Activity Of Culture Filtrates Research Articles

The Effect of Trichoderma harzianum Hypovirus 1 (ThHV1) and Its Defective RNA ThHV1-S on the Antifungal Activity and Metabolome of Trichoderma koningiopsis T-51

Mycoviruses widely exist in filamentous fungi and sometimes cause phenotypic changes in hosts. Trichoderma harzianum hypovirus 1 (ThHV1) and its defective RNA ThHV1-S were found in T. harzianum and exhibited high transmissibility. In our previous study, ThHV1 and ThHV1-S were transferred to an excellent biological control agent T. koningiopsis T-51 to form a derivative strain 51-13. In this study, we assessed the metabolic changes in strain 51-13 and antifungal activity of its culture filtrate (CF) and volatile organic compounds (VOCs). The antifungal activity of CF and VOCs of T-51 and 51-13 was different. Compared with the CF of T-51, that of 51-13 exhibited high inhibitory activity against B. cinerea, Sclerotinia sclerotiorum, and Stagonosporopsis cucurbitacearum but low inhibitory activity against Leptosphaeria biglobosa and Villosiclava virens. The VOCs of 51-13 exhibited high inhibitory activity against F. oxysporum but low inhibitory activity against B. cinerea. The transcriptomes of T-51 and 51-13 were compared; 5531 differentially expressed genes (DEGs) were identified in 51-13 with 2904 up- and 2627 downregulated genes. In KEGG enrichment analysis, 1127 DEGs related to metabolic pathways (57.53%) and 396 DEGs related to biosynthesis of secondary metabolites (20.21%) were clearly enriched. From the CF of T-51 and 51-13, 134 differential secondary metabolites (DSMs) were detected between T-51 and 51-13 with 39 up- and 95 downregulated metabolites. From these, 13 upregulated metabolites were selected to test their antifungal activity against B. cinerea. Among them, indole-3-lactic acid and p-coumaric acid methyl ester (MeCA) exhibited strong antifungal activity. The IC50 of MeCA was 657.35 μM and four genes possibly related to the synthesis of MeCA exhibited higher expression in 51-13 than in T-51. This study revealed the mechanism underlying the increase in antifungal activity of T-51 because of the mycovirus and provided novel insights in fungal engineering to obtain bioactive metabolites via mycoviruses.

Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates

Rapid detection of tuberculosis (TB) infection is paramount to curb further transmission. The gold standard for this remains mycobacterial culture, however emerging evidence confirms the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens. These bacteria do not grow under standard culture conditions and require the presence of culture filtrate (CF), from axenic cultures of Mycobacterium tuberculosis (Mtb), to emerge. It has been hypothesized that molecules such as resuscitation promoting factors (Rpfs), fatty acids and cyclic-AMP (cAMP) present in CF are responsible for the growth stimulatory activity. Herein, we tested the ability of CF from the non-pathogenic bacterium Mycobacterium smegmatis (Msm) to stimulate the growth of DCTB, as this organism provides a more tractable source of CF. We also interrogated the role of Mtb Rpfs in stimulation of DCTB by creating recombinant strains of Msm that express Mtb rpf genes in various combinations. CF derived from this panel of strains was tested on sputum from individuals with drug susceptible TB prior to treatment. CF from wild type Msm did not enable detection of DCTB in a manner akin to Mtb CF preparations and whilst the addition of RpfABMtb and RpfABCDEMtb to an Msm mutant devoid of its native rpfs did improve detection of DCTB compared to the no CF control, it was not statistically different to the empty vector control. To further investigate the role of Rpfs, we compared the growth stimulatory activity of CF from Mtb, with and without Rpfs and found these to be equivalent. Next, we tested chemically diverse fatty acids and cAMP for growth stimulation and whilst some selective stimulatory effect was observed, this was not significantly higher than the media control and not comparable to CF. Together, these data indicate that the growth stimulatory effect observed with Mtb CF is most likely the result of a combination of factors. Future work aimed at identifying the nature of these growth stimulatory molecules may facilitate improvement of culture-based diagnostics for TB.

