acl Bacteriology CZanedia ple xqqziara WANA, arg FeeeN- 110012 ren 1CAR Wrarefviartts Arent gen Pee er) Professor H.S. Gaur ‘Dean & J. Deretor (Edn) FOREWORD Tam happy to know that Dr. Kalyan K. Mondal, Senior Scientist, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi has authored a textbook entitled "Plant Bacteriology". I appreciate Dr. Mondal's effort to bring this compilation in the form of a textbook, which is very much timely and in need for the students studying Agriculture in general and Plant Pathology, in particular. He has covered all important syllabus of the course Plant Bacteriology that is being taught at the Agricultural Universities as well as in Institutes. He has described all the chapters including history, classification, morphology, cell wall chemistry, flagellation, plasmids, growth, survival symptoms and management of important bacterial diseases in a very simplified and understandable manner with appropriate illustrations. T hope this textbook will be very useful for students, teachers as well as researchers studying in Plant Pathology and also serve as reference book. Dated : 15 Jan’ 2011 New Delhi H. S. Gaur Tel. 91-11-25733382 (0) ‘Telegram: KRISHIPUSA. Fax : 91-11-25846420 91-11-25843867 (R) E-mail: hsg_nema@iari.res.inPRE CE In India, the growth of Plant Bacteriology is yet to receive its momentun compared to the other sub-disciplines in Plant Pathology though a number o” crop diseases of bacterial nature have posed serious threat to Indian farmers Moreover, the subject merely kept its popularity among graduate and post graduate students in Agricultural Sciences. One of the reasons to describe this fact that there are very few text books on plant bacteriology whict aimed at to introduce the subject in a more fascinating and systematic way to the students of graduate and post-graduate as well as to the academics, researchers working on plant bacteriology. The book “Plant Bacteriology" is an attempt in this endeavour, The main idea of writing this book is to provide solid foundation with requisite and latest information on the subject in the form of textbook. The book covers the contents of the introductory course Plant Bacteriology that is being taught in all Sate Agricultural Universities and in other organizations. The recent advancement in areas like taxonomy and classification, bacterial secretion system, new emerging diseases has been included. The morphological and structural details of bacterial cell are discussed with the help of schematic diagrams. Special emphasis is given in the chapters including bacterial genetics, plasmids. Few important bacterial diseases are described for ready reference purpose. I hope that this textbook will help students, teachers while studying or teaching the course Bacteriology and will help to generate interest to the subject plant bacteriology. My sincere thanks to Professor H.S. Gaur, Dean and Jt director (Education), Indian Agricultural Research Institute, New Delhi, Professor Anupam Varma, former, Former National Professor, Sr. Scientist, INSA, Dr. RK. Jain, Head, Division of Plant Pathology, Dr. Pratibha Sharma, Professor, Division of Plant Pathology, TART, New Delhi and to all my colleagues for their valuable guidance and inspiration. I pay my humble gratitude to my parents and family for their consent support during writing this book, 15% Jan’ 2011 New Delhi Kalyan K. Mondal. Introduction . History of Bacteriology . Morphology and General Characteristics of Phytopathogenic Bacteria Disease Symptoms Induced by Plant Pathogenic Bacteria Authority and Type Species of Plant Associated Bacterial Genera Structure of Bacteria Flagellar Arrangement, Structure and Movement In Bacteria Classification of Bacteria . Entry, Dissemination and Survival of Bacterial Plant Pathogens |. Growth, Reproduction and Nutrition of Bacteria . Antibiotics and Their Mode of Action . Bacterial Genetics-Chromosome, Plasmid, Replication and Recombination and Variability }. Secretion Systems In Bacteria }. An Introduction to Ricketsia, Mycoplasma, L-form bacteria and Bdellovibrio 102—104 . Bacteriophages: Structure, Life cycle and Uses . Bacterial Spores: Types, Structure and Formation of Endospore |. Important Bacterial Diseases and Their Management Approved List of Plant Pathogenic Bacteria . Bacterial Isolation , Inoculation , Preservation and Culture Media ). Bacterial Culture Collection Centres . List of Journals and e-Resources . Bacterial Genome Structure, Size and Sequence Status Suggestive Objective Type Questions Ls 1-3 430 31-33 34—40-—~ 41437 44—5 55—59 60—69 7018 7983 84—86 87—95 96—101 105—108 109—113 114—124 125—172 173—178 179—179 180—181 182—190cin Chapter Introduction The bacteria are single-celled prokaryotic microorganisms. In prokaryotes the genetic material (DNA) is not delineated by any nuclear membrane and exists freely in the cytoplasm unlike eukaryotes, such as fungi, protista, plants and animals. Prokaryotic cell lacks mitochondria, chloroplast, while the ribosomes are small. However, photosynthetic bacteria contain bacterio- chlorophylls in the cytoplasm or in so called thylakoid membranes. The cell wall of bacteria contains peptidoglycan but never chitin or cellulose. The term eubacteria (eu = true) refers to the true bacterial cell having totally prokaryotic cellular configuration. This terminology is mostly used to differentiate the bacteria (= eubacteria) from archaeabacteria, In archaeabacteria (archaea = false) the cellular configuration though match with prokaryotes but they are most closely related to eukaryotes. The cell walls.of Archaea. donot contain peptidoglycan but pseudopeptidoglycan, polysaccharide, protein or glucoprotein. ‘The characteristic features of bacteria, archaea, and eukaryotes are discussed in tabular form (Table 1). : : % The discipline of biology related to the study of bacteria is called bacteriology. The study which specifically deals with the bacteria associated with plantis termed as plant bacteriology. Thus, plant bacteriology includes the study of both pathogenic as well as beneficial bacteria associated with the plant host. The plant bacteriology is now well established as a sub disciplinary branch of Plant Pathology.Introduction Table 1. The characteristics of bacteria, archaea and eukaryote. Property Living domains