Oil palm is one of the most important agricultural export crops in Indonesia
besides rubber, cocoa, coffee and spices (Stads et al.,
2007). The BSR has been a serious threat to the oil palm industry in Indonesia
because it shortens the productive life of oil palms and causes serious economic
loss. The disease is caused by a white-rot fungi G. boninense and in
the past few decades has been spreading rapidly, for instance, in North Sumatra,
Indonesia, this disease can lead to losses as much as 50% after repeated planting
cycles (25 years) (Corley and Tinker, 2003).
The use of fungicides for fungal control did not produce significant results
yet (Haas and Defago, 2005). This may be due to the fact
that by the time treatment is applied, the palms may already have the disease.
Antifungal-producing bacteria is a promising biocontrol agent (BCA) to overcome
this disease. The BCA does not necessarily be a cure for the disease but to
slowing or even to stop the disease spread by protecting the plant or enhanceing
the plant defense. Endophyte BCAs have more value since, it can live inside
the plant tissue through the plants lifetime.
Endophyte bacteria is bacteria which live inside the plant tissues without
causing apparent harm or symptoms to the host (Munif et
al., 2003). Endophyte as the internal plant habitat provide several
advantages as BCA. It will be in less competition with other microorganisms,
sufficient supply with the nutrients, less exposure to environmental stress
factors and better translocation of bacterial metabolites throughout the host
plant (Hallmann et al., 1997). Applicattion of
endophyte Pseudomonas aeruginosa and B. cepacia could reduce
BSR incidence up to 76% in 8 months oil palm seedling after disease inoculation
(Sapak et al., 2008).
Members of the genus Burkholderia sp. are known for their ability to
suppress soil-borne fungal pathogens by the production of various antibiotic
compounds such as pyrrolnitrin and phenazines (Kirner et
al., 1998). Other different antibiotics such 2,4-diacetylphloroglucinol
(2,4-DAPG) and pyoluteorin has also been found responsible for suppression of
soil-borne fungal pathogens (Subagio and Foster, 2003).
Antibiotic-related genes can be detected from BCAs by using Polymerase Chain
Reaction (PCR) by using antibiotic specific primers which encode phenazine-1-carboxylic
acid, 2,4-DAPG, pyoluteorin and pyrrolnitrin, from Pseudomonas and Bacillus
genome (Zhang et al., 2005).
BCAs have been applied in many ways and on many crops species, for instance,
seed dipping application on rice seeds to control bacterial blight disease caused
by Xanthomonas oryzae was able to reduce the disease incidence (Suryadi
et al., 2012). Besides microbial pathogen, BCA was also reported
able to control plant parasitic nematodes. Munif et
al. (2013) was reported seed dipping application on tomato seeds able
to control Meloidogyne incognita penetration and enhanced the plant growth.
Oil palm germinated seeds dipping application was also conducted by Dikin
et al. (2003) to suppress Schizopyllum commune, causal agents
of brown germ and seed rot in oil palm. In this study, Burkholderia sp.
control activity against G. boninense growth were observed in vitro
and in vivo of oil palm to analyze antifungal encoding gene from Burkholderia
MATERIALS AND METHODS
Burkholderia sp. isolation: Burkholderia sp. were isolated
from soil near the roots and the roots that were taken from a healhty (symtompless)
oil palm tree in an endemic area, in North Sumatera, Indonesia. One gram of
soil was dilluted in sterlie water and poured on Nutrient Agar plates. For the
roots, were surface sterilized with 90% alcohol for 1 min, 70% alcohol for 3
min and washed twice with 50 mL sterile distilled water for 30 sec. The roots
were crushed aseptically and put on NA in plate. The plate were incubated for
24-36 h. Each of the grown colony were subculture and genome extracted for identification.
Purified PCR product of 16S DNA were sent to 1st Base, Singapore.
Antagonist in vitro test: Antagonist test was observed to determine
the Percentage Inhibition of Radial Growth (PIRG) of G. boninense (Bivi
et al., 2010). Six bacterial isolates of Burkholderia sp.
were selected to evaluate their efficacy in enhancing growth and inhibit the
infection of BSR in oil palm in pre nursery. These Burkholderia sp. collection
were isolated from rhizospher and oil palm root tissue. There were five rhizosphere
Burkholderia sp. (B313, B51a, B52c, B51b, B52a) and one endophyte Burkholderia
Burkholderia sp. was streaked into the PDA plate 2.5 cm from the edge
of the petri dish. Agar disc cut diameter 5 mm and 5 day old G. boninense
was placed 2.5 cm from the edge at the opposite side of the same petri dish.
