Art21julysep02

ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) July-September 2002 ACTINOMYCETES ISOLATED FROM SOIL SAMPLES FROM THE
CROCKER RANGE SABAH
C.W. Lo, N.S. Lai, H-Y Cheah, N.K.I. Wong and C.C. Ho1 ABSTRACT
A diversity of actinomycetes was isolated from various sites of top soils throughout the Crocker Range in Sabah. The soils were mainly collected during the expedition (15—25 October 1999) together with 2 soil samples collected on 28 November 1999 under Rafflesia keithii in the Rafflesia Reserve Forest, Gunung Mas. A total of 78 strains of actinomycetes, probably mostly Streptomyces, were obtained from different sites. Amongst these strains 20 have been aerobically grown in shaking liquid cultures. Acetone extracts of these cultures were screened for MAPK Kinase and MAP Kinase Phosphatase in a yeast system in the preliminary screening of novel cancer drugs. This screening system is based on the fact that the MAP kinase pathway is homologues from yeast to human. However, no such inhibitors were found. A few strains with pigmentation were collected from specific locations. INTRODUCTION
Actinomycetes are gram positive bacteria frequently filamentous and sporulating with DNA rich in G+C from 57—75%. Some of their secondary metabolites have employed as useful microbial compounds (Prescott, Harley & Klein, 1993). Examples include streptomycin from Streptomyces griseus for treatment of tuberculosis caused by Mycobacterium tuberculosis and the immunosuppress drug, tacrolimus (FK506) produced by S. tsukubaensis. Actinomycetes of about 100 genera exist in soils (Yokota, 1997). In their natural habitat, such as forests, the actinomycetes interact in various ways with the higher plants. The fallen tress, barks and flowers first provide nutrients both to the microbes and plants through microbial degradation of carbohydrates, lipids and proteins to sugars, fatty acids, glycerol and amino acids and ultimately to mineralisation. Besides providing these nutrients, plant secondary metabolites (such as dipterocarp resins) that are generally toxic to microorganisms, will need to be degraded or detoxified by certain microbes. These degraders (microbes) are selectively pressured and ultimately evolve to produce novel secondary metabolites of their possibly to counteract the toxic plant secondary metabolites (Park et al, 1999 and Ho et al, 2000). In this study, soil samples were collected in different habitats in the Crocker Range National Park to investigate the diversity of actinomycetes. Actinomycetes were then isolated on selective medium humic-acid + modified B vitamins and extracts were screened for biological activities of the secondary metabolites. http://www.arbec.com.my/pdf/art21julysep02.pdf ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) July-September 2002 MATERIALS AND METHODS

Soil samples: Soil samples were collected by sterile method from various locations visited
throughout this scientific expedition to Crocker Range Park (Figure 1), from an area of mist
forest (1400—1500m from sea level), submontane rain forest (Mahua), hill forest (uphill of
Mensalog River, Ulu Senagang) to cultivated areas (of introduced Theobroma cacao and
Tectonia grandis). Soil samples were air-dried under room temperature for about 30 days before
isolation (Table 1). A second set (Table 2) used air-dried soils stored at room temperature over a
long period (9-11 months).
Isolation of actinomycetes: 0.5g of soil samples was suspended in 9.5m1 of sterile distilled
water and was 1000-fold diluted. 0. lml of the dilutions was spread on humic acid + modified B
vitamins agar (HV) medium, pH 7.2, supplemented with cycloheximide. The plates were
incubated at 280C for 2 weeks.
Classification of actinomycetes: Isolated strains were transferred from HV medium onto
oatmeal agar medium, pH 7.2 and incubated at 280C for 14 days. Colouration of aerial mycelium
(on the surface of agar), substrate mycelium (underside of plate) and diffusible pigment were
observed.
Extraction of secondary metabolites: Submerged fermentation of purified cultures were carried
out in liquid medium of 2% mannitol, 2% peptone and 1% glucose, pH 7.2, for 5 days at 280C,
220rpm. Resultant broths were added with equal volume of acetone to extract secondary
metabolites (final concentration of extract in 50% acetone).
Screening:The acetone extracts were tested for inhibitory activity against MAPK kinase and
MAP kinase phosphatase inhibitors into yeast strains Saccharomyces cerevisiae MKKlP386 and S.
cerevisiae MKK1P386— MSG5 respectively.
RESULTS AND DISCUSSION

