Consortium solid formula of bacillus spp. to control bacterial wilt on chili plants

. Due to its high economic worth and ability to be consumed in both fresh and processed forms, chili (Capsicum annum L.) is a fruit that is frequently used as a vegetable. Pests and plant diseases, such as bacterial wilt caused by Ralstonia syzygii subsp. Indonesiensis, are the causes of reduced chili productivity. Plants will eventually perish as a result of symptoms like withering of the leaf tips that eventually spread to the undersides of the leaves. The lower vascular tissue and roots of the affected chili plants appear brown. Bacterial wilt is an important disease of chili caused by Ralstonia syzigii subsp . indonesiensis and can reduce yield by up to 90%. The research aimed to obtain the best solid formula consortium of Bacillus spp. for controlling R. syzygii subsp. indonesiensis on chili peppers. The study consists of 2 stages: 1). Production of a consortium combination of Bacillus spp. in solid waste 2). Test the ability of the consortium of Bacillus spp. in solid waste to control bacterial wilt in chili plants. The study used a completely randomized design that consisted of 19 treatments and four replications. Each treatment was introduced on chili seeds and seedlings, while the inoculation of R. syzygii subsp. indonesiensis was conducted on chili aged 35 days after planting. The results showed that the best solid formula consortium of the Bacillus spp. for controlling bacterial wilt ( R. syzigii subsp. indonesiensis ) on chili plants is the Bran + Bagasse formula for four weeks of storage.


Introduction
Chili (Capsicum annum L.) is a fruit often used as a vegetable because it has high economic value and is consumed in fresh or processed form [1]. Factors causing low chili productivity are pests and plant diseases, including bacterial wilt by Ralstonia syzygii subsp.indonesiensis [2].Symptoms such as wilting of the tops of the leaves then spread to the underside of the leaves, and the plants become wilted; eventually, the plants will die.The attacked chili plants show that the lower vascular tissue and roots look brown [3].
Bacterial wilt control has been carried out through cultural measures, resistant varieties, sanitation, and crop rotation [4], and chemical disease control using bactericidal [5].However, the use of bactericidal is not only economically ineffective.However, it can potentially cause adverse environmental impacts [6], so alternative control is needed by utilizing microorganisms as biological control agents [7].
One currently developed biological agent for controlling plant pathogens is from the Plant Growth Promoting Rhizobacteria (PGPR) group [8].One of the biological agents classified as PGPR is Bacillus spp.[9].The Bacillus spp group.which have been extensively studied as biological agents, namely B. thuringiensis, B. cereus, and B. toyonensis [10].The use of Bacillus sp.singly causes Bacillus sp.cannot to last long and is not optimal either as a biocontrol agent or as a bioactivator [11].Bacillus spp.can inhibit the growth of the fungus Fusarium oxysporum.Furthermore, B. toyonensis strain AGBE1.2.TL has the potential as a growth promoter for chilli plants and can control anthracnose disease [12].
The study aimed to obtain a solid formula for the consortium of Bacillus spp.for controlling bacterial wilt (Ralstonia syzygii subsp.indonesiensis) on chili plants.

Time and place
The research was carried out from January to April 2023.This research was conducted at the Microbiology Laboratory and the Experimental Garden of the Faculty of Agriculture, Universitas Andalas

Confirmation of B. cereus strain SLBE3.1AP and B. toyonensis strain AGBE 1.2.TL
Confirmation of B. cereus strain SLBE3.1AP and B. toyonensis AGBE 1.2.TL strain was carried out by Gram test to determine whether the bacteria were gram-negative or positive.3% KOH solution was dripped on a glass slide, then one single colony of pure cultures of B. cereus strain SLBE3.1AP and B. toyonensis strain AGBE 1.2.TL was taken with an ose needle and mixed [13].Then a hypersensitivity reaction (HR) test was carried out using a suspension of B. cereus strain SLBE3.1AP and B. toyonensis strain AGBE 1.2.TL with a density of 108 CFU/ml infiltrated into the undersurface tissue of Mirabilis jalappa leaves and incubated for 2x24 hours [14].

Propagation of the Bacillus spp. ( B. cereus strain SLBE3.1AP and B. toyonensis strain AGBE 1.2.TL) Consortium
Bacillus spp.multiplied in liquid culture aged 2 x 24 hours was taken, put into 25 ml NB medium in culture bottles (volume 50 ml), and incubated on a rotary shaker for 24 hours.Furthermore, 1 ml of preculture results were transferred into 49 ml sterile coconut water in a culture bottle (100 ml volume).The second stage of the main culture, the Bacillus spp consortium, was created by combining two compatible Bacillus spp.Furthermore, 1 ml of liquid culture of each species of Bacillus spp.(preculture results) was transferred into 23 ml of sterile coconut water in a culture bottle (50 ml) and incubated for 2x24 hours on a rotary shaker with a speed of 150 rpm at room temperature [12].The population density was determined by comparing the turbidity of the bacterial suspension with that of McFarland's scale 8 (estimated population density of 10 8 cells ml-1) [14].

