Isolation and Identification of Antagonistic Bacterium against Pathogens of Bacterial Tuber Rot of Amorphophallus muelleri

Rhizosphere bacteria have the ability to protect the host plants from the infection of pathogenic microorganisms. This study aimed to identify rhizosphere bacteria that were capable of inhibiting the growth of bacterial isolates that cause tuber rot of Amorphophallus muelleri. Rhizosphere bacteria were isolated using Nutrient Agar medium by pour plate method. Isolates were subjected to antagonistic assay against several bacterial isolates from the rotten tuber of A. muelleri using dual culture method. The potential isolate was identified based on 16S rDNA sequence. Isolate R7 showed the strongest inhibition to the growth of bacterial isolates from rotten tuber with an inhibition zone diameter of 19.66 mm. The 16S rDNA sequence of isolate R7 R7 was 99.7% similar to Delftia tsuruhatensis PCL1755. The isolate was potential to be developed as phytopathogen control agent.

INTRODUCTION 1  Tubers of Amorphophallus muelleri Blume.contain a high concentration of glucomannan.Glucomannan is a starch that can be used as a thickener agent in foods such as noodles [1].Glucomannan in A. muelleri tubers has a high economic value and is expected to improve Indonesian economy.Therefore, it is necessary to increase the production of A. muelleri tuber.Nowadays, the problem is pathogenic microorganisms that attack the tubers either under the ground or even post-harvest.The common pathogenic microorganisms that attack the tubers are Erwinia caratovora and Pectobacterium caratovora on Amorphophallus konjac tuber [2,3] and Dickeya dadantii on Amorphophallus rivieri [4,5].
To control the tuber rotting, farmers use chemicals such as pesticides.Continuous application of synthetic pesticides caused negative impacts on the environment [6].The residue of the pesticides in the soil as well as on the plant parts (fruits, leaves, and tubers) [7,8] were indirectly or directly toxic to humans [9,10].
Therefore, biological agents are required as antagonistic agents to control the growth of bacterial rot pathogens.One alternative that can  Correspondence address: Rodiyati Azrianingsih Email : rodiyati@ub.ac.idAddress : Dept.Biology, Faculty of Mathematics and Natural Science, University of Brawijaya, Veteran Malang, Malang 65145.
be used as an ecologically safe and effective antagonistic agent against pathogens is the rhizosphere bacteria [11,12,13].Rhizosphere bacteria are present in the soil around plant roots.They have many benefits for plants such as promoting nitrogen fixation, phosphate and potassium solubilization, production of phytohormones and antibiotics [14,15,16].
Some rhizosphere bacteria that act as antagonistic agents are Bacillus, Pseudomonas, Pantoea, and Lactobacillus.One of the antagonistic bacteria that can inhibit the growth of E. caratovora (one of the causes of tuber rot bacteria) is Bacillus subtilus.It is able to produce antibiotic compounds such as bacitracin, bacillin, bacillomycin B, difficidin, oxydifficidin, lecithinase, and subtilisin.These compounds cause shrink in cells so that bacterial cells of E. carotovora will lose water and experience plasmolysis [17,18].In addition, Bacillus amyloliquefaciens is also able to inhibit the growth of Erwinia bacteria which causes postharvest tuber rot [19].This research aims to analyze the potency of isolated rhizosphere bacteria to inhibit A. muelleri tuber rot bacteria and to identify the potential rhizosphere bacteria based on 16S rDNA sequence.

MATERIAL AND METHOD Isolation of Rhizosphere Bacteria
Soil samples were obtained from the rhizosphere of A. muelleri from Rejosari Village, Bantur City, East Java Province, Indonesia.The soil was taken at a depth of 5-10 cm of topsoil and kept in plastic bags in the isotherm box [20].At each sampling point, abiotic factors including 166 Antagonistic Bacterium against Pathogens of Bacterial Tuber Rot of A. muelleri (Arfani et al) the ambient and soil temperature, and light intensity were measured directly at the field; while moisture, pH, and organic matter of soil were measured in the Laboratory of Microbiology and Laboratory of Ecology, University of Brawijaya.The data of abiotic factors were analyzed using ANOVA and Tukey test with five percent significant differences.
Twenty five grams soil sample was diluted at 10 -1 -10 -7 in sterile physiological saline solution.Each suspension of 0.1 mL was transferred into Nutrient Agar (NA) medium in the Petri dishes according to pour plate method and incubated at room temperature for 72 hours.Each bacterial colony was enumerated and purified according to the spread plate method.The pure culture of rhizosphere bacteria in the NA medium was stored at 4°C [21].The diversity of bacterial communities was determined based on the Simpson Diversity index according to the equation 1 [22][23][24][25].

