UNIVERSITI PUTRA MALAYSIA

DETECTION OF CITRUS GREENING ORGANISM (LIBEROBACTER ASIATICUM)

BY POLYMERASE CHAIN REACTION

MUH. ASAAD

FP 2001 8

DETECTION OF CITRUS GREENING ORGANISM (LIBEROBACTER ASIATICUM)

BY POLYMERASE CHAIN REACTION

MUH. ASAAD

MASTER OF AGRICULTURAL SCIENCE UNIVERSITI PUTRA MALAYSIA

2001

DETECTION OF CITRUS GREENING ORGANISM (LIBEROBACTER ASIA TlCUM) BY POLYMERASE CHAIN REACTION

By

MUH. ASAAD

Thesis Submitted in Fulfilment of the Requirement for the Degree of Master of Agricultural Science in the Faculty of Agriculture

U niversiti Putra Malaysia

September 2001

DEDICATION

To

My parents, Mahmud Sara and Sitti Nurhayati

My wife, Warda Mustafa

My brothers and sisters

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfi lment of the requirement for the degree of Master of Agricultural Science

DETECTION OF CITRUS G REENING ORGANISM (LiBEROBACTER AS/AT/CUM) BY POLYMERASE CHAIN REACTION

By

MUH. ASAAD

September 2001

Chai rman: Associate P rofessor Kamaruzaman Sijam, Ph.D.

Faculty: Agriculture

i i i

Citrus greening disease caused by greening organism (GO; Liberobacter

asiaticum) is one of the most destructive diseases of citrus in Malaysia and

Indonesia. To detect the GO in infected plant tissues, Polymerase Chain

Reaction (PCR) , an accurate, rapid and rel iable detection method was applied

to detect the 1 6S rONA fragments of the GO in leaves showing one of several

typical symptoms of greening collected from GO-infected mandarin trees in

Malaysia and Indonesia.

In GO-infected mandarin trees, four typical symptoms of greening on leaves

were observed, namely mottling (type I) , mild chlorosis with green veins (type

I I) , severe chlorosis with green veins (type I I I ) and vein yellowing (type IV) .

Types I I and I I I symptoms were mostly found in GO-infected mandarin trees in

the field, followed by type I symptom, whi le type IV symptom was rare.

IV

Before PCR was used for the detection of GO in infected plant tissues, several

experiments relating to the optimization of the PCR condition were conducted .

Results indicated that the best sample of citrus tissues for DNA extraction was

the midrib plus the petiole. This can be shown by more intense band observed

after agarose gel electrophoresis. A positive amplification was still visible when

the reaction mixture contained 1 0 ng of total DNA was used. Results of the

optimization of the PCR condition indicated that the optimal PCR buffer for

amplification of GO's DNA was the standard buffer containing 78 mM Tris-HCI

(pH 8 .8), 1 7 m M (NH4hS04, 1 0 mM ()-mercaptoethanol and 200 )1g of Bovine

Serum Albumin (BSA) . The optimal concentrations of MgClz, dNTP, primer and

Taq DNA polymerase to be used in reaction mixture were 1 .5 mM, 0.2 mM,

0.4 J.tM, and 1 Un it, respectively. The optimal anneal ing temperature and

number of cycles of PCR condition were 55°C and 40 cycles, respectively .

The 1 6S rONA fragments of the GO in expected size of 1 1 60 bp were detected

in each typical symptoms. These fragments were ampl ified from DNA extracted

from mandarin cultivars infected with the GO and were not amplified from DNA

extracted from healthy trees. These fragments were also detected in i nsect

vector (Diaphorina citn) collected from GO-infected mandarin trees and were

not amplified from DNA extracted from healthy vector collected from Murayya

paniculata using cetyl trimethyl ammonium bromide (CTAB) method for D NA

extraction .

v

Restriction enzyme analysis of the representative samples of six citrus growing

areas in Malaysia and Indonesia indicated that the 1 68 rONA fragments of GO

were each digested into two fragments of the expected size of 640 bp and 520

bp using restriction enzyme Xbal. Therefore, it was confirmed that GO from

infected mandarin trees in Malaysia and Indonesia was L. asiaticum.

