Alternative methods to describe virulence of Erwinia amylovora and host-plant resistance against fireblight

The evaluation of host-plant susceptibility to Erwinia amylovora and of colonization of host-plant tissue by individual strains was facilitated by labelling the pathogen with green fluorescent protein (GFP). Colonization of apple leaves assayed with a fluorescence microscope was associated with visual disease ratings on plants to describe virulence (= aggressiveness) of the fireblight pathogen. Resistance induced with 2,6-dichloro-isonicotinic acid (INA) and benzo(1,2,3-) thiadiazol-7-carbothioic acid-S-methyl ester (BTH, the active component of BION™) restricted colonization by the pathogen to an area adjacent to the inoculation site. Migration in leaves was associated with symptom formation on pear slices and host plants of mutant strains. Non-virulent E. amylovora mutants did not migrate into the leaf veins and strains with intermediate-to-low virulence moved slowly. To compare the migration efficiency of individual wild-type strains in apple and plum cultivars, a blend of five wild-type E. amylovora strains with specific numbers of short-sequence DNA repeats (SSRs) in the common plasmid pEA29 was applied to distinguish them by PCR. Fast-moving strains identified in the GFP assays were dominant, independent of the apple cultivar. When apple shoots, pear slices or leaves of apple plants were coinoculated with streptomycin (Sm)-resistant strains and the corresponding parent strains, Sm-resistant mutants were able to dominate the wild-type strain for tissue colonization.     

First occurrence of fireblight in Romania

Fireblight was first observed in Romania in 1992-08 near Braila, and shortly after in the pear collection of the Fruit Research Institute in Pitesti-Maracineni. Advanced symptoms indicated that the disease was present at least one year earlier. Symptomatology, fruit pathogenicity, selective culture media, hypersensitivity, agglutination and fatty acid class analysis confirmed the authenticity of the pathogen as Erwinia amylovora. Of the native Romanian fruit cultivars affected, apple cv. Delicios de Voinesti and pear cv. Aromat de Bistrita appear the most susceptible to fireblight.     

Traditional and commercial apple and pear cultivars as sources of resistance to fireblight1

In Hungary, fireblight research programmes were initiated on pear in 1999 and on apple in 2000, with the aim of evaluating the susceptibility or resistance of commercial cultivars. Sources of resistance for future breeding were also sought among traditional apple cultivars collected from Ukraine and pear cultivars in the Hungarian gene bank (Szigetcsép). Experiments were done under secure conditions. Inocula were mixtures of characteristic Erwinia amylovora isolates from pear and apple in Hungary. Host responses (symptom development, disease severity and multiplication rate of bacterial cells in host tissues) were assessed on shoots, flowers and fruits. About 30 pear and 30 apple cultivars, and 35 apple hybrids, were tested and grouped into four categories for pear and three for apple. Of the pear cultivars tested, 50% were susceptible, 30% moderately susceptible and only 10% of low susceptibility. Different plant organs occasionally displayed different responses. Members of the last two groups might serve as useful candidates for growing under IPM conditions. Among the traditional Hungarian varieties tested, we found high resistance in ‘Sikulai’ and ‘Szemes alma’, which could be used as sources of fireblight resistance in breeding programmes and also grown in organic orchards. Furthermore, among the offspring of the apple ‘Prima’ (scab‐resistant), we have found highly resistant lines.     

First outbreaks of fireblight in Slovenia1

Slovenia was recognized as free from Erwinia amylovora until 2001. A low incidence of the disease was reported in 2001 and 2002 in the north‐west uplands of the Gorenjska region. In 2001, only 3 trees in extensive orchards were found positive at one location out of 791 monitoring locations all over the country. In 2002, the same location in Naklo and a second one in a 1‐km radius were found positive, whilst 875 other monitoring locations were fireblight‐free. Despite strict phytosanitary measures implemented after discovery of the first focus, the bacterium spread in 2003 to the entire Gorenjska region, with further spread towards eastern and southern Slovenia. The NPPO registered 184 new foci by the end of the season in Gorenjska. Twenty‐three foci are scattered in other regions. Phytosanitary measures were applied in every focus. Spread of the bacterium was probably enabled by favourable weather conditions, the presence of the inoculum and intensive bee‐hive movement, resulting in flower infections. Since eradication has not been successful, it is concluded that E. amylovora is now present at low prevalence in Slovenia.     

