Types of Rot, Rate of Rotting, and Analysis of Pectic Substances in Apples Rotted by Fungi

The rate at which the fungi grew through apples was determinedin various ways and used to estimate their rate of linear advance.Five fungi were studied;they were Sclerotinia fructigena (firm-browncoloured rot, rapid growth through apples), Botrytis cinerea(soft, light-brown coloured rot, rapid growth through apples),Psyrenochaeta furfuracea (firm to soft rot, variable in colourbut generaly dark, slow growth through apples), Penicilliumexpansion A (soft, white rot, slow growth through apples) andPenicillium expansum B (soft, white rot, medium rate of growththrough apples). S. fructigena which had the highest rate oflinear advance which was about three times that of P. furfuraceawhich had the lowest. Methods for extracting different types of pectic substancesfrom sound and rotted tissues are described, and details aregiven of a rapid and reasonably accurate colorimetric methodof determining the anhydrogalacturonic acid content of theseextracts. The firm-rot fungi reduced the water-insoluble pecticsubstances by 10–20 per cent., but the soft-rot fungicaused much larger changes, up to 70 per cent. being degraded,The firm-rot and soft-rot fungi had different effects on thepectic substances insoluble in dilute acid but soluble in dilutealkali. The soft-rot fungi had little effect on these substances,or reduced their concentration, whereas the firm-rot fungi causedsubstantial increases compared with sound tissue. These resultsare considered in terms of pectic enzyme activity. Analysisof extracts by paper chromatography showed that galacturonicacid, absent from sound tissue, was present in each type ofrotted tissue. Di- and tri-galacturonic acids were present inrots caused by P. expansum, and these rots probably also containedproducts from the break-down of other polysaccharides.     

Studies in the Physiology of Parasitism: XX. The Pathogenicity of Botrytis cinerea, Sclerotinia fructigena, and Sclerotinia laxa, with special reference to the part played by pectolytic enzymes

1. Though Sclerotinia fructigena, S. laxa, and Botrytis cinereacause rotting of apple tissue and death of the protoplasts,little or no pectolytic activity was detectable in extractsof the rotted tissue. 2. Pectic materials were extracted from normal and parasitizedapple tissue in three fractions and precipitated as calciumpectate. There was a loss of total, total insoluble, and solublepectic substances in the invaded tissues. This was most markedwith B. cinerea and S. laxa and least with S. fructigena. 3. Pectolytic activity was measured by methods involving (a)maceration of plant tissues, (b) viscosity and reducing groupdeterminations in pectic substrates, (c) increase in acidityof pectin. By these methods it was shown that pectolytic enzymeswere produced by all three fungi in synthetic media. With S.fructigena, which was the only fungus studied in detail, replacementof glucose by pectin increased the formation of pectolytic enzymes. 4. When various apple extracts were used as culture media, littleor no pectolytic activity was detectable. With all three fungithe presence of apple juice in a culture medium, which by itselfwas suitable for enzyme formation, resulted in the suppressionof pectolytic activity. 5. Oxidized apple juice had a pronounced effect in deactivatingcertain pectolytic enzymes, an effect which was especially markedwith B. cinerea. This points to an interaction between the pectolyticand oxidizing systems and introduces a new line of approachto the study of the biochemical interaction between host andparasite.     

The Effects of Pectic Enzymes and Phosphatidases on Potato Tuber Tissue

Extracts from rots of potato tubers caused by Erwinia atrosepticaand Corticium praticola were fractionated by precipitation withammonium sulphate and by gel filtration. For the various fractionsof the E. atroseptica extracts there was a close relation betweenthe activity of pectate franj-eliminase and capacity to increasethe permeability of protoplasts as assessed by loss of electrolytes.There was no such relation with phosphatidase acting on lecithin. For certain fractions of C. praticola extracts there was a similarclose relation between increase in permeability and activityof a polygalacturonase but for other fractions with low polygalacturonaseactivity there was a better relation with phosphatidase thoughall fractions that caused increase in permeability did havesome polygalacturonase activity. Phosphatidases which probablyplay no part in the killing of cells in E. atroseptica rotsmay, therefore, have some role in the killing of cells in C.praticola rots though they are likely to be less important thanpectic enzymes. Extracts from E. atroseptica rots caused marked increases inuptake of oxygen by tuber discs. Dialysis decreased and heatingeliminated this increase and had corresponding effects on permeability.However, after fractionation with ammonium sulphate, fractionswith high trans-eliminase activity had little effect on oxygenuptake whereas fractions with low trans-eliminase had littleeffect on permeability and greatly increased oxygen uptake. Similar results were obtained with C. praticola rot extracts.In contrast, nigericin and Triton X-100 both increased permeabilityand caused large increases in oxygen uptake The significance of these results is discussed especially inrelation to the killing of protoplasts by extracts from bothtypes of rot.     

