20/09/2011 Young Scientist for Argricultural Development in Vietnam Multifunction Microorganisms in Environmental friendly Agriculture at Highland Centre of Vietnam Dr. Nguyen Van Nam Tay Nguyen University Sep-2011 , Nong Lam University 20/09/2011 1 Contents Selecting of Multifunction 1 1 Microorganism: Nematophagous-Antifungi-PR Protein Machenism and Biotechnology of 2 Bioproducts in Agriculture 20/09/2011 2 1
20/09/2011 The Agriculture of highland centre will deal with Problems at in near future Destroyed Forest Ecology change Drough Agriculture problem 20/09/2011 3 What microorganism Soil microorganism help us function - Support the seed faster germination - Enhence developing crop root - Help plant root to use water more effective - Fix nitrogen from air - Promotion plant growth - Change immobie mineral - Degrade organic materials - Biosynthesis antibiotic - Produce induced system resistance - Help plant resisting to stress 20/09/2011 4 2
20/09/2011 Experimental procedures soil Pure micro-isolates Isolation of fungi, microorganism Clear zone, Screening on function medium nematophagous, antifungi, PR protein Study on machenism Selected fungi, Microorganis Appling on the field Bioproducts 20/09/2011 5 Isolation of Microoganism at Highland centre 20/09/2011 6 3
20/09/2011 Screening of nematophagous fungi Name Infected Second egg (%) -stage juvenil e Auxarthron reticulatum DY-2 73.5 Aspergillus sp1. GR-4 0.0 Aspergillus sp2. GR-5 0.0 Hypocrea lixii DY-16 84.6 + Fusarium sp. DY-43 0.0 Lecanicillium antilanum B-3 100.0 + Paecilomyces variotii DG-3 71.1 Rhizopus microsporus VS-9 0.0 Trichoderma harzianum DY-19 60.1 Trichoderma sp. DY-26 0.0 Trichoderma aureoviride DY-59 0.0 Trichoderma sp. GR-1 0.0 Verticillium saksenae A-1 100.0 + 20/09/2011 7 Antifungal activity of antagonistic fungi Isolate Competition Replacement by F. solani Auxarthron reticulatum DY-2 - + Hypocrea lixii DY-16 +++ - Lecanicillium antilanum B-3 - - Paecilomyces variotii DG-3 +++ - Rhizopus microsporus VS-9 ++ - Trichoderma harzianum DY-19 +++ - Trichoderma aureoviride DY-59 +++ - Verticillium saksenae A-1 - ++ Competitive interaction of chitinolytic fungi and 20/09/2011 8 with Fusarium 4
20/09/2011 H ơ n 50 ch ủ ng Trichoderma đượ c phân l ậ p và l ư u gi ữ t ạ i Đă k L ă k 20/09/2011 9 Các dòng Trichoderma phân l ậ p t ạ i Đă k L ă k 20/09/2011 10 5
20/09/2011 Identified Trichoderma at highland centre Sp. Host Coffee, ruber, black papper, 1. T. Harzianum cacao 2. T. Koningii Coffee, black papper 3. T. Minutisporum Coffee, black papper 4. T. Asperellum Coffee, black papper 5. T. Viride Coffee, black papper 6. T. astroviride Coffee, black papper 7. T. erinaceum 20/09/2011 11 A B C D E F Antagonistic of Tri on plant disease fungi A: T1 -F.solani, B: T7- F.solani , C: Đố i ch ứ ng D: T1-F. oxysporum, E: T7- oxysporum, F: Đố i ch ứ ng 20/09/2011 12 6
20/09/2011 Mutalist of Trichoderma on plant A A B B C D 20/09/2011 13 Degrading organic material in nature 20/09/2011 14 7
20/09/2011 Chitinases digest chitin in fungal cell walls Intact cell wall 7.0 R e d u c in g s u g a r ( u m o l/m l) Digested Reducing sugar cell wall 6.0 5.0 4.0 3.0 2.0 1.0 0.0 1 3 5 7 Time after incubation (h) Reducing sugar from hyphae released by fungal chitinases Fungal chitinases digest cell walls of Fusarium macroconidia 20/09/2011 15 http://altair.chonnam.ac.kr/~nrlgsml/ Glucosamine Saccharide Materials-National Research Lab. (GSM-NRL) Extraction of chitin from cell walls 20 Hyphae 18 PDB medium 16 YPG medium Biomass (ug/ml) 14 12 10 8 6 4 2 0 0 1 2 3 4 5 6 7 Time (day) Fungal biomass in culture medium Alkali insoluble 20/09/2011 16 Fungal biomass Fungal chitin material 8
20/09/2011 Purification of enzym chitinase from antagonistic fungi 20/09/2011 17 Degraded products from enzym chitinase of Trichoderma 20/09/2011 18 9
