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.     

Pectic Enzymes and Phenolic Substances in Apples Rotted by Fungi

The activities of pectic enzymes in extracts from sound applesand from apples rotted by different fungi are described. Sclerotiniafructigenaand Botrytis cinerea rots had little or no polygalacturonaseor macerating enzyme activity, but Penicillium expansum rotswere very active in these respects. Extracts from each of therots had very high pectinesterase activity, and contained galacturonicacid. None of the rots had any cellulase activity. Each of thefungi produced polygalacturonase, macerating enzymes, and pectinesterasein liquid media. The effects of adding extracts of apples tothese media are described. Filtrates from cultures of S. fructigenaand P. expansum liberated galacturonic acid from apple fruitfibre which had been thoroughly extracted with cold water. The phenolic jsubstances present in healthy and rotted tisueswere estimated. B. cinerea and S. fructigena rots containedvery little, but P. expansum rots contained as much as healthytissue which had been allowed to brown. An extract of healthyapple tissue reduced the activity of the polygalacturonase ina culture filtrate of S. fructigena. The substances responsiblefor this were tentatively identified as leuco-anthocyanins whichhad been changed to other compunds following the action of polyphenoloxidase.Thej significance of these results is discussed.     

Infection of potato tubers with soft rot bacteria

Stolons attached to developing potato tubers were inoculated with the soft rot bacterium Erwinia carotovora var. atroseptica. Almost all the stolons rotted, but soft rots developed in less than 10% of new tubers; the bacterium was isolated later from these tubers. No rots developed in the other tubers but the bacterium was later isolated from about half of them. It could not be isolated from tubers attached to inoculated stolons where the rot on them did not extend to the tuber or from tubers attached to stolons that were not inoculated though many of these rotted. The bacterium was reisolated from almost all arrested lesions in tubers inoculated 8 month earlier with E. carotovora var. atroseptica. Blackleg did not develop from plants grown fom these tubers under various soil conditions. It did develop in a large proportion of plants from tubers inoculated shortly before planting and grown in cool, wet soil. Less than 1% blackleg developed in plants grown from tubers from plants with blackleg or from plants immediately adjacent. The presence of pectolytic bacteria and E. caratovora var. atroseptica in seed and new tubers was investigated during June, July and August. Although E. caratovora var. atroseptica was obtained from c. 40% tubers, only c. 0·3% of c. 8400 plants developed blackleg. The bacterium was isolated from only three of 160 new tubers sampled during the summer.     

Some Biochemical Characteristics of Guard Cell and Mesophyll Cell Protoplasts from Commelina communis L.

Guard cell and mesophyll cell protoplasts of Commelina communisL., were isolated and used to investigate their various biochemicalcharacteristics. Contamination of the samples by other celltypes was very low and viability of the protoplasts, assessedby the use of neutral red, Evans blue and fluorescein diacetate,was high (89–98%). Mesophyll cell protoplasts containedmore chlorophyll (x 47), more soluble protein (x 10), more totalN (x 36) and more DNA (x 9) than guard cell protoplasts. Theabsorption spectra of protoplast extracts were similar for bothcell types except that below 400 nm there was a large increasein absorption by the guard cell protoplast extract. In guardcell protoplast extracts, high levels of activity of phosphoenolpyruvatecarboxylase (E.C. 4.1.1.31 [EC] ), NAD malate dehydrogenase (E.C.1.1,1.37), NADP malic enzyme (E.C. 1.1.1.40 [EC] ) and carbonic anhydrase(E.C. 4.2.1.1 [EC] ) were detected while only low levels of pyruvate-orthophosphatedikinase (E.C. 2.7.9.1 [EC] ) activity were detected. Glycollate oxidase(E.C. 1.1.3.1 [EC] ), ribulose-l,5-bisphosphate carboxylase (E.C 4.1.1.39 [EC] ),NADP malate dehydrogenase (E.C. 1.1.1.82 [EC] ) and NAD malic enzyme(E.C. 1.1.1.39 [EC] ) were not detected in guard cell protoplast extracts.High levels of ribulose-1, 5-bisphosphate carboxylase, glycollateoxidase, NAD malate dehydrogenase and carbonic anhydrase weredetected in mesophyll cell protoplast extracts which is typicalof C₃ plants. A pathway of carbon flow during stomatal openingand closing is proposed. Key words: Carbon metabolism, Commelina communis, guard cell protoplasts, mesophyll cell protoplasts, stomata     

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.     

