Enzymes and plant diseases Enzymes Toxins Growth regulators

Enzymes and plant diseases

• • • Enzymes Toxins Growth regulators Polysaccharides antibiotics

• • Disintegration of tissues De Bary-Sclerotina libertiana: Dissolution of tissues Killing of cells

v. Cellulolytic enzymes: • C₁ and Cx: • C ₁ loosens the cellulose fibrils of the crystalline area • It is a large molecule • Inducible

• Cx group- They hydrolyze the cleavage of β 1 -4 glycoside linkages but a prior modifying action of C ₁ is essential for their activity. Cannot attack native cellulose

• Several enzymes have been characterised in the C x group of enzymes which attack the glycoside bonds randomly or terminally and accordingly called Cx endo or exoenzymes respectively

• The endo C x enzymes fragment the long cellulose chains into soluble low molecular weight smaller pieces of 6 or lesser glucose units • The C x exoenzymes by acting on terminal bonds further break these smaller chains into cellobiose which may further be hydrolyzed by another enzyme cellobiose to liberate glucose units

• The C x activity is assayed by measuring loss of viscosity or the production of reducing sugars from the carboxymethyl cellulose • C enzyme has no effect on it

• Native cellulose cellobiose glucose cellulase cellobiase • Native cellulose reactive cellulose Shorter cellulose chains cellobiose glucose

• Hemicelluloses: • Present in the spaces between the cellulose microfibrils of the cell wall. • Complex, heterogenous polysaccharides formed by linear union of smaller homogenous sugar chains like xylans, arbans, mannans and galactans

• Several bacterial and fungal pathogens as well as saprophytic and wood rotting fungi produce hemicellulase enzymes which degrade the polymer by hydrolytic cleavage of the bonds • Since the polymer has several kinds of bonds several enzymes are needed to copewith the variety of bonds

v. Pectic substances and pectolytic enzymes: • Pectic substances form the middle lamella exclusively They form a layer below the cuticle in the epidermal cells and fill the intermicellar spaces of the cellulose fibrils in the primary cell wall. • .

• The different pectic materials and the enzymes concerned with their degradation are detailed: 1. Pectic acid is a linear polymer of D galacturonic acid residues joined laterally by α 1 -4 glycosidic linkages it is water soluble and easily precipitated by calcium and magnesium ions to form insoluble salts

• It occurs mainly as calcium and magnesium pectates chiefly in the middle lamella

• • Pectin: is the methyl ester of pectic acid The esterification maybe to different degree If 75 % or more carboxyl groups are esterified the polymer is referred to as pectin chains with lesser methoxyl content are designated as pectinic acid

• Pectin- water soluble, neutral because most acid groups are esterified and can not be precipitated by polyvalent cations

• Protopectin: • Insoluble native pectic material as occurs in the primary cell wall is called protopectin. • Pectic enzymes : • Pectin methyl esterase: it removes hydrolytically the methyl group from pectin or pectinic acid to give methyl alcohol , pectinic acid of lower methoxyl content and ultimately pectic acid

• Chain length remains unaffected , yet the removal of the methoxyl groups gives new properties, like formation of salts with polyvalent cations. • It also predisposes pectin to cleavage by chain splitting enzymes which otherwise would not degrade pectin

• PME is widely distributed among higher plants but to a much lesser extent in the microorganisms

• Chain splitting enzymes: • Break the α 1 -4 glycosidic bonds forming smaller fragments and ultimately releasing D galacturonic acid residues. • Glycosidases: polygalacturonase and polymethylgalacturonase

• These enzymes break the glycosidic bonds hydrolytically. • If the enzyme shows greater activity on pectic acid the enzyme is called polygalacturonase • If the preferred substrate is pectin it is called polymethylgalacturonase

• The two enzymes are further characterized as endo or exoenzymes • Those attacking terminal bonds are called exo PG or exo PMG • Those attacking linkages at random are designated as endo PG or endo. PMG • The endo PG or PMG are more efficient enzymes as they break the integrity and chainlength

• The endo PG or PMG enzymes thus bring about a loss in viscosity of pectic substrates and are easily assayed viscometrically.