The infection of apples, cv. Bramley's Seedling, by Nectria galligena Bres

In laborator experiments germinating conidia penetrated leticels and wounds but not the intact surfaces of apples. Date of harvest had no significant effect on the numbers of apples infected with Nectria galligena but the earliest picks rotted first in barn store. Inoculations of unpicked apples resulted in small arrested lesions which only developed into progressive rots after a considerable period in store. Rots developed mosy quickly from inoculations made between mid-August and mid-September. The size of rot increased with spore number and many inoculations with 10–100 conidia remained as arrested lesions. Arrested lesions developed 10–15 days after unripe apples were inoculated and consisted of a zone of fungal colonization surrounded by suberized, necrotic cells in which compounds toxic to both N. galligena and Penicillium expansum were detected. No antifungal compounds were found in progressive rots to mature apples or in healthy apples of any age. Antifungal activity, measured by inhibition of P. expansum, was greatest 15–20 days from inoculation of unripe apples with N. galligena but decreased after a total of 35 days incubation at 20 d? C. Much less antifungal activity was produced in ripe or desert apples.     

The use of a surface coating to ameliorate the rate of spread of post-harvest fungal diseases of top fruit

The impact of TAL Pro-long coatings (a sucrose-ester composition) on the development of some common post-harvest fungal rots of top fruit is reported. Coatings were not observed to be fungistatic but did diminish the incidence of fungal infections on Conference pears held in cold store. At higher temperatures coatings modified the spatial distribution and reduced the rate of spread of lesions within apples and pears. In comparison to experimental controls, TAL Pro-long had a greater effect on those rots caused by Botrytis cinerea Fr., Monilinia fructigena (Aberh. & Ruhl.) Honey and Rhizopus nigricans Lind. than those of Alternaria alternata Fr., M. laxa (Aberh. & Ruhl.) Honey and Penicillium expansum Link em. Thom. The efficacy of the treatments was related to the concentration of the preparations applied, the variety of fruit, the timing of application and the temperature to which produce were subsequently exposed.     

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.     

A Role for Pectinase and Protease Inhibitors in Fungal Rot Development in Tomato Fruits

B. cinerea and C. atramentarium rotted wound-inoculated green tomato fruits and wounded or intact ripe fruits while G. cingulata developed rots only in ripe fruits. Pectic en-zymes were extracted from the fruit tissue rotted by B. cinerea and C. atramentarium but no pectic enzymes attributable to the fungus were detected in ripe fruits rotted by G. cingulata. G. cingulata produced endo-PG and endo-PL in vitro, C. atramentarium produced endo-PL in vitro and in vivo and B. cinerea produced exo-PG in vitro and in green fruits but endo-PG and endo-PL in ripe fruits. Well ripened tomato fruits contained high levels of endogenous PG. All three fungi produced proteolytic enzymes in vitro and in vivo. Proteases produced by G. cingulata and C. atramentarium had optimum activity at pH 9 to 10 and were not trypsin-like or chymotrypsin-like in nature. Protease produced by B. cinerea had optimum activity at pH 7 and showed both trypsin and chymotrypsin-like activity. Proteins extracted from the cell walls of tomato fruits inhibited both the endo-PG and endo-PL produced by G. cingulata and the endo-PL produced by B. cinerea but did not in-hibit the activity of PGs produced by B. cinerea, the endo-PL produced by C. atramentarium or the endogenous PG from tomato fruits. The cell wall proteins also contained trypsin and chymotrypsin inhibitor activity which inhibited 70 % of the activity of the protease produced by B. cinerea, but had little effect on the proteases produced by G. cingulata, C. atramentarium or the tomato endogenous protease. Enzymes produced in vitro by G. cingulata macerated green tomato tissue more slowly than enzymes produced in vitro by C. atramentarium and B. cinerea and the rate of mation was further reduced in the presence of added cell wall proteins. Excess inhibitor of the little effect on the rate of maceration by the enzymes produced by C. atramentarium of the cinerea.     

