IDENTIFICATION OF PATHOGEN OF WILT DISEASE IN STRAWBERRY (FRAGARIA SP.)

(1)

IDENTIFICATION OF PATHOGEN OF WILT DISEASE IN STRAWBERRY (FRAGARIA SP.)

AND THE CONTROL POTENTIAL OF

MICROBIAL ANTAGONISTS

(2)

Tim Peneliti :

Dr. Gusti Ngurah Alit Susanta Wirya, SP., M.Agr.

I Putu Sudiarta, SP., M.Si, Ph.D

Prof. Dr. Ir. I Gede Rai Maya Temaja, M.P.

Gusti Ayu Devi Valenia Sari

(3)

Ciwidey

Batu Berastagi

Purbalingga

Bedugul

Production of Strawberries in Indonesia

Strawberries in Indonesia since 1980

Bedugul, 1983

(4)

Wilt Disease on Strawberry

2016

(5)

Reduce

strawberry production

80%

(6)

Symptoms of strawberry wilt disease

(7)

Control of Disease on Strawberry is highly

dependent on the use of synthetic chemical

pesticides

(8)

Purposes

1. To know the species of pathogen of wilt disease on strawberry, based on morphological and molecular analysis

2. To know the antagonistic microbe that potential against the causal agent of

strawberry wilt disease in Bali.

(9)

RESEARCH

METHODS

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The research was conducted at Plant Disease Laboratory, Faculty of Agriculture

Udayana University, and

Pancasari Village, Sukasada District, Buleleng Regency.

From April to September

2017

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Identification of fungi based on

morphological structure

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Phylogeny analisis Sequencing

DNA extraction 4 days old of Miselium kit Wizard Genomic purification, Promega Corp

PCR Amplification

Primer Internal Transkript Spacer (ITS) 1 (5- TCCG TAGGTGAACC GCGG-3) dan ITS 4 (5- TCCTCCGC TTATTGATATGC-3)

+ 800bp

Identification of fungi based on molecular analysis

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level of homology or alignment with the gene sequence (BLAST)

software Molecular Evolutionary Genetics Analysis (MEGA 5.05).

(13)

Isolation of Microbial Antagonists

The antagonistic microbial candidate of the fungus group is cultured from the 10^-3-10-4 dilution level, while the

bacterial group is cultured from 10-5-10-6 dilution.

1 ml

1 ml 1

ml 1

ml

1 ml

500 ml + 50 gr soil

9 ml

Dilution Method

The antagonistic microbial candidate of the fungus group is cultured from the 10^-3-10^-4 dilution level, while the

bacterial group is cultured from 10^-5-10^-6 dilution.

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In Vitro Test of Antagonistic Activity of Biocontrol Agent Againts Pathogen.

A : Pathogen of wilt disease, B : Antagonist microbe, L1 : area of pathogen on treatment (cm²) L0 : area of

pathogen on control (cm²)

P = L0-L1 x 100%

L0

Control

L0

L1

Dual culture

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RESULT AND DICUSSION

Percentage of wilt disease on strawberry production centers

No Village

Percentage of Disease

(%) 1 Pancasari village,

Sukasada District, Buleleng Regency

90 2 Candikuning village,

Baturiti District, Tabanan Regency

82

(16)

Symptoms of

strawberry wilt disease on the roots

Symptoms of

strawberry wilt disease

on stems and leaves

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The growth of the fungi isolated from symptomatic strawberries wilt on the PDA medium

A B

C D

3 dai

7 dai

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Macroconidia and microconidia.

The macroconidia are hyaline, septate, and sickle or banana shaped. microconidia small, unicellular

or bi-cellular, spherical or oval.

Microscopic obseravtion

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Structure of F.

oxysporum. CMI. (1979).

The main branches of conidiofor which produce conidia are

called fialids. Fialid oval shaped. microcidia small,

unicellular or bi-cellular, spherical or oval. Sizes vary

from 5 x 15μm x 2-4 μm.

Macroconidia long, crescent- shaped, pointed tip multi-

septate (3-5 septa)

(20)

To test if isolates obtained from infected strawberry plants are pathogenic strains of F. oxysporum, the

isolate can be subjected to pathogenicity tests to

satisfy Koch's postulates

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Pathogenicity tests

a b

(a) Symptomatic plants wilt disease in the field. (b) Control, plants inoculated with water. (c) Symptomatic plants inoculated with pathogens of wilt disease.

