Crude Culture Filtrate Research Articles

Diagnostic potential of a serological assay for the diagnosis of ulcerans disease based on the putative Mycobacterium ulcerans toxin.

Mycobacterium ulcerans infection is an important and potentially disfiguring disease of man. A rapid diagnostic assay for detection of this organism is required urgently. Serological assays require a species-specific protein to ensure a high level of specificity and thus reduce the occurrence of false positive results. As M. ulcerans had been reported to produce a unique cytotoxin, it was thought that this would provide an ideal antigen on which to base a serological assay for detection of M. ulcerans during infection. Crude culture filtrates, prepared by previously documented methods, were assayed for toxic activity by in-vitro cytotoxicity assays and in-vivo mouse footpad assays. To evaluate the uniqueness of the cytotoxic factor, other species of mycobacteria were also assayed. Analysis of these assays showed that similar biological activity is present in various other mycobacterial species. Furthermore, it was possible to neutralise this activity in all species tested with a polyclonal antiserum raised against M. ulcerans. As the cytotoxic factor was found not to be specific to M. ulcerans, it is unlikely that a serological assay based on such a molecule will be of use.

Selection and Characterization of Resistance in Mango Embryogenic Cultures to Colletotrichum gloeosporioides Penz. Phytotoxin

Anthracnose, caused by Colletotrichum gloeosporioides, is the most serious production and postharvest problem of mango. Most mango cultivars are highly susceptible to this disease. Embryogenic nucellar cultures of two cultivars, `Hindi' and `Carabao', were recurrently selected with either the purified phytotoxin or the crude culture filtrate produced by the fungus. Mycelium growth was suppressed in dual cultures involving the recurrently selected lines. Enhanced extracellular production of proteins was observed in the embryogenic cultures and in regenerants, including a newly expressed acidic chitinase isozyme (`Hindi') and greater expression of two other chitinase isozymes (`Hindi' and `Carabao'). Activity of α-1,3-glucanase was also substantially increased in the recurrently selected lines. Resistant lines were characterized using RAPDs on the basis of polymorphisms generated by eight different primers. In most cases, the differences involved the absence of RAPD markers in resistant lines.

An enzyme from Myrothecium verrucaria that degrades insect cuticles for biocontrol of Aedes aegypti mosquito

Myrothecium verrucaria produced a high activity of extracellular insect cuticle degrading enzymes, chitinases, proteinases and lipases. Both first (I) instar and fourth (IV) instar larvae of a mosquito, Aedes aegypti, a vector of yellow fever and dengue, were susceptible to crude culture filtrate (100% mortality within 48 h at 170 mg/l) . The supplementation of purified M. verrucaria endo-chitinase with commercial lipase decreased the lethal time (LT50) from 48 h to 24 h for I instar and 120 h to 96 h for IV instar. Our results of the larvicidal activity indicate that the cuticle degrading enzyme complex of M. verrucaria has a good potential for the control of mosquitoes.

Formation and activities of xylanhydrolysing enzymes of Humicola grisea var thermoidea

A thermophilic fungus, Humicola grisea var thermoidea, produced in liquid culture two endoxylanases (1,4-β-D-xylan-xylanohydrolase, EC 3.2.1.8) with M r of 95 (Xyl I) and 13 (Xyl II) kDa. PAGE of the crude culture filtrate and of each fraction obtained by gel filtration produced three and one band, respectively. Cross-reaction of the culture filtrate and each fraction with polyclonal antibodies prepared against Xyl II produced two and one precipitin bands, respectively. Hydrolysis of wheat straw and rice husk xylan was maximal using a combination of Xyl I and Xyl II. The products formed after hydrolysis, xylo-oligosaccharides and traces of xylose, indicated an endotype enzyme action and the co-operative activities of the xylanases.

