BACTERIA AS PLANT PATHOGENS

General characteristics of plant pathogenic bacteria Prokaryotic organisms DNA not bound by membrane – not organized into nucleus Single celled, microscopic Possess a rigid cell wall Consists of cytoplasm containing DNA small 70S ribosomes Contain plasmids - additional circular small chromosomes -Replicate independently from bacterial genome -code for resistance to antibiotics or toxin production

Morphology Most plant pathogenic bacteria are rod shaped – except Streptomyces Deviations from the rod shape are club, Y or V and other branched forms in Coryneform bacteria Cell walls of most species enveloped by a viscous, gummy material –called slime or capsule Slime layer or capsule acts as protective covering or reservoir of stored food

Structure Cytosol or cytoplasm with nuclear body Envelope Cell membrane Peptidoglycan Outer membrane Periplasm – space between inner and outer membranes Capsule or glycocalyx

Cell membrane Bilayer unit membrane – present in all prokaryotes. Functions Osmotic barrier Transport of specific solutes (nutrients and ions) Group transport of classes of compounds Active transport Synthesis of membrane lipids Synthesis of wall murein (peptidoglycan) Assembly and secretion of extracytoplasmic proteins Respiratory electron transport Chromosome segregation Chemotaxis (sensing function and motility)

Cell wall 1. Gram negatives have outer membrane – permeability barrier to large molecules (>800 -900 daltons) Structure Lipid bilayer Inner leaflet is standard membrane Outer leaflets is composed of lipopolysaccharides (LPS) and proteins Outer membrane lipoprotein is most abundant protein in the cell. Part of it anchors the outer membrane to the peptidoglycan layer Porins are transmembrane proteins that form channels through the outer membrane. They allow specific nutrients and ions to pass through Porines can allow entry of molecules up to 600 – 700 daltons

Cell wall Peptidoglycan (murein) layer is found in Gram positive and Gram negatives Can be attacked by lysozymes Single molecule of ß 1-4 linked chains of N-acetyl glucosamine and N-acetyl muramic acid cross linked with a tetrapeptide Determines rigidity and shape of cell. Prevents disruption by osmotic pressure and sensitivity to penicillin Penicillin inhibits formation of peptide cross-links Gram positives have multiple sheets of peptidoglycan, which includes teichoic acids (polysaccharrides). Gram negatives have a single thin layer (5%)

Periplasm Space between cytoplasmic membrane and outer membrane in Gram negatives. Same osmolarity as the cell interior. Osmotic concentration maintained by membrane derived oligosaccharides Contains binding proteins, chemotactic receptors and hydrolytic enzymes such as pectic enzymes, proteases, phosphatases and nucleases Binding proteins involved in taking molecules outside the cell into the cell Also detoxify enzymes in antibiotic resistant bacteria Provides a buffer between the inside of the cell and its external

Flagella Involved in motility and chemotaxis which is important to infection Forms of flagella Peritrichous – arising from all over the cell surface e.g Erwinia spp and Agrobacterium (has 4 peritrichous flagella) Polar – This can be a single or tuft of flagella at one end or both ends Monotrichous eg. Xanthomonas, Pseudomonas Amphitrichous eg Pseudomonas Lophotrichous eg Pseudomonas, Corynebacterium Streptomyces has no flagella – consist of non-septate, branched threads, produces exospores

Fimbriae Short fibrous appendages about 0.2 to 2.0µm There are also pili and these are involved in exchange of genetic material and adhesion to plants

Reproduction Binary fission – occurs rapidly Bacteria can divide every 20 to 50 mins One million progeny can be produced within a day Multiplication affect by: Food supply Accumulation of metabolic wastes Other limiting factors

Ecology and spread Referred to as soil invaders Can develop:- in host as parasite On plant surface as epiphytes In plant debris or soil as saprophytes As biofilms in communities attached to each other or a solid surface Erwinia amylovora produce their populations in the host, numbers decline in the soil Populations in soil do not contribute to disease propagation from one season to the other Plant to plant cycles via insect vectors, alternate hosts, vegetative propagating organs Have lost ability to survive in the soil Referred to as soil invaders

Soil invaders Most plant pathogens fall in this category Enter soil in host tissue and have poor ability to compete as saprophytes Survive in soil as long as tissues resist decomposition Survive for varying durations depending on bacterial species and environmental conditions What are the other survival sites for bacteria?

Dissemination ???