Cellular configuration Nuclear membrane Number of chromosomes, DNA covalently closed,,. °| circular Histone proteins present: | Peptidoglycan incell wall ‘Membrane lipids Ribosome size Mitochondria Chloroplasts Cell membrane sterols Cytoplasmic streaming Meiosis and mitosis Amino acid initiating protein synthesis ‘Transcription and translation coupled Protein synthesis inhibited by diphtheria toxin Sensitivity to streptomycin, leanamycin & chloramphenicol Bacteria prokaryotic absent present cster-linked glycerides; unbranched; saturated or monounsaturated 70S absent absent absent absent. Formyl methionin Archaea prokaryotic absent 1 + Fs absent ether-linked branched; saturated 70S absent absent absent absent Methionine Eukaryote eukaryotic present. >l ~ (linear) a absent ether-linked glycerides; unbranched; polyunsaturated 80S (cytoplasmic) present present present + present. Methioninevant Bacteriology The Phylogenetic Relationship of Bacteria with Other Living Domains The three major domains of living organisms include archaea, bacteria, and eukarya. The comparative sequencing data of 16S or 18S RNA revealed the relationship of the three domains. Archaea are apparently more closely related to\cukarya than are the Bacteria. Eukarya consist ofall eukaryotic cell-types, including protista, fungi, plants and animals. Size of Bacteria A typical bacterial cell is about 0.2 -1.5 xm in diameter to 3.0-5.0 um in length. Prokaryotic cells have a much lower volume of cytoplasm and a much higher surface: volume ratio than eukaryotic cells. A typical prokaryotic cell is about the size of a eukaryotic mitochondrion. Thus prokaryotes can only be seen with the aid of a microscope.Pall AN Chapter: History of Bacteriology The historical development is discussed with the pioneering works contributed by eminent researchers in both basic as well as advanced studies in the field of microbiology, general bacteriology and plant bacteriology. Further, an update on recent research advancements in laboratories working on plant associated bacteria is also provided. For reader’s convenience the historical landmarks that provide strong foundation to the subject Plant Bacteriology are divided into six major heads, namely A. Bacterial Etiology ‘Taxonomy, Evolutionary and Systematic Bacteriology . Bacterial Cellular, Genomic and Biochemical Profiling . Bacteriological Stains, Media, Techniques Bacterial Virulence and Molecular Pathogenesis, Plant Growth-Promoting Rhizobacteria (PGPRs) and Biological Control A. Bacterial Etiology Antoni van Leeuwenhock (1632-1723): A Dutch merchant and microscopist, who was the firstto see bacteria in 1683, He observed them in dental plaque. The morphological detail of ihe bacteria was evident from his letter to the British Royal Society dated on 17 September 1683. Louis. Pasteur (1822-1895): A Frenchman who conclusively demonstrated that fermentati lage are the. vity and therel roved the spontaneous generation theory. He is regarded as father of modem bacteriology. Later, he developed swan- poop me SaPlant Bacteriology S neck flask, through which air can pass freely to the flask but not the microbes present in the air. The infusion in the flask was demonstrated to be free of germs. Pasteur published his work in 1861 in the name as Memoir on the Organized Bodies which Exists in the Atmosphere.” Heinrich Hermann Robert Koch (1843-1910): A German physician who is credited for the establishment of etiology of Bacillus anthracis (in 1877), Mycobacterium tuberculosis, (in 1882), and Vibrio cholerae (in 1883). He was awarded the Nobel Prize in physiology or medicine for his tuberculosis findings in 1905. He first used gelatin as liquefiable solid media for bacteriological growth. He developed several important bacteriological techniques including pure culture techniques which have lot of impact in studying bacteria. He is regarded a as father of bacteriological techniques. He developed the well-defined criteria for a microorganism to be called as pathogenic to a given host. These criteria are known as Koch’s Postulates, which has become a universal rule to prove the pathogenecity related study of any microorganisms. The criteria are : 1, The microorganism must be closely associated with the diseased host/lesions. 2. The microorganism must be isolated in pure culture from the diseased host. 3, The microorganism should produce specific diseased symptom when artificially inoculated into a healthy susceptible host. 4, The microorganism must be re-isolated in pure culture from the artificially inoculated host. M. S. Woronin (1838-1903); In 1866, Dr. Woronin was the first who conclusively demonstrated the association of root nodule bacteria with legume plants. Earlier in 1958, Dr. W. B, Leismaan also showed that root nodules in legumes were formed by a specific group of bacteria. M. W. Beijerinck (1888): Dr. Beijerinck from Netherland was the first to isolate root nodule bacteria in pure culture from nodules in legumes and named them as Bacillus radicola. Dr. Beijerinck is considered as father of “Microbial ecology”. Thomas Jonathan Burril (1839-1916): Professor Burril was born at Pittsfield, Mass, on April 25, 1839. He was educated at the Illinois State Normal School, and he was the faculty of University of Illinois. He was always a teacher and a good one. He was first to report (in 1878) bacterial etiology of a plant disease. In 1878, he reported that the fire blight of pear.is caused by Envinia amylovora. He had been president of the Society of American Bacteriologists at the time of his death. Erwin Frink Smith (1854-1927): He was born in the little village of gilberts Mills, New York, on January 21, 1854. He worked on eight bacterial diseases, namely cucurbit wilt, brown rot of potato, black rot of cabbage, bacteriosis of bean, yellows of hyacinth, pear blight, soft rot of hyacinth, and olive tuberculosis. In 1898, Professor Smith disproved the claim of Alfred Fischer, a Botany professor at the University of Leipzig, that “no single example is yet known of bacteria which can insinuate themselves into the closed living cells of a plant”. Professor Smith’s paper in this context was published in 1899 in the prestigious journal Centralblait fur Bacteriologie: History of Bacteriology with the title “Are there Bacterial Diseases of Plants?”