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Internal Transcribed Spacer

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ITS1 : 5' TCCGTAGGTGAACCTGCGG 3' ITS4 : 3' TCCTCCGCTTATTGATATGC 5'

Polymerase Chain Reaction

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Visualization of PCR Product on Agarose Gel electrophoresis

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544 bp

M 1

M: Marker DNA 1 kb (Promega USA); 1, Pathogen isolate

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No Isolat Homologi

1 2 3 4 5 6 7 8 9 10 11 12

1. PLSPs_01 ID 99% 90% 90% 85% 84% 91% 91% 91% 92% 90% 57%

2. DQ452448 99% ID 90% 91% 85% 84% 91% 92% 91% 93% 91% 57%

3. U61679 90% 90% ID 99% 83% 82% 89% 90% 85% 91% 86% 60%

4. U34580 90% 91% 99% ID 84% 83% 90% 91% 86% 91% 86% 59%

5. NR121457 85% 85% 83% 84% ID 96% 84% 85% 81% 90% 81% 52%

6. NR121203 84% 84% 82% 83% 96% ID 83% 83% 80% 86% 80% 53%

7. NR120263 91% 91% 89% 90% 84% 83% ID 99% 86% 88% 86% 56%

8. NR120262 91% 92% 90% 91% 85% 83% 99% ID 87% 88% 87% 56%

9. NR111142 91% 91% 85% 86% 81% 80% 86% 87% ID 88% 99% 58%

10. AY213655 92% 93% 91% 91% 90% 86% 88% 88% 88% ID 89% 57%

11. AY213654 90% 91% 86% 86% 81% 80% 86% 87% 99% 89% ID 58%

12. AB585937 57% 57% 60% 59% 52% 53% 56% 56% 58% 57% 58% ID

PLSPs01 = Fungi Pancasari; DQ452448 = F. oxysporum; U61679 = F. concolor; U34580 = F.

polyphialidicum; NR121457 = F. equiseti; NR121203 = F. boothii; R120263 = F. circinatum;

NR120262 = F. bactridioides; NR111142 =F. acutatum; AY213655 = F. chlamydosporum AY213654 = F. annulatum; AB585937 = Verticillium dahlia var.longisporum

Homology of Internal Transcribed Spacer nucleotide of

pathogen causes wilt disease with other fungi

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Phylogeny of ITS gene of strawberry wilt

disease pathogen

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This result is the first report on F. oxysporum as a pathogen of wilt disease in strawberries

in Bali

The result of Internal Transcribed Spacer sequence (ITS) from F. oxysporum is the first

report of ITS nucleotide from pathogen

causing strawberry wilt disease in Indonesia.

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No Isolat Percentage

1 PsA1 42% Rhizosphere banana Baturiti, Bacteria 2 PsA2 39% Rhizosphere banana Baturiti, Bacteria 3 StP1 48% Rhizosphere strawberry Pancasari, Fungi 4 StP2 51% Rhizosphere strawberry Pancasari, Fungi 5 StP3 25% Rhizosphere strawberry Pancasari, Fungi 6 StP4 37% Rhizosphere strawberry Pancasari, Bacteria 7 StP5 24% Rhizosphere strawberry Pancasari, Bacteria

8 KrB1 48% Rhizosphere Botancal Garden, Fungi

12 CkG1 86% Rhizosphere Clove, Gitgit, Fungi

13 CkG2 40% Rhizosphere Clove, Gitgit, Fungi

16 SpP1 52% Kotoran Sapi, Pancasari, Fungi

20 SpP5 28% Kotoran Sapi, Pancasari, Bacteria

The ability of antagonistic microbes against the causes of strawberry wilt disease on PDA media

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Colony color begins with white, then whiteish green, light green, green and dark green after 7 days old on

PDA media

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Microscopic observation bentuk spora serta fialid Trichoderma menurut Gusnawaty, 2014

1

2

1

2

1. Conidiophore 1. Conidiophore

2. Fialid 2. Fialid

Conidia form:semi-round, conidiofor branched like a pyramid, has a fialid

Conidia and fialid form of Trichoderma according to Gusnawaty, 2014

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CkG1 isolate, obtained from the Clove plant rhizosphere has the highest inhibitory capacity of 86% in 7dai..

Dual culture of microbial antagonist isolates against pathogen of wilt disease. (a)

Fusarium (b) Trichoderma.

a b

Trichoderma is able to suppress the development of pathogens:

microparasites and produce some inhibiting compounds.

Trichoderma spp. also produce various compounds or enzymes that serve to inhibit or degrade the cell walls of plant

pathogens.

Trichoderma has the ability to induce plant resistance

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Kesimpulan

Based on the morphological and molecular identification:

The causal agent of strawberry wilt disease in Bedugul area is F.

oxysporum. This pathogen causes wilt disease in strawberry in Pancasari Village, Sukasada District, Buleleng Regency and Candikuning Village, Baturiti District, Tabanan Regency of 90%

and 82%.

Trichoderma sp. CkG1 isolated from the Clove plant was able to suppress the growth of F. oxysporum, the causes of strawberry wilt disease. Suppression mechanisms of Trichoderma sp. CkG1

to F. oxysporum through its micro-parasitic properties and its ability to produce antifungal compounds.

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IDENTIFICATION OF PATHOGEN OF WILT DISEASE IN STRAWBERRY (FRAGARIA SP.)