Diversity of Streptomyces spp. in desert and savanna soils in Sudan

Objective The purpose of this study was to investigate streptomycete populations in desert and savanna ecozones in Sudan and to identify species diversity on the basis of 16S rRNA gene sequence analysis. Results A total of 49 different Streptomyces phenotypes (23 from sites representing the desert and semi-desert zone; 26 representing the Savanna zone) have been included in the study. The isolates were characterized phenotypically and confirmed using 16S rRNA analysis. The two zones showed similarities and uniqueness in the types of isolates. Shared species were: Streptomyces werraensis , S. enissocaesilis, S. leeuwenhoekii –like, but desert zone revealed unique species such as S. griseostramineus, S. chromofuscus –like and S. prasinosporus . Whereas, the savanna zone revealed unique species such as S. albogriseolus, S. djakartensis, S. chilikensis and S. variabilis . Streptomycetes are widely distributed in both desert and the savannah ecozones and most of them require full descriptions. Extending knowledge on Streptomyces communities and their dynamics in different ecological zones for health and production benefits is needed.

Streptomyces represent the largest taxonomic group within the domain Bacteria which is ubiquitously distributed in both aquatic and terrestrial ecosystems [1,2]. They are found in a wide range of habitats but particularly abundant in soil representing around 1 to 20% of the total viable count [2,3] and known to confer the characteristic earthy smell (geosmin) [4]. Streptomycetes are chemoheteroorganotrophs and distinguished by their tough, leathery colonies and filamentous growth. They have an important ecological role in the turnover of organic material and capable of using complex organic materials like carbon and energy sources and are involved in the breakdown of these products in the soil [5].
The microbial communities including streptomycetes are affected by biotic and abiotic factors notably vegetation, type of soil and climate. Actinomycetes diversity particularly streptomycetes is widely documented for various reasons including the continued generation of environmental isolates for pharmaceutical, biodegradative, and biotechnological screening. Actinomycetes isolated from soil and related substrates show primary biodegradative activity [5].
Exploring new habitats around the globe are becoming increasingly the focus for discovering new taxa of Actinobacteria, namely streptomycetes. These can be a likely new source of metabolites with various biological activities, such as antibacterial, antitumoral and antifungal [6]. Unexplored soil habitats of forest ecosystems in India were found to contain novel Actinobacteria species [7]. The habitat of Sudan has likewise remained untouched except a very few studies. Some areas in Sudan were found rich sources of microbial biodiversity holding within it immense novelty and potentiality of identifying new isolates for production of life-saving drugs such as amphotericin A. Marine habitat of Sudan was found a promising for Streptomyces spp., culture enhancing the authors said may influence the production of secondary metabolites [8] Two actinomycin D producing streptomycetes from Sudanese soil have been characterized and were found different from previously actinomycin producing species [9]. The finding of distinct phylogenetic lineages and the variation in the spatial distribution of clones suggests that selection pressures may vary over the soil-landscape [10].
This study aimed to isolate and identify streptomycetes from in desert and savanna zones in Sudan and to find out the species diversity in the two zones using 16S rRNA gene analysis.

Study site and sampling
Soil samples were collected sites in two ecozones; zone 1 was the desert and the semidesert and zone 2 was the low rainfall woodland savanna. The ecological zones were based on the ecological classification by Harrison and Jackson's [11].
The soil samples were collected during the dry season from the surface as well as 5-10 cm depth. 10g from five sections within each site were collected with a sterilized spatula. Isolation of Streptomyces spp.
Higher nitrogen content (HNC) medium [13] was used as a pretreatment method to assist the extraction and isolation of streptomycetes. HNC medium was prepared and store at 4°C. 0.5 grams of a soil sample was added to 50 ml HNC medium in a sterile Erlenmeyer flask. Flasks were placed on a shaker at 42°C for 1 hour. The suspension was left for 5 minutes and transferred to a clean Falcon tube.
Samples were diluted as 1:0 (no dilution; 1:5; 1:10 and 1:30. From each dilution, 0.1 mL was evenly spread onto the ISP2 medium using sterile Drigalski spatula. Inoculated plates were incubation at 28°C for up to three weeks. Suspected colonies were picked out and streaked onto fresh ISP2 medium to purify streptomycetes colonies [15]. The pure culture were stored on sterile vials containing 20% glycerol solution at -20 o C for further analysis.

