid MGYEA agar at 55°C (7.5 g agar for liter) can be added to the sterilized parts to mimic the inside of the plant. For the surface sterilization test, 100 µl was taken from the last step (washing water) and inoculated on related media at 28°C for 15-30 days.

Plant growth-promoting traits
The experiments were performed in triplicate.

Siderophore production
The technique found by Schwyn and Neilands was modified for the siderophore production test (Schwyn and Neilands 1987). Briefly, ISP2 (Himedia) media' pH was adjusted into 6.8 and was mixed with the blue dye at 55°C. Actively growing organisms in peptone water (PW) (peptone 10 g [Sigma], sodium chloride 5 g [Merck]) media after 14 days incubation (20 µl) at 28°C were inoculated on new blue media (Blue-ISP2) and incubated at 28°C for 14-20 days (Dede et al. 2020). Organisms' colors were transformed into yellow, orange, purple, and magenta after the incubation period was considered positive.

Phosphate solubilization
Actively growing organisms in peptone water media after 14 days incubation (20 µl) at 28°C were inoculated on Pikovskaya agar (Himedia) medium (Pikovskaya 1948) for 14-20 days incubation at 28°C. The formation of transparent zones on the medium of bacteria that were capable of dissolving phosphate was accepted as a positive result.

Indole-3-acetic acid (IAA) production
Incubation of Actinobacteria at 28°C in peptone water medium for 28-30 days, centrifugation was performed at 8000 rpm for 10 min. Two ml supernatant mixed with two drops (⁓40 µl) of orthophosphoric acid (Merck) and 4 ml of Salkowski reagent (1 ml of 0.5 M ferrous chloride [Sigma] in 50 ml of 35% perchloric acid [Fluka]) (Gordon and Weber 1951). The formation of the pink color was regarded as positive.

Ammonia (NH 3 ) production
Bacteria were tested in peptone water for ammonia production. Purified bacteria were incubated at 28°C for 14-20 days after inoculating into 5 ml of growth media. Ammonia production was recorded positive with the formation of brownish-red color after adding Nessler reagent (Sigma, 1 ml) to each tube (Cappuccino and Sherman 2014).

Plant growth-promoting enzymatic production traits
The experiments were performed in triplicate.

Protease production
After Actinobacteria activated in the liquid culture (PW) at 28°C at 14 days were inoculated on (20 µl each) Skim milk agar (SMA) (Himedia), they were incubated for 14-20 days at 28°C. The formation of clear zones around the colonies indicated protease activity and considered positive (Kazanas 1968).

Beta (β) galactosidase production
The experiment performed with the modification by Flores et al. yellow and light yellow color formation visualized as a positive (Flores et al. 1990). Actinobacteria were produced in peptone water medium at 28°C for 14 days. 150 µl mycelium and PW medium mixture transferred in the glass tube which were containing 150 µl of physiological saline (0.9% NaCl [Merck]) solution and one ONPG disk (Sigma), incubated for 3 days at 28°C.

Chitinase production
The conversion of the coarse flake chitin to the form of colloidal chitin was carried out by the method established by Roberts and Selitrennikoff (Roberts and Selitrennikoff 1988). After dissolving the chitin in 37% hydrogen chloride (Sigma), the liquid part was removed by the filtration process, and the chitin was dried in fume hood. Actinobacteria were incubated for 14 days at 28°C in peptone water. After the inoculation (20 µl) to the medium containing ISP2 medium and colloidal chitin (2%) media with adjusted pH to 6,8-7,3 for 14-20 days at 28°C transparent zones around the colonies were recorded as positive.

Antifungal activity tests
Following the growth of pure isolates in the appropriate medium (PW), it was carried out on Sabouraud dextrose agar (SDA) (Difco, USA) of the species with selected plant-pathogens through the 'modified agar well diffusion' method (Magaldi et al. 2004).

Genomic DNA (gDNA) isolation
The pellets of Actinobacteria that possessed higher plant growth and enzymatic traits were collected with centrifugation 14,000 g after 14 days incubation at 28°C in the peptone water medium. Removal of the remaining PW medium with 1 mM Tris (Sigma), and after kept at −80°C for a day, gDNA isolation was performed with Wizard-genomic DNA purification kit (Promega, WI, USA) with manufacturer's recommendations. The presence of gDNA was shown with a 1 Kb gene-ruler marker (Thermo, CA, USA) on a 0.8% agarose (Sigma) gel and stored at −20°C for the future usages within the DNA rehydration buffer.