Ecology and spread Soil inhabitats eg Agrobacterium tumefaciens, Ralstonia solanacearum and Streptomyces scabies Build up populations in the host, in soil populations slowly decline Net increase caused by planting susceptible hosts in successive years

Classification Kingdom: Prokaryote/ Monera Division: Gracilicute (Gram negative bacteria) Class: Proteobacteria – mostly single-celled bacteria Family: Enterobacteriaceae Genus: Erwinia, causing fire blight of pear and apple, Stewart’s wilt in corn Pectobacterium and Dickeya causing soft rots in fleshy fruits and vegetables Pantoea, causing wilt of corn Serratia, S. marcescens, being a phloem-inhabiting bacterium causing yellow vine disease of cucurbits Sphingomonas, causing brown spot of yellow Spanish melon fruit

Classification Family: Pseudomonadaceae Genus: Acidovorax, causing leaf spots in corn, orchids and watermelon Pseudomonas, causing numerous leafspots, blights, vascular wilts, soft rots, cankers and galls Ralstonia, causing wilts of solanaceous crops Rhizobacter, causing the bacterial gall of carrot Rhizomonas, causing the corky root rot of lettuce Xanthomonas, causing numerous leaf spots, fruit spots and blights of annual and perennial plants, vascular wilts and citrus canker Xylophilus, causing the bacterial necrosis and canker of grapevines

Classification Family: Rhizobiaceae Genus: Agrobacterium, cause of crown gall disease, causes hairy root of apple Rhizobium, the cause of root nodules in legumes

Classification Family: still unnamed Genus: Xyllela, xylem - inhabiting, causing leaf scorch and dieback diseases on trees and vines Candidatus liberobacter, phloem inhabiting, causing citrus greening disease Unnamed, laticifer-inhabiting, causing bunchy top disease of papaya

Classification Division: Firmicutes – Gram positive bacteria Class: Firmibacteria – Mostly single cell bacteria Genus: Bacillus, causing rots of tubers, seeds and seedlings, and white stripe of wheat Clostridium, causing rot of stored tubers and leaves and wet wood of elm and poplar

Classification Class: Thallobacteria – Branching bacteria Genus: Arthrobacter, causing bacterial blight of holly Clavibacter, causing bacterial wilts in alfalfa, potato and tomato Curtobacterium, causing wilt in beans and other plants Leifsonia, causing ratooning stunting of sugarcane Rhodococcus, causing fasciation of sweet pea Streptomyces, causing the common potato scab

Classification Mollicutes – Have only cell membrane and lack cell division Division: Tenericutes Class: Mollicutes Family: Spiroplasmataceae Genus: Spiroplasma, causing corn stunt, citrus stubborn disease Family (ies): Still unknown Genus: Phytoplasma, causing numerous yellows, proliferation and decline diseases in trees and some annuals

Specific characteristics for some bacteria Agrobacterium Rod shaped – 0.8 by 1.5 -3 µm Motile 1 – 4 peritrichous flagella Produces abundant polysaccharide slime on carbohydrate-containing media Colonies smooth and non-pigmented Bacteria are rhizosphere and soil inhabitants

Specific characteristics for some bacteria Clavibacter (Corynebacterium) Straight to slightly curved rods 0.5 – 0.9 by 1.5 - µm Sometimes have irregularly stained segments or granules and club-shaped swellings Generally non-motile but some species motile by one or two polar flagella Gram positive

Specific characteristics for some bacteria Erwinia Straight rods, 0.5 – 1.0 by 1.0 – 3.0 µm Motile by means of petrichous flagella Only plant pathogenic bacteria – facultative anaerobes Some produce pectic enzymes and cause necrotic or wilt diseases (amylovora group) Carotovora group – produce pectolytic enzymes (cause soft rots)

Specific characteristics for some bacteria Pseudomonas Straight to curved rods, 0.5 – 1 by 1.5 – 4 µm Motile by means of one or many polar flagella Inhabitants of soil or freshwater and marine environments Most pathogenic species infect plants and few infect animals Produces yellow-green diffusible fluorescent pigments on low content medium Ralstonia - does not produce fluorescent pigments

Specific Characteristics for Some Bacteria Xanthomonas Straight rods 0.4 1 by 1.2 – 3 µm Motile by means of polar flagellum Produces yellow colonies on medium Most species slow growing All species are plant pathogenic, found in association with plant or plant materials

Specific characteristics for some bacteria Streptomyces Slender branched hyphae without cross walls 0.5 – 2 µm in diameter At maturity mycelium forms of three to many spores Many species and strains produce a wide variety of pigments that colour the mycelium and the substrate Produce one or more antibiotics active against bacteria, fungi, algae, viruses and protozoa. All species are soil inhabitants Gram positive

Specific characteristics for some bacteria Xylella Single celled, straight rods, 0.3 by 1 – 4 µm Produces long filamentous strands under some cultural conditions Colonies are small, with smooth or finely undulated margins. Require specialised media, their habitat is xylem vessels Gram positive, non motile, aflagellate, strictly aerobic and non-pigmented

Major types of diseases Blights and cankers Start as local areas of cell death (necrosis) Appear as leaf, pod or fruit spots, blossom death or stem lesions (cankers). Can be localized or spread or spread through the plant. Pathogens call necrogens Vascular wilts Xylem invaded by necrogenic pathogens and wilting is the most obvious symptom. Extracelluar polysaccharides are usually involved in vascular plugging

Major types of diseases Soft rots Macerogens cause host cell separation and death Dead tissue remains soft and watery, damage due to pectic enzymes Galls Host cell proliferation resulting in a cancer-like growth due to growth regulator imbalances Some pathogens transform host cells and are called oncogens

