See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/300636310

Morphological changes of Ganoderma boninense mycelia after challenged by

Trichoderma and Bacillus

Conference Paper · July 2015

DOI: 10.1063/1.4919213

CITATIONS READS

4 1,280

3 authors:

Arnnyitte Alexander Jedol Dayou

Agrifert Marketing Sdn Bhd Universiti Malaysia Sabah (UMS)
30 PUBLICATIONS   129 CITATIONS    162 PUBLICATIONS   1,016 CITATIONS   

SEE PROFILE SEE PROFILE

Khim Phin Chong

Universiti Malaysia Sabah (UMS)
148 PUBLICATIONS   822 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Dye-sensitized solar cell View project

2nd International Congress on Science & Technology 2018 (ICST 2), 6 - 8 December 2018 Phuket, Thailand View project

All content following this page was uploaded by Khim Phin Chong on 22 June 2016.

The user has requested enhancement of the downloaded file.

 Morphological changes of Ganoderma boninense mycelia after challenged
by Trichoderma and Bacillus

Arnnyitte Alexander1, Jedol Dayou2 and Khim-Phin Chong1*

1Sustainable
Palm Oil Research Unit (SPOR), Faculty of Science and Natural
Resources, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah,
Malaysia

2Vibration and Sound Research Group (eVIBS), Faculty of Science and Natural
Resources, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah,
Malaysia.

*Corresponding author: chongkp@ums.edu.my

Abstract

Ganoderma boninense is a fungal pathogen that causes Basal Stem Rot (BSR)
disease in oil palm. This deadly disease has caused major losses in the oil palm
industry and no remedy is reported to date. The more promising control on G.
boninense is the use of biological control agents (BCAs). Despite many attempts
in using BCAs as a control agent but evidence on the colonization of BCAs and
morphological changes of the pathogen is not well documented. We have
investigated the effect of antagonist activity on the combination of Trichoderma
spp. and Bacillus spp. on the morphology of G. boninense. The antagonist activity
was evaluated using agar well diffusion assay. BCAs suppressed the mycelia
growth of G. boninense up to 70%. Observation under Scanning Electron
Microscopy (SEM) shows these BCAs induced stripping of G. boninense hyphal
structure by destroying the cellular structure. Highly disrupted, disaggerated,
shrivelled and lysis of G. boninense hyphal were also observed. The antifungal
activity of Trichoderma spp. and Bacillus spp. observed could be associated with
the production of Cell Wall Degrading Enzymes (CWDE).

Keywords: Ganoderma boninense, Trichoderma, Bacillus, disrupted, lysis

Introduction

Basal Stem Rot (BSR) is a disease in oil palm caused by basidiomycete fungus
Ganoderma boninense. This disease is known as one of the major disease that
continues to inflict considerable yield losses and often death of oil palms. In
Malaysia, the loss caused by this disease is estimated up to RM 1.5 Billion a year
(Ommelna et al., 2012). With no known remedy at present, BSR disease
continues to erode the profitability of the oil palm industry. However, some
promising control of G. boninense using combination of biological control agents
(BCAs) have been reported by several research groups (Guetsky et al., 2011;
Yobo et al., 2011; Alexander and Chong, 2014). Mixture of antagonists is effective
as it broaden the spectrum of control activity against pathogens. Different action
mechanisms have been suggested as being responsible for their biocontrol
activity against various pathogens. However, the evidence on the colonization of
BCAs when comes to interaction with the pathogen and morphological changes
of the pathogen is not well documented. In this paper, we report a thorough
microscopy investigation on the effect of antagonistic mechanisms of the
combination of Trichoderma spp. and Bacillus spp. on G. boninense.

Materials and Methods

Culture of Ganoderma boninense

Pure culture of G. boninense was obtained from the stock culture of Genetic
laboratory in Sustianable Palm Oil Research Unit (SPOR) of Universiti Malaysia
Sabah. The identity of G. boninense had been identified and confirmed before
using molecular technique (Chong et al., 2011). The pure culture was
subcultured and maintained at 25ºC on Potato Dextrose Agar (PDA) until further
use.