Copper induces transcription of BcLCC2 laccase gene in phytopathogenic fungus, Botrytis cinerea

ABSTRACT Laccases are one of many groups of inducible enzymes produced by the filamentous fungus, Botrytis cinerea during colonisation of host plant tissues. While the processes involved in laccase induction are not fully understood, Cupric ions (e.g. CuSO4) and gallic acid (GA) have been reported as laccase inducers. This study investigates laccases activities and the expression of three laccase genes (BcLCC1, BcLCC2, BcLCC3) in three B. cinerea isolates grown in laccase-inducing medium (LIM) supplemented with CuSO4 and GA. Laccase activity in culture filtrates with CuSO4 increased after 48 h of growth in LIM at 24°C. The induction of BcLCC2 transcription was greatest at a concentration of 0.6 mM CuSO4, concentrations greater than 0.6 mM inhibited fungal growth. In contrast, no laccase induction was observed in the presence of GA. Liquid chromatography-mass spectroscopy (NanoLC ESI MS/MS) analysis confirmed the presence of a 63.4 kDa protein, the BcLCC2 isoform in the culture filtrate with 0.6 mM CuSO4. Analysis of mRNA transcripts further showed BcLCC3 was also inducible and the expression of BcLCC2 and BcLCC3 was isolate-dependent. In conclusion, CuSO4 induces a 63.4 kDa laccase in B. cinerea by induced transcription of the BcLCC2 gene.

Secondary metabolites produced by grapevine strains of Lasiodiplodia theobromae grown at two different temperatures

ABSTRACTLasiodiplodia theobromae is a fungal plant pathogen that has been associated with Botryosphaeria dieback of grapevine. Despite several studies on L. theobromae, until now the production of secondary metabolites by strains isolated from grapevines has not been reported. The ability of two strains of L. theobromae isolated from grapevine to produce lipophilic metabolites was studied. Although many typical compounds of low molecular weight were identified from the crude extracts of both strains (e.g., lasiolactols, substituted 2-dihydrofuranones, melleins, jasmonic acid, 3-indolcarboxylic acid, botryodiplodins), (2R/2S,3S,4S)-3-epi-botryodiplodin was isolated for the first time as a natural compound. Furthermore, a comparative study of metabolite production was conducted at 25 and 37 C to understand temperature effects on metabolite profiles. Some metabolites were produced only by one strain (e.g., (3S,4S)-4-acetyl-3-methyl-2-dihydrofuranone produced by LA-SOL3) and others only at a specific temperature (e.g., jasmonic acid at 25 C, botryodiplodins at 37 C). Phytotoxicity and cytotoxicity of pure compounds were evaluated to clarify the influence of lipophilic metabolites on the biological activities of culture filtrates of both strains. The most toxic compound for Vero and 3T3 cells was (2R/2S,3S,4S)-3-epi-botryodiplodin.

Reduction of Extracellular Proteases Increased Activity and Stability of Heterologous Protein in $${ Aspergillus}$$ A s p e r g i l l u s $${ niger}$$ n i g e r

The heterologous protein production in Aspergillus niger is often limited by the activity of the host extracellular proteases. To improve heterologous production, a transcription factor controlling expression of several extracellular protease-encoding genes, prtT, was deleted in A. niger PY11 and the mutant (An\(\Delta \) prtT) characterised. Extracellular proteolytic activity of An\(\Delta \) prtT was reduced as compared to the wild type, a result that was confirmed by RT-PCR analyses that showed reduced expression levels of several protease gene transcripts. To compare the efficiency of the mutant and parental (PY11) strains as hosts for heterologous protein production, the cutinase gene from Glomerella cingulate, under control of the glucoamylase A promoter, was integrated into each genome. The cutinase activity of PY11 and An\(\Delta \) prtT harbouring the G. cingulata cutinase gene was increased 20- and 36-fold higher, respectively, than the untransformed parental strains, suggesting that the ability of the mutant to produce heterologous protein is better than the wild type. Cutinase activity in culture filtrates prepared using both strains was stable at \(4\,^{\circ }\hbox {C}\) for extended periods; however, during incubation at \(25^{\circ }\)C the heterologous cutinase in culture filtrates prepared using An\(\Delta \) prtT retained 80% activity after a two-week incubation versus the less than 3% activity that was retained in culture filtrates prepared using PY11. This indicates that the reduction of extracellular proteases greatly improves the stability of heterologous proteins produced by A. niger.

FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici

Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS–PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars.

