Final Thesis

Sabah Society Journal Vol. 26 (2009) 1–91

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Screening for Potential Microbial Inhibitors against Prokaryotic and Eukaryotic Signal Transduction and

Isocitrate Lyase in Mycobacterium from Danum Valley, Sabah

Ho Wei Loon, Chan Kai Wai, Bernard Tzing Ziang Vui, Lim Siok Har, Ngao Wee Chen, Tong Mei Ling, Teh Soo Chin, Vun Su Chiun, Mak Ken

Hing, Jenifer Rolland L., Celistha Fay J. Ferdinand Deosing, Jessica Peter, Hew Chaw Sen, Lai Ngit Shin, Lee Ping-Chin and Ho Coy Choke*

Biotechnology Programme, School of Science and Technology, Universiti Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu,

Sabah, Malaysia.* E-mail: [email protected]

ABSTRACT

A diversity of actinomycetes and microfungi was isolated from various sites throughout the Danum Valley during expedition I (25/9–27/9/2004) and II (28/10–29/10/2004). A total of 217 soil samples were collected under trees that had been identified to species or genus level in Danum Valley Conservation Area; 535 actinomycetes and 93 microfungi were isolated using selective isolation media to obtain pure cultures. These pure isolates were grown aerobically to produce microbial secondary metabolites. Acetone crude extracts were screened for potential inhibitors against prokaryotic serine/threonine protein kinase (AfsK/AfsR) of Streptomyces griseus, the two-component signal transduction systems (KdpE/KdpD and PhoP/PhoR) and isocitrate lyase (ICL) in glyoxylate shunt of Mycobacterium. This research also screened for potential inhibitors against proteins involved in eukaryotic (yeast and human) signal transduction systems. These include glycogen synthase kinase-3β (GSK-3β) cascade, protein phosphatase 1 (PP1), MAP kinase cascade (MKK1 and MSG5) and Ras/Raf-1 protein-protein interaction. Four fungal strains (H9386, H9387, H9407 and H9409) inhibited AfsK/AfsR of Streptomyces griseus. One actinomycete strain (H11588) was found to be toxic to KdpE/KdpD in M. smegmatis. Actinomycete strain H11855 showed positive inhibitory towards PhoP/PhoR and 15 strains were toxic to M. smegmatis. Meanwhile, 1 fungal strain and 26 actinomycete strains showed toxicity to ICL in glyoxylate pathway in M. smegmatis. In PP1 screening, 37 extracts were toxic to yeast and 3 actinomycete extracts (H11533, H11534 and H11735) were found to be potential inhibitors. In GSK-3β screening, 32 strains were toxic in yeast and 9 actinomycete strains (H11462, H11490, H11526, H11668, H11686, H11695,

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H11720, H11785 and H11809) were found to be positive in the test. Five strains and seven strains of actinomycete showed toxicity in MKK1 and MSG5 respectively, and 37 actinomycete strains were found to be toxic in Ras/Raf-1 protein-protein interaction. No strains were found to inhibit KdpE/KdpD and ICL in M. smegmatis, MKK1, MSG5 and Ras/Raf-1 protein-protein interaction in yeast.

INTRODUCTION

Danum Valley Scientific Expedition was held on the 25–27 September and 28–29 October 2004. The 438-km2 Danum Valley Conservation Area (DVCA) is situated west of the nearest town, Lahad Datu on the southeast coast of Sabah. It is a 81-km drive from Lahad Datu to the main office of Danum Valley Field Centre (DVFC).

The forest in DVCA is dominated by dipterocarp trees with the canopy reaching a height over 70 metres in places. Some 90% of the area is classified as lowland dipterocarp forest with the remaining 10% being low canopy, sub-montane forest mainly found on Mount Danum in the heart of the conservation area. The DVFC is located beside the Segama and Palum Tambun rivers and is truly a remote setting as it is more than 20 km from any other habitation. Research efforts have revealed a tremendous variety of plants and a full range of Sabah’s lowland fauna. Danum Valley was chosen as the study site because of its unique features, which include its undisturbed primary lowland dipterocarp forest, botany, ecology, geology, hydrology, soils and zoology.

Soil samples were collected along the Nature Trail, Tembaling Waterfall Trail, West Trail (Dr. Clive Marsh Trail) and at Newbery Plot, which is two 4-hectare (40,000 m2) plots (plot 1 and 2), where trees greater than or equal to 10-cm girth were enumerated, mapped and identified in 1985–1987 (Newbery et al., 1999). Each plot is 100 m wide (W–E) and 400 m long (S–N) and they lie parallel to one another at ca. 300 m apart. The number of species recorded was 511 (in 164 genera and 59 families) in a total sample of 17,985 individuals. Plots 1 and 2 contain 388 and 387 species respectively, and there are 247 and 242 species of trees greater than or equal to 30-cm girth (Newbery et al., 1992). In plot 1, soil samples were collected under identified trees (30-cm girth and below), which were tagged on the bark with individual numbered aluminium discs.

Actinomycetes and filamentous microfungi were chosen in this study because they are prolific producers of many kinds of bioactive secondary metabolites. The members of these microorganisms produce many commercially important secondary metabolites, notably antibiotics (penicillin, streptomycin and vancomycin), anti-tumour agents (actinomycin D, mitomycin and doxorubicin), immunosuppressive agents (cyclosporin A, rapamycin and tacrolimus) and enzyme inhibitors (clavulanic acid, desferal and acarbose) (Demain, 1999). The actinomycetes and fungi are isolated

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SCREENING FOR POTENTIAL MICROBIAL INHIBITORS

from soils, purified and fermented to produce microbial cell extracts. These cell extracts are then tested on the screening systems developed for potential inhibitors.

One of the crucial conditions in natural products approach to drug discovery is the effective target-directed screening system to identify novel and specific inhibitors in the crude extracts (Kuo & Garrity, 2002). One such convenient screening system is to use the molecular targeted system in yeast (Parsons et al., 2003). In our laboratory, we used the genetically modified yeast systems to screen for inhibitors against protein kinases and phosphatases involved in eukaryotic signal transduction. These screening systems include the protein kinase pathway (MKK1, MSG5 and GSK-3β) as these proteins have mammalian homologues. We also screened for inhibitors against serine/threonine phosphatase (PP1) in yeast system and Ras/Raf interaction using the yeast two-hybrid system. Furthermore, Streptomyces-based screening system targeting at AfsK/AfsR serine/threonine protein kinase system, three Mycobacteria-based screening targeting at KdpE-KdpD, PhoP-PhoR two-component signal transduction systems and glyoxylate pathway in M. smegmatis were also applied in the screening system.

The abilities of Mycobacterium tuberculosis to infect macrophages, establishing a long-term, persistent infection in the host have made it one of the most widespread diseases in the world. During persistent infection, fatty acids are used as the main carbon source and, in response, the glyoxylate shunt is up-regulated. In mice, the disruption of icl gene, which codes for the glyoxylate enzyme isocitrate lyase (ICL), attenuates M. tuberculosis persistent infection (McKinney et al., 2000; Muñoz-Elías & McKinney, 2005). The strategy for survival during the chronic stages of infection entails a metabolic shift in the bacilli utilization of carbon source to C2 substrates, particularly in the form of acetyl-CoA generated by β-oxidation of fatty acids.

A rapid inhibitor screening strategy adapted from Sharma et al. (2000) is being conducted in our laboratory. This screening strategy essentially searches for potential inhibitors of ICL (isocitrate lyase)

Objectives of present study

1. To isolate a collection of diverse strains of soil actinomycetes & fungi from Danum Valley.

2. To screen for potential inhibitor of protein kinases & phosphatases.3. To screen for ICL inhibitor for persistent TB

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MATERIALS AND METHODS

Sampling sites. The study sites were situated along the Nature Trail, Main Trail West, Tembaling Waterfall Trail and at Newbery Plot (also called Sterling-Bern Plot) in DVCA (Figs. 1, 2, 3, 4a, 4b and Appendix).

Collection of soil samples. Soils below the fallen leaves near root areas under identified trees were collected aseptically into sterilized McCartney bottles with sterilized tongue depressors.

Soil pH determination. Ten grams of soil sample was mixed with 25 mL of distilled water. The reading was taken using a calibrated pH meter.

Isolation and purification of actinomycetes. An amount of 0.05 g of soil sample was mixed into 9.95 mL of sterile distilled water in a test tube. The mixture was mixed using a vortex mixer for about 30 seconds. Actinomycetes were isolated by plating out diluted (10-3) soil suspension (100 μL) onto humic acid B vitamins (HV) agar (0.05% humic acid, 0.05% disodium hydrogen phosphate dihydrate, 0.17% potassium chloride, 0.005% magnesium sulphate heptahydrate, 0.001% ferrous sulphate heptahydrate, 0.002% calcium carbonate, 0.5 mg/L each of vitamin B1 hydrochloride, riboflavin, niacin, pyridoxin-HCl, inositol, Ca-pantothenate, p-aminobenzoic acid, 0.25 mg/L biotin, 50 mg/L cycloheximide and 1.8% bacteriological agar, pH 4.4, or 6.7) and incubated at 28ºC for 2–3 weeks (Nonomura and Hayakawa, 1988). Cycloheximide was added as antifungal agent. A single colony was streaked onto oatmeal agar (2% Quaker oatmeal and 1.2% bacteriological agar) for purification.

Isolation and purification of microfungi. Microfungi were isolated by plating 10-3 fold serial dilutions of soil suspension onto potato dextrose agar (PDA) (25% potatoes, 2% dextrose and 1.5% agar, pH 5.5) incorporated with 0.75% NaCl. A concentration of 5 mg/L chloramphenicol was added as antibacterial agent. Colonies of fungi were purified in fresh PDA without NaCl and chloramphenicol and incubated at 28ºC for 1–3 weeks.

Preservation of actinomycetes. The matured (7–14 days) and single colonies of pure actinomycetes strains were stored in sterile 20% glycerol and kept at -20ºC.

Preservation of microfungi. Conidia of single colonies of microfungi were kept in sterile anhydrous silica gel particles at 4ºC (Ogata, 1962). Cultures were preserved by pipetting 0.5 mL conidia suspended in 4% non-fat milk into dry heat sterilized (180°C, 2 hours) 12 x 100 mm tubes half filled with anhydrous silica gel particles. The tubes were left to dry completely in a desiccator, sealed and stored at 4ºC.

Production of microbial secondary metabolites. A loopful of single colony of actinomycetes was aerobically grown in 10 mL of mannitol-peptone broth (2%

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D-mannitol, 2% peptone and 1% glucose monohydrate, pH 4.4 or 6.7) in a 125-mL Erlenmeyer flask and incubated at 28˚C with shaking at 220 rpm for 120 hours. pH 7.2 was chosen if no cells could grow under acidic pH. Fungi were inoculated into 10 mL fungi fermentation liquid medium (1% yeast extract, 1% peptone, 1% sucrose, 0.1% potassium phosphate monobasic anhydrous and 0.03% magnesium sulphate heptahydrate, pH 5.5) and incubated in the same conditions as actinomycetes. An equal volume of acetone was added to the fermented broth to produce crude microbial extract and kept at 4ºC.

Targeted screening systems for specific inhibitors. In all screenings, 20 μL of extract was added to a paper disc (Whatman no. 3, 6-mm diameter) unless stated otherwise. One Streptomyces-based screening system targeting AfsK/AfsR serine/threonine protein kinase screening system, three Mycobacteria-based screening systems targeting KdpE-KdpD, PhoP-PhoR two-component signal transduction systems and glyoxylate pathway in M. smegmatis. Three yeast-based screening systems targeting protein kinases, phosphatases and Ras/Raf-1protein-protein interaction are described below.

(i) AfsK/AfsR serine/threonine protein kinase screening system (Umeyama et al., 1999; Vun, 2005)

Spore suspension of Streptomyces griseus IFO13350 (H10000) was inoculated into 2 sets of YPD (yeast peptone dextrose) media (0.2% yeast extract, 0.6% peptone, 0.5% sodium chloride, 0.2% magnesium sulphate pentahydrate and 1.8% bacteriological agar, pH 7.2), one with the addition of 1.5% of D (+)-glucose monohydrate and another with 1.5% of D-mannitol. Paper discs soaked with 20 μL of extract were arranged on the plates and incubated at 28°C for 1 week. Inhibition of sporulation on glucose plate but not on mannitol plate was scored as positive. Spore absence was confirmed by microscopy observation.

(ii) KdpE/KdpD two-component signal transduction over osmotic stress screening system (Rolland, 2005)

The screening system was designed using wild type M. smegmatis mc2155 (H8000), which was grown using the modified M9 minimal media (0.7% disodium hydrogen phosphate dihydrate, 0.2% monopotassium phosphate, 0.5% ammonium sulphate and 0.01% magnesium chloride heptahydrate, pH 6.7) supplemented with trace elements (zinc sulphate heptahydrate 2 mg/L, calcium chloride dehydrate 1 mg/mL, sodium molybdate dihydrate 0.2 mg/L, cupric sulphate pentahydrate 0.2 mg/L and manganese chloride tetrahydrate 1 mg/mL), 75 μL of 0.18g/mL D (+)-glucose anhydrous and 15 μL of 1 M vitamin B1 hydrochloride with different concentrations of sodium ion or potassium ion (Na+ concentration: low 0.8%, high 4.0%; K+ concentration: low 100 μM, high: 1 mM) at 37ºC for 72 hours without shaking. The autoclaved bottom layer M9 agar (modified M9 minimal medium with 1% bacteriological agar) was poured into each sterile Petri dish (10 mL per plate). A volume of 250 μL of M. smegmatis seed culture grown in different concentrations of sodium or potassium was pipetted

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into 10 mL of top layer agar (modified M9 minimal medium, trace elements and 0.7% bacteriological agar) respectively with 50 μL of 0.18 g/mL D (+)-glucose anhydrous and 10 μL of 1 M vitamin B1 hydrochloride added. The seeded agar was poured onto the bottom layer agar and left to solidify. Paper discs soaked with 20 μL of extract were arranged on upper layer agar. The plates were incubated at 37°C and observation was recorded after 72 hours. High-growth inhibition zone observed only in low sodium or potassium plates, but smaller or no inhibition zone in high sodium or potassium plate indicated potential inhibitor for KdpE/KdpD two-component system.

