Molecular diagnosis of Streptomyces genus and bioactive potential against pathogenic microbes

: This study (40) locally isolated the genus Streptomyces from soil samples collected from different regions of Iraq ( Nineveh , Erbil , Duhok ) and evaluated their antagonistic. The isolates were found to have bioactivity against grampositive and negative bacteria and fungus. Streptomyces were isolated on (S.G. medium), and morphological similarities and the 16 srRNA sequencing were used to characterize them. The results of a polymerase chain reaction (PCR) with eight strands of DNA gene picked from local bacteria isolates in a volume range of (900–1000) base pairs. The nitrogenic base sequence determined the polymerase chain reaction products of DNA samples selected from 6 local isolates. These strands preserved the employed DNA ladder volume. According to DNA Blast NCBI data, the species are Streptomyces atrovirens, Streptomyces SP.S. coeuleroubidus, and Streptomyces bellus.


Introduction
Among the microbe, actinomycetes are one of the critical sources for the production of antibiotics. About two-Streptomyces alone 1 produce thirds of the antibiotics. Streptomyces are filamentous bacteria gram a positive with a high (G+C) content (70%) and found in all environmental 2 .
The genome code includes more than 20 genetic groups for secondary metabolites with high medicinal potential, such as antibiotics, which help fight against microbes 3 .
Many studies have found that Streptomyces has a considerable potential for producing secondary products such as antibiotics, growth factors, and pesticides 4 . Streptomyces can be distinguished from other actinobacteria by their 16 srDNA analysis and DNA -DNA hybridization 5 .

Materials and methods
Collecting of samples (20) Soil samples were gathered from various farms in Nineveh at depths ranging from 5 to 10 cm below the earth's surface. Calcium carbonate (CaCO3) treated the samples at a 1:10 ratio. They were then dried for 4 days at (40-45) degrees centigrade. Samples are collected in plastic bags, closed tightly, and then transferred to a Freezing container until needed 6 .

Isolation of Streptomyces
(1) gram of dirt was well mixed in tubes with 10 ml of distilled water; then, a series of dilutions were performed. produced until the desired concentration was reached.10 -6 , 1 ml was taken from the last dilutions (10 -5 -10 -6 ) and placed on culture medium Starch -casein medium (S.G.) This was done three times for each sample (chilled to 45 degrees centigrade). The inoculum was then disseminated evenly using a sterile glass spreader and incubated for 7 days at 28 C0. Plates with 10-35 colonies were chosen, and the number of solitary colonies was re-cultured in the same medium to generate pure culture 7,8 .

Nutrient agar medium
By melting 23 grams of the medium in (1) liter of distilled water with a pH of (7.2) and sterilizing everything in the autoclave, this medium was created according to (oxoid) the supply firm's recommendations.
ZnSO4. 7H2O, The materials were dissolved in (1) liter of distilled water at pH (7.4) and sterilized in an autoclave 11 .

Isolation and characterization of Streptomyces
The Gram staining, colony morphology, and color of colonies, the starch mineral agar media, and using the slide culture technique been noticed the shape of the aerial and substrate mycelium, as well as the spore arrangement were used to characterize Streptomyces 12 .

Antifungal activity against phytopathogenic fungi
To evaluate the effect of Streptomyces isolates on the growth mycelium of plant pathogenic fungi, the Streptomyces isolates were transferred to the middle of a petri dish, distributed vertically in the petri dish by a sterile loop and incubated at 28-+1 0 C for ( 5 ) days, after the incubation period two discs from the eight-day-old fungal culture were transferred to half dishes ( 1.5 ) cm away from the growth line of the Streptomyces isolates. The control treatment contains fungi discs only, which are incubated 28-+ 1 for 5 days ( the incubation period depends on the arrival of the mycelium fungi to the edges of the dish 13 .

Antibacterial activity against bacteria pathogen
The bioactivity of the Streptomyces was studied against the microbial Agar diffusion method 14 . Asparagine glycerol agar plates were inoculated with Streptomyces isolates and incubated at 28-+ 1 0 C FOR ( 7) days. After the isolates were grown, 8 ( mm ) discs were taken by a sterile cork borer from the developing colonies of Streptomyces and transferred to the nutrient agar medium inoculation with pathogen bacteria.

