Production of Enzymes and Phytotoxins by the Plant Pathogen

8/6/2019 Production of Enzymes and Phytotoxins by the Plant Pathogen

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Plant Microbe Interaction

Production of enzymes andphytotoxins by the plant

pathogen

R.S. SHUKLA

Microbiology & Plant Pathology DivisionCentral Institute of Medicinal and Aromatic Plants,

P.O. CIMAP, Lucknow 226015


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CHEMICAL WEAPONS OF PATHOGENS

Plant pathogens are known to produce toxicsubstances which have been established toplay significant role in pathogenesis.

The main groups of substances secreted bypathogens in plants, which seems to beinvolved in production of diseasesymptoms either directly or indirectlyinclude enzymes, toxins, growthregulators, polysaccharides antibiotics etc


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In soft rot diseases, enzymes seem

to be most important.

In leaf spot and blight diseases e.g. in Victoria blight of oats-primarily the toxin victorin secretedby He lminthosporium, is responsibleprinciple.

In crown gall diseases growthregulators are apparently the main

substances involved.


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Thus enzymes, toxins and growth

regulators are considerably morecommon and important in plantdisease development thanpolysaccharides, antibiotics etc.


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ENZYMES

Most phytopathogenic micro-organismsusually breach the host cell walls duringpenetration and tissue invasion. The ability of the pathogen for the degradation of

polysaccharides in primary wall and thefactors that enable the pathogen to secreatecell wall degrading enzymes are of greatsignificance in pathogenesis.

Plant pathogenic enzymes disintegrate thestructural components of host cells, breakdown inert food substances in the cell or affect the protoplast directly and interfere with

its functioning systems.


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TYPES OF CELL WALL DEGRADING ENZYMES

Pectolytic enzymes Cellulolytic enzymes

PectinMethyl

Esterase

(PME)

Polygalacturonases

( PG )

Endo-PG Exo-PG

Polygalaoturonatetrans-eliminase

(PGTE)Polymethylgal-acturonase

(PMG)

Pectinlyase(PL)

Polymethyl-transeliminase

(PMTE)

CellulaseC1

CellulaseCx


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CELLULOSE DEGRADING ENZYMES

Cellulases degrade cellulose and its derivativesand cause cleavage of b-glucopyranose chain. The cellulase enzyme is constituted of two

components i.e. C1 and Cx.

C e llulas e C1 is responsible for weakening themutual interaction between anhydroglucosechains by breaking H bonds and facilitatingfurther break down of cellulose by the Cx

component into a mixture of glucose andcellobiose. The cellobiose is hydrolized intoglucose and b-glucosidase.C e llulas e CX is unable to solubilize native

cellulose.


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I n vitro PRODUCTION OF PECTOLYTIC AND CELLULOLYTIC ENZYMES

The pathogen was grown on selected medium incorporatingdifferent carbon sources as substrate viz-sucrose, pectin,sodium ploypectate, filter paper pulp and carboxy methylcellulose, 1 percent each.

Erlenmeyer flasks (250 ml) containing 50 ml medium, wereinoculated with the pathogen and incubated for differentperiods at 23+1oC.

At the end of incubation periods, culture filtrates werecollected by filtering through whatman filter paper 1 andagain through bacteria proof membrane filter (0.22 mm,Millipore).

The clear enzyme samples were stored at 5oC for further studies.


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PHYTOTOXIN

Toxins from disease inducing phytopathogens are major factors in the development of a number of destructive plantdiseases.

Three classic cases often cited are (i) Oat production inNorth America from 1946 to 1948, (ii) Rice production in

India (Bengal famine) during 1942-43 and (iii) Maizeproduction in South America during 1970-71, causedgreater economic losses than can be attributed to any other plant disease in recorded history. In all cases toxins werethe major factors in the epidemic and destructionprocesses.

Phytotoxin is a compound produced by a plant pathogen inits host and causes obvious damage to plant tissues at avery low concentration and known to be responsible for

partial/complete symptom development of the disease.


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Toxins are of various chemical types like peptides,

glycoproteins, polysaccharides, organic acids, fatty acids andtheir derivatives, polypeptides terpenoids etc.

Toxins involved in the development of plant diseases areclassified on the basis of their biological activity as hostspecific (host-selective) or non-specific (non-selective).

Host specific-: toxins are produced by fungi that arerestricted to certain plant cultivers, and are toxic only to hosts

of producing pathogen.eg. Victorin, HMT-toxin, HS- toxin.

Non-specific -: toxins are toxic to many plants, whether or notthe plants are hosts of the producing pathogen.eg. Alternaricacid, Altenin, Tenuazonic acid, Fusaric acid,and Colletotin.


