Molecular detection and identification of phytoplasmas in sugarcane in Hawaii, Thailand, Cuba and Near East

Molecular detection and identification of phytoplasmas in sugarcane in Hawaii, Thailand, Cuba and Near East Dissertation Zur Erlangung des Grades Doktor der Naturwissenschaften -Dr. rer. nat.- der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth vorgelegt von Ziad Soufi Aus Latakia, Syrien Bayreuth, Germany Januar 2012 Die vorliegende Arbeit wurde in der Zeit von Juli 2007 bis Januar 2012 am Lehrstuhl für Pflanzenphysiologie der Universität Bayreuth unter der Leitung von Herrn Prof. Dr. Ewald Komor angefertigt. Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.). Dissertation eingereicht am: 11. 01. 2012 Wissenschaftliches Kolloquium: 27. 03. 2012 Amtierende Dekanin: Prof. Dr. Beate Lohnert Prüfungsausschuss: Prof. Dr. Ewald Komor (Erstgutachter) Prof. Dr. Harold L. Drake (Zweitgutachter) Prof. Dr. Gerhard Rambold (Vorsitz) Prof. Dr. Klaus H. Hoffmann PD Dr. Stefan Geimer I Contents 1. Introduction ....................................................................................................... 1 1.1. Plant diseases ............................................................................................................................... 1 1.2. Phytoplasmas as plant pathogens ................................................................................................ 2 1.3. Definition of phytoplasmas .......................................................................................................... 2 1.4. Transmission and spread of phytoplasmal diseases .................................................................... 6 1.4.1. Host cycle of phytoplasmas ................................................................................................... 6 1.4.2. Host Specificity of Phytoplasmas .......................................................................................... 8 1.5. Phylogenetic position of phytoplasmas ....................................................................................... 8 1.6. Management and control of phytoplasma diseases .................................................................... 9 1.6.1. Prevention strategy ................................................................................................................ 9 1.6.2. Control of insect vectors ...................................................................................................... 10 1.6.3. Management strategy .......................................................................................................... 10 1.7. Anatomy of phloem cells ............................................................................................................ 10 1.8. Some phytoplasma diseases of sugarcane ................................................................................. 11 1.8.1. Sugarcane yellow leaf syndrome ......................................................................................... 11 1.8.2. Sugarcane white leaf and sugarcane grassy shoot ............................................................... 12 1.9. Detection of sugarcane phytoplasma infections ........................................................................ 13 1.10. Sugarcane yellow leaf syndrome in Hawaii .............................................................................. 14 2. Material and Methods ...................................................................................... 17 2.1. Material ...................................................................................................................................... 17 2.1.2. Chemicals and Enzymes ...................................................................................................... 17 2.1.2.1. Chemicals ..................................................................................................................... 17 2.1.2.2. Enzymes ....................................................................................................................... 18 2.1.3. Buffers, Solutions ................................................................................................................ 18 2.1.3.1. Buffer and solutions for DNA extraction ..................................................................... 18 2.1.3.2. Buffer for gel electrophoresis ....................................................................................... 19 2.1.3.3. Buffer for PCR ............................................................................................................. 19 2.1.3.4. Buffer for restriction enzymes ...................................................................................... 19 2.1.3.5. Buffer for polyacrylamide gel electrophoresis ............................................................. 19 2.1.4. Kits ...................................................................................................................................... 20 2.1.4.1. Isolation of Nucleic Acids for PCR .............................................................................. 20 2.1.4.2. Nucleic acids purification ............................................................................................. 20 II 2.1.4.3. Q-PCR .......................................................................................................................... 20 2.1.5. Oligonucleotides .................................................................................................................. 21 2.1.6. Software for Gene analysis .................................................................................................. 21 2.2. Methods ..................................................................................................................................... 22 2.2.1. Plant material ....................................................................................................................... 22 2.2.2. DNA extraction strategies ................................................................................................... 22 2.2.3. Polymerase Chain Reaction (PCR) for the detection of phytoplasmas ............................... 24 2.2.3.1. Definition of Nested PCR ............................................................................................. 24 2.2.3.2. Nested PCR Reaction ................................................................................................... 25 2.2.3.3. First round of PCR .................................................................................................... 26 2.2.3.4. Nested round of PCR .................................................................................................... 26 2.2.3.5. Nested-PCR assay (I) ................................................................................................... 26 2.2.3.6. Nested-PCR assay (II) .................................................................................................. 27 2.2.3.7. Nested-PCR assay (III) ................................................................................................. 27 2.2.3.8. Nested-PCR assay (IV) ................................................................................................ 28 2.2.4. Agarose Gel Electrophoresis ............................................................................................... 29 2.2.5. Digestion of nested-PCR products ...................................................................................... 30 2.2.5.1. Inactivation of restriction enzymes .............................................................................. 30 2.2.6. Polyacrylamide Gel Electrophoresis ................................................................................... 31 2.2.6.1. Steps of operation ......................................................................................................... 31 2.2.6.2. Special equipment ........................................................................................................ 31 2.2.6.3. Detection of DNA in polyacrylamide gels by staining ................................................. 31 2.2.7. Sequencing and phylogenetic analysis of ribosomal DNA ................................................. 31 2.2.7.1. Sample Preparation for Value Read Service in Tubes ................................................. 32 2.2.8. Hot water treatment ............................................................................................................. 32 2.2.8.1. Preparation of the plant material prior to the hot water treatment ................................ 33 2.2.9. Sugarcane aphid transmission test ....................................................................................... 33 2.2.9.1. Insect rearing ................................................................................................................ 33 2.2.9.2. Plant material ................................................................................................................ 33 2.2.9.3. Transmission tests ........................................................................................................ 34 2.2.10. Q-PCR (real-time PCR) assay ........................................................................................... 34 2.2.10.1. Methods of monitoring DNA amplification in qPCR ................................................ 34 2.2.10.2. Detection of phytoplasma based on TaqMan qPCR assays........................................ 36 2.2.11. Transmission Electron Microscopy (TEM) ....................................................................... 37 III 2.2.11.1. Preparation of thin sections ........................................................................................ 38 3. Results ............................................................................................................. 39 3.1. Establishment of the test for phytoplasma ................................................................................ 39 3.1.1. PCR for detection of phytoplasma ...................................................................................... 39 3.1.2. Sources of sugarcane samples ............................................................................................. 41 3.2. Phytoplasma in sugarcane in Hawaii, Cuba, Egypt and Syria ..................................................... 42 3.2.1. Phytoplasma detection by nested-PCR assay (I) and identification by RFLP ..................... 42 3.2.2. Phytoplasma detection by nested-PCR assay (II) and identification by RFLP ................... 46 3.2.3. Phytoplasma detection by nested-PCR assay (III) .............................................................. 48 3.2.4. Phytoplasma detection by nested-PCR assay (IV) and identification by RFLP .................. 49 3.3. Phytoplasma in sugarcane in Hawaiian plantations (2009) ....................................................... 52 3.3.1. Phytoplasma detection and identification ............................................................................ 54 3.4. Phytoplasma in sugarcane in Hawaiian former plantation fields (2009) ................................... 55 3.4.1. Phytoplasma detection and identification ............................................................................ 56 3.5. Phytoplasma in sugarcane in Hawaiian breeding station (2010) ............................................... 57 3.5.1. Phytoplasma detection and identification ............................................................................ 57 3.6. Phytoplasma in sugarcane in Hawaiian breeding station and plantations (2011) ..................... 58 3.6.1. Phytoplasma in sugarcane in Hawaiian plantation .............................................................. 58 3.6.1.1. Phytoplasma detection and identification ..................................................................... 59 3.6.2. Phytoplasma in sugarcane in Hawaiian breeding station (2011) ........................................ 61 3.6.2.1. Phytoplasma detection by nested-PCR assay (II) and identification ............................ 61 3.6.2.2. Phytoplasma detection by nested-PCR assay (III) and (IV) and identification ............ 63 3.6.3. Phytoplasma in sugarcane in different areas close to former plantations ............................ 67 3.6.3.1. Phytoplasma detection and identification ..................................................................... 67 3.6.4. Phytoplasma in grass weeds in Sumida water cress farm ................................................... 69 3.7. Phytoplasma in sugarcane in Thailand (2010 – 2011) ................................................................ 70 3.7.1. Phytoplasma in sugarcane in Bang Phra and Khon Kean provinces (2010) ....................... 70 3.7.1.1. Phytoplasma detection and identification ..................................................................... 71 3.7.2. Phytoplasma in sugarcane in Suphan Buri province (2011) ................................................ 75 3.7.2.1. Phytoplasma detection and identification ..................................................................... 75 3.8. Establishment of TaqMan qPCR assay as another test for phytoplasma ................................... 76 3.8.1. Performance characteristics of qPCR .................................................................................. 76 3.8.1.1. Efficiency Measurement ............................................................................................... 77 3.8.1.2. Artificial samples to test sensitivity of qPCR assay ..................................................... 81 IV 3.8.2. Q-PCR results of the sugarcane samples from different sources ........................................ 83 3.8.3. Distribution of phytoplasma in sugarcane ........................................................................... 88 3.10. Hot water treatment in order to get phytoplasma free sugarcane plant ................................ 96 3.10.1. Hot water treatment according to Australian recipe .......................................................... 96 3.10.2. Hot water treatment with various duration ........................................................................ 97 3.11. Insecticide treatment of phytoplasma-infected sugarcane plant ............................................ 99 3.12. Transmission test with sugarcane aphid (Melanaphis sacchari) ............................................ 100 3.13. Transmission electron microscopy for cytological location of phytoplasma ......................... 101 3.13.1. Anatomy of leaf phloem tissue ........................................................................................ 101 3.13.2. Localization of phytoplasma infection ............................................................................ 102 3.13.3. Phytoplasma size and shape ............................................................................................ 106 3.13.4. Ultrastructural changes of the phytoplasma infection on leave anatomy ........................ 108 4. Discussion ..................................................................................................... 110 4.1. Establishment of the test for phytoplasma .............................................................................. 110 4.1.1. Efficiency of PCR amplification ....................................................................................... 110 4.1.2. Carry-over contamination problems .................................................................................. 112 4.1.3. Q-PCR (real-time PCR) ..................................................................................................... 112 4.2. Phytoplasma detection and identification by RFLP analysis .................................................... 115 4.2.1. Phytoplasma types in Hawaiian and Cuban sugarcane ..................................................... 115 4.2.2. Phytoplasma types in Thai sugarcane ................................................................................ 117 4.2.3. Phytoplasma types in Egyptian and Syrian sugarcane ...................................................... 118 4.3. Identification of the phytoplasma strains in sugarcane by phylogentic analysis ..................... 120 4.4. Hot water treatment in order to get phytoplasma free plant ................................................. 121 4.5. Transmission test with sugarcane aphid .................................................................................. 122 4.6. Transmission electron microscopy for cytological location of phytoplasma ........................... 123 5. Summary ....................................................................................................... 125 6. Zusammenfassung ......................................................................................... 127 7. Acknowledgement ......................................................................................... 129 8. References ..................................................................................................... 130 9. List of figures ................................................................................................ 145 10. List of tables ................................................................................................ 148 Erklärung ........................................................................................................... 