“Identification, diversity and detection of Xanthomonas strains associated with pistachio dieback in Australia”
Alireza Marefat
Thesis submitted for the degree of Doctor of Philosophy at The University of Adelaide
Discipline of Plant and Pest Science Faculty of Sciences
December 2005
Dedicated to my son,
‘ Armin’
ABSTRACT...... I
DECLARATION ...... III
ACKNOWLEDGEMENTS ...... IV
ABBREVIATIONS...... VI
CHAPTER 1: REVIEW OF THE LITERATURE ...... 1
1.1 Introduction...... 1
1.2 The pistachio industry...... 2 1.2.1 Origin and history...... 2 1.2.2 World production...... 3 1.2.3 Australian production...... 3
1.3 Pistachio dieback ...... 5 1.3.1 History, distribution and importance ...... 5 1.3.2 Symptoms...... 6 1.3.3 The causal agent ...... 7
1.4 Xanthomonas Dowson 1939 ...... 10 1.4.1 History, distribution and importance ...... 10 1.4.2 Description ...... 10
1.5 Identification and characterisation of xanthomonads...... 11 1.5.1 Non DNA-based methods ...... 11 1.5.1.1 Morphological and structural description...... 12 1.5.1.2 Metabolic testing...... 12 1.5.1.3 Fatty acid methyl ester (FAME) analysis...... 13 1.5.1.4 Gel electrophoresis of whole cell proteins ...... 15 1.5.1.5 Serological techniques...... 15 1.5.2 DNA-based molecular techniques ...... 17 1.5.2.1 PCR-independent methods ...... 18 1.5.2.1.1 DNA-DNA hybridisation ...... 18
1.5.2.1.2 Restriction endonuclease analysis...... 19 1.5.2.1.3 Restriction fragment length polymorphism analysis (RFLP)...... 19 1.5.2.2 PCR-based techniques...... 20 1.5.2.2.1 Multiple arbitrary primed PCR (MAAP) ...... 21 1.5.2.2.2 Amplified fragment length polymorphism analysis (AFLP)...... 21 1.5.2.2.3 Repetitive sequence-based PCR ...... 22 1.5.2.3 PCR-based detection techniques...... 23
1.6 Summary and objectives ...... 25
CHAPTER 2: GENERAL MATERIALS AND METHODS...... 28
2.1 Collection of xanthomonad strains associated with pistachio dieback ...... 28
2.2 Collection of other phytobacteria occurring in and around pistachio orchards ...... 29
2.3 Collection of type and reference strains of Xanthomonas ...... 31
2.4 Propagation and maintenance of bacteria...... 33
2.5 Collection of seeds and other plant materials for pathogenicity tests...... 33
2.6 DNA extraction from bacteria ...... 34
CHAPTER 3: GENETIC DIVERSITY WITHIN THE PISTACHIO DIEBACK PATHOGEN ...... 37
3.1 Introduction...... 37
3.2 Materials and methods ...... 38 3.2.1 Experimental protocol for rep-PCR fingerprinting ...... 38
3.3 Data analysis...... 40
3.4 Results...... 41
3.5 Discussion...... 42
CHAPTER 4: CHARACTERISATION OF XANTHOMONADS CAUSING PISTACHIO DIEBACK ...... 57
4.1 Introduction...... 57
4.2 Materials and methods ...... 58 4.2.1 Biochemical and physiological tests...... 58 4.2.2 Biolog GN2 Microplate System ...... 59 4.2.3 SDS-PAGE of whole cell proteins ...... 59 4.2.4 Sequencing of the 16S-23S rDNA spacer region ...... 61 4.2.5 16S rRNA gene sequencing ...... 62 4.2.6 DNA-DNA hybridisation...... 65 4.2.7 Comparison with pathovars of X. translucens using rep-PCR...... 66
4.3 Results...... 66 4.3.1 Biochemical and physiological tests...... 66 4.3.2 Biolog GN2 Microplate System ...... 70 4.3.3 SDS-PAGE of whole cell proteins ...... 72 4.3.4 Sequencing of the 16S-23S rDNA spacer region ...... 73 4.3.5 Sequencing of the 16S rRNA gene...... 73 4.3.6 DNA-DNA hybridisation...... 74 4.3.7 Comparison with pathovars of X. translucens using rep-PCR...... 78
4.4 Discussion...... 87
CHAPTER 5: PATHOGENICITY OF XANTHOMONADS ASSOCIATED WITH PISTACHIO ....93
5.1 Introduction...... 93
5.2 Materials and methods ...... 94 5.2.1 Pathogenicity to members of the Anacardiaceae ...... 94 5.2.2 Pathogenicity to members of the Poaceae ...... 97
5.3 Results...... 100 5.3.1 Pathogenicity to members of the Anacardiaceae ...... 100 5.3.2 Pathogenicity to members of the Poaceae ...... 101
5.4 Discussion...... 110
CHAPTER 6: DETECTION OF XANTHOMONAD PATHOGENS OF PISTACHIO IN
AUSTRALIA ...... 116
6.1 Introduction...... 116
6.2 Materials and methods ...... 117 6.2.1 Detection of bacteria with X. translucens -specific primers, T1 & T2...... 118 6.2.2 PCR assay for specific detection of the pistachio pathogens...... 119 6.2.2.1 Selection and design of primers...... 119 6.2.2.2 PCR cycling conditions...... 120 6.2.2.3 PCR specificity and sensitivity tests ...... 121 6.2.2.4 PCR efficiency in plant material and verification ...... 121 6.2.2.4.1 Rapid extraction protocols...... 123 6.2.2.4.2 Proteinase-based method...... 123 6.2.2.4.3 CTAB-based method...... 124 6.2.2.4.4 Bio-101 DNA extraction kit ...... 124 6.2.2.4.5 BIO-PCR ...... 125
6.3 Results...... 125 6.3.1 Detection of bacteria with X. translucens -specific primers, T1&T2...... 125 6.3.2 PCR assay for specific detection of the pathogen ...... 127 6.3.2.1 Selection and design of primers...... 128 6.3.2.2 PCR cycling conditions...... 131 6.3.2.3 PCR specificity and sensitivity tests ...... 132 6.3.2.4 PCR efficiency in plant material and verification ...... 137 6.3.2.4.1 Rapid extraction protocols...... 138 6.3.2.4.2 Proteinase-based method...... 138 6.3.2.4.3 CTAB-based method...... 138 6.3.2.4.4 Bio-101 DNA extraction kit ...... 139 6.3.2.4.5 BIO-PCR ...... 139
6.4 Discussion...... 142
CHAPTER 7: GENERAL DISCUSSION ...... 147
APPENDIX A: BUFFERS AND REAGENTS ...... 156
APPENDIX B: MEDIA AND METHODS FOR BIOCHEMICAL AND PHYSIOLOGICAL TESTS ...... 160
APPENDIX C: SEQUENCE ACCESSION NUMBERS IN THE GENBANK...... 167
APPENDIX D: PUBLICATIONS ...... 168
LITERATURE CITED...... 169
Abstract
Bacterial dieback disease was first observed on pistachio ( Pistacia vera ) trees in
Australia in 1992 and has not been reported elsewhere. The disease is characterised by shoot death and dieback, limb and trunk lesions and discolouration of woody tissue in shoots more than one year old. Xanthomonas strains have been the only micro-organism consistently isolated from diseased trees. Characterisation of a small number of strains of the pathogen has already shown close relatedness to X. translucens . At the commencement of this study knowledge about genetic diversity of the pathogen was lacking, characterization of the pathogen and its taxonomic position were rudimentary and there was no efficient, reliable and rapid tool to detect and to recognise the pathogen in planting materials.
In this study, a three-step strategy was undertaken:
First, the genetic diversity of Xanthomonas strains isolated from infected pistachio trees from different geographic regions of Australia over several years was examined by repetitive extragenic palindromic-polymerase chain reaction (rep-PCR). Rep-PCR revealed two distinct genotypes, group A and group B, among the strains and showed some variation within group A. There was an association between the two groups and the geographic origin of the strains.
Second, physiological and biochemical tests, including the Biolog microplate TM System, and polyacrylamide gel electrophoresis (PAGE) of whole cell protein analysis were used to characterise and identify the strains. To obtain a sound taxonomic allocation of pistachio strains within the genus Xanthomonas, the relatedness of pistachio strains to known Xanthomonas species and strains was assessed by comparison of the 16S-23S rDNA internal transcribed spacer (ITS) and 16S rDNA sequences, rep-PCR genomic fingerprints and DNA:DNA homology. Pathogenicity of the strains was assessed on
I
selected members of the Poaceae and Anacardiaceae . Results of physiological and biochemical tests including Biolog analysis and protein profiling confirmed the existence of the two groups within the pathogen. Furthermore, the pathogen was identified as X. translucens . ITS sequencing confirmed two distinct genotypes among the strains and suggested that the pathogen strains were closely related to X. t. pv . poae .
Based on 16S rDNA sequencing, pistachio strains matched most closely X. t. pv . translucens . While DNA:DNA homology studies confirmed that pistachio strains from both groups belong to X. translucens , rep-PCR showed that xanthomonads from pistachio differ from known pathovars of the species . Pathogenicity and host range studies indicated that the two groups were biologically different and suggested that these strains represent two new pathovars of the species.
Third, specific primers for amplification of DNA of the pathogen were developed based on sequences of the ITS region from strains representing groups A and B. Primers were designed for amplification of DNA sequences specific to each group and a multiplex
PCR test was developed that identified and differentiated strains of each group in a single assay. To determine the specificity of the primers, PCR was carried out with
DNA from 65 strains of the pathogen, 31 type and reference strains of Xanthomonas , and from 191 phytobacteria commonly found in and around pistachio orchards. In the multiplex PCR, a 331 bp fragment was amplified from all strains belonging to group A and a 120 bp fragment from all strains in group B. No PCR products were obtained from the other bacteria tested except for the type strain of X. translucens pv. cerealis , which has not been found in Australia. The assay was used to detect strains from both groups of the pathogen in pistachio plant material.
This study will contribute to understanding of the epidemiology and management of pistachio dieback.
II Declaration
This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text.
I give consent to this copy of my thesis, when deposited in the University Library, being available for loan and photocopying.
Signed: [Signature in print copy] Date: 30/12/05
III Acknowledgements
1 This thesis is the result of 3 ⁄2 years of work at The University of Adelaide, Australia, where I have benefited from the stimulating atmosphere and support of many people. I am pleased have the opportunity to express my gratitude.
First, I wish to express sincere appreciation to my supervisors. I would like to express my deep and sincere gratitude to Associate Professor Eileen Scott. Her wide knowledge, enthusiasm for research and logical way of thinking have been of great value for me.
Her understanding, encouraging and personal guidance have made a deep impression on me and have provided a good basis for the present thesis. I owe her much gratitude for having shown me this way of research. My greatest debt is also to Dr Kathy Ophel-
Keller who kept an eye on the progress of my work and always was available when I needed her advice. Her detailed, perceptive comments contributed in no small way to the shape and clarity of the finished thesis. Special thanks are due to Professor Margaret
Sedgley, whose critical view, familiarity with the needs and ideas of the pistachio industry and valuable formulation of the problems in this thesis were always helpful.
I would like to thank Dr. Evelina Facelli for her constant support and encouragement during this research. I also thank Cathy Taylor and Dr Bob Emmett, other members of the pistachio dieback research group, for their useful comments and assistance.
My study at the University of Adelaide would be impossible without assistance from various sections at the University. I thank the administrative staff, the staff of the
International Student Centre, Student Centre, Waite Library, IT help desk, Plant Growth
Services, Waite Campus and the School of Agriculture and Wine, especially staff in the
Discipline of Plant and Pest Science, for their cheerful assistance. I am also grateful to
IV
students and friends in the Plant Pathology Research Group for providing me an excellent work environment during the past years.
Thanks are also expressed to all personnel associated with the Diagnostic Laboratory and Steve Barnett’s lab in SARDI who were very generous and cooperative in supporting the research.
I express sincere appreciation to Dr John Bowman at the Australian Food Safety Centre,
University of Tasmania, for conducting the DNA:DNA hybridization experiments. I also acknowledge Ms Kate Dowling for assistance with experimental design and analysis. In addition, I am grateful to the Institute of Medical and Veterinary Science
(IMVS), Adelaide for the use of the PC3 laboratory.
I would like to thank staff in the Belgium Coordinated Collection of Microorganisms,
Laboratory of Microbiology, University of Ghent, Belgium (BCCM/LMG); Australian
Collection of Microorganisms (ACM), University of Queensland, Brisbane, Australia and the Australian Collection of Plant Pathogenic Bacteria (ACPPB), Agricultural
Institute, Orange, NSW, Australia for providing type and reference strains of bacteria.
Special thanks to Horticulture Australia Limited (HAL, NT02007) and the Pistachio
Growers Association of Australia for providing financial and logistical support.
This course was supported by a doctoral fellowship awarded to me by The Islamic
Republic of Iran. I would like to thank the Ministry of Science, Research and
Technology of Iran for sponsorship of my study abroad.
Finally, I feel a deep sense of gratitude to my father and mother for supporting and encouraging me throughout my education programs.
V Abbreviations
°°°C degree Celsius AFLP amplified fragment length polymorphism ARMS amplification refractory mutation system BSA bovine serum albumin BER-PCR BOX, -ERIC and REP-PCR combined profiles CFU colony forming unit CTAB hexadecyltrimethylammonium bromide cv. cultivar DNA deoxyribonucleic acid DMSO dimethyl sulphoxide dNTP 2’-deoxynucleoside 5’-triphosphate EDTA ethylenediamine tetra acetic acid ERIC-PCR enterobacterial repetitive intergenic consensus-based PCR FAME fatty acid methyl ester g, mg, µµµg, ng gram, milligram, microgram, nanogram h hour(s) ha hectare(s) HR hypersensitive reaction ITS internal transcribed spacer kb kilo base kDa kilo Dalton L, mL, µµµL litre, millilitre, microlitre M, mM molar, millimolar mA milliamp min minute(s) mm millimetre(s) MQ water ultrapure milli-Q water mt metric ton(s) MW molecular weight NSW New South Wales nt nucleotide NT Northern Territory
VI
PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction pv. pathovar Qld Queensland RAPD random amplified polymorphic DNA RNA ribonucleic acid rDNA ribosomal DNA REP-PCR repetitive extragenic palindromic-based PCR rep-PCR repetitive sequence-based PCR RFLP restriction fragment length polymorphism rpm revolutions per minute RT room temperature s second(s) SA South Australia SDS sodium dodecyl sulphate TEMED N-, N'-tetramethylenediamine TAE Tris-acetate-EDTA TE Tris-EDTA tRNA transfer ribonucleic acid UPGMA unweighted pair-group method using arithmetic averages UV ultra violet V volts v volume Vic Victoria w weight
VII Chapter 1: Review of the literature 1
Chapter 1: Review of the literature
1.1 Introduction
Dieback of pistachio ( Pistacia vera ) was first observed in Australian pistachio orchards in 1992 (Edwards & Taylor, 1998) and this was the first report of this bacterial disease on pistachio in the world. The disease causes reduced yield and tree death, and approximately 10% of trees are affected (Anonymous, 2003). Pistachio orchards in the major pistachio growing regions of South Australia, Victoria and New South Wales are infected and the disease has been considered a major threat to the pistachio industry
(Anonymous, 2002).
Based on preliminary studies, Xanthomonas strains have been identified as the causal agent of dieback of pistachio in Australia (Edwards & Taylor, 1998). However, the detailed characterisation of the pathogen, genetic variability among strains, and exact placement of the pathogen within the genus Xanthomonas had not yet been attempted at the commencement of this study. Such information is required for every new disease, to enable subsequent epidemiological studies. Furthermore, there was no sensitive, efficient and reliable method to identify and detect the pathogen. A rapid and reliable
DNA-based detection technique would be useful in epidemiological studies and could enhance management of pistachio dieback.
In this review, the history of pistachio dieback in Australia and the pathogen will be discussed. Methods and techniques which have been used for identification and detection of phytopathogenic bacteria, with emphasis on the genus Xanthomonas , will also be reviewed.
Chapter 1: Review of the literature 2
1.2 The pistachio industry
1.2.1 Origin and history
The pistachio, Pistacia vera L., is native to western Asia and Asia Minor, from
Mediterranean countries in the west to India in the east, and has been introduced from there to other countries such as the United States (Teviotdale et al. , 2002). Pistachio belongs to the family Anacardiaceae and the genus Pistacia. This genus contains 11 species of which only vera produces edible nuts and has economic value (Ferguson,
1995). The tree is dioecious; male and female flowers are borne on different trees, and both trees are required to reproduce. The nut is a drupe, consisting of three parts; an exocarp, a mesocarp and an endocarp which encloses the seed, the edible portion. There are several rootstocks on which the nut-producing species, Pistacia vera, is grown, such as P. terebinthus , P. atlantica , P . integerrima (Pioneer Gold I), as well as Pioneer Gold
II (PGII) and UC Berkeley I (UCB I) two hybrids of P. atlantica and P. integerrima
(Ferguson, 1995). Selection of a rootstock is based mainly on resistance to nematodes and soil-borne diseases such as Verticillium wilt, and also on specific growing conditions (Ferguson, 1995). P. vera has a number of cultivated varieties which are considered as cultivars. Momtaz, Owhadi and Kalehghouchi are the best cultivars in
Iran, Red Aleppo is the common cultivar in Syria and Kerman is the main cultivar in
California. The latter originated from the Kerman region in Iran and was introduced into the United States in 1929 (Ferguson, 1995). Locations with long, hot, dry summers and moderate winters are appropriate for pistachio production. Since the root system is typical of a phreatophyte and mines the soil deeply, the pistachio tree can survive harsh climates without irrigation, however, it needs irrigation for economic production
(Ferguson, 1995).
Chapter 1: Review of the literature 3
1.2.2 World production
The economic viability of the pistachio has been recognised in many countries but, with about 300,000 ha of pistachio orchards, Iran is the largest pistachio producer in the world (Teviotdale et al. , 2002). Large nut size and split shells are good characteristics of the Iranian cultivars (Ferguson, 1995). The second largest producer is the United States of America with about 30,000 ha of pistachio orchards, almost all in California
(Teviotdale et al. , 2002). According to the Food and Agriculture Organization (FAO)
(Anonymous, 2005b), the top four pistachio producers in 2004 were Iran at 275,000 metric tons, the United States of America at 158,000 mt, Syria at 40,000 mt and Turkey at 30,000 mt. Iraq, Greece, Italy and Australia are other producers.
1.2.3 Australian production
Although pistachio was introduced into Australia in 1935, attempts to adapt it to the new environmental conditions did not begin until the 1960s (Robinson, 1997).
Commercial planting commenced in the 1980s and pistachio orchards have been established mainly in the Murray Valley and the Clare Valley in South Australia (see
Figure 1.1), Tamworth/Dubbo in New South Wales, central Victoria, and southwestern
Western Australia (Taylor & Edwards, 2000).
The main female cultivar grown in Australia is Sirora. This cultivar is a selection made by CSIRO, Division of Plant Industry at Merbein, Victoria, from an open pollinated seedling of Red Aleppo (Maggs, 1982; Taylor & Edwards, 2000). Characteristics such as lower chilling requirement and higher nut split rate have made Sirora more suited to
Australian conditions than Kerman. Also, several male cultivars are used to provide a
Chapter 1: Review of the literature 4 suitable window of pollen availability. P. atlantica, P. terebinthus and P. integerrima are the most popular rootstocks
J QLD HA fl
SA
Ð Present productiorr 0 ß Poterrtiirl ¡rr oductiorr
Figure 1.1 Distribution of pistachio orchards and potential production areas in Australia
(Robinson, 1997)
The pistachio industry in Australia is relatively new and consists of about 40 growers with around 700 ha of orchards. The first commercial crop was harvested in 1995.
Australian pistachio production is around 1,000 Llyear and is expected to rise to an
average of 1,100 to 1,200 per annum (Anonymow,2002). Australian consumption is
about 1,500 t annually (Robinson, 1997). Therefore, pistachio nuts are imported, mostly
from Iran and the United States. Chapter 1: Review of the literature 5
1,600 1-400 '1,200 îtL I f ,000 q,UI 800
L 600 I 400 200
sdt fr.","s npe+s +ú +s "d¡ ""+
Figure 1.2 Australian pistachio production, previous and projected (Anonymous, 2002)
1.3 Pistachio dieback
1.3.1 History, distribution and importance
Pistachio dieback is not only a new disease in Australia but also seems to be a new disease for pistachio across the world. The first syrnptoms were observed in Australian pistachio orchards in1992 (Edwards & Taylor, 1998).
To date, two research programs have been undertaken to clarify the cause and the epidemiology of the disease. The first project, funded by the pistachio industry of
Australia, the Horticultural Research and Development Corporation (HRDC) and the
Department of Natural Resources and Environment, Victoria, conìmenced in 1996
(Edwards et al., 1993). The second project was initiated in 2000. This project is also
funded by the pistachio industry, through the Pistachio Growers Association (PGA) and
Horticulture Australia, and is conducted by researchers from the Department of Primary
Industries Victoria, the University of Adelaide and the University of New England. Chapter 1: Review of the literature 6
The development of the disease has been observed in pistachio orchards of South
Australia, Victoria and New South Wales since 1992 (Edwards & Taylor, 1998).
Although no comprehensive study has been conducted to determine losses due to the disease, Edwards & Taylor (1998) reported that the disease has caused the death of more than 10% of trees in some areas. Moreover, they noted that if the disease is not checked, it will seriously affect the economic viability of the Australian pistachio industry.
1.3.2 Symptoms
Symptoms of pistachio dieback have been found on mature male and female trees, however, male trees seem to be more susceptible than females. Juvenile trees less than
4-5 years old rarely show symptoms. The symptoms have been observed on both Sirora and Kerman scions grafted onto the common rootstocks (Taylor & Edwards, 2000).
Symptoms are (Figure 1.3):
A) Shoot death and dieback: meaning the slow collapse and sometimes death of twigs and limbs extending down into the crotch, in some cases, into the trunk and killing the entire tree.
B) Internal staining: discolouration of woody tissue in shoots one year old and older which varies in cross-section from small, dark pin-sized stain to thick, dark rings completely encircling the twig, branch or trunk. By removing strips of the bark from diseased twigs, streaks of brown to black staining can be observed. Internal staining of shoots appears to be associated with lesions in the trunk and limbs.
C) Trunk lesions: lesions on the trunk and major limbs of the diseased trees, black sooty patches with sunken bark can be seen. In some cases, the patch covers half the trunk. The bark in this part is black, due to the growth of saprophytic fungi, such as
Chapter 1: Review of the literature 7
Alternaria alternata and Cladosporium sp. (sooty molds) and the tissue under is dead or dying.
D) Resinous exudates: the excessive oozing of clear and colourless, sometimes milky or bluish, sap from the trunk or major branches. Although this resinous gum is also present in healthy trees, especially in response to wounds, infected trees seem to produce more than healthy trees (Facelli et al. , 2001).
Edwards (1997) reported that the oozing of sap from the bark and the appearance of
"cankers" are the first and second symptoms of the disease, respectively. Thus the disease was initially called "pistachio canker". However, these lesions are not typical cankers, so the disease was renamed “pistachio dieback” (Facelli et al. , 2001).
1.3.3 The causal agent
Although symptoms of pistachio dieback were first observed in 1992, sampling and attempts to identify the probable pathogen(s) did not begin until 1994 (Facelli et al. ,
2001). Edwards (1997) reported that pathogenic viruses, phytoplasmas and nematodes were not associated with the disease. A bacterium identified as a Xanthomonas sp., several weakly pathogenic fungi such as Alternaria sp. and Phoma sp., and one strain of
Verticillium sp. were isolated from the samples. However, the Verticillium isolate differed from V. dahliae , which causes disease on pistachio in other countries. Because the author considered that xanthomonads are usually associated with disease of leaves and not with diseases of wood, the Xanthomonas isolated from diseased pistachio trees
Chapter 1: Review of the literature 8
A B
C D
Figure 1.3 Pistachio dieback symptoms: A: major limbs of the diseased tree showing black sooty patches with sunken bark, B: discoloration of woody tissue in shoot which varies in cross-section from small, dark pin-pricks to thick and dark rings C: the diseased tree showing dieback D: dead pistachio tree infected by the disease
Chapter 1: Review of the literature 9 was initially thought to be a secondary organism and not the primary cause of the disease. Nevertheless, xanthomonads are strongly phytopathogenic bacteria that can cause diseases of wood, such as occurs in citrus and almond bacterial canker. Edwards
(1997) concluded that a new strain of Verticillium or several organisms could be the cause of this disease.
One year later, Edwards & Taylor (1998) reported that X. translucens and X. oryzae , identified by Biolog ® tests and DNA fingerprinting, were the only organisms consistently isolated from diseased pistachio trees and announced that Xanthomonas bacteria caused pistachio canker. Pathogenicity testing of both bacteria was performed on Sirora trees grafted on the rootstock of P. terebinthus . Subsequently, characterisation of a small number of strains of the pathogen indicated a close relationship between the pathogen and X. translucens and two groups were identified within the pathogen
(Facelli et al. , 2005). However, this characterisation was based on limited information about the pathogen and the characteristics reported are not sufficient for a valid definition according to Wayne (1987) and Stackebrandt et al. (2002). Therefore, further research was required to confirm the taxonomic position of the pistachio strains at species and intraspecies levels.
Xanthomonas strains have been reported to cause canker, leaf spot and dieback on one- year old pistachio seedlings in Iran (Tarighi & Rahimian, 2001). However, there is no report of the disease in orchards and identification of strains has been based on a few physiological and biochemical tests only.
Chapter 1: Review of the literature 10
1.4 Xanthomonas Dowson 1939
1.4.1 History, distribution and importance
The first report of a plant disease caused by a yellow bacterium, which is now named
Xanthomonas , goes back to 1881 (Starr, 1983). Subsequently, many similar bacteria isolated from plants with various diseases around the world were named Phytomonas in the first edition of the Manual of Bacterial Plant Pathogens in 1930 and, subsequently,
Xanthomonas by Dowson in 1939 (Starr, 1983). Strains of the genus are distributed worldwide and cause various diseases on different plants. Over 124 monocotyledonous and 268 dicotyledonous plants have been reported to be hosts of the bacterium (Leyns et al. , 1984). Some members of the genus cause severe diseases, such as X. axonopodis pv. citri , the causal agent of citrus bacterial canker, and X. translucens , the causal agent of bacterial diseases of cereals and grasses.
1.4.2 Description
Xanthomonas includes bacteria described as being plant pathogens or associated with plants. Yellow pigments called xanthomonadins and xanthan, an exopolysaccharide which results in a mucoid culture, are characteristic of the genus. Cells are Gram- negative, rod shaped, about 0.4-0.6 × 1-2.9 µm, usually motile by a single polar flagellum, chemo-organotrophic and the G + C content varies from 63.3 to 69.7 mol%
(Vauterin et al. , 1995).
The genus currently comprises 25 species (Anonymous, 2005a) as follows:
Xanthomonas albilineans, X. ampelina, X. arboricola, X. axonopodis, X. bromi, X. campestris, X. cassavae, X. citri, X. codiaei, X. cucurbitae, X. cynarae, X. fragariae, X.
Chapter 1: Review of the literature 11 hortorum, X. hyacinthi, X. maltophilia, X. melonis, X. oryzae, X. phaseoli, X. pisi, X. populi, X. sacchari, X. theicola, X. translucens, X. vasicola and X. vesicatoria .
1.5 Identification and characterisation of xanthomonads
Historically, physiological, biochemical and pathogenicity tests have been used to detect, identify and classify phytopathogenic bacteria. In the past 15 years, DNA-based approaches, especially PCR-based techniques, have revolutionised the characterisation and taxonomy of plant pathogenic bacteria, including xanthomonads. These techniques have facilitated studies to assess the genetic diversity of populations which provides a framework not only to understand the taxonomy, but also to find efficient, sensitive and rapid methods for pathogen detection, disease diagnosis and, subsequently, disease management. Furthermore, these approaches could be used for identification and classification of unknown bacteria. On the other hand, it has been accepted that only polyphasic approaches which are performed based on phenotypic, chemotaxonomic and genotypic data can produce a stable and general purpose classification (Vauterin et al. ,
1995). Therefore, it is necessary to consider in some detail both non-nucleic acid-based
(conventional) and nucleic acid-based methods, with particular reference to
Xanthomonas spp.
1.5.1 Non DNA-based methods
Bacterial diseases caused by various strains of xanthomonads have most often been diagnosed on the basis of typical symptoms and then isolation and characterisation of the pathogen by morphological, physiological and nutritional tests, such as those
Chapter 1: Review of the literature 12 described in popular books and manuals (Klement et al. , 1990; Lelliott & Stead, 1987;
Schaad, 1988; Schaad et al. , 2001) and summarised below.
1.5.1.1 Morphological and structural description
Bacterial shapes, sizes and structures are based on observation made using a microscope and, sometimes, staining procedures. Also, the colour, size and shape of bacterial colonies growing on culture media may be described. Most xanthomonads produce convex, mucoid and yellow colonies on yeast dextrose carbonate agar (YDC) and xanthomonadin, a unique, yellow membrane-bound pigment (Schaad et al. , 2001).
However, morphological descriptions of bacteria lack sufficient details to permit species or even genus level identification.
1.5.1.2 Metabolic testing
Enzymatic reactions, specific requirements for growth and the utilisation of substrates differ among the various bacteria. These variations are assessed via the assimilation of certain carbon sources, the fermentation of the carbon source under anaerobic conditions, the oxidation of the carbon source under aerobic conditions and the detection of specific enzyme activities. Materials and methods have been presented in various books and manuals (Lelliott & Stead, 1987; Schaad et al. , 2001) and complete descriptions of characters for a more precise identification of phytopathogenic bacteria have been presented in Bergey's Manual of Determinative Bacteriology (Holt, 1994).
For instance, some metabolic features that are considered diagnostic of the genus
Xanthomonas are: an alkaline reaction in the litmus milk test, the production of levan from sucrose and H 2S from cysteine, oxidase negative and aerobic growth. In an extensive phenotypic study of the genus Xanthomonas , Mooter & Swings (1990)
Chapter 1: Review of the literature 13 analysed 295 phenotypic characteristics of 266 strains and described eight phena for the genus. Although metabolic testing may provide adequate resolution to permit identifications to genus and species, this often requires different tests, which are laborious and time-consuming. Furthermore, metabolic testing is invaluable for evolutionary, environmental and taxonomic studies. Biolog Microplate TM , a relatively new approach which shows great promise in speed, simplification and standardisation of metabolic testing, was introduced in 1989 (Bochner, 1989). In the use of this commercially available kit, a culture of the test bacterium is inoculated into 95 wells of a 96-well microtitre plate. Each well has been coated with a different organic substrate.
The result (i.e. the utilisation of the 95 different substrates) is determined using a redox colour reaction. The result can be read manually or by an automatic plate reader and can be compared with the databases in relevant libraries (Schaad et al. , 2001). This technique provides a "metabolic fingerprint" to demonstrate metabolic variation among strains of xanthomonads and to identify strains associated with particular diseases (Gent et al. , 2004; Griffin et al. , 1991; Jones et al. , 1993; Mooter & Swings, 1990;
Roumagnac et al. , 2004; Vauterin et al. , 1995; Verniere et al. , 1993). The Biolog GN and GP microtitre plates (Biolog Inc., Hayward, CA) are most commonly used for this purpose. However, this approach is expensive. Furthermore, nutritional profile systems have rarely allowed accurate identification at an infraspecies level (Schaad et al. , 2001).
Verniere et al. (1993) stated that the accuracy of identification based only on this technique is unreliable and other methods are required also.
1.5.1.3 Fatty acid methyl ester (FAME) analysis
Lipids, which comprise fatty acids, are a functional part of the bacterial cell. They have various structures among bacteria and are associated with the cytoplasmic membrane in
Chapter 1: Review of the literature 14
Gram-positive, and with the cytoplasmic and outer membrane in Gram-negative, bacteria. The detailed structure of bacterial lipids has been presented (Neidhardt et al. ,
1990). The diversity in both quality and quantity of bacterial fatty acids provides the basis for an accurate identification method. It should be noted that changes in nutrition, such as growing bacteria on different media, will change the composition of bacterial lipids, therefore, standardised growth conditions are needed for all bacteria under study
(Sasser, 1990). Gas chromatography of the methyl ester form of fatty acids, combined with computer-aided analysis of the profiles, is a rapid and accurate technique to identify and classify bacteria, even at the intraspecies level. Details of the analysis have been reviewed by several authors (Janse et al. , 1992; Komagata & Suzuki, 1987; Sasser,
1990). Stead (1992) described identification of bacteria by fatty acid analysis and recommended a protocol for this purpose. Based upon his results, xanthomonads have very distinct profiles containing as many as 50 fatty acids, many of which are branched chain acids, and all strains possess 12:0 3OH, 11:0 iso 3OH and 13:O iso 3OH.
Variation among the profiles of Xanthomonas strains is great enough to provide an accurate determination even at pathovar level, e.g. for X. campestris pv. oryzae and pv. oryzicola . This approach has been used in research on diagnosis and taxonomy of phytopathogenic xanthomonads (Chase et al., 1992; Stead, 1989; Yang et al. , 1993).
Vauterin et al . (1992) combined this technique with SDS-PAGE and DNA:DNA hybridisation to distinguish xanthomonads which infect members of the Poaceae. In a recent study, Janse et al. (2001) used FAME analysis in a polyphasic study of a new disease on strawberry to show that the unknown strains formed a homogeneous group, with respect to fatty acid analysis, different from X. fragariae , the causal agent of angular leaf spot disease. However, FAME is expensive and requires specialised
Chapter 1: Review of the literature 15 personnel. The analysis seems to be more useful for taxonomic studies rather than routine diagnosis (Stead, 1992).
1.5.1.4 Gel electrophoresis of whole cell proteins
Proteins comprise 55% of the dry mass in bacterial cells (Neidhardt et al. , 1990) and can be separated by electrophoretic techniques such as polyacrylamide gel electrophoresis (PAGE) of whole-cell soluble proteins to achieve a protein electrophoregram. Details of the approach have been published (Kersters, 1990;
Laemmli, 1970; Stead, 1992). The electrophoretic protein patterns can be used to assess similarity among strains at species and subspecies levels. Also, protein profiles combined with computer-aided analysis have potential in phylogenetic and taxonomic studies. In particular, this technique has provided valuable information for differentiation, identification and classification of xanthomonads (Janse et al. , 2001;
Mooter et al. , 1987a; Mooter et al. , 1987b; Vauterin et al. , 1991a; Vauterin et al. ,
1991b). A correlation between groupings obtained by DNA-DNA hybridisation and
PAGE has been found for some Xanthomonas strains (Mooter et al. , 1987a; Vauterin et al. , 1992; Vauterin et al. , 1990). However, protein profiling cannot be used routinely for identification of bacteria and as a detection tool because of the complexity of the profiles and because it is time consuming (Stead, 1992).
