In vitro Evaluation of Actinobacteria against Tomato Bacterial Wilt (Ralstonia solanacearum EF Smith) in West Showa, Ethiopia

Ralstonia solanacearum EF Smith is one of the most important and widespread pathogenic bacterium that causes a wilt disease in vegetable crops including tomato (Lycopersicon esculentum Mill.) in West Showa, Ethiopia, mainly in the off-cropping season. In vitro study was conducted to screen indigenous actinobacteria for their antibacterial activity against tomato bacterial wilt pathogen, R. solanacearum. 210 soil samples were collected from three districts of West Showa Zone, Ethiopia. Of all soil samples, 86 morphologically distinct actinobacterial isolates were isolated. In the preliminary screening test, 52 isolates were selected showing inhibitory activity against the pathogen and further tested for their antibacterial activity under secondary screening test. In the secondary screening test, all the isolates appeared significantly different (P ≤ 0.05) in their inhibition against the target pathogen. Of these isolates, efficient 36 isolates were selected and subjected to the cell free suspension test. All the 36 isolates showed significantly different (P ≤ 0.05) in their inhibition activity in the cell free suspension test. In both dual culture and cell free suspension test, totally 21 isolates showed inhibitory activity against the target pathogen. The actinobacterial isolate, Gosu-qoraS#196-1 had become superior antagonistic isolate against R. solanacearum and followed by Awaro S#174-2, Senkele S#132-5, Awaro S#183-1, Senkele S#133-3, Dhaga file S#113-1, Awaro S#176-4, Gabata S#21-1, Dhaga file S#128-2 and Awaro S#174-3 isolates. This study has concluded that the actinobacterial isolates having antibacterial activity against this target pathogen and also the investigated isolates can be used as one component of integrated disease management practice through detailed further test. Then, the further research on the actinobacterial isolates should be focused on species identification, in vivo evaluation of the isolates against tomato bacterial wilt and further testing of its host range. Citation: Biratu KS, Selvaraj T, Hunduma T (2013) In vitro Evaluation of Actinobacteria against Tomato Bacterial Wilt (Ralstonia solanacearum EF Smith) in West Showa, Ethiopia. J Plant Pathol Microb 4:160. doi:10.4172/2157-7471.1000160


Introduction
Tomato (Lycopersicon esculentum Mill.) is one of the most widely grown vegetables in the world and it is regarded as one of the top priority vegetable. Tomato contributes to healthy, because it is rich in minerals, essential amino acids, sugars, dietary fibers and it is considered to be fairly high in vitamins, vitamin C, lycopene and betacarotene with potential for better quality processing [1]. In Ethiopia, tomato is one of the most important vegetable crop and its production has shown a marked increase since it became the most profitable crop providing a higher income to small scale farmers compared to other vegetable crops [2]. However, the national average of tomato fruit yield in Ethiopia was often low (125 kg/ha), when compared even to the neighboring African countries like Kenya (164 kg/ha) [2]. Current productivity under farmers' condition in Ethiopia is 190 kg /ha whereas yield up to 300 kg/ha recorded on research plots [1].
Farmers get lower yield mainly due to diseases, pests and suboptimal fertilization [3]. However, there are a number of factors which limit tomato yield. These include: the lack of improved well performing varieties, poor fruit setting due to heavy rains and excessively high temperature, pests and diseases. In addition to these, fungal as well as bacterial diseases are considered to be the major constraints to tomato production [2]. Howard [4] reported that 10 to 50% production of tomato were affected by diseases annually. Fungal diseases such as damping-off, crown and root rot, gray mold, blight and powdery mildew and bacterial diseases of wilt and rots are some of the common problems in tomato production. Among those diseases, bacterial wilt (Ralstonia solanacearum EF Smith) of Solanaceous vegetables are the most destructive and widespread disease in tropical and subtropical regions of the world. It is one of the most important and widespread phytopathogenic bacterial wilt pathogen in Ethiopia, mainly in the offcropping season and a destructive disease in vegetable crops including tomato. The percent incidence of tomato bacterial wilt was recorded as high as 55% [5] in major tomato producing areas of Ethiopia.
The management of tomato against this pathogen is important to maximize the crop's yield. Chemicals either not have direct bactericidal or fungicidal activities against the pathogen or converted into non-toxic derivatives by the pathogen. Management of this disease through chemicals and the use of resistant varieties are possible, but the hazardous impact of agrochemicals on the human health and environments, development of resistant mutants, escalating costs of fumigant pesticides and frequent breakdown of resistant varieties; strongly demand a sustainable and alternative disease management approaches [6].
Many bio-control agents (BCA) have been successfully identified for the control of various plant pathogens as alternative disease management approaches. Bio-control of pathogenic bacteria has gained considerable attention in the past two decades. Although several bacterial [7,8] bio-control agents for the control of pest and diseases developed and used in the production of vegetables including tomato crops [7]. Among different BCA, actinomycetes which is otherwise called as Actinobacteria or "higher Bacteria" [9] are known a very effective BCA and pesticide sources throughout the world. Actinobacteria play a quite important role in natural ecological system and they are also profile producers of antibiotics, antitumor agents, enzymes, enzyme inhibitors, vitamins, alkaloids and immune modifiers which have been applied in industry, agriculture, forestry and pharmaceutical industries [10,11].
The biological control of bacterial wilt using bio control agents like actinobacteria have shown a great potential and commercial preparations for the management of such diseases are in use [14]. However, much work still needs to be done in Ethiopia, especially with the bacterial wilt of tomatoes, since the disease is still causing much devastation on the crop. Hence, the present study was carried out to isolate and identify the potential actinobacterial isolates from soils of three different districts of West Showa, Ethiopia, to characterize and screen them to getting some actinobacterial isolates that produce antibiotics and active against tomato bacterial wilt as well as to determine their bio control potential against this test organism through in vitro condition.

