Cup Plate Assay Research Articles

Screening of Actinomycetes from Soil for Antibacterial Activity

Actinomycetes are Gram positive, free living saprophytes which are distributed in soil as one of the major populations and are primary source of antibiotics. This study was carried out with a quest to isolate actinomycetes from soil samples of different places and assess their antibacterial activity. Isolation of actinomycetes was carried out by serial dilution of soil sample followed by spread plate method. The antimicrobial extract was extracted using ethyl acetate. Assessment of antimicrobial activity was performed by using Agar cup plate assay against test organisms (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Salmonella paratyphi, Bacillus subtilis, Staphylococcus aureus). Antibacterial activity was tested against Methicillin Sensitive Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus in the isolates having effective inhibitory activity against Staphylococcus aureus. From 15 soil samples of 12 different locations, 121 actinomycetes isolates were isolated. Among them, 58 (47.9%) isolates were inhibitory against at least 1 test organism in primary screening, of which 22 isolates effective against more than 1 test organism was chosen for secondary screening. Out of them, 8 were inhibitory against 2 test organisms while 14 were inhibitory against 3 test organisms. Staphylococcus aureus was found to be the most susceptible test organism with its susceptibility against 12 actinomycetes isolates. Among 12 isolates effective against Staphylococcus aureus, 10 were found to have an inhibitory effect against Methicillin Susceptible Staphylococcus aureus while 6 were found to have inhibitory effect against Methicillin Resistant Staphylococcus aureus strain. The findings of this study highlight the inhibitory potential of actinomycetes and the need for further investigation for obtaining novel antimicrobial agents from actinomycetes from various unexplored areas.

Novel bio-fabrication of silver nanoparticles using the cell-free extract of Lysinibacillus fusiformis sp. and their potent activity against pathogenic fungi

Green synthesis of nanoparticles is considered an efficient method when compared with chemical and physical methods. Herein, an extracellular green method was adopted to synthesize silver nanoparticles (AgNPs) by using the cell-free extract of a new silver-resistant bacterium, Lysinibacillus fusiformis sp., which was isolated from the sewage treatment plant and identified by 16 S rRNA gene sequencing. Without using any toxic organic solvents and reducing agents, the cell-free extract effectively reduced Ag+ to AgNPs with a conversion rate of 72.21% under the optimal condition of pH 9.0, 50 °C, and 5 mM of silver nitrate solution. The newly-developed AgNPs were characterized by the methods of ultraviolet-visible spectroscopy (UV–vis), Fourier transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), transmission electron microscopy (TEM), zeta potential/size distribution analysis and energy-dispersive x-ray spectroscopy (EDS). Characterization results showed that the as-prepared AgNPs were spherical in shape and well-dispersed with an average size of ∼50 nm. The results of the Oxford cup plate assay used for measuring the antifungal activity indicated a pronounced antifungal activity of the biosynthesized nanoparticles against pathogenic fungi of tinea pedis, i.e., Candida albicans, Trichophyton rubrum, Trichophyton mentagrophytes. In conclusion, the biosynthesized AgNPs might display a high potential as novel antifungal agents in medicine. However, it is indispensable to conduct in vivo studies to explore the biocompatibility of AgNPs for their future therapeutic applications.

Biological control ginseng grey mold and plant colonization by antagonistic bacteria isolated from rhizospheric soil of Panax ginseng Meyer