16S rDNA PCR amplification, purification and sequencing
PCR was performed with two universal primers for bacteria domain during the amplification of the 16S rRNA gene region (27 F: 5ʹ-AGAGTTTGATCMTGGCTCAG-3ʹ and 1492 R: 5ʹ-TACGGYTACCTTGTTACGACTT -3ʹ) (Zhao et al. 2011). Thermal cycle (Veriti, 96-Well thermal cycle, Singapore) conditions were programmed as follows: initial denaturation at 95°C, for 2 min; followed by 35 cycles at 95°C for 30 s, 51.6°C for 30 s, 72°C for 2 min; and final extension at 72°C for 5 min.
The PCR products screened on 1.5% agarose gel (Sigma) (100 ml 1xTAE [Thermo], 1.5 g agarose) with 1 Kb gene-ruler marker (Thermo) on Gel Doc EZ Imager, BIORAD, CA, USA. The ⁓1500 bp region was cut with the sterilized lancet and purification was conducted with GeneJet PCR purification kit, Thermo. Sequencing of the full length of the purified 16S rRNA gene was performed with four universal primers (518 F, 800 R, 1492 R, and 337 F) by Macrogen Inc., Europe with, 23ABI, 3730XLs, capillary sequencer Thermo, USA.

16S rDNA sequence analysis and construction of phylogenetic dendrograms
The most accurate sequenced regions of gene coding 16S rRNA were determined by the BioEdit program. The sequence information of the same organism sequenced with three-four primers combined with the BioEdit program (Hall 1999) and the sequence information of 1500 bp is obtained. Then, comparisons were made with the closest type of organisms using the EzTaxon server (Kim et al. 2012). For the analysis of possible new species, phylogenetic dendrograms prepared with CLUSTAL W option by using the MEGA7 program (Kumar et al. 2016). Phylogenetic dendrograms of 16S rRNA regions created using the neighbor-joining algorithm (Jukes and Cantor 1969). The bootstrap analysis of the phylogenetic trees generated (Felsenstein 1985) and carried out in 1000 replicates using the MEGA7 program.

Fatty methyl esters analysis
The Actinobacteria incubated at 28°C for 14 days on Tryptic soy agar (TSA, Merck). Bacterial mycelium and spores (80 mg) harvested for the extraction of their fatty acid methyl esters (FAMEs). Bacterial fatty acids were extricated and derivatized to FAMEs reported by Sasser (Sasser 2001). FAMEs were divided and separated by the Microbial Identification System (MIDI) (Microbial ID, Inc. Newark, DE, USA) utilizing an Agilent Technologies 6890 N gas-liquid chromatograph with a G2614A autosampler and a 6783 injector. After flame ionization, FAME peaks were identified by MIDI software, version ACTIN1. The analysis was applied to potential new species of isolated Actinobacteria according to EzBioCloud data.

Plant growth promoting tests via hydroponic production
Actinobacteria were inoculated on 5 ml PW at 28°C for 14 days. The organism mycelium parts were dispersed and broke into small pieces with gamma sterilized plastic loops. The surfacesterilized 100 wheat seedlings (same protocol used in part 'Actinobacteria isolation, purification and preservation' and provided from a local seed producer, named as Basribey-95) were inoculated with finely dispersed Actinobacteria within the 30 min and left for the blooming on hydroponic apparatus (in the volume of 250 ml Erlenmeyer flask with sterilized plant growthpromoting formula [Supplementary file 2] or distilled water and the top of the Erlenmeyer with 1.5 mm 2 squared plastic net). The seedlings were treated with 5 ml peptone water and Actinobacteria mycelium mixture and for the negative test, seedling with treated 5 ml PW medium for 10 min. The effect of the special formula on the growth of wheat seedlings compared with distilled water.
In the processing following germination, water addition containing nutrient and non-nutrient media continued until harvesting (for 30 days) the product. After 30 days of germination, the wheat seedlings were collected from the liquid mediums both special and distilled water individually. Their roots and stem lengths were measured separately. Statistical analysis was performed with Jamovi (computer software version 1.1.9.0, The Jamovi Project 2019, https://www.jamovi.org/) via one-way ANOVA test, Tukey variation (Core T 2004).