Isolation of Pathogenic Bacteria from Rotten Tuber of A. muelleri
Bacterial pathogens of A. muelleri tubers were isolated according to Ashmawy et al. [26] with modifications.The rotten of tuber was cut into a dimension of 1.0 x 0.5 x 0.2 cm 3 .It was sterilized by soaking in 1.0% NaOCl solution for two minutes and rinsed two times with sterile ddH2O.The pieces of sterilized rotten tuber were weighed to 25 g.They were blended with 225 mL sterile physiological saline solution and diluted at 10 -1 -10 -7 .Sample suspension of 0.1 mL was inoculated into NA medium according to pour plate method and incubated at room temperature for 48 hours.The bacterial colony was purified according to the spread plate method and pure cultures were stored at 4°C.

Antagonist Assay of Rhizosphere Bacteria Against A. muelleri Tuber Rot Bacteria
The antagonistic assay among bacterial isolates was done using dual culture method [27].The 100 µL suspension of isolated tuber rot bacterium with 10 6 cells.mL - density was spread on NA medium and directly incubated at 4°C for 4 hours.The NA agar plates were perforated to make 6 mm wells.The wells were inoculated with 60 μL of 10 7 cells.mL - density of antagonistic rhizosphere bacteria.The cultures were incubated at room temperature for 72 hours.The growth inhibition of tuber rot bacteria was indicated by the clear zone around the well.The diameter of the inhibition zone were measured and the data was analyzed using ANOVA and Tukey test with five percent significant differences.

Identification of Potential Rhizosphere Bacteria
Rhizosphere bacteria with the highest potency to inhibit tuber rot bacteria was identified based on phenotypic and phylogenetic characters.Phenotypes of bacteria were characterized based on Bergey's Manual of Systematic Bacteriology [28,29].The phenotype of bacteria consists of the colony and cell morphology, biochemical, and physiological characters.Phylogenetically, the bacteria isolate was identified based on 16S rDNA sequence similarity.The genomic DNA of the selected isolate was extracted using Heat Treatment method [30].The sequence of 16S rDNA was amplified using universal primer of: The composition of 50 μL PCR reaction was 25 μL PCR master mix, 19 μL ddH2O, 2 μL of each primer, and 2 μL of DNA template.The components were homogenized and 16S rDNA was amplified using the PCR program at 35 cycles includes: predenaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 90 s; and followed by post extension at 72°C for 5 minutes.
The amplicon of 16S rDNA was verified with electrophoresis on 1.5 % agarose gel.The amplicon of 16S rDNA was purified and sequenced at First Base, Malaysia using Automatic Sequencer Analyzer ABI 3130.The sequence of 16S rDNA was edited using the Sequence Scanner V.1 program and the sequences were combined using the BioEdit V.7.2.5 program.The 16S rDNA sequence of the isolated bacteria was aligned together with 16S rDNA reference that obtained from the NCBI database.The phylogenetic tree was constructed based on Neighbor-Joining with bootstrap 1000 using the MEGA 6.00 program [31,32,30].

RESULT AND DISCUSSION Density and Diversity of A. muelleri Rhizosphere Bacteria
A total of isolates of A. muelleri rhizosphere bacteria were obtained from three locations.Based on Simpson's diversity index, community diversity of the rhizosphere bacteria was in the range of 0.84 -0.87 (Fig. 1).It indicated that the community was highly diverse and there were no dominant species [25].However, the density of rhizosphere bacteria was relatively low in the range of 3.54 -3.56 Log 10 CFU.g -1 (30.0 -59.0 x 10 2 CFU.g -1 soil (Fig. 2).The low density might be caused by the low organic matter and low moisture of the soil.Since those parameters are limiting factors for the growth of several rhizosphere bacteria.Soil bacteria require a minimum of 2% soil organic matter and 60% soil moisture for support of the optimal growth [33,34], while in this experiment the organic matter and soil were less than 0.2 and 32%, respectively.Environmental for the three sampling locations were presented in Table 1.The soil parameters especially plant and soil type, and farm practice affects the diversity and density of soil microorganisms and plants growth [34].The low nutrient and water availability in the soil may inhibit metabolism and growth of micro-organisms.Soil organic matter plays an important role in soil structure and texture, microaggregate stability, soil moisture and pH, nutrient availability, and microorganism density and diversity [25,35].In all locations, soil moisture was low due to low content of organic matter.Organic matter will increase soil moisture and decrease soil pH.The increase of soil organic matter will increase the content of organic carbon which utilized by bacteria as carbon and energy source [36].Soils of A. muelleri plantation were acid, with pH value 3.78 -4.13.In general, bacteria grow in the pH range 5-7 as optimum conditions [37].The acidity of the soil may be caused by contamination of metals derived from the use of chemicals (fungicides and pesticides), pollution, organic fertilizers, and household waste disposal [38].Soil organic matter (%) 0.16 ± 0.02 0.17 ± 0.03 0.16 ± 0.02

