Control of fusarium wilt of <Emphasis Type="Italic">Solanum melongena</Emphasis> by <Emphasis Type="Italic">Trichoderma</Emphasis> spp.

Biological control of wilt of egg plant (Solanum melongena L.) caused by Fusarium solani was made with the application of five Trichoderma species, T. harzianum, T. viride, T. lignorum, T. hamatum and T. reesei. The effect of volatile and non-volatile antibiotics of Trichoderma origin on growth inhibition of the wilt pathogen was studied. T. harzianum showed maximum growth inhibition (86.44 %) of the pathogen through mycoparasitism. The non-volatiles produced by the Trichoderma species exhibited 100 % growth inhibition of the pathogen under in vitro condition. Production of siderophores and fungal cell wall degrading enzymes, chitinase and β-1,3-glucanase were found. Treatments with two most efficient Trichoderma species, T. harzianum and T. viride resulted in the decreasing population of Fusarium solani in soil thereby deterring disease incidence in field condition.     

The properties of l-fucose binding agglutinin associated with the cell wall of Rhizoctonia solani

The adherence of Escherichia coli B cells to cell wall associated-agglutinin of the soil borne plant pathogen Rhizoctonia solani, was inhibited by l-fucose, l-galactose, trypsin, SDS, cycloheximide and Na₂-EDTA. The coiling of the biocontrol agent Trichoderma harzianum around Rhizoctonia hyphae was prevented by SDS, cycloheximide, Na₂-EDTA and methyl--l-fucoside — an inhibitor of Rhizoctonia agglutinin not metabolized by both fungi. The possible role of the agglutinin in Trichoderma-Rhizoctonia interaction is discussed.     

New Insights in Trichoderma harzianum Antagonism of Fungal Plant Pathogens by Secreted Protein Analysis

Trichoderma harzianum ALL42 were capable of overgrowing and degrading Rhizoctonia solani and Macrophomina phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. Hyphae of T. harzianum ALL42 did not show any coiling around Fusarium sp. hyphae suggesting that mycoparasitism may be different among the plant pathogens. In this study, a secretome analysis was used to identify some extracellular proteins secreted by T. harzianum ALL42 after growth on cell wall of M. phaseolina, Fusarium sp., and R. solani. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. A total of 60 T. harzianum ALL42 secreted proteins excised from the gel were analyzed from the three growth conditions. While seven cell wall-induced proteins were identified, more than 53 proteins spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced. Endochitinase, β-glucosidase, α-mannosidase, acid phosphatase, α-1,3-glucanase, and proteases were identified in the gel and also detected in the supernatant of culture.     

A comparison of enzymatic phosphorylation and phosphatidylation of β-l- and β-d-nucleosides

Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates. Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers.     

Growth inhibitory activity of ABA-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucopyranosyl ester and ABA-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidase

Exogenously applied ABA-β-d-glucopyranosyl ester (ABA-GE) inhibited shoot growth of alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), Digitaria sanguinalis L., timothy (Pheleum pratense L.) and ryegrass (Lolium multiflorum Lam.) seedlings at concentrations greater than 0.1 μM. The growth inhibitory activity of ABA-GE on these shoots was 26–40% of that of (+)-ABA. ABA-β-d-glucosidase activities in these seedlings were 11–31 nmol mg⁻¹ protein min⁻¹. These results suggests that exogenously applied ABA-GE may be absorbed by plant roots and hydrolyzed by ABA-β-d-glucosidase, and liberated free ABA may induce the growth inhibition in these plants. Thus, although ABA-GE had been thought to be physiologically inactive ABA conjugate, ABA-GE may have important physiological functions rather than an inactive conjugated ABA form.     

