Fungal Associations: III. The Role of Pectic Enzymes on the Synergistic Relation between Rhizoctonia solani Kuhn and Fusarium solani Snyder and Hansen, in the Rotting of Potato Tubers

The greatest activity of protopectinase obtained from the growthof Rhizoctonia solani and Fusarium solani on autoclaved potatoplugs occurred at pH 6.5, and greatest activity of the ‘lossof viscosity’ enzyme was found at 6–5 for Rhizoctonia,and between 6.5 and 8.3 for Fusarium. Protopectinase enzymeobtained from double infections of the Fusarium spp. with Rhizoctonia,or by mixing the enzymes of individual Fusarium spp. with Rhizoctoniaenzyme, were more active than the enzymes from single inoculations.Cylindrocarpon radicicola enzyme was more active when obtainedfrom a pure culture than from double infection. Similarly, mixingthis enzyme with the enzyme of Rhizoctonia reduced its activity.The evidence indicated that the protopectinase of Rhizoctoniawas similar to that of Cylindrocarpon and differed from thatof the Fusarium spp. Using paper partition chromatography, two bands from Rhizoctoniacrude enzyme had a stimulatory effect on Fusarium enzyme, whileonly one band from Fusarium enzyme stimulated Rhizoctonia enzyme. The purified enzyme of Rhizoctonia degraded pectin to galacturonicacid. Fusarium pure enzyme degraded pectin to an intermediatestage. A mixture of the two enzymes degraded pectin to galacturonicacid, without the intermediate stage formed by Fusarium alonebeing detected. The role played by pectic enzymes upon the synergistic relationof Rhizoctonia solani and Fusarium solani on rotting potatotubers is discussed.     

Studies in the Physiology of Parasitism: XVIII. Pectic Enzymes secreted by Bacterium aroideae

Cell-free filtrates from cultures of Bacterium aroideae on asimple synthetic medium contained an enzyme, provisionally termeddepolymerase, which rapidly reduced the viscosity of pectinsolutions, and protopectinase which macerated slices of potatotuber tissue. These filtrates had little pectin-esterase activity. The activity of depolymerase was directly proportional to enzymeconcentration; the activity of protopectinase was approximatelyproportional to the square root of the enzyme concentration.Crude solutions were partially purified by acetone or ethanolprecipitation; ammonium sulphate was less satisfactory as aprecipitating agent. Enzyme preparations which rapidly reduced the viscosity of pectinand pectate solutions were relatively inactive when assayedfor polygalacturonase. activity by measuring reducing groupsliberated. After prolonged incubation some 20 and 40 per cent.hydrolysis of solutions of pectin and pectate respectively wasobtained. The pH optimum for depolymerase activity was near 9.0, the enzymewas activated by Ca⁺⁺ but not by a number of other cations;the loss of activity following dialysis was largely restoredby adding Ca⁺⁺. The enzyme was rapidly inactivated at temperaturesabove 60° C. and at pH 2.7. The properties of protopectinase generally resembled those ofdepolymerase. Analysis of the breakdown products following enzyme degradationof pectin and pectate solutions by paper chromatography showedthat galacturonic acid was not produced but that a number ofother products were formed, including one of fairly low molecularweight. The differences between the pectic enzymes of B. aroideae andthose from other sources, and the possible identity of depolymerase,polygalacturonase, and protopectinase are discussed.     

Permeability Changes in Tissues and Other Effects of Cell-separating Solutions from Soft Rots Caused by Corticium praticola and Erwinia atroseptica

Cell-free extracts from soft rots of potato tubers caused byErwinia atroseptica and Corticium praticola readily caused cellseparation and loss of electrolytes in discs of potato tubers.Both were most rapid at pH and Ca⁺⁺ ion concentration optimalfor the activity of a pectate trans-climinase in the E. atrosepticaextract and a pectate polygalacturonase in the C. praticolarot extract. Permeability changes and killing of protoplastsbut not cell separation were delayed when solutes at plasmolysingconcentrations were added to the solutions of the cell-separatingenzymes. The role of these enzymes in the permeability changesand killing of protoplasts is discussed.     

A Cell Wall-degrading {alpha}-1,3-arabinofuranosidase Produced by the Potato Dry Rot Pathogen Fusarium caeruleum (Lib.) Sacc.

