Screening of Endophytic Fungi of Melaleuca cajuputi Powell Leaves

as Antibacterial Sources

Hary Widjajanti, Salni, Niken Irfa Nastiti and Elisa Nurnawati

Department of Biology, Faculty of Mathematics and Natural Sciences, Sriwijaya University, Palembang, Indonesia

Keywords: Antibacterial, Endophytic fungi, Melaleuca cajuputi Powell.

Abstract: Melaleuca cajuputi leaves contain high flavonoids, flavonoids are chemical compounds that are

antibacterial. Antibacterial bioactive compounds are found in plants, but the use of plants as an antibacterial

source requires a large amount of biomass. Endophytic fungi that live in plant tissue can produce secondary

metabolites that potentially as an antibacterial compounds. Isolation of the endophytic fungi for antibacterial

sources can reduce the large amount of plant as a source of antibacterial agents. The aims of this research

were obtain endophytic fungi of M.cajuputi leaves as an antibacterial sources. The research stages were

sampling, isolation and purification of endophytic fungi, cultivation and production of secondary

metabolites, antibacterial activity and the minimum inhibitory concentration (MIC) tests, thin layer

chromatography, and characterization and identification of endophytic fungi that potentially as an

antibacterial sources. The results there were 7 isolates of endophytic fungi from Melaleuca cajuputi leaves.

, MC

, and MC

fungal isolates have a strong antibacterial activities. The MIC of MC

and MC

extracts on Escherichia coli were 400 μg/mL and 100 μg/mL. The MIC of MC

and MC

extracts on

Staphylococcus aureus were 200 µg/mL and 700 µg/mL, respectively. The extract of four endophytic fungi

contain phenol and flavonoid that potentially as an antibacterial. Endophytic fungi MC

isolate identified as

Scopulariopsis candida, MC

isolate as Fusarium equiseti, MC

isolate as Fusarium sporotrichoides, and

isolate as Mucor hiemalis.

1 INTRODUCTION

A number of plants have been passed down from

generation to generation used as medicine by almost

every community, including in Indonesia. Plants that

are used as medicinal plants, have now begun to be

explored scientifically by analyzing the content of

active compound that cause medicinal properties.

The active compound in the plant are too few or

minor, therefore if it is to be developed on a large

scale there are constraints from the raw material of

the plant that must be in large quantities.

Antibacterial compounds in the form of crude

extracts produced from plants when purified, the

amount will be even less. These obstacles make it

very difficult if the development of bioactive

compounds from medicinal plants that are rare and

endemic will be carried out. This is because if rare

plants are explored for bioactive compounds to be

taken, then the sustainability of these plants is

threatened. These constraints can be anticipated by

isolating microbes, especially endophytic fungi that

are symbiotic with these plants. The technique used

to take only a few parts of plants commonly used in

traditional medicine, then the isolation of endophytic

fungi from the plant parts is done, so that rare

medicinal plants will remain sustainable and the

development of medicinal compounds from these

plants through endophytic fungi can be developed.

The development of medicinal plants based on

medicinal plants must be considered aspects of

preservation of medicinal plants. One technology

that can be done was isolate endophytic fungi from

parts of medicinal plants that are often used as

traditional medicine and selection of bioactive of

secondary metabolites such as antibacterial from the

culture of endophytic fungi, thus the development of

medicinal plants based on medicinal plants can be

done and the preservation of medicinal plants

especially that are already scarce can be maintained.

Endophytic fungi are fungi that live in plant

tissues in a certain period and are able to live by

forming colonies in plant tissue without endangering

the host. Each higher level plant can contain several

196

Widjajanti, H., Salni, ., Irfa Nastiti, N. and Nurnawati, E.

Screening Endophytic Fungi of Melaleuca cajuputi Powell Leaves as Antibacterial Sources.

DOI: 10.5220/0010139000002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 196-204

ISBN: 978-989-758-556-2

endophytic microbes that produce secondary

metabolites that are suspected as a result of

coevolution or genetic recombination from host

plants to endophytic microbes (Tan and Zou (2001),

Strobel and B.Daisy (2003).

The ability of endophytic fungi to produce

secondary metabolites in accordance with their host

plants is a very large and reliable opportunity to

produce secondary metabolites from endophytic

microbes isolated from these host plants.

