Detection of antifungal activity in Anemarrhena asphodeloides by sensitive BCT method and isolation of its active compound

Antifungal activity was detected from Anemarrhena asphodeloides by the Bio-Cell Tracer (BCT) method. An active fraction was separated by silica gel column chromatography and reverse-phase HPLC. The molecular weight was determined by GC-MS, and the molecular structure was analyzed by IR, (1)H NMR, and (13)C NMR. The isolated compound was found to be identical to nyasol, (Z)-1, 3-bis(4-hydroxyphenyl)-1,4-pentadiene, which formerly appeared in the literature without any remark on the antifungal activity. This compound showed antimicrobial activity against 38 strains of fungi and five strains of bacteria. The minimum inhibitory concentration (MIC) ranged from 12.5 to 200 microg mL(-)(1), except for two strains based on the broth dilution method.     

Substrate specificity of chlorophenoxyalkanoic acid-degrading bacteria is not dependent upon phylogenetically related tfdA gene types

The phenoxyalkanoic acid herbicides constitute a group of chemically related molecules that have been widely used for over 50 years. A range of bacteria have been selected from various locations for their ability to degrade these compounds. Previously reported strains able to utilise 2,4-dichlorophenoxyacetic acid (2,4-D) include, Ralstonia eutropha JMP134, Burkholderia sp. RASC and Variovorax paradoxus TV1 and Sphingomonas sp. AW5 able to utilise 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In addition a novel set of mecoprop-degrading strains including Alcaligenes denitrificans, Alcaligenes sp. CS1 and Ralstonia sp. CS2 are here described. It has been reported recently that TfdA enzymes, initially reported to have a role in 2,4-D catabolism are also involved in the first-step cleavage of related phenoxyalkanoate herbicides. However, a diversity of tfdA gene sequences have been reported. We relate the tfdA gene type to the metabolic ability of these strains. The tfdA-like genes were investigated by polymerase chain reaction amplification using a set of specific tfdA primers. Degradation ability was observed via phenol production from a range of unsubstituted and substituted phenoxyalkanoics including, 2,4-D, 2-methyl 4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop, 2-(2,4-dichlorophenoxy) propionic acid (racemic 2,4-DP), 2,4,5-T, 2,4-dichlorophenoxybutyric acid (2,4-DB), 4-chloro-2-methylphenoxybutyric acid (MCPB) and phenoxyacetate. Mecoprop-degrading strains showed partial tfdA sequences identical to the one described for V. paradoxus TV1 (a strain isolated on 2,4-D). However, substrate specificity was not identical as V. paradoxus exhibited greatest activity towards 2,4-D and MCPA only, whereas the mecoprop-degrading strains showed intense activity towards 2,4-D, MCPA, racemic mecoprop and (R)-mecoprop as substrates. However, Sphingomonas sp. AW5 which has been shown to carry a very different tfdA-like gene was the only strain to utilise the phenoxybutyric acid MCPB as a sole carbon source. In this study, we thus demonstrate that sequence diversity is not related to substrate specificity within the tfdA-like gene family. However, phylogenetically unrelated sequences may govern substrate specific activity.     

Effect of azide resistant mutants of Azotobacter chroococcum on yield and nitrogen content in two cotton genotypes

Seventeen azide resistant mutants were isolated from six strains (103, HT-54, HT-54 (2), 10P, E-12 and MSX 9) of Azotobacter chroococcum. All the parent strains were tested for their minimal inhibitory concentration (MIC) by growing cells in liquid medium containing different concentrations of sodium azide (5 μgml¹ to 200 μgml¹). The strains showed different MIC values indicating that the resistance pattern varied with each individual strain. These azide resistant mutants were assessed for their performance on two cotton cultivars- Gossypium hirsutum var. H-117 and Gossypium herbaceum var. HD-123 under pothouse conditions for various plant parameters. Inoculation of cotton seed with Azotobacter mutants showed better results than inoculation with their respective parental strains. Azide resistant mutants showed increased plant height, early flowering, more yield, high biomass and high total nitrogen content. Better performance by these mutants might be due to high indole acetic acid, ammonia excretion and high nitrogenase activity.     