. In 1890, He showed that crown gall is a case coused by bacteria, He was fascinated by the etiology and control of the disease, as fell as its morpholos ith considered it to be similar to cancerous tumors of humans and animals. In 1913, he received a certificate of honor from the American Medical Association for his work on “Cancer in Plants. In 1903, Professor Smith authored a very illustrious monograph entitled “Bacteria in Relation to Plant Diseases”, Lorenz Hiltner : Dr. Hiltner from Germany coined the term “Rhizosphere” in 1904 to denote that region of soil which is subjected to the influence of plant roots. Mertin B, Waite : Professor Martin, colleague of Professor Erwin F. Smith, was the first to prove that the infection of floral nectarines by bees and other insects in pear blight due to Bacillus amylovora. Makanji Kalyanji Patel (1899-1967) : Professor M.K. Patel laid the foundation of phytobacteriology in India. He established a school of Plant Bacteriologyat College of Agriculture, Pune and first described a ew species Xanthomonas campestris py. uppali in 1948, from the host Jpomea muricota. He first documented that ‘black spot of mango is caused by Pseudomonas ine indicae, however, later the pathogen was redefined ‘Xanthomonas. campestris spv. mangiferaeindicae. He described more than 30 bacterial diseases from India including Xanthomonas desmodii causing bacterial leaf-spot of Desmodium diffusum DC (in 1949), bacterial leaf-spot on Vernonia cinerea Less, (in1968), bacterial leaf-spot of Cyamopsis tetragonoloba (L.) Taub.(in 1953), bacterial leaf spot on Vitis woodrowii Stapf. (in 1951), Xanthomonas badrii sp. nov., on Xanthium strumarium L, (in 1950), Xanthomonas desmodii-gangeticii, sp. nov., Uppal, Patel and Moniz; causing bacterial leaf-spot of Amaranthus viridis (1952), bacterial leaf spot of Pedalium murex L. caused by a new albino species of Xanthomonas (in 1972). M. K, Hingorani : He reported about the complex nature of tundu disease of wheat caused by a bacterium, Cornybacterium tritici (=Rathayibacter tritici) and a nematode, Anguina tritici in 1952 and also he confirmed the causal agent of ring disease of potato as. Pseudomonas (=Ralstonia) solanacerarum. In 1959, Professor Hingorani with his co-worker Proferssor N.J. Singh first reported Xanthomonas punicae sp. nov. on Punica granatum L. causing bacterial blight of pomegranate, M.C. Srinivasan : Professor Srinivasan with his collaborators (MJ Thirumalachar, MK Patel), in 1959, first recorded the occurrence of rice bacterial blight in India from Maharashtra. B. Taxonomy, Evolutionary and Systematic Bacteriology Ernst Haeckel (1834-1919) : a German, first proposed the term Protista to group unicellular organisms, In 1866, he Professor Haekel gave the three kingdoms concept viz. Protista, Plantae, Animalia (Table 2), Edouard Chatton (1883-1947) : In 1937, Professor Chatton grouped the living domains into two major empires, namely prokaryotes and the eukaryotes (Table 2). ————————EeESPlant Bacteriology Table 2.Grouping of bacteria as pI ‘Taxonomists | Groupings Linnaeus 2 Kingdoms (1735) Haeckel 3 kingdoms (1866) Chatton 2 empires (1937) Copeland ‘4kingdoms | Monera Protista [Plantae | Animalia (1956) Whittaker . Skingdoms | Monera [Protista] Fungi Plantae | Animalia (1969) Woeseet al. 6kingdoms | Eubact] Arc|Protista] Fungi] Plantae | Animalia 977) ceria | hae bact| eria Woese etal, | 3domains | Bact | Arc Eukarya (1990) eria_|haea| David Henricks Bergey (1860-1937): Dr. David Hendricks Bergey was an American bacteriologist. He was born in the state of Pennsylvania where he remained his entire life. In his early years, Bergey was a school teacher he taught in schools of Montgomery Country. He left this occupation to attend the University of Pennsylvania, where he obtained his both Bachelor of Sciences and Doctor of Medicine degrees in 1884. He practiced medicine until 1893. He was the first doctor to isolate a bacterium called Actinomyces from a human being in 1907. He then joined as Thomas A. Scott fellow of the university’s hygiene laboratory, where he taught hygiene and bacteriology. He led the laboratory from 1929 until his retirement in 1932, Dr. Bergey was recognized for his prolific and varied researches in the areas of tuberculosis, food preservatives, phagocytosis, and anaphylaxis, He determined the important characteristics to differentiate and identify the organisms of class Schizomycetes. From 1932 until his death in 1937 he remained as director of biological research at the National Drug Company in Philadelphia. In 1923, he and four other bacteriologists “published the first edition of Bergey's Manual of Determinative Bacteriology, a fundamentally _ important reference book for the identification and classification of bacteria. The first three editions of the Manual were published by the American Society for Microbiology. Subsequent editions (fourth to eighth) were published by Bergey’s Manual Trust based at the University of Georgia in Athens, GA, USA. The 9th edition of the manual is replaced by the new title Bergey Manual of Systematic Bacteriology. The 1* edition of this new title published by Williams & Wilkins, Baltimore, MD in 4 volumes [in 1984 (Vol-1), in 1986 (Vol-2) and in1989 (Vol-3 and 4)).Q History of Bacteriology ‘However, the second edition of the new title is published by Springer, New York in five volumes {in2001 (Vol-1), in 2005(Vol-2), in 2009 (Vol-3, and Vol-4) and in 2010 (Vol-5)]. Roger Y. Stanier (1916-1982): Professor Stanier considered prokaryotes as lower protests (blue green algae, myxobacteria and eubacteria) and eukaryotes as higher protests (algae, fungi, protozoa). Robert Harding Whittaker (1920-1980): Professor Whittaker, an American vegetation ecologist, who proposed five kingdom systems for classification of living organisms. Whittaker’s system (1969) includes Animalia, Plantae, Fungi, Protista and Monera (Prokaryotes). D.W. Dye: Professor Dye, a noted plant bacteriologist, established the etiology of many important bacterial diseases. He first reported (in 1958) the host specificity in Xanthomomas. Professor Dyc in collaboration with other internationally acclaimed scientists (J.F. Bradbury, M. Goto, A.C. Hayward, R.A. Lelliott, M.N. Schroth), in 1980, first proposed the “pathovar” concept and postulated the international