Dispersal of Neophilaenus campestris, a vector of Xylella fastidiosa, from olive groves to over-summering hosts

[EN] Neophilaenus campestris is one of the spittlebugs (Hemiptera: Cercopoidea) able to transmit Xylella fastidiosa to olive trees. Considering its vector ability and the wide distribution of this species in Spain, N. campestris should be considered a serious threat to key crops such as olive, almonds and grapevines. Migration and dispersal abilities of insect vectors have profound implications in the spread of vector-borne diseases. Thus, knowledge on the dispersal ability of N. campestris is essential to model, predict and limit the spread of the diseases caused by X. fastidiosa. A mass-mark-recapture technique was developed to track between-field movements of N. campestris during its late spring migration from the ground cover grasses within olive groves to sheltered areas dominated by pine trees. The fluorescent dust used for marking did not affect the survival nor the flying ability of N. campestris. Spittlebug adults captured in olive groves during late spring were dusted with fluorescent colours and released in different locations. Six recapture samplings were performed 23¿42 days after release in 12 different sites located within a maximum distance of 2.8 km from the release point. Results indicated that N. campestris was able to disperse a maximum distance of 2,47 m in 35 days. Furthermore, flight mill studies showed that N. campestris was able to fly long distances, reaching 1.4 km in an 82-min single flight. Altogether, our findings suggest that eradication measures are of limited value because vectors are able to disperse rapidly over distances much longer than expected.Ministerio de Ciencia e Innovacion, Grant/Award Number: AGL2017-89604--R and PRE2018-083307; Comunidad de Madrid, Grant/Award Number: FP19-XYLELLALago, C.; Morente, M.; De Las Heras-Bravo, D.; Martí-Campoy, A.; Rodríguez-Ballester, F.; Plaza, M.; Moreno, A.... (2021). Dispersal of Neophilaenus campestris, a vector of Xylella fastidiosa, from olive groves to over-summering hosts. Journal of Applied Entomology. 145(7):648-659. https://doi.org/10.1111/jen.12888S648659145

Multilocus variable number of tandem repeat analysis reveals multiple introductions in Spain of Xanthomonas arboricola pv. Pruni, the causal agent of bacterial spot disease of stone fruits and almond

Xanthomonas arboricola pv. pruni is the causal agent of the bacterial spot disease of stone fruits, almond and some ornamental Prunus species. In Spain it was first detected in 2002 and since then, several outbreaks have occurred in different regions affecting mainly Japanese plum, peach and almond, both in commercial orchards and nurseries. As the origin of the introduction(s) was unknown, we have assessed the genetic diversity of 239 X. arboricola pv. pruni strains collected from 11 Spanish provinces from 2002 to 2013 and 25 reference strains from international collections. We have developed an optimized multilocus variable number of tandem repeat analysis (MLVA) scheme targeting 18 microsatellites and five minisatellites. A high discriminatory power was achieved since almost 50% of the Spanish strains were distinguishable, confirming the usefulness of this genotyping technique at small spatio-temporal scales. Spanish strains grouped in 18 genetic clusters (conservatively delineated so that each cluster contained haplotype networks linked by up to quadruple-locus variations). Furthermore, pairwise comparisons among populations from different provinces showed a strong genetic differentiation. Our results suggest multiple introductions of this pathogen in Spain and redistribution through contaminated nursery propagative plant material

Análisis de la presencia de Erwinia carotovora y de Pseudomonas cichoriien en agua de riego

Se comparó la eficacia de varios métodos de detección de dichas bacterias: siembra directa en medios comunes y en medios selectivos de erwinias y pseudomonas, enriquecimiento anaerobio en medio liquido selectivo y siembra posterior en medio selectivo, y la filtración por membrana y siembra posterior en medios comunes y selectivos. Se analizaron muestras durante los meses de enero, febrero, junio, septiembre y octubre. Para el aislamiento de E. carotovora subsp. carotovora (Ecc) los mejores resultados se obtuvieron con la siembra directa en medio selectivo y con el enriquecimiento anaerobio en medio selectivo. Con ambas técnicas se aisló dicha bacteria en muestras de agua de acequias de Valencia en cantidades de 0,1 a 1000 celulas/ml. También se identificaron E. c. pv. atroseptica (Eca) y E. chrysanthemi (Ech) en las muestras de agua. No se pudo aislar P. cichorii en las muestras analizadas