• Transeliminases or lyases: • Transeliminative cleavage of the bond involves removal of the proton of C 5 of one residueresulting in the formation of an unsaturated bond between C 4 and C 5

• Classification of glycosidases and lyases: (Bateman and Miller. term lyases has been used instead of transeliminases) A) 1. Random cleavage: a) pectin, preferred substrate endo PMG b) pectic acid endo PG

• 2. Terminal cleavage: • a) pectin- preferred substrate • b) pectic acid Exo PMG endp PG

B) Transeliminative bond cleavage 1. Random cleavage a-pectin endo PL b- pectic acid endo PAL 2. TERMINAL CLEAVAGE a- pectin exo PL b- pectic acid exo PAL

• Wall modifying enzyme: • Modifies native pectic material • Propectinase: the term is used for crude enzyme preparations that attack parent pectic material

• Others: • Proteolytic enzymes • Phosphatidase-lecithin

Toxins and plant disease Classification: Phytotoxin Non specific Incite few or none of the symptoms incited by the pathogen • Lycomarasmin and alternaric acid • •

• Vivotoxin: • Substance produced in the infected host by the pathogen and or/or its host which functions in the production of disease but is not itself the initial inciting agent of disease • The requisites of a vivotoxin are:

1. It must be isolated from diseased plant but should not be present in the healthy host 2. It must be characteried chemically 3. When introduced in pure form into healthy host it must produce the symptoms of disease or a portion of the syndrome eg. Fusaric acid and piricularin

• Pathotoxin: plays a causal role in disease production and production in susceptible plants, symptoms characteristic of the disease • Criteria: • The toxin applied at concentrations which could be reasonably expected in or around the diseased plant produces in a susceptible host all the symptoms characteristic of the disease

• The pathogen and the toxin exhibit similar host specificity • The ability of the pathogen to produce the toxin varies directly with its ability to cause disease • A single toxin is involved

• Victorin, wild fire toxin and toxin produced by Periconia circinata are pathotoxins

• Host specific toxin: • Pringle and scheffer coined and defined host specific toxin as a metabolic product of a pathogenic microorganism which is toxic only to its host • Pathotoxins of wheeler and luke are host specific toxins of pringle and scheffer

• Victorin produced by Helminthosporium victoriae, toxins of Periconia cirinata and Helminthosporium carbonum and some species of Alternaria eg A kekuchiana are host specific toxins

• Sceffer and pringle – divided toxins into two broad categories: 1. Primary determinants( toxins) are essential for pathogenicity and are largely the host specific toxins but may also include some non specific toxic substances

2) Secondary determinants: are not required for pathogenicity but control the virulence scheffer and Pringle treat wildfire toxin secreted by Pseudomonas tabaci as a secondary determinant

• Graniti has advocated the use of only two terms : • Phytotoxins sense stricto for mechanical products that interfere with or alter the metabolism of plant cells • These are produced in the host on its surface or even outside the host

• Vivotoxin: • Phytotoxins whose role in pathogenicity has been demonstrated’ host specific toxins’ and pathogenicity are only host specific vivotoxins

• Victorin: the toxin victorin is produced by Helminthosporium victoriae, the causal organism of Victoria blight disease of oats • Fungus- soil and seed borne • Causes basal stem and root necrosis

• Chemical nature: • Active preparations are obtained from culture filtrates of the pathogen • a seedling root growth bioassay is used to assay the relative toxicity • The toxin is a low molecular weight peptide linked to a base- a nitrogen containing sesquiterpene moiety called victorinine

• Role of victorin in disease development: • The following five evidence suggest victorins key role in development of the disease: 1. Only those strains that produce the toxin are pathogenic non producers are non pathogenic 2. Plants that are susceptible to the fungus are susceptible to the toxin also. Resistant host plants resist the toxin action