Abstrak tesis yang dikemukakan kepada Senat U niversiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains Pertanian

PENGESANAN ORGANISMA PENYAKIT GREENING (LiBEROBACTER AS/AT/CUM) PAD A LlMAU MELALUI TINDAKBALAS RANTAIAN

POLIMERASE

Oleh

MUH. ASAAD

September 2001

Pengerusi: Profesor Madya Kamaruzaman Sijam, Ph.D.

Fakulti: Pertanian

VI

Penyakit 'citrus greening' yang disebabkan organisma 'greening' (GO;

Uberobacter asiaticum) merupakan salah satu penyakit yang paling

merosakkan pokok limau di Malaysia dan Indonesia. Untuk mengesan

kehadiran GO dalam tisu tanaman berpenyakit, tindakba\as rantaian po\ imerase

(peR) , satu teknik pengesanan yang tepat, cepat dan boleh dipercayai telah

digunakan untuk mengesan serpihan 1 6S rONA GO pad a daun-daun l imau

yang menunjukkan beberapa simptom tipika\ penyakit 'greening ' . Oaun-daun ini

diperolehi daripada pokok l imau mandarin yang dijangkiti GO di Malaysia dan

Indonesia.

Pada pokok limau mandarin yang dijangkiti GO, em pat simptom tipikal penyakit

'greening' pada daun-daun telah diperhatikan iaitu 'mottling' Genis I), klorosis

yang tidak ketara (mild chlorosis with green vein , jenis I I) , klorosis yang ketara

vii

(severe chlorosis with green vein , jenis I I I) dan urat daun menguning (vein

yellowing, jenis IV) . Simptom jenis II dan III adalah paling banyak ditemui pada

pokok limau mandarin yang dijangkiti GO di lapangan, diikuti oleh simptom

jenis I, manakala simptom jenis IV adalah simptom yang jarang d itemui .

Sebelu m tindakbalas rantaian polimerase (PCR) digunakan untuk mengesan

kehadira GO dalam tisu tanaman berpenyakit, beberapa kajian berhubung

dengan pengoptimuman keadaan PCR d ijalankan . Keputusan menunjukkan

bahawa tisu l imau yang terbaik digunakan untuk pengekstrakan DNA ada lah

'midrib' dan 'petiole'. In i dapat di l ihat daripada produk PCR yang lebih terang

diperhatikan setelah elektroforesis gel agarose. Amplifikasi yang positif masih

boleh di l ihat apabila campuran PCR mengandungi 1 0 ng DNA yang telah

ditulinkan digunakan. Keputusan pengoptimuman keadaan PCR menunjukkan

bahawa penimbal PCR yang optimum untuk ampl ifikasi DNA GO adalah

pen imbal yang lazim digunakan (standard) yang mengandungi 78 mM Tris-HCI,

1 7 mM (NH4)zS04, 1 0 mM (3-mercaptoethanol dan 200 !J.g Bovine Serum

Albumin (BSA). Kepekatan yang optimum untuk MgCb, dNTP, primer dan Taq

DNA pol imerase yang digunakan dalam campuran PCR adalah masing-masing

1 .5 m M, 0.2 mM, 0.4 !J.M dan 1 unit. Suhu pelekatan primer dan bi langan

kitaran untuk keadaan PCR adalah masing-masing 55°C dan 40 kitaran.

VIII

Serpihan 1 6S rONA GO pada saiz 1 160 bp yang dijangkakan dikesan pada

setiap simptom tipikal penyakit 'greening' . Serpihan in i diamplifikasi daripada

DNA yang diekstrak daripada l imau mandarin yang dijangkiti oleh GO dan tidak

diamplifikasi daripada DNA yang diekstrak daripada pokok limau yang sihat.

Serpihan ini juga boleh dikesan pada vektor serangga (Diaphorina citn) yang

diperolehi daripada pokok limau mandarin yang dijangkiti oleh GO dan tidak

dikesan daripada DNA yang diekstrak daripada vektor sihat.

Keputusan anal isis endonuclease pembatas untuk sampel-sampel daripada

enam kawasan penanaman limau di Malaysia dan Indonesia menunjukkan

bahawa serpihan-serpihan 1 6S rONA GO masing-masing dipotong menjadi

dua serpihan yang d ijangkakan iaitu 640 bp dan 520 bp menggunakan enzim

endonuclease pembatas Xba l . Oleh yang demikian , hasil kajian ini

mengesahkan bahawa organisma 'greening' yang terdapat pada pokok l imau

mandarin di Malaysia dan Indonesia adalah L. asiaticum.

