PLANT-MICROBE INTERACTIONS

SAP 4563
SEMESTER 1 22/23

Lab 3 : Antagonistic activity of volatile compound of

bacteria against phytopathogens: Dual plate assay

LECTURER NAME : DR NUR SABRINA BINTI AHMAD AZMI

NO NAME MATRIC NO.

.

1. MUHAMMAD FAIZ BIN SEKERI 1913565

DATE OF SUBMISSION : 6 / 12 / 2022

1.0) INTRODUCTION

Microbial antagonism is a situation whereby the presence of one microorganism prevents

the growth of another microorganism ( Dukare et al., 2019). It can happen for various reasons
such as competition for resources between the two microbes or the one microbe releasing a
chemical which is toxic to the other. Microbial biological control agents (MBCA) , especially
bacteria mediate various mechanisms against phytopathogens. Secretion of volatile organic
compounds also played a role in biological control activities. Volatile organic compounds (VOC)
basically have easy spreading for a long distance and directly inhibit the growth of the fungi and
induce systemic resistance. The antagonistic activity of volatile compounds of bacteria against
the phytopathogens by dual plate assay method is explained here.

Basal stem rot caused by Ganoderma boninense is the most important and worrisome
disease of oil palm for urgent consideration in plant resistance breeding in southeast Asia,
especially Malaysia and Indonesia , as chemical and cultural control methods have proved to be
inefficient if not ineffective. The disease used to be confined to older palms planted on
ex-coconut or oil palm land with a high water table ( Soh., 2012 ). Thus, there are studies on
potential bacteria as a biocontrol agent for inhibition of Ganoderma boninense ( Ramli et al.,
2016 ; Susanto et al., 2005 ). Bacteria are ubiquitous, mostly free-living organisms often
consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms.
It is important to identify beneficial microbes or bacteria that can produce biocontrol activity for
fighting Ganoderma boninense, thus, can help problems caused by this pathogenic fungi in palm
oil agriculture.

In this experiment, we want to find and observe the antagonistic activity from two
different species of bacteria against fungi, Ganoderma boninense . The bacteria species used for
screening antagonistic activity in this experiment are Pseudomonas oryzihabitans and
Paenibacillus hunanensis.
1.1) OBJECTIVE

1. To assess the relative inhibition efficacy of different bacteria against fungal pathogens,
Ganoderma boninense.
2. To observe the inhibition of the pathogenic fungi by volatile compounds produced from
bacteria.

2.0) MATERIAL & METHODS

2.1 Material

● Potato dextrose agar. l Conical flask.

● Eppendorf tubes.
● Tryptone soya agar.
● Nichrome wire.
● Test tubes.
● pathogenic bacteria.
● Burette, pipettes.

2.2 Method

1. The fungal plant pathogen was isolated and collected from any culture collection center
and maintained.
2. Potato dextrose agar (PDA) was prepared and the fungal culture inoculated in it and
incubated the plate at room temperature/27 degrees Celsius for 5 days. After the growth,
the fungal strain was stored at 4 degrees Celsius.
3. For antagonistic activity, the plate containing the lawn growth of bacteria on the surface
was prepared on the TSA plate.
4. The lid of the petri plate containing antagonistic bacteria was replaced with a 7 cm agar
plate containing active growth of fungal pathogens.
5. The petri plates inoculated with fungal pathogens were inverted over the plates
containing antagonistic bacteria.
6. The plates were sealed together with parafilm and a control plate without bacteria was
maintained in the bottom plate.
7. The petri dish was incubated at room temperature/27 degrees Celsius and observed the
plate at 24 h interval for 72 h.
8. The mycelia growth inhibition were determined by the following formula.

Inhibition (%) = 100 - ((D1 - D2) / D1 x 100 )

where D1 is the diameter of radial growth of pathogenic fungi in control; D2 is the

diameter of radial growth of pathogenic fungi with antagonistic bacteria.

