Biological Control of Fireblight (Erwinia amylovora [Burr.] Winslow et al.) on Ornamentals

Several yellow pigmented, rod-shaped bacterial strains with antagonistic activity against E. amylovora had been isolated from blighted ornamental shrubs. Criteria of the mode of action have been investigated under in-vitro conditions and in inoculation experiments. In contrast to the noninhibitory isolates of the same host origin the active forms obviously produce a substantial principle. This is active only under acid conditions, it is heat-stabile, it can be dialyzed and is solublein water and methanol. It is not a phenolic compound. In disease control experiments using culture filtrates of the antagonistic bacterial isolates fireblight could be reduced to a limited extend following shoot tip inoculation of Cotoneaster bullatus under controlled conditions. Compared to application of the living antagonists in the control experiments disease reduction, however, was considerably less expressed.     

Fireblight Erwinia amylovora and weather: a comparison of warning systems

Warning systems for fireblight Erwinia amylovora developed in New York, Illinois and California, USA, and in south-east England are compared. General principles which might be applicable in the different climates were sought. The consequences of applying threshold temperature values chosen for one area in a different climatic area were examined using Sacramento, California; Rochester, New York; Vlissingen, The Netherlands; Kent, England as examples. A graded system for assessing fireblight risks, derived from all the systems, is suggested. It takes into account both risks of infection and risks of high insect activity and it is best used in conjunction with Billing's incubation period assessment system.     

The nectary as the primary site of infection by Erwinia amylovora (Burr.) Winslow et al.: a mini review

 Erwinia amylovora is the causative agent of fire blight, a bacterial disease existing as an unsolved problem in most countries where pome fruits like apple (Malus domestica) and pear (Pyrus communis) or ornamental plants of Rosaceae are grown. The primary site of colonization is the open flower. As for the establishment of the disease, the importance of various organs within the flowers is considerably different. The usual place for developing a large epiphytic population is the stigma. The actual infection will be attained by the external washing (rain, heavy dew) of bacteria from the stigma to the hypanthium. The bacteria penetrate through the openings of the nectary, so, the nectarthodes are the main entrance sites for them. Nectar is an excellent medium for growth of fire blight bacteria. Most often, however, the incidence of disease is significantly less than the percentage of colonized flowers. Little is known about the interrelationships of free moisture, nectar sugar concentration, ovary water potential, fine-structural characteristics of nectary versus the disease incidence and severity. The aim of this paper is to review the ecology and infection biology of Erwinia amylovora on floral surfaces and in floral tissues. Received July 8, 2002; accepted October 17, 2002 Published online: June 2, 2003     

The Effect of Temperature on the Growth of the Fireblight Pathogen, Erwinia amylovora

S ummary . Growth rates of Erwinia amylovora in yeast extract–peptone broth were assessed, by colony counts and turbidity measurements, at c. 3° intervals over the range 6.5–36.0°. An Arrhenius plot showed a linear relationship between doubling rate and temperatures between 9 and 18°. The slope of this line was comparable to that obtained for Escherichia coli by Ingraham (1958) between c. 12 and 30°. At 18° there was a sharp change in growth rate; between 18 and 28° the doubling time decreased only from 2.1 to 1.3 h (Q₁₀= 1.8) but between 8 and 18° it increased from 2.1 to 14.0 h (Q₁₀= 6.7). This apparently critical temperature is of special interest because maximum air temperatures > 18° appear necessary for epidemic blossom blight in North America.     

Identification of Erwinia amylovora, the Fireblight Pathogen, by Colony Hybridization with DNA from Plasmid pEA29

All strains of Erwinia amylovora characterized carry a medium-size plasmid of 29 kilobases (pEA29). We mapped this plasmid with various restriction enzymes, cloned the whole DNA into an Escherichia coli plasmid, and subcloned restriction fragments. These DNA species were used for identification of E. amylovora after handling of strains in the laboratory and also in field isolates. About 70 strains of E. amylovora and 24 strains from nine other species, mainly found in plant habitats, were checked in a colony hybridization test. Virulent and avirulent E. amylovora strains reacted positively, whereas the other species were negative. Apart from the hybridization assay, the positive strains were additionally tested for ooze production on rich agar with 5% sucrose and on immature-pear slices. Unspecific background hybridization of non-E. amylovora strains found for hybridization with the whole E. amylovora plasmid was almost eliminated when a 5-kilobase SalI fragment from pEA29 was used as a probe and when the washes after the hybridization procedure were done with high stringency. Under these conditions, E. amylovora could be readily identified from field isolates.     

Identification and Characterization of the Erwinia amylovora rpoS Gene: RpoS Is Not Involved in Induction of Fireblight Disease Symptoms

The Erwinia amylovora rpoS gene, encoding the alternative sigma factor RpoS, has been cloned and characterized. Though highly sensitive to a number of environmental stresses, an E. amylovora rpoS mutant was not compromised in its ability to grow or cause disease symptoms within apple seedlings or in an overwintering model.     