standards for naming pathovars of phytopathogenic, bacteria. W.J. Dowson: Professor Dowson is the first who coined (in 1939) the generic name of plant pathogenic bacterium, Xanthomonas. He was the first to outline systematic position of Gram positive and Gram negative bacterial pathogens of crop plants. He described phytopathogenic bacteria in his book “Plant Diseases Due to Bacteria” published in 1957 by Cambridge Univ. Press. WH. Burkholder: Professor Burkholder’s contribution in the taxonomy and nomenclature of phytopathogenic bacteria is well recognized. In 1930, Professor Burkholder, proposed the genus Phytomonas(=Burkholderia). Professor Burkholder enumerated the importance of bacteria as plant pathogens in many scientific articles including a review entitled “Bacteria as Plant Pathogens” published in Annual Review of Microbiology (1948, Vol 2: 389-412). Mortimer P. Starr (....... to 1989): Professor Starr, was a prolific and widely respected plant bacteriologist. He worked at the Department of Bacteriology, University of California Davis, California. His group in 1977 was the first to establish the chemotaxonomic significance of the Xanthomonadins, novel brominated ary-polyene pigments produced by the genus Xanthomonas. Professor Starr was also an esteemed author and editor. His research contributions and valuable concepts on the bacterial plant pathogens including Xanthomonas, Enwinia are published in revered scientific journals, reviews. In 1983, he and his colleagues edited a very useful hand book “The Prokaryotes: A Handbook on Habitats, Isolations and I Identification of Bacteria”, published by Springer-Verlag. He nicely described the important events leading to the development of Phytobacteriology in a review article “Landmarks in Developments of Phytobacteriology” published in Annual Review of Phytopathology (In 1984, Vol. 22, 169-188). He was the founder (in collaboration with publisher Konrad F. Springer) ofa yery popular scientific journal in Microbiology “Current Microbiology” in 1978. He served a founder editor-in-chief of this international journal until his retirement on 1982. Anne K. Vidaver: Professor Vidaver, an illustrious women plant bacteriologist, held several important academic as well as administrative positions in United States. She was the first women. to head a Department of Plant Pathology, University of Nebraska-Lincoln (UNL) and there she was asked to be Director for the Centre for Biotechnology at UNL, from 1988-1989, and 1997- 2000, Stic became first woman Chief Scientist of USDA's National Research Initiative Competitive eePlant Bacteriology 9 Grants Programme, from 2000-2002. During that she initiated a programme on microbial sequencing between USDA and the National Science Foundation. She was also appointed chair of Interagency Working Group (IWG), a Microbe Project, to coordinate the sequencing, functional genomics, and bioinformatics investigations of the 12 agencies involved. In the beginning of her carrier, she took interest to examine the potential for biocontrol of bacterial diseases, using bacteriophages and bacteriocins. Later, at Nebraska, she characterized purple pigment producing bean wilt bacterium, now known as Curtobacterium flaccumfaciens. She also discovered another Gram positive bacterium, Clavibacter michiganensis subsp. nebraskensis causing Goss’ bacterial wilt and blight of com. Later she discovered Xanthomonas campestris py. asclepiadis, which causes a blight of milk weed, Asclepias syriaca. She also unraveled genomic and biological complexity of plant pathogenic and clinical strains in the taxon Burkholderia cepacia. Besides pathogens, her group also found beneficial bacteria associated with plants, including Bradyrhizobium japonicum from soybean, Microbacterium testaceum from maize (endopyte). She first characterized a dsRNA bacteriophage, 09. Her group found a plasmid in Curtobacterium ‘flaccumfaciens pv. oortii, a tulip pathogen, that confers resistance to metals like arsenite, arsenate and antimony. She became Secretary of the American Phytopathological Society (APS) before being nominated to be President of APS. She was the second woman president (1986-1987) after Helen Hart (in 1956). Norberto J. Palleroni: Professor Palleroni, is a noted Microbiologist, worked at Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey, USA. He is recognized for his work on taxonomy, nomenclature, biological and ecological aspects of Pseudomonas. He wrote a substantial number of reviews on the genus Pseudomonas, including a very good editorial review in 2010 entitled “The Pseudomonas Story” in the journal Environmental Microbiology (12: 1377-1383). He also described important biological and ecological properties of Pseudomonas stutzeri, a nonfluorescent denitrifying bacterium widely distributed in the environment. Maso Goto: Professor Goto from Japan is a well-known plant bacteriologist who worked on bacterial blight of rice and bacterial leaf streak of rice. Professor Goto authored a very popular text book entitled “Fundamentals of Bacterial Plant Pathology” published by Academic Press. This book illustrated the current information on bacterial morphology, taxonomy, genetics, host-pathogen interaction, diagnosis of bacterial diseases and ecology. C. Elliot: Professor Elliot characterized the genus Phytomonas. In 1930, Professor Elliot wrote a very illustrative manual entitled “Manual of Bacterial Plant Pathogens, published by Williams and Wilkins, Baltimore (349 pp.). Professor Elliot’s reviewed the status and developments of phytopathogenic bacterial classification in many literatures. Milton N. Schroth: Professor Schroth, at Department of Plant Pathology, University of California, Berkeley, California, is a noted plant bacteriologist, working on both plant pathogenic as well as beneficial plant associated bacteria, Professor Schroth (with Donald C. Hildebrand and Nickolas Panopoulos) céntributed a chapter on Phytopathogenic Pseudomonads and Related Plant-Associated Pseudomonads in the book The Prokaryotes 2006, part 3, section 3.3, 714-740, Springer. Jhon Young: Professor Young, at Department of Plant Pathology, University of Georgia, Millar Plant Sciences Building, Athens is well-known for his work on bacterial taxonomy of different phytopathogenic genera including