Multiplication of solid formula carrier materials for Bacillus spp ( B. cereus strain SLBE3.1AP and B. toyonensis strain AGBE 1.2.TL)
Solid waste carrier materials are bagasse, rice bran, and rice straw, mashed using a blender.Each carrier material was put into a Schott bottle with a volume of 100 ml totaling 9.5 g.Then 0.5 grams of sucrose (5% of the total media weight) was added to each formula and sterilized by autoclaving at 121°C with 1 atm pressure for 15 minutes.After that, the formula ingredients were cooled, and 5 ml of main culture suspension 10 8 cells/ml was added.Bacillus spp.

Inoculum preparation of R. syzygii subsp. indonesiensis
The source of the inoculum was obtained from chili plants with bacterial wilt symptoms.Isolation of R. syzygii subsp.indonesiensis by removing the bacterial mass from the plant's vascular tissue by cutting the base of the plant stem and putting it in sterile distilled water for 10 minutes.The bacterial mass was cultured purely by streaking on a TZC medium to determine the virulent colonies of R. syzygii subsp.indonesiensis, then incubated for 2x24 hours.

Pathogenicity Test
Pathogenicity test of R. syzygii subsp.Indonesiensis, namely wounding, and watering of of R. syzygii subsp.Indonesiensis suspension on chili plant roots aged 35 DAP.Loosening the soil first, the roots are cut on both sides of the plant using scissors.Furthermore, 20 ml of of R. syzygii subsp.Indonesiensis suspension with a density of 107 cells/ml was sprinkled on the roots of the chili plants that had been injured [10].

Propagation of R. syzygii subsp. isolates indonesiensis
Bacteria were propagated using the streaking technique on TZC media, then incubated for 2x24 hours.Isolate R. syzygii subsp.indonesiensis was suspended using sterile distilled water with a density of 10 7 cells/ml then McFarland's solution scale seven was used as a comparison.

Preparation of planting media
The planting medium used was a mixture of soil and manure (2:1 v/v).The planting medium is put in a plastic bag and then sterilized in a boiler for 1 hour at 100 0 C. The planting medium was put 5 g into a pottray for seeding and 10 kg into a polybag for planting [10].

Introduction of formulation for Bacillus spp. Consortium
Formula introduction of Bacillus spp. was carried out two times.Introductions were made to chili seeds and seedlings.

Inoculation of R. syzygii subsp. isolates Indonesia
The bacteria R. syzygii subsp.indonesiensis was inoculated on chili plants aged 35 DAP by cutting the roots on both sides of the plant at a distance of 5 cm from the stem.After that, 30 mL of the bacterial suspension of R. syzygii subsp.indonesiensis, with a population density of 10 7 cells/ml, was sprinkled on the plants [15].

Observation
Observations were made on the viability test of the Bacillus spp consortium solid waste formula, disease development, chili seedling growth, and plant growth.Observation of disease development includes the incubation period, incidence, and severity.Observation of seedling growth included field emergence, seedling height (cm), number of seedling leaves, wet seedling weight, and dry seedling weight.Observations on the growth of chili plants included plant height (cm), number of leaves, and first flower appearance.Data were analyzed using analysis of variance, if significantly different, then continued with Duncan's New Multiple Range Test (DNMRT) at a 5% level.