1.5.1.5 Serological techniques
The different formats of techniques such as Enzyme-Linked Immunosorbent Assay
(ELISA) and Immunofluorescence (IF) have been comprehensively reviewed (Hampton et al. , 1990). The success of any serological technique depends on the specificity and sensitivity of the antiserum and the methods used to amplify and observe the serological
Chapter 1: Review of the literature 16 reaction (Stead, 1992). These are sensitive, relatively specific and rapid tests to identify phytopathogenic bacteria, especially for large numbers of samples. Indeed, these approaches provide powerful tools for epidemiological studies and potentially offer the best methods for detection of bacteria in plant materials compared to other conventional methods (Stead, 1992). Although different formats of serology have been used for detection and identification of Xanthomonas strains (Alvarez & Lou, 1985; Duveiller &
Bragard, 1992), these techniques have some disadvantages. The detection threshold is moderately low, around 1 × 10 5 CFU mL -1 (Alvarez, 2001). In a comparison of ELISA and DIA (immunobinding assay) with isolation on medium and a PCR-based identification technique for detection of X. albilineans , a pathogen of sugarcane, Wang et al. (1999) found that the serological methods had the least sensitivity, at around 10 5-6
CFU mL -1. Furthermore, the quality of the antisera provided by various laboratories may differ. Some antisera, especially polyclonal antibodies, show cross-reactivity, consequently, the greater specificity of monoclonal antibodies is required for a reliable diagnosis (Schaad et al. , 2001). Selection and preparation of monoclonal antibodies also are time-consuming, expensive and require extensive training. Bouzar et al. (1994) reported that their selected monoclonal antibodies, although useful for identification of
X. campestris pv. vesicatoria , reacted with other pathovars and noted that the cross- reaction cannot be avoided for highly heterogeneous pathogens. Likewise, McDonald &
Wong (2001), using a monoclonal antibody, achieved a useful improvement in specificity of serological diagnosis of X. populi . However, this antibody could not distinguish between pv. populi and pv. salicis of the pathogen. Bragard & Verhoyen
(1993) also reported a sensitive identification of xanthomonads pathogenic on small grains with monoclonal antibodies compared to polyclonal antisera, however, monoclonal antibodies could not distinguish between different pathovars.
Chapter 1: Review of the literature 17
1.5.2 DNA-based molecular techniques
Molecular biology and DNA-based techniques have brought about a revolution in the biological sciences. Advances in DNA-based methods, especially the discovery of the polymerase chain reaction (PCR), combined with computer-based analyses, have enabled researchers to analyse genetic diversity in bacterial populations and investigate evolutionary and taxonomic questions. Likewise, this new knowledge has proved valuable in identification and detection of phytopathogenic bacteria by providing powerful DNA markers for quick and precise identification, even at the strain level.
These markers have several advantages compared to phenotypic markers, in that they are often more sensitive, rapid and specific even at the intraspecies levels (Henson &
French, 1993).
This section presents a brief overview of the DNA-based methods currently used in bacteriology, with particular reference to phytopathogenic xanthomonads. First, techniques that are used to investigate genetic diversity among phytobacterial populations are reviewed. Second, the methods used to develop DNA-based markers for the detection of phytopathogenic bacteria and to identify unknown strains are discussed.
Two categories of approaches can be identified based upon whether the technique is
PCR-based or not.
Chapter 1: Review of the literature 18
1.5.2.1 PCR-independent methods
1.5.2.1.1 DNA-DNA hybridisation
The genetic similarity between a type strain and an unknown strain is measured using the re-association of labelled single-stranded DNA from the type strain and single- stranded DNA from the unknown isolate. A 70% value of DNA-DNA homology is considered as the species limit, and greater values are accepted as intraspecies groups
(Murray et al. , 1990). In one of the earliest experiments, Murata & Starr (1973) used this technique to characterise Xanthomonas strains. Their study showed that DNA-DNA homology of up to 50% was characteristic for the strains studied. In another study,
Vauterin et al. (1992) reported that the different strains of Xanthomonas could be divided to five groups based on DNA-DNA homology, and proposed the "translucens group" for xanthomonads pathogenic on cereals and grasses. Furthermore, in a comprehensive study, Vauterin et al. (1995) performed DNA-DNA hybridisation for
183 strains of the genus Xanthomonas and identified 20 DNA homology groups, meaning 20 genomic species. Apart from DNA homology group 9, strains of which had an average homology of 77%, other groups had internal DNA homology values greater than 80%. Also, similarity values between groups were below 40%. Based on the report of the ad hoc committee for the re-evaluation of the species definition in bacteriology
(Stackebrandt et al. , 2002), DNA-DNA homology must be considered as a molecular criterion for species delineation and the technique has been used to provide an accurate taxonomic analysis in several studies (Janse et al. , 2001; Jones et al. , 2000; Palleroni et al. , 1993; Roumagnac et al. , 2004). However, DNA–DNA homology cannot be used for identification because it provides only similarities among strains and cannot describe individual strains. Furthermore, it lacks the necessary sensitivity to recognise close relationships between strains (Vauterin et al. , 2000). Also, it clearly is impractical for
Chapter 1: Review of the literature 19 analysis of large collections of isolates due to experimental limitations such as the need for high quality and quantity of DNA, and specialised equipment (Rademaker et al. ,
2000).
1.5.2.1.2 Restriction endonuclease analysis
Restriction endonucleases are enzymes that recognise short, specific sequences within
DNA molecules and digest the DNA at those sites. Closely related strains may yield fragments of very similar size generated by enzyme digestion, which can then be analysed after separation by polyacrylamide or agarose gel electrophoresis. As an example, Hartung & Civerolo (1987) compared Asian and South American strains of X. c. pv. citri and recognised different geographical groups among the strains. The complexity of the bands due to the large number of fragments generated by the enzymes limits the usefulness of this technique as a routine technique for identification. Rare- cutting restriction enzymes, for obtaining long DNA fragments, in conjunction with pulsed-field gel electrophoresis (PFGE) for separation of large DNA fragments (Chu et al. , 1986) has been shown to be a useful tool for discrimination between closely related strains and has been applied to xanthomonads (Davis et al. , 1997; Quezado-Duval et al. ,
2004; Roberts et al. , 1998). Despite equally good reliability of PFGE and some PCR- based techniques such as AFLP and rep-PCR, PCR-based techniques are more suitable than PFGE and the greater discriminative ability of these methods makes them the preferred methods for genetic diversity studies (Weigel et al. , 2004).
1.5.2.1.3 Restriction fragment length polymorphism analysis (RFLP)
Restriction fragment length polymorphism analysis is an important tool in the study of genetic diversity and diagnosis of phytopathogenic bacteria. Total genomic DNA
Chapter 1: Review of the literature 20 extracted from a culture of a bacterium is subjected to enzyme digestion and the products are resolved on an agarose gel. The separated fragments are transferred to a membrane by Southern blotting (Sambrook et al. , 1989), then hybridised with a radiolabelled probe, which is then detected by autoradiography, or a probe labelled with a chemical such as digoxygenin, which is detected by an immuno-enzyme reaction.
Finally, the visible banding patterns are analysed to distinguish strains or identify isolates. Several authors have studied the use of RFLP for identification of phytopathogenic xanthomonads. Lazo et al. (1987) demonstrated pathovar-specific patterns for the pathovars of X. campestris by means of appropriate probe and restriction enzyme combinations. Alizadeh et al. (1997) also used this method with two pathogenicity-related probes to study variation among Iranian strains of X. campestris from cereals and grasses. The Iranian strains showed three distinct RFLP groups and the authors suggested that RFLP analysis was a useful tool to distinguish the strains tested.
Although this technique is useful for identifying pure cultures of phytopathogenic bacteria and has potential for genetic and taxonomic studies, it requires large quantities of good quality DNA, is time-consuming, generally involves the use of radioactivity and is not appropriate for the detection of pathogens in plant materials (Duveiller et al. ,
1997b). Furthermore, detection of RFLPs by Southern blotting is laborious, so it is unlikely to be used as a routine method for the detection of pathogens.
1.5.2.2 PCR-based techniques
PCR allows a specific target sequence of DNA to be amplified a billion fold in several hours (Innis, 1990). The advantages of PCR-based techniques are speed, throughput and flexibility in quantity and quality of the input DNA. Currently, most techniques for genetic diversity and identification studies are based on the use of both restriction
Chapter 1: Review of the literature 21 enzymes and PCR, and several versions have been designed for routine studies. The various formats are as follows:
1.5.2.2.1 Multiple arbitrary primed PCR (MAAP)
An arbitrary DNA sequence is used as the primer, therefore, the result of PCR is the amplification of many discrete DNA products. Arbitrary primed PCR (AP-PCR) (Welsh
& McClelland, 1990), DNA amplification fingerprinting (DAF) (Caetano-Anolles et al. ,
1991) and random amplified polymorphic DNA (RAPD) (Williams et al. , 1990) are versions of the technique. The latter has been use for fingerprinting of Xanthomonas strains in several studies. Trebaol et al. (2001) employed RAPD to assess the genetic diversity among X. cynarae , which is pathogenic on artichoke, and to identify molecular markers characteristic of the species. In a recent study, RAPD was used to develop a
PCR–based detection method for X. campestris pv . carotae , causal agent of bacterial leaf blight in carrot (Meng et al. , 2004). Louws et al. (1999) stated that RAPD is useful to assess bacterial diversity at the strain and subspecies levels and less useful at the species level. The advantage of RAPD is that there is no requirement for DNA probes or sequence information for primer design. In addition, they do not require blotting or hybridising steps, and are quick, simple, efficient and require small amounts of DNA.
However, it is necessary to screen many primers to achieve the desired results (Louws et al. , 1999) and the reproducibility of the technique has been questioned.
1.5.2.2.2 Amplified fragment length polymorphism analysis (AFLP)
This method, introduced by Vos et al . (1995), has combined the power of RFLP with the flexibility of PCR by ligating primer-recognition sequences (adaptors) to the restricted DNA. Rademaker et al. (2000) compared this technique and rep-PCR (see below) with DNA-DNA hybridisation using Xanthomonas as a model and proposed that
Chapter 1: Review of the literature 22
AFLP and rep-PCR could be used as highly effective methods for phylogenetic and taxonomic studies. Restrepo et al . (2000) showed that this technique was more robust than RFLP to assess genetic diversity of X. axonopodis pv . manihotis in Colombia .
Louws et al. (1999) suggested that AFLP is useful to assess diversity at the species, subspecies and strain levels. However, it is more technically demanding than RAPD analysis, and requires access to expensive equipment.
1.5.2.2.3 Repetitive sequence-based PCR
Primers are used which correspond to specific conserved repetitive sequences distributed throughout the genome of diverse bacteria. Repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences (Versalovic et al. , 1991), and BOX elements (Martin et al. , 1992) are three such sequences which have been studied in detail (de Bruijn, 1992; Louws et al. , 1994).
Having performed a thorough review of PCR-based genomic analysis, Louws et al.
(1999) compared a variety of these techniques, and considered that rep-PCR is more valuable than the other formats for diversity assessment for the following reasons: it is useful to assess diversity at the strain, subspecies and species levels; it is more amenable to whole-cell PCR analysis; it can tolerate a wide range of DNA concentrations; it requires a single set of primers and it uses a unified protocol. This technique is increasingly being applied to study genetic diversity among Xanthomonas strains
(Louws et al. , 1994; Massomo et al. , 2003; Mkandawire et al. , 2004; Restrepo et al. ,
2000; Scortichini et al. , 2001). Also, rep-PCR genomic fingerprint analysis appears to be useful for identification purposes in which the genomic fingerprints of the unknown isolates and a known strain can be compared visually or by computer-assisted pattern analysis for more precise identification. Indeed, the fingerprint of the unknown strains
Chapter 1: Review of the literature 23 can be compared with a database to find a perfect or a near identical match between the unknown and the known strains. Louws et al. (1994), by the use of this technique, were able to identify specific genomic fingerprints for phytopathogenic xanthomonads.
However, it is less useful for routine detection of bacterial contaminants in plant materials because the amplification reaction may be inhibited in tests involving plant material or soil due to the inhibition of polymerase activity (Duveiller et al. , 1997b).
Also, identification of a unique bacterial profile from a mixed sample such as soil or plant is not possible.
1.5.2.3 PCR-based detection techniques
For “detection”, the presence or absence of a target pathogen within a sample is determined (Shurtleff & Averre, 1997). PCR-based techniques have provided rapid and efficient tools for this purpose. The flexibility in quantity and quality of input sample is a major advantage of these techniques. Genomic DNA, whole cells and even a mixture containing target cells can be used directly in the reaction mixture and many target fragments can be rapidly generated. To develop an efficient detection tool, in brief, the
DNA of the target bacterium is isolated and a fragment specific to that pathogen is identified. The sequence of this specific fragment is used to design PCR primers, then an assay is developed which can amplify part or all of that sequence and subsequently can detect the pathogen within a sample. Various components of DNA have been used as an amplification target, as follows.
1.5.2.3.1 Plasmid DNA . Plasmid DNA has been used to design a rapid and sensitive detection method for phytopathogenic xanthomonads (Audy et al. , 1994; Hartung et al. ,
Chapter 1: Review of the literature 24
1993; Hartung et al. , 1996; Verdier et al. , 1998). The stability of the plasmid may be a concern in such detection assays (Louws et al. , 1999).
1.5.2.3.2 Pathogenicity genes. Leite et al. (1994) developed a method for detecting some plant pathogenic strains of X. c. pv . vesicatoria based on hrp sequences, the pathogenicity genes. However, these hrp -specific primers did not amplify genomic
DNA from some phytopathogenic xanthomonads, including X. c. pv . translucens and pv. secalis . Maes et al. (1996b) reported that hrp genes of xanthomonads pathogenic on the Poaceae are of a different type and are not detected with the hrp -specific primers described by Leite et al. (1994). The target pathogenicity gene may be associated with a plasmid-encoded gene. For an example, Verdier et al. (1998) developed primers for
PCR detection of X. axonopodis pv . manihotis based on one fragment of its plasmid which carries the pathogenicity gene as well. Their assay was able to detect pathogenic strains only.
1.5.2.3.3 Specific sequence of unknown function. Anonymous regions of DNA based on RAPD fragments can be used as amplification targets (Manulis et al. , 1994; Meng et al. , 2004; Pooler et al. , 1996; Sulzinski et al. , 1996; Trebaol et al. , 2001). An uncharacterised region of the genome, which comprises a cloned RAPD fragment, is termed a "sequence characterised amplified region" (SCAR) (Paran & Michelmore,
1993). Sulzinski et al. (1996), using a cloned fragment obtained by rep-PCR, designed primers specific for X. campestris pv . pelargonii . Wang et al. (1999) designed specific primers for the detection of X. albilineans based on anonymous sequences of a PCR fragment amplified and cloned from genomic DNA of the pathogen. Such targets are useful when little is known about the molecular biology of the strains under study, because RAPD and rep-PCR techniques do not require prior information about the
Chapter 1: Review of the literature 25
DNA sequences of the strains in question. However, the unknown nature of the fragment may be a concern in the reproducibility of the assay.
1.5.2.3.4 rDNA genes. The ribosomal DNA (rDNA) gene region is controlled by strong evolutionary and functional pressures and includes highly conserved regions containing
16S, Intergenic Transcribed Spacer (ITS) which often includes tRNA, 23S and 5S components (Fox et al. , 1977; Gurtler & Stanisich, 1996). Although the 16S rDNA sequence has proved useful for the delineation of phytopathogenic bacteria at the genus level (DeParasis & Roth, 1990), more variability has been found in the 16S-23S rDNA region, which facilitates the development of species-specific primers (Barry et al. ,
1991). In particular, phylogenetic analysis of the 16S and 16S-23S rDNA sequences of
Xanthomonas species has revealed more variation in the ITS fragment than in the 16S rDNA (Goncalves & Rosato, 2002; Hauben et al. , 1997) and this sequence has been targeted to design PCR primers specific for various Xanthomonas species (Adachi &
Oku, 2000; Honeycutt et al. , 1995; Maes et al. , 1996b; Pan et al. , 1997; Pan et al. ,
1999).
1.6 Summary and objectives
The genus Xanthomonas includes more pathogens than all other genera of phytopathogenic bacteria combined. Consequently, the detection and identification of plant pathogenic Xanthomonas strains has been the subject of many studies. Traditional methods rely on isolating the strains of interest in pure culture and performing determinative physiological and biochemical tests, many of which are laborious and time-consuming. Indeed, these tests may not permit accurate identification at an intraspecies or even species level. Recently approaches such as protein profile and fatty
Chapter 1: Review of the literature 26 acid analyses have provided more accurate identification. However, isolation and purification of the bacteria are still required. Therefore, these tests are not always suitable for routine use. Serological tests have excellent potential for detection of bacteria in plant samples compared to the other conventional methods, but their development and use can be limited. Antibodies may show cross-reaction with other bacteria and may be unable to differentiate specific strains or pathovars.
DNA-based techniques, especially PCR-based methods, have provided researchers with the knowledge to analyse genetic diversity in bacterial populations and, indeed, with rapid and efficient tools to detect and identify phytobacteria. These methods tend to be less time-consuming and more reproducible than conventional methods and some are useful for determining phylogenetic relationship among strains. Currently, most techniques for studies of genetic diversity and identity are based on the combination of restriction enzymes and PCR procedures. Genomic fingerprint methods such as RAPD,
AFLP and rep-PCR are considered to be accurate approaches to assess genetic diversity in bacterial populations. Furthermore, different regions of the bacterial genome, such as pathogenicity genes, plasmid DNA, rDNA fragments including the tDNA region, and anonymous DNA fragments, have been used as amplification targets for the design of specific primers for efficient and rapid detection of phytobacteria by PCR.
Symptoms of pistachio dieback were first observed in Australia in the early 1990s and bacteria associated with the disease have been identified by preliminary studies as
Xanthomonas sp. However, knowledge of genetic diversity of the pathogen was lacking, the exact characterisation of the pathogen and its taxonomic position were unknown and there was no efficient, reliable and rapid tool to detect and to recognise the pathogen in planting materials. Definitive identification of the pathogen, the evaluation of genetic diversity within its population and development of an efficient and rapid method for
Chapter 1: Review of the literature 27 detection of the pathogen would assist in elucidation of epidemiology and the development of improved management strategies.
The aims of the research described in this thesis were to (i) assess genetic diversity of strains belonging to the genus Xanthomonas occurring in pistachio trees diagnosed with dieback in Australia (ii) conduct a comprehensive polyphasic study, using both DNA- independent and DNA-based techniques, to characterise and identify the pathogen and
(iii) develop a PCR-based protocol for the detection of the pathogen.
Chapter 2: General materials and methods 28
Chapter 2: General materials and methods
2.1 Collection of xanthomonad strains associated with pistachio dieback
Symptoms of pistachio dieback have been observed in four regions of Australia, namely
Renmark in South Australia; Kyalite in New South Wales; and Red Cliffs and
Robinvale in Victoria (Figure 2.1). Thirty strains of the pathogen, isolated from diseased pistachio trees at the four locations in 2000 as part of “Pistachio canker epidemiology”, Project NT99004, Horticulture Australia Limited, were used initially.
These strains had been stored at -70 °C at the Waite Campus, University of Adelaide.
Figure 2.1 Geographical locations of major pistachio orchards and also pistachio dieback in Australia., NT: Northern Territory, NSW: New South Wales, Qld:
Queensland, SA: South Australia, T: Tasmania, V: Victoria and WA: Western
Australia.
* from left to right: Renmark, Red Cliffs, Robinvale and Kyalite.
Chapter 2: General materials and methods 29
To collect more strains for various tests and also to assess possible variation within the pathogen population over several years, another sampling was performed in 2003. For this, two orchards were selected in the seriously affected pistachio-growing areas,
Kyalite and Robinvale. Between 15-20 trees, male and female, with dieback were sampled and five 2-year-old shoots with internal staining were collected from each tree.
To avoid cross-contamination, secateurs were sterilised with alcohol (70%) after each cut. Samples were placed in individually zipped plastic bags and kept cool until processing at the Waite Campus, University of Adelaide.
In the laboratory, shoots were rinsed with tap water, dipped into 70% ethanol for 50-60 s and then rinsed three times in sterile distilled water. Segments of 4 × 4 mm were cut from the woody tissue of each sample. The segments were soaked in a few drops of
0.85% sterile NaCl solution for 15 min at room temperature (approximately 22 °C). Two loopfuls of the resulting suspension were streaked on nutrient agar (NA) and yeast dextrose carbonate (YDC) agar (Schaad et al. , 2001; Appendix B). Plates were incubated at 28 °C and inspected for pale yellowish colonies from 2 days onwards, then
35 such colonies were purified on YDC. Strains that had been collected in 2000 and stored at -70 °C, were revived on YDC at 28 °C. The pathogen strains utilised in this study are shown in Table 2.1.
2.2 Collection of other phytobacteria occurring in and around pistachio orchards
To assess the specificity of DNA-based detection methods for the pathogen, other bacteria occurring naturally in and around pistachio orchards were collected. Three orchards were selected, one apparently healthy and one infected orchard in Kyalite and
Chapter 2: General materials and methods 30
Table 2.1 List of Xanthomonas strains isolated from pistachio trees and used in this study
Xanthomonas strain Location of origin a Year isolated Source
2, 3, 4, 5, 6, 7, 8, 9, 10, 20 Kyalite, NSW 2000 NT99004 b
21, 22, 23, 24, 25, 26, 27, 36 Robinvale, Vic 2000 NT99004
11, 13, 14, 15, 16, Red Cliffs, Vic 2000 NT99004
28, 29, 30, 31, 32, 33, 34 Renmark, SA 2000 NT99004
72, 73, 74, 75, 76, 77, 78 Kyalite, NSW 2003 This study
44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71 Robinvale, Vic 2003 This study
a NSW, New South Wales. Vic, Victoria. SA, South Australia b Pistachio canker epidemiology, Project NT99004, Horticulture Australia Limited
one infected orchard in Robinvale. Twenty trees, comprising both female and male, were selected in each orchard in Kyalite and 36 trees in the orchard in Robinvale.
Samples of leaf, shoot, bud and bark tissue were collected from apparently healthy and diseased pistachio trees.
To collect grasses and weeds from the pistachio orchard floor, 10 rows of trees were selected and five samples were collected in each row from all plants at intervals of three trees. Five samples were collected from plants other than grasses or pistachio in the vicinity of each orchard. To account for possible variation in the population of bacteria,
Chapter 2: General materials and methods 31 collections were performed three times, in the autumn (May), winter (August) and summer (December) 2003. Plant material was shaken in 200 mL sterile water containing a drop of Tween 20 wetting agent on a shaker for 20 min, then 100 µL of the suspension was streaked on each of NA, nutrient agar plus 5% (w/v) sucrose (NAS) and
King’s B medium (Schaad et al. , 2001; Appendix B). All plates were incubated at 25°C for 72 h and 191 representative bacteria with diverse morphological characteristics were selected.
2.3 Collection of type and reference strains of Xanthomonas
Thirty reference strains of the genus, including type strains, were obtained from the
Belgium Coordinated Collection of Microorganisms, Laboratory of Microbiology,
University of Ghent, Belgium (BCCM/LMG), under an Australian Quarantine and
Inspection Service (AQIS) permit (200309826). X. albilineans ACM 1733 was obtained from the Australian Collection of Microorganisms (ACM), University of Queensland,
Brisbane, Qld, Australia. X. translucens pv . translucens DAR 35705 from wheat, X. axonopodis pv . malvacearum DAR 26904 and two strains of X. arboricola pv. pruni ,
DAR 64858 and DAR 41287, from stone fruits, all isolated in Australia, were obtained from the Australian Collection of Plant Pathogenic Bacteria (ACPPB), Agricultural
Institute, Orange, NSW, Australia. Bacteria imported from Belgium were revived on
YDC at 28 °C in a PC3 facility at the Institute of Medical and Veterinary Science
(IMVS), Adelaide, South Australia. Purified DNA (see section 2.6) and denatured proteins (see section 4.2.3) extracted from these bacteria were transferred to a PC2 laboratory at the Waite Campus, University of Adelaide. Reference strains of the genus, including type strains, used in this study are shown in Table 2.2.
Chapter 2: General materials and methods 32
Table 2.2 List of type and reference strains of Xanthomonas used in this study and their sources
Strains Accession number Source Xanthomonas translucens pv. poae LMG 728 BCCM a X. translucens pv . phleipratensis LMG 843 BCCM X. translucens pv . hordei LMG 737 BCCM X. translucens pv . phlei LMG 730 BCCM X. translucens pv . undulosa LMG 892 BCCM X. translucens pv . translucens LMG 876 BCCM X. translucens pv . cerealis LMG 679 BCCM X. translucens pv . secalis LMG 883 BCCM X. translucens pv . graminis LMG 726 BCCM X. translucens pv . arrhenatheri LMG 727 BCCM X. pisi LMG 847 BCCM X. vasicola pv . holcicola LMG 936 BCCM X. campestris pv . campestris LMG 568 BCCM X. arboricola pv . corylina LMG 689 BCCM X. arboricola pv . juglandis LMG 747 BCCM X. arboricola pv . pruni LMG 852 BCCM X. hortorum pv . hederae LMG 733 BCCM X. oryzae pv . oryzae LMG 5047 BCCM X. theicola LMG 8684 BCCM X. fragariae LMG 708 BCCM X. cassavae LMG 673 BCCM X. axonopodis pv . axonopodis LMG 982 BCCM X. codiaei LMG 8678 BCCM X. sacchari LMG 471 BCCM X. bromi LMG 947 BCCM X. hyacinthi LMG 739 BCCM X. melonis LMG 8670 BCCM X. cucurbitae LMG 690 BCCM X. vesicatoria LMG 911 BCCM
Chapter 2: General materials and methods 33
X. sp . pv . mangiferaeindicae LMG 941 BCCM X. albilineans ACM 1733 ACM b X. translucens pv . translucens DAR 35705 ACPPB c X. arboricola pv . pruni DAR 64858 ACPPB X. axonopodis pv . malvacearum DAR 26904 ACPPB
a BCCM: Belgium Coordinated Collection of Microorganisms, Laboratory of
Microbiology, University of Ghent, Belgium b ACM: Australian Collection of Microorganisms, University of Queensland, Brisbane,
Qld, Australia c ACPPB: Australian Collection of Plant Pathogenic Bacteria, Agricultural Institute,
Orange, NSW, Australia
2.4 Propagation and maintenance of bacteria
All cultures were grown on NA and YDC at 28 °C for 3 days for use in subsequent tests, unless otherwise stated. Cultures for regular use were sub-cultured every 3-4 weeks and kept at 4 °C. All strains were stored in sterile distilled water at room temperature for short term storage and at -70 °C in nutrient broth containing 30% glycerol for long term storage (Schaad et al. , 2001).
2.5 Collection of seeds and other plant materials for pathogenicity tests
The pistachio dieback pathogen was tested for pathogenicity in two series of tests on plants from the Anacardiaceae and the Poaceae. The Anacardiaceae species used were
Chapter 2: General materials and methods 34
Pistacia vera, P. vera cv . Sirora, P. terebinthus, P. atlantica, P. integerrima, P. chinensis, P. lentiscus, P. palaestina, Rhus leptodictya, R. tripartita, Schinus latifolius,
S. lentiscifolius , S. polygamus , Mangifera indica and Anacardium occidentale . Excised shoots of mature P. terebinthus , 15-20 cm in length, were obtained from CSIRO Plant
Industry, Merbein, Victoria, Australia. Excised shoots of P. vera cv . Sirora, grafted on
P. terebinthus , and P. integerrima were collected from seedlings, 2-3 years old, obtained from Sunraysia Nurseries, Gol Gol, NSW, Australia. Seeds of M. indica and A. occidentale were obtained from the Department of Business, Industry and Resource
Development, Darwin, NT, Australia. Excised shoots of other plants were collected from the current season growth in the Waite Arboretum, The University of Adelaide,
SA.
The poaceous plants used for pathogenicity testing were bread wheat ( Triticum aestivum cv. Frame), durum wheat ( T. durum cv. Arrivato), barley ( Hordeum vulgare cv. Sloop), rye ( Secale cereale ), oat ( Avena sativa cv. Echidna), brome grass ( Bromus inermis ), triticale ( X Triticosecale cv. Tickit), timothy ( Phleum pratense ), cocksfoot ( Dactylis glomerata ), barley grass ( Hordeum leporinum ) and perennial rye grass ( Lolium perenne cv. Victorian). Except for rye grass seed, which was obtained from a pistachio grower at
Kyalite, NSW, seeds were obtained from the South Australian Research and
Development Institute (SARDI), Waite Campus, SA.
2.6 DNA extraction from bacteria
Good quality DNA is a precondition for the application of molecular techniques and it is essential to have DNA that is not contaminated with inhibitors of modifying enzymes or polymerases. To determine the most appropriate method for extracting sufficient
Chapter 2: General materials and methods 35 good quality DNA from the Xanthomonas bacteria, which often produce excessive amounts of polysaccharides, the protocols of Pitcher et al. (1989) as modified by
Rademaker & de Bruijn (1997), Maes et al. (1996b) and Ausubel et al. (1994) were compared. DNA was extracted from two strains from pistachio, 4 and 13, X. arboricola pv. pruni DAR 64858, X. axonopodis pv. malvacearum DAR 26904 and X. translucens
DAR 35705 . The extractions were repeated three times and the quantity and quality of the DNA was assessed following gel electrophoresis. The protocol of Rademaker & de
Bruijn (1997) produced the best quality DNA and using this protocol, DNA was extracted from all strains listed in Tables 2.1 and 2.2, as well as other phytobacteria isolated from plants in and around pistachio orchards. Bacteria were first grown on tryptone soy agar (TSA) (Appendix B) at 28 °C for 48 h. Genomic DNA was extracted following the method of Rademaker & de Bruijn (1997) as follows: cells were removed using a sterile 10 µL disposable loop and re-suspended in 750 µL re-suspension buffer
(RB) (Appendix A) in a sterile 2 mL micro-centrifuge tube. The suspension was centrifuged at 5,000 rpm for 10 min and the supernatant was removed using a 1,000 µL tip. The tube was centrifuged again and the remaining suspension was removed using a
200 µL tip. TE, 100 µL, (Appendix A) was added and mixed using a pipette. GES, 500
µL, (Appendix A) was added and the tube mixed gently. After incubation on ice for 5 min, 250 µL cold (-20 °C) ammonium acetate (7.5 M) was added and the tube was mixed by shaking gently and incubated on ice for 5 min. Chloroform: iso -amyl-alcohol
(24:1 v/v), 500 µL, was added and the tube was shaken vigorously until the solution was homogeneously milky. The mixture was centrifuged at 14,000 rpm for 10 min or until the upper phase was clear. The upper phase (700 µL), DNA-solution, was removed carefully using a 1000 µL tip and added to a tube containing 378 µL iso-propanol (-
20 °C). The mixture was shaken gently until a white cloud of precipitated DNA became
Chapter 2: General materials and methods 36 visible. The DNA was pelleted by centrifugation at 14,000 rpm for 5 min and the supernatant was removed using a 1,000 µL tip. The pellet was washed with 150 µL 70
% ethanol and recentrifuged briefly. Ethanol was removed with a 200 µL pipette. The
DNA pellet was air-dried, 200 µL TE was added and the tube was incubated at 4 °C overnight to dissolve the DNA. RNAse (250 µg mL -1), 25 µL, was added and mixed gently. After incubation at 37°C for 1 h, the DNA concentration was determined using a
-1 -1 spectrophotometer at 260 nm (1 OD 260 = 50 g mL ) and adjusted to 50 ng µL . All
DNA samples were stored at -20 °C.
Chapter 3: Genetic diversity within the pistachio dieback pathogen 37
Chapter 3: Genetic diversity within the pistachio dieback pathogen
3.1 Introduction
In preliminary studies, Xanthomonas strains similar to X. translucens were identified as the causal agent of dieback of pistachio in Australia and two groups were identified based on BOX-PCR fingerprinting of a small number of the pathogen strains (Facelli et al. , 2002; Facelli et al. , 2005). However, little was known about the genetic diversity of the pathogen. It was necessary to determine diversity before a formal bacterial identification could be made.
Historically, DNA-independent methods such as physiological and biochemical tests and protein profiling (see section1.5.1) have been used for characterisation and study of phenotypic variation among xanthomonads (Griffin et al. , 1991; Vauterin et al. , 1991a;
Vauterin et al. , 1991b; Verniere et al. , 1993). Techniques that assess variation in genomic DNA (see section 1.5.2) have since provided more reliable, reproducible and robust tools to evaluate variability (Louws et al. , 1999). DNA fingerprinting based on the presence of repetitive elements dispersed in bacterial genomes (rep-PCR) (Louws et al. , 1994) (see section 1.5.2.2.3) has proved a sensitive and reliable technique to assess diversity of xanthomonads at the species, subspecies and strain levels (Gent et al. , 2004;
Lopes et al. , 2001; Louws et al. , 1995; Mkandawire et al. , 2004; Scortichini & Rossi,
2003; Scortichini et al. , 2001; Scortichini et al. , 2002). The evaluation of genetic diversity within a plant pathogen population improves the understanding of its taxonomy, epidemiology and diagnosis (Milgroom & Fry, 1997).
Chapter 3: Genetic diversity within the pistachio dieback pathogen 38
The work reported in this chapter aimed to assess the genetic diversity of xanthomonads isolated from infected pistachio trees in different geographic regions of Australia over several years using rep-PCR.
3.2 Materials and methods
Pistachio pathogen strains isolated in 2000 were compared using rep-PCR and based on the results, representative strains were selected and compared with the strains isolated in
2003. Sterile distilled water was used as a negative control. Because of the similarity of pistachio strains to X. translucens in preliminary studies (Facelli et al . 2002), X. translucens pv. translucens DAR 35705, the only available reference strain of the species at that time, was used for comparison. Strains were grown on TSA (Appendix
B) at 28 °C for 48 h and genomic DNA was extracted following the method of
Rademaker & de Bruijn (1997) (see section 2.6). The protocol of Rademaker & de
Bruijn (1997) was used for rep-PCR experiments. All reactions were performed using a
Peltier Thermal Cycler model PTC-200 (MJ Research Inc., CA, USA). To check the reproducibility of rep-PCR fingerprints, DNA extraction, rep-PCR and gel analysis were performed three times. For buffers and reagents used see Appendix A.
3.2.1 Experimental protocol for rep-PCR fingerprinting
Three rep-PCR protocols, BOX-PCR, REP-PCR and ERIC-PCR, were used
(Rademaker & de Bruijn, 1997). All strains were subjected to BOX-PCR using the
BOX A1R primer, REP-PCR using the REP 1R and REP 2I primers, and ERIC-PCR using the ERIC 1R and ERIC 2 primers (Table 3.1).
Chapter 3: Genetic diversity within the pistachio dieback pathogen 39
Table 3.1 Primers used in the rep-PCR fingerprinting (Rademaker & de Bruijn, 1997)
BOX A1R 5'- CTA CGG CAA GGC GAC GCT GAC G -3'
REP 1R 5'- III ICG ICG ICA TCI GGC -3'
REP 2I 5'- ICG ICT TAT CIG GCC TAC -3'
ERIC 1R 5'- ATG TAA GCT CCT GGG GAT TCA C -3'
ERIC 2 5'- AAG TAA GTG ACT GGG GTG AGC G -3'
The primers were synthesised by Proligo Pty Ltd, Lismore, Australia. The “master mix” for each PCR protocol was prepared as described in Table 3.2. Sample DNA (1 µL) was added to 24 µL of reaction mixture in 0.2 mL thin-walled tubes and PCR amplification was performed using the program given in Table 3.3.
