Interactions of the tomato with two formae speciales of Fusarium oxysporum

Tomato cuttings were inoculated with Fusarium oxysporum f.sp. lycopersici (FL) and F. oxysporum f.sp. pisi (FP) by standing the cuttings in suspensions of bud-cells of the fungi. FP never induced external symptoms although the fungus persisted in the lower parts of the cutting. FL at concentrations from 10³ to 10⁶ spores per ml induced typical wilt symptoms but there was subsequent recovery of some cuttings with the production of uninvaded side shoots. When the cuttings were inoculated with mixed suspensions of bud cells of the two fungi there was marked reduction of symptoms. The extent of this reduction was related to the proportion of FP/FL bud cells for a fixed inoculum of FL in the mixture and was moderate at a rate of 1/3 and complete at ratios from 4/1 to 9/1. Mixed suspensions of heat-killed bud cells of FP with live bud cells of FL in the ratio of 4/1 induced normal symptoms and it was concluded that the symptom mitigation induced by FP was related to the presence of living cells of the fungus. Root inoculations with mixed suspensions also gave less wilt than with FL alone. Symptom mitigation was apparently associated with a reduction of the extent of invasion of the cuttings but in vitro tests failed to demonstrate that exudates or extracts from normal or invaded tomato tissue induced any reduction of growth of the tomato pathogen.     

Enhanced polygalacturonase production by Aspergillus niger NRRL-364 grown on supplemented citrus pectin

Enhanced levels of extracellular polygalacturonase activity were obtained when Aspergillus niger NRRL-364 was grown on pectic substances as sole carbon sources in a submerged culture. Among the factors affecting enzyme production those of carbon source concentration, nitrogen source, initial pH and time of cultivation were found to be the most important ones. Under optimum growth and activity conditions yields as high as 14.5 U (measured as reducing groups) ml^-1 of growth medium were obtained, comparing favourably with those reported for fungi grown under similar conditions and used in food processes.     

Alteration of cell-wall composition ofFusarium oxysporum by copper stress

A strain ofFusarium oxysporum tolerated copper in the growth medium at concentrations up to 600 mg/L. The optimum growth was obtained at 200 mg Cu/L. The mycelium acquired a blue color in the presence of copper. The copper content of isolated cell walls obtained from mycelium grown in the presence of 600 mg Cu/L was 1.5 times higher than that of cell walls obtained from mycelium grown at 200 mg Cu/L and it contained 2.2 and 3.3% copper at 200 and 600 mg Cu/L, respectively. The amount of protein and total sugars increased in both the mycelium and its isolated cell walls in the presence of copper in the growth medium, chitin was also increased in the cell wall, reaching its maximum amount at 200 mg Cu/L— about 2.4 times higher than without copper. Most of amino acid concentrations in the cell wall were increased in the presence of 200 mg Cu/L and decreased above this concentration. Isoleucine, leucine, tyrosine, phenylalanine, and arginine showed the highest increase at this concentration. The altered cell walls obtained from mycelium grown at 200 and 400 mg Cu/L could rebind individual metals more than the control cell walls could. Rebinding of individual metals was in the order Zn>Fe>Ni>Cu>Co. Rebinding of copper by isolated cell walls depended on pH and temperature.     

Batch and fed batch production of pectin lyase and pectate lyase by novel strain Debaryomyces nepalensis in bioreactor

The effect of various parameters such as pH, agitation and aeration was studied for maximum production of pectin lyase (PL) and pectate lyase (PGL) by a novel yeast strain Debaryomyces nepalensis in bioreactor. The optimal levels of pH, aeration and agitation rate was found to be 7.0, 300rpm and 1vvm, respectively. Under these conditions, D. nepalensis produced 14,200U/L of PL and 12,000U/L of PGL corresponding to a productivity of 600U/Lh and 500U/Lh of PL and PGL, respectively. Fed-batch production was studied by feeding inducer (lemon peel), carbon source (galactose) individually and in combination at 12h of growth for enhanced production of PL and PGL. Combined feeding of inducer and carbon source at 12h was found to be the best strategy for enhanced production of PL and PGL. Under these conditions, production of PL and PGL increased to 23,300U/L and 22,400U/L, respectively which corresponded to a productivity of 728U/Lh of PL and 700U/Lh of PGL, respectively. The production was increased by 1.6- and 1.8-fold and productivity by 1.2- and 1.4-fold for PL and PGL, respectively when compared to batch culture.     