Acidovorax, Agrobacterium, Burkholderia,10 History of Bacteriology Clavibacter, Curtobacterium, Erwinia, Herbaspirillum, Pseudomonas, Rhodococcus, and Xanthomonas that infects crops like Stone-fruit, Kiwifruit, Cucurbits. According to Professor ‘Young phylogenetic classification, which is based on ancestral relationships, is not an adequate method, His view was based on the species and genus concepts. The species concept states that a bacterial species is defined as a population whose strains share >70 % DNA-DNA hybridization. Thus, Professor Young felt Pantoea and Erwinia should not be considered as separate genus when they shares>96 % similarity. Eiko Yabuuchi to Feb’ 2008): Professor Yabuuchi was a Japanese and worked at Osaka City University, Osaka, Japan on bacterial etiology, biochemical profiling of many bacterial groups of clinical significance including Pseudomonas. She was associated with the International Committee on Systematics of Prokaryotes, Subcommittee for Pseudomonas and related organisms (1990-2006). Her life-long contributions to the field of systematic bacteriology were recognized by the award of the “‘Bergey Medal “in 2000 by Bergey's Manual Trust. In 1990, she proposed the genus name Sphingomonas, with it’stype member S. paucimobilis gen. nov. and other members including S. parapaucimobilis sp. nov., S. yanoikuyae sp. nov., S. adhaesiva sp. noy., S. capsulata comb. nov. She identified a novel sphingoglycolipid [chemically galacturonosyl-[(1—51)-ceramide] in Sphingomonas yanoikuyae. This type of sphingoglycolipid was not found in the type species of Sphingomonas, S. paucimobili, but detected in other members of Sphingomonas, suchas S. mali, S. terrae, and S, macrogoltabidus. She rearranged the Pseudomonas homology group II into Burkholderia gen. nov. with the type species Burkholderia cepacia (Palleroni & Holmes 1981) comb. nov. Later, in 1995, Her group suggested the transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov. [Ralstonia Pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb, nov. and Ralstonia eutropha (Davis 1969) comb. nov.]. She, in 1999, proposed the transfer of Rhizomonas suberifaciens to the genus Sphingomonas as S. suberifaciens.She and her associates conclusively established the etiology of brown spots on yellow Spanish melon fruits caused by a-novel pathogen Sphingomonas melonis sp. nov. Ericka Ralston: Professor E. Ralston is a famous plant bacteriologist, who worked in the Department of Bacteriology and Immunology, University of California, Berkeley, California, ‘USA. Inher honour, the plant pathogenic genus Ralstonia is named. She studied DNA homologies of the members of Hydrogenomonas” and found that Pseudomonas facilis(formerly Hydrogenomonas facilis) is closely related to the nonautotrophic species P. delafieldii. P. facilis and Alcaligenese utrophus (often called H. eutropha) are not related to each other or to other hydrogen bacteria and pseudomonads studied. Norman W, Schaad: Dr, Schad isan icon of plantbacteriologiy working at US Department of Agriculture, Agriculture Research Service, Forcign Disease-Weed Science, Research Unit, Ft. Detrick, Maryland. Dr. Schaad is credited with his work on taxonomic grouping based on DNA-DNA hybridization, PCR-based diagnostics of important bacterial plant pathogens. He is regarded as father of molecular taxonomy of plant pathogenic bacteria. He authored several books, scientific reviews, and laboratory manual including a very useful manual entitled “Laboratory Guide for Identification of Plant Pathogenic Bacteria”, Third Edition (eds, 2001) in association with J. B, Jones and W. Chun published by American Phytopathological Society Press [http://Plant Bacteriology it www.scisoc.org], St Paul, USA. He (with Dr. E. Schuenzel) critically reviewed an article on “Sensitive molecular diagnostic assays to mitigate the risks of asymptomatic bacterial diseases of plants”. L, Vauterin: Professor Vauterin is a an esteemed plant bacteriologist working at Microbiology Laboratory, University of Gent, Belgium. He and his co-worker’s comprehensive study on DNA- DNA hybridization of Xanthomonas led to the reclassification of the genus Xanthomonas. They grouped 183 strains of Xanthomonas into 20 DNA homology groups (=genomic species). Four groups corresponded to the previously described species Xanthomonas albilineans, X. fragariae, X. oryzae, and X. populi. However, they divided Xanthomonas campestris (considered to be heterogeneous species) into 16 DNA homology groups. One of these groups exhibited a high level of DNA homology with Xanthomonas axonopodis. The type species of the genus, X. campestris (Pammel 1895) Dowson 1939, is emended to include only the pathovars obtained from crucifets (i.e., X. campestris py. aberrans, pv. armoraciae, pv. barbareae, py. campestris, py. incanae, and py. raphani). X. axonopodis Starr and Garces1950 is emended to include 34 former X. campestris pathovars. They proposed new pathovars of Xanthomonas arboricolasp. nov., including pv. corylina, pv. juglandis, pv. poinsettiicola (type C strainsof the former X. campestris pathovar), pv. populi, and py. pruni; Xanthomonas bromi sp. nov. for strains isolated from bromegrass; Xanthomonas cassavae (ex Wicheand Dowson 1953) sp. nov., nom. rev.; Xanthomonas codiaei sp.nov., including type B strains of the former taxon X. campestris py. poinsettiicola; Xanthomonas cucurbitae (ex Bryan 1926) sp.nov., nom. re Xanthomonas hortorum sp. nov., including pv. hederae, py. pelargonii, and py. vitians; Xanthomonas hyacinthi (ex Wakker 1883) sp. nov,nom. rev; Xanthomonas melonis sp. nov.; Xanthomonas pisi (exGoto.and Okabe 1958) sp. nov., nom. rev.; Xanthomonas sacchari sp. nov. for strains isolated from diseased sugarcane in Guadeloupe; Xanthomonas theicola sp. nov.; Xanthomonas translucens (ex Jones,Johnson, and Reddy 1917) sp.nov., nom. rev,, including pv. arrhenatheri, pv. cerealis, pv. graminis, py. hordei, py. phlei, py. phleipratensis, pypoae, pv. secalis, pv. translucens, and py. undulosa; Xanthomonas vasicola sp. nov., including pv. holcicola and py. vasculorum (type B strains of the former taxon X. campestris py. vasculorum); and Xanthomonas vesicatoria (ex Doidge 1920) sp. nov., nom.rev., which includes the type B strains of the former taxon X. campestris pv. vesicatoria. They differentiated