Occurrence of Banana Blood Disease in Malaysia Presentation

Occurrence of Banana Blood Disease

in Malaysia

Datin Jatil Aliah Timin 1, Irene Lah2 and Saifful Bahri Abd Mutalib3

Plant Biosecurity Division,

Department of Agriculture, Kuala Lumpur, Malaysia

1 Plant Pathology Unit. E-mail: jatilaliah@doa.gov.my; djatilaliah@gmail.com; 2Plant Biotechnology Unit. Email: irene@doa.gov.my; ilw_irene@yahoo.com; 3 Plant Pathology Unit. E-mail: saifulbahari@doa.gov.my; unicormbiru_2002@yahoo.com;

8th ICPPT 8-10 April 2014 K. Lumpur Malaysia 1

INTRODUCTION

• bacterial wilts of banana – Blood Disease, Moko and Bugtok: caused by members of the Ralstonia solanacearum species complex.

• Ralstonia solanacearum is a heterogeneous species, which has been divided into 4 phylotypes (genetic groups).

• Strains causing Moko and Bugtok belong to phylotype II, the blood disease bacterium belongs to phylotype IV.

8th ICPPT 8-10 April 2014 K. Lumpur Malaysia

Bacterial Wilts of Banana & Ralstonia solanacearum species complex

2

World Distrbution of Bacterial Wilt Diseases of Banana: Moko, Bugtok and Blood Disease

Moko: Central and South America, Carribean,Philippines

Blood Disease: Indonesia – Java, Sulawesi, West Kalimantan, Irian Jaya, Sumatera.

Moko & Bugtok: Philippines

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• the causative agent of blood disease (BD)

• belongs to phylotype IV of Ralstonia solanacearum species complex

• a widespread and severe wilt disease of banana and plantain in Indonesia – major cause of production decline of banana in Indonesia.

• Spreads very fast – geographical spread of BD ranges from 100 km/yr in Indonesia and 189 – 203 km/yr in Sumatera (Mairawita et al.,2012)

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Blood Disease Bacterium (BDB)

4

Banana Vascular Wilt in Malaysia

• In 2007, outbreaks of banana wilt and fruit rot symptoms were observed in many banana plantations in the state of Johor, Malaysia.

• The outbreaks of this disease were reported after the December 2006/January 2007 massive flood incidents in Johor.

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8th ICPPT 8-10 April 2014 K. Lumpur Malaysia

Banana Wilt in Johor 2007

• Symptoms consisted of yellowing , wilting and necrosis of leaves, followed by death of the whole plant.

• Bacterial ooze exuded from the cut surface of infected pseudostems.

• Male flowers blackened & shrivelled

• Fruits appeared normal and unaffected but pulp displayed brownish black discolouration & rot 6

• Banana Wilt R. solanacearum in Johor 2007

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7

banana vascular wilt disease status survey in Johor in 2007 60.7 % of 3,212 ha banana orchards were found to be

infected by banana bacteria wilt (Ralstonia solanacearum/ R.s) and 23.5% were infected by panama disease (Fusarium oxysporum f.sp. cubense/ Foc).

Disease severity varied in different varieties

Banana Vascular Wilt in Malaysia

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Disease Status Survey

Based on symptoms, ooze test and BIOLOG

8

1711.6

974.4

201.2

77.95 47.9 48.7

8.8 13.8 72.1

16.4 4.4 1 25.3 7 1 2 0

200

400

600

800

1000

1200

1400

1600

1800

Fig 1: Banana Vascular Wilt Status Survey in Johor: Hectarage Banana Varieties Planted

1. Nipah 2. Berangan 3. Rastali 4. Emas

5. Kapas 6. Nangka 7. Raja 8. Raja Udang

9 Tanduk 10. Boyan 11. Awak 12. Awak Abu

13. Lilin 14. Lemak Manis 15. Masak Hijau 16. Pisang Abu

Ha

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(Source: Che An MJ and Azhar, S. 2008. Laporan Bancian Status Penyakit Layu Fusarium Dan Layu Bakteria Tanaman Pisang Di Johor)

85.1

28.1

64.8

19

40.5

48.9

77.3

21.7

10.1 0

54.5

100

26.1 28.6

100 100

60.7

10.4

47

16.5

35.5 37.4

26.5 22.7

29

20.4

14.6 13.6

0 5.9

42.9

0 0

23.5

4.5

24.9

18.7

45.5

22.1 24.6

0

49.3

69.5

85.4

31.8

0

68

28.6

0 0 0 0

20

40

60

80

100

120

%

I

n

c

i

d

e

n

c

e

Fig 2: Banana Vascular Wilt Incidence in Different Banana Varieties

Bacterial Wilt

Panama Disease

Healthy

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in 2007 to 2009, banana vascular wilt disease status survey throughout Peninsular Malaysia

- a total of 1,976 ha banana were infected by bacterial wilt R.s,

- 882 ha were infected by Foc and

- 27 ha were infected by both diseases

Banana Vascular Wilt in Malaysia

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Disease Status Survey

11

1.Perlis2.