Table 3.2 Composition of the master mix used in rep-PCR (Rademaker & de Bruijn,
1997)
Stock solution Volume (µL) in 25 µL reaction mix
5 × Gitschier Buffer 5 µL
BSA (20 mg mL -1) 0.2 µL
DMSO, 100% 2.5 µL
MQ water, use 13.65 µL for BOX 12.65 µL
dNTP mix (25 mM) 1.25 µL
Primer 1 (0.3 µg µL -1) 1 µL
Primer 2 (0.3 µg µL -1) (not applicable for BOX) 1 µL
Taq DNA polymerase (5 U µL-1, Qiagen) 0.4 µL
Chapter 3: Genetic diversity within the pistachio dieback pathogen 40
Table 3.3 Amplification program for rep-PCR experiments (Rademaker & de Bruijn,
1997)
Program step BOX-PCR ERIC-PCR REP-PCR
1 Taq polymerase activation 95°C for 15 min 95°C for 15 min 95°C for 15 min and initial denaturation 2 30 cycles of:
Denaturation 94°C for 1 min 94°C for 1 min 94°C for 1 min
Annealing 53°C for 1 min 52°C for 1 min 40°C for 1 min
Elongation 65°C for 8 min 65°C for 8 min 65°C for 8 min
3 Final extension 65°C for 16 min 65°C for 16 min 65°C for 16 min
The PCR product, 6 µL, was separated on an agarose gel (1.5%) in a 16 × 35 cm horizontal gel electrophoresis apparatus using 1 × TAE buffer at 70 V constant voltage and at 4 oC for 17 h. Gel was stained with ethidium bromide (0.6 µg mL -1), visualised on a UV transilluminator and photographed with a black and white video camera (SSC-
M370CE, Sony, Japan).
3.3 Data analysis
Captured photographs were subjected to the Gel-Pro Analyzer (Media Cybernetics,
MD, USA) computer program. The banding patterns were determined and DNA fragments which gave an intense band in all three replicates were included in the analysis. Minor bands were scored only when another strain showed an intense band of corresponding molecular weight. Bands were scored in binary form, 1 and 0, indicating the presence and absence of a band, respectively. A similarity matrix was obtained using the Jaccard coefficient and the software package NTSYS-pc (version 2.02K,
Applied Biostatistics, Inc., NY, USA). To determine the relationship among the strains,
Chapter 3: Genetic diversity within the pistachio dieback pathogen 41 cluster analysis was performed with UPGMA (unweighted pair-group method, using arithmetic averages) in the SAHN program of the NTSYS-pc software. Dendrograms were generated for the BOX, REP and ERIC binary matrixes individually and for the three experiments combined (Rademaker et al. , 2000).
3.4 Results
PCR using the BOX primer gave genomic fingerprints with 32 reproducible bands, ranging from about 270 to 3700 bp. This protocol revealed two genotypes within the pathogen. Group A included strains from three areas, Kyalite, Renmark and Red Cliffs, and group B comprised identical strains obtained from Robinvale only. Based on BOX-
PCR there was some variation within group A, separating strains of this group into two subgroups (Figures 3.1 and 3.2). In this experiment, the similarity value, based on
UPGMA, between the two main clusters was about 0.17 (Figure 3.3).
PCR with ERIC primers produced genomic fingerprints with 23 reproducible bands, ranging from about 200 to 5000 bp. This protocol also revealed two genotypes among strains which matched the groups A and B indicated by BOX-PCR (Figures 3.4 and
3.5). ERIC primers also revealed variation within group A, separating strains of this group into two subgroups. Using this protocol, the similarity value, based on UPGMA, between the two main clusters was about 0.26 (Figure 3.6).
PCR using the REP primers gave genomic fingerprints with 29 reproducible bands, ranging from 900 to 5300 bp. This protocol also separated the strains into the groups A and B (Figures 3.7 and 3.8). However, REP-PCR did not define additional genotypes of the pathogen. Based upon REP-PCR fingerprints, the similarity value, based on
UPGMA, between the two main clusters was about 0.14 (Figure 3.9).
Chapter 3: Genetic diversity within the pistachio dieback pathogen 42
Pistachio strains, belonging to both groups, differed from X. translucens pv. translucens
DAR 35705 based on fingerprints generated by all three rep-PCR protocols. There was no difference in profiles of the pathogen strains collected several years apart. A combined binary matrix, prepared from 84 bands generated by the three PCR protocols
(BER-PCR), revealed two main clusters among the pathogen strains that corresponded to group A and group B and four sub-clusters in group A. The similarity value, based on
UPGMA, between the two main clusters was about 0.18 (Figures 3.10 and 3.11).
3.5 Discussion
Genetic diversity was found among 65 Xanthomonas strains isolated from pistachio trees in Australia. The findings confirm and extend the preliminary studies of Facelli et al. (2002, 2005)
BOX-, ERIC- and REP-PCR protocols, which amplify specific conserved repetitive sequences distributed throughout the genome of diverse bacteria, have proved reliable and sensitive in discriminating Xanthomonas strains, especially at the pathovar level
(Louws et al ., 1994). Amplicons produced following these PCR protocols revealed two groups, A and B, among Xanthomonas strains from pistachio. Furthermore, BOX and
ERIC-PCR protocols revealed two subgroups within group A. The similarity value between the two main clusters, based on UPGMA produced by a combined binary matrix generated by the three protocols, was about 0.18, confirming that the two groups of xanthomonads pathogenic on pistachio were genetically distinct.
Chapter 3: Genetic diversity within the pistachio dieback pathogen 43
5090 4072 3054
2036
1636
1018
506
396 344 298
L 2 3 5 7 9 10 4 8 X L 34 28 30 31 32 33 16 14 13 15 11 L 20 21 22 23 27 36 24 25 NC L
A B
Figure 3.1 BOX-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio in 2000 and identified as group A or B. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); X, X. translucens pv. translucens DAR 35705; and NC, negative control (water)
Chapter 3: Genetic diversity within the pistachio dieback pathogen 44
3054
2036
1636
1018
506
396 344 298
L 72 3 7 73 74 75 76 77 78 L 44 45 46 47 48 49 50 25 51 L 52 53 54 55 56 57 58 59 60 61 L 62 63 64 65 66 67 68 69 70 71 L
A B
Figure 3.2 BOX-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio in 2003 and identified as group A or B. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); underlined strains are representatives from the collection established in 2000
Chapter 3: Genetic diversity within the pistachio dieback pathogen 45
7 73 74 75 76 4 5 6 8 9 10 11 13 20 28 32 33 A 34 3 72 77 78 2 14 15 16 29 30 31 25 52 64 60 56 48 27 68 21 26 47 51 55 59 63 67 71 24 66 B 62 58 54 46 36 50 22 69 57 53 49 45 23 70 44 65 61 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Coefficient
Figure 3.3 Dendrogram generated from binary matrix derived from amplicons obtained by BOX-PCR, demonstrating fingerprint groups of 65 strains of Xanthomonas isolated from pistachio in Australia. Numbers on the right denote the strains falling within a particular group, A or B
Chapter 3: Genetic diversity within the pistachio dieback pathogen 46
5090 4072 3054
2036
1636
1018
506
396 344 298
220
L 2 3 5 7 9 10 4 8 X L 34 28 30 31 32 33 16 14 13 15 11 L 20 21 22 23 27 36 24 25 NC L
A B
Figure 3.4 ERIC-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio in 2000 and identified as group A or B. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); X, X. translucens pv. translucens DAR 35705; and NC, negative control (water)
Chapter 3: Genetic diversity within the pistachio dieback pathogen 47
5090 4072
3054
2036
1636
1018
506
396 344 298 220
L 72 28 7 73 74 75 76 77 78 L 44 45 46 47 48 49 50 25 51 L 52 53 54 55 56 57 58 59 60 61 L 62 63 64 65 66 67 68 69 70 71 L
A B
Figure 3.5 ERIC-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio in 2003 and identified as group A or B. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); underlined strains are representatives from the collection established in 2000
Chapter 3: Genetic diversity within the pistachio dieback pathogen 48
A
B
Figure 3.6 Dendrogram generated from binary matrix derived from amplicons obtained by ERIC-PCR, demonstrating fingerprint groups of 65 strains of Xanthomonas isolated from pistachio in Australia. Numbers on the right denote the strains falling within a particular group, A or B
Chapter 3: Genetic diversity within the pistachio dieback pathogen 49
5090 4072 3054
2036
1636
1018
506
396 344 298
220
L 2 3 5 7 9 10 4 8 X L 34 28 30 31 32 33 16 14 13 15 11 L 20 21 22 23 27 36 24 25 NC L
A B
Figure 3.7 REP-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio in 2000 and identified as group A or B. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); X, X. translucens pv. translucens DAR 35705; and NC, negative control (water)
Chapter 3: Genetic diversity within the pistachio dieback pathogen 50
5090
4072
3054
2036
1636
1018
506
396 344 298
L 72 7 73 74 75 76 77 78 25 44 45 46 L 47 48 49 50 51 52 53 54 55 56 57 58 L 59 60 61 62 63 64 65 66 67 68 69 70 71 L
A B Figure 3.8 REP-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio in 2003 and identified as group A or B. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); underlined strains are representatives from strains collected in 2000
Chapter 3: Genetic diversity within the pistachio dieback pathogen 51
A
B
Figure 3.9 Dendrogram generated from binary matrix derived from amplicons obtained by REP-PCR, demonstrating fingerprint groups of 65 strains of Xanthomonas isolated from pistachio in Australia. Numbers on the right denote the strains falling within a particular group, A or B
Chapter 3: Genetic diversity within the pistachio dieback pathogen 52
1 2 3 4 5 6 7 8
7 3 25 7 28 25 7 25
A B A B A B
Figure 3.10 Genomic DNA fingerprinting patterns from representative strains of
Xanthomonas isolated from pistachio, obtained by rep-PCR. Lanes 1, 2 and 3, amplicons generated by BOX-PCR; lanes 4, 5 and 6 , generated by ERIC-PCR; lanes 7 and 8, generated by REP-PCR. Representative strains are underlined and identified as group A or B. Numbers on the left are molecular size markers (bp) (1 kb ladder,
Invitrogen, Australia) corresponding to fragments in the lanes to the left and right
Chapter 3: Genetic diversity within the pistachio dieback pathogen 53
A
B
Figure 3.11 Phenogram showing the relationships among 65 strains of Xanthomonas isolated from pistachio in Australia based on a combined binary matrix derived from amplicons obtained by BOX-, ERIC- and REP-PCR. Numbers on the right denote the strains falling within a particular group, A or B
Chapter 3: Genetic diversity within the pistachio dieback pathogen 54
There was a correlation between the groups and the geographic origin of the strains, in that group A strains originated from Kyalite, Red Cliffs and Renmark whereas group B comprised strains from Robinvale only. The Robinvale orchard is situated between the orchards at Kyalite and Red Cliffs. As growers at all four locations have shared budwood in the past, the origin of this distribution is not clear, although it is possible that the two groups represent different, recent introductions of the pathogen. Sirora is the unique female cultivar grown in all pistachio growing regions of Australia, therefore, there has not been selection for different populations of the pathogen in terms of host genotype.
Linkage between groups, indicated by rep-PCR, and geographical origin has been reported for Xanthomonas strains pathogenic for other plants . For example, Scortichini et al. (2001) reported the existence of genetic diversity in a worldwide collection of X. axonopodis pv . juglandis and showed that the population of the pathogen was unique to each walnut cultivation area. Also, Massomo et al. (2003) observed variation among X. campestris pv . campestris strains isolated from brassica collected from fields in
Tanzania and linked fingerprint patterns to specific geographical areas. They noted that adaptability might be the basis for this phenomenon. Likewise, Mkandawire et al.
(2004) revealed that X. campestris pv . phaseoli strains collected from common bean in
Africa comprised three genotypes, two unique to East Africa and the other associated with strains collected from the New World. Selection for a specialised niche can affect genome organisation and distribution of repetitive sequences in the bacterium genome, resulting in fingerprints unique to a specific pathovar or strain (Louws et al. , 1994).
Because Sirora is the main female cultivar grown in Australian pistachio orchards, adaptation to this host cannot be involved in the genetic diversity of the pathogen. Since pistachio pathogen strains can be pathogenic to some plants of the Anacardiaceae and
Chapter 3: Genetic diversity within the pistachio dieback pathogen 55
Poaceae (see Chapter 5), it would be interesting to see if adaptation of the pathogen with these plants in different areas or climatic factors are involved.
Although each individual rep-PCR genomic fingerprinting procedures (Rademaker & de
Bruijn, 1997) divided the pathogen strains into two groups, A and B, BOX- and ERIC-
PCR were more discriminative than REP-PCR and showed two subgroups in group A.
This might be because of greater similarity of the BOX and ERIC sequences to each other than to the REP element, in terms of size and copy number (Martin et al. , 1992).
However, the subgroups determined by BOX and ERIC–PCR differed. The difference in structure of the two elements may account for the difference in that BOX, unlike
ERIC, has a modular structure with implications for the secondary structure of the sequence (Martin et al. , 1992).
The most reliable result was obtained by combining the data obtained from BOX-,
ERIC- and REP-PCR fingerprints. Combining the data from the three PCR protocols
(BER) increased the number of data points (bands), therefore the genome was more comprehensively covered as was recommended by Rademaker et al. (2000).
Rep-PCR can be used as a highly discriminatory technique to fingerprint closely related strains of the species X. translucens isolated from different hosts (Rademaker et al. ,
2000). Pistachio strains, from both groups, were different from X. translucens pv. translucens DAR 35705, isolated from wheat in Australia, based on fingerprints generated by the three rep-PCR protocols. DAR 35705 was the only authenticated strain of the species available in Australia and more strains would have been tested if they were available. To assess similarity among pistachio strains and other type and
Chapter 3: Genetic diversity within the pistachio dieback pathogen 56 reference strains of the species, based on rep-PCR, further experiments were performed which will be reported in chapter 4.
In summary, the rep-PCR technique indicated that Xanthomonas strains isolated from pistachio in Australia comprise two genetically distinct groups. Good agreement between the fingerprint groups and their geographic origin was obtained. Furthermore, pistachio strains differed genetically from X. translucens isolated from wheat in
Australia.
Chapter 4: Characterisation of xanthomonads … 57
Chapter 4: Characterisation of xanthomonads causing pistachio dieback
4.1 Introduction
Comprehensive diagnosis of a new pathogen is a vital step in the effective control and management of the pathogen and of the disease. Pistachio dieback is a new plant disease and, in preliminary studies, Xanthomonas strains similar to X. translucens were identified as the causal agent (Facelli et al. , 2002; Facelli et al. , 2005). However, the pathogen was not completely characterised or classified. Therefore, there was a need not only to identify the pathogen strains but also to describe their taxonomic status with respect to known xanthomonads.
Arguments have been made that, to perform a comprehensive study on unknown or poorly characterised plant pathogenic bacteria, “polyphasic” taxonomy is needed. This term was first introduced by Colwell (1970) and means the integration of available phenotypic and genotypic information into a multi-dimensional and general-purpose classification. The rationale for this approach is that all biological diversity cannot be encoded in a single bacterium and variability of characters is group dependent, indeed, one bacterium may not express all its characteristics at once. The use of polyphasic identification has provided plant pathologists with a powerful approach for identification and classification of Xanthomonas strains, in particular (Vandamme et al. ,
1996). Multiple phenotypic and genotypic methods have been applied to characterise
Xanthomonas strains associated with several diseases (Gent et al. , 2004; Janse et al. ,
2001; Roumagnac et al. , 2004; Trebaol et al. , 2000).
Chapter 4: Characterisation of xanthomonads … 58
This chapter describes characterisation of Xanthomonas strains associated with pistachio dieback in Australia and their taxonomic position in the genus Xanthomonas .
This comprises a polyphasic study using physiological and biochemical tests, analysis of utilization of carbon sources, polyacrylamide gel electrophoresis (PAGE) of whole cell proteins, internal transcribed spacer (ITS) sequencing, 16S rRNA gene sequencing,
DNA-DNA hybridisation and rep-PCR fingerprinting. Pathogenicity tests, which are usually considered a component of identification, especially in the description of a new pathogen, will be presented in Chapter 5.
4.2 Materials and methods
For buffers, solutions and reagents, see Appendix A. Media and methods used for biochemical and physiological tests are shown in Appendix B.
4.2.1 Biochemical and physiological tests
Twenty-one strains selected randomly from the pathogen collection (Chapter 2) and X. translucens pv. translucens DAR 35705 were tested for: Gram reaction using the KOH solubility test, oxidase reaction, nitrate reduction, urease production, growth at 36 °C and Tween 80 hydrolysis following the methods described by Fahy & Hayward (1983).
Oxidative and fermentative metabolism of glucose in O/F medium, gelatin liquefaction, starch hydrolysis, H 2S production from cysteine, growth in 5% NaCl, digestion of milk proteins, hypersensitive reaction on tobacco, action in litmus milk and mucoid growth on sucrose peptone agar (SPA), glucose yeast-extract carbonate agar (GYCA) and YDC were assessed as described by Schaad et al. (2001). Ice nucleation activity and esculin hydrolysis were assessed according to Lindow (1990) and Sands (1990), respectively.
Chapter 4: Characterisation of xanthomonads … 59
4.2.2 Biolog GN2 Microplate System
Six strains of the pathogen were selected from the clusters generated by rep-PCR (see section 3.4) to test their ability to utilise 95 substrates as sole carbon sources in Biolog
GN2 microplates (Biolog Inc., Hayward, CA, USA). X. translucens pv. translucens
DAR 35705 was included for comparison. A single colony of each strain was cultured on NA and then on TSA for 24 h. The bacteria were swabbed from the surface of the plates. Otherwise, inoculation of the microplates, incubation and analysis were performed as per the manufacturer’s instruction. Colour development was recorded with a MAXLine TM Microplate reader (Molecular Devices Corporation, Sunnyvale, CA,
USA) and the results were checked by visual observation of the colour reaction.
Metabolic fingerprint patterns were compared and identified after 24, 48 and 72 h using the Microlog TM GN 4.01A software. This test was performed twice on separate occasions.
4.2.3 SDS-PAGE of whole cell proteins
Sample preparation for sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) of soluble whole-cell protein extracts was performed for all pistachio strains, by the method of Laemmli (1970) as described by Vauterin et al. (1991a). For this, bacteria were grown on YDC and, after 24 h, cells were harvested with 10-20 mL phosphate buffered saline. The concentration of the suspension was determined by spectrophotometer and adjusted to 1.00 at 600 nm. Cells were washed with the buffer and centrifuged at 15,000 rpm twice. The pellet, 70-80 mg, was re-suspended in 1.5 mL sample treatment buffer (Appendix A) in a sterile 2 mL micro-centrifuge tube. The suspension was mixed and heated at 95 °C for 10 min, then cooled on ice and
Chapter 4: Characterisation of xanthomonads … 60 centrifuged at 10,000 rpm for 15 min. The supernatant was divided into two portions; one was stored at –20 °C for immediate use and another at –80 °C for long-term storage.
Discontinuous, one-dimensional SDS-PAGE was performed on a BRL vertical electrophoresis system (model V16, Bethesda Research Laboratories, MD, USA). The gel was cast and electrophoresis performed as per the manufacturer’s instructions. The concentrations of separating and stacking gels were 10% and 5%, respectively and gels were prepared as Table 4.1.
Table 4.1 Solutions for preparing resolving gels for SDS-polyacrylamide gel electrophoresis
10% gel slab 5% stacking gel
ProtoGel 33.3 mL ProtoGel 16.6 mL
1.5 M Tris-HCl pH 8.8 25 mL 1 M Tris-HCl pH 6.8 12.5 mL
10% SDS 1 mL 10% SDS 1 mL
MQ water 39.6 mL MQ water 68.8 mL
Ammonium persulphate 10% 1 mL Ammonium persulphate 10% 1 mL
TEMED 0.1 mL TEMED 0.1 mL
Total 100 mL Total 100 mL
A Tris–glycine electrophoresis buffer was used for the electrophoresis. Gels were run at
5 mA constant current in a cold room (4 °C) for about 17 h and then stained overnight in
Coomassie Blue solution (0.1% Coomassie Blue dye in 50% methanol and 10% glacial acetic acid). Gels were destained in methanol:acetic acid solution without Coomassie
Blue for 2 h and stored in 7% acetic acid. Gels were analysed visually and scanned
Chapter 4: Characterisation of xanthomonads … 61 images of the gels were analysed using Gel-Pro Analyzer (Media Cybernetics, MD,
USA). To check reproducibility, protein samples were prepared from all strains at three different times and the extracts were separated on three gels.
4.2.4 Sequencing of the 16S-23S rDNA spacer region
The nucleotide sequence of the 16S-23S rDNA spacer region was determined for seven strains selected randomly across the two groups and subgroups distinguished by rep-
PCR (see section 3.4); strains 3, 7, 28 and 30 from subgroups in group A and 25, 36 and
50 from group B. The fragment was amplified in PCR using a forward 20-bp primer
(C1), 5’- AGT CGT AAC AAG GTA AGC CG - 3’, derived from the Escherichia coli
16S rDNA position 1493-1513 and a reverse 20-bp primer (C2), 5’- C(T/C)(A/G)
(T/C)TG CCA AGG CAT CCA CC - 3’, corresponding to the E. coli 23S rDNA sequence position 23-43 (Lane, 1991). PCR was performed in a total volume of 50 µL using 0.5 µL Taq polymerase (5 U µL-1, Qiagen Pty Ltd, Victoria, Australia), 5 µL 10 ×
PCR buffer, 0.4 µL dNTP mixture (25 mM each), 2.5 µL of C1 and C2 primers (10 µM each), 1 µL sample (50 ng µL-1) and 38.1 µL distilled water. PCR was performed as above with the following conditions: 1 × 95 °C for 15 min, 29 × (95 °C for 45 s, 50 °C for
1 min, 72 °C for 2 min) and 72°C for 10 min. Ten µL of PCR product was run on an agarose gel (1%) and stained with ethidium bromide. The Perfectprep Gel Cleanup Kit
(Brinkmann Instruments, Inc., NY, USA) was used to purify the fragment from the gel as recommended by the manufacturer. The purified fragment was directly sequenced in both directions using the Taq DyeDeoxy Terminator Sequencing Kit (Applied
Biosystems, CA, USA) as indicated by the manufacturer and as follows: the template for sequencing was prepared by preparing the reaction mix, comprising 200-500 ng of
Chapter 4: Characterisation of xanthomonads … 62 the DNA, 3.5 µL Big Dye, 3.5 µL sequencing buffer, 2 µL primer (3.2 pmol µL-1) and adjusted to 20 µL with MQ water. Cycle sequencing was performed in a sterile 0.2 mL
PCR tube with 25 cycles of 96 °C for 30 °C s, 50 °C for 15 s and 60 °C for 4 min. For isopropanol precipitation of the extension product, the contents of the PCR tube were transferred to a sterile 1.5 mL micro-centrifuge tube and 80 µL of 75% isopropanol was added. The tube was vortexed and left at room temperature (approx. 25 °C) for 15 min.
The tube was centrifuged at 14,000 for 20 min and the supernatant was removed. The pellet was rinsed with 250 µL of 75% isopropanol and centrifuged in the same direction at 14,000 for 5 min. The supernatant was removed and the sample was dried by placing it, with the lid open, in a heat block at 90 °C for 1 min. The sequence was determined using an Applied Biosystem Model 3700 automated sequencer (Applied Biosystems,
CA, USA) at the Sequencing Centre, Institute of Medical and Veterinary Science
(IMVS), Adelaide, Australia. Chromas (version 2) software (Technelysium Pty Ltd,
Qld, Australia) was used for editing and generating the sequences. Alignment and comparison of the sequences were performed with the programs Clustal X and
GeneDoc available on the Bioinformatics.Net database. Using the Basic Local
Alignment Search Tool (BLAST), the sequences were compared with sequences in
GenBank, EMBL, DDBJ and PDB databases. To check reproducibility, samples were prepared for sequencing twice, each time from different DNA preparations.
4.2.5 16S rRNA gene sequencing
Four strains were selected randomly across the two groups distinguished by rep-PCR; strains 3 and 7 from group A; and 25 and 50 from group B. Primers used for PCR amplification and sequencing are listed in Table 4.2. Primers were the same as those
Chapter 4: Characterisation of xanthomonads … 63 used by Hauben et al. (1997), except the primer 16F434 which was designed in this study. Using 16F27 and 16R1525 primers, DNA of the strains was amplified with the optimised reaction mix and PCR cycling conditions listed in Tables 4.3 and 4.4. The complete PCR product was run on an agarose gel (1%) and stained with ethidium bromide. Purification of the fragment from the gel, preparation of the template for sequencing using all of the sequences listed in Table 4.2, and sequencing were performed as described in section 4.2.4. Alignment and comparison of the sequences were performed with Clustal X and GeneDoc as in section 4.2.4. Sequence database was searched for the nearest neighbours using the Sequence Match tool in the
Ribosomal Database Project (RDP) (Cole et al. , 2005) based on the National Centre for
Biotechnology Information (NCBI) taxonomy database, which includes names and classifications for all of the organisms represented in the protein and sequence databases.
Table 4.2 Oligonucleotide primers used for 16S rDNA amplification and sequencing (Hauben et al. , 1997)
Primer a Sequence Position b Application
Amplification 16F27 AGAGTTTGATCMTGGCTCAG 8-27 and sequencing 16R1087 CTCGTTGCGGGACTTAACCC 1106-1087 Sequencing
16F434 c AGGCCTTCGGGTTGTAAAGC 415-434 Sequencing
Amplification 16R1525 TTCTGCAGTCTAGAAGGAGGTGWTCCAGCC 1525-1496 and sequencing a F, forward primer; R, reverse primer b The numbering of target positions is based on the numbering of the E. coli 16S rRNA sequence (Brosius et al. , 1981) c primer designed in this study
Chapter 4: Characterisation of xanthomonads … 64
Table 4.3 Concentration of PCR reaction mix optimised for 16S rDNA sequencing
Reaction component Volume ( µµµL) in 25 µµµL Final concentration reaction DNA sample (50 ng µL-1) 0.75 37.5 ng
Taq Polymerase (5 U µL-1, Qiagen ) 0.1 0.5 U
10 × Taq Buffer 2.5 1× dNTP s (25 mM each) 0.2 200 µM
Primer 16F27 (10 µM) 1.25 0.5 µM
Primer 16R1525 (10 µM) 1.25 0.5 µM
MQ water 18.95 -
Table 4.4 PCR cycling optimised for amplification of the 16S rRNA gene
Program step Temperature Time
1 Taq polymerase activation and initial denaturation 95°C 15 min
2 30 cycles of:
Denaturation 94°C 45 s
Annealing 59°C 45 s
Elongation 72°C 2 min
3 Final extension 72°C 2 min
To check reproducibility, samples were prepared for sequencing twice, each time from different DNA preparations.
Chapter 4: Characterisation of xanthomonads … 65
4.2.6 DNA-DNA hybridisation
This analysis was performed by Dr. J. Bowman at the Australian Food Safety Centre,
University of Tasmania.
Two pistachio dieback pathogen strains, 7 and 25, were selected as representatives of group A and group B, respectively and their DNA-DNA relatedness was analysed.
Because of the close relatedness of the pathogen strains to X. translucens indicated by biochemical and physiological tests, as well as 16S-23S rDNA and 16S rDNA sequencing, three type and reference strains of the species, X. translucens pv . translucens LMG 876, X. translucens pv. poae LMG 728 and X. translucens pv. graminis LMG 726 were also analysed to evaluate more accurately the genomic homology of the pathogen. Type strains of X. theicola LMG 8684 and X. hyacinthi
LMG 739 were included as out-groups. To determine the percentage DNA homology among the strains, the spectrophotometric renaturation rate kinetic procedure (Huss et al. , 1983) was used. Using sonication, genomic DNA was sheared to a mean size of 1 kb, then dialysed overnight at 4 °C in 2 × SSC buffer and adjusted to approximately 75
µg mL -1. Following denaturation of the DNA, hybridisation was performed at the optimal temperature for renaturation (T OR ) which was 25°C below the DNA melting temperature and was calculated from the following equation: T OR °C = 48.5 + (0.41 × mol %G+C). To calculate DNA hybridisation values, the decline in absorbance of DNA mixtures and control DNA samples over a 40-min interval was measured and the following equation (Huss et al. , 1983) were used: % DNA hybridisation = [4AB-A-
B/2 √(A ×B)] ×100%. A and B represent the change in absorbance for two different DNA samples being compared and AB represents the change in absorbance for equimolar mixtures of A and B. DNA hybridisation values equal to or below 25% are considered to represent background hybridisation and were thus not considered significant (Huss et
Chapter 4: Characterisation of xanthomonads … 66 al. , 1983). Hybridisation between DNA preparations of the two strains of the pathogen was performed four times while hybridisation between DNA of the pathogen strains and the type and reference strains was performed two or three times.
4.2.7 Comparison with pathovars of X. translucens using rep-PCR
Two pistachio pathogen strains, 7 and 25, were selected as representatives of group A and group B, respectively. Using rep-PCR as described in section 3.2.1, the two strains were compared with type and reference strains of the ten pathovars of X. translucens
(Table 2.2) , as well as X. translucens pv. translucens DAR 35705, isolated in Australia.
All strains were analysed using BOX-, ERIC- and REP-PCR individually as described in Chapter 3, and then a combined binary matrix was derived from amplicons obtained by the three protocols. All three protocols were performed three times.
4.3 Results
4.3.1 Biochemical and physiological tests
The results divided the strains from pistachio into two groups, A and B. All strains shared the following features with X. translucens pv. translucens DAR 35705: Gram negative, mucoid growth on SPA and YDC, oxidative metabolism of glucose; positive for H 2S production, growth at 36 °C and hydrolysis of starch, gelatin, esculin and Tween
80, and negative for oxidase, urease and growth in the presence of 5% NaCl (Table 4.5).
Pistachio strains belonging to group A differed from the other bacteria in digestion of milk proteins, ice nucleation activity, reactions in litmus milk and hypersensitive reaction test on tobacco leaves (Table 4.5, Figures 4.1, 4.2 and 4.3).
Chapter 4: Characterisation of xanthomonads … 67
Table 4.5 Comparison of pistachio dieback pathogen strains with Xanthomonas translucens pv. translucens (DAR 35705) by biochemical and physiological tests
Xanthomonads isolated Test from pistachio X. t . pv . translucens Group A Group B DAR 35705 Gram reaction - - - Oxidative/fermentative growth O+, F- O+, F- O+, F- Oxidase - - - Urease production - - -
H2S production from cysteine + + + Nitrate reduction - - - Growth at 36 °C + + + Mucoid on SPA and YDC + + + Growth in 5% NaCl - - - Starch hydrolysis + + + Gelatin hydrolysis + + + Esculin hydrolysis + + + Tween 80 hydrolysis + + + Digestion of milk proteins - + + Ice nucleation at: - 4 °C - + + - 10 °C - or + Action in litmus milk: acid formation - - - peptonisation + + + alkaline reaction w + + reduction - + + Tobacco hypersensitive reaction + - -
+: positive reaction, -: negative reaction, w: weak reaction
Chapter 4: Characterisation of xanthomonads … 68
Figure 4.1 Protein digestion test. A clear zone is observed around positive strains. A, pathogen strain which belongs to group A; B, pathogen strain which belongs to group B and X: X. translucens DAR 35705
Figure 4.2 Action on litmus milk. Change of colour to blue is considered alkaline reaction, white precipitation is a sign of peptonisation and clearing is a sign of reduction. A, pathogen strain which belongs to group A; B, pathogen strain which belongs to group B, X: X. translucens DAR 35705 and C, uninoculated control
Chapter 4: Characterisation of xanthomonads … 69
Figure 4.3 Tobacco hypersensitive test. The complete collapse of the inoculated leaf tissue after 24 h was considered a positive reaction (A). A, pathogen strain which belongs to group A; B, pathogen strain which belongs to group B and X, X. translucens
DAR 35705
Chapter 4: Characterisation of xanthomonads … 70
4.3.2 Biolog GN2 Microplate System
All six strains tested were identified as Xanthomonas campestris pv . translucens , which is the former name of X. translucens pv . translucens , by their metabolic fingerprint. At the first reading, after 24 h, most of the wells were negative whereas more positive results were read at 48 and 72 h and there was no difference between the second and third readings. Metabolic activity was recorded for all strains on the following carbon substrates; dextrin, N-acetyl-D-glucosamine, D-cellobiose, D-fructose, α-D-glucose, D- mannose, D-psicose, sucrose, D-trehalose, pyruvic acid methyl ester, α-keto glutaric acid, D, L-lactic acid, succinic acid, bromosuccinic acid, L-alaninamide, L-alanine, L- alanylglycine, L-glutamic acid, glycyl-L-glutamic acid, L-serine. However, they were unable to metabolise α-cyclodextrin, glycogen, N-acetyl-D-galactosamine, adonitol, L- arabinose, D-arabitol, i-erythritol, D-galactose, gentiobiose, m-inositol, α-D-lactose, lactulose, maltose, D-mannitol, D-melibiose, β-methyl-D-glucoside, D-raffinose, L- rhamnose, D-sorbitol, turanose, xylitol, acetic acid, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γ- hydroxybutyric acid, p-hydroxy phenylacetic acid, itaconic acid, α-keto butyric acid, α- keto valeric acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinamic acid, glucuronamide, D-alanine, L-asparagine, glycyl-L-aspartic acid,
L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L- pyroglutamic acid, D-serine, L-threonine, D,L-carnitine, γ-amino butyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3- butanediol, glycerol, D,L-α-glycerol phosphate, α-D-glucose-1-phosphate, D-glucose-6- phosphate. Strains in groups A and B differed from one another in utilisation of Tween
40, Tween 80, L-fucose, succinic acid mono-methyl-ester and L-aspartic acid (Table
Chapter 4: Characterisation of xanthomonads … 71
4.6). X. translucens pv. translucens DAR 35705 was confirmed as X. c. pv. translucens by the test. This strain did not metabolise α-keto glutaric acid, D, L-lactic acid, bromosuccinic acid, L-alaninamide, L-alanine, L-alanylglycine, L-glutamic acid and L- serine, all of which were utilised by pistachio strains representing both groups (Table
4.6).
