CHAPTER 6

ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF BIOACTIVE

COMPOUNDS FROM GRAMINICOLOUS FUNGI

6.1 Introduction

There is an ever-growing need for new and useful compounds to provide

assistance and relief in all aspects of human health. Drug resistance in bacteria, the

appearance of life-threatening viruses, the recurrent problems of diseases in persons

with organ transplants, and the tremendous increase in the incidence of fungal

infections in the world’s population all underscore our inadequacy to cope with these

medical problems (Trinci, 1992; Kirk et al., 1993, 2002; Moore and Chiu, 2001;

Thaithatgoon et al., 2004; Peláez, 2006). Added to this are enormous difficulties in

raising enough food in certain regions of the earth to support local human populations.

Environmental degradation, loss of biodiversity, and spoilage of land and water also

add to problems facing mankind (Strobel et al., 2004; Peláez, 2003; 2004; Guo et al.,

2008). Recently, over 50 contributions featured in a symposium dedicated to fungal

secondary metabolites (extrolites) at the 8th International Mycological Congress

(IMC8, Cairns, Australia, 2006; Stadler and Keller, 2008). This refers to the many

interesting and benefits of bioactive compounds.

The potential pharmaceutical benefits of secondary metabolites of fungi have

been investigated for about 80 years. The search for new bioactive compounds from

fungi started with the discovery of penicillin (Fleming, 1929), a potent antibiotic

against gram-positive bacteria, which was produced by Penicillium notatum. A further

milestone in the history of fungal products for medicinal use was the discovery of the

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immunosuppressant cyclosporine which is produced, e.g., by Tolypocladium inflatum

and Cylindrocarpon lucidum (Dreyfuss et al., 1976; Csapo et al., 2007; Nisha and

Ramasamy, 2008). It was first discovered as an antifungal metabolite and later

discovered to be immunosuppressive, which made cyclosporine useful for treatment

following organ transplantation (Goodman Gilman et al., 1985; Singh et al., 2006).

The antifungal agent griseoflavin isolated from Penicillium griseofulvum (Rehm,

1980) and the cholesterol biosynthesis inhibitor lovastatin isolated from Aspergillus

terreus (Albert et al., 1980) are two feature examples supporting today’s great interest

in new bioactive compounds from fungi (Gullo et al., 2006; Hoffmeister and Keller,

2007; Stadler and Keller, 2008; Panagiotou et al., 2009).

Many groups of fungi are widely recognized as prolific sources of bioactive

secondary metabolites that might represent useful leads for the development of new

pharmaceutical bioactive compounds (John et al., 1999; Strobel et al., 1999; Brady et

al., 2000; Singh et al., 2000; Yamada et al., 2002; Gullo et al., 2006; Hoffmeister and

Keller, 2007; Shwab and Keller, 2008). So far more than 4000 fungal bioactive

compounds are described (Dreyfuss and Chapela, 1994) and 5000–7000 fungal

species have been examined with respect to their chemical characterization

(Hawksworth, 1991). Hawksworth (1995) approximated the probable number of

existing fungi to be 1.5 million with 80,000 being described. Since more than 1.5 x

106 endophytic fungi are thought to thrive within the estimated 270,000 species of

vascular plants, the prospects for additional discoveries of metabolites from these

fungi are promising (Dreyfuss and Chapela, 1994; ; Stadler and Keller, 2008).

Fungi represent an enormous source for natural products with diverse

chemical characters and activities. Of special interest are creative fungal strains.

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Creativity in this sense is defined as the ability to produce compounds of interest to

humans (Gullo et al., 2006; Hoffmeister and Keller, 2007; Stadler and Keller, 2008;

Shwab and Keller, 2008). Intelligent screening methods have been applied in the

search for novel molecules (Pointing and Hyde, 2001; Damveld et al., 2008; Rosén et

al., 2009). The potential is enormous for the discovery of valuable natural products

resulting from a directed search and screening of fungi from unexplored habitats

(Conception et al., 2001; Strobel and Daisy, 2003; Strobel et al., 2004; Gullo et al.,

2006; Hoffmeister and Keller, 2007). It is expected that new drugs of biotechnological

importance will be discovered with increased focus on tropical endophytic fungi.

Fungal species screened for secondary metabolites using modern techniques are less

than 1% of those that may exist (Nisbet and Fox, 1991; Gullo et al., 2006;

Hoffmeister and Keller, 2007; Rosén et al., 2009).

The natural function of secondary metabolites is often unknown, but it is

assumed that they play an important role in chemical defense and communication

(Krohn, 1996; Gullo et al., 2006). Many of them have been suggested to act as

pheromones, antifeedants or repellents, and as regulators in the development of

organism (Sterner, 1995; Gullo et al., 2006) suggested that the biosynthesis of

secondary metabolites does not occur randomly but is correlated with ecological

factors.

Most fungi studied to date have been isolated from soil and were proven to

have a high creativity index, i.e. new and interesting secondary metabolites could be

isolated. Genera such as Aspergillus, Penicillium, Acremonium, Fusarium, all typical

soil isolates, are known for their ability to synthesize diverse chemical structures.

Dreyfuss (1986), however, described a problem which is often encountered during

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microbiological screening of fungal isolates for their secondary metabolites.

Increasingly, known metabolites are rediscovered making screening programs less

efficient. This may be partially because the same fungal strains are reisolated when

investigating an ecological niche. This could also result in the rediscovery of known

compounds as the same taxa produce the same metabolites with high coincidence

(Loeffler, 1984; Gullo et al., 2006; Hoffmeister and Keller, 2007).

The assumption can be made that certain physical and biological situations in

the natural environment favour the production of a diverse range of secondary

metabolites (Dreyfuss and Chapela, 1994; Gullo et al., 2006; Hoffmeister and Keller,

2007). This indicates that it might be more useful to investigate fungal isolates from

other ecological niches than soil in order to make more direct approach towards

creative and noval fungal taxa. Some relatively enexplored fungal groups derived

from such ecosystems are, endophytic fungi, fresh-water fungi, marine fungi and

saprobic fungi from various substrata (Dreyfuss and Chapela, 1994).

During the investigation of the saprobic and endophytic fungi of grasses in

Thailand, the studies isolated many species of graminicolous fungi including the

novel endophytic species e.g. Periconia siamensis (CMUGE015) (Bhilabutra et al.,

2007), Dactylaria endograminicola and Dactylaria endograminicola (CMUGE125)

(Bhilabutra et al., in press). These were selected for screening for bioactive

compounds that have present antibacterial and antifungal activity. The compounds

were extracted, purified and analyzed for structure elucidation nand identification, as

well as screeing for biological activities. The details of this study are presented in this

Chapter.

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6.2 Materials and Methods

6.2.1 Fungal isolation

Fifty fungi isolated from grasses as described in Chapters 3 and 4 were

selected for further study including Periconia siamensis CMUGE015 (Figure 6.1a)

and Dactylaria endograminicola CMUGE0125 (Figure 6.1b). The isolation methods

used have been described as Chapters 3 and 4. A list of 50 selected fungi and their

geogrphic location , host and mode of life are presented in Appendix F.

b. a.

Figure 6.1 Two fungi selected for screening of bioactive compounds, a. Periconia

siamensis; b. Dactylaria endograminicola.

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6.2.2 Test organisms

6.2.2.1 Bacterial strains (for MIC test)

Five bacterial strains were used as test organisms including Bacillus cereus,

Escherichia coli, Listeria monocytogenes, Staphylococcus aureus (MRSA Methicillin

Resistant) and Pseudomonas aeroginosa (Figure 6.2), and were obtained from the

Department of Biology, Faculty of Science, Chiang Mai University.. All bacterial

strains were separately cultured and maintained on nutrient agar (NA, Appendix A) at

room temperature (27–30 °C).

Figure 6.2 Bacterial strains used as test organisms for screening microbial activities.