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     

Isolation and Identification of Nicotinic Acid as a Flower-Inducing Factor in Lemna

Extracts of flowering plants of the long-day plant Lemna gibbaG3 and the short-day plants Lemna paucicostata 151 and 381 weretested on L. paucicostata 151 for flower-inducing activity.Crude extracts failed to show any activity but after severalpurification steps three fractions with flower-inducing activitywere obtained. One fraction obtained from all three plants wasshown to contain nicotinic acid by mass spectroscopic and NMRspectroscopic analyses. These results raise the possibilitythat nicotinic acid may act to influence the flowering processin Lemna. (Received August 28, 1985; Accepted October 29, 1985)     

Rapid Identification and Differentiation of the Soft Rot Erwinias by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction Fragment Length Polymorphism Analyses

Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the α-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten “atypical” E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two “atypical” E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica.     

Somatic Hybrids between the Forage Legumes Lotus corniculatus L. and L. tenuis Waldst et Kit

A cell suspension of Lotus tenuis was established, as a sourceof protoplasts, from kanamycin resistant callus derived fromroots transformed by Agrobacterium rhizogenes LBA9402 (pRil855-pBinl9).Such protoplasts were treated with a sublethal dose of sodiumiodoacetate prior to their electrofusion with green cotyledonprotoplasts of L. corniculatus. Putative somatic hybrid colonieswere selected on medium containing kanamycin sulphate. The hybridityof plants regenerated from these selected colonies was confirmedby their morphology, esterase banding patterns, the presenceof condensed tannins in leaves and stems, and chromosome complements.The latter approximated to the expected allohexaploid numberof 2n = 6x = 36. Key words: Forage legumes, Lotus corniculatus, L. tenuis, protoplasts, electrofusion, kanamycin resistance, sodium iodoacetate, somatic hybridization     

Acid phosphatase in plant tissues I. Changes in activity and multiple forms in tea leaves and tomato fruit during maturation and senescence

Multiple forms of acid phosphatase varied in tea leaves (Camelliasinensis L.) depending on variety and the age of the leaves.Polyacrylamide gel electrophoresis indicated the presence ofsix multiple forms in extracts of young leaves and seven inextracts of mature leaves of the variety Hatzumomizi. VarietyBenihomare showed five bands in zymograms of young-leaf extractsand six in zymograms of mature-leaf extracts. In addition tothe appearance of the new band, the relative intensity of stainingin two other bands changed during maturation. One decreasedand one increased in intensity. The components were partiallyseparated by CM-cellulose chromatography and isoelectric focusing.Total activity of acid phosphatase did not change greatly ona fresh-weight basis during maturation and aging. Extracts of immature green tomato (Lycopersicon esculentum Mill.)fruit showed six bands on polyacrylamide gels. Isoelectric focusingand subsequent electrophoresis of the fractions obtained atdifferent pH values, produced evidence for seven acid phosphatasecomponents in tomato extracts. Unlike the situation with certainother climacteric fruit, acid phosphatase in tomato extractsdecreased steadily during ripening to a level of activity only25–40% that of immature green fruit extracts. It is thereforeconcluded that the onset of senescence in climacteric fruitsis not dependent on an increase in acid phosphatase. Substratespecificity, response to inhibitors, Km, and pH optima of partiallypurified fractions were similar to those of tea-leaf extracts. (Received November 29, 1972; )     

Virulence and Enzyme Production of Erwinia carotovora ssp. atroseptica on Potato Tuber Tissue

The elicitation of pathogenesis on potato tuber slices by 6 strains of Erwinia carotovora ssp. atroseptica was investigated by neutral red vital staining and has been compared with bacterial growth rate, penetration ability, enzyme production and enzyme spectrum. The induction of enzyme synthesis (particularly, of the extracellular polygalacturonase) elicites the rot attack on tuber tissue and this requires a sufficient bacterial density. Due to wound healing, the inductors of enzyme production are removed, and after 48 h enzymes do not attack tuber tissue any more. Therefore, growth rate and penetration ability (to get the necessary bacterial density and inductor substances) may limit virulence. A similar influence of enzyme production and enzyme spectrum of the strains on the virulence was not detected.     

