Contents Selecting of Multifunction 1 1 Microorganism: - - PDF document

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Sep-2011, Nong Lam University

• Dr. Nguyen Van Nam

Tay Nguyen University

Multifunction Microorganisms in Environmental friendly Agriculture at Highland Centre of Vietnam Young Scientist for Argricultural Development in Vietnam

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Contents

1

Machenism and Biotechnology of Bioproducts in Agriculture

2

Selecting of Multifunction Microorganism: Nematophagous-Antifungi-PR Protein

1

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The Agriculture of highland centre will deal with Problems at in near future

Destroyed Forest

Drough

Ecology change

Agriculture problem

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• 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 Soil microorganism function

What microorganism help us

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Bioproducts Appling on the field soil Screening on function medium Clear zone, nematophagous, antifungi, PR protein Selected fungi, Microorganis Study on machenism

Experimental procedures

Isolation of fungi, microorganism Pure micro-isolates

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Isolation of Microoganism at Highland centre

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Screening of nematophagous fungi

Name Infected egg (%) Second

• 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

Competitive interaction of chitinolytic fungi and with Fusarium 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
• ++

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Hơn 50 chủng Trichoderma được phân lập và lưu giữ tại Đăk Lăk

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Các dòng Trichoderma phân lập tại Đăk Lăk

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Sp. Host

• 1. T. Harzianum
• 2. T. Koningii
• 3. T. Minutisporum
• 4. T. Asperellum
• 5. T. Viride
• 6. T. astroviride
• 7. T. erinaceum

Coffee, ruber, black papper, cacao Coffee, black papper Coffee, black papper Coffee, black papper Coffee, black papper Coffee, black papper

Identified Trichoderma at highland centre

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Antagonistic of Tri on plant disease fungi A: T1-F.solani, B: T7-F.solani, C: Đối chứng D: T1-F. oxysporum, E: T7- oxysporum, F: Đối chứng

A B C F E D

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Mutalist of Trichoderma on plant

A B D C A B

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Degrading organic material in nature

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 1 3 5 7 Time after incubation (h) R e d u c in g s u g a r ( u m o l/m l)

Glucosamine Saccharide Materials-National Research Lab. (GSM-NRL) http://altair.chonnam.ac.kr/~nrlgsml/

Fungal chitinases digest cell walls of Fusarium macroconidia

Reducing sugar from hyphae released by fungal chitinases

Chitinases digest chitin in fungal cell walls

Intact cell wall

Digested cell wall

Reducing sugar

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2 4 6 8 10 12 14 16 18 20 1 2 3 4 5 6 7 Time (day) Biomass (ug/ml) PDB medium YPG medium

Fungal biomass Alkali insoluble material Fungal chitin Hyphae

Fungal biomass in culture medium

Extraction of chitin from cell walls

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Purification of enzym chitinase from antagonistic fungi

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Degraded products from enzym chitinase of Trichoderma

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      [(GlcNAc)2] [(GlcNAc)5] Hydrolysis products from Fusarium solani biomass by crude enzyme DG-3 (GlcNAc) Enzyme Crude chitinase Chi 32 Chi 46

0 5 10 15 20 Retention time (min)

[(GlcNAc)2] [(GlcNAc)2] (GlcNAc)

Chitinases digest chitin in fungal cell walls

Hydrolysis products from Fusarium chitin

0.0 0.1 0.2 0.3 0.4 0.5 Substrate R educing sugar (umol/ml)

1 hour 3 hour 5 hour

Fus chi Fus hyp Fus chi Fus hyp Chi 32 Chi 46

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Bioactivity of Fungus Peacilomyces varioti

Chitinases producer Nematode egg parasitism

We hypothesis that chitinases from DG-3 play a role in parasitic process.

Whether chitinases play a role in this competition and parasitism

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Fungi parasitized on namatode eggs

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The fungus-invaded Meloidogyne eggs by fungus DG-3. The hyphae binded to eggshell by SEM (A) Hyphae in side the egg by a microscope ( x 400).

Bioactivity of Fungus Peacilomyces varioti

Percentage of infected Meloidogyne eggs by chitinolytic fungi DG-3

Rate of parasitized eggs by DG-3 fungi

20 40 60 80 100

1 2 3 4 5

Time (day) Percentage of parasitized egg (%)

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Percentage of infected nematode eggs by crude chitinases Control Treatment with enzymes Enzymes effect on nematode eggshells

Transparency eggs treated by chitinases Intact egg

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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.