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.     

Biological control of postharvest diseases of apples,peaches and nectarines by Bacillus subtilis S16 isolated from halophytes rhizosphere

Penicillium expansum, Botrytis cinerea and Colletotrichum acutatum are the most common postharvest pathogens of apples, peaches and nectarines. In this study, 96 bacteria were isolated from halophytes rhizosphere and assayed for biocontrol activity under in vitro conditions. Among the 96 isolates tested, isolate S16 effectively inhibited the growth of P. expansum, B. cinerea and C. acutatum. The isolate S16 has reduced 78.33±1.53 to 82.98±2.13% of disease severity in apples, peaches and nectarines. Matrix-assisted laser desorption ionisation-time of flight mass spectrometry of the antifungal compounds revealed three lipopeptide complexes, namely surfactins, iturins and fengycins. Lipopeptides and hydrolytic enzymes produced by the isolate S16 play an important role in the antifungal activity. Polymerase chain reaction analysis using ituD, srfAD, fenD and fenE gene-specific primers showed that the isolate S16 carry sequences similar to ituD, srfAD, fenD and fenE genes. Based on the 16S rDNA sequencing, the effective isolate S16 was identified as Bacillus subtilis.     

Role of Cell-wall-degrading Enzymes in the Development of Leaf Spots Caused by Ascochyta pisi and Mycosphaerella pinodes

Extracts of limited and spreading lesions caused by Mycosphaerellapinodes on detached pea leaflets contained proteolytic, cellulolytic,and pectolytic enzymes although only in spreading lesions wasthere much degradation of cell walls. The brown tissue fromlimited M. pinodes lesions was resistant to maceration by enzymesfrom spreading lesions. Limited lesions contained water-soluble,95 per cent ethanol insoluble, partially dialysable, inhibitorsof pectin transeliminase which is probably the macerating enzyme. Green, spreading M. pinodes lesions developed only on leafletsfloating on water. Growth of these lesions was accompanied bycontinous loss of phenolic substances to the water while thephenol content in infected tissue remained similar to that inuninoculated controls. In contrast, the phenol content in mature,limited M. pinodes lesions on leaflets suspended just abovethe water level was about four times that in healthy tissue.It is suggested that loss of phenolics from floating leafletsprevents tissue browning and the development of resistance ofthe cell walls to maceration. But this type of resistance doesnot appear to be a major factor in the limitation of lesionson suspended tissue. Extracts of limited Ascochyta pisi lesions on leaflets floatingon water contained pectolytic and hemicellulolytic enzymes.Some cellulase (Cx) activity was detected although there waslittle evidence of cellulose degradation in cell walls in infectedtissue. The nature of the macerating factor remains uncertainbut it was found that extracts from lesions contained inhibitorsof pectic enzymes and that tissue just beyond that colonizedby the fungus was resistant to maceration; this resistance isprobably important in restricting the growth of the pathogenin the leaf.     

Enzymic analysis of cell wall structure in apple fruit cortical tissue

Galactanase from Phytophthora infestans and an arabinosidase isoenzyme from Sclerotinia fructigena attacked the cortical cell walls of apple fruits liberating galactose and arabinose residues, respectively. Other arabinosidase isoenzymes from S. fructigena attacked cell walls very slowly. A S. fructigena polygalacturonase isoenzyme liberated half of the uronic acid residues with few associated neutral residues, while a second polygalacturonase isoenzyme released more uronic acid with a substantial proportion of arabinose and galactose and lesser amounts of xylose, rhamnose and glucose; reaction products of this enzyme could be further degraded by the first isoenzyme to give high MW fragments, rich in arabinose with most of the xylose, rhamnose and glucose, and low MW fragments rich in galactose and uronic acid. Endoglucanase from Trichoderma viride released a small proportion of the glucose residues from cell walls together with uronic acid, arabinose, xylose and galactose; more extensive degradation occurred if walls were pre-treated with the second polygalacturonase isoenzyme. Endoglucanase reaction products were separated into a high MW fraction, rich in arabinose, and lower MW fractions rich in galactose and glucose residues. The high MW polygalacturonase and endoglucanase products could be degraded with an arabinosidase isoenzyme to release about 75% of their arabinose. Cell walls from ripe fruit showed similar susceptibility to arabinosidase and galactanase to those from unripe apples. Cell walls from fruit, ripened detached from the tree were more susceptible to degradation by polygalacturonase than walls from unripe fruit or fruit ripened on the tree. Endoglucanase released less carbohydrate from ripe fruit cell walls than from unripe fruit cell walls.     