150 V Abbreviations AAY American aster yellows AY aster yellows bp base pair BSA bovine serum albumin °C degree Celsius Ct threshold cycle CTAB hexadecyltrimethylammonium bromide dd double distilled dNTP deoxyribonucleotide triphosphate DNA deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid FAM 6-carboxyfluorescein Fig figure GS grassy shoot HWT Hot water treatment kb kilo base pairs l liter µ micro n-PCR nestedpolymerase chain reaction OY onion yellows PCR polymerase chain reaction PVP polyvinylpyrrolidone Q-PCR Quantitative PCR qPCR Quantitative PCR rRNA ribosomal RNA RFLP restriction fragment length polymorphism RNase ribonuclease rRNA ribosomal RNA VI rpm revolutions per minute RT room temperature RYD rice yellow dwarf S second SCYLP sugarcane yellow leaf phytoplasma SCYLV sugarcane yellow leaf virus SCWL sugarcane white leaf SCGS sugarcane grassy shoots SGS sorghum grassy shoots TAMRA 6-tetramethylrhodamine TBE Tris baseBoric acidEDTA TEM Transmission Electron Microscopy tRNA Transfer RNA U unit UV Ultraviolet YLS yellow leaf syndrom Introduction 1 1. Introduction 1.1. Plant diseases “Plants make up the majority of the earth’s living environment as trees, grass, flowers, etc. Directly or indirectly, plants also make up all the food on which humans and all animals depend. Plants are the only higher organisms that can convert the energy of sunlight into stored, usable chemical energy in carbohydrates, proteins, and fats” (Agrios, 2004). “Plant diseases are very important part of plant protection within the system of plant/crop production. Diseases can significantly lessen the growth and yield or reduce the utility of a plant or plant product. Healthy plants grow and function to the maximum of their genetic potential. However, plants are considered to be diseased when they are negatively affected by a disease-causing agent that lead to interfering with their normal development and physiological functions” (Agrios, 2004). “Correct diagnosis of the cause of a disease is an essential step in order to construct a convenient strategy to manage the plant disease. Usually, the first step includes the determination of the probable cause of the disease: whether the disease is caused by an infectious agent (pathogen) or environmental factor. Since diseases in plants are caused by either non-living (abiotic, non-parasitic, non-infectious, ‘non-pathogenic’) environmental factors or living (biotic, parasitic, pathogenic, infectious) agents. On the other hand, plant diseases are grouped based on the causal agent involved (deficiency diseases, fungal diseases, bacterial diseases, viral diseases, mollicutes diseases, etc.), the plant part affected or the type of symptoms” (Agrios, 2004). In general, plant disease is any growth or developmental condition that is not “normal” to that plant and can usually diminish its economic or aesthetic value. In many cases, the plant pathologists depend on symptoms and signs of the disease for hypothetical diagnosis of diseases in plants. The characteristic internal or external alterations showed by the plant in reaction to the disease-causing agent are called symptoms. Plant pathology is the study of the pathogens and of the environmental factors that cause disease in plants, and the methods of preventing or controlling disease and reducing the damage it causes. Uncontrolled plant diseases may result in less food and higher food prices, or in low-quality food (Agrios, 2004). Over the last decades scientists in molecular plant pathology have also established a new set of diagnostic tools and techniques that are used to Introduction 8 1.4.2. Host Specificity of Phytoplasmas Plant host range for a phytoplasma is dependent upon vectors specificity and feeding habits (behaviours) (monophagous, oligophagous, and polyphagous) of these vectors. For example, North American aster yellows phytoplasmas (16SrI-A,-B) were transmitted experimentally by the polyphagous leafhopper Macrosteles fascifrons and other vectors to 191 plant species belonging to 42 families (McCoy et al., 1989). Not all vectoring insects can transmit all phytoplasmas and there are specific interactions of a particular phytoplasma with its insect vector. Some phytoplasmas, such as peach X-disease phytoplasma, may be transmitted by several species of leafhoppers; others, such as elm yellows phytoplasma, appear to be transmitted by one or only a few species (Lee and Davis, 1992). 1.5. Phylogenetic position of phytoplasmas Phytoplasmas have diverged from gram-positive eubacteria, and belong to the Genus phytoplasma within the Class Mollicutes and order Acholeplasmatales (Figure.1.5.). Currently the phytoplasma is at candidatus status which is used for bacteria that cannot be cultured. Figure.1.5. Phytoplasmas are firmicutes. A. Phylogenetic relationships of several bacterial clades containing bacterial pathogens. B. The 5 phylogenetic groups within the Class Mollicutes. Plant pathogenic/symbiotic bacteria are indicated in green. GL, gene loss; WL, loss of cell wall. Figure taken from Saskias phytoplasma website, http://www.jic.ac.uk/staff/saskia-hogenhout/index.htm. Introduction 9 Recently, phylogenetic analyses based on 16S rRNA and ribosomal protein gene sequences have revealed that the uncultured phytoplasmas form a large discrete monophyletic clade within the class mollicutes (Gundersen et al., 1994). Phytoplasma taxonomic groups are based on differences in the fragment sizes produced by the restriction digest of the 16SrRNA gene sequence (RFLP) or by comparison of DNA sequences from 16S/23S spacer regions (Hodgetts et al., 2007). 1.6. Management and control of phytoplasma diseases Methods of control vary considerably from one disease to another, depending on the kind of pathogen, the host, the interaction of the two, and many other variables (Agrios, 2004). Most serious diseases of crop plants appear on a few plants in an area year after year, spread rapidly, and are difficult to cure after they have begun to develop. Therefore, almost all control methods are aimed at protecting plants from becoming diseased rather than at curing them after they have become diseased (Agrios, 2004). In controlling phytoplasmal diseases, the primary concern is often prevention rather than treatment due there is no known cure for phytoplasmal infections. However, infected plants or dormant propagative tissue can be freed of phytoplasma by heat treatment. 1.6.1. Prevention strategy Propagate from seed or from phytoplasma-free plants, that means select propagating material from sources known to be free of disease or indexed free of disease. Eliminate perennial and biennial weed hosts and eradicate known diseased trees as soon as they occur. Therefore, removal of phytoplasma infected plants eliminates sources of infection. Therefore, early, fast, specific and sensitive detection and diagnosis of phytoplasmas are very important for effective prevention strategies, especially because phytoplasmas may have a very long latency period. However, the most promising strategy for avoiding phytoplasma disease is the identification or development of resistant plant varieties (Welliver, 1999). In order to advance this field of research basic knowledge about the epidemiology, the pathogenicity mechanisms of the phytoplasmas, the effects of environmental factors on disease and symptom development, and the nature of resistance/tolerance in host plants is required Introduction 10 1.6.2. Control of insect vectors “When the pathogen is introduced or spread by an insect vector, control of the vector is as important as and sometimes easier than, the control of the pathogen itself. In the case of viruses, phytoplasmas, and fastidious bacteria, however, of which insects are the most important spreading agents, insect control has been helpful in controlling the spread of their diseases only when it has been carried out in the area and on the plants on which the insects overwinter or feed before they enter the crop. Controlling such diseases by killing the insect vectors with insecticides after they have arrived at the crop has rare proved sufficient. Therefore, in cases where the insect vector is known and the time of its occurrence established, insecticide programs may be of value when directed at the vector before it becomes established on the plants. Typically, insecticide sprays are of limited value since migrating vectors may transmit the phytoplasma before the insecticide kills those” (Agrios, 2004). 1.6.3. Management strategy Because phytoplasmas lack a cell wall , they are resistant against antibiotics that interact with cell wall synthesis like penicillin but other antibiotics with an alternative modes of action like tetracyclines can inhibit their growth (bacteriostatic to phytoplasmas). Therefore, remission of the disease symptoms can be achieved experimentally by injecting the antibiotic tetracycline but without continuous use of this antibiotic, disease symptoms will reappear again (Davies et al., 1968). In addition, antibiotic treatment is expensive and time-consuming. Therefore, the best strategy is to apply an efficient elimination program. As a conclusion, the only dynamic way to control phytoplasma infection has been to prevent the emergence by guaranty that clean planting material is used, or by quest to find and/or breed varieties of crop plants that are resistant or tolerant to the phytoplasma/insect vector. 1.7. Anatomy of phloem cells Phloem cells conduct soluble organic material made during photosynthesis in leaves to rest of the plant. They are alive at maturity and tend to stain green (with the stain fast green). Phloem cells are usually located inside the xylem. The two most common cells in the phloem are the companion cells and sieve tube cells. Introduction 11 The sieve-tube cells lack a nucleus, have very few vacuoles, but contain other organelles. The sieve tube is an elongated rank of individual cells, called sieve-tube members, arranged end to end. The endoplasmic reticulum is concentrated at the lateral walls. Sieve-tube members are joined end to end to form a tube that conducts soluble organic food (photosynthates) materials throughout the plant. The end walls of these cells have many small pores and are called sieve plates and have enlarged plasmodesmata (Esau, 1965). Companion cells retain their nucleus and control the adjacent sieve cells (Figure.1.6). Figure.1.6. Diagram of the longitudinal view of phloem cells. This image is from Purves et al., (1992). Life: The Science of Biology, 4th Edition. 1.8. Some phytoplasma diseases of sugarcane 1.8.1. Sugarcane yellow leaf syndrome Yellows diseases have been known since the early 1900s. One such disease, aster yellows, was first reported in 1902. Before 1967, its causal agent was thought by plant pathologists to be of viral origin because it could not be cultured in artificial media. Sugarcane yellow leaf syndrome (YLS), characterized by a yellowing of the midrib and lamina, (Figure.1.7), was first reported in the 1960s from East Africa (Rogers, 1969) and later from Hawaii (Schenck, 1990), South Africa (Cronje et al., 1998) and Cuba (Arocha et al., 1999). It is now widely distributed in most sugarcane growing countries from all continents. Losses from 30% to over 60% of susceptible varieties have been reported (Schenck et al., 1997; Comstock et al., 1994; Arocha et al., 2000). Symptoms of YLS have been attributed to Introduction 12 many causes, both biotic and a biotic, but the biotic causes are associated with infection by luteovirus or by phytoplasmas in Hawaii, Brazil, Australia, South Africa, Cuba, the USA and Mauritius. Phytoplasmas have been consistently associated with YLS, but latent infections also occur (Bailey et al., 1996; Cronje et al., 1998; Arocha, 2000; Aljanabi et al., 2001). Figure.1.7. Sugarcane yellow leaf syndrome (YLS). Sugarcane yellow leaf syndrome is characterized by a yellowing of the midrib and lamina. Symptoms consist of yellowing leaves with a bright yellow midrib, often when the rest of the lamina is still green. This picture was taken from Komor et al., 2010. 1.8.2. Sugarcane white leaf and sugarcane grassy shoot Sugarcane white leaf (SCWL) and sugarcane grassy shoot (SCGS) occur only in the southeast Asian region and not in the other sugarcane growing areas of the world. Both are caused by a single phytoplasma type that is a member of the SCWL group and appears to infect only sugarcane. The most characteristic symptoms of SCWL are the presence of leaves with total chlorosis, proliferating tillers and pronounced stunting. The leaves are narrower and smaller than those of healthy plants (Figure.1.8). SCWL is naturally transmitted by the leafhopper Matsumuratettix hiroglyphicus organism Matsumura (Matsumoto et al., 1968). Records on mechanically transmission as well as on transmission by aphids have not been confirmed (Rishi and Chen, 1989). Introduction 13 Sugarcane grassy shoot (SCGS) is one of the most important diseases of sugarcane in India. It was first observed in 1949 (Chona, 1958). SCGS has been recorded in most sugarcanegrowing areas of India and is known to occur also in Thailand (Wongkaew et al., 1997; Sdoodee et al., 1999). SCGS disease is characterized by the production of a large number of thin, slender, adventitious tillers from the base of the affected stools. This profuse growth gives rise to a dense or crowded bunch of tillers bearing pale yellow or chlorotic leaves which remain thin, narrow, reduced in size and have a soft texture. The vector(s) responsible for the natural spread of SCGS have not been identified. There are reports on transmission of SCGS by three different species of aphids as well as by the fulgorid Proutista moesta Westwoo (Chona et al., 1960; Edison et al., 1976). However, these reports have not been confirmed (Rishi and Chen, 1989). Figure.1.8. Sugarcane white leaf (SCWL). SCWL disease is caused by phytoplasma in Thailand. The most characteristic symptoms of SCWL are the presence of leaves with total chlorosis, proliferating tillers and pronounced stunting. The leaves are narrower and smaller than those of healthy plants. This picture was taken from Komor et al., 2010. 1.9. Detection of sugarcane phytoplasma infections Sugarcane phytoplasma infections can be detected by microscopic examination of phloem tissue sections stained with the DNA fluorochrome4-6 diamidino-2-phenylindole (DAPI) (Seemüller, 1976; Sarindu and Clark, 1993). This procedure is simple, rapid and not much Introduction 14 expensive. However it is limited when the phytoplasma population is very low and unevenly distributed among the plant host organs, as is often true for sugarcane. For detection and identification of sugarcane phytoplasmas, the powerful PCR technology has widely been employed in several laboratories. It offers several advantages over other methods including versatility, relative simplicity, specificity and high sensitivity. Primers amplifying rRNA gene sequences proved most suitable for PCR. It may be performed as one-round PCR or by reamplifying the DNA fragments obtained in the first amplification using internal primers (nested-PCR). Very often in affected sugarcanes the phytoplasma numbers are so low that infections could be identified only through the highly sensitive nested PCR assay (TranNguyen et al., 2000; Aljanabi et al., 2001). 1.10. Sugarcane yellow leaf syndrome in Hawaii A novel sugarcane disease was observed in Hawaiian sugarcane plantations in the 1990s, characterized by a yellowing of the leaf midrib, which was followed by stunted leaf tops and yield decline (Schenck, 1990, Lehrer et al., 2009). Similar symptoms were reported shortly later from plantations in Brazil, mainland USA and South Africa (Vega et al., 1997; Comstock et al., 1994; Bailey et al., 1996). The disease was called Yellow leaf syndrome (YLS) and classified in 2000 as a “disease of unknown origin” (Lockhart and Cronje, 2000). Research on the nature of the causal agent was controversial among plant pathologists. A RNA-virus was isolated from symptomatic plants and named Sugarcane yellow leaf virus (SCYLV). It was proposed as the causal agent for YLS (Borth et al., 1994; Vega et al., 1997). A survey of YLS-diseased sugarcane plants in Africa failed to reveal a close correlation between SCYLV and symptoms, a better correlation was seen between the presence of a phytoplasma infection and symptom expression (Cronje et al., 1998). The phytoplasma-caused disease was called Leaf yellows (LY) in contrast to the virus-caused disease, which is now called Yellow leaf (YL). The phytoplasma was named Sugarcane yellow leaf phytoplasma (SCYLP). It was found in sugarcane from Australia, South Africa, Cuba, India and Mauritius (Arocha et al., 1999, 2005a; Cronje et al., 1998; Aljanabi et al., 2001; Gaur et al., 2008), sometimes together with SCYLV. Severe sugarcane diseases in South-East Asia and Africa are known to be caused by phytoplasma (Marcone, 2002), for example White leaf (Chen and Kusalwong, 2000), Grassy shoot (Viswanathan, 2000), Green grassy shoot (Pliansinchai and Prammanee, 2000) and Introduction 15 Ramu stunt (Suma and Jones, 2000). Twenty-five different phytoplasma isolates were obtained from North Australian sugarcane plants and none of them was closely related to White leaf and Grassy shoot, although also none of them could be related to sugarcane disease symptoms (Tran-Nguyen et al., 2000). Many publications deal with the Sugarcane yellow leaf virus and the associated disease, for example its worldwide distribution (Abu Ahmad et al., 2006; Komor et al., 2010), its nucleotide sequence (Moonan et al., 2000; Smith et al., 2000), the transmission to the plant (Schenck and Lehrer, 2000; Lehrer et al., 2007) and the physiological effects on the infected plant (Yan et al., 2009). The virus-caused leaf yellowing syndrome is now accepted as an important, worldwide threat for sugar yield (Grisham et al., 2002; Lehrer et al., 2009). Also the South African sugar industry, for which originally the phytoplasmas were thought to be the main reason for YLS, appears to be predominantly infected by SCYLV and not by phytoplasma (Rutherford et al., 2004). However, the YLS-problem is not fully solved yet. The Hawaiian sugarcane cultivars were differentiated into so-called susceptible cultivars which contain relatively high titres of SCYLV, and resistant cultivars with 100 time’s lower virus titres (Zhu et al., 2010). Experiments with infected and virus-free plants of the same cultivar indicated that the viral infection led to higher symptom expression and to yield losses (Lehrer and Komor, 2008). The picture became less clear, when susceptible cultivars (i. e. with high virus-titre) and resistant cultivars (i. e. with low virus titre) were compared. The correlation between symptom expression and SCYLV-presence was not strict, some strongly infected cultivars exhibited relatively little symptoms and some resistant cultivars showed symptoms, although at low intensity (Lehrer and Komor, 2008). Therefore the question arose, whether some of the Hawaiian sugarcane cultivars were also infected by Sugarcane yellow leaf phytoplasma (SCYLP), thus causing leaf yellowing symptoms independent of or together with SCYLV. The simultaneous presence of SCYLV and SCYLP was reported to aggravate the leaf yellowing symptom expression in sugarcane (Aljanabi et al., 2001). So far sugarcane white leaf or sugarcane grassy shoot symptoms had not been reported in Hawaiian plantations; however the presence of a low-symptom pathogen such as SCYLP may have escaped attention of breeders and growers. There are two reports about phytoplasma diseases in Hawaii, one about water cress yellows caused by an Aster yellows type phytoplasma and transmitted by an accidentally introduced leaf hopper (Borth et al., 2002; 2006), the other about a yellows disease of a native tree, Dodonaea viscosa, caused by a Western X-disease phytoplasma (Borth et al., 1995). Introduction 16 We tested Hawaiian cultivars (and for comparison a few cultivars from Cuba, Egypt and Syria) for phytoplasma to reveal whether sugarcane phytoplasma is around in Hawaii and in Hawaiian sugarcane plantations. The main objectives of this project were to determine the following: 1. Possible association of phytoplasma(s) infection with YLS symptoms in sugarcane plants, using of molecular techniques, namely PCR, for a more accurate determination. 