Derivatization of Natural Compound ß-Pinene Enhances Its In Vitro Antifungal Activity against Plant Pathogens.

BACKGROUND: The development of new antifungal agents has always been a hot research topic in pesticide development. In this study, a series of derivatives of natural compound ß-pinene were prepared, and the antifungal activities of these derivatives were evaluated. The purpose of this work is to develop some novel molecules as promising new fungicides. METHODS: Through a variety of chemical reactions, ß-pinene was transformed into a series of ß-pinene-based derivatives containing amide moieties and acylthiourea moieties. The antifungal activities of these derivatives against five plant pathogens including Colletotrichum gloeosporioides, Fusarium proliferatum, Alternaria kikuchiana, Phomopsis sp. and Phytophthora capsici were tested; preliminary structure-activity relationship was discussed. RESULTS: Some derivatives exhibited moderate or significant antifungal activity due to the fusion of the amide moiety or the acylthiourea moiety with the pinane skeleton. The structure-activity relationship analysis showed that the fluorine atom and the strong electron withdrawing nitro group, or trifluoromethyl group on the benzene ring of the derivatives had a significant effect on the improvement of the antifungal activity against Colletotrichum gloeosporioides, Fusarium proliferatum, Alternaria kikuchiana and Phomopsis sp. Meanwhile, the introduction of an ethyl group at the meta-position on the benzene ring of the derivatives could improve the antifungal activity against Phytophthora capsici. Compounds 4e, 4h, 4q, 4r exhibited broad-spectrum antifungal activity against the tested strains. Compound 4o had significant antifungal activity against Phytophthora capsici (IC50 = 0.18 µmol/L). These derivatives were expected to be used as precursor molecules for novel pesticide development in further research.

The DNA-methyltransferase inhibitor 5-aza-2-deoxycytidine affects Humicola grisea enzyme activities and the glucose-mediated gene repression.

Humicola grisea var. thermoidea (Hgvt) is a thermophilic ascomycete that produces lignocellulolytic enzymes and it is proposed for the conversion of agricultural residues into useful byproducts. Drugs that inhibit the DNA methyltransferases (DNMTs) activity are employed in epigenetic studies but nothing is known about a possible effect on the production of fungal enzymes. We evaluated the effect of 5-aza-2'-deoxycytidine (5-Aza; a chemical inhibitor of DNMTs activity) on the secreted enzyme activity and on the transcription of cellulase and xylanase genes from Hgvt grown in agricultural residues and in glucose. Upon cultivation on wheat bran (WB), the drug provoked an increase in the xylanase activity at 96 h. When Hgvt was grown in glucose (GLU), a repressor of Hgvt glycosyl hydrolase genes, 5-Aza led to increased transcript accumulation for the cellobiohydrolases and for the xyn2 xylanase genes. In WB, 5-Aza enhanced the expression of the transcription factor CreA gene. Growth on WB or GLU, in presence of 5-Aza, led to a significant increase in transcripts of the pH-response regulator PacC gene. To our knowledge, this is the first report on the effect of a DNMT inhibitor in the production of fungal plant cell wall degradation enzymes.

Heterologous Expression and Characterization of a GH3 ß-Glucosidase from Thermophilic Fungi Myceliophthora thermophila in Pichia pastoris.

A novel ß-glucosidase of glycoside hydrolase (GH) family 3 from Myceliophthora thermophila (mtbgl3b) was successfully expressed in Pichia pastoris. The full-length gene consists of 2613 bp nucleotides encoding a protein of 870 amino acids. MtBgl3b showed maximum activity at pH 5.0 and remained more than 70 % relative activity at 3.5-6.0. The enzyme displayed the highest activity at 60 °C and kept about 90 % relative activity for 50-65 °C; besides, the enzyme showed psychrophilic trait and remains 51 % relative activity at 40 °C. MtBgl3b exhibited good stability over a wide pH range of 3.0-10.0 and was thermostable at 60 and 65 °C. The enzyme displayed highest activity towards p-nitrophenyl-ß-D-glucopyranoside (pNPG), followed by p-nitrophenyl-D-cellobioside (pNPC), cellotetraose, cellotriose, cellobiose, and gentiobiose. When using 10 % cellobiose (w/v) as the substrate, the enzyme showed transglycosylation activity to produce the cellotriose. The kinetic parametric of K m and V max were 2.78 mM and 927.9 µM mg(-1) min(-1), respectively. Finally, the reaction mode of the enzyme and the substrates were analyzed by molecular docking approach.