Botrytis cinerea Pers.ex Fr. is a common soil borne fungal pathogen that infects economic crops, such as ginseng, worldwide. One ideal strategy for controlling B. cinerea is to develop and use beneficial rhizobacteria. The goal of this study was to assess the potential of rhizospheric bacteria as antifungal agents against ginseng grey mold disease. In this study, 169 bacterial strains isolated from rhizospheric soil of ginseng plants were screened using the K-B paper dispersion method and the Oxford cup plate assay. Subsequently, strain SW-34 was selected as a potential stock for biocontrol due to the broad-spectrum antibiotic activity of the SW-34 fermentation liquor against pathogens. To assess the antifungal efficacy of SW-34 against ginseng grey mold as well as its ginseng growth promotion abilities, pot experiments were performed under plastic greenhouse conditions. The morphology, physiological and biochemical characteristics, 16S rRNA and gyrB gene sequence were assessed to for the taxonomic characterization of strain SW-34. The results showed that, SW-34 (which was identified as Bacillus amyloliquefaciens) exhibited an antagonistic activity against B. cinerea of 85.13% and also provided broad-spectrum antibiotic activity. Inoculation of the SW-34 cell suspension suppressed disease lesions caused by ginseng grey mold with a strong control efficacy of 72.29%, which was significantly higher than that of the Mancozeb 70% WP fungicide treatment (p < 0.05). Moreover, SW-34 exhibited high cell densities in ginseng plants (root, leaf and stem) and promoted ginseng plant growth. Because the B. amyloliquefaciens SW-34 successfully inhibited ginseng grey mold, the results of our study provides a basis for the potential biocontrol of ginseng grey mold caused by B. cinerea.

Studies on Antimicrobial activity of a metabolite produced by Streptomyces malaysiensis isolated from Termite Mound Soil

To discover novel secondary metabolites, our approach is to investigate unexplored regions of the world with the aim of isolating bioactive actinomycetes from the termite mounds soil sample. Actinomycetes are soil microorganisms. They are a type of bacteria. They are the same as each microorganism and fungi, and have characteristics linking them to both groups Actinomycetes square measure typically believed to be the missing biological process link between microorganism and fungi, however they need more characteristics in common with microorganism than they are doing fungi.13 actinomycetes and 3 fungal isolates were screened from the soil sample and out of them 6 actinomycetes showed anti microbial activity by agar-overlay method, Cross streaking method and Cup plate method. By the Agar overlay method we can assure that the isolate TMS 1A is having anti bacterial activity and anti fungal activity by measuring the inhibition zone diameter as 40mm and 35mm respectively. Cross streaking method of the active compound obtained from isolate TMS 1A shows that it is having antimicrobial activity against Gram +ve, Gram-ve bacteria and fungi. Cup plate assay of the active compound obtained from isolate TMS 1A shows that it is having anti bacterial activity. Results from anti microbial activity revealed that our isolate TMS 1 08 09 2010 1A was found to have broad spectrum of anti microbial activity on both (Gram + ve and Gram-ve) bacteria and fungi. Further investigation is required to extract the Bioactive metalbolites from Streptomyces malaysiensis.

Novel pectinase from Piriformospora indica, optimization of growth parameters and enzyme production in submerged culture condition

Abstract Background Pectinases are one of the upcoming enzymes in food processing industries and they hydrolyze pectin substances. Objectives This study was done to examine the production of pectinase by Piriformospora indica in submerged fermentation (SmF) along with growth parameters. Materials and methods The fungus P. indica was cultured on Kafer medium supplemented with pectin for 0–12 days and fungus growth, number of spores, total protein content, and pectinase activity were investigated. Results Firstly, pectinase secretion by P. indica was confirmed by cup-plate assay. The maximum dry cell weight (10.21 g/L), growth yield (0.65 g/g), specific growth rate (0.56 day−1) and pectinase activity (10.47 U/mL) on pectin containing medium (P+) were achieved after 6 day of culture. In the case of pectin free medium (P−) all the parameters were less than P+ medium. The pectinase production by P. indica on P+ was 2.7 times higher than P−. The optimum pH and temperature for maximum polygalacturonase activity were 5 and 50°C, respectively. Conclusions For the first time, the work confirmed pectinase secretion by P. indica fungus and it could be a good source for pectinase production. Moreover, optimum pH 5, make it a potential candidate for future application in fruit juice industries.

