Evaluation of tomato (Lycopersicon Esculentum Miller) varieties for nematode and ralstonia diseases resistance and productivity

Tomato is one of the most remunerable and widely grown vegetables in the world. The objective of this study was to evaluate tomato varieties for nematode and Ralstonia diseases resistance and productivity. The experiment was conducted under a complete randomized design experiment with three replications at Hawassa University shade house. Four varieties of tomato were inoculated with nematode (Meloidogyne incognita), Ralstonia solanacearum and mixed bacteria (Bacillus subtilis, Pseudomonas) in single and in combination of them with a total of six treatments. The multivariate analysis of variance showed a signifi cant effect both for varieties and treatments for most yield and growth parameters except for unmarketable fruit yield per plant and weight of unmarketable fruit per plant. Tomato var. Venise produced the highest total yield (7556.33g) whereas Galilea variety gave the lowest total yield (2656.4g). Based on the yield performance, nematode and ralstonia resistance rating, Venise was found to be the best tomato variety followed by Awassa for disease resistance and productivity. Variety Galilea was severely affected by Meloidogyne incognita as well as by the Ralstonia solanacearum and produced the lowest marketable fruit percentage. None of the tomato varieties was found highly resistant (0 % disease severity) to Meloidogyne incognita and Ralstonia solanacearum. Therefore, there is a need to develop nematode and Ralstonia-resistant tomato varieties. Research Article Evaluation of tomato (Lycopersicon Esculentum Miller) varieties for nematode and ralstonia diseases resistance and productivity Alemu Tsega Alene1* and Zelalem G Mariam2 1Department of Biology, Genetics, Injibara University, Ethiopia 2Department of Biology, Applied Genetics, Hawassa University, Ethiopia Received: 19 November, 2020 Accepted: 08 January, 2021 Published: 12 January, 2021 *Corresponding author: Alemu Tsega Alene, Department of Biology, Genetics, Injibara University, Ethiopia, Tel: 0935477903; E-mail:


Introduction
Tomato is one of the most widely grown vegetables in the world; However the production and productivity of tomato in Ethiopia are often very low as compared to the production in many African countries and the world average. This is because the production is adversely infl uenced by various biotic factors.
The principal biotic constraints in Ethiopia are diseases [1].
Various management practices have been adopted for the control of diseases in tomato varieties such as the application of pesticides, inorganic fertilizers. Although, the application of fertilizers and pesticides have a major role to play for the successful cultivation of tomato crop continuous, excessive and imbalanced use may lead to ill health and ecological hazards resulting depletion of physicochemical properties of the soil and ultimately poor crop yield [2]. Hence, there is a need for alternative sources of safe nutrient fertilizers which may boost crop yield without having adverse effects on soil properties.
So far no research has been done to evaluate quantity and quality of fruit grown in the greenhouse and though it was done in open fi eld area, but also no varieties were screened for protected cultivation systems. There has been lack of information regarding resistance to bacterial wilt and rootknot nematode in locally available tomato varieties.
Citation: Alene  have been shown to produce auxins which help in stimulating plant growth [3]. In view of this study, disease resistance tomato varieties can be screened to solve a disease spread and infection. Cultivation of disease-resistant varieties is effective, economical and safer and it is the most important tool in Integrated Disease Management (IDM). Combining microbial inoculants and resistant varieties are currently becoming well known in the production of healthy seedlings and technology for large-scale production of tomato varieties [4].

Experimental design
Nursery house: Seeds of four tomato varieties were bought from Meki commercial Agriculture Center and grown following standard procedure in a nursery under greenhouse condition.
The nursery was made of four beds consisting of 4m 2 surface areas of each. These corresponded to four varieties of tomato seeds used in this experiment. Seeds were sown in holes of about 2 cm deep in a nursery greenhouse. The distance between neighboring holes was 10cm. The sowing consisted of depositing seeds in the holes and then covered them with soil. The beds were then watered with one watering can full per bed. Care was taken to make sure the nursery was in a good state. This was done by ensuring daily watering two times a day (morning and evening) with 20 L water per bed before germination. were scored on each treatment on a 0-4 basis with 0 = no galls or egg masses, 1= 1 or 2 galls or egg masses, 2= 3-10 galls or egg masses, 3= 11 -30 galls or egg masses, and 4= 31 -100 galls or egg masses.

