Biochemical Characterizations of Selected Indigenous Endophytic Bacteria Potential as Growth Promoters and Biocontrol Agents on Tomato

Nine indigenous endophytic bacteria strains showing the best ability to promote growth and control tomato pathogens had been screened in our previous research. The strains’ biochemical properties, such as nutrition and other traits, must be characterized to design the best formulations for the strains’ biochemical properties. This study aimed to describe the biochemical characteristics of the selected indigenous endophytic bacteria. The variables observed were utilization of carbon sources (glucose, fructose, sucrose, lactose, glycerol, and olive oil), nitrogen sources (peptone, yeast extract, urea, NH4Cl, NH4SO4, and NH4NO3), and citrate, hydrolytic activities (urease, triple sugar iron, starch hydrolysis, gelatin hydrolysis, chitinase, cellulase, protease, lipase, and catalase), oxidative/fermentative assay, salt tolerance, and growth ability at 4° and 44°C. This study showed that all the endophytic bacteria strains characterized had various biochemical characteristics. All strains showed the different ability to utilize nitrogen and carbon source. Some strains survived to grow at 4°C except Bacillus cereus AGBE 1.2 TL. All strains tolerate growth in 4% NaCl concentrations, while some strains can tolerate up to 6%. This result can be used for further studies to develop the most suitable formulations for each strain to get the best results of the growth-promoting and biocontrol activities of the indigenous endophytic bacteria strains.


Introduction
The chemicals used over a long period to control bacterial diseases transmitted through the soil can result in severe environmental contamination [1]. Research on cleaner and safer technological alternatives to humans and the environment has received widespread attention recently. Research conducted shows that microorganism's use does not endanger the environment; therefore, it is safe for food and human health [2].
Naturally, plants can be associated with microorganisms in various ways. Endophytes are microorganisms inside the internal tissue of living plants [3]. According to the widely used definition, endophytic bacteria colonize the host tissue internally, sometimes in high numbers, without damaging the host or raising plant disease symptoms [4]. Some endophytic bacterial species have beneficial effects on plant growth and development by producing growth-promoting substances and/or by fixing nitrogen from the atmosphere and defense against pathogens through disease suppression with regulators encouraging plant growth and antibiotic production [5,6]. Endophytic bacteria can benefit the host plant generally by producing phytohormone, solubilizer phosphate, compounds such as flavonoids and antibiotics, or competence of invasion sites that suppress phytopathogens [7].
They are considering the benefits of intensive agriculture in the present time. The negative impact of chemical fertilizers and pesticides against the environment and usage of Plant Growth-Promoting Rhizobacteria (PGPR) as biofertilizers is one of the most promising biotechnologies used to increase the primary production, eliminating the need for chemical fertilizers [8]. Commercial application of PGPR to control soil-borne diseases depends on the development of commercial formulations in which bacteria can survive for a considerable length of time, the development of a suitable method of application to control pathogen establishment and disease development, and assessment of their efficacy under field conditions [9].
Our previous study had screened nine endophytic bacterial isolates showing the best performance in plant growth-promoting activities [10]. Those isolates also had the potential as biocontrol agents of Ralstonia and Fusarium wilt. Those isolates had been identified as To develop a successful formulation for the endophytic bacteria strains, the study of their biochemical characteristics and nutrient source utilization is necessary to increase the formula's efficacy. This study aimed to characterize the selected indigenous endophytic bacteria's biochemical characteristics for further studies to develop the most suitable formulations for each strain and get the best results of the indigenous's growth-promoting and biocontrol activities endophytic strains.

Biochemical Characterization
The biochemical tests consisting of several enzymatic and physiological activities, or differential growth tests conducted using standard methods [11,12]. Citrate utilization was determined on Simmons citrate agar, while the Oxidative/Fermentative (OF) assay was performed using OF media. Urease activity assay conducted using urea broth medium and phenol red as an indicator. Starch Hydrolysis Activity was tested using starch agar, and Gelatin Hydrolysis Activity was tested using gelatin agar. Meanwhile, Catalase activity was tested by dropping 3% H2O2 to the colony.
Chitinase activity was tested on the agar plates by the method described by Chernin et al. [13]. The semi-minimal medium composed of synthetic media and nutrient broth (3:1) was supplemented with 0.2% colloidal chitin and solidified with 1.5% agar for 48 h. The cellulase activity was tested on M9 medium [14] supplemented with yeast extract (0.12% w/v) and carboxyl-methylcellulose (CMC) (1% w/v) [15]. Isolates surrounded by clear halo after eight days of incubation at 28ºC were considered positive for cellulase production.

Carbon Source Utilization
Carbon source utilization was tested by the growth of the strains on bacto agar added with each carbon source (1%v/w of glucose, fructose, sucrose, lactose, glycerol, and olive oil) incubated for 48 h.

Nitrogen Source Utilization
Nitrogen source utilization was tested by the growth of the strains on bacto agar added with each carbon source (1%v/w of peptone, yeast extract, urea, NH4Cl, NH4SO4, and NH4NO3) incubated for 48 h.