Experience with fireblight in the Czech Republic1

Erwinia amylovora has been spreading in the Czech Republic since 1986 regardless of emergency phytosanitary measures which have been taken. Its spread follows the predominant wind direction (west to east). The infested area now covers two thirds of the area of the country. Wild Crataegus spp. are the most important and widespread host plants. E. amylovora is still regulated as a quarantine pest, and areas free from fireblight are subject to special phytosanitary measures.     

Monitoring fireblight for official phytosanitary legislation in Belgium1

Fireblight has been present in Belgium since 1972. An official monitoring project to prevent the spread of the pathogen started in 1978, combining regulations and large-scale surveys. The most important regulations concern: (a) the concentration of nurseries in officially designated protected zones; (b) prohibition of the growing of highly susceptible Cotoneaster spp.; (c) the supervision of hawthorn hedges; (d) the eradication of diseased host plants. In the surveys, diseased or suspect plant material is diagnosed and Erwinia amylovora populations are monitored on leaves, flowers and buds, sampled from apparently healthy plants during field inspection. The laboratory methods are mainly serological and consist of: (a) direct isolation and testing of suspect colonies by agglutination; (b) screening of samples using indirect immunofluorescence; (c) selective enrichment on immature pear slices. A review of 8 years' work is presented and discussed, illustrated with results of experiments.     

番茄青枯病内生拮抗细菌的筛选

 从广西一些市县采集番茄茎标本分离得到55个细菌菌株,分属为芽孢杆菌(Bacillus spp.)、黄单胞菌(Xanthomonas spp.)、假单胞菌(Pseudomonas spp.)和欧文氏菌(Erwinia spp.),其中芽孢杆菌为优势种群。经回接测试,有36个菌株为番茄植株内生菌。这些内生菌只有7个菌株对番茄青枯病菌有拮抗作用,芽孢杆菌B₄₇菌株对番茄青枯病菌拮抗作用较强,经室内和田间初步防治测定,它对番茄青枯病有较好的防治效果。     

Ten years of experience with fireblight in Austria1

In 1993, fireblight (Erwinia amylovora) was detected for the first time in Austria, in the most westerly part of the country (Vorarlberg). During the following years, especially since 1998, a stepwise migration from west to east was observed. For control, mechanical measures are mostly used. As these measures require early recognition of symptoms, broad information campaigns for the public and extensive surveys of host plants have been set up. Suspicious plant samples are sent to the central laboratory for diagnosis. Regional plant protection services are assisted by trained volunteers. Eradication and pruning measures are performed by trained working teams. Infected plant material is burned. In all pome‐fruit production areas, the disease forecasting system Maryblyt has been established. Regulations concerning the movement of beehives have been adopted, together with prohibition of the planting of certain ornamental host plants (Crataegus, Cotoneaster). Chemical treatments are limited to one copper oxychloride product. In 2003, temporary application of Prohexadione‐Ca for the prevention of shoot infections was authorized. In some areas, the rigorous application of control measures has reduced the incidence of fireblight and possibly slowed down its progress. However these measures are not sufficient to protect against new, unexpected outbreaks.     

Effectiveness of the resistance inducer prohexadione‐Ca against fireblight in shoots of apple trees inoculated with Erwinia amylovora1

Due to the lack of effective, non‐phytotoxic and publicly acceptable materials for controlling fireblight in pome fruit trees, novel strategies against Erwinia amylovora are being sought. Resistance‐inducing compounds, such as prohexadione‐Ca, represent promising alternatives. Prohexadione‐Ca is the active substance of the bioregulator Regalis, currently being introduced in several European countries and overseas. Prohexadione‐Ca reduces shoot elongation due to inhibition of gibberellin biosynthesis. Furthermore, it leads to significant changes in the spectrum of flavonoids and their phenolic precursors in pome fruits, which causes reduced susceptibility to fireblight and other pathogens. In 2002 and 2003, container‐grown apple trees of the cultivars ‘Idared’ and ‘Freedom’ were treated with different dosages of prohexadione‐Ca two weeks before inoculation with E. amylovora. The effect of prohexadione‐Ca against shoot blight was determined by measuring the lengths of necrotic lesions and symptoms on vascular bundles caused by the pathogen. Treatments with prohexadione‐Ca turned out to be much superior to the ones with streptomycin, kasugamycin and a bacterial antagonist, which were used for comparison. Acibenzolar‐S‐methyl (Bion), another resistance‐inducing compound, was included in some of the experiments and gave intermediate results. The simultaneous control of excessive shoot growth and shoot infections by fireblight is seen as a major advantage of using prohexadione‐Ca in pome fruit trees.     