For the control plate, only G. boninense was placed in a similar manner
without bacteria on a fresh petri dish. The plates were incubated at 28°C
for five days. Results shown by measured the radial growth of G. boninense.
The PIRG was calculated using the equation below (Zaiton
et al., 2006):
||Percentage inhibition of radial growth
||Radial growth of G. boninense in the absence of bacteria (control)
||Radial growth of G. boninense in the presence of Burkholderia
sp. The three highest PIRG isolates from in vitro test were used
for in vivo test
In-vivo test: Burkholderia sp. suspensions were prepared
by inoculating 24 h old cultures into Nutrient Broth (NB) and incubated for
20 h and adjusted to 108 CFU mL-1. During the preparation
of mixture, equal volume of the 3 highest PIRG Burkholderia sp. were
mixed. Ganoderma boninense inoculum was prepared on 6x3x15 cm sterilized
oil palm fronds. The fronds were sterlized in a heat resistant plastic each
before inoculated with G. boninense. After sterilization, the fronds
were inoculated with a 0.5 cm diameter disc of G. boninense mycellia
on agar plate. The plastic was sealed and incubated in room temperature for
3 months before used.
The plant material was selected as the most G. boninense susceptible
progeny. Two different progenies were used in this study. Briefly, oil palm
germinated seeds were treated with bacterial suspension (108 CFU
mL-1), dipped for 20 min (seed bacterization) and air dried for 10
min before planted.
Oil palm germinated seeds were planted in polybags (15x20 cm) regarding to
the Standard Operation Prosedure (SOP) in prenursery of oil palm plantation.
There were four treatments in this study. Treatment A and C were not inoculated
with G. boninense and treatments C and D were inoculated with G.
boninense. Treatment B and C were using seedling treated with Burkholderia
sp. Treatments with G. boninense, the seedlings were placed in contact
with radicula. The pots were placed under shead, watered daily and no supplementary
organic fertilizer was applied for 3 months (prenursery). A destructive observation
was conducted after 3 months.
The infection of G. boninense on plant can be scored by observation
on signs and symptom on the treatment plants using Disease Severity Index (DSI)
(Abdullah et al., 2003). The DSI was observed
from the external symtomp from foliar and the roots (destructive method).
The score can be calculated by the equation of Mohd Zainudin
and Abdullah (2008):
||Disease class (0, 1, 2, 3 or 4)
||Number of plants showing that disease class per treatment
||Polymerase chain reaction primers and expected amplification
products from genes encoding enzymes involved in the biosynthesis of several
antibiotics (Zhang et al., 2005)
Detection of antifungal gene: Antifungal gene detection was conducted
by using PCR. Burkholderia sp. was prepared in liquid medium for DNA
isolation by using GeneJET Genome DNA Purification Kit from Thermo Sciencetific.
Specific primers for DAPG (Phl2a-Phl2b), phenazine (PHZ1-PHZ2), pyrrolnitrin
(PRND1-PRND2) and pyoluteorin (PLTC1-PLTC2) were used for detection according
to Zhang et al. (2005) (Table
PCR product was gel extracted from agarose by using Gene JET Gel Extraction
Kit (Fermentas), according to manufacturers instruction. After purification,
the PCR products were sent to 1st Base, Singapore, to be sequenced. The antifungal
genes sequences were aligned by using BioEdit software and searched for sequence
similarity to other sequences which are available in the NCBI database at http://www.ncbi.nlm.nih.gov/
using Basic Local Alignment Search Tool (BLAST) algorithm. Multiple sequence
alignments were performed on the selected closely related sequence accessions
available using CLUSTAL W software in Mega 5.
Statistical analysis: The treatment was repeated into 13 replicates.
This research used factorial design with one factor and the environmental design
is complete randomized design. Statistic analysis was done by using general
method linear model univariate. If there was a significant diferrence further
analysis will be analyzed with á value is 5% by using SAS.
Antagonist in vitro test: All of the Burkholderia sp. isolates
showed an inhibition activity against G. boninense growth in vitro
(Table 2). Scores followed by the same letter indicated that
they were insignificantly different scores. The highest inhibition activity
was shown by isolate B212 with percentage PIRG was 34.38% but not significantly
different with B52a and B52c which were both 27.50%. Burkholderia B313
showed the lowest activity (23.75%) and significantly different with B212 but
still has an antagonist activity against G. boninense growth in vitro.
In vivo test: The necrosis and chlorosis foliar was not seen
in all treatments, though the height of the plants between treatment was seem
different (Fig. 1a). The DSI after 3 months only showed in
treatment C and D (25%) (Table 3). Destructive observation
showed that all plants in treatment C and D were infected with G. boninense.