(iii) PhoP/PhoR two-component signal transduction screening system (Groisman, 2001; Soncini et al., 1996; Tyagi and Sharma, 2004 and Véscovi et al., 1997)

Screening using wild type M. smegmatis mc2155 (H8000) was designed by paper disc susceptibility test method, which targeted Mg2+-dependent protein phosphorylation in PhoP/PhoR two-component system. M. smegmatis was cultured in 15 mL of modified M9 minimal liquid medium (0.7% disodium hydrogen phosphate dihydrate, 0.7% potassium phosphate dibasic, 0.1% ammonium sulphate and 0.25% sodium chloride, pH 6.7) supplemented with trace elements (zinc sulphate heptahydrate 2 mg/L, ferrous sulphate heptahydrate 5 mg/L, sodium molybdate dehydrate 0.2 mg/L, cupric sulphate pentahydrate 0.2 mg/L), 75 μL of 0.18 g/mL D (+)-glucose and 15 μL of 1 M vitamin B1 hydrochloride with different concentrations of magnesium sulphate heptahydrate (Mg2+ concentration: low 100 μM and high 1 mM) at 37ºC for 72 hours without shaking. The autoclaved bottom layer M9 agar (modified M9 minimal medium with 1% bacteriological agar) was poured into each sterile Petri dish (10 mL per plate). A volume of 250 μL of M. smegmatis seed culture grown in different concentrations of magnesium was pipetted into 10 mL of top layer agar (modified M9 minimal medium, trace elements and 0.7% bacteriological agar) respectively with 50 μL of 0.18 g/mL D (+)-glucose and 10 μL of 1 M vitamin B1 hydrochloride added. The seeded agar was poured onto the bottom layer agar and left to solidify. Paper discs soaked with 20 μL of extract were arranged on upper layer agar. The plates were incubated at 37°C and observation was recorded after 72 hours. Inhibition zone observed only in low magnesium plate but not in high magnesium plate indicated potential inhibitor of PhoP/PhoR.

(iv) Glyoxylate pathway screening system (Sharma et al., 2000; Hew, 2006) M. smegmatis mc2155 (H8000) was cultured in 15 mL of modified M9 medium (0.7% potassium phosphate dibasic anhydrous, 0.2% potassium phosphate monobasic anhydrous, 0.01% magnesium sulphate pentahydrate and 0.1% ammonium sulphate) with different carbon sources, D (+)-glucose anhydrous or sodium acetate at 37˚C for 72 hours without shaking. The bottom layer was prepared by pouring modified M9 agar into each sterile Petri dish and left to solidify. A volume of 750 μL of M. smegmatis seed culture grown in sodium acetate or 250 μL of M. smegmatis grown in glucose anhydrous was inoculated into 10 mL of top layer screening agar medium (modified M9 minimal medium and 0.7% bacteriological agar) with 0.02 M sodium

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acetate or glucose respectively with 10 μL of 1 M vitamin B1 hydrochloride added. The seeded agar was poured gently onto the bottom layer screening agar (modified M9 minimal medium, trace elements and 1% bacteriological agar) and paper discs soaked with 40 μL of extracts were arranged onto the agar. The plates were incubated at 37°C and observation was recorded after 72 hours. Inhibition zone observed only in acetate plate but not in glucose plate indicated potential inhibitor of ICL.

(v) GLC7 the catalytic subunit of serine/threonine protein phosphatase type 1 (PP1) in yeast screening system (Andrews and Stark, 2000; Tong, 2005)

The screening system used the temperature-sensitive mutant glc7-10 PAY700-4 (H10017) with cell cycle arrest at 37°C but the lethal effect was suppressed by high osmolarity (1 M sorbitol). The inhibitor acting on GLC7 protein in the wild type PAY704-1 (H10018) should have the same characteristic as that caused by the mutation, namely with growth inhibition at 37°C but with growth with the addition of 1 M sorbitol. A volume of 100 µL yeast culture was added into every 25 mL of YPD medium [1% yeast extract, 2% peptone, 2% D (+)-glucose monohydrate and 1.8% bacteriological agar, pH 7.2] and YPD with 18% D-sorbitol medium. An amount of 20 µL acetone extract was applied to sterile paper discs and tested on the plates. The plates were incubated at 25ºC and 37ºC for 120 hours and observation was scored. The crude acetone extract inhibit PAY704-1 will exhibit a mimic temperature-sensitive mutant PAY700-4 where there is no growth at 37ºC and save by 1 M of sorbitol while growing at 25ºC if the crude acetone extract shown positive inhibitory activity towards PP1.

(vi) GSK-3β screening system based on temperature sensitivity (Andoh et al., 2000; Chan, 2005; Mak, 2005; Teh, 2005)

A gsk-3 null mutant, where all 4 of the yeast GSK3 homologs have been disrupted demonstrates temperature sensitivity at 37°C, is suppressed by the expression of mammalian GSK-3β or its yeast homolog, MCK1 when transformed with pKT10-GSK3b (H10075) or Yep24-MCK1 (H10079), respectively (Andoh et al., 2000). The presence of GSK-3β inhibitor should mimic the phenotype of the gsk-3 null mutant, that is growth defects at 37ºC, as indicated by growth inhibition of transformant of gsk-3 null with pKT10-GSK3b or Yep24-MCK1. YTA003W with genotype MATa his3 leu2 ura3 trp1 ade2 mck1::TRP1 mds1::HIS3 mrk1 yol128c::LEU2 was transformed with pKT10-GSK3b or Yep24-MCK1respectively and grown in 25 mL of SC-Ura medium, shaking incubation, 180 rpm, at 37°C for 2 days. A volume of 400 µL yeast culture was inoculated in 100 mL of SC-Ura agar [0.67% yeast nitrogen base without amino acids and ammonium sulphate, 0.5% ammonium sulphate, 2% D (+)-glucose anhydrate, 1 mL each of 0.03 mg/mL of adenine (hemisulphate salt), 0.03 mg/mL of L-tryptophan, 0.03 mg/mL of L-leucine, 0.03 mg/mL of L-histidine and 1.5% bacteriological agar, pH 5.6]. For H10079 culture, 1.2M sorbitol was added to the culture medium. Paper discs impregnated with microbial extracts were arranged on the screening plates and incubated at both permissive temperature (25ºC) and at high temperature (37°C) for 72 hours. The presence of GSK-3β inhibitor will cause

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growth inhibition on plates incubated at 37°C. H10079 transformant will be rescued on plates incubated at 37°C.

(vii) MAPK Kinase (MKK1) screening system (Watanabe et al., 1995; Heng et al., 2003; Deosing, 2005; Peter, 2005)

The mutant yeast MKK1P386 strain (H10068), which has a GAL promoter incorporated in the MKK1 P386 gene was utilized. The mutant yeast cells were grown aerobically at pH 7.2, 220 rpm at 28ºC for 48 hours in liquid medium containing 0.67% yeast nitrogen base without amino acids and ammonium sulphate, 0.5% casein acid hydrolysate, 1% ammonium sulphate, 2% D (+)-glucose monohydrate. A volume of 100 mL supplement of 75 g/L adenine (hemisulphate salt), 3 g/mL L-histidine HCL, 30 g/L L-leucine and 40 g/L L-tryptophan was added for every 100 mL of liquid medium. Two screening media, medium A (same as above liquid medium with 18% D-sorbitol and 1.8% agar added) and medium B (18% D-sorbitol with 2% D (+)-glucose monohydrate or 2% D-galactose anhydrous) were autoclaved separately and stabilized in a water bath at 48ºC for 1 hour. The yeast cells pellet was obtained by centrifuging at 4,000 rpm for 10 minutes from broth culture. The pellet was washed with 10 mL of phosphate buffer solution (PBS) twice and suspended in 5 mL of PBS. Mediums A and B were mixed thoroughly with 2 mL of the yeast culture added to every 100 mL of screening medium. The agar was poured into sterile Petri dishes (25 mL per dish) and left to solidify. Sterile paper discs (Whatman No. 3) were applied with 20 µL of acetone extract and then left to evaporate in the laminar flow cabinet before being arranged on different plates of glucose and galactose respectively. The plates were incubated at 28ºC for 72 hours and observation was scored. The potential MKK1 inhibitor will allow the yeast cells to grow in galactose plate and would not show inhibition zone.

(viii) MAPK Phosphatase (MSG5) screening system (Watanabe et al., 1995; Ho et al., 2001; Deosing, 2005)

This screening system utilized the mutant yeast MKK1P386-MSG5 strain (H10069). The mutant yeast cells were grown aerobically at pH 7.2, 220 rpm at 28ºC in liquid medium containing 0.67% yeast nitrogen base without amino acids and ammonium sulphate, 0.5% casein acid hydrolysate, 0.5% ammonium sulphate and 2% D (+)-glucose monohydrate. A volume of 100 mL supplement of 75 g/L adenine (hemisulphate salt), 30 g/mL L-histidine HCL, 30 g/mL L-leucine and 40 g/L L-tryptophan was added for every 100 mL of liquid medium. Two screening media, medium A (same as above liquid medium with 18% D-sorbitol and 1.8% agar added) and medium B (18% D-sorbitol with 2% D (+)-glucose monohydrate or 2% D-galactose anhydrous) were autoclaved separately and stabilized in a water bath at 48ºC for 1 hour. The yeast cells pellet was obtained by centrifuging at 4,000 rpm for 10 minutes from broth culture. The pellet was washed with 10 mL of phosphate buffer solution (PBS) twice and suspended in 5 mL of PBS. Mediums A and B were mixed thoroughly with 2 mL of the yeast culture added to every 100 mL of screening medium. The agar was poured into sterile Petri dishes (25 mL per dish) and left to

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solidify. Sterile paper discs (Whatman No. 3) were applied with 20 µL of acetone extract and then left to evaporate in the laminar flow cabinet before being arranged on different plates of glucose and galactose respectively. The plates were incubated at 28ºC and observation was scored after 72 hours. The potential MSG5 inhibitor will show inhibition zone in galactose plate but not in glucose plate.

(ix) Ras/Raf-1 protein-protein interaction screening system (Ki et al., 1998; Mak, 2005; Ngao, 2005)

The genotype of the Saccharomyces cereviasiae reporter strain L40 is MATa trp1 leu2 his3 LYS::lexA-HIS3 URA3::lexA-lacZ. The L40 (H10006) was transformed with both pLexA-RASv12 and pVP16-RAF (strain LZ). LZ (H10014) was able to grow on agar plate lacking histidine and express β-galactosidase once transcription of the reporter genes was stimulated by the formation of the complex of pLexA-RASv12 and pVP16-RAF. These cells were grown at 28ºC for 72 hours in 25 mL culture broth (0.67% of yeast nitrogen base without amino acids and ammonium sulphate, 0.5% ammonium sulphate, 2% D (+)-glucose monohydrate and amino acid supplements 0.01% adenine sulphate, 0.002% each L-uracil, L-arginine, L-isoleucine, L-tyrosine, L-lysine-HCl, L-phenylalanine, L-methionine, L-valine, and L-threonine but lacking L-leucine, L-tryptophan, and L-histidine, pH 5.8). A volume of 400 μL of this culture was inoculated into 100 mL of the medium containing 1.5% of agar supplemented with 130 µM histidine (His+) or without histidine (His-). To increase the drug permeability and sensitivity in the screening, 0.001% SDS and 1 mM 3-aminotriazole were added to the medium. The paper disks soaked in 20 µL of microbial extract were placed on agar plates and incubated at 28ºC and observation was recorded after 72 hours. A potential inhibitor would show a halo zone only on His- plates.

RESULTS AND DISCUSSIONS

Sampling sites and collection of soil samplesA total of 217 soil samples were collected in DVCA. The undisturbed soils were collected just below the leaf litter under identified trees. There are many differences between the leaf litters and the different populations of microbes growing in them (Ho et al., 2003). The soil layer under big tress is also exposed to tree substances like resin which contains the plant’s secondary metabolites e.g., resins of dipterocarps). The resin produced by a tree might have a selective effect on the microbes. Since each of the resins is secreted by different kinds of forest trees, they can also have different effects on the microbes in the production of different kinds of secondary metabolites. This evolution of microbes to produce secondary metabolites enables them to survive in the area. These secondary metabolites might be useful compounds for humans, such as antibiotics, enzyme inhibitors and detoxify agents. Ho et al (2003) proposed the loose association of unique strains of actinomycetes and fungi with leaf litters of specific plants has a number of consequences. One of them is that leaf litters contain cellulose, lignin, proteins and lipids degraded by these microbes to provide nutrients

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and also include toxic plant secondary metabolites to microbes which are degraded by resistant strains. This means that the leaf litter selects the type of strains that can thrive well in it. Therefore, plant diversity is chosen to obtain the microbial diversity. pH determination and isolation of actinomycetes and microfungiThe measurement of soil pH was done a day after the trip to avoid rapid changing of the microbes in the soil samples. The soils collected for pH determination showed acidic to neutral pH (pH 4.00–7.22). This also indicated that the soils in the Natural Trail and Main Trail West areas are around neutral pH, whereas the soils in Newbery Plot are more acidic (Table 1). No soils were collected at Tembaling Waterfall Trail for pH determination. The culturing pH needs to be adjusted according the pH of the sampling area so that the microbes are able to grow well on the media. Two pH (pH 4.4 and 6.7) were chosen to isolate actinomycetes from the soil samples, and pH 5.5 was used for microfungi isolation by using selective media.

Purification and fermentation of actinomycetes and microfungi and production of microbial secondary metabolitesA total of 535 actinomycetes and 93 microfungi were isolated using selective isolation media which were then purified onto oatmeal agar and PDA to obtain pure cultures (Tables 2 and 3). Prior to growth, preservation of actinomycetes and microfungi was done using sterile 20% glycerol and silica gel and kept at -20ºC and 4ºC respectively for long-term storage. These pure isolates were then grown aerobically at 220 rpm, 28ºC for 120 hours to produce bioactive secondary metabolites; pH 7.2 was used if the actinomycetes could not grow on acidic (pH 4.4) fermentation broth. An equal volume of acetone was added to produce the microbial crude extract, which was subsequently tested on designed screening systems.

AfsK/AfsR serine/threonine protein kinase screening systemThe present study clearly demonstrates that a protein serine/threonine kinase AfsK and its target AfsR are involved in the response of aerial mycelium formation to glucose (Umeyama et al., 1999). These proteins appeared to be concerned only with aerial mycelium formation and not with secondary metabolism. Neither the afsK nor afsR disruptants form aerial mycelium and accordingly spores on glucose containing medium, whereas the aerial mycelium formation and sporulation of the disruptants can recover on mannitol containing medium (Umeyama et al., 1999). From the results of the screening test, 1 extract (H9420) was toxic and 4 extracts (H9386, H9387, H9407 and H9409 ) showed inhibition activity (either sporulation or growth of S. griseus was inhibited) on the 1.5% D (+)-glucose plate and none (neither sporulation nor growth of S. griseus was inhibited) on the 1.5% D-mannitol plate in both tests (Table 4). These extracts can only be identified as potential inhibitors which have the possibility to inhibit the AfsK/AfsR pathway in S. griseus IFO13350 (H10000) because no microscopic observation has been carried out for the spores’ absence on the inhibition zones on the glucose plate.

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KdpE/KdpD two-component signal transduction over osmotic stress screening system Two-component KdpE/KdpD system possesses genes annotated as being part of the M. tuberculosis K+ uptake machinery, which are induced by low K+ levels (Gaβel and Altendorf, 2001; Fontan et al., 2004). In this designed mycobacterial osmolarity screening, wild type M. smegmatis mc2155 strain H8000 was used as the screening target against two types of signals—potassium (K+) and sodium (Na+). Growth inhibition on low concentration of respective signals containing medium but no or less inhibition on high concentration is expected to give a positive result. A potential inhibitor that follows the rule will show similar surveillance upon the wild type of M. smegmatis. One extract (H11588) showed anti-mycobacterial activity but later was defined as toxic (Table 5).

