Immature Pear Fruits Research Articles

Erwinia pyrifoliae, an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asian pear trees

Bacteria from necrotic branches of Asian pear trees (Pyrus pyrifolia) in Korea were consistently isolated as white colonies on nutrient agar and formed mucoid, slightly yellow colonies on a minimal medium with copper sulphate. Isolates with this colony morphology were studied in a series of microbiological, molecular and pathological tests. Most isolates allowed the verification of Koch's postulate on P. pyrifolia seedlings and on slices from immature pear (Pyrus communis) fruits and were also positive in hypersensitivity tests on tobacco leaves. They showed characteristics common to species in the genus Erwinia, but were different from Erwinia amylovora, the agent of fire blight. A relationship between the novel pathogen and E. amylovora was found in microbiological and serological tests. Both organisms had similar but not identical protein patterns in 2‐D gel electrophoresis, and in growth morphology the new pathogen produced colonies on MM2 Cu medium that were mucoid and slightly yellow, compared with the clearly yellow colonies of E. amylovora. No similarity was found in the plasmid profiles, and consequently no PCR signal was obtained with primers from the E. amylovora plasmid pEA29. REP‐PCR also produced bands differing for the two organisms.

DETECTION OF BACTERIAL MICROFLORA ANTAGONISTIC TO ERWINIA AMYLOVORA ON APPLE, PEAR AND QUINCE TREES

Naturally occurring epiphytic bacteria were isolated from apple, pear and quince trees from forty five fire blight occuring orchards in the Isparta province of Turkey. The immature pear fruit assay was used to screen the epiphytic bacteria for antagonism against Erwinia amylovora. All antagonistic bacterial isolates were differentiated according to some biochemical properties and tested in-vitro for antibiotic production against Erwinia amylovora, and also screened for copper sensitivity. Fluorescent pseudomonads and yellow bacteria antagonistic to erwinia amylovora were more frequently isolated from apple than pear and quince trees. two gram positive isolates, fifteen fluorescent pseudomonads and three yellow pigmented bacteria produced antibiotics in vitro. The minimum inhibitory concentration of antagonistic isolates differed from 0.08 mM to 0.8 mM CuSO4.

Antibiotic production by Erwinia herbicola Eh1087: its role in inhibition of Erwinia amylovora and partial characterization of antibiotic biosynthesis genes.

Mutants of Erwinia herbicola Eh1087 (Ant-), which did not produce antibiotic activity against Erwinia amylovora, the fire blight pathogen, were selected after TnphoA mutagenesis. In immature pear fruit Ant- mutants grew at the same rate as wild-type strain Eh1087 but did not suppress development of the disease caused by E. amylovora. These results indicated that antibiosis plays an important role in the suppression of disease by strain Eh1087. All of the Ant- mutations obtained were located in a 2.2-kb region on a 200-kb indigenous plasmid. Sequence analysis of the mutated DNA region resulted in identification of six open reading frames, designated ORF1 through ORF6, four of which were essential to antibiotic expression. One gene was identified as a gene which encodes a translocase protein which is probably involved in antibiotic secretion. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasmid proteins produced in Escherichia coli minicells confirmed the presence of proteins whose sizes corresponded to the sizes of the predicted open reading frame products.

Identification of a nonpathogenic Erwinia amylovora guaB mutant

A nonpathogenic Erwinia amylovora transposon mutant that has an insertion in the guaB gene was isolated. The mutation results in a nutritional requirement for guanine or xanthine, and loss of ability to produce ooze on immature pear fruit and to cause symptoms in the apple seedling assay. The mutant expressed other known virulence determinants including extracellular polysaccharide and had an intact hrp/dsp cluster. In addition it was able to grow in host tissue, although the population size in planta was maintained at a considerably lower level than that seen with the parent strain. The inability of the Erwinia amylovora guaB mutant to cause disease indicates that levels of guanine in plant tissue are likely to be insufficient to maintain optimal growth via the purine salvage pathway. This, in turn, appears to compromise the ability of the mutant to develop a sufficiently large population size in planta to overcome host defence mechanisms and cause disease symptoms. This indicates that a functional de novo guanine synthetic pathway is important for Erwinia amylovora to grow on plant tissue and cause disease.