Assessment of growth parameters
The growth parameters considered were:   Disease severity(%), is the relative or absolute areaof plant tissue affected with disease (sometimes called "intensity). Highly Susceptible (76-100%) [7] Table 1.
Average fruit weight per plant (g): The mean number of fruits per each tomato plants.
The number of unmarketable fruit per plant: is the number which was obtained by sorting the diseased, discolored, shrunken shape and small-sized, totally unwanted fruit by consumers from marketable was recorded.

Data compilation and analysis
Both descriptive and inferential statistics were done using SPSS ver. 22. Data were subjected to a two factor multivariate analysis (MANOVA) and means of fruit were compared with SPSS version 22 Software [9]. Analysis of variance was performed on data and means were separated by Levene's test (P≤0.05). F tests were done on individual experiments for both the inoculated seedlings and controlled group. Severity and incidence percentage ratings ranged from 0% to 100% and therefore were calculated. Visual disease ratings (incidence and severity leaves, petioles and stems) were made by visually examining each replication from the non-inoculated varieties and assigning each cultivar a descending number based on the severity of early nematode, ralstonia, and mixed bacterial infection, et al. [10]. Growth and yield parameters were evaluated under collected data after inoculation. The yields were harvested 3 months after transplanting and the yield was determined according to the fresh weight of the tomato fruits per plant [11].

Results and discussion
A multivariate analysis of variance was performed using varieties and inoculants as main factors on growth performance, yield performance and disease resistance percentage. The multivariate analysis of variance showed a signifi cant effect for inoculants, varieties and interaction of inoculants and varieties for growth and yield parameters and disease.

Growth parameters
Estimated marginal means of the interaction effect between varieties and inoculants on growth parameters and yield parameters were presented in Table 2. A two way MANOVA on variety and treatments as main factor showed a signifi cant effect (p ≤ 0.05) for most growth parameters (e.g. number of branches, number of fl owers, number of leaves and days to 100 % fl owering) but indicated a non-signifi cant effect for leaf length and plant height at (p ≤ 0.05). Post-hoc comparisons using Scheffè tests revealed signifi cance difference between Bridget 40 and Galilea. A similar work done by Falak, et al. [12] found a signifi cant effect on growth parameters among the tomato varieties in the battle valley of district Mansehra, Khyber Pakhtunkhwa.
The highest mean number of branches was observed from variety Awassa (Table 2) inoculated with mixed bacteria, whereas the least mean number of branches was recorded  (Table 2)

inoculated with
Nematode, Ralstonia and Mixed Bacteria together but the least leaf length was recorded variety Awassa (Table 2)

Disease incidence percentage of varieties of tomato
The results of the present study showed that the tomato varieties have a signifi cant difference in terms of their disease resistance capacity. A various level of disease severity and incidences were recorded for the different inoculants in single and combination (i.e. Ralstonia, nematode, nematode coupled with Ralstonia, a combination of ralstonia, nematode and mixed bacteria) on the different tomato varieties. Zainab [16] reported that the tomato plants are susceptible to disease at all stage of their growth but yield reductions were determined to great extent by the stage at which tomato plants become infected. Average disease incidence of tomato variety of Awassa, Briget40, Galilea and Venise were 44.44% 38.88, % 55.55% and 22.22% respectively. This indicated that variety Galilea had the highest susceptibility, whereas the least incidence was observed for variety Venise. Furthermore, all varieties were not affected by mixed bacteria only. Based the level of the incidence variety Venise had more resistant but variety Galilea is the least resistant. There was a variation on the magnitude of disease incidence among the different varieties, which may be due to different resistance ability during the growth period of the plant. Several factors (e.g. genetic, environmental or genotype environment integration) could contribute to the susceptibility tomato plant to various pathogens. For instance, María [16] proposed plant infection by bacterial spot is favored by frequent rainfall and high temperatures.
The tomato varieties considered in the present study responded differently to the inoculants applied ( Figure 1).For instance, variety Awassa was more susceptible to the combined application ralstonia and nematode. This was evidenced by the threefold disease of incidence of by the combined treatment of Ralstonia plus nematode (i.e. 37.5%) compared to that of Ralstonia and nematode together with mixed bacteria (i.e. 12.5%).Similarly variety Briget 40 was highly susceptible to the combined treatment of ralstonia plus nematode (i.e.42.8%) whereas treatments of Ralstonia, nematode together with mixed bacteria and nematode has the lowest (14.3%). Zachee [9] showed that disease parameters were the main indices to characterize the resistance of plant species under natural conditions.