IX

ACKNOWLEDGEMENTS

Praises to almighty Al lah, whose blessing have enabled the author to complete

this project .

The author expresses his sincere appreciation to Assoc. Prof. Dr.

Kamaruzaman Sijam, the chairman of the supervisory committee for his active

guidance, valuable advice, and generous help during the research work and

preparation of this thesis. Gratitudes are also due to Dr. Suhaimi Napis, and

Assoc. Prof. Dr. Mohd. Idris Zainal Abidin , the members of the supervisory

committee for their fruitful suggestions and effective corrections in order to

improve the quality of manuscript.

The author gratefully acknowledges the ARMP-I I Project for the financial

support for the study under Program of the Master of Agricultural Science .

A very special appreciation is due to Dr. Ir . Endang Setyawati T. , MSc. (ARMP

Project Leader) who is in charge of this project, for her generosity and help in

overcoming l iving problems in Malaysia. Thanks are also extended to UPM's

technical and support staff notably Encik Yusuf, Encik Zawawi , Cik J unaina,

Mr. Ong , and many others for providing the author with moral encouragement

and homely atmosphere. Special thanks are due to al l the staff members of the

Faculty of Agriculture of UPM for their help in one way or other.

x

The author is indebted to Agency for Agricultural Research and Development

(AARD) for approving a full-time deputation and for encouraging the author to

continue his postgraduate study abroad .

Last but not least, the author is extremely grateful to his mother Sitti Nurhayati ,

h is father Mahmud Sara, and his wife Warda Mustafa, for their sacrifice,

patience and endless prayers without which this project could never have been

completed .

xi

I certify that an Examination Committee met on 27th September 2001 to conduct the final examination of MUh. Asaad on his Master of Agricultural Science thesis entitled "Detection of Citrus Greening Organism (Liberobacter asiaticum) by Polymerase Chain Reaction" in accordance with U niversiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 198 1 . The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination committee are as follows:

Sariah Meon, Ph .D . Professor F acuIty of Agriculture U niversiti Putra Malaysia (Chairperson)

Kamaruzaman Sijam, Ph.D . Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Member)

Mohd. Jdris Zainal Abidin , Ph. D . Associate Professor F acuIty of Agriculture Universiti Putra Malaysia (Member)

Suhaimi Napis, Ph .D. Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

MO . GHAZALI MOHAYID IN, Ph. D. Professor/Deputy Dean of Graduate School. Universiti Putra Malaysia

Date: 9 OCT 2001

XII

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfi lment of the requirement for the degree of Master of Agricultural Science.

AIN I IDERIS, Ph .D . Professor Dean of Graduate School Universiti Putra Malaysia

Date: '1 3 DIe 2001

xiii

DECLARATION

hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

TABLE OF CONTENTS

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL SHEETS DECLARATION FORM LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER

1

2

3

INTRODUCTION 1.1. Background of the Study 1.2. Objective of the Study

LITERATURE REVIEW 2.1. Citrus Greening Disease

2.1.1. The Occurrence and the Economic Importance 2.1.2. Symptomology 2.1.3. Types and Strains 2 .1.4. Varietal Susceptibil ity and Host range 2.1.5. The Causal Agent and Transmission 2.1.6 . Detection

2.2. Polymerase Chain Reaction (PCR) 2.2.1. I ntroduction 2.2 .2. Optimization of the PCR Condition 2.2.3. The Use of the PCR

2.3. Gel Electrophoresis of DNA and Staining

MATERIALS AND METHODS 3.1. Collection of Samples 3.2. Preparation of Genomic DNA

3.2 . 1 . DNA Extraction from Different Plant Samples 3.2.2. DNA Extraction from Insects

3.3. Measurement of DNA Purity and Quantity 3.4. Amplification of GO's DNA from Different Citrus Tissues 3.5. Electrophoresis and Staining 3.6. Optimization of PCR Condition for Detection of GO's DNA