Approximately 300,000 species of plants scattered

on this earth, each plant contains one or more

endophytic microbes consisting of bacteria and fungi

(Strobel and Daisy, 2003). If the endophytes

isolated from a medicinal plant can produce the

same alkaloids or secondary metabolites as the

original plants or even in higher amounts, then we

don't need to cut down the original plants to be taken

as simplicia, which is likely to take decades to be

harvested (Radji,2005). According to Stierlie et al.

(1995) the use of endophytic microbes in producing

active compounds has several advantages, including

(1) faster producing of uniform quality, (2) can be

produced on a large scale, and (3) the possibility of

obtaining new bioactive components by providing

conditions that are new different.

M. cajuputi can live on land that has limited

nutrients from fertile soil and is rich in nutrients.

This plant is also able to live in soil with critical

conditions with little nutrient elements. M.cajuputi

leaves in Indonesia are widely used as raw materials

in the production of eucalyptus oil (Widiana et al,

2014). M.cajuputi leaves has many benefits, one of

which can be used as an antibacterial source (Al-

Abd et al., 2015). Melaleuca cajuputi has potential

as an antibacterial, antioxidant, and even potentially

as an antifilaria. (Al-Abd

et al., 2016). M.cajuputi

has been used traditionally for the treatment of

diseases such as coughing, stomach cramps, burns,

and influenza. This plant also exhibits anti-

inflammatory, antidengue, anticancer and

anticonvulsant activities. The active compound of

M.cajuputi leaves were trans caryophyllena, β-

selinene, germacrena (C15H26O), neopitadiene,

cyclohexakarboksal dehide, β-caryophyllena, 3-

methoxy benzoic acid trimethylsilan; limonene; 1,4

naphthoquinone-5,8-dihydroxy-2-methoxy; 3

carena; α-caryophylena; cineol; patchulin; ethyl

benzene; benzene 1-metil-3 (metiletyl), and 1.3%

volatile oil [10]. Essential oils are one of the

compounds found in plants that have antibacterial

activity (Ajizah, 2014).

M. cajuputi leaves also contain high flavonoids,

flavonoids are chemical compounds that are

antibacterial (Al-Abd et al., 2015). The aims of the

research were 1). Get endophytic fungi isolates from

Melaleuca cajuputi Powell that are able to produce

secondary metabolites that are antibacterial and

antioxidant, 2). Conducting in vitro tests to verify

the antibacterial and antioxidant properties of

secondary metabolites of the plant's endophytic

fungi of Melaleuca cajuputi Powell, 3). Identify the

isolates of endophytic fungi of Melaleuca cajuputi

Powell which have high potential to produce

antibacterial.

2 MATERIALS AND METHOD

2.1 Isolation and Purification of

Endophytic Fungi

The sterile leaves are cut 2x1 cm aseptically, then

placed in a commercial Potato Dextrose Agar

powder (PDA)(20 g dextrose, 15 agar, and 4 g

potato starch) medium. Incubated at room

temperature until the fungi appeared to grow. The

different fungal colonies on PDA medium are

further purified on new PDA medium.

2.2 Cultivation of Endophytic Fungi

and Secondary Metabolite

Extraction

Twenty agar plug of endophytic fungi isolates

inoculated into 500 mL Potato Dextrose Broth

(PDB) medium and incubated at room temperature

for ± 30 days. After a change in the color of the

medium which indicates the production of secondary

metabolites, the medium was extracted using an

ethyl acetate solvent (1: 1) and concentrated with a

rotary evaporator to obtain a crude extract of

secondary metabolites (Jamal et al., 2008).

2.3 Antibacterial Test

A 0.5 McFarland standard is prepared by mixing

0.05 mL of 1.175% barium chloride dihydrate

(BaCl

.2H

O), with 9.95 mL of 1% sulfuric acid

). The 0.5 Mc Farland solution measured the

absorbance using a UV-Vis spetrophotometer (625

nm). The absorbance value obtained is equivalent to

1.5x10

bacterial cells/mL (Fatisa, 2013).

Escherichia coli, Staphylococcus aureus,

Salmonella typhimurium, and Shigella dysentriae

were inoculated into a physiological saline solution

(0.85% NaCl) then homogenized and measured their

Screening Endophytic Fungi of Melaleuca cajuputi Powell Leaves as Antibacterial Sources

197

absorbance using a UV-Vis spectrophotometer (625

nm). The absorbance of the suspension test bacteria

was prepared similar to the McFarland standard

solution to reach the density of 1.5×10

CFU/mL

(Kumala, 2014).

