Phenyl methyl ethers: novel electron donors for respiratory growth of<Emphasis Type="Italic"> Desulfitobacterium hafniense</Emphasis> and<Emphasis Type="Italic"> Desulfitobacterium</Emphasis> sp. strain PCE-S

Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S grew under anoxic conditions with a variety of phenyl methyl ethers as electron donors in combination with fumarate as electron acceptor. The phenyl methyl ethers were O-demethylated to the corresponding phenol compounds. O-demethylation was strictly dependent on the presence of fumarate; no O-demethylation occurred with CO₂ as electron acceptor. One mol phenyl methyl ether R-O-CH₃ was O-demethylated to R-OH per 3 mol fumarate reduced to succinate. The growth yields with vanillate or syringate plus fumarate were approximately 15 g cells (dry weight) per mol methyl moiety converted. D. hafniense utilized vanillate or syringate as an electron donor for reductive dehalogenation of 3-Cl-4-hydroxyphenylacetate, whereas strain PCE-S was not able to dechlorinate tetrachloroethene with phenyl methyl ethers. Crude extracts of both organisms showed O-demethylase activity in the O-demethylase assay with vanillate or syringate as substrates when the organism was grown on syringate plus fumarate. Besides the homoacetogenic bacteria, only growing cells of Desulfitobacterium frappieri PCP-1 have thus far been reported to be capable of phenyl methyl ether O-demethylation. This present study is the first report of Desulfitobacteria utilizing phenyl methyl ethers as electron donors for fumarate reduction and for growth.Abbreviations PCE Tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - ClOHPA 3-Cl-4-Hydroxyphenylacetate - OHPA 4-Hydroxyphenylacetate - FH₄ Tetrahydrofolate     

Utilization of methoxylated benzoates and formation of intermediates by Desulfotomaculum thermobenzoicum in the presence or absence of sulfate

Desulfotomaculum thermobenzoicum strain TSB (DSM 6193) was found to utilize some methoxylated benzoates as carbon and energy source with or without sulfate. 3- or 4-Methoxybenzoate, vanillate (4-hydroxy-3-methoxybenzoate), syringate (3,5-dimethoxy-4-hydroxybenzoate) and 3,4,5-trimethoxybenzoate were converted to corresponding hydroxybenzoates. However, neither 2-methoxybenzoate nor 2,6-dimethoxybenzoate was utilized. The organism grew acetogenically on each of the methoxylated benzoates in the absence of sulfate.3,4-Dihydroxy-5-methoxybenzoate was detected during conversion of syringate, and syringate and 3,4-dihydroxy-5-methoxybenzoate were detected during conversion of 3,4,5-trimethoxybenzoate as intermediates.These findings indicate that 4-methoxyl-group is most readily cleaved, whereas 2-methoxyl-group is not utilized by the organism.     

Vanillic acid metabolism by selected soft-rot,brown-rot,and white-rot fungi

Metabolism of vanillic acid, a product of lignin degradation, has been studied in selected representatives of soft-rot, brown-rot and white-rot fungi. All of the brown-and white-rot species examined decarboxylated vanillate to methoxyhydroquinone oxidatively. Mycelium extracts of all these fungi, except Pleurotus ostreatus contained high levels of an NAD(P)H-dependent vanillate hydroxylase. P. ostreatus also released ¹⁴CO₂ from ¹⁴COOH-vanillate but by a different mechanism possibly involving phenoloxidases. Most of these fungi also contained a dioxygenase which catalysed the intra-diol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to form maleylacetate. No 3-O-demethylase activity was detected, and data indicate that in some of the fungi examined cleavage of the aromatic ring occurs without prior removal of the methoxyl group. None of the soft-rot fungi tested contained vanillate hydroxylase or hydroxyquinol 1,2-dioxygenase, but very low levels of protocatechuate 3,4-dioxygenase were detected in mycelium extracts. Vanillate catabolism among members of this group occurs via a different route which may involve ring demethylation although no 3-O-demethylase activity was detected in this study. The enzyme NAD(P)H-quinone oxidoreductase was demonstrated to exist in all the studied groups of fungi.     

