A study on the potential of Bacillus thuringiensis AK08 to control pathogenic fungi associated with chili plant

Colletotrichum sp. and Fusarium sp. are significant fungal plant pathogen that affect chili plants. Various techniques are employed to manage the plant pathogens, including the application of biological agents such as Bacillus thuringiensis (Bt). This study aims to evaluate the efficacy of Bt AK08 in managing Colletotrichum sp. and Fusarium sp. Bt AK08 was subjected to dual-culturing experiments with the pathogens individually. Positive controls were employed in this study, consisting of Colletotrichum sp. and Fusarium sp. that were cultivated separately on pure potato dextrose agar. Propineb supplemented growth medium was serves as a negative control. The study involved the assessment of colony diameter of Colletotrichum sp. and Fusarium sp., as well as the evaluation of the antagonism activity exhibited by Bt AK08 through antibiosis activity and hyphal lysis. The findings indicated that Bt AK08 did not exhibit any antibiosis action since it did not result in a reduction in the colony diameter of the plant pathogenic fungus. However, we observed the breakdown of the hypha of the fungi. The present discovery suggests that Bt AK08 has promise as a potential agent for the management of Colletotrichum sp. and Fusarium sp. Hence, additional investigation is necessary to maximize its potential.


Introduction
Colletotrichum capsici and Fusarium oxysporum are the main fungal pathogens that attack chili plants.The pathogen C. capsici is responsible for the development of anthracnose disease, affecting various plant parts such as seeds, leaves, branches, and fruits, both prior to and after harvest According to the Ministry of Agriculture [1], the incidence of anthracnose disease in chilli crops can result in significant yield reductions, ranging from 20% to 90%, particularly in periods of increased rainfall.
In a comparable manner, the fungal species Fusarium oxysporum, known for its ability to induce fusarium wilt disease, has a pathogenicity that spans from the initial stages of germination to the mature phase, so leading to substantial reductions in crop productivity, with potential losses of up to 50% or complete crop failure, thereby imposing considerable economic burdens on agricultural practitioners [2,3].
Several strategies have been identified as efficient means of mitigating the severity of anthracnose and fusarium wilt diseases.These include the utilisation of synthetic fungicides, implementing crop rotation practises, and employing resistant cultivars [4,5].As for biological control, employing antagonistic agents like Bacillus thuringiensis, has been the subject of substantial investigation as an additional control strategy.Bacillus thuringiensis (Bt) is a bacterium that forms spores and is commonly found in soil, water, and on plant surfaces.Bt has been utilised as a means of pathogen control due to its ability to synthesise many chemicals, including antimicrobial substances such as beta-exotoxin, antibiotics, degradative enzymes, bacteriocins, and signal molecules involved in the bacterial quorum-sensing system [6].The inhibitory impact of Bt on phytopathogenic fungi can be attributed to the secretion of chitinase enzymes, which have the ability to degrade the cell walls of these fungi.The efficacy of Bt has been the subject of significant research in the control of many diseases, including Rhizoctonia solani, F. oxysporum, Meloidogyne, C. capsici, Phytophthora palmivora, and Erwinia caratovora [6][7][8][9][10].
A study conducted by Maulidia et al. [11] documented the isolation of Bt from the roots of cocoa plants (Theobroma cacao L.).This particular isolate was assigned the code AK08 and was registered at the Plant Pathology Laboratory.The efficacy of Bt AK08 in managing the fungal disease Synchitrium pogostemonis on patchouli and F. oxysporum on tomato plants has been examined, resulting in inhibitory suppression rates ranging from 50% to 70% [12,13].
Based on the findings of prior investigations, it is apparent that Bt AK08 exhibits promising characteristics for further development as a biological agent.Hence, it is imperative to conduct further research on the efficacy of BT AK08 in pathogen control.This study aims to evaluate the efficacy of Bt AK08 in managing two significant fungal diseases, namely Colletotrichum sp. and Fusarium sp., in chili plants.