Plant Growth Inhibitory Compounds from Aqueous Leachate of Wheat Straw

When seedlings of lettuce, cress, rice and wheat were incubated with the leachate of wheat straw, the roots growth of lettuce and garden cress were particularly inhibited. The leachate of wheat straw (100 g eq./l) showed 80.5 and 79.4% inhibition for lettuce and cress roots, respectively. The inhibitory activity was stronger as the concentration of wheat straw leachate was greater. This result indicates that allelochemical(s) inhibiting the roots growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of the wheat straw and identified as syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan by spectral analyses. Syringoylglycerol 9-O-β-d-glucopyranoside inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 10.0 μM, respectively. On the other hand, l-tryptophan inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 1.0 μM, respectively. The content of syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan in the leachate of wheat straw (100 g eq./l) was 18.4 ± 0.7 and 6.2 ± 0.6 μM, respectively. Syringoylglycerol 9-O-β-d-glucopyranoside (18.4 μM) showed 21.5 and 13.5% inhibition in the lettuce and cress roots assay, respectively. On the other hand, 6.2 μM of l-tryptophan showed 47.5 and 35.0% inhibition in the lettuce and cress roots assay, respectively. These results suggested that l-tryptophan may be a major contributor to the allelopathy in aqueous leachate of wheat straw and syringoylglycerol 9-O-β-d-glucopyranoside may be a minor contributor.     

Degradation of rice bran hemicellulose by <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. strain HC1: gene cloning,characterization and function of β-D-glucosidase as an enzyme involved in degradation

A bacterium (strain HC1) capable of assimilating rice bran hemicellulose was isolated from a soil and identified as belonging to the genus Paenibacillus through taxonomical and 16S rDNA sequence analysis. Strain HC1 cells grown on rice bran hemicellulose as a sole carbon source inducibly produced extracellular xylanase and intracellular glycosidases such as β-d-glucosidase and β-d-arabinosidase. One of them, β-d-glucosidase was further analyzed. A genomic DNA library of the bacterium was constructed in Escherichia coli and gene coding for β-d-glucosidase was cloned by screening for β-d-glucoside-degrading phenotype in E. coli cells. Nucleotide sequence determination indicated that the gene for the enzyme contained an open reading frame consisting of 1,347 bp coding for a polypeptide with a molecular mass of 51.4 kDa. The polypeptide exhibits significant homology with other bacterial β-d-glucosidases and belongs to glycoside hydrolase family 1. β-d-Glucosidase purified from E. coli cells was a monomeric enzyme with a molecular mass of 50 kDa most active at around pH 7.0 and 37°C. Strain HC1 glycosidases responsible for degradation of rice bran hemicellulose are expected to be useful for structurally determining and molecularly modifying rice bran hemicellulose and its derivatives.     

High-level expression of a truncated 1,3-1,4-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase from <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by optimization of codons and fermentation

1,3-1,4-β-d-glucanase is an important endoglycosidase in the brewing and animal feed industries. To achieve high-level expression of recombinant glucanase in Pichia pastoris, we designed sequences encoding the α-factor signal peptide from Saccharomyces cerevisiae and the truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes as a whole. The codons encoding the 52 amino acids of the signal peptide and 106 residues of the glucanase protein were optimized for expression in P. pastoris; 189 nucleotides were changed. The G + C content was adjusted to 48–49%, and AT-rich stretches were eliminated to avoid premature termination. The messenger ribonucleic acid secondary structure near the AUG start codon was also adjusted to ensure efficient translation; the resulting glucanase production was twofold higher compared with that achieved with gene structure optimization alone. We also propose a new fermentation strategy for the induction phase, in which 5/95% glycerol/methanol mixed feed was used in days 1–3 and 100% methanol was used on days 4–6. By comparison with methanol feed and glycerol/methanol-mixed feed alone, the yield of recombinant glucanase increased by 38.5 and 16.5%, respectively. The expressed optimized recombinant 1,3-1,4-β-d-glucanase constituted ~90% of the total secreted protein, reaching up to 3 g l⁻¹ in the medium.     