PhoP/PhoR two-component signal transduction screening systemA screening system was designed through the bacterial pathogenesis study on Salmonella typhimurium PhoP/PhoR system. Growth of S. typhimurium in micromolar concentration of Mg2+ promotes transcription of PhoP-activated genes in PhoP/PhoR dependent manner, whereas growth in millimolar concentration of Mg2+ represses expression of PhoP-activated genes (Groisman, 2001). So in a low Mg2+ environment, the PhoP/PhoR system upregulates transcription of high-affinity Mg2+ transport systems allowing the bacteria to overcome Mg2+ deprivation (Vescovi et al., 1997). In addition, a mutant in the PhoP/PhoR system caused S. typhimurium to become avirulent (Miller et al., 1989). A mutant in M. tuberculosis H37Rv PhoP/PhoR system was also attenuated in macrophages and mice. In vitro growth experiments using a mutant in the M. tuberculosis PhoP/PhoR system revealed an inability to grow in low Mg2+-containing medium, indicating this system may be sensing Mg2+ and is the functional homologue of the S. typhiumurium PhoP-PhoR system (Fontan et al., 2004). In this test, 15 acetone extracts (H11482, H11588, H11612, H11615, H11724, H11764, H11781, H11782, H11818, H11857, H11865, H11888, H11900, H11911 and H11949) showed toxicity in the screening system using M. smegmatis mc2155 strain H8000. One strain (H11855) showed greater inhibition in a low concentration (25 mM) of Mg2+ but less inhibition in a high concentration (1 mM) of Mg2+-containing medium was subjected to further study (Table 6–11).

Glyoxylate pathway screening systemThis system screened for inhibitors against ICL as detected by growth inhibition of M. smegmatis in acetate but non-inhibition on glucose medium (Hew, 2006). No potential ICL inhibitor was obtained. Twenty-six crude actinomycetes acetone extracts [H11469 (data not shown), H11482, H11484, H11499, H11526, H11542, H11588, H11600, H11612, H11689, H11735, H11745, H11764, H11781, H11782, H11818, H11826, H11855, H11856, H11857, H11865, H11867, H11876, H11888, H11900 and H11911] and one crude fungus acetone extract (H9448) showed toxic (inhibition zones on glucose and acetate plates) against M. smegmatis (Table 12).

12


12

GLC7 the catalytic subunit of serine/threonine protein phosphatase type 1 (PP1) in yeast screening systemIn yeast PP1 screening, two types of yeast were used as the screening targets against protein phosphatase 1; wild type yeast which carried GLC7, and mutant yeast which carried a high temperature-sensitive glc7 allele (glc7-10). The mutant caused cell cycle arrest and cell wall integrity defect at 37ºC but was rescued by the addition of 1 M sorbitol. A potential inhibitor that acted on the wild type Glc7p will show similar characteristics to the mutant strain. Thirty-seven extracts (H11428, H11449, H11462, H11463, H11477, H11478, H11479, H11482, H11483, H11490, H11532, H11535, H11536, H11537, H11544, H11547, H11548, H11555, H11557, H11559, H11739, H11743, H11745, H11757, H11764, H11772, H11781, H11821, H11822, H11837, H11844, H11853, H11856, H11860, H11865, H11875 and H11876) were toxic against type 1 serine/threonine phosphatase (GLC7) screening in yeast, while 3 extracts (H11533, H11534 and H11735) were potential inhibitors against this GLC7 screening system (Table 13).

GSK-3β screening systemIn the screening of inhibitors against GSK-3β, two strains of yeast were used. The first strain was H10075, which was constructed by transforming gsk-3 null mutant with pKT10-GSK3β. The second yeast, known as H10079, was the transformant yeast of gsk-3 null mutant with Yep24-MCK1. Both of the yeast transformants have the ability to suppress temperature-sensitive phenotype of gsk-3 null mutant. The presence of a potential inhibitor will lead to growth inhibition of both transformants at 37ºC but is rescued by 1.2 M sorbitol. In the screening, 9 actinomycete extracts (H11462, H11490, H11526, H11668, H11686, H11695, H11720, H11785, H11809) were showing potential inhibitors and 32 extracts were toxic (H11477, H11482, H11483, H11520, H11536, H11537, H11544, H11555, H11557, H11559, H11588, H11612, H11635, H11647, H11722, H11739, H11757, H11764, H11810, H11821, H11822, H11843, H11844, H11853, H11855, H11856, H11857, H11860, H11866, H11888, H11900 and H11911) against mammalian GSK-3β in mutant yeast strain H10075 (Table 14). In the following test, inhibition by H11490 was not rescued by 1.2 M sorbitol (Table 15). The remaining 8 extracts (H11462, H11526, H11668, H11686, H11695, H11720, H11785 and H11809) were tested in the next screening system using H10079 (Table 16), only 3 of the extracts (H11462, H11785, and H11809) were found to be positive (Table 17).

MAPK Kinase (MKK1) screening systemThe MAPK kinase (MKK1) inhibitor screening system utilized the mutant yeast MKK1P386 strain, which has a GAL promoter incorporated in the MKK1P386 gene (Watanabe et al., 1995). In the glucose plate, the mutant yeast utilizes glucose and the GAL1 promoter is not induced, leading to yeast growth on the whole glucose plate. In the galactose plate, GAL1 promoter is induced and overexpressed, which results in the growth inhibition (cell lysis) of yeast on the galactose plate, even in the presence of 1 M sorbitol as an osmoprotectant. A potential inhibitor of MKK1 is present without

1313


an inhibition zone appearing on the glucose plate but with yeast growth around the disc on the galactose plate. The toxicity against the yeast system is showed when an inhibition zone appears around the disc on the glucose plate. Five actinomycete extracts (H11428, H11588, H11612, H11635 and H11813) showed toxicity against the MKK1P386 mutant yeast and by coincidence all of them were also toxic against the MSG5 screening system (Table 18).

MAPK Phosphatase (MSG5) screening systemIn the MAPK phosphatase (MSG5) inhibitor screening system, the mutant yeast MKK1P386–MSG5 strain was utilized (Watanabe et al., 1995). In the glucose plate, yeast growth is seen on the whole plate because of the utilization of glucose by the mutant yeast and the GAL1 promoter is not induced. The galactose plate also has yeast growth wholly on it due to the introduction of MSG5 into the MKK1P386 system and overexpression under induction by galactose utilizing the GAL1 promoter of MKK1P386–MSG5 mutant yeast, which then suppresses the growth inhibition effect of the overexpression of MKK1P386 gene. A potential inhibitor of MSG5 is present with an inhibition zone around the disc only on the galactose plate. The toxicity against the yeast system is shown when inhibition zones appear around the discs on both the glucose and galactose plates. Seven actinomycete extracts (H11428, H11588, H11610, H11612, H11635, H11640 and H11813) showed toxicity against the MKK1P386 -MSG5 mutant yeast (Table 19). In comparison, both H11610 and H11640 showed no activity against MKK1 screening system.

Ras/Raf-1 protein-protein interaction screening systemIn this screening system, the yeast S. cerevisiae reporter strain L40 which transformed 2 plasmids of pLexA-RASv12 and pVP16-RAF was used. The transformed yeast strain LZ was able to grow on agar medium lacking histidine and express β-galactosidase. If the interaction between oncogenic Rasv12 and Raf-1 was inhibited in LZ yeast, the cells should grow in the presence of histidine but not in its absence (Ki et al., 1998). A total of 37 extracts (H11499, H11515, H11518, H11520, H11525, H11526, H11530, H11532, H11533, H11534, H11535, H11536, H11537, H11544, H11548, H11550, H11555, H11557, H11559, H11661, H11669, H11670, H11671, H11677, H11739, H11750, H11809, H11810, H11822, H11855, H11856, H11857, H11858, H11861, H11867, H11878 and H11879) showed toxicity and no extract showed potential inhibitor against the Ras/Ras protein-protein interaction (Table 20).

GENERAL CONCLUSIONS

In conclusion, this study indicates that the forest soils of the lowland dipterocarp forest in the Danum Valley Conservation Area possess a rich microbial diversity, particularly of actinomycetes and microfungi that produce a variety of secondary metabolites which act as inhibitors against signal transduction of prokaryotes and eukaryotes. The potential inhibitors identified as against proteins involving signal

14


14

transduction, particularly PP1 and GSK-3β, are of interest for drug discovery in cancer and neurological disease research. Similarly the inhibitor identified in the actinomycetes strain H11855 (PhoP/PhoR) in M. smegmatis is of interest in tuberculosis research. Thus, the primary lowland dipterocarp forest in Danum Valley is an ideal site for non-destructive bio-prospecting for novel drug discovery, research in tropical biology and ecotourism. The area deserves to be perpetually conserved.

ACKNOWLEDGEMENTS

This paper is dedicated to Prof. Arnold L. Demain for his contributions to industrial microbiology. We would also like to express our appreciation to Prof. Dr. David M. Newbery for allowing us to conduct sampling at Newbery Plot in Danum Valley Conservation Area, Dr. Glen Reynolds and Dr. Marcus Lingenfelder for their precious help and advice. We are greatly indebted to Danum Valley senior field ranger Mr. Bernadus Bala Ola, field guides Mr. Herman Francis, Mr. Johar Aribin and Mr. Nasir Abdul Majid for their field assistance and identification of plants. We thank Mr. Leopard Madani and Mr. Postar @ Jaiwit Miun from Forest Research Centre (FRC), Sandakan and Mr. Johnny Sigil from Institute for Tropical Biology and Conservation (ITBC), UMS for confirmation of leaf identification. We thank Mr. Foo Sek Hin, Mr. Ong Si Mon and Mr. Tan I @ Tan Yee for critical reading of the manuscript and technical assistance. This project is supported by Royal Society-Danum Valley (95500-35), Universiti Malaysia Sabah Fundamental Research grant (B-0901-PR/U011) and Intensive Research Priority Area grant (01-0210-0038-EA0036) from Malaysian Ministry of Science, Technology and Innovation.

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Table 1. Location and pH of soil samples.

Soil samples pH LocationDV1 7.01 Nature Trail DV5 6.59 West Trail DV7 6.39 West TrailDV8 4.09 Newbery Plot 1DV15 7.22 West TrailDV95 4.74 West TrailDV232 3.47 Newbery Plot 1DV236 4.01 Newbery Plot 1DV280 4.01 Newbery Plot 1 DV282 4.00 Newbery Plot 1

Distilled water 6.27

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Table 2. Actinomycetes isolated from Danum Valley with aerial mycelia colour, substrate mycelia colour, extracellular pigmentation and sporulation (Colour description follows Nippon paint colour chart)

No. Strain Soil sample

Characterization on oatmeal agar mediumAerial

mycelia colour

Substrate mycelia colour

Extracellular pigmentation

1 H11782

DV1Parashorea tomentella

Brown White 02 H11783 Green Light orange Light green3 H11784 Pink Light orange 04 H11785 White Light yellow 05 H11786 Dark brown Light brown 06 H11787

DV2Dysoxylum undulatum

Grey Beige 07 H11788 Grey Beige 08 H11789 Grey Beige 09 H11790 White Black 010 H11791


Light grey Light beige 011 H11792 Grey Light yellow 012 H11793 Light grey Light beige 013 H11794 Grey Light grey 014 H11795 White Light brown 015 H11796


Green Beige 016 H11797 Dark brown White 017 H11798 Brown White 018 H11799 Dark brown White 019 H11800 Green Beige 0

20 H11801

DV5Shorea johorensis

Brownish pink Beige Light yellow

21 H11802 White White 022 H11803 White Light beige 023 H11804 Light beige Light beige 024 H11805 White Light beige 025 H11806

DV6Shorea parvifolia

Beige Light yellow 026 H11807 Light grey Beige 027 H11808 Brown Light brown 028 H11809 DV7

Parashorea tomentellaLight brown White 0

29 H11810 Grey Light grey 030 H11811 DV8

UnidentifiedWhite Beige Light beige

31 H11812 White White 0

20


20

32 H11813DV9

Alangium javanicum

Grey Beige 033 H11814 White Light beige 034 H11815 Grey Yellow Yellow35 H11816 DV10

Magnolia candollei var. singapurensis

Grey Light grey 036 H11817 White Beige Light beige37 H11818 Light grey Light beige 038 H11657


Light brown Brown 039 H11658 Light beige Light yellow 040 H11659 Light beige Light yellow 041 H11660 Light beige Light yellow 042 H11661 Greyish blue Brown 043 H11662

DV12Blumeodendron tokbrai

Light brown Light yellow 044 H11663 Brown Light beige 045 H11664 Light beige Light beige 046 H11665

DV13Aglaia odoratissima

Dark grey Grey 047 H11666 White White 048 H11667 White White 0

49 H11668 DV14Knema cinerea Violet Dark purple 0

50 H11669DV15

Parashorea tomentella

Greyish blue Golden brown 051 H11670 Crystal blue Brown 052 H11671 Peach Light beige 053 H11672


Light grey Light grey 054 H11673 Black Black 055 H11674 Black Black 056 H11675


Light grey Beige 057 H11676 Dark grey Grey 058 H11677 Grey Grey 059 H11678 Black Brown 060 H11679 Grey Beige 061 H11680

DV18Pentace laxiflora

Grey Cream 062 H11681 Light grey Cream 063 H11682 Grey Cream 0

64 H11683 DV19Shorea parvifolia Light grey Marble grey 0

65 H11684DV20

Aglaia tomentosa

White Light yellow 066 H11685 White White 067 H11686 Grey Light grey 0

2121


68 H11527


Marble grey Bone white 069 H11528 Marble grey Bone white 070 H11529 Marble grey Light grey 071 H11530 Marble grey Beige 072 H11531 Marble grey Bone white 073 H11532

DV22Alangium ebenaceum

Grey Black 074 H11533 Grey Black 075 H11534 Grey Black 076 H11535 Grey Black 077 H11536 Grey Black 078 H11537 DV23

Diospyros elliptifoliaWhite Amber white 0

79 H11538 Marble grey Black 080 H11539

DV24Memecylon

appendiculatum

White Milky yellow 081 H11540 Brown Dark brown 082 H11541 White Bone white 083 H11542 Grey Blue 0

84 H11543DV25

Parashorea malaanonan

White Yellow 0

85 H11544DV26

Homalanthus populneus

Grey Black 086 H11545 Grey Green 087 H11546 Grey Black 088 H11547 Grey Dark grey 089 H11548 Grey Dark grey 090 H11549 DV27

Shorea johorensisMarble grey Black 0

91 H11550 Smoke grey Amber white 092 H11551

DV28Chisocheton sarawakanus

White Yellow 093 H11552 White Yellow 094 H11553 White Amber white 095 H11554 White Beige 096 H11555 DV29

Chisocheton polyandrus

Grey Black 0

97 H11556 White Milky yellow 0

98 H11557DV30

Baccaurea tetrandra

White Yellow 099 H11558 Yellowish Light yellow 0

100 H11559 Aqua white Bluebell white 0

22


22

101 H11731DV31


Venus Golden sand 0102 H11732 Primrose Jasmine 0

103 H11733 Lavender grey Primrose 0

104 H11734 Venus Primrose 0105 H11735

DV32Nothaphoebe

canescens

Papier grey Off white 0

106 H11736 Lavender grey Maple 0

107 H11737 Papier grey Slate 0108 H11738 Papier grey Calm green 0

109 H11739 Lavender grey Pearl white 0

110 H11740

DV33Parashorea malaanonan

Lavender grey Ash grey 0

111 H11741 Slate Ivory 0112 H11742 White Honey cream 0

113 H11743 Lavender grey Marigold 0

114 H11744 White Off white 0115 H11745 Sweet dream Primrose 0116 H11746

DV34Dimocarpus longan

Pearl white Primrose 0117 H11747 Ash grey Ash grey 0118 H11748 Pearl white Pearl white 0

119 H11749 Brown velvet Lavender grey 0

120 H11750 Ash grey Off white 0121 H11751 DV35


White Copper tone 0122 H11752 White Honey cream Off white123 H11753 Ivory Off white 0124 H11754


White Barbary gold 0125 H11755 Pearl white Pearl white Off white126 H11756 White Jasmine Primrose127 H11757 Jute Ivory 0128 H11758 Marigold Maple Primrose129 H11759


Sakura Pearl white Marigold

130 H11760 Lavender grey Barbary gold Marigold

131 H11761 Congo brown Mahosary 0132 H11762 White Straw cream 0

133 H11763 DV38Phoebe macrophylla Venus Oak 0

2323


134 H11764 DV39Vatica oblongifolia

Secret Pearl white 0135 H11765 Primrose Pearl white 0136 H11766

DV40Aglaia silvestris

White Primrose 0137 H11767 White Ivory 0138 H11768 Primrose Pearl white 0139 H11491

DV41Eugenia sp.