Diagnosis of selected samples of bacteria Streptomyces
The selected Streptomyces was diagnosed to the species level using the diagnostic tests 12,[15][16][17] .

Preparation of inoculation
The elements in the inoculation medium are the same as those in the production media. They were cultivated in autoclave-sterilized 250 mL flasks with 50 mL each. They were inoculated by transferring a tip of Streptomyces cultured on Glycerol asparagine agar to the inoculum. The flask was then shaken in a shaker incubator for 3 days at 140 rounds per second at (28-+1) Celsius.

Cultural Condition
In a 250 mL flask, 50 mL of antibiotic manufacturing media was created. The flasks were tightly sealed, and sterilization in the autoclave was conducted. They were all allowed to cool before injecting inoculum prepared from the 3-day-old chosen isolation at a 2:1 (v/v) ratio. The flaks were kept in an incubator vibrator for 7 days at (28+-1) degrees Celsius and 240 round minutes.

Purification and acquisition of DNA from Streptomyces
The DNA from the Streptomyces samples was extracted using Geneaid's analysis kit.

PCR reactions: Genomic DNA Extraction
The T.E. (the solution was used to optimize the DNA concentration in all of the samples used in this experiment, and it was successful in obtaining the desired concentration for the PCR reaction to continue ( 50 nanograms per microliter ).
In a (0.2 ml) Eppendorf tube provided by the British business, the DNA sample was mixed with the specific primer for each gene to make the master reaction for each PCR reaction ( bio labs ). Using distilled water, the reaction volume was set to 20 microliters, and the reaction was placed in a microfuge for 3-5 seconds. The tubes were then placed within a heat cycler, which used a customized program for each reaction to achieve the polymer reactions. The samples were then electrophoresed using a bio labs ladder in a well of a 2 percent agarose gel for 60-70 minutes, and the gel was filmed using Gel documentation.

Streptomyces molecular diagnostics based on 16srRNA amplification
To identify the amplification area, Add 4 microliters (100 nanograms) of DNA template and 1 microliter (10 picomols) of each gene primer were added to the final mix Primer Forward: AAGCCC TGG AAACGGGGT Primer Reverse : CGTGTGCAGCCC AAGACA After that, the reaction tubes were placed in the thermal cycler, and the polymers reaction was carried out according to the program listed in the table. (1).

DNA Sequencing analysis
DNA sequencing is the most frequently and commonly utilized technique for finding mutations and SNP variants in DNA samples. The sequence of amplified portions of DNA is used to determine, and PCR reactions usually determine research mutations. DNA sequencing results have gotten more precise in determining mutations in recent years 18,19 . If the PCR reaction produces many strands, they are purified, and the desired section of the DNA is recovered from the gel; however, if the reaction produces just one strand, it will be the predominant strand and will be used to identify the sequence 20 .

Using DNA sequencing to determine the nucleotides of the amplifier section
The genetic sequences of the samples were determined using the PCR technique outlined previously. Used in the study, and the primers were analyzed with the Hitachi 3130 Genetic Analyzer device. The results were analyzed using the BLAST tool, which compared the genetic sequences to those in the National Center for Biotechnology Information) NCBI( database.

Diagnosis
The Streptomyces samples were identified using the slide culturing technique, one of the best techniques (on a genus level) to display substrate and hyphae, which are the differentiating traits that identify thread-like bacteria 16 .
The substrate hyphae are spore-free, well-branched, and unsegmented. While the aerial hyphae appeared to be a darker, thicker, and less branched thread than the substrate mycelium, the aerial hyphae have a sporophore containing chain of spores, which can be erect (rectus), spiral (spiral), or Crict with waves (rectus -flexible) depending on how these spores are arranged21. figure (1) .
When the isolates were cultivated on different media types, they revealed a variety of colors. They could not produce melanin and other colors, with gray colonies being the most common 21 .

