BIOTEKNOLOGI DALAM BIDANG PERLINDUNGAN

TANAMAN

Dr. Noor Istifadah, Ir., MSc. Prof. Dr. Hj. Hersanti, Ir., MP.

Dr. Danar Dono, Ir., MSi. Endah Yulia, SP., MSc., PhD.

Fitri Widiantini, SP., MBtS., PhD. Rika Meliansyah, SP., MSi.

Kompetensi Dasar

Setelah mengikuti perkuliahan ini, mahasiswa mampu menjelaskan tentang:

Perlunya perlindungan tanaman

Penggunaan bioteknologi dalam bidang perlindungan tanaman

Metode untuk meningkatkan kinerja biokontrol

Feeding the hungry world

What we are hoping

The reality

Yield losses

Plant pests and diseases: 20-40% (FAO, 2013).

Weeds

Natural disaster Drought

Excessive rain

Hurricane

Earthquake

Volcano eruption

Fires

etc

Pemanfaatan bioteknologi dalam perlindungan tanaman

Bioteknologi bidang perlindungan

tanaman

Peningkatan kinerja agen biokontrol

Deteksi dan identifikasi patogen

Pembuatan tanaman resisten OPT

Peningkatan Kinerja Agen Biokontrol

Mutasi gen

Kultur jaringan

Modifikasi gen dengan rekayasa genetik

http://evolution.berkeley.edu/evolibrary/article/0_0_0/mutations_04

Peningkatan Kinerja Agen Biokontrol-Mutasi

Substitution Insertion Deletion

Apakah mutasi itu?

Perubahan secara permanen pada susunan DNA yang menyusun suatu gen

Peningkatan Kinerja Agen Biokontrol-Mutasi

Peningkatan Kinerja Agen Biokontrol-Mutasi

www.nature.com

Somatic mutation

Germ-line mutation

Peningkatan Kinerja ABK-Mutasi

Bagaimana mutasi bisa terjadi?

Inherited

Acquired

Penyebab mutasi:

Random mistakes

Environmental

Radiation

Chemical

Transposon insertion

Type of Radiation

Non-ionizing radiation

Ionizing radiation

http://www.epa.gov/rpdweb00/understand/

Mutation-Radiation

Radiation

Mutation-Chemical

Bahan kimia penyebab terjadinya mutasi-mutagen, umumnya carcinogenic

Golongan utama penyebab mutasi: DNA Reactive chemicals-directly reactive

with DNA or when involved in metabolism process in cells is able to produce mutagens Alkylating agents-chemicals that add an alkyl

group (CnH2n+1) to another molecule. Alkylation of a base may change the normal base pairing

Base analogs-mengganti susunan basa pada susunan DNA

Intercalating agent-able to insert itself into the stacking of DNA bases EtBr

Mutation-Transposon element

First found by Barbara McClintock 1940an-colour pattern on corn

Small piece of DNA

Capable to move and to integrate into different location within genome

http://www.broadinstitute.org/education/glossary/transposable-elements

Seleksi Mutant-positive selection

Seleksi Mutant-negative selection

RODAC-replicate organism direct agar contact

Peningkatan Kinerja Agen Biokontrol-Mutasi Cheng et al., 2003

Pseudozyma flocculosa

BCA pada powdery mildew

WT mode of action-antibiosis (A)

Insertion mutagenesis

Some loss their activity

Some have higher activity compared to WT

MeOH extraction P. flocculosa Phomopsis sp.

Pseudomonas fluorescens F113 Mutant with Enhanced Competitive Colonization Ability and Improved Biocontrol Activity

against Fungal Root Pathogens

Barahona et al., 2011

Directed mutagenesis, insertion of target gene fragment

Controlling pest insect

Three strategies:

Manipulating the genes of entomopathogenic bacteria, viruses or fungi to produce more efficient bio-pesticides

Cloning the gene encoding insecticidal protein and incorporate it into plants, resulting transgenic plant producing its own bio-pesticides

Genetic modification of insect pest to target it for biocontrol

Sterile Insect Technique

British Colombia Sterile Insect Release Program

Launch in 1992

Sterilised by radiation

Successful to keep the moth population low

http://pmtp.wsu.edu/newslettersV4I7.html

Sterile Insect Technique by TE

Mycoherbicides-Gressell et al. 2007

Peningkatan Kinerja ABK-Kultur Jaringan

Tissue culture can be used to increase the BCA agent through the following:

The use of fusion protoplast to enhance the biological control activities

Maintaining obligate biological control agent

Production of secondary metabolite

Fusion Protoplast

Type of genetic modification in plant as a resulted from fusion of two distinct species of plants with the characteristics of both

http://www.eplantscience.com/

Fusion Protoplast

Type of genetic modification in plant as a resulted from fusion of two distinct species of plants with the characteristics of both

http://www.eplantscience.com/

Gene transfer Trichoderma and Aspegillus through protoplast fusion to increase production of citric acid and cellulase

El-bondkly A.M. (2006)