Bacillus subtilis sp. B1, Chitinase Producing Isolated from Indonesian Tropical Shrimp Pond Waste Water

Twenty-five bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Bacillus subtilis sp. B1 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Bacillus subtilis sp. B1 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Bacillus subtilis sp. B1 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 4 and 40◦C, with values specific activity of chitinase to be 324.56 U/mg and 259.70 U/mg, respectively. In addition, the study of the morphological alteration of chitin treated by enzyme, indicating a potential application of this enzyme in several industries.

&lt;b&gt;Use of agro-industrial wastes as substrates for α-amylase production by endophytic fungi isolated from &lt;i&gt;Piper hispidum&lt;/i&gt; Sw

Millions of tons of corn, pineapple, sugarcane and wheat are annually produced and their industrial processing generates large amounts of wastes. Current study evaluated the use of corncob (CC), pineapple peel (PP), sugarcane bagasse (SB) and wheat bran (WB) as substrates for α-amylase production by submerged cultures of nine endophytes from Piper hispidum Sw. Initially, fungi were grown on a starch medium and the cup plate assay showed that five strains were amylase-positive: Bipolaris sp. JF767001, Marasmius cladophyllus JF767003, Phlebia sp. JF766997, Phyllosticta capitalensis JF766988 and Schizophyllum commune JF766994, with enzymatic halos ranging between 6.30 ± 0.08 and 8.87 ± 0.68 mm in diameter. Further, the use of agro-industrial wastes was evaluated by the cup plate assay, where the highest halo diameters were obtained from fungi grown on PP or SB: up to 15.00 ± 0.16 mm ( Phlebia sp.) and 14.80 ± 0.18 mm ( S. commune ), respectively. Submerged cultures with PP or SB showed the highest levels of α-amylase activity according to the starch-iodine assay, or rather, up to 4.14 ± 0.02 U mL -1 ( Bipolaris sp.) and 4.09 ± 0.02 U mL -1 ( Phlebia sp.), respectively. Results give an in-depth knowledge on tropical endophytes that might be α-amylase sources, and indicate the suitability of these agro-industrial wastes as substrates for fungal enzymatic production.

An investigation of Panax ginseng Meyer growth promotion and the biocontrol potential of antagonistic bacteria against ginseng black spot

BackgroundGinseng black spot disease resulting from Alternaria panax Whuetz is a common soil-borne disease, with an annual incidence rate higher than 20–30%. In this study, the bacterial strains with good antagonistic effect against A. panax are screened. MethodsA total of 285 bacterial strains isolated from ginseng rhizosphere soils were screened using the Kirby–Bauer disk diffusion method and the Oxford cup plate assay. We analyzed the antifungal spectrum of SZ-22 by confronting incubation. To evaluate the efficacy of biocontrol against ginseng black spot and for growth promotion by SZ-22, we performed pot experiments in a plastic greenhouse. Taxonomic position of SZ-22 was identified using morphology, physiological, and biochemical characteristics, 16S ribosomal DNA, and gyrB sequences. ResultsSZ-22 (which was identified as Brevundimonas terrae) showed the strongest inhibition rate against A. panax, which showed 83.70% inhibition, and it also provided broad-spectrum antifungal effects. The inhibition efficacies of the SZ-22 bacterial suspension against ginseng black spot reached 82.47% inhibition, which is significantly higher than that of the 25% suspension concentrate azoxystrobin fungicide treatment (p < 0.05). Moreover, the SZ-22 bacterial suspension also caused ginseng plant growth promotion as well as root enhancement. ConclusionAlthough the results of the outdoor pot-culture method were influenced by the pathogen inoculum density, the cropping history of the field site, and the weather conditions, B. terrae SZ-22 controlled ginseng black spot and promoted ginseng growth successfully. This study provides resource for the biocontrol of ginseng black spot.

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization.

Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and Vmax values of chitinase to be 5.6 mg/mL and 0.87 μmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

Evaluation and Comparison of the Antimicrobial Activity Of Bifidobacteria bifidum and Bifidobacteria infantis as Probiotic Bacteria against Salmonella enterica Serotype Enteritidis

1.1. Background: Using the antimicrobial assays for determining the inhibitory effects of many compounds and also microorganisms has been ordinary in researching food and drug laboratories. During the last decades, the prevalence of foodborne diseases due to contaminated food as well as demand for natural and healthy foods has increased. Using probiotics for this purpose and for inhibiting growth of food pathogens is an interesting topic. 1.2. Objective: The aim of this study was to investigate three different methods namely spot on lawn assay, agar well diffusion assay(Cup plate assay) and agar disk diffusion assay and to compare the methods by the means of SPSS program for the antibacterial effects of Bifidobacterium bifidum and Bifidobacterium infantis against salmonella enterica serotype Enteritidis. 1.3. Material and methods: Supernatant and sediment of the two probiotic bacteria culture was tested in three different assays (spot-on-lawn, well diffusion and disk diffusion) against salmonella. 1.4. Results: In this study Well diffusion assay (Cup plate assay) was the best method to identify the antagonism of microorganisms and spot- on lawn method was the worse. Also in all three methods, the supernatant was significantly more effective than sediment in inhibiting the pathogen.Results showed that in all three assays, sediment and supernatant of Bifidobacterium infantis culture had a greater inhibition effect on salmonella than Bifidobacterium bifidum but the difference was not significant from statistical analyses point of view. 1.5. Discussion: Better functioning of Well diffusion assay (Cup plate method) could be explained by high accuracy and volume of supernatant used in this method. The inhibition zone in all three methods could be related to metabolites such as Acids, Diacetyle, Hydrogen proxide, Bacteriocins,…produced by probiotics.

Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw.

Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter) suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

Cellulolytic and Pectinolytic Enzymes of Some Selected Heat Resistant Fungi

Ten heat resistant fungi, Arthrinium sp., Aspergillus cejpii, A. nidulans, A. spinosus, Byssochlamys nivea, Hamigera avellanea, Talaromyces trachyspermus, T. barcinensis, T. ucrainicus, and Trichoderma asperellum were tested on their ability to produce cellulolytic and pectinolytic enzymes. Except for B. nivea and A. cejpii, all species secreted cellulolytic enzymes in the used cup plate assay. All isolates produced considerable levels of pectinolytic enzymes, when investigated on a liquid pectin medium. The appearance of unexpected blue color in the pectin medium for some species was discussed, but further investigation is required to elucidate this phenomenon.

Antimicrobial Activity of Oleiyl Glucoside Isolated from the Traditional Medicinal Plant, Capparis decidua

Ethnopharmacological relevance: In the traditional system of medicine, the plant Capparis decidua is well known for curing a variety of ailments such as toothache, cough, asthma, intermittent fever, rheumatism and inflammation. Aim of the study: The aim of the current study was to isolate and characterize the constituent responsible for the antimicrobial activity of the ethanolic extract of C decidua. Materials and methods: The isolated compound was extracted from ethanolic extract & characterized by spectroscopic methods. The antimicrobial activity of isolated compound was carried out against gram positive strains (S aureus, M luteus, B subtilus) and gram negative ones (P aeruginosa, E coli), and Fungi like C albicans and A niger by the cup-plate assay method and minimum inhibitory concentrations (MICs). Results: The active principle Oleiyl glucoside (n-octadec-9-enyl- � -D- glucopyranoside) was isolated from the ethanolic extract. The MIC values of the compound against gram positive bacteria were 62.5� g/ml, 62.5 � g/ml, 125� g/ml and against gram negative ones 31.25� g/ml, 250� g/ml. The compound was found to be active against C albicans 250 � g/ml only.