Diverse mechanisms adopted by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> PGC2 during the inhibition of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Rhizoctonia solani and Phytophthora capsici are two of the most destructive phytopathogens occurring worldwide and are only partly being managed by traditional control strategies. Fluorescent Pseudomonas isolates PGC1 and PGC2 were checked for the antifungal potential against R. solani and P. capsici. Both the isolates were screened for the ability to produce a range of antifungal compounds. The results of this study indicated the role of chitinase and β-1,3-glucanase in the inhibition of R. solani, however, antifungal metabolites of a non-enzymatic nature were responsible for inhibition of P. capsici. The study confirmed that multiple and diverse mechanisms are adopted by the same antagonist to suppress different phytopathogens, as evidenced in case of R. solani and P. capsici.     

The α-d-mannan core of a complex cell-wall heteroglycan of Trichoderma reesei is responsible for β-glucosidase activation

A heteroglycan responsible for the binding of the enzyme β-1,4-d-glucosidase (EC 3.2.1.21) to fungal cell walls was isolated from cell walls of the filamentous fungusTrichoderma reesei. The heteroglycan, composed of mannose, galactose, glucose, and glucuronic acid, also activated β-1,4-d-glucosidase, β-1,4-d-xylosidase andN-acetyl-β-1,4-d-glucosaminidase activity in vitro. The structural backbone of this heteroglycan was prepared by acid hydrolysis and gel filtration. The molecular structure of the core of the heteroglycan was determined by NMR studies as a linear α-1,6-d-mannan. The mannan core obtained by acid degradation stimulated the β-glucosidase activity by 90%. Several glycosidases fromAspergillus niger were also activated by theT. reesei heteroglycan. The β-glucosidase ofTrichoderma was activated by mannan fromSaccharomyces cerevisiae to a comparable extent.     

Relationship between indoor and outdoor airborne fungal spores, pollen, and (1→3)-β-D-glucan in homes without visible mold growth

In this exploratory study, indoor and outdoor airborne fungal spores, pollen, and (1→3)-β-D-glucan levels were determined through long-term sampling (24-h) using a Button Personal Inhalable Aerosol Sampler. The air samples were collected in five Cincinnati area homes that had no visible mold growth. The total count of fungal spores and pollen in the collected samples was conducted under the microscope and Limulus Amebocyte Lysate (LAL) chromogenic assay method was utilized for the determination of the (1→3)-β-D-glucan concentration. For the combined number concentration of fungal spores and pollen, the indoor and outdoor geometric mean values were 573 and 6,435 m⁻³, respectively, with a geometric mean of the Indoor/Outdoor (I/O) ratio of .09. The geometric means of indoor and outdoor (1→3)-β-D-glucan concentrations were .92 and 6.44 ng m⁻³, respectively, with a geometric mean of the I/O ratio equal to .14. The I/O ratio of (1→3)-β-D-glucan concentration was found to be marginally greater than that calculated based on the combined number concentration of fungal spores and pollen. This suggests that (1→3)-β-D-glucan data are affected not only by intact spores and pollen grains but also by the airborne fragments of fungi, pollen, and plant material, which are ignored by traditional enumeration methodologies. Since the (1→3)-β-D-glucan level may elucidate the total exposure to fungal spores, pollen, and fungal fragments, its I/O ratio may be used as a risk marker for mold and pollen exposure in indoor environments.     

Structural characterisation and biological activities of a unique type β-<Emphasis Type="SmallCaps">d</Emphasis>-glucan obtained from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A β-d-glucan obtained from Aureobasidium pullulans (AP-FBG) exhibits various biological activities: it exhibits antitumour and antiosteoporotic effects and prevents food allergies. An unambiguous structural characterisation of AP-FBG is still awaited. The biological effects of β-d-glucan are known to depend on its primary structures, conformation, and molecular weight. Here, we elucidate the primary structure of AP-FBG by NMR spectroscopy, and evaluate its biological activities. Its structure was shown to comprise a mixture of a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every two residues (major structure) and a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every three residues (minor structure). Furthermore, this β-d-glucan exhibited immunostimulatory effects such as the accumulation of immune cells and priming effects against enterobacterium. To our knowledge, 1-3-β-glucans like AP-FBG with such a high number of 1-6-β-glucopyranosyl side branching have a unique structure; nevertheless, many 1-3-β-glucans were isolated from various sources, e.g. fungi, bacteria, and plants.     