Sample collection and isolation of tomato bacterial wilt pathogen
In this study, the target pathogen was obtained from preserved culture of Ambo Plant Protection Research Center (APPRC), Ambo and the samples of diseased tomato plants were collected from tomato growing areas of the country, mainly around Ziway area of West Showa. For isolation of the pathogen, about 1 g of the diseased sample was surface sterilized by 1% hypochlorite and macerated in sterile distilled water and the filtrate was further diluted using sterile distilled water. From the appropriate dilutions, 0.1 ml of an aliquot was spread-plated in duplicates on pre-dried surfaces of Triphenyl Tetrazolium Chloride (TTC) agar medium [15]. After 48 h of incubation at 30°C, the typical mummified fluid, slightly red-tinted colonies of R. solanacearum was easily distinguished, picked and further purified through repeated streak plating on the same medium. The biochemical characterization such as cytochrome oxidase, catalase [16], KOH solubility [17], Tween 80 hydrolysis, fatty acid esterase activity [18], starch hydrolysis [18] and pathogenicity (Koch's postulates test) of the target pathogen was tested.

Soil sample collection
The soil samples were collected from three different districts viz., Tikur Incini, Toke kutaye and Ambo of West Showa, Oromia, Ethiopia (Table 1). In each district, four different kebeles were selected. Nano, Bere, Gabata and Garmama kebeles were selected from Tikur Incini district, Malka Dera, Mutulu-Odobari, Kilinto and Gorfo kebeles were selected from Toke kutaye district and Sankele, Faris, Awaro and Gosu-kora Kebeles were selected from Ambo district. The soil samples were collected from forests, virgin lands, cultivated and grazing lands as stratified random sampling options. Soil samples were randomly collected from top 4 cm soil profile at an interval of about half a km between the sampling spots. Soil samples (approx. 200 g) were collected in sterile polythene bags, stored in iceboxes and transported to APPRC laboratory, Ambo for further analysis. From all sampling spots, totally about 210 soil samples were collected.

Morphology:
The actinobacteria were characterized morphologically by the following methods given in the International Streptomyces Project (ISP) [20]. The characters including colony morphology of the isolates such as the color of aerial mycelium (on the surface of agar), reverse side color, production of diffusible pigments [11,12], melanoid and soluble pigments [20] were observed after incubation at 28°C for 7-10 days on SCA. Gram staining [21] and KOH solubility test [17] were detected on the 7-day old cultures. The microscopic morphology of isolates such as formation of aerial and substrate mycelium and spore arrangement, which are highly characteristic and useful in the identification of actinobactera, were observed by cover slip technique [22] with light microscopy. The color of the colony was compared with standard color chart for identification [23].

Screening of antibacterial activity of actinobacteria
Preliminary screening: Antibacterial activity of actinobacteria was subjected to primary screening by cross streak plate technique [24]. The pure preserved actinobacterial isolates were reinitiated and used for anti-bacterial activity screening against tomato bacterial wilt. The

No.
Location The biodiversity of actinobacterial population density is less common in terrestrial and temperate soils. The actinobacteria are resources under extreme environments (including extreme high and low temperature, extreme high or low pH, high salt concentration etc.) and have received comparatively little attention from pathologists and microbiologists in Ethiopia. The difference in physical and chemical characteristics and the occurrences in the terrestrial and temperate environments of these actinobacteria may helps for their population density, diversity and potential which provide opportunities to a greater extent for the control of bacterial plant diseases [11]. Many studies have been attempted on the isolation of actinobacteria from terrestrial, tropical, sub-tropical and marine sediments [11][12][13] but not in temperate high mountain soils of Ethiopia.

