Investigating the impact of root-knot nematode double infection on bacterial wilt of tomato

To check the possibility of double infection of tomato plants by root-knot nematodes (RKN), Meloidogyne spp., and tomato wilt bacterium, Ralstonia solanacearum, pot experiments was conducted in the green house, department of Plant Pathology, the University of Agriculture Peshawar. Four treatments consisting bacteria and/or nematode (alone or in combination) were investigated on five tomato varieties i.e. Lyreka, Money-maker, Red-stone, Rachna and Reo-Grand. Results stated that when RKN was inoculated together with R. solanacearum , severity of bacterial wilt enhanced while the yield was reduced significantly. Maximum number of plants (96%) survived when plants were not inoculated with bacterium. However, with double-inoculation 77.33% plant survived. Similarly, maximum (3.33) disease ratings were recorded when plants were double-inoculated. Yield was reduced from 2.44 kg.plant -1 to its minimum (0.54 kg.plant -1 ) when plants were double-inoculated. It was concluded that that RKN having a synergistic effect on bacterial wilt diseases, if inoculated together with R. solanacearum and it is recommended RKN should be managed properly to reduce the impact of bacterial wilt disease in tomato.


Preparation of Nematode inoculum
Nematode inoculum was papered with help of the procedures of Atamian et al. [17]. Root knot nematode infected roots of tomato, potato and okra (alternate hosts for RKN) plants ware collected from naturally infected field in a survey of Malakend areas of Khyber Pakhtunkhwa, used for nematode inoculum. One day before inoculation, the roots were kept in sterilized distilled water for overnight at room temperature and then chopped with a needle. Nematode egg masses were carefully isolated from the roots under stereo-microscope, and transferred to an eppendorf tube (ten in each) in sterilized 0.85% saline solution and stored it at 4 o C for incoulation to plants later.

Preparation of bacterial inoculum
Due to high virulence based on colony morphology (i.e., white mucoid colonies with pink centers) [18] PCR confirmed (with RS-specific primers) bacterial isolate RS14-PES1 (Bacterial culture bank, Deptt. Plant Pathology, The University of Agriculture Peshawar) was grown on Nutrient Agar for 24 hours, scrapped off the plates and stored in solution of 100g skimmed milk and 10g Na-glutamate per litter at -20˚C for (24 hours) to avoid possible development of non-pathogenic mutants. The stock culture was thawed, streaked on the TTC (2,3,5-triphenyltetrazolium chloride) medium and incubated at 30˚C for 48 hours. Bacteria were collected with sterilized scalpel and suspended into 0.85% saline sterilized distilled water. The concentration was adjusted to 10 7 cell.ml -1 (using photospectrometer with OD600) for inoculation to plants.

Preparation of plant material
Seedlings of each variety were separately raised in peat soil in green house at 25±5˚C. After 30 days of sowing, seedlings were transplanted to the earthenpots (with diameter 40cm). Environmental and soil condition were kept same for all varieties (as all the varieties used in the experiment were local). Similarly, plant nutrition, water requirements and cultural practices were also kept constant.

Inoculation of bacteria and Nematode to plants
Before transplanting seedling to the earthen pots.
Ten nematode eggmasses (approximately 5000 eggs) were applied to each earthen pot and watered it well in order to provide hatching conditions to the nematode eggs. After three days of nematode inoculation to soil the seedling were transplanted to earthen pots. However each seedling was dipped in the bacterial suspension (10 7 cell.ml -1 ) for 30 seconds before transplantation.  Figure 2&4). All treatments were found highly significant. For treatments maximum (3.33) disease ratings were recorded for treatment 3 where plants were inoculated with bacterium and nematode combined. It was followed by treatment 2 with 1.80 disease ratings, where plants were inoculated with the bacterium only, while treatment 1 (control) and treatment 4 (inoculated only with nematode) also showed some disease ratings i.e. 0.27 and 0.33 respectively but it was negligible and might be due to infected soil or inoculums spread during cultural practices. On the other hand behavior of varieties to the disease severity was nonsignificant. Yield (kg.plant -1 ) Data regarding yield in kg.plant -1 are presented in (Figure 3&4). All the treatments, varieties and interactions were found highly significant. For treatments, maximum (2.44 kg.plant -1 ) yield was obtained for treatment 1 (control) where plants were keptnon-inoculated with bacterium and/or nematode. It was followed by treatment 2 with 1.74 kg.plant -1 , where plants were inoculated with the bacterium only. While treatment 3 (inoculated with bacteria and nematode both) showed minimum (0.54 kg) yield.plant -1 . On the other hand variety M. maker was best yielding variety with 1.78 kg.plant -1 yield, followed by R. stone with 1.56 kg.plant -1 , whereas, variety Rachna was lowest (1.38kg.plant -1 ) yielding variety. It was also observed that the treatment showing low disease score gave more yield and vice-versa ( Figure 5).  . In our studies we found that when RKN was inoculated together with R. solanacearum, severity of bacterial wilt enhanced significantly. Our results are in agreement with previous findings [14,15] which reported that double infection of RKN and R. solanacearum resulted in significant increase in severity of bacterial wilt even on resistant tomato cultivars. However, in survival percentage both the un-inoculated and inoculated with only bacteria showed statistically similar results indicated that RKN needs more time for its infection and establishment within the roots and root zone of the plants. RKN predispose tomato plants to bacterial wilt by severely injuring and making them week. Wounding reduces the time needed for bacterial invasion in the root system [16]. Moreover, when a plant is severely infected by RKN, the normal root system is reduced to a limited number of severely galled roots, with a completely disorganized vascular system. These roots are seriously hampered in their main functions of uptake and transport of water and nutrients [20]. Some previous findings [21] suggest that the role of RKN in augmenting bacterial wilt severity might be more than just producing wounds for the entry of the bacterial pathogen into plant tissues and causing the root system to disfunction. These researchers were of the view that RKN-induced stress was more important as a wilt-triggered factor than the wounding. Other researchers [22,23] found that RKN infection caused considerable biochemical changes in the diseased plant resulting in weakening of its chemical resistance mechanisms that led to enhancement of wilt. Further research is needed, especially on finding new, environment friendly components of the IDM against bacterial wilt of tomato.

Conclusion and recommendation
From the present research it can be concluded that that Root-knot nematode having a synergistic effect on bacterial wilt diseases, if inoculated together with R. solanacearum and it is recommended rootknot nematodes should be managed properly to reduce the impact of bacterial wilt disease in tomato.