Table 4.6 Differences among xanthomonads isolated from pistachio and Xanthomonas translucens pv. translucens DAR 35705 in utilisation of carbon substrates in the Biolog
GN2 microplate test
Xanthomonads isolated from X. t. pv . Carbon source pistachio translucens Group A Group B DAR 35705 Tween 40 + - -
Tween 80 + - -
L-fucose + - -
Succinic acid mono-methyl-ester + - +
L-aspartic acid - + -
α-keto glutaric acid + + -
D, L-lactic acid + + -
Bromosuccinic acid + + -
L-alaninamide + + -
L-alanine + + -
L-alanylglycine + + -
L-glutamic acid + + -
L-serine + + -
+ metabolised the carbon substrate - negative reaction
Chapter 4: Characterisation of xanthomonads … 72
4.3.3 SDS-PAGE of whole cell proteins
The strains fell into two groups based on analysis of the patterns of total proteins following SDS-PAGE. These groups were identical to those indicated by biochemical tests. Protein profiles comprised 25 and 27 reproducible bands, ranging from about 4 kDa to more than 90 kDa, for group A and B, respectively. Although strains in each group appeared to be homogeneous in protein profiles, the two groups differed with respect to low molecular weight proteins, about 8-14 kDa, in that three bands were apparent for group A only whereas four different bands were apparent for group B.
Furthermore, an intense band was present at about 4 kDa for group A but not for group
B, whereas two weak bands were present in this region for group B only (Figure 4.4).
1 2 3 4 5 6 7 8 9 10 11 12
Figure 4.4 SDS-PAGE profiles of total proteins of Xanthomonas isolated from pistachio. Protein profiles show that there are two groups among the strains. Group A: lanes 1-6 and group B: lanes 7-12. Brackets show discriminating bands and molecular weight (kDa) is indicated on the right
Chapter 4: Characterisation of xanthomonads … 73
A few strains randomly selected from the pathogen collection had already been compared with the SDS-PAGE database of plant pathogenic xanthomonads at Ghent
University, Ghent, Belgium, and identified as xanthomonads similar to X. translucens
(Facelli et al. , 2005). Since the strains used in the Ghent study included strains from both groups identified in this study and we found there to be no variation within the two groups based on SDS-PAGE, further comparison between the pistachio pathogen strains and reference strains was not performed.
4.3.4 Sequencing of the 16S-23S rDNA spacer region
Sequencing of the complete 16S-23S rDNA spacer region yielded a fragment of 558 bases for the four strains representing group A and a fragment of 557 bases for the three strains representing group B. No sequence difference was observed within each group.
However, the two groups differed in 1, 6 and 5 bases in ITS1, ITS2 and ITS3, respectively. The 16S-23S rDNA spacer region, in all strains studied, contained tRNA Ala and tRNA Ile genes (Figure 4.5). Comparison of the sequences with sequences in GenBank, EMBL, DDBJ and PDB databases revealed that the 16S-23S rDNA sequence of strains in group A and group B most closely matched the region in
Xanthomonas translucens pv . poae, with 98% and 97% similarity, respectively. The nucleotide sequences determined for the pathogen strains have been deposited in the
Genbank database under the accession numbers given in Appendix C.
4.3.5 Sequencing of the 16S rRNA gene
Nearly complete 16S rDNA sequences, 1500 bp, were determined for four strains selected from the two groups indicated by rep-PCR. The two groups differed in one nucleotide only, at position 1111 (Figure 4.6) and there was no variation between the
Chapter 4: Characterisation of xanthomonads … 74 two strains of each group. Based on comparison of the sequences with the database, group A and group B strains matched X. translucens pv . translucens LMG 876 with
100% and 99.6% similarities, respectively. The nucleotide sequences determined for the pathogen strains have been deposited in the Genbank database under the accession numbers given in Appendix C.
4.3.6 DNA-DNA hybridisation
The degree of hybridisation between the two strains of the pathogen, representing group
A and group B, was 84%. The average G+C content for both strains was consistent at
70 ± 0.5 mol%. When DNA from the pathogen strains was hybridised with DNA from three type and reference pathovars of X. translucens, as well as X. theicola and X. hyacinthi , they reassociated at a level of 70% and above with pathovars of X. translucens . The pathogen strain of group A had the greatest relatedness with the genome of X. translucens pv. poae LMG 728, with 84% similarity, whereas the group B representative showed the greatest relatedness with the genome of X translucens pv. graminis LMG 726, with 90% similarity. Both strains clearly differed from X. theicola
LMG 8684 and X. hyacinthi LMG 739 (Table 4.7).
Chapter 4: Characterisation of xanthomonads … 75
* 20 * 40 * 60 * 80 * 100 * 120 * 140 3 : ...... A...... : 140 7 : ...... A...... : 140 28 : ...... A...... : 140 30 : ...... A...... : 140 25 : ...... G...... : 140 36 : ...... G...... : 140 50 : ...... G...... : 140 GGCTGGATCACCTCCTTTTGAGCATGACA CTACGCCTACAGGCGTCCTCACAAGTAACCTGCATTCAGAGAGTTCCGCCACAGGGCGGAGCACCCCGATTTCGGGGCCATAGCTCAGCTGGGAGGGGGCCATAGCTCAGCTGGGAGGGGGCCATAGCTCAGCTGGGAGAGCACCTGCTTTGCAAGCACCTGCTTTGCA tRNA ala
* 160 * 180 * 200 * 220 * 240 * 260 * 280 3 : ...... T...... T..C...... G....C...... A...... : 280 7 : ...... T...... T..C...... G....C...... A...... : 280 28 : ...... T...... T..C...... G....C...... A...... : 280 30 : ...... T...... T..C...... G....C...... A...... : 280 25 : ...... -...... C..T...... A....A...... G...... : 279 36 : ...... -...... C..T...... A....A...... G...... : 279 50 : ...... -...... C..T...... A....A...... G...... : 279 AGCAGGGGGTCGTAGCAGGGGGTCGTCGGTTCGATCCCGACTGGCTCCACCACGGTTCGATCCCGACTGGCTCCACCACGGTTCGATCCCGACTGGCTCCACCAGATT GCAGATCCCTCTGCAAACGC CG ACCTGCGTGTGCGGAC GTCT AGGGACCTGC AGAGCCAAGACTTTGGGTCTGTAGCTCAGGTGGTTAGAG
* 300 * 320 * 340 * 360 * 380 * 400 * 420 3 : ...... : 420 7 : ...... : 420 28 : ...... : 420 30 : ...... : 420 25 : ...... A...... : 419 36 : ...... A...... : 419 50 : ...... A...... : 419 CGCACCCCTGATAAGGGTGAGGTCGGTGGTTCGAGTCCTCCCAGACCCACCCGCACCCCTGATAAGGGTGAGGTCGGTGGTTCGAGTCCTCCCAGACCCACCAACTCTGAATGTAAGAAGCACACTAAGAATTTAAGATGCGCCAGCAGTGAGGA CTGGGGTATGTTCTTTTAAAATTTGTGACGTAGCGAGC tRNA ile
* 440 * 460 * 480 * 500 * 520 * 540 * 3 : ...... C...... CG...... T...... : 558 7 : ...... C...... CG...... T...... : 558 28 : ...... C...... CG...... T...... : 558 30 : ...... C...... CG...... T...... : 558 25 : ...... T...... TT...... C...... : 557 36 : ...... T...... TT...... C...... : 557 50 : ...... T...... TT...... C...... : 557 GTTTGAGATCAAACTATCTTGACGTGTCGTTGTGGCTAAGGCGGGGAC TCGAGTCCCTAGAAATTGAGTCGTTATAGTTCGCGTCCGGG TTGTACCCCCGGACTCAGCATGACCT GAGGCAACTTGAGGTTATA
Figure 4.5 Aligned sequences of the 16S-23S rDNA region of xanthomonads isolated from pistachio trees. Two groups, A and B, were identified. Strains placed in group B and their sequence differences with group A strains are shaded. Dash denotes deletion in the sequence. tRNA ala and tRNA ile genes are in bold type
Chapter 4: Characterisation of xanthomonads … 76
* 20 * 40 * 60 * 80 * 100 * 120 * 140 3 : ...... : 140 7 : ...... : 140 25 : ...... : 140 50 : ...... : 140 AGTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGCAGCACAGTGGTAGCAATACCATGGGTGGCGAGTGGCGGACGGGTGAGGAATACATCGGAATCTACCTTTTCGTGGGGGATAACGTAGGGAAACTTAC
* 160 * 180 * 200 * 220 * 240 * 260 * 280 3 : ...... : 280 7 : ...... : 280 25 : ...... : 280 50 : ...... : 280 GCTAATACCGCATACGACCTTAGGGTGAAAGCGGAGGACCTTCGGGCTTCGCGCGGATAGATGAGCCGATGTCGGATTAGCTAGTTGGCGGGGTAAAGGCCCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGAT
* 300 * 320 * 340 * 360 * 380 * 400 * 420 3 : ...... : 420 7 : ...... : 420 25 : ...... : 420 50 : ...... : 420 CAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGGGTGAAGAAGGCCTTCGGGTTGTAAAGCCCTTTTGTTGGGA
* 440 * 460 * 480 * 500 * 520 * 540 * 560 3 : ...... : 560 7 : ...... : 560 25 : ...... : 560 50 : ...... : 560 AAGAAAAGCAGTCGGTTAATACCCGATTGTTCTGACGGTACCCAAAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGTGCGTAGGTG
* 580 * 600 * 620 * 640 * 660 * 680 * 700 3 : ...... : 700 7 : ...... : 700 25 : ...... : 700 50 : ...... : 700 GTTGTTTAAGTCCGTTGTGAAAGCCCTGGGCTCAACCTGGGAATTGCAGTGGATACTGGGCAACTAGAGTGTGGTAGAGGATGGCGGAATTCCCGGTGTAGCAGTGAAATGCGTAGAGATCGGGAGGAACATCTGTGGCG
* 720 * 740 * 760 * 780 * 800 * 820 * 840 3 : ...... : 840 7 : ...... : 840 25 : ...... : 840 50 : ...... : 840 AAGGCGGCCATCTGGACCAACACTGACACTGAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGCGAACTGGATGTTGGGTGCAACTTGGCACGCAGTATCGAAGCTAAC
* 860 * 880 * 900 * 920 * 940 * 960 * 980 3 : ...... : 980 7 : ...... : 980 25 : ...... : 980 50 : ...... : 980 GCGTTAAGTTCGCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAC
Chapter 4: Characterisation of xanthomonads … 77
* 1000 * 1020 * 1040 * 1060 * 1080 * 1100 * 1120 3 : ...... C...... : 1120 7 : ...... C...... : 1120 25 : ...... A...... : 1120 50 : ...... A...... : 1120 TTTCCAGAGATGGATTGGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCCAGCACGT ATGGTGGGA
* 1140 * 1160 * 1180 * 1200 * 1220 * 1240 * 1260 3 : ...... : 1260 7 : ...... : 1260 25 : ...... : 1260 50 : ...... : 1260 ACTCTAAGGAGACCGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTACTACAATGGTGAGGACAGAGGGCTGCAAGCTCGCGAGAGTAAGCCAATCCCA
* 1280 * 1300 * 1320 * 1340 * 1360 * 1380 * 1400 3 : ...... : 1400 7 : ...... : 1400 25 : ...... : 1400 50 : ...... : 1400 GAAACCTCATCTCAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGT
* 1420 * 1440 * 1460 * 1480 * 1500 3 : ...... : 1500 7 : ...... : 1500 25 : ...... : 1500 50 : ...... : 1500 TGCACCAGAAGCAGGTAGCTTAACCTTCGGGAGGGCGCTTGCCACGGTGTGGCCGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGC
Figure 4.6 Aligned sequences of the 16S rRNA gene of xanthomonads isolated from pistachio trees. Two groups, A and B, were identified. Strains placed in group B and their single sequence difference with group A strains are shaded
Chapter 4: Characterisation of xanthomonads … 78
Table 4.7 Results of DNA-DNA hybridisation analysis between pistachio dieback
Xanthomonas strains and the type strains of selected Xanthomonas species
Group A Group B Strain strain 7 strain 25
Group A strain 7 (pistachio pathogen representing group A) 100*
Group B strain 25 (pistachio pathogen representing group B) 84±5 100
X. tanslucens pv. translucens LMG 876 81±7 86±5
X. translucens pv. poae LMG 728 84±10 77±6
X translucens pv. graminis LMG 726 78±4 90±8
X. theicola LMG 8684 45±8 54±10
X. hyacinthi LMG 739 47 52
* values are means of two-four hybridisations
4.3.7 Comparison with pathovars of X. translucens using rep-PCR
BOX-PCR gave genomic fingerprints with 52 reproducible bands, ranging from about
250 to 3700 bp (Figure 4.7). The dendrogram of relationships among the pistachio strains and the type and reference strains of the species derived by cluster analysis of similarities based on BOX-PCR fingerprints, using UPGMA, is shown in Figure 4.8.
Based on BOX fingerprints, the group A representative (strain 7) was most similar to X. translucens pv . translucens LMG 876 and both were placed in a cluster with X. translucens pv . translucens DAR 35705, X. t. pv . undulosa LMG 892 and X. t. pv . secalis LMG 883. The group B representative (strain 25) was more similar to a cluster comprising X. t. pv . phleipratensis LMG 843, X. t. pv . arrhenatheri LMG 727, X. t. pv . phlei LMG 730 and X. t. pv . cerealis LMG 679.
Chapter 4: Characterisation of xanthomonads … 79
ERIC-PCR created genomic fingerprints with 31 reproducible bands, ranging from about 200 to 5000 bp (Figure 4.9). The relationships among the pistachio strains and the other strains, based on ERIC-PCR fingerprints (Figure 4.10), showed that the group A representative (strain 7) was closer to other pathovars of the species than the group B representative (strain 25).
REP-PCR formed genomic fingerprints with 63 bands, ranging from about 500 to 5600 bp (Figure 4.11). The dendrogram of relationships among the strains tested, based upon
REP-PCR fingerprints, is shown in Figure 4.12. Based on REP-PCR patterns, again, the group A representative (strain 7) was more similar to the other strains than the group B representative (strain 25).
A dendrogram of relationships was derived via a combined binary matrix from 146 bands generated by the three rep-PCR protocols (Figure 4.13). This dendogram revealed that pistachio strains, from both groups, differed from X. translucens pv. translucens
DAR 35705 isolated from wheat in Australia and from all pathovars of the species. The pistachio pathogen strain representing group A (strain 7) was closer to the other strains than the group B representative (strain 25). X. t. pv. translucens DAR 35705 most closely matched X. t. pv . secalis LMG 883 . However, neither BER-PCR nor the separate rep-PCR fingerprints resulted in an exact match for the representatives of the pistachio strains tested.
Chapter 4: Characterisation of xanthomonads … 80
5090 4072
3054
2036
1636
1018
506
396 344 298
L 1 2 3 4 5 6 7 8 9 10 L 11 12 13
Figure 4.7 BOX-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio, and type and reference strains of X. translucens pathovars. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); 1, Xanthomonas translucens pv. translucens DAR 35705; 2, pistachio dieback pathogen strain 7, representing group A; 3, pistachio dieback pathogen strain 25, representing group B; 4, X. t. pv . undulosa LMG 892; 5, X. t. pv . translucens LMG 876; 6, X. t. pv . cerealis LMG 679; 7, X. t. pv . secalis LMG 883; 8, X. t. pv . poae LMG 728; 9, X. t. pv . phleipratensis LMG 843; 10 , X. t. pv . arrhenatheri LMG 727; 11 , X. t. pv . graminis LMG 726; 12 , X. t. pv . phlei LMG 730 and 13 , X. t. pv . hordei LMG 737
Chapter 4: Characterisation of xanthomonads … 81
X.t.Australia
X.t.secalis
X.t.undulosa
X.pistachioA7
X.t.translucens
X.t.poae
X.pistachioB25
X.t.cerealis
X.t.arrhenather
X.t.phleipraten
X.t.phlei
X.t.graminis
X.t.hordei
0.5 0.6 0.8 0.9 1.0 Coefficient
Figure 4.8 Dendrogram generated from binary matrix derived from amplicons obtained by BOX-PCR fingerprints of Xanthomonas strains isolated from pistachio in Australia, and type and reference strains of X. translucens pathovars. X.t.Australia , Xanthomonas translucens pv. translucens DAR 35705 isolated from wheat in Australia; X.t.secalis, X. t. pv . secalis LMG 883; X.t.undulosa, X. t. pv . undulosa LMG 892; X.pistachioA7, pistachio dieback pathogen strain 7 representing group A; X.t.translucens, X. t. pv . translucens LMG 876; X.t.poae, X. t. pv . poae LMG 728; X.pistachioB25, pistachio dieback pathogen strain 25 representing group B; X.t.cerealis, X. t. pv . cerealis LMG
679; X.t.arrhenather, X. t. pv . arrhenatheri LMG 727; X.t.phleipraten, X. t. pv . phleipratensis LMG 843; X.t.phlei, X. t. pv . phlei LMG 730; X.t.graminis, X. t. pv . graminis LMG 726 and X.t.hordei, X. t. pv . hordei LMG 737
Chapter 4: Characterisation of xanthomonads … 82
5090
4072
3054
2036
1636
1018
506
396 344 298
L 1 2 3 4 5 6 7 8 9 10 L 11 12 13
Figure 4.9 ERIC-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio, and type and reference strains of X. translucens pathovars. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); 1, Xanthomonas translucens pv. translucens DAR 35705; 2, pistachio dieback pathogen strain 7, representing group A; 3, pistachio dieback pathogen strain 25, representing group B; 4, X. t. pv . undulosa LMG 892; 5, X. t. pv . translucens LMG 876; 6, X. t. pv . cerealis LMG 679; 7, X. t. pv . secalis LMG 883; 8, X. t. pv . poae LMG 728; 9, X. t. pv . phleipratensis LMG 843; 10 , X. t. pv . arrhenatheri LMG 727; 11 , X. t. pv . graminis LMG 726; 12 , X. t. pv . phlei LMG 730 and 13 , X. t. pv . hordei LMG 737
Chapter 4: Characterisation of xanthomonads … 83
X.t.Australia
X.t.secalis
X.t.undulosa
X.t.translucens
X.t.poae
X.t.phlei
X.t.phleipraten
X.t.arrhenather
X.t.cerealis
X.t.hordei
X.t.graminis
X.pistachioA7
X.pistachioB25
0.4 0.6 0.7 0.8 1.0 Coefficient
Figure 4.10 Dendrogram generated from binary matrix derived from amplicons obtained by ERIC-PCR fingerprints of Xanthomonas strains isolated from pistachio in
Australia, and type and reference strains of X. translucens pathovars. X.t.Australia,
Xanthomonas translucens pv. translucens DAR 35705 isolated from wheat in Australia;
X.t.secalis, X. t. pv . secalis LMG 883; X.t.undulosa, X. t. pv . undulosa LMG 892;
X.pistachioA7, pistachio dieback pathogen strain 7 representing group A;
X.t.translucens, X. t. pv . translucens LMG 876; X.t.poae, X. t. pv . poae LMG 728;
X.pistachioB25, pistachio dieback pathogen strain 25 representing group B;
X.t.cerealis, X. t. pv . cerealis LMG 679; X.t.arrhenatheri, X. t. pv . arrhenatheri LMG
727; X.t.phleipraten, X. t. pv . phleipratensis LMG 843; X.t.phlei, X. t. pv . phlei LMG
730; X.t.graminis, X. t. pv . graminis LMG 726 and X.t.hordei, X. t. pv . hordei LMG
737
Chapter 4: Characterisation of xanthomonads … 84
5090 4072
3054
2036
1636
1018
506
396 344 298
L 1 2 3 4 5 6 7 8 9 10 L 11 12 13
Figure 4.11 REP-PCR fingerprinting patterns of Xanthomonas strains isolated from pistachio, and type and reference strains of X. translucens pathovars. L, molecular size markers (bp) (1 kb ladder, Invitrogen, Australia); 1, Xanthomonas translucens pv. translucens DAR 35705; 2, pistachio dieback pathogen strain 7 representing group A; 3, pistachio dieback pathogen strain 25 representing group B; 4, X. t. pv . undulosa LMG 892; 5, X. t. pv . translucens LMG 876; 6, X. t. pv . cerealis LMG 679; 7, X. t. pv . secalis LMG 883; 8, X. t. pv . poae LMG 728; 9, X. t. pv . phleipratensis LMG 843; 10 , X. t. pv . arrhenatheri LMG 727; 11 , X. t. pv . graminis LMG 726; 12 , X. t. pv . phlei LMG 730 and 13 , X. t. pv . hordei LMG 737
Chapter 4: Characterisation of xanthomonads … 85
X.t.Australia
X.t.secalis
X.t.cerealis
X.t.poae
X.t.undulosa
X.t.hordei
X.t.phlei
X.t.translucens
X.t.phleipraten
X.t.arrhenather
X.t.graminis
X.pistachioA7
X.pistachioB25
0.6 0.7 0.8 0.9 1.0 Coefficient
Figure 4.12 Dendrogram generated from binary matrix derived from amplicons obtained by REP-PCR fingerprints of Xanthomonas strains isolated from pistachio in
Australia, and type and reference strains of X. translucens pathovars. X.t.Australia,
Xanthomonas translucens pv. translucens DAR 35705 isolated from wheat in Australia;
X.t.secalis, X. t. pv . secalis LMG 883; X.t.undulosa, X. t. pv . undulosa LMG 892;
X.pistachioA7, pistachio dieback pathogen strain 7 representing group A;
X.t.translucens, X. t. pv . translucens LMG 876; X.t.poae, X. t. pv . poae LMG 728;
X.pistachioB25, pistachio dieback pathogen strain 25 representing group B;
X.t.cerealis, X. t. pv . cerealis LMG 679; X.t.arrhenather, X. t. pv . arrhenatheri LMG
727; X.t.phleipraten , X. t. pv . phleipratensis LMG 843; X.t.phlei, X. t. pv . phlei LMG
730; X.t.graminis, X. t. pv . graminis LMG 726 and X.t.hordei, X. t. pv . hordei LMG
737
Chapter 4: Characterisation of xanthomonads … 86
X.t.Australia
X.t.secalis
X.t.undulosa
X.t.translucens
X.t.poae
X.t.cerealis
X.t.phleipraten
X.t.arrhenather
X.t.graminis
X.t.phlei
X.t.hordei
X.pistachioA7
X.pistachioB25
0.5 0.6 0.8 0.9 1.0 Coefficient
Figure 4.13 Phenogram showing the relationships among Xanthomonas strains isolated from pistachio in Australia, type and reference strains of X. translucens pathovars and
X. translucens DAR 35705, based on a combined binary matrix derived from amplicons obtained by BOX, ERIC and REP PCR. X.t.Australia, Xanthomonas translucens pv. translucens DAR 35705 isolated from wheat in Australia; X.t.secalis, X. t. pv . secalis
LMG 883; X.t.undulosa, X. t. pv . undulosa LMG 892; X.pistachioA7, pistachio dieback pathogen strain 7 representing group A; X.t.translucens, X. t. pv . translucens
LMG 876; X.t.poae, X. t. pv . poae LMG 728; X.pistachioB25, pistachio dieback pathogen strain 25 representing group B; X.t.cerealis, X. t. pv . cerealis LMG 679;
X.t.arrhenather, X. t. pv . arrhenatheri LMG 727; X.t.phleipraten , X. t. pv . phleipratensis LMG 843; X.t.phlei, X. t. pv . phlei LMG 730; X.t.graminis, X. t. pv . graminis LMG 726 and X.t.hordei, X. t. pv . hordei LMG 737
Chapter 4: Characterisation of xanthomonads … 87
4.4 Discussion
DNA-independent methods, viz. physiological and biochemical tests including carbon substrate utilization, and SDS-PAGE profiling, and DNA-based analyses comprising
16S-23S rDNA and 16S rDNA sequencing, DNA-DNA hybridisation and rep-PCR fingerprinting were used to describe the phenotypic and genotypic characteristics of
Xanthomonas strains causing dieback of pistachio in Australia. The relationship of these strains to previously described Xanthomonas strains, particularly pathovars of X. translucens , was also evaluated. The pistachio dieback pathogen was found to be phenotypically and genetically heterogeneous. Two distinct groups were identified which matched group A and group B of the pathogen indicated by rep-PCR, as described in Chapter 3. The pathogen strains belong to the X. translucens species but are clearly distinct from known pathovars of the species.
Physiological and biochemical tests supported the existence of two groups within the pathogen collection and the phenotypic properties of the pathogen were similar to those of X. translucens . However, it should be noted that, whereas positive reactions have been reported for X. translucens in digestion of milk proteins and ice nucleation tests
(Schaad et al. , 2001), group A strains were negative for milk digestion and most of these strains did not exhibit ice nucleation activity . Among xanthomonads only strains of X. translucens have been shown to exhibit ice nucleation activity (Lindow, 1990).
Since this phenotype is considered a virulence factor which can suit invasion and multiplication of a pathogen (Lindow, 1982; Lindow, 1990), the negative reaction of most group A strains to this test may relate to differences in their virulence determinants. On the other hand, bacteria capable of ice nucleation have an important role in certain plant diseases. For example, Klement et al. (1974) showed that canker
Chapter 4: Characterisation of xanthomonads … 88 and dieback on apricots, caused by Pseudomonas syringae , need ice formation, and that canker and dieback occur in areas where frost is created by the pathogen. Although frost is not common in pistachio growing areas, possible damage to plant tissues caused by ice produced by pistachio strains and also the sensitivity of Sirora to frost should be investigated.
The Biolog GN MicroPlate system identified pistachio strains, as well as X. translucens isolated from wheat in Australia, to species level, however, it was not accurate for differentiation at pathovar or strain level. In this study, the Microlog TM GN 4.01A database, the only available version identified all strains as X. campestris pv . translucens , the former name of X. translucens pv . translucens . The suitability of this technique as a rapid and reliable diagnostic tool for the pistachio pathogen should be investigated using other versions of the database. It must be added that the accuracy of the technique for identification of Xanthomonas strains at subspecies level has been criticised (Massomo et al. , 2003; Sahin et al. , 2003). In this test, we used the data obtained 48 h after inoculation of the plates because colour reactions at 48 h were considered more accurate than those at 24 h, due to the slow growth of the bacteria, as was reported by Vauterin et al. (1995) and Verniere et al. (1993). Group A and B strains differed in utilisation of L-L-aspartic acid which was utilised by group B but not by group A strains, and the substrates Tween 40, Tween 80, L-fucose and succinic acid mono-methyl-ester which were metabolised by group A but not by group B strains.
These discriminative substrates could be used as the sole source of carbon in media for diagnostic tests and also may be useful in designing a selective medium where strains of each group are capable of growing by metabolism of a certain substrate.
Chapter 4: Characterisation of xanthomonads … 89
Although type and reference strains of Xanthomonas , including the ten pathovars of X. translucens , were imported from Belgium, the Australian Quarantine and Inspection
Service (AQIS) permit under which these strains were imported precluded any tests involving living bacteria. Therefore, only X. translucens pv. translucens DAR 35705, isolated from wheat in Australia and the single representative of this species available in
Australia, was included in physiological and biochemical tests.
SDS-PAGE protein analysis revealed profiles characteristic for each group, A and B, but not variation within the groups. This technique has already been used to compare several strains of the pathogen with a wide range of Xanthomonas strains and identify them as X. translucens at Ghent University, Ghent, Belgium (Facelli et al ., 2005), supporting the more comprehensive findings of the present study.
ITS sequencing also supported the existence of two groups within the pistachio pathogen and placed the pistachio strains closer to X. translucens pathovars than other known xanthomonads. In a comprehensive study, Goncalves & Rosato (2002) analysed the phylogenetic relationships among species of the genus Xanthomonas based upon their ITS sequences and placed them in six clusters, where X. translucens forms cluster
IV. Their study also revealed that five species, namely X. translucen s, X. codiaei , X. theicola , X. melonis and X. hyacinthi , have a long sequence (78-85 bp) in the ITS2 region (ITS2-L), whereas other species have only 19 bp in this region (ITS2-S). Both groups of the pistachio pathogen exhibited ITS2-L and included substitution of bases in the sequence similar to those of X. translucens . The 16S-23S rDNA sequence differences between the two groups of the pathogen (12 bp) provided the basis for the development of a PCR-based assay for the specific detection of each group for use in pathogen recognition and disease diagnosis, and will be discussed in Chapter 6.
Chapter 4: Characterisation of xanthomonads … 90
Compared to ITS sequences, little sequence variability was found between the two groups in terms of the 16S rDNA sequences. Hauben et al. (1997) showed that diversity within the 16S rDNA regions of xanthomonads is limited, and is the basis on which all
Xanthomonas species are placed in only three clusters; cluster 1, X. campestris ; cluster
2, X. sacchari ; and cluster 3, X. albilineans . The latter cluster comprises X. albilineans ,
X. translucens , X. hyacinthi and X. theicola. These four species show only 0.3% difference in the 16S rDNA sequences and clearly are separated from other
Xanthomonas species, with 97.8 and 98.2% similarity with clusters 1 and 2, respectively. Showing 99.6-100% similarity with X. translucens pv . translucens LMG
876, pistachio strains are, therefore, placed in cluster 3. The phylogenetic relatedness within and among the clusters was demonstrated previously by Hauben et al. (1997).
The close identity based on 16S rDNA sequences (99%) indicated that the two groups of the pathogen are likely to be members of the same species and DNA-DNA homology results corroborated this.
DNA-DNA similarity value is a standard criterion for species delineation (Stackebrandt et al. , 2002) and the phylogenetic definition of a species includes strains with approximately 70% or greater DNA-DNA relatedness (Vauterin et al. , 1995). Overall genomic homology between the two groups of the pathogen based on DNA-DNA hybridisation was 84%, which showed that they belong to the same species. The similarity (77-90%) of group A and group B strains to the three reference pathovars of
X. translucens tested, demonstrated that they are members of this species, approximately equidistant from the three reference pathovars. The results also confirmed that the pistachio strains are not members of X. theicola and X. hyacinthi , the two other type species included in the analysis, with DNA hybridisation values of less
Chapter 4: Characterisation of xanthomonads … 91 than 70%. Vauterin et al. (1995) also demonstrated that while the average levels of
DNA homology between different Xanthomonas DNA homology groups are generally low (less than 40%), higher levels of homology (around 50%) are found between some groups such as X. translucens , X . theicola and X. hyacinthi which confirms our results.
Pistachio strains had an average G+C content of about 70 mol%. Hauben et al. (1997) found this level of G+C only in xanthomonads belonging to the X. albilineans cluster, which includes X. translucens , and emphasised that other Xanthomonas species have average G+C content of 65-66 mol%.
Rep-PCR demonstrated that Xanthomonas strains from pistachio differed from the ten pathovars of X. translucens tested. Louws et al. (1994) reported that rep-PCR has potential to discriminate among closely related but distinct Xanthomonas strains.
Likewise, Rademaker et al. (2000) found that while the pathovars comprise one DNA-
DNA homology group (Vauterin et al. , 1995), this group is heterogenous based on rep-
PCR and they demonstrated that strains representing the ten pathovars of the species differed in genomic fingerprints generated by the technique. In a more recent study,
Rademaker et al. (2005) again reported that combined data from the three rep-PCR protocols (BER) can differentiate X. translucens from other species of the genus and more discrimination can be revealed at subspecies level for this species by the separate rep-PCR patterns. No exact match for pistachio strain representatives (strains 7 and 25) was found among known pathovars of the species by rep-PCR. This may suggest that pistachio strains may be new pathovar/s of X. translucens . A host range study of pistachio strains was performed for this purpose (Chapter 5).
Despite the high level of genetic diversity within the pathogen based on rep-PCR and despite the differences between the pathogen strains and known pathovars of X. translucens , the high sequence similarities in ITS and 16S rDNA among the strains
Chapter 4: Characterisation of xanthomonads … 92 suggested that a close phylogenetic relationship exists within Xanthomonas strains associated with pistachio and also between these strains and pathovars of X. translucens . DNA-DNA hybridisation conclusively demonstrated that all of these strains are members of the same species.
Although multiple phenotypic and genotypic methods identified pistachio strains as X. translucens , the identification at subspecies level differed for each technique and strains most closely matched to different pathovars of the species. One explanation for this is that information about all known pathovars is not available in the databases.
Furthermore, this may confirm that strains of X. translucens cannot be reliably identified at the pathovar level based on phenotypic and genotypic characteristics and their pathogenicity to host plants must be determined (Dye et al. , 1980).
In summary, ITS sequencing and DNA-DNA hybridisation confirmed the existence of two distinct genotypes, A and B, within Xanthomonas strains associated with pistachio dieback in Australia. These groups align with the rep-PCR groupings described in
Chapter 3. Biochemical and physiological tests as well as protein profile analysis verified phenotypic differences between the two groups. As shown by ITS sequencing,
DNA-DNA hybridisation and phenotypic properties, the pistachio pathogen belongs to the species X. translucens. This species includes closely related pathovars causing bacterial leaf streak (BLS) in small grains and grasses, as well as several pathovars also isolated from the Poaceae that are not pathogens on small grains (Vauterin et al. , 1992).
Rep-PCR, however, showed that the pistachio strains differ from other pathovars of X. translucens . As a pathovar is assigned exclusively based on distinctive pathogenicity to one or more plant hosts (Dye et al. , 1980), two series of pathogenicity tests were performed and are discussed in Chapter 5.
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 93
Chapter 5: Pathogenicity of xanthomonads associated with pistachio
5.1 Introduction
Strains of Xanthomonas associated with pistachio dieback belong to X. translucens based on phenotypic and genotypic characteristics (Chapter 4). This species comprises closely related pathovars causing bacterial leaf streak (BLS) of small grains and grasses
(Vauterin et al. , 1992), along with several pathovars also isolated from the Poaceae that are not pathogenic on small grains (Vauterin et al. , 1995).
Because of the high degree of homogeneity within this species, especially among strains pathogenic on small grains, X. translucens pathovars cannot be reliably distinguished based on fatty acid analysis and SDS-PAGE of proteins (Stead, 1989; Vauterin et al. ,
1992), polyclonal antibodies (Azad & Schaad, 1988) or monoclonal antibodies (Bragard
& Verhoyen, 1993). Also, despite several attempts to differentiate the pathovars by
DNA-based techniques (Alizadeh et al. , 1997; Bragard et al. , 1995; Bragard et al. ,
1997; Rademaker et al., 2005), pathogenicity tests are required to assign new strains to a pathovar.
Since pistachio dieback is the first report of a bacterial disease caused by X. translucens on a plant other than the Poaceae , and because a pathovar is defined solely on the basis of distinctive pathogenicity to one or more plant hosts (Dye et al., 1980), pistachio strains were tested for pathogenicity in two series of tests on plants representing the
Anacardiaceae and Poaceae to clarify if group A and B strains represent different pathovars of the translucens species. Since the AQIS permit under which type and
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 94 reference strains of Xanthomonas were imported specifically precluded any testing on plant material, we were unable to include any of these strains in pathogenicity tests.
However, X. translucens pv. translucens DAR 35705, isolated from wheat in Australia and the only representative of this species available in Australia at the time, was used in the tests.