Disease severity percentage of nematode
The nematode resistances ratings of tomato varieties (percentage of tomato plants damaged by nematode) were presented in Table 4. None of the tomato variety was found highly resistant to the nematode inoculation. Tomato var. Galilea had the highest severity (i.e. 66.66%) affected by nematode followed by Briget40 (i.e. 33.33%). The least percentage of plants that were affected by nematodes was observed in variety Venise (i.e. 8.33%). Jaiteh, et al. [17] reported that nematode resistance in host plants was manifested by reduced rates of nematode reproduction, egg masses and consequently, low nematode population densities than that of a susceptible one.

Disease severity percentage of Ralstonia (Ralstonia solanacearum)
The ralstonia resistance degrees of tomato varieties (percentage of tomato plants damaged by Rralstonia) were presented in Table 5. None of the tomato variety under evaluation was highly resistant to Ralstonia. Variety Venise was the least affected (i.e. 16.66%) whereas as variety Galilea was highly affected (i.e. 58.33%) by Ralstonia. Development of disease resistant tomato genotypes is the most effi cient and environmentally friendly way to control the diseases when suffi cient genetic variation for resistance is available and therefore plant breeders can upgrade the existing tomato varieties through cross-breeding. Jaiteh, et al [17], demonstrated that at moderate to high initial population densities, ralstonia reached their maximum levels on a susceptible cultivar. Whereas on partially resistant cultivars had less damage by the nematodes, the population densities were still increasing.

Disease Severity percentage of Ralstonia solanacearum combined Meloidogyne incognita
The Ralstonia and nematode resistance rating of tomato varieties (percentage of tomato plants damaged by Ralstonia combined nematode) is presented in Table 6. The results of the present study indicated that var. Awassa was resistant whereas Galilea was highly affected by ralstonia plus nematode treatment.

Disease Severity percentage of Bacillus subtilis and Pseudomonas, Ralstonia solanacearum, Meloidogyne incognita
Average nematode, ralstonia and mixed bacteria resistance rating of tomato varieties were given in Table 7. Variety Galileahad highly affected with combined treatment the nematode, ralstonia and mixed bacteria (i.e. 100%). The highest percentage of plants with low damage by nematode, Ralstonia and mixed bacteria (16.66%) was observed in Venise variety and therefore, Venise was found to be the least affected tomato variety.

Yield performance of four tomato varieties
In the present study, signifi cant differences were observed among the varieties of tomato, as revealed by the analysis of variance (Table 9). The varieties had signifi cant effect on the total number of fruit per plant, the total weight of fruit per plant, the weight of a single fruit per plant, the number of marketable fruit per plant, the weight of marketable fruit per plant and average fruit weight per plant but a non-signifi cant difference on number of unmarketable fruit per plant and weight of unmarketable fruit per plant. MANOVA revealed all tomato varieties differ signifi cantly for most yield parameters (p ≤ 0.05), likewise there was a signifi cant effect on inoculants but number of unmarketable fruit per plant and weight of unmarketable fruit per plant were not signifi cantly different between varieties of tomato. Post-hoc comparisons using Scheffè tests showed signifi cantly different between tomato varieties.
The low marketable yield obtained for some tomato varieties used might be due to non-development of fl owers into fruits as about 50% only of the fl owers developed into fruits. Varieties with high numbers of fruits harvested might have successfully developed more fl owers into fruits possibly as a result of better genetic components. Jalloh, et al. [17] indicated that only 50% of fl owers produced developed into fruits, therefore sink size (genetically controlled) infl uences     fruit production in tomato and perhaps higher capacities to convey photosyntheticmaterials towards economicyield.