20/09/2011 Chitinases digest chitin in fungal cell walls Enzyme (GlcNAc) (GlcNAc) [(GlcNAc) 5 ] [(GlcNAc) 2 ] Crude chitinase [(GlcNAc) 2 ] Hydrolysis products from Fusarium solani biomass by crude enzyme DG-3 Chi 32 0.5 educing sugar (umol/ml) 0.4 1 hour 3 hour 5 hour [(GlcNAc) 2 ] 0.3 0.2 Chi 46 0.1 R 0 5 10 15 20 0.0 Retention time (min) Substrate Fus chi Fus hyp Fus chi Fus hyp Hydrolysis products from 20/09/2011 19 Chi 32 Chi 46 Fusarium chitin Bioactivity of Fungus Peacilomyces varioti Nematode egg Chitinases producer parasitism Whether chitinases play a role in this competition and parasitism We hypothesis that chitinases from DG-3 play a role in 20/09/2011 20 parasitic process. 10
20/09/2011 Fungi parasitized on namatode eggs 20/09/2011 21 Bioactivity of Fungus Peacilomyces varioti Percentage of infected Meloidogyne eggs by chitinolytic fungi DG-3 Percentage of parasitized 100 The fungus-invaded 80 Meloidogyne eggs by fungus egg (%) 60 DG-3. 40 The hyphae binded to eggshell 20 by SEM (A) 0 1 2 3 4 5 Hyphae in side the egg by a microscope ( x 400). Time (day) 20/09/2011 22 Rate of parasitized eggs by DG-3 fungi 11
20/09/2011 Enzymes effect on nematode eggshells Control Treatment with enzymes Intact egg Transparency eggs treated by chitinases 20/09/2011 23 Percentage of infected nematode eggs by crude chitinases Chi32 and Chi46 were purified from P. variotii DG-3 Anion exchange (DEAE-Sephadex A-50), filtration chromatography (Sephadex G-100) and chitinase activity staining (0.01% glycol chitin as a substrate in gel) of purified chi32 (F-1) and chi46 (F-3) separated on 12% SDS-PAGE gel under reducing and non-reducing condition. 40µl of proteins were loaded in each lane. The gels were stained with Coomassive brilliant blue R-250 and with Calcofluor white M2R. Lane A (F-1 and F-3), molecular weight marker (Amersham Biosciences); lane B (F-1), crude enzyme; lane C (F-1) flow thought protein; lane D (F-1) anion exchange column; lane F (F-1), filtration column; lane F, G, H (F-1), chitinase activity-stained of crude enzyme (F), anion exchange column (G); filtration column (H). B, C (F-3), anion exchange and filtration chromatography; D (F- 3) chitinase activity-stained in non-denaturing sample buffer. 20/09/2011 24 12
20/09/2011 F-3 chitinase is similar to family 18, class 3. three domains, enzyme structure from Trichoderma sp. Chi32 Chi46: D D P A Y X Q X T Y N V R V S Y V T A G Q Y D F F V V S N P W D A L F 20/09/2011 25 Catalytic of Chi32 and Chi46 on chitin oligomers Enzymatic hydrolysis products of chitin oligomers by Chi32 (on the left) and Chi46 (on the right). Trimmer (GlcNAc)n, Standard chitin oligomer, Hydrolysis products by Tetramer HPLC from: Trimer (A) Pentamer Tetramer (B) Pentamer (C) Hexamer Hexamer (D) . Endo-type Exo-type 20/09/2011 26 13
20/09/2011 Time course of catalytic of Chi32 and Chi46 on chitin oligomers • Time course of chitin Trimer (A, E) oligomer degradation and product formation by Chi32 (on the left) and Chi46 (on the right) • Mixture reactions were Tetramer (B, F) performed in 50 mM citrate buffer (pH 4) at 37 o C for 0, 30, 60, 90 and 120 min. • The products in 3 µl of reaction were separated by Petamer (C, G) HPLC in the 20 min run. - ■ -: (GlcNAc)1 - -: (GlcNAc)2 - ▲ -: (GlcNAc)3 Hexamer (D, H) - ● -: (GlcNAc)4 - ○ -: (GlcNAc)5 20/09/2011 27 - -: (GlcNAc)6. Effective rate of nematode eggs by enzymes Enzyme After treatment 1 day 2 day 3 day 4 day Control 5.1 ± 1.9 c 10.3 ± 1.3 b 12.3 ± 3.3 c 13.3 ± 0.3 d Crude chitinase 6.5 ± 0.5 c 15.8 ± 3.1 b 11.7 ± 0.3 c 20.0 ± 2.0 c Chi32 14.1 ± 1.1 b 14.6 ± 2.4 b 18.3 ± 1.7 b 22.5 ± 2.5 c Chi46 25.6 ± 2.6 a 25.4 ± 2.7 a 30.0 ± 1.0 a 32.6 ± 1.4 a Chi32+Chi46 13.8 ± 0.2 b 18.8 ± 0.2 ab 18.5 ± 0.5 b 29.5 ± 1.5 ab Serratia Chitinase 8.4 ± 1.9 c 14.4 ± 1.9 b 19.5 ± 4.5 b 25.0 ± 3.6 bc 20/09/2011 28 14
20/09/2011 Visualization of effect of enzyme on nematode eggs C B A M. incognita eggs damaged by Chi46. (A): The intact egg, untreated with enzyme (B): The enzyme-treated egg under light microscope. (C): The intact egg, untreated with enzyme D E F (D): The enzyme-treated egg under fluorescence microscope, staining with 0.01% Fluorescent Brightener 28. 10µm 20/09/2011 29 Purification of chitinases from other fungi B-3 Peak 4 ? Chitinase Glucanase Protease Protein and chitinase activity of B-3 culture supernatant from DEAE- 20/09/2011 30 Sephadex column chromatography. 15
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