Actinobacteria isolation, purification and preservation
A total of 422 Actinobacteria were selected, purified and stocked from 12 different plant parts and 20 different soil regions. Thirty-three of them were endophytic, and 389 of them were free-living Actinobacteria. Numerical representations according to the regions where Actinobacteria isolated as walnut fruits 2, walnut shoots 4, walnut leaves 4, olive roots 23, olive surface soil 113, olive root soil 58, walnut surface soil 101, walnut root soil 117 and the total isolate number was found 422. After the isolation process, all isolates inoculated onto ISP3 and ISP4 media, and purity controls were performed based on colony characteristics. Isolates were stored in 20% glycerol solution at −86°C ultra-freezer. All isolated organism' regions were also given in Supplementary file 3. The bacterial growth and inoculation patterns are shown in Supplementary file 4.

Isolation of free living actinobacteria
Serial dilutions were carried out during the isolation of free-living Actinobacteria from different soil samples. After isolation on MGYEA medium, 171 isolates were isolated from the surface and rhizosphere of olive trees and 218 isolates from the soil surface and rhizosphere of walnut trees. Walnut Actinobacteria were isolated from five different walnut trees, 101 of them from the surface soils and 117 of them were from the different rhizospheric zones. Olive Actinobacteria were isolated from five different olive trees, 113 of them from surface soils and 58 of them were isolated from different rhizospheric zones.

Isolation of endophytic actinobacteria
During the study, the number of endophytic Actinobacteria obtained into the pure cultures after the surface sterilization from different parts of leaves, shoots, roots, and fruit parts were recorded as 33. Purified organism's numbers obtained as 23 from the olive trees, ten from the walnut trees.

Siderophore production
For the determination of siderophore production, samples that changed their color into yellow, orange, purple, and magenta colors recorded as positive. 195 of them were positive after the activity on MCAS agar (Supplementary file 3). Some of the Actinobacteria capable of producing siderophore were shown in Supplementary file 5.A.

Phosphate solubilization
Transparent zones identified on the Pikovskaya agar were positively recorded for indication of phosphate solubilization, and 165 isolates were recorded as positive (Supplementary file 3). Representative of the phosphate soluble Actinobacteria were shown in Supplementary file 5.B.

IAA production
Production of indole acetic acid was observed by formation of pink color. A total of 214 isolates were recorded as positive (Supplementary file 3). Examples of the Actinobacteria capable of producing IAA were shown in Supplementary file 5.C.

NH 3 production
The brownish-red color formed after adding the Nessler reagent to the test tube was recorded as positive and 376 isolates produced ammonia (Supplementary file 3). Some of the ammonia producing Actinobacteria were shown in Supplementary file 5.D.

Protease production
The appearance of transparent zones that occurred after a week of inoculation of Actinobacteria on SMA medium was accepted as positive for protease production to break down proteins. A total of 228 Actinobacteria produced protease (Supplementary file 3). Some of the Actinobacteria capable of producing protease were shown in Supplementary file 5.E.

β galactosidase production
Actinobacteria, producing a yellow and light-yellow color, were recorded as positive for betagalactosidase. A total of 221 Actinobacteria showed the ability to produce beta-galactosidase (Supplementary file 3). Some of the Actinobacteria capable of producing beta-galactosidase are shown in Supplementary file 5.F.

Chitinase production
Transparent zones formed by adding insoluble chitin onto ISP2 medium showed a typical example of Actinobacteria which can produce chitinase. A total of 230 Actinobacteria produced chitinase (Supplementary file 3). Some of the Actinobacteria capable of producing chitinase are shown in Supplementary file 5.G.

Antifungal activities of the actinobacteria
After 5 days of incubation on SDA medium, Actinobacteria which form transparent zones on plant pathogenic fungi were determined to have antagonistic activity. In total, 89 Actinobacteria had antigrowth effects (Supplementary file 3). The total anti-growth effect counts were A. parasiticus (64), A. fumigatus (39), G. graminis (31), Fusarium moniliforme (21), A. niger (22), P. notatum (38). Some of the isolates had anti-growth abilities with antifungal activity as shown in Supplementary file 6.

Combining the raw 16S rDNA sequence data
Unprocessed data of 91 isolates with the usage of four primers (337 F, 518 F, 800 R, and 1492 R) to obtained accurate sequence readings with the BioEdit program. The sequences were compared and determined with the closest relatives in the database of EzTaxon. The 16S rDNA gene of identified isolates uploaded on NCBI Genbank depositories and Genbank accession numbers were given in Supplementary file 7.