3.1.Result
Based on the Simmon citrate agar assay, six strains showed positive results (Table 1). All the isolates showed the ability to grow under aerobic (oxidative) and anaerobic (fermentative) conditions. Biochemical characterization indicated that the endophytic bacteria showed different characteristics ( Table 1). Protease and catalase enzyme productions were detected in all strains. All the isolates had no urea degradation ability, indicated by urease production and ammonium ions' release into the medium.  Table 2).
The carbon and nitrogen source utilization assay (Table 3 and 4) was performed to determine the strains' growth only with sole nutrition source. The information about nutrition source utilization of the endophytic bacteria strains is necessary for developing the strain formulations. The formulations will act as the nutrition source of the strains before used for commercial biocontrol agents. All the strains were shown to have various patterns of both carbon and nitrogen source utilization. Still, all the isolates grew on the sole carbon source of glucose and fructose and a single nitrogen source of peptone and yeast extract. However, not all strains could use a mineral source of nitrogen from urea and ammonia salt.

Discussion
In the last decade, most agricultural research has been aimed at studying rhizosphere microorganisms [19]. Among these organisms, bacteria are the most technologically advanced in terms of both production and the use of microbial preparations in agriculture. It opens up the prospects for beneficial prokaryotic microorganisms for plant development under different conditions [20].
Endophytic bacteria are considered one of the options to control vascular wilt disease because of their ability to live and colonize within plants' roots. Our previous research had screened nine selected indigenous endophytic bacteria isolates that can promote growth rate [10]. Further research also confirmed that those strains suppressed R. syzigii subsp. indonesiensis, without developing any symptoms, five isolates could suppress symptoms of Foc [21]. The current study was conducted to characterize the biochemical characteristics of the selected indigenous endophytic bacteria. This present study can be used to develop the strains' ability for its new bio formulations development.
PGPR produces hydrolytic enzymes (chitinase, glucanase, protease, and cellulase). These enzymes have responsible for phytopathogens' lysis process through hyper-parasitism. The antagonistic characteristic of hydrolytic enzymes against various phytopathogens plays an essential role in biocontrol [22,23]. Our current study found that all the strains showed various hydrolytic activity ( Table 1). The hydrolytic action may directly impact the endophytic bacteria strain's ability to control pathogens [21].
Chitinase [EC 3.2.1.14] plays an essential role in the biocontrol of many plant diseases by lysing phytopathogenic fungal cell walls through the degradation of chitin polymers in cell walls [24]. Protease [E.C. 3.4.24] plays a vital role in phytopathogenic fungal cell wall lysis because chitin and/or β-glucan fibrils are embedded in the protein matrix, and proteolytic activity is a prerequisite for all fungal cells lysis [25]. Protease enzymes break down major proteins into chains of peptides and/or amino acids, making up phytopathogens and causing phytopathogenic ability to act on plant cells to be lost [24]. Bacillus sp. produce extracellular proteases. Several species of Bacillus such as Bacillus cereus, Bacillus stearothermophilus, Bacillus mojavensis, Bacillus megaterium, and Bacillus subtilis are known to produce proteases [26][27][28][29]. Cellulase [EC 3.2.1.4] catalyzes the hydrolysis of 1,4-β-D-glycosidic linkages in cellulose and plays an essential role in nature by recycling polysaccharides. Cellulose is a linear polymer unit of β-D-glucose linked through a 1,4-β relationship with polymerization rates ranging from 2,000 to 25,000 [30]. Cellulase causes cell wall degradation and further penetration into the host mycelium [31].
Several approaches have explained the optimization of biocontrol agents' formulations, including applying appropriate carriers and formulation additives. Ideal formulation additives must enhance biocontrol agents' ability without supporting pathogen growth or damaging host plants [32]. The formulation is a crucial step to develop microbes into biocontrol agents for plant pathogens. Burges and Jones [33] define biocontrol agents' formulation as a technique to preserve and send antagonists to their targets and increase their activity. Wiyono et al. [32] investigated the role of defined nutrients (Cand N-source) as formulation additives in Pf B5 ad planta's efficacy. In this study, it expanded in current research through the use of nitrogen compounds and trace elements. The use of nitrogen compounds in formulations has been reported for B. thuringiensis Berliner with D-L-tryptophan and L-tyrosine [34]. Tailor and Joshi [35] further state that urea as a nitrogen and tyrosine source as a carbon source are the most suitable for Siderophore production by S-11. Schmidt et al. [36] also suggested that peptone (1%) can increase the efficacy of B. subtilis (Ehrenberg) Cohn and Pantoea agglomerans against Eutypa lata in grapes (Vitis vinifera L.). Nitrogen fertilizer containing a mixture of NO3 and NH4 + can also increase the capacity of P. fluorescens to enhance plant growth and inhibit Fusarium growth in wheat (S. cereale) [37].

Conclusion
This study showed that all the endophytic bacteria strains characterized had various biochemical characteristics. All strains showed the different ability to utilize nitrogen and carbon source. Some strains survived to grow at 4 o C except Bacillus cereus AGBE 1.2 TL. All strains tolerate growth in 4% NaCl concentrations, while some strains can tolerate up to 6%. This study suggests promising