2. Which type(s) of phytoplasma(s) are associated with YLS symptoms in sugarcane plants from Hawaii breeding station, Hawaii plantations, Cuba, Middle East and areas in Thailand? 3. How does this type(s) compare to other known phytoplasma types by phylogenetic analysis? 4. Could it be that Hawaiian plantations have phytoplasma after hot water treatment? 5. Can sugarcane aphid (Melanaphis sacchari) transmit the detected phytoplasma to sugarcane plants? 6. Ultrastructural changes of the leave anatomy and cytology by phytoplasma infection and cytological location of phytoplasma. Material and Methods 17 2. Material and Methods 2.1. Material Balance (Mettler P1210) Centrifuges, Type centrifuge 5403 (Eppendorf) Centrifuge, Type Mikro 20 (Hettich) Centrifuge, Type UNIVERSAL 32R (Hettich) Diamond knife (type 35°, Diatome, Biel, Switzerland) Electron microscope, Type ZEISS 902 (Zeiss, Oberkochem) Gel Electrophoresis, Type GNA 100 (Pharmacia LKB) Gene power supply, type GPS 200/400 (Pharmacia) Mini-Vertical Gel Electrophoresis, Type SE 250 and SE 260 (Mighty small II) Thermomixer comfort (Eppendorf) Thermal cycler, Type PTC100 (MJ Research) Thermal cycler, Type Mastercycler personal, with heated lid and 1 personal card, 115 V/60 Hz (Eppendorf) Thermal cycler, type MyiQ qPCR detection system for single-colour experimentation (Biorad) Spectrophotometer, Type 650 (Beckman) Ultra cut microtome (Leica Microsystems, Wetzlar, Germany) Vortexer, Type REAX-1R (Heidolph) Heated magnetic stirrer, Type MR 82 (Heidolph) Microwave oven, Type KOR6115 (Alaska) Nanophotometer, Type UV/Vis spectrophotometer (Implen) pH-mV-meter , Type 531 (Knick) UV-SYSTEME (NTAS) 2.1.2. Chemicals and Enzymes 2.1.2.1. Chemicals Agarose NEOO (Carl Roth GmbH) 30% Acrylamide 10% Ammonium Persulfate Material and Methods 24 2.2.3. Polymerase Chain Reaction (PCR) for the detection of phytoplasmas Symptomatology had been one the major criteria for diagnosing the phytoplasma disease before molecular-based methods become available. It remains the important clue used for preliminary identification of putative phytoplasmal diseases. The polymerase chain reaction (PCR) is a rapid procedure for in vitro enzymatic amplification of a specific segment of DNA (Donald et al., 2006) . PCR has been used during the last years for the detection of large number of microorganisms, also including phytoplasmas. Several universal primer pairs designed for the amplification of the 16SrRNA gene of phytoplasmas were tested. The method found to give consistent results was the nested PCR (Heinrich et al., 2001; Srivastava et al., 2005). 2.2.3.1. Definition of Nested PCR Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment. The first pair of PCR primers amplifies a fragment similar to a standard PCR. However, a second pair of primers called nested primers (as they lie) are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one (Pérez de Rozas et al., 2008), (Figure.2.1). Material and Methods 25 Figure.2.1. A diagram illustrating of the method of nested PCR. Figure taken From Wikipedia, the free encyclopaedia. The advantage of nested PCR is that if the wrong PCR fragment was amplified, the probability is quite low that the region would be amplified a second time by the second set of primers. Thus, nested PCR is a very specific PCR amplification. Furthermore, the double amplification in the nested-PCR increases the sensitivity of PCR reaction in 2-3 logarithmic units when compared with conventional PCR (Lindqvist, 1999; Marsilio et al., 2005). 2.2.3.2. Nested PCR Reaction Nested PCR requires two sets of primers which are used to amplify a specific DNA fragment using two separate runs of PCR. A standard reaction mixture of 25 µl consisted of the following: Material and Methods 26 10X Taq buffer dNTP mix (200 µM each dNTP) Forward and reverse primers (0.4 µM) Taq DNA polymerase (5 U/µl) Template DNA (100 ng) dd H 2 O to final volume 25 µl 2.2.3.3. First round of PCR Nucleic acid samples were diluted in sterile distilled water to give a final concentration of 100 ng/ul and in some cases DNA concentrations were not adjusted after extraction, but used as isolated, 1 µl of DNA solution was used per reaction tube. 2.2.3.4. Nested round of PCR One micro litre of diluted (1:30 or 1:20) PCR products from the first round was used as the template in the second amplification. In most cases first PCR products were used with any dilution. The PCRs (30 cycles) were done with an automatic thermal cycler in 25µl reaction tubes. Several universal primer pairs, which were previously designed, based on the phytoplasma rRNA operon, for the amplification of phytoplasmal DNA were tested (Figure.2.2 and Figure.2.3). The method found to give consistent results was the nested PCR. However, it was amazing when some primer pairs didn’t work continually and we had to test in this case other primer pairs to check if the negative results were false due the primer pairs or due the phytoplasmas disappeared from our greenhouse sugarcane plants. Since this phenomenon may occur especially in our greenhouse when there are no insect vectors for phytoplasmas and the plants reproduce by vegetative propagation therefore, the titre of phytoplasma would be lower generation by generation. 2.2.3.5. Nested-PCR assay (I) The primer pair combination used in the first round was P1/P7 while the nested primer pair was R16F2n/ R16R2 (Table.2.3). Parameters of the PCR assays using external primer pair (P1/P7) were: denaturation step at 94°C for 30 s (4 min for the first cycle), annealing for 1.5 min at 55°C and primer extension for 1.5 min (10 min in final cycle) at 72°C. Material and Methods 27 Parameters of the PCR assays using internal (nested) primer pair (R16F2n/R16R2) were: denaturation step at 94°C for 30 S (4 min for the first cycle), annealing for 1.5 min at 56°C and primer extension for 1.5 min (10 min in final cycle) at 72°C. Table.2.3. Oligonucleotide primers used for nested-PCR assay I. Primer Location Type of PCR P1 (Forward) 16S First P7 (Reverse) 23S First R16F2n (Forward) 16S Nested R16R2 (Reverse) 16S Nested 2.2.3.6. Nested-PCR assay (II) The primer pairs combinations used in the first and nested rounds of nested-PCR assay (II) are indicated in (Table.2.4). Parameters of the PCR assays using external primer pair SN910601/P6 were: denaturation step at 94°C for 30 s (4 min for the first cycle), annealing for 1 min at 54°C and primer extension for 1.5 min (10 min in final cycle) at 72°C. Parameters for PCR using internal primer pair R16F2n/R16R2 ,which amplifies 1250bp DNA fragment, were: denaturation step at 94°C for 30 s (4 min for the first cycle), annealing for 1 min at 56°C, and primer extension for 1.5 min (10 min in final cycle) at 72°C. Table.2.4. Oligonucleotide primers used for nested-PCR assay II. Primer Location Type of PCR SN910601 (Forward) 16S First P6 (Reverse) 16S First R16F2n (Forward) 16S Nested R16R2 (Reverse) 16S Nested 2.2.3.7. Nested-PCR assay (III) The primer pairs combinations used in the first and nested rounds of nested-PCR assay (III) are indicated in (Table.2.5). Material and Methods 28 The parameters of the PCR assays using external primer pair MLO-X/MLO-Y were: denaturation step at 94°C for 30 s (4 min for the first cycle), annealing for 1 min at 58°C and primer extension for 1.5 min (10 min in final cycle) at 72°C. The parameters for PCR using internal primer pair P1/P2, which amplifies 210 bp DNA fragment, were: denaturation step at 94°C for 30 s (4 min for the first cycle), annealing for 45 s at 62°C, and primer extension for 1 min (10 min in final cycle) at 72°C. Table.2.5. Oligonucleotide primers used for PCR assay III. Primer Location Type of PCR MLO-X (Forward) 16S First MLO-Y (Reverse) spacer region (near 23S) First P1 (Forward) 16S (near the spacer region) Nested P2 (Reverse) "tRNA-Ile" (near the spacer region) Nested 2.2.3.8. Nested-PCR assay (IV) The primer pairs combinations used in the first and nested rounds of nested-PCR assay (IV) are indicated in (Table.2.6). Parameters of the PCR assays using external primer pair U-1/ MLO-7 were: denaturation step at 94°C for 30 s (4 min for the first cycle), annealing for 1 min at 56°C and primer extension for 1.5 min (10 min in final cycle) at 72°C. Parameters for PCR using internal primer pair MLO-X/MLO-Y, which amplifies 700bp DNA fragment, were: denaturation step at 94°C for 30 s (4 min for the first cycle), annealing for 1 min at 60°C, and primer extension for 1.5 min (10 min in final cycle) at 72°C. Table.2.6. Oligonucleotide primers used for PCR assay IV. Primer Location Type of PCR U-1 (Forward) 16S First MLO-7 (Reverse) 23S First MLO-X (Forward) 16S Nested MLO-Y (Reverse) spacer region (near 23S) Nested Material and Methods 29 Figure.2.2. Diagrammatic representation of location of used primer pairs and expected size of their amplified products based on phytoplasma rRNA operon. Figure.2.3. Diagrammatic representation of a phytoplasma rRNA operon and genomic location of primers used for phytoplasma detection. 2.2.4. Agarose Gel Electrophoresis PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide and DNA bands visualized using a UV transilluminator Agarose gel electrophoresis was used to visualize and isolate DNA molecules following PCR amplification. Agarose (1%) was dissolved in TBE buffer by heating in a microwave. After cooling, 1 µ of a 1 mg/ml ethidium bromide solution was added per 50 ml gel and the gel was poured. Gels were run at 80-100 V for 1 hour. PCR III PCR IV PCR II PCR I Material and Methods 30 2.2.5. Digestion of nested-PCR products By RFLP analysis of PCR-amplified 16S rRNA gene, the phytoplasmas detected can be differentiated and classified (Lee et al., 1993). The basic technique for detecting RFLPs involves fragmenting the samples of DNA or (PCR products) by the restriction enzymes. Restriction enzymes recognize specific nucleotide sequences and cleave DNA molecules at a position either within or outside their recognition site (Roberts and Kenneth, 1976). These enzymes are important tools for numerous applications, including restriction fragment length polymorphism (RFLP) analysis of PCR products .The resulting DNA fragments are then separated by their length through gel electrophoresis. RFLP analysis of PCR-amplified 16S rRNA gene sequences with a number of restriction enzymes was used by Lee et al., (1993) and Schneider et al., (1993) to differentiate various phytoplasmas by their distinct RFLP patterns. This procedure proved to be simple, reliable, and practical. Our nested PCR products were analyzed by single enzyme digestion with different restriction endonucleases HpaIII, HinfI, KpnI, MesI and RsaI (MBI Fermentas). The reaction mixture (30 µl) consisted of the following: Reagents: 10 µl PCR products 2 µl 10X recommended buffer for restriction enzyme 1-2 µl (10-20 u) restriction enzymes 17 µl water nuclease free The reaction mixtures were incubated in the incubator at 37°C for 3-16 h. 2.2.5.1. Inactivation of restriction enzymes Inactivation of restriction enzymes following a digestion reaction is often required for downstream applications. Thermal inactivation is a convenient method used to terminate enzyme activity. The majority of restriction enzymes can be heat-inactivated at 65°C or 80°C in 20 min. Digested products were separated by electrophoresis on 5% polyacrylamide gels. Next RFLP patterns were compared with those previously published. Material and Methods 31 2.2.6. Polyacrylamide Gel Electrophoresis Nondenaturing polyacrylamide gels are used for the separation and purification of fragments of double-stranded DNA. 2.2.6.1. Steps of operation Assembling the apparatus and preparing the gel solution Casting the gel Loading the samples and running the gel 2.2.6.2. Special equipment The SE 250 Mighty small II is a miniature vertical slab gel unit intended for rapid electrophoresis of nucleic acid samples of small volume. 2.2.6.3. Detection of DNA in polyacrylamide gels by staining Unlike agarose gels, polyacrylamide gels cannot be cast in the presence of ethidium bromide because the dye inhibits polymerization of the acrylamide. However, ethidium bromide can be used to stain the polyacrylamide gel after electrophoresis. In order to detect of DNA the gels were gently submerged in the appropriate staining solution. We used just enough staining solution to cover the gel completely and the gels were stained for 30 min at room temperature.Then the gels were removed from the staining solution and placed on the UV transilluminator and photographed. 2.2.7. Sequencing and phylogenetic analysis of ribosomal DNA By direct sequence analysis or RFLP analysis of PCR-amplified products, the phytoplasmas detected can be differentiated and classified. Several classification systems have been proposed either directly based on sequence analysis or indirectly, by RFLP analysis of PCRamplified 16S rRNA gene. In order to amplify the 16S/23S spacer region we used P1/P7 for first PCR and P4/P7 for second PCR (Smart et al., 1996). P4/P7 PCR product was purified from agarose gels using Agarose Gel DNA Extraction Kit (Roche Diagnostics GmbH). The DNA sample was sequenced in one direction using P4 primer. Unfortunately, this primer pair didn’t work well Material and Methods 32 for some cultivars. Therefore, we used MLO-X/MLO-Y for first PCR and P1/P2 for nestedPCR to amplify partially the 16S/23S spacer region. In order to amplify the 16SrRNA we used R16F2n/R16R2 for nested PCR. Nested-PCR products were cleaned up using High pure PCR products purification Kit (Roche). DNA samples were sequenced in both directions using nested primer pairs. 2.2.7.1. Sample Preparation for Value Read Service in Tubes The value read is the service of choice for fast and reliable standard sequencing reactions. It is highly automated to allow rapid processing of plasmids or PCR products. We used 1.5 ml tubes (no additional sealing with Parafilm) for samples and primers and we used one tube per sequencing reaction. The DNA samples (purified PCR products) were dissolved in the elution buffer (10 mM Tris-HCl, pH 8.5) and the concentrations of these purified PCR products were adjusted to get the final concentration 10 ng/µl or 2 ng/µl in a minimum volume of 15 µl and the required primer concentrations were 2 pmol/µl with minimum total volume 15 µl. The DNA samples (purified PCR products) were direct-sequenced in an ABI 3730XL automated sequencer using the sequencing service of Eurofins MWG Operon Ebersberg, Germany (http://www.eurofinsdna.com/ ) . Next, sequences were compared with others in GenBank database using BLAST program.The sequence data were deposited in GenBank. 2.2.8. Hot water treatment “When a pathogen is excluded from the propagating material (seed, tubers, bulbs, nursery stock, grafts, and cuttings) of host, it is often possible to grow the host free of that pathogen for the rest of its life” (Agrios, 2004).Vegetative propagating material free of pathogens that are systemically distributed throughout the plants (viruses, viroids, and phytoplasmas) is obtained from mother plants that had been tested and shown to be free of the particular pathogen or pathogens. Furthermore, the new plants must be grown in pathogenand vectorfree soil and then be protected from airborne vectors. Phytoplasma may be transmitted by the propagation of scions and or cutting collected from diseased plant. Therefore, in vegetatively propagated crops like sugarcane; phytoplasma can be readily spread to new locations through infected stem cutting if suitable precautions are not taken. These precautions include coldand hot water treatment and tissue culture (Parmessur et al., 2002). Material and Methods 33 A hot water treatment is an effective method for the control of number of plants pests and diseases (plant pathogens) including phytoplasmas. Hot water treatment (HWT) has been proposed since 1966 by Caudwell at 30°C for 72 h in order to cure dormant woody plant material from phytoplasmas. Afterward other works showed the effectiveness of the treatment against these pathogens (Lherminier et al., 1990; Tassart-Subirats et al., 2003). However, (HWT) must be carefully applied because may interfere with the vitality of plant material. Thus, (HWT) of dormant canes or plants aims at phytoplasma elimination without any alteration in their vegetative development capacity. In addition the treatment demonstrates a positive effect of sanitation against several bacterial diseases, pests and insects (including eggs) which may be present on plant material. 