Potentials of Indigenous Bacillus thuringiensis Isolates from the soil in controlling Fusarium wilt of Cucumber cause by Fusarium oxysporum f.sp cucumerinum

Cucumber (Cucumis sativus L.) production is generally low in Nigeria due to continuous soil nutrient limitation and diseases. However, the persistence in the use of agrochemicals for cucumber production in Nigeria is associated with high cost and deleterious effects on man, animal and the environment. This study was conducted to investigate the potentials of indigenous Bacillus thuringiensis (Bt), a spore-forming bacterium known for its insecticidal properties in controlling Fusarium wilt of cucumber. Bacillus thuringiensis strains were isolated from soil samples collected from different farm sites in Abeokuta, Nigeria, and identified phenotypically and molecularly. The in-vitro antagonistic activity of B. thuringiensis strains on F. oxysporum f.sp. cucumerinum was evaluated by dual culture method, followed by pot experiment in the screen house. 16S rRNA gene sequencing was performed on the antagonistic B. thuringiensis to confirm Bt species. The results of the in-vitro antagonistic activity revealed that most indigenous B. thuringiensis strains showed significant growth inhibition of Fusarium oxysporium f. sp. cucumerinum. Similarly, application of B. thuringiensis A and C isolates significantly suppressed the incidence of Fusarium wilt of cucumber in the screen house when compared to the control. The 16S rRNA gene sequencing technique identified the isolates A and C as Bacillus thuringiensis strain LTS-209 and Bacillus thuringiensis strain VITSJ-01, respectively. Hence, indigenous B. thuringiensis A and C isolates should be incorporated into cucumber cultivation for controlling Fusarium wilt disease of cucumber.


Introduction
Modern agriculture heavily depends on the application of agrochemicals for fertilizing the soil and for disease control. However, the over-use of these chemical compounds poses potential risks to human health and the environment, and can also lead to the chemical resistance in the disease-causing agents (Akintokun and Taiwo, 2016). These effects had led to a total ban or restricted use of most chemical pesticides. Therefore, the need for biocontrol agents as an alternative to reduce synthetic chemicals usage is now being encouraged (Wen-Kun et al., 2014). Fusarium oxysporum f. sp. cucumerinum is an economically important wilting pathogen of cucumber which can cause significant yield loss (Gamal, 2010). Biological control of Fusarium wilt by beneficial microorganisms are complex but most studies conducted previously have focused on using non-pathogenic fusarium spp. or other antagonists and their antagonistic activities could be through mechanisms such as competition for nutrients, competition for infection sites on roots and production of antibiotics (Gamal, 2010). The ability of members of the Gram-positive genus Bacillus to form spores is advantageous for controlling a variety of soil-borne phytopathogenic fungi (Koumoutsi et al., 2007;Arguelles-Arias et al., 2009;Chowdhury, 2013) and some are commercially marketed as biopesticides, biofertilizers and soil amendments because of their easy colonization, good competition and broad antimicrobial spectrum (Cazorla et al., 2007;Baysala et al., 2008;Chung et al., 2008;Choudhary and Johri, 2009). Bacillus thuringiensis (BT) is an ubiquitous, Grampositive, spore-forming bacterium that produces parasporal crystals during the stationary phase of its growth cycle. The crystals comprise one or more crystal proteins (encoded by cry or cyt genes) that show specific toxicity against several orders of insects, including Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera, and Mallophaga, as well as nematodes, mites, and protozoa (Weixing et al., 2012).
Bacillus thuringiensis was initially characterized as an insect pathogen and its insecticidal activity was ascribed largely or completely to the parasporal protein crystals.
Apart from crystal proteins, Bt strains are able to produce exoenzymes, such as proteases and αamylases (Smitha, 2010) as well as β-exotoxins which are able to inhibit the growth of phytopathogenic fungi (Guo et al., 2008).
However, several studies have been conducted on the insecticidal activity of Bacillus thuringiensis, less attention has been paid to exploiting its antifungal activity for biological control of Fusarium oxysporum f. sp. cucumerinum. The present study was therefore undertaken to evaluate the ability of indigenous Bacillus thuringiensis isolated from cultivated and uncultivated soils in suppressing Fusarium wilt disease of cucumber.