Phylogenetic dendrograms
After obtaining the 16S rDNA gene sequences, there were at least 45 different Actinobacteria (Supplementary file 8). The comparison was made with the type species in the EzBioCloud database. All sequences aligned with MEGA7 software with CLUSTAL. The neighbor-joining algorithm was used to determination of obtaining phylogenetic trees. Phylogenetic tree was shown on iTOL, a webbased drawing program (Figure 1). The comprehensive phylogenies were made with the MEGA7 software program. As a result, four main genera were encountered within the scope of the study, namely, Streptomyces spp. (70)

Fatty methyl esters identifications
The differences with the organisms via determination of fatty methyl ester analysis were also conducted within the study. As a result, a total of 35 isolates were analyzed and the major fatty acids were found as C 16:0 , isoC 15:0 and anteiso C 17:0 . Also c15:0, C16:0, iso C 16:0 and anteiso C 17:0 were Neighbor-joining method was used to determine the evolutionary history of Actinobacteria (Saitou and Nei 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) (Felsenstein 1985). The interactive phylogenetic tree was reformed with web-based tool iTOL (Letunic and Bork 2019).

Hydroponic production of wheat seedlings
A total of six Actinobacteria were selected to perform as their plant growth promoting traits (Table 1) with hydroponic production of wheat seedlings in terms of root elongation, seed germination and stem length. The EA12, EA22 and Z109 Actinobacteria resulted in statistically positive root elongation when compared to negative controls (Supplementary file 10).
In the results evaluated over the germination of 100 seeds, the highest seed germination rate (89 out of 100) was observed with EA22 (Amycolatopsis spp.) Actinobacteria. In statistical analysis, EA12 (Amycolatopsis spp.) and Z109 (Streptomyces spp.) significantly increased root length, while a significant growth relationship in terms of stem length was observed only with Z109 (Table 2, Supplementary file 11).

