Bacillus subtilis from Potato Rhizosphere as Biological Control Agent and Chili Growth Promoter

Bacteria associated with plants may have a role as growth promoters and improve plant health. PGPR (plant growth–promoting rhizobacteria) is a root colonizing rhizosphere bacteria that is able to increase plant growth. The ability of bacteria as PGPR directly is to stimulate plant growth, and indirectly to control pathogens. Biological control is to reduce the use of synthetic pesticides that have a negative impact on the environment, non–target microorganisms and may cause pathogenic resistance. Compant et al. (2010) stated that the bacterial-plant association in the natural ecosystem plays a role in improving plant health and growth. Some rhizosphere bacteria (rhizobacteria) can pass the roots and live as endophytic populations by producing IAA (Indole-3-Acetic Acid) to stimulate plant growth (Dawwam et al., 2013; Dwimartina et al., 2017) and the others bacteria can pass through the endodermis barrier by the root cortex to the vascular system, and then become endophytic in the stems, leaves, tubers, and other plant organs (Compant et al., 2005). The roles of rhizobacteria according to Kesaulya et al. (2015) are as bioprotectant (suppress plant diseases), biofertilizer (improve the absorption of nutrients for plants), and biostimulant (produce phytohormone).Rhizobacteria can suppress disease by antagonistic mechanisms against soil infectious pathogens or induce plant systemic resistance to root and leaf pathogens. This resistance is generally not specific to certain pathogens, yet under natural conditions of several pathogens simultaneously. According to Ahemad & Kibret (2014), siderophore pseudomonine can induce plant resistance associated with salicylic acid. The number of rhizobacteria that able to spur plant growth is only 2–5% of all the population (Chaiharn et al., 2008). The mechanism of rhizobacteria directly to improve the plant growth are by obtaining phosphate ABSTRACT


INTRODUCTION
Bacteria associated with plants may have a role as growth promoters and improve plant health. PGPR (plant growth-promoting rhizobacteria) is a root colonizing rhizosphere bacteria that is able to increase plant growth. The ability of bacteria as PGPR directly is to stimulate plant growth, and indirectly to control pathogens. Biological control is to reduce the use of synthetic pesticides that have a negative impact on the environment, non-target microorganisms and may cause pathogenic resistance. Compant et al. (2010) stated that the bacterial-plant association in the natural ecosystem plays a role in improving plant health and growth. Some rhizosphere bacteria (rhizobacteria) can pass the roots and live as endophytic populations by producing IAA (Indole-3-Acetic Acid) to stimulate plant growth (Dawwam et al., 2013;Dwimartina et al., 2017) and the others bacteria can pass through the endodermis barrier by the root cortex to the vascular system, and then become endophytic in the stems, leaves, tubers, and other plant organs (Compant et al., 2005). The roles of rhizobacteria according to Kesaulya et al. (2015) are as bioprotectant (suppress plant diseases), biofertilizer (improve the absorption of nutrients for plants), and biostimulant (produce phytohormone).Rhizobacteria can suppress disease by antagonistic mechanisms against soil infectious pathogens or induce plant systemic resistance to root and leaf pathogens. This resistance is generally not specific to certain pathogens, yet under natural conditions of several pathogens simultaneously. According to Ahemad & Kibret (2014), siderophore pseudomonine can induce plant resistance associated with salicylic acid.
The number of rhizobacteria that able to spur plant growth is only 2-5% of all the population (Chaiharn et al., 2008). The mechanism of rhizobacteria directly to improve the plant growth are by obtaining phosphate (dissolve phosphate), fixating N 2 , and producing phytohormones such as IAA (Adesemoye et al., 2009). The rhizobacteria may have one or more mechanisms, either sequentially or simultaneously actively affecting different phases of plant growth (Saharan & Nehra, 2011). The mechanism of rhizobacteria indirectly is as a biological control agent of plant pathogens by producing antibiotics, lytic enzymes, hydrogen cyanide, and siderophore, or through nutritional competition and space that significantly improve plant health and stimulate plant growth by increasing germination, vigor and crop yields (Chaiharn et al., 2008;Mishra & Kumar, 2012). Therefore, this study was aimed to evaluate the potential of B. subtilis isolates B209, B211, and B298 as biological agents to control anthracnose and as PGPR to increase the growth of chili plants.

