Sodium Polypectate Research Articles

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel β-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4–5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL−1 and Vmax 690 μmol·min−1.mg−1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Substrate utilization by endophytic bacteria Paenibacillus polymyxa P2b-2R that may facilitate bacterial entrance and survival inside diverse plant hosts

Bacterial endophytes are thought to enter plants either through pre-existing openings in plant tissues or by creating openings by hydrolyzing major plant cell wall components. A lodgepole endophyte, Paenibacillus polymyxa P2b-2R, consistently formed endophytic colonies when inoculated in diverse plant hosts, viz., lodgepole pine, western red cedar, corn, canola, and tomato. We were interested to know, whether or not this bacterial strain possesses enzymes that can hydrolyze three major plant cell wall components namely cellulose, xylan, and pectin to facilitate entrance into the host plants. Using a BIOLOG assay, we also tested this bacterial strain’s ability to utilize carbon sources that might facilitate its entrance and hence its survival inside host plants. Paenibacillus polymyxa P2b-2R hydrolyzed sodium carboxymethylcellulose, beechwood xylan, and sodium polypectate and utilized 39 of the 95 carbon sources (41%) tested. Of the 39 carbon substrates oxidized by P2b-2R, the “carbohydrates” group represents the largest source of utilizable carbon (23 out of 39). Thus, it can be concluded that P. polymyxa P2b-2R is able to degrade major cell wall components (cellulose, xylan, and pectin) and utilize some of the available carbon substrates, possibly to gain entry and survive inside the plant and form endophytic colonies thereafter.

Differential expression of pectolytic enzyme genes in Xanthomonas citri subsp. citri and demonstration that pectate lyase Pel3 is required for the formation of citrus canker

Bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. The pectolytic enzymes produced by phytobacteria are important virulence factors involved in tissue maceration, electrolyte loss and cell death of host plants. In this study, the promoter activity of the pectolytic enzyme genes pel1, pel2, pel3, pglA, and peh-1 were investigated in Xcc XW19 strain using the β-glucuronidase (GUS) gene as a reporter. GUS activity expressed under the control of the pel1, pel3, pglA, and peh-1 gene promoters positively correlated with bacterial growth. These gene promoters displayed high GUS activity in the presence of sodium polypectate. In addition, the four genes were induced in XVM2 minimal medium. However, only pel1 was subjected to catabolite repression by glucose. GUS activity was significantly enhanced in the XW19-derived reporter strains after they were inoculated into the leaves of Mexican lime and grapefruit, suggesting the involvement of the pel1, pel3, pglA, and peh-1 genes in XW19 pathogenesis. The pel3 promoter produced the highest GUS activity under all test conditions, whereas no GUS activity was detected using the pel2 promoter in vitro and in planta. In comparison with wild type Xcc, a pel3 mutant generated from Xcc XW19 using unmarked mutagenesis displayed reduced growth and induced smaller canker lesions on the leaves of Mexican lime, demonstrating that Pel3 of Xcc strain XW19 is a virulence factor.

Functional properties of a manganese-activated exo-polygalacturonase produced by a thermotolerant fungus Aspergillus niveus

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40°C, presenting protein levels almost twofold higher than at 25°C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7% carbohydrate, molecular mass of 102.6kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50°C and 4.0-6.5, respectively. The enzyme was stable at pH3.0 to 9.0 for 24h. The DEAE-cellulose derivative was about sixfold more stable at 60°C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232% activated by Mn(2+). The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K m, V max, K cat, and K cat/K m values were 6.7mg/ml, 230U/mg, 393.3/s, and 58.7mg/ml/s, respectively. The N-terminal amino acid sequence presented 80% identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.

The influence of carbon source and calcium on the production of proteases by Erwinia chrysanthemi

Erwinia chrysanthemi (Ec) is the causative agent of soft rot diseases in many plants. In order to cause disease, it produces hydrolytic enzymes, which are involved in the degradation of plant tissue. However, the role of proteases in its pathogenicity to plants is not well understood. E. chrysanthemi targets the host’s middle lamella, which was mainly made of pectic substances containing calcium, for colonization. In this study, it is reported that a pectic substance, such as sodium polypectate (NaPP) may be preferable, not only for the production of hydrolytic enzymes, but for the production of protease as well. Furthermore, since the middle lamella contains high levels of calcium, it was thought that it may play an important role in the production of proteases by this organism. Both predictions were supported by the results presented in this study. When E. chrysanthemi subsp. chrysanthemi (Ecc) was grown in HXC medium supplemented with NaPP and calcium, it produced the highest levels of protease as compared to NaPP alone. These results suggest that proteases produced by E. chrysanthemi may be induced in part by calcium. Key words: Erwinia chrysanthem i, protease, phytopathogenicity, extracellular enzyme.

