Pectin Methylgalacturonase Research Articles

Characterization and Pathogenicity of Colletotrichum truncatum Causing Hylocereus undatus Anthracnose through the Changes of Cell Wall-Degrading Enzymes and Components in Fruits.

Anthracnose is one of the destructive diseases of pitaya that seriously affects the plant growth and fruit quality and causes significant yield and economic losses worldwide. However, information regarding the species of pathogens that cause anthracnose in pitaya (Hylocereus undatus) fruits in Gansu Province, China, and its pathogenic mechanism is unknown. Thus, the purposes of our present study were to identify the species of pathogens causing H. undatus fruits anthracnose based on the morphological and molecular characteristics and determine its pathogenic mechanism by physiological and biochemical methods. In our present study, forty-six isolates were isolated from the collected samples of diseased H. undatus fruits and classified as three types (named as H-1, H-2, and H-3), according to the colony and conidium morphological characteristics. The isolation frequencies of H-1, H-2, and H-3 types were 63.04%, 21.74%, and 15.22%, respectively. The representative single-spore isolate of HLGTJ-1 in H-1 type has significant pathogenicity, and finally we identified Colletotrichum truncatum as the pathogen based on the morphological characteristics as well as multi-locus sequence analysis. Moreover, the H. undatus fruits inoculated with C. truncatum had a significantly increased activity of cell wall-degrading enzymes (CWDEs) cellulase (Cx), β-glucosidase (β-Glu), polygalacturonase (PG), and pectin methylgalacturonase (PMG), while having a decreased level of cell wall components of original pectin and cellulose in comparison to control. The average increased activities of Cx, β-Glu, PG, and PMG were 30.73%, 40.40%, 51.55%, and 32.23% from day 0 to 6 after inoculation, respectively. In contrast, the average decreased contents of original pectin and cellulose were 1.82% and 16.47%, respectively, whereas the average increased soluble pectin content was 38.31% in comparison to control. Our results indicate that C. truncatum infection increased the activities of CWDEs in H. undatus fruits to disassemble their cell wall components, finally leading to the fruits' decay and deterioration. Thus, our findings will provide significant evidence in the controlling of pitaya anthracnose in the future.

Activities of cell‐wall‐degrading enzymes produced by Pestalotiopsis guepinii

AbstractPestalotiopsis guepinii is a pathogenic fungus that causes grey blight on Camellia pitardii. In this study, we investigated the enzyme activity and kinetics of these cell‐wall‐degrading enzymes (CWDEs) produced by Pestalotiopsis guepinii in both C. pitardii leaves and culture medium. Our enzyme activity experiments revealed that the activities of xylanase, pectin methyl‐galacturonase (PMG), β‐1,4‐endoglucanase (Cx), and β‐glucosidase were high in both C. pitardii leaves and culture medium. These enzymes played a significant role in the pathogenic process. However, the activity of laccase was found to be very low and had a minor impact on the pathogenic process. Furthermore, our enzyme dynamics experiments demonstrated that the optimal reaction temperature for PMG and Cx was 50°C, while for β‐glucosidase and xylanase, it was 60°C. The optimal reaction pH for Cx, β‐glucosidase, and xylanase was 5.0, whereas for PMG, it ranged from 5.0 to 6.0. This indicates that these four enzymes prefer acidic conditions. Moreover, we observed that the activities of Cx, PMG, and xylanase decreased with increasing reaction time. On the other hand, the activity of β‐glucosidase initially increased sharply and then decreased slowly. The maximum reaction rates of the four cell‐wall‐degrading enzymes were ranked as follows: xylanase > PMG > β‐glucosidase > Cx. Additionally, the affinities of these enzymes with substrates were ranked as follows: PMG < Cx < xylanase < β‐glucosidase.

Efficacy and Mechanism of Thymol/KGM/LG Edible Coating Solution on Inhibition of Mucor circinelloides Isolated From Okra.