2.2.8.1. Preparation of the plant material prior to the hot water treatment Infected plants or dormant propagative organs can be totally freed of phytoplasmas by heat treatment. Infected plants are kept in growth chambers at 30°C to 37°C for several days, weeks, or months; but dormant organs are immersed in hot water (Agrios, 2004). Soaking induces a thermal shock susceptible of modifying the physiological state of the plant material (breaking of bud dormancy, inducing storage losses). Therefore, in order to prevent a poor vegetative development, the plant material should be thermally prepared to the treatment by storage for 12 to 48 hours at room temperature in a humid and aerated chamber. Furthermore, the temperature after immersion and the treatment duration should be respected and after treatment, the plant material should be left to set back to room temperature (avoid direct contact with cold water). 2.2.9. Sugarcane aphid transmission test 2.2.9.1. Insect rearing Melanaphis Sacchari (Sugarcane aphid) insects were provided from Hawaii Island. Colonies of Melanaphis Sacchari were established on phytoplasma-infected sugarcane plants. 2.2.9.2. Plant material All test plants raised from single-eye setts that had received coldand hot-water treatment or hot-water treatment were negative (phytoplasma free) when tested by nPCR prior to being Results 40 In another experiment, DNA of the positive control was diluted by increasing quantities of DNA which had been extracted from sugarcane leaves of plants which were known to be phytoplasma-free. The purpose was to test, whether compounds from sugarcane leaves may possibly inhibit the amplification of phytoplasma 16S rRNA gene. The sugarcane extract by itself did not give an amplicon. The positive control sample always yielded a positive signal, even when diluted up to 40-fold by sugarcane DNA, the 50-fold dilution with sugarcane DNA did not give an amplicon anymore (Figure.3.1). Figure.3.1. Nested PCR-products of positive and negative controls and of a positive control, which was mixed with increasing amounts of sugarcane DNA. The positive control (pos. c.) was a sample of American aster yellows phytoplasma grown in periwinkle (obtained from Dr. Bertaccini, Bologna). The water control 1+2 was with water instead of DNA in the first PCR round and further amplified in the second PCR round. Water control 2 contained water instead of first round amplicon. The sugarcane DNA was from Egyptian cultivar ( Ph8013) which had been shown to be phytoplasma-free (sugarcane c.) using the primer pairs combination (P1/P7 and R16F2n/R16R2). The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). The arrows point to the phytoplasma-specific band of 1.2 kbp. Though PCR analysis is routine technique for phytoplasma detection, it’s still meeting some difficulties, at least with some primers: several primer pairs and their combination are recommended (Heinrich et al., 2001). In our lab, PCR assay was carried out with different primer pairs combination. To amplify region that includes the 16S rRNA gene, the spacer region and the start of 23S rRNA gene of the phytoplasma genome. Therefore, each sugarcane Results 41 sample was investigated for phytoplasma by using four nested-PCR assays which were numbering as following: (I), (II), (III) and (IV). The primer pairs and their sequences which used in each assay were mentioned at material and methods chapter. 3.1.2. Sources of sugarcane samples Sugarcane samples, which were investigated in our lab, were obtained from different areas and different dates. Some of them were obtained as stem cuttings and grown in the greenhouse while others were harvested and conserved as air-dried leaves. Most of them are showing sugarcane yellow leaf syndrome symptoms whereas others were symptomless (Table.3.1). Table.3.1. Original sources of sugarcane samples. Most of sugarcane samples were obtained from Hawaiian Islands while others from Thailand. In addition, some sugarcane samples were taken from Cuba and Middle East area including Egypt and Syria. Some of them were obtained as stem cuttings and grown in the greenhouse whereas others were collected and conserved as air-dried leaves. Original source Location Date of getting sugarcane samples Type of sugarcane sample Hawaii Breeding station of HARC in Maunawili 2003 Stem cuttings Hawaii Plantations (Maui and Kauai) 2009 Sun-dried leaves Hawaii Former plantation fields (Maui, Kauai and Hawaii) 2009 Sun-dried leaves Hawaii Breeding station of HARC in Maunawili 2010 Sun-dried leaves Hawaii Close to former plantation fields in Hawaii 2011 Sun-dried leaves Hawaii Breeding station of HARC in Maunawili 2011 Sun-dried leaves Hawaii plantation (Maui) 2011 Sun-dried leaves Thailand Farmer fields (Bang Phra) 2010 Sun-dried leaves Thailand Breeding station (Khon Kean) 2010 Sun-dried leaves Thailand Farmer fields (Suphan Buri) 2011 Sun-dried leaves Cuba Breeding station 2005 Stem cuttings Egypt Breeding station 2008 Stem cuttings Syria Farmer fields (Baniyas) 2008 Stem cuttings Results 42 3.2. Phytoplasma in sugarcane in Hawaii, Cuba, Egypt and Syria Six sugarcane cultivars from Hawaii were obtained in 2003 as stem cuttings from the breeding station of HARC in Maunawili, Oahu , three SCYLV-susceptible cultivars (H874094, H73-6110, H65-7052) and three SCYLV-resistant cultivars (H78-7750, H78-4153, H87-4319). These stem cuttings were grown in greenhouse of university of Bayreuth. In addition, cultivars from Cuba were obtained from Dr. Ortega, Habana, in 2005 also as stem cuttings and grown beside Hawaiian samples. Cultivars from Egypt were obtained as stem cuttings from the University of Gizah in 2008. The cultivar from Syria was obtained as stem cuttings in 2008 from a farmer´s field near Baniyas. The question was as the following: Are these obtained sugarcane samples infected with phytoplasma? 3.2.1. Phytoplasma detection by nested-PCR assay (I) and identification by RFLP DNA was extracted from source leaves and tested for phytoplasma by nested-PCR assay (I) with primer pairs (P1/P7 and R16F2n/R16R2) in 2008. All cultivars contained phytoplasma showing an amplicon at 1.2 kbp, although apparently at different titres, for example H736110, a strongly SCYLV-infected cultivar, had only a low SCYLP-titre (Figure.3.2). The Cuban cultivars (C10-5173, CP43-62, JA60-5) and one cultivar from Egypt (G84-47) was also infected by phytoplasma, although apparently at a low titre, not however the cultivar Gt954 and Ph-8013 from Egypt and the plant from Syria (Figure.3.3). The results with cv. Gt954, Ph-8013 and the Syrian cultivar thus were an important negative control, showing that there is no DNA sequence in the sugarcane genome which gives a false positive signal with this primer pair. Results 43 Figure.3.2. Phytoplasma in Hawaiian and Cuban sugarcane cultivars. DNA prepared from leaves of the indicated cultivars was tested with primers P1/P7 and R16F2n/R16R2. The positive control (pos. c1) was phytoplasma aster yellows from periwinkle obtained from Dr. Seemüller, Dossenheim, pos.c2 was phytoplasma aster yellows from periwinkle obtained from Dr. Bertaccini, Bologna. The water control 1+2 was with water instead of DNA in the first PCR round and further amplified in the second PCR round. The marker M1 is DNA ladder FastRuler Middle range (MBI Fermentas, fragment sizes: 4, 2, 1, 0.5 kbp).The arrows point to the phytoplasma-specific band of 1.2 kbp. Figure.3.3. Phytoplasma in Egyptian and Syrian sugarcane cultivars. DNA prepared from leaves of the indicated cultivars was tested with primers P1/P7 and R16F2n/R16R2. Re-amplification of aliquot of first PCR water control with nested primer combination is in lane W. The marker M2 DNA GeneRuler 1kb (MBI Fermentas). The arrows point to the phytoplasma-specific band of 1.2 kbp. G84 - 47 G t - 954 Ph - 8013 Syrian cv. M2 W Results 44 Restriction fragment analysis had been successfully applied to differentiate between the phytoplasma strains (Kirkpatrick et al., 1994; Lee et al., 1998; Valiunas et al., 2007). The amplicons of the second round of PCR (I) were subjected to three restriction enzymes which were diagnostic for the phytoplasma strains. The obtained RFLP patterns were compared with those previously published by Lee et al., 1998. The restriction patterns identified the phytoplasma from Hawaiian cultivars and from one Cuban cultivar as belonging to the Aster yellows phytoplasma “Ca. Phytoplasma asteris”, whereas the phytoplasma from the Cuban cultivar CP4362, which originally had been bred in Canal Point, Florida and from JA605, belonged to the Western X-disease phytoplasma “Ca. Phytoplasma pruni”. However, a second profile was clearly visible in the gel in some Hawaiian sugarcane cultivars that indicate to the possible presence of phytoplasmas related to rice yellow dwarf group (16SrXI), “ Ca. Phytoplasma oryzae” (Figure.3.4 and Table.3.2). a Results 45 Figure.3.4. Restriction fragment analysis of PCR products from Hawaiian and Cuban sugarcane cultivars containing phytoplasma. The nested-PCR products were amplified with primers R16F2n/R16R2 following digestion with RsaI (a), HpaII (b) or KpnI (c) and separated on 5% polyacrylamide. The positive controls (Aster yellows and Western X-disease) were used as references. The black arrows indicate to second profile which reveals possible presence of rice yellow dwarf (16SrXI) phytoplasmas as mixed infection .The marker (M) is Mass Ruler DNA Ladder, low range (MBI Fermentas), fragment sizes 1031, 900, 800, 700, 600, 500, 400, 300, 200, 100, and 80 bp. b c Results 46 Table.3.2. Results of nested-PCR assay (I) and identification of phytoplasmas based on RFLP analyses. Two phytoplasmas were identified in mixed infection in some Hawaiian sugarcane cultivars: one related to aster yellows group (16SrI) while the other tentatively related to rice yellow dwarf group (16SrXI). +, phytoplasma detected. Sugarcane varieties Original source Detection of phytoplasma in 2008 based on PCR assay (I) Phytoplasma group based on RFLP analyses of 16S rRNA gene H8740 94 Hawaii + Aster yellows C1051 73 Cuba + Aster yellows H7877 50 Hawaii + Aster yellows and rice yellow dwarf Cp43 62 Florida + X-disease H7841 53 Hawaii + Aster yellows and rice yellow dwarf H7361 10 Hawaii + Aster yellows and rice yellow dwarf H6570 52 Hawaii + Aster yellows and rice yellow dwarf JA60 5 Cuba + X-disease H8743 19 Hawaii + Aster yellows and rice yellow dwarf H8740 94 VF Hawaii + Aster yellows 3.2.2. Phytoplasma detection by nested-PCR assay (II) and identification by RFLP Oligonucleotide primers used for nested-PCR assay (II) were (SN910601/P6) for first-PCR and (R16F2n/R16R2) for nested-PCR. Hawaiian, Egyptian and Syrian sugarcane samples grown in greenhouse were tested by this PCR assay. According to this analyse four Hawaiian cultivars, two Egyptian cultivars and Syrian cultivar were negative for phytoplasma (Figure.3.5, Table.3.3 and 3.4). Results 47 Figure.3.5. Nested-PCR assay (II) products (1.2kb) amplified with primers (SN910601/P6, R16F2n/R16R2). (a): Hawaiian sugarcane samples grown in greenhouse. (b): Egyptian and Syrian sugarcane samples. The marker M was GeneRuler 100 bp plus (MBI Fermentas). The arrows point to the phytoplasmaspecific band of 1.2 kb. According to this analysis two Hawaiian sugarcane cultivars H78-4153 and H87-4319 and one Egyptian cultivar G8447 were positive for phytoplasmas. Products of nested-PCR assay (II) were analyzed by RFLP analysis using single enzyme digestion with restriction endonucleases (HpaII and MseI). The obtained RFLP patterns were compared with those previously published by Lee et al., 1998. According to this digestion the Hawaiian cultivars H78-4153 and H87-4319 contain phytoplasmas fall in aster yellows group (Figure.3.6; a and Table.3.3) whereas Egyptian cultivar G8447 infected with phytoplasma belongs to rice yellow dwarf group (Figure.3.6; b and Table.3.4). However, further RFLP analysis is required to differentiate if this phytoplasma belongs to sugarcane white leaf strain (SCWL) or sugarcane grassy shoot one (SCGS). 1,2kb G8447 M Ph8013 GT549 Syria 1,2kb 4153 7052 4319 6110 7750 4094 M a b b a Results 48 3.2.3. Phytoplasma detection by nested-PCR assay (III) Oligonucleotide primers used for nested-PCR assay III were (MLO-X/MLO-Y) for first-PCR and (P1/P2) for nested-PCR. According to this analyse all Hawaiian cultivars, one Egyptian (G8447) and Syrian cultivar were positive for the presence of phytoplasma but not the other two Egyptian cultivars (Figure.3.7, Table.3.3 and 3.4). MseI MseI HpaII H87 - 4319 H87 - 4319 H78 - 4153 H78 - 4153 HpaI I M M M G8447 G8447 HpaI I MseI M M a b a a b Figure.3.6. RFLP profiles of nested-PCR assay (II) products. These products amplified with primer pair (SN910601/P6, R16F2n/R16R2) of Hawaiian sugarcane samples (a) and Egyptian sugarcane samples (b) grown in greenhouse following single enzyme digestion with (HpaII and MseI) and separation on 2% agarose gel. The marker M was GeneRuler 100 bp plus (MBI Fermentas). According to this digestion the Hawaiian cultivars H78-4153 and H874319 contain phytoplasmas fall in aster yellows group while in Egyptian cultivar G8447 falls in rice yellow dwarf group. Results 49 Figure.3.7. Nested-PCR assay (III) products (0.2kb) amplified with primer pair (MLO-X/MLO-Y, P1/P2). (a): Hawaiian sugarcane samples. (b): Egyptian sugarcane samples. (c): Syrian sugarcane sample grown in greenhouse. Re-amplification of aliquots of first PCR water controls with nested primer combination are in lanes W. The marker M was GeneRuler 100 bp plus (MBI Fermentas).The arrows point to the phytoplasma-specific band of 0. 2 kb. 3.2.4. Phytoplasma detection by nested-PCR assay (IV) and identification by RFLP Oligonucleotide primers used for nested-PCR assay IV were (U-1/MLO-7) for first-PCR and (MLO-X/MLO-Y) for nested-PCR. According to these data the primers used in this assay could not detect phytoplasmal DNA present in all Hawaiian cultivars which were positive for phytoplasma as mentioned above (Table.3.3). Phytoplasmal DNA in Syrian and one Egyptian cultivar (G8447) was detected with this primer pair (Figure.3.8 and Table.3.4) 210bp 4153 7052 4319 6110 7750 4094 0,2kb G844 7 0,2kb Syrian M M Ph801 G t 549 W W W W a b c M W W Results 56 al. 2010) and they may have become infected by phytoplasma in case that the appropriate insect vectors were present. Samples of the uppermost fully unfolded leaves of plants found in former plantation fields were collected, sun-dried and tested for phytoplasma. 3.4.1. Phytoplasma detection and identification The extracted DNA from these samples gave amplification only with primer pairs of PCR assay (III). The obtained amplicons were sequenced in order to identify the phytoplasma (Figure.3.13 and Table.3.6). Figure.3.13. phytoplasma in former Hawaiian plantations sugarcane samples (2009). Nested-PCR assay (III) products (0,2kb) amplified with primers (MLO-X/MLO-Y, P1/P2). The samples M10 to M13 were from Maui plantation, H1 to H4 were from Hawaii plantation, K2 to K5 from Kauai, all as air-dried and then ovendried. Re-amplification of aliquots of first PCR water controls with nested primer combinations are in lanes W. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). M1 0 H1 H2 H3 H4 K 2 K3 K4 K5 210 bp M M W W W W M1 3 M1 2 M1 1 0.2kb Results 57 Table.3.6. Phytoplasma in sugarcane samples from former Hawaiian plantations (2009). Results of phytoplasma detection based on nested-PCR assays and identification based on DNA sequencing of nested-PCR assay (III) using P1/P2. +, phytoplasma detected; -, phytoplasma not detected. The samples (H1 to H4) were from Hawaii while the samples (M10 to M13) were from Maui and the samples (K2 to K5) were from Kauai. Island and collection site phytoplasma detection based on PCR assay (I), (II) and (IV) phytoplasma detection based on PCR assay (III) Phytoplasma group based on DNA sequencing of PCR assay (III) (P1/P2) Hawaii H1 - + Aster yellows H2 - + Aster yellows H3 - + Aster yellows H4 - + Aster yellows Maui (H65-7052) M10 - + M11 - + Rice yellow dwarf M12 - + Rice yellow dwarf M13 - + Rice yellow dwarf Kauai (H65-7052) K2 - + Aster yellows K3 - + Aster yellows K4 - + K5 - + 3.5. Phytoplasma in sugarcane in Hawaiian breeding station (2010) Six sugarcane cultivars from Hawaiian breeding station of HARC in Maunawili, Oahu were sent from Dr. Zhu in 2010 as sun-dried leaves in order to test for phytoplasmas. 3.5.1. Phytoplasma detection and identification Only the primers of PCR assay (III) amplified DNA in these sugarcane cultivars. However, the obtained bands were very weak, therefore; only two of them were sequenced (Figure.3.14 and Table.3.7). 0,2kb 4153 7052 4319 6110 7750 4094 M W Figure.3.14. phytoplasma in Hawaiian breeding station sugarcane samples (2010). Nested-PCR assay (III) products (0.2kb) amplified with primers (MLO-X/MLO-Y, P1/P2) obtained from Hawaiian sugarcane breeding station (2010) as air-dried samples. Reamplification of aliquot of first PCR water control with nested primer combination is in lane W. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). Results 58 Table.3.7. Phytoplasma in sugarcane plants from Hawaiian (Maunawili, HARC) breeding station (2010). Results of phytoplasma detection based on nested-PCR assays and identification by DNA sequencing of nestedPCR assay (III) using P1/P2. +, phytoplasma detected; -, phytoplasma not detected. Sample name phytoplasma detection based on n-PCR assay (I)& (II)& (IV) phytoplasma detection based on n-PCR assay (III) Phytoplasma group based on DNA sequencing (P1/P2) H65-7052 - + Rice yellow dwarf H73-6110 - + Aster yellows H78-7750 - + H78-4153 - + H87-4094 - + H87-4319 - + 3.6. Phytoplasma in sugarcane in Hawaiian breeding station and plantations (2011) In February 2011, sugarcane leaf samples were harvested from different areas in Hawaiian Islands including plantation HC&S, Maui and Maunawili breeding station. Then, these sugarcane samples were sun-dried in order to test the presence of phytoplasm in our lab. Most of these samples were taken from sugarcane plants are showing sugarcane yellow leaf syndrome symptoms (Figure.3.15). 3.6.1. Phytoplasma in sugarcane in Hawaiian plantation These plantations obtained the cultivars from the breeding station of HARC in Maunawili, Oahu, followed by several cycles of field testing and multiplication. The question was if phytoplasmas can be responsible for YLS in this plantation field and if there is significant correlation between the presence of phytoplasma and showing sugarcane yellow leaf syndrome since some samples were strongly or slightly symptomatic while others asymptomatic. Samples from uppermost fully unfolded source leaves from plants of different cultivars were collected in the plantation fields and sun-dried. The samples from Maui were from HC&S plantation (fields 702, 500, 608 and 809, all south-east of Puunene). In each case 3 leaf samples from 3 different plants were tested. Results 59 3.6.1.1. Phytoplasma detection and identification DNA was extracted from source leaves and each sugarcane sample was investigated for the presence of phytoplasma by using four nested-PCR assays (I), (II), (III) and (IV) with different primer pair combinations as was clarified before. Only nested-PCR assay (III) gave positive reactions. Our results showed that symptomatic and nonsymptomatic plants contain phytoplasma ((Figure.3.16 and Table.3.8). A B C Figure.3.15. Sugarcane leaves showing symptoms of infection with yellow leaf syndrome (A) and (B), compared with an uninfected green leaf (C). Sugarcane yellow leaf syndrome symptoms are caused by several agents including phytoplasma. Results 60 Table.3.8. Phytoplasma in sugarcane plants from Hawaiian HC&S plantation of Maui island, close to Puunene (2011). Results of phytoplasma detection based on nested-PCR assays and identification based on DNA sequencing of products of nested-PCR assay (III) using P1/P2. According to the DNA sequencing analysis the sugarcane samples contain rice yellow dwarf phytoplasma. +, phytoplasma detected; -, phytoplasma not detected. Order of Sugarcane varieties Sugarcane cultivar and Leaf condition phytoplasma detection based on PCR assay (I), (II), (IV) phytoplasma detection based on PCR assay (III) Phytoplasma group based on DNA sequencing (P1/P2) 1 H65-7052, 6 months Nonsymptomatic - + 2 H65-7052, 6 months symptomatic - - - 3 H73-3567, 4 months Nonsymptomatic - - - 4 H87-4319, 9 months Nonsymptomatic - + Rice yellow dwarf 5 H87-4319, 9 months slightlysymptomatic - + 6 H86-3792, 6 months Nonsymptomatic - + Rice yellow dwarf 7 H87-5794, 9 months Nonsymptomatic - + Rice yellow dwarf 210bp 1 2 3 4 5 6 7 M Figure.3.16. Phytoplasma in Hawaiian HC&S plantation of Maui island, close to Puunene (2011). Nested-PCR assay (III) products (0.2kb) amplified with primers (MLO-X/MLO-Y, P1/P2) of sugarcane samples obtained from Hawaiian plantation HC&S, Maui, close to Puunene; (2011) as sun-dried leaves. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). Results 61 3.6.2. Phytoplasma in sugarcane in Hawaiian breeding station (2011) Samples from uppermost fully unfolded source leaves from 10 cultivars of sugarcane plants were collected from HARC breeding station in Maunawili (fields A, B and P which are widely distant from each other) and sun-dried for phytoplasma investgation. Names of these sugarcane cultivars are indicated in (Table.3.9). These cultivars were previously tested for sugarcane yellow leaf virus (SCYLV) by Lehrer et al., 2001.The results of this test are indicated in (Table.3.11). 3.6.2.1. Phytoplasma detection by nested-PCR assay (II) and identification DNA was extracted and tested for phytoplasma by nested-PCR assay (II). Most of these sugarcane cultivars produced an amplicon at 1.2 kbp, although apparently at different titres (Figure.3.17). Products of nested-PCR assay (II) were digested with restriction endonucleases (HpaII and MseI). The obtained RFLP patterns were compared with those previously published by Lee et al., 1998. According to this digestion these Hawaiian sugarcane samples contain phytoplasmas fall in rice yellow dwarf group (Figure.3.18 and Table.3.9). However, further RFLP analysis is required to differentiate if this phytoplasma belongs to sugarcane white leaf strain (SCWL) or sugarcane grassy shoot one (SCGS) as mentioned before. Results 62 Table.3.9. Phytoplasma in Hawaiian (Maunawili, HARC) breeding station sugarcane samples (2011). Results of phytoplasma detection based on nested-PCR assay (II) and identification based on RFLP analysis using single enzyme digestion with HpaII and MseI. Sample numbers indicate to the order of samples in next figures (3.17) and (3.18). +, phytoplasma detected; -, phytoplasma not detected. Samples number Samples name phytoplasma detection based on PCR assay (II) Phytoplasma group based on RFLP with HpaII and MseI 1 H87-4094 field A11 + 2 H78-3567 “ + Rice yellow dwarf 3 H87-4319 “ + 4 H65-7052 “ + 5 H50-7209 “ + Rice yellow dwarf 6 H78-4153 “ + Rice yellow dwarf 7 H77-4643 “ + Rice yellow dwarf 8 H73-6110, field A22 + 9 H32-8560, field B31 10 H78-7750, field B62 + 11 H87-4319 “ + Rice yellow dwarf 12 H78-3606 “ - - 13 H77-4643, field P11a + 14 H78-3606, field P11 - - 15 H50-7209, field P12 - - 16 H87-4319 “ + 17 H65-7052 “ - - 18 H78-7750 “ + 19 H73-6110, field P13 + Results 63 Figure.3.17. Phytoplasma detection in sugarcane samples from Hawaiian (Maunawili, HARC) breeding station (2011). Nested-PCR assay (II) products (1.2kb) amplified with primers (SN910601/P6, R16F2n/R16R2). Samples numbers and names are indicated in table (3.9) above. Re-amplification of aliquots of first PCR water controls with nested primer combinations are in lanes W. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). Figure.3.18. Phytoplasma identification in sugarcane samples from Hawaiian (Maunawili, HARC) breeding station (2011). RFLP profiles of nested-PCR assay (II) products (1.2kb) amplified with primers (SN910601/P6, R16F2n/R16R2) following single enzyme digestion with HpaII (a) and MseI (b). The marker M was GeneRuler 100 bp plus (MBI Fermentas). 3.6.2.2. Phytoplasma detection by nested-PCR assay (III) and (IV) and identification According to PCR assay (III) results most of these sugarcane cultivars were positive for phytoplasma (Figure.3.19). This is also true for PCR assay (IV) (Figure.3.20). Products of PCR assay (IV) were digested with restriction endonuclease (HinfI). According to this digestion these Hawaiian sugarcane samples contain phytoplasma strain of sugarcane white leaf (SCWL) (Figure.3.21 and Table.3.10). 1,2kb 3567 7209 4153 4643 6110 M M 4319 1 2 3 4 5 6 7 10 8 11 12 13 14 15 16 17 18 19 W W W W 3567 M 7209 4153 4643 a b Results 64 Table.3.10. Phytoplasma in sugarcane samples from Hawaiian (Maunawili, HARC) breeding station (2011). Results of phytoplasma detection based on nested-PCR assays (III) and (IV) and identification based on RFLP analysis of (IV) products using single enzyme digestion with (HinfI). Sample numbers indicate to the order of samples in next figures (3.19 and 3.20). +, phytoplasma detected; -, phytoplasma not detected. Sample number Sample name Phytoplasma detection based on PCR assay (III) Phytoplasma detection based on PCR assay (IV) Phytoplasma strain based on RFLP with (HinfI) 1 H87-4094 field A11 + + Sugarcane white leaf 2 H78-3567 + + “ 3 H87-4319 + + “ 4 H65-7052 + + “ 5 H50-7209 + + “ 6 H78-4153 + + “ 7 H77-4643 - + “ 8 H73-6110 field A22 + + “ 9 H32-8560 field B31 - - - 10 H78-7750 field B62 + + “ 11 H87-4319 - - - 12 H78-3606 + + “ 13 H77-4643 field P11a + + “ 14 H78-3606 field P11 + + “ 15 H50-7209 field P12 - + - 16 H87-4319 + - “ 17 H65-7052 - + - 18 H78-7750 + + “ 19 H73-6110 + + “ Results 65 Figure.3.19. Phytoplasma detection in sugarcane samples from Hawaiian (Maunawili, HARC) breeding station sugarcane samples (2011). Nested-PCR assay (III) products (0.2kb) amplified with primers (MLOX/MLO-Y, P1/P2). Samples numbers and names are indicated in (Table.3.10). Re-amplification of aliquots of first PCR water controls with nested primer combination is in lanes W. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). Figure.3.20. Phytoplasma detection in sugarcane samples from Hawaiian (Maunawili, HARC) breeding station (2011). Nested-PCR assay (IV) products (0.7kb) amplified with primers (U-1/MLO-7, MLO-X/MLO-Y). Samples numbers and names are indicated in (Table.3.10). Re-amplification of aliquots of first PCR water controls with nested primer combination is in lanes W. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). 0,7kb 210bp 1 2 3 4 5 6 7 10 9 8 11 12 13 14 15 16 17 18 19 W M W 1 2 3 4 5 6 7 10 8 11 12 13 14 15 16 17 18 19 W M W 9 Results 72 Table.3.14. Phytoplasmas in sugarcane samples from provinces of Bang Phra and Khon Kean in Thailand in 2010; based on nested-PCR assays. Samples (F1 to F8) were from farmer fields in province of Bang Phra while the samples (S1 to S13) were from breeding station also in province of Bang Phra. Samples (KK1 to KK38) were from province of Khon Kean. Sugarcane sample Desiease Symptoms PCR assay (III) PCR assay (IV) Phytoplasma strains based on RFLP with (HinfI) F1 White fly + + Sugarcane white leaf F2 Leaf spot + + “ F3 rust + + “ F4 Yellow spot + + “ F5 Mosaic virus + + “ F6 Curly spindle + + “ F7 Stunted leaf + + “ F8 White leaf + + “ S1 Spotted mosaic - - S2 Spotted mosaic + + Unknown S3 - - S4 rust - + Unknown S5 rust - + Unknown S6 - - S7 - - S8 - - S9 + - S10 Streak mosaic + + Sugarcane white leaf S11 Streak mosaic + + Unknown S12 Streak mosaic - - S13 Streak mosaic - - KK1 + + Sugarcane white leaf KK2 Mosaic + + “ KK3 Grassy shoot + + “ KK4 Yellow midrib + + “ KK11 Yellow midrib + - KK12 Leaf scalel + + “ KK13 Erianthus cross yellow midrib + - KK14 mosaic + + “ KK17 mosaic + + “ KK18 Erianthus + - KK20 mosaic + + “ KK21 Spot - - KK32 white leaf + + “ KK33 white leaf + + “ KK34 white leaf + + “ KK35 Stripe + + “ KK36 Stripe + + “ KK37 Yellow midrib + + “ KK38 Yellow midrib + + “ Results 73 Figure.3.25; a, b and c. Phytoplasma detection in Thia sugarcane samples from Bang Phra and Khon Kean. Nested-PCR assay (III) products (0.2kb) of sugarcane samples obtained from farmer fields (F1 to F8) and breeding station (S9 to S11) in Bang Phra and Khon Kean (KK1 to KK38) as sun-dried leaves. Samples numbers and names are indicated in (Table 3.14). Re-amplification of aliquots of first PCR water controls with nested primer combinations are in lanes W. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). 210 bp F1 F2 F3 F7 F6 F5 F4 F8 M W W 210 bp Kk1 2 4 3 11 12 13 14 17 M Kk18 20 32 35 36 37 38 S9 S10 S11 M W W 0,2kb a b c Results 74 Figure.3.26; a, b, c and d. Phytoplasma detection in Thia sugarcane samples from Bang Phra and Khon Kean. Nested-PCR assay (IV) products (0.7kb) of sugarcane samples obtained from farmer fields (F1 to F8) and breeding station (S10 to S11) in Bang Phra and Khon Kean (KK1 to KK38) as sun-dried leaves. Samples numbers and names are indicated in (Table 3.14). Re-amplification of aliquots of first PCR water controls with nested primer combination is in lanes W. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). 0,7kb F1 F2 F3 F4 F5 F6 F7 F8 W M kk1 3 4 11 12 14 17 20 18 32 35 M W W 36 38 S10 S11 M M W a b c d 0,7kb 2 13 Results 75 Figure.3.27; a, b, c and d. Phytoplasma identification in Thia sugarcane samples from Bang Phra and Khon Kean. RFLP profiles of nested-PCR assay (IV) products (0.7kb) of sugarcane samples obtained from farmer fields (F3 to F8) and breeding station (S2 to S11) in Bang Phra and Khon Kean (KK1 to KK35) as sundried leaves following single enzyme digestion with (HinfI). The marker M was GeneRuler 100 bp plus (MBI Fermentas). 3.7.2. Phytoplasma in sugarcane in Suphan Buri province (2011) Sugarcane samples were sent from farmer fields in province of Suphan Buri as sun-dried leaves. Four samples were taken from sugarcane plants showing yellow leaf syndrome symptoms and one sample was taken from plant shows sugarcane grassy shoot symptoms. 3.7.2.1. Phytoplasma detection and identification The extracted DNA from these samples gave amplification only with primer pairs of PCR assay (III). Some products of this assay were sequenced for the identification (Figure.3.28 and Table.3.15). F4 F3 F5 F7 F6 F8 M M M Kk1 Kk3 Kk14 Kk32 Kk35 M S10 S11 Kk2 Kk20 S2 S4 S5 M M a b c d 0.5 kb 0.2 kb 0.5 kb 0.2 kb 0.5 kb 0.2 kb Results 76 Table.3.15. Outlines of phytoplasmas in Thai sugarcane samples from Suphan Buri. Sugarcane sample phytoplasma detection based on PCR assay (III) phytoplasma detection based on PCR assay (IV) Phytoplasma group based on DNA sequencing (P1/P2) 1 + - Rice yellow dwarf 2 + - „ 3 + - " 4 + - " Graasy shoot + - " 3.8. Establishment of TaqMan qPCR assay as another test for phytoplasma Most universal as well as specific phytoplasma diagnostic protocols rely on nested PCR, which, although extremely sensitive, is also time-consuming and possess risks in terms of carry-over contamination between the two rounds of amplification (Weintraub and Jones, 2010). Recently, direct qPCR has replaced the traditional PCR in efforts to increase the speed and sensitivity of detection and to improve techniques for mass screening (Weintraub and Jones, 2010). 3.8.1. Performance characteristics of qPCR Performance characteristics which include efficiency, limit of detection and sensitivity of amplicons were determined by amplifying three separately prepared sets of dilution series of three standard samples in water which include 1phytoplasma-infected periwinkle 1 2 3 4 M M W Grassy shoot 0,2kb a b Figure.3.28; a and b. Phytoplasma in Thia sugarcane samples from Suphan Buri. Nested-PCR assay (III) products (0.2kb)) of sugarcane samples obtained from farmer fields in Suphan Buri as sun-dried leaves. Samples numbers and names are indicated in (Table.3.15). Re-amplification of aliquot of first PCR water control with nested primer combination is in lane W. The marker (M) was DNA GeneRuler 100 bp plus (MBI Fermentas). Results 77 (phytoplasmal DNA) 2phytoplasma-infected sugarcane (phytoplasmal DNA) 3phytoplasma-free sugarcane (plant DNA). Since the copy number of target genes in the standard samples is unknown, the standard curves are helpful for the evaluation of PCR efficiency and sensitivity, but not for absolute quantification. 3.8.1.1. Efficiency Measurement In this study, efficiency (E) values were measured using the Ct slope method. This method involves generating a dilution series of the target template and determining the Ct value for each dilution. A plot of Ct versus log DNA concentration is constructed (Figures.3.29, 30 and 31). Amplification efficiency was calculated from the slope of this graph using the equation: Ex = 10^ (-1/slope) – 1. The effect of efficiency is exponentially dependent on cycle number. If E=1, amplicon quantity is duplicated every cycle. If E=0.8 amplicon quantity is only duplicated every 1. 2 cycle. The squared regression coefficient after the linear regression (R²) was also determined (Table.3.16 and 17). Results 78 Figure.3.29. A: Standard curve. Standard curve determined at six concentration levels (ranging from 10^0 to 10^-5) using 10-fold dilution series of the reference sample (phytoplasma-infected periwinkle). The threshold numbers of PCR cycles (CT value; means of triplicates) are plotted against the dilution (log scale).B: Logview of standard curve chart. Threshold; is an arbitrary level of fluorescence chosen on the basis of the baseline variability. Ct; is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold.∆Rn; is an increment of fluorescent signal at each time point. The ∆Rn values are plotted versus the cycle number. 15 17 19 21 23 25 27 29 31 33 35 -4 -3 -2 -1 0 1 2 Threshold Cycle Log starting quantity A B Amplification chart Ct Threshold ∆ Rn Standard curve of qPCR Results 79 Figure.3.30. A: Standard curve. Standard curve determined at six concentration levels (ranging from 10^0 to 10^-5) using 10-fold dilution series of the reference sample (phytoplasma-infected sugarcane). The threshold numbers of PCR cycles (CT value; means of triplicates) are plotted against the dilution (log scale). B: Logview of standard curve chart. ∆Rn; is an increment of fluorescent signal at each time point. The ∆Rn values are plotted versus the cycle number 12 14 16 18 20 22 24 26 28 30 32 -3 -2 -1 0 1 2 3 Threshold Cycle log starting quantity A B Amplification chart ∆ Rn Standard curve of qPCR Results 80 Figure.3.31. A: Standard curve. Standard curve determined at six concentration levels (ranging from 10^0 to 10^-5) using 10-fold dilution series of the phytoplasma-free sugarcane plant sample. The threshold numbers of PCR cycles (CT value; means of triplicates) are plotted against the dilution (log scale). B: Logview of standard curve chart. ∆Rn; is an increment of fluorescent signal at each time point. The ∆Rn values are plotted versus the cycle number 8 10 12 14 16 18 20 22 24 26 28 -4 -3 -2 -1 0 1 2 Thresholed Cycle Log strating quantity A B Amplification chart ∆ Rn Standard curve of qPCR Results 81 3.8.1.2. Artificial samples to test sensitivity of qPCR assay Performance characteristics were also evaluated for the serial dilution of phytoplasmainfected sugarcane (phytoplasmal DNA) mixed with sugarcane DNA, instead of water, isolated from phytoplasma-free sugarcane leaves to imitate real infected sugarcane samples. Thus, PCR sensitivity was evaluated for potential effects of host-material inhibition. This uninfected sugarcane material had already been tested and confirmed to be phytoplasma-free sugarcane. Artificial samples imitating infected sugarcane samples were prepared by serial dilutions of phytoplasma-infected sugarcane DNA mixed with phytoplasma-free sugarcane DNA. A plot of Ct versus log DNA concentration is also constructed as above ( Figure.3.32). Results 88 The Ct values differed considerably among samples in the phytoplasma assay, while Ct values obtained in the plant assay were different slightly. This result indicated that phytoplasma titer was variable. 