(www.magma.ca/~scimat/b_cereus.htm; www3.niaid.nih.gov/.../image_library.htm;

http://www.gasdetection.com/news2/health_news_digest84.html; http://aleas.exteen.

com/page/2; http://scienceblogs.com/mikethemadbiologist/2007/10/staphylococcus_

aureus_dont_pic.php).

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6.2.2.2 Fungal strains (for MIC test and dual culture test)

Candida albicans (ATCC90028, obtained from the American Type Culture

Collection) and Penicillium avellaneum (obtained from Kunming Botanical Garden,

Kunming, China), Colletotrichum musae (anthracnose which cause of anthracnose

disease in banana ) and Fusarium oxysporum (Fusarium wilt is the one of the most

destructive and notorious diseases of plants) were cultured and maintained at room

temperature on malt peptone agar (MPA, Appendix A) and/or yeast glucose agar

(YGA, Appendix A). C. musae and F. oxysporum were obtained from stock culture of

the Biology Department, Faculty of Science, Chiang Mai University.Test organisms

for screening of antifungal activities are illustrated in Figure 6.3.

Figure 6.3 Fungal strains used as test organisms for screening antifungal activities.

(www.christinas-home-remedies.com; www.biology.ed.ac.uk; www.bspp.org.uk;

http://botit.botany.wisc.edu).

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6.2.3 Preparation of assay plates

Cells of C. albicans were scraped off the agar and inoculated into 5 ml of malt

peptone broth. The tubes were then incubated for 16 h at room temperature on a

reciprocal shaker (130 rpm). For P. avellaneum, 0.1% of Tween 80 was added before

inoculation with one loop of conidia. The mixture was then vortexed, and

immediately used for assay plates. One ml of inoculum of each test organism was

added to 200 ml of appropriate medium at 55 °C, poured into assay plates, and

allowed to solidify. Test organisms were subcultured into potato dextrose agar (PDA,

Appendix A) plates and prepared for dual culture test.

6.2.4 Cultivation of fungi for production of antibacterial substance

For initial screening various media (F1, F2, F3, F4 and F5, see Appendix A,

Cheeptham et al., 1999) were used for culture fermentations. All selected fungi were

subcultured on to PDA plates and incubated for 1 week. After incubation, 8 mm discs

of mycelium were transferred, one per flask, to the five fermentation media. The

cultures were incubated at room temperature for 7 days on a reciprocal shaker at 130

rpm. The supernatant was then filtered by passing through Whatman No. 4 filter paper

and used immediately to test for inhibitory activity. The results of screening for

antibacterial substances are presented in Table 6.2. The supernatant was also used to

examine for inhibitory antifungal activity against C. albicans and P. avellaneum. The

results of this study are presented in Table 6.3.

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6.2.5 Screening of antifungal activities using dual culture test

Bioactivity against Colletotrichum musae and Fusarium oxysporum was

screened by inoculating each pathogen with each potential inhibitor producing strain

in dual cultures on PDA in 9 cm Petri dishes. The agar medium was inoculated with a

5 mm diameter agar disk of the actively growing selected mycelium positioned at the

opposite side of a 5 mm diameter agar disk of the actively growing pathogen. The

distance between discs was approximately 5 cm. The trial was replicated five times

for each pairing. Mycelial plugs of the selected fungi were taken from the margins of

young colonies growing on PDA. The two colonies were allowed to grow towards

each other. Plates inoculated with the test fungi only were used as controls. All plates

were then incubated at 30 °C. The diameter of the colony in the control, and each

treatment was recorded 1 week after inoculation (Figure 6.4). Percentage inhibition

was measured using the following formula.

The details of results of this study are presented in Table 6.3.

Percentage of inhibition = Radius of pathogen in dual culture plate (D) x 100 Radius of pathogen in control plate(C)

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D

C

Figure 6.4 Dual culture test used for antifungal activities test. Radius of pathogen in

control plate (C) and radius of pathogen in dual culture plate (D) were used to

calculate the percentage inhibition.

6.2.6 Selected strains for antimicrobial test

The strains exhibiting the best inhibition against the test fungi were Dactylaria

endograminicola (CMUGE1125), Periconia siamensis (CMUGE015) and

Eupenicillium sherii (CMUGE1047). These were inoculated from the stock PDA

slants onto PDA plates and incubated at 30 °C for 7 days to their selected suitable

production medium (F1, F2, F3, F4 and F5) were observed for optimization of

antibacterial and antifungal productions. The result of optimization of their media is

presented in Tables 6.4a and 6.4b.

6.2.7 Optimization of antibacterial production

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Optimal fermentation conditions have to be identified to achieve maximum

productivity of antibacterial producers, therefore it was decided to study the effect of

various factors on antibacterial production in liquid culture by the selected strains of

Periconia siamensis CMUGE015, Dactylaria endograminicola CMUGE1125 and

Eupennicillium sherii CMUGE1047.

6.2.7.1 Fermentation media

Various media (F1, F2, F3, F4, F5 [Cheeptham et al., 1999], PDB and malt

extract) were used as fermentation media. Periconia siamensis CMUGE015 was

subcultured from stock culture on the PDA plates and incubated for 7 days. After

incubation, one fungal disk (bored with cork borer 8 mm diam) was transferred to 50

ml of each fermentation media (Appendix A) in 250 ml Erlenmeyer flasks. The flasks

were incubated at room temperature for 7 days on a reciprocal shaker at 130 rpm. A

paper disc agar diffusion assay method was used to check inhibitory effect against the

test organisms.

6.2.7.2 C and N sources

The effects of carbon and nitrogen sources on antibacterial production were

studied. Various carbon sources (fructose, glucose, lactose, maltose, manitol and

sucrose) and nitrogen sources (malt extract, peptone, polypeptone, soy bean meal and

yeast extract) were added to 50 ml of a basal medium, in 250 ml Erlenmeyer flasks

and incubated at room temperature on a reciprocal shaker at 130 rpm. The incubation

time was studied for the production of antibacterial compound. The crude extracts of

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cultures were obtained and a paper disc agar diffusion assay method was used to

check inhibitory effects.

6.2.7.3 pH effect on antibacterial production

One disc of Periconia siamensis strain CMUGE015 was inoculated into 250

ml Erlenmeyer flasks containing 50 ml of basal medium with optimal C and N source.

The pH of the fermentation media was adjusted to 5.5, 6.0, 7.0, 7.5 and 8.0 by adding

HCl and /or NaOH. Flasks were incubated at room temperature on a reciprocal shaker

at 130 rpm. The optimum incubation time for production of the antibacterial

metabolite(s) was 14 days. The crude extract of cultures was obtained and a paper

disc agar diffusion assay method was used to check inhibitory effects.

6.2.7.4 Temperature effect on antibacterial production

The temperature effect for antifungal production was tested by fermenting the

fungal strain with optimum pH and incubation at different temperatures (25, 30, 37,

and 45 °C). The crude extract of cultures was obtained and paper disc agar diffusion

assay method was used to check inhibitory effects.

6.2.7.5 Optimum time for antibacterial production

The optimum time needed for antibacterial production was established by

inoculating the fungal strain in the best basal medium, then incubating at room

temperature on a reciprocal shaker at 130 rpm. Some extract of the culture was

removed daily for 13 days. A paper disc agar diffusion assay method was used to

check for highest antifungal activity.