MATERIALS AND METHODS
This research was conducted in the Laboratory of Plant Protection, Faculty of Agriculture, Universitas Jenderal Soedirman, in Purwokerto and the experimental field in Rempoah Village, Baturraden District, Banyumas Regency over 7 months. The inhibitory test of anthracnose pathogenic fungi, characteristics of three B. subtilis isolates as PGPR were conducted in the laboratory while testing the ability of B. subtilis to suppress anthracnose and increase the growth of chili plants were conducted in the field.

B. subtilis as a Biological Control Agent for Anthracnose Pathogenic Fungi Test
B. subtilis was tested for its ability as a biological control of plant pathogenic fungi in vitro using the dual culture method by Wang et al. (2013) by growing both in a 9 cm diameter petri dish. Inoculation of B. subtilis antagonist bacteria was carried out after the pieces of the mycelium Colletotrichum gloeosporioides (chili anthracnose pathogen) were cultured. This plate which contained two microbes was incubated at room temperature (28 ± 2) o C for 7 days to observe its inhibition. The percentage of inhibition was calculated by measuring the growth of fungal colonies (Wang et al., 2013): I: inhibition; C: the growth of control fungus mycelium (in the opposite direction to antagonistic bacteria); Q: fungal mycelium growth that leads to antagonists.

Phosphate Solvent Activity Test
The ability of Bacillus isolates to dissolve phosphate was conducted using potato-dextrose yeast extract agar (PDYA), containing 50 ml 10% (w/vol) K 2 HPO 4 and 100 ml 10% (w/vol) CaCl 2 . A liter of sterile PDYA was added to produce CaHPO 4 precipitation. Each bacterial culture was inoculated in the line middle of the PDYA-CaP at 3 points and incubated at room temperature for 10 days. Phosphate solvents were assessed by measuring the clear zone. Zone calculation is the total clear zone reduced by the diameter of the bacterial colony (de Freitas et al., 1997).

IAA Production Test
IAA production test was employed according to Shrivastava & Kumar (2011). The three isolates of B. subtilis were grown on YPGA medium with and without L-Tryptophan (0.5%) at room temperature for 48 hours, dropped with 1 ml of Salkowski reagent A [1:50 from 0.5M FeCl 3 and 35% perchloric acid (HClO)], then observed until a reddish-pink was formed which indicates diffusion of the agar medium. The variation in color-changing becomes reddish-pink showed the difference in the amount of IAA produced. This test was repeated 3 times to obtain consistency of IAA produced by the isolate.

Nitrogen Producing Activity Test
This test was carried out by growing five isolates of B. subtilis on a nitrogen-free broth medium (Xie et al., 2003) by shaking for 24 hours at room temperature. The total nitrogen in culture was calculated using the micro Kjeldahl method, which is based on the principle of sample digestion, distillation, and titration. Digestion is the decomposition of organic samples using a solution of sulfuric acid to produce a solution of ammonium sulfate, distillation by adding a base thus changing NH 4 + to NH 3, followed by boiling and condensing NH 3 gas in the solution. Furthermore, titration that produces ammonium with changes in color and total N concentrations can be measured using autoanalysers (Amin & Flowers, 2004).

Antibiotic Resistance Test
Antibiotic resistance test was conducted to determine whether B. subtilis is susceptible or resistant to antibiotics. One plate containing the suitable medium was incubated by spreading with different density bacteria, a paper-disc containing different concentrations of antibiotics was placed on the plate, then incubated at room temperature for 3 days. The presence of inhibition zones around the paper-disc at different antibiotic concentrations was recorded. Each treatment was repeated 3 times. The antibiotics used were Chloramphenicol 10 and 30 µg/ml, Streptomycin 10 µg/ml, Kanamycin 5 and 30 µg/ml, Penicillin 10 µg/ml, and Tetracyclin 30 µg/ml, and Rifampicin 10 and 30 µg/ml.

