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Mutations affecting the development of root symbiosis between legume plants ( Fabaceae) and nodule bacteria (rhizobia) are often associated with pleiotropy. This might either primarily be caused by the mutation or develops as a physiological consequence of a changed nodule structure, number and activity. Three pleiotropic traits were revealed in the pea ( Pisum sativum L.) mutant RisfixC which is of supernodulation/nitrate-tolerant symbiosis (Nts) type. They comprise shortened internodes, reduced shoot dry mass accumulation and increased nitrogen content in the root tissues when compared to the wild type. The changes were expressed in the same degree in asymbiotic and nodulated plants when the effect of symbiotic nitrogen on plant growth was abolished with a saturating nitrate level. Consequently, the pleiotropic traits are inherently associated with the mutation. In RisfixC, the pleiotropy coincided with the presumed absence of the systemic feedback factor regulating nodule number. However, no differences were detected in the comparison of nonnodulating mutant Risnod27 ( sym8) with the wild type and of inoculated with noninoculated wild-type plants although these pairs also differ in the presence of the systemic factor. Therefore, the pathway leading from the RisfixC mutant product to pleiotropic changes appears to be independent of systemic nodule number regulation. Implications for the genetic improvement of growth and yield parameters of supernodulating breeding lines are discussed. [ABSTRACT FROM AUTHOR]

Abstract: The macro- and micro-morphological features, mycelial extension rate, enzymatic activities and possible genetic changes were studied in 30 selected strains of basidiomycetes after 10-year cryopreservation on perlite in liquid nitrogen (LN). Comparisons with the same strains preserved by serial transfers on nutrient media at 4°C were also conducted. Production of ligninolytic enzymes and hydrogen peroxide was studied by quantitative spectrophotometric methods, whereas semiquantitative API ZYM testing was used to compare the levels of a wide range of hydrolytic enzymes. Our results show that cryopreservation in LN did not cause morphological changes in any isolate. The vitality of all fungi was successfully preserved and none of the physiological features were lost, even though the extension rate and enzyme activity were slightly affected. Moreover, sequence analysis of eight strains did not detect any changes in their genetic features after cryopreservation. These findings suggest that the perlite-based freezing protocol is suitable for long-term preservation of large numbers of basidiomycetes. [Copyright &y& Elsevier]

Abstract: Mycelial basidiomycete cultures on perlite in cryovials survived successfully three successive cycles of freezing, storage in liquid nitrogen (LN) and thawing without noticeable changes. This indicates that using perlite as a carrier for cryopreservation could in most cases overcome difficulties caused by interrupted supply of LN or electric power during the storage. Cultures on perlite can also be reused for successive inoculations. [Copyright &y& Elsevier]

Abstract: A new cryopreservation method using perlite as a carrier was evaluated on a large set of mycelial cultures of basidiomycetes. The viability and some other characteristics—growth, macro- and micromorphology, and laccase production—of 442 strains were tested after 48-h and then after 3-year storage in liquid nitrogen using a perlite protocol (PP). All (100%) of them survived successfully both 48-h storage and 3-year storage in liquid nitrogen without noticeable growth and morphological changes. Also laccase production was unchanged. The viability and laccase production of a part (250) of these strains were compared with those of the strains subjected to an original agar plug protocol (OP). Using OP, 144 strains (57.6%) out of 250 survived a 3-year storage in liquid nitrogen. The results indicate that the cryopreservation protocol used significantly influences survival of the strains. Markedly better results were achieved using the PP. [Copyright &y& Elsevier]

The root exudates of 24 pea (Pisum sativum L.) non-nodulating mutants, originating from cv. ′Finale′, were tested for Rhizobium nodulation ( nod) gene-inducing activity to reveal potential differences from the wild type. The interaction with flavonoid receptor NodD of R. leguminosarum bv. viciae strain 248 was monitored as the activity of the regulated operon nodABC fused to the reporter gene lacZ. In the uninoculated plants, the only decreased exudate activity was found in the line Risnod17 and ascribed to the impaired root growth in this line. Consequently, constitutive deficiency in the flavonoid nod gene inducers is not a cause of the asymbiotic phenotypes in the studied set. On the other hand, line Risnod5 had constitutively higher nod gene-inducing activity which might be responsible for its conditional nodulation and Fix– phenotype when the nodules are present. Monitoring of the response of wild-type plants to inoculation revealed an increase in nod gene-inducing activity (Ini response) starting first day after inoculation. The chosen mutant lines Risnod22 (putative locus sym9) and Risnod25 (putatively sym8) showed Ini as well indicating that the flavonoid burst can be mutationally dissociated from nodule initiation. On the other hand, absence of Ini in Risnod20 (assumed sym19) suggests branching of the signal transduction pathway downstream of this mutation and places it upstream of the two former mutations in the transduction of the bacterial Nod factor signal. More intense Ini response observed in the supernodulating mutant RisfixC, included in the study for its contrasting symbiotic phenotype, suggests common regulation of nodulation and Ini response via systemic shoot factor. The time shift in the exudation of isoflavonoid phytoalexin pisatin compared to Ini, its increased level in Risnod25 and lowered level in RisfixC are consistent with the regulatory role of plant defence in nodule initiation, which might be mediated directly by isoflavonoids as negative regulators. [ABSTRACT FROM AUTHOR]