Discussion
Organic contents released from plant roots which is surrounding the soil create a living environment for microorganisms. Indirectly, branches and leaves left from plants to the soil surface cause the amount of organic matter to be also another reservoir for saprophytic organisms (Saatchi et al. 2007). Since the amount of organic matter alone is not sufficient for plant growth in ecosystems with very dense vegetation, bacteria-plant interaction studies are drawing attention for future studies and different research branches of scientific areas.
Actinobacteria produce different antibiotics due to their remarkable antagonistic activities. Apart from antibiotic production, they are responsible for the production of herbicides, antifungals, antitumors, and others (Purushotham et al. 2018). Actinobacteria were isolated from different soils (Hamdali et al. 2008) and plant parts (Shimizu 2011) to reveal the Actinobacteria-plant relationship. However, different studies were conducted with the walnut and the olive trees (Rincón et al. 2006;Müller et al. 2015;Pham et al. 2017;Gao et al. 2019). These studies were performed on soil or plantoriginated waste products which organism populations were generally tried to be determined by total genomic DNA studies. In our study, Actinobacteria isolation was differentiated with various plant parts (roots, shoots, leaves, and fruits) and soils.
ISP2 (Yang and Song 2018), ISP4 (Anwar et al. 2016), starch casein agar (Yamei et al. 2018), modified starch casein agar from soil enriched with yeast extract (Zhu et al. 2014) were regularly used as a growth media in literature. MGYEA medium was preferred in our study to isolate different species or different organisms. When our study and previous studies were compared, it was shown that the number of isolates obtained after the studies on ISP2 medium (55 isolates) (Yang and Song 2018) and ISP4 medium (four) (Anwar et al. 2016) did not reach the number 416 isolated from our study (the list of the bacteria were given at Supplementary file 3). Actinobacteria isolation agar (AIA) APT: ammonia production test; PPT: protease production test; CPT: chitinase production test: β-GPT; beta galactosidase production test. (Gopalakrishnan et al. 2014) was used by different researchers for the isolation of endophytic Actinobacteria from walnut fruit. Endophytic Actinobacteria that can be cultured from olive and walnut trees are revealed for the first time in our study. Isolation of diverse Actinobacteria from different plants can also be interpreted as an indicator of Actinobacteria-plant specificity. The rhizosphere and plant parts of fruit trees are a group that is meagerly studied than other perennial plant groups, and annual herbs (tomato, potato, etc.) (Andreote et al. 2009;Buchholz et al. 2019). Endophytic bacteria are isolated from various plant groups. For example, Pseudomonas, Roseomonas, Acinetobacter, Moraxella and Brevundimonas were isolated after inoculation on bacterial nutrient agar from the embryonic structures of the hybrid walnut tree. Also, after total gDNA isolations performed from plant parts, it has been shown that organisms such as Proteobacteria, Firmicutes, Actinobacteria were found in branches (shoots). However, there is no genus-level information in the analysis after gDNA isolation (Pham et al. 2017). Considering the abundance of plant parts examined (five different olive and seven different walnut plant parts were examined) within the scope of the study, the number of Actinobacteria that can be isolated was only 33. However, Amycolatopsis were only found in endophytically without the preselection method of freeliving soil Actinobacteria with the usage of CaCO 3 .
In terms of growth characteristics of Actinobacteria on different media, ISP3 medium, which is a medium in which colony morphologies were examined, was used for separation after isolation in 'new species studies' (Oubaha et al. 2019). However, due to the possibility that the differences that may occur in a single medium are not distinctive determinant, ISP4 medium, in which spore formation was encouraged, need to use during the purity control of the isolates.
Since the plant growth regulatory-enhancing tests mostly contain bacteria groups other than Actinobacteria, most of the tests performed in our study were modified according to the growth characteristics of Actinobacteria required optimization. As plant growth regulatory-enhancing tests were siderophore production, phosphate solubility, IAA production, ammonia production (Dede et al. 2020), protease production, beta-galactosidase production and chitinase production optimized for Actinobacteria (all experiments were represented in Supplementary file 5).
For phosphate solubility tests, methods based on determining the solubility properties of insoluble phosphate molecules such as rock phosphate (Hamdali et al. 2008), tri-calcium phosphate (Nautiyal 1999) have been developed for phosphate solubility tests (Bashan et al. 2013). In our study, Pikovskaya medium (Rodríguez and Fraga 1999) was preferred because it does not prevent the development of different Actinobacteria on it, and calcium phosphate was shown to be soluble. The duration of the experiment requires an incubation period of at least 14 days. The medium recommended for the siderophore production test (Cappuccino and Sherman 2014) was not sufficient for the growth of Actinobacteria in our study. Therefore, with the modification, the ISP2 medium was put into analysis after confirming with positive controls. In a study, the medium was modified to determine the production potential of organisms after 7 days of incubation to perform siderophore analysis (Khamna et al. 2009). The incubation period was chosen as 14 days (Dede et al. 2020) since developmental characteristics of organisms can be observed accurately following the use of MCAS.
In our study, a non-tryptophan-dependent IAA production test was carried out, and in some Actinobacteria IAA production could not be determined due to the high pigment production of the organism. As a result of experimenting with different media, the peptone water was preferred because of the low pigmentation levels. Takizawa et al. reported that IAA production occurred in 138 out of 210 Actinobacteria from Egypt with the use of L-tryptophan (Takizawa et al. 1993). In our study in which non-tryptophan dependent IAA production was tested 214 out of 422 isolates were positive.
