Polymethylgalacturonase Research Articles

Effects of iron on the asexual reproduction and major virulence factors of Curvularia lunata

Curvularia lunata (Wakker) Boed, the causal agent of Curvularia leaf spot, is an important fungal pathogen in maize. Iron deficiency or excess plays a crucial role in mycelial growth and virulence of plant pathogenic fungi. In this study, the following pathogenic characteristics of C. lunata under different Fe3+ concentrations were investigated: mycelial growth, conidiation, conidium germination, virulence in maize, toxin and cell wall degrading enzymes (CWDEs) activity. The results showed that iron had a limited impact on the mycelial growth, while it had an obvious impact on mycelial biomass, conidiation, and the conidial germination at a concentration of 40–50 μM Fe3+. Exogenous supplementary of Fe3+ also significantly enhanced the pathogenicity of C. lunata CX-3 strain on maize inbred line Huangzaosi. However, toxin activity was independent of Fe3+ concentration. Exogenous iron application improved polygalacturonase (PG) activity in the early stages of infection, and the activity of cellulase (Cx), pectin polymethyl galacturonase (PMG), polygalacturonic acid transeliminase (PGTE) and pectin methyl transeliminase (PMTE) after 72 h of infection in maize.

Cell-wall-degrading enzymes produced in vitro and in vivo by Rhizoctonia solani, the causative fungus of peanut sheath blight.

Rhizoctonia solani causes the disease peanut sheath blight, involving symptoms of maceration and necrosis of infected tissue, mainly caused by cell-wall-degrading enzymes (CWDEs). This study investigated the production of CWDEs including polygalacturonase (PG), polymethyl-galacturonase (PMG), cellulase (Cx) and β-glucosidase by R. solani in vitro (in liquid culture) and in vivo (in peanut plants). Significant PG, PMG, Cx and β-glucosidase activities were detected in infected tissues including stalk and leaves of Baisha and Silihong peanut cultivars. Extracts of healthy tissue showed little or no such activities. In shaken liquid cultures of R. solani in medium containing pectin or pectin plus carboxymethyl cellulose (CMC) as the carbon source(s), PG and PMG were notably active. Significant Cx activity was detected in cultures with CMC or pectin plus CMC as the carbon source(s). However, only a very low level of β-glucosidase activity was observed in cultures with any of the tested carbon sources. An increase of pH was recorded in decayed peanut tissues and liquid culture filtrates; the filtrate pH and fungal growth positively correlated. The fungal growth and/or pH were important factors for the production of PG, PMG and Cx in culture with pectin plus CMC as the carbon source. A single active PG isozyme with isoelectric point around 9.2 was detected in culture filtrates and in infected peanut tissues by the method of isoelectric focusing electrophoresis. The crude enzymes extracted from liquid culture of R. solani induced decay of healthy peanut leaves.

Action of selenium against Sclerotinia sclerotiorum: Damaging membrane system and interfering with metabolism

Selenium (Se) in soil is beneficial for environmental stress tolerance of plants, and it has widespread toxic effects on pathogens. Based on the fact that Se significantly inhibited the growth of Sclerotinia sclerotiorum, we set experiments with different concentrations of Se to investigate the action of Se against S. sclerotiorum in this study. The results showed that Se (>0.5 mg L−1) changed the morphology of S. sclerotiorum mycelia, and higher Se concentrations severely damaged mycelial structures. Fourier transform infrared spectroscopy (FTIR) analysis indicated that Se treatment induced the chemical composition of mycelia with much abundance of functional groups such as alcohols, ketones, ammonium and esters, and 0.5 mg L−1 Se maximized their concentrations. Under Se treatments, the electrical conductivity of mycelia increased in a time-dependent manner, and osmolyte concentrations of mycelia increased as well. Se supplementation significantly reduced polymethylgalacturonase (PMG) and carboxymethylcellulase (Cx) activities, which protecting plants from infection, and increased the energy expenditure in S. sclerotiorum. Combined action of Se damage on membrane system, osmoregulation, reduction of cell wall degrading enzymes activities and improvement of energy expenditure resulted in the inhibition of S. sclerotiorum growth. Findings in this study provided evidences for using Se as a potential fungicide to control S. sclerotiorum.