Growth of Lesions Caused by Botrytis fabae on Field Bean Leaves in Relation to Foliar Bacteria, Non-enzymic Phytotoxins, Pectic Enzymes and Osmotica

Extracts from spreading chocolate spot lesions contained a heat-stable,water-soluble, ether- or ethanol-insoluble phytotoxic fraction.Elution from Sephadex G75 indicated that the molecular weightsof the toxic compounds were between 10000 and 30000 daltons.The toxins were adsorbed on DEAE Sephadex and eluted from itwith 0.1–0.2 M NaCl. Toxic activity was enhanced in solutionswith low osmotic potentials similar to those found in lesions.Controlling the growth of contaminating bacteria in lesionswith antibiotics appeared to reduce the levels of heat-stabletoxins extracted and to reduce the rate of increase in lesionsize. There were high levels of polygalacturonase (PG) activity inlesion extracts; large quantitites of PG, but little or no trans-eliminases,were produced by Botrytis fabae in liquid culture. A pectolyticstrain of Bacillus lentus was associated with trans-eliminasesin lesion extracts, and produced transeliminases, but not PG,in liquid culture. Activities of both heat-stable phytotoxins and of pectic enzymesmay depend on fungal isolate and types and populations of contaminatingbacteria. Botrytis fabae L., Bacillus, Vicia faba L., phytotoxins, pectic enzymes, chocolate spot     

Changes in Ulrastructure following Fungal Invasion and the Possible Relevance of Extracellular Enzymes

Changes in fine structure of fruit tissues were examined byelectron microscopy for the following host-parasite relationships:Sclerotinia fructigena on apple and pear, S. sclerotiorum oncucumber, and Phytophthora palmivora on egg plant. While S.fructigena caused localized degradation of the wall region,S. sclerotiorum caused extensive wall degradation, and infectionby P. palmivora was characterized by disintegration of plasmalemmaand tonoplast. These effects appeared to be correlated respectivelywith pectolytic enzyme activity in vivo with S. fructigenaycellulolytic activity with S. sclerotiorum, and the in vitrolysis of membranes by culture filtrates of P. palmivora.     

Multiplicity of Cell Wall Degrading Glycosidic Hydrolases Produced by Apple Infecting Botrytis cinerea

It was shown that Botrytis cinerea, an isolate infecting apples, secreted in vivo and in culture a variety of glycosidic hydrolases with substrate specificity towards the polysaccharides of cell walls. The following enzymes were partially separated by column chromatography on DEAE-Sepharose CL-6B: two cellulases, three xylanases, one arabinanase, polygalacturonase, β-glucosidase, β-xylosidase, β-galactosidase, β-mannosidase and α-galactosidase. The activity of glycosidic hydrolases tested was strongly inactivated by NBS and weaker by PCMB, tetranitromethan, dibromoacetophenon and Fe³+, The results indicate synergistic action of the obtained cellulase, xylanase, polygalacturonase and arabinanase in apple cell wall degradation.     

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.     

Effect of pectin on pectinases in autolysis of Botrytis cinerea

Pectic activity in autolyzed cultures of Botrytis cinerea in a medium with and without pectin was similar, but in the medium with pectin maximal activities occurred in younger cultures. The pectic activities found were polygalacturonase, polymethylgalacturonase, endo activity (pectin as substrate) and pectin lyase. The molecular weights of polygalacturonase, polymethylgalacturonase and endo activity (pectin as substrate) were 36000, 33000 and 30200 daltons respectively, and the molecular weight of pectin lyase was 18200 daltons. By gel electrophoresis four different pectic activities were detected, three in the top of the gel and one in the bottom. Two enzymes were characterized, the polygalacturonase activity (first band in the top) inhibited by Ca⁺⁺ and the pectin lyase activity (in the bottom) which was not inhibited by Ca⁺⁺. These enzymes are not induced by the presence of pectin in the medium during degradation of Botrytis cinerea.     