3.8.3. Distribution of phytoplasma in sugarcane Q-PCR assay of phytoplasma 16S DNA was used to determine the distribution of the phytoplasma within infected sugarcane plant. The relative distribution of sugarcane white leaf phytoplasma in different parts of the plant was quantified using the comparative Ct method. Three leaf samples of phytoplasma infected sugarcane including white, variegated and green; and root samples were analysed ( Figure.3.33) . The phytoplasma was detected in all tested organs including leaves and roots. It seems there is correlation between titer of phytoplasma and symptoms expression where the titer of phytoplasma in white leaf was higher than variegated and green leaves. Lower Ct values correspond to higher initial quantities of phytoplasma DNA template (Table.3.28). Table.3.28. Q-PCR results of the distribution of the phytoplasma in sugarcane plant. CT is a threshold cycle number of qPCR assay. +,phytoplasma detected. Sugarcane sample CT mean value (16S) Phytoplasma CT mean value (18S) Sugarcane Phytoplasma detection White leaf 11.56 08.31 + Variegated leaf 12.97 08.76 + Green leaf 14.02 08.79 + Root 12.28 08. 82 + Results 89 Figure.3.33. A: Thai sugarcane plant infected with sugarcane white leaf phytoplasma where some leaves are totally bleaching whereas others are variegated and some green leaves also exists. This picture was taken three months post germination comparison with non-infected sugarcane plants in (B). 3.9. Phylogenetic analysis of the phytoplasma strains in sugarcane Restriction fragment analysis had been successfully applied to differentiate between the phytoplasma strains (Kirkpatrick et al., 1994; Lee et al., 1998; Valiunas et al., 2007). The products of the second round PCR were subjected to restriction enzymes which were diagnostic for the phytoplasma strains. The restriction patterns identified the phytoplasma from Hawaiian cultivars and from one Cuban cultivar as belonging to the Aster yellows phytoplasma, whereas the phytoplasma from the Cuban cultivar CP4362, which originally had been bred in Canal Point, Florida and from JA605, belonged to the Western X-disease phytoplasma (Figure.3.4 and Table. 3.2). This classification was supported by sequence comparison. The complete sequence of R16F2n/R16R2-amplified fragments was determined for three different sugarcane cultivars which are infected by three different phytoplasma isolates, two cultivars are from Cuba and one from Egypt (Figure.3.34). The complete sequence of 16S/23S intergenic spacer region was determined for one Hawaiian sugarcane cultivar using the primer pair P4/P7 (Figure.3.34). The partial sequence of 16S/23S intergenic spacer region was also determined for other two Hawaiian sugarcane cultivar and for one cultivar from Thailand using the primer pair P1/P2 (Figure.3.34). A B Results 90 Figure.3.34. Diagrammatic representation of genomic location of primers used for DNA sequencing. The obtained nucleotide sequences were compared with sequences of phytoplasmas and acholeplasmas from GenBank using the BLASTN program. Multiple alignments of near-fulllength 16S rRNA gene sequences from 22 phytoplasma and one Acholeplasma species and multiple alignments of 16S/23S intergenic spacer region from 13 phytoplasma and two Acholeplasma species were examined using MUSCLE software. Phylogenetic trees of both sequence parts were constructed to reveal the position of the isolated phytoplasma strains from Hawaiian, Cuban, Egyptian and Thai sugarcane, relative to phytoplasma strains which had been isolated from sugarcane and other plants. Figure.3.35 presents the two phylogenetic trees that were constructed by maximum likelihood estimation with geneious program through the PhyML software (Guindon and Gascuel, 2003). Bootstrap analysis was performed 1.000 times to evaluate branch supports in a sound statistical framework. The phytoplasma isolate (HM804282) from Cuban sugarcane cultivar Ja605 clustered together with other strains of X-disease group, among them already reported sugarcane yellows phytoplasma strain found in South Africa (AF056095) with a bootstrap value of 48.7 and shared 99% sequence identity (Figure.3.35 and Table.3.29). Other Cuban sugarcane cultivar C10-5173 was infected with phytoplasma strain (HQ116553) clustered to the aster yellows group, closely together with sugarcane yellows phytoplasma from Brazil (EU423900) and maize bushy stunt phytoplasma from Colombia (HQ530152) with a bootstrap value of 49.6 and shared 99% sequence identity (Figure.3.35 and Table.3.29). Egyptian sugarcane cultivar G8447 contains phytoplasma strain (JN223446) clustered to the rice yellow dwarf group, closely together with sorghum grassy shoot phytoplasma from Australia (AF509324) with a bootstrap value of 81.1 and shared 99% sequence identity (Figure.3.35 and Table.3.29). Results 91 The phylogenetic tree of the 16S/23S spacer region contained less phytoplasma entries in GenBank. The Hawaiian sugarcane phytoplasma isolate (HQ116554) from cultivar H84-4094 and another Hawaiian sugarcane phytoplasma isolate (JN223447) from unknown cultivar obtained from Hawaiian former plantations as a different original source clustered to the aster yellows group, closely together with water cress yellows from Hawaii (AY665676) and Russian potato purple top phytoplasma (EU333399) with a bootstrap value of 69.6 and shared 99% sequence identity (Figure.3.35 and Table.3.29). Hawaiian sugarcane phytoplasma isolate (JN223448) from cultivar H78-7750 which obtained from Hawaiian breeding station of HARC in Maunawili, Oahu clustered to the rice yellow dwarf group, closely together with sugarcane white leaf phytoplasma from Taiwan (AY139874) with a bootstrap value of 86.6 and shared 98% sequence identity (Figure.3.35 and Table.3.29). Thai sugarcane phytoplasma isolate (HQ917068) from unknown sugarcane cultivar obtained from province of Khon kaen clustered to the rice yellow dwarf group, closely together with sugarcane white leaf phytoplasma from Myanmar (AB646271) with a bootstrap value of 64.2 and shared 100% sequence identity (Figure.3.35 and Table.3.29). Results 92 Table.3.29. Phytoplasma strains and their GenBank accession numbers used in this study for the phylogenetic trees (Figure.3.35). 16S rRNA gene and 16S/23S intergenic spacer region sequences of phytoplasmas determined in this study are in bold. Phytoplasma strains of monocotyledonous plants and strains which showed close sequence similarity to the Hawaiian, Cuban, Egyptian and Thai sugarcane phytoplasma were selected for construction of the trees. The sequences from Acholeplasma axanthum and Acholeplasma palmae were used as out groups. Phylogenetic tree of 16S rRNA (a) Accession number Phytoplasma strain Group AF056095 Sugarcane yellows phytoplasma type I (South Africa) X-disease AF411592 Erigeron witches'-broom phytoplasma Ash yellows AF509324 Sorghum grassy shoot phytoplasma variant I (Australia) Rice Yellow Dwarf AF498307 Coconut lethal yellowing phytoplasma Coconut lethal yellowing AJ550984 Bermuda grass white leaf phytoplasma (Southern Italy) Bermuda white leaf AM261831 Sugarcane grassy shoot phytoplasma (India) Rice Yellow Dwarf AY197652 Spartium witches'-broom phytoplasma Apple proliferation AY736374 Napier grass stunt phytoplasma (Kenya) Rice Yellow Dwarf EF413055 Sorghum verticilliflorum phytoplasma (Mauritius) X-disease EF413056 Sugarcane yellows phytoplasma clone SC245 (Mauritius) X-disease EU294011 Malaysia Bermuda grass white leaf phytoplasma Bermuda white leaf EU423900 Sugarcane yellows phytoplasma type I (Brasil) Aster yellows FM208260 Sugarcane white leaf (Thailand) Rice Yellow Dwarf GQ336993 Kidney bean little leaf phytoplasma clone Z16 16S Peanut WB GQ850122 Coconut root wilt phytoplasma isolate RD3 (India) Rice Yellow Dwarf GU565959 Candidatus Phytoplasma pyri isolate 932801 Apple proliferation HM804282 Sugarcane Ja60-5 yellow leaf (Cuba) X-disease HQ116553 Sugarcane C1051-73 yellow leaf (Cuba) Aster yellows HQ530152 Maize bushy stunt phytoplasma strain MBSColombia (Colombia) Aster yellows HQ589200 Milkweed yellows phytoplasma strain MWI(USA) X-disease JF508514 Sesame phyllody phytoplasma strain Seph2 Peanut WB JN223446 Sugarcane grassy shoot phytoplasma (Egypt) Rice Yellow Dwarf NR_029152 Acholeplasma palmae strain J233 Out group Results 93 Phylogenetic tree of 16S/23S intergenic spacer region (b) Accession number Phytoplasma strain Group AB243298 Sugarcane grassy shoot phytoplasma (India) Rice Yellow Dwarf AB646271 Sugarcane white leaf phytoplasma (Myanmar) Rice Yellow Dwarf AF434989 Texas Phoenix palm phytoplasma Coconut lethal yellowing AY139874 Sugarcane white leaf phytoplasma (Taiwan) Rice Yellow Dwarf AY665676 Aster yellows phytoplasma “Watercress” (Hawaii) Aster yellows DQ004923 Acholeplasma palmae Out group DQ400425 Acholeplasma axanthum Out group EU294011 Malaysia Bermuda grass white leaf phytoplasma Bermuda white leaf EU333399 Russian potato purple top phytoplasma (Russia) Aster yellows FN562932 Candidatus Phytoplasma vitis Elm yellows HQ116554 Hawaiian sugarcane H87-4094 yellow leaf phytoplasma Aster yellows HQ589192 'Psammotettix cephalotes' flower stunt phytoplasma Rice Yellow Dwarf HQ917068 Sugarcane white leaf phytoplasma (Thailand) Rice Yellow Dwarf JN223447 Hawaiian sugarcane Phytoplasma Aster yellows JN223448 Sugarcane white leaf phytoplasma (Hawaii) Rice Yellow Dwarf Results 94 a Rice yellow dwarf X-disease Aster yellows Results 95 Figure.3.35. Position of the phytoplasma strains from Hawaiian, Cuban, Egyptian and Thai sugarcane in a phylogenetic tree together with other phytoplasma isolates (Table.3.29). a: Phylogenetic tree constructed using 16S rRNA sequences from 22 phytoplasma and one Acholeplasma species, b: Phylogenetic tree constructed using 16S/23S spacer sequences from 13 phytoplasma and two Acholeplasma species. Bar represents phylogenetic distance of 2%. Numbers on branches are confidence percentage obtained from 1.000 bootstrap replicates. b Rice yellow dwarf Aster yellows Results 96 3.10. Hot water treatment in order to get phytoplasma free sugarcane plant Hot water treatment had been proposed as a cure for phytoplasma in dormant woody plant material (Edison and Ramakrishnan, 1972; Caudwell et al., 1997), because phytoplasmas have only limited heat tolerance. Treatment of infected sugarcane stalks with moderately high temperatures such as 50°C for 2 h were reported to successfully eliminate grassy shoot disease and white leaf phytoplasma from stem material. Hot water treatment of stem cuttings together with immersion in a fungicide solution is a routine practice in Hawaiian sugarcane plantations to prevent fungal rot of planted seed pieces. The question was which temperature regimes and which incubation durations are needed to eliminate SCYLP from sugarcane stems and whether the routine hot water-treatment against fungi had unintentionally also cured from phytoplasma. One-eye stem cuttings were immersed in hot water of defined temperature and for defined period, then planted in sterile soil in pots and kept in insect-tight mesh cages for germination and growth. Indeed, incubation of seed pieces at 50°C for 30 min or longer was sufficient to eliminate phytoplasma (Table.3.30), irrespective whether it was from Aster yellows or from Western X-disease type. The incubation in hot water for 3 h had a detrimental effect on seed piece viability unless the hot water treatment was preceded by 10°C incubation for 48h, a procedure routinely used in the Australian and Cuban sugar industry. 3.10.1. Hot water treatment according to Australian recipe Two sugarcane cultivars (H65-70 52 and H78-77 50) were used as material in the hot water treatments. Stalks were cut into single-eye sets and treated by immersion for 48 h in cold water (10°C) followed by 3h in hot water (50°C). Next, setts were planted in sterile soil and placed in mesh cage to protect them against insects. Subsequently, these plants which rose from these cuttings were tested for the presence of phytoplasma after 2 and 6 months and one year post germination (Figure.3.36). Results 97 Figure.3.36. Nested PCR results of the test plants, which received cold and hot-water treatment after 2 months post germination. Lane (1) contains PCR product obtained from untreated sugarcane (without hot water treatment) (H6570 52). Lane (2) contains PCR product obtained from untreated sugarcane (H7877 50). Lane (3) test plant (H6570 52) after coldand hot-water treatment (Australian recipe). Lane (4) test plant (H7877 50) after coldand hot-water treatment (Australian recipe). Re-amplification of aliquot of first PCR water control with nested primer combination is in lane (5). DNA ladder is FastRuler Middle Range (MBI Fermentas). Figure.3.37. Nested-PCR results of test plants, which received cold and hot-water treatment after 6 months (a) and 1 year (b) post germination. Lane (1) treated plant (H65-70 52); lane (2) treated plant (H7877 50); lane (3) is positive control; re-amplification of aliquots of first PCR water control with nested primer combination are in lanes (4-5); lane (M) GeneRuler DNA ladder (MBI Fermentans). 3.10.2. Hot water treatment with various duration These tests were carried out in order to investigate what is the minimum immersion time at 50°C can eliminate the phytoplasma in infected sugarcane plants. Furthermore, in these tests we didn’t soak the cuttings in cold water at 10°C before hot water treatment in order to check the influence of lacking of cold water treatment on the vegetative development. Thus, the 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5 M a b Results 104 Figure.3.42. Comparison between phloem sieve elements of white, variegated and green leaves of white leaf phytoplasma-infected sugarcane. A: white leaf phenotype of sugarcane white leaf phytoplasma infection, where phytoplasmas are clearly visible in sieve elements (arrow). B: variegated leaf phenotype of sugarcane white leaf phytoplasma infection. Phytoplasmas are obvious present in sieve elements in increased numbers in comparison with white leaf (arrow). C: green leaf phenotype of sugarcane white leaf phytoplasma infection. Phytoplasmas are more abundant in some sieve elements than others (arrow). D: green leaf of uninfected sugarcane plant where it is used as control plant for comparison. It is obvious that no phytoplasma is present in sieve elements. Infected and uninfected leaves show the typical sieve anatomy. Vacuoles and cytoplasm are fused to a so called mictoplasm (Esau et al., 1965). Most of the organelles are absent, only typical round shaped sieve elements plastids are present. Sieve element plastids show the typical phenotype with crystal inclusions which are common for poaeceae (arrow Fig. 53 D). Sieve tube (S), companion cell (CC), parenchyma cell (PC). Bar = 2 µm. S S S CC CC CC CC PC S CC S S S S Results 105 Figure.3.43. Comparison between phloem companion cells of phytoplasma infected and uninfected sugarcane. A: white leaf phenotype of phytoplasma-infected sugarcane where surrounding companion cell of sieve elements contains different organelles including vacuoles and mitochondria but they don’t show any phytoplasma. B: variegated leaf phenotype of phytoplasma-infected sugarcane show typical companion cells connected to each other by plasmodesmata. Despite of these companion cells surround sieve element which contain phytoplasmas but these cells are phytoplasma-free. C: green leaf phenotype of phytoplasma infection where companion cell is also lack phytoplasma. D: green leaf of uninfected sugarcane which used as control and show partially one companion cell surrounds phytoplasma-free sieve element .Mitochondria have a similar size to phytoplasma but could be clearly distinguished due to the presence of Crystal of the inner mitochondrial membrane. Sieve tube (S), Companion cell (CC), vacuoles (V), plastid (P). Bar = 1 µm. Results 106 Figure.3.44. Ultrathin sections of bundle sheath and mesophyll tissues of phytoplasma-infected sugarcane. A and B: variegated leaf phenotype of phytoplasma-infected sugarcane show that phytoplasmas are absent in the bundle sheath tissues. C and D: mesophyll tissues of variegated leaf phenotype of phytoplasma-infected sugarcane are also phytoplasma-free. Parenchyma cells (PC), (A), (C) bar = 5 µm. (B), (D) bar = 2 µm. 3.13.3. Phytoplasma size and shape Size of the phytoplasma bodies varied from 200 nm to 800 nm (0.2 µm to 0.8 µm) in diameter. Our transmission electron microscopic studies of white leaf phytoplasma-infected sugarcane leaf showed spherical bodies which were bounded by a poorly defined membrane; (Figure.3.45). Sieve tubes filled with numerous phytoplasmas were seen particularly in variegated leaves of diseased sugarcane; (Figure.3.46). Results 107 Figure.3.45. Transmission electron micrograph of typical membrane-bound phytoplasma bodies which present in sieve tube and contain resembling