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The Potency of Bacillus siamensis LDR as Biocontrol Agent Against Fungal Phytopathogen

Bacillus spp. was recognized as biocontrol agent because could produce antifungal towards many phytopathogenic fungi. In relation to study the potency of B. siamensis LDR as biocontrol, antagonistic activity of the bacilli was evaluated against Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228. The antagonist assay was carried out by dual culture method using streak and pour plate technique. Result of antagonistic activity assay with streak technique showed that growth inhibition rate of Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228 were 36.83%, 55.20%, and 33.78%, respectively. Antagonist assay using pour plate technique showed that percentage growth of inhibition for Fusarium sp. (93.96%), Ganoderma sp. (100%), and Chaetomium globosum InaCC F228 (76.64%) were higher than the results from streak method. Furthermore, results from antibiosis assay showed that growth inhibition rate of Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228 were 10.85%, 73.31%, and 30.32% respectively.


INTRODUCTION
Fungal phytopathogen was most devastating problem in agriculture industry. It could reduce the productivity and also quality of the plant products and further more can affect its economic value. Some of the common fungal phytopathogen which usually infect plant agriculture were Fusarium sp., Ganoderma sp. and Chaetomium sp. Infection of Fusarium sp. on agricultural plant causes vascular wilts, root rots, and yellowing disease [1]. Ganoderma sp. can infect stem of plantation plant, such as oil palm (Elaeis guineensis) and coconut plant (Cocos nucifera) cause rot of the plant [2]. Susanto et al. [3] stated infection of Ganoderma boninense cause basal stem rot disease which is most destructive disease due to more than 50% productive plant have died. Another fungal phytopathogen, Chaetomium sp. was known firstly cause leaf spot on Hevea brasilliensis [4] and Punica granatum by Guo et al. [5].
Therefore, infection of those fungal phytopathogen should be managed and controlled. Nowadays, biocontrol agent was more common to be used to overcome plant fungal infection. It is because the biocontrol agent is more eco-friendly compare to chemical pesticide.
The genus Bacillus has been known as one of potential biocontrol agent. The Bacillus spp. have more advantages such as rapid growth, can grow in simple nutrition, have thick peptidoglycan, and produce endospore. The thick peptidogliycan and endospore will make cells are more resistant to the environmental stress [6]. Bacillus are reported to produced several bioactive compounds including antifungal. Bacillus siamensis JFL15 was reported can produce lipopeptide antifungal compound which inhibit Colletotrichum gloeosporioides [7]. Bacillus siamensis LDR has been isolated from cocopeat [8]. Putri et al. [9] reported B. siamensis LDR could inhibit growth of Aspergillus niger ABP and ART. Moreover, Pertiwi et al. [10] reported B. siamensis LDR have antagonist activity against A. clavatus, A. fumigatus, and A. tamarii. The aim of this research is to explore the potency of antifungal activity from B. siamensis LDR against fungal phytopathogen, Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228.

Microorganisms
The microorganisms used in this research were Bacillus siamensis LDR and fungal phytopathogen such as Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228. Bacillus siamensis LDR, Fusarium sp. and Ganoderma sp. were provided by Microbiology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Indonesia. Chaetomium globosum InaCC F228 was obtained from LIPI, Cibinong, Bogor. The micoorganisms were purified using quadrant streak method on Potato Dextrose Agar (PDA) medium. The single colony of each isolate was maintained in PDA medium slant.