Marigold Marigold 0140 H11492 Honeydew Honeydew 0141 H11493 Marigold Marigold 0

142 H11494DV42

Paranephelium nitidum

Woodland olive White 0

143 H11495 Sweet dream Ivory 0144 H11496 Grey Primrose Primrose145 H11497

DV43Xanthophyllum affine

Light brown Peach 0146 H11498 White Peach 0147 H11499 White Marigold 0148 H11500

DV44Baccaurea stipulata

Pink Yellow 0149 H11501 Light purple Marigold 0150 H11502 Sweet dream Ivory 0151 H11503

DV45Polyalthia sp.

White Marigold 0152 H11504 Grey Jasmine Jasmine153 H11505 Grey Jasmine Jasmine154 H11506

DV46Lithocarpus sp.

Ash grey Off white 0155 H11507 White Off white 0156 H11508 Honey Tulip 0157 H11509

DV47Lithocarpus sp.

White Marigold 0158 H11510 Grey Primrose Primrose159 H11511 Grey Primrose Primrose160 H11512

DV48Gonystylus keithii

Grey Primrose Primrose161 H11513 White Pumpkin 0162 H11514 White Pumpkin 0163 H11515

DV49Syzygium tawaense

Grey Jasmine Jasmine164 H11516 Grey Jasmine Jasmine165 H11517 Grey Jasmine Jasmine166 H11518

DV50Neesia strigosa

Grey Jasmine Jasmine167 H11519 White Cream 0168 H11520 Brown Marigold 0169 H11457


Light brown Light beige 0

170 H11458 Greyish brown Greenish grey 0

171 H11459 Light grey Orange Yellow

24


24

172 H11460 DV52Nephelium sp. Brown Brownish 0

173 H11461DV53

Lansium domesticum

Greyish brown Orange Pink

174 H11462 Greyish brown

Yellowish grey 0

175 H11463

DV54Dimocarpus longan

Greenish grey Light green 0

176 H11464 Dark greenish grey

Greyish brown 0

177 H11465 Dark grey Brownish yellow Yellow

178 H11466 Light grey White 0179 H11467 DV55


White Milky 0180 H11468 White Yellowish Light brown181 H11469 White Yellowish 0

182 H11470 DV56Parashorea tomentella

Grey Greyish brown 0

183 H11471 Light grey Milky 0

184 H11472 Grey Yellowish green 0

185 H11473DV57


Greenish grey Greenish grey 0186 H11474 Greenish grey Greenish grey 0187 H11475 White Milky 0

188 H11476 Grey Yellowish green Yellow

189 H11477DV58

Santiria tomentosa

Light brown Yellowish brown 0

190 H11478 Brown Light grey 0191 H11479 Brown Light grey 0192 H11480 Brown Light grey 0193 H11481 DV59

Xanthophyllum vitellinum

White Pink 0

194 H11482 Grey Milky 0

195 H11483 DV60Madhuca korthalsii

White Yellowish 0196 H11484 White Milky 0

197 H11426DV61


Dark brown Yellowish grey 0

198 H11427 Grey Marigold 0199 H11428 White White 0200 H11429 Grey Brown 0

2525


201 H11430 DV62Baccaurea tetrandra

Greyish brown Light grey 0

202 H11431 Grey Milky white 0203 H11432 DV63

Uncaria sp.Maroon Light pink 0

204 H11433 Light grey Milky yellow 0205 H11434

DV64Tetracera akara.

White Grey Grey206 H11435 Yellow Yellow 0207 H11436 Light brown Milky white 0208 H11437 White Milky white 0209 H11438 Grey Milky white 0210 H11439


Light green Yellow 0211 H11440 Maroon Bright yellow Yellow212 H11441 Turquoise Light green 0213 H11442 Light blue Marigold 0214 H11443 Pink Light yellow Yellow215 H11444 DV66

Ficus sp.Light brown Marigold 0

216 H11445 Brown Light red Light maroon

217 H11446 DV67Shorea johorensis

Greyish brown Yellow 0

218 H11447DV68

Dysoxylum cyrtobotryum

Brown White 0219 H11448 Brownish red Grey 0220 H11449 Grey Brown Light brown221 H11450 Brown Whitish grey 0222 H11451 Brown Grey 0

223 H11452 DV69Phaeanthus sp. 1 Grey White 0

224 H11690DV81


Woodland olive Ivory 0

225 H11691 White Jasmine 0226 H11692 White White 0227 H11693 White Pink 0228 H11694

DV82Alangium ebenaceum

White slate Off white 0229 H11695 Ivory white Jasmine 0230 H11696 White jute Honey cream 0231 H11697 White slate Jasmine 0232 H11698 Light brown Yellow Primrose233 H11699


Honey blue Jasmine Primrose234 H11700 Jasmine white Sunflower 0235 H11701 Honey blue Jasmine Primrose

26


26

236 H11702

DV84Brownlowia peltata

White venus Primrose 0

237 H11703 White sweet dream White 0

238 H11704 Primrose sweet dream Sunflower 0

239 H11705 White slate White 0

240 H11706 White papier grey Orange Jasmine

241 H11707


White lavender grey Black Slate

242 H11708 White black White 0243 H11709 White Primrose 0

244 H11710 White lavender grey Black Slate

245 H11711 White papier grey Jasmine 0

246 H11712 DV86Parashorea malaanonan

White brown velvet

Woodland olive 0

247 H11713 Beige Primrose 0

248 H11714


White congo brown Brown velvet Woodland

olive249 H11715 Mandarin Mandarin 0

250 H11716 White congo brown Brown velvet Woodland

olive


olive


olive

253 H11719DV88

Beilschmiedia sp. A

White brown Brown velvet Woodland olive

254 H11720 Pearl white Off white 0

255 H11721 White primrose White 0

256 H11722 DV89Polyalthia sumatrana

White lavender grey Honey cream Beige

257 H11819

DV91Dysoxylum undulatum

Grey Yellow Yellow258 H11820 Grey Dark beige Dark beige259 H11821 Light pink Light beige 0260 H11822 Grey White 0

261 H11823 Brownish grey

Greenish beige 0

2727


262 H11824


Light beige Beige 0263 H11825 White Red Red264 H11826 Brown Beige 0265 H11827 Light pink Light beige 0266 H11828 Beige Light beige 0267 H11829


Dark brown Dark brown Brown268 H11830 Beige Dark beige 0269 H11831 Light pink Beige 0270 H11832 White Light grey 0271 H11833 Beige Brown 0272 H11834

DV94Aglaia sp.

Dark brown Beige 0273 H11835 Grey Light yellow 0274 H11836 Grey Light beige 0

275 H11837 Grey Yellowish brown

Yellowish brown

276 H11838DV95


Grey Beige 0277 H11839 Grey Yellow Yellow278 H11840 Light pink Beige 0279 H11841 Light pink Dark beige 0280 H11842


Beige Light beige 0281 H11843 Brown Light beige 0282 H11844 Grey White 0

283 H11845 Grey Yellowish beige 0

284 H11846

DV97Polyalthia sp.

Grey Light grey 0285 H11847 Grey Light grey 0286 H11848 Grey Brown 0287 H11849 Grey Light grey 0288 H11850 Grey Red 0289 H11851

DV98Gonystylus keithii

Grey Yellow 0290 H11852 Greenish grey Light grey 0291 H11853 Grey Light grey 0292 H11854 White White 0293 H11855 Grey Beige 0294 H11856

DV99Shorea agamii

Dark brown White 0295 H11857 Grey Beige 0296 H11858 Grey Dark brown 0

28


28

297 H11859DV100

Gonystylus keithii

Light grey Beige 0298 H11860 Grey Light grey 0299 H11861 White Light beige 0300 H11862

DV101Eugenia sp.

White White 0301 H11863 Dark brown Light brown 0302 H11864 Light grey Brown 0303 H11687 DV102

Alangium sp.Smoke grey Beige 0

304 H11688 Dark brown Cream 0305 H11769

DV104Shorea sp.

Off white Pearl white 0306 H11770 Wooden olive Beige 0307 H11771 Brown velvet Barbary gold 0308 H11772 Secret Pearl white 0309 H11773 Maple Marigold 0310 H11774 Off white Pearl white 0311 H11775 Sakura Sweet dream 0312 H11521


Grey Jasmine Jasmine313 H11522 Grey Jasmine Jasmine314 H11523 White White 0

315 H11485 DV106Shorea agamii Brown Light grey 0

316 H11453 DV107Aglaia sp. White White 0

317 H11723


White brown velvet Off white 0

318 H11724 Primrose white Off white 0

319 H11725 White brown velvet Off white 0

320 H11726 White lavender grey White 0

321 H11865

DV110Goniothalamus sp.

Brown White 0322 H11866 Grey Light grey 0323 H11867 Light grey Light beige 0324 H11868 Grey Beige 0325 H11869 Light grey White 0326 H11870


Light grey Light beige 0327 H11871 Green Light beige 0328 H11872 White Beige 0329 H11873 White Beige 0330 H11874 White White 0

2929


331 H11689 DV112Neesia synandra Smoke grey Beige 0

332 H11560 DV113Sympetalandra

borneensis

White Yellow 0333 H11561 Pink Yellow 0334 H11562 Orchid white Bone white 0335 H11563 Orchid white Bone white 0336 H11776

DV114Eugenia sp.

Sunflower Jasmine Primrose337 H11777 White Primrose 0338 H11778 Mandarin Orange 0339 H11779 Sakura Golden sand 0340 H11780 Off white White 0341 H11781 White Primrose 0342 H11524

DV115Aglaia odoratissima

White Off white 0343 H11525 White Marigold 0344 H11526 Pink Off white 0

345 H11486

DV116Litsea sp.

Grey Yellow orange Yellow

346 H11487 Brownish grey Light grey 0

347 H11488 Light brown Light brownish 0

348 H11489 Brownish grey Grey 0

349 H11490 Grey Yellow orange 0

350 H11454DV117

Canarium denticulatum

Turquoise Light green 0351 H11455 White White 0352 H11456 Turquoise Light green 0

353 H11727DV119

Lithocarpus sp.

Lavender grey white Jasmine 0

354 H11728 White grey White 0355 H11729 Honey cream Brown 0356 H11730 White grey White 0357 H11875

DV120Ficus sp.

Greenish grey Beige Beige358 H11876 Greyish white Light yellow 0359 H11877 White Dark brown Dark brown360 H11878 Brown Light brown 0361 H11879 Greenish grey Beige 0

30


30

362 H11880DV200

Dacryodes rostrata

White Beige 0363 H11881 White White 0364 H11882 White White 0365 H11883 DV201

Dysoxylum cyrtobotryum

White Beige 0366 H11884 Grey Beige 0367 H11885 Grey Beige 0368 H11886 DV202

Shorea argentifoliaWhite White 0

369 H11887 Dark grey Light grey 0370 H11888

DV203Shorea argentifolia

Grey Beige 0371 H11889 White Light green 0372 H11890 White Beige 0373 H11891

DV204Lithocarpus gracilis

Grey Grey 0374 H11892 White Light grey 0375 H11893 Dark brown Dark brown 0376 H11894

DV205Syzygium chrysanthum

White Light beige 0377 H11895 White Light yellow 0378 H11896 Black Black 0379 H11897 DV206

Mallotus wrayiWhitish grey White 0

380 H11898 White Light yellow Light yellow381 H11899 DV207

Buchanania insignisLight grey Light beige 0

382 H11900 White Yellow Light yellow383 H11901 White Light beige 0384 H11902

DV208Dysoxylum rigidum

Light pink Dark beige Dark beige385 H11903 Grey Yellow 0386 H11904 Light brown Beige 0

387 H11905 DV209Shorea fallax Dark brown Dark brown 0

388 H11610DV210

Shorea johorensis

Steel grey Langkawi melon Buttercup

389 H11611 White Fresh lemon 0390 H11612 Steel grey Jonquil 0391 H11613

DV211Dialium

hydnocarpoides

White Buttercup 0392 H11614 White Malt white 0

393 H11615 Stonewall Buttercup Langkawi melon

394 H11616DV212

Syzygium castaneum

White Light yellow 0395 H11617 White Sundew 0396 H11618 Malt white Buttercup 0

3131


397 H11619DV213

Shorea parvifolia

White Scroll 0398 H11620 White Scroll 0399 H11621 White Scroll 0400 H11622

DV214Quercus argentata

Windstar Cream 0401 H11623 Floral white Malt white 0402 H11624 Windstar Jonquil 0

403 H11625 DV215Barringtonia

lanceolata

Windstar Sahara gold Light sahara gold

404 H11626 Stonewall Lotus white 0405 H11627 Stonewall Lotus white 0406 H11628

DV216Shorea leprosula

Windstar Cream 0407 H11629 Stonewall Cream 0408 H11630 Windstar Off white 0409 H11631