9. The results were recorded.

3.0) RESULTS

For this experiment, there are 3 treatments. First, control sample were there are only Ganoderma
boninense incubated for the growth, Second treatment is P. hunanensis - G. boninense , Third
treatment is P. oryzihabitans - G. boninense. In each treatment, there are 3 replicates.

Treatment Inhibition activity

Ganoderma boninense -
control

P. hunanensis - G.
boninense

P. oryzihabitans - G.
boninense

Table 1; All of the figures show the condition of pathogenic fungal growth after several days.
The antagonistic activity observed in this experiment is inhibition of the growth of agar contain
pathogenic fungi, Ganoderma boninense.
 Treatment Rep 1 (cm) Rep (2cm) Rep 3 (cm) mean(cm)

Ganoderma boninense - control 1.30 1.20 1.00 1.17

P. hunanensis - G. boninense 0.60 0.40 0.50 0.50

P. oryzihabitans - G. boninense 0.00 0.00 0.00 0.00

Table 2. The growth of G. boninense measured in each plates (cm) after several days.

By using formula Inhibition (%) = 100 - ((D1 - D2) / D1 x 100). Thus, we get inhibition ( % )
of pathogenic fungi, Ganoderma boninense from the treatment ( table 3).

Treatment Inhibiton ( %)

P. hunanensis 42.74

P. oryzihabitans 00.00
Table 3. inhibition ( % ) of pathogenic fungi, Ganoderma boninense by the treatment
4.0) DISCUSSION

In this experiment, The bacteria species used for screening antagonistic activity are
Pseudomonas oryzihabitans, Paenibacillus hunanensis. Pseudomonas oryzihabitans is a
nonfermenting yellow-pigmented, gram-negative, rod-shaped bacterium that can cause sepsis,
peritonitis, endophthalmitis, and bacteremia. This bacteria are characterized as pathogenic
bacteria as it is able to cause infections in individuals that usually have compromised immune
systems (Freney et al., 1988). For second bacteria, Paenibacillus is a genus of facultative
anaerobic, endospore- forming bacteria. The genus is capable of fixing nitrogen, so is used in
agriculture and horticulture and often have symbiosis relationships with the plants.

For this experiment, from the result in Table 3, there are no inhibitions of pathogenic
fungi, Ganoderma boninense by P. oryzihabitans . This shows that P. oryzihabitans does not
produce any biocontrol that can inhibit this fungal pathogenic growth. As for Paenibacillus
hunanensis, there is inhibition of G. boninense growth for about 42.74 %. From the previous
study, there are reported that the bateria from Paenibacillus sp can produces VCs with
antagonistic activity, being able to inhibit the growth of 9 out of 10 fungi analyzed and the two
oomycetes ( Costa eet al., 2022), have antifungal activity and produced lytic enzymes with
fungicidal potential which decreased biomass production of several pathogenic fungi by
45%-75%. The inhibition was accompanied by degradation of fungal cell walls and alterations in
hyphal morphology ( Aktuganov et al., 2008). .Although there are not 100% inhibition of
pathogenic fungal from P. hunanensis , but by identifying the protein or enzyme produced from
this bacteria, that have the antagonistic activity that helps inhibit the growth of pathogenic
fungal, Ganoderma boninense , we can use this protein or enzyme to help fight the disease from
Ganoderma boninense in oil palm field.
5.0) CONCLUSION

In conclusion, In many distinct areas of microbiology, the ability of bacteria to produce

functional biocontrol activity such as antagonistic activity that helps inhibit the growth of
pathogenic fungal such as Ganoderma boninense has important applications. In this experiment,
qualitatively, we managed to get the positive result for the inhibition of the growth of G.
boninense by using P. hunanensis. P. oryzihabitans does not have the ability to produce
antagonistic activity for the inhibition of G. boninense. After identifying the beneficial biocontrol
compounds secreted from the specific bacteria, we can use this information to help inhibit the
growth of G. boninense in future, thus, reduce or fix the basal stem rot caused by Ganoderma
boninense, which is the most important and worrisome disease of oil palm in southeast Asia.

6.0) REFERENCES

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