Kedah3. P. P

4.Perak

5.Selang

or6. N. S

7.Melaka

8.Johor

9.Pahang

10.T'ganu

11.K'tan

Bacterial wilt 0 0.4 0 0.6 0 9.8 4.81 1,951 0 0 10

Fusarium/ Panama disease 0 1 6 14.8 0.4 11.2 9.51 756 75 1 6.8

Both 0 0 0 12 15 0 0 0 0 0 0

0

500

1000

1500

2000

2500

Are

a af

fect

ed

(h

a)

Banana Vascular Wilt Incidence in Different States

Bacterial wilt

Fusarium/ Panama disease

Both

[Source: Laporan Status Serangan Penyakit Layu Bakteria (bakteria Ralstonia solanacearum ras 2) dan Layu Fusarium atau 'Panama Disease’ (kulat Fusarium oxysporum f.sp cubense) di Semenanjung Malaysia 2010 (Mengikut laporan diagnostik dan pemantauan1 / bancian status2 di negeri-negeri tahun 2008-2009) - Laporan Unit Patologi Tumbuhan, Bahagian Biosekuriti Tumbuhan, Kuala Lumpur]

0

500

1000

1500

2000

2500

Are

a af

fect

ed

(h

a)

State

Fig 3: Banana Vascular Wilt Incidence in Different States

Bacterial wilt

Fusarium/ Panama disease

Both

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• banana wilt disease in Johor , N. Sembilan & Malacca

- ProMED mail July 2009: Professor Emeritus Ivan Buddenhagen, University

of California, Davis suggested that BDB may be the possible cause of the

banana disease that appears to be emerging in south-western Malaysia.

However confirmation by molecular methods would be needed for a conclusive

diagnosis.

Banana Vascular Wilt in Malaysia

• In 2012-2013 plants with bacterial wilt symptoms were reported in Negeri Sembilan, Selangor, Penang, Pahang, Johor and Sabah. Infected plants were tested for R. solanacearum spesies complex and BDB using molecular methods.

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In 2007- 2009, identification of banana bacterial wilt was done using ooze test and BIOLOG system –BIOLOG identification only to R. solanacearum level thus cannot determine the exact phylotype or race.

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Laboratory Analysis : Identification of Ralstonia solanacearum species complex

A phylotype multiplex PCR protocol was used to verify the phylotypes of Ralstonia solanacearum species complex and BDB in infected banana samples.

All R. solanacearum strains should produce a band (282 bp) with the 759/760 primers, as well as another band for each of the Phylotypes

I = 144 bp II = 372 bp III = 91 bp IV = 213 bp

The Blood Disease Bacterium (BDB) diagnostic protocol uses the BDB primer set: 121F CGT ATT GGA TGC CGT AAT GGA and 121R AAG TTC ATT GGT GCC GAA TCA – amplifies a 317 bp amplicon from BDB strains.

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Location

No of

infected

samples

analysed*

No of positive samples

Ralstonia solanacearum species complex Blood Disease

Bacterium (BDB) R. s

General Phylot I Phylot II Phylot III Phylot IV

Sabak Bernam,

Selangor 6 3 1 0 0 4 4

Gemas, N.S 6 6 0 0 0 6 6

Putrajaya, WP 4 4 0 0 0 4 4

Tuaran, Sabah 18 6 0 0 0 6 4

Sg. Burong,

Selangor 4 2 0 0 0 2 0

Batu Pahat, Johor 4 4 0 0 0 4 4

Jerantut, Pahang 3 3 0 0 0 3 3

Serdang, Selangor 3 3 0 0 0 3 3

Sekinchan

Selangor. 3 3 0 0 0 3 3

S. Prai, Penang. 5 5 0 0 0 5 5

Laboratory Analysis : RESULTS Identification of Ralstonia solanacearum species complex

