Population , morphological and chemotaxonomical characterization of diverse rare actinomycetes in the mangrove and medicinal plant rhizosphere

Actinomycetes populations in rhizosphere soils of mangrove forests in Cox’s Bazar and medicinal plant in Dhaka, Bangladesh were examined by simple dilution and an agar plate method. Actinomycetes populations (colony forming units/g, soil samples) ranged from 1x10 3 to 157x10 3 among 20 mangrove rhizosphere soil samples and 22x10 3 to 168x10 3 in 12 medicinal plant rhizosphere soil samples of Bangladesh. Total population and distribution of rare genera of actinomycetes were varied with the different rhizosphere samples and populations in mangrove rhizosphere soil were lower compared to medicinal plant rhizosphere soil. Strains under the genus Micromonospora were observed as major isolates in both mangrove and rhizosphere soil samples. About 17 genera of rare actinomycetes were observed in mangrove rhizosphere soil and 11 genera in medicinal plant rhizosphere soil with 20 or 40% unknown isolates. The further chemotaxonomic data of 19 unidentified randomly selected actinomycetes from mangrove rhizosphere soil indicated that the isolates belonged to the rare genera Micromonospora, Catellatospora, Nonomuraea, Actinomadura, Microbispora and 4 other unknown genera in the family Micromonosporaceae, Streptosporangiaceae and Thermomonosporaceae. This is the first intensive study and we confirmed that the mangrove and medicinal plant rhizosphere areas of Bangladesh are good sources for the isolation of diverse rare actinomycetes.


INTRODUCTION
Actinomycetes have been well known as the major producers of antibiotics and 61 to 70% of the novel bioactive metabolites were derived from actinomycetes (Moncheva et al., 2002;Miyadoh, 1993).In view of the microbiological aspect, one of the historical study in antibiotic screening was Weinstein's discovery of gentamicin (Weinstein et al., 1963) from a Micromonospora strain.After this, the isolation of rare and slow grower, uncommon actinomycetes (nonstreptomycetes) have become an increasingly important part of novel natural product discovery.Many ecological studies have been conducted on plants and animals *Corresponding author.E-mail: drmoonismet@gmail.com or iara@ksu.edu.sa.Tel: +966 1478 9585 or +966534509242. in mangrove forest, but very few studies have addressed the actinomycete community (Vijayakumar et al., 2007;Ara et al., 2002Ara et al., , 2004;;Hatano, 1997;Nakagaito and Hasegawa, 1991).Mangrove are woody trees or shrubs that grow in mangrove habitats generally located in subtropical and exclusively tropical regions, formed complex environments under the influence of tidal flow and soils in the environments are muddy, anoxic due to intermittent inundation, although it is known that mangrove roots that supply oxygen to rhizosphere soil (Mitsch and Gosselink, 1993).Mangrove forests in Cox's Bazar and medicinal plants garden in Dhaka city are one of the unexploited sampling sites of Bangladesh in terms of isolation of actinomycetes.Moreover, Cox`s Bazar is the world`s longest natural sea beach (120 km) and located 152 km south of Chittagong, Bangladesh.In this study, it has been emphasized that marine sediments may be valuable for the isolation of novel strains of actinomycetes, which could potentially yield useful new products.Humic acid-vitamin agar, containing soil humic acid as the sole carbon and nitrogen source, is used in our isolation method, as it reportedly enables the efficient recovery of rare actinomycetes while restricting the growth of non-filamentous bacteria (Hayakawa and Nonomura, 1987a, b).Subsequently, employing pretreatments of soil by drying and heating stimulated the isolation of rare actinomycetes (Nolan and Cross, 1988;Kim et al., 1995).Therefore, our main purposes for this study were to gain preliminary insight into the population, diversity of rare actinomycetes and to determine the tentative number of actinobacteria genera from whose representative can be isolated in the mangrove and medicinal plant rhizosphere soil samples of Bangladesh.