Purification of a toxin from Fusarium oxysporum f.sp. lycopersici race 1

Leaf protoplasts from near-isogenic tomato ( Lycopersicon esculentum) cultivars, differing only at the I 1 resistance gene, showing differential cell death in response to culture filtrates of Fusarium oxysporum f.sp. lycopersici race 1 were used to quantify the toxic activity of crude and partially purified culture filtrates. Toxic activity was partially lost after treatment with protease or storage at pH values above neutrality and was completely destroyed by autoclaving. Fractions with toxic activity were separated by gel filtration. Following acid native polyacrylamide gel electrophoresis and electro-elution of a desalted fraction precipitated by 20% saturation with ammonium sulphate from crude culture filtrates, toxic activity was found to be associated with a single proteinaceous band with an R F value of 0·32. Electrophoresis under denaturing conditions revealed that the toxin was comprised of two bands, with apparent molecular weights 56 and 61 kDa, which stained positively for protein. Protoplasts of cv. Ace, lacking resistance genes, were sensitive to the toxin, with a LD 50 of 55 ng protein ml -1 compared with >22 μg protein ml -1 against cv. Royal Ace protoplasts, possessing the I 1 resistance gene to F. oxysporum f.sp. lycopersici race 1. Injection of the toxin into tomato plants induced symptoms similar to those of natural infection in susceptible cv. Ace, but caused no symptom development in resistant cv. Royal Ace. This corresponded to the cultivar race interactions observed under field conditions.

529 PB 525 DETERMINING RESISTANCE IN STRAWBERRY TO ANTHRACNOSE USING TOXINS PRODUCED IN FUNGUS CULTURE

We have determined in tests conducted both at Beltsville and Poplarville that several strawberry isolates of Colletotrichum acutatum, C. gloeosporioides and C. fragariae produce toxin-like compounds in culture. Crude culture filtrates (CFI elicited general and specific responses in tomato and strawberry plants. Tomato plants initially were used because they are highly responsive to toxins in general, whereas the reaction of strawberry plants apparently is greatly affected by environmental and nutritional growing conditions of the test plant. Toxin symptoms included leaf chlorosis and wilting, leaf midvein darkening, and plant death when CF was applied to leaves or if seedlings or petioles were immersed into CF. Juvenile tissues appear to be more susceptible to the effects of the toxins than mature tissue. No differences in response to culture filtrates were apparent among those from the Colletotrichum isolates. The putative toxins appear to act differentially with susceptible or resistant strawberry germplasm.

Cloning and targeted gene disruption of EXG1, encoding exo-beta 1, 3-glucanase, in the phytopathogenic fungus Cochliobolus carbonum

The phytopathogenic fungus Cochliobolus carbonum produces an extracellular enzyme capable of degrading beta 1,3-glucan in an exolytic manner. On the basis of partial amino acid sequences of the purified enzyme, two degenerate oligonucleotides were synthesized and used as PCR primers to amplify a 1.1-kb fragment of corresponding genomic DNA. The PCR product was used to isolate the genomic copy of the gene, called EXG1. Partial sequencing of the genomic DNA confirmed that the PCR product corresponded to EXG1. A strain of the fungus specifically mutated in the EXG1 gene was constructed by homologous integration of an internal fragment of EXG1. In the mutant, enzymatic activity and the corresponding peak of UV absorption during high-pressure liquid chromatography purification were reduced by at least 98%. However, crude culture filtrates of the mutant retained 44% of the wild-type beta 1,3-glucanase activity. This residual activity was due to two additional activities which were chromatographically separable from the product of EXG1 and which were coeluted with beta 1,3-beta 1,4-glucanase activity. Growth of the EXG1 mutant was normal on sucrose and oat bran but was reduced by 65% on pure beta 1,3-glucan. The EXG1 mutant was still pathogenic to maize.

Isolation and Characterization of Cell Lines Resistant to Crude Culture Filtrate of Fusarium in Chickpea

Cell lines resistant to Fusarium oxysporum f. sp. Ciceri crude culture filtrate have been selected In Cicer arietinum cv C235. Phenol content and the activities of tyrosine ammonia lyase (TAL), phenylalanine ammonia lyase (PAL) and s-1, 3-glucanase were Increased by the elicitor In callus cultures of Fusarium susceptible cultlvar C235, resistant cultlvar WR315 and a stable Fusarium resistant cell clone FC-3. The Increase In phenols and glucanase activity, was more In selected cell line than In ev C235 and ev WR315, while the increase In activities of TAL and PAL showed reverse correlation.