Potential biocontrol activity of a strain of Pichia guilliermondii against grey mold of apples and its possible modes of action

The efficacy of Pichia guilliermondii strain M8 against Botrytis cinerea on apples was evaluated under storage conditions, and its possible modes of action were investigated both in vitro and in vivo experiments. After storage at 1 °C for 120 days, M8 reduced grey mold incidence from 45.3% (control) to 20.0%. In apple juice medium (AJM) and in wound-inoculated apples, M8 at 10⁹ and 10⁸ cells ml⁻¹ inhibited the spore germination of B. cinerea and the grey mold development. When co-culturing B. cinerea in vitro or in vivo in the presence of the yeast, neither inactivated cells nor culture filtrate of the yeast had any effect on spore germination or germ tube elongation. In AJM, the spore germination was significantly recovered by the addition of 1% glucose, sucrose and fructose, or 0.5% and 1% of (NH₄)₂SO₄, phenylalanine and asparagine. When the pathogen and the yeast were co-incubated in apple wounds with addition of the same nutrients, the inhibition of rots was significantly reduced by the supplemental nutrients. Light microscopy revealed that the yeast strongly adhered to the hyphae and spores of B. cinerea. M8 produced hydrolytic enzymes, including β-1,3-glucanase and chitinases in minimal salt media with different carbon sources. Pretreatment with M8 at 10⁸ cells ml⁻¹ followed by washing, significantly reduced grey mold lesions, suggesting an induction of defense responses. Direct attachment, competition for nitrogen and carbon sources, secretion of hydrolytic enzymes and induction of host resistance play a role in the biocontrol mechanism of P. guilliermondii M8 against B. cinerea.     

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.     

The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases

Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. PGIPs display differential inhibition towards PGs from different fungi, also towards different isoforms of PGs originating from a specific pathogen. Recently, a PGIP-encoding gene from Vitis vinifera (Vvpgip1) was isolated and characterised. PGIP purified from grapevine was shown to inhibit crude polygalacturonase extracts from Botrytis cinerea, but this inhibitory activity has not yet been linked conclusively to the activity of the Vvpgip1 gene product. Here we use a transgenic over-expression approach to show that the PGIP encoded by the Vvpgip1 gene is active against PGs of B. cinerea and that over-expression of this gene in transgenic tobacco confers a reduced susceptibility to infection by this pathogen. A calculated reduction in disease susceptibility of 47–69% was observed for a homogeneous group of transgenic lines that was statistically clearly separated from untransformed control plants following infection with Botrytis over a 15-day-period. VvPGIP1 was subsequently purified from transgenic tobacco and used to study the specific inhibition profile of individual PGs from Botrytis and Aspergillus. The heterologously expressed and purified VvPGIP1 selectively inhibited PGs from both A. niger and B.␣cinerea, including BcPG1, a PG from B. cinerea that has previously been shown to be essential for virulence and symptom development. Altogether our data confirm the antifungal nature of the VvPGIP1, and the in vitro inhibition data suggest at least in part, that the VvPGIP1 contributed to the observed reduction in disease symptoms by inhibiting the macerating action of certain Botrytis PGs in planta. The ability to correlate inhibition profiles to individual PGs provides a more comprehensive analysis of PGIPs as antifungal genes with biotechnological potential, and adds to our understanding of the importance of PGIP:PG interactions during disease and symptom development in plants.Dirk A. Joubert and Ana R. Slaughter contributed equally to this work.     

Effects of ozone treatment on the quality of kiwifruit during postharvest storage affected by Botrytis cinerea and Penicillium expansum

The objective was to reveal the effects of ozone treatment on quality maintenance and resistance to Botrytis cinerea and Penicillium expansum in kiwifruit during postharvest storage. Kiwifruits were treated with 79.44 ppm gaseous ozone for 1 hr once a day for 7 day at 0°C to determine the effects of ozone treatment on the quality and disease incidence caused by B. cinerea and P. expansum in vivo and the growth of B. cinerea and P. expansum in vitro. Ozone treatment significantly reduced the disease incidence of kiwifruit and inhibited the mycelial development and spore germination of B. cinerea and P. expansum. High levels of fruit firmness and titratable acidity were maintained in the ozone‐treated kiwifruit, and the activities of the defence‐related enzymes were remarkably enhanced. Therefore, ozone treatment may be an effective method to maintain the quality of kiwifruit and control its decay during postharvest storage.