In-vitro endophytic bacteria activity against Ralstonia syzygii subsp. indonesiensis, the causal agent of bacterial wilt disease in tomato (Solanum lycopersicum) to achieve sustainable agriculture

The bacterial wilt disease transmitted through soil, identified as Ralstonia syzygii subsp. indonesiensis, is a substantial vascular disease and major constraint in various Solanaceae plants. The use endophytic bacteria-as Arthrobacter sp. and Bacillus thuringiensis-is one approach to control this pathogen. In vitro test using the disc paper method was conducted to evaluate the compatibility of Arthrobacter sp. and B. thuringiensis, along with their inhibitory capabilities through secondary metabolite production, in suppressing the growth of R. syzygii subsp. indonesiensis. The results showed incompatibility among isolates of endophytic bacterial, consequently, both endophytic bacteria were capable suppressing the growth of the R. syzygii subsp. indonesiensis significantly. Arthrobacter sp. formed a larger inhibition zone compared to B. thuringiensis, and both endophytic bacteria can be candidate biological control agents to reduce pesticide use to control R. syzygii subsp. indonesiensis, which therefore achieving more environmentally agriculture sustainability.


Introduction
The bacterial wilt disease transmitted through soil, identified as Ralstonia syzygii subsp.indonesiensis, is substantial vascular disease and major constraint in various Solanaceae plants [1].Controlling R. syzygii subsp.indonesiensis presents considerable challenges due to its ability to endure within residual host material and remain dormant in soil.Furthermore, it effortlessly propagates through water currents and exhibits mobility on the water's surface layer [2].A crucial element of a comprehensive pest management strategy is biological intervention.This approach involves leveraging plant defense mechanisms, such as stimulating plant resistance against pathogens [3].Induction of resistance by the provision of elicitors as external triggers in plants stimulates and activates the physiological conditions of the plant defense system [4].
The endophytic bacteria were one of solution for the management of agricultural productivity and has the ability to achieve agricultural sustainability given the current state of the environment [5].Utilized to enhance the plant resistance [6], enhance biodegradation [7], stress [8] and production of compounds for plant growth without degrading the environment [9] and thus reduced pesticide use in agriculture activity and protect the environment from hazardous chemicals.Looking for an eco-friendly IOP Publishing doi:10.1088/1755-1315/1302/1/012042 2 way to control plants pathogen is the major concern.The possible use of endophytic bacteria as biocontrol represents an alternative strategy motivated by a concern for sustainability [10].
Extensive research and development have been focused on utilizing microorganisms as agents for bio control [11].Endophytic bacteria are believed to have the capacity to bolster the plant's defense system against disruptions caused by plant diseases.This is due to their capability to generate antimicrobial substances, enzymes, salicylic acid, ethylene, and other secondary compounds that contribute to the stimulation of plant resistance [12].Certain rhizobacteria carry out the task of suppressing multiple phytopathogens through various mechanisms.They achieve this by competing for resources and habitat, elevating bacteriocin production, generating lytic enzymes, and synthesizing antibiotics such as Bacillus, Enterobacter, Herbaspirillum, Staphylococcus, Pseudomonas, Serratia, Burkholderia, Stenotrophomonas.and Ochrobactrum.These bacteria can effectively hinder the proliferation of phytopathogens [13].Hence, there exists the possibility to harness endophytic bacteria as biological control agents against plant pathogens.In this context, we conducted tests to assess the impact of Arthrobacter sp. and B. thuringiensis, both of which display antagonistic properties, on the inhibition of R. syzygii subsp.indonesiensis growth in in-vitro conditions.

Endophytic bacterial preparation
Isolates of Arthrobacter sp. and B. thuringiensis bacteria were acquired from the collection of plant disease laboratory, Universitas Syiah Kuala, Aceh.Isolates collected from agar plates were cultivated on nutrient agar (NA) media and incubated at 28 o C for 2 x 24 hours.Single colonies from the culture were grown in 10 mL of nutrient broth media and shaker (125rpm) for 2 x 24 hours.The resulting suspension was measured using a spectrophotometer, yielding a concentration of 10 8 cfu/mL [14].

Preparation of
Ralstonia syzygii subsp.indonesiensis pathogen R. syzygii subsp.indonesiensis were isolated from infected tomato plants in Barus Jahe village, Dolat Rayat subdistrict, Karo Regency, North Sumatra.The isolation process involved immersing the stems in sterile water until ooze appeared and streaked on Triphenyl Tetrazolium Chloride (TZC) medium.The medium contained 10 g amino acid, 1 g casein hydrolysate acid, 5 g glucose, in 1 L distilled water and added 12 g of agar powder.It was sterilized by autoclaving at 121 o C for 20 minutes.Five milliliter 1% TZC were added to the sterilized medium before pouring into plates.The culture was observed for parameters such as colony shape, color in TZC media, cell shape, gram strain test, KOH 3%, bacterial growth at 27 o C and 40 o C, starch hydrolysis, catalase test, and acid production from TSIA media as given by [1] to identify the R. syzygii subsp.indonesiensis.