Colonization of Olive Inflorescences by Verticillium dahliae and its Significance for Pathogen Spread

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is the most severe disease affecting this crop in most olive growing countries. In this study, the presence of viable structures of V. dahliae in dried inflorescences from wilted olive shoots was investigated. The pathogen was found inside peduncles and flowers, by assessing the number of typical star-shaped microsclerotial colonies formed onto the modified sodium polypectate agar medium. Microsclerotia of V. dahliae were observed inside the peduncles under the stereoscopic microscope. The presence of microsclerotia in these easily decomposable olive tissues shows that infected inflorescences can act as a source of inoculum for Verticillium wilt epidemics.

First Report of Verticillium Wilt of Pistachio Caused by Verticillium dahliae in Spain.

Pistachio (Pistacia vera L.) trees in the Castilla La Mancha and Andalusia regions of central and southern Spain are grown close to olive orchards, which are often severely affected by Verticillium dahliae. During the last decade, wilt and death of one or several branches have been observed on pistachio (cv. Kerman) scions grafted on rootstock (P. terebinthus). Discoloration of vascular tissue was occasionally observed. In five surveyed orchards, incidence of affected trees was less than 2%. Wood chips with the bark removed from symptomatic trees were washed in running tap water, surface disinfested in 0.5% sodium hypochlorite for 1 min, and placed onto potato dextrose agar plates incubated at 25°C in the dark. Isolates were identified as V. dahliae on the basis of the characteristics of microsclerotia, conidiophores, and conidia. V. dahliae isolate V117 from olive was used as reference (1). The fungus was also isolated from soil in pistachio orchards using wet sieving and a modified sodium polypectate agar medium (1). Inoculum density varied from 0 to 4.73 microsclerotia per gram of soil. P. terebinthus seedlings were inoculated with two isolates of V. dahliae from pistachio trees by injecting the stems with 50 μl of a conidial suspension (107 conidia per ml) (2). Wilt symptoms of varying severity developed in 12 and 15 seedlings of the 20 pistachio seedlings inoculated with each of two isolates. No symptoms developed on the control seedlings. The pathogen was recovered from stem tissues of inoculated plants. To our knowledge, this is the first report of Verticillium wilt of pistachio in Spain. This study demonstrates the susceptibility of certain rootstocks to V. dahliae and the importance of using resistant rootstocks, such as UCBI (2), in pistachio plantations established on soils infested by V. dahliae.

Effects of culture ages on the production and activities of polygalacturonase and cellulase (CX) enzymes produced by <i>Pythium aphanidermatum</i> (Edson Fitzpat.) Isolated from soft stem rot disease of cowpea.

Effect of culture ages was investigated on the production and activities of polygalacturonase (PG) and Cx cellulase enzymes. Pythium aphanidermatum was isolated from cowpea seedlings infected by soft stem rot disease and prepared into pure culture through successive culturing. Two discs (2 mm each) of two-day-old culture of P. aphanidermatum were put into 21 Erlenmeyer flasks containing 50 ml of Reese and Levinson’s solution, consisting of Sodium Polypectate (NaPP) as carbon source, and incubated for 7 days at 38oC. At one-day interval, content of 3 flasks was independently centrifuged at 10,000g for 30 min. The PG enzyme activity was determined viscometrically and expressed as the Relative Viscometric Unit (RVU). Data collected were analysed using analysis of variance (Anova) and the means separated by the Duncan Multiple Range Test. Activity of Cx cellulase enzyme was also evaluated by the same method used for PG enzyme but using, carboxylmethyl cellulose (CMC) as the carbon source. The results showed that there was significant difference in the production and activities of PG and Cx enzymes in the various days of the incubation. The PG and Cx enzymes were produced progressively from the 1st day of incubation and reached their peak on the 5th day for PG (RVU 105) and 4th day for Cx (RVU 49), after which, production and activities of the enzymes started declining. Results suggested that soft stem rot disease in cowpea is a monocyclic disease, whereby any attacked plant that is able to withstand the disease is capable of surviving after the peak of the infection. Key words: Pythium aphanidermatum, polygalacturonase, Cx cellulose,enzymes, viscometric, rot, disease.