Data analyses
Data were analyzed using SAS version 9.0 (Statistical Analysis System for Windows, 2001-2004, SAS Institute, USA). GLM procedure was followed for data analysis. Means were compared at 5% significance level, using Duncan's Multiple Range (DMR) test.

Isolation and characterization of the target pathogen
The target bacterial pathogen was isolated from freshly collected samples at farmers tomato cultivated fields and APPRC isolate culture was compared on the basis of their fluidity, color, and morphological

Biochemical characterization of R. solanacearum
All the targeted bacterial pathogen isolates were catalase positive, oxidase positive and KOH solubility but none of the bacterial isolates showed clear zone during starch hydrolysis test. All the isolates were negative to starch hydrolysis test and were positive to Tween 80 hydrolysis test and also showed an opaque zone of crystals around the colony. The pathogenicity on the artificially inoculated test pieces and symptoms of the disease were also proved.

Isolation and characterization of actinobacteria
Among 210 soil samples, a total of 86 morphologically distinct and diverse actinobacterial isolates were isolated. Each isolate showed different morphological characteristics based on aerial mass color, reverse side pigments, melanoid and soluble pigments production ( Table 2). The colors were recorded using standard color chart as recommended by ISP [20]. Among them, 8 isolates from Tikur Incini district soils which were 9.3%; 14 isolates from Toke Kutaye district soils which covers 16.28% and 64 isolates from Ambo district soils encompassing 74.42% of the total actinobacterial isolates. Common actinobacterial species at all the three different districts were not observed. The identified species (86) were morphologically distinct and were studied upon the aerial, reverse side color, diffusible pigment productions, melanoid and soluble pigments, formation of mycelium and sporophore morphology under microscopic examinations. Similar findings were also previously reported by Kim et al. [26] and Vijayakumar et al. [27]. The soil samples collected from the typical highland of Tikur Incini was almost covering sandy-loam soil types the low land soils collected from Toke kutaye and sand to clay-loam of black soils were stands for soil samples of the Ambo district mid high land i.e. more actinobacterial isolates were isolated from the soils collected from Ambo district. This indicated that actinobacterial isolates are very diverse and occur in diversified terrestrial habitats of cultivated, grazing and forest soils of all lowland, midland and high land of different soil types which were similarly stated by Okami and Hotta [10], as actinobacterial population density was less common in marine sediments relative to terrestrial soils. The soil samples were collected from forests, virgin land, cultivated and grazing lands as stratified actinobacterial isolates were streaked on half part of pre-dried surfaces of modified nutrient agar plates and incubated at 30°C for 3-5 days. After observing good growth of the actinobacteria, the target pathogen was streaked at right angles to the streaked growing actinobacteria as close as possible and incubated at 30°C for about three days. This screening was done in triplicates for each isolate on Completely Randomized Design (CRD). The inhibitory activity was observed visually daily and actinobacterial isolates showing inhibition to the growth of the target pathogen was selected for further next secondary screening.
Secondary screening: Actinobacterial isolates showing antibacterial activity during preliminary screening was used for the secondary screening step. The actinobacterial isolates were again further tested for their antibacterial activity using modified dual culture technique [25]. In dual culture method, the actinobacterial isolates were spot inoculated on pre-dried surfaces of nutrient agar plates and the plates were incubated at 30°C for 3-5 days. After good growth of actinobacteria, 1 ml (about10 7 cfu/ml) of 24 h old culture of the test organism from nutrient broth was inoculated on the plates and carefully distributed by rocking. Excess inoculum was carefully removed by tilting the plate at about 45°C. Standard antibiotic disc, Chloramphenicol (30 µg/disc) was used for comparison. The plates were then incubated at 30°C and inhibitory activity of the actinobacteria and the standard antibiotic discs were continuously observed and recorded after 24-72 h. Visually observed zone of inhibition (both diameter and annular radii) was measured from the back side of the plates using calipers. All the plates were replicated three times in CRD. In the same manner as the preliminary screening, actinobacterial isolates showing better/equivalent inhibition zone as visually compared to the standard antibiotic discs were selected for further cell free suspension screening test.
Actinobacterial isolates selected during dual culture were inoculated into SCB and allowed to grow at ambient temperature on a shaker (120 rpm) for about a week until high turbidity was observed. After incubation, the broths were centrifuged twice at 6000 rpm for 30 min each and then the filtrates were again filtered using microbial filter of 0.22 μm. Finally, the extracted antimicrobial compounds were aseptically loaded onto 6 mm diameter sterile disc (50 µl/disc). Then the discs were placed on nutrient agar plates which were pre-seeded with the test organism. Standard Chloramphenicol discs (30 µl/disc) were used as comparison. The plates were then incubated at 30°C and inhibitory activity of the actinobacteria and the standard antibiotic discs were continuously observed and recorded after 24-72 h. Visually observed zone of inhibition (both diameter and annular radii) was measured from the back side of the plates using calipers.
structures. Both isolates produced mummified fluidal of irregular bacterial colonies with the pink color on the TTC agar medium after 48 h incubation at 30°C and were observed for rod shaped after examined under compound microscope. The cultures showed highly mummified fluidal viscous and slight creamy to white color on the TTC medium ( Figure 1) and colonies color were showed brown color after 48 h of incubation at 30°C. All the isolates produced characteristic whitish on TTC medium and this observation was confirmed with the observation of Kelman [15].
with black color; sandy soil with red to brown color which represents