Partial characterization of an inhibitory strain of Erwinia herbicola with potential as a biocontrol agent for Erwinia amylovora, the fire blight pathogen

Erwinia herbicola Eh1087 isolated from apple blossom inhibits development of Erwinia amylovora in immature pear fruit and produces a broad spectrum antibiotic activity in vitro that is bactericidal for Erw. amylovora. The antibiotic activity is present in cell‐free culture supernatant fluids of late log‐early stationary phase cultures of Eh1087. This antibiotic activity is not inhibited by proteases, excess ferric ions or essential amino acids. It is stable to acidic and basic pH and is inactivated at high temperature. The antibiotic activity is inactivated by β‐lactamase digestion.

Incidence of bacteria inhibitory to Erwinia amylovora from blossoms in New Zealand apple orchards

Populations of total bacteria and Erwinia herbicola were monitored on apple blossom in selected orchards at two different geographical regions in New Zealand (Canterbury and Hawke's Bay). In all four orchards surveyed E. herbicola populations remained negligible (less than 50cfu/blossom) throughout flowering, increasing rapidly at petal drop to reach levels of 1 × 103 cfu/blossom (Hawke's Bay) to 1 × 105 cfu/blossom (Canterbury). Total bacterial populations increased 100‐fold at petal drop in both locations. Pseudomonas populations were predominant in Canterbury throughout all flowering stages and in Hawkes Bay after early flowering. When screened for inhibitory activity to E. amylovora, 26% of all isolates from Canterbury showed some inhibition in vitro, but none of the isolates from Hawke's Bay showed inhibition of E. amylovora in vitro. Two Canterbury isolates with strong in vitro inhibitory activity also inhibited E. amylovora in immature pear fruit. One isolate was identified as E. herbicola and one isolate as Pseudomonas fluorescens.

Erwinia amylovora hrpN mutants, blocked in harpin synthesis, express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco

Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting many rosaceous plants and especially pear tree and apple tree. A protein named harpin, secreted through the Hrp secretion pathway and able to elicit an hypersensitive reaction (HR) on tobacco has recently been isolated. Mutants inhrpN, the gene encoding harpin were described as non pathogenic on immature pear fruit and unable to elicit an HR on tobacco [Weiet al., 1992; Wei and Beer, 1993]. In this paper, the phenotype on plant ofhrpN mutants was carefully determined.hrpN mutants expressed a weak but significant virulence on host plants. Furthermore, when infiltrated into tobacco leaf mesophyll, thehrpN mutants elicited varied responses that fluctuated from null reaction to full necrosis of the infiltrated area. These results show that harpin is not absolutely required neither for pathogenicity on host plant nor for elicitation of an hypersensitive reaction on tobacco. Furthermore, in all the tests performed, mutant blocked in harpin secretion remained non pathogenic and unable to elicit an HR on tobacco. This suggests that factor(s), different from harpin, involved both in pathogenicity and HR eliciting ability is (are) secreted through the Hrp secretion pathway.

Mechanisms of inhibition of Erwinia amylovora by Erw. herbicola in vitro and in vivo