Comparison of the virulence and cognate virulence factors of multinucleate, binucleate and uninucleate Rhizoctonia isolates, causing sheath blight on maize plants

Maize sheath blight is a destructive disease of maize world-wide. The causal pathogens of the disease include multinucleate, binucleate and uninucleate Rhizoctonia isolates according to the latest reports. In this study, the virulence and virulence factors of one uninucleate, two binucleate and nine multinucleate Rhizoctonia isolates were compared. The results showed that there was significant difference in the virulence of the tested Rhizoctonia isolates on maize, with multinucleate Rhizoctonia isolates the most virulent, binucleate Rhizoctonia isolates moderate and uninucleate Rhizoctonia isolate the lowest. The cell wall-degrading enzymes (CWDEs) and phytotoxins of the tested isolates were extracted and their enzymatic or biological activities were detected. The correlation coefficients between the enzymatic activity of polymethylgalacturonase (PMG), polygalacturonase (PG) and endo-β-1,4-glucanase (Cx) and pathogenicity of the isolates were 0.8210, 0.7314 and 0.8497, respectively. The correlation coefficients between the inhibition rate of corn seed germination and root growth caused by the phytotoxins and pathogenicity of the isolates were 0.9257 and 0.9690, respectively. These results indicated that CWDEs and phytotoxins likely act as virulence factors during infection. In conclusion, the lowest virulence of the uninucleate Rhizoctonia isolate JN on maize is possibly due to the low activities of CADEs and phytotoxins.

Factors Affecting the Production of Poly Methyl Galacturonase Enzyme by Sclerotium rolfsii Sacc

The aim of this work was to investigate the effects of different culture conditions on the production of poly methyl galacturonase enzyme bySclerotium rolfsii and their optimization. Activity of poly methyl galacturonase enzyme was assayed by viscometric method. Culture conditionslike culture media, incubation period, temperature and pH greatly influenced the production of poly methyl galacturonase enzyme. Out of tenculture media, only six media were proved to be producer of poly methyl galacturonase enzyme. Amongst them, Elliot’s medium was foundto be the best for the maximum production of poly methyl galacturonase enzyme. Analyzing the different incubation period, the production ofpoly methyl galacturonase enzyme was found rapid (3 days incubation) in Sclerotium rolfsii. Whereas, production of poly methyl galacturonaseenzyme, gradually increased with increasing the length of incubation period up to 18 days and further increase in length of incubation up to 10days, did not show any effect on the production of poly methyl galacturonase. The nine days incubation has found to be the best incubationperiod for the maximum production of poly methyl galacturonase enzyme. The Sclerotium rolfsii was also able to produce the poly methylgalacturonase enzyme at wide range of temperatures (from 15ºC to 35ºC), but 30ºC temperature was found to be the best suitable for themaximum production of poly methyl galacturonase enzyme. Among the wide range of pH tested (pH 3.0 to 9.0), the pH 5.0 was proved to bethe best for the maximum production of poly methyl galacturonase enzyme in Sclerotium rolfsii when it was cultured in the same medium. Onthe basis of overall experimental results it could be concluded that Sclerotium rolfsii has potential to produce poly methyl galacturonase enzymeconstitutively.DOI: http://dx.doi.org/10.3126/ijasbt.v3i1.12200 Int J Appl Sci Biotechnol, Vol. 3(1): 89-95

Isolation and Partial Characterization of a Bacterial Thermostable Polymethyl Galacturonase from a Newly Isolated Bacillus sp. strain BR1390