DNA in sieve tube of white leaf phytoplasma-infected sugarcane leaf. Sieve tube (S), phytoplasma (P). Bar = 0.2 µm. Figure.3.46. Ultrathin sections of phloem tissue of phytoplasma-infected sugarcane. A: Ultrathin section in variegated leaf phenotype shows that phytoplasmas (arrows) fill a phloem sieve element with a large number which is approximately more than 100 phytoplasma cells in one sieve tube. Phytoplasmas block the downward translocation photosynthates and passing through a sieve-plate pore lined with callose. B: higher magnification of the same last ultrathin section. Sieve cell (S), companion cell (CC), parenchyma cell (PC), sieve plate (sp), plastid (P), callose (CA). (A): bar = 2µm. (B): bar = 1µm. S P P P CA Results 108 3.13.4. Ultrastructural changes of the phytoplasma infection on leave anatomy Several ultrastructural changes were observed on ultrathin sections of the vascular tissues of affected sugarcane plants under transmission electron microscope ( TEM). Paranchymatic cells of bundle sheath and mesophyll tissue of affected leaves showed some alterations comparing to uninfected leaves. In these cells accumulations of starch granules and plastoglobuli were observed in white leaf phytoplasma-infected sugarcane comparing to control uninfected one; (Figure.3.47). Our electron microscopic studies are in agreement with literature where phytoplasma infections led to a significant increase of starch in source leaves (Lepka et al. 1999). These data are consistent with ultrastructural observations reporting starch accumulation in chloroplasts associated with a severe disorganization of thylakoids and a reduction in chlorophyll content (Musetti, 2006). Figure.3.47. Ultrastructural comparison between paranchymatic bundle sheath cells of infected and uninfected sugarcane. A: variegated leaf phenotype of phytoplasma-infected sugarcane, where ultrastructural observations indicate accumulation of starch granules (arrows) and plastoglobuli (head arrow) in chloroplasts of bundle sheath cells of infected sugarcane. B: green leaf of uninfected sugarcane where it is used as control plant for comparison. It is clearly that accumulation of starch granules (arrows) in chloroplasts of bundle sheath cells is less than infected sugarcane. In addition, plastoglobuli are not accumulated in uninfected sugarcane. Parenchyma cell (PC), plastid (P), nucleus (N), vacuoles (V). Bar = 2µm. Results 109 Figure.3.48. Comparison between chloroplasts structure of mesophyll cells in infected and uninfected sugarcane. A: variegated leaf of phytoplasma-infected sugarcane where an increase of plastoglobuli number and size in disorganized chloroplasts was found in mesophyll paranchymatic cells. B: green leaf of uninfected sugarcane which used as control, where mesophyll paranchymatic cells contain normal chloroplast with lower formation of plastoglobuli in comparison with infected sugarcane. Plastid (P), plastoglobuli (arrows). Bar = 1µm. Discussion 110 4. Discussion 4.1. Establishment of the test for phytoplasma Yellow leaf syndrome (YLS) of sugarcane has been associated with several biotic and abiotic causes during the past four decades. Lute viruses and phytoplasmas are two types of plant pathogens that are typified as causing symptoms of yellowing in their hosts (Jones, 2002). It is hardly surprising that in sugarcane the pathogen cannot be distinguished by symptoms alone (Cronje et al., 1998; Arocha et al., 1999). In the present study we have employed molecular-based tools for detection and identification of the putative causal agent of yellow leaf syndrome and report for the first time the presence of phytoplasma in Hawaiian sugarcane cultivars. The exceptional sensitivity of PCR offers many advantages for detection of plant pathogens (Herson and French, 1993). Application of this technique for detection and investigation of phytoplasmas seems particularly appropriate due to the small size of these plant pathogens and inability to culture them in vitro. 4.1.1. Efficiency of PCR amplification Phytoplasma diagnostics and phylogenetics have historically been based on the 16S rRNA gene and the 16-23S rRNA spacer region because of the availability of universal primers for this region (Hodgetts and Dickinson, 2010). Numerous PCR primer combinations have been designed for diagnostics and phylogenetics. However, diagnostics based on these primers can be problematic, with occasional false positives, particularly through amplification of any bacillus spp. that might be present in a plant sample (Harrison et al., 2002). However, based on our investigation we never found bacillus spp. in sugarcane plant samples which was confirmed by RFLP analysis and DNA sequencing of nested-PCR products. Though PCR is a routine technique for phytoplasma detection, there still meet some difficulties, at least with some primers which in some cases can induce dimmers, bands of non specific sizes. In these cases, false positive results can be expected. Two types of control were therefore routinely applied in each PCR run to test possible generation of false positive amplicons. One was the implementation of water control to test the generation of primer dimmers. However, in our hands, nested-PCR with all primer combinations used didn’t amplify products from water used as template. The other control experiment was with sugarcane DNA from sugarcane plants which were phytoplasma-free, namely plants from Discussion 111 Egypt (Gt549, Ph8013) and plants which had undergone hot water treatment. These preparations did not give a phytoplasma-specific amplicon which indicated that the sugarcane DNA does not contain nucleotide sequences which bind to the phytoplasma-specific primers. In contrast, in many cases the same phytoplasma-infected sugarcane samples amplified with some primer pairs never reacted with other primer pairs, despite the fact that the used primers were universal. In the case of no visible products were obtained from phytoplasma positive samples, a higher dilution of DNA is used to dilute the plant inhibitors which may exist. However, these phytoplasma positive samples have shown false negative results after dilution too. It seems that in the case of phytoplasma positive samples, the primers preferentially amplified phytoplasma sequence of expected size. For example, (P1/P7, R16F2n/R16R2) and (R16mF2/R16mR1, R16F2n/R16R2) have been widely used for the detection of phytoplasma and are probably the most thoroughly investigated. They detect all strains of phytoplasmas whereas the DNA of non-infected plants does not react. Many phytoplasma positive samples were false negatives with these assays. Therefore, each sugarcane sample was investigated for phytoplasma by different nested-PCR assays (I), (II), (III) and (IV) with different primer pair combinations. Our tests have showen significant differences in the results of the PCR assays due of some weak or no amplification using particular primer combinations. According to our tests the primer pairs used for nested-PCR assay III, (MLO-X/MLO-Y) and (P1/P2), which amplified 16S-23S rRNA spacer region, was demonstrated to be the most reliable one to detect the phytoplasma in sugarcane plants due to the high efficiency of PCR amplification, high annealing temperature and low or no non-specific bands; in addition to the high sensitivity of these primer pairs where yielded standard products visualized in bands of a strong intensity. Our analysis demonstrated difficulties with the detection ability of phytoplasma in sugarcane plants. In order to explain the different result patterns obtained with particular primer combinations, a subliminal amount of template DNA, a presence of PCRinhibiting substances in DNA preparations and sequential variability of primer target sites can be taken into account (Skrzeczkowski et al., 2001; Heinrich et al., 2001). For example, use of the 16S-23S rRNA spacer region in our investigation was more reliable than 16S rRNA gene region. It was more powerful than the 16S rRNA gene because it yielded standard products visualized in bands of a strong intensity as mentioned above. As a consequence, in the case of critical samples, different primer pair combinations and also sequencing should be used for elucidation of phytoplasma presence (Franova, 2011). Discussion 112 4.1.2. Carry-over contamination problems The ability of the PCR to amplify minute amounts of template has the disadvantage that small quantities of contaminating DNA may be a problem for some applications like pathogens detection. In general, the titre of phytoplasma in sugarcane plants is very low and the standard method is nested PCR, which enhances the sensitivity of the test by two successive rounds of amplification. It is a very sensitive method and the risk of false positive signals is high. Two types of water control were therefore routinely applied to test for possible generation of false positive amplicons. One was the implementation of water control, where water was included in the first PCR-round instead of DNA from sugarcane leaves and then the hypothetical amplicon was transferred to the second PCR-round. In parallel the second PCR-round was also performed with water instead of the first-round amplicon and several water controls were used in each PCR round. On the other hand, it is important to have a designated clean area for setting up PCR reactions from which other DNA samples, especially PCR products, are excluded. 4.1.3. Q-PCR (real-time PCR) Performance characteristics of used qPCR assay were determined by amplifying three separately prepared sets of dilution series of three standard samples in water. All systems gave good values as far as R², Ct, efficiency, limit of detection and sensitivity of amplicons which showed a broad dynamic range (five log orders of magnitude). These parameters were also evaluated for the artificial samples that imitate infected sugarcane samples. The calibration curve of these artificial samples was very important to check sensitivity of realtime PCR assay and to explain the false negative results of qPCR for most our sugarcane samples. The irregular signals at the sixth dilution of artificial samples look similar to the signals of experimental sugarcane samples (Figure 4.1). It is most likely that at sixth concentration level (10¯⁵), using a 10-fold dilution series of the phytoplasma-infected sugarcane (phytoplasmal DNA) mixed with sugarcane DNA to imitate real sugarcane samples, the titer of phytoplasmal DNA is very low. It could be that sugarcane samples, which contain low titer of phytoplasmas cannot be detected sensitively by this direct qPCR assay due to the influence of host-material and that may be true for the sugarcane plants which show yellow leaf syndrome and contain low titer of phytoplasma. Discussion 113 Figure.4.1. A: Logview of standard curve chart. Standard curve determined at six concentration levels (ranging from 10⁰ to 10¯⁵) using 10-fold dilution series of the phytoplasma-infected sugarcane (phytoplasmal DNA) mixed with sugarcane DNA to imitate real sugarcane samples. Threshold; is an arbitrary level of fluorescence chosen on the basis of the baseline variability. Ct; is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold.∆Rn; is an increment of fluorescent signal at each time point. The ∆Rn values are plotted versus the cycle number. B: Logview of amplification chart of qPCR results. On the other hand, it is essential that the nucleic acid is sufficiently pure for qPCR analysis. Template contamination (i.e., protein, carbohydrates or organic solvents) can have a huge impact on assay reliability and reproducibility. We used high pure PCR template preparation kit. Then the template DNA quality was determined by Nanophotometer. Since, diagnosis of pathogen in the plants including sugarcane is often hampered by the presence of PCR inhibitors such as polyphenolics, polysaccharides and other molecules that may produce falsenegative results even sometimes from heavily infected samples (Weintraub and Jones, 2010). To prove that the absence of a signal is not due to such causes, protocols for control amplification and detection of the host plant DNA have been developed such as 18S rRNA gene (Christensen et al., 2004). According to our plant 18S rRNA gene analysis, however, sugarcane samples were sufficiently pure for qPCR analysis. It appears that PCR inhibitors can hamper diagnosis of phytoplasma only when phytoplasmas exist in very low titer as most of our sugarcane samples. Amplification chart Amplification chart A B PCR cycle number Threshold Ct ∆ Rn PCR cycle number ∆ Rn Plant DNA phytoplasma DNA Signals of sixth dilution Discussion 120 4.3. Identification of the phytoplasma strains in sugarcane by phylogentic analysis Due to the inability to cultivate phytolasmas in cell-free media, molecular analyses of conserved gene sequences have become rational means for phytoplasma taxonomy and classification. Use of DNA sequences to build up phylogenetic trees is widespread and recognized as a valid approach for identifying taxonomic relationships between organisms (Hodgetts and Dickinson, 2010). Following decisions for phytoplasma taxonomy taken by the Phytoplasma Working Team during the 13th International Organization of Mycoplasmology held in Fukuoka, Japan (14 to 19 July 2000) in general, a strain can be described as a new “Candidatus Phytoplasma species” if its 16S rRNA gene sequence has less than 97.5% identity to any previously described “Candidatus Phytoplasma species.” A BLAST search for the 16S rRNA gene sequences reported in this study showed that they shared 99 to 100% sequence identity with those of other phytoplasmas in the aster yellows, Xdisease and rice yellow dwarf groups. This confirmed that the detected phytoplasmas belong to these groups of ‘Candidatus phytoplasma’. For example, Egyptian sugarcane cultivar G8447 contains phytoplasma strain (JN223446) clustered to the rice yellow dwarf group, closely together with sorghum grassy shoot phytoplasma from Australia (AF509324) with a bootstrap value of 81.1 and shared 99% sequence identity (Table.3.29 and Figure.3.35.a). Furthermore, it was previously reported that the more distantly related to SCGS agent, is the sorghum grassy shoot (SGS) (Rao et al., 2007). Therefore, the Egyptian sugarcane cultivar G8447 contains phytoplasma strain (JN223446) belongs to the rice yellow dwarf group ‘Candidatus phytoplasma oryzae’ and this strain cannot be described as a new “Candidatus Phytoplasma species” due of its 16S rRNA gene sequence has more than 97.5% identity to any previously described “Candidatus Phytoplasma species.”. That is also true for phytoplasma isolate (HM804282) from Cuban sugarcane cultivar Ja605 clustered together with other strains of X-disease group, among them already reported sugarcane yellows phytoplasma strain found in South Africa (AF056095) with a bootstrap value of 48.7 and shared 99% sequence identity (Table.3.29 and Figure.3.35.a). Therefore, the Cuban sugarcane cultivar Ja605 contains phytoplasma strain (HM804282) belongs to the X-disease group ‘Candidatus phytoplasma pruni’ and can’t be described as a new “Candidatus Phytoplasma species”. Other Cuban sugarcane cultivar C10-5173 was infected with phytoplasma strain (HQ116553) clustered to the aster yellows group, closely together with sugarcane yellows phytoplasma Discussion 121 from Brazil (EU423900) and maize bushy stunt phytoplasma from Colombia (HQ530152) with a bootstrap value of 49.6 and shared 99% sequence identity (Table.3.29 and Figure.3.35.a). Therefore, the Cuban sugarcane cultivar C10-5173 contains phytoplasma strain (HQ116553) belongs to the Aster yellows group ‘Candidatus phytoplasma asteris’ and this strain can’t be also described as a new “Candidatus Phytoplasma species”. 4.4. Hot water treatment in order to get phytoplasma free plant When a pathogen is excluded from the propagating material of a host, it is often possible to grow the host free of that pathogen for the rest of its life (Aslam, 2001). Hot water treatment has been proposed to cure dormant woody plant material from phytoplasmas. While tissue culture techniques are routinely used for virus eradication; few reports have been published on their potentiality in phytoplasma elimination (Dai et al., 1997; Parmessur et al., 2002; Chalak et al., 2005). The effectiveness of the method is based on the fact that dormant plant organs can withstand higher temperatures than those their respective pathogens can survive for a given time (Agrios, 2004). The first aim of our hot water treatment was to get negative control (phytoplasma-free plant) Therefore, we used an Australian recipe (Arocha, 2005b), as long duration treatment (48 h in cold water (10°C), followed by 3h in hot water (50°C)).The cuttings (approx. 30 mm average diameter) were kept in cold water before the hot water treatment was applied. It seems that, immersion at 50°C for 30 min was not effective to eliminate the phytoplasma totally from the cuttings but it could be effective in smaller diameters. Furthermore, it could be that also depends on the titer of phytoplasmas in the plant material. Our tests showed that the appropriate hot water treatment, which recommended for phytoplasma elimination, is immersion at 50°C for at least 60 min. Furthermore, our tests showed that the plant material (cuttings) should be thermally prepared to the treatment by storage for 48 hours at 10°C in order to prevent a poor vegetative development especially for long duration treatment at 50°C for 3 hours. Due it could be that this treatment could lead to high mortality rates. The hot water treatment which is practiced by the Hawaiian plantations for their seed cane fields (3 h at 50°C) is sufficient to eliminate phytoplasma, whereas the duration of the hot water treatment for seed pieces which are planted in the fields (20 min at 52°C) may be at the margin of successful bacteria elimination. Discussion 122 Therefore field plants were tested for phytoplasma with emphasis on the comparison of green plants with YLS-symptomatic plants, standing side-by-side. Our results showed that these field plants were in the case of a mostly phytoplasma-infected and it seems that there is no clear association between phytoplasma and symptoms due to some green sugarcane plants were also positive for phytoplasma. Asymptomatic sugarcane was frequently phytoplasmapositive; this has also been reported by other workers (Cronje et al., 1998a). One explanation for the poor correlation between phytoplasma and symptoms is that some phytoplasmas can exist in plants without ever causing disease or having only a minor impact, as is the case for ash yellows in velvet ash (Sinclair et al., 1994) and phytoplasmas in alders (Lederer and Seemuller, 1991), apricots (Kirkpatrick et al., 1990) and almonds (Uyemoto et al., 1992). Such associations suggest that the host plant is either tolerant or resistant to phytoplasma infection. 