Effects Of Actinomycetes (Micromonospora) From Marine Distributionon Bursaphelenchus Xylophilus

Rare marine actinomycetes samples used for medical antibiotics in this study were separated and screened from deep sea soil near Minjiang River,Fujian Province.In order to broaden the targets of these strains,with high biological activities from 196 marine actinomycete samples were screened for against Bursaphelenchus xylophilus.The fermentation conditions were also optimized.The isolate No.11 with higher nematicidal activity,was screened and identified with morphological and molecular biological methods.1.In order to screen other more fungi for culturing pinewood nematode,several kinds of fungi were tested.It was firstly found that Alternaria,Peronophythora litchi and Mycogone perniciosa magn would also be used for the nematode culture,with after cultured 10 days in PDA culture medium at 28?,the breeding rate of P.litchi reached to 64.50,and that of M.perniciosa magn came to 63.20.2.Using fermentation crude extracts,bioassay of B.xylophilus was carried out with 96 well-plate assay under different time and concentrations.It was found that the nematicidal activity was relatively high,which over 80%samples reached the mortalities of 50%and 77.94%when treated with low concentration of 5%in 12 h and 24 h respectively.Therefore,6 active strains were obtained for further screening.After using organic and inorganic medium for simulative fermention test,the isolate No.11 was detemined for futher experiments.3.The isolation No.11 was identified as Micromonospora chalcea,according to 16S rDNA gene sequence analysis and phylogenetic tree,along with the morphological,physiological and biochemical characteristics.16S rDNA sequencing was found mostly similar to M.chalcea(1464-217 L)with the similarity of 99%.4.Through Single experiment,uniform design,as well as response surface methodology,the best fermentation conditions were as follows:(1)According to single experiment,the best conditions of carbon,nitrogen,fermentation time,inoculum size,volume of culture medium,initial pH for this isolate strain was soluble starch,yeast powder,96 h fermentation time,12%inoculum size,60 mL/250 mL volume of culture medium,and initial pH with 7.2-7.5 respectively.(2)The most significant factors influencing the activity of the isolate were CaCO3 concentration(0.29 g/L),culture medium volume(55 mL),and nitrogen concentrations(7g/L)respectively when analysed with Plackett-Burman design.(3)Order orthogonal regression design and response surface methodology analysis were both adopted to analyse the mutual interactions between these variables.After optimization,the best cultural and nutritional condition was shown as follows:soluble starch(20 g/L),yeast powder(25 g/L),NaCl(0.5 g/L),K2HPO4(0.5 g/L),MgSO4(0.5 g/L),CaCO3(0.38 g/L),temperature(28?),initial pH(7.2),fermentation time(96 h),inoculum size(10%)and culture medium volume(72 mL/250 mL).It reached 99.2%and 97.54%mortality to B.xylophilus with the active substance in supernatant and intracellular respectively.The biomass of mycelium was 2.16 g,coinciding with the predicted value.Meanwhile,concentrations of soluble starch,yeast powder,K2HPO4,MgSO4,NaCl,CaCO3 were optimized by uniform design.The results showed that the optimal concentrations of nutritional condition were 10g/L soluble starch,1 g/L yeast powder,0.1 g/L NaCl,0.1 g/L MgSO4.The lethality rate of supernatant and intracellular cultured in the optimal fermentation conditions against B.xylophilus were 98.06%and 86.35%,respectively.The biomass of the isolate No.11 was 0.9236 g,Compared with the RSM and UD.The optimization effect of RSM was better than UD,but raw material costs of the former was higher than the later.All results showed that it was potential to develop this isolate as bio-nemeticidal product in the future.