Treatment C and D which were inoculated with G. boninense, showed a brown-blackening
roots especially on the parts which colonized with the G. boninense (Fig.
1b). The roots in treatment A and C, without G. boninense, were cream-brown
(a) Plants treatment left to right (A-D)
and (b) Healthy root with no appearance of fungal mycellia (left). Appearance
of fungal mycellia (red arrow)
|| Percentage of inhibition ratio from Burkholderia sp.
against Ganoderma boninense growth in vitro
|| Disease Severity Index (DSI) of treated plant with Ganoderma
Progeny give a significant different mostly to the shoot lenght and dry weight
(Table 4). Total number of roots between progeny also showed
a significant difference. However, there is no correlation between progenies
and treatments though in some parameter that were observed was show a significant
Burkholderia sp. consortia application on inoculated seeds showed significantly
lowest in shoot growth compare to control and other treatments. In all parameters
that were measured, the treatment decrease the plant growth and significantly
decrease shoot lenght, shoot dry weight, total number of shoot and root (Table
5). However, Burkholderia sp. consortia application on un-inoculated
seeds showed the highest in shoot and root growth compare other treatments.
The shoot lenght and dry weight was slightly different compare to control. The
root lenght did not showed a significant different among all the treatment.
Antifungal gene detection: Primers PRND1 and PRND2 amplified the predicted
790 bp fragment from DNA of B212, B313, B51a and B52c. Primers PLTC1 and PLTC2
amplified the predicted 438 bp from DNA of Burkholderia B313, B51a and
B52c (Fig. 2). Primers for phenazine and DAPG did not yield
a PCR product from all isolates. This may be concluded that these isolates do
not contain phenazine and DAPG biosynthesis gene.
||Effect of different progeny on root and leaf total number,
lenght and dry weight after treatment with Ganoderma boninense and
|*Means within a column with the same letter are not significantly
different at p<0.05 using DMRT
||Effect of dipping treatment of antagonistic bacteria on seedling
of oil palm inoculated with Ganoderma boninense in prenursery at
3 months after planting
|*Means within a column with the same letter are not significantly
different at p<0.05 using DMRT
||PCR product of pyrrolnitrin and pyoluteorin gene amplification.
M: Marker, 3. prn primer, 4. plt primer, a: B212, b: B313, c: B51a , d:
B52c, e: B51b, f: B52a. Red arrows showed the expected band size
|| Phylogenetic tree of PrnD gene from B212 and B51a
The PCR product were BLAST and showed a 99% identity with B. cepacia
partial prnD gene, strain ESR63 (Fig. 3). It is confirmed
B212 has a potency to produce pyrolnitrin and assumed pyrolnitrin is the responsible
compound in inhibiting Ganoderma growth in vitro.
Ganoderma boninense has been the most threatening disease in oil palm
plantation. Ganoderma boninense has the ability to degrade lignin in
the plant and decay the lower stem and sometimes the root system, leading to
severe symptoms such as flattening of the crown and unopened spear leaves (Cooper
et al., 2011). Many attemps have been made to control this disease
including using BCAs such as bacteria.
Bacteria have various mechanism of antagonistic such as synthesizing antibiotic
compounds, production of hydrolytic enzymes, siderophore production, competition
for substrates and also induction of systemic resistance in the host plant will
increase the plant resistance to a broad spectrum of pathogens (Kloepper
and Ryu, 2006). Many soil-borne plant diseases caused by fungi and oomycetes
can be controlled by strains of the genus Burkholderia (Kirner
et al., 1998).
In this study endophyte Burkholderia B212 showed the highest antagonist
activity against G. boninense growth (Table 2). Several
strains of Pseudomonas and Burkholderia species can produced a
broad-spectrum of antibiotics which play an important role in the suppression
of multiple plant pathogenic fungi (De Souza and Raaijmakers,
In vivo test showed a different result from the in vitro test.
In comparison to Mohd Zainudin and Abdullah (2008)
and Sapak et al. (2008), the external symptom
of G. boninense infection in oil palm appeared after 4 months after planting
even though the disease has been invested from seedling or after 3 months planting.
However, external symptom may also occur after 2 months planting with the disease.
This diferences may be caused by many factors such as pathogenicity level of
G. boninense, inoculum source of G. boninense, plant tolerance
and climate. White-rot fungi has been classified to the ligninolytic enzymes
they express and able developed unspecific ligninolytic systems consisting of
peroxidases and laccases which employ an oxidative process to degrade the wood
cells (Paterson, 2007). It can be assumed that these
enzymes activity can be related to G. boninense pathogenicity level.
