Tomato (Lycopersicon esculentum Mill) is one of the most important vegetable crop for improving nutrition and income globally1. Nutritionally, tomato contains a substantial amount of protein and essential vitamins, minerals and trace elements to enhance human diet2,3. The bacterial wilt disease (BWD) caused by Ralstonia solanacearum species complex (RSSC)4 significantly affect production of crops such as tomato worldwide1,5. RSSC is a soil-borne gram-negative bacterium with the ability to infect about 450 plant species from 54 different botanical families such as tomato, potato, eggplant and many native plant species causing huge yield losses6. Losses caused by BWD are known to be enormous but cannot be accurately estimated as it varies with cultivar, soil, environment and bacterial isolates. Approximately more than 80 countries are affected by BWD which causes economic loss of more than $1billion annually7,8. The bacterium normally invades plant roots from the soil through wounds or natural openings, colonizes the intercellular space of the root cortex, vascular parenchyma and eventually enters the xylem vessel and spreads up into the stem and leaves. Infected plants stunt, wilt and die rapidly while young leaves remain green.
Various management strategies are used to manage BWD1 but the diversity of RSSC challenges effectiveness of the management strategies9. Use of resistant cultivars for instance is considered the main management strategy but the stability of resistance is highly affected by the biology of RSSC10. Being a species complex plant pathogen, Ralstonia has a broad pathological, genetic and physiological diversity. For example by using host range, pathological, biochemical and molecular descriptors, RSSC was classified into races11-13, virulent/avirulent and pathogenic/non-pathogenic, biovarsor phylotypes14-20.
New strains of RSSC may be introduced in uninfected environments from various sources such as planting materials, water, soil, insects and nematodes21. In 2016 and 2017, tomato growing seasons, two outbreaks of bacterial wilt disease occurred in Tanzania. Tomato plants in different fields were reportedly wilting without yellowing at flowering growth stage22. Severe symptoms with complete plant death were commonly observed. Recently, there has been a regular outbreak of bacterial wilt disease in tomato fields in Tanzania. However, there is little information on the characteristics of the causative pathogens. The causative agent of tomato bacterial wilt disease in the coastal zone of Tanzania belong23 to RSSC biovar 3. However, this information is limited in terms of its coverage and management, therefore this study was undertaken to determine Biovars of RSSC causing tomato BWD in Tanzania to alert the plant quarantine officers to embark on necessary phytosanitary measures.
MATERIALS AND METHODS
Survey and sampling: A field survey was conducted from September, 2017 to December, 2018 to determine biovars of RSSC causing BWD in Tanzania. Purposive sampling was adopted by selecting ten districts namely Arumeru, Babati, Manyoni, Nyamagana, Kilolo, Temeke, Chake Chake, Mbeya Urban, Kibondo and Kongwa to cover the major agro-ecological zones of Tanzania (Fig. 1). A multistage random sampling procedure was used in selecting the wards, villages and the farms. Four wards were selected at random in a district and five farmers’ fields were sampled from each village. Within the farm, five plots of 50 m2 were sampled by critically observing symptoms of BWD. For a quick field diagnosis, the streaming of milky white masses of bacterial cells distinguished BWD caused by RSSC from vascular wilts caused by fungal pathogens and nematodes.
Assessment of disease incidence and severity: Data on wilt incidence were recorded in five plots within a field by counting number of plants with BWD symptoms in a plot15,22. Then the percent wilt incidence was calculated by the following equation:
Disease severity was recorded based on a 1-5 scale15,22. Briefly, 1: No symptom, 2: Top young leaves wilted, 3: Two leaves wilted, 4: Four or more leaves wilted and 5: Plant dies.
Average disease severity per district was calculated using a equation:
where, A is the number of plants on scale 5, B is the number of plants on scale 4, C is the number of plants on scale 3, D is the number of plants on scale 2, E is the number of plants on scale 1 and N is the total number of plants evaluated. Twenty samples of tomato stems with typical bacterial wilt symptoms were collected from each district (200 samples in total) and sent to the laboratory for the isolation and characterization of RSSC.