The antibacterial activity test was performed

using disc diffusion method (Kirby-Bauer). The

bacterial suspension was inoculated as much as 0.1

ml into a Mueller-Hinton Agar (MHA) medium with

a spread plate methods. Crude extracts of secondary

metabolites made concentrations of 400 μg/disc

(4%) and standard antibiotic tetracycline 30 μg/disc

(0.3%) as positive control. The disc paper saturated

in a solution of secondary metabolite extract and

tetracycline antibiotics then inoculated on the

surface agar containing bacterial suspension and

incubated atroom temperature for 24 hours (Islam et

al., 2013). The criteria for each concentration of

antibacterial compounds tested against standard

antibiotics were determined by weak : A/B x 100%

<50%; medium : 70% <A/B x 100%>50%; strong :

A/Bx100% >70%, with A was clear zone diameter

of extract and B was clear zone diameter standard

antibiotic (Chan, 2007).

2.4 Determination of Minimum

Inhibitory Concentration (MIC)

Secondary metabolite extracts that had strong

antibacterial activity determined the minimum

inhibitory concentrations. Determination of KHM

using disc diffusion methods. The concentrations to

be used were 4%, 3%, 2%, 1% and 0 (as a control).

Paper discs that have been saturated with extracts of

each of the existing concentrations and tetracycline

as a standard antibiotics are placed on the surface of

MHA medium that has been inoculated with the

suspension of the test bacteria. Incubated at room

temperature for 24 hours. Inhibition zones are

measured and a minimum concentration value that

can inhibit bacteria was determined.

2.5 Thin Layer Chromatography

Analysis

Endophytic fungal extracts which have high

antibacterial activity then analyzed by TLC. Thin

layer chromatography analysis using silica gel 60

plates with ethyl acetate and n-hexane solvents with

the eluent ratio used (ethyl acetate: n-hexane) were

1: 4; 3: 2; 2: 3 and 4: 1. The formed spots were seen

by using UV light with a wavelength of 366 nm, and

the Rf value of the compound formed was

determined (Jamal et al., 2008).

2.6 Characterization and Identification

of Endophytic Fungi

Fungal endophytes were growth on Potato Dextrose

Agar (PDA), Czapek Dox Agar (CDA), Czapek

Yeast Extract Agar (CYEA), and Malt Extract Agar

(MEA) media at room temperature (28

C) for 7

days. Fungal colonies grown in each medium

observed about colony color, colony diameter,

medium color around the colony, and colony reverse

color (Srikandace, 2007). Microscopic morphology

observed were hypha (septate or not), hyaline

(colorless) or dark pigmented hyphae (greenish or

blackish brown, dark black, grayish black), spores

(asexual and sexual) with Henrici's Slide Cuture

(HSC) methods (Gandjar et al., 1999).

3 RESULTS AND DISCUSSION

3.1 Antibacterial Activity of Secondary

Metabolite of Endophytic Fungi

Isolation and purification of endophytic fungi of

Melaleuca cajuputi Powell leaves obtained 7

isolates, 5 isolates from the young leaves and 2

isolates from the old leaves. The antibacterial

activity test of Melalleuca cajuputi endophytic fungi

extract on Escherichia coli showed that there were

two endophytic fungi isolates had the highest

antibacterial activity, i.e MC3 and MC5 fungi

isolates with 11 mm and 10 mm clear zone diameter.

Five isolates did not have antibacterial activity on E

coli namely MC1, MC2, MC4, MC6, and MC7

isolates. The antibacterial activity of a secondary

metabolite extract varies with bacteria depending on

the components of the bioactive compound in the

extract.

Extracts of secondary metabolites of endophytic

fungi that have the highest antibacterial activity on

Staphylococcus aureus were extract of MC1 and

MC7 fungal isolates, with 10 mm and 11 mm clear

zone diameter.

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Table 1: Antibacterial activity of endophytic fungi of M cajuputi leaves against E coli, S aureus, S typhi, S. dysenteriae

using the disc-diffusion methods.