There are two type of G. boninense inoculum source used in G. boninense
research, wood block and oil palm fronds. Oil palm fronds are more easy to get
since it is abundance in oil palm plantation. Different climate may also effect
lignin degradation which lignin degradation is less efficient at 37°C compare
to 25°C (Paterson et al., 2008).
Application method of Burkholderia sp. may also effect the result of
infection. Burkholderia sp. has been reported that its antifungal producing
is related to quorum-sensing which is mean that the antifungal activity depends
on Burkholderia sp. biomassa (Chapalain et al.,
2013). Ganoderma boninense infection were found in all plants which
were treated with the disease including the Burkholderia sp. consortia
treatment. Burkholderia sp. consortia were expected to inhibit the disease
infection but the result showed on the contrary. It is very different to other
report which is using soil drenched application and the result showed that application
with Burkholderia and Pseudomonas able to reduce G. boninense
incidence (Sapak et al., 2008). Different method
of application and number of Burkholderia sp. applied may come to a different
Burkholderia sp. consortia application on inoculated seeds was expected
to have the highest plant growth compare to plants without Burkholderia
sp. and control. However, the treatment showed the lowest plant growth compare
to all other treatments. Burkholderia sp. as endophyte has the ability
to penetrate the plant by using cellulase enzyme (Reinhold-Hurek
et al., 2006). It can be assumed that the Burkholderia sp.
has a potency to open a way for G. boninense to infect the plants. Biomass
of Burkholderia sp. held an important key in suppress G. boninense
infection, since, the antifungal compound is regulated by quorum sensing. This
fenomena could explain the infected plants without Burkholderia sp. consortia
application is higher than the infected plants with Burkholderia sp.
If compared with the shoot and root ratio (S/R) among the treatment, infected
plants with Burkholderia sp. consortia shown the lowest number. Which
means that in this treatment, the root has the higher biomass compare to the
shoot. Burkholderia sp. consortia application gave a more affect to the
roots than the shoots on infected plants. Burkholderia sp. also could
acted as PGPR which can lead an indirect biocontrol agent, since it could enhanced
plant growth. Burkholderia has reported as a plant growth promoting rhizobacteria
(PGPR) (Compant et al., 2008).
Burkholderia sp. consortia application on inoculated seed was significantly
higher on root dry weight compare to other treatment. Ganoderma boninense
as pathogen could induced plant defense by forming lignin as its first defence
system. Lignification or cell thickness is the form of plant defense against
pathogen (Xu et al., 2011) and could increase
Total number of Burkholderia applied also play an important role in
proper activity of this biocontrol agent. Schmidt et
al. (2009) has reported that some of antifungal such as pyrrolnitrin
is regulated by quorum sensing. Quorum sensing is a mechanism to regulate the
production of antimicrobial compounds by population-density-dependent (Liu
et al., 2007). Different results between in vitro and in
vivo test may be caused by many factors such as plant ages by the time artificially
inoculated, concentration of microbes applied and application technique.
Many research showed that Burkholderia could decrease G. boninense
infection on oil palm but yet a correct application technique of this bacteria
also important to get the best result. Antifungal compound is a secondary metabolite
which is produce by bacteria in their stationary phase. Further research need
to be conducted for the proper technique application of this Burkholderia
for its optimum action.
Pyrrolnitrin encoded gene was amplified and showed that Burkholderia
B212 has a potential in producing antifungal agent such as pyrrolnitrin. Pyrrolnitrin
has been implicated as an important mechanism of biological control of fungal
plant pathogens by several Pseudomonas strains (Hasan
and Turner, 1998). Pyrollnitrin is a chlorinated phenylpyrrole antibiotic
that was first isolated from Burkholderia pyrrocinia (Kloepper
and Ryu, 2006) and later from other microorganisms, including Pseudomonas
fluorescens, P. chlororaphis, P. aureofaciens, B. cepacia,
Enterobacter agglomerans, Myxococcus fulvus and Serratia species
(Hammer et al., 1999).
Pyrrolnitrin is synthesize by four protein encoded by 4 gene, prna,
prnb, prnc and prnd. The Prnd gene was the final
protein to form an active pyrrolnitrin compound. The prnD catalyzes the
oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin
(Kirner et al., 1998). In some strains of Burkholderia
species pyrrolnitrin biosynthesis was shown to be regulated by quorum sensing
(Schmidt et al., 2009).
The authors would like to thank the valuable technical assistance of staff
from the PT. SMART Tbk. in Microbiome Technology laboratory and Biotechnology
laboratory. The authors also thank to PT. SMART Tbk for the research funding.