Study areas visited during the bacterial wilt disease survey in Tanzania
Preparation of bacterial suspension: From the 200 samples of wilted tomato stems originating from the agro ecological zones, eight from each zone were randomly picked to represent a ward in the study area and coded accordingly. A total of forty samples were thus selected for the biovar (s) determination. Selected samples were washed with running water from the tap to remove soils and then immersed in 70% ethanol for 2-3 min to remove any saprophytic or epiphytic bacteria from stem surfaces. Surface-sterilized stems of each isolate were macerated in sterile water to obtain a bacterial suspension.
Protocol adopted for selected pathological and biochemical descriptors
+: Pathogenic, G: Greenish, Y: Yellow
Protocol development: The virulence, pathogenesis and biovars of RSSC isolates were tested by using the pathological and biochemical approaches as shown in Table 1. Based on virulence, isolates of RSSC can be characterized as virulent which are those isolates which produced typical pink center colonies with white margins while avirulent isolates produce red off white margins colonies. Through the pathogenicity test, the pathogenic isolates of RSSC cause wilting of tomato plants as opposed to the non-pathogenic isolates. In addition, oxidation of different types of carbohydrates is used to characterize isolates of RSSC into biovar(s). This is indicated by a color change of medium from green to yellow after inoculating with the isolate of RSSC.
Preparing media, disaccharides and sugar alcohols solution: Solution of TTC and mineral media, disaccharides and sugar alcohols were prepared as detailed in Table 2. D-Trehalose, L-Tryptophan and D-Ribose were included in the test to differentiate between the sub-phenotypes 2A and 2T of biovar 2. Negative controls were set up without any carbohydrate, where salicin and sterile water were used. Dextrose, the most commonly utilized carbohydrate by all biovars was included as a positive control.
Identification of RSSC isolates by using pathological descriptors
TTC medium: The bacterial suspension was streaked into the TTC medium agar plates and incubated at 28°C for 48 h. Single growing colonies were picked and sub-cultured onto a fresh medium to obtain pure cultures. Identification of presumptive colonies were made when typical colonies of virulent showed a characteristics light red colored center and whitish margin while those of avirulent isolates were smaller, off-white and non-fluidal14. The virulent isolates were selected for the subsequent experiment.
Pathogenicity test: Seeds of tomato variety called Tanya, a commonly cultivated but susceptible variety to BWD in Tanzania were used in this experiment. The seeds were sown in a 1 L pot filled with forest soil and sand at 3:1. The pots were placed in green-house at the average temperature of 25°C and watering were conducted after every other day. After two weeks, seedlings were inoculated with the inoculum of the isolates which had presumptive RSSC colony appearance on TTC medium15. Three seedlings in replicate were inoculated with suspension of isolates at the rate of 109 CFU mL1 with punctures made with a sterile needle in stem between two cotyledons. Three replicates per bacterial suspension were used so that a total of nine seedlings were inoculated with each bacterial isolate. Seedlings inoculated with sterile water were included as negative control. Prior to inoculation, seedlings were not irrigated for 24 h.
The experiment was designed in a completely randomized design with three replications and held at 25°C in screen-house. Development of wilting symptoms was observed and severity was recorded weekly13,24 on a 0-5 scale where, 0: No symptoms, 1: Leaf above inoculation wilted, 2: Two leaves wilted, 3: Three leaves wilted, 4: Four or more leaves wilted and 5: Plant died.
Preparation of media, disaccharides and sugar alcohols solution
Score of BWD severity were related with the BWD severity recorded in the field by computing a correlation coefficient by using CoStat data analysis software.
When typical symptoms were observed, re-isolation of the bacteria was made on TTC medium. After 48 h incubation at 28°C, presence of RSSC looking colony was examined and recorded. Isolates with RSSC colony characteristics were subjected to biovar(s) determination.
Identification of biovars of RSSC by carbohydrates oxidation test
Preparing inoculum: Ten isolates identified as the most virulent according to Koch’s rule (culturing and pathogenicity test) were used. Single colony of each isolate was streaked on TTC medium and incubated for 48 h at 28°C14. A loop-full of each isolate was taken and mixed into 1 mL sterile water in a 2.0 mL centrifuge tube (Eppendorf) to make suspension containing about 109 CFU mL1.
