STUDIES ON THE EFFICACY OF SOME BACTERIA AND FUNGI FOR CONTROL OF GLOBODERA RESTOCHIENSIS

Biocontrol potential of B. thuringiensis var. berliner (Kustom larvo WT-1 ml/pot), var. kurstaki (Kostar 76 LW-1 ml/pot), Saccharopolyspora spinosa (Treisr 480 EC-1 ml/pot), Fusarium spp. (1; 2; 4 g mycelium/pot) and Trichoderma spp. (1; 5; 10 g mycelium/pot) against G. rostochiensis on potato was examined in glasshouse. They were applied to the soil with the planting. All treatments improved the plant growth and yield of potatoes, being greatest with Kostar 76 LW. The greatest increase in the yield occurred in the Oxamyl application (16.6%) followed by Kostar 76 LW or Fusarium spp. (9.9%), Kustom larvo WT (8.2%) and Trichoderma spp. (6.6%). The percentage of infected females was from 40.5% to 48.9%, from 29.6% to 49.3% and from 28.1% to 31.5% when applied fungi Fusarium spp. Trichoderma spp. and bacterial preparations respectively. The bioagents caused significant inhibitory effect on multiplication of the nematode. Reproduction rates decreased from 27.7% to 33.3% by bacterial preparations and from 36.0% to 44.4% by both fungi in comparison with the control.


Introduction
The potato cyst nematode (PCN) Globodera rostochiensis is an important quarantine pest in Bulgaria and it can cause severe losses in the yield (Trifonova, 1995(Trifonova, , 2000)).The absence of crop rotation and growing of susceptible cultivars for a long time led to heavy infestation of private fields with PCN.Methods presently recommended for integrated control-longer rotation with non-hosts, resistant cultivars and nematicides.Each technique has certain disadvantages (Heinicke and Maykuhs, 1982;Behringer, 1988).In the wake of acceptance of eco-friendly approaches, potato cyst nematode management tactics have come to depend on the use of organic amendments (Kantharaju and Reddy, 2001), antagonistic plants (Franco et al., 1999) and biocontrol agents (Mani et al., 1998;Mayer et al., 1990).
There has been growing interest in the use of biopesticides as a substitute for chemicals in nematode disease management.Soils are reservoir for microfloras that are highly varied in composition and activity.Species as Fusatium, Penicillium, Paecilomyces, Phoma and Bacillus may be able to colonize nematodes (Sharma, 2003;Banna and Horani, 2007;Nagesh and Reddy, 2004).Natural parasitism of cyst nematodes by Fusarium and Trichoderma spp.are well-known (Sharon et al., 2001;Suazez et al., 2004;Nagesh and Reddy, 2004;Randey, 2005;Khan et al., 2005).Endoparasitic fungi which infect nematodes with their conidia have been suggested as possible biocontrol agents, although very little is known about their biology and isolation in pure culture.Bacillus spp. was reported to be effective in suppressing phytonematodes in many crops (Davies et al., 1988;Noel, 1990).
The objective of this research was to study the effect of biopreparations of B. thuringiensis var.berliner and kurstaki, Sacharopolyspora spinosa, Fusarium and Trichoderma spp.for the control of potato cyst nematode G. rostochiensis.

Nematode
The population of G. rostochiensis was obtained from soil samples collected from heavily infested potato fields in Smolyan region.The cysts of G. rostochiensis were produced on susceptible potato cultivars cv.Nadejda in glasshouse at 18-22 o C.They were extracted from the soil by wet-sieve technique (Southey, 1986).
Isolates of Fusarium spp.and Trichoderma spp.(local isolates accession number R4-134BPI and T-9) were obtained from roots and lower stems of naturally diseased potato plants (Vatchev, 1995).The inoculums of the two fungal pathogens were developed in pure cultures on barley (Hordeum vulgare L.) grains, preliminary placed in Petri dishes (200 kernels in each) soaked in distilled water for 24 hrs, drained and autoclaved at 121 o C. Each dish was inoculated individually with a 5 mm-agar disc of the fungus from margins of the actively growing 7 and 10-day-old OA cultures, respectively.Dishes were incubated at 25 o C until barley grains were completely colonized.The fungi were used at rates -1; 2 and 4 g mycelium/pot for Fusarium spp.and 1; 5 and 10 g mycelium/pot for Tichoderma spp.

