Plant calcium can modulate a particular plant-pathogen interaction and have a decisive role in disease development. Enhanced resistance to the phytopathogenic enterobacterium Erwinia carotovora, the causal agent of bacterial soft rot disease, is observed in high-calcium plants. One of the main virulence determinants of E. carotovora, the PehA endopolygalacturonase, is specifically required in the early stages of the infection. Production of PehA was found to be dependent on the calcium concentration in the bacterial environment. An increase in extracellular calcium to mM concentrations repressed pehA gene expression without reducing or even enhancing expression of other extracellular enzyme-encoding genes of this pathogen. An increase in plant calcium levels could be correlated to enhanced resistance to E. carotovora infection and to an inhibition of in planta production of PehA. Ectopic expression of pehA from a calcium-insensitive promoter allowed E. carotovora to overcome this calcium-induced resistance. The results imply that plant calcium can constitute an important signal molecule in plant-pathogen interaction, which acts by modulating the expression of virulence genes of the pathogen.

Salicylic acid (SA) has been inferred to be an endogenous signal in the systemic acquired resistance response of plants. In this study, we demonstrated that exogenously added SA can enhance plant resistance to the phytopathogenic enterobacterium Erwinia carotovora. Addition of SA to the growth medium of axenically growing tobacco seedlings made them almost fully resistant to subsequent infection by the soft rot pathogen E. c. subsp. carotovora. Both the development of soft rot symptoms (tissue maceration) and the in planta proliferation of E. c. subsp. carotovora were inhibited in SA-treated plants. The observed effect was not caused by direct action of SA on growth, nor on extracellular enzyme production or activity of the pathogen at the physiological SA concentrations, but was rather a consequence of induction of plant defense response. This was suggested by the development of hypersensitive-like reactions in SA-treated Erwinia-infected plants, by the temporal pattern of resistance development, and by the parallel increase in pathogenesis-related proteins. The plants reacting hypersensitively to E. c. subsp. carotovora showed a further increase in endogenous SA levels, indicating that SA and SA-controlled processes such as systemic acquired resistance are involved in Erwinia-plant interaction. The molecular mechanism of the SA-induced resistance to E. c. subsp. carotovora is not clear but appears to involve inhibition of plant cell wall-degrading enzymes secreted by this pathogen.

Erwinia carotovora subsp. carotovora, the etiological agent of bacterial soft rot, produces a variety of plant cell wall-degrading exoenzymes that are the main virulence factors of this pathogen. To determine the role of these enzymes in the plant-bacterium interaction, individual exoenzymes were produced in Escherichia coli harboring cloned exoenzyme encoding genes from E. c. subsp. carotovora and applied to tobacco plants. The plant response was monitored by following the expression of a plant gene encoding a pathogenesis-related beta-1,3-glucanase. The transcript for beta-1,3-glucanase was shown to rapidly accumulate in plants treated with pectic enzymes, but not with a cellulase from E. c. subsp. carotovora. Both pectate lyase (Pel) and polygalacturonase (Peh) were shown to increase the host beta-1,3-glucanase mRNA levels. In addition, a similar plant response could be elicited by the application of polygalacturonase-treated polypectate. In planta analysis of tobacco seedlings inoculated with reduced virulence mutants of the pathogen that still produced pectic enzymes resulted in accumulation of beta-1,3-glucanase mRNA. However, no accumulation of beta-1,3-glucanase mRNA was observed in plants inoculated with exoenzyme-negative mutants. These results indicate that pectic enzymes of E. c. subsp. carotovora probably elicit the plant defense response by releasing pectic fragments from the plant cell wall that may function as endogenous elicitors. Interestingly, infection of plants by the wild-type pathogen induced the plant response only weakly and transiently, suggesting that the wild-type bacteria are able to suppress the plant response. Induction of the plant defense by exoenzyme treatment conferred increased resistance against subsequent infections by E. c. subsp. carotovora.

A simple technique for rapid isolation of mitochondrial DNA (mtDNA) from fish tissues is described. The method, which uses the commercially available Qiagen Plasmid Isolation Kit, can be applied to both fresh and liquid-nitrogen-frozen tissues. The yield of mtDNA is comparable to that obtained by standard techniques. The mtDNA is sufficiently pure for restriction fragment length polymorphism (RFLP) analysis by the end-labeling procedure.

Mitochondrial DNA (mtDNA) was purified from the Arctic charr, Salvelinus alpinus, the brook charr, Salvelinus fontinalis, and the lake charr, Salvelinus namaycush, and digested with restriction enzymes Ava II, Hinf I, EcoR V, Pst I and XbaI. Two Arctic charr samples were from natural populations and they represented two different morphotypes of Arctic charr. All other studied populations were hatchery maintained. Eight additional restriction enzymes and double digestions were employed to study morphotypes of Arctic charr. We distinguished two morphotypes with restriction enzyme Nci I. Sequence divergence among mtDNA types was 2.9–3.8% between S. alpinus and S. fontinalis, 3.4–4.6% between S. alpinus and S. namaycush, and 4.7–5.3% between S. fontinalis and S. namaycush. lntraspecific variation was lowest in Arctic charr, the average of nucleon diversity for three populations being 0.179, while for brook charr and for lake charr nucleon diversity was 0.334 and 0.550, respectively. According to the number of mtDNA types, it is obvious that introduction to Finland and hatchery propagation have not greatly affected the mtDNA variation of brook charr or lake charr.

Two hatchery-maintained populations of non-anadromous and anadromous European (Finland) Atlantic salmon, Salmo salar L., were compared by restriction enzyme analysis of mitochondrial DNA (mtDNA). These two widely used stocks could be identified by their distinct restriction fragment patterns with HinfI and HaeIII. The stocks appeared identical with regard to other enzymes tested, but differed from previously analyzed North American (Canadian) S. salar populations.

Restriction endonuclease analysis of mitochondrial DNA (mtDNA) from four breeding stocks of rainbow trout, Salmo gairdneri Richardson, revealed extensive polymorphism. Two different restriction morphs were observed by HindIII digestion and three different morphs by BglII. All of the variant mtDNA types (A–D) could be explained by restriction site gains or losses.

A simple technique for rapid isolation of mitochondrial DNA (mt DNA) from animal cells is described. The method is based on the selective alkaline denaturation procedure of Birnboim and Doly [(1979) Nucleic Acids Res. 7, 1513-1523] and avoids the use of CsCl gradient centrifugation. The yield of mtDNA is comparable to that obtained by standard techniques. This DNA is sufficiently pure for restriction analysis and cloning of mtDNA fragments.