The effects of exogenous sucrose, lactose. ᴅ-glucose, ᴅ(-)fructose, ᴅ-galactose, ᴅ-mannose, ւ-sorbose, ւ-arabinose and ᴅ-xylose on nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) levels, on anaerobic nitrite production and on respiratory O2 consumption were studied in excised roots of pea (Pisum sativum L. cv. Raman). Sucrose, glucose and fructose increase NR and GS levels and decrease GDH level (when compared with roots cultures without any sugar) at all concentrations used, but the extent of this effect varies. NR induction is enhanced by all sugars within the concentration range studied. Precultivation of roots with mannose and galactose results in an increase in anaerobic nitrite production in a medium consisting of phosphate buffer and KNO3. GS reaches its maximum at lower sugar concentrations, this fact being especially clear-cut with galactose. The decrease in GS level observed in roots cultured without sucrose is enhanced by higher sorbose concentrations. The increase in GDH level occurring in roots cultured without sucrose is depressed by low galactose and mannose concentrations but enhanced by high galactose, mannose, xylose and a wide range of sorbose concentrations. Lactose exerts only slight influence on the enzymes. The effects of sugars are in no case consistent with their effect on respiratory O2 consumption which is most pronounced with NR. The above results show that the effects of sugars on NR, GS and GDH are not mediated by one universal mechanism. [ABSTRACT FROM AUTHOR]

Abstract: Thirty different white rot strains were screened for Orange G and Remazol Brilliant Blue R (RBBR) decolorization on agar plates. Three promising strains, Dichomitus squalens, Ischnoderma resinosum and Pleurotus calyptratus, selected on the basis of this screening, were used for decolorization study in liquid media. All three strains efficiently decolorized both Orange G and RBBR, but they differed in decolorization capacity depending on cultivation conditions and ligninolytic enzyme production. Two different decolorization patterns were found in these strains: Orange G decolorization in I. resinosum and P. calyptratus was caused mainly by laccase, while RBBR decolorization was effected by manganese peroxidase (MnP); in D. squalens laccase and MnP cooperated in the decolorization processes. [Copyright &y& Elsevier]

The strain Pleurotus ostreatus Florida f6, its 45 basidiospore-derived isolates (both monokaryons and dikaryons prepared in our laboratory), Trametes versicolor strain CCBAS 614 and 22 other T. versicolor isolates obtained from the sporocarps collected in distant localities were successfully preserved for 12 y using perlite and straw cryopreservation protocols. All tested isolates survived a 12-year storage in liquid nitrogen (LN) and their laccase production and Poly B411 decolorization capacity was preserved. Also mycelium extension rate and the types of colony appearance of individual isolates remained unchanged. Different cryopreservation techniques were also tested for the short time (24 h) and the long time (6 m) storage of the culture liquid with extracellular laccase produced by T. versicolor strain CCBAS 614. The results showed that 10 % glycerol was the most suitable cryopreservant. The absence of the cryopreservant did not cause high loss of laccase activity in the samples; the presence of DMSO (5 or 10 %) in LN-stored samples caused mostly a decrease of laccase activity. For the preservation of laccase activity in the liquid culture the storage in the freezer at −80 °C is more convenient than the storage in liquid nitrogen. [ABSTRACT FROM AUTHOR]

Abstract: Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1mM TBBPA within 4days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2mM TBBPA within 5h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5mM within 3h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2′,6,6′-tetrabromo-4,4′-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity. [Copyright &y& Elsevier]

Production of extracellular enzymes participating in the degradation of biopolymers was studied in 29 strains of nonbasidiomycetous microfungi isolated from Quercus petraea forest soil based on the frequency of occurrence. Most of the isolates were ascomycetes and belonged to the genera Acremonium, Alternaria, Cladosporium, Geomyces, Hypocrea, Myrothecium, Ochrocladosporium, and Penicillium (18 isolates), and two isolates were zygomycetes. Only six isolates showed phenol oxidation activity which was low and none of the strains were able to degrade humic acids. Approximately half of the strains were able to degrade cellulose and all but six degraded chitin. Most strains produced significant amounts of the cellulolytic enzymes cellobiohydrolase and β-glucosidase and the chitinolytic enzymes chitinase, chitobiosidase, and N-acetylglucosaminidase. The highest cellulase activities were found in Penicillium strains, and the highest activity of chitinolytic enzymes was found in Acremonium sp. The production of the hemicellulose-degrading enzymes α-galactosidase, β-galactosidase, and α-mannosidase was mostly low. The microfungal strains were able to produce significant growth on a range of 41-87, out of 95 simple C-containing substrates tested in a Biolog™ assay, monosaccharides being for all strains the most rapidly metabolized C-sources. Comparison with saprotrophic basidiomycetes from the same environment showed that microfungi have similar cellulolytic capabilities and higher chitinase activities which testifies for their active role in the decomposition of both lignocellulose and dead fungal biomass, important pools of soil carbon. [ABSTRACT FROM AUTHOR]

Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv. viciae were tested for their effect on symbiosis formation with garden pea as the host. A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation. The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30. A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 μg ml–1 nutrient solution. At 10 μg ml–1, the highest concentration tested, naringenin was already inhibitory. By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 μg ml–1, as well as by another tested nod gene inducer, hesperetin. The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth. Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules. The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms. Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin‐conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity. By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity. Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 μg ml–1, however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation‐inhibiting high flavonoid concentrations. The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations. This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes. The losses were 99, 96.4, and 90% of the initial concentration of 10 μg ml–1 for naringenin, hesperetin, and quercetin, respectively. [ABSTRACT FROM PUBLISHER]