Chi32 and Chi46 were purified from P. variotii DG-3

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F-3 chitinase is similar to family 18, class 3. three domains, enzyme structure from Trichoderma sp.

Chi32 D P Y Q T N V V Y T G Q D F V S P D L F Chi46: D A X X

Y R S V A Y F V N W A

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Trimmer Tetramer Pentamer Hexamer Enzymatic hydrolysis products of chitin

• ligomers by Chi32 (on

the left) and Chi46 (on the right). (GlcNAc)n, Standard chitin oligomer, Hydrolysis products by HPLC from: Trimer (A) Tetramer (B) Pentamer (C) Hexamer (D) .

Catalytic of Chi32 and Chi46 on chitin oligomers

Exo-type Endo-type

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• Time course of chitin
• ligomer degradation and

product formation by Chi32 (on the left) and Chi46 (on the right)

• Mixture reactions were

performed in 50 mM citrate buffer (pH 4) at 37oC for 0, 30, 60, 90 and 120 min.

• The products in 3 µl of

reaction were separated by HPLC in the 20 min run.

• ■-: (GlcNAc)1
• -: (GlcNAc)2
• ▲-: (GlcNAc)3
• ●-: (GlcNAc)4
• ○-: (GlcNAc)5
• -: (GlcNAc)6.

Time course of catalytic of Chi32 and Chi46 on chitin oligomers

Trimer (A, E) Tetramer (B, F) Petamer (C, G) Hexamer (D, H)

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Effective rate of nematode eggs by enzymes

Enzyme After treatment 1 day 2 day 3 day 4 day Control 5.1 ± 1.9c 10.3 ± 1.3b 12.3 ± 3.3c 13.3 ± 0.3d Crude chitinase 6.5 ± 0.5c 15.8 ± 3.1b 11.7 ± 0.3c 20.0 ± 2.0c Chi32 14.1 ± 1.1b 14.6 ± 2.4b 18.3 ± 1.7b 22.5 ± 2.5c Chi46 25.6 ± 2.6a 25.4 ± 2.7a 30.0 ± 1.0a 32.6 ± 1.4a Chi32+Chi46 13.8 ± 0.2b 18.8 ± 0.2ab 18.5 ± 0.5b 29.5 ± 1.5ab Serratia Chitinase 8.4 ± 1.9c 14.4 ± 1.9b 19.5 ± 4.5b 25.0 ± 3.6bc

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A B C D E F 10µm

• 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): The enzyme-treated egg under fluorescence microscope, staining with 0.01% Fluorescent Brightener 28.

Visualization of effect of enzyme on nematode eggs

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Protein and chitinase activity of B-3 culture supernatant from DEAE- Sephadex column chromatography. Chitinase Protease Glucanase Peak 4 ? Purification of chitinases from other fungi B-3

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Effect of B-3 purified enzymes from DAED Sephadex column chromatography on eggshell structure (% clear eggs)

Treatment 1 day incubation 3 day incubation 5 day incubation Protease 19.3a ± 5.2 22.2ab ± 4.8 25.4a ± 2.1 Chitinase 18.9a ± 3.1 26.3a ± 4.0 29.4a ± 5.1 Protease plus chitinase 24.6a ± 8.9 26.1a ± 0.9 36.0a ± 4.4 Glucanase 0.0b ± 0.0 11.5bc ± 3.4 12.6b ± 2.7 Peak 4 0.0b ± 0.0 8.1c ± 0.4 8.6b ± 0.8 Water (control) 0.0b ± 0 7.9c ± 8.3 8.4b ± 6.8 Rate (%) of damaged eggs by B-3 enzymes fractionated from DEAE- Sephadex chromatography.

20/09/2011 31 20 40 60 80 100 120 140 160 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Fractions (5 ml) P rotein (m g/m l)

1 2 3 4 5 6 7 8 E n z ym e activity (U/m l)

Protein Chitinase Chitosanase Protease

Protein, Chitinase, Protease and chitosanase activity of A-1 supernatant from Sephadex G-150 column chromatography. The protein was eluted with in 20 mM sodium acetate buffer (pH 5).