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COMMONLY USED CRITERIA TO EVALUTETOXINS AS FACTORS IN PATHOGENISIS

Host specificity :- Host specific or non hostspecific.Presence in infected plants.Production at a key step in disease

development.Induction of typical disease symptoms.Correlation of virulence with quantity of toxins produced in vitro . It is influenced by a

number of factors and environmentalcondition.High toxin producer isolate under one set of culture condition may be low toxin producer

under another set of conditions.


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Yoder and Scheffer (1969) studied infection of susceptible and resistance oat leaves by H.Victoriae using toxin producing and non- toxin

producing isolate (mutant). The non toxin producingisolate behaved on both leaves in the same way asa toxin producer on resistant leaves.However, whenVictorin was added to infection droplets, the mutant

freely colonized susceptible but not resistantleaves.

VICTORIN AS A PATHOGENESTICITY FACTOR


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PRODUCTION OF TOXIN

Once the involvement of a toxin is suspectedin a plant disease, subsequent researchdepends on reliable methods for the

production, isolation and characterization of the toxic compounds.

Production of toxin by pathogenic andnonpathogenic isolates is compared todetermine if a toxin is a pathogenicity factor.Production by isolates with higher or lowvirulence is compared to evaluate a virulence

factor.


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The pathogen was grown in 250 mlErlenmeyer flask containing 50 ml of liquidmedia and incubated at 23+1 oC.

At the end of incubation period, fungal massfiltered off through whatman filter paper 1and bacteria proof membrane filter (0.22mm) and culture filtrates were autoclavedfor ten minuts to inactivate enzymes activity,

if any. These heated culture filtrates were used astoxin samples


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TOXIN BIOASSAY

The phytotoxicity of the culture filtrate was testedon the host leaves, shoot cuttings and germinatingseeds, to find out its effect on lesion formation,wilting and inhibition of the root elongation.

Leaf spot (leaf-puncture bioassay): was carriedout by placing a drop of culture filtrate on healthyleaves of the host and an injury was made gentlywith a sterile needle.

The development of lesion around the pricked spotwas observed at the end of designated periods andrated on an arbitrary scale.


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Phytotoxic reactions on leaves of geranium cv. Algerian after 72 hrs.

of toxin treatment at various conc.


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Wilt inducing test: was carried out by host leaves or

shoot cutting bearing 2-3 leaves. The cut ends wereinserted in to the culture filtrate filled in 10 ml vials andwilting symptoms were observed at various intervals.Root growth inhibition test:- was made on germinatingwheat seeds, having approximately 5 mm roots, wereselected and five such selected germinating grains inpetriplates (17x60 mm) containing 5 ml of culture filtrate,were placed and incubated for 48 hours at 23+1oC. Thelongest root of each seedling was measured.

Assay end point was determined after 48 hoursincubation by evaluating the maximum dilution whichrequired for 50% inhibition of seedling root growth.


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Wilting reactions on leaves of geranium after 24 hrs. of toxintreatment at various conc.


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Phytotoxic reactions on geranium cuttings and wheat seedlings

root growth after 48 hrs. of toxin treatment at various conc.


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SENSITIVITIES OF BIOASSAYS

Some times, few toxins may go undetectedbecause more sensitive assays are required,e.g. the toxin produced by H. maydis race T .H. maydis race T. has long been speculated toproduce a toxin that accounts for its specificitytowards certain corn lines.

Most attempts employed assays such asseedling root growth, is known to be relativelyinsensitive to HM T-toxin whereas protoplastsare highly sensitive.


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Table I: Sensitivities of Bioassays for T-toxin a.Bioassay Sensitivity Elapsed

Time

Protoplast death (Earle et al 1978) 20 d 1-2 days

Mitochondrial respiration (Yoder et al . 1977) 30-60 Sec.

Succinate 70 e

NADH 70 d

Malate 140 e

Dark Co 2-fixation (Bhullar et al. 1975) 100e 1-2 hr

Seedling root growth (Yoder et al . 1977) 700 e 2 days

Leaf whorl infection (Yoder and Gracen 1975) 1000 c 2-3 days

Leaf puncture (Karr et al . 1974) 18000 d 2-3 days

a All assays were performed with single lot of T-toxinc Toxin concentration in ng/mld Detectable levele ED 50 levelf. Approximate toxin exposure time.


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BIOASSAY FOR PHYTOTOXINS

SL

NO.