With the increasing demand and quality requirement for the natural nutritious food in modern society, okra has attracted much attention because of its high nutritional value and remarkable functionality. However, the occurrence of postharvest diseases of fresh okra severely limited the application and the value of okra. Therefore, in this study, the dominant pathogens causing postharvest diseases such as soft rot were isolated from naturally decaying okra. It was identified as Mucor circinelloides by its morphological characteristics and standard internal transcribed spacer ribosomal DNA sequence. Furthermore, the biological characteristics of M. circinelloides were studied, and the inhibitory effect of thymol/KGM/LG (TKL) edible coating solution on M. circinelloides and its possible mechanism was discussed. In addition, TKL edible coating solution had a dose-dependent inhibitory effect on M. circinelloides, with a 50% inhibitory concentration (EC50) of 113.55 mg/L. The TKL edible coating solution at 960 mg/L of thymol completely inhibited mycelial growth and spore germination of M. circinelloides. The results showed that the best carbon source of M. circinelloides was maltose, the best nitrogen source was beef extract and potassium nitrate, the best pH was 6, the best temperature was 28°C, the best NaCl concentration was 0.5%, and the light was conducive to the growth of M. circinelloides. It was also observed by scanning electron microscope (SEM) that TKL was more likely to destroy the cell wall integrity of M. circinelloides, inhibit spore morphology and change mycelium structure. Meanwhile, the activity of chitinase (CHI), an enzyme related to cell wall synthesis of M. circinelloides, was significantly decreased after being treated by TKL with thymol at 100 mg/L (TKL100). The content of Malondialdehyde (MDA) in M. circinelloides decreased significantly from 12 h to 48 h, which may cause oxidative damage to the cell membrane. The activity polygalacturonase (PG), pectin methylgalacturonase (PMG), and cellulase (Cx) of M. circinelloides decreased significantly. Therefore, the results showed that TKL had a good bacteriostatic effect on okra soft rot pathogen, and the main bacteriostatic mechanism might be the damage of cell membrane, degradation of the cell wall, inhibition of metabolic activities, and reduction of metabolites, which is helpful to further understand the inhibitory effect of TKL on okra soft rot pathogen and its mechanism.

The Antifungal Activity and Action Mechanism of Lemongrass (Cymbopogon flexuosus) Essential Oil Against Fusarium avenaceum

Fusarium avenaceum is distributed worldwide and directly causes considerable crop losses. The compositions of lemongrass essential oils (EOs) were analysed by gas chromatography-mass spectrometry (GC-MS). The antifungal activity of lemongrass essential oils (EOs) and the main compounds against F. avenaceum were evaluated in vitro, and the underlying mechanism was investigated. Geranial, geranyl acetate, β-citronellol, citronellal, limonene, and citral were demonstrated to be the major constituents of the EOs. Lemongrass EOs and the main compounds possessed the antifungal effect against F. avenaceum, and citral exerted the strongest antifungal effects against F. avenaceum compared with other compounds, with IC50 values of 0.087 µl/mL. Lemongrass EOs and their major constituents increased the permeability of the F. avenaceum plasma membrane, resulting in increased conductivity, protein and soluble sugar leakage. The activity of pectin methyl galacturonase (PMG) in F. avenaceum was suppressed after treatment with lemongrass EOs. The results indicated that lemongrass EOs inhibited F. avenaceum growth by targeting its plasma membrane and reduced its pathogenicity by inhibiting enzyme activity.

