Comparison of Techniques for the Production of Sclerotinia trifoliorum Ascospores in the Laboratory for Forage Legumes Resistance Tests

This study aimed to improve the production, under controlled conditions, of Sclerotinia trifoliorum ascospores to be used for selection tests on forage legumes. Sclerotia of this fungus, produced in the laboratory, were buried 1–2 cm deep in permanently soaked vermiculite, at T = 15°C, 12/24 h of fluorescent light, air moisture >80% in order to differentiate apothecia. The ascospores were harvested by aspiration and collected on a membrane filter. Various types of apparatus containing sclerotia have been tested for their ability to maintain live apothecia and for the facility of spore catching. The aspiration method has been found to be much more efficient than the previous method of cutting off apothecia. Moreover, it was observed that using this harvesting technique the best apparatus presented small and independent compartments (truncated plastic bottles). The ascospores can be stored, on the membrane filter at 5°C and in dry conditions in order to preserve their germinating abilityfor, a period of 3 months at least. This spore harvesting method permits avaibility of large quantities of a cheap inoculum for S. trifoliorum resistance test on forage legumes all year round.     

Oxidative stress responses in transgenic tobacco containing altered levels of glutathione reductase activity

A pea glutathione reductase cDNA was expressed in tobacco. Three classes of construct were used which gave a range of elevated levels of glutathione reductase (GR) activity in the cytosol (GR32), chloroplasts (GR36), or in both chloroplasts and mitochondria (GR46). In some transgenic progeny (T₂) from self-fertilized GR32 and GR36 primary transformants, having approximately twofold elevation of GR activity as compared with recessive siblings, there was an amelioration of the effect on leaf discs of up to 15 µM paraquat. However, lines with similarly elevated levels of GR activity showed no decreased sensitivity to the herbicide. None of the GR32 and GR36 lines was less sensitive to ozone. Conversely, T₂ progeny of GR46 lines, with greater than 4.5-fold elevations of GR activity, showed no reduced sensitivity to paraquat but two out of four of these lines were less sensitive to ozone fumigation. The differential response to stress co-segregated with the presence of the transgene but there was no relationship between the degree of stress response and the level of GR activity. There was an elevation in the total glutathione pool in all lines showing increased GR activity but there was no change in the ratio of oxidized to reduced glutathione. These results demonstrate that the mechanisms of protection against ozone and paraquat are different although both can be mediated by elevated GR activity.     

Oxidative Protective Mechanisms and Resistance to the Dicarboximide Fungicide, Iprodione, in Alternaria alternata

The free radical scavengers α-tocopherol and butylated hydroxytoluene, but not ascorbate, diminished the growth-inhibiting effects of the dicarboximide fungicide, iprodione in Alternaria alternata. Growth of A. alternata in the presence of iprodione increased the activities of superoxide dismutase and glutathione reductase while catalase was unaffected. Four iprodione sensitive and four iprodione resistant isolates of A. alternata were compared for activity of free radical enzymes. The isolates of A. alternata resistant to iprodione had more catalase activity than those which were sensitive, but did not differ in superoxide dismutase of glutathione reductase, activities. 3-Amino-1.24.-triazole, a specific inhibitor of catalas, reduced the ability of DAR 69775, a dicarboximide resistant isolate of A. alternata. to grow in the presence of iprodione. In A. alternata dicarboximide resistance appeats to be at least partially mediated by enhanced activitiesof, catalase.     

Repression of lipopolysaccharide biosynthesis in Escherichia coli by an antisense RNA of Acetobacter methanolicus phage Acm1

Lysogenic Acetobacter methanolicus strains carrying the prophage Acm1 were found to be unable to synthesize both the capsutar polysaccharide (CPS) and the O-specific side-chain of lipopolysaccharide (LPS) and to represent rough variants of the host bacterium. A 262 bp DNA fragment of phage Acm1, obviously required for interference with LPS biosynthesis, was cloned and expressed in Escherichia coli Independently of the O-type, transformation of various E. coli strains with the recombinant DNA resulted in a suppression of biosynthesis of the O-specific chains. The DNA fragment of phage Acm1 contained three very short open reading frames of 21, 24, and 36 bp. However, attempts to express phage-encoded peptides were not successful. Instead, the Acm1-derived DNA fragment was shown to code for the synthesis of a trans-acting RNA molecule of 97 nucleotides, designated lbi (L PS b iosynthesis-i nterfering) RNA. This RNA contains sequence complementarity to E. coli target RNA sequences and appears to have the ability to form intracellularly RNA hybrid duplexes with mRNA. The data presented in this study support the hypothesis that the phenotypic effect of conversion to rough-type LPS is accompanied by the expression of an antisense RNA of phage Acm1.     

Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: Comparison with other Butyrivibrio plasmids and application in the development of a cloning vector

Abstract A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79 bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM β1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens .