Chiang Mai J. Sci. 2021; 48(3) : 793-807http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Antimicrobial Potential of Fungi Isolated from Soils of Dry Dipterocarp Forest in Northeast ThailandPanjamaphon Chanthasena [a,b], Watsana Penkhrue [a], Phimpha Khowangklang [a], Pishyaporn Sritangos [a] and Nawarat Nantapong*[a][a] School of Preclinical Sciences, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima

30000, Thailand. [b] Faculty of Medical Science, Nakhonratchasima College, Nakhon Ratchasima 30000, Thailand.

*Author for correspondence; e-mail: nawarat@sut.ac.thReceived: 13 December 2020

Revised: 17 March 2021Accepted: 24 March 2021

ABSTRACT A total of sixteen antimicrobial-producing fungal isolates were isolated from forest soil

collected from Suranaree University of Technology (SUT), Thailand. These fungal isolates exhibited antimicrobial activity against test pathogenic Gram-positive bacteria (methicillin-resistant Staphylococcus aureus (MRSA), S. aureus, S. epidermidis, Bacillus subtilis, and B. cereus), Gram-negative bacteria (Escherichia coli, Enterobacter aerogenes, Salmonella typhimurium, and Proteus mirabilis), and yeasts (Candida albicans, C. tropicalis, and Saccharomyces cerevisiae). The fungal isolates that exhibited the highest antimicrobial activity against MRSA, S. aureus, S. epidermidis, B. subtilis, B. cereus, E. coli, En. aerogenes, Sal. typhimurium, P. mirabilis, C. albicans and C. tropicalis are PKF125, PKF60, PKF125, PKF6, PKF127, PKF104, PKF152, PKF152, PKF152, PKF105 and PKF105, respectively. The highest antifungal activity toward Sac. cerevisiae was observed from PKF77 and PKF105. An initial molecular classification of fungal strains was conducted by sequencing the internal transcribed spacer (ITS) region. The ITS region of fungal isolates were analyzed by BLAST search and phylogenetic analysis. The BLAST search revealed that 16 fungal isolates were members of the genus Aspergillus (9 isolates), Penicillium (3 isolates), Clonostachys (2 isolates) and Talaromyces (2 isolates). The obtained fungal isolates in the genus Aspergillus were separated into three sections including Flavipedes (PKF116, PKF145, and PKF105), Nigri (PKF161), and Flavi (PKF59, PKF38, PKF6, PKF60, and PKF61). On the otherhand, isolates PKF121, PKF125, and PKF127 were placed in Penicillium section Citrina. Isolate PKF152 and PKF124 were assigned to Talaromyces section Talaromyces and Islandici, respectively. Isolate PKF77 and PKF104 were deemed as Clonostachys rogersoniana. The current study suggests that unexplored forest soil represents a rich reservoir for the search of new antimicrobial compounds. Fungal isolates in this study exhibited the ability to inhibit MRSA, providing insights into pharmaceutical targeting against drug-resistant microorganisms.

Keywords: antimicrobial activity, antimicrobial-producing fungi, drug-resistant microorganisms, MRSA, soil fungi

1. INTRODUCTIONInfectious diseases caused by microorganisms

have been a major global health problem for decades [1]. Key microorganisms which commonly cause infectious diseases are S. aureus, S. epidermidis,

Chiang Mai J. Sci. 2021; 48(3)794

Pseudomonas aeruginosa, and C. albicans. These microorganisms can cause various minor health issues to life-threatening diseases in humans, including food poisoning, skin infections, burn infections, and genital yeast infections [2-5].

Antibiotics have been widely used for the treatment of infectious diseases since the discovery of penicillin in 1928. Since then, over a thousand antibiotics were discovered and were excessively used for therapeutic, agriculture and veterinary [6]. These widespread use and misuse of antibiotic drugs lead to the worldwide emergence of drug-resistant organisms. Key drug-resistant traits, particularly resistance to methicillin (e.g. methicillin-resistant S. aureus (MRSA)), lead to the resistance of several essential antibiotic drugs, including β-lactams, quinolones, and aminoglycosides [7-10]. Infections caused by these drug-resistant microorganisms are difficult to treat with currently available antibiotics. Therefore, MRSA-associated infections became responsible for most morbidity and mortality, especially in elderly and immunocompromised patients.

