Isolation, Identication, biocontrol activity and plant growth promoting of a superior strain Streptomyces tricolor strain HM10

Purpose: Streptomyces are familiar with biocontrol activity and producing a broad range of biologically active substances. Our goal was to isolate a local Streptomyces bacteria that can promote plant growth via siderophore, indole acetic acid (IAA) production and phosphate solubilization and biocontrol the phytopathogens to reduce the heavy usage of chemical fertilizers and fungicides. Methods: The designated strains were isolated from local soil samples in Saudi Arabia via the standard serial dilution method, identied morphologically, by scanning electron microscope (SEM) and 16S rDNA sequencing and screened against many soils borne fungi. Siderophore, IAA production and phosphate solubilization were detected and measured. Results: A strain designated Streptomyces tricolor strain HM10 was isolated and showed aerial hyphae in SEM. Selected isolate showed highly homolog revealed to 99.69 % with the type strain Streptomyces tricolor LMG 20328. In vitro experiments showed a growth inhibition of ten phytopathogenes by strain HM10 fermentation broth and production of plant growth promoting compounds such as siderophore, IAA and solubilize phosphate. S. tricolor strain HM10 exhibited high antagonism effect against tested fungi (i.e. Colletotrichum gloeosporides with inhibition zone exceed 18 mm whereas the lowest effect was against Alternaria solani (8 mm)). Furthermore, the highest siderophore production was recorded to strain HM8 followed by strain HM10 with 64 and 22.56 h/c (halo zone area/colony area). With regard to IAA production, Streptomyces strain HM10 was the highest producer with value 273.02 µg/ml. Conclusion: Streptomyces tricolor HM10 is an important biological agent that can be used to promote plant growth and control of phytopathogen.


Background
In the search for new and active natural resources and to nd friendly environmental solutions for yield increase and crop protection, Actinomycetes (especially Streptomyces) are gaining a great interest in agriculture as biological control agents and/or plant growth promoting bacteria. In which the production of secondary metabolites is synchronized with the aerial hyphae development and sporulation (Kunova, et al. 2016;Vurukonda, et al. 2018a). Two-thirds of the known antimicrobials produced by microorganisms are indeed synthesized by actinobacteria. Streptomyces produce 80% of the secondary metabolites with biological activities from the total production of actinomycetes (Waksman, et al. 2010; Barka, et al. 2015;Takahashi and Nakashima 2018). Soil borne bacteria, notably, Streptomyces are known from long time as the main antibiotics producer in the world. At least, more than 5000 documents stated by scientists mentioned that bioactive compounds are produced by the Streptomycetes genus. Actinomycetes that have been isolated from soil are able to inhibit the growth of plant pathogens such as Pantoae dispersa, Ralstonia solanacearum, Fusarium palmivora ( Streptomyces, the gram-positive bacteria, have been reported from every ecosystem. They show a lamentous and complex cycle resembling fungi. Streptomyces differentiation morphologically into a layer of hyphae that can differentiate into spore's chain. Furthermore, the genus has capability for complex multicellular development, since their spores germinate to form hyphae with aerial multinuclear mycelium (de Lima Procópio, et al. 2012 ;Jung, et al. 2018; Olanrewaju and Babalola 2019). The Streptomyces genus is ubiquitous in nature, which can be live in symbiosis with eukaryotic organisms ranging from marine animals to insects and plants to fungi or free-living in soil (Seipke, et al. 2012).
Many reported cited that Streptomyces species are able to promote plant growth and some of them suppress plant pathogens. By inhibiting fungal pathogens, they can protect plant roots via the production of antifungal compounds and lytic enzymes (Doumbou, Hamby Salove, et al. 2001;Palaniyandi, et al. 2013;Bonaldi, et al. 2014). Moreover, through the siderophore or auxin production, plant growth promotion has been observed. The positive effects of Streptomyces application were documented on root and shoot growth and on seed germination. The combination of streptomycetes capability for producing a wide variety of biologically active substance and their abundance in soil suggested that this class of organisms can play a signi cant role in microbe-microbe and plant-microbe interactions. Streptomycetes have been used as biological control agents against a variety of plant pathogens and considered a promising agent as biofertilizers (Sadeghi, et al. 2012;Law, et al. 2017;Jung, et al. 2018;Vurukonda, et al. 2018b).
The biological control agent selection usually starts with an in vitro screening by a dual culture assay between a selected group of strains against a group of pathogens. A wide variety of antibiotics and enzymes as extracellular production protect plants against pathogens of plant diseases. Several properties associated with actinomycetes might explain their ability to act as biocontrol tools. Those properties are the ability to colonize on plant surface, the antibiosis against plant pathogens, extracellular proteins synthesis in particular, and the phytotoxins degradation of antagonistic Yekkour, et al. 2012;Singh, et al. 2018) which can be quanti ed as clear zone as a result to growth inhibition of the pathogen's mycelium.
Upon the beginning of sporulation and development of aerial hyphae, the production of Streptomyces secondary metabolites is induced. As a biological agent, Streptomyces ma.FS-4 is an important microbial resource for the control of plant pathogenic fungi in banana (Trejo-Estrada, et al. 1998;Boukaew, et al. 2011;Pliego, et al. 2011;Schrey, et al. 2012;Ji, et al. 2014;Duan, et al. 2020).
On the other hand, some fungal pathogens require iron (Fe) for their pathogenicity whereas bene cial rhizobacteria that produce siderophores are scavenging ferric iron from the environment and subsequently may inhibit the growth of pathogen via iron competition (Expert, et al. 2012). In the same time, these bacteria make the iron available for growth of plant and serve as plant inducers.
Otherwise, the environment contaminated too much by the heavy use of agrochemicals such as pesticides and/or fertilizers. Some opponents raised the trepidation from the intensive use of pesticides which drove to signi cant change in people's situation against pesticides usage in both surrounding environment and agriculture circumstances (Yoon, et