A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction

A simple and rapid method for extracting DNA from plants based on the use of an extraction buffer and precipitation with isopropanol was assayed to see its usefulness in detecting pathogenic bacteria in plant material. The method was compared with a phenol-chloroform standard procedure obtaining higher sensitivity levels of detection. The protocol developed was efficient for detecting a Gram-positive bacterium, Clavibacter michiganensis subsp. sepedonicus and several Gram-negative pathogenic bacteria (Ralstonia solanacearum, Erwinia amylovora, Xanthomonas axonopodis pv. citri) with a sensitivity of 10(2)-10(3) cfu/ml in spiked samples. It was also efficient to specifically identify such bacteria in naturally infected plant material. This procedure is proposed as a routine tool for detection of plant pathogenic bacteria, as well as in environmental microbiology and biotechnology studies

Advantages of an integrated approach for diagnosis of quarantine pathogenic bacteria in plant material

The accurate and reliable diagnosis of quarantine bacteria and their detection in asymptomatic material requires the use of an integrated approach based on the use of several techniques. This strategy is more expensive and time consuming but it combines conventional, serological and molecular tests in order to get better results. Bacterial isolation is required for having pure cultures necessary for target identification and to perform pathogenicity tests. However, its results can be negative, especially for slow growing pathogens, or when the bacteria in the samples are in the viable but non culturable state. Serological tests, especially indirect immunofluorescence (IIF) and ELISA are very useful for routine testing. Nevertheless, both can have specificity problems due to the poor quality of some available antibodies. The first one has usuallyan acceptable sensitivity and can be employed as a screening test. On the opposite, ELISA sensitivity is, in general, poor and a previous enrichment step is recommended. The use of PCR has improved the sensitivity and specificity of the diagnosis and in many different variants, is a very efficient method for rapid screening of samples. However, there are manycases where the presence of inhibitors gives false negative results, or for which the positive ones by PCR cannot be confirmed with anyother technique. Bioassays have also proved their usefulness, especially for recovering stressed or low populations of bacteria. Theyrequire few weeks for being performed when doing the biological enrichment in planta, but other possibilities such as in vitro growing plants or detached leaves, can also be used. The experience in applying this integrated methodology for diagnosis (isolation, serological tests, PCR and bioassays) has allowed to detect more accurately Erwinia amylovora, Ralstonia solanacearum and Xanthomonas axonopodis pv. citri in plant material

MDS representation of the distances among 264 strains of <i>X</i>. <i>arboricola</i> pv. <i>pruni</i>.

<p>Countries of origin are represented by different symbols. MDS axes 1–2, 1–3 and 1–4 described 59.3%, 47.4% and 44.4% of the total variation, respectively.</p

Genetic differentiation of Spanish strains of <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> from seven provinces (n ≥ 12) estimated by F_ST (above the diagonal) and R_ST (below the diagonal) pairwise comparisons based on MLVA data.

<p>Genetic differentiation of Spanish strains of <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> from seven provinces (n ≥ 12) estimated by F_ST (above the diagonal) and R_ST (below the diagonal) pairwise comparisons based on MLVA data.</p

Categorical minimum spanning tree from MLVA data (239 strains and 119 haplotypes) representing the genetic diversity of the Spanish strains of <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> in relation with its province of origin.

<p>Dot diameter represents the number of strains per haplotype. Numbers represent the 18 genetic clusters identified. Haplotypes in the same genetic cluster are up to quadruple locus variants. Thick links indicate single locus variants and thin links indicates double locus variants. Shaded areas show different clonal complexes in genetic clusters 1 and 2 and are identified with letters (A-E). Genetic clusters not enclosed in dashed lines are formed by a unique clonal complex or singleton. *Indicates the primary founder haplotype.</p

Genetic diversity estimated from MLVA data of <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> for the strains from 11 sampled Spanish provinces.

<p>Genetic diversity estimated from MLVA data of <i>X</i>. <i>arboricola</i> pv. <i>pruni</i> for the strains from 11 sampled Spanish provinces.</p