The role of pectinase enzyme in the development of soft rot caused by Pseudomonas fluorescens in the purple variety of onions (Allium cepa)

Pectic enzyme production was induced in vitro by culturing the pathogen (P flourescens) in minimal salt medium containing citrus pectin as the only carbon source and in vivo by inoculating the pathogen into healthy onion bulbs. The spent broth and onion rot extract were obtained and enzyme detection was done using viscometric method and cup plate assay. The crude enzyme was partially purified by precipitating with ammonium sulphate and dialyzed against water at 4oC for 24 h before use. The activity of the purified enzyme was determined based on the macerating ability on Onion and Potato discs and later by estimation of reducing sugars (galacturonic acid) level. The type of cleavage reaction of the enzyme was determined using the thiobarbituric acid reaction and the presence of antimicrobial substance in the rot tissue was also investigated. The pathogen produced a hydrolytic polygalactu-ronase in vitro and in vivo. The purified enzyme showed appreciable activity with all the assay methods used. Activity was highest at pH 5 in the culture filtrate sample and at pH 4 in the rot extract. Maceration was observed in potato and onion discs. The detection of increasing concentration of polyphenols in the rot tissue during rot development demonstrated the plant’s response to the pathogen. Key words: Onion, maceration, pectinase, polyphenol, rot.

Isolation and Characterization of Polygalacturonase Produced by Tetracoccosporium sp.

Thirty five fungal strains which isolated from vegetable wastes, were screened for the use of polygalacturonic acid as the sole carbon source. Twenty five isolates were positive for polygalacturonase activity in cup-plate assay, as evidenced by clear hydrolysation zones. The most productive strain was determined by measuring clear zones formed around colonies stained with ruthenium red. The highly pectinolytic fungal strain was tentatively identified as Tetra- occosporium sp. according to morphological characterization. The cultivation of the selected strain (Tetracoccosporium sp.) in liquid media resulted in high quantities of polygalacturonase enzyme. Maximum polygalacturonase activity was reached in 48 h of growth in the pectate medium. The collected polygalacturonase had optimum activity at pH 5.0 and maximal activity of the enzyme was determined at 35 °C. Mn 2+ , Ag 3+ and surface active detergents such as tween 20 and triton X- 100 increased the polygalacturonase activity by 37 % and EDTA enhanced the activity up to 125 %.

Screening of fungal strains for pectinolytic activity: endopolygalacturonase production by Peacilomyces clavisporus 2A.UMIDA.1

Forty-six fungal strains, isolated from soil and samples of cacao ( Theobroma cacao L.) jelly, were screened for pectinase production. All the strains were positive for pectinase activity in a cup-plate assay, as evidenced by clear hydrolysation halos. Twenty-eight percentages presented considerable pectinolytic activity. The cultivation of the best strain ( Peacilomyces clavisporus 2A.UMIDA.1) by submerged and semi-solid fermentation, resulted in high quantities of endopolygalacturonase enzyme. The optimal temperature for fungal growth and endopolygalacturonase production by P. clavisporus 2A.UMIDA.1 in submerged culture was 30 °C. Formulations containing cará–roxo ( Dioscorea alata) were also employed as substrates for endopolygalacturonase production, but results were poor. Solid state fermentation proved to be a better process than submerged fermentation for endopolygalacturonase production, giving the highest enzyme production rate in 96 h. An attempt to scale up the solid state fermentation resulted in a decrease in enzyme production.

Postharvest calcium chloride treatments do not help to increase shelf-life of bananas

Introduction. Calcium chloride (CaCl2 ) treatment has been shown to increase the shelf-life of fruits, mainly through making cell walls less accessible to pathogens and softening enzymes. Materials and methods. bananas of four cultivars [`Ambon' (AAA), `Embul' (AAB), `Kolikuttu' (AAB) and `Seenikehel' (ABB)] were dipped in or pressure infiltrated with 4% CaCl2 . To determine the effect of exogenous ethylene on treated fruits, they were ripened with exogenous ethylene. Ca2+ in cell wall fractions were monitored by atomic absorption spectrophotometry. Cup plate assays were performed to determine pectinase activity. Results and discussion. Pressure infiltration accelerated ripening and disease, and reduced firmness (P ). However, when exposed to ethylene, CaCl2 pressure-infiltrated bananas were insignificantly firmer than distilled water-infiltrated and ethylene-ripened bananas, showing a significant interaction (P ) between infiltration treatments and ethylene ripening. There was no consistent increase in covalently bound pectin of cell walls as seen in fruits that respond positively to CaCl2 . Firmness reduction and ripening acceleration by Ca2+ treatment cannot be explained if polygalacturonase (PG) (known to be inhibited by Ca2+ ) was the dominant pectinase. Enzyme assays gave evidence of PG activity. When ammonium oxalate (known to bind Ca2+ ) was eliminated from the test medium, pectinase activity increased with increasing pH (pH 5 to 9). The presence of a pectinase enzyme which exhibits activity in the presence of Ca2+ is apparent. Conclusion. Ca2+ does not appear to influence cell wall structure of bananas but appears to influence ripening physiology.