Total fruit weight per plant
Total fresh fruit weight per plant was signifi cantly different (P ≤0.05) among the varieties inoculated by diseases (Table  8). Venise had mean maximum in fresh fruit weight (701.32 g) inoculated with mixed bacteria that promote the growth of the plant by fi xing inorganic phosphorus and nitrogen while Galilea tomato variety had a minimum in fresh fruit weight (90.5 g) inoculated with ralstonia (see Figure 2 and Appendix Table 1). The highest weight of fresh fruit obtained from Venise over other varieties investigated may be attributed to the possibility of possession of higher stomatal conductance, better partitioning of photosynthetic materials towards economic yield, better genetic structure and higher potential to transport photosynthetic materials within plants. Similarly, Baliyan and Roa [7] showed six tomato varieties contributed differently to the total yield of tomato. Further, the four tomato varieties do exhibit an adverse effect in terms of the quality of tomato produced. This study is in agreement with Gebisa Benti, et al. [13] indicated that the varietals differences in yield might be attributed to the differences in inoculation of tomato varieties with diseases.

Number of marketable fruits per plant
The highest mean number of marketable fruit was obtained from varieties Awassa with uninoculated and Venise inoculated with mixed bacteria (7.667) while the least from Galilea inoculated with nematode plus Ralstonia (see Figure 3. The recorded variations of varieties in marketable yield could be due to their differences in genetic make-up and/or disease resistance.

Unmarketable number of fruits per plant
Interaction effect of variety by inoculants showed had no a signifi cant difference (p ≤ 0.05) on unmarketable yield (see Table 8. The highest mean number of unmarketable fruit was obtained from Galilea (1.667) inoculated with Ralstonia and nematode together while the least was obtained from Briget 40 (0.0011g) inoculated with mixed bacteria only (see Figure 4 and Appendix Table 1). This unmarketable number was recorded based on shrunken shaped fruits, small sized, and discolored fruits that were estimated to be due to the differences in the inherent characters of the varieties, those lacked uniformity when drying, and or due to physiological disorders (bleaching) during the fruit set or due to the inoculants effect. Abdulazeez [18] indicated that fertilization infl uenced the nonmarketable yield in all the years at Samaru and fertilizer produced signifi cantly highest nonmarketable fruit yield than the control treatments in all the years and combined data while at Kadawa.

The weight of unmarketable of fruits per plant
The highest unmarketable mean fruit weight per plant was obtained from Awassa inoculated by ralstonia(46.133g) while the least unmarketablefruit weight per plantwas gained from Briget40 inoculated with mixed bacteria only (0.003553g), see Figure 5 and Appendix Table 1). This unmarketable yield was recordedthrough subjective judgment based on shrunken   shaped fruits, small sized, and discolored fruits that were estimated to be due to the differences in the inherent characters of the varieties, those lacked uniformity when drying, and or due to physiological disorders (bleaching)during the fruit set or due to the inoculants effect.The results of this study showed that the yield performance for the varieties wasbelow theaverage productivity of tomato as reported by Meseret,et al. [19], who found variations in theperformance of tomato varieties and heirloom varieties under tropical conditions.

Weight of marketable fruits per plant
The highest marketable mean yield (455.2 g) was obtained for variety Venise inoculated with mixed bacteria while the least weight of marketable fruit (60.867g) was obtained from Galilea inoculated with nematode plus Ralstonia ( Figure 6 and Appendix Table 1).The recorded variations of varieties in marketable yield could be due to their differences in genetic make-up and/or disease resistance ability. This study is in agreement with that of Zishan, et al. [20] who analyzed data for fruit weight (grams) showed that there were signifi cant difference among all the lines at p ≤ 0.05.