Treatments
The treatments were made in a glasshouse at 20-22 o C and 13 hr day length.Clay pots of 12 cm diameter were filled with 600 g autoclaved soil.Seed tubers of cv.Nadejda were used in all tests.Each pot contained one potato seedling inoculated with 50 cysts of G. rostochiensis (put in muslin bags) poured in the root zone of the plants and covered with soil.They were irrigated to maintain optimum moisture level.Oxamyl (Vidate 10 G) at the rate of 1.5 mg/g soil was applied through irrigation before seedling planting.There were four replicates of each treatment.Non-inoculated plants were used as a control.The experiments were terminated after 90 days or after completion of the nematodes life-cycle.Final cyst density was determined by the wet-sieve decantation technique.The number of cysts were counted and eggs and juvenile numbers determined by separating the eggs from cysts by crushing with a hand.The reproduction index (R=Pf/Pi), plant and root length, fresh shoot and root weight, yield of potato tubers and percentage of infected eggs were recorded.
Then females were selected randomly from each pot and the presence of infection determined by squashing the females and observing the body contents under X 400 magnification (Kerry and Crump, 1977).The following categories were used: infected egg (those developing a fungal colony or bacterial spores), distorted eggs (those whose contents were not identifiable as juveniles orembryos) and normal eggs (containing embryos or second stage juveniles) (Lopez-Llorca and Boag, 1993).
Then cysts from each treatment were removed gently, surface sterilized with 0.5% sodium hypochlorite for 30 secs rinsed thrice in sterile water and placed over medium (0.5% water agar) in Petri-plates.After being incubated at 21 o C for 7 days the fungal infection was observant on a microscope.

Statistical analysis
The data were analysed statistically by analysis of variance and Dunkan's multiple range test (Minkevich and Zakharova, 1977).

Results and Discussion
The soil application of B 1 , B 2 and B 3 significantly improved plant growth and increased the yield of potatoes by 8.2% to 9.9% compared with the untreatedinoculated plants (C).The plants were higher, fresh weight of the shoot of the treated plants was significantly larger compared to infested-untreated plants (N).The best results were obtained in the case of B 2 -49.2 cm length of shoot, 2.7 cm fresh weight and 10.7 cm root length.Infection by G. rostochiensis caused a significant decrease in the fresh weight of potato compared to the uninoculated control (C).Treatment of inoculated plants with Bacillus spp.suppressed nematode reproduction thus helping to increase plant fresh weight and plant length.Data in Table 1 showed that the root fresh weight and length of inoculated-treated with B 1 , B 2 and Fusarium spp.plants were not significantly different from the untreated-inoculated plants (P ≤ 0.05).
Fungal bioagents infected larger number of females than the bacterial.The percentage of infection was from 40.5% to 48.9% and from 29.6% to 40.3% in the soil application of Fusarium spp.and Trichoderma spp.respectively.B 2 infected larger number of females (31.5%) than B 1 (28.1%) or B 3 treatments (27.9%).The degree of efficacy on G. rostochiensis eggs by applying these bioagents increased significantly over the control (P ≤ 0.05).Our results indicate that commercial insecticide-Kystom larvo WT, Kostar 76 LW and Treisr 480 EC reduced the population density of G. rostochiensis in potato.Under the conditions of this experiment, application of the bioagents-B.turingiensis, S. spinosa, Fusarium spp.and Trichoderma spp. in used rates may lead to reducing nematode level and could be an alternative control option for the management of cyst nematode G. rostochiensis.
The results are in agreement with those reported for the efficacy of B. turingiensis to the plant parasitic nematodes-Heterodera glycine, Tylenchulus semipenetrans and Meloidogyne javanica (Banna and Horani, 2007;Noel, 1990).According to Sayre and Wergin (1977) spores of B. thuringiensis attached to the cuticle of second-stage juveniles in the soil, germinate and produce metabolites that are toxic to the nematodes.S. spinosa is effective grain protectant against Rhyzopertha dominica (Daglish et al., 2008).
Earlier studies also reported that the endophytic fungi Fusarium spp.and Trichoderma spp.may colonise eggs and juveniles of G. rostochiensis (Saifullah and Thomas, 1996) and consume the egg contents (Wronkowska, 1989;Janowicz et al., 1997).Trichoderma spp. was reported to mineralise phosphorus in soil (Sharma, 2003) which may have been responsible for the significant increase in the yield when the fungi were applied in the soil.According to Negesh and Reddy (2004) these fungi association with the root system of the plant secrete some enzymes or other metabolites that lower the multiplication of root-knot and cyst nematodes in the rhizosphere.More investigation is needed in order to provide sufficient information about efficacy of these bioagents for control of G. rostochiensis under field conditions.

Conclusion
The results obtained from the research of the soil treatments with the biopreparations -Kustom larvo WT, Kostar 76 LW, Treisr 480 EC and fungi like Trichoderma spp.and Fusarium spp.for control of G. rostochiensis allowed us to make the following conclusions: The bacterial preparations and the fungi Fusarium spp.and Trichoderma spp.caused significant inhibitory effect on multiplication of G. rostochiensis.Reproduction rates decreased by 27.7% to 33.3% by bacterial preparations and by 36.0% to 44.4% by both fungi in comparison with control.
The percentage of infected females is from 40.5% to 48.9%, from 29.6% to 49.3% and from 27.9% to 31.5% when applied the fungi Fusarium, Trichoderma or bacterial preparations respectively.
All treatments improved the plant growth and yield of potatoes, being greatest with Kostar 76 LW.

Table 1 .
Effect of soil application of B. thuringiensis, Fusarium spp.and Trichoderma spp. on plant growth and yield of potato plants inoculated with Globodera rostochiensis.