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Abstract
One isolate ATS of Steinernemae spp. and three isolates of Heterorhabdities spp. 20M, 56Mn and 91Mn were isolated from soil samples in Regha, Giza Governorate. Three isolates C1, C2 and C3 of Metarhizium anisopliae and three isolates of Beauveria bassiana Bb1, Bb2 and Bb3 were isolated from cadavers of the adult stage of the red palm weevil and soil samples, respectively.
Heterorhabdities spp. isolate 20M was more pathogenic to all stages of S. gregaria, whereas the LT50 values were 2 days with 3rd nymphal instar. On another hand Steinernemae spp. isolate AT4 was more pathogenic to all stages of S. gregaria, whereas the LT50 values were 3.4 days with adult stage.
M. anisopliae isolate C3 was more pathogenic to all stages of S. gregaria, whereas the LT50 values were 1.8 days with 3rd nymphal instar. On another hand B. bassiana isolate Bb3 was more pathogenic to all stages of S. gregaria, whereas the LT50 values were 2.7 days with adult stage.
The 2nd and 3rd nymphal instars were more susceptible to infection with both pathogens than 4th, 5th nymphal instar and adult stage.
Heterorhabdities spp. isolate 20M was more pathogenic than Steinernemae spp. isolate AT4, while the M. anisopliae isolate C3 was more pathogenic than B. bassiana isolate Bb3.
The interaction of entomopathogenic nematodes and entomopathogenic fungi in vitro showed that the Heterorhabdities spp. (isolates 91Mn) inhibit the germination rates of conidiospores of all isolates of both fungi B. bassiana and M. anisoplia, while Steinernematidae spp isolates (AT4, ATS and SFN.27) inhibit M. anisoplia isolates (C1 and C2) and B. bassiana isolates (Bb1 and Bb2).
The studying of the effect of the combination of entomopathogenic nematodes and fungi on S. gregaria showed that the mortalities of 3rd nymphal instar and adult stages were zero% one day after treated simultaneously at low concentration of fungus (105 spores / ml) and nematode (60IJs /ml). However the mortalities was increased when the combination of both pathogens were used at high concentrations. Mortality of 3rd nymphal instar and adult stage reached to 100% one day after treated in sequential combination (insects exposed to nematode 2 days after fungus) with two pathogens at high concentration of all isolates of nematode (500 IJs) and high concentration of all isolates of fungus (107 spores/ ml), whereas the LT50 values were 0.1 day.
The studying of the effect of dual infection on pathogen development appeared that the simultaneously exposure treatment of Steinernema sp. (AT4) and M. anisopliae (C3) encourage nematode development and inhibit fungal mycoses. Increasing of nematode concentrations increased the percentage of insects appeared nematode development. However increasing of fungal concentration decreased the percentage of insects which appeared nematode development. While, the sequential exposure treatment of Steinernema spp. (AT4) and M. anisopliae (C3) to nymphal and adult stages of S. gregaria encourage fungal mycoses and inhibit nematode development. Increasing of nematode concentrations increased the percentage of S. gregaria appeared nematode development exposure to nematode one day only after treatment with fungus while, the percentage of S. gregaria appeared nematode development was decreased as a result of exposure to nematode two days after treatment with fungus. The concentration of fungal spores at 2.5X106 and 107 spores / ml in sequential combination (exposure to nematode after 2 days after fungus treatment disappeared development of nematode and fungus together on treated 3rd and adult stage of S. gregaria.
Examination of treated S. gregaria 3rd nymphal instar with combination of entomopathogenic fungus M. anisopliae and /or B. bassiana and nematode Steinernema sp. by Scanning Electron Microscope (SEM) showed that the spores developed a germ tube that able to penetrate the cuticle 24h after treatment. The examination showed that the nematode was able to penetrate the insect cuticle 24 h. after treatment.
Inner examination of treated insect showed that extensive fungal hyphae and nematode larvae were evident in the cavity (hemocoel) within 48 and 72 hours after the exposure to combination.
The studying of the combined effect of fungus M. anisopliae and nematode Steinernema spp. on adult stage (male and female) of S. gregaria under cages conditions appeared that the all treatments resulted in a significant mortality in S. gregaria adults compared to the control. The faster mortality was occurred when adults (male and female) exposed to treated sand with combination of fungus and nematode followed by fungus alone and nematode alone. The highest mortality was observed in the combination of fungus and nematode (100 %) 7 days after application.