To convert ground rice straw into citric acid and cellulose

T. reesei NRRL 18670, Trichoderma harzianum NRRL 13879, and T. viride strain as cellulases producers

A. niger NRRL 599 as citric acid producer

Protoplasts were prepared through enzymatic hydrolysis of mycelium suspension

Protoplasts prepared from each of the two strains were mixed

Gene transfer Trichoderma and Aspegillus through protoplast fusion to increase production of citric acid and cellulase

Fusants produced citric acid three fold more than A. niger

Transfer of tuber soft rot and early blight resistances from Solanum brevidens into cultivated potato

Tek et al., 2004

Non cultivated S. brevidens shows significant resistance to tuber soft rot (Erwinia carotovora) and early blight (Alternaria solani)

A somatic hybrid between potato (S. tuberosum) and S. brevidens was developed by protoplast fusion

Clone C75-5+297 has consistently out-yielded common varieties under disease stress

Using both molecular and cytogenetic approaches demonstrated that a single copy of chromosome 8 from S. brevidens replaced a potato chromosome 8 in C75-+297.

Maintaining obligate biological control agent

vasicular arbuscular mycorrhiza pada akar inangnya atau dg teknik transformasi Agrobacterium rhizogenes membuat akar rambut (hairy roots)

Compiled by Noor Istifadah-Danar Dono

Production of secondary metabolite

Medicinal plants are most exclusive life-saving drug sources Taxol-taxus tree-an anticancer

Morphine and codein-opium poppy-analgesic

Hairy root culture for secondary metabolite production

Based on the inoculation with Agrobacterium rhizogenes

Production of secondary metabolite

No System Mechanism of action Compound Plant source

1 Cholinergic system

Inhibition of acetylecholinestrase (AChE)

Essential oils Azadirachtina indica, Mentha spp., Lavendula spp.

Cholinergic acetylcholine nicotinic receptor agonist/antagonist

Nicotine Nicotiana spp., Delphinium spp., Haloxylon salicornicum, Stemona japonicum

2 GABA system GABA-gated chloride channel Thymol, Silphinenes

Thymus vulgaris

3 Mitochondrial system

Sodium and potassium ion exchange disruption

Pyrethrin Crysanthemum cinerariaefolium

Inhibitor of cellular respiration (mitochondrial complex I electron transport inhibitor or METI)

Rotenone Lonchocarpus spp.

Affect calcium channels Ryanodine Ryania spp.

Affect nerve cell membrane action Sabadilla Schoenocaulon officinale

4 Octopaminergic system

Octopaminergic receptors, Essential oils Cedrus spp., Pinus spp., Citronella spp., Eucalyptus spp.

Block octopamine receptors by working through tyramine receptors cascade

Thymol Thymus vulgaris

Miscellaneous Hormonal balance disruption Azadirachtin Azadiractina indica

Rattan, 2010

Peningkatan Kinerja ABK-Rekayasa Genetik

Identifying and characterizing the gene of interest Entomopathogenic nematodes

Fungal biocontrol for insect pest

Improving efficacy by genetic engineering (gene modification) Fitness, persistence, host range, enhance pathogenicity,

improve environmental tolerance, and alter host range

Characterization of the gene expression

Gene insertion, either on the original organism or into other organism such as plant to develop transgenic plant

Peningkatan Kinerja ABK-Rekayasa Genetik

Ryder et al., 2012 – genetic modification of chitinase-encode gene in Trichodema hamatum, impaired saprotrophic competitiveness against Rhizoctonia solani and promote the plant growth.

Segal D., and Glazer I, 2000 – genetic engineering entomopathogenic nematode (EPN) Heterorhabditis bacteriophora towards environmental extremes , selection for heat tolerance and resistance to nematicides

Reference

El-bondkly A.M. (2006) Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulases. Applied Biochemistry and Biotechnology 135:117-32.

Tek A.L., Stevenson W.R., Helgeson J.P., Jiang J. (2004) Transfer of tuber soft rot and early blight resistances from Solanum brevidens into cultivated potato. Theoretical and Applied Genetics 109:249-54.

Barahona E., Navazo A., Martinez-Granero F., Zea-Bonilla T., Perez-Jimenez R.M., Martin M., Rivila R. (2011) Pseudomonas fluorescens F113 mutant with enhanced competitive colonization ability and improved biocontrol activity against fungal root pathogens. Appl. Environ. Microbiol. 77:5412-5419.

Gressel J., Meir S., Herschkovitz Y., Al-Ahmad H., Greenspoon I., Babalola O., Amsellem Z. (2007) Approaches to and successes in developing transgenically enhanced mycoherbicides, in: M. Vurro and J. Gressel (Eds.), Novel Biotechnologies for Biocontrol Agent Enhancement and Management, Spinger, Dordrecht.

Grigliatti T.A., Meister G., Pfeifer T.A. (2007) TAC-TICS: transposon-based biological pest management systems, in: M. Vurro and J. Gressel (Eds.), Novel Biotechnologies for Biocontrol Agent Enhancement and Management, Spinger, Dordrecht.

Cheng Y., McNally D.J., Labbe C., Voyer N., Belzile F., Belanger R.R. (2003) Insertional mutagenesis of a fungal biocontrol agent let to discovery of a rare cellobiose lipid with antifungal activity. Applied and Environmental Microbiology 69:2595-2602.