To prevent further drug-resistance, global efforts have been made to limit the unnecessary prescription of antibiotics. Currently, vancomycin is reserved as the last-line drug for the treatment of MRSA infections [11]. Unfortunately, the case of vancomycin-intermediate S. aureus (VISA) was first reported in Japan in 1997 [12-14]. Since then, vancomycin-resistant S. aureus (VRSA) was isolated from several countries, including the USA, France, Korea, South Africa, Brazil and Scotland [15]. Therefore, the search for new and effective antibiotic drugs become necessary to overcome this problem.

Soil microorganisms serve as an important resource of bioactive compounds such as insecticides, herbicides, anticancer drugs, and antimicrobial drugs [16]. For instance, Penicillium sp. isolated from Brazilian Cerrado soil was reported to produce a major secondary metabolite as methyl 6-acetyl-4-methoxy-5, 7, 8-trihydroxynaphthalene-

2-carboxylate (0.3175 mg/L), which exhibited antibiotic effects against C. albicans, Listeria monocitogenes, and B. cereus [17]. In another study, Makut and Qwolewa collected soil samples from Keffi Metropolis, Nigeria, and isolated ten fungal species, including Absidia corymbifera, Alternaria alternata, A. flavus, A. fumigatus, A. niger, Cladosporium herbarum, Curvularia lunata, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. All these fungal isolates inhibited the growth of pathogens such as E. coli, S. aureus, Ps. aeruginosa, and C. albicans [18]. Aside from antibiotics properties, bioactive compounds derived from microorganisms could also exert antitumor effects. Antitumor antibiotic GKK 1032B was reported to be produced by Pen. citrinum [19,20]. Therefore, discovering new microorganism strains may facilitate the discovery of novel potent bioactive compounds

Despite the efforts to search for new antibiotics, it was estimated that only 1% of antimicrobial-producing bacteria and 5% of fungi were isolated and characterized [21]. Thus, an immense number of microbial species remains to be discovered. Antibiotic-producing soil microorganisms included actinobacteria (70%), fungi (20%), and other eubacteria (10%) [18]. Soil microorganisms, particularly fungi, produce many useful secondary metabolites such as antibiotics, organic acids, and enzymes [22, 23]. Fungi also produce essential antibiotics, including penicillin, fusidic acid, cephalosporin, and lovastatin [24, 25].Previous works reported that forest soil in Suranaree University of Technology (SUT) contained high biodiversity of antimicrobial-producing actinobacteria. The actinobacteria isolated from SUT soil showed antimicrobial activity against various opportunistic pathogens, including MRSA [26]. However, the biodiversity of antimicrobial-producing fungi in SUT has never been reported. In this present study, we described the phylogenetic diversity of antibiotic-producing fungi isolated from forest soil in SUT, Nakhon Ratchasima province, Thailand.

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2. MATERIALS AND METHODS2.1 Media and Reagents

Potato dextrose broth (PDB) and potato dextrose agar (PDA) were used for the isolation and cultivation of fungal strains. Muller-Hinton agar (MHA) and Sabouraud dextrose agar (SDA) were used for the evaluation of antimicrobial activity. All fungal culture reagents were purchased from Hi-Media (India). According to the manufacturer’s datasheet, PDA contained (per liter) 200 g of potatoes infusion powder, 20 g of dextrose, and 10 g of agar (pH 5.6). MHA (per liter) contained HM infusion powder (equivalent to Meat Infusion powder) 300 g, AcicaseTM 17.5 g, starch 1.5 g, and agar 17 g (pH 7.3). SDA medium contained (per liter) 10 g of peptone, 40 g of glucose, and 15 g of agar (pH 5.6) [27].

Reagents used for fungal DNA extraction were lysis buffer and TE buffer. Lysis buffer contained 60 mM EDTA pH 8.0, 400 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1% (w/v) sodium dodecyl sulfate [28]. TE buffer consisted of 10 mM Tris-HCl and 0.1 mM EDTA pH 8.0 [29].