Number of fruits per plant
The highest mean number of fruits per plant (8.667) was observed in Venise variety inoculated with mixed bacteria only while the lowest number of fruits per plant (3) was obtained from Galilea variety (Figure 7 and Appendix Table 1). This difference may be attributed to the higher number of fl owers that developed into fruits. Unlike other varieties whose 50% of their fl owers dried up and fell off or formed tiny fruits which shriveled up and fell off without further development, fl owers of variety Venise successfully developed in to more fruits possibly because of the better genetic constitution. Produced Flowers developed into fruits, thus sink size (genetically controlled) infl uences fruit production in tomato and it may also be attributed to better genetic structure and higher potentials to transport photosynthetic materials towards economic yield. This result is supported by Joyce [21] who worked on combining micro-propagation and AMF inoculation increased the potato seed yield and quality of root colonization signifying that the extent of root colonization did correlate with the benefi ts of the host plant. Similar results were reported as studies Moustaine [22] who showed that application of Bacillus can stimulate yield and quality parameters in sugar beet, barley, apricot and apple. Inoculation based Bacillus bacterial strains have resulted in more effective results in terms of growth and yield compared to other applications (Mycobacterium) and compared to the control. These results are in agreement with the fi nding of Nguyen [23] who, indicated that fruit number per plant was also signifi cantly infl uenced by plant diseases, the low plant diseasesresulting insignifi cantly more fruit number as compared to high plant density diseases [24][25][26][27][28][29][30][31][32].

Average fruit weight per plant
Awassa had maximum (82.731g) fruit mean weight without inoculation while Galilea variety had a minimum (27.981) in fruit weight (Figures 8,9). Fruit weight migt be attributed due to varietal genetic make and attributed poor tomato yieldtonon-developmentof fl owers into fruits and foundthat only 50% of the fl owers produced developed into fruits and limited the size and weight of fruits. The maximum fruit weight could be varietal differences in growth and yield which might be attributed to the differences in inoculants effect on tomato varieties. Besides the differences in varietal genetic makeup, the low marketable yield obtained for some tomato varieties used might be due to non-development of fl owers into fruits as about 50% of the fl owers develop into fruits. The result was supported by Zishan, et al. [21] who showed average number of fruits per plant showed signifi cant difference among the lines at p ≤ 0.05.

Total marketable yield of varieties
Variety Venise gave the highest marketable yield (7224.13 g)    followed by Awassa (5793.6 g). The percentage of marketable fruit (by weight) varied from 95.6% (Venise) to 802.8% (Awassa). The variety Galilea produced the lowest total yield (2656.4g) as well as the lowest marketable yield (2373g) among the four varieties under evaluation. Variety Venise performed well as it produced a total yield of 7556.33 g and produced the highest marketable percentage (95.6%) fruits.
The highest marketable percentage fruits produced by Venise can be attributed to very high resistance to the nematode and Ralstonia disease and therefore, Venise was a good variety (see Table 9). This suggests that this variety was better able to resist the inoculated disease and yield constraints encountered during the growing period than any of the other four varieties evaluated in this study.

Conclusions
The results obtained from this study revealed that tomato varieties performed better at inoculation of mixed bacteria. The study, in general, indicates that diseases and varietal differences affect agro-morphological traits, yield, quality characteristics of tomato fruit and resistance to disease. The results of the experiment indicated that Venise was the best yielding and disease resistance tomato variety followed by Awassa. It was concluded that Venise tomato varieties was highly resistant to nematode, ralstonia or to the combined effect of ralstonia and nematode. Galilea was found to be the most severely attacked variety (55.55%) by nematode and ralstonia where as Venise was least attacked variety by nematode and ralstonia (22.22%).
As indicated in the results there were signifi cant differences among the varieties, treatments and the interactions between inoculants and tomato varieties for most parameters growth, disease resistance and severity yield except leaf length per plant. Venise and Awassa were increased fruit yield per plant over the other varieties of tomato. The results obtained showed that variety Venise was superior in yield parameters and disease resistance percentage. Therefore host resistant and bio control agents might be applied both for small scale and large scale production to boosting the production in the future. Therefore Based on the current fi ndings of the study, it was recommended that, Varietal selection for sustainable production and productivity enhancement and A need to develop and evaluate new tomato varieties which are resistant to diseases. Besides, the use of resistant varieties, biocontrol methods shouldalso beadopted for disease control, growth and yield enhancement with respect to being eco-friendly, non-hazardous and nontoxic reducing the cost of tomato production.