Studies of the survival ofFusarium oxysporum conidia produced by submerged culture

Summary To study the survival of conidia ofFusarium oxysporum produced by submerged culture on malt extract, a harvesting process and different packaging and storage conditions have been tested. Conidia dried with talc and stored at +4°C preserve their viability after about 4.5 months.     

Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X _m) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108–113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X _m values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.     

The production and properties of a crude pectin lyase from Lachnospira multiparas

The production of pectin lyase (PL) was dependent on medium pH with two peaks of production, at pH 61 and 7-2, that at pH 61 showing considerably higher activity. Crude PL activity was stable for at least 3 h at both 30 and 40C. At 50C there was a 20%, reduction in activity after 1 h; at 60C there was a 40% reduction after 30 min yet > 50% activity was retained for a further 2 h. The crude enzyme exhibited macerating ability against carrot tissue commensurate with that of a currently available commercial pectinase enzyme.     

Induction of polygalacturonase and the formation of oxalic acid by pectin in brown-rot fungi

Extracellular polygalacturonase (PG) production was estimated in vitro, using liquid cultures of three species of brown-rot decay fungi (Postia placenta, Gloeophyllum trabeum and Serpula incrassata), by cup-plate assay, assay of reducing sugars, and decrease in viscosity. Although all three experimental assays demonstrated that PG was induced by pectin in all three fungi, decrease in viscosity gave the best correlation with decay capacity in soil block tests. PG activity, determined as an increase in reducing sugar activity, was greatest in G. trabeum and weakest in S. incrassata. The optimum pH for PG activity was between pH 2.5 and 4.5. Oxalic acid production was also enhanced by pectin and functioned synergistically with PG activity. We conclude that these fungi produce PG that is best induced by pectin and that PG activity exceeds production of xylanase and endoglucanase activity in vitro. Polygalacturonase is likely to act synergistically with oxalic acid to solubilize and hydrolyse the pectin in pit membranes and middle lamellae. Thus, production of PG and oxalic acid should facilitate early spread of hyphae and enhance the lateral flow of wood-decay enzymes and agents into adjacent tracheids and the wood cell wall, thus initiating the diffuse decay caused by brown-rot fungi.The Forest Products Laboratory is maintained in co-operation with the University of Wisconsin. This article was written and prepared by US Government employees on official time, and it is therefore in the public domain and not subject to copyright.     

Clarification of apple juices by pectin lyase from Penicillium paxilli

Summary Pectin lyase EC 4.2.2.10 from Penicillium paxilli was able to clarify apple juices with a low level of polyphenols. Enzymatic clarification of these juices was stimulated by Ca⁺⁺ions in a concentration of 10^-2M.     

Regulation of the production of polygalacturonase by Aspergillus terreus

The synthesis of polygalacturonase (PG) (EC 3.2.1.15) by a strain of Aspergillus terreus was induced by polygalacturonic acid and repressed by glucose, galactose or fructose even in the presence of the inducer. The production of PG increased when the mycelium was washed free of glucose and incubated in a glucose-free medium containing the inducer, a fact that indicated the reversibility of the repression mechanism. When Actinomycin D and cycloheximide were added to the culture medium, the synthesis of PG ceased. PG synthesis increased 43% with the addition of methionine and 64% both with leucine and with tyrosine. Specific productivity with leucine was 210% higher than that of the control as against 149% with methionine and 70% with tyrosine. The results obtained suggest that PG synthesis is regulated by leucine.     

Regulation of the production of polygalacturonase by Aspergillus niger

Synthesis of ethylene in static cultures as well as the effect of endogenous and exogenous ethylene on the synthesis of polygalacturonase byAspergillus niger were determined. This strain produced maximum ethylene amounts when cultured at 30 °C for 3 d. The effect of adding ethylene precursors (citrate-cycle intermediates) on ethylene production was investigated. Best intracellular and extracellular polygalacturonase production was obtained with 2-oxoglutaric, pyruvic and fumaric acids, and with glutamic acid too. Addition of ethylene to the culture medium also increased the synthesis of polygalacturonase, although to a lower degree than when glutamic acid was added.     