2.2 Strain of Test MicroorganismsThe pathogenic microorganisms used for

the determination of antimicrobial activity were obtained from the Thailand Institute of Scientific and Technological Research (TISTR) and the Department of Medical Sciences Thailand (DMST). Gram-positive bacteria purchased were methicillin-resistant S. aureus DMST20654 (MRSA), S. aureus TISTR1466, S. epidermidis TISTR518, B. subtilis TISTR008, and B. cereus TISTR687. Gram-negative bacteria purchased were E. coli TISTR780, En. aerogenes TISTR1540, Sal. typhimurium TISTR292, and Proteus mirabilis TISTR100. Yeasts purchased were C. albicans TISTR5779, C. tropicalis TISTR5174, and Sac. cerevisiae TISTR5049.

2.3 Collection of Soil and Isolation of Fungal Isolates

Forest soil samples were collected from SUT, Thailand (14.8818°N, 102.0207°E). Soil samples

were stored in plastic bags and transferred to the laboratory in an icebox. A gram of each soil samples was suspended in 99 mL of sterile distilled water and incubated on a shaker for 30 minutes at room temperature. Suspended soil samples were then diluted using the ten-fold dilution method. Samples diluted at 10-3, 10-4, and 10-5 of the initial concentration were spread on PDA containing 50 µg/mL chloramphenicol. The PDA plates were incubated at 30°C for 7-14 days. Suspected fungal colonies were isolated and sub-cultured onto new PDA plates. After 5-7 days of incubation at room temperature, the culture plates were stored at 4oC until further study. Selected isolates were cultured on PDA and the size and texture of fungal colonies were recored after cultivation at 30°C for 7-14 days. Microscopic characters of selected strain such as spore shape and size were observed under light microscope.

2.4 Determination of Antimicrobial Activity Using the cross-streaking technique, fungal

isolates were tested for bacterial and yeast sensitivity on MHA and SDA, respectively [30]. Fungal isolates were incubated at 28oC for five days. Then, the test pathogens were perpendicularly streaked to the line of fungal colonies and incubated at 37°C for 24-48 h. The zone of inhibition in millimeter against test pathogens was measured and recorded. The inhibition zones of each fungal isolates against test pathogens were statistically evaluated. One-way ANOVA with Dunnett’s post-hoc test was performed. The p-value<0.05 indicated statistical significance.

2.5 Extraction of Fungal DNAChromosomal DNA was extracted from

fungal mycelia grown in 10 mL of PDB, at 28°C, for 5 days under 200 rpm shaking condition [31]. The mycelia and 500 µL lysis buffer were mixed in a mortar and grounded with glass beads and pestle. The suspension was transferred to a 1.5 mL microcentrifuge tube, and 165 µL of 5 M NaCl was added. The suspension was mixed by

Chiang Mai J. Sci. 2021; 48(3)796

repeatedly inverting the tube then centrifuged at 13,000 rpm for 10 min. The supernatant was transferred to a new tube and mixed with 800 µL of chloroform:isoamyl alcohol (1:1). The suspension was gently mixed until the solution became milky before centrifugation at 13,000 rpm for 20 min. The aqueous layer was removed and extracted with an equal volume of chloroform and centrifuged again for 20 min at 13,000 rpm. After the supernatant was transferred to a new microcentrifuge tube, DNA was precipitated using absolute ethanol, at -20°C for 15 min. The DNA pellet was washed three times, using 800 µL of 70% cold ethanol, then air-dried. The extracted DNA was dissolved in 50 µL of TE buffer and stored at -20°C.