Regulation of the utilization of 4-hydroxybenzoate and vanillate in batch and continuous cultures of Pseudomonas acidovorans

In Pseudomonas acidovorans, the pathways of 4-hydroxybenzoate and vanillate metabolism converge on the early intermediate, protocatechuate, which undergoes meta-cleavage. The methoxyl group of vanillate is almost completely oxidized, as shown by an experiment with (¹⁴C-methoxyl) vanillate. In batch cultures, 4-hydroxybenzoate and vanillate are simultaneously oxidized. Simultaneous oxidation was explained above all by the fact that both substrates mutually repress the ability of the cells to utilize the partner substrate.If P. acidovorans is growing in a turbidostat on one of the two substrates and is suddenly exposed to an equimolar mixture of both substrates, the respiration rates for the two substrates reciprocate, the for the substrate utilized first passing through a transient minimum, that for the added substrate passing through a transient maximum. Finally, a balance appears to be established, the for 4-hydroxybenzoate being slightly above that for vanillate. Transient phenomena also occur if a chemostat culture with both substrates is suddenly operated as a turbidostat culture or if cells not adapted to either substrate are suddenly exposed to a mixture of both substrates in the turbidostat.If a chemostat culture of P. acidovorans, growing at the expense of an equimolar mixture of 4-hydroxybenzoate and vanillate, is operated under conditions of increasing oxygen deficiency, the utilization ratio of the two substrates increases in favour of 4-hydroxybenzoate. However, if the culture is operated under conditions of increasing nitrogen deficiency, the utilization ratio increases in favour of vanillate.Abbreviations 4HB 4-hydroxybenzoate - VA vanillate - OD optical density     

Vanillate Metabolism in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.     

Degradation 1,2-dimethylbenzene by Corynebacterium strain C125

In an attempt to obtain bacteria growing on 1,2-dimethylbenzene as sole carbon and energy source two different strains were isolated. One was identified as an Arthrobacter strain, the other as a Corynebacterium strain. Corynebacterium strain C125 was further investigated. The organism was not capable to grow on 1,3- and 1,4-dimethylbenzene. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 3,4-dimethylcatechol-2,3-dioxygenase activity was found in cell extracts. When 3,4-dimethylcatechol was added to cell extract of 1,2-dimethylbenzene-grown cells, first a compound with the spectral properties of 2-hydroxy-5-methyl-6-oxo-2,4-heptadienoate was formed and subsequently acetate was produced. It is proposed that dioxygenases are involved in the initial steps of 1,2-dimethylbenzene degradation, and ring opening proceeds via meta-cleavage.     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Regeneration of <Emphasis Type="Italic">Aeschynanthus radicans</Emphasis> via direct somatic embryogenesis and analysis of regenerants with flow cytometry