3.6.1. The Use of Different PCR Buffers 3.6 .2 . The Use of Different MgCb Concentrations

XIV

Page

II III

VI IX XI xiii XVII

xvii i xx

1.1 1 .1 1.6

2 .1 2 .1 2 .1 2 .3 2 .4 2.6 2.8 2.10 2 .13 2 .13 2 .16 2.24 2.25

3 . 1 3 . 1 3.5 3.5 3.7 3 . 7 3 .8 3 .10 3 .12 3.12 3 .13

4

5

6

xv

3.6 .3 . The Use of Different dNTP Mixture Concentrations 3 . 1 4 3.6.4. The Use of Different Specific Primer Concentrations 3 . 1 4 3 .6 .5 . The Use of Different Taq DNA Polymerase 3 . 1 5 3.6 .6 . The Use of Different Annealing Temperatures 3 . 1 6 3 .6 .7 . The Use of Different Number of Cycles 3. 1 7

3.7. The Use of the Optimized PCR Condition for Detection of GO's DNA 3 . 1 8

3.8. Determination of Liberobacter Species by Restriction Enzyme Analysis of Amplified DNA 3 .20

RESULTS 4. 1 . Symptoms of Citrus Greening Disease 4 .2 . The Use of Different Citrus Tissues 4 .3. The Use of Different Total DNA Concentrations 4 .4. Optimization of PCR Condition for Detection of GO's DNA

4.4. 1 . The Use of Different PCR Buffers 4.4.2 . The Use of Different MgCI2 Concentrations 4.4.3 . The Use of Different dNTP Mixture Concentrations 4 .4.4. The U se of Different Specific Primer Concentrations 4 .4 .5. The Use of Different Taq DNA Polymerase 4.4.6. The Use of Different Annealing Temperatures 4.4 .7 . The Use of Different Number of Cycles

4 .5 . Detection of Greening Organism DNA in Citrus Leaves 4 .6. Detection of Greening Organism DNA in Psyll id 4 .7 . Characterization of the Amplified DNA

DISCUSSION 5. 1 . Symptoms of Citrus Greening Disease 5 .2 . The Use of D ifferent Citrus Tissues 5 .3 . The Use of Different Total DNA Concentrations 5.4. Optimization of PCR Condition for Detection of GO DNA 5 .5 . Detection of Greening Organism DNA in Citrus Leaves 5 .6 . Detection of Greening Organism DNA in Psyllids 5 .7 . Characterization of the Amplified DNA

CONCLUSION

4 . 1 4 . 1 4 . 5 4 . 6 4 .7 4 .7 4 .8 4 .9 4 . 1 0 4. 1 1 4 . 1 2 4. 1 3 4 . 1 4 4 .20 4 .21

5 . 1 5 . 1 5 . 5 5 .5 5 .6 5 . 1 2 5 . 1 6 5 . 1 7

6 . 1

REFERENCES R . 1

APPENDICES A. 1 A 1 Preparation of Stock solution A. 1 A2 Preparation of Extraction buffer A. 1 A3 Preparation of Chloroform : Isoamyl alcohol A.2 A4 Preparation of Tris Borate-acid EDTA and Tris EDTA A.2

A5 Preparation of Load ing Dye and Ethidium Bromide Stain Solution

A6 Preparation of 5x Standard peR Buffer A 7 Preparation of Primers A8 Composition of Some PCR Buffers

BIODATA OF THE AUTHOR

XVI

A 3 A.3 A4 A5

B. 1

xvii

LIST OF TABLES

Table Page

2. 1 The general reaction of citrus cultivars to greening 2.7

2 .2 Range of separation in gels containing different amounts of agarose 2 .26

3 . 1 Location of citrus orchards and type of symptoms of leaves col lected in Malaysia and Indonesia 3.3

3 .2 Primer sequences and size of amplified DNA fragment 3 .8

3 . 3 The volumes of components for a peR reaction 3 . 1 9

4 . 1 Distribution of symptom types of GO-infected mandarin trees in each citrus growing areas in Malaysia and Indonesia 4.4