2.6 PCR Amplification and Sequencing The extracted fungal DNA was used as a

template for PCR amplification using a thermal cycler (BIORAD, USA). The ITS regions of fungal isolates were amplified. The ITS universal primer, ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) [32], was used for PCR amplification. The cycling parameters were as follows: initial denaturation at 95°C for 5 min, 30 cycles of amplification (denaturation at 95°C for 30 s, annealing at 50oC for 1 min, extension at 72°C for 1 min), and a final extension at 72°C for 7 min. The NucleoSpin® Gel and PCR clean-up kit (MACHEREY-NAGEL, Germany) were used for the purification of the amplified fragments. The purified DNA fragments were submitted for a sequencing at Macrogen Inc. (Korea). 2.7 Construction of Phylogenetic Tree

The sequences of ITS region were compared with references sequences from GenBank database (https://blast.ncbi.nlm.nih.gov/). The sequences were aligned using Clustal W. The Molecular Evolutionary Genetics Analysis software version 7.0 (MEGA 7.0) was used for the construction of phylogenetic trees, using the maximum likelihood

approach. The tree topologies were evaluated by using bootstrap analysis (1,000 replications). The distances matrix between sequences was generated by using Kimura’s two-parameter model [33].

3. RESULTS3.1 Isolation and Identification of Antimicrobial-Producing Fungi

Twenty-one forest soil samples were randomly collected from SUT, Thailand. Soil samples were used for the isolation of fungal isolates by serial dilution and spread plate techniques on PDA medium. One hundred-seventy seven fungal isolates were selected based on the difference of colony morphologies. Fungal isolates were coded as PKF1 to PKF177. Their antimicrobial activity against S. aureus TISTR1466, methicillin-resistant S. aureus DMST20654, S. epidermidis TISTR518, B. subtilis TISTR008, B. cereus TISTR687, E. coli TISTR780, En. aerogenes TISTR1540, Sal. typhimurium TISTR292, P. mirabilis TISTR100, C. albicans TISTR5779, C. tropicalis TISTR5174, and Sac. cerevisiae TISTR5049 were examined by using the cross-streak method. The antimicrobial-producing fungal isolates were then identified based on their morphological characteristics and internal transcribed spacer (ITS) sequence. Out of the 177 fungal isolates, 16 isolates (PKF6, PKF38, PKF59, PKF60, PKF61, PKF77, PKF104, PKF105, PKF116, PKF121, PKF124, PKF125, PKF127, PKF145, PKF152, and PKF161) exhibited antimicrobial activity against tested pathogens. The colony morphologies of these isolates grown on PDA are shown in Figure 1. Macroscopic features of strained isolated colonies were diversely varied. Some isolates were mainly white with various morphologies such as fluffy texture (PKF6, PKF38, PKF59, PKF60, and PKF61), and raised colony (PKF104, PKF105, and PKF145). Other isolates produced bluish-green colonies with coloration and varied morphologies such as velvety to powdery and encircled with a white border (PKF121, PKF125, and PKF127), yellowish edges (PKF124 and PKF152), and olive

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green plane with abundant white mycelium on the margins (PKF161). PKF77 colony was pinkish-white and produced diffusible brownish-red to wine-red pigment. Fungal isolate PKF116 was the only colony that exhibited yellow color on PDA. The microscopic characteristics of the PKF isolates under light microscopy were also recorded. From the isolates investigated, the spores observed were grouped into two groups based on their distinct generic features. The first group exhibited biseriate with philiades radiating from all sides, globose conidia with varying sizes, and unbranched conidiophore (PKF6, PKF38, PKF59,

PKF60, PKF61, PKF105, PKF116, PKF145, and PKF161). The second group exhibited spherical conidia with a smooth surface, baring long and well-defined columns (PKF77, PKF104, PKF121, PKF124, PKF125, PKF127, and PKF152).

The ITS region is used for the molecular identification of fungi since the region demonstrates a higher degree of variation, enabling relatively accurate fungal species identification. The ITS region (650-750 bp) consists of ITS1 (5.8S rRNA gene) and ITS2, located between 18S rRNA and 28S rRNA genes [34]. The sequenced ITS regions of the 16 antimicrobial-producing fungi

Figure 1. Colony features of 16 fungal isolates, grown on PDA at room temperature for 7-14 days (Scale bar = 1 cm).

Chiang Mai J. Sci. 2021; 48(3)798

Tabl

e 1.

Ant

imic

robi

al a

ctiv

ity o

f fu

ngal

isol

ates

aga

inst

test

pat

hoge

ns u

sing

the

cros

s-st

reak

met

hod.

Fung

al

isol

ates

Inhi

bitio

n zo

ne o

f an

timic

robi

al a

ctiv

ity (m

m.)