Plant regeneration through direct somatic embryogenesis in Aeschynanthus radicans ‘Mona Lisa’ was achieved in this study. Globular somatic embryos were formed directly from cut edges of leaf explants and cut ends or on the surface of stem explants 4 wk after culture on Murashige and Skoog (MS) medium supplemented with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA), TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D), or 6-benzylaminopurine (BA) or kintin (KN) with 2,4-D. MS medium containing 9.08 μM TDZ and 2.68 μM 2,4-D resulted in 71% of stem explants producing somatic embryos. In contrast, 40% of leaf explants produced somatic embryos when induced in medium containing 6.81 μM TDZ and 2.68 μM 2,4-D. Somatic embryos matured, and some germinated into small plants on the initial induction medium. Up to 64% of stem explants cultured on medium supplemented with 9.08 μM TDZ + 2.68 μM 2,4-D, 36% of leaf explants cultured on medium containing 6.81 μM TDZ and 2.68 μM 2,4-D had somatic embryo germination before or after transferring onto MS medium containing 8.88 μM BA and 1.07 μM NAA. Shoots elongated better and roots developed well on MS medium without growth regulators. Approximately 30–50 plantlets were regenerated from each stem or leaf explant. The regenerated plants grew vigorously after transplanting to a soil-less substrate in a shaded greenhouse with more than a 98% survival rate. Three months after their establishment in the shaded greenhouse, 500 plants regenerated from stem explants were morphologically evaluated, from which five types of variants that had large, orbicular, elliptic, small, and lanceolate leaves were identified. Flow cytometry analysis of the variants along with the parent showed that they all had one identical peak, indicating that the variant lines, like the parent, were diploid. The mean nuclear DNA contents of the variant lines and their parent ranged from 4.90 to 4.99 pg 2C⁻¹, which were not significantly different statistically. The results suggest that the regenerated plants have a stable ploidy level, and the regeneration method established in this study can be used for rapid propagation of ploidy-stable Aeschynanthus radicans.     

Utilization of benzylpenicillin as carbon,nitrogen and energy source by a Pseudomonas fluorescens strain

A bacterium which utilizes benzylpenicillin as carbon, nitrogen and energy source was isolated from a lake sediment. The organism was identified as a strain of Pseudomonas fluorescens with a GC content of 59.71 Mol %. After growth of the organism on a mineral salts medium containing benzylpenicillin, the derivatives benzylpenicilloic acid, benzylpenilloic acid and benzylpenicillenic acid were found in culture media. There was no indication that the phenylacetate side chain of benzylpenicillin is decomposed. In uninoculated culture media benzylpenicillin, benzylpenicilloic acid and benzylpenicillenic acid were demonstrable. The following compounds were found to be absent from inoculated or uninoculated culture fluids: d-penicillamine, l-valine, l-cysteine, benzylpenillic acid and 6-aminopenicillanic acid. The organism possesses penicillinase. Penicillin acylase was not demonstrable. The reaction product of penicillinase, benzylpenicilloic acid, supports only little growth. There is no growth on 6-aminopenicillanic acid with or without NH₄Cl. Relatively little growth occurs on 6-aminopenicillanic acid in the presence of phenylacetic acid.The data indicate that the nucleus of the benzylpenicillin molecule is utilized as carbon, nitrogen and energy source. During growth a part of the substrate is destroyed into scarcely usable benzylpenicilloic acid; hereby the antibiotic is detoxified.Abbreviations TLC thin-layer chromatography - DNPH 2,4-dinitrophenylhydrazine     

Isolation,identification and removal of filamentous organism from SND based SBR degrading nitrophenols

Four identical lab scale sequencing batch reactors R, R1, R2, and R3, were used to assess nitrophenol biodegradation using a single sludge biomass containing Thiosphaera pantotropha. Nitrophenols [4-Nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP) and 2,4,6-trinitrophenol (2,4,6-TNP)] were biotransformed by heterotrophic nitrification and aerobic denitrification (SND). Reactor R was used as background control, whereas R1, R2, and R3 were fed with 4-NP, 2,4-DNP, and 2,4,6-TNP, respectively. The concentration of each nitrophenol was gradually increased from 2.5 to 200 mg/l along with increase in COD, during acclimation studies. The final COD maintained was 4,500 mg/l with each nitrophenolic loading of 200 mg/l. During late phase of acclimation and HRT study, a filamentous organism started appearing in 2,4-DNP and 2,4,6-TNP bioreactors. Filaments were never found in 4-NP and background control reactor. Biochemistry and physiology behind filamentous organism development, was studied to obtain permanent solution for its removal. The effect of different input parameters such as COD loading, DO levels, SVI etc. were analyzed. The morphology and development of filamentous organism were examined extensively using microscopic techniques involving ESEM, oil immersion, phase contrast, and dark field microscopy. The organism was grown and isolated on selective agar plates and was identified as member of Streptomyses species.     

