| Plant-parasitic nematodes cause serious crop losses worldwide.Conventional insecticides are often limited to control these parasites because of their unavailability since the target nematodes mostly live in plant rhizospheres or parasitize inside the tissues during their life cycles;however,the biocontrol of these parasites by the nematode-pathogenic microbes are considered as an alternative means because these microorganisms are able to colonize in the plant rhizosphere or even to be endophytic.The project intended to study the antagonistic mechanism of B.cereus against M.incognita.The paper studied the induced defense enzymes,biomass,root activity,control efficiency,isolation and identification of the active substance.The main results are as follows:1.Detection of the control effects brought by Bacillus cereus.The extracellular supernatants of B.cereus had highly nematicidal activity and the total mortality of C.elegans and M.incognita reached to 77.89 % and 90.96 % at 48 h after processing by it.The control efficiency of B.cereus’ culture medium to M.incognita could reach81.4 %.Control efficiency of B.cereus’ supernatant to M.incognita was 67.4 %,which was lower than bacterial culture medium.2.Isolation and identification of active substances from B.cereus.The broth of B.cereus were studied by polarity gradient extraction,thin layer chromatography(TLC)and HPLC.The active substances including two compounds were identified as C16 sphingosine and phytosphingosine via LC-MS.The LC50 median lethal concentration(LC50)of sphingosine was 0.64 μg/mL.By ultrafiltration,protease detection method showed that there was protein substances in strain supernatant S2 which could kill nematodes.By DEAE-52 anion exchange column chromatography found that the protein had a low nematocidal than sphingosine.3.We measured the content change of defense enzymes in the root of tomatoes.The activities of POD,PPO and PAL all were higher than control plants after 60 d.The PAL,POD and PPO activities of tomatoes inoculated B.cereus culture medium at 0 grade disease and tomatoes inoculated B.cereus supernatant at 1 grade or 0 grade all significantly higher than that of control group.It suggested that induced systemic resistance was an antagonistic mechanisms of the B.cereus against M.incognita.4.Researches on death mechanism.By detecting apoptosis,DNA testing and acridine orange staining found that it was irrespective between lethal effects of phytosphingosine and apoptosis to C.elegans.Treated nematode could produce ROS,while ROS was not detected in control group.It was speculated that ROS induced by sphingosine might have lethal effect on nematodes.The structure of nematode in experimental group had significantly difference with control group.There are some bubbles in nematode of experimental group and its genital area could not be observed.While in control group nematode had no such changes,and nematode genital area could be obviously observed.It suggested that the sphingosine could destroy genital area of nematodes,accordingly inhibited nematodes’ reproduction even killed nematodes.5.Inhibiting reproduction of C.elegans by sphingosine.The effects of sphingosine(0.64 μg/mL)was tested on reproductive C.elegans in different times(24 h,48 h,72 h).The sphingosine could obviously inhibit reproduction of nematode,and inhibition rate reached 42.72 %,38.72 %,38.66 % on 24 h,48 h,72 h,respectively,which indicated that sphingosine exhibited reproduction and fertility of nematoda.In summary,as a new type of biological control nematode method,it has great potential.The B.cereus could induce defensive activity of tomatoes and have better control effects on M.incognita.The activity subsances(sphingosine)have good prospects in biological control.B.cereus is a significant strain on M.incognita in biocontrol,it can reduce the damage to agricultural production brings by nematode and replace chemical pesticides.Thereby,the nematicidal activity of B.cereus and its metabolic product opening new perspectives in the biocontrol of nematodes. |
