In-Vitro Antagonistic Effect of Bacillus thuringiensis on Ralstonia solanacearum, the Causal Agent of Bacterial Wilt Disease of Tomato

Tomato (Lycopersicon esculentum, Mill) is a rich source of vitamins, minerals and lycopene, which has many health benefits. However, its production is hampered by bacterial wilt caused by Ralstonia solanacearum resulting in significant yield losses. Use of chemicals in the control of plant pathogens has detrimental effects on humans and the environment in terms of leaving residues in soil which later find their way into underground waters. Therefore, it is desirable to find an alternative to chemical control of this bacterial pathogen. This study investigates the potential of native Bacillus thuringiensis (Bt) for biological control of Ralstonia solanacearum (Rs) under laboratory conditions. B. thuringiensis was isolated from cultivated soil, noncultivated soil, stagnant water, sawdust, horse dung, grain dust, dead leaves and poultry manure. R. solanacearum was isolated from stem exudates of bacterial wilt infected plants and its pathogenicity assay was carried out using 2-week-old seedlings of Beske tomato variety. The Bt and R. solanacearum isolates were then characterized phenotypically. Bt isolates were further identified using endospore and parasporal staining techniques. All the Bt isolates were tested for in-vitro antagonistic activity on R. solanacearum using agar well diffusion method. Isolates Bt2, Bt16, Bt17, Bt32 and Bt34 were confirmed as Bacillus thuringiensis while isolate Rs was confirmed as R. solanacearum. Beske showed wilting symptoms from the fourth day of inoculation and eventual death of seedlings. The zone of inhibition exhibited ranged from 0.0 mm to 20.0 mm.


In-Vitro Antagonistic Effect of Bacillus thuringiensis on Ralstonia solanacearum, the Causal Agent of Bacterial Wilt Disease of Tomato (Lycopersicon esculentum Mill). Introduction
Tomato (Lycopersicon esculentum Mill) belongs to the Solanaceae family (Khokar, 2013). It is widespread throughout the world and represents the most economically important vegetables even in Nigeria (Olaniyi et al., 2010;Bergougnoux,2013).Tomatoes contribute to a healthy, well-balanced diet. They are rich in minerals, vitamins, essential amino acids, sugars and dietary fibers. Tomato contains vitamin B and C, iron and phosphorus; its fruits are consumed fresh in salads or cooked in sauces, soup and meat or fish dishes.
They can be processed into purées, juices and ketchup. Yellow tomatoes have higher vitamin A content than red tomatoes, but red tomatoes contain lycopene, an antioxidant that may contribute to protection against carcinogenic substances (Khokar, 2013).
Recently, it has been estimated that huge proportions of vegetable crops get deteriorated annually during growth or post-harvest storage, owing to diseases caused by fungi, nematodes, bacteria, and viruses. This is one of the major limiting factors influencing food production and human development over thousands of years (Dun-chun et al., 2016).Bacterial wilt is a common bacterial disease in tropical, subtropical and some temperate regions of the world (Fegan and Prior, 2005). It is endemic in most tomato-growing areas of Nigeria, causing 60 to 100% yield losses (Popoola et al., 2015).
For the past 50 years, application of chemical pesticides has been the prevailing control measure for disease management in vegetables and other crops production. The continuous exposure to chemical pesticides adversely affects productivity, soil texture, nutritional content of vegetables, as well as the health of human beings (Singh et al., 2017).
Due to the hazards associated with chemically synthesized herbicides and pesticides, management of diseases via biological control method is the novel emerging technology gaining importance in better agricultural sustainability (Singh et al ., 2017). which are popularly used in plant protection (Keswani et al., 2015;Mishra et al., 2015).
Bacillus thuringiensis (Bt), a spore-forming bacterium is well known for its insecticidal properties, associated with its ability to produce crystal inclusions during sporulation. These inclusions are proteins encoded by cry genes and have shown to be toxic to a variety of insects and other organisms like nematodes and protozoa (Koneckaet al., 2007). Formerly, only the insecticidal properties of B. thuringiensis attracted extensive attention. However, in recent years, the roles of B. thuringiensis in plant disease control have been discovered. Apart from crystal protein and other insecticidal substances, B. thuringiensis also produces other active components with good prospects for application as the following: zwittermicin A, which is extremely efficient at preventing the damping-off of alfalfa caused by Phytophthor amedicaginis (Silo-Suhet al., 1994), and acyl homoserine lactone (AHL) lactonases, which can quench bacterial pathogenicity (Dong et al.,2002). Therefore, there is a need to assess the in-vitro antagonistic effect of Bacillus thuringiensis on Ralstonia solanacearum.