Effects of fructose on human fibroblast metabolism: the application of DNA measurements as a basis for interpretation

Summary A fluorometric procedure for measuring DNA was used to study growth and metabolic responses of eight cell strains of human foreskin fibroblasts. In preliminary studies this procedure gave more precise specific activity changes inN-acetyl-β-d-glucosaminidase (NAG) than did a protein activity basis, when changes in this enzyme's specific activity were investigated as a function of experimental cell manipulation. When fibroblast growth in eight cell strains was compared in 134 mM d-fructose vs. 13.4 mM glucose-supplemented minimum essential media, a significant increase in cellular DNA (50%) and protein (45%) occurred over an 11-d period. No significant differences in media pH change, lactate production, or carbohydrate uptake occurred on a DNA basis when cell metabolism was compared over the last 24 h of culture in the two media. Cells grown in fructose-containing media tended to show a reduction in NAG specific activity when compared with those grown in glucose-containing media.     

Characterization of an acid-labile, thermostable β-glycosidase from Thermoplasma acidophilum

A recombinant putative β-galactosidase from Thermoplasma acidophilum was purified as a single 57 kDa band of 82 U mg⁻¹. The molecular mass of the native enzyme was 114 kDa as a dimer. Maximum activity was observed at pH 6.0 and 90°C. The enzyme was unstable below pH 6.0: at pH 6 its half-life at 75°C was 28 days but at pH 4.5 was only 13 h. Catalytic efficiencies decreased as p-nitrophenyl(pNP)-β-d-fucopyranoside (1067) > pNP-β-d-glucopyranoside (381) > pNP-β-d-galactopyranoside (18) > pNP-β-d-mannopyranoside (11 s⁻¹ mM⁻¹), indicating that the enzyme was a β-glycosidase.     

High accumulation of dehydrodiconiferyl alcohol-4-β-d-glucoside in free and immobilized Linum usitatissimum cell cultures

As flaxseed mainly accumulates lignans (secoisolariciresinol diglucoside and matairesinol), these compounds were barely or not detected in plant cell suspensions initiated from Linum usitatissimum. In contrast, these cell suspensions were shown to accumulate substantial amounts of a neolignan identified as dehydrodiconiferyl alcohol-4-β-d-glucoside (DCG) (up to 47.7 mg g⁻¹ DW). The formation of this pharmacologically active compound was evaluated as a function of cell growth and in relation to phytohormone balance of the culture media. After establishment of efficient culture conditions, production of DCG was investigated in immobilized plant cell suspensions initiated from plantlet roots of L. usitatissimum. The results indicate that immobilization enhances the DCG production up to 60.0 mg g⁻¹ DW but depresses the cell growth resulting in no improvement of the total DCG yield. Nevertheless, with immobilized cell suspensions, a release of DCG into the medium is observed allowing an easier recovery.     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.     

Four glycoside hydrolases are differentially modulated by auxins, cytokinins, abscisic acid and gibberellic acid in apple fruit callus cultures

α-l-Arabinofuranosidase, α- and β-d-xylosidase, and β-d-glucosidase activity was detected in the soluble fraction (S-F) extracted with water and in the NaCl-released fraction (NaCl-F) extracted with a high-salt concentration buffer from apple callus cultures. The activity was found to be differentially modulated by the addition of various plant growth regulators (PGRs) to calluses that had lost their requirement for specific PGRs (“habituation” phenomenon). α-l-Arabinofuranosidase activity was 93%, 130%, 126% and 186% higher in the NaCl-F from IAA-, IBA-, ABA- and GA₃-treated callus than in that extracted from untreated callus while S-F α-l-arabinofuranosidase activity was only 71%, 24%, 55% and 66% higher, respectively. α-d-Xylosidase displayed low activity levels in both S-F and NaCl-F but 2iP-treated callus showed higher α-d-xylosidase activity in both fractions than the control. 2,4-D increased α-d-xylosidase activity by 110% in the NaCl-F but decreased it by 40% in the S-F. β-d-Xylosidase activity increased by 99% in S-F from 2iP-treated callus but slightly decreased in the NaCl-F. In GA₃-treated callus, NaCl-F β-d-xylosidase activity increased by 188%. S-F and NaCl-F from Picloram-treated callus showed undetectable or only slightly noticeable α-l-arabinofuranosidase, α-d-xylosidase and β-d-xylosidase activity. Interestingly, β-d-glucosidase activity rose 28-fold in the S-F extracted from Picloram-treated callus. β-d-glucosidase was the only enzyme assayed that greatly increased its NaCl-F activity after 10 subcultures, and the addition of any PGR to the callus culture –except for Picloram and ABA– decreased its activity, suggesting that this enzyme may be associated with certain stress conditions, such as PGR starvation or Picloram addition. This is the first report on glycoside hydrolases from fruit callus as modulated by different PGRs.     