Soil samples and treatments
A total of 32 soil samples were taken from the mangrove sampling sites of Cox's Bazar, Chokoria, Kasturi Ghat, Maheshkhali mangrove forests and different medicinal plant rhizosphere of Dhaka city, Bangladesh.Moisture content and pH were measured for all samples and then dried in air at room temperature (25-28°C) for seven days and then dried soil and fine roots were removed by use of sieve.Isolation method was modified from Nonomura and Ohara (1971a, b).Moist heat treatment was performed by holding the 1 g soil samples mixture (1 g in 10 ml sterile normal saline) in a water bath at 70°C for 60 min for reducing the number of first growing non-filamentous bacteria.Heat-treated soil suspension in 10 ml of sterile normal saline (0.9% NaCl) again treated with an Ultrasonic Cleaner (Branson,5510) for 20 min at 40°C, as recommended by Hayakawa andNonomura (1987b, 1989).

Selection of rare actinomycetes in mangrove and medicinal plant rhizosphere soil
Strains were grown on different agar medium at 28°C for 21 days and then selected agar media such as, HV agar, ISP medium 2 and ISP medium 3 were observed by light and scanning electron microscopy (model S-2400 Hitachi, Tokyo, Japan).The samples for scanning electron microscopy were prepared as described by Itoh et al. (1989) and using simple freeze-dry technique (Ara et al., 2008).For the freeze-dry technique, the selected agar blocks were fixed in osmium tetroxide vapor overnight.The prepared specimen was rapidly frozen using liquid N2 and immediately freeze-dried for 5 h to overnight.The specimen was gold coated and examined under the microscope.Selected and purified rare actinomycetes were tentatively grouped under different genera on the basis of morphological characteristics and the isomers of cell wall diaminopimelic acid (DAP) detected by thin-layer chromatography (TLC) (Staneck and Roberts, 1974;Lechevalier and Lechevalier, 1980).

Classification of unknown rare actinomycetes from mangrove rhizosphere
The unknown group of rare actinomycetes in mangrove rhizosphere soil samples were further classified on the basis of morphological and chemotaxonomic characteristics.Chemical compositions of cells were analyzed by several standard methods.The freeze-dried cells used for chemotaxonomic analyses were obtained from cultures grown in yeast-starch broth (JCM medium no.61) on a rotary shaker at 30°C.The isomers of diaminopimelic acid (DAP) in the cell wall peptidoglycan were determined by using TLC as described by Staneck and Roberts (1974).Reducing sugars from whole-cell hydrolysates were analyzed by the HPLC method of Mikami and Ishida (1983).The N-acyl group of muramic acid in peptidoglycan was determined by the method of Uchida and Aida (1984).Phospholipids in cells were extracted and identified by the method of Minnikin et al. (1984).Methyl esters of cellular fatty ac ids were prepared and analyzed according to the instructions of the Microbial Identification System (MIDI) (Sherlock Microbial Identification System; MIDI, Hewlett Packard, Palo Alto, CA, USA) (Sasser, 1990).Isoprenoid quinones were extracted by the method of Collins et al. (1977Collins et al. ( , 1984) ) and were analyzed by a HPLC equipped with a Cosmosil 5C18 column (4.6 by 150 mm; Nacalai Tesque, Kyoto, Japan).Preparation and detection of methyl esters of mycolic acids were carried out as described by Tomiyasu (1982).