4.2 Relationship between typical symptoms and amount of pathogen in leaves of GO-infected mandarin trees 4 . 1 9

xviii

LIST OF FIGURES

Figure Page

2. 1 The schematic diagram of the PCR process 2 . 1 5

3 . 1 A typical greening-infected mandarin trees i n the field 3 .2

3 .2 Location map of sampling sites in Malaysia and I ndonesia 3 .4

3 .3 A microcentrifuge for DNA extraction 3 .6

3 .4 A thermocycler (eppendorf mastercycler personal) used for the amplification of DNA 3 .9

3 .5 Apparatus for agarose gel electrophoresis 3.11

3 .6 Gel Doc 2000 video gel documentation system (Biorad) 3 .11

3 .7 A waterbath for incubating the digested PCR product 3 .20

4 . 1 Typical symptom of greening disease on mandarin trees 4 .2

4.2 Typical symptom of greening disease on leaves of mandarin trees 4 .3

4 .3 The use of different citrus tissues on amplification of 1 6S rONA of GO using specific primers 4 .5

4 .4 The use of different total DNA concentrations on amplification of 1 6S rONA of GO using specific primers 4.6

4 .5 The use of different PCR buffers on amplification of 1 6S rONA of GO using specific primers 4.7

4 .6 The use of different MgCI2 concentrations on ampl ification of 1 6S rONA of GO using specific primers 4 .8

4 .7 The use of d ifferent dNTP-mix concentrations on amplification of 1 6S rONA of GO using specific primers 4.9

4 .8 The use of different specific primer concentrations on amplification of 16S rONA of GO using specific primers 4 .10

XIX

4.9 The use of different Taq DNA polymerase concentrations on ampl ification of 1 6S rONA of GO using specific primers 4 . 1 1

4 . 1 0 The use of different annealing temperatures on amplification of 1 6S rONA of GO using specific primers 4 . 1 2

4. 1 1 The use of different number of cycles on ampl ification of 1 6S rONA of GO using specific primers 4 . 1 3

4 . 1 2 Agarose gel electrophoresis of DNA amplified with specific primers , 01 1 and 012c from GO-infected mandarin trees at Bertam Valley (Malaysia) 4 . 1 5

4 . 1 3 Agarose gel electrophoresis of DNA amplified with specific primers , 0 1 1 and 012c from GO-infected mandarin trees at Serdang (Malaysia) 4. 1 5

4. 1 4 Agarose gel electrophoresis of DNA amplified with specific primers , 0 1 1 and 012c from GO-infected mandarin trees at Terengganu (Malaysia) 4 . 1 6

4 . 1 5 Agarose gel electrophoresis of DNA ampl ified with specific primers , 0 1 1 and 012c from GO-infected mandarin trees at Jeneponto (Indonesia) 4 . 1 7

4 . 1 6 Agarose gel electrophoresis of DNA amplified with specific primers , 01 1 and 012c from GO-infected mandarin trees at Sidenrreng Rappang ( Indonesia) 4 . 1 8

4 . 1 7 Agarose gel electrophoresis of DNA amplified with specific primers , 0 1 1 and 012c from GO-infected mandarin trees at Selayar (Indonesia) 4 . 1 8

4 . 1 8 Agarose gel electrophoresis of DNA amplified with specific primers , 0 1 1 and 012c from psyll ids 4 .20

4 . 1 9 Electrophoresis on 1 .5% agarose gel of Xbal digested DNA amplified from several citrus growing areas in Malaysia and Indonesia 4 .22

LIST OF ABBREVIATIONS

Chemicals / Units

CTAB

dNTP

EDTA

MgCh

rONA

TBE

TE

Tris-HCI

bp

hr

kb

mg

min

mM

ng

00

pmol

sec

Cetyl tnmethyl ammonium bromide

deoxyribonucleotide triphosphate

Ethylene diamine tetraacetic acid

Magnesium chloride

ribosomal DNA

Tris-Borate- EDT A

Tris-EDTA

Tris (hydroxymethyl) aminomethane hydrochloric acid

base pair

hour

kilobase

milligram

minute

millimolar

nanogram

optical density

picomole

second

microliter

micromolar

xx

CHAPTER 1

INTRODUCTION

1 .1 . Background of the Study

Citrus (Citrus spp . ) is one of the major world trade commodities both

as fresh or processed fruits. Citrus fruits comprising oranges, mandarins,

lemons, limes, grapefruits, and pomelos are widely grown in al l tropical and

subtropical areas of the world . In I ndonesia and Malaysia particularly ,

mandarin cultivar (Citrus reticu/ata Blanco) is widely cultivated by farmers

because of its early vigour, well-adapted in the lowland, and offering a good

income (Aubert, 1 992; Ko, 1 992; Sastijati, 1 992; Sugiyanto, 1 992).