南美天胡荽及其根际土壤水浸提液化感成分分析

为探讨南美天胡荽对其他植物种子萌发的影响以及筛选影响其他植物的主要化合物,该文采用种子萌发试验、气相色谱-质谱联用以及液相色谱-质谱联用的方法,分析了南美天胡荽不同溶剂浸提液对种子萌发的影响、南美天胡荽植株及其根际土壤浸提液成分。结果表明:(1)南美天胡荽不同溶剂浸提物均具有一定程度的抑制种子萌发作用。(2)气相色谱-质谱分析下,南美天胡荽植株水浸提液中共分离鉴定了35种化合物,其中,邻苯二甲酸二丁酯(15.2%)、10,15-十八烷二元酸(8.58%)、2,4-二叔丁基苯酚(6.81%)相对含量最高; 根际土壤水浸提液中共分离鉴定了17种化合物,其中,油酸酰胺(26.47%)、正二十七烷(9.63%)、十六酸乙酯(4.83%)相对含量最高。(3)液相色谱-质谱分析下,南美天胡荽植株水浸提液共分离鉴定了109种化合物,ESI⁺模式下,L-苯丙氨酸(3 483.99 ng·mg^-1)、木犀草素(2 306.64 ng·mg^-1)含量最多,ESI^-模式下,右旋奎宁酸(21 827.71 ng·mg^-1)、绿原酸(12 589.25 ng·mg^-1)含量最多; 根际土壤水浸提液中共分离鉴定了93种化合物,ESI⁺模式下,丁酸(7 660.53 ng·mg^-1)、棕榈酰胺(3 200.36 ng·mg^-1)含量最多,ESI^-模式下,正二十八酸(18 605.35 ng·mg^-1)、蔗糖(12 183.23 ng·mg^-1)含量最多。(4)南美天胡荽的潜在化感物质主要为脂肪酸类、酰胺类、酯类、芳香酸类化合物,而土壤中直接起化感作用的物质可能为丁酸、正二十八酸、羟基乙酸、油酸酰胺、棕榈酰胺、十六酸乙酯、苯甲酸,其中脂肪酸类化合物输入可能来源于南美天胡荽、土壤微生物和土壤动物,酰胺类、酯类、芳香类化合物则更可能来源于南美天胡荽植株。     

Acetogenesis coupled to the oxidation of aromatic aldehyde groups

Vanillin cultures of Clostridium formicoaceticum produced higher cell densities than did vanillate cultures. During growth at the expense of vanillin, vanillate was the predominat intermediate formed; 3,4-dihydroxybenzaldehyde was not a significantly detectable intermediate. Acetate and protocatechuate were both produced in equimolar ratio relative to vanillin consumption. 4-Hydroxybenzaldehyde was a growth-supportive aromatic compound for both C. formicoaceticum and Clostridium aceticum (doubling times approximated 5 h), was oxidized stoichiometrically to 4-hydroxybenzoate, and was not appreciably toxic at concentrations up to 15 mM. Acetate was (i) the major reduced end product detected concomitant to growth and to benzaldehyde oxidation and (ii) formed in close approximation to the following stoichiometry: 4 4-hydroxybenzaldehyde + 2CO₂+2H₂O4 4-hydroxybenzoate + CH₃COOH. We conclude that these two acetogens are capable of benzaldehyde-coupled acetogenesis and growth.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

Degradation of veratric acid and other lignin-related aromatic compounds by the white-rot fungus Pycnoporus cinnabarinus