The mechanisms by which Erwinia herbicola inhibits Erwinia amylovora, the fire blight pathogen, were investigated. The optimum pH for growth of Erw. amylovora strain Ea273 in nutrient‐yeast extract‐glucose broth (NYGB) was 7.0 and growth was markedly reduced at pH values below 6.0. In contrast, the growth rates of Erw. herbicola strains Eh141 and Eh112Y were only slightly reduced at pH levels as low as 4.5, relative to pH 6‐8. When Ea273 was grown in NYGB in the presence of Eh141 or Eh112Y, the media became acidic and lower populations of Ea273 were recovered, compared with populations from buffered NYGB. Acidification of plant tissue as a consequence of growth of Erw. herbicola did not occur, however, and thus acid‐based inhibition of growth in planta is unlikely. The growth rates of nine strains of Erw. herbicola and their abilities to reduce the pH of NYGB did not correlate with their different abilities to prevent development of fire blight incited by Ea273 in a research apple orchard. When grown in mixed culture, Eh114 and Eh112Y grew to higher populations than Ea273 due to depletion of a nitrogen source needed by Ea273. The ability of 12 strains of Erw. herbicola to produce antibiotics inhibitory to Ea273 on a glucose‐asparagine medium correlated with the effectiveness of the strains in suppressing fire blight. A crude preparation of the Eh318 antibiotic delayed development of disease in immature pear fruits incited by Ea273 but not by strain Ea273R318, which is resistant in vitro to the Eh318 antibiotic. Cells of Eh318 protected immature pear fruits more effectively from infection by Ea273 than from the resistant strain Ea273R318.

Biological Control of Fire Blight of Hawthorn (Crataegus monogyna) with Fluorescent Pseudomonas spp. under Protected Conditions

AbstractNaturally‐occurring epiphytic fluorescent pseudomonads were isolated and characterized in terms of their potential to control fire blight infection of hawthorn, caused by Erwinia amylovora.Preliminary testing and selection of antagonists using an immature pear fruit assay gave some inconsistency in the amount of pathogen suppression on the pear tissue and also in the prediction of biocontrol effectiveness on the intact plant.Selected antagonists provided significant but variable control of fire blight under protected (polythene tunnel and glasshouse) conditions, with isolates HL83 and HL99 giving control of both blossom‐blight and shoot‐blight. In some cases the degree of control was equal to that of chemical treatments, including agrimycin 17 and experimental bactericides, and was achieved without any numerical advantage of applied control agent over pathogen. The timing of pseudomonad application in relation to pathogen inoculation was found to have a significant effect on the level of control of blossom‐blight.

Identification and Spread ofErwinia amylovoraon Pear in Turkey

Diagnostic and pathogenicity tests of two isolates of bacteria from blighted pear trees in Turkey were compared with tests of known isolates of Erwinia amylovora. Turkish isolates formed orange-yellow colonies with deep orange centers and clear margins on modified Miller-Schroth medium; they did not fluoresce under ultraviolet light when grown on King's medium B. Bacterial ooze was produced on immature pear fruit, and susceptible pear shoots were blighted in 6 days. Based on these tests, the two Turkish isolates were positively identified as Erwinia amylovara (.)

The Effect of Bacillus subtilis on In Vitro Growth and Pathogenicity of Erwinia amylovora

Five isolates of Bacillus subtilis were found to be antagonistic to 3 isolates of Erwinia amylovora in vitro on sucrose agar medium. However, B. subtilis or its culture filtrate did not exhibit any activity against E. amylovora in planta tests in the laboratory on immature green pear fruits or in a commercial pear orchard under natural growing conditions.

Gene for pathogenicity and ability to cause the hypersensitive reaction cloned from Erwinia amylovora

A genomic library of Erwinia amylovora isolate T was constructed in the cosmid pLAFR3 and maintained in Escherichia coli. Clones were transferred individually by conjugation into the non-pathogenic isolate P66 of E. amylovora. Transconjugants were screened for restoration of pathogenicity to pear by stab inoculation into sections of immature pear fruits. Three clones complemented P66 restoring pathogenicity and ability to cause the hypersensitive reaction (HR) in Phaseolus vulgaris. Restriction mapping and hybridization experiments showed that the three clones had a common 3·7 kb fragment of E. amylovora DNA. Sub-cloning and insertion mutagenesis with Tn5- lac confirmed that a determinant of pathogenicity and ability to cause the HR ( hrp gene) was located on a 2·1 kb HindIII/ BamHI fragment within the common DNA. Hybridization experiments using the 2·1 kb HindIII/ BamHI fragment as a probe demonstrated that the hrp gene was located in the chromosome of isolate T and that homologous sequences were present in the non-pathogenic isolates P66 and S. Clones which restored hrp function did not affect the growth of isolate P66 in minimal or nutrient-rich media. Transconjugants of Pseudomonas syringae pv. phaseolicola race 1 harbouring the hrp gene(s) cloned from E. amylovora did not cause the HR in susceptible cultivars of bean but symptoms developed more slowly than in the absence of the clones or with pLAFR3 alone.