An Inhibitor of Mitochondrial Oxidations in Extracts Prepared from Senescing Leaves

Aqueous extracts were prepared from leaves of Ricinus Communis,L. by centrifuging homogenates at 18000 g for 20 min. Extractsprepared from yellow or brown leaves inhibited the oxidationof succinate by mitochondria prepared from Ricinus endosperm.Extracts became less inhibitory when dialysed or treated withbovine serum albumen. The inhibitory properties of the extractsare attributed to their fatty acid content.     

Field biology and control of Phytophthora parasitica on papaya (Carica papaya) in Hawaii

Epidemic attacks of Phytophthora parasitica on papaya fruits developed during wet, windy weather, but declined rapidly when the weather became dry. During rain, abundant sporangia were liberated from the mycelial mats on infected fruits, but none was trapped in dry air. Collar rots and tree collapse caused by root rot occur irrespective of spray treatments and previous aerial infections on the same tree: they are no more prevalent in blocks with abundant aerial infections than in blocks with none. Basal rots occurred earlier and more frequently on plantings near abandoned diseased fields: they were scattered randomly and appeared before aerial infections. It is suggested that new outbreaks might be initiated from seeds of infected fruit, in which chlamydospores have been found, or from infected nursery seedlings. Drosophila spp. were found to be occasional vectors of sporangia. Control of aerial infections was obtained with maneb, basic copper sulphate, captan or difolatan. Spraying only the upper half of the acropetally developing fruit cluster gave much better control than spraying only the lower fruits.     

Compartmentation of Aldolase Isoforms in Maize Leaves

The leaves of maize seedlings contain two principal isozymesof fructose 1,6-bisphosphate aldolase (E.C. 4.1.2.13 [EC] ), one chloroplasticand one cytosolic (Gasperini and Pupillo, 1982). Mesophyll protoplastswere separated from bundle sheath (BS) strands of both light-grownand dark-grown maize leaves. Aldolase isozymes were separatedfrom extracts of chloroplasts, etioplasts, protoplasts and BSstrands by column isoelectric focusing. The major isozyme ofgreen leaves (pI 4.2) was exclusively in BS chloroplasts, andthere was no evidence of other isozymes occurring in BS tissue.The cytosolic isozyme (pI 6.7) was present in protoplasts ofmesophyll cells, where it may limit the synthesis of hexose-phosphates(estimated activity of 9.4 µmol h^–1 g^–1 fr.wt.) together with lower activities of an acidic form (pI 4.6).Etiolated leaves contained significant amounts of the pI 6.7isozyme in both mesophyll and BS cells, but also minor activitiesof one or more acidic forms with pI values of 4.4–4.7(average pI 4.6) which appear to be located partly in BS etioplasts.The main developmental events for maize leaf aldolase afterillumination were a moderate decrease of cytosolic isozyme (pI6.7) which disappears from the BS within hours and a large,gradual increase of the BS plastid isozyme (pI 4.2). The isoformwith a pI 4.6 also increased rapidly to a low, steady activityin greening mesophyll protoplasts. Key words: C₄, fructose 1,6-bisphosphate, aldolase, Zea mays     

Facultative parasites and host respiration. I. Healthy tissue adjacent to the site of infection

Summary The respiration, as measured by oxygen uptake, was higher for healthy tissue adjacent to soft rots of apple (var. Bramley) caused byPenicillium expansum Link andThom. andBotrytis cinerea Fr. and of potato tuber (var. Arran Banner) caused byErwinia aroideae Townsend, than tissue at a more distant site. The respiration of tissue adjacent to a physiological rot caused by bruising was not affected. It was concluded that there is diffusion of a substance(s) from the site of infection which causes an increase in the rate of respiration of the adjacent tissue.This work formed part of a Ph. D. thesis submitted to the University of London, July 1961.     