Cellulolytic and hemicellulolytic enzymes of sclerotium rolfsii: Optimization of the culture medium and enzymatic hydrolysis of lignocellulosic material

A culture medium for a wild strain of Sclerotium rolfsii was optimized with respect to cellulase formation by employment of response surface methodology. The medium components that were found to show the strongest influence on cellulase production were selected for the optimization process, and included α-cellulose and peptone from meat. The optimal concentrations for these two factors were determined in an experiment planned according to an orthogonal central composite design, in which a mathematical model was fitted to the experimental data. The optimal concentrations for the two variables cellulose and peptone, as predicted from the model equation, were 37.6 g/liter and 80.2 g/liter, respectively. The optima, as predicted by the model, were verified in shaken flask experiments. When S. rolfsii was cultivated on the optimized medium, a maximum cellulase activity of 5.72±0.39 FPU/ml was obtained. Additionally, high levels of β-glucosidase and various hemicellulolytic enzyme activities, including xylanase and mannanase, were produced. A crude culture filtrate of S. rolfsii was used for the hydrolysis of various lignocellulosic materials, including agricultural residues.

Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay.

A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum. The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay. In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E. Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution. Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase. Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C. botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins. ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed. This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m3 fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5.

Changes in Pattern of Phenolic Acids Induced by Culture Filtrate of Fusarium oxysporum in Alfalfa Plants Differing in Susceptibility to the Pathogen

Plants of alfalfa ( Medicago sativa L.) differing in their susceptibility to Fusarium oxysporum Schlecht were cultivated in hydroponic solution with crude culture filtrate of this pathogen. Susceptibility of plant seedlings to the pathogen positively correlated with the sensitivity of seedlings to culture filtrate treatment. The 6-week old susceptible control seedlings accumulated higher amounts of free and bound phenolic acids than was determined in resistant control seedlings (122.54 and 98.37 μg g −2 fresh mass, respectively). In spite of the higher content of total phenolics, salicylic and caffeic acids were not detected in the susceptible genotype. Culture filtrate treatment caused significant changes in the total content and fraction composition of phenolic acids in both studied plants. In susceptible seedlings the increase in the level of free and cell wall-bound phenolic acids was compensated for by a decrease in the content of phenolic glycosides. In resistant seedlings all the determined fractions (free, ester-, glycoside- and cell wall-bound phenolic acids) participated in the rise of total content of phenolics. The level of cinnamic acid derivatives increased earlier and more significantly in resistant seedlings. A relatively high level of salicylic acid was detected in seedlings of both genotypes challenged for 72 h.

Histopathologic effects of culture filtrates of Clostridium septicum in rabbit intestinal loops.

Clostridium septicun is pathogenic for humans and animals and is one of the clostridia capable of potentiating myonecrosis following traumatic injury. In recent years, this organism has been implicated in nontraumatic infections including spontaneous myonecrosis, neutropenic enterocolitis, and bacteremia [1-3]. Such nontraumatic infections are increasingly being diagnosed in individuals suffering from malignancy, neutropenia, diabetes, or severe atherosclerosis, and they tend to be fulminant and associated with high rates of mortality unless treated promptly and aggressively with surgical measures and antimicrobial agents. In patients with C. septicumn bacteremia, the most frequently implicated portal of entry has been the intestinal tract, with or without concomitant neoplastic disease. Autopsies have revealed necrotizing inflammation of the terminal ileum and cecum, and some neutropenic patients with C. septicum bacteremia have died with necrotizing enterocecitis (author's unpublished data) [1]. C. septicum is known to elaborate a variety of potential virulence factors, including two hemolysins, deoxyribonuclease, hyaluronidase, neuraminidase, and fibrinolysin. Previous studies performed in our laboratory showed that crude cell-free culture filtrates of C. septicurn were cytotoxic for Vero and other cell lines. The object of this preliminary study was to survey the responses of rabbit intestinal loops to cell-free culture filtrates of the organism and to compare the histopathologic changes that it causes with those produced by Clostridium difficile. In this preliminary study, we found that the majority of the C. septicum isolates tested produced significant fluid accumulation and histological changes within the rabbit intestinal loop model that were similar to those produced by C. difficile. All 17 strains of C. septicum used in this study were selected from the culture collection of the Anaerobe Labora-

Purification and characterization of three endo-(1,4)-β-xylanases and one β-xylosidase from Aspergillus awamori

Three endo-(1,4)-β-xylanases (endo-xylanase I, II, and III) and one β- d-xyloside xylohydrolase (β-xylosidase) were purified from a crude culture filtrate of Aspergillus awamori CMI 142717, grown on milled oat straw as carbon source. Aspergillus awamori xylanases differ in some characteristics of known xylanases. The optimum pH for the endo-xylanases were between 4.0 and 5.5 and the optimum temperature between 45°C and 55°C; β-xylosidase was optimal around pH 6.5 and 70°C. All endo-xylanases were able to degrade xylan to xylobiose and xylotriose. Endo-xylanase I also produced small amounts of xylose. The molecular weights of endo-xylanase I, II, and III were, respectively, 39 000, 23 000, and 26 000. The molecular weight of β-xylosidase was 110 000. The specific activities of endo-xylanase I, II, and III towards water-soluble oat spelts arabinoxylan were, respectively, 69.6 U mg −1, 68.6 U mg −1, and 16.3 U mg −1. The specific activity of β-xylosidase towards p-nitrophenyl-β-xylopyranoside was 34.1 U mg −1. The activity of these enzymes was significantly inhibited by Hg 2+, Pb 2+, and Ag +.