Background: Pectinases are pectin degrading class of enzymes including polygalacturonase (PG), polymethyl galacturonase (PMG), pectate lyase (PEL), and pectin esterase (PE) that are commonly used in processes involving the degradation of plant materials, such as speeding up the extraction of fruit juices. Objectives: A highly methylated pectin degrading bacterium from soil covered with fruit waste was isolated and its extracellular pectinase as a novel polymethyl galacturonase was characterized. Materials and Methods: Pectin-degrading microorganism screening was performed using agar plates containing pectin as the sole carbon source. The biochemical studies were used to characterize the enzyme. Results: Bacterium with greater PMG activity was a Bacillus sp. based on 16S rDNA sequence analysis and named as a Bacillus sp. strain BR1390. Two steps column chromatography showed a dimeric protein with apparent molecular masses of 104 and 56 kDa, evident by gel filtration and SDS-PAGE. Substrate specificity analysis using various polygalacturonic acid compounds revealed its polymethyl galacturonase (PMG, EC 3.2.1.-) activity. Biochemical studies represent the thermophilic characteristics and reasonable pH stability of the polymethyl galacturonase when using pectin as substrate. The PMG activity significantly enhanced in the presence of most divalent cations such as Ca2+ and Mg2+, but Hg2+ and Fe3+ served as strong inhibitor. Conclusions: Overall, regarding to have suitable activity in acidic conditions and high operational stability of the purified pectinase, the introduced PMG can be an ideal functional substitute for applications in the fruit juice industry, especially in citrus fruits extraction and clarification.

English

The production of polygalacturonase (PG), polymethylgalacturonase (PMG) and cellulase (Cx) enzymes have been studied at different incubation period. The fruit rot pathogen of tomato Alternaria solani was cultured at 28°C on semi ripe tomato fruit broth medium for 2, 4, 6,  8, 10 and 12 days. Isolated pathogen A. solani has a capability of producing PG, PMG and Cx enzyme within short incubation period, that is, 2 days incubation. Production of these enzymes gradually increased by increasing the length of incubation period up to 6 days and further increase in length of incubation up to 12 days, did not show any effect on the production of PG, PMG and Cx. The 6 days incubation was found to be the best incubation period for the production of all these three enzymes. The production of PG and Cx was also more in comparison with PMG enzyme. The enzyme activity in healthy and diseased semi ripe tomato fruits was also assayed at different incubation periods and it was found to be maximum in diseased tomato fruits in comparison with healthy tomato fruits. It was also found that PG and PMG enzymes were produced in higher concentration than the Cx enzyme. Among six incubation periods, 6 days incubation period was found best for maximum production of all these three enzymes.   Key words: Alternaria solani, semi ripe tomato fruit medium, semi ripe tomato fruit, incubation period. &nbsp

In vitro antagonistic efficacy of plant extracts on the enzyme activity of Colletotrichum gloeosporioides

Anthracnose fungus, Colletotrichum gloeosporioides was isolated in culture from the diseased fruit samples of chilli (Capsicum annuum). The fungus was identified by morphological and molecular analysis. Plant root extracts of A. precatorius and R. tetraphylla and chemical fungicide, dithane-M45 were tested for their inhibitory effects on the cell wall degrading enzymes of C. gloeosporioides under in vitro condition. The activity of cell wall degrading enzymes viz. polymethyl galacturonase (PMG), pectin transeliminase (PTE) and the carboxymethyl cellulase (CMC) was assayed. Root extracts of A. precatorius was found to be more efficient in inhibiting both the pectinolytic (PMG and PTE) and cellulolytic (CMC) enzymes than dithane-M45. The inhibitory effects of enzyme might be due to the presence of fungitoxic compounds such as phenolics, proteins and flavonoids in the root extracts of A. precatorius. On the contrary, extract of R. tetraphyllawas found to induce enzyme activity. Key words: Plant extract, botanical pesticide, pectinolytic enzyme, cellulolytic enzyme.