5.2 Materials and methods
5.2.1 Pathogenicity to members of the Anacardiaceae
The pathogenicity of two strains representing each group of the pathogen, as indicated in Chapters 3 and 4, viz. strain 7 from group A, and 25 from group B, was first determined by inoculation of 2-year-old P. vera cv. Sirora grafted on P. terebinthus in the glasshouse. X. t. pv. translucens DAR 35705 and sterile distilled water were used for negative controls. Inocula were prepared from 48-h-old cultures on YDC and photometrically adjusted to 1.2 × 10 8 colony forming units (CFU) mL -1 sterile distilled water. There were three replicate plants per treatment. Using a sterile syringe with a 25- gauge needle, bacterial suspension (0.1 mL) was injected at three locations per plant, in the trunk 5-7 cm above the graft union, at the junction of the trunk and the oldest branch, and at the junction of a petiole and the oldest branch. Inoculation was performed in spring (November) 2004. Following inoculation, plants were watered, incubated individually in transparent plastic bags at 24/17 °C, day/night temperature and
12-h photoperiod for 72 h, and monitored for symptoms at 7-9-day intervals for 90 days after inoculation. At the end of the experiment, internal symptoms were assessed and leaf and discoloured stem tissues were collected for pathogen re-isolation. Plant tissues were macerated in sterile distilled water and the suspensions plated on YDC after 15
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 95 min. Plates were monitored for yellow, mucoid colonies after 48 h and such bacteria were purified on YDC then compared with the inoculum strains using BOX-PCR.
To compare the host range of groups A and B, detached shoots of Anacardiaceae were inoculated with a mixture of two strains from each group of the pathogen, viz. strains 7 and 72 from group A, and 25 and 50 from group B. A suspension of killed bacteria (a mixture of strains 7, 25, 50 and 72), viable X. t. pv. translucens DAR 35705 and distilled water were used as negative controls. Bacteria were collected from 48-h old cultures on YDC in 3 mL MQ water and suspensions were adjusted to 1.2 × 10 7 colony forming units (CFU) mL -1 sterile distilled water by spectrophotometry.
Based on availability for the experiment, the following plants were used: pistachio,
Pistacia vera, P. vera cv . Sirora, P. integerrima, P. terebinthus, P. atlantica, P. chinensis, P. lentiscus and P. palaestina ; sumac, Rhus leptodictya and R. tripartita ; pepper tree, Schinus latifolius, S. lentiscifolius and S. polygamus ; mango, Mangifera indica and cashew, Anacardium occidentale (see section 2.5). For the last two plants, seedlings were grown in pasteurised potting mix in a growth room with 30/24 °C, day/night temperature, pots containing one seedling at the 3-leaf stage were transferred to the glasshouse and seedlings were used for the experiment. For P. vera cv . Sirora and
P. integerrima , excised shoots, 15-20 cm in length, were collected from the current season growth in November 2005. For the other plants, excised shoots, 15-20 cm in length, were collected from the current season growth in November and December
2004.
Three replicates were used for each inoculum-plant combination and three leaves and one bud were inoculated per replicate. Before inoculation, shoots were surface sterilised
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 96 using 70% alcohol and rinsed three times with sterile distilled water. Using a syringe with a 26-gauge needle, about 0.2 mL of the bacterial suspension was infiltrated into the leaf lamina and major veins until a water-soaked area was visible (Klement, 1990).
Also, 0.1 mL of the suspension was injected into the bud using a 25-gauge needle.
Inoculated shoots were placed in autoclaved Sigmaware TM Culture Tubes (C5916,
Sigma-Aldrich, NSW, Australia) containing 55 mL Murashige and Skoog basal medium
(MS) (M5519, Sigma-Aldrich, NSW, Australia). Each treatment was individually placed in an autoclaved transparent Sun Bag (B7026, Sigma-Aldrich, NSW, Australia) containing sterile, wet paper towel (Figure 5.1). Treatments were randomly distributed on the bench in a glasshouse with 24/17 °C, day/night temperature and natural light.
Plants were checked for symptoms 3, 5, 7, 9 and 14 days after inoculation. After 2 weeks, infected leaves and buds were collected for pathogen re-isolation. Leaves were macerated in a few drops of sterile distilled water and the suspensions were plated on
YDC after 15 min. The plates were incubated at 28 °C and inspected for pale yellowish colonies from 2 days onwards. Such colonies were purified on YDC and compared with the inoculum strains using BOX-PCR. The entire experiment was performed three times during November and December 2004 except for testing of P. vera cv . Sirora and P. integerrima which was performed twice in November and December 2005.
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 97
Figure 5.1 Pathogenicity test on the Anacardiaceae , showing humid chambers made by placing each inoculated, excised shoot in an autoclaved transparent Sun Bag containing sterile, wet paper towel
5.2.2 Pathogenicity to members of the Poaceae
Strains 7 and 25 were selected as representative of group A and group B, respectively.
Additionally, a mixture of five strains from each group (3, 7, 11, 28 and 73 from group
A and 20, 24, 27, 50 and 70 from group B) was used for comparison. X. t . pv . translucens DAR 35705 was inoculated as a positive control and two negative controls were used, distilled water and a suspension of killed bacteria (a mixture of the two groups as above). The pathogenicity of the bacteria was tested on bread wheat ( Triticum aestivum cv. Frame), durum wheat ( T. durum cv. Arrivato), barley ( Hordeum vulgare cv. Sloop), rye ( Secale cereale ), oat ( Avena sativa cv. Echidna), brome grass ( Bromus inermis ), triticale ( X Triticosecale cv. Tickit), timothy ( Phleum pratense ), cocksfoot
(Dactylis glomerata ), barley grass ( Hordeum leporinum ) and perennial rye grass
(Lolium perenne cv. Victorian) (see section 2.6). Most of these plants have been used in host range study of X. translucens pathovars (Duveiller et al. , 1997a). Rye grass cv.
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 98
Victorian was included in the experiment because it is grown close to pistachio orchards at Kyalite. Seeds were sorted for uniformity and surface sterilised with 1% sodium hypochlorite for 10 min, 70% ethanol for 5 s, 2% sodium thiosulphate for 5 min and rinsed three times with sterile distilled water after each stage. Seedlings were grown in pasteurised potting mix in a glasshouse with 22/17 °C, day/night temperature in natural light in December 2004. Pots containing three seedlings at the 3-leaf stage were transferred to a growth room with 25/17 °C and a 12-h photoperiod light (Osram
Powerarc 400W Metal Halide Lamps) (Figure 5.2). A randomised block design (RBD) was performed. Three replicates were used for each treatment-plant combination and three leaves were inoculated in each replicate. Bacterial suspensions were prepared from
48-h cultures on YDC and adjusted to 1.2 × 10 7 CFU mL -1 sterile distilled water by spectrophotometry. The suspension (0.1 mL) was infiltrated into the intercellular spaces using a syringe with a 26-gauge needle and two soft rubber stoppers (Klement, 1990)
(Figure 5.3). Inoculated plants were sealed in plastic chambers with a humidifier to produce 100% relative humidity for 4 days and then transferred to the glasshouse with conditions as described previously. Inoculated plants were watered from below and monitored for symptom development 3, 5, 7 and 10 days after inoculation. Observation of greasy and water-soaked streaks covered with exudate 3-5 days after inoculation was considered a compatible reaction (Bragard et al. , 1997) and further development of symptoms was recorded for the plants individually. Diseased leaves were collected for re-isolation of bacteria 10 days after inoculation. Leaves were macerated in a few drops of sterile distilled water and the suspensions were plated on YDC after 15 min as above.
Plates were monitored for yellow, mucoid colonies after 48 h and such bacteria were purified on YDC and compared with the inoculum strains using BOX-PCR. The experiment was performed once.
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 99
Figure 5.2 Growth room used for the pathogenicity test on plants from the Poaceae , showing incubation chambers and humidifier to supply a mist of water in the chambers
Figure 5.3 Method and device used for injecting bacterial suspensions into leaves of plants from the Poaceae (from Klement, 1990)
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 100
5.3 Results
5.3.1 Pathogenicity to members of the Anacardiaceae
Strains 7 and 25 induced symptoms on young pistachio plants. Necrosis developed at the branch-petiole junction 7-10 days after inoculation and at the trunk-branch junction
20-25 days after inoculation. The necrotic areas tended to enlarge in plants inoculated with strains 7 and 25. No symptoms were visible at the inoculation site near the graft union on the trunk. In plants inoculated with the pistachio strains, necrosis and dieback began on treated branches and foliage arising from them about 45 days after inoculation
(Figure 5.4), while untreated branches and their shoots remained healthy. At the end of the experiment, stained wood was observed in two treated branches, as well as one untreated branch adjacent to a branch inoculated with strain 7, while only one branch inoculated with strain 25 showed staining. No symptoms, internal or external, were observed on controls except for tiny necrotic areas at injection points. Inoculation of detached shoots with the pistachio strains resulted in water-soaked spot on leaves of excised shoots of all species of the Anacardiaceae tested, except R. leptodictya (Table
5.1), 3-4 days after inoculation with strains from groups A and B. A necrotic area expanded from the middle of this spot 5-7 days after inoculation. Such symptoms were identical for all three replicates for each treatment. Spots were surrounded by a yellowish halo on some inoculated leaves. Complete necrosis and collapse of some infected leaves were observed 14 days after inoculation (Figure 5.4). The spread of discolouration along the veins was clearly observed in some hosts (Figure 5.5).
Although not quantified, strains from group A seemed to be more aggressive than those from group B, because appearance and progress of lesions appeared to be more rapid in leaves inoculated with group A strains. Furthermore, white-grey bacterial ooze was
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 101 observed from some buds inoculated with strains from group A but not group B (Figure
5.6). Strains from group A were pathogenic to P. lentiscus whereas strains from group B failed to induce any symptoms on inoculated leaves of this species (Figure 5.7). Leaves treated with sterile distilled water, killed bacterial cells or X. t. pv . translucens DAR
35705 did not show water-soaked lesions. From all samples which showed the symptoms, bacterial strains were recovered on YDC culture medium and assigned to group A or B of the pathogen by BOX-PCR (data not shown).
5.3.2 Pathogenicity to members of the Poaceae
Pistachio pathogen strains belonging to both groups induced water-soaked and greasy areas with bacterial exudates within 3-5 days of inoculation on bread wheat, durum wheat, barley, rye, oat, brome grass, triticale, barley grass and cocksfoot (Table 5.2).
Streaks with a greasy appearance developed after 7 days. A translucent lesion with a yellow to light brown halo occurred on some leaves. Typical symptoms caused by pistachio dieback pathogen strains are shown in Figure 5.8. Strains from group A failed to induce lesions on timothy whereas strains from group B produced water-soaked streaks 3-5 days after inoculation, followed by necrosis and collapse 7-10 days after inoculation (Figure 5.9). Likewise, strains from group B did not induce any symptoms on rye grass whereas strains from group A produced symptoms as described above
(Figure 5.10). X. t . pv. translucens DAR 35705 was pathogenic on all plants tested except cocksfoot and rye grass. Leaves treated with water or killed bacteria remained healthy. From all samples which showed the symptoms, bacterial strains were recovered on YDC culture medium and assigned to group A or B of the pathogen by BOX-PCR
(data not shown).
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 102
Table 5.1 Differences among xanthomonads isolated from pistachio and Xanthomonas translucens pv. translucens DAR 35705 from wheat, based on pathogenicity to detached leaves of representatives of the Anacardiaceae
Pistachio strains a X. t. pv . translucens Host Group A Group B DAR 35705 Pistacia vera + + _
P. vera cv . Sirora + + _
P. integerrima + + _
P. atlantica + + _
P. terebinthus + + _
P. chinensis + + _
P. lentiscus + _ _
P. palaestina + + _
Rhus leptodictya _ _ _
R. tripartita + + _
Schinus latifolius + + _
S. lentiscifolius + + _
S. polygamus + + _
Mangifera indica _ _ _
Anacardium occidentale _ _ _
a Inoculum comprised a mixed suspension of two strains representing group A or two strains representing group B. +: Positive reaction (water-soaked area on leaf lamina within 4 days of inoculation, followed by development of necrosis), _: negative reaction or chlorotic symptoms restricted to inoculated areas only. Symptoms were identical in three replicates for each treatment. There were no symptoms on controls treated with sterile distilled water or killed bacteria (data not shown)
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 103
A B
C D
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 104
E F
Figure 5.4 Symptoms caused by pistachio dieback pathogen strains on members of the
Anacardiaceae . A and B, necrosis and dieback on branches and foliage of 2-year-old
Pistacia vera cv. Sirora grafted on P. terebinthus caused by the pathogen strains belonging to groups A and B, respectively, 60 days after inoculation; C, necrosis and collapse of leaves of P. vera cv. Sirora 14 days after inoculation by group A strains; D, necrosis of bud and young stem of P. vera 9 days after inoculation by group A strains;
E, water-soaked and necrotic lesions on leaves of P. terebinthus 7 days after inoculation by group A strains; F, necrotic lesions on leaf of P. terebinthus 14 days after inoculation by group A strains.
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 105
A B
Figure 5.5 The spread of discolouration along the veins caused by the pistachio dieback pathogen strains, belonging to group A, on inoculated leaves of Schinus latifolius ( A) and its close up ( B) 7 days after inoculation
Bacterial ooze
Figure 5.6 A fresh white-grey mass of Xanthomonas cells, belonging to group A, oozing from the inoculated bud of Pistacia terebinthus 7 days after inoculation
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 106
A
B
Figure 5.7 Pathogenicity of pistachio dieback pathogen strains on excised shoots of
Pistacia lentiscus . A, leaf necrosis caused by group A strains 7 days after inoculation; and B, leaves inoculated with group B strains
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 107
Table 5.2 Differences among xanthomonads isolated from pistachio and Xanthomonas translucens pv . translucens DAR 35705 isolated from wheat based on pathogenicity to representatives of the Poaceae
Plant Pistachio strains a X. t. pv . translucens A B DAR 35705 Wheat (bread) + + +
Wheat (durum) + + +
Barley + + +
Rye + + +
Oat + + +
Brome grass + + +
Triticale + + +
Barley grass + + +
Cocksfoot + + -
Timothy - + +
Rye grass + - -
a Inoculum comprised one strain or a mixture of five strains representing group A, or one strain or a mixture of five strains representing group B.
+: compatible reaction (water-soaked and greasy area covered by exudates on leaf lamina within 3-5 days of inoculation, followed by necrosis), -: incompatible reaction
(without any symptoms or chlorotic symptoms restricted to inoculated areas only).
There was no difference between treatments inoculated by one strain or a mixture of five strains representing each group. Symptoms were identical in three replicates for each treatment and there were no symptoms on controls treated with sterile distilled water or killed bacteria (data not shown)
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 108
A
B
C
D
E
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 109
F
G
Figure 5.8 Symptoms caused by xanthomonad strains associated with pistachio dieback on plants of the Poaceae , A, water-soaked areas caused by strains of group A on wheat
(cv. Frame) leaves; B, bacterial streak (with yellow halo) on oat leaf caused by strains of group B; C, from top to bottom: yellow lesions with expanding necrosis from the middle of lesion, translucent streaks and necrosis on wheat (cv. Frame) leaves caused by strains of group A; D, greasy areas surrounded by yellow halo caused by strains belonging to group B on wheat (cv. Arrivato) leaves; E, streak, with yellow halo, on wheat (cv. Arrivato) caused by X. t . pv. translucens DAR 35705; F, translucent and necrotic leaf streaks on cocksfoot leaves caused by strains of group A; and G, top: greasy area on leaf lamina of brome grass caused by strains of group B and bottom: purple stripes on brome grass leaf caused by strains of group A
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 110
A
B
Figure 5.9 Reaction on timothy leaves 7-9 days after inoculation with A, strains of group A, and B, strains belonging to group B
A
B
Figure 5.10 Response on rye grass leaves 7-9 days after inoculation with A, strains of group A, and B, strains belonging to group B
5.4 Discussion
The assignation of the pistachio dieback pathogen to Xanthomonas translucens (Chapter
4) was confirmed by pathogenicity. Furthermore, differential host ranges in
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 111 pathogenicity tests on representatives of the Anacardiaceae and Poaceae revealed a biological difference between the two phenotypic and genetic groups of the pathogen.
The virulence determination of a new pathogen, especially one which causes shoot death and dieback, using the hypersensitive reaction (HR) test is critical. After determining the HR response, further pathogenicity tests can be conducted to determine the host range and pathogenic variation. Strains in group A induced a typical HR on tobacco leaves, indicating that they were pathogenic, whereas strains in group B did not
(see section 4.3.1). An important point in the HR test is that not all plant pathogenic bacteria can induce HR in tobacco leaves (Klement, 1982). Furthermore, in some host- pathogen combinations, particularly in the case of Xanthomonas strains, specific criteria such as high concentration of inoculum and incubation of plants at a certain temperature before and after inoculation are needed to achieve a consistent result for the test
(Klement, 1982). Group B strains may require such specific conditions to induce the HR response.
Groups A and B could also be distinguished by pathogenicity tests on representatives of the Anacardiaceae and Poaceae , which revealed the two groups differed in host range.
In X. translucens , a single strain can have a wide host range variation (Duveiller et al. ,
1997a) and it has been shown that transposon and chemical mutagenesis can change the host range (Mellano & Cooksey, 1988). Attempts were made to quantify the differences in disease severity caused by strains of each group by measuring the percent leaf area affected or the lesion length. However, there was a considerable degree of variation in the data, mainly because we could not standardise the inoculum concentration infiltrated into the leaf blade and it was not possible to estimate percentages accurately over the entire experiment. Likewise, the evaluation of disease caused by X. translucens strains
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 112 in cereal leaves always has been difficult; various scales have been used to measure disease severity and estimates vary considerably from person to person (Duveiller et al. ,
1997a). Nevertheless, group A appeared to be more aggressive than group B, in terms of the HR response, development of symptoms in inoculated plants and producing bacterial ooze in inoculated buds. Further studies are needed to compare disease severity caused by the two groups, to determine the structure and function of the genes involved in pathogenicity and host range determination, and also to investigate virulence determinants in the pistachio strains.
The term “pathovar” has been described as “a strain or set of strains with the same or similar characteristics, differentiated at the infrasubspecific level from other strains of the same species or subspecies on the basis of distinctive pathogenicity to one or more plant hosts” (Dye, 1980). X. translucens includes several pathovars which have been reported as pathogens of the Poaceae only, such as X. t. pv. cerealis , X. t. pv. graminis ,
X. t. pv. arrhenatheri , X. t. pv. phlei , X. t. pv. phleipratensis , X. t. pv. poae , X. t. pv. secalis , X. t. pv. translucens , X. t. pv. undulosa and X. t. pv. hordei (Vauterin et al. ,
1995). Based on the most recent International Society for Plant Pathology (ISPP) names of plant pathogenic bacteria (Young et al. , 1996), X. t. pv. hordei is no longer a valid name and is considered a synonym of X. t . pv. translucens for strains which are pathogenic on barley only (Duveiller et al. , 1997a). In this study, pistachio strains were pathogenic to several members of the Anacardiaceae and Poaceae , which complicates assigning pistachio strains to one of the known pathovars of the species. Based on the results, xanthomonads pathogenic on pistachio differ from known pathovars of X. translucens and the differences between the groups in terms of host range suggests that they are two new pathovars of the species.
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 113
Both the scion ( Pistacia vera ) and the most popular rootstocks in Australia ( P. atlantica , P. terebinthus and P. integerrima ), showed symptoms following artificial inoculation of leaves. A pistachio scion grafted on to an aggressive rootstock may be more susceptible to the disease than that same scion grafted on to a less aggressive rootstock, because aggressive rootstocks usually result in retardation of shoot growth. It has been shown that scion/rootstock combination has a significant influence on the incidence and severity of bacterial diseases (Leite & Santos, 1988; Sayler et al. , 2002;
Spotts & Mielke, 1999). Therefore, it would be important to assess and compare the susceptibility of pistachio cultivar/rootstock combinations to the disease.
Previous attempts to confirm the pathogenicity of one Xanthomonas strain isolated from pistachio by injection of bacteria into the trunks of 2-year-old grafted trees in pots in autumn resulted in discolouration of wood and re-isolation of the bacteria from various parts of the plants (Facelli et al. , 2002; Facelli et al. , 2005), and monitoring of inoculated trees for trunk and limb lesions continues on the basis that such symptoms are observed in trees 6-7 years old (Pistachio canker epidemiology, Project NT99004,
Horticulture Australia Limited). In our study, however, multiple inoculation points, especially close to the apex, inoculation in spring and providing a moist environment for several days after inoculation resulted in necrosis and dieback. X. t . pv . translucens
DAR 35705 was not pathogenic to pistachio, demonstrating that despite the close relatedness of pistachio and cereal strains they differ in pathogenicity.
Inoculation of excised shoots, as a manageable and less time-consuming method, was used to compare groups A and B with respect to host range. Strains representative of both groups induced lesions on leaves of shoots excised from trees belonging to the
Anacardiaceae , whereas such symptoms have not been observed in pistachio orchards.
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 114
This requires further study, especially for group A strains that are HR positive, because the work of Klement (1982) suggests that all xanthomonad species and pathovars which create local lesions on their host plant have the potential to induce HR on a non-host plant. The appearance of symptoms on excised shoots may reflect optimal conditions for infection in the bioassay, or leaves on excised shoots incubated in moist conditions may be more susceptible than those on the tree. Pathogenicity of Xanthomonas and
Pseudomonas species on excised plant material has been correlated with results obtained following inoculation in the field (Randhawa & Civerolo, 1985; Yessad et al. ,
1992) which suggests further pathogenicity tests, with the pathogen strains, in pistachio orchards are warranted. Using detached plant parts in pathogenicity tests is more convenient than the inoculation of plants in the field because excised plant material can easily be manipulated in laboratory conditions and inoculation of small parts under optimal controlled conditions often shows a rapid response (Randhawa & Civerolo,
1985). Detached plant material has been used to demonstrate the pathogenicity of various xanthomonads (Fahy & Hayward, 1983; Goncalves & Rosato, 2000; Randhawa
& Civerolo, 1985; Sharon et al. , 1982). Pathogenicity of pistachio strains on other plants of the Anacardiaceae suggests the possibility that Xanthomonas belonging to groups A and B infect foliage of plants other than cultivated pistachio in nature, albeit rarely, and that the pathogen may have been introduced from other members of the
Anacardiaceae to pistachio. Further study and survey of species of the Anacardiaceae existing in Australia, especially those growing close to pistachio orchards, is needed to assess this possibility.
Pistachio strains were pathogenic to small grains and grasses. This suggests that the pathogen strains may have been introduced to pistachio from grasses, and the two groups may have evolved from different ancestors (e.g., X. translucens strains
Chapter 5: Pathogenicity of xanthomonads associated with pistachio 115 indigenous and exotic to Australia) or have been introduced on two different occasions.
X. translucens strains have already been reported in Australia (Moffett & McCarthy,
1973; Noble, 1935) and X. translucens strains, although different from the pistachio pathogen strains in rep-PCR fingerprint and ITS sequence (see section 6.3.1), have been found regularly on grasses collected from pistachio orchards. It would be of interest to determine if these strains may have crossed from grasses to pistachio. Also, it is important to determine if pistachio strains can colonise grasses in and around pistachio orchards or cereal fields close to the orchards. These plants may act as alternative hosts and may serve as a reservoir of the pathogen.
It should be noted that the Australian pistachio industry is based on the female cultivar,
Sirora, which was selected to suit Australian conditions and currently is the major female cultivar grown in all pistachio orchards. The sensitivity of this cultivar to the disease should be compared with that of cultivars grown in other countries.
Chapter 6: Detection of xanthomonad pathogens of pistachio 116
Chapter 6: Detection of xanthomonad pathogens of pistachio in Australia
6.1 Introduction
While pistachio dieback is considered to be a serious threat to pistachio orchards in
Australia (Anonymous, 2002), an efficient and reliable method to identify the pathogen in pure culture or plant material was lacking at the beginning of this project. The presence of the two groups within the pistachio pathogen (Chapters 3, 4 and 5) complicated the detection of the pathogen. Routine disease diagnosis depended upon isolation of bacteria from infected woody tissues on agar media followed by physiological and biochemical tests to identify the pathogen. This process was laborious, unreliable and time-consuming, often requiring several weeks to complete.
Therefore, development of a faster PCR-based method was a priority in the study of the disease, with the ability to distinguish the two groups an important component of any detection method. Detection of the pathogen in plant material, especially asymptomatic infected plants, would assist in selection of diseased-free planting material and minimise risk from the pathogens.
PCR-based methods are being used increasingly for the diagnosis of phytopathogenic bacteria and have proved to be fast, sensitive and reliable for detection and diagnosis
(see section 1.5.2.3). As reported in chapters 4 and 5, pistachio dieback pathogen strains belong to X. translucens . A primer set has been reported for specific detection of X. translucens (Maes et al. , 1996b). These primers, T1 & T2, were designed to target the
ITS region, to detect Xanthomonas pathogens that cause cereal leaf streak in seed.
Chapter 6: Detection of xanthomonad pathogens of pistachio 117
However, the assay detects ten pathovars of X. translucens and does not distinguish between them. Xanthomonas strains isolated from pistachio differ from X. translucens in certain phenotypic, genotypic and pathogenic features (Chapters 4 and 5), therefore, methods used for X. translucens associated with cereals may be unsuitable for the specific detection of the pistachio pathogens.
As shown in Chapter 4, the 16S-23S rDNA sequences of strains in group A and group B of the pistachio dieback pathogen were identical within each group and the two groups differed in terms of a few bases in this region. This provided the opportunity for the design of primers specific for each group.
The objectives of the work reported in this chapter were to (i) evaluate published X. translucens -specific primers in PCR for the identification of the pistachio dieback pathogen and, subsequently, to (ii) develop a PCR protocol for the simultaneous detection of both groups of the pathogen.
6.2 Materials and methods
All pistachio dieback pathogen strains listed in Table 2.1, as well as other bacteria, including type and references strains of Xanthomonas (Table 2.2) and bacteria isolated from plants in and around pistachio orchards (see section 2.2), were grown on TSA
(Appendix B) at 28 °C for 48 h. Genomic DNA was extracted following the method of
Rademaker & de Bruijn (1997) as outlined in section 2.6. All amplification reactions were performed on a Peltier Thermal Cycler model PTC-200 (MJ Research Inc., CA,
USA).
Chapter 6: Detection of xanthomonad pathogens of pistachio 118
6.2.1 Detection of bacteria with X. translucens -specific primers, T1 &
T2
Published X. translucens -specific primers, T1 (5’-CCGCCATAGGGCGGAGCACC
CCGAT-3’) and T2 (5’-GCAGGTGCGACGTTTGCAGAGGGATCTGCAAATC-3’)
(Maes et al. 1996b) were tested to determine if they could be used for the specific amplification of DNA from the pistachio pathogen. DNA from all pathogen strains and phytobacteria isolated from plants in and around pistachio orchards was tested including strains of Xanthomonas isolated from orchard floor grasses. X. translucens DAR 35705 was used as a positive control and X. axonopodis pv. malvacearum DAR 26904 and X. arboricola pv. pruni DAR 64858 were used as negative controls. Primers were synthesised by Proligo Pty Ltd, Lismore, Australia and PCR amplification was performed as described by Maes et al. (1996b) with minor modification. The reaction mixture was prepared in a total volume of 20 µL, comprising 0.2 µL Taq polymerase (5
U µL-1, Invitrogen Pty Ltd, Victoria, Australia), 2 µL 10 × PCR buffer (200 mM Tris-
HCl (pH 8.4), 500 mM KCl), 0.6 µL MgCl 2 (50 mM), 0.16 µL dNTP mixture (25 mM each), 0.8 and 0.6 µL of T1 and T2 primers (10 µM each), respectively, 1 µL of sample
(50 ng µL-1) and 14.64 µL distilled water. PCR was performed with the following conditions: 1 × 90 °C for 2 min, 35 × (93 °C for 30 s, 60 °C for 45 s, 72 °C for 1 min) and
72 °C for 10 min. The PCR product (5 µL) was analysed by electrophoresis on a 2% agarose gel in TAE buffer (Appendix A) at 10 V cm –1 for 70 min, staining with ethidium bromide (0.6 µg mL -1) and visualising under UV light.
Chapter 6: Detection of xanthomonad pathogens of pistachio 119
6.2.2 PCR assay for specific detection of the pistachio pathogens
The complete 16S-23S rDNA spacer region sequence was determined for X. translucens
DAR 35705 and one X. translucens strain obtained from orchard floor grasses and detected by the T1 & T2 primer set, as described in section 4.2.4. Then, sequences of the 16S-23S rDNA spacer region determined for pathogen strains from group A and group B (see sections 4.2.4 and 4.3.4) were compared to those of X. translucens DAR
35705 and the grass strain, to design specific primers. Corresponding sequences of the closely related bacteria, X. translucens strain LMG 876, X. translucens pv. graminis and
X. translucens pv. poae , available in the GenBank database under accession numbers
AF209764, AY247064 and AY253329, respectively, were included for comparison.
Alignment and comparison of the sequences were performed with the programs Clustal
X and GeneDoc available on the Bioinformatics.Net database.
6.2.2.1 Selection and design of primers
Sequences in the ITS which diverged between group A and group B strains, and between pistachio strains and other xanthomonads, were identified as sites for the design of primers. Several primers were designed for specific identification of group A and B strains. The design of some primers was based on the Amplification Refractory
Mutation System (ARMS) (Newton et al. , 1989), in which a primer is designed so that it can discriminate between templates which differ in a specific single nucleotide. The oligonucleotides were synthesised by GeneWorks Pty Ltd, Adelaide, Australia.
Chapter 6: Detection of xanthomonad pathogens of pistachio 120
6.2.2.2 PCR cycling conditions
For the initial assessment of the primers, a 25-µL reaction volume was used, comprising
20 ng DNA (representative strain of group A or group B), 0.1 µL Taq polymerase (5 U
µL-1, Qiagen Pty Ltd, Victoria, Australia), 2.5 µL 10 × PCR buffer, 0.2 µL dNTP mixture (25 mM each); and 1.25 µL of forward and reverse primers (10 µM each). The volume was adjusted to 25 µL with sterile distilled water. The preliminary PCR was carried out with the following program: 1 × 95 °C for 15 min, 30 × (94 °C for 45 s, 60 °C for 45 s, 72 °C for 1 min) and 72 °C for 5 min. Six µL of the PCR product were subjected to electrophoresis on an agarose gel (2%) using Tris-acetate EDTA (TAE) buffer at 10
V cm –1 for 70 min, stained with ethidium bromide (0.6 µg mL -1) and visualised under
UV light. The suitability of the primers was assessed using DNA from representative strains from the two groups of the pathogen, 10 type and reference pathovars of X. translucens , X. translucens DAR 35705 and the X. translucens -like bacterium isolated from pistachio orchard grasses as template in the PCR. Further optimisation of the PCR mixture, especially MgCl 2, Taq polymerase and primer concentrations, and optimisation of cycling conditions, especially annealing temperature and number of cycles, was carried out to achieve the best conditions for specific identification of the pathogen strains. Two forward primers, hereafter named PAf (specific for groups A strains) and
PBf (specific for groups B strains), and one reverse primer, for both groups (PABr) were selected. The comparison of these primers with DNA sequences in GenBank,
EMBL, DDBJ and PDB databases, using the BLAST program, revealed that the primers would be highly specific for pistachio pathogen strains.
To distinguish group A and B strains from one another and from other bacteria in a single assay, a multiplex PCR, in which both forward primers and the reverse primer are
Chapter 6: Detection of xanthomonad pathogens of pistachio 121 used simultaneously, was developed and conditions were optimised by changes in both the PCR mixture and the cycling program, as described by Henegariu et al. (1997).
6.2.2.3 PCR specificity and sensitivity tests
To assess the specificity of the designed primers, the PCR was carried out with DNA from all pathogen strains (Table 2.1), type and reference strains from the genus
Xanthomonas (Table 2.2) and various bacteria collected from in and around pistachio orchards (section 2.2). To determine the detection limit of the primers, 10-fold serial dilutions were made from both representative cultures of the pathogen (strains 7 and 25 representatives of group A and group B, respectively) and 5-µL aliquots from each dilution were added directly to the PCR mixture. The number of cells in each series was assessed by counting CFU following plating on YDC agar medium. This assessment was performed for each strain individually and repeated three times.
6.2.2.4 PCR efficiency in plant material and verification
Samples comprising leaves and woody tissue were collected from shoots, 2-3 years old, from symptomatic and asymptomatic trees in pistachio orchards naturally infected by group A (Kyalite) or B (Robinvale) in February, March and April 2004. Shoot samples were also collected from a healthy tree in a non-infected area at Waite campus, The
University of Adelaide, as a negative control. In addition, samples from woody tissue,
2-3 years old, from artificially infected trees were provided by Dr. Evelina Facelli in
April and May 2004. The trees were infected with the pathogen strains from group A and group B, individually, 20 months earlier or infected with strains from both groups 5 weeks before in a glasshouse. Several DNA extraction protocols and detection methods were tested in order to develop a suitable assay which could detect xanthomonad
Chapter 6: Detection of xanthomonad pathogens of pistachio 122 pathogens of pistachio in plant tissue and could be used as a tool in epidemiological studies of the disease.
A preliminary experiment indicated that pistachio plant material, especially woody tissue, had a significant inhibitory effect in PCR, because no amplification was achieved even when a mixture comprising plant tissue plus pathogen genomic DNA was used as template in PCR. Polyvinylpyrrolidone (PVP) has potential to enhance the amplification by adsorbing inhibitory components such as polyphenolics and can be used during extraction of bacterial DNA from plant material or directly in the PCR (Fegan et al. ,
1998; Koonjul et al. , 1999, Maes et al. , 1996a; Meng et al. , 2004). In a further experiment, woody tissue (500 mg) of a sample collected from a healthy tree in March
2004 was chopped and soaked in 1 mL MQ water. After 20 min, 5 µL of the resulting water extract was used in the PCR mixture along with DNA of the pathogen (10 ng).
PVP (MWt 44,000) was included in the reaction mixture at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4,
4.5 and 5% w/v. This experiment was performed three times in March and April 2004.
For initial screening, 10 samples with internal staining, characteristic of the disease, collected from orchards in Kyalite and Robinvale, and from the glasshouse, plus one sample from a healthy tree, were selected. The samples were checked for the presence of the pathogen by soaking woody tissue (500 mg) in distilled water for 15 min and plating 100-µL aliquots from a 10-fold serial dilution of the extract on YDC plates
(Appendix B). The plates were incubated at 28 °C and inspected for pale yellowish colonies from 2 days onwards. Such colonies were purified on YDC and their identity was confirmed by comparing them with representatives of groups A and B using BOX–
Chapter 6: Detection of xanthomonad pathogens of pistachio 123
PCR (see section 3.2.1). Also, using these samples, the efficiency of the following methods was evaluated.
6.2.2.4.1 Rapid extraction protocols
A simple and rapid protocol based on those developed by Pan et al. (1997) for the detection of X. albilineans in sugarcane and by Verdier et al. (1998) for X. axonopodis in cassava tissue was tested. Woody tissue, 500 mg, was excised from shoots, chopped by a scalpel and soaked in 1 mL sterile MQ water in a 1.5-mL microcentrifuge tube.
After 15 min, it was vortexed and 5 µL of the liquid was used in PCR. Also, soaked samples were kept at room temperature overnight, the suspension was then transferred to a fresh tube and spun at 4,000 rpm for 10 min. The pellet was washed twice and resuspended in 20 µL water. Each sample was serially diluted in 10-fold increments in sterile distilled water and 5 µL of each sample was used in the PCR assay. A replicate reaction was conducted using PVP (MWt 44,000) in the reaction mixture at final concentration of 1% (w/v).