Evaluation of Organisms Antagonistic to the Sclerotioid Organs ofDrechslera teres,the Causal Agent of Barley Net Blotch

Twenty-seven microorganisms were evaluated for their ability to reduce sclerotioid organ formation and myceliogenesis in barley straw when applied prior to and following colonization by Drechslera teres. Ten of them were able to partly or totally inhibit the formation of sclerotioid organs on barley straw when applied as a precolonization treatment. All antagonists inhibited sclerotioid organ morphogenesis, whether through direct application or via the crude culture filtrates. Form maculata was more sensitive than form teres to culture filtrates of the antagonists

Comparative investigation of cellulose-degrading enzyme systems produced by different strains of Myceliophthora thermophila (Apinis) v. Oorschot

Several strains of Myceliophthora (syn. Sporotrichum, Chrysosporium) thermophila (Apinis) van Oorschot were investigated with the aim of comparing the cellulase systems. Strain-to-strain variations, in response to growth substrate, were observed at different levels, such as cultural behavior, amount of enzyme secreted, or enzyme composition. All strains grew well on glucose and cellobiose. The majority of strains, however, did not thrive on lactose, which was also inefficient in triggering the secretion of cellulase enzymes. A cellulase system was typically formed during growth on cellulose (Avicel), but some systems were also induced, although less efficiently, by cellobiose. The composition of the cellulase system produced by different strains was analyzed by anion exchange chromatography, gel filtration, and chromatofocusing and found to be strikingly similar, though not identical. The exocellulase component of the systems was assayed directly in the crude culture filtrate by a procedure involving the hydrolysis of p-nitrophenyllactoside and by an electroimmunoassay performed with a specific rabbit antiserum raised against the pure component from strain ATCC 42 464, as reference strain. Enzyme from all strains of Myceliophthora thermophila cross-reacted with the antiserum in a manner that exactly paralleled the enzymatic detection of exocellulase: these observations showed that the different exocomponents had similar antigenic determinants.

Studies on the cellulase of the rumen anaerobic fungus Neocallimastix frontalis, with special reference to the capacity of the enzyme to degrade crystalline cellulose

By using cotton fiber, carboxymethylcellulose (CM-cellulose), and o -nitrophenyl-β- d -glucoside as substrates, it was possible to demonstrate that there were at least three different types of enzyme present in culture filtrates of Neocallimastix frontalis RK21. The activity to crystalline cellulose (cotton fiber) resided in a high-molecular-weight complex that comprised endoglucanase activity, β-glucosidase activity, and another enzyme. However, synergism between the components in the high-molecular-weight complex and between the complex and low-molecular-weight endoglucanases and β-glucosidases was also apparent in the solubilization of crystalline cellulose. The composition of the complex varied according to the growth conditions: it was, however, between 750 and 1000 kDa in size. Cultures containing rumen fluid contained only small amounts of the high-molecular-weight complex. Cultures grown on defined medium were rich in high-molecular-weight complex, but only when the concentration of the carbon source was less than 1.0%. Treatment of the crude culture filtrates with chitinase under conditions that had little effect on the activity of the enzyme to endoglucanase (CM-cellulose) or β-glucosidase completely destroyed the activity to crystalline cellulose. It is tentatively suggested that an enzyme crucial for the activity to crystalline cellulose may be cell wall-bound and may be dependent on its association with the cell wall for the maintenance of its conformation for attacking crystalline cellulose. The enzyme involved in degrading crystalline cellulose is much more thermolabile than the CM-cellulase or the β-glucosidase: activity is optimal at pH 6.0 and 40°C.