Utilization of trehalose, benzoate, valerate, and seed and root exudates by genotypes of 2,4-diacetylphloroglucinol producing Pseudomonas fluorescens

Isolates of Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are effective biocontrol agents against soilborne pathogens. A previous study showed that the superior (“premier”) root colonizer P. fluorescens Q8r1-96 (genotype D) utilized trehalose, benzoate and valerate as sole carbon sources but average colonizers Q2-87 (genotype B) and 1M1-96 (genotype L) did not. We tested the utilization of these three carbon sources by a collection of 55 2,4-DAPG-producing P. fluorescens strains from 17 genotypes and found no correlation between a strain's ability to utilize these carbon sources and superior rhizosphere competence on wheat and pea. Of the strains tested, 73%, 48% and 69% were able to utilize trehalose, benzoate and valerate as sole carbon sources, respectively. With some exceptions, we found a correlation between the utilization of these compounds and previous groupings of these strains by BOX-PCR; genotype D strains utilized all three compounds. Twenty-three strains grew efficiently on root and seed exudates from wheat and pea, with doubling times between 0.9 and 1.6 h generation⁻¹ and lag phases between 5 and 8 h, comparable to growth on glucose as a sole carbon source. Only 10 strains, including those with “premier” (Q8r1-96) and “average” (Q2-87) rhizosphere competence, showed slower growth in wheat root exudates, with lag phases between 16 and 22 h. Results were the same when soil was added to the culture medium. Growth of four strains in media containing glucose or wheat or pea seed exudates as a sole carbon source was not influenced by whether the bacterial cells used as inoculum were harvested from wheat seeds or broth culture. We conclude that the superior ability of some strains to colonize the roots of certain crops cannot be explained by the utilization of the carbon sources tested in our study.     

Synthesis and fungistatic activity of new groups of 2,4-dihydroxythiobenzoyl derivatives against phytopathogenic fungi

Twenty-six compounds, derivatives of amides, hydrazines, hydrazides, hydrazones, and semicarbazides, with a 2,4-dihydroxythiobenzoyl moiety, were synthesized from sulfinyl-bis(2,4-dihydroxythiobenzoyl). The compositions and chemical structures of these compounds were confirmed by IR, (1)H NMR, EI-MS, and elemental analysis. Antifungal properties of chemicals under in vitro conditions against five phytopathogenic fungi were estimated. In vivo studies against Erisiphe graminis were also carried out. The compounds N-substituted with an 2,4-dihydroxythiobenzamide group proved to be the most active. N-2-(1-Cinnamylbenzene ester)-2,4-dihydroxythiobenzamide, under in vitro conditions, showed activity at the level of 80-100% development of most pathogens at a concentration of 20 microg/mL and partially at a concentration of 200 microg/mL. For compounds with -HN-NH- or -NH-N= moiety, weak or no fungistatic properties were found at the concentrations studied.     

白僵菌抗多菌灵突变株的诱变及生物学特性研究

采用菌丝生长速率法测定了球孢白僵菌对多菌灵的敏感性和最低完全抑制浓度(MIC)。结果显示,在供试的球孢白僵菌菌株中,Bb06菌株对多菌灵最敏感,其EC₅₀值为1.0688μg/ml,多菌灵对球孢白僵菌菌丝生长的MIC为9μg/ml。通过菌丝块诱变法和分生孢子诱变法获得球孢白僵菌抗多菌灵突变株,并测定球孢白僵菌不同抗性突变株对多菌灵的抗性水平。结果表明,在抗性突变株中,BC-4菌株的EC₅₀值最大,为258.7711,对多菌灵的抗性水平最高,达到242.11;BC-3菌株的EC₅₀值最小,为18.6311,抗性水平最低。同时对球孢白僵菌抗多菌灵突变株的菌丝生长速率和产孢能力差异的研究表明,Bb06菌株(亲本菌株)的生长速率最大,为1.78mm/d,BC-8菌株的生长速率最小,为1.41mm/d。球孢白僵菌不同抗性突变株的产孢能力均比对照敏感菌株强,但不同菌株间存在一定差异,在抗性突变株中,BC-4菌株的分生孢子产生量最大。     