A total of 78 isolates of actinomycetes were isolated from 22 soil samples (Table 1 & 2) while 16
other soil samples without any isolate (Table 3). Some strains were isolated from soil under
Theobroma cacao, Rhododendron sp. and particularly Rafflesia kethii (Figure 2) and R. pricei
(Figure 3). Most of the isolates were presumed to be of the genera Streptomyces as they showed
good sporulation with compact, chalk-like dry colonies of different colours. A few pigmented
strains, unique to individual sites were observed.
Table 1. Strains isolated from air-dried soils collected in Crocker Range Sabah - isolated within one month after collection (Lo & Ho, 2001)
Type of forest
Collection
Soil from under
Location
Laboratory
http://www.arbec.com.my/pdf/art21julysep02.pdf ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) July-September 2002 tree/ plant
number of
isolates
• All of the isolates were recovered from humic acid + B vitamins* agar plates (pH 7.2) which http://www.arbec.com.my/pdf/art21julysep02.pdf ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) July-September 2002 had been incubated for 14-30 days at 280C. *B vitamins: thiamine-HC1, pyridoxin-HC1 and inositol. • The isolates from E1-El1 were recovered from humic acid + B vitamins** agar plates (pH7.2) which had been incubated for 14-30 days at 280C. *B vitamins: thiamine-HC1, pyridoxin-HC1, ribiflavin, niacin, inositol, Ca-pantothenate & p-aminobenzoic acid. • Isolated by ‘L’-Lo, C.W; ‘C’- Cheah, H-Y; ‘W’-Wong, N.K. ‘E’- Lai, N.S. Eric Table 2: Strains isolated from air-dried soils collected in Crocker Range, Sabah — after long storage, 9 to 11 months, at room temperature (Lo&Ho,2001) Laboratory
Collection
Soil from under
Type of forest
Location
Isolates
tree/plant
number of
• All of the isolates were recovered from humic acid + B vitamins** agar plates (pH 7.2) which had been incubated for 14-30 days at 280C. Isolated by Lo.C.W. ** B vitamins: thiamine-HC1, pyridoxin-HC1, ribiflavin, niacin, inositol, Capantothenate, p-aminobenzoic acid and biotin http://www.arbec.com.my/pdf/art21julysep02.pdf ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) July-September 2002 Table 3: List of soils without isolate of actinomycetes on humic acid + B vitamins agar plates
Soil from
Collection
Type of forest
Location
tree/plant
Park All the isolates were grouped into 3 colour groups (white series, grey series and brown series) based on the colour of aerial mycelium on oatmeal agar, after 14 days incubation at 280C (Figure 4). Majority of the strains were of the grey series, followed by white series and brown the least http://www.arbec.com.my/pdf/art21julysep02.pdf ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) July-September 2002 (Table 4). The grey series include pale grey, light grey, medium grey and dark grey; white colour group includes yellowish white, milky white and orange white while brown colour group includes greyish orange, brownish orange and greyish brown. As description of colour is quite subjective, a colour chart from Nippon 9000(1997) was used for standardization. A few strains varied according to sites are as follow: L-28 exhibited red pigmentation all over the agar medium, while L-27 exhibited orange colour extracellular pigment. Both strains were isolated from soil samples obtained under Theobroma cacao and Tectonia grandis respectively (Table 5). In the MAP kinase screening, 20 extracts were screened but none was found to be inhibitory. Table 4: Colour group pf isolated actinomycetes as determined in oatmeal medium Colour of aerial mycelium

Table 5: Strains with unique sporulation and pigmentation
Strain
Uniqueness
Sampling site
Secondary forest/cultivated area, Crocker Range The search for novel metabolites especially from actinomycetes requires a large number of isolates (over thousands) in order to discover a novel compound of pharmaceutical interest. The search will be more promising if diverse actinomycetes are sampled and screened. For this reason, soils were specifically collected under identified trees. This is based on the hypothesis that actinomycetes diversity may be influenced by the diversity of plant species as these bacteria grow profusely in the humus and leaf litter layer. Furthermore, different plants produce different type of secondary metabolites and some of these chemical compounds are toxic to soil microorganisms including actinomycetes. However, adaptation has in turn lead the actinomycetes to produce their own secondary metabolites. Although the collection sites have mainly been limited to fairly disturbed forests in the fringes of Crocker Range, yet they possess many actinomycetes in the leaf-litter humus layer. The conservation of this park will ensure the survival of these commercially important industrial microbes of biotechnological and pharmaceutical importance together with striking Rafflesia, orchids and Rhododendron. http://www.arbec.com.my/pdf/art21julysep02.pdf ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) July-September 2002 REFERENCES
Ho, C.C, G.Y.A Tan, I. Seow, N. Ajam, E.I. Tan, M. Goodfellow, A.C. Ward, R. Brown,
N.K. Wong, C.W. Lo, H-Y. Cheah, N.S. Lai and K.E. Suzuki.
(2000). Isolation, characterisation and biological activities of actinomycetes isolated from
dipterocarp rain forest soils in Malaysia. In: B.H. Nga, H.M.Tan & K.I. Suzuki (Eds)
Microbiology diversity in Asia. World Scientific, Singapore (in press).
Lo, C.W. and C.C. Ho.
(2001). Bioprospecting and conservation of rich microbial resources in rain-forests of Sabah,
Borneo for pharmaceutical industries. Proceedings of International Conference on “In-situ and
Ex-situ Conservation in the New Millennium” Kota Kinabalu, Sabah, 19-21 June 2000.
Park, D-J., S.H. Lee, C-I. Kim and M. Uramoto.
(1999). Isolation of rare actinomycetes from soil samples in specific micro environments-natural
lime cave and plant rhizosphere soil. Proceedings of International Conference on Asian Network
on Microbial Research, Chiang Mai, Thailand 29 Nov. —1 Dec. 1999. 728-737pp.
Prescott, L.M., J.P. Harley and D.A. Klein.
(1993). Microbiology (2nd Ed.) Wm. C. Brown Publishers, Dubuque.
Yokota, A.
(1997). Phylogenetic relationship of actinomycetes. Atlas of Actinomycetes. The Society for
Actinomycetes Japan, 194pp.
School of Science & Technology, University Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah, Malaysia. http://www.arbec.com.my/pdf/art21julysep02.pdf

Source: http://www.arbec.com.my/pdf/art21julysep02.pdf