Metabolism of veratric acid and other aromatic compounds has been studied in two strains of Pycnoporus cinnabarinus. In non-agitated cultures which contained cellulose as an additional carbon source, veratric acid was demeth(ox)ylated to vanillic acid which accumulated in the medium. Under these conditions, ¹⁴CO₂ evolution from [4-O¹⁴CH₃]-veratric acid preceded that from [3-O¹⁴CH₃]-veratric acid in the case of both strains. ¹⁴CO₂ evolution was markedly accelerated and increased when 100% oxygen was employed instead of air. Oxygen had not so strong effect on the decarboxylation of ¹⁴COOH-labelled vanillic and p-hydroxybenzoic acid but it did increase decarboxylation of ¹⁴COOH-labelled veratric acid, indicating the effect of oxygen on the preceding demeth(ox)ylation. There were indications, for example rapid demethylation of veratric acid in early stages of growth when apparent phenol oxidase (laccase) activity was zero, for an existence of a separate demethylase enzyme. However, the participation of phenol oxidases in demeth(ox)ylation cannot be ruled out. Degradation pattern of vanillic acid was basically similar in P. cinnabarinus compared to Sporotrichum pulverulentum (Phanerochaete chrysosporium). Also the effect of carbon source was similar: cellulose as a carbon source enhanced degradation of vanillic acid through methoxyhydroquinone whereas in glucose medium, vanillic acid was reduced to the respective aldehyde and alcohol.Non-standard abbreviations CBQ cellobiose: quinone oxidoreductase - MHQ methoxyhydroquinone     

Metabolic production of a novel polymer feedstock, 3-carboxy muconate,from vanillin

Vanillin can be produced on a commercial scale by depolymerising renewable lignin. One product of microbial metabolism of vanillin by common soil microbes, such as Acinetobacter baylyi, is a tricarboxylic acid with a butadiene backbone known as 3-carboxy muconate (3CM). Three enzymes, 4-hydroxy benzaldehyde dehydrogenase, vanillate monooxygenase and protocatechuate 3,4-dioxygenase, catalyse the biotransformation of vanillin to 3CM. These three enzymes were metabolically engineered into an Escherichia coli host, giving a biocatalyst that converted vanillin into 3CM. The biocatalyst was found to give 100% yield of 3CM from 1 mM of vanillin after 39 h. The rate-limiting reaction was identified as the conversion of vanillate to 3,4-dihydroxybenzoate catalysed by vanillate monooxygenase. Low expression of the reductase subunit of this enzyme was identified as contributing to the reduced rate of this reaction. Proof of principle of a novel application for 3CM was demonstrated when it was converted into a trimethyl ester derivative and copolymerised with styrene.     

Somatic embryogenesis and plant regeneration from callus cultures of several species in the genus <Emphasis Type="Italic">Tricyrtis</Emphasis>

Summary The liliaceous perennial plants, Tricyrtis spp., are cultivated as ornamental plants in Japan. Natural populations of several Japanese Tricyrtis spp. are severely threatened by indiscriminate collection and habitat destruction. In this study, a plant regeneration system based on somatic embryogenesis has been developed for efficient clonal propagation of T. hirta, T. hirta var. albescens, T. formosana, T. formosana cv. Fujimusume, T. flava ssp. ohsumiensis, and T. macrantha ssp. macranthopsis. Flower tepal explants of these genotypes were cultured on media containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) alone or in combination with N-(1,2,3-thiadiazol-5-yl)-N′-phenylurea (thidiazuron, TDZ). Calluses induced on media containing 2,4-D produced somatic embryos following their transfer to a plant growth regulator-free medium, indicating that these calluses were embryogenic. A combination of 4.5μM2,4-D and 0.45 μM TDZ was most effective for inducing embryogenic calluses from tepal explants. Among various explant sources, filaments were most suitable for inducing embryogenic calluses on a medium containing 4.5μM 2,4-D and 0.45 μM TDZ. Embryogenic calluses were only obtained from filament explants for T. macrantha ssp. macranthopsis. Embryogenic calluses could be maintained by subculturing monthly onto the same medium, and a 1.5–3.5-fold increase in fresh weight was obtained after 1 mo. of subculture. Depending on the plant genotype, 50–500 somatic embryos per 0.5g fresh weight of embryogenic callus was obtained 1 mo. after transfer to a plant growth regulator-free medium. Most of the embryos developed into plantlets, and they were successfully acclimatized to greenhouse conditions. Regenerated plants showed no alteration in the ploidy level as indicated by chromosome observation and flow cytometric analysis.     