Synthesis, characterization, and antifungal activity of novel inulin derivatives with chlorinated benzene.

A group of novel inulin derivatives containing benzene or chlorinated benzene were synthesized by reaction of chloracetyl inulin (CAIL) with the Schiff bases of 4-amino-pyridine, including (2-pyridyl)acetyl inulin chloride (PAIL), 2-[4-(2-chlorobenzylideneamino)-pyridyl]acetyl inulin chloride (2CPAIL), 2-[4-(4-chlorobenzylideneamino)-pyridyl]acetyl inulin chloride (4CPAIL), and 2-[4-(2,4-dichlorobenzylideneamino)-pyridyl]acetyl inulin chloride (2,4DCPAIL). Their antifungal activity against three kinds of phytopathogens was estimated by hypha measurement in vitro. Of all the synthesized chitosan derivatives, 2,4DCPAIL inhibited the growth of the tested phytopathogens with inhibitory indices of 67%, 47%, and 43% against Colletotrichum lagenarium (Pass) Ell.et halst, Phomopsis asparagi (Sacc.) Bubak and Fusarium oxysporum (schl.) F.sp. niveum (F. oxysporum) respectively at 1.0 mg/mL. The results indicate that all the inulin derivatives have better antifungal activity than inulin, and the inhibitory index is affected by the chlorine atom grafted to the inulin derivatives.

Combinatorial synthesis of benzimidazole-azo-phenol derivatives as antifungal agents.

A chemically diverse library of benzimidazole-azo-phenol derivatives was efficiently prepared and screened for their antifungal activities against five phytopathogenic fungi. Some compounds exhibited potent antifungal activities. As compared with a commercially available agricultural fungicide, hymexazol, especially compound V-5 showed the most promising broad-spectrum antifungal activities against five phytopathogenic fungi. The EC50 values of V-5 against F. graminearum, A. solani, V. mali, B. cinerea, and C. lunata were 0.09, 0.08, 0.06, 0.07, and 0.11 µmol/mL, respectively.

Diplogelasinospora grovesii IMI 171018 immobilized in polyurethane foam. An efficient biocatalyst for stereoselective reduction of ketones.

Diplogelasinospora grovesii has been reported as a very active biocatalyst in the reduction of ketones. Along the text, the properties of this filamentous fungus as an immobilized catalyst are described. For this purpose, several immobilization supports as agar and polyurethane foam were tested. Experimental assays were also performed to test different co-substrates for the regeneration of the required enzyme cofactor. The fungus immobilized in polyurethane foam lead to the most stable and active catalyst. This derivative, using i-PrOH as co-substrate, could be reused at least 18 times without appreciable activity loss (>90% activity remains). Kinetic runs experiments shown that the reduction of cyclohexanone, selected as model substrate, followed a pseudo-first kinetic order and that the rate controlling step was the mass transfer through the cell wall. The deactivation kinetic constants were also determined. The reduction of different chiral ketones showed that the ketone reductase activity followed the Prelog's rule.

Invasive Myceliophthora thermophila infection mimicking invasive aspergillosis in a neutropenic patient: a new cause of cross-reactivity with the Aspergillus galactomannan serum antigen assay.

Myceliophthora thermophila is a thermophilic mould widely found in the environment but rarely responsible for human infections. We describe a case of invasive Myceliophthora thermophila infection mimicking invasive aspergillosis in a neutropenic patient with haematological malignancy. Cross-reactivity with Aspergillus galactomannan assay (GM) was demonstrated by repeated positive results and confirmed by cross-reaction between the fungal isolate and the GM assay. The patient was successfully treated with voriconazole. Potential GM cross-reactivity must be considered in future studies including patients categorized as having probable invasive aspergillosis using the GM as the only mycological criterion.

Effect of dsRNA on growth rate and reproductive potential of Monosporascus cannonballus.