Actinobacteria population in mangrove and medicinal plant rhizospheres
Heat treatment, and antifungal, antibacterial antibiotics  : calculated by loss of sample weight after drying.
which was often used as pretreatment of soil, marine sediments, highly reduced the numbers of Gram-negative bacteria that commonly occur in soil samples and overrunning the isolation plates (Pisano et al., 1986;Barcina et al., 1987;Jensen et al., 1991).Tables 1 and 3 show the occurrence and distribution of rare actinomycetes in the 20 mangrove and 12 medicinal plant rhizosphere soil collected at different sampling sites of Bangladesh.Actinomycetes population ranged from 1 x 10 3 to 157 x 10 3 colony forming units (cfu/g of dry soil sample) in mangrove rhizosphere soil samples.Population and distribution of genera were varied with the different mangrove sampling sites (Tables 1 and 2).Based on the diversity and population of rare genera, the highest number of isolates was isolated from Chokoria (55%), Cox's Bazar (28%) and the lowest was from Maheshkhali (9%) and Kasturighat (8%) (Table 2).Total 279 actinomycetes were isolated based on colony morphology on HV agar, yeast extract-malt extract agar (ISP medium 2) and oatmeal-nitrate agar and but the presence of meso-DAP was observed in 241 isolates.Tentative generic classification of more than 80% of the total 241 isolates was achieved by morphological and chemotaxonomic properties.
On the other hand, actinomycete populations in rhizosphere soil samples of different medicinal plants ranged from 22 x 10 3 to 168 x 10 3 cfu/g of soil (Table 3).
The highest number of strains was isolated from the rhizosphere soil of Abroma augasta (Ulat Kambal, local name) (15%) and the lowest was from Adhatoda vasica (Basak, local name) (2%) (Table 3).Total population and distribution of actinomycetes were varied with different rhizosphere samples.In this study, total actinomycete populations in mangrove rhizosphere soil were lower compared to medicinal plant rhizosphere soils except for those of mangrove sample numbers 3, 4, 11 and 16 (Tables 1 and  3).In mangrove rhizosphere soils, actinomycete populations were 100 to 1000 times smaller than those of medicinal rhizosphere soil.It was reported by Cross (1981), in rhizosphere soils, actinomycete populations were 1000 to 10,000 times smaller than those of arable lands.However, the number of actinomycetes is generally estimated to be over 10 7 /g of dried soils collected from a normal environment (Alan and Stanley, 1990;Goodfellow and Williams, 1983).Therefore, our data showed that the number of actinomycetes in mangrove and medicinal plant rhizosphere soils was lower than ordinary soils.In our study, antifungal, antibacterial antibiotics may affect the growth intensity of some actinomycetes.Results also suggest that even oxygen is supplied to the mangrove rhizosphere through fine roots; these environments are not favorable for the easy survival of some actinomycetes like rhizosphere

Sample no. a
A* soil.On the other hand, the population of actinomycetes at sampling sites 3, 4, 11 and 16 were about 64 to 157x10 3 cfu/g, being similar to that of medicinal rhizosphere soil, which suggests that these sites are not highly anoxic, probably due to the absence of tidal influence.

Diversity of rare actinomycetes in genus level isolated from mangrove and medicinal plant rhizosphere soil
The genus diversity of isolates differed according to the sampling sites as shown in Tables 2 and 4. In general, Micromonospora was the major genus of rare actinomycetes in both mangrove and medicinal plant rhizosphere soil samples.The result was very similar to those of Ara et al. (2002), Hatano (1997), Jiang and Xu (1996) and Cross (1981); Micromonospora was the major genus in isolates from soggy soil.Among the total 241 isolates, about 17 genera of rare actinomycetes were observed and identity of the 20% isolates is unknown.In this study, Micromonospora (37.3%) was the dominant genus including other genera Actinomadura (15.8%),Microbispora (8.3%), Nocardiopsis (3.7%), Streptosporangium (3.3%),

Conclusions
This study shows successful isolation of diverse rare actinomycetes from mangrove and medicinal plant rhizosphere soils using simple heat pretreatment techniques with suitable nutrient media supplemented with antibacterial and antifungal agents.
The results may provide valuable information about the rare actinobacteria diversity in the region and offer an excellent source for the discovery of novel bioactive compounds.In addition, several unknown isolates in mangrove rhizosphere were classified chemotaxonomically and therefore conclude that mangrove rhizosphere is a good source for isolating new and diverse actinomycetes.

Table 1 .
Total population of rare actinomycetes in mangrove rhizosphere soil.

Table 2 .
Diverse genera of rare actinomycetes in mangrove rhizospheres.

Table 3 .
Total population of rare actinomycetes in medicinal plant rhizosphere soil.

Table 4 .
Diverse genera of rare actinomycetes in medicinal plant rhizosphere soil.

Table 5 .
Chemotaxonomic characteristics of the selected unknown rare actinomycetes isolated from mangrove rhizosphere soil.