Production of Polygalacturonase and Increase of Longitudinal Gas Permeability in Southern Pine by Brown-Rot and White-Rot Fungi

SummaryHydrolysis of bordered and pinoid pits may be a key event during colonization of wood by decay fungi. Although pits are numerous, studies of pectin-hydrolyzing enzymes in wood decay fungi are scarce, probably because of the relatively low content (less than 4 %) of pectin in wood and because of the primary focus on understanding the degradation of lignified components. Endopolygalacturonase (endo- PG) activity was estimated by cup-plate assay and viscosity reduction of pectin from liquid cultures of fifteen brown-rot and eight white-rot basidiomycetous fungi using sodium polypectate as the carbon source. Oxalic acid was estimated in liquid culture and related to mycelial weight of each fungus. Changes in longitudinal gas permeability of southern pine cores exposed to selected decay fungi in liquid culture were measured to determine the extent of hydrolysis of bordered pits. Twelve of fifteen brown-rot and six of eight white-rot fungi tested were positive for at least one of the polygalacturonase test methods. Accumulation of oxalic acid was detected in thirteen of fifteen brown-rot isolates and none of the white-rot fungi tested. Gas permeability of pine cores increased approximately fourfold among brown-rot fungi tested and eighteenfold among white-rot fungi tested. Scanning electron microscopy revealed bordered pit membrane hydrolysis in cores colonized by white-rot fungi, but only torus damage, weakening and tearing of the pit membranes, was observed in cores exposed to brown-rot fungi. We conclude that both brown- and white-rot decay fungi have the enzymatic capacity to hydrolyze pectin, damage bordered pit membranes, and increase wood permeability during colonization and incipient decay.

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.

Activity of polygalacturonase produced by Cryphonectria parasitica in chestnut bark and its inhibition by extracts from American and Chinese chestnut

A purified polygalacturonase (PG) from culture filtrates of a virulent strain of Cryphonectria parasitica, Ep 155, caused browning of the inner bark of American chestnut. The enzyme also hydrolysed isolated cell wall materials from both American and Chinese chestnut bark in vitro. PG activity was detected in infected chestnut bark by a cup-plate assay, and by isoelectric focusing (IEF) and overlay-gel activity staining. A hypovirulent strain of C. parasitica, Ep 713, induced small cankers on American chestnut, and produced small amounts of PG in vivo as compared to its isogenic virulent strain Ep 155. Lower PG activity was also detected in cankers on resistant Chinese chestnut as compared to those on susceptible American chestnut, IEF and immunoblotting of the PG from infected bark against antibodies raised against the purified PG produced in vitro indicated that these enzymes were the same. Tannin isolated from the bark of American chestnut inhibited this PG more than tannin isolated from Chinese chestnut bark. A proteinaceous extract from Chinese chestnut bark, however, was about 15 times more inhibitory to the C. parasitica PG than a similar protein extract from American chestnut. This PG inhibitor was active also against the PG of C. lindemuthianum, but not the PG from Rhizopus sp. The high level of this nominally specific PG inhibitor in the resistant host lends further support to the hypothesis that PG is a virulence factor for C. parasitica and that its inhibition may contribute to the resistance of Chinese chestnut.