4.5. Transmission test with sugarcane aphid Our tests showed that the sugarcane aphids (Melanaphis sacchari) are able to acquire the phytoplasmas because DNA extracted from these insects produced an expected size of nestedPCR product. In addition, DNA sequencing of these PCR products confirmed that. Our tests showed that these sugarcane aphids are able to acquire the phytoplasmas but they are unable to transmit the phytoplasmas into the sugarcane plants because all target plants (phytoplasmafree plants) were negative for phytoplasma infection after three months post inoculation. In fact, many aphids, whiteflies and mealy bugs are phloem-feeders on plant species infected with phytoplasmas, but so far none of them has been found to be a vector of phytoplasmas. Recently, apple aphids were found to be positive in PCR assays for apple proliferation phytoplasmas and were suspected to be vectors, but the results of transmission experiments seem to exclude this possibility (Cainelli et al., 2007). A phloem-feeding habit is thus necessary but insufficient for phytoplasma transmission. It was an expected result that sugarcane aphids are unable to transmit the phytoplasmas (SCYP) because thus far, there has been no report of phytoplasma or spiroplasma transmission by a phloem-feeding aphid. The reasons for lack of transmission by aphids are not known. Sites of mollicute attachment to insect tissues and other pathogen-insect interactions can be cited in a general sense to explain transmission specificities. But what molecular mechanisms, that are present in leafhoppers and presumably absent in aphids, Discussion 123 account for the differences in mollicute transmission between these major insect groups? (Mishra, 2004). As a consequence, interestingly, aphids apparently do not serve as phytoplasma vectors. 4.6. Transmission electron microscopy for cytological location of phytoplasma Phytoplasmas are transferred with saliva of infected insect vectors into the pierced sieve element, from which they spread systemically in the plant using the continuous sieve tube system due they are pleiomorfic and sufficiently small to pass freely through sieve pores. Our transmission electron microscopic studies revealed the presence of sugarcane white leaf phytoplasma only in phloem sieve tubes of diseased sugarcane leaves but not in cells adjacent to the sieve elements including companion cells and phloem parenchyma, although in many cases the phytoplasmas have been reliably documented in companion cells and phloem parenchyma cells by electron microscopy, as well as in sieve elements (Siller et al., 1987). Several ultrastructural changes were observed under transmission electron microscope (TEM). Parenchymatic cells of bundle sheath and mesophyll tissue of infected leaves showed some alterations compared to uninfected leaves. In these cells accumulations of starch granules and plastoglobuli were observed in white leaf phytoplasma-infected sugarcane compared to uninfected control ( Figure.3.47). Our electron microscopic studies are in agreement with literature, where phytoplasma infections led to a significant increase of starch in source leaves (Lepka et al., 1999). These data are consistent with ultrastructural observations reporting starch accumulation in chloroplasts associated with a severe disorganization of thylakoids and a reduction in chlorophyll content (Musetti, 2006) due to the decrease of both Chl a and Chl b in leaves. “A decrease in photosynthetic pigments has been observed in maize plants infected with maize bushy stunt (Junqueira et al., 2004), apples infected with apple proliferation and grapevine infected with the bois noir phytoplasma. This is probably the result of enhanced chlorophyllase activity in infected leaves (Bertamini et al., 2002b) and it has been suggested that phytoplasmas have a role in the inhibition of chlorophyll biosynthesis in plant host leaves (Bertamini et al., 2002a).” “The descent of photosynthesis is the result of phytoplasma infection on photosynthetic electron transport and enzymatic activities, due to the loss of several thylakoid membrane proteins and to the reduction of leaf soluble proteins. These changes are similar to those induced by leaf ageing, so an interference of phytoplasmas with plant hormones that regulate senescence processes in leaf tissues could be hypothesized. In all kinds of diseases in which Discussion 124 there is destruction of leaf tissue like sugarcane white leaf phytoplasma, photosynthesis is reduced because the photosynthetic surface of the plant is lessened. Most viruses, mollicute diseases induce varying degrees of chlorosis and stunting. In the majority of such diseases, the photosynthesis of infected plants is reduced greatly. In diseases caused by phytoplasmas, bacteria exist and reproduce in the phloem sieve tubes, thereby interfering with the downward translocation of nutrients” (Musetti, 2006). An increase plastoglobuli number and size in disorganized chloroplasts was found in mesophyll paranchymatic cells of variegated leaf of white leaf phytoplasma-infected sugarcane compared to green leaf of uninfected sugarcane which was used as control ( Figure.3.48) . It is known that characteristics of plastids in senescent cells include reduced size, rounded shape and larger plastoglobuli (Thomson and Platt-Aloia, 1987; Biswal and Biswal, 1988; Noode´n, 1988). As a consequence, the phytoplasma diseases are complex and their progress is also highly variable and depends on many factors including the state of the host plants, the pathogen and its different biotypes, the tendency for mutation, the presence and dynamics of the vectors, the titer of the phytoplasma, the environmental conditions as well as the agronomical practices being used (Ciancio and Mukerji, 2008). Summary 125 5. Summary The Yellow leaf syndrome (YLS) had been first detected and described in Hawaiian sugarcane plantations. The polerovirus Sugarcane yellow leaf virus was identified as a causal agent of the syndrome; however there was no strict correlation between the degree of symptom expression and the virus titre. Therefore several surveys on breeding station sugarcane plants in Hawaiian Islands were done for Sugarcane yellow leaf phytoplasma (SCYLP), a bacterium which had been hypothesized to be also a causal agent of YLS. Two types of phytoplasmas were found in Hawaiian sugarcane cultivars mainly sugarcane white leaf phytoplasma (SCWL) which is a member in rice yellow dwarf group, in addition to aster yellows group. This was also true for sugarcane plants from Hawaiian plantations, which routinely use hot water-treatment for the seed cane cuttings. Sugarcane samples were obtained also from other countries including Cuba, Egypt, Syria and Thailand where sugarcane plants are also showing symptoms of yellowing or whiting. Aster yellows and X-disease phytoplasmas were found in Cuban cultivars whereas one sugarcane cultivar from Egypt contains grassy shoot phytoplasma that is a member in rice yellow dwarf group, but the other two Egyptian ones were phytoplasma-free. Syrian sugarcane was infected by phytoplasma that identified preliminary in rice yellow dwarf group. To our knowledge, this is the first report for the detection and identification of phytoplasma in sugarcane plants from Hawaii, Egypt and Syria. Our investigation on Thai sugarcane plants was in agreement with previous literature where sugarcane white leaf (SCWL) phytoplasma is associated with white leaf disease (Nakashima et al., 1994; Wongkaew et al., 1997). Q-PCR (real-time PCR) offers the opportunity to detect the phytoplasma in a sensitive, specific and quick manner, but that is not true for sugarcane plants with a very low titer of phytoplasma. Therefore, nested-PCR is better than qPCR for low titer phytoplasma detection and that is true for sugarcane yellow leaf phytoplasma disease. A BLAST search for the 16S rRNA gene sequences reported in this study showed that they shared 99 to 100% sequence identity with those of other phytoplasmas in the Aster yellows, X-disease and Rice yellow dwarf groups. However, no one of these identified strains can be described as a new “Candidatus Phytoplasma species”. On the other hand, Hawaiian sugarcane cultivar H787750 as a representative of Hawaiian breeding station sugarcane contains phytoplasma clustered to strain sugarcane white leaf (SCWL) phytoplasma, closely together with sugarcane Summary 126 white leaf phytoplasma from Taiwan (AY139874). It is possible to explain the occurrence of (SCWL) phytoplasma in Hawaiian Islands, by insect vectors or by infected stem cuttings which were obtained from other countries. Thai sugarcane contains phytoplasma isolate closely together with sugarcane white leaf phytoplasma from Myanmar. The transmission electron microscopic (TEM) studies revealed the presence of sugarcane white leaf phytoplasma only in phloem sieve tubes of diseased sugarcane leaves but not in adjacent cells to the sieve elements including companion cells and phloem parenchyma as well. According to ultrastructural observations under TEM, parenchymatic cells of bundle sheath and mesophyll tissue of affected leaves showed some alterations including accumulations of starch granules, increase plastoglobuli number and size in disorganized chloroplasts. Insect vectors of phytoplasmas are phloem feeders. Thus far, none of aphid species has been found to be a vector of phytoplasmas. Our tests showed also that black sugarcane aphids (Melanaphis Sacchari) were unable to transmit the phytoplasmas from infected sugarcane into the phytoplasma-free one. Hot water treatment has been proposed to cure plant material from phytoplasmas. Our tests showed that the appropriate hot water treatment, which recommended for phytoplasma elimination, is immersion of the sugarcane stem cuttings at 50°C for 60 min. Zusammenfassung 127 6. Zusammenfassung Das Yellow Leaf Syndrom (YLS) bei Zuckerrohr wurde zuerst in Plantagen Hawaiis entdeckt und von dort beschrieben. Das Polerovirus Sugarcane Yellow Leaf Virus konnte als verursachendes Agens des Syndroms identifiziert werden, jedoch gab es keinen strikten Zusammenhang zwischen der Intensität der Symptome und dem Virustiter. Deshalb wurden Analysen an Zuckerrohrpflanzen aus der hawaiianischen Zuchtstation durchgeführt, um die Pflanzen auf Sugarcane yellow leaf phytoplasma (SCYLP) zu testen, einem Bakterium, das ebenfalls als möglicher Auslöser von YLS vermutet wurde. Zwei Typen von Phytoplasma wurden in den hawaiianischen Zuckerrohrkultivaren entdeckt, nämlich Sugarcane White Leaf Phytoplasma (SCWL), ein Stamm der Rice Yellow Dwarf Gruppe, und ein Stamm der Aster Yellows Gruppe. Dies galt auch für Zuckerrohrpflanzen aus hawaiianischen Plantagen, obwohl bei diesen routinemäßig eine Heißwasser-Behandlung ihrer Setzlinge, welche Phytoplasma eliminieren könnte, durchgeführt wird. Proben von Zuckerrohrpflanzen anderer Länder (Kuba, Ägypten, Syrien und Thailand), in denen Pflanzen mit Vergilbungsoder Bleichungssymptomen festgestellt werden, konnten ebenfalls getestet. Aster Yellows und X-Disease Phytoplasmen fand man in kubanischen Kultivaren, während ein ägyptisches Kultivar Grassy Shoot Phytoplasma (ebenfalls ein Stamm der Rice Yellow Dwarf Gruppe) enthielt. Zwei andere Kultivare aus Ägypten waren phytoplasmafrei. Auch das syrische Zuckerrohr war von einem Phytoplasma der Rice Yellow Dwarf Gruppe infiziert. Unseres Wissens sind das die ersten Nachweise von Phytoplasma in Zuckerrohr aus Hawaii, Ägypten und Syrien. Die Analysen an thailändischen Pflanzen bestätigten publizierte Ergebnisse, dass mit Sugarcane White Leaf (SCWL) Phytoplasma infizierte Pflanzen mit White Leaf Disease in Zusammenhang stehen (Nakashima et al., 1994; Wongkaew et al., 1997). Q-PCR (real-time PCR) gilt als empfindliche, spezifische und rasche Methode um Phytoplasma in Pflanzenmaterial zu messen, dies erwies sich aber offensichtlich nicht für Zuckerrohr mit niedrigem Phytoplasma-Titer. Deshalb wurde nested-PCR als die sensitivere Methode, um Phytoplasma-Infektion niedrigen Titers bei Zuckerrohr festzustellen, angewandt. Ein BLAST-search zeigte, dass die 16S rRNA der gefundenen PhytoplasmaStämme 99-100% Sequenzidentität mit Phytoplasmen der Aster Yellows, X-Disease und Rice Yellow Dwarf Gruppen aufweisen, sodass keiner davon als neue “Candidatus phytoplasma Zusammenfassung 128 Art” beschrieben werden kann. Das Phytoplasma aus dem kommerziellen hawaiianischen Kultivar H78-7750 gruppierte sich in Sugarcane White Leaf Phytoplasma (SCWLP) ein, zusammen mit einem Stamm aus Taiwan. Es erscheint also möglich, dass über Insekten als Vektoren oder infizierte Setzlinge Phytoplasma aus Taiwan nach Hawaii kam oder umgekehrt. Das thailändische Phytoplasma steht am nächsten dem White Leaf Phytoplasma aus Myanmar. Gewebeschnitte im Transmissions-Elektronenmikroskop (TEM) zeigten, dass Phytoplasma ausschließlich in den Siebröhren der Leitbündel zu finden ist, nicht in Geleitzellen, Phloemparenchym oder anderen Blattzellen. Die normalerweise grünen Gewebe der infizierten Blätter (Bündelscheide und Mesophyll) zeigten starke zytologische Veränderungen wie Akkumulation von Stärkekörnern, eine große Anzahl von Plastoglobuli und desorganisierte Strukturen in Chloroplasten. Vektoren für Phytoplasma sind Phloemsauger, jedoch wurde bisher keine Blattlaus als Vektor nachgewiesen. Es konnte gezeigt werden, dass die schwarze Zuckerrohrlaus Melanaphis sacchari, die der wichtigste Vektor für Sugarcane Yellow Leaf Virus ist, Phytoplasma nicht übertragen kann. Heißwasser-Behandlung war als Methode zum Abtöten von Phytoplasma in Pflanzenteilen beschrieben worden. Dies konnte bestätigt werden und eine 60-minütige Behandlung in 50° heißem Wasser kann für die Eliminierung von Phytoplasma in Zuckerrohrsetzlingen empfohlen werden. Acknowledgement 129 7. Acknowledgement First of all, I would like to acknowledge my supervisor Prof. Dr. Ewald Komor, who gave me the acceptance to come to University of Bayreuth and to work in his lab. Thanks to all people in my department (plant physiology) for their help, especially Prof. Dr. Stephan Clemens, Christiane Meinen and Ursula Ferrera. I would like to thank Dr. Eric Hummel, Daniel Souza, Christian Seybold, Philipp Gasch and Thomas Liebenstein; they are not only colleagues but nice friends who kindly helped me with all kinds of situations. I appreciate the assistance from Dr. Alfons Weig to do phylogenetic analysis. I am grateful to Rita Grotjahn (technician in the laboratory of electron microscopy) for preparation of all ultrathin sections. This is the right place to thank Prof. Dr. A. 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Zhu, Y. J., Lim, T. S., Schenck, S., Arcinas, A., Komor, E. (2010). RT-PCR and quantitative real-time RT-PCR detection of Sugarcane Yellow Leaf Virus (SCYLV) in symptomatic and asymptomatic plants of Hawaiian sugarcane cultivars and the correlation of SCYLV titre to yield. European Journal of Plant Pathology 127, 263-273. List of figures 145 9. List of figures 1. Introduction 1 Figure.1.1. Comparison of sizes of some eubacteria. 3 Figure.1.2. Pleomorphic phytoplasmas in sieve tubes. 4 Figure.1.3. Phytoplasmas and their diseases are worldwide. 6 Figure.1.4. Host cycle of phytoplasmas. 7 Figure.1.5. Phytoplasmas are firmicutes. 8 Figure.1.6. Diagram of the longitudinal view of phloem cells. 11 Figure.1.7. Sugarcane yellow leaf syndrome (YLS). 12 Figure.1.8. Sugarcane white leaf (SCWL). 13 2. Material and Methods 17 Figure.2.1. A diagram illustrating of the method of nested PCR. 25 Figure.2.2. Diagrammatic representation of location of used primer pairs and expected size of their amplified products based on phytoplasma rRNA operon. 29 Figure.2.3. Diagrammatic representation of a phytoplasma rRNA operon and genomic location of primers used for phytoplasma detection. 29 Figure.2.4. Diagram illustrating of SYBR Green during PCR amplification. 35 Figure.2.5. Diagram illustrating of TaqMan probe chemistry mechanism. 36 Figure.2.6. Diagrammatic representation of genomic location of qPCR primers and probe used for phytoplasma detection. 37 3. Results 39 Figure.3.1. Nested PCR-products of positive and negative controls and of a positive control, which was mixed with increasing amounts of sugarcane DNA. 40 Figure.3.2. Phytoplasma in Hawaiian and Cuban sugarcane cultivars. 43 Figure.3.3. Phytoplasma in Egyptian and Syrian sugarcane cultivars. 43 Figure.3.4. Restriction fragment analysis of PCR products from Hawaiian and Cuban sugarcane cultivars containing phytoplasma. 45 Figure.3.5. Nested-PCR assay (II) products (1.2kb) amplified with primers (SN910601/P6, R16F2n/R16R2). 47 Figure.3.6. RFLP profiles of nested-PCR assay (II) products. 48 Figure.3.7. Nested-PCR assay (III) products (0.2kb) amplified with primer pair (MLO-X/MLO-Y, P1/P2). 49 Figure.3.8. Nested-PCR assay (IV) products. 50