RESULTS AND DISCUSSION
The role of B. subtilis B298 as a biological control agent of C. capsici and C. gloeosporioides was shown in Figure 1, with inhibition percentages of 57.6 and 64.6%, respectively against C. capsici and C. gloeosporioides. In vitro test for controlling of Colletotrichum sp. chemically with 800 ppm mancozeb using poisoned food technique showed inhibition of colony growth by 98%, while with cymoxanil 2000 ppm only suppressed 37.2% (Paramita et al., 2014). These results showed that B. subtilis B298 could be used as a biological control agent of plant pathogenic fungi through the mechanism of antibiosis by producing the enzyme chitinase to inhibit the growth of pathogenic fungi whose cell walls consist of chitin. B. subtilis B298 is able to produce the chitinase enzyme as study conducted by Lestari et al. (2017), with an activity of 6.937 U/ml at 15 hours incubation, 5.764 U/ml at 40 o C, and 6.813 U/ml at pH 5. B. amyloliquefaciens SAHA 12.07 showed chitinase activity at pH incubation 5 with an activity of 1.158 U/ml (Azizah et al., 2015).
The characteristics of B. subtilis as PGPR are its ability to be able to produce secondary metabolites (consisting of enzymes and antibiotics), IAA, and nitrogen; and as a phosphate solvent bacterium. The observation of the ability of B. subtilis as PGPR (Table 1) revealed that the three B. subtilis isolates capable as PGPR, which are proven to be able to dissolve phosphate and produce IAA, nitrogen and antibiotic resistance. B. subtilis B298 showed the strongest isolate as phosphate solvent (the widest clear zone), the highest producer of IAA (dark red color) than other isolates. The total N activity produced by B. subtilis B298 isolate was 10.34 µg/ml which was produced based on micro Kjeldahl nitrogen analysis.
The three isolates of B. subtilis (B209, B211, and B298) which were explored from the rhizosphere of healthy potatoes showed their ability as phosphate solvents by forming the celar zones around the B. subtilis colonies on Pikovskaya with zone sizes varying from 1.2 to 4 mm ( Figure 2). P is an important nutrient for plants, abundantly available in the soil, for example in Ultisol or red-yellow podsolic soil or as a result of the use of phosphate fertilizer. However, P is in the form of minerals that are slowly available to plants. According to Gupta et al. (2014), the phosphate would bind to Al and Fe in acid soil and Calcium phosphate in alkaline soil. The presence of rhizosphere bacteria can dissolve phosphate from the insoluble P to be dissolved which is characterized by its capacity to decrease pH by secretion of organic acids such as gluconate, citrate, lactate, succinate, and as protons during NH 4 + assimilation. These rhizosphere bacteria are Bacillus, Burkholderia, Enterobacter, Klebsiella, Kluyvera, Streptomyces, Pantoea, and genus Pseudomonas.
The ability of B. subtilis as IAA-producing was shown in Figure 3, with a red discoloration after adding Salkowski reagent to pure culture of B. subtilis, and B. subtilis B298 isolate showed the strongest reaction. Antibiotic resistance was tested to justify the resistance of B. subtilis to certain antibiotics hence the antibiotic can be used as a marker to test the presence of B. subtilis after application. Three B. subtilis isolates are resistant to Rifampicin compared to 4 other antibiotics, i.e. Kanamycin, Streptomycin, and Chloramphenicol. Resistance to antibiotics is indicated by the absence of zones around the paperdisc have been added 10 µl antibiotic in an agar plate contained B. subtilis with was grown by the pour plate method (Figure 4).    Table 3. The disease intensity of anthracnose in the field after application of Bacillus subtilis and fungicide Remarks: Values followed by the same letters in the same column in the same column were not significantly different according to LSD (α = 5%); DAP = day after planting.
anthracnose disease with the highest effectiveness of 80.36% by the treatment of B. subtilis B209.

ACKNOWLEDGEMENT
The author would like to thank to DRPM Kemenristekdikti for funding this research in 2016 with the contract number of 1967/UN23.14/PN/2016.