Inorganic phosphate solubilization by phosphate solubilizing fungi isolated from acidic soils

The experiment was conducted to evaluate the native phosphate solubilizing fungi on inorganic phosphate solubilization in the acidic soils of Jharkhand, India. Mineral phosphate solubilizing activities of three fungal isolates were tested in tricalcium phosphate and ferric phosphate medium by analyzing the possible phosphorus release and phosphatase activity from 5th, 10th and 15th day of incubation. Maximum P release was observed on 5th and 10th day of incubation in ferric phosphate and tricalcium phosphate medium, respectively, by all the isolates. Among the isolates, Aspergillus nigerinduced highest P release of 635 and 695 µg ml-1 and resulted in 75 and 77% P solubilisation in tricalcium phosphate and ferric phosphate medium, respectively. The pH of the culture filtrate medium gradually decreased with the progress of incubation and maximum decrease was observed between 5-10th day of incubation by all the fungal species. The highest peak of acid phosphatase activity in culture filtrates of fungal isolates were observed on 5th and 10th day of incubation in ferric phosphate and tricalcium phosphate medium, respectively. The highest acid phosphatase activity of 73 U was induced both by Aspergillus niger and Trichoderma viride in tricalcium phosphate medium. However, the highest acid phosphatase activity in ferric phosphate medium was 51 U induced by A. niger. Among the isolates, A. niger was more efficient in solubilising the inorganic P followed by T. viride and Penicillium chrysogenum. Key words: Acid phosphatase, fungi, phosphorus, phosphate solubilization.

An experimental system to study responses of Medicago truncatula roots to chitin oligomers of high degree of polymerization and other microbial elicitors

A fully acetylated, soluble CO preparation of mean DP of ca. 7 was perceived with high sensitivity by M. truncatula in a newly designed versatile root elicitation assay. The root system of legume plants interacts with a large variety of microorganisms, either pathogenic or symbiotic. Understanding how legumes recognize and respond specifically to pathogen-associated or symbiotic signals requires the development of standardized bioassays using well-defined preparations of the corresponding signals. Here we describe the preparation of chitin oligosaccharide (CO) fractions from commercial chitin and their characterization by a combination of liquid-state and solid-state nuclear magnetic resonance spectroscopy. We show that the CO fraction with highest degree of polymerization (DP) became essentially insoluble after lyophilization. However, a fully soluble, fully acetylated fraction with a mean DP of ca. 7 was recovered and validated by showing its CERK1-dependent activity in Arabidopsis thaliana. In parallel, we developed a versatile root elicitation bioassay in the model legume Medicago truncatula, using a hydroponic culture system and the Phytophthora β-glucan elicitor as a control elicitor. We then showed that M. truncatula responded with high sensitivity to the CO elicitor, which caused the production of extracellular reactive oxygen species and the transient induction of a variety of defense-associated genes. In addition, the bioassay allowed detection of elicitor activity in culture filtrates of the oomycete Aphanomyces euteiches, opening the way to the analysis of recognition of this important legume root pathogen by M. truncatula.

Antagonistic Activity of Plant Growth Promoting Rhizobacteria Isolated from Tomato Rhizosphere Against Soil Borne Fungal Plant Pathogens

The use of single strain inoculum with multiple plant growth promoting activities offers a new concept to address mode of action by combined use of multi-strain inoculum of plant growth promoting rhizobacteria (PGPR) each with specific capability and function. In the present work, bacterial isolates with plant growth promoting activities like P-solubilization, IAA production, siderophore production and broad spectrum antifungal activity against Fusarium oxysporum, Rhizoctonia solani and Sclerotinia sclerotiorum were isolated from the rhizosphere soil of tomato seedlings. In vitro antifungal antibiotic study revealed that among eleven isolates N11 showed maximum inhibition against F. oxysporum (82.85%), R. solani (76.45%) and S. sclerotiorum (74.71%) after seven days of incubation. The per cent growth inhibition increased with increase in bacterial cell density from O.D 0.25 to 1.50. The novelty of the isolates is that the antibiotic production is induced only in the presence of fungal host and lack of antifungal activity in culture filtrate. Bacterial isolate N11 showing maximum plant growth promoting activities and was identified to species level by biochemical characterization and 16S rRNA sequencing. Preliminary identification of bacterial isolate was made on the basis of morphological and biochemical characters and confirmed by partial 16S rRNA gene sequencing, which validated N11 as Bacillus subtilis CKT1. Thus, the present study concludes that these bacterial isolate could serve as a proficient biocontrol PGPR inoculant in the integrated management of soil borne diseases of tomato.