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6.2.7.6 The effect of pH, temperature and duration on antifungal experiment

Dactylaria endograminicola CMUGE1125 was selected as the best strain to

inhibit the growth of C. albicans, P. avellaneum, C. musae and F. oxysporum in liquid

culture. One agar disc of D. endograminicola CMUGE1125 mycelium was inoculated

into 250 ml Erlenmeyer flask containing 50 ml of fermentation media at various pH

(5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) and the flask was incubated for 14 days at room

temperature on a reciprocal shaker set at 220 rpm. The optimum temperature for

antifungal production was tested by fermenting the fungal production strain at the

optimum pH and incubation at different temperatures (25, 30, 37 and 45 °C). The

paper disc agar diffusion assay method was used to check for inhibitory effects. The

optimum time for fermentation was established by cultivation of the producing strain

at the optimum pH and temperature and then some of the crude extract from the

culture was removed daily for 13 days with sterile Pasture pipettes, and centrifuged at

4000 rpm for 15 minutes. The supernatant were used to test antifungal activity. The

paper disc agar diffusion assay method was used to check for inhibitory effects.

6.2.8 Thin layer chromatography (TLC)

Preliminary characterization of the antibacterial and antifungal compounds

from the crude extract of liquid fermentation of Periconia siamensis CMUGE015 and

Dactylaria endograminicola CMUGE1125 were determined by thin layer

chromatography (TLC). Ten microliters of the dried fungal extract dissolved in a

small amount of ethyl acetate, were spotted on a silica gel plate (Merck aluminum

sheet, 60 F254). The compounds were separated using a dichloromethane:methanol

solvent system. Separated components were detected by iodine vapor and

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bioautography was carried out to identify those components that had antibacterial

and/or antifungal activities. The TLC plate was placed in assay plates, as previously

described for the paper disc assay, except that after one hour the TLC plate was

removed and the assay plates were incubated overnight at room temperature. The

positions of the inhibitory zones of antibacterial and antifungal activities was

measured to determine a Rf value for the extract component. These were compared

with the band which appeared following exposure to iodine vapor.

6.2.9 Extraction and concentration

At the end of the fermentation, the cultures were harvested by filtration. The

culture broths were extracted twice with an equal volume of ethyl acetate (EtOAc).

The extracts were pooled and dried in a rotary evaporator (BÜCHI Switzerland) at

45ºC under reduced pressure. The extract residue was dissolved in dimethyl sulfoxide

(DMSO) and stored at 4ºC until the bioassays (described below) could be conducted.

6.2.10 Characterization of bioactive compounds from Periconia siamensis

6.2.10.1 Culture conditions and extraction

Periconia siamensis CMUGE015 was grown on 100 plates of F1 medium for

2 weeks at 28°C until it completely covered each plate. The colonies formed on each

plate were cut into small pieces (0.5 x 0.5 cm) using a sterilized cutter. The agar

pieces were placed in a 3L Erlenmeyer flask and ethyl acetate (2L) was used to

extract the metabolites produced. The flasks were shaken on a reciprocal shaker at

180 rpm for 30 min. The process was repeated 5 times. The combined extracts (10L)

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were dried by flash evaporation at 40°C and the recovery yield of dry material was

about 2.8 g.

6.2.10.2 Fractionation and purification of the compounds

The dried extract was mixed with silica gel 60 (size 0.015-0.040 mm, Merck)

and packed into a chromatography column. Fractionation using increased

concentrations of Hexane:EtOAc:MeOH (100:0:0, 80:20:0, 60:40:0, 40:60:0,

20:80:0, 0:100:0, 0:99:1, 0:98:2, 0:96:4, 0:92:2, 0:90:10, 0:80:20, 0:60:40, 0:40:60,

0:20:80 and 0:0:100) produced fifty five 25ml fractions. These were evaporated to

dryness, weighed and fractionated again by column chromatography (silica gel 60 size

0.063-0.200 mm, Merck). A white crystalline material (compound 1, Fig.6.14a) was

recovered in fraction 7 and a yellow powder (compound 2, Fig. 6.14b) found in

fraction 5 were active in the initial antibacterial assay.

The white crystals were washed in cold EtOAc for purification (120 mg). The

material from fraction 5 (194 mg dry weight) was subjected to repeated fractionation

by column chromatography, using, Hexane:EtOAc (60:40) as eluant, and the product

(105 mg) was recovered in fractions 12-16. These were combined and checked by

TLC. The two compounds were screened again for their antibacterial activity against

the test organisms as decribed below, using the paper disk method (Venugopal and

Venugopal, 1994).

6.2.10.3 Structure elucidation of compounds

1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were analysed at the

Department of Chemistry, Faculty of Science, Silpakorn University, Thailand using a

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Bruker DPX300 spectrometer, with TMS (δH 0 ppm) and CDCl3 (δ13C 77.0 ppm) as

internal references. Melting points were determined using an electrothermal melting

point apparatus. Optical rotations were measured with a JASCO J-810

spectropolarimeter. IR spectra were recorded on an FT-IR system 2000 (Perkin-Elmer)

spectrometer. Mass spectra were recorded on a JEOL JMS- X505WA mass

spectrometer. The method for elucidation of compounds is presented in Figure 6.5.

6.2.11 Bioassays

6.2.11.1 Screening for antibacterial metabolite production

Ethyl acetate extracts of culture filtrates, and column chromatography eluants

of similar extracts of agar plates, were assayed for the presence of metabolites and for

antibacterial activity using a paper disk diffusion assay method. Sterile paper disks (8

mm diam., Advatec, Toyo Roshi Kaisa, LTD., Japan) were soaked in the extracts and

allowed to air dry, before they were placed on seeded assay plates. Controls were

disks impregnated with solvents, used for extraction of culture filtrates and in column

chromatography, which had been allowed to air dry. All assay plates were then

incubated at 37ºC for 24 hours for all bacterial strains. Nutrient broth cultures of the

test bacteria organisms were used to produce inocula for the bioassay plates.

Screening for antibacterial metabolite production was conducted in triplicate and data

generated was analyzed using the SPSS v.10 package for one-way analysis of

variance (ANOVA). For the statistic calculated for this study see Appendix G.

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UV

MS

NMR

IR

Figure 6.5 Spectroscopic analysis methods used in this study.

6.2.11.2 Minimum inhibitory concentration (MICs)

Purified compounds 1 and 2 were assayed to determine the MICs of these

compounds against Bacillus cereus, Listeria monocytogenes, MRSA and

Pseudomonas aeroginosa. Antibacterial activity was determined by the 2-fold

microtiter broth dilution method (Kim et al., 2002). Dilutions of the test compound

dissolved in dimethyl sulfoxide (DMSO) were added to each well of a 96-well

microtiter plate containing a fixed volume of standard methods broth (SM broth,

Difco) (final 0.5% DMSO). Each well was inoculated with bacteria (105 CFU/mL)

and incubated at 37°C for 24 h. The MIC was calculated as the concentration at which

no growth of bacteria was observed. An overview of minimum inhibitory

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concentration (MIC) test is illustrated in Figure 6.6. The results of MIC are presented

in Figure 6.7.

Figure 6.6 Minimum inhibitory concentration (MIC) test.

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b

a.

Figure 6.7 Minimum inhibitory concentration (MIC) test for purification compound

from this study, in different concentrations, against selected pathogenic organisms a:

before test, b: after incubation at 37°C over night, showing the clear zone in different

concentration with different test organism.

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6.3 Results

6.3.1 Screening of isolates as potential sources of antibacterial and antifungal

agents

One hundred and twenty three fungal isolated stains from grasses were

examined for their potential antibacterial activities against pathogenic

microorganisms. Sixty-five strains were isolated as saprobic fungi from both T.

latifolia and S. spontaneum While 58 strains were isolated as endophytic fungi from

Thysanolaena latifolia and V. zizanioides. Thirty-three strains are ascomycetes while

90 strains are anamorphic fungi (75 hyphomycetes and 15 coelomycetes). All taxa are

listed in Appendix F.

The definition of the best strains were those that produced zones of inhibition

>15mm. The most interested isolates were Periconia siamensis CMUGE015,

Dactylaria endograminicola CMUGE1125 and Eupenicillium sherii CMUGE1047.