Streptomyces isolation and cultural characteristics
Cultural characteristics for isolated strains (i.e. Pigmentation, Opacity of Colony, Colony consistency and Growth under liquid media surface) were achieved. The distinguished pigments for isolated strains were cream, yellow to brown with sediment of balls in liquid culture (Table 1). Otherwise, aerial hyphae was detected in strain Actino12 (Streptomyces tricolor strain HM10) by scanning electron microscope (SEM), (Fig 1). Based on pigment production, morphological, physiological and 16S rDNA sequence, the isolated strains were identi ed. Out of eleven isolated strain, eight strain were selected to identify with sequence of 16S rDNA. The identi ed strains and their similarity with published Streptomyces strains at NCBI website (https://www.ncbi.nlm.nih.gov/) were listed in table 2.

Fungal PathogensScreening Streptomyces isolates with biocontrol activity
The eleven isolated Streptomyces strains were tested against 10 selected soil borne phytopathogens fungi via dual plate assay. Streptomyces tricolor strain HM10 and Streptomyces thinghirensis strain HM3 exerted inhibitory effects on all tested pathogenic fungi i.e. Fusarium oxysporum, F. graminearum, F. solani, F. Moniliforme, Colletotrichum gloeosporides, Alternaria solani, Thielaviopsis basicola, Botrytis cinerea, Myrothecium roridum and Rhizoctonia solani ( Table 3). The highest antagonism effect was observed from Streptomyces tricolor strain HM10 against all tested fungi (i.e. Colletotrichum gloeosporides growth was inhibited with inhibition zone exceed 18 mm whereas the lowest inhibition effect was against Alternaria solani (8 mm) (Fig 2). The second superior strain was Streptomyces thinghirensis strain HM3 which showed antagonistic activity against all the tested fungi with inhibition activity ranged from 3 to 15 mm. On the other hand, four identi ed strains include Streptomyces sp. strain HM2, Streptomyces sp. strain HM6, Streptomyces panayensis strain HM7 and Streptomyces sp. strain HM8 showed no secondary metabolites activity or antagonistic effect with tested fungi.