The Occurrence of 1,3-{beta}-Glucanase in Healthy and Verticillium-infected, Resistant and Susceptible Tomato Plants

Leaf, stem, and root extracts of near-isogenic tomato plantscv. Craigella, resistant and susceptible to Verticillium albo-atrum,showed constitutive 1,3-ß-glucanase activity whichincreased following inoculation with the pathogen. Partiallypurified enzyme extracts were obtained by dialysing a 30–80%ammonium sulphate fraction of the tissue brei. The enzyme hadpH and temperature optima of 5?5 and 44 ?C respectively, withhigh activity between 50 and 60 ?C. The response to laminarinconcentration was linear between 1?2 and 7?5 mg ml^–1.Root inoculation of susceptible plants with 10⁶ propagules ml^–1V. albo-atrum led to a umform 300 per cent increase in all steminternodes except the terminal one, which was 500 per cent ofthe controls. No spatial relationship of enzyme activity tothe localization of fungus within the stem was apparent. Petioles,leaves, and roots of susceptible infected plants similarly showedan increase in activity but less than that in stems. Changedlevels of stern enzyme activity at different times after inoculationwere associated with reductions in the number of vessels containinghyphae. Extracts of plants of the resistant isoline showed increasedglucanase activity over controls, but this was substantiallylower than that in susceptible plants and was associated withthe greatly reduced mycelial colonization in resistant plants. It is concluded that single gene resistance in tomato to Verticilliumis not associated with innately higher levels of 1,3-ß-glucanasein healthy plants. The increased activity in infected plantsis proportional to the overall quantity of pathogen in the plantor of pathogenic metabolites.     

Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I

Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1×10⁶ colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 g/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35S promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atroseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.Abbreviations AP acidic carboxyl terminal polypeptide - Eca Erwinia carotovora ssp. atroseptica - Ecc E. carotovora ssp. carotovora - Ech E. chrysanthemi - IF intercellular fluid - SP signal peptide - TPNI (tpnI) tachyplesin I     

Comparative Studies of Leaf Tissue 4: III. EFFECTS OF WALL-DEGRADING ENZYMES AND OSMOTIC STRESS

Commercially available cell wall-degrading enzymes frequentlyused for protoplast isolation inhibited CO₂ fixation and photosyntheticO₂ evolution, and stimulated dark respiration by leaf tissueand isolated mesophyll protoplasts of Nicotiana tabacum L. andAntirrhinum majus L. They also depolarized the membrane potentialof cells of leaf tissue, inhibited uptake of ⁸⁶Rb by tobaccoleaf tissue and isolated mesophyll protoplasts, and stimulated³⁶CI uptake by tobacco leaf tissue. Where studied, these effectswere found to be reversible. The depolarization effect on Antirrhinumleaf cells occurred even when the enzyme preparations had beendenatured, dialysed, or desalted, and the effect was greatestin those fractions of the enzyme preparation which showed thehighest cellulase activity. Plasmolysis of tobacco leaf tissue inhibited photosyntheticO₂ evolution, CO₂ fixation, and ⁸⁶Rb uptake to levels belowthose exhibited by isolated protoplasts in media of the samecomposition and osmolarity. The implications of these resultsfor work with leaf tissue and isolated protoplasts are discussed.     

Topographical Studies on Subunit Polypeptides of the PS I Reaction Center Complex in the Thylakoid Membranes of the Thermophilic Cyanobacterium Synechococcus sp.

The permeability to protein molecules of the outer limitingmembranes and the thylakoid membranes in hypotonically shockedprotoplasts of the thermophilic cyanobacterium Synechococcussp. was studied by examining the effects of NaBr-washing andpronase E-digestion on phycobiliproteins and a 35 kDa proteinwhich are associated with the outer and inner surface of thethylakoid membranes, respectively, and by measuring photooxidationof added cytochrome c. All the results obtained indicate thatthe shocked protoplasts are in essence a homogenous right side-outthylakoid membrane preparation; the outer limiting membranesare leaky to protein molecules, whereas the thylakoid membranesare still impermeable to proteins. The thylakoid membranes becamepermeable to proteins when the protoplasts were mechanicallydisrupted. Following on from these findings, the membrane topology of subunitpolypeptides of the photosystem I reaction center complex wasstudied. Proteolytic digestion of shocked protoplasts with trypsinand pronase E indicated that four of the five subunit polypeptidesof the PS I reaction center complex are exposed at the stromalsurface of thylakoid membranes; two subunits of 14 and 13 kDawere selectively digested by trypsin, whereas two chlorophyll-bindingsubunits of 62 and 60 kDa were preferentially attacked by pronaseE. However, a 10 kDa subunit appears to be strongly resistantto the proteases. Experiments with mechanically disrupted protoplastsfailed to provide evidence for a uniform transmembrane organizationof the PS I subunits. (Received March 31, 1986; Accepted August 18, 1986)