23kDa Chi 23 kDa

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10 20 30 40 50 60 70 80 90 100 Không nhúng M1 M2 M3 M4 M6 B Dòng nấm

Hiệu lực diệt côn trùng Hiệu lực diệt côn trùng

Screening insect-parsitized fungi

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Developing bioproducts for biocontrol

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Control of Meloidogyne on Cucumber by chitinolytic fungi

15 galls/root 11 galls/root 13 galls/root 20galls/root 0 galls/root A-1+Ne B-3+Ne A-1+B-3+Ne Ne Control

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Applying

• n control

Nematode

• n black

papper

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• A. T7, B. T22, C. no treatment
• D. C07
• E. DY16 F. Comecial products

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Applying on control Nematode on coffee

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Antifungal activity from Cinnamon compounds

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• 2. Materials and Methods

Preparation of crude extracts from C. cassia bark

99% MeOH (2 L) at 30 oC for 7 days Whatman No.2 At 40 oC

• C. cassia bark (DW 500g)

Extraction Filtration Evaporation

Dissolve in MeOH

• C. cassia crude extracts

(CCE) 100 mg/ml Crude extracts (DW 20g)

Cut into 3 cm

Evaporator

Effects of C. cassia crude extract against root-knot nematode

• EXP. I

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Name of plant species Plant parts tested Phytopathogenic fungus Phy Rhi Fus Col Bot Perilla ocymoides Leaf

• Leucaena glauca

Leaf

• Artemisia

capillaris thumb Leaf

• +
• Cymbopogon

nardus Leaf

• Aster sp.

Seed

• Phyllanthus

urinaria Plant

• Azadirachta indica

Seed

• Mentha spicata

Leaf

• Mentha gracilis

Leaf

• Senecio aureus

Leaf +

• +
• Cassia glauca

Plant

• Tagetes erecta

Plant

• Name of plant

species Plant parts tested Phytopathogenic fungus Phy Rhi Fus Col Bot Mimosa pudica Flower

• Leucaena glauca

Leaf

• Cassia angustifolia

Leaf

• Albizia lebbeck

seed

• Allium fistulosium

Seed

• Zingiber officinale

Bulb

• ++

+ +

• Alpinia galanga L.

Bulb + ++ ++ + ++ Glycyrrhiza uralensis Bulb ++ + + + + Polygonum multiflorum Root

• Ganoderma lucidum

Root

• Lonicera japonica

Root

• Chrysanthemum

Flower

• Luffa cylindrica

Fruit

• Cinnamomum verum

Bark

• +++

+

• In vitro antifungal activity of crude methanolic extracts from medicinal

plants against five important plant pathogens under agar diffusion assay

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Antifungal activity of solvent fraction of cinnamon compound

Solvent Compound concentration Mycelial inhibition after treatment (% ± SD) At 10 h At 30h n-Hexane 5.0% 100.0 ± 0.0a 100.0 ± 0.0a 2.5% 94.5 ± 4.7a 98.5 ± 0.2a 1.0% 65.5 ± 11.0b 59.4 ± 4.0b Chloroform 5.0% 89.6 ± 5.8a 67.3 ± 7.4b 2.5% 56.5 ± 6.9bc 55.9 ± 3.0b 1.0% 45.0 ± 1.6cd 30.3 ± 8.4c Crude extract 5.0% 38.5 ± 3.3de 14.5 ± 2.3de 2.5% 26.8 ± 1.9ef 11.4 ± 2.6de 1.0% 8.5 ± 1.5gh 7.0 ± 3.3de Ethyl acetate 5.0% 21.2 ± 1.6fg 18.1 ± 1.7cd 2.5% 19.1 ± 1.6fg 11.4 ± 1.8de 1.0% 8.4 ± 1.8gh 3.7 ± 0.9e n-Butanol 5.0% 2.4 ± 1.7h 12.0 ± 1.6de 2.5% 0.6 ± 0.1h 8.7 ± 1.2de 1.0% 0.6 ± 0.1h 7.3 ± 2.0de

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1 2 3 4 A B C 1, 0% 2, 1% 3, 5% 4, 10% A, normal hyphae B, effected hyphae C, hyphal tip swollen

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F 4 F 3 F 2 F 1

1 2 3 4 5 F-1 F-2 F-3 F-4

A B

F-1 F-2 F-3 F-4

TLC of compounds from different solvent fractions of cinnamon extract (A), crude compound in methanol (1), hexane fraction (2), ethyl acetate fraction (2), cloroform fraction (4) and n-butanol fraction (5), major compounds (F-1, F-2, F-3 and F-4) were separated in mobile phase of hexane:ethylacetate (80:20, v/v) and detected at 253 nm UV light. Separated compound in methanol after TLC (B), separated compounds in silica gel plates were cut and dissolved again in methanol and evaporated by vacuum at 40oC and then dissolved in methanol with final concentration of 10 % compound