Isolates

Zone of inhibition in mm (inhibition %)

E coli S aureus S typhi S dysenteriae

MC1 0 10(90.9)

+++

0 8,13(42.5)

MC2 0 0 7(63.4)

12.04(62.9)

MC3 11(100)

+++

9(81.8)

+++

6,12(55.4)

9.14(47.8)

MC4 0 0 0 9,02(47.2)

MC5 10(90.9)

+++

9(81.8)

+++

0 0

MC6 0 0 0 0

MC7 0 11(100)

+++

7,2(65.2)

8,08(42.3)

Tetrasicline 11 11 11.04 19.12

Figures in parentheses are inhibition percentages compared to tetracyclin. Antibacterial activity is categorized as strong +++ for

inhibition ≥70%, moderate ++ for inhibition 50<70%, and weak + for inhibition 50% (Chan et al., 2007).

The extract of MC3 and MC5 endophytic fungal

isolates have antibacterial activity against E. coli

with 11 mm and 10 mm clear zone diameter, also

have antibacterial activity against S. aureus with

both 9 mm clear zone diameter (Table 1). This

means that the extract of secondary metabolites of

MC3 and MC5 isolates have a broad spectrum of

antibacterial activity, because it has antibacterial

activity against Gram-negative and Gram-positive

bacteria. This shows the differences in the content of

bioactive compounds found in endophytic fungi

isolates that act as antibacterial. Research conducted

by Ayepola and Adeniyi (2008), showed that

secondary metabolites from leaf extracts of

Eucalyptus camaldulensis including the plant family

Myrtaceae have a broad spectrum of antibacterial

activity in inhibiting bacterial growth, both Gram

positive and Gram negative bacteria.

3.2 Minimum Inhibitory Concentration

(MIC) of Secondary Metabolite

Extract of Endophytic Fungi

The MIC of secondary metabolite extract of

endophytic fungi of Melaleuca cajuputi leaves on

Eschrichia coli. MIC of MC

fungal isolates was

400 µg/mL with inhibition zone diameter 6,16 mm.

The MIC of secondary metabolite extract of MC

endophytic fungal isolates was 100 µg / mL with an

inhibition zone diameter 6.02 mm. Based on the

MIC value, it showed that MC

and MC

endophytic

fungi isolates had moderate antibacterial activity,

when compared with Batos et al. (2016) where the

Eugenia calycina plant extract has a MIC value of E.

coli was 500 µg / mL, where the range of values of

500 µg / mL - 1000 µg / mL means that it is slightly

active, a value of 100 µg / mL - 500 µg / mL being,

and KHM value less than 100 µg / mL means it is

very active.

The MIC of the secondary metabolite extract of

MC1 isolates on Staphylococcus aureus were 200

µg/mL with 6.1 mm inhibitory zone diameter of 6,1

mm. The MIC of MC

endophytic fungi isolate was

700 µg/mL with a inhibition zone diameter of 6.02

mm. The concentration 700 µg/mL means that it is

large enough to obtain a MIC value, compared with

the results of a study conducted by Banhos et al.

(2014) where extracts of the metabolites of the

endophytic fungi of the Myrcia guianensis plant

produced a MIC of S. aureus was 25 μg/mL. It is

suspected that the bioactive compounds in MC

fungal isolates are not strong enough to inhibit the

growth of S. aureus at low concentrations, besides it

is suspected that S. aureus bacteria have experienced

resistance to bioactive compounds contained in MC7

fungal isolates. According to Vuong et al. (2015), S.

aureus is one of the bacteria with a wide spread of

antibiotic resistance. This bacterium has been

resistant to penicillin, methicillin, even vancomycin,

one of the antibiotics that are still commonly used.

3.3 Thin Layer Chromatography

(TLC) Analysis

The results show that the color spot formed on the

TLC plates for the three extracts of Melaleuca

cajuputi endophytic fungal isolates which have

relatively strong antibacterial activity, the isolates

MC1, MC5, and MC7 were yellow. Based on the

results of research conducted by Fadel et al. (2018),

phenolic compounds when tested using thin layer

chromatography plates will be yellow, blue or pink.

Screening Endophytic Fungi of Melaleuca cajuputi Powell Leaves as Antibacterial Sources

199

The color spot formed for MC3 isolate extract

are yellow which shows phenol and brownish yellow

compounds, showing flavonoid compounds,

according to research conducted by Nugrahaningtyas

et al., (2005) that flavonoid compounds have a

brownish-yellow color to a red color. The color spot

formed on the plates of each different endophytic

fungi isolates showed the components of chemical

compounds contained in each different endophytic

fungi isolates.