There was no need for any modification on the medium and reagent used in determining ammonia production (Nimnoi et al. 2010). The only modification was made by extension of the time.
It is recommended that the chitin used in the production of chitinase be converted to the suspended chitin (colloidal chitin) format for easier degradation by organism (Roberts and Selitrennikoff 1988). In a study (Gopalakrishnan et al. 2013), colloidal chitin was formed with methanosulfonic acid. In our study, the usable form of the chitin was prepared with concentrated hydrochloric acid (HCl, 37%). It was then shown that chitinase activities could be determined in colloidal chitin with ISP2 medium for 14 days incubation.
The Actinobacteria required longer periods for detecting beta-galactosidase production, they were incubated for at least 3 days, and the test volume was increased from 100 µl to 300 µl to prevent loss of liquid by evaporation. Thus, it has been shown once again that the ONPG disks were usable (Flores et al. 1990).
The Actinobacteria with 16S rDNA gene sequence information in our study were identified as Micromonospora, Amycolatopsis, Nocardiopsis, and Streptomyces. Streptomyces spp. and Micromonospora spp. were belonged to both soil and plant parts while Amycolatopsis spp. were isolated endophytically from various plant parts and Nocardiopsis spp. was only isolated from the soil. Streptomyces has been identified as the most encountered genera members like the other studies (Shimizu et al. 2000;Vurukonda et al. 2018;Dede et al. 2020). According to Figure 1, Supplementary file 12 and 13 with usage of maximum parsimony tree, and neighbor-joining tree, there was no significant difference in organisms between plants and plant-soil dispersion. If the soil Actinobacteria selection was performed like the endophytic isolation processes, that would have isolated the other genera members.
Since Actinobacteria are the most known secondary metabolite producer bacteria group (Jensen et al. 2007), antifungal activity tests were included in the study to examine their antifungal properties. In our study, experimental analyses were created with plant pathogenic fungi. Endophytic Actinobacteria isolated from walnuts and olives were found to have surpassing antagonistic effects than free-living Actinobacteria, and these samples were included in Streptomyces and Amycolatopsis genera. A Nocardiopsis isolate inhibited the growth of a single pathogenic fungus. Unfortunately, Micromonospora members (El-Tarabily et al. 2009;Martínez-Hidalgo et al. 2015), known for their various antagonistic activities, did not show any antifungal effect in our study. Antagonistic efficacy tests can be performed in the form of actinomycete mycelium disks (Shimizu et al. 2000) or by planting fungus in the empty parts of the Actinobacteria planted in the middle of the Petri dish (Aouar et al. 2012). In our study, the effectiveness was investigated by dropping 20 µl of Actinobacteria in suspension.
The fatty acid profile is an important chemotaxonomic character in microbial identification particularly for classification of novel isolates. In our study, it was determined that the fatty acid profile of the EA18-coded isolate was different from other organisms, and even it contained C 12:0 3OH, and 'sum in feature 7 fatty acids, which were not seen in any actinomycete (Supplementary file 8). While the cultural characteristic of this sample showed an actinomycete-like colony morphology (agar penetrating micelle-like structure), it was determined that it was a Pseudomonas species after 16S rDNA sequence was examined. It shows that the analysis with morphology is not enough to pre-identification processes of the microorganisms.
A. endophytica (Miao et al. 2011), A. suaedae (Chantavorakit et al. 2019), A. jiangsuensis (Xing et al. 2013) have been isolated from plant structures endophytically in previous studies and have been introduced to the literature as a new type species. In our study, six endophytic members belonging to the genus Amycolatopsis were identified supporting the endophytic character of the genus Amycolatopsis.
The hydroponic production prevents root losses that may occur in experimental stages involving soil and removes the possibility of non-homogeneous distribution of soil contents used in pots.
Although plant growth had been demonstrated with the use of Actinobacteria in different studies (Shimizu 2011;Anwar et al. 2016), all of the experiments were designed with soil-based studies. In our study, wheat seed applications with Actinobacteria in the hydroponic medium have been carried out for the first time as far as it is known. The Actinobacteria that have been isolated in our study have the plant growth promoting abilities and therefore have the potential to be used as microbial fertilizer particularly in Turkey. This procedure has prevented the loss of the root mass of plants and could perform on various fast-growing plant species, specifically Poaceae or Gramineae family (Supplementary file 11).

Conclusion
In our study, a total of 422 which are 389 Actinobacteria from the rhizosphere and root surface soil samples of olive and walnut trees, and 33 from various plant parts (root, stem, and fruit) were isolated. A total of 195 isolated Actinobacteria were found to be capable of producing siderophore, 165 isolates dissolved phosphate, 214 isolates produced IAA, 376 isolates produced ammonia, 228 isolates produced protease, 221 isolates produced beta-galactosidase, 230 isolates produced chitinase to regulate and increase plant growth in different ways. Only 56 out of 389 (⁓14.4%) free-living Actinobacteria had antagonistic activity, whereas 16 out of 33 (⁓48.4%) endophytic Actinobacteria had antagonistic activity. 16S rDNA analysis revealed that the most common free-living Actinobacteria were Streptomyces and most common endophytic Actinobacteria were Amycolatopsis. EA12 (Amycolatopsis spp.), EA22 (Amycolatopsis spp.), and Z109 (Streptomyces spp.) Actinobacteria increased wheat root elongation and Z109 sample increased wheat stem growth with the usage of the hydroponic systems.

Type of contribution
AD and KG planned the project and experiment designs. AD performed all experiments, data arrangements, drafting the manuscript and writing processes. KG also checked and corrected the writing processes with suggestions.