Herbicidal activity of culture filtrates of Trichoderma spp. against two problematic weeds of wheat

The herbicidal potential of culture filtrates of four Trichoderma spp., namely Trichoderma harzianum Rifai, Trichoderma pseudokoningii Rifai, Trichoderma reesei Simmons and Trichoderma viride Pers., was evaluated against two problematic weeds of wheat, Phalaris minor L. and Rumex dentatus L. In laboratory bioassays, generally, metabolites of all four Trichoderma species significantly reduced various root and shoot growth parameters of the two target weed species. The original concentrations of the culture filtrates of all Trichoderma spp., except T. harzianum, significantly reduced various parameters of root and shoot growth of wheat seedlings. In a foliar spray bioassay, the culture filtrates of all four Trichoderma spp. significantly diminished root and shoot biomass of R. dentatus. The effect of these filtrates on the shoot growth of P. minor and wheat was not significant. Culture filtrates of the four Trichoderma species were successively extracted with butanol, n-hexane, chloroform and ethyl acetate. In detached leaf injection bioassays, n-hexane fractions (3 mg mL−1) of T. pseudokoningii, T. reesei and T. viride, and ethyl acetate fractions of T. horzianum and T. pseudokoningii were found to be toxic against R. dentatus. This study concludes that the culture filtrates of Trichoderma species have herbicidal potential in the control of R. dentatus.

Overexpression of the Victoriocin Gene inHelminthosporium(Cochliobolus)victoriaeEnhances the Antifungal Activity of Culture Filtrates

We have previously reported the isolation and characterization of the broad-spectrum antifungal protein, victoriocin, from culture filtrates of a virus-infected isolate of the plant-pathogenic fungus Helminthosporium (teleomorph: Cochliobolus) victoriae. We predicted that the 10-kDa mature victoriocin is derived in vivo from a preprotoxin precursor that is processed by a signal peptidase and kexin-like endopeptidase. We also presented evidence that the victoriocin precursor is encoded by a host gene, designated the victoriocin (vin) gene. In the present study, an H. victoriae genomic DNA library was constructed in the cosmid vector pMLF-2, and a cosmid clone carrying the vin gene and flanking sequences was isolated and used to generate constructs for transformation of virus-free and virus-infected H. victoriae isolates with the vin gene. Culture filtrates of the virus-free vin transformants exhibited high levels of antifungal activity compared with that revealed by the nontransformed virus-free wild-type strain, which exhibited little or no antifungal activity. Moreover, transformation of the wild-type virus-infected H. victoriae strain with the vin gene resulted in still higher production of victoriocin and higher antifungal activity in the culture filtrates of the vin transformants compared with the virus-infected wild-type strain. As previously predicted, the presence in the vin transformants of the preprovictoriocin and its post-translationally generated products, the provictoriocin and the mature victoriocin, was clearly demonstrated. Processing of the victoriocin preprotoxin requires eukaryotic host factors because no processing occurred in an in vitro translation system or in bacteria. It is of interest that some of the virus-free isolates transformed with the vin gene exhibited some features of the virus-induced disease phenotype, including moderate stunting and sectoring. Present data suggests that victoriocin may play an indirect role in disease development. Taken together, these results indicate that victoriocin is the primary protein responsible for the antifungal activity in culture filtrates of virus-infected H. victoriae isolates and that virus infection upregulates the expression of victoriocin. Overproduction of victoriocin may give the slower-growing virus-infected fungal strains some competitive advantage by inhibiting the growth of other fungi.

