Erwinia amylovora: Características generales. métodos de diagnóstico de la enfermedad e identificación de E. amylovora

El género Erwinia, que debe su nombre a la memoria del fitopatólogo Erwin F. Smith, se creó inicialmente para agrupar a las enterobacterias asociadas a las plantas, Gram negativas, bacilares, no formadoras de esporas y móviles (Winslow et al., 1920). Por ello, los miembros de este género incluían además enterobacterias saprofitas ecológicamente asociadas a plantas, así como patógenos oportunistas del hombre y los animales (Brenner, 1984). Esta heterogeneidad de especies fue la causa de que el género Erwinia fuera objeto de varias reclasificaciones. Finalmente, gracias al avance de las técnicas moleculares, las especies del género Erwinia se clasificaron en cuatro grupos filogenéticos basados en la comparación de secuencias del ADN ribosómico 16S (Hauben et al., 1998). El grupo I (género Erwinia) representa a las verdaderas erwinias e incluye diversas especies, que producen necrosis o marchitamientos en plantas, o que pueden ser epifitas. Erwinia amylovora es la especie tipo de este género. El grupo II (actuales géneros Pectobacterium y Dickeya) agrupa especies que originan podredumbres blandas en un amplio rango de hospedadores debido a su gran actividad pectolítica. El grupo III (actual género Brenneria y la especie Dickeya paradisiaca) incluye varias especies que afectan a plantas leñosas produciendo generalmente chancros y exudados. El grupo IV (género Pantoea) contiene especies que son saprofitas o patógenos más o menos frecuentemente oportunistas de plantas, animales y del hombre. Entre ellas destaca la antigua Erwinia herbicola, actualmente denominada Pantoea agglomerans, frecuentemente asociada en rosáceas a E. amylovora

Strategies for improving serological and molecular detection of plant pathogenic bacteria

Serological and molecular techniques are the most appropriate for rapid detection of plant pathogenic bacteria in plant material. They can reach high specificity when selected probes are used and high sensitivity after optimisation of the most important steps of each technique. ELISA and PCR are techniques widely used for developing simple protocols useful for processing large number of samples in routine. As they are based in different principles and probes, their combination allows accurate, rapid and specific detection of the bacteria in plant material. Several aspects of their optimisation for detecting some phytopathogenic bacteria are described here

ACTA HORTICULTURAE

Different reagents and methods have been reviewed, selected, improved standardized and validated for detection and diagnosis of Erwinia amylovora. The validated methods and protocols will be published on an Internet web-site for comments and will be included as the European Union official scheme for the detection and identification of the bacterium. A combination of two different methods based on serology and PCR in addition to isolation followed by a pathogenicity test, will be required to officially confirm the E. amylovora presence. The detection of the bacterium in symptomless plant material must be assayed after a validated enrichment step

ACTA HORTICULTURAE

The EU-funded project DIAGPRO has developed diagnostic protocols for 18 quarantine pests including Erwinia amylovora. The protocol for this organism includes a diagnosis scheme for detection of the bacterium in symptomatic and asymptomatic plants with details of the different techniques as isolation in three media, direct tissue print-ELISA, enrichment DASI-ELISA, immunofluorescence, conventional PCR and nested-PCR in a single closed tube (before and after enrichment of the samples in two liquid media). A ring test was conducted to evaluate and standardize some techniques and protocols. Testing was performed independently at 10 plant health laboratories analysing 15 samples (healthy, spiked or inoculated). The reliability of the diagnosis across all laboratories decreased with inoculum concentration. The accuracy of the different techniques, defined as (true positive diagnosis + true negatives)/total number of samples, was: 0.88, 0.92 and 0.92 respectively for isolation on King’s B, SNA and CCT media; 0.70, 0.72 and 0.66 for immunofluorescence performed with one monoclonal and two polyclonal antibodies commercially available; 0.79 and 0.83 for enrichment-ELISA with a commercial kit after enrichment in two media; 0.51 for conventional PCR and 0.69 for nested-PCR in a single tube, both techniques increasing their accuracy up to 0.78 and 0.86, respectively, after enrichment. The designed protocol is freely available at the web site: www.csl.gov.uk/science/organ/ph/diagpro