Biological control of fire blight of hawthorn (Crataegus monogyna) with Erwinia herbicola under protected conditions

Isolates of Erwinia herbicola, obtained from flowers and leaves of hawthorn (Crataegus monogyna), were screened as potential control agents of fire blight disease (caused by Erwinia amylovora) using an immature pear fruit assay. Selected isolates were subsequently tested for disease control by infection of hawthorn blossom in the laboratory, and by shoot infection of hawthorn plants grown under controlled (glasshouse) and fluctuating (polythene tunnel) environmental conditions.Although the immature pear fruit assay provided a general screen for the selection of antagonists for the control of both blossom and shoot blight, it had two major limitations when quantitatively applied. Firstly there were inconsistencies in the relative effects of different isolates on the pear‐slice surface, with some isolates being more suppressive than the standard antagonist Eh252 in the first screening and less in the second. Secondly the assay was not able to predict accurately the level of control in the intact plant‐as no correlation occurred between the level of control in the pear fruit assay and the percentage control of either blossom blight or shoot blight.Two isolates of E. herbicola, WL9 and WL40, reduced both blossom‐ and shoot‐blight. WL9 provided over 80% control of blossom blight, equivalent to that provided by chemical agents, and also gave total control of shoot blight when applied at a WL9: pathogen ratio of 10:1.

ThercsAGene fromErwinia amylovora: Identification, Nucleotide Sequence, and Regulation of Exopolysaccharide Biosynthesis

RcsA is a positive activator of extracellular polysaccharide synthesis in the Enterobacteriaceae. A cosmid clone containing the rcsA gene from Erwinia amylovora was identified by its ability to restore mucoidy to an E. stewartii rcsA mutant. The rcsA gene was subcloned on a 2.2-kilobase HindIII-PstI fragment that hybridized with an E. stewartii rcsA probe and complemented E. stewartii and Escherichia coli rcsA mutants. In addition, the cloned E. amylovora rcsA gene stimulated expression of cps::lac fusions in E. coli and E. stewartii. The rcsA region was sequenced, and one open reading frame of 211 amino acids was found. The predicted protein sequence specified by this open reading frame was 55% homologous with that of the Klebsiella pneumoniae RcsA protein. Highly conserved regions in the 3' and 5' ends of the two proteins were observed. An E. amylovora rcsA mutant was constructed by Tn5 mutagenesis of the cloned gene followed by recombination of the mutation into the chromosome of wild-type strain Ea1/79. The synthesis of both amylovorin and levan was reduced by more than 90% in this mutant, indicating common regulation of the two polysaccharides by rcsA. Virulence of the rcsA mutant on immature pear fruit was diminished but not completely abolished.

Effect of Increased β-Glucosidase Activity on Virulence of Erwinia amylovora

Plant tissues often contain beta-glucosides that can be enzymatically hydrolyzed to produce toxic aglycones. It has been suggested that the low beta-glucosidase activity found in Erwinia amylovora contributes to bacterial virulence by allowing the bacteria to infect plants that contain beta-glucosides without inducing the formation of toxic aglycones. To test this suggestion, we created strains of E. amylovora which had high beta-glucosidase activities and studied the ability of these strains to cause fire blight disease in pears (Pyrus communis). We isolated spontaneous mutants that were able to utilize beta-glucosides as the sole carbon source and showed that one class had about 10 times as much beta-glucosidase activity as the wild-type strain. In addition, we constructed several plasmids that carry the Escherichia coli bgl operon under the control of a transposon Tn5 promoter that is expressed in E. amylovora. These plasmids were introduced in E. amylovora by transformation. Pathogenesis studies in immature Bartlett pear fruits, etiolated sprouts, and young shoots showed that a 100-fold increase in beta-glucosidase activity does not interfere with normal development of fire blight disease in these model systems.