Materials and methods
The research was conducted at Laboratory of Plant Pathology, Faculty of Agriculture Universitas Syiah Kuala.Six treatments were arranged to study the efficacy of Bt AK08 to control Colletotrichum sp. and Fusarium sp. as shown in Table 1.The treatments P1, P2, P3, and P4 were arranged on potato dextrose agar (PDA) media.While dual culture assay (P5 and P6) were conducted on PDA and Nutrient Agar (NA) combined media.Propineb was employed as positive control.The chili fruit segment exhibiting symptoms was rinsed using a continuous flow of water and subsequently sectioned into 1 cm 2 fragments using a sterilised knife.The disinfection procedure was extended by immersing the object in a solution of 5% sodium hypochlorite (NaOCl) for a duration of one minute, followed by three subsequent rinses with distilled water (aquades).The specimen was subjected to desiccation using sterile tissue paper, followed by inoculation onto Potato Dextrose Agar (PDA) medium.The PDA medium was produced by combining 6 g of PDA powder with 150 ml of distilled water in an erlenmeyer flask.The mixture of PDA powder and aquades was placed in an erlenmeyer flask, which was thereafter covered with cotton, plastic wrap, and aluminium foil.The flask was then subjected to sterilisation in an autoclave operating at a temperature of 121°C for a duration of 30 minutes, under a pressure of 1 atm.A quantity of 0.07 mg of chloramphenicol was introduced into the sterilised media solution as a means of inhibiting bacterial proliferation inside the medium.Following the mixing process, the media was carefully transferred into sterile petri plates and subsequently solidified within a controlled laminar airflow environment.The specimen was subjected to incubation under ambient conditions at a temperature of 28°C.Daily monitoring was conducted to observe the progressive development of the colonies until the entire surface of the PDA medium was completely colonised by the mycelia of the Colletotrichum sp.fungus.Fusarium sp.employed in this study was a collection of the Laboratory of Plant Pathology, Agriculture Universitas Syiah Kuala.Regeneration of Fusarium sp. was also carried out on PDA media.The technique for preparing PDA media, inoculation, and incubation of the isolate is the same as for the Colletotrichum sp.fungus.

Bacillus thuringiensis AK08 (BT AK08) preparation
Bt AK08 was regenerated on nutrient agar (NA) media.A quantity of 28 g of NA powder was measured and afterwards transferred into an erlenmeyer flask.Subsequently, a volume of 1000 ml of distilled water was introduced and subjected to sterilisation using autoclaving at a temperature of 121 o C for a duration of 30 minutes, under a pressure of 1 atm.Following the process, the media solution was let to remain undisturbed for a duration of 10 minutes within a controlled laminar airflow environment.The solution was carefully transferred into a petri dish with a diameter of 9 cm and afterwards left undisturbed to harden within a controlled laminar airflow environment.The Bt AK08 isolate was cultured on the specified media by aseptically transferring bacterial colonies from the original culture using a sterile inoculation loop, and afterwards streaking them in a zigzag pattern.The Bt AK08 isolate was thereafter subjected to an incubation period of 48 hours at a temperature of 28°C.

Dual culture test
The medium utilised for this test is a mixture of PDA and NA with a ratio of 1:1.Based on the initial test findings, it has been determined that the combination of these two media is capable of facilitating the proliferation of both Bt AK08 and pathogenic fungi.Media preparation is conducted in accordance with the guidelines provided on the packaging label.The dual culture test involves the placement of a 3 mm diameter isolate of Bt AK08 on one side of a petri dish that has been prepared with growing media.The Colletotrichum sp.isolate is introduced in close proximity to Bt AK08, with a separation distance of 3 cm.The identical approach is employed to assess the antagonistic effects of Bt AK08 on Fusarium sp.Colletotrichum sp. and Fusarium sp. are cultivated on PDA medium only for the purpose of serving as negative controls.Propineb, common synthetic fungicides, was introduced to the media used as positive control.
Daily observations were conducted to monitor the progression of fungal growth, antibiosis activity, and hyphae lysis.Fungal growth was examined by measuring the diameter colonies of Colletotrichum sp. and Fusarium sp. on the growth media.Antibiosis activity was studied by observing a clear zone that appear between pathogenic fungi inoculum and Bt AK08.The observation under microscope was carried out to analised the hyphae lysis of the pathogens.All the experiments were replicated 3 times.