* Ooze test positive 8th ICPPT 8-10 April 2014 K. Lumpur Malaysia 15

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Bil Sample R. solanacearum species complex Notes

Ref Sample General Phylotype IV BDB

1. SP3048 Petiole Positive Positive Positive R. solanacearum sp. complex

Blood Disease of Banana

2. SP3050 Petiole Positive Positive Negative R. solanacearum sp. complex

unknown

3. SP3055

Pseudostem (inner

part)

Positive Positive Positive R. solanacearum sp. complex

Blood Disease of Banana

4. Pulp Positive Positive Positive

5. Fruit skin Positive Positive Positive

6. SP3056

Pseudostem (inner

part)

Positive Positive Negative R. solanacearum sp. complex.

7. Pulp Positive Positive Positive R. solanacearum sp. complex

Blood Disease of Banana

8. Fruit skin Positive Positive Negative R. solanacearum sp. complex.

9. SP3057

Pulp Positive Positive Negative R. solanacearum sp. complex

unknown 10. Fruit skin Positive Positive Negative

11. SP3058

Pulp Positive Positive Positive R. solanacearum sp. complex

Blood Disease of Banana

12. Fruit skin Positive Positive Negative R. solanacearum sp. complex.

Laboratory Analysis : RESULTS - SABAH Identification of Ralstonia solanacearum species complex

• BD in Sg Burung, Selangor 2012

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• BD in Sabak Bernam, Selangor 2012

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• BD in Sabak Bernam, Selangor 2012

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• BD in Federal Territory Putrajaya 2012

Amplication of 317 bp amplicon from banana samples infected with BDB. Lane 1-mol wt marker, lane 2- negative control, 3-14 banana samples, 15 positive control

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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• BDB in Batu Pahat, Johor 2013

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Amplication of 317 bp amplicon from banana samples infected with BDB. Lane 1-mol wt marker, lane 2- negative control, 3-14 banana samples, 15 positive control

Amplication of 282 bp and 213 bp amplicon from banana samples infected with R. s and phylotype IV. Lane 1-mol wt marker, lane 2- negative control, 3-14 banana samples, 15 positive control

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• BDB in Raub Pahang 2013

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• Banana Wilt R. solanacearum in Johor 2007

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Date

Disease/ pathogen Detection Method

Location

2007-2010 Bacterial Wilt Ralstonia solanacearum Ooze test and BIOLOG

Johor, N. Sembilan, Malacca, Kedah, Kelantan, Perak

2011-2014 Bacterial Wilt Ralstonia solanacearum species complex , BDB

Ooze test, PCR

Selangor, Johor, Sabah, Pahang, Penang, W.P Putrajaya, N. Sembilan

Banana Bacterial Wilt in Malaysia ● ● ● Summary ● ● ●

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Infected banana samples from Peninsular Malaysia and Sabah were found to be infected by:

(2007-2009) R. solanacearum ??

– Kedah, Perak, N.Sembilan, Johor, Kelantan, Malacca

(2009-present) R. solanacearum phylotype IV and BDB

– N. Sembilan , Johor, Selangor, WP Putrajaya,

Penang, Pahang, Sabah

Banana Bacterial Wilt in Malaysia ▪ ▪ ▪ CONCLUSION ▪ ▪ ▪

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R. solanacearum

R. s phylotype IV + BDB

Banana Bacterial Wilt in Malaysia ▪ ▪ ▪ CONCLUSION ▪ ▪ ▪

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• Blood disease of banana which previously limited to Indonesia has spread to Peninsular Malaysia and Sabah.

• To the best of our knowledge, this is the first confirmation of Banana Blood Disease in Malaysia.

• Department of Agriculture has developed a Standard of Procedure which stated the action plans and various control measures to mitigate this disease from affecting the banana industry in Malaysia. DOA imposed new import regulation for importation of banana fruits into Malaysia.

Banana Bacterial Wilt in Malaysia ▪ ▪ ▪ CONCLUSION ▪ ▪ ▪

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We would like to thank the following people for their support and contribution in making this Banana Vascular Wilt and BDB detection program possible.

• collegues and staffs of Plt Patho Unit & Plt Biotech Unit, Plant Biosecurity Division HQ and State Plant Biosecurity Division

• Ms Julia Lamdin & Ms Evenni Poili (DOA Sabah),Dr Lily Eng (DOA Sarawak), Dr Noor Hana Hussain (UiTM)

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Life without banana……

Thank You

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