Compatibility test among endophytic bacterial isolates
Isolates of endophytic bacteria were streaked on a petri dish containing Mueller Hinton Agar (MHA) medium.The medium contained 2 g beef extract, 1.5 g starch, 17.5 g casein hydrolysate acid in 1 L distilled water and added 17 g of agar powder then incubated for 24 hours at 28 o C, and the formation of inhibition zones between the two streaks of endophytic bacteria was observed.The observation indicator is a bacterial consortium is said to be incompatible if there is a zone of inhibiting between microbes, and in the contrast it is compatible if forming a clear zone [15] (Figure 1).

In-vitro antagonism test of endophytic bacteria against Ralstonia syzygii subsp. indonesiensis
The test was carried out using the Agar disk-diffusion method [16].Bacterial test suspension was inoculated onto MHA media using 0.1 mL (10 8 cfu/mL), spread and allowed to dry.Discs soaked in an endophytic bacteria suspension with a concentration of 10 8 cfu/mL for 15 minutes were placed on the media surface under aseptic conditions and repeated for 6 experimental replicates.Subsequently, an inhibition zones were observed around the disc paper.Subsequently, inhibition zones around the disc paper were observed according to [17], namely the diameter of the inhibition zone formed minus the diameter of the filter paper, observations were made every 12 hours for 2 days with formula (Figure 2):

Results and discussion
The results of this identification revealed that R. syzygii subsp.indonesiensis formed convex, round colonies with smooth edges, white periphery, and a red center on TZC media.This indicates that the bacterium falls within the virulent bacterial category [18].Based on the results of morphology, physiology and biochemistry test, R. syzygii subsp.indonesiensis was identified as a gram-negative bacterium, acid was produced oxidatively from glucose, sucrose, and lactose, and the species was able to utilize carbon sources and formed FeS in TSIA medium.R. syzygii subsp.indonesiensis grew well at a temperature of 27°C and exhibited catalase activity by breaking down hydrogen peroxide into water and oxygen (Table 1).The testing outcomes indicated similarities in characteristics between the tested bacterial isolates and R. syzygii as described by [19] and R. syzygii subsp.indonesiensis by [1] except hydrolysis test result.Hydrolysis test in this study was positive.This results was supported by the 4 description of R. syzygii subsp.indonesiensis by [1] who tested several strains of the R. solanacearum species complex bacteria which expressed the bacterial group phylotype IV R. syzygii subsp.indonesiensis which causes bacterial wilt in various Solanaceae plants.3a).The compatibility test results among endophytic bacteria revealed antagonistic activity, proven by the formation of inhibition zones observed between isolates (Figure 3b).Different bacterial isolates are considered compatible if no inhibition zones appear at the point of contact between the two endophytic bacteria isolates, indicating a synergistic interaction [20].Microorganism grow with various types of associations with other organisms, one of which is the antagonistic nature involving diverse mechanisms to impede the growth of other microorganisms.According to [21], the presence of inhibitory zones occurs due to extracellular metabolic reactions produced by one or both of the bacteria.These mechanisms can involve the production of toxins, antibiotics, or siderophores [22].

Antagonistic test of endophytic bacteria against Ralstonia syzygii subsp. indonesiensis
The antagonistic test of endophytic bacteria Arthrobacter sp and B. thuringiensis against R. syzygii subsp.indonesiensis was observed direct interaction and inhibition zones by endophytic bacteria around the disc paper against R. syzygii subsp.indonesiensis.0.00c 0.00c 0.00c The numbers followed by the different letters in the same column were significantly different according to the Duncan test at the 5% level.
Each endophytic bacterium had the ability to inhibit the R. syzygii subsp.indonesiensis pathogen.However, the most effective treatment observed was the treatment with Arthrobacter sp. in 10.12 mm where it showed a significantly larger inhibition zone (Figure 4b) compared to B. thuringiensis only 5.53 mm (Figure 4c).The inhibition zones formed by both endophytic bacteria were clearly visible and distinct at the 24 hours observation timepoint, but there was no increase in zone size at 48 and 72 hours Arthrobacter sp.