Results and discussion
All consortia of Bacillus spp. in different solid waste storage extended the incubation period compared to the negative control (Table 1) The Bacillus spp.consortium in different storage solid wastes suppressed the incidence of bacterial wilt disease and significantly differed from the negative control (Table 2).Treatment of Bran + Rice Straw 4 and 6 weeks storage, Bran + Bagasse + Rice Straw 4 weeks storage, and Bran + Bagasse 4 weeks storage are formulas that have the potential to reduce the incidence of bacterial wilt disease and show significantly different effects compared to negative control and streptomycin with a disease incidence rate of 0.00%.The highest incidence of bacterial wilt was found in the negative control and streptomycin, namely 100% (Table 2).are treatments that have the potential to reduce the severity of bacterial wilt and show significantly different effects compared to negative controls with a disease severity value of 0.00-19.14%.Negative control was the treatment with the highest bacterial wilt disease severity, with a disease severity value of 100% (Table 3).The Bacillus spp consortium in different storage solid wastes increased the height of chili plants and significantly differed from the positive control (Table 4).treatment Bran + Bagasse +Rice Straw 4 weeks storage, Bran + Rice Straw 4 weeks storage, Bran + Bagasse +Rice Straw 0 weeks storage, Sugarcane Bagasse + Rice Straw 4 weeks storage, Bran + Rice Straw 6 weeks storage, Bran + Rice Straw Sugarcane dregs stored for 4 weeks, Bagasse + Rice Straw stored for 8 weeks, and Bran + Rice Straw stored for 8 weeks are treatments that have the potential to increase plant height with seedling heights ranging from 11.20-11.55cm.The lowest chili plant seed height was found in the positive control, which was 9.33 cm (Table 4).The Bacillus spp.consortium in different storage solid wastes increased the number of chili seedling leaves and significantly differed from the positive control (Table 5).Treatment of Bran + Rice Straw storage for 4 weeks, Bran + Bagasse + Rice Straw without storage, 4 weeks, and 8 weeks had the potential to increase the number of leaf seedlings.It significantly differed from the positive control and 39 streptomycin with a leaf number range of 6,25-7, 00 strands.The lowest number of chili plant seed leaves was found in the positive control, namely 5.25 leaves (Table 5) The Bacillus spp.consortium in different solid waste storage on chili plants was able to extend the incubation period and reduce the incidence and severity of bacterial wilt disease compared to the negative control (inoculated with Ralstonia syzigii subsp.indonesiensis) and Streptomycin.Four treatments that have the potential to control Ralstonia syzigii subsp indonesiensis until the end of the observation (42 dai), namely Formula Bran + Bagasse stored for 4 weeks, Bran + Rice Straw stored for 4 weeks and 6 weeks, and Bran + Bagasse + Rice Straw stored for 4 weeks.It was allegedly due to the nutrient content of the carrier material supports the bacteria in maintaining its population during storage, which completeness of nutrition is the key to the success of each treatment in controlling plant pathogens and Bacillus spp.also produces secondary metabolites, such as antibiotics, siderophores, bacteriocins, and extracellular enzymes so that they can inhibit the growth of Ralstonia syzigii subsp.indonesiensis.Bacillus spp.can also induce plant resistance compounds and act as a Plant Growth Promoting Rhizobacteria (PGPR) [16].Four formulations of Bacillus spp.capable of inhibiting the growth of the pathogen Xanthomonas oryzae pv.oryzae, which shows the occurrence of an antibiosis mechanism in which Bacillus spp., in the process of metabolism, produces antibiotic compounds that are secreted when bacteria form a stationary phase and produce enzymes such as chitinase enzymes, mycobacillin, bacitracin and others [17].Furthermore, tofu dregs and tofu dregs + corn cobs are the best formulae for reducing root rot disease (Sclerotium rolfsii) in tomato plants [18].
Introduction to the Bacillus spp.consortium in different storage solid waste on chili seeds can increase the seedlings' height and the number of leaves of chili seedlings compared to the positive control.The best treatment to increase the growth of chili seedlings was the Bran + bagasse treatment which was stored for 4 weeks.It was thought to be due to the content of the carrier material, which provided nutrients for Bacillus spp. in interactions with plants.It is in accordance with the statement regarding solid waste containing organic carbon (carbohydrates) and organic nitrogen (proteins and amino acids), which are both used as a source of energy and for the growth of Bacillus spp.further supported by the statement of Asmin and Karimuna [19], Rice straw is an organic material that can bind Fe 2+ ion solution, so K + has a great opportunity to be absorbed by plant roots and become a source of plant nutrients.Utilizing bagasse as organic material has the potential to become compost which can replace inorganic fertilizers and is beneficial for plant growth [20].Bran provides nutrients that are very important for plant growth [21].Bacillus spp. reported that as a bacteria inducing plant resistance and as a Plant Growth Promoting Rhizobacteria (PGPR), which can increase plant growth [22].

Conclusion
Bacillus spp..consortiumformula. in solid waste, the best for controlling bacterial wilt by Ralstonia syzigii subsp.indonesiensis and increasing the growth and yield of chili plants, namely the Bran + Bagasse formula for 4 weeks storage.

Table 1 .
The incubation period of bacterial wilt on chili peppers introduced by the consortium of Bacillus spp. in solid waste with different storage times.

Table 2 .
The incidence of bacterial wilt in chili plants introduced by the Bacillus spp.consortium. in solid waste with different storage times.The Bacillus spp.consortium in different storage solid wastes suppressed the severity of bacterial wilt disease and significantly differed from negative controls.Treatment Rice Bran + Rice Straw without storage, 4 weeks, 6 weeks and 8 weeks, Bran + Bagasse without storage, 4 weeks, 6 35 weeks and 8 weeks, Bagasse + Rice Straw without storage, 4 weeks, 6 weeks and 8 weeks, Bran + Bagasse + Rice Straw without storage, 4 weeks, 6 weeks and 8 weeks

Table 3 .
The severity of bacterial wilt in chili plants introduced by the Bacillus spp.consortium on solid waste with different storage times (42 dai)

Table 4 .
Height of chili seedlings introduced to the Bacillus spp.consortium on solid waste with different storage times(21 das) * Numbers followed by the same lowercase letter in the same column are not significantly different according to DNMRT at the 5% level.

Table 5 .
The number of chili seedlings introduced to the Bacillus spp consortium on solid waste with different storage times (21 das).