Antagonistic Potency of Rhizosphere bacteria
Rhizosphere bacteria consisting of nine isolates with a density of 10 7 CFU.mL - were tested for their inhibition against three tuber rot bacteria of A. muelleri (PT4, PL9, and PR11).The rhizosphere bacterial isolates had different potency in inhibiting and only four isolates can inhibit tuber rot bacteria (Fig. 3).Isolate R3 was not able to inhibit isolate PT4 but it inhibited isolate PL9 and PR11 with inhibition zone diameter 12 and 10 mm, respectively.Isolate R5 was able to inhibit PT4, PL9, and PR11 with inhibition zone diameter of 2.07, 5.93, and 6.96 mm respectively.Isolate R7 was able to inhibit the three isolates of tuber rot bacteria, PT4, PL9, and PR11 isolate with inhibition zone diameter of 19.66, 11.24, and 12.42 mm.Isolate R9 was only able to inhibit isolate PT4 and PL9 with inhibition zone diameterof 2.00 and 6.21 mm, respectively.
Isolate R7 had the highest inhibition potency among the other A. muelleri rhizosphere bacteria.The isolate was able to inhibit the three isolates of tuber rot bacteria and categorized in the high potential with inhibition zone more than > 10 mm [39].Based on previous experiment [40], Bacillus circulans rhizosphere bacterium was able to inhibit Escherichia coli, Bacillus subtilis, and Serratia marcescens with inhibition zone diameter of 11, 12, and 6 mm, respectively.Antagonistic bacteria have a mechanism to inhibit the growth of pathogens.The inhibition is performed by producing antimicrobial compounds such as enzymes capable of attacking the main cell components of pathogens [41,42].Antimicrobial compounds produced by antagonistic bacteria cause damage to the cell membrane and shrink the cell.Furthermore, the activity of the bacteria becomes disturbed and causes it to die.Another antibiotics compounds are responsible for inhibition of protein synthesis process.The synthesis is inhibited when exposed to antibiotic compounds and cause cell death of pathogens [43,44].The inhibition mechanism of tuber rot bacteria by isolate R7 was antibiosis.Antibiosis is the ability of antagonistic isolates to produce secondary metabolites of antibiotics, siderophores, and several enzymes such as chitinase, protease, and cellulase enzymes that inhibit the growth of target cell [45,46].The activity of antibiosis was determined by clear zones; it proved that rhizosphere bacteria could produce antibiotics that inhibited the growth of tuber rot bacteria [47,48].In addition, inhibition potency of antagonistic bacteria may also be caused by antagonism such as competition of root colonization and nutrient [49][50][51][52].Antagonism is the ability of antagonistic microorganisms to produce antibiotics that can kill pathogenic microorganisms [53].Competition of space and nutrients cause limitation of nutrition and space for growth of pathogens [37,54,55,56].

Identification of A. muelleri Rhizosphere Bacterium as Antagonist of Tuber Rot Bacteria
The isolate R7 (Fig. 4) had a 99.7% similarity of 16S rDNA sequence with Delftia tsuruhatensis PCL1755.The phenetypic data of isolate R7 was used as additional data to support the results of phylogenetic identification (Table 2).The strain is isolated from various soil types such as eggplant, tomato, pepper, and avocado and it showed a widespread inhibition to the growth of Fusarium oxysporum f. sp.radicis lycopersici [57,58].
The D. tsuruhatensis is one of the rhizosphere bacteria that act as a biocontrol agent or plant growth promoting rhizobacteria (PGPR) [59].Some strains of these bacteria have the ability to degrade the inorganic pollutants [60].The natural habitat of these bacteria are dispersed in soil, activated sludge, and also in contaminated environments.Bacteria D. tsuruhatensis was first isolated from active sludge and acted as degradation of terephthalate or plastic (environmental pollutants) [57,58,61].One strain of Delftia is D. tsuruhatensis HR4 had the ability to control disease in rice caused by Xanthomonas oryzae, Rhizoctonia solani, and Pyricularia oryzae [60,62,63].In some studies, although it has the ability as an antagonist agent against pathogens, the mechanisms of synthesis and antimicrobial compounds owned by these bacteria is still unclear.This is due to the lack of research on these bacteria.The strain of D. tsuruhatensis Pathogens of Bacterial Tuber Rot of A. muelleri (Arfani et al)

Figure 1 .
Figure 1.The Bacterial Diversity at rhizoSphere of A. muelleri Plantation.The same notation show diversity index does not significantly different among the sampling location (p> 0.05).

Figure 2 .
Figure 2. The Bacterial Density at Rhizosphere of A. muelleri Plantation.The same notation show cell density does not significantly different among the sampling location (p> 0.05) Pathogens of Bacterial Tuber Rot of A. muelleri (Arfani et al)

Figure 3 .
Figure 3.The Potency of A. muelleri Rhizosphere Bacteria to inhibit tuber rot bacteria (PT4.PL9, and PR11).The same notation show diversity index does not significantly different among the sampling location (p> 0.05) MTQ3 has the ability to inhibit the growth of Ralstonia solancearum and Phytophtohora nicotinae [58].

Figure 4 .
Figure 4. Phylogeny Tree of Rhizosphere Bacteria and Reference Isolates Based on 16S rDNA Sequence according to Neighbor-Joining Algorithm

Table 1 .
Enviromental Parameters at Sampling Locations