In vitro susceptibility of Erwinia amylovora (Burrill) Winslow et al. to geraniol and citronellol

The in vitro antimicrobial activity of geraniol and citronellol towards seven strains of Erwinia amylovora , the causal agent of 'fire blight'of Rosaceous plants, was assessed in tube cultures. All of the strains tested at 1 × 10⁵ cfu/ml were inhibited for 24 h by geraniol in the range 600–1500 mg/1, whereas its minimum bactericidal concentration was 800–1700 mg/1. Citronellol was less effective, being bactericidal for only two of seven strains. RIF-NY, isolated from apple orchards, was relatively resistant to geraniol; 1700 mg/1 of the chemical only reduced the growth of an inoculum of 1 × 10⁷ cfu/ml. In general, such terpenoids commenced exerting a bactericidal effect 6 h after addition to the suspensions, even if geraniol added at 1700 mg/1 to 1 × 10³ cfu/ml of five strains, commenced its bactericidal activity earlier than 6 h.     

Effect of Increased beta-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.     

Preliminary in vitro evaluation of the antimicrobial activity of terpenes and terpenoids towards Erwinia amylovora (Burrill) Winslow et al.

Extracts of black tea exhibited bactericidal activity against Vibrio cholerae O1. The tea extract inhibited the haemolysin activity of V. cholerae O1, El Tor and the morphological changes of Chinese hamster ovary cells induced by cholera toxin. Tea extract also reduced fluid accumulation induced by cholera toxin in sealed adult mice and by V. cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea has protective activity against V. cholerae O1.     

Occurrence of Erwinia amylovora on Insects in a Fire Blight Orchard

In order to screen for natural dissemination of fire blight under field conditions, insects were caught in an experimental orchard and assayed on semi-selective agar plates and by BIO-polymerase chain reaction for their contamination with Erwinia amylovora , the causative agent of the bacterial disease. The pathogen was detected on 15 out of 348 insects collected (4.3%). The insects contaminated with E. amylovora have different living habits and belong to four different orders (Homoptera, Coleoptera, Hymenoptera and Diptera) and at least eight insect families (Aphididae, Psyllidae, Coccinellidae, Curculionidae, Ichneumonidae, Formicidae, Apidae, and Muscidae) and to other Brachycera. After contact of insects with E. amylovora , the pathogen could survive at least 5 days in/on the green lacewing ( Chrysoperla carnea ) and 12 days on aphids ( Aphis pomi ).     

De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.)

Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine max L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N₂ per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N₂ per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate.     

The genome of cowpea (Vigna unguiculata [L.] Walp.)

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub‐Saharan Africa, that is resilient to hot and drought‐prone environments. An assembly of the single‐haplotype inbred genome of cowpea IT97K‐499‐35 was developed by exploiting the synergies between single‐molecule real‐time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination‐poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high‐recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS‐LRR and the SAUR‐like auxin superfamilies compared with other warm‐season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.     

The leaf tannin of willow-herb [Chamaenerion angustifolium (L.) Scop.]

1. The leaf tannin of willow-herb [Chamaenerion angustifolium (L.) Scop.] has been isolated and separated into two fractions of differing solubility. 2. The tannin contains a penta-O-galloyl-β-d-glucose core to which further galloyl groups are depsidically bound. 3. The unfractionated tannin contains an average of 10·5 galloyl groups/glucose molecule; the soluble fraction has on average 7·6 galloyl groups/glucose molecule and the less soluble fraction has 12·4. 4. The tannin is a mixture of molecules ranging at least from hepta- to trideca-galloyl-β-d-glucose. 5. The tannin forms complexes with proteins and the fact that it is a hydrolysable gallotannin has a bearing on the release of nitrogen from the protein of the dead leaf.     

Oidium albicans Robin [Mycotorula albicans (Rob.) Lang. et Tal.] e Monilia pseudotropicalis Castellani [Mycocandida pseudotropicalis (Cast.) Cif. et Red.]

Riassunto Gli AA., sulla base dello studio colturale, micromorfologico, biochimico e biologico di un ceppo isolato dalle feci di un bambino, rivedono la diagnosi e la posizione sistematica di Mycocandida pseudotropicalis (Cast.). Cif. et Red., insistendo sui suoi caratteri differenziali nei confronti di Mycotorula albicans (Robin) Lang, et Tal. e di Candida tropicalis (Cast.) Red.

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Summary The AA. through a Cultural, micromorphological, biochemical and biological study of a yeast isolated from the feces of a child, revised the diagnosis and the systematic position of Mycocandida pseudotropicalis (Cast.) Cif. et Red., insisting on the differential characters in relation to Mycotorula albicans (Robin) Lang. et Tal., and to Candida tropicalis (Cast.) Red.

     

Molecular analysis of sucrose metabolism of Erwinia amylovora and influence on bacterial virulence

Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon source was cloned. By transposon mutagenesis, the scr regulon of E. amylovora was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genes scrK, scrY, scrA, scrB, and scrR, had high homology to genes of the scr regulons from Klebsiella pneumoniae and plasmid pUR400. scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) of E. coli, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a K(m) of 125 mM. Binding of a MalE-ScrR fusion protein to an scrYAB promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose. Expression of the scr regulon was studied with an scrYAB promoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose. The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry. Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by E. amylovora.