The infection of grapes by Talaromyces rugulosus O1 and the role of cell wall-degrading enzymes and ochratoxin A in the infection

Talaromyces rugulosus O1 is an ochratoxin A (OTA)-producing pathogen from grapes. The mechanisms involved in the infection of postharvest grapes by T. rugulosus O1 are not yet clear. In this study, the infection of grapes by T. rugulosus O1 and OTA production in grapes were investigated, and the role of cell wall degrading enzymes of T. rugulosus O1 and OTA in the infection of grapes by T. rugulosus O1 was explored. The results indicated that T. rugulosus O1 showed significant infection effect on grapes at 20 °C, and accumulated OTA in the wound of grapes in the infection progress. In comparison with the control, the activities of polygalacturonase (PG), pectin methylgalacturonase (PMG), exo-1,4-β-glucanase (C1) and β-glucosidase (β-G) secreted by T. rugulosus O1 in grapes were all increased in the infection process. Furthermore, the pectinase (PG and PMG) activities were higher than cellulase (C1 and β-G) activities. The expression levels of genes encoding PG and β-G in T. rugulosus O1 infecting grapes increased as the time prolong. Inoculation of T. rugulosus O1 in combination with addition of OTA in the wound of grapes resulted in higher decay incidence and lesion diameter of treated grapes compared with the treatment with T. rugulosus O1 or OTA alone. This indicated that OTA can promote the infection of grapes by T. rugulosus O1. This study provides useful information about the role of cell wall degrading enzymes and OTA in the infection of grapes by T. rugulosus O1.

Application of sodium silicate retards apple softening by suppressing the activity of enzymes related to cell wall degradation.

During the storage of apples, apple softening is one of the main problems. Sodium silicate has been used to enhance disease resistance and maintain quality of fruits. In the present study, apple fruit (cv. Golden delicious) were treated with 100 mmol L-1 sodium silicate for 10 min and stored at 20 °C to investigate its effects on weight loss, flesh firmness, and the activity of cell wall-degrading enzymes. The results indicated that 100 mmol L-1 of sodium silicate treatment delayed the increase of weight loss and decrease of the flesh firmness in apples. Sodium silicate treatment also suppressed the activity of polygalacturonic acid transeliminase and pectin methyltranseliminase, pectin methylgalacturonase, polygalacturonase, cellulase and β-galactosidase in the fruit. Delaying apple softening by sodium silicate treatment is closely related to the inhibition of the activity of cell wall-degrading enzymes and weight loss. © 2018 Society of Chemical Industry.

Inhibitory effects of sodium silicate on the fungal growth and secretion of cell wall‐degrading enzymes by Trichothecium roseum

AbstractTrichothecium roseum causes decay in muskmelons, apples, tomatoes and mangoes, which leads to economic losses. In this study, we investigated the effect of sodium silicate on the growth of T. roseum and the cell wall‐degrading enzymes (CWDEs) secreted by the hyphae. The results indicated that sodium silicate significantly inhibited mycelial growth and spore germination of T. roseum. The sodium silicate treatment also retarded the secretion of several CWDEs, including pectate lyase (PL), polygalacturonic acid transeliminase (PGTE), pectin methyltranseliminase (PMTE), pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β‐glucosidase. These results suggest that sodium silicate exerts its effects on T. roseum through direct inhibition of its growth and secretion of CWDEs.

Control Effect and Possible Mechanism of the Natural Compound Phenazine-1-Carboxamide against Botrytis cinerea.