Germination-Arrest Factor (GAF): 3. Determination that the herbicidal activity of GAF is associated with a ninhydrin-reactive compound and counteracted by selected amino acids

A novel, naturally-occurring herbicide (Germination-Arrest Factor, GAF), produced by Pseudomonas fluorescens WH6 and several related isolates of rhizosphere bacteria, irreversibly arrests germination of the seeds of a wide range of graminaceous species, including a number of important grassy weed species. GAF activity has been shown previously to be associated with a hydrophilic, low molecular weight compound that contains an acid group. In the present study, thin-layer chromatography (TLC) of extracts of WH6 culture filtrate demonstrated that GAF activity migrates on TLC plates with a particular ninhydrin-reactive compound. This compound was found to be present in GAF-producing P. fluorescens isolates and absent in P. fluorescens strains that lack the ability to produce GAF. Treatments, including mutagenesis, which resulted in the loss of GAF activity in culture filtrates from P. fluorescens WH6 were shown to result in the disappearance of this ninhydrin-reactive compound from extracts of WH6 culture filtrates or in alteration of its appearance on TLC chromatograms. The ninhydrin-reactivity of GAF indicates that it probably contains an amino group, as well as the acid group previously demonstrated, and suggests that GAF may be a small peptide or amino acid analog. Biological investigations motivated by this conclusion demonstrated that the effects of GAF in inhibiting the germination of seeds of annual bluegrass ( Poa annua L.) could be counteracted by treatment with alanine or glutamine and, to lesser extent, by several other amino acids, suggesting that this compound may act by interfering with some aspect of amino acid metabolism or function.

ROLE OF MICROORGAINSMS AND THEIR ENZYMES IN COMPOSTING PROCESS FROM PLANT RESIDUES

Newly reclaimed sandy soils are considered one of the main items of agricultural expanding policy in Egypt. Therefore we need to continuous addition of organic matter to increase soil fertility and agricultural production. For this reason, eight heaps were prepared each heap involves four plant residues. One set of four heaps was supplemented with organic manure. Another set was supplemented with cellulose decomposers. Some physical, chemical and microbiological properties were determined during composting process. The important obtained results are as follows: temperature inside the compost heaps began to rise from the third day of composting and reached maximum values at the third week of the process. From the fourth week there was a gradual decrease in temperature till the end of composting. The pH values of the eight heaps tended slightly to acidic at early intervals of composting process till the 20th day, then the pH values increased to reach neutral or slightly alkaline by the end of the process. The Gradual increases in EC and bulk density values during the process. The obtained results showed a gradual decrease in organic carbon and organic matter by time and increase in total-N of all composted heaps during the different intervals of composting process. The results showed remarkable decrease in C/N ratio with the progress of the composting process. At the beginning of the composting, total number of mesophilic bacteria was increased, however it was markedly decreased in the third week of composting. Then it was highly increased at the end of composting. The same pattern of the results was also observed with mesophilic fungi and actinomycetes. On the other hand, thermophilic bacteria were sharply increased at the third week and then obviously decreased at the end of the composting process (cooling phase). The same trend was also observed with thermophilic fungi and actinomycetes. It has been also observed that no marked differences were obtained in the total microbial counts of different compost heaps for both mesophilic and thermophilic organisms. In concerning the enzymes activities of common selected fungal, bacterial and actinomycets strains the obtained results were: In regarding to Cellulase enzyme activity, it was obvious that Trichoderma reesei, Bacillus stearothermophilius and Streptomyces antibioticus recorded the highest activity in culture filtrates. In regarding to dehydrogenase enzyme activity, Trichoderma reesei, Bacillus subtilis and streptomyces roseus exhibited the highest activity. In case of protease activity and also soluble protein the fungus Trichoderma ressei, Bacillus subtilis, and streptomyces antibioticus showed high protease activity and also high soluble protein content.

Factors effecting chitinase activity of Rhizobium sp. from Sesbania sesban

AbstractTwenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.

Pectin Lyase Activity in Culture Filtrate of Penicillium Species

Pectin Lyase Activity in Culture Filtrate of Penicillium Species

The role of GlnD in ammonia assimilation in Mycobacterium tuberculosis

SummaryThe control of ammonia assimilation in Mycobacterium tuberculosis is poorly understood. We have been investigating a regulatory cascade predicted to control the activity of glutamine synthetase (GS). We previously demonstrated that the GS-modifying protein, GlnE (an adenylyl transferase), is essential for M. tuberculosis growth. GlnD, a uridylyl transferase, is involved in the control of GlnE activity in other bacteria. In M. tuberculosis, glnD is arranged in an apparent operon with amt and glnB; all three genes are up-regulated in a low-ammonia medium. We constructed an in-frame deletion of glnD by homologous recombination. The mutant had no growth defect in media containing different nitrogen sources. Total GS activity in culture filtrates was markedly reduced in the mutant, although activity in cell-free extracts remained normal. Virulence was unaffected in both in vitro and in vivo model systems of infection, indicating that the presence of extra-cellular GS is not critical for virulence and that the residual intra-cellular GS activity is sufficient. Thus although GlnD does play a role in the control of ammonia assimilation, it is not required for virulence.