Allocation of disaccharides and sugar alcohol solution into mineral medium, inoculation and data collection: After autoclaving, the media was cooled to 65°C. Ten milliliter of carbon source was each mixed with 90 mL of the mineral medium to make sugar/alcohol amended medium. Three hundred microliter of sugar/alcohol-amended medium were dispensed in each 2.0 mL centrifuge tube and inoculated with 10 μL of suspension of each isolate then incubated at 28°C. Observations were recorded on changing pH as indicated by color change for 7 days incubation25.
Statistical analysis: Data of disease incidence and severity were pooled together by calculating the average of incidence and severity of each ward in a district, this resulted into forty samples which were thereafter subjected to the analysis of variance (ANOVA). The mean separation were carried out by using the least significance difference (LSD) procedure at p = 0.05. The Costat data analysis software program facilitated analyses.
BWD incidence and severity in main agro-ecological zones of Tanzania: The results indicated that on average bacterial wilt disease was present in 55% of the visited farmers’ tomato fields. The mean of disease incidence and severity in the study area ranged from 12-37 and 22.47-89.43%, respectively (Table 3).
Virulence and pathogenesis of isolates of RSSC: Results showed that out of forty isolates of RSSC evaluated for virulence from different zones29 produced typical colonies of virulent isolates on TTC medium (Table 4).
Bacterial wilt disease incidence and severity in the main agro-ecological zones of Tanzania
*p<0.01, **p<0.05, means with the same letter(s) within the column are not significantly different based on LSD (p = 0.05)
Irregular and fluidal colony appearance with pink center and white margins color were consistently observed (Fig. 2).
Figure 2 represents colony appearance of RSSC on TTC medium: Virulent (Left) and avirulent (right). In pathogenicity test, 19 isolates showed typical wilting (Fig. 3), whereas the remaining 11 were non-pathogenic and could be rated as saprophytic although they had similar colony appearance with RSSC. Subsequently, when the isolates were re-isolated on TTC medium, they produced virulent colonies of RSSC.
Virulence and pathogenesis reaction of isolates of Ralstonia solanacearum species complex (RSSC)
+: Virulence/pathogenic, -: Avirulence/non-pathogenic
Furthermore, wilting severity in tomato seedlings relatively differed among isolates. RSSC isolates from the Southern zone were significantly (p<0.05) more pathogenic than the isolates from other agro ecological zones (Table 5). The scatter graph indicated positive correlation between disease severity and pathogenesis of RSSC (Fig. 4).
Biovars of RSSC: Results showed that all the tested bacterial isolates were able to oxidize the four basic carbon sources (Dextrose, sucrose, mannitol and lactose) in 3 days (Table 6). Furthermore, the result of the biovar test showed that nine isolates oxidized disaccharides (sucrose, lactose and maltose) and sugar alcohols (mannitol, sorbitol and dulcitol) while an isolate SIK4 was not able to utilize the sugar alcohols (Table 7).
Ralstonia solanacearum colony appearance on TTC medium: Virulent (Left) and avirulent (right)
Pathogenicity test of bacterial wilt disease on tomato seedlings
A scatter graph for severity in the field and pathogenesis of BWD isolates
Differences in wilting severity among the pathogenic isolates of Ralstonia solanacearum species complex
*Significant at p<0.05, mean with the same letter(s) within the column are not significantly different based on LSD test (p = 0.05)
Utilization reaction of the basic carbon source
s by isolates of Ralstonia solanacearum
+: Positive reaction, -: Negative reaction, RSSC: Ralstonia solanacearum species complex, DW: Distilled water
Differentiation of isolates of Ralstonia solanacearum species complex into biovar(s)
+: Positive reaction, -: Negative reaction, 2T: Sub-group of biovar 2, DW: Distilled water
The isolate coded as XYZ was isolated from the wilted round potato stems and was include as an out group, it behaved similarly as isolate SIK4. On the other hand, all the control plates of carbon sources (dextrose and salicin) and DW remain unchanged (Table 7 and Fig. 5).