Agar well diffusion assay

The bioassays between Ganoderma and BCAs were carried out using agar well
diffusion technique. Using a 6 mm cork borer, four wells were dug on a PDA
plate. A mycelia plug was taken from the edge of a seven day old culture G.
boninense and placed on the centre of the PDA petri, 2 cm away from each well.
Approximately 0.2 mL of the BCAs mixture (a commercial product consists of
Bacillus spp. and Trichoderma spp.) was then inoculated in each well. The plates
were replicated five times and left at ±25oC for 2 h to allow diffusion from the
wells to occur. They were then incubated face upwards at this temperature for
12 d. The diameters of zones of growth were measured daily. Evaluation of the
microbial interaction against G. boninense was assessed based on the pathogen’s
radius growth.

Measurement of the Percentage Inhibition Radius Growth (PIRG)

In vitro evaluation of the percentage inhibition of radial growth of G. boninense

was assessed using the formula as described by Siddiquee et al., (2009).
R1 – R2
PIRG = x 100
R1
Where, PIRG = Percent inhibition of radial growth
R1 = Radial growth of G. boninense in the absence of BCAs
R2 = Radial growth of G. boninense in the presence of BCAs

Sample preparation for Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) was used to examine the morphological

changes of G. boninense hyphae with or without BCAs treatment. The zones of
hyphal interaction between Ganoderma and BCAs used in the agar well diffusion
assays were prepared for examination with SEM following the procedure as
described by Chaiyasut et al. (2010). Agar plates with fungal mycelia were first
excised using scalpel and trimmed to approximately 10 mm x 10 mm in size, and
as thin as possible, to reduce the moisture. The samples were dried at 60°C for 3
h. The samples were mounted on carbon formvar coated with gold-palladium in
an Emitech K550x carbon coater for 1 min and viewed under a Zeiss EVO® 15LS
SEM.

Results and Discussion

Inhibition of Ganoderma by Trichoderma spp. and Bacillus spp. in well

diffusion assay

The antagonist activity of BCAs (Trichoderma spp. and Bacillus spp.) against G.
boninense was shown in Figure 1. There was a significant difference in the PIRG
of G. boninense treated with BCAs, compared with the growth of G. boninense in
the control plates without BCAs. The inhibitory effect on G. boninense was
observed started from day 6 and the PIRG values was up to 70% after 12 d of
incubation. This result has shown a positive relationship between the BCAs in
producing a synergistic antagonistic effect against G. boninense. This is in
agreement with an observation by Guetsky et al. (2002) which suggests
combination of BCAs with different mechanisms of disease control will have an
additive or synergistic effect and resulted in enhanced disease control compared
to their individual application.

Day(s)
Treatment 1 6 12

Control
(GB)

GB+BCAs

Figure 1: Antagonistic assay of G. boninense on PDA medium with or

without the presence of BCAs at 1, 6 and 12 days of incubation. GB denotes
G. boninense; BCAs: Biological Control Agents. Bar size: 2 cm

SEM images of G. boninense excised from the interaction zones of the agar well
diffusion assay showed severe morphological abnormalities of the pathogen in
hyphal structures compared with the control. Figure 2 (A) (control plate) shows
a healthy, dense and branched mycelium of G. boninense which is free from any
abnormality or disruption. The mycelium packed with branched hyphal strands
appears healthy, with no deformity. However, the hyphal of G. boninense when
challenged with BCAs were entirely colonized with spores of Trichoderma spp.
(Figure 2 (B)). The BCAs caused the hyphal structure of the pathogen to become
highly disrupted, disaggregated, flattened and shrivelled to a looser mass (Figure
2 (C) and (D)). The damage formed on the mycelium structure will eventually
inhibit the growth of G. boninense.

Antagonistic effects of BCAs on G. boninense mycelium in agar well diffusion

assay at day 12. (a) The healthy and dense branching mycelium of G. boninense
from a control plate cultured in the absence of BCAs treatment. (b) Spores of
Trichoderma spp. from BCAs attached to G. boninense hyphae. (c) Hyphal
branches of G. boninense were highly disrupted and disaggregated in the
presence of BCAs. (d) Highly shrivelled and flattened/lysis mycelium of
G. boninense in the presence of BCAs. Scale bars: (a)-(b): 10 µm. (c)-(d): 2 µm.