However, some other strains were more specific displaying either antibacterial or

antifungal activities only against some test microorgansims e.g. Cladosporium

cladosporioides CMUG1040, Dactylaria dimorphospora CMUGS1055,

Eupenicillium sp. CMUGE3048, Periconia bissoides CMUGS1003, Periconia

digitata CMUGS2049, Periconia sp. CMUGS2006, Periconia sp. CMUGE3016,

Pestalotiopsis sp. CMUGE1025, and Pyricularia sp. CMUGS1021. The results of the

antibacterial activities tests are presented in Table 6.1.

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Table 6.1 List of selected fungi and their antibacterial activities. Strin no. Taxa Microbial activities tests BCa ECb LMc MRSAd PAe CMUGE1004 Pestalotiopsis sp. ++ - + - + CMUGE1007 Fusarium sp. ++ - + - + CMUGE1008 Gaeumannomyces sp. + - + - + CMUGE1009 Zygosporium sp. + - - - - CMUGE1011 Bactrodesmium longisporum ++ - ++ - - CMUGE1012 Alternaria tenuis + - - - - CMUGE015 Periconia siamensis*# ++ ++ ++ ++ ++ CMUGE1016 Cladosporium cladosporioides# ++ + + ++ + CMUGE1018 Neotyphodium coenophialum + - - - - CMUGE1019 Nigrospora oryzae + - - + - CMUGE1023 Arthrinium euphorbiae - - + - - CMUGE1028 Humicola grisea + - + - - CMUGE1033 Gilmaniella humicola + - - - - CMUGE1038 Helicosporium phragmitis - - - + - CMUGE1043 Xylaria sp. + - - - - CMUGE1046 Trichoconis sp. + - - - - CMUGE1047 Eupenicillium sheri# ++ + ++ ++ + CMUGE1050 Eupennicillium sp. ++ + - - ++ CMUGE1057 Thermomyces sp. ++ - + - - CMUGE1066 Acremonium polychrome + - + - - CMUGE1077 Balansia sp. ++ - + - CMUGE1107 Scolecobasidium sp. + - + - - CMUGE1123 Phomopsis sp. + - - - - CMUGE1131 Stachybotrys sp. + - - - - CMUGE1134 Nodulisporium gregarium - - - + - CMUGE1159 Paecilomyces sp. 1 + - + - - CMUGE1078 Idriella lunata + - - - - CMUGE1220 Colletotrichum gloeosporioides + - + + - CMUGE1222 Curvularia alternata. ++ - + + - CMUGE1125 Dactylaria endograminicola *# ++ + ++ ++ + CMUGE3002 Periconia sp. 1 ++ - + - + CMUGE3005 Fusarium semitectum ++ - + - + CMUGE3016 Periconia sp. 2 ++ + - ++ + CMUGE3017 Helminthosporium sp. 1 + - - - - CMUGE3023 Gelasinospora sp. - - - + - CMUGE3025 Arthrobotrys sp. 1 + - - - - CMUGE3027 Monacrosporium sp. ++ - + + - CMUGE3029 Geotrichum sp. - - - + - CMUGE3034 Phoma sp. # ++ - ++ + + CMUGE3037 Glomomerella sp. + - - - - CMUGE3042 Nigrospora sacchari + - - - - CMUGE3043 Nigrospora sp. 2 ++ - - - - CMUGE3045 Ellisembia vaga + - + - - CMUGE3048 Eupenicillium sp. 1 ++ + - ++ - CMUGE3054 Dactylella sp. 1 + - + - - CMUGE3055 Dactylaria sp. 3# + + ++ - + CMUGE3057 Dactylaria sp. 1 ++ - + - - CMUGE3060 Pearepichloe sp. - - + - - CMUGE3067 Acremonium sp. 1 + + - - CMUGE3074 Acremonium sp. 2 ++ - + - - CMUGE3087 Bactrodesmium sp. + - + - -

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Table 6.1 (Continued). Strin no. Taxa Microbial activities tests BCa ECb LMc MRSAd PAe

CMUGE3101 Colletotrichum sp. 1 + - - - CMUGE3123 Cochliobolus sp. 1 ++ - + - - CMUGE3125 Cochliobolus bicolor ++ - - - CMUGE3127 Colletotrichum sp. 2 - + + - - CMUGE3155 Nodulisporium sp. - - - - - CMUGE3158 Paecilomyces sp. - - + - - CMUGE3170 Papulaspora sp. 1 - - - - - CMUGS1001 Tetraploa aristata + - - - - CMUGS1003 Periconia byssoides# ++ + + + - CMUGS1004 Verticillium sp. + - - - - CMUGS1005 Acremonium kiliense ++ - ++ - - CMUGS1007 Stachylidium bicolor - - - - - CMUGS1008 Periconia cookei + - + - - CMUGS1010 Arthrinium sp. + - - - - CMUGS1011 Pestalotiopsis versicolor + - - + - CMUGS1012 Chaetophoma sp. + - - - - CMUGS1014 Periconia macrospinosa + - - - - CMUGS1015 Xylaria sp. + - - - - CMUGS1017 Drumopama monoseta* ++ - - - - CMUGS1018 Oxydothis sp. - - + - - CMUGS1021 Pyricularia sp.# ++ - ++ + - CMUGS1022 Phoma sp. 2 + - + - - CMUGS1023 Guaemannomyces graminis ++ - - - - CMUGS1025 Pestalotiopsis sp. ++ - ++ + + CMUGS1026 Stachybotrys echinata ++ + - - - CMUGS1027 Nigrospora oryzae + - - - - CMUGS1029 Magnaporthe salvinii + - - - - CMUGS1030 Chaetosphaeria lentomita + - - - - CMUGS1032 Curvularia lunata + - + + - CMUGS1033 Phomopsis sp. + - - - - CMUGS1034 Spegazzinia deightonii + - + - - CMUGS1035 Fusarium oxysporum ++ - + - - CMUGS1036 Massarina phragmitocola - - - - - CMUGS1037 Stilbella sp. + - - - - CMUGS1038 Dactylaria triseptata ++ - + + - CMUGS1040 Cladosporium cladosporioides ++ + - + - CMUGS1041 Sporidesmium cookei + - - - - CMUGS1042 Dactylella ellipsospora ++ - - - - CMUGS1043 Dendrographium sp.* + - ++ - - CMUGS1044 Pleurophragmium simplex - - - - - CMUGS1045 Lophiosphaeria sp. + - - - - CMUGS1047 Pycnothyriopsis sp.* - - + - - CMUGS1049 Didymella sp. + - - - - CMUGS1051 Dactylaria sp. + - + - - CMUGS1055 Dactylaria dimorphospora ++ + + + - CMUGS1057 Lophaeostoma sp. + - + + - CMUGS2001 Phoma sp. 1 + - - - - CMUGS2006 Periconia sp. 1 ++ + + + - CMUGS2007 Coelomycete (RP)* + - - - - CMUGS2009 Dictyosporium sp. 1 + - - + - CMUGS2011 Massarina sp. 4* ++ - + + - CMUGS2013 Acrodictys sacchari + - - - - CMUGS2014 Myrothecium sp. 1 + - - - -

217

Table 6.1 (Continued). Strin no. Taxa Microbial activities tests BSa ECb LMc MRSAd PAe