Gra

m-p

ositi

ve b

acte

riaG

ram

-neg

ativ

e ba

cter

iaYe

asts

MRSA DMST20654

S. aureus TISTR1466

S. epidermidis TISTR518

B. subtilis TISTR008

B. cereus TISTR687

E. coli TISTR780

En. aerogenes TISTR1540

Sal. typhimuriumTISTR292

P. mirabilis TISTR100

C. albicans TISTR5779

C. tropicalis TISTR5174

Sac. cerevisiae TISTR5049

PKF6

-16

.27±

0.46

a20

.00±

0.50

a21

.00±

0.00

a-

--

--

--

-

PKF3

8-

13.1

7±0.

29b

20.0

0±0.

00a

15.2

7±0.

31b

--

--

--

--

PKF5

9-

10.3

0±0.

26bc

16.2

7±0.

25b

8.00

±0.

00cd

--

--

--

--

PKF6

0-

17.0

0±0.

10a

20.1

7±0.

29a

15.0

0±0.

00bc

7.00

±0.

00b

--

--

--

-

PKF6

1-

10.1

3±0.

23bc

21.0

0±0.

00a

18.3

3±0.

58a

--

--

--

--

PKF7

7-

5.00

±0.

00c

20.2

0±0.

35a

13.0

0±0.

00c

--

--

-18

.00±

0.00

a10

.00±

0.00

b15

.00±

0.00

a

PKF1

0411

.17±

0.29

a9.

00±

0.00

c12

.03±

0.25

c14

.00±

0.00

bc-

6.00

±0.

00a

--

-9.

07±

0.21

b8.

00±

0.00

b-

PKF1

05-

-5.

00±

0.00

d-

--

--

-20

.23±

0.40

a20

.00±

1.00

a15

.00±

0.00

a

PKF1

16-

--

--

--

--

17.4

3±0.

76a

8.00

±0.

00b

10.1

3±0.

23b

PKF1

2110

.13±

0.23

a5.

00±

0.00

c20

.20±

0.35

a14

.00±

0.00

bc6.

00±

0.00

b-

--

-10

.00±

0.00

b5.

00±

0.00

b3.

00±

0.00

c

PKF1

2410

.17±

0.76

a3.

00±

0.00

c23

.00±

0.00

a-

--

--

--

--

PKF1

2512

.13±

0.32

a7.

00±

1.00

c25

.00±

1.00

a13

.00±

0.00

c14

.00±

0.00

ab-

--

--

--

PKF1

2711

.33±

0.58

a11

.00±

1.00

b20

.00±

0.00

a20

.00±

0.00

a15

.30±

0.52

a-

--

--

--

PKF1

45-

--

--

--

--

5.33

±0.

58c

5.00

±0.

00b

10.1

7±0.

29b

PKF1

52-

--

9.00

±0.

00cd

5.00

±0.

00b

-7.

00±

0.00

a11

.00±

0.00

a7.

00±

0.00

a5.

40±

0.69

c-

3.00

±0.

00c

PKF1

615.

00±

0.00

b5.

00±

0.00

c15

.00±

0.00

b7.

07±

0.38

d5.

00±

0.00

b-

--

--

-5.

00±

0.00

c

(-) n

o ef

fect

. The

exp

erim

ent w

as p

erfo

rmed

in th

ree

repl

icat

es. V

alue

s ar

e gi

ven

in m

ean

± S

D m

m. V

alue

s in

eac

h co

lum

n w

ith th

e sa

me

lette

r ar

e no

t sta

tistic

ally

diff

eren

t (p

< 0

.05)

.