Screening for antibacterial activity
Preliminary screening: The preliminary screening was conducted for all 86 actinobacterial isolates against the test pathogen, R. solanacearum. Of all the tested isolates, 52 isolates showed inhibition effect to R. solanacearum ( Figure 4) and were selected for further screening in the secondary test as recommended by Martin and French [28].

Secondary screening:
The 52 actinobacterial isolates showing inhibition to the target pathogen during the preliminary screening were further tested for their inhibition using dual culture technique. From the actinobacterial isolates screened by dual culture method, 36 of them were performed effective against tested pathogen in their inhibition activity ( Figure 5 and Table 3). Both diameter and annular radius of the inhibition zone were measured and analyzed. All the isolates appeared significantly different (P ≤ 0.05) in their inhibition against R. solanacearum pathogen. According to the analysis made, there was very highly significance difference among actinobacterial isolates; Gosu qoras#196-1 which was the superior isolate under this experiment, Awaros#174-2, Senkeles#132-5 and Awaros#183-1 were second, third and fourth antagonistic isolates exhibited more inhibition activity against R. solanacearum in their order of inhibition importance (Table 3). Finally, 36 isolates having relatively higher/equal mean of annular radii inhibition zone than control or standard antibiotic disc of Chloramphenicol were selected for their supernatant activity test.
Cell free suspension test screening: From the top performing isolates, 36 of them were again tested using their cell free suspension ( Figure 6). Cell free suspension from all the 36 isolates showed significantly different inhibition activity. The actinobacterial isolate, random sampling options simply to extend the chance of success of obtaining very distinct actinobacterial isolates. Actinobacteria isolated from forest soil samples were covered 26.74%, cultivated land for 56.98% and grazing land for 16.28% (Figure 2), in which 23, 49 and 14 isolates were isolated from forest, cultivated and grazing lands, respectively.
In both dual culture and cell free suspension test, 21 of isolates such as isolate Gosu qoras#196-1, Awaros#174-2, Senkeles#132-5, Awaros#183-1 and others showed significant differences between actinobacterial isolates (Table 5) and have showed inhibitory activity against the target pathogen and this shows that their inhibitory activity comes from secretion of extracellular antimicrobial compounds as stated by Brock [29]. However, 15 isolates showed very minimal inhibitory activity against the target pathogen and this may indicate that the inhibitory activity during the dual culture test might have come from the competition effect than the secretion of extracellular antimicrobial compounds as the similar work of Brock [29]. Key: R=replication, Means with the same letter are not significantly different Table 3: Mean comparison of inhibition zone of actinobacterial isolates against R. solanacearum using dual culture method. This study concluded that the actinobacterial isolate, Gosu-qoraS#196-1 had become superior antagonistic isolate against R. solanacearum followed by Awaro S#174-2, Senkele S#132-5, Awaro S#183-1, Senkele S#133-3, Dhaga file S#113-1, Awaro S#176-4, Gabata S#21-1, Dhaga file S#128-2 and Awaro S#174-3 isolates. Therefore, R. solanacearum has been controlled successfully by actinobacterial isolates under in vitro condition. In this study, the levels of bio control achieved in laboratory condition by the various isolates of actinobacteria has provide reliable, effective, and alternative management approach to the R. solanacearum. This study has investigated actinobacterial isolates having antibacterial activity against this target pathogen, from these very few samples indicating that actinobacteria from Ethiopian ecology can be a good potential for investigation of bioactive metabolites for More research is also needed on the formulation and delivery of the actinobacteria bio control agent's preparations. Further research on the actinobacterial isolates should be focused on species identification, in vivo evaluation and further testing of its host range and disease controlling capabilities in other pathogens. The strong inhibition of actinobacteria to bacterial pathogen, Ralstonia solanacearum justifies the need for evaluating its potential as bio control agents for controlling a wider range of pathogens. Means with the same letters are not significantly different