Purification and Partial Characterization of an Exo-polygalacturonase from Paecilomyces variotii Liquid Cultures

An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K(m) and V(max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 micromol/min/mg, respectively. PG was found to have temperature optimum at 65 degrees Celsius and was totally stable at 45 degrees Celsius for 90 min. Half-life at 55 degrees Celsius was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).

Effect of Cultural Conditions on the Commercial Production of Polygalacturonase by <i>Rhizoctonia bataticola</i>

Polygalacturonase (PG) activity was detected in culture filtrate and Rhizoctonia-infected tissues of potato tubers (Solanum tuberosum). Culture produced PG formed more reducing groups that PG from diseased tissue. PG production and pH of the culture medium increased with incubation period and attained optimal values between the 4th and 6th day. The ability of Rhizoctonia bataticola to produce PG varied with the carbon and nitrogen sources in the medium; relatively large amounts of PG were produced in media supplemented with citrus pectin, sodium polypectate and potato slices as carbon sources and asparagine and D-L-_-phenylalanine as nitrogen sources. The industrial use of the enzyme to separate cellulose fibres and the maceration of vegetables are discussed. Keywords:Rhizoctonia bataticola, Solanum tuberosum, polygalacturonase, nitrogen and carbon sources. Discovery and Innovation Vol. 19 (4) 2007: pp. 264-268

Effect of cultural conditions on the production of polygalacturonase by Rhizoctonia bataticola

Polygalacturonase (PG) activity was detected in culture filtrate and Rhizoctonia-infected tissues of potato tubers (Solanum tuberosum L.). Culture-produced PG formed more reducing groups than PG from diseased tissue. PG production and pH of the culture medium increased with the incubation period and attained optimal values between the 4th and 6th days. The ability of R. bataticola to produce PG varied with the carbon and nitrogen sources in the medium; relatively large amounts of PG were produced in media supplemented with citrus pectin, sodium polypectate and potato slices as carbon sources and asparagines and D-L-β-phenylalanine as nitrogen sources. The industrial use of the enzyme to separate cellulose fibres and the maceration of vegetables are discussed.

Physicochemical and Gelling Characterizations of Pectin Extracted from Sweet Potato Pulp

A novel pectin was extracted from sweet potato (Ipomoea batatas) pulp (SPP) using 50 mM disodium phosphate solution. Uronic acids in the pectin extract were almost completely removed from the supernatant after the addition of 0.3 volumes 95% ethanol. The sweet potato pectin consisted of 64.8% galacturonic acid and small amounts of neutral sugars. The ash concentration was 21.6% and sodium comprised 6.1 g per 100 g of the pectin. The degree of esterification of the pectin was 1.4%. The infrared spectrum of the pectin was similar to that of sodium polypectate. These results indicate that most of the carboxyl groups of the pectin are replaced by sodium. The pectin had average molecular-weights of approximately 7.85×105 and 2.42×105 Da. The viscosity was of the pectin (2%) the 130 mm2/s at 5°C. The effect of extrinsic parameters, such as pH and concentrations of pectin, calcium and sucrose, on the breaking pressure of the pectin gel was evaluated. The results of the breaking pressure analysis of the pectin gel indicate that the pectin has properties typical of low methoxyl pectin.

A semi-selective medium for detection, isolation and enumeration ofErwinia carotovora ssp.carotovora from plant materials and soil

Using a modified crystal violet sodium polypectate (CVPB) double layer medium with 2,3,5-triphenyltetrazolium chloride and L-asparagine in the basal medium and bromthymol blue in the upper, both containing tryptone, gave red to pink colonies of Erwinia carotovora ssp. carotovora on a blue green background with cavity formation. The addition of 1.2 μg/ml polymyxin B sulphate to the upper layer inhibited the growth of Erwinia carotovora ssp. atroseptica, while 32 μg/ml erythromycin in the upper layer inhibited the growth of E. chrysanthemi. The addition of 35 μg/ml novobiocin inhibited the growth of Bacillus sp. Non-target bacteria such as Pseudomonas fluorescens, P. marginalis, Ralstonia solanacearum and E. herbicola formed pink, red, yellowish or white colonies without cavity formation. The growth of non-target bacteria on the semi-selective medium was one tenth of that obtained using CVPB medium. Copyright © 2005 Whurr Publishers Ltd.