Chiang Mai J. Sci. 2021; 48(3) 799

were blasted and identified via the GenBank database (https://blast.ncbi.nlm.nih.gov/). The closest relative strains of the 16 fungal isolates are shown in Table 2. Based on the GenBank database, the ITS1-5.8S-ITS2 sequence was also used to determine the taxonomic relationships of the fungal isolates and their relative reference strains. The phylogenetic tree showed that isolates PKF116, PKF145, PKF105, and PKF 59 were assigned to the same clade with their closest relative type strains obtained from blast search (Figure 2). Isolates PKF116, PKF145, and PKF105 revealed 100% identities to A. frequens (Table 2), thus, could be classified as A. frequens. As PKF59 closely resembled A. nomius with 99.75% identity (Table 2). PKF59 was classified as A. nomius. In addition, five fungal isolates (PKF161, PKF38, PKF6, PKF60, and PKF61) closest to Aspergillus

spp. could not be identify at the species level. Hence, they were classified into two sections based on a phylogeny of ITS gene (Figure 2). Isolate PKF161 was classified to Aspergillus section Nigri, while isolates PKF38, PKF6, PKF60, and PKF61 were assigned to Aspergillus section Flavi.

We also identified three fungal isolates (PKF121, PKF125, and PKF127) that formed a phylogenetic clade with type strains from genera Penicillium (Figure 3). PKF121, PKF125, and PKF127 showed 100% resemblance to their reference relatives and were classified as Pen. citrinum (Table 2). Isolates PKF152 and PKF124 exhibited 98-100% identities to T. purpureogenus and T. allahabedensis, respectively (Table 2). Our constructed maximum-likelihood phylogenetic tree (Figure 4) also showed that these two fungal isolates (PKF152 and PKF124) were assigned to the same clade with their closest

Table 2. The closest relative type strains of 16 antimicrobial-producing fungi based on ITS region. The most closely related species search was performed using basic local alignment search tool (BLAST) from GenBank database.

Fungal isolates /GenBank Accession No. Closest to type strains (GenBank Accession No.) Similarity

(%)

PKF6 /MF583166 Aspergillus flavus var. flavus ATCC 16883 (KU729026) 100.00

PKF38 /MF583165 Aspergillus sojae CBS 100928 (MH862715) 99.48

PKF59 /MF583163 Aspergillus nomius DTO 321 F2 (D84354) 99.75

PKF60 /MF583168 Aspergillus minisclerotigenes CBS 117635 (KY937925) 100.00

PKF61 /MF583169 Aspergillus minisclerotigenes CBS 117635 (KY937925) 99.80

PKF77 /MF583170 Clonostachys rogersoniana LS227 (MK715296) 100.00

PKF104 /MF583164 Clonostachys rogersoniana LS227 (MK715296) 100.00

PKF105 /MF583167 Aspergillus frequens NRRL 4578 (NR137473) 100.00

PKF116 /MF583192 Aspergillus frequens NRRL 4578 (NR137473) 100.00

PKF121 /MF583194 Penicillium citrinum CBS 139.45 (MH856132) 100.00

PKF124 /MF583171 Talaromyces allahabadensis CBS 453.93 (MH862430) 100.00

PKF125 /MF583193 Penicillium citrinum CBS 139.45 (MH856132) 100.00

PKF127 /MF583195 Penicillium citrinum CBS 139.45 (MH856132) 100.00

PKF145 /MF583172 Aspergillus frequens NRRL 4578 (NR137473) 100.00

PKF152 /MF583196 Talaromyces purpureogenus CBS 286.36 (JX315671) 98.98

PKF161 /MF583197 Aspergillus foetidus CBS 121.28 (NR163668) 99.21

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Figure 2. A maximum-likelihood phylogenetic tree of fungal isolates belonging to the genus Aspergillus and their related taxa from the GenBank database. The number at the nodes indicate bootstrap support levels based on 1,000 replications. Only bootstrap values ≥ 50% are shown. The scale bar indicates 0.01 substitutions per nucleotide position.

Chiang Mai J. Sci. 2021; 48(3) 801

Figure 3. A maximum likelihood phylogenetic tree of fungal isolates belonging to the genus Penicillium and their related taxa from GenBank database. The number at the nodes indicate bootstrap support levels based on 1,000 replications. Only bootstrap values ≥ 50% are shown. The scale bar indicates 0.02 substitutions per nucleotide position.

relative type strains (Figure 4). Therefore, PKF152 and PKF124 were classified as T. purpureogenus and T. allahabedensis, respectively. Two fungal isolates, PKF77 and PKF104, showed 100% identities to Cl. rogersoniana (Table 2). The phylogenetic tree (Figure 5) of these isolates (PKF77 and PKF104) exhibited a monophyletic clade with their closet type strain. Thus, PKF77 and PKF104 could be classified as Cl. rogersoniana.