Microbial degradation of piperonylic acid

Several organisms were isolated for their ability to utilize piperonylate as a sole carbon source for growth and aPseudomonas species (Ps. PP-2) was selected for a study of the degradation of this substrate. Only vanillate, isovanillate,p-hydroxybenzoate and protocatechuate, of several possible catabolities, served as growth and oxidation substrates for the organism. Detailed analysis of the culture fluid from piperonylate-grown cells revealed the presence of vanillate and protocatechuate but isovanillate,p-hydroxybenzoate andm-hydroxybenzoate were not detected. The evidence presented suggests that piperonylate is metabolized first to vanillate by methylenedioxy ring cleavage and next to protocatechuate by direct demethylation of vanillate.     

Methoxyhydroquinone, a Growth Inhibitor of Ophiobolus graminis, in Leaves of Oat Seedlings

A methanol extract of leaves of oat seedlings grown in sand cultures in the dark contained a compound which inhibited the growth of Ophiobolus graminis. The inhibitory factor was isolated and proved to be present in the plant as methoxyhydroquinone glucoside. The glucoside was readily hydrolysed to the corresponding aglucone. The methoxyhydroquinone, or possibly its oxydation product, methoxy-P-benzoquinone, was inhibitory to both Ophiobolus graminis var. graminis and Ophiobolus graminis var. avenae, whereas Fusarmm oxysporum var. lycopcrsici was not affected. Synthetic methoxyhydroquinone at 80 mg/l gave a 100% inhibition of Ophiobolus graminis var. graminis. After being exposed to 80 mg/l of the inhibitor for 24 h the mycelium was unable to initiate growth when transferred to a fresh nutrient solution. Only extracts from young leaves showed inhibitory activity, extracts from mature leaves giving no inhibition. The hydroquinone, or its glucoside, was not detected in roots of young seedlings, where avenacin was the only antifungal compound present.     

Clonal propagation of mesquite tree (<Emphasis Type="Italic">Prosopis laevigata</Emphasis> Humb. &; Bonpl. ex Willd. M.C. Johnston). I. Via cotyledonary nodes

Plantlet regeneration in Prosopis laevigata (Humb. & Bonpl. ex Willd.) Johnston (Fabaceae), a multipurpose tree, has been achieved from cotyledonary nodes excised from in vitro grown seedlings. The explants were cultured on MS media containing different concentrations of N-6 benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-d) and a mixture of organic components. The highest number (3.37 + 0.51) of multiple shoots was observed in MS media containing 2,4-d (9.05 μM) + BA (6.62 μM). The regenerated shoots were then transferred onto half-strength MS medium containing a plant growth regulator that was either: indole-3-butyric acid, 1-naphthaleneacetic, indole-3-acetic acid, or 2,4-d as well as phytagel or vermiculite for adventitious root initiation. Best rooting efficiency of 44.0% was obtained when NAA (16.11 μM) and vermiculite were used. After rooting, the cloned plantlets were successfully hardened to ex vitro conditions. This work may help to reduce the devastation caused by the overexploitation of this species.     

Somatic embryogenesis and plant regeneration from seeds of wild<Emphasis Type="Italic"> Dicentra spectabilis</Emphasis> (L.) LEM

Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro.Abbreviations BAP 6-Benzylaminopurine - 2.4-D 2,4-Dichlorophenoxyacetic acid - KIN Kinetin - SEM Scanning electron microscopyCommunicated by H. Lörz