DV217Microcos elmeri

White Chiffon 0410 H11632 Stonewall Off white 0

411 H11633 Dawn Golden ray Langkawi melon

412 H11634DV218

Shorea pauciflora

White Eggshell white 0

413 H11635 Windstar Charcoal Light grey414 H11636 Dawn Chiffon 0415 H11637


Windstar Scroll 0416 H11638 Broken white Sherbet 0417 H11639 Broken white Scroll 0418 H11564


Grey Light brown 0419 H11565 Grey Dark yellow 0420 H11566 Grey Buttercup 0

421 H11567DV221

Syzygium tawaense

Creamy white Yellowish brown

Brownish white

422 H11568 Bright white Sahara gold 0423 H11569 Grey Yellow 0424 H11570

DV222Syzygium tawaense

Grey Sahara gold 0425 H11571 White Yellow 0426 H11572 Grey Light brown 0427 H11573

DV223Lithocarpus

nieuwenhuisii

White Buttercup 0428 H11574 Grey Greyish white 0

429 H11575 Grey Light greyish white 0

32


32

430 H11576DV224

Syzygium chrysanthum

Beige White 0431 H11577 Grey Light yellow 0432 H11578 White Yellow 0

433 H11579 DV225Syzygium chrysanthum

White Brownish white 0

434 H11580 Grey White 0435 H11581

DV226Gironniera celtidifolia

White Light brown 0436 H11582 White Yellow 0437 H11583 Light grey Grey 0

438 H11584DV227

Mallotus wrayi

White Yellowish white 0

439 H11585 White Yellowish white 0

440 H11586 Light grey Yellow 0

441 H11587DV228

Glochidion elmeri

Dark grey Yellowish white 0

442 H11588 Grey Yellowish white 0

443 H11589 Grey Sahara gold 0444 H11590

DV229Parinari oblongifolia

White Yellow 0445 H11591 Light grey Primrose 0

446 H11592 White Yellowish white 0

447 H11939DV250

Madhuca korthalsii

Brown White 0448 H11940 Grey Light yellow Light yellow449 H11941 Light grey Yellow Light yellow450 H11942

DV251Lithocarpus gracilis

Brown Light yellow 0451 H11943 Brown Light yellow 0452 H11944 Light brown Light yellow 0453 H11945

DV252Mallotus wrayi

Grey Yellow Yellow 454 H11946 Light grey Light brown Light yellow 455 H11947 Grey Light yellow Light yellow456 H11948 DV255

Mallotus wrayiDark grey Light yellow 0

457 H11949 Light grey Light brown 0

458 H11950 DV256Shorea johorensis Light grey Light yellow 0

459 H11951 DV257Reinwardtiodendron

humile

Light orange Brown 0460 H11952 Light grey Light yellow 0461 H11953 White Milky white 0

3333


462 H11954DV258

Sindora irpicina

Light grey Yellow Yellow 463 H11955 White Light grey 0464 H11956 Light grey White 0465 H11957 DV259

Cryptocarya teysmanniana

Light grey Yellow Dark yellow

466 H11958 White Light orange 0

467 H11906DV260

Aglaia silvestris

Brown Brown 0468 H11907 White White 0469 H11908 Light beige Light beige 0470 H11909

DV261Aporusa falcifera

White White 0471 H11910 White Light beige 0472 H11911 Brown Light beige 0

473 H11912DV262

Polyalthia sumatrana

White Yellowish beige

Yellowish beige

474 H11913 Dark brown Dark brown 0475 H11914 Grey Beige 0476 H11915

DV263Drypetes sp. E

White Light beige 0477 H11916 Grey Light brown 0478 H11917 White White 0

479 H11918DV264


White Blackish brown Dark beige

480 H11919 White Light beige 0481 H11920 Light grey Light beige 0482 H11921 DV265

Lithocarpus nieuwenhuisii

Dark brown Dark brown 0483 H11922 White Dark beige Dark beige484 H11923 Light grey Light beige 0

485 H11640DV266

Mallotus wrayi

Battleship grey French grey 0

486 H11641 White Scroll 0487 H11642 White Fresh lemon 0488 H11643

DV267Garcinia forbesii

Maize Matador 0489 H11644 White Burgundy 0490 H11645 Windstar Melon 0491 H11646

DV268Dysoxylum rigidum

Dawn Golden ray 0

492 H11647 Steel grey Langkawi melon 0

493 H11648 Windstar Cream 0494 H11649 DV269

Lithocarpus nieuwenhuisii

Windstar Jonquil 0495 H11650 White Buttercup 0496 H11651 White Fresh lemon 0

34


34

497 H11652 DV270Trigoniastrum hypoleucum

Pigeon white Jonquil 0498 H11653 Magnolia Sherbet 0499 H11654 White Chiffon 0500 H11655 DV271

Quercus argentataWhite Fresh lemon 0

501 H11656 Light grey Cream 0502 H11593 DV272

Shorea fallaxGreenish grey White 0

503 H11594 Grey Light brown 0

504 H11595

DV273Neesia strigosa

White Yellowish white 0

505 H11596 White Mahogany 0

506 H11597 Lavender grey Greyish white 0

507 H11598 White Dark brown 0508 H11599

DV274Dillenia excelsa

White Sahara gold 0509 H11600 White Yellow 0510 H11601 White Magnolia 0

511 H11602

DV275Shorea fallax

Light Grey Yellowish white 0

512 H11603 Brownish white Yellow 0

513 H11604 Brownish grey Jasmine 0

514 H11605 DV276Parashorea malaanonan

Bright white White 0

515 H11606 Grey Brown velvet Reddish

516 H11607DV277

Mallotus wrayi

Brown Tangerine 0517 H11608 Grey Light brown 0518 H11609 Brown Cream 0519 H11959 DV290

Polyalthia xanthopetala

White White 0520 H11960 White White 0521 H11961 White Light yellow 0522 H11962


Light blue Light yellow 0523 H11963 Grey Yellow Light yellow524 H11964 Grey Light yellow Light yellow525 H11965

DV292Baccaurea macrocarpa

Grey Black Grey526 H11966 Light yellow Light yellow 0527 H11967 Orange Light orange 0528 H11968

DV293Knema latericia

White Light yellow 0529 H11969 White Light yellow 0530 H11970 White Yellow 0

3535


531 H11971 DV294Antidesma

neurocarpum.

Light orange Light yellow 0532 H11972 Orange Yellow Yellow 533 H11973 White Light yellow 0534 H11974 DV295

Mallotus wrayiWhite Light yellow 0

535 H11975 White Light yellow 0

0 = No extracellular pigmentation

Characterization on OA mediumAll strains sporulated on oat medium except for strains H11692, H11693, H11940, H11942, H11945, H11946, H11947, H11951, H11953, H11955, H11956, H11958, H11960, H11961, H11963, H11964, H11966, H11968, H11969, H11970, H11971, and H11974.

36


36

Table 3. Microfungi isolated from Danum Valley with aerial mycelia colour, substrate mycelia colour and extracellular pigmentation. (Colour description follows Nippon paint colour chart)

No Strain Soil sampleCharacterization on PDA

Aerial mycelia colour

Substrate mycelia colour

Extracellular pigmentation

1 H9386


Marigold white Black manila beige

Deep leather black

2 H9387 Woodland olive Congo brown Mahogany

3 H9388 Marigold primrose

Marigold sunflower 0

4 H9389 Slate Primrose 05 H9390 Slate Marigold 06 H9391 DV72

Blumeodendron tokbrai

Slate Primrose 07 H9392 Slate Sunflower 08 H9393 Slate Sunflower 09 H9394

DV73Diospyros sp.

Sakura Honey cream 010 H9395 Sakura Jute 0

11 H9396 Sakura Congo brown maple 0

12 H9397 White sweet dream

Dark congo brown

Light congo brown

13 H9398DV74

Baccaurea trunciflora

Sakura Jute 014 H9399 Sweet dream Sakura 015 H9400 Slate Sunflower 016 H9401 Lavender grey Sunflower 017 H9402 Slate Primrose 018 H9403 DV75


Slate Jasmine 019 H9404 Slate Primrose 020 H9405 Papier grey Jasmine 0

21 H9406DV76Shorea

johorensisWhite Pearl white 0

22 H9407DV77

Shorea leprosula

Pearl white Barbary gold Light barbary gold

23 H9408 Slate Marigold 024 H9409 Pearl white Mahogany Maple25 H9410 DV78

Shorea johorensis

Slate Primrose 026 H9411 Slate Primrose 027 H9412 Slate Primrose 0

3737


28 H9413 DV79Aglaia

tomentosa

Slate Primrose 029 H9414 Slate Primrose 030 H9415 Slate Primrose 0

31 H9416DV80


Slate Primrose 0

32 H9417 DV108Nephelium

ramboutan-ake

Slate Primrose 033 H9418 Slate Primrose 034 H9419 Slate Primrose 035 H9420 DV118

Paranephelium xestophyllum

Slate Barbary gold 0

36 H9421 Lavender grey Sunflower 0

37 H9422 DV230Madhuca korthalsii

Green white Milky 0

38 H9423 Light green White 0

39 H9424 DV231Shorea

parvifolia

Green Orange 0

40 H9425 Green Yellowish 0

41 H9426DV232

Shorea leprosula

Dark green Dark green 042 H9427 Beige, green White 043 H9428 White Yellowish 044 H9429 White Yellowish 045 H9430

DV233Madhuca korthalsii

White Milky 046 H9431 Green Dark brown 0

47 H9432 Green Yellowish brown 0

48 H9433 Green Yellowish brown 0

49 H9434

DV234Buchanania

insignis

White yellowish Light yellow 050 H9435 White Beige 051 H9436 White Light brown 052 H9437 White Light yellow 053 H9438 Yellowish white Brownish 054 H9439 White White 0

55 H9440 Green, red, white, yellow Beige 0

56 H9441DV235

Glochidion rubrum

White Yellowish 0

38


38

57 H9442

DV236Aporusa falcifera

Green, red, white Milky 0

58 H9443 White Milky 059 H9444 White Milky 060 H9445 Brown Greenish brown 061 H9446 White Brownish 062 H9447 Yellow Beige 063 H9448

DV237Magnolia

candollei var. candollei

Black Black 064 H9449 Yellow Orange 065 H9450 White Yellowish 066 H9451 Yellow Beige 067 H9452 Black Black green 068 H9453 Green Beige 069 H9454 DV238

Fordia splendidissima

Brown White greenish 070 H9455 Brown Dark brown 071 H9456 White pinkish White 0

72 H9457DV239Shorea

parvifoliaBrown Brown 0

73 H9458DV278

Polyalthia xanthopetala

Green White 0

74 H9459 DV279Microcos reticulata

White Orange Orange

75 H9460 Beige Yellow 0

76 H9461

DV280Mallotus wrayi

White yellowish Yellowish 0

77 H9462 Green, red, white, yellow Yellowish 0

78 H9463 Green, white, yellow Beige 0

79 H9464 Green, red, white Yellowish 0

80 H9465 White Greyish 081 H9466 Green White 082 H9467 Dark green Dark green 083 H9468 DV281

Aporusa falcifera

White Yellowish 0

84 H9469 Dark green Dark green 0

85 H9470 DV282Aporusa falcifera

White Brownish 0

86 H9471 Dark green Dark green 0

3939


87 H9472

DV283Mallotus wrayi

Green Yellowish 088 H9473 Green, white Yellowish 089 H9474 White greyish Yellow cream 090 H9475 White Yellowish 091 H9476 White Yellowish 092 H9477 White Yellowish 093 H9478 White Yellowish 0

0 = No extracellular pigmentation

40


40

Table 4. Results of the screening test against AfsK/AfsR in Streptomyces griseus IFO13350 (H10000).

Fungal extract(20 μl)

Diameter of inhibition zones (mm)

StatusFirst test Second test1.5% D (+)-

Glucose1.5%

D-Mannitol1.5% D

(+)-Glucose1.5%

D-Mannitol

H9386 ( 18 ) 0 ( 15 ) 0 Potential inhibitor




H9391 ( 12 ) 0 0 0 No activityH9420 ( 7 ) 0 0 0 Uncertain H9388–H9390, H9392–H9406, H9408, H9410–H9419, H9421–H9478

0 0 0 0 No activity

( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm); 0 = no inhibition zone around the paper disc

4141


Table 5. Results of the screening test against anti-mycobacterial (KdpE/KdpD) in wild type Mycobacterium smegmatis mc2155 strain H8000.

Actinomyceteextract (20 μl)

Diameter of inhibition zones (mm)Status100 μM

KH2PO4

1 mM KH2PO4

0.8%NaCl

4.0%NaCl

H11588 30 27 24 20 ToxicH11426–H11427, H11429–H11431, H11564–H11581,H11583–H11587, H11589–H11638, H11640–H11644, H11646, H11648–H11656, H11813, H11873

0 0 0 0 No activity

0 = no inhibition zone around the paper disc

42


42

Table 6. Results of the actinomycetes and fungal extracts screening test against Salmonella (PhoP/PhoR) in wild type Mycobacterium smegmatis mc2155 strain H8000.

Fungal 20 uL extract

Diameter of inhibition zones (mm)Remark100 µM

MgSO4.7H2O1 mM MgSO4.7H2O

H9386–H9478 0 0 No activity

Actinomycete20 uL extract

Diameter of inhibition zones (mm)Remark100 µM

MgSO4.7H2O1 mM MgSO4.7H2O

H11855 (14) (9) Potential inhibitor

H11482, H11588, H11612, H11615, H11724, H11764, H11781, H11782, H11818, H11857, H11865, H11888, H11900, H11911, H11949

(30) (30) Toxic

H11426–H11481, H11483, H11485–H11518, H11520–H11525, H11527–H11574, H11576–H11581, H11583–H11587, H11589–H11611, H11613–H11614, H11616–H11696, H11698–H11723, H11725–H11744, H11746–H11763, H11765–H11771, H11773–H11780, H11783–H11817, H11819–H11854, H11856, H11858–H11864, H11866–H11887, H11889–H11892, H11894–H11899, H11901–H11910, H11913–H11923, H11939–H11948, H11950–H11975

0 N.T. No activity

H11519, H11526, H11772 0 0 No activity

( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm)0 = no inhibition zone around the paper discN.T. = Not tested ____ = representative strain with data

Table 6. continues on Page 43

4343


Remark: The screening test was first carried out on both low and high Mg2+-containing media. For convenience, only a low Mg2+-level test was performed. If an inhibition zone appeared, a high Mg2+-level test was performed.

