Screening And Identification Of Biocontrol Fungi Against Plant Parasitic Nematodes

Biological control of plant parasitic nematodes has been an important research field.Screening and activity detection of biocontrol fungi are the crucial step.Screening system of biocontrol fungi,active detection,fermentation condition optimizing of high activity strains and species identification of strains based on the previous research were investigated in this study.The results clarified the method of the surface sterilization on the root-knot nematode eggs,appropriate sampling time for high hatching ratio of eggs;In vitro screening of the biocontrol fungi on Meloidogyne incongnita,Heterodera glycines,Bursaphelenchus xylophilus;the pots experiment for detecting the infection and reproduction of the eggs and J2 s after treated with fermentation filter of strains XN-2,XO-2,CLR-3,CQE-4 and FCE-2;fermentation condition optimizing for high nematicide activity of the strains XN-2,XO-2,CLR-3,CQE-4 and FCE-2;and morphological and molecular biological identification of the 9 potential biocontrol strains.The main results as follows:1.Established the screening system of plant parasitic nematodes biocontrol fungi and screened several potential fungi strains on Meloidogyne incongnita,Heterodera glycines,Bursaphelenchus xylophilus.The results showed that 70% alcohol could give a good surface sterilization on the Meloidogyne incongnita eggs,and the eggs hatching ratio had no significant difference with the sterile distilled water control(P<0.05).The best sampling time for the eggs from tomato roots was the period from 28 d to 38 d after inoculation(DAI)with eggs,and there were higher hatching rate from 28 and 38 DAI than that of other time.The hatching ratio was 60.94% at 28 d after inoculation,and the highest hatching ratio occurred at 32 DAI with 79.13%.The results showed that the XO-2 and CLR-3 stains fermentation filtrate could significantly inhabit the hatching of eggs.The hatching of eggs treated by strains XN-2,FCH-1,CLK-2,FCD-2,CLS-2,CLR-1 and XS-5 were significantly lower than sterile distilled water control(P<0.05).XO-2 strain fermentation filtrate could inhabit significantly the hatching of Heterodera glycines eggs(P<0.05).The hatching of eggs treated by strains FCD-2,CLS-2,CLR-1,FCH-1,CLK-2,XI and CLR-3 were significantly lower than that of sterile distilled water control(P<0.05).The knocking down ratio of root-knot nematode J2 s were more than 90% at 72 h after treated with 10 times fermentation filtrate of strains CLQ-3,CLV-2,XN-2,CQE-4,CLK-2 and FCE-2,and the mortality ratio were 100% at 72 h after treated XN-2 and FCE-2 strains fermentation filtrate,and the J2 s were all died at 24 h after treated with FCE-2 strain fermentation filtrate.100% Bursaphelenchus xylophilus were knocked down at 72 h after treated with 2 times dilution of FCC-4,CLV-2,XI,XN-2,CQE-4,CLK-2 and XK-1 strains fermentation filtrate,and mortality ratio of the nematode reached 100% at 72 h after treated with XN-2 and CQE-4 strains fermentation filtrate.The mortality ratio of the nematode treated with FCC-4,CLV-2,XI,CLK-2 and XK-1 strains fermentation filtrate were 98.81%,87.53%,97.82%,98.41% and 88.07% respectively.100% Bursaphelenchus xylophilus were knocked down at 72 h after treated with XI,XN-2,CQE-4 and CLK-2 strains fermentation filtrate 5 times dilution,and the mortality ratio were all more than 90%.Under 10 times dilution condition,only XI,CLK-2 and XN-2 strains fermentation filtrate had the high lethal activity on Bursaphelenchus xylophilus and the mortality ratio were 91.53%,93.73% and 76.05% respectively.2.The pot experiment of fermentation filtrate activityXN-2 and XO-2 fermentation filtrate had better inhibition effect on the eggs hatching of root-knot nematode than other strains and hatching ratio were significantly lower than sterilize distilled water control(P<0.05),and the fermentation filtrate have no effect on growing of tomato plants.CQE-4 strain fermentation filtrate showed the highest nematicide effect,the number of galls were 2.3 average and were significantly lower than sterilized water control(P<0.05).The next was FCE-2 strain.3.Fermentation condition optimization for strains of inhibiting eggs hatching and toxic J2sBase on the above experiment,5 strains were selected for fermentation condition optimization.The result showed that hatching ratio was lower in fermentation 2 weeks than that of 1 and 3weeks for XO-2 and XN-2 strains,and 60% died eggs and J2 s were degraded.The eggs hatching ratio was lower than 10% in 5,10 and 20 times dilution of CLR-3 strain fermentation 1 week,and significantly lower than sterilized distilled water control(P<0.05).However,hatching rate was 53.67% in 2 times dilution,and higher than that of 30 times(18.71%)and 40 times(34.66%),they were significantly lower than sterilized water control(P<0.05).The phenomenon of there was high hatching rate in high concentration fermentation filtrate should be further study in future.For XN-2 strain,The nematicidal activiey of fermentation 1 and 2 weeks was higher than that of 3 weeks and 100% J2 were killed in 2,5 and 10 times dilution.The mortality ratio of J2 was 96.03% treated with 3 weeks fermentation 10 times dilution.The mortality ratio of J2 was 90.54% and 99.56% respectively at 72 h after treated with 20 times dilution of 1 and 2 weeks fermentation,and significantly higher than 3 weeks fermentation by 56.16%(P<0.05).For CQE-4 strain,The mortality ratio of J2 was 100% at 72 h after immersion in 2,5,10 and 20 times dilution of 2 and 3 weeks fermentation filtrate and were significantly higher than that of 1 week fermentation filtrate(P<0.05).The FCE-2 strain showed better nematicidal activity when incubate 4? for 30 d.Nematicidal activity of the FCE-2 strain was higher in 3 weeks fermentation filtrate than that of 1 and 2 weeks.The FCE-2 strain showed better nematicidal activity when incubate 4? for 30 d.All J2 s died at 2 h after immersion in 2 times dilution of 3 weeks fermentation filtrate.Degraded died J2 s body was observed at 6 h,and all the J2 bodies were degraded.The mortality ratio of J2 was 100% at 12 h after immersion in 2,5 and 10 times dilution.For 3 weeks fermentation filtrate,2,5 and 10 times dilution showed degraded nematode body function,20,30 and 40 times dilution have no this phenomenon,and they have lower nematicidal activity than that of the former.4.Identification of bio-control strainsThe method of the morphological and molecular biology was used,and CLR-1 strain was identified as Trichoderma harzianum;CLR-3 and XI were both Penicillium janthinellum;CLS-2 was Trichoderma harzianum Hypocrea lixii,CLV-2 was Fusarium oxysporum;CQE-4 was Myrothecium verrucaria;FCE-2 was Aspergillus japonicas;XN-2 was Alternaria spp.;XO-2 was Aspergillus niger.