Fusarium caeruleum produced an arabanase which showed maximalactivity at pH 3·5 and which liberated arabinose andits oligomers from -l,3-arabinofuranan and isolated potato tuberparenchyma cell walls. This enzyme was most active in dry rotlesions produced in susceptible potato tubers by a fast-growingisolate of F. caeruleum. This arabanase is not of primary importancein causing tissue maceration, protoplast death or electrolyteloss but it may play a secondary role in these processes.     

An Analysis of the Mussell and Morre Quantitative Bioassay for Polygalacturonases using Pectate trans-eliminase from Erwinia atroseptica

The Mussell and Morré method for estimating the activityof chain-splitting pectic enzymes was tested with sections ofparenchyma from cucumber fruit and potato tubers. It provedto be a very sensitive test for the pectate trans-eliminaseof Erwinia atroseptica, between 10 and 100 times as sensitiveas a viscometric method based on degradation of polypectate.Loss of fresh weight was readily detected within a few minutesafter immersion in enzyme solution and continued until the sectionof tissue had lost coherence. For some time after immersion,loss of weight was mainly due not to loss of cells from thesurface of the section but to loss of water and solutes, presumablybecause of the effect of the trans-eliminase in decreasing thepermeability of the protoplasts. In the later stages loss ofcells became more important. Potato sections lost weight atabout half the rate of cucumber sections but the rate of increaseof absorbance at 235 nm of the ambient solution was about athird higher for potato than for cucumber. Potato sections lostelectrolytes much more rapidly than did cucumber sections. Thesedifferences probably reflect, at least in part, the much higherwater content of cucumber sections. The advantages of the Mussell and Morré method are itssensitivity and the fact that the natural substrates of chain-splittingenzymes can be used for assessing activity.     

PECTIC ENZYMES SECRETED BY VERTICILLIUM DAHLIAE AND THEIR ROLE IN THE DEVELOPMENT OF THE WILT DISEASE OF COTTON

An isolate of Verticillium dahliae obtained from Uganda was highly virulent to young cotton plants under greenhouse conditions. A hyaline variant which often appeared in culture was as virulent as the parent isolate, but preliminary experiments indicated that it did not survive as long in unsterile soil. The parent isolate grew rapidly in cotton plants after root inoculation and was isolated from stems and leaves well before the appearance of disease symptoms visible to the naked eye.
Protopectinase was produced in the absence of pectic materials, but more active preparations were obtained when media contained pectic substances. In general, there was a close correspondence between the protopectinase activity of culture filtrates and the toxicity of these filtrates to parenchymatous cells. Some separation of the two activities was obtained by heating enzyme solutions or by plasmolysing the test tissue.
Protopectinase solutions had little pectinesterase activity but rapidly reduced the viscosity of solutions of pectic substances. In general, the properties of protopectinase and the viscosity-reducing enzyme were similar.
Young cotton shoots wilted rapidly when placed in cell-free filtrates from cultures of the pathogen. Wilting was delayed under conditions unfavourable for transpiration. Evidence was obtained which showed that wilting was caused by the uptake of thermostable compounds of high molecular weight which impeded the upward flow of the vascular sap. Pronounced vascular browning was obtained only when solutions containing protopectinase were used. Wilting and vascular browning were obtained with solutions having little pectinesterase activity; in contrast, a solution having high pectinesterase activity produced relatively little vascular browning.     

An isoelectric focusing study of the effect of methyl-esterified pectic substances on the production of extracellular pectin isoenzymes by soft rot Erwinia spp.