3.2 Antimicrobial Activity of Fungal IsolatesSince only 16 fungal isolates were antibiotic-

producers, this study focused on the antimicrobial activity of these isolates (Table 1). These isolates were separated into four groups based on the narrow to broad spectra antimicrobial activity. Group I (PKF6, PKF38, PKF59, PKF60, PKF61, PKF124, PKF125, and PKF127) was defined as isolates with narrow antimicrobial activity

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Figure 4. A maximum likelihood phylogenetic tree of fungal isolates belonging to the genus Talaromyces their related taxa from GenBank database. The number at the nodes indicate bootstrap support levels based on 1,000 replications. Only bootstrap values ≥ 50% are shown. The scale bar indicates 0.02 substitutions per nucleotide position.

against only Gram-positive bacteria. Group II isolates, consisted of PKF116 and PKF145, exhibited antifungal activity against only yeasts. Group III isolates (PKF77, PKF105, PKF121, and PKF161) showed antimicrobial activity against both Gram-positive bacteria and yeasts. The final group IV, which included PKF104 and PKF152, exhibited broad antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and yeasts. Interestingly, we found that

six fungal isolates (PKF104, PKF121, PKF124, PKF125, PKF127, and PKF161) were effective against MRSA (S. aureus DMST20654) (Table 1).

4. DISCUSSIONAntibiotic drugs are essential bioactive

compounds indispensable for the treatment of most infectious diseases. The widespread and uncontrolled use of antibiotics led to the emergence of antibiotic-resistant microorganisms, which

Chiang Mai J. Sci. 2021; 48(3) 803

Figure 5. A maximum-likelihood phylogenetic tree of fungal isolates belonging to the genus Clonostachy and their related taxa from the GenBank database. The number at the nodes indicate bootstrap support levels based on 1,000 replications. Only bootstrap values ≥ 50% are shown. The scale bar indicates 0.02 substitutions per nucleotide position.

caused most commercially available antibiotics to become ineffective for clinical treatment [35, 36]. Therefore, there is an urgent need to search for new, safe, and effective antibiotics to replace the invalidated agents. Our previous work reported that antibiotic-producing actinobacteria could be isolated from the soil sample in dry dipterocarp forest at SUT [26]. However, the study on antimicrobial-producing fungi in this area has never been documented. Therefore, the present study focused on identifying and investigating antimicrobial-producing fungi from soil in SUT. In this study, 16 antibiotic-producing fungi were successfully isolated from forest soil in SUT.

Classification and identification of fungi can be achieved by various strategies, including morphological characters, physiological characters, and ITS sequence analysis [37]. It is well-established that the differences in ITS region could be exploited for fungal identification [38,39]. The 16 antibiotic-producing fungi isolates were genotyped by DNA sequencing of the ITS region and were compared with GenBank DNA database. Comparisons against ITS databases and phylogenetic analysis are considered practical combinations suitable for fungi identification [40]. However, it should be noted that the ITS region of the 5.8 rRNA gene could only provide a preliminary identification at the genera level. Other more accurate methods

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for fungi identification that should be included in future study is the sequencing of housekeeping genes such as calmodulin gene (CaM), beta-tubulin gene (BenA), and RNA polymerase II second largest subunit (RPB2) [41]. The blast search results from GenBank and phylogenetic analysis revealed that 16 antimicrobial-producing fungi isolated from soil in SUT were A. frequens, A. nomius, Pen. citrinum, T. allahabadensis, T. purpureogenus, and Cl. rogersoniana. The dominant genus found in this area was the genus Aspergillus (56.25%). Others were Penicillium (18.75%), Clonestachys (12.50%), and Talaromyces (12.50%). In agreement with a previous study, Aspergillus spp. was determined to be the dominant genera of antimicrobial-producing soil fungi [42]. Aspergillus spp. possesses several vital abilities such as salt-tolerance, rapid reproduction, and the capability to grow on several types of substrate [42]. Terrestrial Aspergillus spp. serve as an important source of antibiotics, including helvolic acid, claviformin, aspergillin, fumagillin, dihydrogeodin, 3,4-dimethoxyphenol, 1,3,5-trimethoxybenzene, rubrofusarin B [42].