According to Reaves and Crawford. (1994), antagonistic characteristics of

Trichoderma spp. such as terminal and intercalary spores or circular coiling or
both, usually occurred at the interaction zones between the microbes. However,
hyphal coiling was not observed under SEM in this study. However, SEM
observation on the morphology of the hyphae on agar well diffusion assay
showed spores of Trichoderma adhered to the G. boninense hyphae, just as other
parasitic hyphae did. Adhesion of fungal spores to the host surface is followed by
germination of the fungal mycoparasite’s spores and establishment of a
successful parasitic interaction (Kubicek and Harman, 1998). When spores
germinated, they utilized the contents of the host hyphae as nutrient source and
parasitized the host. Lectin present in the cell wall of the host are suggested to
play a major role in the recognition of host hyphae by Trichoderma spp. to its
host (Motlagh and Samimi, 2013), suggesting that lectin could also be involved in
the spores attachment. Severe morphological abnormalities obeserved also could
be due to the production of antibiotics and secretion of cell wall degrading
enzymes from Bacillus spp. and Trichoderma spp respectively. This antimicrobial
mechanisms has led to the membrane damage causing the cell wall weakened
and undergoes lysis. Some possible mechanisms of antagonism employed by
Trichoderma spp. and Bacillus spp. include mycoparasitism, competition of
nutrients and space or the production of inhibitory compounds from the
Trichoderma, thus, modifying the rhizospere to a condition where G. boninense
cannot strive (Alexander and Chong, 2014).

Conclusion
Synergistic antifungal effect from the combination of Trichoderma spp. and
Bacillus spp. has led to major distruption on G. boninense mycelia. This
combination of BCAs could give a promising suppression of G. boninense. Further
study on the antifungal compounds produced from the combination of
Trichoderma spp. and Bacillus spp. would be needed.

References
Alexander, A. and Chong, K. P. 2014. Combination of Biological Agents in
Suppressing Colonization of Ganoderma boninense of Basal Stem Rot. American-
Eurasian Journal of Sustainable Agriculture, 8(7): 1-7

Chaiyasut, C., Kruatama, C. and Sirilun, S. 2010. Breaking the spores of

Ganoderma lucidum by fermentation with Lactobacillus plantarum. African
Journal of Biotechnology. 9(43): 7379-7382

Chong, K. P., Foong, C. P., Wong, C. M .V. L., Rossall, S.and Atong, M. 2011. First
identification of Ganoderma boninense isolated from Sabah based on PCR and
sequence homology. African Journal of Biotechnology. 10(66): 14718-14723.

Guetsky, R., Shtienberg, D., Elad, Y., and Dinoor, A. 2001. Combining biocontrol
agents to reduce the variability of biological control. Phytopathology. 91:621-
627.

Guetsky, R., Steinberg, D., Elad, Y., Fischer, E. and Dinoor, A. 2002. Improving
biological control by combining biocontrol agents each with several mechanisms
of disease suppression. Phytopathology. 92: 976-985

Kubicek, C. P. and Harman, G. E. 1998. Trichoderma and Gliocladium. Vol. 1. Basic

Biology, Taxonomy and Genetics. Taylor & Francis, London.

Motlagh, M. R. S and Samini, Z. 2013. Evaluation of Trichoderma spp., as

biological agents in some of plant pathogens. Annuals of Biological Research,
4(3): 173-179.

Ommelna, B. G., jennifer, A. N. and Chong, K. P. The potential of chitosan in

suppressing Ganoderma boninense infection in oil palm seedlings. Journal of
Sustainable Science Management, vol 7(2),pp. 186-192.
 Reaves, J. L. and Crawford, R. H. 1994. In vitro colony interactions among species
of Trichoderma with inference toward biological control. U.S. Department of
Agriculture, Forest Service, Pacific Northwest Research Station. Portland,
Oregon.

Siddiquee, S., Yusuf, U. K., Hossain, K., and John, S. 2009. In vitro studies on the
potential Trichoderma harzianum for antagonistic properties against Ganoderma
boninense. Journal of Food, Agriculture and Environment. 7(3&4): 970-976

Yobo, K. S., Laing. M. D. and Hunter, C. H. 2011. Effects of single and combined
inoculation of selected Trichoderma and Bacillus isolates on growth of dry bean
and biological control of Rhizoctonia solani damping-off. African Journal of
Biotechnology. 10(44): 8746-8756

View publication stats