Identification of an Erwinia sp different from Erwinia amylovora and responsible for necrosis on pear blossoms

Necrotic pear blossoms (NPB) were found in several pear orchards of ‘Ercolini’ (‘Coscia’) and ‘Tendral’ in Turís, Valencia (Spain), in April 1999, during a routine survey, in an area free of Erwinia amylovora. The symptoms resembled those of fire blight [E. amylovora] but affected only blossoms and did not progress to other parts of the tree. Erwinia-like colonies were isolated from the necrotic blossoms that year and the following 2 years, and the morphology of the colonies on CCT medium of Ishimaru and Klos, King’s B medium, and sucrose nutrient agar was similar to that of Erwinia amylovora. The isolates were identified as an Erwinia sp. by their microbiological characteristics and showed API 20E, API 20NE, API ZYM, API 50CH patterns and fatty acid profiles similar, but not identical, to those of E. amylovora and Erwinia pyrifoliae. The isolates reacted as E. amylovora in immunofluorescence with several antisera and one monoclonal antibody (MAb) employed for E. amylovora detection, but did not react in the enzyme-linked immunosorbent assay against specific E. amylovora monoclonal antibodies. Polymerase chain reaction with primers from 23S rDNA sequences of E. amylovora were positive, but no signal was obtained with primers from plasmid pEa29 or from chromosomal DNA sequences of E. amylovora. The isolates were able to elicit the hypersensitive reaction on tobacco and tomato leaves and to induce necrosis in pear flowers, but were unable to develop typical fire blight symptoms in other organs of pear trees, and on various host plants of E. amylovora. The isolated Erwinia sp. strains are pathogenic and different from E. amylovora and other described bacterial species affecting pear trees

Laboratory-scale evaluation of composite spiked samples for erwinia amylovora detection in asymptomatic plants

Erwinia amylovora, the fire blight pathogen, can survive in plants and produce latent infections that can lead to false negative results in routine diagnostic procedures, since the pathogen can be present at very low numbers under the detection limit of available techniques. A protocol combining different techniques included in the EPPO diagnostic standard for E. amylovora (2013) was designed in order to determine the minimum amount of plants necessary for detecting the pathogen in asymptomatic plant material. To this aim, single samples of flowers and shoots from pear, apple, and loquat were spiked with an E. amylovora strain at different concentrations, and then analyzed by isolation, real-time PCR and ELISA-DASI. Thereafter, using the single spiked samples that were positive for any of the techniques used, composite spiked samples were prepared by adding to single spiked samples increasing amounts of plant material. Interestingly, whole results show that the most robust technique, less affected by the amount of plant material, was the real-time PCR after enrichment, since 101 -102 cfu ml-1 of E. amylovora could be detected in most cases, both in flowers and in shoots, regardless of the amount of simulated plants analyzed, 3, 5, 10 or 20. The enrichment was confirmed as a necessary step in the analysis of bulked samples to achieve a greater sensitivity. This study is a relevant contribution for programs of extensive screening surveys for prevention of fire blight disease, since it shows that samples can be processed as a pool, improving the detection of E. amylovora in asymptomatic samples

ACTA HORTICULTURAE

Fire blight was first detected in Spain in 1995. In the following years the disease was identified in seven out of the seventeen Spanish regions, in the Northeastern and Central parts of the country. Typical symptoms were observed in pears, apples, loquats, quinces and in ornamental hosts susceptible to fire blight. The spatial and temporal distribution of the foci suggest that plant material has probably been responsible of the introduction of Erwinia amylovora in some of the areas. Eradication measures have been taken consisting of intensive surveys, analysis and destruction of diseased trees. These measures have been very effective so far because, in most of the zones, the number of outbreaks have not increased exponentially. Consequently, six years after the first detection, Spain is still considered a protected fire blight country in the European Union, because E. amylovora has only been found in a few areas and has subsequently been eradicated. The characterization of 40 Spanish strains of E. amylovora was performed by conventional bacteriological tests, carbon utilization in miniaturized tests, serology, amplified fragment length polymorphism(AFLP) and macrorestriction fragment length polymorphism of genomic DNA followed by PFGE (MRFLP-PFGE). The obtained data support the hypothesis that E. amylovora has been introduced into Spain by infected plant material