U. Mazzucchi

The inhibition of susceptible and hypersensitive reactions by protein-lipopolysaccharide complexes from phytopathogenic pseudomonads: relationship to polysaccharide antigenic determinants

Abstract Protein-lipopolysaccharide (pr-LPS) complexes were purified from a virulent strain of Pseudomonas tabaci (NCPPB 1427) and two incompatible strains ( P. aptata NCPPB 2664 and P. lachrymans NCPPB 1436). They all contained two major polypeptides with mol. wts of 20 000 and 40 000. Slide agglutination tests with O- or pr-LPS-antisera produced against antigens from P. aptata NCPPB 2664 showed that the surface antigens of P. aptata had common determinants with P. tabaci NCPPB 1427, but not with P. lachrymans NCPPB 1436. Precipitin ring tests of heat-stable antigens (121 °C) in the presence of pr-LPS antiserum indicated that most determinants were carried by lipopolysaccharides. Pr-LPS complexes from the three strains, pre-injected into tobacco leaves, prevented hypersensitive confluent necrosis evoked by seven incompatible pseudomonads, and delayed or inhibited the development of the susceptible reaction induced by five compatible strains. These effects were unrelated to the degree of virulence of compatible strains and to the type of surface antigenic determinants of the whole bacterium, as recognized by the rabbit immune system. This suggests a common mechanism of interaction in the protected tissue, causing plant tolerance to various degrees towards different strains. Pr-LPS complexes of P. tabaci NCPPB 1427 were most effective in inducing protection, consistent with the possibility that compatible strains may produce surface substances having higher tolerance-inducing activity.

Prevention of confluent hypersensitive necrosis in tobacco leaves by a bacterial protein-lipopolysaccharide complex

Abstract A protein-lipopolysaccharide (pr-LPS) complex has been selectively detached from washed living cells of Erwinia chrysanthemi NCPPB 454 by treatment with disodium ethylene diamine tetraacetate (EDTA). The material was partially purified by ultrafiltration, then chemically characterized, and its purity was checked by spectrophotometry and gel chromatography. The method yielded a protein-lipopolysaccharide complex of the outer membrane containing about 15% protein. The protein moiety was characterized by a major polypeptide of 40 000 daltons. Dispersions of the complex were injected into interveinal areas of mature tobacco leaves kept at 25°C. A slight chlorosis slowly developed in most treated areas. After 48 h, treated and control areas were injected with 4 × 107 cells/ml Pseudomonas syringae NCPPB 2664. Whereas the control areas developed confluent hypersensitive necrosis, the areas pretreated with the complex did not. Prevention of hypersensitivity was consistent at concentrations of 50 to 500 μg/ml; complete or partial prevention was obtained in the range of 3 to 50 μg/ml. Substances immunologically identical to the purified pr-LPS complex were spontaneously released by E. chrysanthemi during growth in broth cultures. The results thus suggest that pr-LPS complexes of bacterial outer membranes may play a role in modifying the response of plants to phytopathogenic bacteria.

Pectic lyase isozymes produced by Erwinia chrysanthemi Burkh. et. al. in polypectate broth or in Dieffenbachia leaves

Abstract Exocellular pectate lyases produced by three strains of Erwinia chrysanthemi in polypectate broth or in infiltrated Dieffenbachia leaves have been separated by isoelectric focusing. The proteins can be classified into three main groups on the basis of their immunological and enzymological properties and isoelectric points: “alkaline” (pI 9·7 to 9·0), “central” (pI 8·7 to 7·8) and “acid” (pI 4·7 to 5·0) isozymes. Strain NCPPB 454 (from Philodendro ) produced four isozymes (one “alkaline”, two “central”, one “acid”) when grown either on polypectate as the sole carbon source, or in Dieffenbachia leaves. The other two strains when grown on polypectate, did not produce the full complement of isozymes characteristic for the species. Strain NCPPB 2308 (from Dieffenbachia ) produced three isozymes: one “alkaline” (pI 9·7) and two “central” ones (pI 8·35 and 7·8). Strain ICPB-EC 172 (from Chrysanthemum ) produced one “alkaline” (pI 9·7) and one “central” (pI 8·7) isozyme. However, the whole complement of isozymes could be detected in Dieffenbachia leaves infiltrated with either strain NCPPB 2308 or strain ICPB-EC 172. Production of the acid isozyme appears therefore to have been induced during bacterial growth in the leaves.

Encapsulation of Pseudomonas syringae pv. tabaci in relation to its growth in tobacco leaves both pretreated and not pretreated with protein-lipopolysaccharide complexes

Abstract Protein-lipopolysaccharide (pr-LPS) complexes from Pseudomonas aptata NCPPB 2664 were purified and used to pretreat tobacco leaves, thus inducing protection against P. syringae pv. tabaci NCPPB 1427. Protection consisted in the delaying of the susceptible reaction. The growth of P. syringae pv. tabaci was approximately the same in the control as in the pretreated tissue during the first hours of the experiment, but the number of bacteria became significantly greater in the control tissue after 24 h. This peak was followed by a population decline. In contrast, the maximum level of growth was reached after 48 h in the pretreated tissue, and was followed by a gradual decline. P. syringae pv. tabaci appeared to be encapsulated, both in the control and in the protected tissue, during a period which fell between the lag phase and the maximum of the growth curves. When the population reached their maximum, the bacteria appeared to be entrapped within a network of fibrillar material in the intercellular spaces. The encapsulation of P. syringae pv. tabaci leaves is interpreted as a critical phase which enables the bacteria to survive until achieve is established.

MOLECULAR ANALYSIS OF THE dspEF LOCUS IN ERWINIA AMYLOVORA WILD STRAINS OF NORTHERN ITALY

In the years following the severe 1997 epidemic in Emilia-Romagna (northern Italy), fire blight cases were mainly found on pear and the AFLP analysis showed that the E. amylovora (Ea) populations belonged to the same clone (Ea1994). From 1997 to 2001, there has been an increase in outbreaks on apple and hawthorn. The introduction of novel bacterial strains or the modification of virulence factors in the existing clonal populations was hypothesized, as well as the involvement of the dspEF locus of clone Ea1994 under immune pressure by different hosts. The AFLP analysis of serial isolates of Ea1994 during and after 6 passages on pear, apple and hawthorn showed the same genomic profiles. Modifications of the dspEF locus were investigated by means of dspE gene restriction analysis and of gene sequence analysis of dspF in 7 strains with virulence higher than Ea1994 and of the dspE gene of Ea1994. Restriction analysis revealed indistinguishable profiles, and sequence analysis showed that the sequences of the dspEF locus totally correlated (99.9- 100%) with those of Ea321, a North-American Ea strain. In the Po Valley the existence of more virulent strains and the increase of fire blight cases in apple do not seem attributable to changes in the dspEF locus. The results confirmed that the DspE effector and its chaperon DspF, are highly conserved. Thus, the increase of the fire blight cases on apple in this area might be the result of agronomic factors (i.e. higher plant density, new training forms, new cultivars more susceptible to fire blight, higher presence of 1-7 year plantings) associated with a higher inoculum potential in the environment.