Nematicidal activity of (E,E)-2,4-decadienal and (E)-2-decenal from Ailanthus altissima against Meloidogyne javanica

Methanol extracts of various plant parts of Ailanthus altissima were tested against the root knot nematode Meloidogyne javanica . Extracts of bark (ABE), wood (AWE), roots (ARE), and leaves (ALE) from A. altissima were investigated against freshly hatched second-stage juveniles (J(2)). AWE was the most active extract, with EC(50/3d) of 58.9 mg/L, while ALE, ARE, and ABE did not show nematicidal activity. The chemical composition of the extracts of A. altissima was determined by gas chromatography-mass spectrometry, and (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-decenal, hexanal, nonanal, and furfural were the most prominent constituents. (E,E)-2,4-Decadienal, (E)-2-decenal, and furfural showed the highest nematicidal activity against M. javanica , with EC(50/1d) = 11.7, 20.43, and 21.79 mg/L, respectively, while the other compounds were inactive at the concentrations tested. The results obtained showed that AWE and its constituents (E,E)-2,4-decadienal and (E)-2-decenal could be considered as potent botanical nematicidal agents.     

Importance of 2,4-DAPG in the biological control of brown patch by Pseudomonas fluorescens HP72 and newly identified genes involved in 2,4-DAPG biosynthesis

The Pseudomonas fluorescens strain HP72 used as biocontrol agent was isolated from the roots of creeping bentgrass on brown patch-suppressive soil. This strain can suppress brown patch disease caused by Rhizoctonia solani. The analysis of secondary metabolites from strain HP72 revealed that it produced known antifungal compounds, 2,4-diacetylphloroglucinol (2,4-DA-PG), HCN, and a fluorescent siderophore. In the present study, the Tn5inserted mutants of strain HP72, which did not show any antifungal activity, were selected. None of the mutants produced 2,4-DA-PG but they produced a fluorescent siderophore, while some strains produced HCN. Therefore, it is suggested that 2,4-DA-PG plays a major role in the biological control of brown patch disease caused by R. solani. In the genomic region where Tn5 was inserted, two open reading frames (ORFs A and B), which are not included in the 2,4-DAPG gene cluster of HP72, were detected. It was demonstrated that ORFs A and flare involved in the regulation of 2,4-DAPG biosynthesis.     

Antibacterial activity of the plant-derived compounds 23-methyl-6-O-desmethylauricepyrone and (Z,Z)-5-(trideca-4,7-dienyl)resorcinol and their synergy with antibiotics against methicillin-susceptible and -resistant Staphylococcus aureus

The present study investigated the antibacterial activity of two plant-derived compounds, 23-methyl-6-O-desmethylauricepyrone (1) and (Z,Z)-5-(trideca-4,7-dienyl)resorcinol (2), and their synergistic effects with erythromycin and gentamicin against methicillin-susceptible (MSSA) and gentamicin- and methicillin-resistant Staphylococcus aureus (MRSA). Studies of the individual antibacterial activity of each plant-derived compound and synergy experiments were carried out, by the microdilution test in agar and by the checkerboard method, respectively. Compound 1 showed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 2 and 8 μg/mL, respectively, against both strains of S. aureus, while compound 2 exhibited anti-MSSA and anti-MRSA activity with MICs and MBCs of 4 and 8 and 2 and 8 μg/mL, respectively. Time-kill curves showed that, while compound 1 produced complete killing of both strains at 24 h from the beginning of the experiment, 2 produced the same effect in the first hour. Combinations of 1 with erythromycin or gentamicin showed a notable synergism against MSSA, which enabled the antibiotic concentration to decrease by up to 300 or 260 times, respectively. When the aminoglycoside was placed together with compound 2, only an additive effect was observed. The assayed compounds did not produce erythrocyte hemolysis or genotoxicity and they did not affect macrophage viability at the effective or higher concentrations. These results suggest that both compounds could be considered as promising antibacterial agents while compound 1 could be used in combinatory therapies with erythromycin and gentamicin.     