To develop new agents against strawberry grey mould and to aid in the development of biological pesticides, we investigated the inhibitory effect of a natural compound, phenazine-1-carboxamide (PCN), against Botrytis cinerea using a growth rate assay. Additionally, indoor toxicity and the in vitro control effect of PCN were further studied to determine its potential mechanisms of action on B. cinerea. PCN was inhibitory against B. cinerea with a 50% effective concentration (EC50) of 108.12 μg/mL; the toxicity of PCN was equivalent to that of carbendazim (CBM). The best in vitro control effect of PCN against grey mould in strawberry (fruit) reached 75.32%, which was slightly higher than that of CBM. The field control effect of PCN against grey mould reached a maximum of 72.31% at a PCN concentration of 700 μg/mL, which was 1.02 times higher than that of CBM. Fungistatic activity was observed at low concentrations of PCN, while high concentrations of PCN resulted in fungicidal activity against B. cinerea. This natural compound strongly inhibited both spore and sclerotium germination of B. cinerea, with the best relative inhibition rates of 77.03% and 82.11%, respectively. The inhibitory effect of PCN on mycelial growth of B. cinerea was significant and reached levels of 87.32%. Scanning electron microscopy observations revealed that after 48 h of PCN treatment, the mycelia appeared loose, locally twisted, and folded, with exudation of contents; the mycelia was withered and twisted, with edge burrs, deformations, ruptures and a sheet-like structure. Transmission electron microscopy observations revealed that after 48 h of PCN treatment, the structure of the cell nucleus was unclear and the vacuoles had ruptured; additionally, various organelles exhibited disordered structures, there were substantial non-membrane transparent inclusions, the cells were plasmolysed, the cell walls were collapsed in some cases, and the hyphal tissue was essentially necrotic. A PCN dosage of 35–140 μg/mL had no effect on the cell membrane permeability of the mycelia, while a PCN dosage of 700 μg/mL resulted in significant permeability. PCN inhibited B. cinerea toxin; the mycotoxin level was approximately 0.41 of the value recorded for the control at a PCN dosage of 700 μg/mL. PCN affected the activity of pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β-glucosidase (BG); the lowest activities of PMG, PG, BG and Cx reached 0.3 U/mg, 0.62 U/mg, 0.64 U/mg, and 0.79 U/mg, respectively, after treatment with 700 μg/mL PCN.

Influence of native carbon sources on the production of pectolytic and cellulolytic enzymes by Fusarium oxysporum and Fusarium moniliforme

Summary Pectin in the case of F. oxysporum and sodium polypectate in the case of F. moniliforme were proved to be the best native carbon sources for polygalacturonase (PG) production. Maximal pectinmethyl-galacturonase (PMG) production was recorded with pectin in both the test pathogens. Cotton supported maximal cellulase production in F. oxysporum whereas CMC in F. moniliforme . Sucrose which served as control was inferior to all the native carbon sources for the production of pectic and cellulolytic enzymes. Both the species of Fusarium were capable of producing various pectic and cellulolytic enzymes constitutively and the increased production in presence of certain native (pectic/cellulosic) substances can be considered merely the stimulation of these enzymes synthesis rather than actual induction. No exact correlation could be drawn between biomass and enzyme production. All the native carbon sources used in the present studies were found to be inferior than sucrose for biomass production.

Phenolic compounds: Inhibitory agents of cell wall degrading enzymes by Fusarium oxysporum and Fusarium moniliforme in vitro

All the phenolics decreased the synthesis of pectic (PG abd PMG) and cellulase (Cx) enzymes. The degree of inhibition in each case was, however, found to vary with the type of its concentration. Usually, inhibitory effect increased with the increase in concentration of each phenolic substance in both the pathogens. Total repression of polygalacturonase (PG) enzyme production was observed by vanillin (100 ppm) in both the Fusarium species, α-naphthol and pyrocatechol, both at 1,000 ppm concentration, completely checked the PG enzyme production in F. oxysporum and F. moniliforme , respectively. Pyrocatechol, phloroglucinol and vanillin (1,000 ppm) checked the pectin methyl galacturonase (PMG) production in F. moniliforme , whereas in F. oxysporum , vanillin and α-naphthol (1,000 ppm) were effective. None of the phenolic compunds could check the cellulase production in F. oxysporum , but in F. moniliforme , vanillin (500 ppm) could check the cellulase production. No exact correlation could be drawn between fungal growth and enzyme production. Alle phenolischen Verbindungen reduzierten die Bildung von Pectinase- und Cellulaseenzymen. Der Grad der Hemmung variierte in jedem Falle mit der Konzentration. Gewöhnlich nahm die Hemmwirkung mit zunehmender Konzentration der Phenole bei beiden Pathogenen zu. Eine totale Unterdrückung der Polygalcturonaseproduktion wurde durch Vanillin (100 ppm) bei beiden Fusarium -Arten beobachtet. α-Naphthalin und Pyrocatechol in Konzentrationen von 1,000 ppm hemmten ganz die PG-Enzymproduktion bei F. oxysporum und F. moniliforme . Pyrocatechol, Phloroglucin und Vanillin (1,000 ppm) hemmten die Pectinmethylgalcturonasebildung bei F. moniliforme , und bei F. oxysporum waren es Vanillin und α-Naphthol (1,000 ppm). Keine der phenolischen Verbindungen konnte die Cellulasebildung bei F. oxysporum unterdrücken; bei F. moniliforme wurde sie allerdings durch Vanillin (500 ppm) gestoppt. Es konnte keine strenge Korrelation zwischen dem pilzlichen Wachstum und der Enzymproduktion festgestellt werden.