Materials and methods
Study area and collection of soil samples

Isolation from soil
Bacillus thuringiensis was isolated using the method described by Palma (2015) with slight modifications. Vegetative cells from sporulated bacteria were isolated by homogenizing 3 g of each soil sample in 10 ml of sterile distilled water, intensely vortexed and mixed for 1 minute and incubated at 70±2°C for 15 minutes. After which the samples were vortexed and heated again. Each sample was then subjected to ten-fold dilutions and 20µl (from 10 -3 to 10 -5) was dispensed on nutrient agar. Plates were incubated at 28±2°C for at least 72 hrs (Suguna et al., 2011). Isolation from stagnant water Ten milliliter (10.0 ml) of each stagnant water sample was dispersed in 90 ml of saline solution. This was left to equilibrate for 20 minutes and then subjected to heating in a water bath at 80±2°C for 10 minutes (Saadeldin, 2007). Each sample was then subjected to ten-fold dilutions and 20µl (from 10 -3 to 10 -5) was dispensed on nutrient agar. Plates were incubated at 28±2°C for at least 72 hrs (Suguna et al., 2011). Isolation from insects, grain dust, animal dung and dead leaves Approximately 5 g of dead insects, saw dust, grain dust, animal dung and dead leaves were weighed separately into 95 ml sterile distilled water in 250 ml conical flasks and incubated in an orbital shaker set at 25±°C for 5 hr. One milliliter (1 ml) aliquot was transferred into 5ml pre-warmed boiling tubes. The tubes were given heat-shock treatments at 80±°C for 15 minutes, in a water bath. Each sample was then subjected to ten-fold dilutions and 20µl (from 10 -3 to 10 -5) was dispensed on nutrient agar. Plates were incubated at 28±2°C for at least 72 hrs (Suguna et al., 2011).

Isolation of Ralstonia solanacearum
Ralstonia solanacearum was isolated as described by Shew and Lucas (1991) with slight modifications. A stem section was cut from diseased plants with vascular discoloration using a sterile sharp blade. The section was swabbed with 70% ethanol and subsequently placed against the inside wall of a water-filled clear test-tube so that the end of the section slightly touches the water surface. Milky white strands containing bacteria and extracellular polysaccharide streaming from the cut ends of the xylem were then cultured on nutrient agar plates incubated at 28±2°C for at least 24 hrs (Jayesh et al., 2014). Smooth, circular, raised and dirty-white colonies were sub-cultured to obtain pure cultures. The isolates were kept on nutrient agar slants maintained at 4°C.

Characterization of Bacterial isolates
The bacterial isolates were subjected to standard microbiological methods such as morphological characteristics of the colony (shape, size, elevation, colour) and Gram staining to differentiate Gram negative and positive bacteria. Biochemical tests including catalase, oxidase, citrate utilization, Voges-Proskauer and methyl-red were carried out on the isolates (Fawole and Oso, 1998;Cheesbrough, 2006). The morphological and biochemical characteristics were examined according to the Bergey's Manual of Determinative Bacteriology (Holt et al., 1994).