In the world, the main citrus production countries are Brazi l , USA,

China, Spain , Mexico, I ran and Italy (FAa, 200 1 ) . Whereas, in South East

Asia are Thailand, Indonesia and Philippines (Xu et a I . , 1 99 1 ; FAa, 200 1 ) .

During the period of1 990 to 1 998, the total harvested area and production of

citrus i n I ndonesia averaged at 44,433 ha and 508,780 t, respectively

(Ministry of Agriculture, Republ ic of I ndonesia, 2001 ) .

Production of citrus i n Asian countries, particularly China and South

East Asian countries decreased sharply since 1 980's because of the

presence of several major diseases. One of the most destructive diseases of

citrus is citrus greening disease (CGD) that caused crop and tree loss i n

1.2

many parts of Asia and Africa. Before it was identified as one disease, it was

known by various other names such as yellow shoot (huang lungbin) in

China (Lin and Lin, 1 990) , l ikubin (decline) in Taiwan (Su and Huang, 1 990),

dieback in India, leaf mottle in Phil ippines (Gonzales, 1 987) , citrus vein

phloem degeneration (CVPD) in Indonesia, and yellow branch or greening in

South Africa (da Graca, 1 99 1 ) . As it became clearer that all these were

similar d isease, the name "greening" was widely adopted.

Losses from greening to the citrus industry in detail have not been

much published , but some data of the severity of the disease in several

citrus-growing areas have been published . For example, in China the citrus

production in Yangcun farm (one of the largest citrus farms) dropped to

5 ,000 t in 1 982 from 450,000 t in 1 977 because of huanglungbin (greening)

disease (Ke, 1 987), In I ndonesia, estimated more than 8 mill ion trees were

infected and caused an annual loss of US$ 22,000 (Nurhadi et aI . , 1 992)

because of greening disease.

In Malaysia, evidence of the occurrence of greening d isease was

known after efforts on greening disease research were intensified by

Malaysian government with FAO/UNDP since 1 988 (Lim et aI . , 1 990) . Based

on the typical greening symptoms, the presence of the vector (Diaphorina

citn) , and the positive results of transmission tests, greening disease was

found in Kuala Terla (Cameron H ighland), Jelebu, Kuala Jerangau, Serdang

1.3

and Klang (Lim et aI. , 1 990; Aubert, 1 992; Osman and Lim, 1 992) . In

I ndonesia the greening disease, known by the name of Citrus Vein Phloem

Degeneration (CVPD), has apparently penetrated the Indonesian

archipelago in the 1 940s (Aubert et aI. , 1 985) and seriously affected citrus

production in Indonesia since 1 950 (Setyobudi et aI . , 1 992) .

Greening disease is caused by a nonculturable, phloem-limited

bacterium, which was proposed by Jaqoueix et al . ( 1 994) the name

Liberobacter asiaticum in Asia and L. africanum in Africa, after the nucleotide

sequences of the 1 6S rONA of the Indian and African isolates of the GO

have been determined. According to Jagoueix et al. ( 1 996) , Xbal restriction

enzyme should hydrolyze the 1 6S rONA of L. asiaticum into two fragments

(640 bp and 520 bp) and that of L. africanum into three fragments (520 bp,

506 bp and 1 30 bp) . Greening disease was initially considered that the

causal agent of greening d isease was a mycoplasma-l ike organism (MlO).

However, this organism was soon found to be enclosed by a 25 nm thick

envelope, which was much thicker than the unit membrane envelope

characteristic of MlOs (7 to 1 0 nm thickness) . By analogy with MlO, this

organism was called Bacterium-l ike Organism (BlO) or greening organism

(Nakashima et aI . , 1 996) . This organism is transmitted by two insect vectors,

the psyll id Diaphorina citri in Asia and Trioza erytreae in Africa (Martinez and

Wallace, 1 969; McClean et aI . , 1 969).