ISSN No. 2321-5488
February 2019
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35
ISOLATION OF ENDOPHYTIC FUNGI FROM AGRICULTURE FIELD OF
NAGPUR REGION AND ASSESSMENT OF ISOLATED FUNGI FOR
ANTIQUORUM SENSING ACTIVITY
Ashok Y. Dawande*1, Vijay N. Charde2, Yogesh S. Banginwar3
1,2
Department of Microbiology, Taywade College, Koradi, Nagpur
3
Department of Microbiology, Arts and Science College, Pulgaon, Wardha
Abstract
The aim of present study was to isolate the endophytic fungi from agriculture field of Nagpur
region and evaluate the isolated fungi for the inhibition of quorum sensing activity. Initially,
samples were collected for isolation of fungal endophytes. Fungal endophytes was isolated
from the healthy, symptomless leaves, stem and root of various plants such as mustard,
Linseed, Bengal gram, safflower, Pigeon pea and Orange. The identification of isolated
endophytic fungi was done on the basis of their morphological and microscopic structures.
The Slide culture technique was performed using isolated fungi and temporary slides were
prepared in Lactophenol cotton blue stain for identification purpose. For quorum sensing
inhibitors production, first Liquid- state fermentation and then extraction of inhibitors were
carried out. The extract from each endophytic fungal isolate was assayed for the presence of
inhibitors using biosensor strains such as Chromobacterium violaceum wild type MTCC
2656.
Keywords: Endophytic fungi, quorum sensing inhibitors, Chromobacterium violaceum
Introduction:
Endophytic fungi reside within host plants exclusive of causing any noticeable
symptoms of infection (Pimental et al., 2006).The endophytic fungal hyphae present internal
cellular aerial plant parts such as leaf sheaths, sometimes even within the stem and root
system also. Association with plants, the endophytic fungi can reduce the damage from the
pathogens by inhibiting pathogen infection and propagation within the host. In vitro study
showed that endophytic fungi capable of producing various enzymes necessary for colonizing
their plant hosts. Enzymes such as amylase, pectinase, xylanase, proteinase, and cellulose
have been produced by fungal endophytes. (Rajesh and Rai, 2013).
Endophytic fungi have been considered as biofactories of novel bioactive molecules,
among the extracted molecules and purified substances derived from the broth or fungal
biomass, some have possessed antibacterial activity with moderate to powerful effect when
assayed on the bacterial strains resistant to the antibiotics which are currently in use (Radic,
N., Strukelj, B., 2012).
As endophytes are serving as the richest source of enzymes and secondary
metabolites, such metabolites can be used as quorum sensing inhibitors (antimicrobials)
directing possible new method for industrially significant substance production and
utilization (Rajesh and Rai, 2013).
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In the search for these inhibitors, studies have documented that many plants, fungi
and even bacteria themselves produce anti-QS substances. Fungal survey on more than 20
years have documented that endophytic asymptomatic infection of land plants by fungi is
everwhere. One of the important products produced naturally from endophytic microbes are
antibiotics.
Pestalotiopsis microspora (a rainforest fungal endophyte) is known to produce many
secondary metabolites. Ambuic acid, a secondary metabolite serve as an antifungal agent.
Also Colletotric acid, from Collectotricum gloeosporioides, known to show antimicrobial
activity.
Agriculture field grown plants are supposed to known for presence of various type of
fungal endophytes. Such endophytes are capable of producing antiquorum sensing inhibitors.
At low cell population, bacteria behave as single cellular organisms, as soon as their
population density jumped a threshold level, bacteria may change their behaviour to
‗multicellular‘ type by sensing it. This show kind of cell to cell communication through small
signaling molecules responsible expression of genes for certain phenotypes like their virulent
behaviour. This is termed as bacterial Quorum Sensing (QS) and the signals molecules are as:
Oligopeptides (in Gram-positive bacteria), N-acyl homoserine lactones (AHLs produced by
Gram-negative bacteria) , autoinducer Furanosyl borate etc. There are different mechanism of
disruption of QS, but among the different possibilities, enzymatic degradation of QS signal
molecules (AHLs) has been studied the most (Kalia, V.C, 2013) (Waters, C. M., Bassler, B.
L., 2005).
The present study mainly focus on isolation and identification of the endophytic fungi
and detection of quorum sensing activity inhibitor.
Methods:
Sample Collection
A total of 6 healthy field-grown plants were randomly sampled, the plants collected
were mustard, Linseed, safflower (from agriculture field of College of Agriculture, Nagpur),
Bengal gram, Pigeon pea and Orange (from nearby agriculture field of Taywade College,
Koradi, Nagpur). All samples were utilized immediately after collection.