Antifungal activity of Plumbago species against anthracnose fungus Colletotrichum gloeosporidodes (Penz.) of chilli

The efficacy of aqueous leaf extracts of three Plumbago (Plumbago indica, P. capensis, P. zeylanica) species on anthracnose fungus Colletotrichum gloeosporioides were assessed. Out of the extract of three plants screened, that of Plumbago indica showed the best result both in inhibition of conidial germination up to (98.9%), and radial mycelial growth (98.75%) of the pathogen over the control and in the pectinolytic enzyme studies also P. indica showed a minimum activity of Polymethyl esterase (1.91U), Polymethyl galacturonase (endo PMG-2.77%; exo PMG-0.028U) and Pectin trans eliminate; the least amount of sugar (endo PTE-17.14%; exo PTE-11.11U) was released in P. indica. In cellulolytic enzyme studies, the amount of viscosity loss due to the activity of 1,4-β endoglucanase was remarkably reduced (3.57%) in P. indica compared to the other two species. P. capensis showed moderate results; P. zeylanica showed minimum inhibition of the enzyme activity of the pathogen. Results show that among all three plumbago species tested, Plumbago indica was reported to be an effective plant extract against anthracnose fungus.

Pectinolytic enzyme production by Colletotrichum truncatum, causal agent of soybean anthracnose

Background Colletotrichum truncatum is the most common pathogenic fungus associated with soybean anthracnose, a prevalent disease in Argentina. Pectinolytic enzymes are involved in the pathogenicity of a wide range of plant pathogenic fungi. Objectives To explore pectinolytic enzyme production in Argentinian Colletotrichum strains isolated from diseased soybean plants from different geographic locations, as a preliminary step to establish the biological role of the pectinolytic enzymes in the Colletotrichum spp.–soybean system, yet unknown. Methods Ten strains were screened for in vitro pectinolytic enzyme production on a defined medium based on pectin as carbon source. Results All isolates were able to grow in this medium and polymethylgalacturonase (PMG), polygalacturonase (PG) and pectin lyase (PL) activities were detected. On the whole, the peak of polygalacturonases activities preceded the day of maximum growth, while PL activity reached its highest level afterwards. Strain BAFC 3097 (from Santa Fe province) yielded high titles of the three enzymes (1.08 U/ml PG, 1.05 U/ml PMG, 156 U/ml PL), after a short incubation period (7–10 days). Low synthesis of polygalacturonases in cultures containing glucose as unique carbon source suggests that these enzymes are constitutive in contrast with PL, which was not detected. Conclusions The disparity observed in enzyme production among strains cannot be related to fungal growth, since no major differences in mycelial yield were found; it was not connected with their geographic origin, but might be associated with differences in virulence among strains not yet evaluated.

Induction of systemic resistance against fusarial wilt in pigeon pea through interaction of plant growth promoting rhizobacteria and rhizobia

A Bacillus cereus strain BS 03 and a Pseudomonas aeruginosa strain RRLJ 04 were studied for their effect on induction of systemic resistance against Fusarium udum wilt in pigeon pea, both individually and in combination with a rhizobial strain RH 2. Split root experiments confirmed that plants with combination treatment of PGPR and Rhizobium can survive longer than individual treatments and control. An increased level of defense-related enzymes, viz., l-phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO), were also recorded in co-inoculated plants. Production of β-1,3-glucanase and polymethyl galacturonase by the pathogen in culture medium was also sharply reduced in presence of both the PGPR strains. An increased level of phenol content with a decreased level of nitrogen content was recorded in the leaves of bacterized plants grown in pathogen infested soil. Presence of BS 03 in the culture medium stimulated the production of chlamydospore-like structures in the fungal mycelium. Such structures were not observed in case of RRLJ 04 treatment but the dry weight of mycelium was reduced significantly. Fusaric acid production by the pathogen was also drastically reduced in the presence of the PGPR strains. The results promise the combined use of PGPR and rhizobia for induction of systemic resistance against fusarial wilt in pigeon pea.