Evidence for Two Virulence Determinants in the Fireblight Pathogen Erwinia amylovora

Seven avirulent strains of Erwinia amylovora were mixed in various combinations and tested for virulence. Only one combination, where the capsulated avirulent strain P66 was combined with the non-capsulated avirulent strain 4S2, induced fireblight symptoms in immature pear fruits and apple shoots. Growth studies in vivo strongly suggested that these strains were acting synergistically and that the non-capsulated strain possessed another, as yet unknown, virulence factor.

Conjugational transfer of genes determining plant virulence in Erwinia amylovora.

A stable virulent donor strain (EA 178R1-99) of Erwinia amylovora can transfer, by conjugation during a 3-h mating period, the gene or genes which determine(s) plant virulence to avirulent recipient strains (EA178-M64S1 and EA178-M173S1) of Escherichia amylovora. The virulence of over 200 recombinant clones was tested; they all were as virulent on immature Bartlett pear fruits (and, in the smaller series of strains tested, also, on Pyracantha twigs) as was the parent donor strain. Although the avirulent recipeint strains are amino acid auxotrophs, addition of the required amino acids to the inocula in plant virulence trials does not of itself restore virulence. Two small series of prototrophic revertant clones were selected from the auxotrophic avirulent recipient strains; only nine of the 21 prototrophic revertant clones regained virulence, whereas the other 12 prototrophic revertant clones remained avirulent, again suggesting a lack of parallelism between nutritional status and virulence in this system. Preliminary interrupted mating trials, carried out at 15-min intervals over 3 h, show that ser is transferred during the first 15 min, that pro starts entering at about 75 min (and with a higher frequency later), and that lac (originating from an integrated Escherichia coli F'lac) enters toward the end of the 3-h mating period and at a reduced frequency compared to the other markers. The gene or genes which determine(s) plant virulence in this Escherichia amylovora donor strain appear(s) to be transferred readily and seemingly completely to recipient strains during the first 15 min of a 3-h mating period. Exposure of the virulent donor strain to acridine orange or ethidium bromide does not result in loss of virulence, suggesting (but, of course, not proving conclusively) that the determinant(s) of virulence in Escherichia amylovora might be chromosomal rather than extrachromosomal.

Influence of Succinic Acid-2,2-Dimethylhydrazide and Ethylene on Respiration and Ethylene Production by Developing ‘Bartlett’ Pear Fruits1

Abstract Ethylene-induced endogenous ethylene production was substantial in immature pear fruits, attaining a max rate in samples harvested 60 days after full bloom and declining thereafter. Ethylene production was not dependent on the occurrence of a respiratory climacteric nor related to the rate of respiration. Field applications of succinic acid-2,2-dimethylhydrazide (SADH) suppressed both ethylene production and fruit growth.

Arbutin and a Related Glucoside in Immature Pear Fruit

SUMMARY– Arbutin (p‐hydroxyphenyl‐β‐D‐glucoside) was separated from other phenols in an extract of immature fruit of Pyrus C.V. Kieffer by preparative paper chromatography and isolated as the penta‐acetyl derivative. A monoacetylarbutin (p‐hydroxyphenyl‐6‐0‐acetyl‐β‐D‐glucoside) was identified in the same pear extract. In addition, arbutin was found in both mature and immature pears, but at much higher levels in the immature fruit.

Chlorogenic Acids in Fruit Trees

WORK on the chemical composition of the various parts of fruit trees, particularly apple and pear, is proceeding at Long Ashton aud East Malling Research Stations and the Ditton Laboratory. The problems being studied are not quite the same in each laboratory, but discussions of common interests have disclosed certain overlapping findings. For this reason, we wish jointly to report the presence of chlorogenic acid in both mature and immature apple and pear fruits, and of this substance and a substance similar to, or identical with, isochlorogenic acid of coffee1 in leaves and shoots of pear.