Collection of samples
Soil samples were collected aseptically from three farm sites (Isolu, Federal University of Agriculture Abeokuta (FUNAAB) and Osiele) in Abeokuta, Nigeria where there was no previous record of application of Bacillus thuringiensisbased pesticides. The soil samples were taken 2 cm to 5 cm below the surface, after scraping the surface with sterile spatula. Soil samples were then transported immediately to the laboratory on ice box and stored in sterile plastic bags at 4°C until their processing.
The phytopathogenic Fusarium oxysporum f.sp cucumerinum was collected from the Department of Crop Protection, Federal University of Agriculture, Abeokuta, Abeokuta and confirmed at the laboratory of Department of Microbiology.

Isolation of indigenous Bacillus thuringiensis
Bacillus thuringiensis were isolated from the soil samples using the selective sodium acetate heat treatment method as described by Rathinam et al. (2007). Briefly, 10.0 g of each soil sample was suspended in 90 ml sterile distilled water and vortexed for 1 minute. The suspension was heated in a water bath at 80ºC for 30 minutes. One milliliter (1.0 ml) of each suspension was added to 10 ml of nutrient broth buffered with 0.25 M Sodium acetate, pH 6.8. The inoculated broth was incubated at 30ºC for 4 hours, followed by heating at 80ºC for 3 min. Suspensions were serially diluted to 10 -6 and 1.0ml of 10 -6 dilution was inoculated on nutrient agar plates using pour plate method. The sample dilution and agar medium were thoroughly mixed and allowed to solidify. The plates were incubated at 30ºC for 24 hrs. The number of colonies on the surfaces of the agar plates were counted, recorded and expressed as Colony forming units per gram (CFU/g) of soil. Pure bacterial isolates were obtained by subculturing on nutrient agar plates, incubated at 30°C. All pure cultures of bacterial isolates were maintained on nutrient agar slants and stored at 4°C for further studies.
Phenotypic characterization of Bacillus thuringiensis isolates The Bt-like isolates were characterized by observing their colony morphologies (shape, size, elevation, surface, margin, pigmentation), endospore staining, parasporal body staining, Gram staining, motility and subjecting them to a series of biochemical tests. The identification of the isolates was then done using the Bergey's Manual of Determinative Bacteriology (Holt et al., 1994).

In vitro antagonistic assay of indigenous Bacillus thuringiensis
isolates against Fusarium oxysporum f.sp cucumerinum The indigenous Bt isolates were screened against Fusarium oxysporum f. sp cucumerinum (causing Fusarium wilt of cucumber) using the dual culture method as described by Sharma et al. (2012). Fresh mycelium of F. oxysporum f. sp. cucumerinum was inoculated at the centre of each potato dextrose agar (PDA) plate. On the opposite of the fungus, about 3.00cm from the fungus, a loop full of each Bt isolate was streaked. The Bt isolate was replaced with sterile distilled water in control treatments. The plates were then incubated at room temperature (25 ± 2 o C) for 3 days. The barrier between the fungus and the Bacillus thuringiensis strains indicated the antagonistic interaction between them. The percentage inhibition of growth over control was calculated using the formula: Growth inhibition = 1 − (a / b) x 100% a = radial growth of fungus interacting with antagonistic bacteria; b = radial growth of the fungus only in the control plate.
In vivo antagonistic assay of indigenous Bacillus thuringiensis isolates against Fusarium oxysporum f.sp cucumerinum A beneficial bacterial-phytopathogen fungi interaction study was conducted using the method described by Baniasadi et al. (2009). The experiment was conducted in the screen house at Federal University of Agriculture, Abeokuta.
Seeds of cucumber (susceptible to infection by Fusarium oxysporum f.sp cucumerinum) were surface-sterilized with 5% Sodium hypochlorite solution for 2 minutes, washed three times in sterile distilled water and then air-dried at room temperature (25±2 o C). Four milliliter (4.0 ml) of Fusarium oxysporum f. sp cucumerinum inoculum was mixed with 50.0 ml sterile distilled water and directly inoculated on the sterile soil, after which planting bags were immediately covered with black polyethylene bags for 48 hours. Five Bttreated cucumber seeds and control seeds were planted per planting bag, and immediately covered with soil. All plants were kept in the experimental screen house under normal light and temperature conditions, and watered with sterile water at regular intervals. The seedlings were observed after 8 weeks to determine the disease incidence (DI). The percentage of cucumber seedlings in a bag that showed visible signs of infection was also calculated (Michel et al., 1997). Disease severity was also recorded by visual observation of the disease symptoms with reference to the untreated infected controls. Disease index data were obtained and recorded using the scale of 0 to 4 (Soonthompoct et al., 2001). Symptom severity was graded into five disease classes as follows: 0 (No disease or wilt i.e. apparently healthy seedlings), 1 (one to two leaves infected), 2 (three to five leaves infected or traces of stem rot), 3 (all leaves infected/stunted growth/stem rot) and 4 (damping off/ wilting/ seedling death). The disease reduction percentage (DRP) was also calculated as: DRP = 100 x [1 -(DI of treatment / DI of controls)]. (5'-AGAGTTTGATCMTGGCTCAG -3') and 1492R (5' -ACCTTGTTACGACTT -3') primers (Blackwood et al., 2005). The amplified fragments were resolved by electrophoresis on a 1% agarose gel. The PCR amplicons were purified and directly sequenced by using the ABI 3730 Genetic Analyzer at STAB VIDA Technologies, Portugal. Sequence assembly and alignment were carried out using CLC 6.8.4 bio software. To identify the isolates, the gene sequences were compared with the GenBank sequences at the National Centre for Biotechnology Information (NCBI) database using BLASTn search tool.