Disease Source of Toxins Effect on Plant Type of assay

1 Southern cornleaf blight

H .maydis race-1 Yellowing necrosis a). lesions on leaveb.) Dark CO2 fixationc.) Mitochondrialrespiration &SRGId.) Protoplast Death

2 Victorian blightof oats

H elimthosporiumbictoriae

Death of plant a) Seedling Root Growthinhibition (SRGI).b). Root cap cellc). protoplasts

3 Eye-spot diseaseof sugar cane

H . sacchar1 Streaks on leaves,necrosis

a) Streaks onLeavesb) .Electrolite Leakage

4 Stem canker of tomatao

Alternariaalternate

Sp. Lycopersici

Canker on stemsandleaf clorosis

chlorosis

5 Leaf blight of Costus

Drechslera maydis Yellowing &necrosis

SRGI


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FACTORS AFFECTING THE PRODUCTION OFTOXINS

Selection of strainsScheffer et.al (1967) examined 16 strain of H.carbonum in which toxin production varied over 100- fold , and Scheffer et al.(1964) and Nelsonet.al(1963) demonstrated that victorin production byH.victoriae could be enhanced by culturingascospore progeny resulting from crossed of certainstrain of the fungus


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Nutritional factors1. Culture media

i) Carbon sourceii) Nitrogen source

2. Growth factors ( mostly Yeast extract)

3. Culture conditionsi) Solid verses liquidii) Still verses shake

4. Incubation period5. Environmental factors

i) Temperatureii) pH

iii) Light


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PHYTOTOXINS PRODUCED BY FUNGAL PATHOGENS

Toxins Pathogen

A. Alternaria toxinsI)Alternaric acidII)ZinnolIII)AlteninIV)TentoxinV)AltenueneVI)AlternariolVII)Alternariol monomethyl ether VIII)Tenuazonic acid

Alternaria solani A.zinnae A. kikuchiana A.alternata (A. tenuis) A. tenuis A. tenuis A. tenuis A. alternata & A. longepes

B. Fusaria toxinsI)Naphtazarin pigmentsII)Fusaric acidIII)LycomarasminIV)Phytonivein

F usarium solani f. S p. pisi F . oxysporum f. Sp. lini F . oxysporum f. Sp. lycopersici F . oxysporum f. Sp. niveum


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C. Helmenthosporium/Drechslera Toxins

I) Hv-toxin (VictorinC)II)HC-toxinIII)H S -toxin AB&CIV)HMT-toxin Band 1,2,3V)HelminthosporalVI)Drechslerol A&C

VII)Drechslerol BVIII)Triticone A&B

H. victoriaeH. carbonumH. sacchariH. maydisH. sativumD. maydis (H. maydis)

D. maydis (H. maydis)D. Triticirepentis

Toxins Pathogen


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D. Other PhytotoxinsI)PC-toxinII)PM-toxinIII)FusicoccinIV)Piricularin

V)AscochytinVI)DiaporthinVII)Cercospora toxinVIII)ColletotinIX)Ceratocystis ulmi toxin

X)Phytophthora toxinXI)Verticillium toxin

P ericonia circinata P hyllosticta maydis F usicoccum amygdali P iricularia oryzae

Ascochyta fabae Endothia parasiticaCercospora beticolaColletotrichum fuscumC.ulmi P . nicoticana var. P arasiticaV. alboatrum


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PHYTOTOXINS PRODUCED BY BACTERIAL PATHOGENS

S.no. Toxins Pathogen

1.2.3.4.5.

6.

Tabtoxin (Wildfire toxin)PhaseotoxinRhizobiotoxinS yringomycinPolysaccharides

Glycopeptide

P seudomonas tabaci P . phaseolicola Rhizobium japonicum P seudomonas syringae P . solanacerum Xanthomonas phaseoli X. campestrisCorynebacterium

sepedonicumC. michiganese


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EXTRACTION OF TOXIC METABOLITES

The techniques for the separation of toxins from thecomplex mixtures of culture filtrates, microbial cells, or infected plants are based on the same principles as for

the extraction and isolation of other natural products. All methods depend upon the selective distribution of thedesired components of the mixture into one phase whileleaving the unwanted materials in another phase. Thesephases may be solid, liquid, or gas.

During extraction a known volume of culture filtrate isshaken successfully with one third volume of the organicsolvent, in the separating funnel.


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During extraction the solvent may always be regardedas being associated with the solute and the relativevolumes appears in two phases i.e. upper and lower phases respectively.

The organic substances can be separated frominorganic impurities by shaking an fermentedsuspension with immiscible organic solvents of increasing polarity such as benzene, chloroform, ethylether, acetone, ethanol, methanol etc.

After several such extractions the combined organicphase is dried over anhydrous Na2SO4 and the solventis evaporated by Buchi-Flash rotary evaporator at 40oCunder reduced pressure.