Protein synthesis inhibitory activity in culture filtrates from new strains of Streptomyces isolated from Brazilian tropical soils.

To investigate the effect of the culture supernatants from three newly isolated Streptomyces strains, 221, 235 and 606 on eukaryotic cells. Cell lines were treated with the culture filtrates and assayed for protein synthesis by metabolic labelling, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. RNA synthesis was investigated by [5-3H]uridine incorporation. The three culture filtrates presented a strong inhibitory activity, reducing total protein synthesis of different eukaryotic cell lines by more than 85%. No effect on cellular RNA synthesis was detected. The culture filtrates did not affect the growth of the prokaryotic cells tested. These new Streptomyces strains, recently isolated from Brazilian tropical soils, produce molecule(s) with inhibitory activity specific to eukaryote protein synthesis. Streptomyces strains 221, 235 and 606, probably representing new species, might produce new bioactive compound(s), and can be used as valuable tools to study the protein synthesis pathway in eukaryotes.

Mutational analysis of beta-glucanase genes from the plant-pathogenic fungus Cochliobolus carbonum.

Two new beta-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-beta1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (beta1,3-beta1,4) glucanase with low activity against beta1,4-glucan and no activity against beta1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total beta1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.

Induction of Systemic Resistance in Cucumber against Several Diseases by Plant Growth-promoting Fungi: lignification and Superoxide Generation

Five fungal isolates (Trichoderma, Fusarium, Penicillium, Phoma and a sterile fungus) from zoysiagrass rhizosphere that promote plant growth were tested for their ability to induce systemic resistance in cucumber plants against Colletotrichum orbiculare. Roots of cucumber plants were treated with these fungal isolates using barley grain inocula (BGI), mycelial inocula (MI) or culture filtrate (CF). Most isolate/inoculum form combinations significantly reduced the disease except BGI of Trichoderma. These fungal isolates were also evaluated for induction of systemic resistance against bacterial angular leaf spot and Fusarium wilt by treatment with BGI. Penicillium, Phoma and the sterile fungus significantly reduced the disease incidence of bacterial angular leaf spot. Phoma and sterile fungus protected plants significantly against Fusarium wilt. Roots treated with CFs of these fungal isolates induced lignification at Colletotrichum penetration points indicating the presence of an elicitor in the CFs. The elicitor activity of CFs was evaluated by the chemiluminescence assay using tobacco callus and cucumber fruit disks. The CFs of all isolates elicited conspicuous superoxide generation. The chemiluminescence activity of the CF of Penicillium was extremely high, and its intensity was almost 100-fold higher than that of other isolates. The chemiluminescence activity was not lost following treatment with protease or autoclaving or after removal of lipid. The MW 12,000 dialyzed CF fraction was highly effective in eliciting chemiluminescence activity. Chemiluminescence emission from cucumber fruit disks treated with Penicillium was the same as that obtained from tobacco callus, except that the lipid fraction also showed a high activity. Both the MW 12,000 fraction and the lipid fraction induced lignification in the epidermal tissues of cucumber hypocotyls.

Molecular and biochemical evidence for manganese-dependent peroxidase activity in Tylospora fibrillosa

Gel-based and spectrophotometric assays were used to demonstrate expression of a manganese-dependent peroxidase (MnP) activity in culture filtrates of the ectomycorrhizal basidiomycete Tylospora fibrillosa. PCR amplification using a primer pair specific for a 260 bp fragment from the 5′-end of a gene encoding an H3-like MnP isozyme in Phanerochaete chrysosporium, produced single amplification products of 260 bp from DNA extracted from two isolates of T. fibrillosa. The amplified fragment from T. fibrillosa had a 93.7% nucleotide overlap (over 201 bases) with a published sequence for a 260 base amplification product from the P. chrysosporium H3 MnP isozyme gene, strongly suggesting the presence of a homologous gene in the ectomycorrhizal fungus. The results are discussed in the context of lignin degradation by ectomycorrhizal fungi.