The production of extracellular pectic isoenzymes by seven strains of soft rot bacteria, Erwinia carotovora subsp. carotovora, E.c. atroseptica and E. chrysanthemi , when grown in media containing four different pectic substances with different degrees of methylation or with potato tuber cell-wall extract was examined by isoelectric focusing activity staining. In addition to the isoenzymes of pectate lyase, polygalacturonase and pectin methyl esterase produced constitutively or following induction by polygalacturonic acid (PGA) and coded by known genes, between two and seven novel isoenzymes of the three enzymes with a wider pI range were apparently induced by the pectins and cell-wall extract. Pectin lyase, which is induced in vitro by DNA-damaging agents, was not produced in the absence of mitomycin C in a medium containing PGA but up to two isoenzymes were found with pectin or cell-wall extract. In contrast, cellulase isoenzyme production was not affected by pectin or cell-wall extract. A greater number of novel isoenzymes of all pectic enzymes except pectin lyase tended to be produced in media containing Link pectin, which is PGA methylated to 98%, than the other pectic substances and cell-wall extract. Pectate lyase and polygalacturonase were induced by pectin lyase-degraded products of highly methylated pectin but not by PGA in an E. chrysanthemi strain with all its known pei and peh genes mutated. The results suggest that the production of novel pectic isoenzymes could be related to the presence of CH⁺₃ groups and that their induction differs from that for isomers induced by PGA-degraded products and DNA-damaging agents or produced constitutively.     

Substrate Degradation Patterns of Polygalacturonic Acid Lyase from Erwinia carotovora and Bacillus polymyxa and Release of Phytoalexin-eliciting Oligosaccharides from Potato Cell Walls

The patterns of substrate degradation by purified pectate lyase(PGL) (E.C. 4.2.2.2 [EC] ) from Erwinia carotovora and Bacillus polymyxawere compared. Reaction products released by both enzymes frompotato cell walls, sodium polypectate and citrus pectin wereseparated by anion exchange chromatography using a TSK DEAE-5PWcolumn and measured quantitatively. The relative amounts ofoligomers released by both enzymes varied, especially the levelof unsaturated tetramers. Degradation patterns also varied accordingto the substrate used and results with citrus pectin suggestedthat methylation reduced the ability of E. carotovora PGL torelease wall fragments. Oligomers released from potato cell walls by E. carotovora PGLwere pooled separately and assayed for phytoalexin elicitoractivity using the soybean cotyledon bioassay. Fractions containingdeca- and undecagalacturonides had the highest elicitor activitywhen tested at 5.0µg of uronic acid per cotyledon. Key words: Pectic enzyme, elicitor, phytoalexin     

Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties

A mung bean (Vigna radiata) pectin acetyl esterase (CAA67728) was heterologously expressed in tubers of potato (Solanum tuberosum) under the control of the granule-bound starch synthase promoter or the patatin promoter in order to probe the significance of O-acetylation on cell wall and tissue properties. The recombinant tubers showed no apparent macroscopic phenotype. The enzyme was recovered from transgenic tubers using a high ionic strength buffer and the extract was active against a range of pectic substrates. Partial in vivo de-acetylation of cell wall polysaccharides occurred in the transformants, as shown by a 39% decrease in the degree of acetylation (DA) of tuber cell wall material (CWM). Treatment of CWM using a combination of endo-polygalacturonase and pectin methyl esterase extracted more pectin polymers from the transformed tissue compared to wild type. The largest effect of the pectin acetyl esterase (68% decrease in DA) was seen in the residue from this extraction, suggesting that the enzyme is preferentially active on acetylated pectin that is tightly bound to the cell wall. The effects of acetylation on tuber mechanical properties were investigated by tests of failure under compression and by determination of viscoelastic relaxation spectra. These tests suggested that de-acetylation resulted in a stiffer tuber tissue and a stronger cell wall matrix, as a result of changes to a rapidly relaxing viscoelastic component. These results are discussed in relation to the role of pectin acetylation in primary cell walls and its implications for industrial uses of potato fibres.     

Pectic enzymes associated with Penicillium digitatum decay of citrus fruit

Pectin methylesterase was the only pectic enzyme detectablein extracts from rind of sound or Penicillium digitatum-infectedoranges. No pectic enzymes were detected in juice from soundor infected fruit. Extracts from infected rind, and juice frominfected fruit, had macerating activity. Chromatographic analysesof rind extracts, and juice from infected fruit, showed galacturonicacid as a possible product of the degradation of pectic substances.Orange juice contained a thermo labile inhibitor of pectic ‘chain-splitting’,and macerating enzymes.     