Isolation of endophytic fungi
For isolation of endophytic fungi, healthy, symptomless leaves, stem and root were
selected from plant species such as mustard, Linseed, Bengal gram, safflower, Pigeon pea
and Orange at random, washed several times in running tap water. The samples were
fragmented into small pieces and surface-sterilization done by sequentially dipping into 0.1%
HgCl2 for 1 min, again cleaned with sterilized distilled water and then placed into distilled
water. Then, pieces of each plant were placed on a Potato Dextrose Agar Petri dish
supplemented with 0.5 g/l streptomycin sulphate. Plates were incubated at 30 oC and checked
for fungal growth and sporulation. Fungi coming out of the plant pieces were then transferred
to other PDA plates, and incubated at 30 oC for 7 days. After fungal purity check it is
transferred to another PDA medium plate. Endophytic fungal identification was based on
morphological characters on PDA plates and microscopic characters of colonies like asexual
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or sexual reproductive structures using optical microscopy (Pimentel et al., 2006 and Rajesh
& Rai 2013).
Quorum sensing inhibitors from endophytic fungi
Bacterial strains
The bacterial strain used for QS inhibition assays was Chromobacterium violaceum
MTCC JCM 1249 (wild type strain). Bacterial strain was kept in 40% glycerol stocks at -20
o
C and was cultured in Luria–Bertani (LB) broth prior to use.
Fermentation
50 ml of sucrose broth was prepared in a 250 ml Erlenmeyer conical flask. All the
isolated endophytic fungi was inoculated and then incubated at 30 0C for 24 hours. After
incubation the same culture (2% v/v) transferred into 50 ml sucrose broth and incubated at 30
0
C in a orbital shaker incubator at 140 rpm for 5 days incubation.
Extraction of quorum sensing inhibitors
After fermentation, the cells were filtered using 1 MM Whatman filter paper and the
filtrate was extracted with an equal amount of ethyl acetate. Then, the contration of organic
phase was done by evaporation and the residue was dissolved in sterile DMSO and kept in a
deep freezer for further use (Ueda et al., 2007).
Bioassay
The extract from each isolates was assayed for the detection of QS inhibitors. First the
LB agar plates were flooded with 1ml of C. violaceum wild type culture to prepare bacterial
lawn. One 10 mm diameter well were created in each agar LB plate using a sterile Borer.
Then the well of 10 mm was loaded with fungal extract (40µL) and Distilled water and two
antibiotic discs namely Piperacillin/Tazobactum (PIT 10mcg) and Aztreonam (AT 30mcg)
were used as negative and positive controls, respectively. Then, it was incubated at 28 oC for
24 h. The presence of a clear zone i.e. violet color pigment inhibition around the wells
indicates the presence of antiquorum sensing activity.
Results and Discussion
From Fig. 1, a total of 19 endophytic fungi were isolated from the 53 healthy,
symptomless leaves, Stem and root fragments analyzed giving a percentage colonization rate
of 48.7% from various plants such as Mustard, Linseed, Bengal gram, Safflower, Pigeon pea
and Orange (Table 1).
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Table 1 - number of isolates of endophytic fungi obtained from various plants leaf, stem
and root fragments
Total
number of
isolates
Growth
Number of Colonization
by organ
environment
rate *(%)
Plant
Organ fragments
Mustard
Root
5
60.00
3
Linseed
Root
7
42.85
3
Bengal
gram
Root
14
7.14
1
Root
10
0
0
Field-grown plants Safflower
Pigeon pea Leaf
5
80
4
Stem
4
75
3
Orange
Leaf
4
75
3
Stem
4
50
2
Total plants
6
Total
fragments
53
taken
Percentage
colonization rate
48.7
Total isolates
19
*Percentage colonization- the total number of pieces colonized by fungi in relation to the
total number of pieces x 100.
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Among 19 isolates, only 10 isolates were found to be different from each other and were
considered for study (Fig.3). Morphological characteristics of isolated endophytic fungi were
shown in Fig.3
Fig. 3- Isolated endophytic fungi cultivated on PDA. Fungal code: F1 to F10 (SPP).
Remaining 9 fungi showed morphologically and microscopically similar characteristics with
rest of fungi. Endophytic fungi were identified as Pestalotiopsis sp., Colletotrichum sp,
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Aspergillus sp1, Penicillium sp, Fusarium sp.1 Phoma sp. Aspergillus sp2 Fusarium sp.2
Trichoderma sp. and Phomopsis sp.