the new species on the basis of metabolic activity on a range of carbon substrates by using the BiologGN microplate system. Jeffrey B. Jones: Professor Jones, from Department of Plant Pathology, University of Florida, Gainesville, Florida, is a recognized plant bacteriologist working on taxonomy and evolutionary relationships among members of the genus Xanthomonas associated with tomato, pepper, citrus. Professor Jones first identified four phenotypic xanthomonad groups that are pathogenic to pepper, tomato, or both hosts. These include Xanthomonas axonopodis pv. vesicatoria (A and C group), Xanthomonas vesicatoria (Bgroup) and Xanthomonas gardneri (D group). Later, Prof. Jones’s group suggested that strain C should given a separate species status as strain C shares less than 70% DNA relatedness with strain A, with the type strain of X. axonopodis, and with the currently classified species within Xanthomonas axonopodis. The name X. perforans sp. nov is proposed for the C group of strains previously designated as X. axonopodis pv. vesicatoria (ATCC BAA-983" = NCPPB 4321"). They observed that A strains most closely resemble the strains originally isolated by Doidge in 1921 and to avoid confusion in12 History of Bacteriology Nomenclature A strains of X. axonopodis pv. vesicatoria be renamed as X. euvesicatoria (ATCC11633" = NCPPB2968" = ICMP 109" = ICMP 98"). They also proposed that Rardneri’, which has less than 70% DNA relatedness with any of the Xanthomonas species and which has never had taxonomic status, be named X. gardneri (ATCC 19865 = NCPPB 8817) to reflect the specific epithet proposed by Sutic in 1957. : Jacob Dirk Janse: Dr. Janse, is a noted plant bacteriologist, at Plant Protection Service, Wageningen, The Netherlands is working on taxonomy, nomenclature of bacterial plant pathogens. In 1982, Professor Janse reported Pseudomonas syringae subsp. savastanoi (ex Smith) subsp. ov., nom, rev., the bacterium causing excrescences on Oleaceae and Nerium oleander L. In Janse along with co-workers reclassified Pseudomonas syringae pv. avellanae snae (spec, nov.), the bacterium causing canker of hazelnut (Corylus several books in phytobacteriology including a very useful book ) entitled “Phytobacteriology: Principles and Practice”. In this book, ‘lity of complex bacteria, their characterization, identification se and how plants react to such disease. Professor filing techniques for bacterial pathogens in another 2 Tool for Classification of Pseudomonas (CABI Dean W. Gabriel: Dr. Gabriel, Professor of Plant Pathology, University of Florida, is a noted plant bacteriologist working on population structure, epidemiology, taxonomy, and detection of Xanthomonas. Professor Gabriel’s laboratory provided a detailed insight into the structure, fimetion and expression of cloned virulence genes of citrus canker, cotton blight and common bean blight bacteria. C. Bacterial Cellular, Genomic and Biochemical Profiling Hans Ris (1914-2004): Dr. Hans Ris was born in Bern, Switzerland, in 1914. He was professor of biology at the University of Wisconsin in Madison, USA. He coined the term genophore to describe the bacterial nucleus and chromosome. F, Jacob, S. Brenner and F. Cuzin (1963): Professor Jacob along with Professor Brenner and Cuzin proposed the rolling circle model for replication of bacterial chromosome. Oswald T. Avery (1877-1953): Professor Avery’s group in 1944 demonstrated that DNA is the genetic material of bacteria, M. Webb (1948): Professor Webb first showed conclusive evidence that the Gram positiveness in Gram +ve bacteria is lost when treated with lysozyme. W. Weidal, H. Frank, H.H. Martin (1960): Professor Weidal in collaboration with Professor Frank, and Professor Martin first reported the presence of murcin in bacterial cell wall. M. R. J, Salton (1963): Professor Salton first demonstrated that the higher concentration ofalcohol used in Gram staining is the key factor in achieving the Gram differentiation. Professor Salton monitored the release of *P-labelled compounds was more or less same at lower alcohol concentrations. But at 95% and above the release was much more in Gram —ve than Gram +ve bacteria, Alcohol has got two modes of actions, i.e, dehydrating and solvent action, In Gram-tve bacteria, where lipid contentis less and murein is multilayered, the alcohol as a dehydrating agent reduces the pore size resulting in low permeability of cell wall and trapping the primary stain. In ——_ —_—_——_ LTPlant Bacteriology 13 Gram —ve bacteria, where lipid (lipo-protein & lipopolysaccharides) content is high murein is monolayered, the alcohol through its solvent action dissolves the cell wall lipids and makes more porous. The resultant increase in permeability even after dehydration action of alcohol causes the leakage of primary stain. Selman Abraham Waksman (1888-1973): Professor Waksman, an American Scientist who in 1944 discovered the antibiotic streptomycin produced by the Actinomycete Streptomyces griseus. He received Nobel prize in 1952 for this contribution. J. F. Daniel: Professor Daniel, at IRD, Plant pathology Unit, Montpellier, France, studied the pathiogenic and genetic diversity of Xanthomonas campestris pathovar malvacearum, X. c. pv. manihotis infecting cotton, cassava and other xanthomonads especially on weeds. R. D. Fleischmann: Professor Fleischmann and his associates from Johns Hopkins University School of Medicine, Baltimore, MD, USA, the first in 1995 sequenced complete genome of a bacterial pathogen, Haemophilus influenza RAKW20. The complete genome of H. influenzae Rd KW20 is of 1,830,137 base pairs. A.J. Simpson: Professor Simpson from Brazil with other collaborators in 2000first sequenced the full genome of a bacterial plant pathogen, Xylella fastidiosa clone 9aSc, causing citrus variegated chlorosis, a serious disease of orange trees. The complete genome sequence of X. Jfastidiosa clone 9a5¢ is comprised of a 52.7% GC rich 2,679,305 bp circular chromosome and two plasmids of 51,158 bp and 1,285 bp. They also observed that at least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer. Christian A. Boucher: Professor Boucher from France along with collaborators (Professor Marcel Salanoubat, France) sequenced full genome of Ralstonia solanacearum strain GMI1000 in 2001. Professor Boucher used shotgun