Expression of Bacterial l-aspartate-α-decarboxylase in Tobacco Increases β-Alanine and Pantothenate Levels and Improves Thermotolerance

l-Aspartate-α-decarboxylase catalyzes the decarboxylation of l-aspartate to generate β-alanine and carbon dioxide. This is an unusual pyruvoyl-dependent enzyme unique to prokaryotes that undergoes limited self-processing. The Escherichia coli panD gene encoding l-aspartate-α-decarboxylase was expressed under a constitutive promoter in transgenic tobacco. Transgene expression was verified by assays based on RNA blots, immunoblots and enzyme activity in vitro. The panD lines had increased levels of leaf β-alanine (1.2- to 4-fold), pantothenate (3.2- to 4.1-fold) and total free amino acids (up to 3.7-fold) compared to wild-type and vector controls. Growth of homozygous lines expressing E. coli l-aspartate-α-decarboxylase was less affected than that of the control lines when the plants were stressed for 1 week at 35 °C. When transferred from 35 to 30 °C for 3 weeks, the PanD transgenic lines recovered significantly (P ≤ 0.001) better than the controls: PanD lines had on an average 54% and 84% greater fresh and dry weights respectively, compared to the controls. Homozygous lines expressing E. coli l-aspartate-α-decarboxylase had significantly greater thermotolerance (P ≤ 0.05) during germination. At 42 °C, 95% of two T3 PanD transgenic line seeds germinated after 12 days compared to 73% for the wild-type seeds. Our results indicated that E. colil-aspartate-α-decarboxylase was correctly processed and active in the transgenic eukaryotic host and its expression resulted in increased thermotolerance in tobacco. This is Florida Agricultural Experiment Station journal series number R-10355. W.M.F. was supported by the Egypt Development Training fellowship and by the UF College of Agriculture and Life Sciences assistantship.     

Gene cloning and expression in Escherichia coli of a bi-functional beta-D-xylosidase/alpha-L-arabinosidase from Sulfolobus solfataricus involved in xylan degradation

An open reading frame encoding a putative bi-functional β-d-xylosidase/α-l-arabinosidase (Sso3032) was identified on the genome sequence of Sulfolobus solfataricus P2, the predicted gene product showing high amino-acid sequence similarity to bacterial and eukaryal individual β-d-xylosidases and α-l-arabinosidases as well as bi-functional enzymes such as the protein from Thermoanaerobacter ethanolicus and barley. The sequence was PCR amplified from genomic DNA of S. solfataricus P2 and heterologous gene expression obtained in Escherichia coli, under optimal conditions for overproduction. Specific assays performed at 75°C revealed the presence in the transformed E. coli cell extracts of this archaeal activity involved in sugar hydrolysis and specific for both substrates. The recombinant protein was purified by thermal precipitation of the host proteins and ethanol fractionation and other properties, such as high thermal activity and thermostability could be determined. The protein showed a homo-tetrameric structure with a subunit of molecular mass of 82.0 kDa which was in perfect agreement with that deduced from the cloned gene. Northern blot analysis of the xarS gene indicates that it is specifically induced by xylan and repressed by monosaccharides like d-glucose and l-arabinose.