6.2.2.4.2 Proteinase-based method
The method of Hartung et al. (1993) was used with slight modification. Woody tissue
(approximately 500 mg) was chopped in 750 µL sterile MQ water and left at room temperature for 15 min. The resulting extract (5 µL) was used as template in PCR. The mixture was incubated at 95 °C for 5 min, then proteinase K (Sigma-Aldrich, P-8044) was added (final concentration: 30 µg mL -1) and the mixture was incubated at 55 °C for
15 min to lyse any bacteria. Proteinase K was inactivated by incubation of the mixture at 95 °C for 10 min and the amplification was performed. To check the effect of PVP on
Chapter 6: Detection of xanthomonad pathogens of pistachio 124
PCR amplification, a replicate reaction was included using PVP (MWt 44,000) in the reaction mixture at a final concentration of 1% (w/v).
6.2.2.4.3 CTAB-based method
DNA was extracted from 10 samples of diseased wood and the sample collected from a healthy tree by the method of Green et al. (1999) and Hartung et al. (1993) with the following modification: small pieces of woody tissue (500-750 mg) were ground in liquid nitrogen, and then transferred to 5 mL centrifuge tubes. CTAB extraction buffer
(5 mL, Appendix A) was added and the sample was incubated at 60 °C for 45 min with occasional mixing. After centrifugation at 6,000 rpm for 10 min, the supernatant was removed and mixed with 0.5 volume chloroform: iso -amyl alcohol (24:1) with vigorous shaking until the solution was homogeneously milky. The suspension was centrifuged at
6,000 rpm for 15 min, the supernatant was removed, and again extracted by centrifuging with an equal volume of chloroform: iso -amyl alcohol. The upper phase was carefully removed using a 1,000 µL tip and added to a fresh tube containing 0.6 volume of iso - propanol (-20 °C). The suspension was shaken gently and centrifuged at 3,000 rpm for 3 min and then at 5,000 rpm for 3 min. The supernatant was removed and the pellet was washed with 150 µL ethanol (70%) twice. The DNA pellet was air-dried and redissolved in 100 µL TE buffer (Appendix A). Five µL of a 1:10 and 1:100 dilution of the suspension were subjected to amplification using PCR.
6.2.2.4.4 Bio-101 DNA extraction kit
DNA was extracted from 10 samples from diseased wood and one sample collected from a healthy tree, using the Bio-101 (Bio-101, USA) extraction method as recommended by the manufacturer. Aliquots (2 µL) of a 1:10 and 1:100 dilution of the
Chapter 6: Detection of xanthomonad pathogens of pistachio 125 original DNA extract were subjected to PCR. A replicate reaction was carried out by the inclusion of PVP (MWt 44,000) in reaction mixtures at a final concentration of 1% w/v.
6.2.2.4.5 BIO-PCR
BIO-PCR combines biological and enzymatic amplification of target bacterial cells. It has been useful for the detection of phytopathogenic bacteria in plant materials when the pathogen is present in low numbers and enrichment is required (Fatmi et al. , 2005;
Sakthivel et al. , 2001; Schaad et al. , 1995; Wang et al. , 1999). The following method was developed for detection of xanthomonad strains isolated from pistachio.
Woody tissue (500 to 750 mg) was removed from 10 shoot samples which showed internal staining, as well as from one healthy sample, and soaked in 10 mL of nutrient broth overnight in a shaking-incubator at 27 °C. The resulting suspension was transferred to a fresh tube and centrifuged at 5,000 rpm for 5 min. The pellet was resuspended in 100 µL sterile distilled water and a 5-µL aliquot was used as template in
PCR. A parallel series of PCR was conducted using PVP (MWt 44,000) in each reaction mixture (1% w/v final concentration).
6.3 Results
6.3.1 Detection of bacteria with X. translucens -specific primers, T1&T2
A 139-bp fragment, specific for X. translucens pathovars (Maes et al. , 1996b), was amplified using primers T1 & T2 from all pathogen strains, including strains of group A and B, X. translucens DAR 35705, the positive control, and 15 X. translucens -like
Chapter 6: Detection of xanthomonad pathogens of pistachio 126 bacteria isolated from grasses in pistachio orchards. In contrast, no amplicon was obtained from the negative controls (Figure 6.1).
1000 800
600
400
300
200
100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 6.1 . Electrophoretic analysis of PCR–amplified DNA using Xanthomonas translucens -specific primers, T1 & T2. Lanes: 1 and 20 , molecular weight markers (bp)
(100 bp ladder, Invitrogen, Australia); lanes 2-8, pistachio pathogen strains from group
A; 9-14 pathogen strains from group B; lane 15 , X. translucens pv. translucens DAR
35705; lane 16 , X. translucens (strain 61W) isolated from pistachio orchard grasses; lane 17 , X. axonopodis pv . malvacearum DAR 26904; lane 18, X. arboricola pv. pruni
DAR 64858 and lane 19 , the PCR mixture without DNA
Xanthomonas strains isolated from grasses in pistachio orchards in Kyalite and
Robinvale, which were detected by T1 & T2, yielded identical fingerprinting profiles using BOX-PCR (data not shown). One representative of these strains, strain 61W, was selected and compared with representatives of groups A (strain 7) and B (strain 25) of the pistachio pathogen using BOX-, REP- and ERIC-PCR (see section 3.2.1). This demonstrated that Xanthomonas strains commonly found on grasses in pistachio orchards differed genetically from xanthomonads causing dieback in pistachio (Figure
6.2).
Chapter 6: Detection of xanthomonad pathogens of pistachio 127
1 2 3 4 5 6 7 8 9
5090 4072
3054
2036
1636
1018
506
396 344 298
A B G A B G A B G
Figure 6.2 Comparison of genomic DNA fingerprinting patterns from Xanthomonas isolated from grasses in pistachio orchard floor with those from representative strains of
Xanthomonas isolated from pistachio, obtained by rep-PCR. Lanes 1, 2 and 3, amplicons generated by BOX-PCR; lanes 4, 5 and 6, generated by ERIC-PCR; lanes 7,
8 and 9, generated by REP-PCR. A, pistachio pathogen strain belonging to group A
(strain 7), B, pistachio pathogen strain belonging to group B (strain 25) and G, grass strain (strain 61W). Numbers on the left are molecular size markers (bp) (1 kb ladder,
Invitrogen, Australia)
6.3.2 PCR assay for specific detection of the pathogen
Alignment of the complete 16S-23S rDNA spacer region obtained from pistachio strains with those obtained from X. translucens pv . translucens DAR 35705 and X. translucens
Chapter 6: Detection of xanthomonad pathogens of pistachio 128
(strain 61W) isolated from grasses in pistachio orchards, and from X. translucens pv . graminis (Genbank, AY247064), X. translucens pv . poae (Genbank: AY253329) and
Xanthomonas translucens pv . translucens (Genbank: AF209764), sequences of three closely related bacteria in the GenBank database, is shown in Figure 6.3.
6.3.2.1 Selection and design of primers
Based on the 16S-23S rDNA sequencing data shown in Figure 6.3, 15 primers were designed for specific detection of the pistachio dieback pathogens. The specificity and sensitivity of the primers varied when their capability was assessed using DNA from representative strains from the two groups of the pathogen, 10 type and reference pathovars of X. translucens , X. translucens DAR 35705 and X. translucens isolated from pistachio orchard grasses as template in the PCR. Two forward primers, PAf and
PBf, specific for group A and group B respectively, and PABr, a single reverse primer specific to the sequence conserved between the two groups, were selected as the most suitable primers (Table 6.1). PAf, an ARMS primer (Newton et al. , 1989), is complementary to the corresponding sequence of the group-A pathogen strains except for one additional deliberate mismatch, G/C, at the third nucleotide from the 3’-OH terminus of the primer. However, there are two mismatched nucleotides, A/G at the 3’- end and G/C three bases from the 3’-end, between this primer and the corresponding sequence of the other bacteria, including group B. The deliberate mismatch was introduced to enhance discrimination between group A strains and other bacteria. Using the primers, a multiplex PCR assay was developed to amplify a 331-bp group A- specific DNA fragment and a 120-bp group B-specific DNA fragment in a single test.
Chapter 6: Detection of xanthomonads pathogens of pistachio 129
* 20 * 40 * 60 * 80 * 100 * 120 * 140 X.g : ...... : 140 X.gr : ...... : 140 X.p : ...... : 140 X.t.Au: ...... T...... : 140 X.t : ...... G...... : 140 PB : ...... : 140 PA : ...... A...... : 140 GGCTGGATCACCTCCTTTTGAGCATGACAGCTACGCCTACAGGCGTCCTCACAAGTAACCTGCATTCAGAGAGTTCCGCCACAGGGCGGAGCACCCCGATTTCGGGGCCATAGCTCAGCTGGGAGAGCACCTGCTTTGCA CCTCCTTTTGAGCATGAGAA
* 160 * 180 * 200 * 220 * 240 * 260 * 280 X.g : ...... CT.T...TT...... : 280 X.gr : ...... T.T...TT...... : 280 X.p : ...... G.A.C...... : 280 X.t.Au: ...... T...... : 280 X.t : ...... T...... : 280 PB : ...... -...... T...... A....A...... G...... : 279 PA : ...... T...... : 280 AGCAGGGGGTCGTCGGTTCGATCCCGACTGGCTCCACCAGATTTGCAGATCCCTCTGCAAACGCCCGCACCTGCGTGTGCGGACGGTCTCAGGGACCTGCAAGAGCCAAGACTTTGGGTCTGTAGCTCAGGTGGTTAGAG
* 300 * 320 * 340 * 360 * 380 * 400 * 420 X.g : ...... A...... C...... : 420 X.gr : ...... C...... : 420 X.p : ...... C...... A...... : 420 X.t.Au: ...... : 420 X.t : ...... ----...... -....-...... -...... G...... : 413 PB : ...... A...... : 419 PA : ...... : 420 CGCACCCCTGATAAGGGTGAGGTCGGTGGTTCGAGTCCTCCCAGACCCACCACTCTGAATGTAAGAAGCACACTAAGAATTTAAGATGCGCCAGCAGTGAGGCTGGGGTATGTTCTTTTAAAATTTGTGACGTAGCGAGC
* 440 * 460 * 480 * 500 * 520 * 540 * X.g : ...... : 558 X.gr : ...... : 558 X.p : ...... : 558 X.t.Au: ...... T...... : 558 X.t : ...... -...... -...... -...-.....C--...... CT...... -...... -...... -...... G.ACT...... C.A.T...... --C.T...... : 540 PB : ...... T...... TT...... : 557 PA : ...... T...... : 558 GTTTGAGATCAAACTATCTTGACGTGTCGTTGTGGCTAAGGCGGGGACCTCGAGTCCCTAGAAATTGAGTCGTTATAGTTCGCGTCCGGGCGTTGTACCCCCGGACTCAGCATGACCTCGAGGCAACTTGAGGTTATA
Chapter 6: Detection of xanthomonads pathogens of pistachio 130
Figure 6.3 ITS sequences used to design primers specific for PCR amplification of pistachio dieback pathogen rDNA. X. g: Xanthomonas translucens pv . graminis (Genbank, AY247064.1), X. gr: X. translucens (strain 61W) isolated from pistachio orchard floor grasses (Genbank: AY994099), X. p: X. t. pv. poae (Genbank: AY253329), X. t. au: X. t. pv . translucens DAR 35705 isolated from wheat in Australia (Genbank: AY994098), X. t: X. t. pv . translucens (Genbank: AF209764.1), PB: pistachio strain 25 representative of group B (Genbank: AY579379) and PA: pistachio strain 7 representative of group A (Genbank: AY579378). Dash denotes deletion in the sequence. Sequences of the primers are shaded and arrows indicate the direction of priming of the primers. The ARMS primer used in this study is shown below the sequence and the underlined letter indicates the nucleotide alteration introduced
to enhance the 3’ mismatch effect
Chapter 6: Detection of xanthomonad pathogens of pistachio 131
Table 6.1 Oligonucleotide primers for specific detection of pistachio dieback pathogens
Primer Direction Sequence
PAf Forward for group A 5’-CCTCCTTTTGAGCATGAGAA-3’
PBf Forward for group B 5’-ACAGTCTAAGGGACCTGCG-3’
PABr Reverse for both groups 5’-TCACTGCTGGCGCATCTTA-3’
6.3.2.2 PCR cycling conditions
The optimised reaction mixture and PCR cycling conditions for a multiplex assay to distinguish groups A and B strains from one another and from other bacteria in a single assay are listed in Tables 6.2 and 6.3.
Table 6.2 Concentration of PCR mixture optimised for the multiplex detection of xanthomonad pathogens of pistachio in Australia
Reaction component Volume ( µµµL) in 25 µµµL reaction Final concentration
DNA sample (50 ng µL-1) 0.25 12.5 ng
Taq Polymerase (5 U µL-1, Qiagen) 0.1 0.5 U
10 × Taq Buffer 2.5 1× dNTP s (25 mM each) 0.2 200 µM
Primer PAf (10 µM) 1.5 0.6 µM
Primer PBf (10 µM) 1.25 0.5 µM
Primer PABr (10 µM) 1 0.4 µM
MQ water 18.2 _
Chapter 6: Detection of xanthomonad pathogens of pistachio 132
Table 6.3 PCR cycling optimised for the multiplex detection of xanthomonad pathogens of pistachio in Australia
Program step Temprature Time
1 Taq polymerase activation and initial denaturation 95°C 15 min
2 30 cycles of:
Denaturation 94°C 45 s
Annealing 54°C 45 s
Elongation 72°C 2 min
3 Final extension 72°C 10 min
The PCR product (6 µL) was subjected to electrophoresis on an agarose gel (2%) using
Tris-acetate EDTA (TAE) buffer at 10 V cm –1 for 70 min, stained with ethidium bromide (0.6 µg mL -1) and visualized under UV light.
6.3.2.3 PCR specificity and sensitivity tests
All strains of the pathogen belonging to group A produced a unique amplification product of 331 bp when DNA from these bacteria was used as a template for PCR with the designed primers. All strains of the pathogen belonging to group B produced a unique product of 120 bp. In contrast, no PCR products were obtained from other bacteria tested except for the type strain of X. translucens pv. cerealis (Figures 6.4 and
6.5, Table 6.4).
Chapter 6: Detection of xanthomonad pathogens of pistachio 133
Table 6.4 List of bacterial strains used in this study, results of PCR using the X.
translucens -specific primer set, T1&T2 (Maes et al. , 1996b), and primers designed in
this study
PCR a Strains and accession numbers Source T1&T2 PAf&PABr PBf&PABr
Pistachio pathogen strains, group A (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, 14, 15, 16, 20, 21, 22, 23, This study 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 36, 72, and 73, 74, 75, 76, 77, 78) NT99004 b + + _ Pistachio pathogen strains, group B (44, 45, 46, This study 47, 48, 49, 50, 51, 52, 53, 54, 55, 6, 57, 58, 59, and 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71) NT99004 + _ + Xanthomonas translucens pv. poae LMG 728 BCCM c + _ _ X. translucens pv . phleipratensis LMG 843 BCCM + _ _ X. translucens pv . hordei LMG 737 BCCM + _ _ X. translucens pv . phlei LMG 730 BCCM + _ _ X. translucens pv . undulosa LMG 892 BCCM + _ _ X. translucens pv . translucens LMG 876 BCCM + _ _ X. translucens pv . cerealis LMG 679 BCCM + _ + X. translucens pv . secalis LMG 883 BCCM + _ _ X. translucens pv . graminis LMG 726 BCCM + _ _ X. translucens pv . arrhenatheri LMG 727 BCCM + _ _ X. pisi LMG 847 BCCM N _ _ X. vasicola pv . holcicola LMG 936 BCCM N _ _ X. campestris pv . campestris LMG 568 BCCM N _ _ X. arboricola pv . corylina LMG 689 BCCM N _ _ X. arboricola pv . juglandis LMG 747 BCCM N _ _ X. arboricola pv . pruni LMG 852 BCCM N _ _ X. hortorum pv . hederae LMG 733 BCCM N _ _ X. oryzae pv . oryzae LMG 5047 BCCM N _ _ X. theicola LMG 8684 BCCM N _ _ continued
Chapter 6: Detection of xanthomonad pathogens of pistachio 134 table 6.4 (continued) X. fragariae LMG 708 BCCM N _ _ X. cassavae LMG 673 BCCM N _ _ X. axonopodis pv . axonopodis LMG 982 BCCM N _ _ X. codiaei LMG 8678 BCCM N _ _ X. sacchari LMG 471 BCCM N _ _ X. bromi LMG 947 BCCM N _ _ X. hyacinthi LMG 739 BCCM N _ _ X. melonis LMG 8670 BCCM N _ _ X. cucurbitae LMG 690 BCCM N _ _ X. vesicatoria LMG 911 BCCM N _ _ X. sp . pv . mangiferaeindicae LMG 941 BCCM N _ _ X. albilineans ACM 1733 ACM d N _ _ X. translucens pv . translucens DAR 35705 ACPPB e + _ _ X. arboricola pv . pruni DAR 64858 ACPPB _ _ _ X. axonopodis pv . malvacearum DAR 26904 ACPPB _ _ _ 191 phytobacteria isolated from plants in and around pistachio orchards This study 15 f _ _
a detection of bacteria with T1&T2: specific primers for X. translucens (Maes et al.
1996b), and PAf&PABr and PBf&PABr: primers designed in this study. +: amplicon of
expected size obtained, -: no amplicon and N: not determined.
b pistachio canker epidemiology, Project NT99004, Horticulture Australia Limited
c BCCM: Belgium Coordinated Collection of Microorganisms, Laboratory of
Microbiology, University of Ghent, Belgium
d ACM: Australian Collection of Microorganisms, University of Queensland, Brisbane,
Qld, Australia
e ACPPB: Australian Collection of Plant Pathogenic Bacteria, Agricultural Institute,
Orange, NSW, Australia
f the number of bacterial strains detected
Chapter 6: Detection of xanthomonad pathogens of pistachio 135
1000
400
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Figure 6.4 Specific amplification of target DNA from the pistachio pathogen by PCR using primers PAf, PBf and PABr. Lanes: 1-11 , strains belonging to group A; 12-19 , strains belonging to group B; and 20 , control (no DNA). Numbers and lane on the left are molecular size markers (bp) (100 bp ladder, Invitrogen, Australia)
1000
300
100
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Figure 6.5 Specific amplification of target DNA from the pistachio pathogen by PCR using primers PAf, PBf and PABr. Lanes: 1, control (no DNA); 2, a mixture of representative strains of groups A and B; 3, group A representative (strain 7); 4, group
B representative (strain 25); 5, X. translucens isolated from grasses on pistachio orchard floor; 6, X. t. pv . translucens DAR 35705 isolated from wheat in Australia. 7, X. t. pv . undulosa ; 8, X. t. pv . translucens ; 9; X. t. pv . cerealis ; 10 , X. t. pv . secalis ; 11 , X. t. pv . poae, 12 , X. t. pv . phleipratensis; 13 , X. t. pv . arrhenatheri ; 14 . X. t. pv. graminis ; 15 ,
X. t. pv. phlei ; 16 , X. t. pv. hordei. Numbers and lane on the left are molecular size markers (bp) (100 bp ladder, Invitrogen, Australia)
Chapter 6: Detection of xanthomonad pathogens of pistachio 136
The minimum number of bacterial cells detected by the PCR based on analysis of
ethidium bromide-stained agarose gel and plating of suspension on YDC, was 20 to 50
cells per amplification (25 µL). In some cases, faint bands of the expected size (120 or
331 bp) could be detected under UV light in products from lower concentrations of the
pathogen cells. Agarose gel electrophoresis of PCR products from a dilution series of
the pathogen cells is shown in Figure 6.6.
1000
400 300 200 100
A 1 2 3 4 5 6 7
1000
200 100
B 1 2 3 4 5 6 7
Figure 6.6 Agarose gel electrophoresis of PCR products from a dilution series of the
pistachio dieback pathogens. A: representative from group A (strain 7), B:
representative from group B (strain 25). Lanes 1-5 are the PCR products from 3 ×10 4,
3×10 3, 3 ×10 2, 3 ×10 1 and 0 CFU. Lanes 6 and 7 are negative and positive controls,
respectively. Numbers and lane on the left are molecular size markers (bp) (100 bp
ladder, Invitrogen, Australia)
Chapter 6: Detection of xanthomonad pathogens of pistachio 137
6.3.2.4 PCR efficiency in plant material and verification
Pistachio tissue contains compounds which inhibit PCR. However, this inhibition was
reversed by the inclusion of PVP (MWt 44,000). PVP was most effective when used at
a final concentration of 1 to 2.5% (w/v) (Figure 6.7).
1000
300 200 100
1 2 3 4 5 6 7 8 9 10 11 12 13
Figure 6.7 Effect of addition of PVP to PCR in neutralising PCR-inhibiting factors of
pistachio tissue. Lanes: 1, PCR mixture with pathogen genomic DNA (positive control);
2, mixture comprising plant tissue and the pathogen genomic DNA (10 ng, group A)
without PVP; 3, negative control (sample prepared from healthy plant tissue, no DNA
and no PVP); 4-13 , samples prepared from plant tissue and seeded with the pathogen
DNA (10 ng, group A) plus PVP (MWt 44,000) at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and
5% w/v final concentration, respectively. Numbers and lane on the left are molecular
size markers (bp) (100 bp ladder, Invitrogen, Australia)
Chapter 6: Detection of xanthomonad pathogens of pistachio 138
The pathogen strains from both groups, A and B, were isolated from the 10 samples tested, purified on YDC plates and their identity was confirmed using BOX-PCR (data not shown).
6.3.2.4.1 Rapid extraction protocols
No visible amplicons were produced from infected wood when samples were soaked in water for 15 min and the resulting extract used in the PCR. Likewise, no product was obtained from samples soaked in water overnight. The inclusion of PVP also did not result in visible bands in the gel. Bands of the expected sizes were amplified from the positive control comprising plant tissue extract seeded with 10 ng genomic DNA of group A and B representatives and 1% (w/v) PVP (data not shown).
6.3.2.4.2 Proteinase-based method
There was no PCR amplification from DNA extracted using proteinase, even in the presence of PVP. However, controls yielded amplicons of the expected size (data not shown).
6.3.2.4.3 CTAB-based method
No PCR products were detected following PCR amplification of DNA extracted from infected pistachio samples and the healthy shoot using the CTAB method. However,
PCR amplification of the positive control, DNA extract seeded with bacterial genomic
DNA, resulted in amplification products of the expected sizes (data not shown).
Chapter 6: Detection of xanthomonad pathogens of pistachio 139
6.3.2.4.4 Bio-101 DNA extraction kit
No PCR products were amplified from DNA extracted from infected and the healthy pistachio shoots following extraction using the Bio-101 DNA extraction kit. The inclusion of PVP also did not result in amplification. However, PCR amplification products of expected sizes were visible for the positive control and DNA extract seeded with bacterial genomic DNA (data not shown).
6.3.2.4.5 BIO-PCR
The predicted DNA fragment was amplified from all symptomatic samples of pistachio wood from both artificially and naturally infected trees. These products were detectable only when PVP was added to the reaction mixture. No product was detectable from a mixture comprising plant tissue and the pathogen genomic DNA without PVP, indicating that there was significant inhibition of PCR by pistachio tissue. No product was observed from the healthy plant tissue, nor were there non-specific PCR products.
The pathogen strains from both groups were detected in pistachio plant material by the assay. Detection of the pathogen belonging to group A in a naturally infected pistachio sample is shown in Figure 6.8.
All shoots, symptomatic and asymptomatic (72 samples), collected from both artificially and naturally infected pistachio trees were assessed using this assay. The assay detected the pathogen in four samples of asymptomatic wood, one from an artificially infected tree and three from naturally infected trees (Figure 6.9). From all samples which showed a positive signal in the assay, bacterial strains were recovered on
YDC culture medium and assigned to group A or B of the pathogen by BOX-PCR, reflecting the inoculum applied or the group expected from the source of the material
(data not shown).
Chapter 6: Detection of xanthomonad pathogens of pistachio 140
1000
300 200 100
1 2 3 4 5 L
Figure 6.8 Detection of pistachio dieback pathogen, belonging to group A, in naturally infected plant material. Samples prepared for the PCR by soaking pistachio woody tissue in nutrient broth overnight, centrifuging and resuspending the pellet in sterile distilled water. DNA was amplified using primers PAf, PBf and PABr. Lanes: 1, PCR mixture with pathogen genomic DNA (positive control); 2, mixture comprising infected plant tissue and the pathogen genomic DNA without PVP; 3, sample prepared from infected plant tissue without PVP; 4, sample prepared from infected plant tissue plus
PVP (MWt 44,000, 1% w/v final concentration); 5, sample prepared from a healthy plant (negative control). Numbers and lane on the right are molecular size markers (bp)
(100 bp ladder, Invitrogen, Australia)
Chapter 2: General materials and methods 141
L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 L 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 L 34 35 36 37 38 39 40
Figure 6.9 Detection of pistachio dieback pathogens, belonging to groups A and B, in naturally and artificially infected plant material. Pistachio woody tissue prepared for the PCR by soaking in nutrient broth overnight, centrifuging and resuspending the pellet in sterile distilled water. Lanes: L, molecular size marker (bp) (100 bp ladder, Invitrogen, Australia); 1, sample prepared from a healthy plant (negative control); 2, 3, 8, 9, 34 , 35 , 36 , 37 ,
38 and 39 , samples prepared from asymptomatic samples from which no bacterial cells were recovered on YDC plates; 4, 5, 6, 7, 10 , 11 , 12 , 13 , 14 , 15 ,
19 , 20 , 21 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 and 31 , samples prepared from symptomatic samples from both naturally and artificially infected trees from which the pathogen cells were recovered on YDC plates; 16 , 17 and 18 samples prepared from asymptomatic samples collected from the orchard in
Kyalite and 32 , from asymptomatic sample collected from glasshouse experiments from which bacterial cells were recovered by plating on YDC; 22 and 33 , samples prepared from shoots inoculated with pathogen strains from both groups in glasshouse experiments; and 40 , PCR mixture with pathogen genomic DNAs (positive control). The recovered bacterial cells were assigned to groups A and B by BOX-PCR
Chapter 6: Detection of xanthomonad pathogens of pistachio 142
6.4 Discussion
For the first time, a rapid, sensitive and highly specific method has been developed for the identification of xanthomonad pathogens of pistachio in Australia. This method uses a unique primer design (ARMS) in a multiplex-PCR and can be used to detect and to identify the pathogen in culture and directly from plant material. It can differentiate between the two groups of the pathogen (A and B) in a single assay.
Although the PCR-based protocol developed by Maes et al. (1996b) for detection of X. translucens amplified the expected fragment of 139 bp from DNA of the pistachio strains, the assay did not distinguish groups A and B. This PCR amplified a fragment of
139 bp from the ten pathovars of X. translucens tested, all of which are known pathogens of cereals and grasses (Maes et al. , 1996b), as well as from the X. translucens -like bacterium which is genetically distinct from pistachio pathogens and has been regularly isolated from grasses in pistachio orchards. This demonstrated that the published primers were not suitable for detection of pistachio pathogens.
Sequence variation in the intergenic spacer flanked by 16S and 23S rRNA genes between pistachio dieback strains in group A and B and between these two groups and other xanthomonads studied, provided an opportunity to design specific primers for use in a multiplex PCR assay. The Amplification Refractory Mutation System (ARMS)
(Newton et al. , 1989) was used for primer design to distinguish strains belonging to group A from other bacteria including group B. In ARMS-PCR, a primer is designed so that it can discriminate between templates which differ at a specific single nucleotide at the 3’-terminal base of the primer. The basis of the technique is that Taq polymerase lacks a 3’ to 5’ exonuclease activity, therefore, oligonucleotides with a mismatched 3’-
Chapter 6: Detection of xanthomonad pathogens of pistachio 143 end cannot function as primers in the PCR. Although a single base mismatch located at the 3’-end of the primer is sufficient for preferential amplification of the perfectly matched oligonucleotide, incorporating an artificial mismatch at the residue 1 or 2 bases from the 3’-end of the primer can enhance primer specificity under appropriate conditions. This deliberate mismatch has no significant influence on the yield of PCR products. The ARMS primer designed in this study, PAf, generates a deliberate mismatch (G/C, primer/sequence) three bases from the 3’-end with the corresponding sequence of strains belonging to group A. Besides this mismatch, PAf forms another mismatch (A/G, primer/sequence) at the 3’-end with other bacteria including group B.
The type and the position for introduction of the deliberate mismatch was optimised by comparing results of several ARMS primers with various alterations (data not shown) and, of these, PAf gave the most effective discrimination. This confirms that a strong mismatch (purine/purine or pyrimidine/pyrimidine) at the 3’-terminus of a nucleotide– specific primer will likely need a weak second mismatch (purine/pyrimidine) (Ye et al. ,
2001). ARMS-PCR is increasingly being applied in medicine and genetics to detect point mutations and deletions/insertions in the genomic DNA (Fan et al. , 2003; Ye et al. , 2001) and has recently been utilised for reliable differentiation between closely related plant pathogenic fungi (Jayne & Taylor, 2001; Mach et al. , 2004). The study described here is thought to be the first description of the use of ARMS-PCR for the detection of a plant pathogenic bacterium.
In order to develop a multiplex-PCR to detect and distinguish both groups simultaneously, the specific forward primer for strains belonging to group B, PBf, was selected so that the primer annealing condition was roughly similar to that of PAf, and so that the amplified DNA fragments were of different length using a common reverse primer (PABr). The development of a multiplex-PCR to detect more than one target
Chapter 6: Detection of xanthomonad pathogens of pistachio 144 simultaneously needs extensive optimisation (Henegariu et al. , 1997). The best result for the triple primer pair multiplex-PCR designed in this study was achieved with optimisation of dNTPs, primer concentration and annealing temperature which allows detection of either or both of two groups of the pathogen simultaneously. Multiplex-
PCR has been used for concurrent detection of two plant pathogenic bacteria in other studies. Fegan et al. (1998) designed a multiplex PCR assay for identification of
Clavibacter xyli to the subspecies xyli and cynodontis using a single forward and two reverse primers specific for each subspecies. Likewise, Glick et al. (2002) developed a two primer pair multiplex PCR assay for detection of either or both of X. campestris pv. pelargonii and Ralstonia solanacearum , two important pathogens of geraniums, using primer pairs specific for each pathogen.
In developing a specific PCR test for a pathogen, specificity and sensitivity of the test are two important factors. The specificity of the assay reported here is adequate for reliable identification of pistachio Xanthomonas strains. The predicted products were amplified from all strains belonging to groups A and B, and no amplicons were obtained from the other bacteria isolated from plants in and around pistachio orchards or from type and reference strains of the genus, except for the type strain of X. translucens pv. cerealis. The latter has not been reported in Australia. In experiments performed on numerous plant samples, no non-specific PCR signal produced by plant material or other plant-associated organisms has been observed so far. Xanthomonads were recovered from plant samples which were positive in the PCR assay and not from any specimens that were negative. The identity of recovered strains was confirmed by the
BOX-PCR.
Chapter 6: Detection of xanthomonad pathogens of pistachio 145
Specific amplification of target DNA from infected plant material was successful only when an enrichment method was conducted and PVP was added to the PCR. This demonstrates that the failure of the reaction using other methods was due to PCR- inhibiting factors as well as low concentration of pathogen cells in plant tissues. It is believed that phenolic terpenoids and tannins present in plant material can bind to RNA and DNA upon cell lysis (John, 1992). To minimise the effect of these compounds, which can inhibit the PCR, PVP has been used either during extraction of bacterial
DNA from plant material (Maes et al. , 1996a; Meng et al. , 2004) or directly in the PCR
(Fegan et al. , 1998; Koonjul et al. , 1999).
Specific bands were observed on ethidium bromide-stained agarose gel when using 20 to 50 cells per PCR (25 µL). This sensitivity corresponds to an average detection limit of 0.8-2 × 10 3 CFU of the pathogen per mL. Conventional methods for the detection of plant pathogenic bacteria often use plating on agar media and serological techniques.
Plating on medium has the potential to detect single, viable cells, however, the percentage recovery of target bacteria from plant tissue depends on several factors, such as the optimal release of the target from tissue, the minimisation of competition by saprophytic microbiota and the minimisation of interference by inhibitory plant compounds and debris (Saettler et al. , 1989). Indeed, for pistachio dieback, isolation into culture is not enough to identify the pathogen and further tests are required, especially to distinguish the two groups. Labour costs and time are other disadvantages of culturing techniques. Immunodiagnostic methods, such as enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF), both popular and successful formats, provide rapid detection and identification of bacteria, however, the detection limit is normally around 1 ×10 5 CFU mL -1 (Alvarez, 2001). Furthermore,
Chapter 6: Detection of xanthomonad pathogens of pistachio 146 techniques based on polyclonal and monoclonal antibodies cannot differentiate among the various pathovars of X. translucens because of the high degree of serological similarities within this group (Azad & Schaad, 1988; Bragard & Verhoyen, 1993).
The PCR test developed in this study has several advantages, as it works well with genomic DNA, can be applied directly to colonies on agar media, and can be used directly on plant material to detect the pathogen and identify it to group simultaneously.
Detection of infection in plant material can be achieved within 1 day. The assay was able to detect the pathogen in some asymptomatic samples collected from artificially and naturally infected trees from which the pathogen was recovered on agar medium.
Detection of the pathogen in plant material, especially in asymptomatic, infected planting material may be important in minimising the spread of the disease. Thus, this technique will contribute to understanding of the epidemiology of the disease in order to reduce risk.
In summary, a PCR-based assay based on the 16S-23S internal spacer region was developed for identification of the xanthomonad pathogens of pistachio in Australia.
The assay was successfully used to detect the pathogen in culture and in plant material.
Furthermore, the two groups of the pathogen (A and B) could be distinguished in a multiplex PCR.
Chapter 7: General discussion 147
Chapter 7: General discussion
This study has investigated diversity amongst bacterial strains, belonging to the genus
Xanthomonas, found in pistachio trees with dieback in Australia. The pathogen strains were characterised and a PCR-based assay was developed for rapid and specific detection.
BER-PCR analysis of 65 strains from pistachio trees in different locations of South
Australia, Victoria and New South Wales revealed significant genetic variation amongst these strains. These differences in the genomic structure of the pathogen population, and the relationship between the two genotypes of the pathogen and their geographic regions, cannot be attributed to selection by their host plant, because Sirora is the main female cultivar grown in all areas. However, it is possible that different pathogen strains are capable of causing dieback of pistachio in different climatic regions or geographic locations. Evidence for correlation between groups of bacterial pathogens obtained by rep-PCR fingerprinting and ecological adaptation has been reported by other researchers
(Massomo et al. , 2003; Mkandawire et al. , 2004; Scortichini et al. , 2001). It would be important to investigate if pistachio growing areas differ in attributes such as climate and rainfall conditions or in orchard management strategy. There was no variation in rep-PCR fingerprinting patterns of strains isolated over several years. This finding confirms the proposal of Versalovic et al. (1991) that the repetitive elements are conserved and natural selection may constrain variation in these sequences.