Genomic DNA polymers reaction
Using the forward primer, the following DNA,s reaction was performed on pure DNA acquired from organisms gathered from samples depending to the Geneaid protocol: (AAGCCCTGGAAACGGGGT) And the revers:(CGT-GTG CAGCCCAAGACA). (Maleki et al.,2013) In Figure 3, it can be seen that strands of pure DNA from the samples are of the same length (1000-9000) base pairs generated from the Streptomyces DNA specified polymer process. It has been discovered that they have the same length, indicating that there are nitrogenous base sequences in DNA that are mutual.
These isolates' genomic DNA can link with the primer and continue the reaction, resulting in additional DNA strands of the same length. These results are similar to what was found 22 When utilizing the polymers reaction approach to identify local bacterial samples and acquire a positive result, the length of the DNA created was (1000) nitrogenous base pairs, and so on. When using the polymers reaction method to analyze a bacterial local sample identification, the length of the DNA obtained was 1000 nitrogenous base pairs and so on (µ) marker evaluating and assessing the sequence of nitrogenous bases obtained from particular PCR., from local samples.
The findings of the specific polymer process on isolated DNA from local samples were forwarded to the NCBI, where they were compared to sequences in the gene bank figure (4).

Antifungal activity against phytopathogenic fungi
In this study of the ability of Streptomyces isolates to inhibit plant pathogenic fungi Table ( The Streptomyces can be biologically controlled by resistance / or killing fungi and bacteria pathogens; they are often Bioactive before the pathogen thoroughly infects its host. Recently a focus has been placed on different methods of developing biocontrol strategies against soil-borne fungi 23 . For example, the seed of the Arabidopsis thaliana.
Molecular diagnosis of Streptomyces genus and bioactive potential against pathogenic microbes Table 1. Shows the PCR reaction inside the thermocycler device. Before sowing, treatment with Streptomyces and Micomonospora SP was protected from infection by Erwinina carotovra and F. oxysporum.
It has been observed that Streptomyces have an antagonistic ability to stimulate the defense mechanisms in the plant 24 .
In this study 25 . Indicated a signification inhibition of mycelium growth in dual culture, using three strains of Streptomyces. S.P.ALP 07R, Streptomyces S.P. MRA 1W, against six types of plant pathogenic fungi Sclerotinia sclerotiorum, Fusarium oxysporm, f. S.P. Lactuca, Rhizoctonio solani Thielvopsis basicola, Phytophthora S.P. Pythium ultimum. According to (26), one strain of Streptomyces was found among Streptomyces' one hundred seven isolates. In vitro and in vivo, S.P. SI RO3 reduced the growth of mycelium Pestalotipsis theae by a percentage percent 86.15 percent 93.85, respectively.

Antibacterial activity against bacteria pathogen
In this study, the biological activity of Streptomyces against some pathogenic bacteria was also evaluated. The Streptomyces Bellus give inhibition against all pathogenic bacteria used in this study, while the Streptomyces SP. and S.atrovirence were effective against all bacteria except Klebis-la Penumonia Table (3). The researchers 27 . Showed that the inhibition extracted by acetate was effective against E SBLproducing E-coli. Numerous studies have indicated that the metabolites produced by actinomycetes showed high activity against Gram-negative, including E. coli 28 . The Streptomyces SMO1 gives a high inhibition against bacteria Staphylococcus aureus at a concentration ( 5 ml ) when grown on medium AIA at P.H. ( 7 ) for 7 days compared with Streptomycin ( 5 mg ) ampicillin ( 5 mg ) also indicated that among 8 isolates, one isolates S MO1 give inhibition against six bacteria strains except for K. pneumonia and S. typhimurium 29 . However, due to a double membrane barrier and brown transmembrane efflux, Gram-negative bacteria are more resistant to antimicrobial agents than Gram-positive bacteria 28 .

Conclusions
Among the results obtained from both aspects, the molecular study proved its effectiveness and usefulness for plant breeders and the building block for the success of any plant breeding program and the shortening of time in determining the appropriate varieties to conduct crosses, improving the characteristics of the yield.

Acknowledgments
In this section, we acknowledge any person who supports us to complete this project.

Conflicts of Interest
There is no conflict.