Fluorescent caged compounds of 2,4-dichlorophenoxyacetic acid (2,4-d): photorelease technology for controlled release of 2,4-D

A novel controlled-release formulation (CRF) of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was developed to reduce its negative environmental impacts by improving its herbicidal efficacy. The 2,4-D was chemically caged by coupling with photoremovable protecting groups (PRPGs) of coumarin derivatives. Photophysical studies of caged compounds showed that they all exhibited strong fluorescence properties. Controlled release of 2,4-D was achieved by irradiating the caged compounds using UV-vis light (310, 350, and 410 nm). The effect of various factors such as pH, solvent, and different substituents at the seventh position of coumarin moiety on the rate of photorelease was studied. The herbicidal activity of caged compounds and 4-(hydroxymethyl)-7-substituted coumarins was studied against Vigna radiata . The new formulation provided greater control over the release of 2,4-D by UV-vis light and also demonstrated the potential of the PRPGs not only to act as a delivery device but also to possess herbicidal activity after photorelease.     

Diversity of rhizobia isolated from an agricultural soil in Argentina based on carbon utilization and effects of herbicides on growth

Seventy-six rhizobial isolates belonging to four different genera were obtained from the root nodules of several legumes (Vicia sativa, Vicia faba, Medicago sativa, Melilotus sp., Glycine max and Lotus corniculatus). The action of five commonly used herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate (GF), dicamba, atrazine and metsulfuron-methyl] on the growth of rhizobial strains was assessed. Subsequently, GF and 2,4-D were tested in a minimum broth as C and energy sources for 20 tolerant strains. The ability of these strains to metabolize different carbon sources was studied in order to detect further differences among them. Tolerance of the bacteria to agrochemicals varied; 2,4-D and GF in solid medium inhibited and diminished growth, respectively, in slow-growing rhizobial strains. Among slow-growing strains we detected Bradyrhizobium sp. SJ140 that grew well in broth + GF as the sole C and energy source. No strain was found which could use 2,4-D as sole C source. The 20 strains studied exhibited different patterns of C sources utilization. Cluster analysis revealed three groups, corresponding to four genera of rhizobia: Rhizobium (group I), Sinorhizobium (group II) and Mesorhizobium–Bradyrhizobium (group III). On the basis of the results obtained on responses to herbicides and C sources utilization by the isolates investigated, it was possible to differentiate them at the level of strains. These results evidenced a considerable diversity in rhizobial populations that had not been previously described for Argentinean soils, and suggested a physiological potential to use natural and xenobiotic C sources.     

Synthesis and antifungal activity of a series of N-substituted [2-(2,4-dichlorophenyl)-3-(1,2,4-triazol-1-yl)]propylamines

A series of N-mono- or N, N-disubstituted [2-(2,4-dichlorophenyl-3-(1,2,4-triazol-1-yl)]propylamines and N-[2-(2,4-dichlorophenyl-3-(1,2,4-triazol-1-yl)propyl]amides were synthesized and tested for their fungicidal activity in vitro and in vivo against a group of plant pathogenic fungi. Some compounds exhibited a fairly good in vitro activity. The replacement of the ether group of tetraconazole with a secondary or tertiary amino group leads to compounds that maintain the antifungal activity on several phytopathogenic fungi, provided that the substituents are not too bulky or lipophilic. The allyl, propargyl, and cyclopropyl groups appear particularly suitable. Although these compounds have some structural similarities with terbinafine and naftifine, which act as squalene epoxidase inhibitors, they maintain the usual mechanism of action of the other triazoles.     