CMUGS2015 Massarina sp. 2 + - - - - CMUGS2016 Spegazzinia tessarthra + - - - - CMUGS2017 Dictyosporium oblongum ++ - + - - CMUGS2022 Cercospora koepkei ++ - ++ - + CMUGS2024 Annulatascus sp. 1 + - - - - CMUGS2025 Drechslera stenospila ++ - + - - CMUGS2028 Ascomycetes* + - - - - CMUGS2033 Arecophila sp. 5* + - - - - CMUGS2034 Phialophorophoma sp. 1 + - - - - CMUGS2036 Fusarium sp. ++ - + + - CMUGS2037 Apiospora montagnei - - - - - CMUGS2039 Periconia echinochloae# ++ + + - + CMUGS2040 Ascomycetes* + - - - - CMUGS2041 Dictyoarthrinium saccharii - - - - - CMUGS2043 Arecophila sp. 1 + - - - - CMUGS2044 Lophiostroma sp.2 - - - - - CMUGS2049 Periconia digitata# ++ + ++ + + CMUGS2052 Ophiobolus leptosporus + - - - - CMUGS2060 Colletotrichum sp. 1 + - - - - BCa = Bacillus cereus; ECb= Escherichia coli; LMc = Listeria monocytogenes; MRSAd = Staphylococcus aureus; PAe = Pseudomonas aeruginosa * new species/ or probably new species in this study. # highly microbial activities. ++ = clear zone more than 15 mm; + = clear zone less than 15 mm; - = no clear zone

The same 123 fungal strains were used for antifungal activities test against C.

albicans, P. avellaneum, C. musae and F. oxysporum as target organisms. The

antagonism of selected fungi against C. albicans and P. avellaneum growth is

reported as diameter of clear zone, while percentage inhibition zone is reported

against C. musae and F. oxysporum. The most significant isolate in these tests was D.

endograminicola CMUGE1125 showing 85.54% and 64.67% inhibition against C.

musae and F. oxysporum, respectively. The following strains were less active, but still

potentially interesting were E. sherii CMUGE1047 (70.33% and 60.00% in

inhibition), Eupenicillium sp., CMUGE1050 (69.23% and 60.00%), Phoma sp.

CMUGS102 (67.19% and 50.94%), Periconia siamensis CMUGE015 (69.30% and

54.18%), Pestalotiopsis sp. CMUGE1004 (69.23% and 60.00%), Pyricularia sp.

218

CMUGS1021 (70.15% and 69.36%) and Verticillium sp. CMUGS1004 (69.23% and

51.04%). Data of their antifungal activities are presented in Table 6.2. The highly

potential antifungal activities led to the selection of Periconia siamensis CMUGE015

and Dactylaria endograminicola CMUGE1125 for more detailed investigations to

optimize suitable media that favour the production of their bioactive compounds.

Table 6.2 List of selected fungi and their antifungal activities. Strin no. Taxa Antifungal activities tests CAa1 PAb1 CMc2 FOd2

CMUGE1004 Pestalotiopsis sp. ++ - 69.23 60.00 CMUGE1007 Fusarium sp. - - 55.67 17.14 CMUGE1008 Gaeumannomyces sp. + + 62.00 37.60 CMUGE1009 Zygosporium sp. - - 7.69 2.13 CMUGE1011 Bactrodesmium longisporum - - 49.54 64.67 CMUGE1012 Alternaria tenuis - - 51.53 45.83 CMUGE015 Periconia siamensis*# ++ + 63.30 51.18 CMUGE1016 Cladosporium cladosporioides ++ - 0.00 1.60 CMUGE1018 Neotyphodium coenophialum + + 66.15 23.81 CMUGE1019 Nigrospora oryzae - - 57.23 41.82 CMUGE1023 Arthrinium euphorbiae - - 67.19 32.36 CMUGE1028 Humicola grisea - - 53.67 30.83 CMUGE1033 Gilmaniella humicola - - 55.69 43.75 CMUGE1038 Helicosporium phragmitis + - 45.83 28.33 CMUGE1043 Xylaria sp. - + 60.31 50.63 CMUGE1046 Trichoconis sp. - - 64.92 47.50 CMUGE1047 Eupenicillium sherii# ++ + 70.33 60.00 CMUGE1050 Eupennicillium sp. + + 69.23 60.00 CMUGE1057 Thermomyces sp. - - 53.85 41.25 CMUGE1066 Acremonium polychrome + - 55.69 44.53 CMUGE1077 Balansia sp. - - 64.62 49.38 CMUGE1107 Scolecobasidium sp. + - 64.92 47.50 CMUGE1123 Phomopsis sp. + - 62.00 46.67 CMUGE1131 Stachybotrys sp. - - 67.19 43.00 CMUGE1134 Nodulisporium gregarium - - 55.69 20.95 CMUGE1159 Paecilomyces sp. 1 - - 64.00 48.75 CMUGE1178 Idriella lunata - - 57.54 56.73 CMUGE1220 Colletotrichum gloeosporioides - - 0.00 5.00 CMUGE1222 Curvularia alternata. + - 58.46 30.83 CMUGE1125 Dactylaria endograminicola *# ++ ++ 85.54 64.67 CMUGE3002 Periconia sp. 1 + - 67.19 51.04 CMUGE3005 Fusarium semitectum ++ - 59.70 44.00 CMUGE3016 Periconia sp. 2 ++ + 53.00 35.25 CMUGE3017 Helminthosporium sp.1 - - 65.67 49.82 CMUGE3023 Gelasinospora sp. - - 7.69 12.45 CMUGE3025 Arthrobotrys sp. 1 - - 24.61 36.67 CMUGE3027 Monacrosporium sp. - - 51.33 45.83 CMUGE3029 Geotrichum sp - - 65.31 39.00

219

Table 6.2 (Continued). Strin no. Taxa Antifungal activities tests CAa1 PAb1 CMc2 FOd2

CMUGE3034 Phoma sp.# + + 62.00 54.18 CMUGE3037 Glomomerella sp. - - 50.15 49.82 CMUGE3042 Nigrospora sacchari - - 49.54 44.73 CMUGE3043 Nigrospora sp. 2 - - 56.00 12.45 CMUGE3045 Ellisembia vaga - - 57.54 45.83 CMUGE3048 Eupenicillium sp. 1 - - 66.15 30.00 CMUGE3054 Dactyrella sp. 1 ++ + 65.31 35.00 CMUGE3055 Dactylaria sp. 3 + - 64.62 43.75 CMUGE3057 Dactylaria sp. 1 - + 54.46 42.50 CMUGE3060 Pearepichloe sp. - - 60.31 37.27 CMUGE3067 Acremonium sp. 1 + - 58.77 35.25 CMUGE3074 Acremonium sp. 2 + + 53.85 18.87 CMUGE3087 Bactrodesmium sp. + - 60.93 12.45 CMUGE3101 Colletotrichum sp. 1 + + 69.23 51.04 CMUGE3123 Cochiobolus sp. 1 + - 65.94 45.83 CMUGE3125 Cochliobolus bicolor + - 69.03 38.91 CMUGE3127 Collectotrichum sp. 2 + + 56.42 28.33 CMUGE3155 Nodulisporium sp. + - 7.69 50.6 CMUGE3158 Paecilomyces sp. - - 49.54 5.00 CMUGE3170 Papulaspora sp. 1 - - 57.54 41.46 CMUGS1001 Tetraploa aristata - - 55.69 17.74 CMUGS1003 Periconia bissoides + - 65.31 44.00 CMUGS1004 Verticillium sp.# + ++ 69.23 51.04 CMUGS1005 Acremonium kiliense + - 59.38 18.87 CMUGS1007 Stachylidium bicolor - - 62.00 36.67 CMUGS1008 Periconia cookei + - 65.33 12.45 CMUGS1010 Arthrinium sp. - - 65.83 36.00 CMUGS1011 Pestalotiopsis versicolor + + 67.19 28.33 CMUGS1012 Chaetophoma sp. ++ - 66.63 50.94 CMUGS1014 Periconia macrospinosa - + 64.17 39.62 CMUGS1015 Xylaria sp. + - 0.00 1.60 CMUGS1017 Drumopama monoseta - - 7.96 0.00 CMUGS1018 Oxydothis sp. - - 57.54 17.14 CMUGS1021 Pyricularia sp.# ++ + 70.15 69.36 CMUGS1022 Phoma sp. 2 - ++ 67.19 50.94 CMUGS1023 Guaemannomyces graminis# ++ + 53.85 49.82 CMUGS1025 Pestalotiopsis sp. + - 55.94 37.60 CMUGS1026 Stachybotrys echinata - - 55.69 32.36 CMUGS1027 Nigrospora oryzae + - 57.23 20.00 CMUGS1029 Magnaporthe salvinii - - 69.23 50.94 CMUGS1030 Chaetosphearia lentomita - - 59.69 30.00 CMUGS1032 Curvularia lunata - - 55.67 23.81 CMUGS1033 Phomopsis sp.# ++ + 65.33 51.04 CMUGS1034 Spegazzinia deightonii - + 68.92 35.32 CMUGS1035 Fusarium oxysporum - - 62.00 50.94 CMUGS1036 Massarina phragmitocola + - 65.63 32.00 CMUGS1037 Stilbella sp. - - 7.69 1.60 CMUGS1038 Dactylaria triseptata ++ - 69.23 44.53 CMUGS1040 Cladosporium cladosporioides + - 0.00 2.13 CMUGS1041 Sporidesmium cookei - - 57.54 17.14 CMUGS1042 Dactyrella ellipsospora + + 59.10 13.33 CMUGS1055 Dactylaria dimorphospora - - 51.38 20.95 CMUGS1043 Dendrographium sp.* + - 64.62 47.50