Fungal Assciations: II. Cultural Studies on Rhizoctonia solani Kuhn, Fusariutn solani Snyder and Hansen, and Other Fungi, and Their Interactions

Several types of interaction between Rhizoctonia solani, Fusariumsolani, and Phoma foveata were found when these fungi were grownon potato-dextrose agar. After being used by Rhizoctonia a potatomash medium gave better growth of Rizoctonia and Fusarium thanit did when the medium was initially used by Fusarium; and thiswas so whether the reaction of the spent medium was readjustedor not. It is suggested that potato mash medium used by Fusariumcontains a thermostable factor(s) affecting the subsequent growthof Rhizoctonia or Fusarium. The range of pH values suitable for Rhizoctonia growth was narrowerthan that for Fusarium, optimum values being approximately 5•9for the former and 7•8 for the latter. In mixed culturesof the two fungi on potato-dextrose agar adjusted to differentpH values, the fungus for which the reaction of the medium wasmore suitable usually became visually predominant after sometime. A study of various carbon sources showed that poor growth ofRhizoctonia was obtained when pectin was used as the sole sourceof carbon. On a pectin-agar medium, the rate of growth of aRhizoctonia colony increased on the sector which lay towardsan adjacent Fusarium colony; also, after the two fungi camein contact, there was more rapid growth of Rhizoctonia roundthe Fusarium colony than elsewhere. On a synthetic liquid mediumwith pectin as the carbon source better Rhizoctonia growth wasobtained when Fusarium-spent medium was added to it than whenRMzoctoma-spent medium was added. Rhizoctonia showed partial deficiencies in thiamine, biotin,and inositol. Both the extract of Fusarium mycelium, grown onvitamin-free medium, and the Fusarium-spent medium, stimulatedthe growth of Rhizoctonia on vitamin-free medium.     

Distribution of methoxyl groups in apple pectic substances

The distribution of methoxyl groups in apple pectic substances was investigated by means of fractionation on ion-exchange and gel-filtration columns and by means of degradation of pectin fractions by pectin lyase and pectate lyase. Pectin fragments thus obtained were fractionated by gel-permeation chromatography and high-pressure liquid chromatography. It was concluded that a heterogeneous intermolecular distribution of the methoxyl groups exists with peaks at degrees of esterification of about 50%, 70% and 95%. The intramolecular distribution of the methoxyl groups cannot be distinguished from a random distribution. Since plant pectin esterases cause a blockwise de-esterification, it is unlikely that the biosynthesis of apple pectic substances passes through a stage of 100% esterification after which partial de-esterification by pectin esterase occurs.     

Physiological Basis for a Cycloheximide-induced Soft Rot of Potatoes by Pseudomonas fluorescens

Cycloheximide renders discs of potato tissue (Solanum tuberosum,cultivar Kennebec) susceptible to soft rot by a non-pathogenicisolate of Pseudomonas fluorescens. Pectate lyases (E.C. 4.2.99.3 [EC] )are the dominant extracellular macerating agents produced bythe test organism. Potato discs aged 24 h become resistant tomaceration by purified lyase preparations. Cycloheximide blocksthe development of resistance by inhibiting suberization. Thesite of inhibition is thought to be the cycloheximide-sensitivesynthesis of phenylalanine ammonia-lyase (E.C. 4.3.1.5 [EC] ) in potatodiscs. This enzyme is necessary for production of phenolic precursorsof suberin. Comparison of tissue from a number of potato cultivarscorrelates the synthesis of phenylalanine ammonia-lyase withresistance of discs to attack by the Pseudomonad. Resistance of potato tissue to pectate lyase is also affectedby intrinsic reactions not involving suberization. Resistanceincreases in fresh unsuberized discs when tubers are transferredfrom cold storage to room temperature before use. Resistancedecreases rapidly when tubers are transferred back to the cold.The intrinsic resistance appears to increase in the surfacelayer of cells in ageing discs. It is estimated that intrinsicreactions and suberization contribute equally to resistanceof aged discs to pectate lyase maceration.     