水稻无侧根突变体RM109的氧化还原代谢

比较无侧根突变体RM1 0 9和原品种大力根琥珀酸脱氢酶 (SDH)的活性发现 :RM1 0 9为大力的 60 %～ 70 % ,还原剂处理下大力的SDH活性显著增加 ,而RM1 0 9无变化。RM1 0 9的该酶受单显性基因控制。RM1 0 9对NAA、TIBA的耐性明显增加 ,对H2 O2 的耐性明显下降。结果表明 :氧化还原代谢有突变     

鸵鸟异嗜白细胞抗菌肽的分离纯化及部分性质研究

目的：分离纯化出鸵鸟异嗜白细胞抗菌肽,并对其部分特性进行研究,为开发新一代高效肽类抗菌药提供依据。方法：使用氯化铵裂解、超声波破碎、醋酸提取、CM-Sepharose F F弱酸性阳离子交换层析和反相高效液相色谱（RP-HPLC）等方法,从鸵鸟异嗜白细胞中分离纯化出了抗菌肽,使用微量琼脂糖弥散法进行了抗菌活性与最小抑菌浓度（MIC）的测定,应用二级质谱（MS/MS）对抗菌肽的分子量进行测定。结果：在鸵鸟异嗜白细胞中存在抗菌肽,其对金黄色葡萄球菌S.aureus 1056MRSA、鸡大肠杆菌E.coli O78和白色念珠菌C.albicans ATCC10231具有较强的抑制作用;抗菌肽具有阳离子性质;热稳定性良好;经RP-HPLC纯化得到峰6、峰16和峰24,峰6对白色念珠菌的MIC为0.065μg/mL,峰16的分子量为4012.472Da,对鸡大肠杆菌和金黄色葡萄球菌的MIC分别为3.971μg/mL和5.245μg/mL,峰24的分子量为3542.479Da,对鸡大肠杆菌和金黄色葡萄球菌的MIC分别为26.472μg/mL和21.561μg/mL。结论：本研究得到的抗菌肽与已报道的鸵鸟抗菌肽ostricacins相比显示出更强的抑菌活性,是否为同一物质需要进一步的验证。     

Factors impacting the activity of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens against take-all of wheat

Take-all, caused by Gaeumannomyces graminis var. tritici, is an important soilborne disease of wheat worldwide. Pseudomonas fluorescens producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are biocontrol agents of take-all and provide natural suppression of the disease during wheat monoculture known as take-all decline. To identify factors that could contribute to the effectiveness of 2,4-DAPG producers in take-all suppression, P. fluorescens strains Q8r1-96 (genotype D) and Q2-87V1 (genotype B; reduced antibiotic production) were tested against three pathogen isolates differing in sensitivity to 2,4-DAPG (LD5, ARS-A1 and R3-111a-1) and two wheat cultivars (Tara and Buchanan). The antibiotic sensitivity of the pathogen and cultivar significantly affected the level of take-all suppression by Q8r1-96 and Q2-87V1; suppression was greatest with LD5 and Tara. Q8r1-96 suppressed ARS-A1 and R3-111a-1 on Tara but not Buchanan, and Q2-87V1 failed to suppress either pathogen isolate on either cultivar. Q8r1-96 colonized the rhizosphere of Tara and Buchanan grown in soil similarly, but 2,4-DAPG accumulation was higher on the roots of Buchanan than Tara. 2,4-DAPG at 7.5 μg mL⁻¹ reduced the growth of roots of both cultivars, and 10 μg mL⁻¹ caused brown necrosis and tissue collapse of seedling roots and reduced root hair development. The half-life of 2,4-DAPG in the rhizosphere was estimated to be 0.25 days. These results suggest that several interconnected factors including sensitivity of G. graminis var. tritici to 2,4-DAPG, wheat cultivar, fluctuations in populations of 2,4-DAPG producers, and antibiotics accumulation in the rhizosphere will impact the robustness of take-all suppression by P. fluorescens in the field.     