This is consistent with the statement of Sharma

et al. (2016), different endophytic fungi in a plant

can produce different secondary metabolites. Based

on the result of Rf value it is known that there are

endophytic fungi isolates that have the same Rf

value also have the same color spots. This shows

that the possibility of chemical compounds

contained in the extract of secondary metabolites of

endophytic fungi isolates may be the same.

According to Ahamed et al. (2017), the Rf was a

general characteristic value that can change

depending on the polarity of the mobile phase and its

fixed phase. The Rf value indicates important

information regarding the polarity of the chemical

compounds to be identified. In addition, the Rf value

can also be used in determining good solvents as a

mobile phase in thin layer chromatography tests.

3.4 Characterization and Identification

of Endophytic Fungi of Melaleuca

cajuputi

The macroscopic and microscopic characteristics

endophytic fungi isolates of Melaleuca cajuputi leaf

are presented in Figure 1, 2 and Table 2.

Microscopic observations of MC1 endophytic fungi

isolates based on Figure 2 shows that hyphal fungi

isolates of MC1 endophytes are septate and hyaline-

colored, have short conidiophores and there are

conidia-forming cells in the cylindrical conidiophore

terminal, the conidia are rounded with smooth

surfaces.

Based on the microscopic characteristics

obtained it is suspected that MC1 endophytic fungi

isolates belong to the type of Scopulariopsis

candida. According to Gandjar et al. (1999), S.

candida has a colony diameter of 3-4 cm within 7

days. Reverse color beige to light brown. Its

conidiophores are short, have conidial-forming cells

that are cylindrical in shape, conidia in the form of

rounded to wide ovals, smooth-walled, and white to

beige.

The macroscopic and microscopic characteristics

endophytic fungi isolates of Melaleuca cajuputi leaf

are presented in Figure 3, 4 and Table 2. Based on

microscopic characters of MC3 endophytic fungi

isolates obtained that MC3 endophytic fungi isolates

was identified as Fusarium sporotrichiodes, with

characteristic hyphae and insular hyphae, there were

two-branched conidia, oval-shaped, three-sided

macroconidia, rounded microcondidia, and

hyaluronic hyphae. According to Samson et al.

(1995), F. sporotrichiodes has a white colony to

yellow and finally brown. Microconidia is

commonly found in aerial mycelia in the form of

ovoid, piriform or fusoid, 3-5 macroconidias.

Based on macroscopic and microscopic

characters of MC5 endophytic fungi isolates on

Figure 5, 6, and Table 6. Type of hyphae: septate;

hyphae color: hyaline; conidia color: hyaline;

clamydospore color: brownish. Based on

macroscopic and microscopic characters, MC5

endophytic fungi isolate was identified as Fusarium

equiseti. According to Samson et al., (1995),

Fusarium equiseti on PDA medium has a whitish

color and becomes creamy to brown in color, has a

lot of chlamydospores pale brown when old,

smooth-walled or rough, in chains or clumps, in

hyphae or conidia. The macroscopic and

microscopic characteristics endophytic fungi MC7

isolates of Melaleuca cajuputi leaves were presented

in Figure 7, 8 and Table 7.

Based on macroscopic and microscopic

characters of MC7 endophytic fungi isolates based

on Figure 7, 8 and Table 7 obtained results, MC7

endophytic fungi isolates had hyaline-colored

hyphae and insulated hyphae, there were rounded

and hyaline-colored columns, and had zigospores

with rough surfaces. Based on the characteristics

obtained, MC7 endophytic fungi isolate is suspected

to be a type of Mucor hiemalis. The microscopic

and macroscopic characteristics are in accordance

with Gandjar (1999), Mucor hiemalis has a rather

creamy yellow colony, a yellowish white reverse

color. The columns are round and hyaline, the

zigospores are semipurate or round in shape and the

surface is rough.

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Figure 1: Colony images of MC1 endophytic fungal isolate of Melaleuca cajuputi on different medium. (a) PDA, (b) MEA,

Table 2: Colony characteristics of MC1 endophytic fungal isolate of Melaleuca cajuputi on different medium.