Studies in the Physiology of Parasitism: XVII. Enzyme Secretion by Strains of Bacterium carotovorum and other Pathogens in relation to Parasitic Vigour

1. When tested on potato tubers and roots of turnip, swede,and carrot, the organisms Bacterium aroideae, B. carotovorum(i), B. carotovorum (ii), and Phyto-monas carotae show diminishingpathogenicity in the order named. Attack on potato is greatestat 25–35° C., and falls off gradually from 20°to 10°, below which it ceases. 2. The susceptibility of potato tubers rises as they grow tofull size but falls during the dormant period. With the activeparasite B. avoideae there is a considerable rise in susceptibilityas the tuber reaches the storage of sprouting. 3. Susceptibility of potato tubers is increased by storage forsome time at an elevated temperature (c. 35° C.), or byrasing their water-content, either by soaking or by direct injection,and especially by the latter. Similar responses to these treatmentsare given by enzymic preparations of the bacteria. 4. The greater pathogenicity of B. avoideae than of the carotovorumstrains is associated with a greater rate of multiplicationof the former under comparable conditions and with a greatertendency of teh latter to an acid-forming type of metabolismwhich is unfavourable to multiplication of the organism andto the activity of its enzyme.     

Pectic substances,pectic enzymes and haze formation in fruit juices

The pectic substances, located primarily in the middle lamella between cells in higher plant tissues, are complex polysaccharides. They include the negatively charged rhamnogalacturonans, and the neutral arabinogalactans I and II and l-arabinans. These polysaccharides add viscosity to juices but may also form hazes and precipitates and retard maximum recovery of juices from the fruit. The rhamnogalacturonans are degraded by the enzymes pectin methylesterase and polygalacturonase normally present in plant tissues and by these enzymes and pectate lyase in microbially derived commercial pectic enzymes added during processing. The presence of a?abinofuranosidase, which degrades l-arabinans, in commercial pectic enzyme preparations, can cause haze formation in juices such as apple and pear.     

Characterization of pectin methyltransferase from soybean hypocotyls

Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycinemax Merr.). The enzyme was maximally active at pH 6.8 and 35–40 °C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 μg · ml⁻¹, respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [¹⁴C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo. Received: 10 September 1999 / Accepted: 11 October 1999     

Cell elongation and metabolic turnover of the cell wall as affected by auxin and cell wall degrading enzymes

A cell wall fraction (pectic substances) of oat coleoptile segmentsfed with ¹⁴C-glucose contained more radioactivity under theeffect of auxin than did the control. When labeled segmentswere grown for 6 hr in auxin or glucanase solution the labelin the hemicellulose fraction decreased as growth increased.ß-1,3-Glucanase prepared from the culture of a fungus,Sclerotinia libertiana, induces elongation of segments of thepea stem and the oat coleoptile. Traces of cellulase and pectinmethylesterase contaminating the enzyme preparation are notresponsible for the stimulatory effect. Cellulase seemed tobe rather inhibitory and pectin methylesterase showed only aslight effect on coleoptile elongation. A possible relationshipbetween the metabolic turnover of hemicellulosic polysaccharideand cell wall extension is suggested. (Received February 5, 1968; )     

Studies in the Physiology of Parasitism: XXV. Some Propertles of the Pectic Enzymes secreted by Pythium debaryanum

The pectic enzymes of Pythium debaryanum have been comparedwith those from two other soft-rot causing organisms, Erwiniaaroideae and Botrytis cinerea, by their effects (viscosity reduction,acid production, and reducing power) on 1 per cent, solutionsof (a) high methoxyl pectin, (b) sodium polypectate, and (c)sodium pectate (2 types). The Pythium debaryanum preparationdiffered particularly in giving no increase in reducing poweror evidence of galacturonic-acid-like derivatives. It maceratedthe walls of potato-tissue freely but had nopolygalacturonaseor pectinesterase activity.     

Metabolism of S-methylcysteine and pectin esterification in Chinese cabbage, Brassica pekinensis Rupr.

S-Methyl-L-cysteine was actively metabolized in Chinese cabbageand carbon from its methyl group was distributed into both thesoluble and insoluble fractions. The high incorporation of ¹⁴Cfrom the methyl group into the insoluble fraction after administeringof S-methyl-L-cysteine-¹⁴CH₃, and our previous results thatS-methyl-L-cysteine is demethylated to give cysteine, suggestthat S-methyl-L-cysteine might act as a methyl donor in Chinesecabbage. To obtain evidence for this possibility, incorporationof the methyl-¹⁴C of S-methyl-L-cysteine into methyl estersof pectic substances was investigated. Most of the ¹⁴C incorporatedinto pectic substances was liberated by treatment with dilutealkali and pectin esterase. The results show that S-methyl-L-cysteineacts as a methyl donor to form pectin ester. (Received October 12, 1971; )