Physiological tests characterised pistachio strains of Xanthomonas to be more and less similar to a range of X. translucens pathovars reported as pathogens of cereals and
Chapter 7: General discussion 148 grasses, such as pv. translucens . The strong correlation observed between the groups obtained by rep-PCR genomic fingerprinting and phenotypic characteristics of each group (Chapters 3 and 4) may reflect the suggestion of Versalovic et al. (1991) that naturally occurring repetitive elements may represent sites of essential protein:DNA interactions.
Considering that the host range of X. translucens has been limited to cereals and grasses in the Poaceae only (Vauterin et al. , 1995), the unexpected identification of pistachio strains as members of this species based on phenotypic characteristics necessitated gathering essential genomic information about the pathogen. ITS sequencing revealed that groups A and B differed from one another in 15 bp (out of 558 bp). Furthermore, group A and group B showed 98% and 97% similarity to Xanthomonas translucens pv . poae, respectively, indicating a close phylogenetic relationship among pistachio strains and X. translucens . Strains of both groups shared a common ITS structure, including
ITS1, tRNA Ala , ITS2, tRNA Ile and ITS3, with all Xanthomonas species as described by
Goncalves & Rosato (2002). Sequencing an approximately complete 16S rDNA fragment (>1300 nt) is one of the criteria for species delineation (Stackebrandt et al. ,
2002). The entire sequence of the 16S rDNA fragment, 1500 nt, was determined for the two groups. Based on the 16S rDNA sequences data, a close genetic relationship was found among the pathogen strains of group A and group B and X. translucens . The phylogeny of Xanthomonas species based on 16S rDNA sequences has been studied by
Hauben et al. (1997). Based on their study, X. translucens along with X. albilineans, X. hyacinthi and X. theicola grouped together as an ‘ X. albilineans core’. The differences in 16S rDNA sequences clearly differentiated members of this core from other xanthomonads, however, it could not discriminate these species, confirming that 16S rDNA sequencing cannot replace DNA-DNA hybridisation for species delineation. Of
Chapter 7: General discussion 149 the four species in the ‘ X. albilineans core’, only X. theicola is an exception in its pathogenicity to dicotyledonous plants (Bradbury, 1986; Hauben et al. , 1997) and pistachio strains may be considered as another exception. DNA-DNA hybridisation is an important standard criterion for species designation (Stackebrandt et al. , 2002) and
70% DNA-DNA relatedness is the limit for species definition (Wayne, 1987). Using this approach, xanthomonad strains from pistachio were shown to belong to X. translucens .
In order to differentiate strains below the species level, BER-PCR was applied to compare the pathogen strains with known pathovars of X. translucens . This technique has shown potential for differentiation of these pathovars (Rademaker et al. , 2000;
Rademaker et al. , 2005) and distinguished clearly pistachio strains from the ten pathovars. This suggested that a host range study of pistachio strains might lead to a more accurate classification at the pathovar level.
The pathogenicity of pistachio strains to members of the Poaceae suggests that these strains may have originated from or share a common lineage with X. translucens from cereals and grasses. X. translucens has already been reported in Australia (Moffett &
McCarthy, 1973; Noble, 1935). Furthermore, Xanthomonas strains belonging to X. translucens , although different from pistachio strains, are commonly found on grasses in pistachio orchards. Mellano & Cooksey (1988) showed that the pathogenicity of X. c. pv . translucens towards certain hosts apparently is determined by positive factors and may be inactivated by single point mutations. The converse has not been shown, i.e. where members of a pathovar turn into a pathogen of a new plant via mutation.
However, other mechanisms such as introduction of plasmids containing host range determinants (Vivian et al. , 2001) could be considered as a hypothesis for the origin of
Chapter 7: General discussion 150
Xanthomonas strains pathogenic to pistachio. Understanding the molecular basis of host specialisation by pistachio strains that has enabled them to cause dieback in pistachio is an important area for future research.
Group A strains were HR positive, in accordance with the hypothesis that all
Xanthomonas strains which create local lesions on their host plant have the potential to induce a hypersensitive reaction (HR) on a non-host plant (Klement, 1982). The fact that group B strains produced spots and necrosis on pistachio leaves similar to those produced by group A strains, while these strains were HR negative, suggests that group
B strains may need specific conditions (such as high concentration of inoculum or certain temperatures before and after inoculation) to induce HR response in tobacco, as has been reported for other bacteria including xanthomonads (Klement, 1982).
Differences in pathogenicity characteristics of the two groups, such as HR, ice nucleation activity and, more importantly, differences in the host range suggest that certain, currently unknown, conditions and mechanisms modulate the activity of strains of each group and the host response. Future studies should therefore focus on understanding and comparing regulatory mechanisms that define the pathogenicity of each group.
Atypical symptoms of the disease could be artificially produced in pistachio and some other plants from the Anacardiaceae following syringe-inoculation with the pathogen strains. Such symptoms, however, have not been observed under natural conditions.
This may be attributable to the optimal conditions provided for the infection in these experiments or the increased susceptibility of detached material to the pathogen, as has been reported by other researchers for other plant pathogenic bacteria (Brisset et al. ,
1988; Yessad et al. , 1992). In the same way, the host range of X. translucens pathovars
Chapter 7: General discussion 151 among members of the Poaceae determined by artificial inoculation with the bacteria is wider than their natural host range (Bradbury, 1986). It is also possible that environmental factors such as high temperature and lack of moisture on the leaf surface in pistachio orchards may contribute. One could also speculate that in order to produce such symptoms on pistachio leaves and buds in pistachio orchards, a large population of the pathogen is needed. It is possible that other bacteria commonly found in the pistachio phylloplane may inhibit the pathogen during stages of infection such as epiphytic growth, penetration, initial events in pathogenesis and multiplication of the pathogen. Significant inhibition of the pathogen strains by other bacteria isolated from pistachio material on culture media was regularly observed during this study. It would be useful to identify such bacteria, investigate their population changes during the growing season and determine mechanism/s of the inhibition in vitro and in vivo . It has been shown that bacterial antagonists can significantly suppress populations of X. translucens strains and subsequent bacterial leaf streak (BLS) severity on wheat
(Marefat & Rahimian, 1998; Stromberg et al. , 2000; Stromberg et al. , 2004).
Full appreciation of the range of symptoms that pistachio trees with dieback may experience, and changes in disease patterns that may occur with changes in climatic factors and increasing host age, is a problematic issue that would require extensive resources, and could take months to obtain. Therefore, in this study, inoculation of excised shoots, as a more manageable and less time-consuming method, was conducted to evaluate pathogenicity of the bacteria. Although this simple test was efficient and could be used for rapid diagnosis of the pathogen, further studies should focus on artificial inoculation of branches on mature trees in the orchard to monitor symptoms caused by the pathogen.
Chapter 7: General discussion 152
Where do the pathogen strains come from? If the pathogen has crossed from cereals and grasses to pistachio, why has this disease been reported in Australia only, whereas X. translucens is a pathogen of cereals and grasses worldwide (Duveiller et al. , 1997a) and exists in countries where pistachio has been grown for hundreds of years? The lack of a thorough survey may account for this. Canker and leaf spot, along with dieback, have been reported on one-year old pistachio seedlings in Iran and, based on a few physiological and biochemical tests, Xanthomonas sp. has been identified as the causal agent (Tarighi & Rahimian, 2001). Some characteristics of this Xanthomonas , such as ice nucleation activity, tobacco hypersensitive reaction and use of some carbon sources, were similar to those of strains belonging to group A in this study. Unfortunately, because Xanthomonas strains from Iran could not be obtained, we could not compare the bacteriological properties and pathogenicity of strains from Australia with those of strains reported from Iran. Also, pistachio dieback and discolouration in woody tissues, albeit as an unknown disorder, recently have been reported in Turkey (Sarpkaya & Can,
2005). Further studies are needed to compare the disease and the causal agent(s) in
Australia with those in other countries. Furthermore, it is possible that the adaptation of pistachio to Australian conditions, especially the selection made to produce Sirora, the major cultivar in Australia, has allowed the pathogen strains to adapt to pistachio or changed certain mechanism/s in order to provide a new plant-pathogen interaction. This raises questions regarding how this specialisation might be accomplished and how this would be reflected in the activity of bacterial cells. This is an area in which a thorough study is needed.
The distinct differences between the two groups of the pathogen and among pistachio strains and known pathovars of X. translucens in terms of host range strongly suggest that the two groups of the pathogen are two new pathovars. Pathovar ‘ pistacia’ and
Chapter 7: General discussion 153 pathovar ‘ vera’ are proposed for group A and group B strains, respectively. Strains 7 and 25, representative of groups A and B based on all tests described in this study, have been accessioned in the International Collection of Micro-organisms from Plants,
Auckland, New Zealand, as ICMP 16316 and ICMP 16317, respectively.
Some Xanthomonas pathovars, such as X. campestris pv. mangiferaeindicae , X. campestris pv. phaseoli , X. campestris pv . campestris and X. oryzae pv . oryzae , have been further differentiated into pathogenic variants (pathotypes or races) based upon their interaction with differential host genotypes (Dayakar & Gnanamanickam, 1996;
Mazzola et al. , 1994; Mew, 1992; Opio et al. , ; Vicente et al. , 2001). However, there are conflicting reports on the existence of races for X. translucens pathovars. Bradbury
(1986) has referred to various pathogenic races for pv. cerealis , whereas Milus &
Chalkley (1994) examined 81 strains of X. c. pv. translucens isolated from wheat, triticale, rye and barley for virulence to 19 wheat cultivars and found no significant evidence for races among the strains. A race structure may exist within the population of each group of the pistachio pathogen. It would be useful to screen various cultivars of pistachio with more strains of the pathogen to find if there are pathogenic variants among pistachio strains. One of the most interesting directions for future research would be parallel studies of the disease based on climatic conditions and pistachio resources in
Australia and those in other countries. This would require a close collaboration between research groups involved. Understanding of genetic and pathogenic variation in the pathogen population, effect of environment on the disease and investigation of any source for disease resistance in pistachio cultivars would be useful for management strategies.
Chapter 7: General discussion 154
Problems confronting the detection of strains of the pistachio dieback pathogen, especially in plant material, by conventional methods based on culturing, necessitated the development of a PCR-based detection assay. The evaluation of a previously published PCR procedure for detection of X. translucens strains from cereals and grasses (Maes et al. , 1996b) showed that this procedure did not permit the unambiguous detection of pistachio strains (Chapter 6). In contrast, the primers designed in this study provided the required degree of specificity to differentiate pistachio strains from pathovars of the species and Xanthomonas strains commonly found on grasses in pistachio orchards. For the first time, we used an ARMS method for identification of a plant pathogenic bacterium to differentiate group A strains from those of group B. The assay was combined with the biological amplification of the pathogen strains (BIO-
PCR) for the detection of pathogen strains in plant tissue. The ability of this assay to detect the pathogen strains in asymptomatic infected plants would assist in selection of disease-free planting material and would be important in minimising the spread of the disease. This procedure can be used for indexing pistachio planting material without previous bacterial isolation and the contamination of pistachio material by the pathogen can be determined in less than 24 h.
At present, the assay is capable of detecting the pathogen strains and assigning them to groups based on the presence or absence of the specific band for each group in an agarose gel. However, the pathogen cannot currently be quantified, and future study should focus on the development of a quantitative assay. It is possible that a real-time
PCR assay, which can measure the amplified PCR product at each cycle throughout the reaction (Freeman et al. , 1999), may be appropriate. Such a technique, along with a reliable sampling strategy, and in combination with epidemiological studies, would shed light on a number of issues, some of which are important to an understanding of the
Chapter 7: General discussion 155 pathogen and of the disease. As an example, simultaneous infection of a tree by strains of the two groups has not so far been reported. A quantitative assay would be useful to investigate if there is competition between the two genotypes, or if strains from one group can affect the population size of the other group. Also such a technique would be useful to monitor the pathogen population in different parts of the host plant or to compare the population size in response to management strategies.
In conclusion, Xanthomonas strains isolated from pistachio trees in Australia were identified and differentiated according to phenotypic, genotypic and pathogenic characteristics. The results confirmed that these strains belong to X. translucens and showed that they represent two pathovars distinct from the known pathovars of the species. A PCR-based detection assay was developed, is now being used in epidemiological studies of pistachio dieback, and will provide the pistachio industry with a powerful and specific molecular tool for the detection of the dieback pathogens, contributing to the development of disease management strategies.
Appendix A: Buffers and reagents 156
Appendix A: Buffers and reagents
Bovine serum albumin (BSA) BSA, nuclease free (Boehringer, #711454), a 850 µg mL -1 solution was divided into 20- µL aliquots and stored at -20 °C
Coomassie blue solution Coomassie blue 0.1 g Methanol 45 mL MQ water 45 mL Acetic acid 10 mL Filter through a Whatman No.1 filter paper
CTAB extraction buffer (Green et al., 1999) Tris-HCl, pH 8 100 mM NaCl 1.4 M EDTA, pH 8 50 mM CTAB 2.5% β-mercapto-ethanol 0.2% PVP (MW40,000) 1% Sterilise by autoclaving
Dimethyl sulphoxide (DMSO)
DMSO, 100% (Fluka, #41640), the solution was divided in 0.5-mL aliquots and stored at -20 °C, one working solution was kept at RT
Appendix A: Buffers and reagents 157 dNTP Ultra pure dNTP set, 100 mM each (Invitrogen, #272035-1), 100 mM solutions were mixed 1:1:1:1 to obtain a solution with 25 mM of each nucleotide, the solution was divided in 100 µL aliquots and stored at -20 °C
EDTA 0.5 M (pH 8)
Add 186.1 g Na 2EDTA to 800 mL MQ water. Stir vigorously on magnetic stirrer. Adjust to pH 8 with NaOH pellets to dissolve the EDTA. Make up to 1L with MQ water. Sterilise by autoclaving
5 ××× Gitschier buffer (Rademaker & de Bruijn, 1997)
1 M (NH 4)2SO 4 16.6 mL 1 M Tris-HCl (pH 8.8) 67 mL
1 M MgCl 2 6.7 mL 1:100 dilution of 0.5 M EDTA (pH 8.8) 1.3 mL β-mercapto-ethanol 2.08 mL (14.4 M commercial stock stored at 4 °C) Adjust to 200 mL with approximately 106 mL MQ water and mix. Store at -20 °C in 1 mL aliquots
Guanidine thiocyanate-EDTA-Sarkosyl (GES) solution (Rademaker & de Bruijn, 1997) Guanidine thiocyanate 60 g 0.5 M EDTA pH 8 20 mL Sterile water 50-60 mL Dissolve all components by heating to 65ºC, cool down and add N-lauroyl sarcosine 1 g
Adjust to 100 mL with water, filter using a 0.45 µm filter and store at RT
Appendix A: Buffers and reagents 158
Phosphate buffered saline
Na 2HPO 4 4.26 g
KH 2PO 4 2.27 g NaCl 8 g MQ water 1,000 mL
ProtoGel 30% (w/v) Acrylamide:0.8% (w/v) Bis-acrylamide stock solution (37.5:1), Order No. EC-890. National Diagnostic Inc., Itlings Lane, Hessle, Hull HU13 9LX, UK
Resuspension buffer (RB) (Rademaker & de Bruijn, 1997) NaCl 4.383 g 0.5 M EDTA (pH 8) 10 mL Water 490 mL Sterilise by autoclaving
Sample treatment buffer Tris 7.5 g β-mercapto-ethanol 50 mL Glycerol 100 mL SDS 20 g Bromophenol blue 1 g Make up to 1,000 mL with MQ water. Adjust pH to 6.8 with HCl
2×××SSC buffer NaCl 0.3 M
Sodium citrate 0.03 M
TAE electrophoresis buffer (50 ×××) Tris (free base) 242 g Glacial acetic acid 57.1 mL
Appendix A: Buffers and reagents 159
Na 2 EDTA 18.61 g Make up to 1 L with MQ water and store at RT
TE buffer (pH 8) (Rademaker & de Bruijn, 1997) 1 M Tris HCl pH 8 1 mL 0.5 M EDTA pH 8 200 µL MQ water 99 mL
5 ××× Tris-glycine electrophoresis buffer Glycine 94 g Tris base 15.1 g MQ water 900 mL Sodium dodecyl sulphate (SDS) 10% solution 50 mL Adjust the volume to 1,000 mL with MQ water
Appendix B: Media and methods for biochemical and … 160
Appendix B: Media and methods for biochemical and physiological tests
Action in litmus milk Use Difco dehydrated litmus milk and prepare as recommended by manufacturer. Adjust pH to 7 by 1N NaOH before sterilisation. Acid production turns the milk red whereas alkaline production turns the milk blue. Peptonisation will make a white precipitate and reduction will make the milk transparent
Anaerobic growth (Hugh and Leifson 1953) (Schaad et al. , 2001) Peptone (Difco) 2 g NaCl 5 g
KH 2PO 4 0.3 g Agar (Difco) 3 g MQ water 1,000 mL Bromothymol blue (1% aqueous solution) 3 mL Dissolve all ingredients, adjust to pH 7.1 and add 5 mL of the medium to test tubes and autoclave. Prepare a 10% aqueous solution of glucose and sterilise by filtration. Add 0.5 mL of the glucose to each tube. Inoculate two tubes with each strain of the pathogen to be tested. Cover one tube with a layer, 5-10 mm, of sterile paraffin. A colour change from blue to yellow in both tubes is recorded as positive for anaerobic growth
Esculin hydrolysis (Sands, 1990)
NH 4H2PO 4 0.5 g
K2HPO 4 0.5 g
MgSO 4.7H 2O 0.2 g NaCl 5 g Yeast extract (Difco) 5 g Ferric ammonium citrate 50 mg Esculin 1 g Agar (Difco) 15 g MQ water 1,000 mL
Appendix B: Media and methods for biochemical and … 161
Adjust to pH 6.8, autoclave and pour into Petri dishes. Grow the strain to be tested on the plate and incubate for 2 weeks. Development of a dark brown colour around the colony is regarded as indicating utilisation of esculin
Gelatin liquefaction (Schaad et al. , 2001) Nutrient agar (Difco) 23 g Gelatin (Sigma) 4 g MQ water 1,000 mL Autoclave and pour into Petri dishes. Grow the strain to be tested on the plate and incubate for 3-5 days. Flood the plate surface with 5 mL of mercuric chloride reagent
(12 g HgCl 2 in 16 mL HCl and 80 mL MQ water). A clear zone surrounding bacterial growth indicates reaction gelatin hydrolysis
Glucose yeast-extract carbonate agar (GYCA) (Schaad et al. , 2001)
Yeast extract (Difco) 5 g Glucose 5 g Calcium carbonate, light powder 40 g Agar (Difco) 15 g MQ water 1,000 mL Adjust to pH 7 and autoclave at 10 PSI for 1 h, cool the medium to 50 °C in a waterbath and suspend CaCO 3 by swirling before pouring the plates
Growth at 36 °°°C (Fahy & Hayward, 1983) Inoculate 5-10 mL YS broth in a test tube with the strain to be tested and incubate the culture in a water bath, with stirring, at 35 °C for 10-12 days. Observe for turbidity
H2S production from cysteine (Schaad et al. , 2001)
NH 4H2PO 4 0.5 g
K2HPO 4 0.5 g
MgSO 4.7H 2O 0.2 g NaCl 5 g Yeast extract (Difco) 5 g
Appendix B: Media and methods for biochemical and … 162
Cysteine hydrochloride 0.1 g MQ water 1,000 mL Strips of filter paper moistened with a 5% solution of neutral lead acetate are held in place over the medium to maintain the lower end of the paper about 5 mm above the surface of the liquid medium. A blackening of the paper indicates the presence of H 2S
Hypersensitive reaction on tobacco (Schaad et al. , 2001)
Prepare a suspension (approximately 10 9 CFU mL -1 water) from a 48 h-old agar culture of the bacterium to be tested. Inject the suspension into the intercellular spaces of a leaf of tobacco cv. Burley using a 25-gauge needle and syringe. Complete collapse of the tissue after 24 h is considered as positive.
Ice nucleation activity test (Lindow, 1990) Coat smoothly an aluminium foil sheet with a thin layer of paraffin by spraying with a saturated solution of paraffin in xylene (1-5% w/v). Heat the sheet to about 60 °C to melt the paraffin on the sheet and to remove the xylene. Cool the sheet at room temperature. Fold the sheet into a boat and cool it to –4°C or –10 °C on the surface of a methanol- water coolant in a cooling bath. Place small droplets, 10 µL, of bacterial suspensions (>10 8 CFU mL -1) on the surface of the foil and observe for quickly frozen droplets
King B medium agar (KB) (Schaad et al. , 2001) Proteose peptone #3 (Difco) 20 g
K2HPO 4 1.5 g
MgSO 4.7H 2O 1.5 g Glycerol 15 mL Agar (Difco) 15 g MQ water 1,000 mL Autoclave and pour in plates Although this medium is suitable for general isolation and growth of Pseudomonas strains and observation of their fluorescein pigments, most bacteria will grow on it
Appendix B: Media and methods for biochemical and … 163
KOH solubility test (Fahy & Hayward, 1983) Prepare 3% KOH solution in MQ water. Mix a loopful of the strain to be tested with one-two drops of the solution. Gram-negative bacteria will become gummy upon mixing with a loop for 5-10 min, while Gram-positive bacteria will not
Nitrate reduction (Fahy & Hayward, 1983)
KNO 3 1 g Peptone (Difco) 5 g Yeast extract (Difco) 4 g Oxoid ion agar No. 2 3 g MQ water 1,000 mL Adjust pH to 7-7.2 and autoclave. Seed 4 mL broth with inoculum and grow 24-48 h, examine for foam, which indicates gas production. Test tubes on various days: add 1 mL of a 0.6% (v/v) solution of N, N-dimethyl-1 naphthylamine and 1 mL of a 0.8% (v/v) solution of sulphanilic acid, both in 5N acetic acid, to each tube. A distinct pink or red colour in the broth indicates the prescence of nitrite and is considered positive. If within 1 h a pink or red color does not appear, add zinc dust to the tube. The zinc will react with the nitrate, if present, and will produce a pink colour. This will confirm a negative test. If the tube does not turn pink then the complete denitrification has occurred and the test is considered positive
Nutrient agar (NA) (Schaad et al. , 2001) Beef extract (Difco) 3 g Peptone (Difco) 5g Agar (Difco) 15g MQ water 1,000 mL Sterilise by autoclaving
Oxidase test (Fahy & Hayward, 1983) Place a Whatman No. 1 filter paper in a Petri dish and add 3-4 drops of a freshly prepared 1% aqueous solution of tetramethyl-p-phenylenediamine dihydrochloride. Rub a small loopful of the 24-hour-old culture of the target strain on the paper. The strain is considered oxidase-positive if a purple colour develops within 10 s
Appendix B: Media and methods for biochemical and … 164
Proteins digestion (Schaad et al. , 2001) Nutrient agar (Difco) 11.5 g Yeast extract (Difco) 2.5 g MQ water 350 mL After autoclaving add skim milk solution 150 mL (50 g skim milk in 150 mL MQ water, sterilised by steaming for 30 min on three successive days) Autoclave and pour over the surface of a thin layer of nutrient agar in Petri dishes. Grow the target bacterium and observe for a clear zone around the colonies after 3, 5 and 7 days
Starch hydrolysis (Schaad et al. , 2001) Soluble starch 2 g Nutrient agar (Difco) 23 g MQ water 1,000 mL Autoclave and pour into plates. Streak the plate with the strain to be tested and incubate for 2-7 days depending on growth. Flood with iodine solution (1 g iodine and 2 g potassium iodide in 100 mL MQ water). Starch stains dark-blue and a clear zone is present where starch hydrolysis has occurred
Sucrose peptone agar (SPA) (Schaad et al. , 2001) Sucrose 20 g Peptone (Difco) 5 g
K2HPO 4 0.5 g
MgSO 4.7H 2O 0.25 g Agar (Difco) 15 g MQ water 1,000 mL Adjust pH to 7.2-7.4, autoclave and pour in plates
Tryptone soy agar (TSA) (Schaad et al. , 2001) Tryptone (Difco) 15 g Soy peptone 5 g NaCl 5 g
Appendix B: Media and methods for biochemical and … 165
Agar (Difco) 15 g MQ water 1,000 mL Adjust to pH 7.3, autoclave and pour into plates
Tween 80 hydrolysis (Fahy & Hayward, 1983) Peptone (Difco) 10 g NaCl 5 g
CaCl 2.2H 2O 0.1 g Agar (Difco) 15 g Tween 80* 10 mL MQ water 1,000 mL * autoclave separately Adjust to pH 7.4, autoclave and pour into plates. Grow the target strains on the plates for 2-7 days depending on growth. The development of opaque zones, crystals of the calcium soap around the bacterium, indicates that Tween hydrolysis has occurred
Urease production (Fahy & Hayward, 1983)
NH 4H2PO 4 0.5 g
K2HPO 4 0.5 g
MgSO 4.7H 2O 0.2 g NaCl 5 g Yeast extract (Difco) 1 g Cresol red 16 mg MQ water 800 mL After autoclaving add urea stock solution 200 mL (Stock solution: Dissolve 20 g urea in 180 mL MQ water and filter sterilise) Add 5 mL of the medium to sterile test tubes and inoculate with the target strain. A marked increase in alkalinity is considered as positive for urease production
Yeast extract dextrose carbonate agar (YDC) (Schaad et al. , 2001) Yeast extract (Difco) 10 g Dextrose 20 g Calcium carbonate, light powder 20 g
Appendix B: Media and methods for biochemical and … 166
Agar (Difco) 15 g MQ water 1,000 mL Adjust to pH 7 and autoclave at 10 PSI for 1 h, cool the medium to 50 °C in a waterbath and suspend CaCO 3 by swirling before pouring the plates
YS medium (Fahy & Hayward, 1983)
NH 4H2PO 4 0.5 g
K2HPO 4 0.5 g
MgSO 4.7H 2O 0.2 g NaCl 5 g Yeast extract (Difco) 1 g MQ water 1,000 mL Sterilise by autoclaving
Appendix C: Sequence accession numbers 167
Appendix C: Sequence accession numbers in the Genbank
Accession Fragment Length (bp) Strain number AY579378 16S-23S ribosomal RNA 558 Pistachio dieback intergenic spacer, tRNA ala and pathogen strain 7 tRNA ile genes, complete (ICMP 16316), sequence representative of group A AY579379 16S-23S ribosomal RNA 557 Pistachio dieback intergenic spacer, tRNA ala and pathogen strain 25 tRNA ile genes, complete (ICMP 16317), sequence representative of group B AY994098 16S-23S ribosomal RNA 558 X. translucens pv . intergenic spacer, tRNA ala and translucens DAR tRNA ile genes, complete 35705 sequence AY994099 16S-23S ribosomal RNA 558 X. translucens (strain intergenic spacer, tRNA ala and 61W) isolated from tRNA ile genes, complete orchard floor grasses sequence in Australia AY994100 16S rRNA complete sequence 1500 Pistachio dieback pathogen strain 7 (ICMP 16316), representative of group A AY994101 16S rRNA complete sequence 1500 Pistachio dieback pathogen strain 25 (ICMP 16317), representative of group B
Appendix D: Publications 168
Appendix D: Publications
1. Marefat A, Scott E, Ophel-Keller K, Sedgley M, 2004. Diversity within a new Xanthomonas pathogen of pistachio in Australia. In: Proceedings of the 15th International Plant Protection Congress. Beijing, China: Foreign Languages Press, 433.
2. Marefat A, Scott E, Ophel-Keller K, Sedgley M, 2005. Polyphasic characterisation of a new Xanthomonas pathogen of pistachio in Australia. In: Proceedings of the IV International Symposium on Pistachios and Almonds. Tehran, Iran: Iranian Pistachio Research Institute, 187.
3. Marefat A, Ophel-Keller K, Scott ES, Sedgley M, 2005. A PCR-based protocol for the detection of a new Xanthomonas translucens pathovar, causal agent of pistachio dieback in Australia. In: Proceedings of the 15th Biennial Australasian Plant Pathology Society Conference. Geelong, Victoria, Australia: 77.
4. Marefat A, Scott ES, Ophel-Keller K, Sedgley M, 2006. Genetic, phenotypic and pathogenic diversity among xanthomonads isolated from pistachio ( Pistacia vera ) in Australia. Plant Pathology DOI: 10.1111/j.1365-3059.2006.01437.x .
5. Marefat A, Ophel-Keller K, Scott ES, Sedgley M, 2006. The use of ARMS PCR in detection and identification of xanthomonads associated with pistachio dieback in Australia. European Journal of Plant Pathology DOI: 10.1007/s10658-006-9038-z.
Literature cited 169
Literature cited
Adachi N, Oku T, 2000. PCR-mediated detection of Xanthomonas oryzae pv. oryzae by
amplification of the 16S-23S rDNA spacer region sequence. Journal of General
Plant Pathology 66, 303-309.
Alizadeh A, Arlat M, Sarrafi A, Boucher CA, Barrault G, 1997. Restriction fragment
length polymorphism analyses of Iranian strains of Xanthomonas campestris
from cereals and grasses. Plant Disease 81, 31-35.
Alvarez A, 2001. Serological techniques. In: Schaad NW, Jones JB, Chun W, eds.
Laboratory Guide for Identification of Plant Pathogenic Bacteria. St. Paul,
Minnesota, USA: The American Phytopathological Society, 338-339.
Alvarez AM, Lou K, 1985. Rapid identification of Xanthomonas campestris pv.
campestris by an enzyme-linked immunosorbent assay ELISA. Plant Disease
69, 1082-1086.
Anonymous, 2002. Pistachio Growers Association Incorporated. Pistachio Growers
Association Strategic Plan . Sydney, Australia: Horticulture Australia Limited.
Anonymous, 2003. Pistachio annual industry report 2002-2003. Horticulture Australia
Limited. Online. [http://www.horticulture.com.au/industry/annualreports.asp ].
Anonymous, 2005a. Bacterial Nomenclature Up-to-Date. DSMZ - Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH. Online.
[http://www.dsmz.de/microorganisms/main.php?contentleft_id=14 ].
Anonymous, 2005b. FAOSTAT data. Food and Agriculture Organization. Online.
[http://faostat.fao.org/faostat/collections?subset=agriculture ].
Audy P, Laroche A, Saindon G, Huang HC, Gilbertson RL, 1994. Detection of the bean
common blight bacteria, Xanthomonas campestris pv . phaseoli and X. c.
Literature cited 170
phaseoli var. fuscans , using the polymerase chain reaction. Phytopathology 84,
1185-1192.
Ausubel FM, Brent R, Kingston RE, Moor DD, Seidman JG, Smith JA, Struhl K, 1994.
Current Protocols in Molecular Biology . New York, USA: John Wiley & Sons.
Azad H, Schaad NW, 1988. Serological relationships among membrane proteins of
strains of Xanthomonas campestris pv . translucens . Phytopathology 78, 272-
277.
Barry T, Colleran G, Glennon M, Dunican LK, Gannon F, 1991. The 16S/23S
ribosomal spacer region as a target for DNA probes to identify eubacteria. PCR
Methods and Applications 1, 51-56.
Bochner BR, 1989. Sleuthing out bacterial identities. Nature 339, 157-158.
Bouzar H, Jones JB, Stall RE, Hodge NC, Minsavage GV, Benedict AA, Alvarez AM,
1994. Physiological, chemical, serological, and pathogenic analyses of a
worldwide collection of Xanthomonas campestris pv. vesicatoria strains.
Phytopathology 84, 663-671.
Bradbury JF, 1986. Guide to Plant Pathogenic Bacteria . Surrey, UK: C.A.B.
International.
Bragard C, Singer E, Alizadeh A, Vauterin L, Maraite H, Swings J, 1997. Xanthomonas
translucens from small grains: diversity and phytopathological relevance.
Phytopathology 87, 1111-1117.
Bragard C, Verdier V, Maraite H, 1995. Genetic diversity among Xanthomonas
campestris strains pathogenic for small grains. Applied and Environmental
Microbiology 61, 1020-1026.
Literature cited 171
Bragard C, Verhoyen M, 1993. Monoclonal antibodies specific for Xanthomonas
campestris bacteria pathogenic on wheat and other small grain, in comparison
with polyclonal antisera. Journal of Phytopathology 139, 217-228.
Brisset MN, Paulin JP, Duron M, 1988. Feasibility of rating fire blight susceptibility of
pear cultivars ( Pyrus communis ) on in vitro microcuttings. Agronomie 8, 707-
710.
Brosius J, Dull TJ, Sleeter DD, Noller HF, 1981. Gene organization and primary
structure of a ribosomal RNA operon from Escherichia coli . Journal of
Molecular Biology 148, 107-127.
Caetano-Anolles G, Bassam BJ, Gresshoff PM, 1991. DNA amplification fingerprinting
using very short arbitrary oligonucleotide primers. Biotechnology 9, 553-557.
Chase AR, Stall RE, Hodge NC, Jones JB, 1992. Characterization of Xanthomonas
campestris strains from aroids using physiological, pathological, and fatty acid
analyses. Phytopathology 82, 754-759.
Chu G, Vollrath D, Davis RW, 1986. Separation of large DNA molecules by contour-
clamped homogeneous electric fields. Science 234, 1582-1585.
Cole JR, Chai B, Farris RJ, Wang Q, Kulam SA, McGarrell DM, Garrity GM, Tiedje
JM, 2005. The Ribosomal Database Project (RDP-II): sequences and tools for
high-throughput rRNA analysis. Nucleic Acids Research 33, D294-296.
Colwell RR, 1970. Polyphasic taxonomy of the genus Vibrio : numerical taxonomy of
Vibrio cholerae , Vibrio parahaemolyticus , and related Vibrio species. Journal of
Bacteriology 104, 410-433.
Davis MJ, Rott P, Warmuth CJ, Chatenet M, Baudin P, 1997. Intraspecific genomic
variation within Xanthomonas albilineans , the sugarcane leaf scald pathogen.
Phytopathology 87, 316-324.
Literature cited 172
Dayakar BV, Gnanamanickam SS, 1996. Biochemical and pathogenic variation in
strains of Xanthomonas campestris pv. mangiferaeindicae from Southern India.
Indian Phytopathology 49, 227-233. de Bruijn FJ, 1992. Use of repetitive (repetitive extragenic palindromic and
enterobacterial repetitive intergeneric consensus) sequences and the polymerase
chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and
other soil bacteria. Applied and Environmental Microbiology 58, 2180-2187.
DeParasis J, Roth DA, 1990. Nucleic acid probes for identification of phytobacteria:
identification of genus-specific 16S rRNA sequences. Phytopathology 80, 618-
621.
Duveiller E, Bragard C, 1992. Comparison of immunofluorescence and two assays for
detection of Xanthomonas campestris pv. undulosa in seeds of small grains.
Plant Disease 76, 999-1003.
Duveiller E, Bragard C, Maraite H, 1997a. Bacterial Leaf Streak and Black Chaff
Caused by Xanthomonas translucens . In: Duveiller E, Fucikovski L, Rudolph K,
eds. The Bacterial Diseases of Wheat: Concept and Methods of Disease
Management. Mexico: D.F. CIMMYT, 25-47.