Pathogenicity and Virulence Pathogenicity – ability to produce disease Virulence – ability to increase disease severity Outcome of infection Compatible – pathogen grows in susceptible host and causes symptoms Incompatible – pathogen elicits responses in resistant host which blocks its multiplication. HR – hypersensitive response Neutral (nonhost) – pathogens may or may not grow, but high populations never achieved, symptoms are not produced and host does not respond

Toxins Characteristics Microbial metabolites deleterious to the host in small doses (often enzyme inhibitors) Characteristics Most produced by pathovars of P. syringae All are nonhost specific Commonly considered to be virulence rather than pathogenicity factors Pathogenicity for P. s. tabaci on bean Virulence for all other P. syringae pathovars (toxin increases symptoms, but not requred for lesion formation) Loss of toxin – smaller lesions, less bacterial growth, lack of stunting and chlorosis and inability to infect some cultivars of a host

Advantages of toxins Suppress resistance – by killing the cells, extend host range Release nutrients Cause host to release degradative enzymes Facilitate movement through plant tissues Inhibit other microorganisms

Examples of Pseudomonas toxins Syringomycin (SR) Produced by P.s. pv syringae (bacterial cankers of stone fruits, brown spot of bean) Primary determinant of pathogenicity Syringotoxin (ST) Produced by citrus strains of P. s. syringae Similar structure to SR but has different amino acids Effect on pathogenicity not reported

Examples of Pseudomonas toxins Coronatine Produced by P.s. pvs atropurpurea, glycinea, tomato and morsprunorum Produces chlorosis, stunting and hypertrophy of plant tissue The toxin stimulates the production of ethylene and this could cause some of the symptoms Phaseolotoxin P. s. pv phaseolicola Causes halos around lesions and systemic chlorosis Produced only at cool temp. (18 0C but not 26 C) Not primary determinant of pathogenicity Tox-mutants cause lesions without halos

Examples of Pseudomonas toxins Tabtoxin P.s. pv tabaci (wildlife on tobacco and bean) P. s. pv. Coronafaciens (halo blight of oats) Primary determinant of pathogenicity on bean but not tobacco. Tox-mutant grow initially in the plant but do not cause lesions and eventually die out

Why are bacteria not killed by their own toxins???

An inactive form of the toxin is excreted The target enzymes in the bacterium is resistant They do not take toxin back up after it is excreted

Extracellular polysaccharides (EPS) and wilt diseases EPS – slime (not attached to the cell, washed off with water) Capsule – attached to the cell, removed by washing in high salt) Pathogens Vascular in all genera of phytopathogenic bacteria except Agrobacterium Best studied: E. amylovora, E. stewartii, R. solanacearum, X. c. pv campestris

EPS involvement in wilting Two theories Physical plugging of xylem by cells and EPS (or formation of tyloses in response to infection Vascular blockage Any large molecule (bacterial slime) – problems going through pit membranes and cause wilting in cut plant bioassays EPS may interact with plant products to form gums and gels

Why do bacteria produce EPS It has roles in survival, dissemination and pathogenesis Enhances water- soaking in lesions. This creates a favourable environment for bacteria in leaves by holding water and nutrients in the intercellular spaces Hydroscopic and holds water, protects the cell from desiccation Binds charged nutrients and holds them in the vicinity of the cell (retention of nutrients) Highly charged, prevents entry of antibiotics and heavy metals Shields the cell from phages, bacteriocins and plant defenses Aids in attachment to surfaces and hosts. Could also affect insect transmission of pathogens EPS causes swelling of the vascular tissue and enable bacteria to rupture pit membrane and walls to escape from vessel. Aids in systemic movement

Degradative enzymes Functions Soft rot bacteria – provide a mechanism for movement of bacteria through plant tissue can kill cells resulting in release of nutrients Non soft rot bacteria – also produce the enzymes but their role in pathogenesis is not clear Pectic enzymes are most the most common degradative enzymes Other extracellular enzymes are proteases and nucleases but have not been shown to be virulence factors yet

Pectic enzymes Classified according to: Substrate –pectin polygalacturonic acid Mode of cleavage – hydrolase (Polygalacturonase) (Pectic lyase, trans-eliminase) Point of attack – endo or exo Soft rotting Erwinias produce endo and exo PG and multiple isozymes of PL. PLs are the most important for virulence In vitro, pectic enzymes synthesis is induced by pectin. Pectic enzymes are repressed by glucose

Growth regulators and abnormal growth Effect of growth regulators Indole acetic acid Cell enlargement Adventitious roots Cambial activity Cytokinins Increase cell division Loss of apical dominance Lateral bud and branch development

Diseases Olive and oleander knot P. syringae pv savastanoi Bacterium produces IAA and cytokinins which result in gall formation. The gall provides a niche for the bacterium. Presence of the pathogen required for gall maintenance Witches Broom of cacoa Rhodococcus fasciens Loss of apical dominance and bud formation is due to cytokinin production by the pathogen