Statistical analysis of data
Data were analyzed using the statistical package for social sciences (SPSS) version 16.0 for Windows (SPSS, Chicago IL, U.S.A). Means were separated using Tu key-Kramer HSD test at α = 0.05.

Results and Discussion
Isolation of indigenous Bacillus thuringiensis from soil samples Table 1 shows total Bacillus counts of soil samples from three farm sites in Abeokuta. The results showed that there were significant differences in total Bacillus counts of cultivated and uncultivated soil samples from Isolu and FUNAAB farm sites, but there were no significant differences in the counts between Osiele and Isolu soil samples. Cultivated and uncultivated soil samples from FUNAAB farm sites recorded the highest and lowest counts respectively (Table  1). In general, soil samples from uncultivated farm sites had lower Bacillus counts than soil samples from the cultivated farm sites. These results are in agreement with Meihiar et al. (2014) who reported high counts of Bacillus thuringiensis from cultivated areas compared to uncultivated natural areas and interior arid areas.
Also, Ralte et al. (2016) reported high counts of Bacillus species from agricultural land compared to non-agricultural land. This may be as a result of high organic and moisture contents as reported by Obeidat et al. (2004) and Meihiar et al. (2014) that soil with high organic matter and moisture content are very rich in Bt due to high levels of nutrients and insect activity.  Table 2. All the Bt isolates were able to ferment glucose, lactose, mannitol and sucrose sugars, but were unable to ferment maltose sugar.  Bacillus thuringiensis G had the least percentage inhibition (0%) (Figure 1). This is in concordance with Batista-Junior et al. (2002) who reported that Bacillus thuringiensis kurstaki HD1 strains inhibited the growth of Fusarium solani, Fusarium oxysporum and Colletotrichum sp phytopathogens. The inhibiting effect of Bacillus thuringiensis strains on phytopathogenic fungi can be associated with enzyme production that can act against the fungal cell wall (Figueiredo et al., 2010).
Percentage disease incidence and reduction of Phytopathogenic Fusarium oxysporum f.sp cucumerinum growth by Bacillus thuringiensis isolates under screen house.
The control seedlings exposed to Fusarium oxysporum f.sp cucumerinum but not inoculated with any Bacillus thuringiensis strains expressed disease symptoms like leaf wilting, leaf curl, stem rot, leaf blight, root rot and were not able to survive till the fourth week of planting. In contrast, seedlings inoculated with Bacillus thuringiensis strains showed outstanding antifungal effects. The percentage disease incidence for the fungus was highest in the control and Bacillus thuringiensis isolate G (100%) while the least (6%) was recorded in Bacillus thuringiensis C. The percentage disease reduction was highest in Bacillus thuringiensis isolate C (95%) and lowest was recorded in control and Bacillus thuringiensis isolate G (0%) as shown in Figure 2. This is similar with the report of Ahmed et al. (2006)

Conclusion and Recommendation
This study confirmed that Bacillus thuringiensis strains LTS-209 and VITSJ-01 are highly effective against the Fusarium oxysporum f. sp cucumerinum. The implication of this is that the potent danger that may occur through application of pesticides to the environment is averted, hence, the stability of ecosystem is guaranteed in the use of the above strains. However, continuous study with these strains is now needed on the field trials, under different local environmental conditions in order to enhance the sustainability of the crops in Nigeria by making use of the bacteria as bio-fungicides.