Characteristics PDA medium MEA medium CDA medium CYA medium

Colony colo

White/ yellowish Cream Turbid white Turbid white

Colony reverse

colo

Turbid white/

yellowish

Cream Turbid white Turbid white

Medium color

around colony

Turbid white Turbid white Turbid white Turbid white

Colon

surface Smooth Smooth Smooth Smooth

Colony

diamete

4.75–5.7cm in 7 days 3.35–3.9cm in 7 days 3.05–3.25cm in 7 days 1.85–2.7cm in 7

Figure 2: Microscopical characteristics of MC1 isolate.

Figure 3: Colony images of MC3 endophytic fungal isolate of Melaleuca cajuputi on different medium. (a) PDA, (b) MEA,

Table 3: Colony characteristics of MC3 endophytic fungal isolate of Melaleuca cajuputi on different medium.

Characteristics PDA medium MEA medium CDA medium CYA medium

Colony color White White White White

Colon

reverse colo

White Brownish white White White

Medium color around

colony

Turbid white Yellowish Turbid white Turbid white

Colon

surface Granular and notche

Powder

Floccose Floccose

Colony diameter 3.4-4.05 cm in 3 days 1.8-2.2cm in 7 days 3.4-4.05 cm in 3 days 2.35-5cm in 3 days

Screening Endophytic Fungi of Melaleuca cajuputi Powell Leaves as Antibacterial Sources

201

Figure 4: Microscopical characteristics of MC3 isolate.

Figure 5: Colony images of MC5 endophytic fungal isolate of Melaleuca cajuputi on different medium. (a) PDA, (b) MEA,

Table 4: Colony characteristics of MC5 endophytic fungal isolate of Melaleuca cajuputi on different medium.

Characteristics PDA medium MEA medium CDA medium CYA medium

Colony color Greys white White Yellowish turbid white Cream

Colony reverse color Brownish white Brownish white Yellowish turbid white Cream

Medium color around colony Yellowish Yellowish Turbid white Turbid white

Colony surface Powdery Powdery Smooth Powdery

Colony diameter 1.8-2.5 cm in 7 days 2.95-3.75 cm in 7 days 3.15-3.95 cm in 7 days 1.75-2.35 cm in 7 days

Figure 6: Microscopical characteristics of MC5 isolate.

Figure 7: Colony images of MC7 endophytic fungal isolate of Melaleuca cajuputi on different medium. (a) PDA, (b) MEA,

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Table 5: Colony characteristics of MC7 endophytic fungal isolate of Melaleuca cajuputi on different medium.

Characteristics PDA medium MEA medium CDA medium CYA medium

Colony color Turbid white/yellowish Turbid white/yellowish Turbid white Turbid white

Colony reverse color White greyish brown Turbid white/yellowish Turbid white Turbid white

Medium color around colony Yellowish Turbid white Turbid white Turbid white

Colony surface Cottony Cottony Cottony Cottony

Colony diameter 4.65-5.25 cm in 3 days 4.85-5.55 cm in 3 days 5-5.75 cm in 3 days 5.5-6.05 cm in 3 days

Figure 8: Microscopical characteristics of MC7 isolate.

4 CONCLUSION

Endophytic fungi obtained from Melaleuca cajuputi

Powell obtained 7 isolates, 5 isolates obtained from

young leaves, and 2 isolates from old leaves. Ethyl

acetate extract of secondary metabolites endophytic

fungi of Melaleuca cajuputi Powell which classified

as strong and has a high percentage of antibacterial

activity, namely isolates of endophytic fungi MC1,

MC3, MC5, and MC7 against Escherichia coli and

Staphylococcus aureus. Minimum inhibitory

concentration (MIC) of secondary metabolite

extracts of MC5 and MC3 endophytic fungi isolates

against Escherichia coli were 400 µg / mL and 100

µg / mL respectively, while secondary metabolite

extracts of MC1 and MC7 endophytic fungi isolates

against Staphylococcus aureus bacteria were 200

mg / mL and 700 mg / mL. MC1 fungi isolates were

identified as Scopulariopsis candida, MC3 fungi

isolates were identified as Fusarium

sporotrichiodes, MC5 fungi isolates were identified

as Fusarium equiseti, , and MC7 fungi isolates were

identified as Mucor hiemalis.

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