220

Table 6.2 (Continued). Strin no. Taxa Antifungal activities tests CAa1 PAb1 CMc2 FOd2

CMUGS1044 Pleurophragmium simplex - - 45.54 22.86 CMUGS1045 Lophiosphaeria sp. - - 17.14 0.00 CMUGS1047 Pycnothyriopsis sp.* + - 58.46 1.60 CMUGS1049 Didymella sp.# + + 57.54 18.13 CMUGS1051 Dactylaria sp. - - 60.94 51.04 CMUGS1057 Lophaeostoma sp. - + 24.60 2.13 CMUGS2001 Phoma sp. 1 + - 66.79 30.83 CMUGS2006 Periconia sp. 1 - - 59.10 17.14 CMUGS2007 Coelomycete 1* - - 0.00 0.00 CMUGS2009 Dictyosporium sp. 1 - - 60.93 43.00 CMUGS2011 Massarina sp. 4* + - 57.84 23.81 CMUGS2013 Acrodictys sacchari + - 69.38 32.50 CMUGS2014 Myrothecium sp. 1 - - 59.10 29.00 CMUGS2015 Massarina sp. 2 - - 69.30 26.67 CMUGS2016 Spegazzinia tessarthra - - 61.33 45.60 CMUGS2017 Dictyosporium oblongum - - 24.61 4.00 CMUGS2022 Cercospora koepkei - - 7.67 0.63 CMUGS2024 Annulatascus sp. 1 - - 24.61 23.44 CMUGS2025 Drechslera stenospila - - 60.93 50.63 CMUGS2028 Ascomycetes* - - 53.00 18.13 CMUGS2033 Arecophila sp. 5* + - 24.61 17.14 CMUGS2034 Phialophorophoma sp. 1 - - 50.00 29.17 CMUGS2036 Fusarium sp. + + 53.67 45.60 CMUGS2037 Apiospora montagnei - - 56.56 18.13 CMUGS2039 Periconia echinochloae# + ++ 67.97 60.00 CMUGS2040 Ascomycetes - - 0.00 13.33 CMUGS2041 Dictyoarthrinium saccharii + - 65.94 49.00 CMUGS2043 Arecophila sp. 1 - - 7.67 16.19 CMUGS2044 Lophiostroma sp.2 - - 60.90 26.67 CMUGS2049 Periconia digitata + - 62.99 35.00 CMUGS2052 Ophiobolus leptosporus - - 53.00 29.00 CMUGS2060 Colletotrichum sp. 1 + + 51.33 60.00 CAa = Candida albicans PAb= Penicillium avellaneum CMc = Colletotrichum musae FMd = Fusarium oxysporum * new species/ or probably new species in this study. # highly microbial activities. ++ = clear zone more than 15 mm; + = clear zone less than 15 mm; - = no clear zone

6.3.2 Production of antibacterial and antifungal compounds by Periconia

siamensis CMUGE015 and Dactylaria endograminicola CMUGE1125

Periconia siamensis CMUGE015 and Dactylaria endograminicola

CMUGE1125 were tested for their antibacterial and antifungal activities.

Fermentation media (F1, F2, F3, F4 and F5) were examined for optimization of

growth and metabolite production by Dactylaria endograminicola CMUGE1125 and

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Periconia siamensis CMUGE015. The result for selected fungi in fermentation media

are shown in Table 6.3 and 6.4. The suitable media for Periconia siamensis

CMUGE015 was F1 (glucose as C source, Figure 6.8, and polypeptone as N source,

Figure 6.9) and medium F2 for Dactylaria endograminicola CMUGE1125 (fructose

as C source, Figure 6.8, and soy bean meal as N source, Figure 6.9).

Table 6.3 The inhibition zone (diameter, mm) of crude extract from Periconia

siamensis against pathogenic bacteria and fungi in fermentation media F1, F2, F3, F4

and F5.

a Means of inhibition clear zones surrounding each application point, three replicates

Inhibition Zone (mm)aPathogenic organisms F1 F2 F3 F4 F5

Bacillus cereus 15.0±0.15 12.9±0.15 8.9±0.10 9.2±0.10 8.5±0.11Canida albicans 11.1±0.05 9.2±0.11 7.8±0.05 8.2±0.05 6.5±0.11Listeria monocytogenes 13.1±0.11 10.7±0.05 7.1±0.05 8.3±0.10 6.4±0.05Penicillium avellaneum 10.1±0.11 8.6±0.05 6.4±0.05 8.0±0.05 7.5±0.05Pseudomonas aeroginosa 10.1±0.11 8.4±0.05 6.8±0.05 8.1±0.10 6.8±0.05Staphylococcus aureus 14.3±0.05 10.8±0.11 7.4±0.17 8.0±0.05 7.5±0.05

Table 6.4 The Inhibition zone (diameter, mm) of crude extract from Dactylaria

endograminicola against pathogenic bacteria and fungi in fermentation media F1, F2,

F3, F4 and F5.

Inhibition Zone (mm)aPathogenic organisms F1 F2 F3 F4 F5

Bacillus cereus 12.0±0.15 14.3±0.05 9.2±0.10 8.5±0.11 8.9±0.10Canida albicans 10.1±0.11 10.1±0.11 8.3±0.10 6.4±0.05 7.8±0.05Listeria monocytogenes 11.7±0.05 13.0±0.15 8.3±0.10 7.5±0.05 7.1±0.05Penicillium avellaneum 8.6±0.05 11.7±0.05 8.0±0.05 6.8±0.05 6.4±0.05Pseudomonas aeroginosa 8.4±0.05 9.8±0.11 8.1±0.10 6.8±0.05 6.8±0.05Staphylococcus aureus 10.8±0.11 10.6±0.11 8.0±0.05 7.4±0.17 7.4±0.17

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0

5

10

15

20

25

Lac to s e Fructo s e Malto s e Gluco s e Sucro s e Manito l

C-sources

Gro

wth

zon

e (m

m)

P. siamensisD. endograminicola

Figure 6.8 Effect of C-sources on antimicrobial agent production by Periconia

siamensis CMUGE015 and Dactylaria endograminicola CMUGE1125.

0

2

4

6

8

10

12

Malt extrac t Yeas textrac t

So ybeanmeal

P o ypepto ne P epto ne

N-sources

Gro

wth

zon

e (m

m)

P. siamensisD. endograminicola

Figure 6.9 Effect of N-sources on antimicrobial agent production by Periconia

siamensis CMUGE015 and Dactylaria endograminicola CMUGE1125.