Bacillus thuringiensis
(Table 2).This suggests that these bacteria might have entered a stationary phase, wherein bacterial growth remains relatively constant or becomes indistinct.Some of physical and molecular changes take place during this phase and bacterial growth was stops but metabolically active [22] Consequently, the inhibition zone measurements between 48 and 72 hours became indistinct covered with the R. syzygii subsp.indonesiensis growth (Table 2).The small size of inhibition zones suggests that each bacterium possesses a different ability to produce a variety of antibiotic compounds or inhibitor substances as proposed by [23].Antagonistic microbes have inhibitory mechanisms that are not the same as each other, whether they originate from organic matter or the rhizosphere.Endophytic bacteria can prevent pathogens growth by producing secondary metabolites, which include hormones, toxins, enzymes, and volatile substances.Additionally, endophytic bacteria may act as a plant resistance determinant.The mechanism through which endophytic bacteria prevent pathogens growth is influenced by multiple factors.These factors include competing for resources and habitat, generating antibiotic substances, and producing lytic enzymes to impede pathogen growth and enhance plant resistance [24] through mediated ethylene and jasmonic acid transduction pathways [25] Bioactive compounds such as corticosteroids, alkaloids, isoprenoids, peptides, polyketones, flavonoids, quinone, and phenolic are produced in large quantities by B. thuringiensis [26].B. thuringiensis related studies report for toxicity against insect pests due to its entomopathogenic properties [27].Meanwhile B. thuringiensis shows effects on plant growth [28] and against various phytopathogens [29].In the case of plants colonized by B. thuringiensis interactions between plant tissues and B. thuringiensis trigger plant defense responses [30].These abilities suggest that B. thuringiensis can be evolved into a dual-purpose biopesticide for ecofriendly plant disease management.The mechanism of Arthrobacter sp involves the production of siderophore metabolite compounds [31].Siderophores such as salicylic acid and pyochelin (Pch) have involved in the ability to induced plant resistance and its produced by endophytic bacteria.The bacteria have a strong affinity for binding iron and can selectively block other opposing microorganisms [32] there by limiting pathogen R. syzygii subsp.indonesiensis growth.The difference was brought about by both the genetic material of the endophytic bacteria isolates that were employed and the composition of the chemical metabolites.The quantity of metabolites produced varies amongst microorganisms.Brader et al. [33] determined that the generation of broad-spectrum antimicrobial compounds by the bacteria and the concentration of the active ingredient determine variances in the degree of antagonism.
Antimicrobial metabolites compounds produced by endophytic bacteria can suppresses pathogen growth.Endophytic bacteria are considered as an effective biological control agent way to reduce pesticide use and can be an eco-friendly approach in sustainable agricultural practices.According to [6] Such bacteria can be inducers of plant defense mechanism in addition to providing direct antagonistic effects on pathogens.Bioactive substances produce by endophytic bacteria has been directly associated with the host.Some researchers reported the endophytic bacteria ability in inducing basic properties plant changes become more resistant.Another affect is that the host plant can early recognize of the pathogen through intercellular reactions and activated induced systemic resistance response (ISR) mechanism mediated ethylene and jasmonic acid transduction pathways [25].This allowed them to perform certain functions, such as protection from various types of pathogens and better adapt to the host plant [34].
Currently, chemical pesticides use without measuring the economic threshold of pests or diseases on agricultural land is an important problem in agriculture production and environmental health.Therefore, it takes effort to find environmentally friendly control solution to support sustainable agricultural subjects.For this purpose, endophytic bacteria are promising biological control agents, including Arthrobacter sp.[7], B. thuringiensis [27,28] or other well-known endophytic bacteria with their respective roles in this sector.As a result, these studies underscore the significance of specific host bacteria and also assert that these protective endophytic bacteria can exert antagonistic activities against various pathogens by utilizing well-known, safe endophytic bacteria to spread sustainable farming methods.

Conclusions and suggestions
The presence of antagonistic activity was evidenced by the formation of inhibition zones observed between isolates of Arthrobacter sp. and B. thuringiensis.Consequently, both endophytic bacteria were capable of significantly suppressing the growth of the R. syzygii subsp.indonesiensis pathogen treatment where Arthrobacter sp.formed a larger inhibition zone as compared to B. thuringiensis.Arthrobacter sp. and B. thuringiensis can be candidates as biological control agents to reduce pesticide use, to control pathogen R. syzygii subsp.indonesiensis for more environmentally agriculture sustainability.Further research is needed to know more and explore the effect of endophytic bacteria to controlled R. syzygii subsp indonesiensis in the field

3 Figure 1 .
Figure 1.Pattern of placement of in-vitro compatibility test (a) Antagonism (indentation and narrowing of inhibition Zone at) (b) Synergy (appearance of inhibition zone).

Figure 2 .
Figure 2. Pattern of placement of in-vitro antagonism test for endophytic bacteria and R. syzygii subsp indonesiensis.

Figure 3 .
Figure 3. Endophytic bacteria compatibility test (a) Placement patterns of endophytic bacteria in petri dishes; (b) Formed inhibition zones.
Figure 4a represents R. syzygii subsp.indonesiensis only as an untreated control.

Table 1 .
The results of morphology, physiology and biochemistry test for Ralstonia syzygii subsp.indonesiensis isolates.
3.1.Endophytic bacteria compatibility testIn culture compatibility studies, each plate carried both the test bacteria with Arthrobacter sp. in the center and B. thuringiensis streaks radiating from the center (Figure