Uniformly (14)C-ring-labeled 2,4-dichlorophenoxyacetic acid: a metabolism study in bluegill sunfish, Lepomis macrochirus.

The fate of 2,4-dichlorophenoxyacetic acid (2,4-D), a mixture of [phenyl(U)-(14)C]-2,4-D and unlabeled 2,4-D, in bluegill sunfish was investigated after exposure to approximately 11 ppm under static conditions for 4 days. Total radioactive residues (TRR) in whole fish increased from 0.41 ppm on day 1 to 0.60 ppm on day 3. TRR levels in fillet (edible) and viscera (nonedible) of treated fish on day 4 were 0.41 and 1.9 ppm, respectively. Most residues in both matrices were acetonitrile soluble; small amounts were hexane soluble or unextractable with solvents. Acid and base hydrolyses with ethyl acetate partitioning were used to release the fillet unextractable residues. The identification of 2,4-D and 2,4-dichlorophenol (2,4-DCP) in the fillet was conclusively confirmed by GC-MS analysis. On the basis of the experimental data from this study, a metabolic pathway for 2,4-D in bluegill sunfish in which the 2,4-D is metabolized to 2,4-DCP and conjugates of 2,4-D and 2,4-DCP is proposed.     

Formation of volatile compounds in model experiments with crude leek (Allium ampeloprasum Var. Lancelot) enzyme extract and linoleic acid or linolenic acid

Three continuous assays are described for lipoxygenase (LOX), hydroperoxide lyase (HPL) and alcohol dehydrogenase (ADH) in leek tissue. The catalytic activity of LOX showed significant difference (significance level 5%) between linolenic acid (9.43 x 10(-)(4) katals per kg protein) and linoleic acid (2.53 x 10(-)(4) katals per kg protein), and the pH-optimum of LOX was 4.5-5.5 against linoleic acid. The catalytic activity of HPL was statistically the same for 9-(S)-hydroperoxy-(10E,12Z)-octadecadienoic acid (1.01 x 10(-)(2) katals per kg protein) and 13-(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid (7.69 x 10(-)(3) katals per kg protein). ADH showed a catalytic activity of 5.01 x 10(-)(4) katals/kg of protein toward hexanal. Model experiments with crude enzyme extract from leek mixed with linoleic acid or linolenic acid demonstrated differences in the amount of produced aroma compounds. Linoleic acid resulted in significantly most hexanal, heptanal, (E)-2-heptenal, (E)-2-octenal, (E,E)-2,4-decadienal, pentanol, and hexanol, whereas linolenic acid resulted in significantly most (E)-2-pentenal, (E)-2-hexenal, (E,Z)-2,4-heptadienal, (E,E)-2,4-heptadienal, and butanol. Leek LOX produced only the 13-hydroperoxide of linoleic acid and linolenic acid.     

Uptake and metabolic fate of [14C]-2,4-dichlorophenol and [14C]-2,4-dichloroaniline in wheat (Triticum aestivum) and soybean (Glycine max)