223

6.3.3 The effect of pH, temperature and time on antibacterial and antifungal

production

Optimized of fermentation conditions for antibacterial and antifungal

production by Periconia siamensis CMUGE015 and Dactylaria endograminicola

CMUGE1125 were pH 6.5 (Figure 6.10) and 30°C (Figure 6.11). The widest zone of

inhibition occurred after 14 days of incubation (Figure 6.12). There no significant

difference in the inhibition between 25 and 30 °C, but at 30 °C the largest inhibition

zone was observed.

02

46

810

1214

16

5.5 6 6.5 7 7.5 8

pH

Gro

wth

zon

e (m

m)

P. siamensisD. endograminicola

Figure 6.10 Effect of pH on antimicrobial productions by Periconia siamensis

CMUGE015 and Dactylaria endograminicola CMUGE1125.

224

0

2

4

6

8

10

12

14

25 30 37 45

Temperature (°C)

Gro

wth

zon

es

P. siamensisD. endograminicola

Figure 6.11 Effect of temperature on antimicrobial productions by Periconia

siamensis CMUGE015 and Dactylaria endograminicola CMUGE1125.

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16 18 20

Time (days)

Gro

wth

zon

e (m

m) y

P. siamensisD. endograminicola

Figure 6.12 Effect of incubation time on antimicrobial productions by Periconia

siamensis CMUGE015 and Dactylaria endograminicola CMUGE1125.

225

6.3.4 The characterization of bioactive compounds from Periconia siamensis

CMUGE015.

6.3.4.1 Screening for antibacterial metabolite production

Preliminary tests for antibacterial activity showed that Periconia siamensis

CMUGE015 produced metabolites that are inhibitory against the growth of Bacillus

cereus, Listeria monocytogenes, Staphylococcus aureus and Pseudomonas aeroginosa

on all media. F1 medium containing a combination of carbon source (fructose) and

soybean meal (as N-source) was identified as the most effective for the production of

antibiotics active against the test bacteria (Appendix F). Data analysis using SPSS

v.10 package for one-way analysis of variance (ANOVA) showed that F1 medium

was significantly better than the other media used (Appendix G).

226

a.

b. c.

Figure 6.13 a: X-ray crystallography of compound 1, b: compound 1, c: compound.

2 (Fun et al., 2006).

6.3.4.2 Extraction and concentration

A crude extract (1500 mg) of P. siamensis CMUGE015 was subjected to silica

gel-column chromatography and 16 fractions were obtained. Each fraction was tested

for antibacterial activity. Fractions 5 and 7 were markedly effective in inhibiting the

growth of the test microorganisms in this experiment indicating that they contained

bioactive compounds. Fraction 7 (hexane:ethyl acetate = 99:1) was precipitated with

cold ethyl acetate and designated as the hyaline white, pin-shaped crystal, Compound

227

1 (120 mg). Fraction 5 was then subjected to repeated chromatography in a mixture of

hexane and ethyl acetate (6:4), and then designated Compound 2 (105 mg).

Purification of fraction 5 also resulted in other compounds (ii) (43 mg), (iii) (32 mg)

and (iv) (14 mg).

6.3.4.3 Characterization of bioactive compounds from Periconia siamensis

CMUGE015

(a)

Modiolide A

Figure 6.14 Structure of antibacterial compound from Periconia siamensis

CMUGE015. This compound is known as Modiolide A.

Compound 1 (Figure 6.14) was fractionated from fraction-7 and crystallized

from solvent system EtOAc:MeOH (99:1), identified by IR, NMR and mass spectra

data as 5,8-dihydroxy-10-methyl-5,8,9,10-tetrahydro-2H-oxecin-2-one (Modiolide A),

MW 198 and the molecular formula was shown to be C10H14O4 by HREIMS. IR

absorption bands at 3292 and 1716 cm-1 were attributed to OH and carbonyl group-

(s), respectively 1H and 13C NMR data disclosed the existence of an ester carbonyl

(äC 170.9), four sp2 methines (äC 139.4, 138.7, 131.8, and 123.7), three oxymethines

(äC 73.6, 73.0, and 70.9), one sp3 methylene (äC 44.7), and one methyl group (äC

22.4). Since three out of four unsaturations were accounted for, Compound 1 was

228

inferred to contain one ring. The 1H-1H COSY and HMQC spectra revealed

connectivities from C-2 to C-10 (Fig. 6.15). The HMBC correlation from H-2 to C-1

suggested that the ester carbonyl group was attached to C-2. The relatively lower-field

resonance of H-9 (äH 5.25) suggested that C-9 was involved in an ester linkage to C-1.

The existence of two hydroxyl groups at C-4 and C-7 was determined by a lower-field

shift of H-4 and H-7 (äH 5.44 2H, m) by esterification with p-methoxycinnamoyl

chloride (vide infra). This observation supported the presumption that the compound

was a 10-membered macrolide. Geometries of two disubstituted olefins at C-2- C-3

and C-5-C-6 were assigned as Z and E, respectively, by 1H-1H coupling constants

[J(H-2/H-3), 12.3 Hz; J(H-5/ H-6), 15.8 Hz] and the NOESY correlation for H-2/H-3.

These results are similar with the previous report of Modiolide A compound.

Modiolide A D +42° (c 0.25, MeOH); UV (MeOH) ìmax 204 nm (6400); IR (KBr)

îmax 3292 and 1716 cm-1; 1H and 13C NMR; EIMS m/z 180 (M - H2O)+ and 198

(M+); HREIMS m/z 198.0892 (M+, calcd for C10H14O4 198.0891 (Tsuda et al., 2003).

4-Chromanone, 6-hydroxy-2-methyl- (5CI)

Figure 6.15 Structure of antibacterial compound from Periconia siamensis

CMUGE015. 4-Chromanone, 6-hydroxy-2-methyl- (5CI).

229

Compound 2 (Figure 6.15) was fractionated from fraction-5, identified by IR,

NMR and mass spectra data as 4-Chromanone, 6-hydroxy-2-methyl- (5CI). The

molecular formula was revealed to be C10H10O3 (Fig. 1b) by HREIMS. MW 178 and

IR absorption bands at 3292 and 1716 cm-1. Compound 2 was a yellow, amorphous

powder: melting point 214–215 °C (MeOH); UV lmax (MeOH) nm (log ε): 256

(4·04), 324 (4·22);); UV lmax (sodium methoxide) nm: 256, 368; 1H NMR

(CD3COCD3): d8·50 (1H, s, exchangeable D2O, OH-4'), 7·14 (2H, d, J=8·5 Hz, H-2',

H-6'), 6·82 (2H, d, J=8·5 Hz, H-3', H-5'), 6·51 (1H, d, J=2·5 Hz, H-8), 6·37 (1H, d,

J=2·5 Hz, H-6), 5·80 (1H, s, H-3), 3·91 (3H, s, OMe-7), 3·53 (3H, s, OMe-5);

Δδ=δC5D5N2-δCD3COCD3 =H-2'+H-6' (+0·19), H-3'+H-5' (+0·32), H-8 (+0·11), H-6

(+0·01), H-3 (+0·38) OMe-7 (20·16), OMe-5 (20·23); IR νmax (CHCl3) cm-1: 1708,

1612, 1598, 1512, 1159, 1112, 1054, 952, 870, 860, 832; MS m/z (relative intensities):

178 [M]+ (100), 270 [M–CO]+ (82), 255 [M–MeCO]+ (29), 227 [M–43–CO]+ (15).

6.3.5 Minimum inhibitory concentration (MICs)

Modiolide A and 4-Chromanone-6-hydroxy-2-methyl-(5IC) showed

antibacterial activity against the five test bacteria used. Modiolide A was generally the

more effective of the two (Table 6.5), however, the high MIC values with S. aureus

and E.coli rule out further investigation. Both compounds were compared with

standard antibiotic, Penicillin G with Modiolide A being most similarly effective

(Table 6.5).

230

Table 6.5: Antibacterial activity (MIC μg/mL) of Modiolide A and 4-Chromanone,

6-hydroxy- 2-methyl- (5CI).