Statictical analysis
The data obtained were statistically analysed for Analysis of Variance (ANOVA) on Microsoft Excel 2016.Variance analysis was performed on all experimental data and significant differences (P < 0.05) between individual means (three replicates) was analysed using a post hoc Least Significant Difference (LSD) test.

Colony diameter of Colletotrichum sp. and Fusarium sp.
The observation of the growth of Colletotrichum sp. and Fusarium sp. was conducted through daily measurements of the colony diameter.The results of the observation indicated that Bt AK08 did not exhibit the ability to inhibit the growth of the two pathogenic fungi that were investigated.Figure 1. and Figure 2. display the average diameter colonies growth of Colletotrichum sp. and Fusarium sp., individually, grown on propineb-supplemented media, and dual cultured with Bt AK08.These findings contradict a prior work that demonstrated the inhibitory capacity of Bt AK08 on the growth of F. oxysporum isolated from melon.Researchers suggested that antifungal potential of Bacillus sp.influenced by the strain, culture conditions, nutrient, and host specificity of pathogen/endophyte [14][15][16].Mojica-Marin et al. [8] reported that out of 64 strains of B. thuringiensis, only 8 strains significantly reduced the mycelial growth of F. oxyporum.In fact, it has been observed that B. thuringiensis isolates are generally less potent in terms of antifungal activity compared to other Bacillus species such as B. amyloliquefaciens and B. subtilis [17].
While there is limited research specifically on the ability of B.thuringiensis to control Colletotrichum sp., the ability of B. thuringiensis to control Fusarium sp. has been investigated in several studies.For example, Fatima et al. [18] focused specifically on the ability of B. thuringiensis strain CHGP12 to suppress F. oxysporum f. sp.ciceris and enhance the biomass of chickpea plants.They found that Bt CHGP12 exhibited high inhibition of the pathogen.

Antibiotic activity
The antibacterial activity of B. thuringiensis on an agar plate can be observed by the presence of a clear zone surrounding the antimicrobial agent.A greater extent of inhibition signifies a more potent antimicrobial agent, whereas a lesser or non-existent extent of inhibition signifies a less potent or inactive antimicrobial action, respectively.Our study revealed that Bt AK08 did not exhibit antibiotic action, as there was no clear zone of inhibition (Figure 3).Researchers suggested that the ability of Bacillus species to produce antibiotics is mediated by various factors such as gene expression [19,20].We did not analyse gene expression of Bt AK08 that employed in this study, hence we cannot provide a definitive justification.

Conclusions
The growth of Colletotrichum sp. and Fusarium sp. was not inhibited by Bacillus thuringiensis (Bt) AK08.There was no observable clear zone on the dual culture plate, indicating that Bt AK08 did not exhibit antibiotic production capable of suppressing the growth of both harmful fungi.Nevertheless, Bt AK08 has the capability to disrupt the hyphae of Colletotrichum sp. and Fusarium sp.Based on this discovery, it may be inferred that Bt AK08 exhibits promising efficacy in the management of Colletotrichum sp. and Fusarium sp.

Figure 3 .
Figure 3.The growth of Colletotrichum sp. and Fusarium sp.dual cultured with Bt AK08

Figure 4 .
Figure 4. Hyphae of Colletotrichum sp. and Fusarium sp. after treated with Bt AK08

Table 1 .
The experimental treatments arrangement to study the efficacy of Bt AK08 to control Colletotrichum sp. and Fusarium sp.