Purification and characterization of pectin lyase secreted by Aspergillus flavus MTCC 10938

An indigenously isolated fungal strain Aspergillus flavus MTCC 10938 was subjected to pectin lyase (PNL) production under submerged fermentation conditions. The enzyme was purified to homogeneity from the culture filtrate of the fungus involving concentration by ultrafiltration, anion exchange chromatography on DEAE cellulose and gel filtration chromatography on Sephadex G-100. The purified PNL gave a single protein band in SDS-PAGE analysis with a relative molecular mass corresponding to 50 kDa. Using citrus pectin as the substrate the K _m and k _cat values of the enzyme were obtained as 1.7 mg/ml and 66 s^?1, respectively. The optimum pH of the purified PNL from A. flavus MTCC 10938 was 8.0 and up to 90% of its activity retained in the pH range from 3.0 to 11.0 after 24 h incubation. The optimum temperature of the purified enzyme was revealed at 55°C and it was completely stable up to 40°C when exposed for 30 min. The purified A. flavus MTCC 10938 PNL showed efficient retting of Crotalaria juncea fibres.     

Pectin lyase production by Penicillium griseoroseum: Effect of tea extract, caffeine, yeast extract, and pectin

Summary Mycelial growth and production of extracellular pectin lyase by Penicillium griseoroseum at different concentrations of inducers were investigated. The fungus was cultured in mineral medium using sucrose as a carbon source and caffeine, yeast extract, tea extract or pectin as inducers. Caffeine, yeast extract and tea extract in the presence of sucrose, and tea extract alone were capable of inducing pectin lyase in P. griseoroseum, even at low concentrations.     

Selective and sensitive detection of pectin lyase activity using a colorimetric test: application to the screening of microorganisms possessing pectin lyase activity

Several methods have been described for the detection and quantification of polygalacturonase (PG) and pectin lyase (PL) activities. The most frequently used tests are the Nelson method using copper(II) and an arsenomolybdate reagent to detect PG activity, and the colorimetric method using thiobarbituric acid (TBA) to detect PL activity. We observed that none of these methods are suitable to differentiate between these two enzymatic activities. Therefore, we optimized the test conditions of the TBA method. As a result, the detection of the enzymatic beta-elimination (PL activity) became sensitive and selective. A basic pretreatment at 80 degrees C for 5 min of the solution which contains the pectin fragments of the PL activity furnished aldehydes which were condensed with TBA or its derivatives. After acidification of the medium, a pink fluorescent dye was detected spectrophotochemically (lambda = 550 nm). The interference of galacturonic acid or oligomers resulting from PG activity was completely eliminated. The most sensitive reagent was N-(pyridin-2-yl)-thiobarbituric acid. The application of this method with the new reagent was extended to the screening of microorganisms possessing the PL activity. The obtained results confirm that Aspergillus niger strain and a Saccharomyces cerevisiae SCPP strain possess this activity.     

Temperature-dependent production of carotovoricin Er and pectin lyase in phytopathogenic Erwinia carotovora subsp. carotovora Er

The production of pectin lyase (Pnl) and carotovoricin (Ctv), as well as cell lysis in the plant-pathogenic bacterium Erwinia carotovora subsp. carotovora Er are induced by mitomycin C. Here, Pnl and Ctv production and cell lysis were found to be temperature-dependent. The optimal temperature for Pnl production was 30 degrees C. However, the optimal temperature for both Ctv production and cell lysis was 23 degrees C, at which Pnl production was reduced to 47% of the maximum. These data suggest the possible existence of novel regulation system(s) for the production of Pnl and Ctv, and cell lysis, in addition to the well-documented regulation system of recA, rdgA, and rdgB genes.     

Viscosity decrease of pectin and fruit juices catalyzed by pectin lyase from Penicillium italicum in batch and continuous-flow membrane reactors

Summary The extracellular pectin lyase (PNL, E.C. 4.2.2.10) from Penicillium italicum was utilized in batch and confined in a continuous-flow ultrafiltration membrane reactor. The enzyme catalyzed the decrease in viscosity of pectin solutions at pH 6.0 as well as of different fruit juices at their respective pH. PNL decreased the viscosity of pectin solutions in the membrane (60% after 30 min) more than in the batch (46% after 70 min) reactors, but similar viscosity reduction of fruit juices was achieved in both reactors. The enzyme decreased the viscosity of grape, peach and melon juices to different extents, but failed to do so with apple or pear juices. It can be concluded that the utilization of PNL in a membrane reactor appears of interest for the clarification of fruit juices.     

Partial purification and properties of pectin lyase from Penicillium expansum

A pectin lyase, poly(methoxygalacturonide) lyase, EC 4.2.2.10, from a culture filtrate of Penicillium expansum was partially purified 33-fold with 7.3% yield. The enzyme was monomeric with a molecular mass of 36.5 kDa. The enzyme did not contain pectate lyase activity and degraded citrus and apple pectin best at pH 7.0 and 40 to 45°C. The K _m for citrus pectin was 9 mg ml^-1.