Microbial characterization
The recognized Streptomyces isolates were studied for morphological and microscopic properties using light microscopy [2]. Strains that showed mutual characteristics were clustered to form preliminary Streptomyces color groups for further analysis.
The 16S rRNA was amplified from genomic DNA samples using eubacterial universal primers 27 F 5 ′ -AGAGTT TGA TCC TGG CTC AG-3 ′ and 1492R 5 ′ -GGT TAC CTT GTT ACG ACT T-3 ′ [16]. Amplification reactions were performed in a final volume of 20 μl, containing Promega Green Mix. The thermal cycling conditions were as follows: initial denaturation at 94°C for 5 min; 31 cycles at 95°C for 30 s, 54°C for 90 s and 72°C for 120 s; and a final extension at 72°C for 5 min. The amplification reaction was performed by Bio-Rad thermal cycler (MyCycler, Bio-Rad, USA) and the amplified products were examined by 1% agarose gel electrophoresis.
Obtained DNA sequences were aligned with other reference sequences available in the GenBank database using BLAST. Alignments were inspected visually. Then sequences were analyzed phylogenetically by the neighbor-joining method [19] using MEGA software. [20] Results A total of 49 different Streptomyces phenotypes (23 from sites representing the desert and semi-desert zone; 26 representing the Savanna zone) were recovered and identified.
Various bacterial colonies appeared on ISP2 agar after 3 days of aerobic incubation at 28 o C (Fig. 1). Separation and purification of colonies by using pick spot technique on ISP2 agar master plates were used to purify and store colony mass for further analysis.

Discussion
Streptomycetes are widely distributed in nature especially in soils with different structures and chemistry. This study was undertaken to highlight the presence of streptomycetes in two types of ecosystems and identify isolates to species levels. The results indicate the similarity in the types of isolates and the uniqueness of each ecosystem as Savanna revealed species that are not present in the desert soil. These findings emphasize the value for more analysis towards getting novel antimicrobial agents from these streptomycetes from either desert or savanna zones. Few scientific works have been carried out in this field notably in the distinctive ecosystems of Sudan.
In many sub-Saharan African countries including Sudan, have considerable diverse soils, such as clay in the east-central area and sand dunes in the west and north with variable climatic conditions [22]. Comparing the isolates, site-dependent patterns related to the ecoregion-specific soil type and annual rainfall were noticed. S. griseostramineus, S. Israel, which were related to specific environmental factors rather than to geographic distances and spatial distribution patterns [23].
These findings are similar to those reported from Atacama Desert in Chile where distinguished clades of Streptomyces pp. These were found with a widespread range of antibacterials and differing modes of action have been isolated from the Atacama desert [24]. The forces controlling desert ecosystem degradation rates are poorly understood, particularly concerning relevance of the arid-soil microbiome. Neilson et al. [25] distinguished important environmental and geochemical factors that model the arid soil microbiome along aridity and vegetation gradients spanning over 300 km of the Atacama Desert, Chile. Decreasing average soil relative humidity and increasing temperature explain significant reductions in the diversity and connectivity of these desert soil microbial communities and lead to significant reductions in the abundance of key taxa typically associated with fertile soils [25] as we have seen in the savanna soils examined in the present study.
In summary, we have effectively isolated and identified several streptomycetes from two different ecozones. A notable percentage of these isolates fit in uncommon species and some may represent new species. We believe the information provided will be of use to the Streptomyces researchers.

Limitations
Microorganisms in soils are exposed to different stress factors: chemical, physical and biological. Of these variable conditions, sometimes nutrients can be limited, preventing growth [1].
Given the unusual climatic conditions, the desert has been believed to home for unique potential Streptomyces which are mostly yet to be discovered. Our finding indicates the richness of the desert soil in revealing many species in major clusters but sample sites were limited. MAIH and MME supervised and participated in manuscript writing; MEH analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

Funding
This work was supported by a fellowship from Alexander von Humboldt Foundation, Bonn, Germany (MEH).

Availability of data and materials
All data generated or analyzed during this study are included in this published article and DNA sequence data have been kept in GenBank of the National Centre for Biotechnology Information (NCBI) as and accessions numbers are available in the article table.

Ethics approval and consent to participate
This research did not involve human participants, or the collection of human data or human tissue, and it did not involve animals. Accordingly, this research was not subject to ethics approval and consent to participate.

Consent for publication
Not applicable.

Figure 1
Colony morphology of Streptomyces spp. isolated from desert and savanna soils in Sudan using ISP2 medium, incubated at 28oC for 15 days (plat number indicated soil sits and letter indicates phenotype).

Figure 2
Phylogenetic relationships based on 16S rRNA sequences amongst 49 Streptomyces strains in relationship o closely related validly described species.
Evolutionary analysis was based on the Neighbor-joining method using MEGA7 software [21].