Studies on pH and thermal deactivation of pectolytic enzymes from Aspergillus niger

Pectolytic enzymes, viz., polymethylgalacturonase (PMG), polygalacturonase (PG), and pectinlyase (PL) were deactivated at various combinations of pH and temperature. The deactivation process was modelled as first-order kinetics and the deactivation rate constant was found to be minimum at pH 2.2 and 23 °C, respectively for PMG, 4.8 and 28 °C, respectively for PG and 3.9 and 29 °C, respectively for PL. Thermodynamic parameters like Δ G ∗, Δ H ∗, Δ S ∗, and activation energies were evaluated for crude and partially purified enzymes. It was found that entropy values are negative for all the three enzymes. The partially purified enzymes were found to be less stable than the crude enzymes. Hence, the interaction effect among the enzymes and also the effect of protein (BSA) on deactivation were studied. The addition of protein doubled the half-life times of partially purified PMG, PG I and PG II. The interaction effect of pectolytic enzymes on deactivation was found to be significant.

Physiological aspects of immobilised Aspergillus niger cells producing polymethylgalacturonase

Conidia of Aspergillus niger 26 spores were immobilised in 3% Ca-alginate beads and some intrinsic kinetic characteristics including growth, polymethylgalacturonase (PMG) production and specific oxygen uptake rates determined. A negative correlation between the specific growth rate and specific PMG production indicated a non-growth-associated enzyme formation. Immobilisation did not change the model of PMG synthesis even in repeated replacement cultures and leads under certain conditions to an enhanced PMG production. The immobilised mycelium was concentrated at the surface layer of the alginate beads and the fungal hyphae could not be found in the central part of the gel. The dense growth of the mycelia on the outer bead surface allowed the formation of a fur-like mycelium coat, which had a large surface for oxygen uptake. This growth behaviour is a reason why the effectiveness factor increased during incubation from 0.54 to 0.93.

Induction of polymethylgalacturonase biosynthesis by immobilized cells of Aspergillus niger 26

The involvement of the regulatory mechanism induction in the biosynthesis of polymethylgalacturonase (PMG) by immobilized cells of Aspergillus niger 26 was studied. The immobilization altered the spectrum of pectin-degrading enzymes. The free cell cultures produced four pectinolytic enzyme activities, namely PMG, polygalacturonase (PG), pectinesterase (PE) and pectinylase (PL), while the entrapped mycelium synthesized PMG and PG only. The best inducer for PMG synthesis was highly esterified apple pectin. The effects of various pectin concentrations and time of incubation with inducer showed the induction model of PMG biosynthesis. The deinduction experiments provided evidence that the presence of the inducer in every cycle was obligatory for the PMG biosynthesis during repeated replacement cultivation.

The controlled fermentation of cocoa (theobroma cacao L) using yeasts: Enzymatic process and associated physico‐chemical changes in cocoa sweatings

Starter culture fermentation of cocoa may become a novel approach to the effective utilization of some waste by‐products in the cocoa industry. Cocoa sweatings could be rapidly collected for use in food product development when some strains of yeasts are used as starter cultures for the fermentation. The action of the yeasts in degrading cocoa pulp which is 76–80% esterified to cocoa sweatings is most likely due to presence of polymethyl galacturonase, since pectin esterase activity was completely absent in all the yeast strains tested. The pectin content of the resulting cocoa sweatings could be used to regulate the period of sweating collection depending on its intended use in food product development.

Immobilization of polymethylgalacturonase producing Aspergillus niger on Luffa sponge material.

The vegetable sponge of Luffa cylindrica was studied as a matrix for the immobilization of Aspergillus niger 26, producer of polymethylgalacturonase (PMG). Entrapped spores could grow and multiply within the lattice of the sponge. The influence of loofa sponge inoculum content, initial spore inoculum content, and duration of the growth cycle on the enzyme activity and mycelium growth was studied. The best yield of PMG was reached with 1 piece of loofa sponge (approx. 0.10 g dry weight), 10(9) spores per g carrier and 48 h duration of one cycle. Data obtained during long-term semicontinuous cultivation showed that production capacity increased significantly and the production period was extended more than 10 times compared with the free cell culture.