Table 7. Dependency of extract volume from strain H11855 in 100 µM and 1 mM of magnesium sulphate media

MgSO4.7H2ODiameter of inhibition zones (mm)

Volume of extract20 µL 40 µL 60 µL 80 µL 100 µL

100 µM ( 10 ) (pi) ( 13 ) ( 14 ) ( 17 ) ( 18 )1 mM ( 7 ) (pi) ( 10 ) (pi) ( 12 ) ( 13 ) ( 15 )

( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm pi = partial inhibition

Table 8. Dependency of extract volume from strain H11855 in 25 µM, 50 µM, 100 µM and 1 mM of magnesium sulphate medium



25 µM ( 8 ) (pi) ( 14 ) ( 16 ) ( 19 ) ( 25 )50 µM ( 9 ) ( 16 ) ( 19 ) ( 20 ) ( 23 )100 µM ( 11 ) ( 15 ) ( 19 ) ( 21 ) ( 24 )1 mM ( 8 ) (pi) ( 10 ) ( 15 ) ( 19 ) ( 20 )

( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm); pi = partial inhibition

44


44

Table 9. Dependency of extract volume from strain H11855 in 25 µM, 50 µM, 100 µM and 1 mM of magnesium sulphate media



25 µM ( 10 ) (pi) ( 13 ) ( 14 ) ( 15 ) ( 16 )50 µM ( 9 ) ( 12 ) ( 13 ) ( 14 ) ( 15 )100 µM ( 9 ) (1 2 ) ( 12 ) ( 12 ) ( 14 )1 mM ( 8 ) (pi) ( 10 ) ( 11 ) v12 ) ( 13 )

( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm); pi = partial inhibition

Table 10. Dependency of extract volume from strain H11855 in 25 µM, 50 µM, 100 µM and 1 mM of magnesium chloride medium

MgClDiameter of inhibition zones (mm)


25 µM ( 11 ) ( 12 ) ( 13 ) ( 16 ) ( 17 )50 µM ( 10 ) ( 12 ) ( 13 ) ( 14 ) ( 17 )100 µM ( 10 ) ( 12 ) ( 13 ) ( 13 ) ( 16 )1 mM ( 9 ) ( 11 ) ( 13 ) ( 15 ) ( 15 )

( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm)

Table 11. Dependency of extract volume from strain H11855 tested with 20 µg/mL of streptomycin.

MgClDiameter of inhibition zones (mm)

20 µg/mL of streptomycin20 µL 40 µL 60 µL 80 µL 100 µL

25 µM ( 10 ) ( 11 ) ( 17 ) ( 21 ) ( 23 )2 mM ( 7 ) ( 10 ) ( 11 ) ( 19 ) ( 21 )

( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm)Streptomycin showed the same pattern of growth inhibition.

4545


Table 12. Results of the actinomycetes and fungal extracts screening test against ICL in wild type M. smegmatis mc2155 strain H8000.

Extract (40μl)Diameter of inhibition zones

(mm) StatusGlucose Acetate

H9448*, H11482*, H11484*, H11499*, H11526*, H11542*, H11588*, H11600*, H11612*, H11689*, H11735*, H11745*, H11764*, H11781*, H11782*, H11818*, H11826*, H11855*, H11856*, H11857*, H11865*, H11867*, H11876*, H11888*, H11900*, H11911*

(19) (13)

Toxic

H9386–H9447, H9449–H9478, H11483*, H11485–H11498, H11500–H11525, H11527–H11539, H11540*, H11541* H11543–H11581, H11583–H11587, H11589–H11599, H11601–H11611, H11613–H11614, H11615*, H11616–H11688, H11690–H11696, H11698–H11721, H11723–H11734, H11736–H11744, H11746–H11763, H11765–H11780, H11783–H11793, H11794*, H11795–H11798, H11799*, H11800–H11806, H11807*, H11808–H11817, H11819, H11820*, H11821–H11825, H11827–H11853, H11858–H11864, H11866*, H11868*–H11870, H11872–H11875, H11878*–H11879, H11881–H11887, H11889–H11892, H11894–H11899, H11901–H11904, H11905*, H11906–H11909, H11910*, H11913–H11929, H11930*, H11926–H11938 H11931–H11938

N.T. 0

No activity

( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm)0 = no inhibition zone around the paper disc

____ = representative strain with data* = extract tested twice

N.T. = not tested

46


46

Table 13. Results of the screening test against type 1 protein phosphatase (PP1) inhibitor in wild type yeast PAY704-1 strain H10018 and mutant PAY700-4 strain H10017

ActinomyceteExtract(20 uL)


StatusWild type PAY704-1 Mutant PAY700-4

YPD YPD + Sorbitol YPD YPD +

Sorbitol25 °C 37 °C 25 °C 37 °C 25 °C 37 °C 25 °C 37 °C

H11533* 0 11 0 0 0 – 0 0 Potential inhibitor

H11534* 0 12 0 0 0 – 0 0 Potential inhibitor

H11735* 0 12 0 0 0 – 0 Weak Potential inhibitor

H11428*, H11449*, H11462, H11463, H11477, H11478, H11479, H11482, H11483, H11490, H11532*, H11535*, H11536*, H11537*, H11544*, H11547*, H11548*, H11555*, H11557*, H11559*, H11739*, H11743*, H11745*, H11757*, H11764*, H11772*, H11781*, H11821, H11822, H11837, H11844, H11853, H11856, H11860, H11865, H11875, H11876

(12) (17) (8) (9) (14) – (11) (12) Toxic


4747


H11426*–H11427*, H11429*–H11448*, H11450*–H11456*, H11457–H11461, H11464–H11476, H11480–H11481, H11484–H11489, H11527*–H11531*, H11538*–H11543*, H11546*, H11549*–H11554*, H11556*, H11558*,H11560*–H11563*, H11813–H11817, H11819–H11820, H11823–H11836, H11838–H11843, H11845–11852, H11854–H11855, H11857–H11859, H11861,H11863–H11864,H11866–H11874,H11877–H11878,H11880–H11882,H11884–H11886

0 0 0 0 0 - 0 0 No activity

* 40 mL of extract applied ( ) = inhibition zone diameter (diameter of paper disc ＝ 6 mm)0 =no inhibition zone around the paper disc

– = no yeast growth on plate____, = representative strain with data

48


48

Table 14. Results of the screening test against mammalian GSK-3β in mutant yeast strain H10075.

Actinomycete extract(20 μl)

Diameter of inhibition

zones (mm) Status

25ºC 37ºCH11462 (6) (19) Potential inhibitorH11490 0 (20) Potential inhibitorH11526 0 (12) Potential inhibitorH11668 0 (8) Potential inhibitorH11686 0 (7) Potential inhibitorH11695 0 (13) Potential inhibitor

H11720 (12) (30) Potential inhibitor

H11785 0 (17) Potential inhibitor H11809 (10) (27) Potential inhibitor

H11477, H11482, H11483, H11520, H11536*, H11537*, H11544*, H11555*, H11557*, H11559*, H11588**, H11612, H11635, H11647, H11722, H11739*, H11757, H11764, H11810**, H11821, H11822, H11843, H11844, H11853, H11855, H11856, H11857, H11860, H11866, H11888, H11900, H11911

(30) (30) Toxic


4949


H11426–H11456, H11457**–H11460**, H11461, H11463, H11464**, H11465, H11466**, H11468**–H11469**, H11470–H11471, H11472**–H11474**, H11475–H11476, H11478–H11479, H11481, H11484–H11486, H11487**–H11488**, H11489, H11491–H11519, H11521–H11525, H11527*–H11535*, H11538*–H11543*, H11545*–H11554*, H11556*, H11558*, H11560*–H11563*, H11564–H11578, H11580–H11581, H11584, H11585**–H11587**, H11589**–H11591**, H11592–H11600, H11601**, H11602–H11604, H11605**–H11609**, H11610–H11611, H11613, H11615–H11634, H11636, H11638–H11644, H11646, H11648–H11667, H11669–H11685, H11687–H11694, H11696–H11719, H11721, H11723–H11730, H11731*

, H11732, H11733*, H11734, H11735*, H11736–H11737, H11738*, H11740*, H11742*–H11743*, H11744–H11745, H11746*, H11747*, H11748, H11749*, H11750, H11751*, H11752, H11754*, H11755–H11756, H11758–H11759, H11760*–H11763*, H11765–H11766, H11768–H11770, H11771* , H11772, H11774*–H11776*, H11777, H11778*–H11779*, H11780, H11781*, H11782**–H11784**, H11786**–H11808**, H11811**, H11819–H11820, H11823–H11842, H11845–H11852, H11854, H11858–H11859, H11861, H11865, H11867–H11869, H11875–H11879, H11887, H11889, H11891–H11892, H11894–H11897, H11899, H11901–H11905, H11907, H11909–H11910, H11913–H11915, H11917–H11920, H11922–H11923

0 0 No activity

* 30 mL of extract applied ** 15 mL of extract applied


____ = representative strain with data

50


50

Table 15. Results of the screening test against mammalian GSK-3β in yeast mutant strain H10075 with and without sorbitol rescue.

Actinomycete extract (20 μl)

Diameter of inhibition zones (mm)Status

without sorbitol 1.2 M sorbitolH11462 15 6 Potential inhibitorH11490 7 7 (pi) ToxicH11526 7 0 Potential inhibitorH11668 13 7 Potential inhibitorH11686 7 0 Potential inhibitorH11695 16 8 Potential inhibitorH11720 18 8 Potential inhibitorH11785 10 8 Potential inhibitorH11809 35 20 Potential inhibitor

Diameter of paper disc = 6 mmpi: partial inhibition

Table 16. Results of the screening test against yeast transformant gsk-3 null with Yep24-MCK1 mutant strain H10079.

Actinomycete extract (20 μl)


25oC 37oCH11462 10 18 Potential inhibitorH11526 7(w) 8 Toxic H11668 0 0 No activityH11686 0 0 No activityH11695 10 13 ToxicH11720 0 0 No activityH11785 0 8 Potential inhibitorH11809 16 24 Potential inhibitor

Diameter of paper disc = 6 mmw: weak inhibition zone

5151


Table 17. Results of the screening test against yeast transformant gsk-3 null with Yep24-MCK1 mutant strain H10079 with and without sorbitol rescue.

Actinomyceteextract (20 μl)


Without sorbitol 1.2 M sorbitol

H11462 18 12 Potential inhibitor



Diameter of paper disc = 6 mm

52


52

Table 18. Results of the fungal and actinomycete extracts screening test against MAPK kinase (MKK1P386) in mutant yeast strain H10068.

Fungal extract (20 μl)Diameter of inhibition zones

(mm) StatusGlucose plate Galactose plate

H9386–H9478 β, – λ, – No activity

Actinomycete extract (20 μl)Diameter of inhibition zones

(mm) StatusGlucose plate Galactose plate

H11428* β, 9 λ, – ToxicH11588* β, 8 λ, – ToxicH11612* β, 18 λ, – ToxicH11635* β, 11 λ, – ToxicH11813* β, 10 λ, – ToxicH11426*–H11427*, H11429*–H11433*, H11457*–H11460*

, H11463*–H11466*, H11527*–H11534*, H11564*–H11581*, H11583*–H11587*, H11589*–H11592*, H11610*–H11611*, H11613*–H11634*, H11636*–H11664*, H11690*–H11695*, H11814*–H11817*, H11863*–H11864*, H11870*–H11874*, H11877*, H11880*–H11882*, H11884*–H11886*

β, – λ, – No activity

Diameter of paper disc = 6 mm– = No growth around paper disc

β = Growth of yeast on the whole plate λ = No growth of yeast on the whole plate * 40 µL of extract applied

5353


Table 19. Results of the screening test against MAPK phosphatase (MKK1P386- MSG5) in mutant yeast strain H10069.



StatusGlucose

PlateGalactose

PlateH11428* β, 13 β, 12 ToxicH11588* β, 9 β , 8 ToxicH11610* β, 15 β , 15 ToxicH11612* β, 16 β, 15 ToxicH11635* β, 10 β, 10 ToxicH11640* β, 7 β,7 ToxicH11813* β, 19 β, 18 ToxicH11426–H11427, H11429–H11433, H11457–H11460, H11463–H11466, H11527–H11534, H11564–H11581, H11583–H11587, H11589–H11608, H11611, H11613–H11634, H11636–H11639, H11641–H11664, H11690–H11695, H11814–H11817, H11863–H11864, H11870–H11874, H11877, H11880–H11882, H11884–H11886

β, – β, – No activity

Diameter of paper disc = 6 mm* 40 µL of extract applied – : No inhibition β : Growth of yeast on the whole plate

54


54

Table 20. Results of the screening test against Ras/Raf-1 protein-protein interaction in LZ yeast strain H10014.



Status Without histidine

130 µM Histidine

H11499, H11515, H11518, H11520, H11525, H11526, H11530, H11532*, H11533*, H11534*, H11535*, H11536*, H11537*, H11544*, 11548*, H11550*, H11555*, H11557*, H11559*, H11661, H11669, H11670, H11671, H11677* H11739, H11750, H11809**, H11810**, H11822, H11855, H11856, H11857, H11858, H11861, H11867, H11878, H11879

(12) (12) Toxic

H11426–H11498, H11500–H11514, H11516–H11517, H11519, H11521–H11524, H11527*–H11529*, H11531*, H11538*–11543*, H11545*–H11547*, H11549*, H11551*–H11554*, H11556*, H11558*, H11560*–H11564*, H11657–H11660, H11662–H11668, H11672–H11676, H11678–H11683, H11684**–H11686**, H11687–H11688, H11690–H11730, H11731–H11738, H11740–H11749, H11751–H11770, H11782**–H11808**, H11811**–H11812**, H11816–H11818, H11819–H11821, H11823–H11854, H11859–H11860, H11865–H11866, H11868–H11869, H11875–H11877

0 0 No activity

* 30 µL of extract applied** 15 µL of extract applied


____ = representative strain with data

5555


Fig. 1. Sampling sites along the Dr. Clive Marsh Trail.

56


56

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60


60

Appendix: Danum Valley soil list with location and tree description.

No. Soilsample

Date ofcollection Location Tree description

1 DV1 25/9/2004

21.53 m from the Palum Tambun River suspension bridge. Right side of the Danum Valley Nature Trail.

Family : DipterocarpaceaeName : Parashorea tomentellaLocal name : Urat mata beluduC : 0.86 mH : 18 mOther info : Soil pH 7.01

2 DV2 25/9/2004Right side of the Nature Trail. 5.3 m from DV91.

Family : MeliaceaeName : Dysoxylum undulatumLocal name : -C : 3.02 mH : 30 mOther info : -

3 DV3 25/9/2004Left side of the Nature Trail.16 m from DV92.

Family : DipterocarpaceaeName : Parashorea tomentellaLocal name : Urat mata beluduC : 2.8 mH : 30 mOther info : -

4 DV4 25/9/2004Left side of the Nature Trail.5.7 m from DV93.


5 DV5 26/9/2004

Near Segama River suspension bridge.Left side of the West Trail.10.5 m from Dr. Clive Wallis Marsh memorial.

Family : DipterocarpaceaeName : Shorea johorensisLocal name : Seraya majauC : 2.35 mH : 40 mOther info : Soil pH 6.59

6 DV6 26/9/2004

Right side of the West Trail. 18.97 m from DV95.

Family : DipterocarpaceaeName : Shorea parvifoliaLocal name : Seraya punaiC : 2.15 mH : 38 mOther info : Big buttress

6161


7 DV7 26/9/2004

Left side of the West Trail.Approximately 20 m from DV96.