The influence of carbon nutrition on activation and activities of pectolytic enzyme in cultures of Xanthomonas campestris pv. vignicola

Summary In Xanthomonas campestris pv. vignicola the two hydrolases PMG (pectin methyl galacturonase) and PG (polygalacturonase) were best activated at 35°C with 60 min of enzyme-substrate incubation. Both of them were adaptively synthesized with pectin and NaPP. Addition of glucose to either pectin or NaPP enhanced the production of PMG and PG, respectively. Maltose and CMC had adverse influence on PG development. In young cultures pectin transeliminase (PTE) was adaptively synthesized with pectin as the source. In old cultures it was best produced constitutively with CMC as the source. While NaPP increased the capacities of glucose or maltose to activate PTE, it retarded the constitutive ability of CMC to synthesize this enzyme. The other transeliminase, PGTE, was constitutively synthesized in young culture with glucose as the choice carbon source. The addition of NaPP to glucose considerably lowered the constitutive ability of 24-h-old filtrate for activating this enzyme. It had separate synergistic influence on maltose, pectin, or CMC for very prominent level production of PGTE. In young and old cultures the endo-PG activity had preponderance over exo-PG, since NaPP substrate was hydrolysed with more of random cleavage. However, in the older filtrates exo-PG activity, too, picked up gradually.

Influence of various nitrogen and carbon sources on the production of pectolytic, cellulolytic and proteolytic enzymes byAspergillus niger

Three isolates ofAspergillus niger produced polygalacturonase (PG) and pectin methyl galacturonase (PMG) in the presence of organic and inorganic nitrogen sources. Complete inhibition of PG PMG cellulase (Cx) and proteinase synthesis was found in the presence of cystine in all isolates. Maximum biomass was found in sodium nitrate whereas no isolate could grow in the presence of cystine. A correlation between biomass and enzyme production could be obtained when sodium nitrate and cystine were added to the medium separately. All isolates produced pectic cellulolytic and proteolytic enzymes in the presence of various native carbon sources. Sodium polypectate was found to be the best carbon source for the production of PG and PMG; pectin inhibited completely the production of PG and PMG. Maximum cellulase production was brought about by cotton in all three isolates. Maximum proteinase production was observed with gelatin which served as poor substrate for fungal growth. Sucrose supported maximum fungal growth in comparison with all other native carbon sources. The increased production of pectolytic cellulolytic and proteolytic enzymes in the presence of sodium polypectate reflected a stimulation rather than an induction of synthesis of these enzymes.

Production of pectolytic and cellulolytic enzymes byfusarium oxysporum andF. moniliforme under different cultivation conditions

Six-day incubation was most suitable for production of pectolytic and cellulolytic enzymes byFusarium on different culture media. Czapek’s medium favoured maximum production of polygalacturonase (PG) and cellulase (Cx), peptone dextrose gave highest yields of pectin methyl galacturonase (PMG) withF. oxysporum. Cole’s medium was found to be poor for the enzyme production by both organisms. A positive correlation was observed between the growth rate of the pathogenic forms and their enzyme production. InF. oxysporum the PG secretion was maximum at pH 4.5 and inF. moniliforme at pH 5.0. PMG production optimum was at pH 5.5. No PG and PMG were produced above pH 7. InF. oxysporum the Cx activity was highest at pH 5.5 and inF. moniliforme at pH 4.5. Maximum PG and PMG activities were recorded at 35 °C in both pathogens. The Cx activity of both organisms was maximum at 45 °C but some carboxymethyl cellulose hydrolysis was found even at 60 °C.