Bacterial Strains
The inhibitory effect of fermentation broth that comes from identi ed strains against three kinds of bacteria is shown in Table 4. The fermentation broth of Streptomyces thinghirensis strain HM3 had better inhibitory effect on E. coli (gramnegative) than B. subtilis (gram-positive) but no inhibitory effect on Pseudomonas putida was observed (Fig 3). The Streptomyces griseorubens strain HM1 fermentation product exhibited inhibitory effect with Pseudomonas putida tester strain. Meanwhile, six strains showed no inhibitory effect with the tested bacterial strains.

Screening isolated Streptomyces strains with Plant Growth Promoting
Siderophore production All identi ed Streptomyces strains have the ability to produce siderophores and chelate the iron ions from the CAS medium (Table 5, Fig 4). The largest clear zone was recorded to strain Streptomyces sp. strain HM8 followed by Streptomyces tricolor strain HM10 with 64 and 22.56 h/c (halo zone area/colony area), respectively, and Streptomyces thinghirensis strain HM3 showed the lowest value with 1.67 h/c.

Phosphate solubilization
Four of eight (50%) Streptomyces strains had clear ability to solubilize phosphate with nearly the same capability ( Table  5). The Other two strains (25 %) had traces of soluble phosphate whereas two more strains had no ability to solubilize phosphate (Streptomyces sp. strain HM4 and Streptomyces sp. strain HM6).

Phylogenetic Analysis
For phylogenetic classi cation of bacteria, sequencing of encoding 16S rRNA gene were aligned against identi ed strains using ClustalW in MEGA X software. The phylogenetic analysis of the identi ed eight strains was conducted with related species their accession numbers were obtained from (Kaur, et al. 2019b). To show their phylogenetic relationships ( Figure 5) the evolutionary history was inferred by using the Maximum Likelihood method, Tamura-Nei model and Neighbor-Joining method for initial tree. This analysis involved 29 nucleotide sequences and the evolutionary analyses were conducted in MEGA X. Consistently, the phylogenetic tree also con rmed that these eight isolates belonged to genus Streptomyces. Two groups were constructed in the tree, group I contained six identi ed Streptomyces strains while the strain S. tricolor HM10 and Streptomyces sp. strain HM8 belongs to group II. Moreover, the closest relatives to strain Streptomyces tricolor HM10 (MN527236) was the type strain S. tricolor LMG 20328 (AJ781380), and S. fradiae NBRC 12773 T (AB184134), respectively. Furthermore, the second superior strain Streptomyces thinghirensis HM3 (MN527231) belonged to the group I, showed relationship with Streptomyces griseorubens HM1 (MN527229).

Discussion
Streptomyces are known for biocontrol activity against plant and animal pathogens. For a wide variety of plant pathogens, Actinomycete-fungus antagonism has been demonstrated. S. tricolor HM10 (MN527236) and S. thinghirensis strain HM3 (MN527231) had a signi cant effect against ten tested soil borne fungi with broad spectrum antifungal activity.
Moreover, Streptomyces sp. 9p had broad spectrum antifungal effects against four phytopathogens: Collectotrichum gleosporioides OGC1; Alternaria brassiceae OCA3; Phytophthora capsici; and Rhizoctonia solani MTCC 4633 (Shivakumar, et al. 2012). Streptomyces hygroscopicus strain SRA14 exhibited in vitro antagonism and inhibition growth against Colletotrichum gloeosporioides and Sclerotium rolfsii fungi due to extracellular antifungal metabolites present in culture ltrates when culture ltrates was collected from the exponential and stationary phases; whereas isolated Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, from grapevine plants reduce the fungal pathogens infection rate. Streptomyces albireticuli MDJK11 and S. albo avus MDJK44 showed robust inhibition on the F. solani growth and the biocontrol activity of MDJK44 was more stronger than that of MDJK11 (Prapagdee, et al. 2008 Based on 16S rRNA, eight Streptomyces strains were identi ed and phylogenetically analyzed. Streptomyces strain HM8 and HM10 located in group II while the rest six identi ed strains located in group I which means more differentiation from those six strains. In Pakistan, (Anwar, et al. 2016

Conclusions
S. tricolor HM10 (MN527236) and S. thinghirensis strain HM3 (MN527231) had a signi cant effect against ten tested soil borne fungi with broad spectrum antifungal activity. Strain HM10 showed high value of siderophore and IAA production with ability to solubilize phosphate which can help to promote plant growth.