Microscopic characters of isolated endophytic fungi were shown in Fig. 2
Quorum sensing inhibitors from endophytic fungi
The plant defense machinery uses bioactive compounds produced from the plant or
endophytic microorganism. So the production of QS inhibitor from such isolates was detected
by bioassay with Chromobacterium violaceum wild type MTCC 2656. Fig. 4 represents the
capability of endophytic fungal extract for QS inhibition by inhibiting the violacein pigment
production by C. violaceum without inhibiting the bacterial growth. The isolates of Fusarium
sp.2 (F9SF) and Phomopsis sp.(F6MP) possess quorum sensing inhibitor by inhibiting the
synthesis of violacein pigment.
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Table 3- Antiquorum sensing activity of fungal
endophytes
Inhibition
of
Isolated endophytic quorum sensing
activity
Code
fungi
F1
Aspergillus sp1
F2
Penicillium sp
F3(J1)
Aspergillus sp2
F4(PP)
Pestalotiopsis sp.
F5(SPT) Trichoderma sp.
F6(MP)
Phomopsis sp.
+
F7(GF)
Fusarium sp.1
F8(GC)
Colletotrichum sp
F9(SF)
Fusarium sp.2
+
F10(SPP) Phoma sp
+ Good activity,- No activity
Fig.4. Result of anti-QS screening for fungal extract F1 to F10 (SPP) and the controls.
Among all, the fungal extracts of Fusarium sp.2 (F9SF) and Phomopsis sp.(F6MP) caused
observable inhibition of violacein pigment produced by C. Violaceum wild type as opposed to
complete clearing of bacterial growth around the well for the antibiotic control (+ve C)
(Aztreonam, AT 30mcg and Piperacillin/Tazobactum, PIT 10mcg). (C) Showing no clearing
is for the negative control using sterile distilled water.
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With combination to antibiotics, antiquorum sensing molecules could be one of the effective
therapeutic tool in future, serving as the alternative to multidrug resistant pathogens.
References
Bulman, Z., Le, P., Hudson, A. O., Savka M. A., 2011. A novel property of propolis (bee
glue): Anti-pathogenic activity by inhibition of N-acyl-homoserine lactone mediated
signaling in bacteria. J. Ethanopharm. 138, 788-797.
Kalia, V.C, 2013. Quorum sensing inhibitors: An overview. Biotech. Adv. 31, 224-245
McClean, K.H., Winson, M.K., Fish, L., Taylor, A., Chhabra, S.R., Camara, M., Daykin,
M., Lamb, J.H., Swift, S., Bycroft, B.W., Stewart, G.S.A.B., Williams, P., 1997. Quorum
sensing and C. violaceum: exploitation of violacein production and inhibition for the
detection of N-acylhomoserine lactones. Microbiology 143, 3703–3711.
Pimentel, I.C., Glienke-Blanco, C., Gabardo, J., Stuart, R.M., Azevedo, J.L., 2006.
Identification and colonization of endophytic fungi from soybean (Glycine max (L.) Merril)
under different environmental conditions. Braz. Arch. Biol. Technol. 49, 705–711.
Radic, N., Strukelj, B., 2012. Endophytic fungi—the treasure chest of antibacterial
substances. Phytomed. 20, 337-342.
Rajesh, P. S., Ravishankar Rai, V. 2013. Hydrolytic Hydrolytic enzymes and quorum sensing
inhibitors from endophytic fungi of Ventilago madraspatana Gaertn. Biocatal. Agri.
Biotech. 2, 120-124.
Taganna, J.C., Quanico, J. P., Perono, R.M.G., Amor, E. C., Rivera, W. L., 2011. Tannin-rich
fraction from Terminalia catappa inhibits quorum sensing (QS) in Chromobacterium
violaceum and the QS-controlled biofilm maturation and LasA staphylolytic activity
in Pseudomonas aeruginosa. J. Ethanopharm. 134, 865-871.
Ueda, M., Kubo, T., Miyatake, K., Nakamura, K., 2007. Purification and characterization
of fibrinolytic alkaline protease from Fusarium sp. BLB. Appl. Microbiol. Biotechnol. 74,
331–338.
Uroz, S., Heinonsalo, J., 2008. Degradation of N-acyl homoserine lactone quorum sensing
signal molecules by forest root-associated fungi. FEMS Microbiol. Ecol. 65, 271–278.
Waters, C. M., Bassler, B. L., 2005. Quorum Sensing: Cell-to-Cell Communication in
Bacteria. 21, 319-346.
Winters, A.L., Minchin, F.R., 2005. Modification of the Lowry assay to measure proteins and
phenols in covalently bound complexes. Anal. Biochem. 346, 43–48.
232
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