Family : DipterocarpaceaeName : Parashorea tomentellaLocal name : Urat mata beluduC : 4 mH : 40 mOther info : Soil pH 6.39

8 DV8 26/9/2004 Northern part of NB, plot 1.

Family : Unidentified Name : -Local name : -C : 1.078 mH : 20 mOther info : Soil pH 4.09


Family : AlangiaceaeName : Alangium javanicumLocal name : -C : 0.357 mH : 15 mOther info : NB1380


Family : MagnoliaceaeName : Magnolia candollei var.

singapurensisLocal name : CempakaC : 0.48 mH : 15 mOther info : NB1407

11 DV11 25/9/2004

At the campsite (facing downstream of the Segama River, on the right-hand bank).1.5 m south of campsite situated downstream. 10 m northwest of DV1.

Family : DipterocarpaceaeName : Parashorea tomentellaLocal name : Urat mata beluduC : 3.24 mH : 30 mOther info : Produces resin, height of

buttress: 3.8 m

12 DV12 25/9/2004

Facing uphill, on the right side of the Nature Trail.5 m west of DV2.

Family : Euphorbiaceae Name : Blumeodendron tokbraiLocal name : GangulangC : 0.86 mH : 12 mOther info : -

62


62

13 DV13 25/9/2004

Facing uphill, on the left side of the Nature Trail.3.7 m west of DV3.

Family : MeliaceaeName : Aglaia odoratissimaLocal name : Langsat-langsatC : 0.57 mH : 8 mOther info : -

14 DV14 25/9/2004

Facing uphill, on the right side of the Nature Trail.3.4 m southwest of DV4.

Family : MyristicaceaeName : Knema cinereaLocal name : Darah-darahC : 0.95 mH : 16 mOther info : -

15 DV15 26/9/2004

Facing uphill, on the right side of the West Trail.17 m northwest of the Clive Marsh memorial.

Family : DipterocarpaceaeName : Parashorea tomentellaLocal name : Urat mata beluduC : 5.37 mH : 40 mOther info : Height of buttress: 3 m, soil

pH 7.22

16 DV16 26/9/2004

Facing uphill, on the left side of the West Trail.23.2 m southwest of the first small bridge after the Clive Marsh memorial.

Family : DipterocarpaceaeName : Shorea johorensisLocal name : Seraya majauC : 2.69 mH : 25 mOther info : Bamboos grow around the

tree, height of buttress: 1.7 m

17 DV17 26/9/2004

Facing uphill, on the left side of the West Trail.11 m southwest of the W3 checkpoint.

Family : DipterocarpaceaeName : Shorea johorensisLocal name : Seraya majauC : 3.90 mH : 40 mOther info : Fungi milky white in colour

grow all around the tree trunk starting from above the buttress to nearly half the height of the tree. Each fungus is about 4–5 cm in length, height of buttress: 2.3 m

18 DV18 26/9/2004

Northern part of NB, plot 1.8 m east of DV8. 3 m south of DV28.

Family : TiliaceaeName : Pentace laxiflora Local name : Takalis daun halusC : 0.89 mH : 22 mOther info : NB0389

6363


19 DV19 26/9/2004

Northern part of NB, plot 1.4.5 m northwest of DV79.

Family : DipterocarpaceaeName : Shorea parvifoliaLocal name : Seraya punaiC : 0.48 mH : 23 mOther info : NB1328

20 DV20 26/9/2004Northern part of NB, plot 1.1.5 m south of DV10.

Family : MeliaceaeName : Aglaia tomentosaLocal name : KalambiaoC : 0.38 mH : 11 mOther info : NB1498

21 DV21 25/9/2004Left side of the Nature Trail. 9.95 m from DV1.


22 DV22 25/9/2004Left side of the Nature Trail. 4 m from DV12.

Family : Alangiaceae Name : Alangium ebenaceumLocal name : Kondolon C : 0.72 mH : 12 mOther info : -

23 DV23 25/9/2004

Left side of the Nature Trail. 0.10 m from DV13.

Family : Ebenaceae Name : Diospyros elliptifoliaLocal name : Kayu malamC : 1.68 mH : 27 mOther info : -

24 DV24 25/9/2004

Left side of the Nature Trail.3.3 m from observation tower.

Family : Melastomataceae Name : Memecylon appendiculatumLocal name : Nipis kulit C : 0.40 mH : 8 mOther info : -

25

DV25 26/9/2004

Left side of the West Trail.16.28 m from Dr. Clive Marsh memorial.

Family : DipterocarpaceaeName : Parashorea malaanonanLocal name : Urat mata daun licinC : 0.90 mH : 25 mOther info : -

64


64

26 DV26 26/9/2004Left side of the West Trail. 5.3 m from DV16.

Family : EuphorbiaceaeName : Homalanthus populneusLocal name : Ludai susuC : 2.70 mH : 18 mOther info : -

27 DV27 26/9/2004

Right side of the West Trail.18.4 m from W3 marker.

Family : DipterocarpaceaeName : Shorea johorensisLocal name : Seraya majauC : 3.1 mH : 20 mOther info : -


Family : MeliaceaeName : Chisocheton sarawakanusLocal name : -C : 1.45 mH : 16 mOther info : NB0418


Family : MeliaceaeName : Chisocheton polyandrusLocal name : -C : 1.32 mH : 15 mOther info : NB0476


Family : EuphorbiaceaeName : Baccaurea tetrandraLocal name : Kunau-kunauC : 0.42 mH : 9 mOther info : NB1434

31 DV31 25/9/2004

Left side of the Nature Trail.10.62 m from DV21.


32 DV32 25/9/2004Right side of the Nature Trail.5.6 m from DV22.

Family : LauraceaeName : Nothaphoebe canescensLocal name : MedangC : 0.78 mH : 12 mOther info : -

6565


33 DV33 25/9/2004

Right side of the Nature Trail.12.82 m from DV23.


34 DV34 25/9/2004

Left side of the Nature Trail.5.5 m from the observation tower.

Family : SapindaceaeName : Dimocarpus longanLocal name : Mata kucingC : 0.64 mH : 15 mOther info : -

35 DV35 26/9/2004

Right side of the West Trail.15.13 m from Dr. Clive Marsh memorial.


36 DV36 26/9/2004

Right side of the West Trail.12.01 m from the W2 marker.


37 DV37 26/9/2004

Right side of the West Trail.Beside small river. 21.51 m from the W3 marker.



Family : LauraceaeName : Phoebe macrophyllaLocal name : Medang ladaC : 1.24 mH : 23 mOther info : NB0442


Family : DipterocarpaceaeName : Vatica oblongifoliaLocal name : Resak daun panjangC : 1.17 mH : 20 mOther info : NB0484

66


66


Family : MeliaceaeName : Aglaia silvestris Local name : -C : 0.512 mH : 15 mOther info : NB1360

41 DV41 25/9/2004

Checkpoint to observation tower. On the right of DV31. 6.4 m from DV31.

Family : MyrtaceaeName : Eugenia sp.Local name : ObahC : 2.12 mH : 25 mOther info : -

42 DV42 25/9/2004

On the left of DV32. 2.705 m from DV32.

Family : SapindaceaeName : Paranephelium nitidumLocal name : MembuakatC : 0.57 mH : 10 mOther info : -

43 DV43 25/9/2004

On the left of DV33. 1.84 m from DV33.

Family : Polygalaceae Name : Xanthophyllum affineLocal name : Minyak berokC : 0.6 mH : 15 mOther info : -

44 DV44 25/9/2004

Opposite observation tower.On the right of DV34. 5.8 m from DV34.

Family : EuphorbiaceaeName : Baccaurea stipulataLocal name : Kunau-kunauC : 0.56 mH : 12 mOther info : -

45 DV45 26/9/2004

On the right of Dr Clive Marsh memorial. 15.6 m from DV35.

Family : AnnonaceaeName : Polyalthia sp.Local name : KaraiC : 1.45 mH : 15 mOther info : -

46 DV46 26/9/2004On the left of DV36. 3 m from DV36.

Family : FagaceaeName : Lithocarpus sp.Local name : MempeningC : 2.4 mH : 15 mOther info : -

6767


47 DV47 26/9/2004

On the left of W3 marker. 20.1 m from DV37.

Family : FagaceaeName : Lithocarpus sp.Local name : MempeningC : 2.8 mH : 20 mOther info : -


Family : ThymelaeaceaeName : Gonystylus keithiiLocal name : -C : 0.45 mH : 15 mOther info : NB0459


Family : MyrtaceaeName : Syzygium tawaenseLocal name : -C : 0.42 mH : 20 mOther info : NB1370


Family : BombacaceaeName : Neesia strigosaLocal name : Durian monyetC : 0.35 mH : 8 mOther info : NB1439




Family : SapindaceaeName : Nephelium sp.Local name : -C : 0.54 mH : 9 mOther info : -

53 DV53 25/9/2004Left side of the Nature Trail. 0.5 m from DV43.

Family : MeliaceaeName : Lansium domesticumLocal name : LangsatC : 0.23 mH : 10 mOther info : -

68


68


Family : Sapindaceae Name : Dimocarpus longanLocal name : Mata kucingC : 1.2 mH : 23 mOther info : -

55 DV55 26/9/2004Right side of the West Trail. 1.6 m from DV45.

Family : DipterocarpaceaeName : Parashorea tomentella Local name : Urat mata beluduC : 4.5 mH : 40 mOther info : -

56 DV56 26/9/2004Right side of the West Trail. 1.6 m from DV46.

Family : DipterocarpaceaeName : Parashorea tomentella Local name : Urat mata berluduC : 4.5 mH : 40 mOther info : -

57 DV57 26/9/2004Right side of the West Trail. 115 m from DV47.

Family : DipterocarpaceaeName : Parashorea malaanonan Local name : Urat mata daun licinC : 1.36 mH : 20 mOther info : -


Family : BurseraceaeName : Santiria tomentosaLocal name : KerantaiC : 0.88 mH : 20 mOther info : NB0348


Family : PolygalaceaeName : Xanthophyllum vitellinumLocal name : -C : 0.6 mH : 25 mOther info : NB1383


Family : SapotaceaeName : Madhuca korthalsii Local name : Nyatoh madhucaC : 0.35 mH : 12 mOther info : NB1420

6969


61 DV61 25/9/2004


Family : Dipterocarpaceae Name : Parashorea tomentellaLocal name : Urat mata beluduC : 0.89 mH : 18 mOther info : -


Family : EuphorbiaceaeName : Baccaurea tetrandraLocal name : Kunau-kunauC : 0.302 mH : 12 mOther info : -

63 DV63 25/9/2004


Family : Rubiaceae Name : Uncaria sp.Local name : LiaraC : 0.70 mH : 20 mOther info : -

64 DV64 25/9/2004

Behind the observation tower. Right side of the Nature Trail.6.2 m from DV54.

Family : DilleniaceaeName : Tetracera akara.Local name : -C : 0.57 mH : 20 mOther info : -

65 DV65 26/9/2004Left side of the West Trail.35 m from DV55.


66 DV66 26/9/2004

Left side of the West Trail. 8.14 m from DV56.

Family : MoraceaeName : Ficus sp.Local name : -C : 2.5 mH : 25 mOther info : -

67 DV67 26/9/2004

Left side of the West Trail.16.9 m from DV57.

Family : DipterocarpaceaeName : Shorea johorensisLocal name : Suraya majauC : 4.02 mH : 37 mOther info : High buttress

70


70


Family : MeliaceaeName : Dysoxylum cyrtobotryumLocal name : -C : 0.61 mH : 20 mOther info : NB0344


Family : AnnonaceaeName : Phaeanthus sp. 1Local name : -C : 0.724 mH : 20 mOther info : NB0381


Family : EuphorbiaceaeName : Baccaurea tetrandraLocal name : Kunau-kunauC : 0.32 mH : 8 mOther info : NB1378

71 DV71 25/9/2004



72 DV72 25/9/2004


Family : EuphorbiaceaeName : Blumeodendron tokbraiLocal name : GangulangC : 0.58 m H : 15 mOther info : -

73 DV73 25/9/2004


Family : EbenaceaeName : Diospyros sp.Local name : Kayu malamC : 1.39 mH : 21 mOther info : -

74 DV74 25/9/2004


Family : EuphorbiaceaeName : Baccaurea truncifloraLocal name : -C : 0.499 mH : 15 mOther info : -

7171


75 DV75 26/9/2004



76 DV76 26/9/2004


Family : Dipterocarpaceae Name : Shorea johorensisLocal name : Seraya majauC : 5.459 mH : 40 mOther info : -

77 DV77 26/9/2004

Right side of the West Trail.13.36 m from DV57.

Family : DipterocarpaceaeName : Shorea leprosulaLocal name : Seraya tembagaC : 2.968 mH : 40 mOther info : -


Family : DipterocarpaceaeName : Shorea johorensisLocal name : Seraya majauC : 0.71 mH : 18 mOther info : NB0351


Family : MeliaceaeName : Aglaia tomentosaLocal name : KalambiaoC : 0.579 mH : 12 m Other info : NB1326


Family : BurseraceaeName : Canarium denticulatumLocal name : KedondongC : 0.52 mH : 15 mOther info : NB1417

81 DV81 25/9/2004 16.7 m south of DV61.


72


72

82 DV82 25/9/2004 3.69 m south of DV72.

Family : AlangiaceaeName : Alangium ebenaceumLocal name : KondolonC : 0.53 mH : 15 mOther info : Open area, sunlight can pass through

83 DV83 25/9/2004 6.97 m south of DV73.


84 DV84 25/9/2004 3.1 m southwest of DV74.

Family : TiliaceaeName : Brownlowia peltataLocal name : Pinggau-pinggauC : 0.18 mH : 10 mOther info : -

85 DV85 26/9/2004 17.0 m southwest of DV65.


86 DV86 26/9/2004 12.2 m west of DV76.


87 DV87 26/9/2004 11.2 m north of DV77.



Family : LauraceaeName : Beilschmiedia sp. ALocal name : -C : 1.82 mH : 35 mOther info : NB0413

7373



Family : AnnonaceaeName : Polyalthia sumatranaLocal name : Karai putihC : 0.62 mH : 22 mOther info : NB0420



91 DV91 25/9/2004

Left side of Nature Trail.8.35 m from DV81.

Family : MeliaceaeName : Dysoxylum undulatumLocal name : -C : 0.785 mH : 20 mOther info : Low buttress, smooth bark


Family : EuphorbiaceaeName : Baccaurea stipulataLocal name : Kunau-kunauC : 0.4 mH : 4 mOther info : -

93 DV93 25/9/2004


Family : EuphorbiaceaeName : Baccaurea stipulataLocal name : Kunau-kunauC : 0.33 mH : 4.5 mOther info : -

94 DV94 25/9/2004


Family : Meliaceae Name : Aglaia sp.Local name : LantupakC : 1.926 mH : 15 mOther info : -

95 DV95 26/9/2004

Right side of the West Trail.Cross 2 small streams.Next to old tree stump.9.08 m from W1 marker.