Streptomyces isolation and media composition.
A total of ve soil samples were collected from10-20 cm depth of cultivated lands, Qassim region, Saudi Arabia.

Observation of morphological characteristics
The morphological properties of isolated Streptomyces strains were characterized such as colony characteristics, pigment color, areal hyphae, opacity of colony, colony consistency, fragmentation pattern and growth under surface of liquid media. For aerial hyphae and hypha characteristics in scanning electron microscope (SEM), Actino12 strain was grown for 48 h in growth medium. Harvested cells by centrifugation at 6.000 rpm for 10 min were processed via a critical point drying method (Dhanjal and Cameotra 2010); With phosphate buffered saline (PBS, pH 7.4), the cells were washed three times and xed by incubation in modi ed Karnovsky's xative solution (2.5 ml of 50% Glutaraldehyde, 2 gm Paraformaldehyde) for four hours. Again, cells were washed with PBS, followed by a distilled water wash and dehydrated by critical point drying through a series of alcohol dehydration steps (30%, 50%, 70%, 90% and 100%). The dehydrated samples were layered with t-butyl alcohol for freeze drying, sputter coated with titanium and viewed at 1,000 to 5,000 X magni cation on a scanning electron microscope (AMRAY 3300FE).

Physiological and biochemical characteristics
The isolated Streptomyces were cultured at 28 °C for 7 days in Tryptone Soya Agar medium. The soluble pigments color, the hyphae color and airborne hyphae were detected in Glucose Soya bean meal agar.
PCR ampli cation of 16S rRNA and Phylogenetic relationships DNA was extracted according to the simple method of DNA extraction with little modi cations (Cook and Meyers 2003). Isolated Streptomyces strains were grown in TSB (tryptone soya broth) at 30 ºC for 48 h. Cells were collected with centrifugation at 12.000 rpm for 3 min. and washed once with TE buffer (pH 7.7). Cells were resuspended again in 0.5 ml TE buffer, heated in boiling water bath for 10 min and allowed to cool then centrifuged (12000 rpm for 5 min). Extracted DNA was transferred to a clean tube and stored at 4 C for PCR ampli cation. PCR ampli cation was conducted using GoTaq® green master mix (Promega, USA) for 16S rDNA in 50 µl volumes by universal primers 27 F 5′-AGA GTT TGA TCA TGG CTC AG-3 and 1492 R 5′-TACG GTT ACC TTG TTA CGA CTT-3. PCR products were electrophoresed on 1% agarose gels to ensure the fragment of the correct size had been ampli ed. Products were puri ed and sequences (Capillary Electrophoresis Sequencing (CES), ABI 3730xl System, Macrogen Company, South Korea). A phylogenetic tree was inferred with a maximum likelihood method with following parameter: Tamura-Nei model, Neighbor-Joining method for initial tree, Bootstrap method approach, Uniform Rates. Evolutionary analyses were conducted in MEGA X (Kumar, et al. 2018).

Antimicrobial Activity Assays
Isolated Streptomyces strains were grown for 3 days in GSB liquid media. Antifungal activities against 10 fungal plant pathogens were measured according to (Kanini, et al. 2013). For the previous assay, potato dextrose agar (PDA) plates were used for the fungal strains grown for 3 days at 30 ºC, then a 6 mm mycelium disk from each selected phytopathogenic fungi were then placed in a new PDA plate center. On the other hand, bacterial inoculations were put into the opposite sides of each PDA plate, containing 50 µl of a 5-day culture from each tested Streptomyces strain. The inoculated plates with fungi and Streptomyces were incubated for ve days at 28 °C. The antagonistic activity was observed via measuring the inhibition zone distance. On the other side, antibacterial assay was measured via ve-day cultures ltrates from grown Streptomyces tested strain against selected bacteria by using the Kirby-Bauer agar well-diffusion method with modi cations (Institute 2011), Brie y, each tested strain was grown overnight in LB media, an inoculum from each tested strain (about 2 ml) were added to 25 ml from new LB media before solid (at nearly 50 ºC). Six mm (diameter) wells were perforated in the agar medium, and 50 µl of each 5-days Streptomyces culture were placed into the well and incubated at 30 or 37 ºC (according to the best temperature for each bacteria). Inhibition zones were measured after 24 h of incubation.