Studies on glycosidases: Effect of carbohydrates and amino acids on the production of pectin methyl galacturonase by Alternaria species

Summary Among various carbohydrates, glucose and galactose were found to be the best promotors for pectin methyl galacturonase (PMG) production in Alternaria solani and A. tenuis , starch caused maximum inhibitory effect in both the Alternaria species. Histidine and asparagine could produce maximum amount of PMG in case of A. solani and A. tenuis . Tyrosine in the case of A. solani and serine in the case of A. tenuis were also effective for this enzyme production. No exact correlation could be drawn between fungal growth and enzyme production.

Inhibitory effect of some plant growth regulators, phenolics and fungicides on the production of pectin methylgalaturonase byAlternaria solani andA. tenuis

Secretion of pectin methylgalacturonase (PMG) byAlternaria solani was suppressed in the following sequence of active agents (100 ppm): kinetin > indolebutyric acid > vanillin > gibberellic acid > ferulic acid > 3-hydroxybenzaldehyde > phloroglucinol > indolepropionic acid > indoleacetic acid. InA. tenuis the sequence was kinetin=gibberellic acid=indoleacetic acid=indolebutyric acid (all completely inhibitory) > phloroglucinol > indolepropionic acid > ferulic acid. Of ten fungicides tested, most potent in both species were thiram, brassicol, blimix and sultaf (at 1000 ppm). Mycelial growth was relatively less affected by the active agents and fungicides inA. tenuis (range of % inhibition from 10 to 21) than inA. solani (range from 25 to 67%).

The Pectolytic Enzymes and Their Activities in Cultures of Xanthomonas malvacearum (Smith) Dowson

Summary The pectolytic enzymes, pectinmethyl galacturonase (PMG), polygalacturonase (PG), polygalacturonate trans-eliminase (PGTE), and pectin methylesterase (PME), were sensitized differentially by varied carbon sources in culture of Xanthomonas malvacearum . PG activities ensured release of mono- and poly-galacturonides from NaPP-substrate. Potato maceration too was effectuated. Enzyme-substrate incubation at 30 °C for 60 min was optimum for PMG and PG elaboration. PME was activated best with 4 h of incubation. PMG and PME were pectin-inducible. PG and PGTE were NaPP-adaptive. However, pectin was equally effective for PG secretion. Addition of glucose to pectin repressed PGTE, PMG, and PG extrusion when added separately to NaPP. Glucose and CMC had retardatory effect on PMG and PGTE excretion. CMC, in combination with pectin, also reduced PME and PG activities. Individually glucose and maltose were poor carbon sources for production of these enzymes by the pathogen. Exo-PG activity, leading to NaPP-substrate break-down and release of galacturonides in varying proportions, was evident in filtrate of 24- and 48-hour-old cultures. Additionally, endo-PG activity picked up well in 72-hour-old culture. Such situations were uniformly true of enzymes obtained from NaPP-, glucose-, maltose-, and pectin-supplied culture filtrates. Separate addition of glucose to NaPP and to pectin, and of CMC to pectin exhibited synergic influence on filtrate which engineered more of galacturonide liberation from the substrate. Individually, maltose was a better source than glucose for inducing exo-PG activity. Potato disc maceration was best effectuated by 48-hour-old culture filtrate. It was optimum for enzyme obtained from NaPP-supplied medium. The next best in this regard was pectin + glucose-supplied culture filtrate enzyme.