In Thailand, the genera Aspergillus, Clonostachys, Penicillium and Talaromyces have been found in terrestrial soil. However, the antimicrobial activity of these soil isolated species remains uninvestigated [43-50]. Previous works suggested that SUT soil might serve as a rich reservoir for the screening of novel antimicrobial drugs [26]. This is because the dry dipterocarp soil sample collected from SUT exhibited dry, sandy, highly acidic, and low nutrients characteristics. These severe soil conditions induced a competitive environment for the growth and reproduction of microorganism, facilitating antimicrobial production. This present finding also showed that six fungal isolates (PKF104, PKF121, PKF124, PKF125, PKF127, and PKF161) isolated from SUT soil exhibited antimicrobial activity against MRSA.

Among the identified isolates, PKF121, PKF125, and PKF127 were classified as Pen. citrinum. Multiple antimicrobial-producing strains of Pen. citrinum have been reported worldwide,

mostly from marine samples and an extreme environment such as permafrost [51-57]. These extreme environments often serve as a promising source of novel bioactive secondary metabolites [58]. Although a previous study already reported that Pen. citrinum could be isolated from Thai soil, no data regarding its antimicrobial activity was documented [50]. In this study, the soil-derived Pen. citrinum PKF121, PKF125, and PKF127 strains were isolated from a non-extreme habitat but exhibited antimicrobial activities comparable to the marine-derived Pen. citrinum PSU-F51, isolated from “sea fan”, Southern Thailand [58]. Another potentially important species of antimicrobial-producing microorganism is T. allahabadensis. In 2018, Rui-Huan Huang reported that marine-derived fungi, T. allahabadensis D21 could be isolated from coastal marine habitats of the Yellow Sea and South China Sea, China. The isolated T. allahabadensis D21 exhibited anti-phytopathogenic activity against Pseudomonas syringae pv. Lachrymans, and Acidovorax avenae [59]. However, the anti-MRSA activity of T. allahabadensis remains to be investigated. This study, thus far, provides the first evidence that T. allahabadensis PKF124 isolated from terrestrial soil exerted anti-MRSA activities.

Therefore, the current study provided evidence that the soil could serve as a good source of novel antimicrobial-producing microorganisms and biological compounds potentially useful for antibiotic-resistant pathogens.

5. CONCLUSIONSThis current study demonstrates the isolation

and identification of antimicrobial-producing fungi from dry dipterocarp soil in Northeast Thailand. The soil in this area is dry, sandy, highly acidic, and low in nutrients establishing a severe environment for the growth and reproduction of living organisms. This drastic condition has been known to activate the production of antimicrobial agents of antibiotic-producing microorganisms. MRSA infections are difficult to treat and become a major global health concern. The current study

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identified 6 strains of fungal isolates that exhibited antimicrobial effects against MRSA in which fungal isolate PKF125 could be the best candidate for the study of anti-MRSA agent. We believe that our study provided beneficial insights for the discovery and development of new antimicrobial drugs effective against antibiotic-resistant pathogens. Future study may be required to provide more accurate identification of fungi, e.g. using β-tubulin and calmodulin genes as genetic markers. The toxicity of bioactive compounds and toxins produced by PKF strains should also be evaluated.

ACKNOWLEDGMENTSThis work was supported by research grant

of Suranaree University of Technology through National Research Council of Thailand (NRCT) for research support plans to strengthen capacity and develop new researchers, according to the strategic direction research and innovation: the graduate- level year 2019. We also wish to thank Suranaree University of Technology for the facilities. The authors also thank to Suranaree University of Technology for the funding through the plant genetic conservation project. This project was operated under the Royal initiative of her Royal highness princess Maha Chakri Sirindhorn-Suranaree University of Technology (RSPG-SUT).

CONFLICT OF INTEREST STATEMENTThe authors have no conflict of interest to

declare.

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