Pathogenicity test
Pathogenicity assay of the Ralstonia solanacearum isolates was carried out under screen house conditions by inoculating susceptible Beske tomato seedlings. Bacterial isolates were grown on nutrient agar medium for two days at 28±2°C. The cells were removed by centrifugation at 7,000 rpm for 10 minutes; pellets were re suspended in sterile distilled water and adjusted to a final density of 1 × 10 7 / ml (Elsharkawy et al., 2015). Tomato seeds were nursed in a plastic tray and transplanted into disposable plastic cups 10 days after planting, while the inoculum was introduced into healthy seedlings 2 days after, by drenching the soil with 10ml each of the suspected pathogens (Hyakumachi et al., 2013).
Seedlings inoculated with sterile water served as negative control. Wilt intensity was calculated after 21 days of inoculation using the method described by Abeer and Hend (2013).
, where I = wilt intensity, ni = no of plants with respective disease rating, v1= disease rating (following scale : 1= no symptoms, 2= 1 leaf wilted, 3=2 or 3 leaves wilted, 4 = four or more leaves wilted, 5= plant dead), V= the highest disease rating and N= the no of plants observed.

In-vitro activity
In-vitro antagonistic activity on the pathogen was determined using agar well diffusion method in which about 10µl of pathogen suspension (1 × 10 7 cfu/ml) was evenly spread on nutrient agar plates. Subsequently, with the aid of a sterile cork borer (6mm in size), wells of 20 mm apart and diameter of about 6mm were punched aseptically. Antagonist suspension (1.8 × 10 8 cfu/ml, about 100µl), was added into each well. Wells inoculated with sterile distilled water served as control (Elsharkawy et al., 2015;Mounyr et al.,2016). This was carried out as follows: antagonist and pathogen were inoculated at the same time, pathogen was inoculated first followed by antagonist applied 48 hrs after, antagonist inoculated first while pathogen was applied 48 hrs after. The plates were then incubated for 24hrs at 30±2°C after which zones of inhibition were measured (Cleidson et al, 2007, Mounyr et al.,2016, Marissa et al., 2016.
The conditions for Polymerase Chain Reaction (PCR) in the thermal cycler (GeneAmp PCR System 9600) were as follows: 94˚C for 5 minutes followed by 36 cycles of denaturation at 94˚C for 30 sec, annealing at 56˚C for 30 sec, extension at 72˚C for 45 sec, and final extension at 72˚C for 7 minutes. The amplified fragments were resolved by electrophoresis on a 1.5% agarose gel prepared in 0.5X TBE, stained with Gel red using a 50bp ladder. The gel ran for 50 minutes at 100V and was visualized under UV transilluminator. The PCR product of the amplified 16S rRNA amplified region was purified using ethanol precipitation method.
The PCR product was used for another PCR reaction (sequencing reaction), using the big Dye Terminator method with 3130xl genetic analyzer from Applied Biosystems. Amplification and sequencing were done at International Institute for Tropical Agriculture (IITA) Biosciences Laboratory, Ibadan, Oyo State.

Sequence editing and database matching
The sequences were edited, assembled and aligned using BioEditsoftware (version 7.1.9). Gene sequences were compared at the Genbank database of NCBI (National Centre for Biotechnology Information) using BLASTn search tool to identify the isolates. Phylogenetic analysis was done using Molecular Evolutionary Genetics Analysis (MEGA) version 6 (Tamura et al., 2013).

Morphological and Biochemical characteristics of bacterial isolates
All the bacterial isolates were rod-shaped and motile. Isolate T3 had dirty -white color while others were cream colored. Some were large while others were small in size. Some of the bacteria had raised elevation while others had flat elevation (Table 1). The bacterial isolates were Gram positive except T3 (Table 2).

+ Positive reaction; -Negative reaction
Endospore and parasporal crystal staining for Bacillus thuringiensis identification Isolates Bt2, Bt16, Bt17, Bt32, and Bt34 were positive for endospore and crystal staining (Table 3).  Figure 1.  Wilting of varying degrees were observed on seedlings inoculated with pathogens while seedlings inoculated with sterile water showed no sign of wilting as shown in Table 3.

