Potential of Local Bacillus spp. Isolates as Wilt Disease Biocontrol Agents for Fusarium (Fusarium oxysporum f. sp. cepae) on Wakegi Onions (Allium × wakegi Araki)

The use of biological agents as a biocontrol against plant pathogens was often ineffective because it worked slowly. The objective of this research was to examine the potential of local isolates of Bacillus spp. as a biocontrol agent in suppressing Fusarium wilt disease (Fusarium oxysporum f. sp. cepae) on wakegi onions. The research was designed using a completely randomized design with the treatment of rhizosphere bacterial isolates. The treatments consisted of control (without isolate application), isolates KP17, KP5, DB9, DB12, DB18, DG4, and DG11 so that the number of treatments was eight. Each treatment was repeated 5 times and each replication consisted of 10 wakegi onion plants. This research was divided into 2 stages, namely laboratory research which included isolation, characterization of colony morphology of rhizosphere bacterial isolates, and in vitro testing of the inhibitory power of biocontrol agents against pathogens. The test in the greenhouse was in the form of a disease case suppression test. The results obtained seven candidate isolates of biocontrol from 46 isolates obtained from the rhizosphere of the wakegi onion. These isolates had similarities with Bacillus spp. based on colony morphology, physiology, and biochemistry characteristics. Among the isolates found, the DB12 isolate had the potential to be developed as a biocontrol agent compared to other isolates.

124 Isolation of antagonistic bacteria was done using the serial dilution method and was specific 125 for Bacillus spp. according to [17,18,19]. Cultures were incubated at room temperature for 24 126 to 48 hours. The grown colonies were then purified using the same medium and tested for 127 hypersensitivity reactions, colony morphology characterization, as well as physiological and 128 biochemical properties testing.

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Hypersensitivity reaction test 130 A hypersensitivity reaction (HR) test of antagonistic bacteria isolates to tobacco plants was 131 done according to Asrul et al. [20]. The isolates that caused a negative reaction (no symptoms 132 of necrosis but the leaves remained green) in the inoculated tobacco leaf area were then tested 133 for antagonists to determine their inhibitory power.  146 The inhibition power test of rhizosphere bacteria against Foc pathogenic fungi (laboratory 147 collection) was done using dual culture technique according to Jimtha et al. [26]. The 149 bacterial colonies. Cultures were incubated at room temperature and antagonism interactions 150 were observed at 7 days after inoculation (dai). Percentage of rhizosphere bacteria inhibition 151 was calculated by the formula [27]: P = (R -r)/R × 100%, where P is the percentage of 152 antagonist inhibition power against pathogens, R is the maximum radius of the pathogenic 153 colony stay away from the rhizosphere bacterial colony (cm), and r is the radius of the 154 pathogenic colony opposite with the rhizosphere bacterial colony (cm).

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Isolate candidates of rhizosphere bacteria that showed inhibition power greater than 60% 156 were antagonist isolates classified as having effective inhibition activity.

Preparation of Antagonist Bacterial Suspension
160 The suspension was done aseptically utilizing colonies of rhizosphere bacteria on rejuvenation 161 media aged 48 hours taken using an Ose needle and suspended in a tube containing 10 ml of 162 sterile water. Furthermore, the suspension was made into the mother liquor to calculate the 163 density of the colony population, which was done by the spread dish method. A series of culture 164 tubes containing 9 ml of sterile distilled water were taken, and serial dilutions of 10 -5 , 10 -6 , and 165 10 -7 were made. A total of 1 ml from each dilution series was pipetted and grown on NA 166 medium to which 100-ppm cycloheximide was added to suppress fungal growth. Each dilution 167 series was repeated three times. Bacterial colonies that grew were also counted to determine 168 their population density. Next, the suspension was diluted again to obtain a population density 169 of 108 CFU/ml and the bacterial suspension was ready to be applied.    Figure 5B, it could be seen that   Based on the physiological and biochemical characteristics, the local isolates were gram-361 negative bacteria, aerobic or fermentative anaerobic, with positive oxidation, positive catalase, 362 did not produce fluorescent pigments, and were able to grow at a temperature of 65°C (Table  364 that (Table 4).

Preparation of Pathogenic Fungal Suspension
376 In Table 4, it can be seen that the application of local isolates gave a significantly longer 381 incubation period than the control. In the control, the incubation period required for the 382 appearance of the first symptoms was 5.45 dai, while in the application of local isolates there