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O O

• Fig. 10. The 1H NMR (A), 13C NMR (B) spectra, and the chemical structure (C) of compound purified from CCE

1H (proton) NMR (300MHz, CDCl3):

δ = 2.04 (s, 3 H), 4.67 (d, J = 6.4 Hz, 2 H), 6.24 (dt, J = 16.0, 6.4 Hz, 1 H), 6.60 (d, J = 16.0 Hz, 1 H), 7.19–7.35 (m, 5 H) (Fig. 13A);

13C NMR (75MHz, CDCl3): δ = 1.22,

65.29, 123.35, 126.81 (2 C), 128.28, 128.81 (2 C), 134.41, 136.38, 171.05 (Fig. 13B); Barbero et al. (2008)

A B C

C11H12O2 Cinnamyl acetate

Plant Resources Science Lab.

Chemical structure of cinnamyl acetate

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Antifungal activity from TLC fraction

Concentration 1, 0% 2, 0.5% 3, 1% 4, 2%

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Antifungal activity of compounds from TLC- based fractions at concentration

• f 1%, and

2%

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Effects of fungicidal compound I separated by silica gel plates on hyphal morphology of Rhizoctonia solani was observed by a microscope (at 40 × magnification). (A) Healthy hyphae growing on control place, (B) lightly affected hyphae with transparency cytoplasm at 0.5 % compound concentration, (C) shrinkage hyphae treated with 1% compound concentration, and (D) swollen and abnormality of hyphal tips (solid arrow) and collapsed hyphae (spared arrow) at 2% compound concentration at 1 hour after treatment by contact phase.

• Comp. I

20/09/2011 49 Fig 12. Morphology of M. incognita treated for 40 min with 1.0% methanol as control (A) and 125 µg/ml of cinnamyl acetate isolated from C. cassia bark (B). The pictures were taken under a microscope at 100x magnification.

A B Nguyen Van Nam Nematicidal activities of cinnamyl acetate Control cinnamyl acetate

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• 1. Nguyen VN, Kim YJ, Oh KT, Jung WJ, Park RD. 2007. The

role of chitinase from Lecanicillium antillanum B-3 in parasitism to root-knot nematode Meloidogyne incognita eggs. Biocontrol Science and Technology 10: 1047-1058.

• 2. Nguyen VN, Kim YJ, Oh KT, Jung WJ, Park RD. 2008. The

antifungal activity of chitinases from Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9. Current Microbiology 56:28-32.

• 3. Van-Nam Nguyen, In-Jae Oh, Young-Ju Kim, Kil-Yong Kim,

Young-Cheol Kim, Ro-Dong Park. 2008. Purification and characterization of chitinases from Meloidogyne incognita egg-parasitic fungus Paecilomyces variotii DG-3. Journal of Industrial Microbiology and Biotechnology. (2009) 36:195- 203

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• 4. Van-Nam Nguyen, Dang-Minh-Chanh Nguyen, Dong-

Jun Seo, Ro-Dong Park, Woo-Jin Jung. 2009. Antimycotic activities of Cinnamon-derived compounds against Rhizoctonia solani in vitro. Biocontrol, DOI 10.1007/s10526-009-9220-2

• 5. Dang-Minh-Chanh Nguyen, Van-Nam Nguyen, Dong-

Jun Seo, Ro-Dong Park, Woo-Jin Jung Nematicidal activity of extracted compounds from medicinal plants against pine wood nematode (Bursaphelenchus xylophilus), 2009. Nematology.

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SLIDE 27

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Harman, Plant disease, 2000 Glucosamine Saccharide Materials-National Research Lab. (GSM-NRL) http://altair.chonnam.ac.kr/~nrlgsml/ Carmen et al. phytopathology, 1999

Future development chitinase and chitinolytic fungi in biological control

Recombinant chitinase gene microorganisms

Chitinase products Resistance plant Antagonists

Chitinolytic fungi Cht

• 3. Plant compound

for biocontro

• 2. Systemic resistence

from Fungal chitin

• ligomer

1

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Glucosamine Saccharide Materials-National Research Lab (GSM-NRL) Homepage : http://altair.chonnam.ac.kr/~nrlgsml/

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