In vitro Assessment of Plant Growth Promotion (PGP) Traits in Streptomyces
Three parameters related to plant growth promotion were evaluated in isolated Streptomyces strains.

Siderophores production
The CAS (Chrome Azurol S) general assay to detect siderophore production according to (Schwyn and Neilands 1987) was applied. Iron (III) solution was prepared by mixing 1 mM FeCl3 in 10 ml of 10 mM HCl. In another conical ask, 60.5 mg of CAS was dissolved in distilled water (50 ml). The orange color mixture was then added to the previously prepared solution of the iron (10 ml) which turned the solution color into purple. While stirring, the previous purple solution was slowly poured into HDTMA (hexadecyltrimethylammonium) (72.9 mg) dissolved in 40 ml of distilled water which turned the color into blue dark after mixing. Growing Streptomyces cells on PDA liquid medium were taken with approximately the same OD 600 and put into succinate medium mixed with CAS dye and incubated for 72-96 h. A clear to orange halo around growing cells were detected.
Color intensity and size of detected halos is directly related to chelating strength and the amount of produced siderophore.
Extra cellular indole-3-acetic acid (IAA) production Isolated Streptomyces strains were grown in nutrient broth medium for one day of incubation at 28 ºC. Cells were diluted up to (10 8 cfu/ml) in NB medium supplemented with L-tryptophane (500 µg/ml) and grown with shaking for 5 days at 28 ºC. Cells were centrifuged at 12,000 rpm for 10 min and the supernatant was collected. Using Salkowski reagent (1 ml of 0.5 FeCl3 in 50 ml of 35% HCLO4), IAA concentration was measured by colorimetric assay (Bano and Musarrat 2003) and the pink color of individual assays was measured after 25-30 min using spectrophotometer at 530 nm. Standard curve was achieved to evaluate the IAA concentration.

Phosphate solubilization
Pikovskaya agar (PKV) medium were prepared and Ca 3 (PO 4 ) 2 was added separately after autoclaving to agar plates. A 50 µl of each strain containing approximately (10 8 cfu/ml) was added to agar plates and incubated for 5 days at 28 ºC. Bacterial colonies with clari cation halos around them were considered as phosphate solubilizers (Donate-Correa, et al.

Availability of data and materials
All data generated or analyzed in this study are presented within this manuscript. All materials used in this study including raw data shall be available upon reasonable request. The 16S rRNA nucleotide sequences for selected eight Streptomyces strains were deposited in GenBank (NCBI) under the accession numbers MN527229 -MN527236.

Competing interests
The authors declare that they have no competing interests.   Streptomyces tricolor strain HM10 ---0 1, + A thin yellow area surrounding the colony (about 1 mm width), ++ less than 5 mm width of yellow area surrounding the colony, +++ more than 5 mm width of yellow area surrounding the colony. D = Diameter. h/c = halo zone area/colony area.
2, + A thin transparent area surrounding the colony (about 1 mm width), ++ less than 5 mm width of transparent area surrounding the colony, +++ more than 5 mm width of transparent area surrounding the colony.
3, Quantitative estimation of IAA as microgram per ml according the equation: y = 185.8 x + 41.05. Figure 1 Scanning electron microscopy of Streptomyces tricolor strain HM10 hyphae grown on GSA medium  Antibacterial activity of some selected isolated Streptomyces against two gram negative bacteria E, coli and P. putida.

Figure 4
Iron cheating of isolated eleven Streptomyces strains in CAS general assay to detect siderophore production according to (Schwyn and Neilands 1987)