A brief review of the status of fireblight in UK1

Fireblight (due to Erwinia amylovora) has continued to spread northwards and westwards in UK. In SW England, the disease has been particularly severe in perry pear orchards, causing the loss of many thousands of mature trees, and in cider apple orchards. Cider apple trees are not rapidly killed but crop losses have been severe in some years. The disease has become established in the NW midland counties of England and also in Wales. Outbreaks have occurred as far north as Lancashire just above the administrative line which separates the affected area from the disease-free (clear) area. Spread has occurred through infections in Crataegus hedges, especially along motorways, and also through movement of infected ornamental species. New legislation is envisaged which will redefine the clear area and increase the protection given.     

Results of the detection survey for fireblight in Slovakia1

Erwinia amylovora did not occur in Slovakia until 2003, as confirmed by the results of detection surveys and numerous tests. On this basis, Slovakia was recognized as a pest‐free area for E. amylovora by the EU. In the first half of 2003, the Slovak NPPO confirmed the first occurrence of this bacterial disease, in the south‐eastern part of the country. This article describes the detection survey which was then undertaken and the measures that were taken with the aim of eradicating the disease.     

Practice of applying streptomycin to control fireblight in Hungary1

The conditions under which streptomycin may be used for fireblight control in Hungary are described. Authorizations for individual limited treatment programmes are given to registered growers only.     

Present status of fireblight in the Federal Republic of Germany1

The three phases of spread of fireblight in FRG are described, from its first appearance in 1971 to the present day. The disease is now well established in Schleswig-Holstein and Niedersachsen and scattered outbreaks have occurred over the whole of the rest of the country (but only one in Bayern). The control strategy now centres on destruction of diseased plants within 500 m of any outbreak found in an orchard or nursery (object protection).     

Experiences with the control of fireblight in Norway during 1986/20031

Fireblight was detected for the first time in Norway in 1986, in a limited outbreak on ornamentals, in particular Cotoneaster. An organization for the eradication and containment of the disease was rapidly established, and given comprehensive statutory powers and resources to do surveys and to remove diseased plants and highly susceptible plants from infested areas. The activities have so far been successful. The disease has remained within a restricted area on the West Coast, and has not moved into important fruit‐growing areas or nurseries. Since 2000, fireblight has spread to some adjoining areas, mainly due to uncontrolled movement of beehives. New statutory powers prohibit movement of beehives out of infested areas from May to October.     

Prohexadione‐Ca induces resistance to fireblight and other diseases1

Prohexadione‐Ca is primarily used for the control of shoot growth in pome and other fruit trees. During its development, treated apple and pear trees were found to be significantly less affected by fireblight (Erwinia amylovora) and other pathogens, although prohexadione‐Ca is inactive as a bactericide or fungicide. Prohexadione is a structural mimic of 2‐oxoglutaric acid, and the distinct dioxygenases involved in gibberellin biosynthesis that require this compound as a cosubstrate are blocked. As a result, less growth‐active gibberellins are formed and treated plants remain compact. 2‐Oxoglutaric acid‐dependent dioxygenases are also involved in flavonoid metabolism. In shoots of apples and pears, prohexadione‐Ca causes considerable changes in the formation of flavonoids and their phenolic precursors by inhibiting flavanone 3‐hydroxylase. Convincing evidence is available that prohexadione‐Ca triggers pathogen resistance primarily by inducing the formation of 3‐deoxyflavonoids, in particular luteoforol, with phytoalexin‐like properties. Morphoregulatory effects caused by prohexadione‐Ca are only of secondary relevance. The simultaneous control of excessive shoot growth and shoot infections by fireblight is seen as a major advantage of using prohexadione‐Ca in pome fruit trees.     

Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 10³ cells mL^−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.