In-vitro activity
There were no zones of inhibition when either antagonist or pathogen was applied first. When pathogen and antagonist were applied at the same time, isolate Bt34 had the highest zone of inhibition (20 mm) while isolate Bt17 had the least zone of inhibition (0 mm) (Table 4).

Molecular Characterization of the Bacterial Isolates
Purity and concentration of DNA extracted from the bacterial isolates ranged from 1.74 to 1.92 and 32 ng/µl to 160.50 ngµl, respectively, as shown in Table 5. Similarity of sequences of the bacterial isolates with sequences obtained from NCBI is shown in Table 6.
Gel images of genomic DNA are shown in Plate 1 while Plate 2 shows the gel image of amplified 16S rRNA gene of the isolates. Phylogenetic relationship of the bacterial isolates is shown in Figure 3.

Discussion
Biocontrol is particularly desirable because it is sustainable, environment friendly, cost-effective and could be used in integrated pest management programs (Elshakawy et al, 2015).
In this study, 17 bacterial isolates were isolated from cultivated and non-cultivated soils, stagnant waters, dead leaves and dead insects. This corroborates the findings of Argôlo-Filho and Loguercio, (2013), El-kersh et al., (2016), and Denane et al., (2017), that were able to isolate Bt from different sources. One bacterial isolate was also isolated from diseased tomato plants showing symptoms of bacterial wilt. Based on their morphological and biochemical characteristics, 5 out of the 17 bacterial isolates (Bt2, Bt16, Bt17, Bt32 and Bt34) recovered from the different sources (cultivated soils, dead insects and stagnant waters) were tentatively identified as Bacillus thuringiensis (Bt).
Endospore and crystal staining further confirmed the Bt isolates. This corroborates the works of Mohsina et al., (2013), Palma (2015) and Meihiaret al., (2015). They were able to establish Bt as crystal protein and endospore producers.
Ammons et al., (2016) and Neethu et al.,(2015) also reported that crystal proteins account for their pesticidal and insecticidal activities. Out of the 17 different Bacillus species isolated from the different sources, Bacillus pumilus occurred most, this could be as a result of their spores' extreme resistance to radiation, desiccation, and hydrogen peroxide treatment as reported by Link et al., (2004) and Kempf, et al., (2005). As a result, B. pumilus has been classified as an extreme microorganism according to the planetary protection standards (Vaishampayan et al., 2012). Bacillus thuringiensis had the highest percentage occurrence in cultivated (agricultural) soil samples; this contradicts the findings of Kassougue et al., (2015) but agrees with that of Ralte et al., (2016). Kassogue found low Bt strains in cultivated soil in Mali while Ralte found higher frequency of Bacillus thuringiensis in agricultural soils than non-agricultural soils. This could be as a result of plants serving as a form of protection to the soil from the harsh ultraviolet rays of the sun and as such the soil organisms remain intact. The bacterial isolate from diseased tomato plants was also tentatively identified as Ralstonia solanacearum. Pathogenicity assay of Ralstonia solanacearum isolate on Beske tomato variety showed wilting of varying degrees while the control showed no sign of wilting. This is in agreement with Popoola et al., (2015) who reported Beske variety as susceptible to bacterial wilt of tomato. In-vitro activity of the Bt isolates (Bt2, Bt16, Bt17, Bt32 and Bt34) against Ralstonia solanacearum revealed isolate Bt34 as having the highest zone of inhibition with whole cell (20.00 cm) and supernatant (18.00 cm) while isolate Bt17 had the least zone of inhibition and whole cell. Similarly, isolates Bt 16, Bt 17 and Bt 32 had the least zones of inhibition (0.00 cm) only. This corroborates the work of Abeer and Hend (2013), reporting that Bacillus thuringiensis was able to significantly reduce the growth of Ralstonia solanacearum under laboratory conditions with a zone of inhibition of 8.2 mm.
This result shows that native Bacillus thuringiensis may be used to control bacterial wilt of tomato under laboratory conditions.