Biochemical reaction of R. solanacearum isolates from different agro ecological zones of Tanzania
BWD is widely spread in Tanzania affecting more than 55% of farmers’ field at significantly (p<0.05) different levels of incidence and severity. The level of virulence and pathogenesis among isolates significantly varied within and across the agro ecological zones. Variations in the incidence and severity of BWD in the agro ecological zones may be attributed to factors such as diversity of RSSC isolates soil types and the production environment26. In terms of production environment, hot weather and with high (80-90%) relative humidity are suitably favor survival of RSSC increasing the incidence and severity of bacterial wilt disease. Such environmental conditions are common in most screen-houses especially in developing countries and therefore prevention measures are critically important to avoid introduction of RSSC to such environments. RSSC survives well in moist soils with a pH values from 6-7. Since soils in the agro ecological zones of Tanzania are not homogenous, variations of bacterial wilt disease incidence and severity may be caused by differences in soil moisture and pH levels. Higher (>85%) relative humidity and changes in soil pH increase severity and incidences of bacterial wilt disease22.
There was a strong association (coefficient of correlation r = 0.84) of BWD severity in the field and pathogenesis of the causing isolates which supported Koch’s postulates. The pathological descriptors adopted were able to distinguish virulent from avirulent isolates and further pathogenic from non-pathogenic isolates. Moreover, the use of biochemical descriptors successfully differentiated isolates of RSSC into biovars as previously reported by He et al.13, Hayward16 and Kumar et al.27 that biovar 3 oxidizes both disaccharides and sugar alcohols, biovar 2 oxidizes only disaccharides whereas biovar1utilizes hexose alcohols and biovar 4 oxidizes only alcohols. Isolates of RSSC utilizes various carbon sources for maintenance and growth28.
Out of ten isolates of RSSC used in this study, 90% were identified as biovar 3, while the remaining (10%) were recognized as biovar 2 (2-T). Biovar 3 was recorded from all the zones while biovar 2 was present in the Southern zone of Tanzania. To the best of our knowledge, this is the first report of the prevalence of biovar 2 of RSSC in Tanzania and hence is an alarm to design management strategies to prevent spread to other geographical locations.
Biovar 2 of RSSC infects both tomato and potatoes and thus considered to be of more economic importance. The prevalence and survival of biovar 2 of RSSC in the Southern agro ecological zones of Tanzania could be associated with the continuous cultivation of host plants such as round potato, tomato, pepper and eggplants21. This could indicates that the farmers are not aware of the demerits of continuous cultivation of the same host plant in the same piece of land every season. It therefore call upon the extension service providers to help farmers to adopt plant protection practices such as crop rotation for improved disease management. RSSC biovar 2 is distributed worldwide, occurring in temperate regions, subtropical areas and at higher altitudes in the tropics, because of its lower optimum temperature. Results suggest that biovar 2 of RSSC was recently introduced in Tanzania, although its origin is unknown. Generally, biovar 2 strains around the world appear to be spreading rapidly into previously uninfected areas and implementation of stronger standard phytosanitary measures are recommended to prevent further spread.
The study has investigated and discovered that two major biovars of Ralstonia solanacearum species complex are associated with tomato BWD in Tanzania. Biovar 3 was previously known as the only RSSC biovar responsible for tomato BWD in Tanzania. However, findings from this study have discovered that RSSC biovar 2 also causes tomato BWD in Tanzania. This alerts plant health regulators to implement necessary phytosanitary measures to prevent further spread and/or introduction of the disease considering its quarantine status in different countries.
This research was funded by the German Academic Exchange Service Program (DAAD) through the In-country/ In-Region Scholarship Programme Tanzania 2016 (grant number 91637162) and Centre for Research, Agriculture Advancement, Teaching Excellence and Sustainability (CREATES) in Food and Nutrition Security (grant number 02090107-048-301-4001-P044-J01S01-C42) of the Nelson Mandela African Institution of Science and Technology (NM-AIST).
This study discover the existence of biovar 2 of RSSC as one of the pathogens of bacterial wilt disease in Tanzania that can be beneficial for plant health regulators to take stronger phytosanitary measures to prevent further spread and /or introduction to uninfected areas. This study will help the researcher to uncover management strategy of tomato BWD based on the characteristics of prevailing pathogen strain in a given agro-ecological region that many researchers were not able to explore. Thus, an effective and sustainable management strategy of BWD that is pathogen targeted may be developed.