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Various method are available for isolationand purification by chromatography, either as liquid chromatography or as vapour

phase (Gas chromatography).One of the most widely used method isconventional column chromatography. Themost frequently used are adsorption,

partition, ion- exchange, gel-permeation andmore recently affinity chromatography.

Isolation and purification of toxins


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Thin or Thick Layer Chromatography (TLC)

In TLC the solvent creeps up the stationary phase by capillarity in aclosed tank. The silica gel is spread on a rectangular glass plate(silica gel/ water 1:2) and plate is activated by heating at 100 to110C for 1 hrs. the compounds are spotted about 2.5 cm from thebottom of the plate and allow to dry. The plate is then placed up rightin a tank containing eluting solvent and elution is completed with in

three hrs. The thickness of the plates could be between 0.2 mm to 2mm or more.The thicker plates are used for preparative work. The separatedspots are easily scrapped off from the plate and eluted with solventthese spots can be observed on the plates by UV light or by sprayingwith a reagent that gives coloured product with the spot (Iodinevapour give brown colour with amines) or dilute sulphuric acid(Organic compound become coloured or black and plates are heatedat 100C).


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Isolation of toxins from infected plants

Toxin production is usually much lower in the plants thanin culture filtrate even if high producing isolates areused. Another problem is that extracts of infected plantscontain many soluble plants constituents that couldinterfere with purification. In general the isolation of Drechslerol-B toxin from infected leaves wasdemonstrated by Shukla, et al; (1990) involvedpulverizing the infected tissue by grinding or

homogenization in a suitable solvent, removal of theplant tissue debris by centrifugation or filtration andpurification by the same procedures used for theisolation of toxin from culture filtrate.


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Table:- Some important toxins and their Application

Toxin designation Producing organism Applications

HV- toxin H elminthosporium victoriae S creening & Genetics

HC- toxin H. carbonum race-I S creening & Genetics

HS - toxin H. sacchari S creening

HMT- toxin H. maydis race-T S creening & Genetics

HO- toxin H. oryzae S creening

PC- toxin Periconia circinata S creening & Genetics

AB-toxin Altenaria brassicae S creeningHelminthosporal H. sativum Growth regulater

Fusicoccin Fusicoccum amygdali Metabolic probe

Rhizobitoxine Rhizobium japonicum Growth regulater

Xanthon Xanthomonas compestries Commerce

Tentoxin Alternaria alternata Metabolic probe, Bioherbicide

Moniliformin Fusarium moniliforme Bioherbicide

Bipolaroxin Bipolaris cynodontis -do-

Alteichin Alternaria eichorniae -do-

Bilaphos S treptomyces viridochromogenes -do-

Tab toxin Pseudomonas syringae Pv. tabaci -do-

Endo toxin Bacillus thuringiensis Bioinsecticide


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Disease resistant plants of various crops obtained by in vitro selection

using phytotoxins

Crop Pathogen Selective agent Selection level Level of resistant observed

Alfafa F usarium oxysporum f.sp.medicagines

Culture filtrate Callus Increased

Barley H elminthosporium sativum Crude toxin Callus Resistance

Brinjal Verticillium dahliae Culture filtrate Cell suspension Increased resistance

Maize H elminthosporium maydis race-T Hm-T toxin Callus Resistance

Oats H .victoriae Victorin Callus Resistance to victorins

Peach Xanthomonas campestria Fractionated Culturefiltrate

Callus Increased resistance

Potato P hytophthora infestans Culture filtrate Callus Resistance to culture filtrate

Mustard P homa lingam Culture filtrate Cell suspension embryoculture

Increased resistance

Mustard A. brassicae Crude toxin Callus Increased resistancePaddy H elminthosporium oryzae Crude toxin Callus Increased resistance

S ugarcane H elminthosporium sacchari Hs-toxin Callus Increased resistance

Tobacco F usarium oxysporum Culture filtrate Cell suspension Enhanced tolerance

Tomato P seudomonas solanaceum Culture filtrate Callus Delayed symptoms

Wheat H elminthosporium sativum Crude toxin Callus Resistance


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Isolation of A . alternata toxin resistant calliclones of geranium cv. Algerian in Ist selection cycle


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Regeneration of A .alternata toxin tolerantcalli (2 nd cycle) of geranium on toxinincorporated and toxinfree modified MSmedium

Hardening of toxin tolerantcalliclones


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Reactions of geraniumcalliclones to leaf blight diseasecaused by A . alternata


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Documents

Production of Enzymes and Phytotoxins by the Plant Pathogen