3) Spores contain the toxin which is released during germination A trace of toxin is shown to be present at the point of contact of the germinating spore with the host If a drop of toxin is added to the infection drop the non pathogenic strain behaves like a pathogenic strain

4) The toxin shows extremely high biological activity 5) The toxin causes the same biochemical changes in the host as are caused by the pathogen

• Mode of action: • The primary effect of the toxin is on the permeability of the host plasma membrane

• Resistant varieties of oats inactivate the toxin • Resistant cells appear to lack the receptor site as such cells do not respond in any observable way

• • • Lycomarasmine: F lycopersici Chemical structure and mode of action: Dipeptide Mol wt 277

• H₃N-CI-CH₂ CH₃ HOOC-CH-NH-CO-CH₂-NH-C-OH COOH solution of lycomarasmine is capable of causing wilting and necrosis of excised tomato leaves at minimum concentration of 5 X 10⁻⁴ molar

• Structural analogue of the yeast growth factor – strepogenin anoligopeptide containing glutamic acid against aspartic acid of lycomarasmine • Toxin action is reversed by adding streptogenin externally • Thus it is believed that lycomarasmine causes wilting action by interfering with streptogenin metabolism resulting in its deficiency

• It chelates iron ions or dislodges them from essential sites and transports the toxin – Fe complex to the leaves, where the unstable complex is decomposed under the influence of light

• Fe accumulates in toxic concentration in the leaves which results in damage to the permeability of the leaf cells • Excessive transpiration results and flaccidity of leaves occurs

• Fusaric acid: • Vivotoxin • Produced by several genera and species of fungi • Some of these fungi also produce toxic metabolites, closely related to fusaric acid or different from it

• Plays a role in wilt of tomato, cotton and bakane disease of rice

• Chemistry and mode of action: • 5, n butyl pyridine -2 -carboxylic acid • Fusaric acid causes injury to the plasma membrane bringing about a dysfunction of osmoregulation and the concomitant ionic imbalance

• Linskens detected leakage of ions and amino acids from fusaric acid treated tomato leaf tissues • The water permeability of protoplasts and the water balance of whole plant is altered

• It also increases apparently the rate of respiration and inhibits polyphenol oxidase activity • Like lycomarasmine fusaric acid chelates iron causing its deficiency

• The two parts of the toxin molecule are essential for toxicity • The carboxyl group at α position is associated with the inhibition of respiration rate and polyphenol oxidase enzyme. • The aliphatic side chain in 3 position is necessary for damage to water permeability of cells, severity of damage increasing with the length of the chain from methyl to butyl

• Of the two the first appears to be more critical in pathogenesis • A volatile decarboxylation product of fusaric acid, 3 -n. butyl pyridine is more injurious to water permeability,

• Wild fire toxin: • It is a bacterial toxin produced by Pseudomonas tabaci causal agent of the wild fire disease of tobaco • Resembles victorin in that it reproduces all the symptoms of the disease yet differs in being non specific

• Chemistry and mode of action: • Characterized as a new type of amino acid. APPEARS TO BE A STRUCTURAL ANALOGUE OF METHIONINE • CAUSING methionine deficiency by competing for active enzyme sites

• α picolinic acid, piricularin and pyriculol were implied by Tamari and Kaji in the blast disease of rice caused by Piricularia oryzae • Both toxins isolated fom culture filtrates in crystalline form and detected in extracts fro infected tissues

• α picolinic acid interferes with respiration by chelating iron of respiratory enzymes • Its specific effect on catalase is made good by adding iron externally • Piricularin also inhibits respiration and several respiratory enzymes

• Pyriculol was isolated from P oryzae • Causes natural blast lesion and also inhibits the growth of rice seedlings • 3, 4 dihydro 3, 4, 8 trihydroxy-1(2 H) naphthalenone and Tenuazonic acid