Accuracy and precision of population estimates of Verticillium dahliae on growth media in quantitative soil assays

Verticillium dahliae Kleb. is a serious pathogen of many plant species. Growth media used to measure population density of V. dahliae in soil were evaluated for high recovery of the pathogen, as well as accuracy and precision of population estimates from naturally and artificially infested sandy loam soil using the soil dilution method. Recovery of V. dahliae from naturally infested field soil was highest on soil pectate Tergitol agar (SPT), soil extract agar + sodium polypectate (SEAP), modified pectate agar (MPA), potato dextrose agar + streptomycin sulphate (PDAS), and Talboys' prune lactose agar (TPA); however, PDAS and TPA were overgrown with contaminating fungi, making enumeration difficult. Use of SPT medium resulted in the most precise population estimate with a standard error (SE) of 12.6% of the mean followed by use of pectate agar (PA) (SE = 14.8%) and SEAP (SE = 19.1%). Ethanol, biotin, and Dox salts enhanced recovery of V. dahliae from naturally infested soil, but combining Dox salts with ethanol and biotin significantly reduced population density. Soil extract had no significant effects on population density. Accuracy of V. dahliae population estimates from sterile artificially inoculated soil was highest on modified soil extract agar (MSEA) (64%) followed by SPT (58%). However, accuracy of V. dahliae population estimates from nonsterile artificially inoculated soil was highest on SPT (36%). Soil extract is not an essential ingredient and biotin may increase recovery of V. dahliae from soil.Key words: Verticillium dahliae, verticillium wilt, population density, recovery, accuracy, precision.

Characterization of Erwinia Populations from Nursery Retention Ponds and Lakes Infecting Ornamental Plants in Florida.

Water shortages in Florida are occurring due to intense utilization of the aquifer system by municipalities and agriculture, and because of continued deficits in annual rainfall. Water districts therefore, are, recommending the use of recycled irrigation water, stormwater runoff, reclaimed municipal sewage water, and lakes for agricultural use. With recycled water, however, there is potential for both introducing and concentrating plant pathogens. In Florida, Erwinia soft-rot bacteria (synonym Pectobacterium) cause extensive crop losses in ornamental plant production. To determine Erwinia spp. population levels, samples were taken monthly for 1 year from four hypereutropic lakes and eight nursery retention ponds. Seventy-seven Erwinia strains were collected by both direct plating and by an enrichment process. With the direct plating method, 0 to 29 CFU/ml were detected on sodium polypectate medium. Significantly higher populations of Erwinia were detected in retention ponds of nurseries that were actively reutilizing their water. Erwinia strains were identified to species by fatty acid analysis and biochemical tests. Strains were further characterized by repetitive element-polymerase chain reaction (rep-PCR) and compared with 120 strains of Erwinia collected from ornamentals over a 17-year period in Florida. Using rep-PCR, most strains were clustered into two heterogeneous populations of E. chrysanthemi and E. carotovora subsp. carotovora in a 1:2 and a 1:4 ratio for isolates from ornamentals and from water, respectively. Within each population of E. chrysanthemi and E. carotovora, genetically different subpopulations could be identified that contained high percentages of Erwinia strains from water sources. Even though genetic differences exist, 99% of the strains from water sources were found to be pathogenic on dieffenbachia. Without water treatment of irrigation and stormwater runoff, there is a potential for both introducing and concentrating Erwinia populations within these water sources.

Pectic Enzymes Produced In vitro and In vivo by Erwinia spp. Isolated from Carrot and Pepper in Egypt

AbstractThirty soft‐rot Erwinia strains were isolated from diseased carrot (Daucus carrota) roots and pepper (Capsicum annuum) fruits. Pathogenicity tests showed that these strains are able to induce soft‐rot disease symptoms in different vegetable species indicating a wide host range. Although pectin methyl esterase and polygalacturonase activities were not detected in culture supernatants, they were found in extracts of carrot roots and pepper fruits infected with three strains representing three soft‐rot Erwinias. On the other hand, culture supernatants and extracts of infected plant tissues contained pectic lyase activity. The enzyme from either sodium polypectate or pectin medium degraded the former more actively than the latter indicating pectate rather than pectin lyase activity. In contrast, the enzyme produced in infected tissue showed higher activity on pectin than sodium polypectate. Moreover, the enzyme activity on pectin was not reduced in the absence of Ca2+, which might suggest that, in addition to pectate lyases, pectin lyases could be produced in plant tissues by Erwinia species.

