Mark S. Mount

Influence oF Pseudomonas putida on nodulation of Phaseolus vulgaris

Abstract The effect of Pseudomonas putida (isolate M17) on Rhizobium phaseoli nodulation of the common bean, Phaseolus vulgaris , was investigated under field and greenhouse conditions. The results indicated that P. putida markedly increased nodulation compared to R. phaseoli controls. Furthermore, 2-ketogluconic acid, a phosphate-solubiliring compound, was detected in P. putida M17. This could imply an increased P supply to roots of P. vutgaris , which may function to increase nodules. Bean yields and shoot fresh weight were not significantly altered by the addition of P. putida M17.

Complementation of an Erwinia carotovora subsp. carotovora protease mutant with a protease-encoding cosmid

SummaryWe report the complementation of a genetic lesion in the genome of Erwinia carotovora subsp. carotovora (Ecc), a pathogenic bacterium that incites soft rot of plants. A Sau3AI genomic library of Ecc was constructed using the conjugal cosmid pLAFR-3 as a vector. Sixteen cosmid clones encoding various plant tissue-degrading enzymes were identified, including a proteolytic clone, five cellulolytic clones, and ten pectolytic clones. We detected a mutant of Ecc with no proteolytic activity following transposon mutagenesis with an unstable Tn5-carrying plasmid. Conjugal transfer of the protease-encoding cosmid to this mutant restored near-wildtype extracellular protease production. Further manipulation and study of genes encoding pathogenic determinants in Ecc will be possible using this system.

Erwinia Carotovora: Molecular Cloning of a 3.4 Kilobase DNA Fragment Mediating Production of Pectate Lyases

(1986). Erwinia Carotovora: Molecular Cloning of a 3.4 Kilobase DNA Fragment Mediating Production of Pectate Lyases. Canadian Journal of Plant Pathology: Vol. 8, No. 1, pp. 17-27.

Biocontrol of the chestnut blight fungus Cryphonectria parasitica.

Summary Chestnut blight disease has devastated the American chestnut [Castanea dentata (Marsh.) Borkh.] in eastern forests of North America. Biocontrol efforts on European chestnut (Castanea sativa Mill.) using hypovirulent strains of the blight fungus Cryphonectria parasitica (Murr.) Barr have been successful, while similar efforts on C. dentata in North America have been unsuccessful. This study explores the use of endogenous bark fungi from healthy American chestnut as potential biocontrol organisms against C parasitica. An antagonistic isolate of Trichoderma spp. with good biocontrol potential against C. parasitica was found and evaluated on American chestnut. Such antagonistic bark fungi may be useful as components of biocontrol strategies against the chestnut blight disease both in North American and in Europe.

Molecular cloning of an endo-pectate lyase gene from Erwinia carotovora subsp. atroseptica

Abstract We report the construction of a clone library of the Erwinia carotovora subsp. atroseptica genome and the isolation of a gene for endo -pectate lyase. The library, inserted into the Pst I site of plasmid pBR322, contains approximately 1700 clones. Five of these produce pectolytic enzymes as detected by a plate screening assay. Using a cloned endo -pectate lyase gene from the related bacterium, E. carotovora subsp. carotovora , as a probe, we found that one pectolytic E. carotovora subsp. atroseptica clone had strong homology to the probe. We characterized that clone by restriction endonuclease mapping and studied its pectolytic protein product. The purified enzyme is an endo -pectate lyase with a cofactor preference for Co 2+ . The molecular weight of the protein is 31 000 and it has an isoelectric focusing point of 9·2, corresponding to an endo -pectate lyase produced by E. carotovora subsp. atroseptica , but not to the protein product of the E. carotovora subsp. carotovora probe DNA, which has a pI of 9·5. Restriction endonuclease site polymorphisms in the two cloned endo -pectate lyase genes suggest substantial sequence divergence between these two loci.

BACTERIA FOUND ON AMERICAN CHESTNUT BARK AND THEIR POTENTIAL IN BIOCONTROL OF CHESTNUT BLIGHT

Summary The American chestnut (Castanea dentata [Marsh.] Borkh.) became susceptible to a blight fungus, Cryphonectria parasitica [Murr.] Bar, in the 1870s. This was imported on seedings from Asia, and American chestnut had no resistance to it. Since then, all attempts to eradicate or control the disease have met with very limited success or failure. This paper briefly reviews the history of the biocontrol of chestnut blight, from the use of ‘hypovirulent’ strains of C. parasitica to the use of a bacterium of Castanea spp. that are (1) antagonistic to C. parasitica and (2) can survive for an extensive time period on the bark of C. dentata. The study reveals that some Bacillus megaterium isolates from C. dentata bark may have potential in biological control. The results also provide an explanation for the success of mud packs in ‘healing’ cankers.

Molecular Cloning of Two Erwinia Carotovora Subsp. Carotovora DNA Fragments which Mediate the Production of Similar Pectate Lyases

Erwiniacarotovora subsp. carotovora strain EC14 produces pectate lyases that elute in the void volume from DEAE cellulose DE53 columns. Two pBR322 derivatives containing shotgun-cloned EC14 DNA mediate the production of pectate lyases in Escherichia coli strain HB101 that elute in the void volume from these columns. The EC14 DNA insert in pDRl is 3.4 kilobases in length and contains no internal BamHI sites. Plasmid pDR30 contains an EC14 DNA insert which is 2.4 kilobases in length and is flanked by BamHI sites. There is no hybridization between the EC14 DNA inserts of these two plasmids. Therefore two separate EC14 chromosomal DNA fragments mediate the production of these pectate lyases. Evidence indicates the possibility that plasmid pDR30 also mediates the production of a polygalacturonase.