Antagonistic Activity Assay
The antagonistic activity was done using dual culture method with streak [11,12] and pour plate technique [13,14] on PDA medium. In streak technique, B. siamensis LDR was streaked on PDA medium then the fungal phytopathogen, Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228 were inoculated 4 cm beside the bacteria. The pour plate technique was perfomed by inoculating cells suspension of B. simaensis LDR (200 µl) into melted PDA medium, then the medium was poured into petri dish. After the medium has solidified, cell suspension of fungal phytopathogen was inoculated in to paper disc and then was placed on the center of medium in petri dishes. Control was prepared same as treatment, but the PDA medium was not inoculated with bacterial cells.
The result of antagonist assay was observed after 5 days incubation at 28°C. The radius of fungal colony was measured from center of inoculated fungal colony up to the edge colony which is facing the bacterial colony. Meanwhile for pour plate technique the diameter of fungal colony was measured. The inhibition growth then calculated to obtained Growth Inhibition Rate (GIR) of fungal phytopathogen [11].
(1) C= width mycelial of control fungal T= width mycelial of treatment fungal

Antibiosis Activity Assay
Antibiosis assay was performed with filtrate fermentation of B. siamensis LDR against fungal phytopathogen according to Putri et al. [9]. The Potato Dextrose Broth (PDB) medium (200 mL) was inoculated by suspension cell of B. siamensis LDR (1% v/v, 10 7 CFU/mL). The medium was incubated for 14 days at 28°C with still culture method. After fermentation, the growth medium was centrifuged at 3000 g for 20 minutes to obtain the filtrate free of cells. The filtrate was used to dissolved PDA medium. As a control PDA was dissolved using aquadest. Cell suspension of fungal phytopathogen, Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228 were inoculated into paper disc (10 µL). The inoculated paper discs, then were placed on the center of PDA filtrate and PDA control.
The antibiosis activity was presented by inhibition growth of fungal phytopathogen after 7 days incubation at 28°C. The diameter of fungal phyopahtogen was measured using caliper and the data were represented as growth inhibition rate which were calculated according the formula of Li et al. [11].

Antagonistic Activity Assay
Antagonistic activity assay showed Bacillus siamensis LDR can inhibit fungal phytopathogen, Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228. The inhbition was represented as percentage of growth inhibition rate. Growth inhibition of fungal phytopathogen can be seen in streak and pour plate technique.
Antagonistic activity assay using pour plate technique also showed B. siamensis LDR have antagonistic activity against fungal phytopathogen (Table 2).

Antibiosis activity assay
The result of antibiosis activity assay showed Bacillus siamesis LDR can inhibit all fungal phytopathogen tested (Table 3). The inhibition also respresented as percentage inhibition growth of fungal phytopathogen. Bacillus siamensis LDR can inhibit Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228 with percentage value 10.85%, 73.31%, and 30.32% respectively. Nevertheless, the percentage inhibtion of fungal phytopathogen were lowest than antagonist assay either in streak or pour plate technique. Growth inhibition of fungal phytopathogen can be seen in Figure 3.

Discussion
Bacillus siamensis LDR has antagonistic and antibiosis activity against fungal phytopathogen such as Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228. The antagonistic activity was assumed that B. siamensis LDR produces antifungal compound which inhibit growth of the fungi tested. Result of antagonistic activity with pour plate technique showed in Table 2 was higher than streak technique in Table 1. In pour plate technique, the bacterial cell will spread over the medium and will dominate the space and nutrient uptake besides produce antifungal compound. The double effect of B. siamensis LDR will give more suppression to the growth of all fungal tested.
Result of antibiosis assay exhibited lower inhibition percentage value compared to antagonist assay. It indicated that the hyphae of Fusarium sp. were more resistant to antifungal produces by B. siamensis LDR. Probably the most essential antagonist effect was due to the fast growth of B. siamensis LDR cells. This will compete with the fungal cell, dominate the space and nutrient uptake as can be observed in pour plate technique. Alabouvette et al. [17] stated tnhat one of the mechanisms of antagonistic activity is through the competition of the nutrient.

CONCLUSION
In conclusion, Bacillus siamensis LDR can inhibited growth of fungal phytopathogen, Fusarium sp., Ganoderma sp., and Chaetomium globosum InaCC F228. The inhibition growth showed in result of antagonist and antibiosis assay. The antagonistic activity assay using pour plate technique showed highest inhibition of fungal phytopathogen compared to streak technique and antibiosis assay.