Duveiller E, Bragard C, Rudolph K, Fucikovsky L, 1997b. General concepts and
methods for the identification of plant pathogenic bacteria of wheat. In:
Duveiller E, Fucikovski L, Rudolph K, eds. The Bacterial Diseases of Wheat:
Concept and Methods of Disease Management. Mexico: D.F. CIMMYT, 5-24.
Dye DW, Bradbury JF, Goto M, Hayward AC, Lelliott RA, Schroth MN, 1980.
International standards for naming pathovars of phytopathogenic bacteria and a
list of pathovar names and pathotype strains. Review of Plant Pathology 59, 153-
168.
Literature cited 173
Edwards M, 1997. Pistachio canker and dieback. In: Proceedings of Seventh Australian
Nut Industry Council Conference. Victoria, Australia: Australian Nut Industry
Council Ltd, 58-61.
Edwards M, Tassone L, Moran J, Constable F, Griffin T, Taylor C. 1998. Pistachio
canker and dieback, final report for HRDC project (NT608) . Mildura, Australia:
Agriculture Victoria.
Edwards M, Taylor C, 1998. Pistachio Canker: The story so far. In: Proceedings of
Eighth Australian Nut Industry Council Conference. Victoria, Australia:
Australian Nut Industry Council Ltd, 31-32.
Facelli E, Taylor C, Emmett R, Scott E, Sedgley M, 2001. Pistachio dieback - a
devastating problem for the industry. Australian Nutgrower 15, 12-14.
Facelli E, Taylor C, Scott E, Emmett R, Fegan M, Sedgley M, 2002. Bacterial dieback
of pistachio in Australia. Australasian Plant Pathology 31, 95-96.
Facelli E, Taylor C, Scott E, Fegan M, Huys G, Emmett R, Noble D, Sedgley M, 2005.
Identification of the causal agent of pistachio dieback in Australia. European
Journal of Plant Pathology 112, 155-165.
Fahy PC, Hayward AC, 1983. Media and methods for isolation and diagnostic tests. In:
Fahy PC, Persley GJ, eds. Plant Bacterial Diseases, a Diagnostic Guide.
Sydney, Australia: Academic Press, 337-378.
Fan XY, Hu ZY, Xu FH, Yan ZQ, Guo SQ, Li ZM, 2003. Rapid detection of rpoB gene
mutations in rifampin-resistant Mycobacterium tuberculosis isolates in Shanghai
by using the amplification refractory mutation system. Journal of Clinical
Microbiology 41, 993-997.
Literature cited 174
Fatmi M, Damsteegt VD, Schaad NW, 2005. A combined agar-absorption and BIO-
PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrus.
Plant Pathology 54, 1-7.
Fegan M, Croft BJ, Teakle DS, Hayward AC, Smith GR, 1998. Sensitive and specific
detection of Clavibacter xyli subsp. xyli , causal agent of ratoon stunting disease
of sugarcane, with a polymerase chain reaction-based assay. Plant Pathology 47,
495-504.
Ferguson L, ed 1995. Pistachio Production. Davis, USA: Center for Fruit and Nut Crop
Research and Information.
Fox GE, Pechman KR, Woese CR, 1977. Comparative cataloging of 16S ribosomal
ribonucleic acid: Molecular approach to procaryotic systematic. International
Journal of Systematic Bacteriology 27, 44-57.
Freeman WM, Walker SJ, Vrana KE, 1999. Quantitative RT-PCR: pitfalls and
potential. BioTechniques 26, 112-125.
Gent DH, Schwartz HF, Ishimaru CA, Louws FJ, Cramer RA, Lawrence CB, 2004.
Polyphasic characterization of Xanthomonas strains from onion. Phytopathology
94, 184-195.
Glick DL, Coffey CM, Sulzinski MA, 2002. Simultaneous PCR detection of the two
major bacterial pathogens of geranium. Journal of Phytopathology 150, 54-59.
Goncalves ER, Rosato YB, 2000. Genotypic characterization of xanthomonad strains
isolated from passion fruit plants ( Passiflora spp.) and their relatedness to
different Xanthomonas species. International Journal of Systematic and
Evolutionary Microbiology 50, 811-821.
Literature cited 175
Goncalves ER, Rosato YB, 2002. Phylogenetic analysis of Xanthomonas species based
upon 16S-23S rDNA intergenic spacer sequences. International Journal of
Systematic and Evolutionary Microbiology 52, 355-361.
Green MJ, Thompson DA, MacKenzie DJ, 1999. Easy and efficient DNA extraction
from woody plants for the detection of phytoplasmas by polymerase chain
reaction. Plant Disease 83, 482-485.
Griffin DE, Dowler WM, Hartung JS, Bonde MR, 1991. Differences in substrate
utilization among isolates of Xanthomonas oryzae pv . oryzae and X. campestris
pv . oryzicola from several countries. Phytopathology 81, 1222.
Gurtler V, Stanisich VA, 1996. New approaches to typing and identification of bacteria
using the 16S-23S rDNA spacer region. Microbiology 142, 3-16.
Hampton R, Ball E, De Boer S, 1990. Serological Methods for Detection and
Identification of Viral and Bacterial Plant Pathogens, a Laboratory Manual . St.
Paul, Minnesota, USA: The American Phytopathological Society Press.
Hartung JS, Civerolo EL, 1987. Genomic fingerprints of Xanthomonas campestris pv .
citri strains from Asia, South America and Florida. Phytopathology 77, 282-285.
Hartung JS, Daniel JF, Pruvost OP, 1993. Detection of Xanthomonas campestris pv.
citri by the polymerase chain reaction method. Applied and Environmental
Microbiology 59, 1143-1148.
Hartung JS, Pruvost OP, Villemot I, Alvarez A, 1996. Rapid and sensitive colorimetric
detection of Xanthomonas axonopodis pv . citri by immunocapture and a nested-
polymerase chain reaction assay. Phytopathology 86, 95-101.
Hauben L, Vauterin L, Swings J, Moore E, 1997. Comparison of 16S ribosomal DNA
sequences of all Xanthomonas species. International Journal of Systematic
Bacteriology 47, 328-335.
Literature cited 176
Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH, 1997. Multiplex PCR:
critical parameters and step-by-step protocol. BioTechniques 23, 504-511.
Henson JM, French R, 1993. The polymerase chain reaction and plant-disease
diagnosis. Annual Review of Phytopathology. 31, 81-109.
Holt JG, ed 1994. Bergey's Manual of Determinative Bacteriology. Baltimore,
Maryland, USA: Williams & Wilkins.
Honeycutt RJ, Sobral BWS, McClelland M, 1995. tDNA intergenic spacers reveal
polymorphisms diagnostic for Xanthomonas albilineans . Microbiology 141,
3229-3239.
Huss VAR, Festl H, Schleifer KH, 1983. Studies on the spectrophotometric
determination of DNA hybridization from renaturation rates. Systematic Applied
Microbiology 4, 184-192.
Innis MA, 1990. PCR Protocols : A Guide to Methods and Applications. San Diego,
California, USA: Academic Press.
Janse JD, Derks JHJ, Spit BE, Van der Tuin WR, 1992. Classification of fluorescent
soft rot Pseudomonas bacteria, including P. marginalis strains, using whole cell
fatty acid analysis. Systematic and Applied Microbiology 15, 538-553.
Janse JD, Rossi MP, Gorkink RFJ, Derks JHJ, Swings J, Janssens D, Scortichini M,
2001. Bacterial leaf blight of strawberry ( Fragaria (x) ananassa ) caused by a
pathovar of Xanthomonas arboricola , not similar to Xanthomonas fragariae
Kennedy & King. Description of the causal organism as Xanthomonas
arboricola pv . fragariae (pv. nov., comb. nov.). Plant Pathology 50, 653-665.
Jayne AB, Taylor EJA, 2001. Scorpion ARMS primers for SNP real-time PCR
detection and quantification of Pyrenophora teres . Molecular Plant Pathology 2,
275-280.
Literature cited 177
John ME, 1992. An efficient method for isolation of RNA and DNA from plants
containing polyphenolics. Nucleic Acids Research 20, 2381.
Jones J, Bouzar H, Stall R, Almira E, Roberts P, Bowen B, Sudberry J, Strickler P,
Chun J, 2000. Systematic analysis of xanthomonads (Xanthomonas spp.)
associated with pepper and tomato lesions. International Journal of Systematic
and Evolutionary Microbilogy 50, 1211-1219.
Jones JB, Chase AR, Harris GK, 1993. Evaluation of the Biolog GN Microplate System
for identification of some plant-pathogenic bacteria. Plant Disease 77, 553-558.
Kersters K, 1990. Polyacrylamide gel electrophoresis of bacterial proteins. In: Klement
Z, Rudolph K, Sands DC, eds. Methods in Phytobacteriology. Budapest,
Hungary: Akademiai Kiado and Nyomda Vallalat, 191-198.
Klement Z, 1982. Hypersensitivity. In: Mount MS, Lacy GH, eds. Phytopathogenic
Prokaryotes. New York, USA: Academic Press, 149-177.
Klement Z, 1990. Inoculation of plant tissues. In: Klement Z, Rudolph K, Sands DC,
eds. Methods in Phytobacteriology. Budapest, Hungary: Akademiai Kiado and
Nyomda Vallalat, 103-104.
Klement Z, Rozsnyay DS, Arsenijevic M, 1974. Apoplexy of apricots. III. Relationship
of winter frost and the bacterial canker and die-back of apricots. Acta
Phytopathologica Academiae Scientiarum Hungaricae 9, 35-45.
Klement Z, Rudolph K, Sands DC, eds, 1990. Methods in Phytobacteriology. Budapest,
Hungary: Akademiai Kiado and Nyomda Vallalat.
Komagata K, Suzuki K, 1987. Lipid and cell-wall analysis in bacterial systematics.
Methods in Microbiology 19, 161-207.
Literature cited 178
Koonjul P, Brandt W, Farrant J, Lindsey G, 1999. Inclusion of polyvinylpyrrolidone in
the polymerase chain reaction reverses the inhibitory effects of polyphenolic
contamination of RNA. Nucleic Acids Research 27, 915-916.
Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227, 680-685.
Lane DJ, 1991. Small subunit ribosomal RNA sequences and primers. Large subunit
ribosomal RNA sequences and primers. In: Goodfellow E, ed. Nucleic Acid
Techniques in Bacterial Systematics. Chichester, England: John Wiley & Sons,
148-175.
Lazo GR, Roffey R, Gabriel DW, 1987. Pathovars of Xanthomonas campestris are
distinguishable by restriction fragment-length polymorphism. International
Journal of Systematic Bacteriology 37, 214-221.
Leite RP, Minsavage GV, Bonas U, Stall RE, 1994. Detection and identification of
phytopathogenic Xanthomonas strains by amplification of DNA sequences
related to the hrp genes of Xanthomonas campestris pv . vesicatoria . Applied and
Environmental Microbiology 60, 1068-1077.
Leite RP, Santos SD, 1988. Susceptibility of Sicilian lemon (Citrus lemon) grafted on
different rootstocks to citrus canker caused by Xanthomonas campestris pv.
citri. Fitopatologia Brasileira. 13, 353-358.
Lelliott RA, Stead DE, 1987. Methods for the Diagnosis of Bacterial Diseases of Plants .
Oxford, UK: British Society of Plant Pathology/Blackwell Scientific
Publications.
Leyns F, De Cleen M, Swings J, De Ley J, 1984. The host range of the genus
Xanthomonas . Botanical Review 50, 308-356.
Literature cited 179
Lindow SE, 1982. Epiphytic ice nucleation active bacteria. In: Mount MS, Lacy GH,
eds. Phytopathogenic Prokaryotes. New York, USA: Academic Press, 335-362.
Lindow SE, 1990. Bacterial ice-nucleation activity. In: Klement Z, Rudolph K, Sands
DC, eds. Methods in Phytobacteriology. Budapest, Hungary: Akademiai Kiado
and Nyomda Vallalat, 428-34.
Lopes SA, Damann KE, Grelen LB, 2001. Xanthomonas albilineans diversity and
identification based on rep-PCR fingerprints. Current Microbiology 42, 155-
159.
Louws FJ, Fulbright DW, Stephens CT, de Bruijn FJ, 1994. Specific genome
fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and
strains generated with repetitive sequences and PCR. Applied and
Environmental Microbiology 60, 2286-2295.
Louws FJ, Fulbright DW, Stephens CT, de Bruijn FJ, 1995. Differentiation of genomic
structure by rep-PCR fingerprinting to rapidly classify Xanthomonas campestris
pv. vesicatoria . Phytopathology 85, 528-536.
Louws FJ, Rademaker JLW, de Bruijn FJ, 1999. The three Ds of PCR-based genomic
analysis of phytobacteria: diversity, detection, and disease diagnosis. Annual
Review of Phytopathology 37, 81-125.
Mach RL, Kullnig-Gradinger CM, Farnleitner AH, Reischer G, Adler A, Kubicek CP,
2004. Specific detection of Fusarium langsethiae and related species by DGGE
and ARMS-PCR of a [beta]-tubulin (tub1) gene fragment. International Journal
of Food Microbiology 95, 333-339.
Maes M, Garbeva P, Crepel C, 1996a. Identification and sensitive endophytic detection
of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA
sequences and the polymerase chain reaction. Plant Pathology 45, 1139-1149.
Literature cited 180
Maes M, Garbeva P, Kamoen O, 1996b. Recognition and detection in seed of the
Xanthomonas pathogens that cause cereal leaf streak using rDNA spacer
sequences and polymerase chain reaction. Phytopathology 86, 63-69.
Maggs DH, 1982. An Introduction to Pistachio Growing in Australia . Adelaide,
Australia: Commonwealth Scientific and Industrial Research Organization.
Manulis S, Valinsky L, Lichter A, Gabriel DW, 1994. Sensitive and specific detection
of Xanthomonas campestris pv . pelargonii with DNA primers and probes
identified by random amplified polymorphic DNA analysis. Applied and
Environmental Microbiology 60, 4094-4099.
Marefat A, Rahimian H, 1998. Identity and population dynamic of epiphytic bacteria of
wheat in Mazandaran, Isfahan and Tehran provinces and preliminary studies on
the antagonistic effects of representative strains on biological control of leaf
streak caused by Xanthomonas translucens pv. cerealis. In: Proceedings of the
13th Plant Protection Congress of Iran. Karaj, Iran: College of Agriculture, 58.
Martin B, Humbert O, Camara M, Guenzi E, Walker J, Mitchell T, Andrew P,
Prudhomme M, Alloing G, Hakenbeck R, 1992. A highly conserved repeated
DNA element located in the chromosome of Streptococcus pneumoniae . Nucleic
Acids Research 20, 3479-3483.
Massomo SMS, Nielsen H, Mabagala RB, Mansfeld-Giese K, Hockenhull J, Mortensen
CN, 2003. Identification and characterisation of Xanthomonas campestris pv .
campestris strains from Tanzania by pathogenicity tests, Biolog, rep-PCR and
fatty acid methyl ester analysis. European Journal of Plant Pathology 109, 775-
789.
Literature cited 181
Mazzola M, Leach JE, Nelson R, White FF, 1994. Analysis of the interaction between
Xanthomonas oryzae pv. oryzae and the rice cultivars IR24 and IRBB21.
Phytopathology 84, 392-397.
McDonald JG, Wong E, 2001. Use of a monoclonal antibody and genomic
fingerprinting by repetitive-sequence-based polymerase chain reaction to
identify Xanthomonas populi pathovars. Canadian Journal of Plant Pathology
23, 47-51.
Mellano VJ, Cooksey DA, 1988. Development of host range mutants of Xanthomonas
campestris pv . translucens . Applied and Environmental Microbiology 54, 884-
889.
Meng XQ, Umesh KC, Davis RM, Gilbertson RL, 2004. Development of PCR-based
assays for detecting Xanthomonas campestris pv. carotae , the carrot bacterial
leaf blight pathogen, from different substrates. Plant Disease 88, 1226-1234.
Mew TW, Vera-Cruz CM, Medalla ES, 1992. Changes in race frequency of
Xanthomonas oryzae pv. oryzae in response to rice cultivars planted in the
Philippines. Plant Disease 76, 1029-1032.
Milgroom MG, Fry WE, 1997. Contributions of population genetics to plant disease
epidemiology and management. Advances in Botanical Research 24, 1-30.
Milus EA, Chalkley DB, 1994. Virulence of Xanthomonas campestris pv . translucens
on selected wheat cultivars. Plant Disease 78, 612-615.
Mkandawire ABC, Mabagala RB, Guzman P, Gepts P, Gilbertson RL, 2004. Genetic
diversity and pathogenic variation of common blight bacteria ( Xanthomonas
campestris pv . phaseoli and X. campestris pv . phaseoli var. fuscans ) suggests
pathogen coevolution with the common bean. Phytopathology 94, 593-603.
Literature cited 182
Moffett ML, McCarthy JP, 1973. Xanthomonas translucens on Japanese millet
(Echinochloa crus-galli var. frumentacea ). Australian Journal of Experimental
Agriculture and Animal Husbandry 13, 452-454.
Mooter MVd, Maraite H, Meiresonne L, Swings J, Gillis M, Kersters K, Ley Jd, 1987a.
Comparison between Xanthomonas campestris pv . manihotis (ISPP list 1980)
and X. campestris pv. cassavae (ISPP list 1980) by means of phenotypic, protein
electrophoretic, DNA hybridization and phytopathological techniques. Journal
of General Microbiology 133, 57-71.
Mooter MVd, Steenackers M, Maertens C, Gossele F, De Vos P, Swings J, Kersters K,
Ley Jd, 1987b. Differentiation between Xanthomonas campestris pv . graminis
(ISPP list 1980), pv. phleipratensis (ISPP list 1980) emend., pv. poae Egli and
Schmidt 1982 and pv. arrhenatheri Egli and Schmidt 1982, by numerical
analysis of phenotypic features and protein gel electrophoregrams. Journal of
Phytopathology 118, 135-156.
Mooter MVd, Swings J, 1990. Numerical analysis of 295 phenotypic features of
Xanthomonas strains and related strains and an improved taxonomy of the
genus. International Journal of Systematic Bacteriology 40, 348-369.
Murata N, Starr MP, 1973. A concept of the genus Xanthomonas and its species in the
light of segmental homology of deoxyribonucleic acids. Phytopathologische
Zeitschrift 77, 285-323.
Murray RGE, Brenner DJ, Colwell RR, De Vos P, Goodfellow M, Grimont PAD,
Pfennig N, Stackebrandt E, Zavarzin GA, 1990. Report of the ad hoc committee
on approaches to taxonomy within the Proteobacteria. International Journal of
Systematic Bacteriology 40, 213-215.
Literature cited 183
Neidhardt FC, Ingraham JL, Schaechter M, 1990. Physiology of the Bacterial Cell: a
Molecular Approach . Massachusetts, USA: Sinauer Associates, Inc.
Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith
JC, Markham AF, 1989. Analysis of any point mutation in DNA. The
amplification refractory mutation system (ARMS). Nucleic Acids Research 17,
2503-2516.
Noble RJ, 1935. Notes on plant diseases recorded in New South Wales for the year
ending 30th June, 1935. International Bulletin of Plant Protection 12, 270-273.
Opio AF, Allen DJ, Teri JM, Pathogenic variation in Xanthomonas campestris pv.
phaseoli , the causal agent of common bacterial blight in Phaseolus beans. Plant
Pathology 45, 1126-1133.
Palleroni NJ, Hildebrand DC, Schroth MN, Hendson M, 1993. Deoxyribonucleic acid
relatedness of 21 strains of Xanthomonas species and pathovars. Journal of
Applied Bacteriology 75, 441-446.
Pan YB, Grisham MP, Burner DM, 1997. A polymerase chain reaction protocol for the
detection of Xanthomonas albilineans , the causal agent of sugarcane leaf scald
disease. Plant Disease 81, 189-194.
Pan YB, Grisham MP, Burner DM, Legendre BL, Wei Q, 1999. Development of
polymerase chain reaction primers highly specific for Xanthomonas albilineans ,
the causal bacterium of sugarcane leaf scald disease. Plant Disease 83, 218-222.
Paran I, Michelmore RW, 1993. Development of reliable PCR-based markers linked to
downy mildew resistance genes in lettuce. Theoretical and Applied Genetics 85,
985-993.
Pitcher DG, Saunders NA, Owen RJ, 1989. Rapid extraction of bacterial genomic DNA
with guanidium thiocyanate. Letters in Applied Microbiology 8, 151-156.
Literature cited 184
Pooler MR, Ritchie DF, Hartung JS, 1996. Genetic relationships among strains of
Xanthomonas fragariae based on random amplified polymorphic DNA PCR,
repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic
consensus PCR data and generation of multiplexed PCR primers useful for the
identification of this phytopathogen. Applied and Environmental Microbiology
62, 3121-3127.
Quezado-Duval AM, Leite-Junior RP, Truffi D, Camargo LEA, 2004. Outbreaks of
bacterial spot caused by Xanthomonas gardneri on processing tomato in
Central-West Brazil. Plant Disease 88, 157-161.
Rademaker JLW, de Bruijn FJ, 1997. Characterization and classification of microbes by
rep-PCR genomic fingerprinting and computer-assisted pattern analysis. In:
Caetano-Anolles G, Gresshoff PM, eds. DNA Markers: Protocols, Application
and Overviews. New York, USA: John Wiley & Sons, 151-171.
Rademaker JLW, Hoste B, Louws FJ, Kersters K, Swings J, Vauterin L, Vauterin P, de
Bruijn FJ, 2000. Comparison of AFLP and rep-PCR genomic fingerprinting with
DNA-DNA homology studies: Xanthomonas as a model system. International
Journal of Systematic and Evolutionary Microbiology 50, 665-677.
Rademaker JLW, Louws FJ, Schultz MH, Rossbach U, Vauterin L, Swings J, de Bruijn
FJ, 2005. A comprehensive species to strain taxonomic framework for
Xanthomonas . Phytopathology 95, 1098-1111.
Randhawa PS, Civerolo EL, 1985. A detached-leaf bioassay for Xanthomonas
campestris pv . pruni . Phytopathology 75, 1060-1063.
Restrepo S, Velez CM, Verdier V, 2000. Measuring the diversity of Xanthomonas
axonopodis pv. manihotis within different fields in Colombia. Phytopathology
90, 683-690.
Literature cited 185
Roberts PD, Hodge NC, Bouzar H, Jones JB, Stall RE, Berger RD, Chase AR, 1998.
Relatedness of strains of Xanthomonas fragariae by restriction fragment length
polymorphism, DNA-DNA reassociation, and fatty acid analyses. Applied and
Environmental Microbiology 64, 3961-3965.
Robinson B, 1997. Pistachio nuts. The New Rural Industries. A Handbook for Farmers
and Investors. RIRDC . Online.
[http://www.rirdc.gov.au/pub/handbook/ostrich.html ].
Roumagnac P, Gagnevin L, Gardan L, Sutra L, Manceau C, Dickstein ER, Jones JB,
Rott P, Pruvost O, 2004. Polyphasic characterization of xanthomonads isolated
from onion, garlic and Welsh onion ( Allium spp.) and their relatedness to
different Xanthomonas species. International Journal of Systematic and
Evolutionary Microbiology 54, 15-24.
Saettler AW, Schaad NW, Roth DA, eds, 1989. Detection of Bacteria in Seed and Other
Planting Material. St. Paul, Minnesota, USA: The American Phytopathological
Society Press.
Sahin F, Abbasi PA, Ivey MLL, Zhang J, Miller SA, 2003. Diversity among strains of
Xanthomonas campestris pv. vitians from lettuce. Phytopathology 93, 64-70.
Sakthivel N, Mortensen CN, Mathur SB, 2001. Detection of Xanthomonas oryzae pv .
oryzae in artificially inoculated and naturally infected rice seeds and plants by
molecular techniques. Applied Microbiology and Biotechnology 56, 435-441.
Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning: A Laboratory Manual .
New York, USA: Cold Spring Harbor Laboratory Press.
Sands DC, 1990. Physiological criteria-determinative tests. In: Klement Z, Rudolph K,
Sands DC, eds. Methods in Phytobacteriology. Budapest, Hungary: Akademiai
Kiado and Nyomda Vallalat, 133-143.
Literature cited 186
Sarpkaya K, Can C, 2005. Dieback: an unknown disorder in pistachio. In: Proceedings
of the IV International Symposium on Pistachios and Almonds. Tehran, Iran:
Iranian Pistachio Research Institute, 192.
Sasser M, 1990. Idenatification of bacteria through fatty acid analysis. In: Klement Z,
Rudolph K, Sands D, eds. Methods in Phytobacteriology. Budapest, Hungary:
Akademiai Kiado and Nyomda Vallalat, 199-204.
Sayler RJ, Southwick SM, Yeager JT, Glozer K, Little EL, Kirkpatrick BC, 2002.
Effects of rootstock and budding height on bacterial canker in French prune.
Plant Disease 86, 543-546.
Schaad NW, 1988. Laboratory Guide for Identification of Plant Pathogenic Bacteria .
St. Paul, Minnesota, USA: The American Phytopathological Society Press.
Schaad NW, Cheong SS, Tamaki S, Hatziloukas E, Panopoulos NJ, 1995. A combined
biological amplification (BIO-PCR) technique to detect Pseudomonas syringae
pv . syringae in bean seed extracts. Phytopathology 85, 243-248.
Schaad NW, Jones JB, Chun W, eds, 2001. Laboratory Guide for Identification of Plant
Pathogenic Bacteria. St. Paul, Minnesota, USA: The American
Phytopathological Society Press.
Scortichini M, Marchesi U, Di Prospero P, 2001. Genetic diversity of Xanthomonas
arboricola pv . juglandis (synonyms: X. campestris pv . juglandis; X. juglandis
pv . juglandis ) strains from different geographical areas shown by repetitive
polymerase chain reaction genomic fingerprinting. Journal of Phytopathology
149, 325-332.
Scortichini M, Rossi MP, 2003. Genetic diversity of Xanthomonas arboricola pv.
fragariae strains and comparison with some other X. arboricola pathovars using
Literature cited 187
repetitive PCR genomic fingerprinting. Journal of Phytopathology 151, 113-
119.
Scortichini M, Rossi MP, Marchesi U, 2002. Genetic, phenotypic and pathogenic
diversity of Xanthomonas arboricola pv. corylina strains question the
representative nature of the type strain. Plant Pathology 51, 374-381.
Sharon E, Okon Y, Bashan Y, Henis Y, 1982. Detached leaf enrichment: a method for
detecting small numbers of Pseudomonas syringae pv. tomato and Xanthomonas
campestris pv. vesicatoria in seed and symptomless leaves of tomato and
pepper. Journal of Applied Bacteriology 53, 371-377.
Shurtleff MC, Averre CWI, 1997. Glossary of Plant-Pathological Terms . St. Paul,
Minnesota, USA: The American Phytopathological Society Press.
Spotts RA, Mielke EA, 1999. Resistance of pear cultivars in Oregon to natural fire
blight infection. Fruit Varieties Journal 53, 110-115.
Stackebrandt E, Frederiksen W, Garrity GM, Grimont P, Kampfer P, Maiden M, Nesme
X, Rossello-Mora R, Swings J, Truper HG, Vauterin L, Ward AC, Whitman
WB, 2002. Report of the ad hoc committee for the re-evaluation of the species
definition in bacteriology. International Journal of Systematic and Evolutionary
Microbiology 52, 1043-1047.
Starr MP, 1983. Phytopathogenic Bacteria . New York, USA: Springer-Verlag.
Stead DE, 1989. Grouping of Xanthomonas campestris pathovars of cereals and grasses
by fatty acid profiling. Bulletin OEPP/EPPO Bulletin 19, 57-68.
Stead DE, 1992. Techniques for detecting and identifying plant pathogenic bacteria. In:
Duncan JM, Torrance L, eds. Techniques for the Rapid Detection of Plant
Pathogens. Oxford, UK: Blackwell Scientific Publications, 76-111.
Literature cited 188
Stromberg KD, Kinkel LL, Leonard KJ, 2000. Interactions between Xanthomonas
translucens pv. translucens , the causal agent of bacterial leaf streak of wheat,
and bacterial epiphytes in the wheat phyllosphere. Biological Control 17, 61-72.
Stromberg KD, Kinkel LL, Leonard KJ, 2004. Quantifying the effect of bacterial
antagonists on the relationship between phyllosphere population sizes of
Xanthomonas translucens pv. translucens and subsequent bacterial leaf streak
severity on wheat seedlings. Biological Control 29, 58-65.
Sulzinski MA, Moorman GW, Schlagnhaufer B, Romaine CP, 1996. Characteristics of
a PCR-based assay for in planta detection of Xanthomonas campestris pv .
pelargonii . Journal of Phytopathology 144, 393-398.
Tarighi S, Rahimian H, 2001. Canker and leafspot of pistachio caused by Xanthomonas
sp. Iranian Journal of Plant Pathology 37, 161-162.
Taylor C, Edwards M. 2000. Managing canker and dieback in pistachios, final report for
Horticulture Australia Limited project (NT97002) . Mildura, Australia:
Agriculture Victoria.
Teviotdale BL, Michailides TJ, Pscheidt JW, eds, 2002. Compendium of Nut Crop
Diseases in Temperate Zones. St. Paul, Minnesota, USA: The American
Phytopathological Society Press.
Trebaol G, Gardan L, Manceau C, Tanguy JL, Tirilly Y, Boury S, 2000. Genomic and
phenotypic characterization of Xanthomonas cynarae sp. nov., a new species
that causes bacterial bract spot of artichoke (Cynara scolymus L.). International
Journal of Systematic and Evolutionary Microbiology 50, 1471-1478.
Trebaol G, Manceau C, Tirilly Y, Boury S, 2001. Assessment of the genetic diversity
among strains of Xanthomonas cynarae by randomly amplified polymorphic
DNA analysis and development of specific characterized amplified region for
Literature cited 189
the rapid identification of X. cynarae . Applied and Environmental Microbiology
67, 3379-3384.
Vandamme P, Pot B, Gillis M, De Vos P, Kersters K, Swings J, 1996. Polyphasic
taxonomy, a consensus approach to bacterial systematics. Microbiological
Reviews 60, 407-438.
Vauterin L, Hoste B, Kersters K, Swings J, 1995. Reclassification of Xanthomonas .
International Journal of Systematic Bacteriology 45, 472-489.
Vauterin L, Rademaker J, Swings J, 2000. Synopsis on the taxonomy of the genus
Xanthomonas . Phytopathology 90, 677-682.
Vauterin L, Swings J, Kersters K, Gillis M, Mew TW, Schroth MN, Palleroni NJ,
Hildebrand DC, Stead DE, Civerolo EL, Hayward AC, Maraite H, Stall RE,
Vidaver AK, Bradbury JF, 1990. Towards an improved taxonomy of
Xanthomonas . International Journal of Systematic Bacteriology 40, 312-316.
Vauterin L, Swings JG, Kersters K, 1991a. Grouping of Xanthomonas campestris
pathovars by SDS-PAGE of proteins. Journal of General Microbiology 137,
1677-1687.
Vauterin L, Yang P, Hoste B, Pot B, Swings J, Kersters K, 1992. Taxonomy of
xanthomonads from cereals and grasses based on SDS-PAGE, fatty acid analysis
and DNA hybridization. Journal of General Microbiology 138, 1467-1477.
Vauterin L, Yang P, Hoste B, Vancanneyt M, 1991b. Differentiation of Xanthomonas
campestris pv . citri strains by sodium dodecyl sulphate polyacrylamide gel
electrophoresis of proteins, fatty acid analysis, and DNA-DNA hybridization.
International Journal of Systematic Bacteriology 41, 535-542.
Literature cited 190
Verdier V, Mosquera G, Assigbetse K, 1998. Detection of the cassava bacterial blight
pathogen, Xanthomonas axonopodis pv. manihotis , by polymerase chain
reaction. Plant Disease 82, 79-83.
Verniere C, Pruvost O, Civerolo EL, Gambin O, Jacquemoud-Collet JP, Luisetti J,
1993. Evaluation of the Biolog substrate utilization to identify and assess
metabolic variation among strains of Xanthomonas campestris pv. citri . Applied
and Environmental Microbiology 59, 243-249.
Versalovic J, Koeuth T, Lupski J, 1991. Distribution of repetitive DNA sequences in
eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids
Research 19, 6823-6831.
Vicente JG, Conway J, Roberts SJ, Taylor JD, 2001. Identification and origin of
Xanthomonas campestris pv . campestris races and related pathovars.
Phytopathology 91, 492-499.
Vivian A, Murillo J, Jackson RW, 2001. The roles of plasmids in phytopathogenic
bacteria: mobile arsenals? Microbiology 147, 763-780.
Vos P, Hogers R, Bleeker M, Reijans M, Lee TV, Hornes M, Frijters A, Pot J, Peleman
J, Kuiper M, Zabeau M, 1995. AFLP: a new technique for DNA fingerprinting.
Nucleic Acids Research 23, 4407-4414.
Wang ZK, Comstock JC, Hatziloukas E, Schaad NW, 1999. Comparison of PCR, BIO-
PCR, DIA, ELISA and isolation on semiselective medium for detection of
Xanthomonas albilineans , the causal agent of leaf scald of sugarcane. Plant
Pathology 48, 245-252.
Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Moore
LH, Moore WEC, Murray RGE, Stackebrandt E, Starr MP and Truper HG,
1987. Report of the ad hoc committee on reconciliation of approaches to
Literature cited 191
bacterial systematics. International Journal of Systematic Bacteriology 37,
463–464.
Weigel RM, Qiao BZ, Teferedegne B, Suh DK, Barber DA, Isaacson RE, White BA,
2004. Comparison of pulsed field gel electrophoresis and repetitive sequence
polymerase chain reaction as genotyping methods for detection of genetic
diversity and inferring transmission of Salmonella . Veterinary Microbiology
100, 205-217.
Welsh J, McClelland M, 1990. Fingerprinting genomes using PCR with arbitrary
primers. Nucleic Acids Research 18, 7213-7218.
Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV, 1990. DNA
polymorphisms amplified by arbitrary primers are useful as genetic markers.
Nucleic Acids Research 18, 6531-6535.
Yang P, Vauterin L, Vancanneyt M, Swings J, Kersters K, 1993. Application of fatty
acid methyl esters for the taxonomic analysis of the genus Xanthomonas .
Systematic and Applied Microbiology 16, 47-71.
Ye S, Dhillon S, Ke X, Collins AR, Day IN, 2001. An efficient procedure for
genotyping single nucleotide polymorphisms. Nucleic Acids Research 29, E88.
Yessad S, Manceau C, Luisetti J, 1992. A detached leaf assay to evaluate virulence and
pathogenicity of strains of Pseudomonas syringae pv . syringae on pear. Plant
Disease 76, 370-373.
Young JM, Saddler GS, Takikawa Y, Boer SHd, Vauterin L, Gardan L, Gvozdyak RI,
Stead DE, 1996. Names of plant pathogenic bacteria 1864-1995. Review of Plant
Pathology 75, 721-763.