Use of Pathogen Filtrate to Differentiate Physiological Resistance of Dry Bean to White Mold Disease

White mold, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is major concern to the dry bean (Phaseolus vulgaris L.) industry because it reduces yield. Incorporating partial physiological resistance (PPR) into bean is one strategy to combat the disease, but this requires a reliable screening technique. The objective of this work was to develop a reliable screening method to identify bean germplasm that express PPR to S. sclerotiorum. An in vitro method that assayed weight of calli formed on a medium amended with crude culture filtrate of S. sclerotiorum was evaluated. Hypocotyl explants of eight bean genotypes (six cultivars, a breeding line, and one plant introduction), taken from 7‐d‐old seedlings, were incubated for 15 d on a solid Murashige and Skoog‐based medium, containing 5.0 μM/ kinetin and 2.5 μM picloram, and amended with 200 mL L−1 culture or control filtrate. Callus fresh weight, expressed as a percent of the control (CWPC), was analyzed over two runs. Lack of a genotype × run interaction, and a genotypeffect (P &lt; 0.05) for CWPC indicated that this screening method gave repeatable results and was able to differentiate PPR among the eight genotypes. The field resistant ‘Sierra’ and ‘C‐20’ had the greatest PPR, as indicated by high C‐%VPC values, and the field susceptible ‘UI‐114’ and ‘Upland,’ the least PPR. These results to fungal metabolites parallel field resistance of the genotypes, implicating PPR as an important component of the field reaction of a given genotype. The callus‐weight assay may provide a way to differentiate dry bean germplasm for partial physiological resistance to S. sclerotiorum.

A non-haemagglutinating form of Clostridium difficile toxin A.

Analysis of crude culture filtrate of Clostridium difficile by Mono Q-anion exchange fast protein liquid chromatography (FPLC) demonstrated that toxin A had distinct peaks of activity for cytotoxicity and haemagglutination, as also did highly purified toxin A obtained by thyroglobulin affinity chromatography (TG) followed by two sequential anion-exchange chromatographic steps with Q-Sepharose FF and Mono Q. From TG unbound fractions a highly cytotoxic but weakly haemagglutinating variant (toxin A') of toxin A was obtained by Q-Sepharose FF and Mono Q chromatography. Analysis of toxins A and A' from cultures of C. difficile in a chemically defined medium, and of toxin A dialysed against brain heart infusion broth, indicated that A' was not merely toxin A coupled to a component of the growth medium. Polyacrylamide gel electrophoresis under non-denaturing conditions showed that toxins A and A' had the same Mr. Immunoblotting with mouse monospecific A antitoxin showed that five bands larger than the major 240-Kda band were more strongly developed in toxin A than in A' in denaturing but non-reducing conditions, and in reducing conditions eight bands (38-175 Kda) were seen in toxin A but not A'. Immunoblotting with a monoclonal antibody (PCG-4) showed that, in both reducing and non-reducing conditions, two bands of 160 and 155 Kda were more prominent in toxins A and A' respectively, and four bands (195, 180, 175 and 125 Kda) were detected only in toxin A'.

Bioherbicidal Potential ofFusarium moniliformeand its Phytotoxin, Fumonisin

An isolate ofFusarium moniliforme(Sheldon) (JW #1) was discovered on greenhouse-grown jimsonweed in Mississippi, U.S.A., in 1990. Applications of aqueous suspensions of spores/mycelia (&gt;1 × 107propagules ml−1) grown on potato-dextrose agar to the potted soil in which 2- to 4-wk-old jimsonweed plants were grown caused local lesions or mosaic-like patterns on the leaves and inhibited growth. Application of lesser doses to the leaves only produced no visual effects. When the fungal suspensions were applied at higher dosages (&gt;1 × 107propagules ml−1) either by soil drench or subirrigation, symptoms such as local lesions or a mosaic-like pattern on the leaves appeared and plant growth was inhibited. When the fungus was grown on an autoclaved rice medium and the ground mixture applied to jimsonweed plants, the plants wilted within 24 h and died after 48 h. Crude and cell-free filtrates of the fungal-rice culture also killed 1- to 2- and 3- to 4-week-old plants after 24 to 72 h. A major toxin identified was fumonisin B1, isolated from fermented rice at 400 μg g−1. Fumonisin B1applied in water at 2.5 μg 100 μl−1to excised jimsonweed leaves caused the same symptomology (i.e., soft rot diffusing along leaf veins) within 24 h as the cell-free extract or the crude culture filtrates. Similar damage occurred to intact plants treated with crude or cell-free filtrates or purified aqueous fumonisin B1solutions.