The uptake and metabolism of [14C]-2,4-dichlorophenol (DCP) and [14C]-2,4-dichloroaniline (DCA) were investigated in wheat and soybean. Seeds were exposed to a nutrient solution containing 50 microM of one of two radiolabeled compounds, and plant organs were harvested separately after 18 days of growth. In wheat, uptake of [14C]-2,4-DCP was 16.67 +/- 2.65 and 15.50 +/- 2.60% of [14C]-2,4-DCA. In soybean, uptake of [14C]-2,4-DCP was significantly higher than [14C]-2,4-DCA uptake, 38.39 +/- 2.56 and 18.98 +/- 1.64%, respectively. In the case of [14C]-2,4-DCP, the radioactivity absorbed by both species was found mainly associated with roots, whereas [14C]-2,4-DCA and related metabolites were associated with aerial parts, especially in soybean. In wheat, nonextractable residues represented 7.8 and 8.7% of the applied radioactivity in the case of [14C]-2,4-DCP and [14C]-2,4-DCA, respectively. In soybean, nonextractable residues amounted to 11.8 and 5.8% of the total radioactivity for [14C]-2,4-DCP and [14C]-2,4-DCA, respectively. In wheat, nonextractable residues were nearly equivalent to extractable residues for [14C]-2,4-DCP, whereas they were greater for [14C]-2,4-DCA. In soybean, the amount of extractable residues was significantly greater for both chemicals. However, in both species, nonextractable residues were mainly associated with roots. Isolation of soluble residues was next undertaken using excised shoots (wheat) or excised fully expanded leaves including petioles (soybean). Identification of metabolite structures was made by comparison with authentic standards, by enzymatic hydrolyses, and by electrospray ionization-mass spectrometric analyses. Both plant species shared a common metabolism for [14C]-2,4-DCP and [14C]-2,4-DCA since the malonylated glucoside conjugates were found as the final major metabolites.     

Characterization of volatile compounds from the reaction of 3-hydroxy-2-butanone and ammonium sulfide model system

The reactions between 3-hydroxy-2-butanone and ammoniun sulfide at 25, 50, 75, 100, 125, and 150 degrees C were studied. Four well-known flavor compounds, 2,4,5-trimethyloxazole, 2,4, 5-trimethyl-3-oxazoline, 2,4,5-trimethylthiazole, and 2,4, 5-trimethyl-3-thiazoline, were identified. Another four interesting intermediate compounds, 2-(1-hydroxyethyl)-2,4, 5-trimethyl-3-oxazoline, 2-(1-mercaptoethyl)-2,4, 5-trimethyl-3-oxazoline, 2-(1-hydroxyethyl)-2,4, 5-trimethyl-3-thiazoline, and 2-(1-mercaptoethyl)-2,4, 5-trimethyl-3-thiazoline, were also identified by GC-EIMS and GC-CIMS. All these intermediate compounds were formed at 25 degrees C. On the other hand, tetramethylpyrazine was the major product with a reaction temperature higher than 100 degrees C.     

Effect of molecular complexity and acidity of earthworm faeces humic fractions on glutamate dehydrogenase,glutamine synthetase,and phosphoenolpyruvate carboxylase in Daucus carota α II cells

Carrot cells were grown in cultures supplemented with two hormones [2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6BAP)] and two humic fractions extracted from earthworm faeces, one with high acidity and a low apparent molecular size (<3500) and the other with low acidity and a large molecular size. 2,4-D stimulated growth through an effect on cell enlargement, while the strongly acidic humic fraction (0.2 mg l^-1) and the weakly acidic fraction (1 mg l^-1) were both less effective. With 4–16 h of pre-incubation, the highly acid humic fraction, mainly alone, induced the best increase in protein content; the effect of the weakly acid humic fraction and the hormones was generally less important. The two humic fractions also differed in their influence on glutamate dehydrogenase activity. After 2 h of pretreatment, the highly acidic fraction increased glutamate dehydrogenase activity, while the other fraction did not affect it. After 4–16 h of pre-incubation, the activity of this enzyme was still not influenced by these humic fractions. The presence of the two hormones did not interfere with the humic matter effects. Glutamine synthetase activity was not affected by a pre-incubation of up to 4 h with the two humic fractions, but it was stimulated after 8–16 h of pre-incubation. A 2,4-D+6BAP mixture stimulated glutamine synthetase activity (from +12 to +50%). Again, the presence of the hormones did not interfere with the effects induced by the humic fractions. After 16 h of pre-incubation, phosphoenolpyruvate carboxylase activity was increased by the highly acidic humic fraction (+93%) and by both humic fractions together (+34%). An explanation of the different incubation times necessary for the humic fractions to exert stimulatory effects on these enzymes is proposed here. The regulatory properties of the strongly acidic humic fraction appeared to depend on the combination of high acidity (expecially carboxylic C) with low molecular size.