Family : DipterocarpaceaeName : Parashorea tomentellaLocal name : Urat mata beluduC : 1.18 mH : 30 mOther info : Small buttress, soil pH 4.74

74


74

96 DV96 26/9/2004Left side of the West Trail.3.4 m from DV86.

Family : DipterocarpaceaeName : Parashorea malaanonanLocal name : Urat mata daun licinC : 1.82 mH : 40 mOther info : Low buttress

97 DV97 26/9/2004

Right side of the West Trail.29.52 m from DV87.

Family : AnnonaceaeName : Polyalthia sp.Local name : KaraiC : 1.84 mH : 15 mOther info : -


Family : ThymelaeaceaeName : Gonystylus keithiiLocal name : -C : 056 mH : 7 mOther info : NB1346


Family : DipterocarpaceaeName : Shorea agamiiLocal name : Melapi agamaC : 0.123 mH : 20 mOther info : NB1346


Family : ThymelaeaceaeName : Gonystylus keithiiLocal name : -C : 0.38 mH : 10 mOther info : NB1346

101 DV101 27/9/2004

Near observation tower.Left side of the Nature Trail.14.2 m from Tembaling waterfall signboard.

Family : Myrtaceae Name : Eugenia sp.Local name : ObahC : 1.10 mH : 15 mOther info : -

102 DV102 27/9/2004

Facing uphill, on the right side of the trail.12.8 m west of DV101, 2.6 m north of DV103

Family : Alangiaceae Name : Alangium sp.Local name : KondolonC : 1.15 mH : 20 mOther info : -

7575



Family : Dipterocarpaceae Name : Shorea sp.Local name : SerayaC : 1.75 mH : 20 mOther info : -

104 DV104 27/9/2004


Family : DipterocarpaceaeName : Shorea sp.Local name : SerayaC : 1.14 mH : 18 mOther info : -

105 DV105 27/9/2004 17.5 m from DV104.

Family : DipterocarpaceaeName : Shorea parvifoliaLocal name : Seraya punaiC : 0.81 mH : 20 mOther info : -

106 DV106 27/9/2004


Family : DipterocarpaceaeName : Shorea agamiiLocal name : Melapi agamaC : 1.36 mH : 20 mOther info : -

107 DV107 27/9/2004


Family : MeliaceaeName : Aglaia sp.Local name : LantupakC : 0.862 mH : 12 mOther info : -

108 DV108 27/9/2004


Family : SapindaceaeName : Nephelium ramboutan-akeLocal name : MeritamC : 2.302 mH : 18 mOther info : -

109 DV109 27/9/2004 8.2 m south of DV108.

Family : DipterocarpaceaeName : Parashorea tomentellaLocal name : Urat mata beluduC : 1.00 mH : 20 mOther info :

76


76

110 DV110 27/9/2004


Family : AnnonaceaeName : Goniothalamus sp.Local name : LimpanasC : 0.50 mH : 10 mOther info : -

111 DV111 27/9/2004


Family : DipterocarpaceaeName : Parashorea malaanonanLocal name : Urat mata daun licinC : 2.2 mH : 50 mOther info : High buttress

112 DV112 27/9/2004

Facing uphill, on the left side of the trail.11.2 m southeast of DV111, 4.6 m west of DV113.

Family : BombacaceaeName : Neesia synandraLocal name : Durian monyetC : 2.03 mH : 20 mOther info : -

113 DV113 27/9/2004


Family : LeguminosaeName : Sympetalandra borneensisLocal name : Merbau lalatC : 2.6 mH : 25 mOther info : -

114 DV114 27/9/2004Left side of the Nature Trail.

Family : MyrtaceaeName : Eugenia sp.Local name : ObahC : 0.93 mH : 18 mOther info : -

115 DV115 27/9/2004 6.43 m from DV114.

Family : Meliaceae Name : Aglaia odoratissima Local name : Langsat-langsatC : 0.74 mH : 12 mOther info : -

116 DV116 27/9/2004


Family : LauraceaeName : Litsea sp. Local name : MedangC : 0.52 mH : 10 mOther info : -

7777


117 DV117 27/9/2004


Family : BurseraceaeName : Canarium denticulatumLocal name : KedondongC : 0.86 mH : 11 mOther info : -

118 DV118 27/9/2004


Family : Sapindaceae Name : Paranephelium xestophyllum Local name : MembuakatC : 0.491 mH : 12 mOther info : -

119 DV119 27/9/2004 7.24 m southeast of DV118.

Family : Fagaceae Name : Lithocarpus sp.Local name : mempeningC : 1.66 mH : 15 mOther info : -

120 DV120 27/9/2004


Family : MoraceaeName : Ficus sp.Local name : -C : 5.5 mH : 23 mOther info : -


Family : BurseraceaeName : Dacryodes rostrataLocal name : KedondongC : 0.368 mH : 12 mOther info : NB2006


Family : MeliaceaeName : Dysoxylum cyrtobotryumLocal name : -C : 0.85 mH : 15 mOther info : NB2348


Family : Dipterocarpaceae Name : Shorea argentifoliaLocal name : Seraya daun masC : 0.402 mH : 10 mOther info : NB2853

78


78


Family : Dipterocarpaceae Name : Shorea argentifoliaLocal name : Seraya daun masC : 0.634 mH : 10 mOther info : NB2829


Family : Fagaceae Name : Lithocarpus gracilisLocal name : MempeningC : 0.634 mH : 10 mOther info : NB3641


Family : Myrtaceae Name : Syzygium chrysanthum Local name : - C : 0.482 mH : 8 mOther info : NB3672


Family : EuphorbiaceaeName : Mallotus wrayiLocal name : Mallotus sagar-sagarC : 0.35mH : 9 mOther info : NB3564


Family : AnacardiaceaeName : Buchanania insignisLocal name : -C : 0.652 mH : 23 mOther info : NB3592


Family : MeliaceaeName : Dysoxylum rigidumLocal name : -C : 0.36 mH : 5 mOther info : NB3543


Family : DipterocarpaceaeName : Shorea fallaxLocal name : Seraya daun kasarC : 1.332 mH : 20 mOther info : NB4443

7979





Family : LeguminosaeName : Dialium hydnocarpoidesLocal name : -C : 1.038 mH : 19 mOther info : NB2337


Family : MyrtaceaeName : Syzygium castaneumLocal name : -C : 0.432 mH : 10 mOther info : NB2836




Family : FagaceaeName : Quercus argentataLocal name : -C : 0.445 mH : 12 mOther info : NB3628


Family : LecythidaceaeName : Barringtonia lanceolataLocal name : -C : 0.427 mH : 8 mOther info : NB3661


Family : DipterocarpaceaeName : Shorea leprosulaLocal name : Seraya tembagaC : 0.760 mH : 25 mOther info : NB3551

80


80


Family : TiliaceaeName : Microcos elmeriLocal name : -C : 0.730 mH : 20 mOther info : NB3582


Family : DipterocarpaceaeName : Shorea paucifloraLocal name : Oba sulukC : 0.740 mH : 22 mOther info : NB3611










Family : FagaceaeName : Lithocarpus nieuwenhuisiiLocal name : MempeningC : 0.74 mH : 15 mOther info : NB2863

8181



Family : MyrtaceaeName : Syzygium chrysanthumLocal name : -C : 0.43 mH : 11 mOther info : NB3627


Family : MyrtaceaeName : Syzygium chrysanthum Local name : -C : 0.36 mH : 7 mOther info : NB3667


Family : UlmaceaeName : Gironniera celtidifoliaLocal name : -C : 0.43 mH : 10 mOther info : NB3547


Family : EuphorbiaceaeName : Mallotus wrayiLocal name : Mallotus sagar-sagarC : 0.56 mH : 13 mOther info : NB3280


Family : EuphorbiaceaeName : Glochidion elmeriLocal name : -C : 0.49 mH : 19 mOther info : NB3596


Family : ChrysobalanaceaeName : Parinari oblongifoliaLocal name : MerbatuC : 0.445 mH : 15 mOther info : NB4416


Family : SapotaceaeName : Madhuca korthalsiiLocal name : Nyatoh madhucaC : 0.98 mH : 17 mOther info : NB2240

82


82


Family : DipterocarpaceaeName : Shorea parvifoliaLocal name : Seraya punaiC : 1 mH : 25 mOther info : NB2344






Family : AnacardiaceaeName : Buchanania insignisLocal name : -C : 0.92 mH : 18 mOther info : NB3644


Family : EuphorbiaceaeName : Glochidion rubrum Local name : Obah nasiC : 0.6 mH : 10 mOther info : NB3632


Family : EuphorbiaceaeName : Aporusa falciferaLocal name : -C : 0.78 mH : 15 mOther info : NB3569


Family : MagnoliaceaeName : Magnolia candollei var.

candolleiLocal name : Cempaka C : 0.32 mH : 7 mOther info : NB3579

8383



Family : LeguminosaeName : Fordia splendidissimaLocal name : -C : 0.34 mH : 7 mOther info : NB3570








Family : AnnonaceaeName : Phaeanthus sp. 1Local name : -C : 0.364 mH : 13 mOther info : NB2813


Family : LauraceaeName : Litsea sp. 1Local name : -C : 0.346 mH : 9 mOther info : NB2860



84


84


Family : EuphorbiaceaeName : Aporusa elmeriLocal name : PenatanC : 0.56 mH : 12 mOther info : NB3658




Family : LauraceaeName : Cryptocarya teysmanniana Local name : -C : 0.738 mH : 25 mOther info : NB3540


Family : OlacaceaeName : Scorodocarpus borneensis.Local name : Bawang hutanC : 0.50 mH : 18 mOther info : NB3536


Family : MyrtaceaeName : Syzygium chrysanthumLocal name : -C : 0.588 mH : 12 mOther info : NB4518




Family : FagaceaeName : Lithocarpus gracilisLocal name : MempeningC : 1.035 mH : 23 mOther info : NB2303

8585





Family : MyrtaceaeName : Syzygium sp. NLocal name : -C : 0.364 mH : 10 mOther info : NB2322






Family : DipterocarpaceaeName : Shorea johorensisLocal name : Saraya majauC : 0.696 mH : 20 mOther info : NB3553


Family : MeliaceaeName : Reinwardtiodendron humileLocal name : -C : 0.500 mH : 25 mOther info : NB3599


Family : LeguminosaeName : Sindora irpicinaLocal name : SepetirC : 0.360 mH : 12 mOther info : NB8990

86


86


Family : LauraceaeName : Cryptocarya teysmanniana Local name : -C : 0.414 mH : 9 mOther info : NB4448


Family : MeliaceaeName : Aglaia silvestris Local name : -C : 0.59 mH : 15 mOther info : NB4515


Family : Euphorbiaceae Name : Aporusa falciferaLocal name : -C : 0.808 mH : 8 mOther info : NB4980




Family : Euphobiaceae Name : Drypetes sp. ELocal name : -C : 0.93 mH : 15 mOther info : NB5008


Family : BurseraceaeName : Canarium denticulatumLocal name : Kedondong C : 1.208 mH : 20 mOther info : NB4978


Family : Fagaceae Name : Lithocarpus nieuwenhuisiiLocal name : MempeningC : 0.671 mH : 8 mOther info : NB5830

8787





Family : GuttiferaeName : Garcinia forbesiiLocal name : BebataC : 0.605 mH : 10 mOther info : NB4974


Family : MeliaceaeName : Dysoxylum rigidumLocal name : -C : 0.372 mH : 15 mOther info : NB5054


Family : FagaceaeName : Lithocarpus nieuwenhuisiiLocal name : MempeningC : 0.390 mH : 9 mOther info : NB5035


Family : TrigoniaceaeName : Trigoniastrum hypoleucumLocal name : -C : 0.460 mH : 5 mOther info : NB5781


Family : FagaceaeName : Quercus argentata Local name : -C : 0.998 mH : 20 mOther info : NB5734



88


88


Family : BombacaceaeName : Neesia strigosaLocal name : Durian monyitC : 0.47 mH : 8 mOther info : NB4893


Family : DilleniaceaeName : Dillenia excelsaLocal name : Simpoh lakiC : 0.465 mH : 7 mOther info : NB4990




Family : DipterocarpaceaeName : Parashorea malaanonanLocal name : Urat mata daun licinC : 0.33 mH : 6 mOther info : NB5787




Family : AnnonaceaeName : Polyalthia xanthopetalaLocal name : Angilan hutanC : 0.30 mH : 6 mOther info : NB4430


Family : TiliaceaeName : Microcos reticulataLocal name : -C : 0.65 mH : 21 mOther info : NB4963

8989









Family : EuphorbiaceaeName : Mallotus wrayi Local name : Mallotus sagar-sagarC : 0.31 mH : 10 mOther info : NB5811


Family : TrigoniaceaeName : Trigoniastrum hypoleucumLocal name : -C : 0.352 mH : 16 mOther info : NB4417




Family : AnnonaceaeName : Polyalthia congestaLocal name : -C : 0.646 mH : 15 mOther info : NB4991

90


90


Family : LauraceaeName : Litsea machilifoliaLocal name : -C : 0.642 mH : 9 mOther info : NB5031


Family : TiliaceaeName : Pentace laxifloraLocal name : Takalis daun halus C : 0.56 mH : 7 mOther info : NB5070


Family : CombretaceaeName : Terminalia foetidissimaLocal name : TalisaiC : 1 mH : 22 mOther info : NB5807


Family : MeliaceaeName : Chisocheton sarawakanusLocal name : -C : 0.988 mH : 23 mOther info : NB4511




Family : EuphorbiaceaeName : Baccaurea macrocarpaLocal name : Tampoi merahC : 0.944 mH : 18 mOther info : NB5053


Family : MyristicaceaeName : Knema latericiaLocal name : -C : 0.413 mH : 10 mOther info : NB5777

9191



Family : EuphorbiaceaeName : Antidesma neurocarpum.Local name : Sumping gudaunC : 0.334 mH : 9 mOther info : NB5813



Abbreviation: DV - Danum ValleyNB - NewberyW - West C - CircumferenceH - HeightINFO - Information

Soil collected by: DV1-10, DV101, DV 111 Ho Wei LoonDV11-20, DV102, DV 112 Chan Kai WaiDV21-30, DV103, DV 113 Bernard Tzing Ziang VuiDV31-40, DV104, DV 114 Lim Siok HarDV41-50, DV105, DV 115 Ngao Wee Chen DV51-60, DV106, DV 116 Tong Mei LingDV61-70, DV107, DV 117 Teh Soo ChinDV71-80, DV108, DV 118 Vun Su ChiunDV81-90, DV109, DV 119 Mak Ken HingDV91-100, DV110, DV 120 Prof. Dr. Ho Coy ChokeDV200-209, DV260-265 Ho Wei LoonDV210-219, DV266-271 Celistha Fay J. Ferdinand DeosingDV220-229, DV272-277 Jenifer Rolland LDV230-239, DV278-283 Jessica PeterDV240-249, DV284-289 Mohammad Ridzuan Bin BakeriDV250-259, DV290-295 Yahya Bin Jalal

Documents

Final Thesis