A selective medium for the detection ofAlternaria dauci andAlternaria radicina

A selective medium to detectAlternaria dauci andA. radicina on seed, plant debris and other substrates was developed. Growth and sporulation by most undesired organisms was reduced, but adequate mycelial growth and sporulation byA. dauci andA. radicina was maintained so they could be identified by their unusual mycelial growth or characteristic spores. The medium is based upon carrot leaf extract, which promotes profuse sporulation by both pathogens. Glucose, sodium polypectate and mineral salts further enhanced spore and mycelial production. Streptomycin sulfate and metalaxyl (or mefenoxam), combined with either benomyl or thiophanate-methyl, reduced growth and sporulation of unwanted organisms. There were strong interactions among the fungicides, bactericide, and most other medium components. There were also significant effects on the type of mycelial growth produced by some medium components. If the unique, dark mycelia produced by both pathogens is the desired detection method, the medium components must be optimized for eitherA. dauci orA. radicina because the two fungi responded differently in this regard. All medium configurations allowed both fungi to be identified by their characteristic spores. The sensitivity of theAlternaria dauci-radicina selective medium (ADRSM) to detectA. radicina on carrot seeds was similar to other methods presently in use, but ADRSM was more sensitive than the other methods for detectingA. dauci on infested carrot seeds.

Purification and partial characterization of exopolygalacturonase I from Penicillium frequentans

A polygalacturonase with a molecular mass of 74 kDa, an isoelectric point around pH 4.2 and pH--and temperature optima of 3.9 and 50 degrees C, respectively, was purified from a culture fluid of Penicillium frequentans. The enzyme was characterized as an exo-alpha-1,4-polygalacturonase (exo-PG I). Km and Vmax for sodium polypectate hydrolysis were 0.68 g/l and 596.8 U x mg(-1), respectively. The enzyme, a glycoprotein with a carbohydrate content of 81%, is probably the main pectinase of Penicillium frequentans responsible for cleaving monomer units from the non-reducing end of pectin.

Evidence for carbon source regulated protein kinase A and protein kinase C signaling in the duplication cycle, polarization and septum formation in Aspergillus nidulans

The effects of glucose and of a pectic substrate in the duplication cycle, spore polarization and septation of Aspergillus nidulans were tested in poor and rich media. Growth on poor conditions and on sodium polypectate slowed nuclear duplication and reduced the coupling of polarization to mitosis. Coupling of septation to the third mitosis was also reduced by changing growth conditions. When protein kinase A (PKA) and protein kinase C (PKC) activators were added to the media the results suggested a role for PKA in slowing the duplication cycle, while allowing polarization. Addition of a PKC activator to poor media uncoupled the first septum formation from the third mitosis in a carbon source-regulated manner, suggesting a role for PKC in coordinating cell cycle signals, growth and cytokinesis.

Modified crystal violet pectate medium (CVP) based on a new polypectate source (Slendid) for the detection and isolation of soft rot erwinias

The selective-diagnostic crystal violet pectate (CVP) medium for the detection and isolation of soft rot erwinias was modified and improved to allow the use of a new source of sodium polypectate (Slendid type 440), as the previous polypectate (Bulmer) is no longer available. Two formulations were developed: CVP-S1 medium was less transparent but otherwise similar to the Bulmer polypectate-based CVP medium (CVP-B) except that NaOH was added and CaCl2 concentration reduced. CVP-S2 medium was prepared by mixing equal volumes of two double strength preparations containing 1) polypectate and NaOH, and 2) all other ingredients, both sterilised separately. Although erwinia cavity formation was slower, it showed a number of advantages over CVP-B and CVP-S1 media, thereby facilitating colony/cavity detection and enumeration. These included the absence of precipitate, greater firmness and a reduced risk of liquefaction in the presence of large erwinia numbers, and a reduction in the number of saprophytic bacteria.