strategy to sequence complete genome. It’s genome is composed of two replicons of 3.6 and 2.1 megabase pair as estimated from pulsed gel electrophoresis. Ana CR da Silva: Dr. Ana CR da Silva from Alellyx Applied Genomics, Rua James Clerk Maxwell, Campinas, Brazil in collaboration with other scientists first sequenced full genome of two bacterial pathogens, Xanthomonas axonopodis py. citri str. 306 and Xanthomonas campestris py. campestris ATCC 33913 in 2002. B.M. Lee: Professor Lee at National Institute of Agricultural Biotechnology, Rural Development Administration Suwon, Korea, with other collaborators first sequenced full genome of Xanthomonas oryzae pathovar oryzae (Xoo) strain KACC10331, the bacterial blight pathogen ofrice. The full genome is comprised ofa single, 4,941,439 bp, circular chromosome that is GC tich (63.7%). The genome includes 4637 open reading frames of which 3340 (72.0%) could be assigned putative function. The genome analysis further revealed that 245 genes were found to be specific to Xoo while 80% of the predicted Xoo genes were found in the previously reported X.axonopodis py. citri and X. campestris py. campestris genomes. They suggested that the presence of Xoo specific genes may control the interactions of this pathogen with its gramineous host. Steven Salzberg : Dr. S. Salzberg from USA (at Center for Bioinformatics and Computational Biology, Biomolecular Sciences Bldg, University of Maryland, College Park, MD, USA) in collaboration with other scientists in 2006 first deposited the full genome sequence of14 History of Bacteriology Xanthomonmas oryzae py, oryzicola, the causal bacterium of leaf streak of rice. The full ‘genome is composed ofa sirtgle circular chromosome of 4.83 Mb size with 64.05 % GC content. V.P. Bhide: Professor Bhide is renowned for his contribution in plant bacteriology teaching and research in India, He was specially deputed to USA to receive training in general bacteriology. G. Rangaswami: Professor Rangaswami described the importance of phytobacterial diseases in India, He authored a book entitled “Bacterial Plant Diseases in India”, Asia Publishing House, Bombay 1. D.N. Srivastava (1925-2000): Professor Srivastava is mostly remembered for his tremendous contribution (\with his colleague Dr. ¥.P Rao) on bacterial blight of rice. GS. Shekhawat: Professor Shekhawat, a plant bacteriologist from India is acclaimed for hisresearch contribution on bacterial diseases of rice, and potato, He authored a book on “Bacterial Disease on Potato”. ¥.S. Kulkarni (1910-2008): Professor Kulkarni was one of the few microbiologists in India, who got world-wide acceptance for his ideas and concept. Professor Kulkarni worked on bacterial leaf spot of castor, stem canker of pigeon pea, leaf spot of grapes. Professor Kulkarni (in association with and S.G. Abhyankar) in 1950 documented a new bacterial leaf-spot and stem canker of pigeon pea. In 1951, He (along with Professor M.K Patel and G.W Dhande) reported a new bacterial disease of Cassia tora L. caused by Xanthomonas cassia. He developed a rapid method of protein profiling to distinguish closely related rhizobacterial strains associated with cowpea. He also first time demonstrated the biological control of bacterial wilt of potato using antagonistic microbes. PIN. Patel: Professor PNY Patel contributed (\with Prof. J.K. Jindal) in the field of bacterial diseases of legumes in India including common and fuscous blight of bean, bacterial pustules of soybean, bacterial blight of guar, bacterial leaf spot of green gram, halo blight of green gram, bacterial blight of cowpea, bacterial blight of moth bean, bacterial leaf spot of Dolichos lablab, bacterial leaf spot of urd bean, bacterial leaf spot of stem canker of pigeon pea. Professor Patel edited a well-illustrated book entitled “Plant Bacteriology” written into 3 volumes: Vol I dealt with “Bacterial Diseases of Plants in India”, Vol Il dealt with “Phytobacteriological Techniques and Demonstration” and Vol III dealt with “Bibliography of Plant Bacteriology in India”. This book provided detailed descriptions of important bacterial diseases occurring in India with pictures. Stevens MBrumbley: Professor Brumbley, a noted plant pathologist is working on molecular biology of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease, at Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, Brisbane, Australia. Heand his coworkers studied genetic uniformity and developed PCR-based detection technique for Leifsonia xyli subsp. xylt. Futher, his group is credited with the establishment of functional genomics for Leifsonia xyli subsp. xyli. He was also associated with the genome sequence programme for Leiftonia xyli subsp. xyli. Michael J. Davis: Professor Davisof Citrus Research and Education Center, University of Florida, Homestead, Florida, USA is known for contribution on bacterial pathogens, Clavibacter and Xylella. Bruce C. Kirkpatrick: Professor Kirkpatrick is recognized for his research on diseases caused by phytoplasmas and Xylella fastidiosa from Department of Plant Pathology, University of California at Davis, USA: His group developed and used molecular diagnostic assays to studyPlant Bacteriology 18 the etiology and epidemiology of important woody plant diseases including Pierce’s disease of grapevines. Professor Kirkpatrick's group has studied genetic diversity of pierce’s disease strain and other pathotypes of X. fastidiosa, and sequenced two phytoplasma plasmid genomes. His group demonstrated transposon mutagenesis in X. fastidiosaby electroporation of Tn5 synaptic complexes. Further, he and his coworkers identified avirulence genes in X. fastidiosa that contribute to biofilm maturation, colonization and attenuate virulence in X. fastidiosa. They also characterized a unique immune dominant membrane protein gene from the Western X-disease Phytoplasma. Y. Duan : Dr. Y, Duan at USDA, Fort Pierce, FL, USA and his collaborators first reported (in 2009) complete genome sequence of citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’, an uncultured alpha-proteobacteria that is both an intracellular plant pathogen and insect symbiont. This bacteriais spread by citrus psyllids and is phloem-limited. They sequenced complete circular genome by metagenomics, using the DNA extracted from a single ‘Ca. L. asiaticus’-infected psyllid. The full genome is of 1.23-Mb with a low 36.5% GC content. Their study revealed that ‘Ca. L. asiaticus? lacks type III and type IV secretion systems as well as typical free-living or plant-colonizing extracellular degradative enzymes. However, ‘Ca. L. asiaticus’ possesses type I secretion system genes needed for both multidrug efflux and toxin effector secretion. Anne M. Alvarez: Professor Anne M. Alvarez is an illustrious plant bacteriologist at the Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, USA. She worked on diverse bacterial genera including Acidovorax, Burkholderia, Clavibacter, Curtobacterium, Erwinia, Pseudomonas, Ralstonia, Xanthomona and Mycobacterium, with reference to the development of serological detection techniques, biochemical and molecular characterization. She also did study with an emerging bacterial blight disease of Anthurium caused by Xanthomonas axonopodis pv. dieffenbachiae. Professor Alvarez (with her collaborators) developed two monoclonal antibodies specific for lipopolysaccharide antigens of Xanthomonas campestris py. begoniae and py. pelargonii. Her group also determined specificity of monoclonal antibodies to strains of Dickeya sp. that cause bacterial heart rot of pineapple. In another study she with her coworkers characterized polycyclic aromatic hydrocarbon (widespread environmental contaminants)-degrading species of Mycobacterium from Hawaiian soils including M. crocinum, M. pallens, M. rutilum, M. rufum and M. aromaticivorans. She is currently the member of APS (American Phytopathological Society) foundation board. Kemal Benlioglu: Dr. Kemal Benlioglu, Professor at Adnan Menderes University, Faculty of Agriculture, Plant Protection Department, Aydin, Turkey is a noted plant bacteriologist working on bacterial diseases caused by Agrobacterium, Erwinia, Pseudomonas, Burkholderia, Clavibacter, Xanthomona affecting crops like tomato, potato, pome fruits with reference to biological control, serological and molecular detection techniques. Rosemary Loria: Professor Loria is the former chair of the Plant Pathology Department at Comell University. Her lab is recognized for research on the molecular genetic analysis of the Gram-positive plant pathogenic Streptomyces species. Cloning of highly conserved virulence gene nec/ by her team led to the discovery of a large, mobile pathogenicity island (PAI. Mobilization of the PAI was demonstrated in a newly emerged pathogen, S. turgidiscabies. Her team characterized the biosynthetic pathway for the phytotoxin, thaxtomin and discovered thatate History of Bacteriology niric oxide synthase is involved in nitration of this dipeptide. Dr. Loria’s lab is now actively involved in genomic and proteomic analysis of pathogenicity using 10 Mb S. scabies genome. Alan Jones: Professor Alan Jones at Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan is famous for research on Erwinia amylovora, which causes fire blight of plants in the Rosaceae family, In 2000, Professor Alan Jones’s team sequenced PEA29 plasmid genome from E. amylovora strain Fa88. The complete sequence of plasmid PEA29 consists of 28,185 bp with a 50.2% G+C content. They also observed that plasmid-cured strains exhibited reduced virulence and modified colony morphology on minimal medium without thiamine, indicating that some of the genes in pEA29 play a role in the physiology or metabolism of E. amylovora. Later, Professor Jones sequenced another two plasmids, pEU30 and pE160 from E. amylovora and found that the gene content of pEU30 resembled plasmids found in plant-associated bacteria, while that of pEL60 is most similar to INCL/M plasmids inhabiting enteric bacteria. Theo H. M. Smits: Dr. Smits (from Division of Plant Protection, Swiss National Competence Center for Fire Blight, Wadenswil, Switzerland) along with other collaborators, in 2010, first sequenced full genome of the fire blight pathogen Erwinia amylovora CFBP 1430 as well as Pantoea vagansstrain C9-1, commercially registered biological control agent for fire blight. The genome of Z. amylovora CFBP 1430 is composed of one circular chromosome of 3.8 Mb with 53.6%.GC. The 4,89-Mb total genome of P. vagans is composed of one circular chromosome of 4.03 Mb and three circular plasmids, pPagl, pPag2, and pPag3 of 0.17 Mb, 0.16Mb, and 0.53 Mb, respectively. D. Development of Bacteriological Stains, Media, Techniques Paul Ehrlich (1854-1915): Professor Paul Ehrlich was a German chemist who discovered the aniline dyes. Professor Ehrlich was fascinated by the reactions of cells and tissues to dyes. ‘Using aniline dyes, he investigated white blood cells. In the process, he developed new ways of staining cells for research, including the methylene blue stain for bacteria. His field of study was the effects of chemicals in the human body. He is regarded as the father of the fields of chemotherapy and hematology (the study of blood). He also made important contributions to the understanding of immunity and discovered Salvarsan, the first effective treatment for syphilis. Carl Weigert (1845-1904): Professor Carl Weigert was a German pathologist who introduced a number of bacteriological and tissue stains, and methods of staining eg., methyl violate. In 1871 he was the first to stain bacteria and was able to demonstrate the presence of bacteria in tissue sections. This advance was of the greatest importance for the subsequent work of Robert Koch. The problem of the selective action of dyes on biological materials (microchemical reactions), which led Ehrlich to develop chemotherapy, led Weigert to make revolutionary advances in histological techniques. These advances made it possible for researchers to gain fundamental insights into the fine structure of the nervous system. Weigert is thus closely associated with brain and spinal cord research and with neurology and psychiatry. Hans Christian Gram (1853-1938): Dr, Hans Christian Gram, a Danish scientist who developed a staining technique called “Gram staining” in 1884 to discriminate between two groups of bacteria based on the chemical and physical properties of their cell walls. Using this technique Dr. Gram successfully discriminated Pneumococci and Klebsiella pneumoniae bacteria. Today this staining protocol is extensively used to differentiate and characterize the