The effect of double stranded RNA (dsRNA) infection on growth rate and the reproductive potential of Monosporascus cannonballus was studied in 21 isolates collected in cucurbit growing areas of Spain and Tunisia. The isolates were incubated on potato dextrose agar (PDA) under different conditions of temperature, pH, and water potential (Ψ(s)). They showed optimal growth temperatures over the range of 27-34°C and perithecia formation was obtained mainly at 25 and 30°C, although some isolates were able to produce perithecia at 35°C. All isolates were able to produce perithecia in a broad range of pHs (4-8). Regarding the effect of Ψ(s,) the isolates were more tolerant to grow on KCl than on NaCl. For each solute, radial growth decreased progressively as Ψ(s) decreased and was severely limited at -5.0 to -6.0MPa. Perithecia formation was highest at -0.5MPa, decreased at -1.0MPa and occurred just in some isolates at -2.0MPa. Nine of the M. cannonballus isolates harboured dsRNA with 2-6 bands each and a size range of 1.9-18.0Kb. Phenotypical data were subjected to multivariate factorial analysis. Most of the isolates clustered in two groups corresponding with the presence/absence of dsRNA elements. Isolates without detectable dsRNA produced more perithecia. However, isolates with dsRNA produced lower number of perithecia depending on the pH, Ψ(s,) or solute used. These results improve our understanding of the behaviour and growth of this pathogen in soil, and can be useful to implement effective disease control.

MST12 regulates infectious growth but not appressorium formation in the rice blast fungus Magnaporthe grisea.

In the rice blast fungus Magnaporthe grisea, a mitogen-activated protein kinase gene, PMK1, is known to regulate appressorium formation and infectious hyphae growth. Since PMK1 is homologous to the FUS3 and KSS1 genes that regulate the transcription factor STE12 in yeast, we functionally characterized the STE12 homologue in M. grisea (MST12). A polymerase chain reaction-based approach was used to isolate the MST12 gene that is homologous to yeast STE12. Four mst12 deletion mutants were isolated by gene replacement. No obvious defect in vegetative growth, conidiation, or conidia germination was observed in mst12 mutants. However, mst12 mutants were nonpathogenic on rice and barley leaves. In contrast to pmk1 mutants that did not form appressoria, mst12 mutants produced typical dome-shaped and melanized appressoria. However, the appressoria formed by mst12 mutants failed to penetrate onion epidermal cells. When inoculated through wound sites, mst12 mutants failed to cause spreading lesions and appeared to be defective in infectious growth. These data indicate that MST12 may function downstream of PMK1 to regulate genes involved in infectious hyphae growth. A transcription factor or factors other than MST12 must exist in M. grisea and function downstream from PMK1 for appressorium formation.

High mortality in a large-scale zebrafish colony (Brachydanio rerio Hamilton & Buchanan, 1822) associated with Lecythophora mutabilis (van Beyma) W. Gams & McGinnis.

Zebrafish (Brachydanio rerio) have become an important model system for studying vertebrate embryonic development and gene function through manipulation of genotype and characterization of resultant phenotypes. An established research zebrafish colony without substantial disease problems for more than 7 years of operation began experiencing appreciable mortalities in November of 1997. Young fish (fry), from five to 24 days after hatching, spontaneously developed elongate strands of organic material protruding from the mouth, operculum, and anal pore, leading workers in the laboratory to describe the infected fish as "bearded." Unlike typical freshwater fish fungal infections, the skin surface did not have evidence of fungal colonization. The disease was associated with progressive lethargy, reduced feeding, and subsequent mortality. From 10 to 100% of the fry in a given tank were affected. Initial examination indicated that the biofilm around the head of affected fry consisted of bundles of septate fungal hyphae, large numbers of mixed bacterial populations, and protozoans. Environmental samples of air and water in the laboratory were obtained to ascertain the source of the infective agent and to isolate and identify the fungus. A fungus identified as Lecythophora mutabilis was isolated repeatedly from infected fish and water samples from infected fish tanks, and from the main laboratory water supply tanks, but not from laboratory air. Some biofilm beards on fish were found to consist of relatively pure bacterial populations, and beards on occasional fish examined in the later part of the study consisted of hyphae and spores of the oomycete genus Aphanomyces. Lecythophora mutabilis did not invade tissues; however, elimination of the epizootic correlated with reduction in the number of L. mutabilis conidia in the water following modification of the laboratory water system by use of new filtration and sterilization systems. We conclude that the dense hyphal strands of L. mutabilis composing the predominant biofilm type, along with mixed bacteria and protozoa, contributed to the die-off in young fry by occluding the oral cavity and/or gills, leading to starvation and/or asphyxiation.