Enhanced polymethylgalacturonase production from Aspergillus niger 26 by calcium alginate immobilisation

Spores of Aspergillus niger 26 were immobilised in calcium alginate and the immobilised growing mycelium reused in batch mode for the production of polymethylgalacturonase (PMG). The influence of alginate concentration, initial inoculum gel volume, spore concentration, and duration of the fermentation cycle on enzyme activity and mycelium growth are reported. The best yield of PMG was reached with 5 ml initial gel volume 3% alginate concentration, 10 9 spores/ml gel and a cycle time of 72 h. The optimisation of these factors, which affect the stability and productivity of the immobilised system, resulted in an increase in enxyme activity and the possibility of semicontinuous cultivation.

Investigations on the Activity of Cell Wall‐degrading Enzymes in Young Wheat Plants After Infection with Pseudocercosporella herpotrichoides (From) Deighton

AbstractIn earlier investigations, it has been demonstrated that Pseudocercosporella herpotrichoides (Fron) Deighton is capable of producing pectolytic and cellulolytic enzymes as well as hemicellulases in vitro. The investigation of enzyme activity in extracts from wheat plants infected with P. herpotrichoides (isolates 21e and R6) and from non‐infected plants revealed the activity of the following enzymes: pectin methylesterase (PME), polymethylgalacturonase (PMG), pectin lyase (PL), carboxymethylcellulase (CMCase), xylanase and arabanase. Compared to non‐infected plants, the enzyme activity in infected plants was considerably higher; in some experiments, only traces of enzyme activity could be found in control plants. The difference in the enzyme activity in infected as compared to non‐infected plants was, in most cases, statistically significant, especially beginning at the end of the second week after inoculation.The enzyme activity depended on the temperature during plant cultivation; with the exception of pectin methylesterase (PME), the activity of all investigated enzymes increased with temperature and the highest activity was found in plants grown at 20°C. The highest PME activity was measured in plants grown at 10°C; the activity of this enzyme was generally lower at 15 and 20°C.

In Vitro Secretion of Pectolytic Enzymes by Xanthomonas campestris

Summary Xanthomonas campestris (Pammel) Dowson produces pectic enzymes under the influence of different carbon sources. Polymethyl galacturonase (PMG) was a pectin-inducible enzyme in X. campestris , being best extruded with pectin as carbon source. Addition of glucose to pectin had practically no effect, to a smaller extent NaPP (sodium polypectate) effectuated PMG secretion. Addition of CMC to NaPP lowered the PMG secretion, glucose and maltose were found to be poor carbon sources for PMG induction. Polygalacturonase (PG), too, was adaptively produced in the culture filtrate of the pathogen, grown with NaPP as the carbon source. Addition of glucose to NaPP enhanced PG secretion and that of CMC retarded it. Glucose, pectin, and maltose individually caused PG liberation, but in a very minor way. 30 °C and 60 min of enzyme-substrate incubation were found to be optimum physical conditions for the secretion of PMG and PG. Polygalacturonase trans-eliminase (PGTE) was an exo-cellular enzyme, produced by X. campestris under adapted condition with NaPP as the best inducible carbon source. Separate addition of glucose and CMC to NaPP reduced PGTE extrusion. Pectin, glucose, and maltose separately effected PGTE liberation, but to a very limited extent, being much less than with NaPP. Addition of glucose to pectin suppressed whatever PGTE the pectin was alone able to produce.

Pectolytic and cellulolytic activity in Rhizopus-infected rin and nor tomato mutants

Polygalacturonase (PG) and polymethylgalacturonase (PMG) activity was recorded in the two non-ripenng mutants of tomato devoid of these enzymes, rin and nor fruits, following Rhizopus stolonifer infection. Activity increased markedly with disease development.