MIC (μg/mL) Microorganism Modiolide

A 4-Chromanone, 6-hydroxy- 2-methyl- (5CI)

Penicillin G

Bacillus cereus 3.12 6.25 6.25 Listeria monocytogenes 6.25 12.5 6.25 Staphylococcus aureus (MRSA) 25 50 25 Pseudomonas aeroginosa 12.5

12.5 12.5 Escherichia coli 50 100 50

6.4 Discussion

6.4.1 Bioactive compounds from fungi

According to Dreyfuss and Chapela (1994), an estimated 4000 secondary

metabolites that prossess bioactive activities have been characterized from fungi. To

date, various species of Aspergillus, Fusarium and Penicillium, in particular are well

known for producing several secondary metabolites e.g. cyclosporine (Rodriguez et

al., 2006), paxilline (Fox and Howlett, 2008), 6-methylsalicylic acid (Panagiotou et

al., 2009). Fungi produce a diverse range of secondary metabolites which have no

effect on the growth of the producer strain but can be strongly inhibitory against other

microorganisms and many of these have important chemotherapeutic and other uses

(Bhadury et al., 2006; Boustie and Grube., 2007). Endophytic fungi are widely

recognized as prolific sources of bioactive secondary metabolites that might represent

useful leads in the development of new pharmaceutical agents (John et al., 1999;

Strobel et al., 1999; Brady et al., 2000; Singh et al., 2000; Yamada et al., 2002;

Bhadury et al., 2006; Misiek and Hoffmeister, 2007).

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6.4.2 Potential of graminicolous fungi for antibacterial and antifungal

productions

The present study has shown that all of fungal isolates tested produce

antimicrobial agents and some endophytic fungi from grasses have a promising

prospect for production of useful bioactive compounds. The selected fungal strains,

Periconia siamensis CMUGE015 and Dactylaria endograminicola CMUGE1125

showed the highest activities for producing antibacterial and antifungal substances,

respectively. The compounds investigated in this study have a broad spectrum of

activity against several bacteria and fungi including B. cereus, E. coli, P. aeroginosa,

C. albicans and P. avellaneum. Strobel et al. (1999) found that cryptocandin produced

by Cryptosporiopsis quercina, a fungal endophyte from Tripterigenum wilfordii, has

antifungal activity against the human pathogens C. albicans and Trichophyton spp.

and the plant pathogens Sclerotinia sclerotiorum and Botrytis cinerea.

Cryptosporiopsis quercina also produces cryptocin, a tetramic acid with potent

activity against Pyricularia oryzae and a number of other plant pathogenic fungi (Li

et al., 2000; 2001). Cryptocin was generally ineffective against an array of human

pathogenic fungi. Nevertheless, with a minimum inhibitory concentration against P.

oryzae of 0.39 μg/ml, this compound is being examined as a natural chemical control

agent for rice blast and is being used as a model to synthesize other antifungal

compounds (Strobel et al., 2004).

Species of Periconia are known to produce secondary metabolites, including

the periconicins, which are the fusicoccane diterpenes produced by an endophytic

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species isolated from the inner bark of Taxus cuspidate (Kim et al., 2002). In this

study Periconia siamensis, also found as endophyte produced antibacterial activities.

A discussion of the characterization of bioactive compounds from this fungus will be

presented in section 6.4.4.

6.4.3 Effect of pH, fermentation media and temperature of antibacterial and

antifungal productions

The initial pH of the culture medium is one factor which influences production

of antibacterial and antifungal activities (Te Dorsthorst, 2004). In this study, a pH of

6.0–7.0 and temperature of 25–30°C were optimal for the production of bioactive

compounds produced by the selected fungal strains. This pH is generally in the range

for optimal fungal growth. Krier et al. (1998) also reported the effect of temperature

and pH on the production of two bacteriocins by Leuconostoc mesentriodes and the

optimal temperature was 20–25°C and pH between 5.5–6.5. They found that

temperature and pH had a strong effect on the production of these agents, which are

stimulated by slow growing rates.

The carbon and nitrogen sources used in a fermentation medium can have a

significant effect on the expression of secondary metabolite biosynthetic pathways. In

this study, glucose and fructose were used as the C-sources and polypeptone and

soybean meal were used as N-sources. C and N sources showed the highest repression

of synthesis, while Cheeptham et al. (1999) found that a mixture of glucose and

glycerol presented the high repressive effects and the mixture of yeast and

polypeptones also showed the highest repression. Sucrose may be an excellent of C-

source for antifungal production in fermentation of producing strain, while glucose

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only promoted growth for producing strains. Peptone is good as the N-source, it is

more nutrition complex than soybean meal. This study supported the results from

earlier work with C-sources, however, for N-sources soybean meal and polypeptone

presented the highest repression than the previous report. Different fungi clearly

utilise different nutrients for their growth and production of secondary metabolites as

controlled by their genomes. The chemical nature (C and N sources), pH and

temperature also were found to have a profound influence on bioactive compounds

production in liquid culture of fungi.

6.4.4 Characterization of antibacterial activities of Periconia siamensis

CMUGE015

From this research on new biologically active metabolites from fungi two

antibacterial compounds from P. siamensis CMUGE015, an endophytic fungus from

the grass, Thysanolaena latifolia (Bhilabutra et al., in press) have been characterised.

Two antibacterial compounds were identified as Modiolide A (C10H14O4) and 4-

Chromanone-6-hydroxy-2-methyl-(5IC) (Figure 6.14 and 6.15).

Modiolide A was recently reported as a novel compound from the marine

fungus, Paraphaeosphaeria sp., however, no antibacterial activity was described

(Tsuda et al., 2003). Recently, the structure of this compound was reported using X-

ray crystallography (Fun et al., 2006) (Fig. 6.13a). In this study, it has been

demonstrated that Modiolide A has potent activity against Bacillus cereus and

Listeria monocytogenes, MRSA (Methicillin Resistance Staphylococcus aureus) and

Pseudomonas aeroginosa the causative agents of food borne disease, listeriosis, skin

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infections and lung disease, respectively. The compound was compared with standard

antibiotic, Penicillin G. The results show that Modiolide A inhibits gram positive

bacteria rather than gram negatives (Table 6.6).

Modiolide are classified into the macrolide group, a group of drugs (typically

antibiotics) whose activity stems from the presence of a Mcrolide ring, a large

macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and

deesosamine, may be attached. The lactone rings are usually 14, 15 or 16-membered.

Macrolides belongs to the polyketide class of natural products (Shiomi and Ōmura,

2002). The mechanism of action of the macrolide is inhibition of bacterial protein

biosynthesis by binding reversibly to the subunit 50S of the bacterial ribosome,

thereby inhibiting translocation of peptidyl tRNA. This action is mainly

bacteriostatics, but can also be bactericidal in high concentration (Keicho and Kudoh,

2002; Schultz, 2004).

Compound 2 (Fig. 6.15) or 4-Chromanone-6-hydroxy-2-methyl-(5IC) is

similar to Tuckolide (C10H16O5, compound in Lactones group, Figure 6.16) that has

been reported as an inhibitor of cholesterol synthesis in liver cells (Andrus and Shih,

1996). However, 4-Chromanone is compound of Flavonoids group. Flavonoids are

most commonly known for their antioxidant activity. However, it is now known that

the health benefits they provide against cancer and heart disease are the result of other

mechanisms (Cushnie and Lamb, 2005). There has never been reported as having

antibacterial activities. It showed a similar spectrum of activity as Modiolide A and 4-

Chromanone-6-hydroxy-2-methyl-(5IC), however, the latter was more potent and

could have potential as a lead compound for the development of antibacterial agents

for many gram positive bacterial strains.

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Figure 6.16 Structure of Tuckolide (C10H16O5), similar compound of 4-Chromanone,

6-hydroxy-2-methyl- (5CI).