Phage-based biocontrol strategies can be an effective alternative to control Psa-induced bacterial canker of kiwifruit. The global production of kiwifruit has been seriously affected by Pseudomonas syringae pv. actinidiae (Psa) over the last decade. Psa damages both Actinidia chinensis var. deliciosa (green kiwifruit) but specially the susceptible Actinidia chinensis var. chinensis (gold kiwifruit), resulting in severe economic losses. Treatments for Psa infections currently available are scarce, involving frequent spraying of the kiwifruit plant orchards with copper products. However, copper products should be avoided since they are highly toxic and lead to the development of bacterial resistance to this metal. Antibiotics are also used in some countries, but bacterial resistance to antibiotics is a serious worldwide problem. Therefore, it is essential to develop new approaches for sustainable agriculture production, avoiding the emergence of resistant Psa bacterial strains. Attempts to develop and establish highly accurate approaches to combat and prevent the occurrence of bacterial canker in kiwifruit plants are currently under study, using specific viruses of bacteria (bacteriophages, or phages) to eliminate the Psa. This review discusses the characteristics of Psa-induced kiwifruit canker, Psa transmission pathways, prevention and control, phage-based biocontrol strategies as a new approach to control Psa in kiwifruit orchards and its advantages over other therapies, together with potential ways to bypass phage inactivation by abiotic factors.

Kiwifruit bacterial canker: an integrative view focused on biocontrol strategies.

Pinus pinaster
 Ait. susceptibility to pinewood nematode significantly differed among provenances, and the two Atlantic provenances of the Iberian Peninsula being the most affected. However, significant provenance × environment interaction was found. Provenance susceptibility was related to basal diameter, number of branches and oleoresin flow, and some climatic parameters.
 The pinewood nematode Bursaphelenchus xylophilus, native to North America, is an important pest affecting pine forests throughout Eurasia. In Europe, it has been detected in Portugal and Spain and is primarily associated with Pinus pinaster, an important Mediterranean tree species. We have investigated the differences in susceptibility among several P. pinaster provenances in the Iberian Peninsula and France, as well as their relationship to certain growth traits and physiological parameters. Three independent inoculation tests were performed on 3 to 4-year-old trees, followed by assessment of growth traits and physiological variables, along with time course destructive sampling for nematode quantification. The results showed significant differences among provenances for almost all growth traits, wilting, and mortality, though a significant provenance × environment interaction was also detected. Two Atlantic provenances, Noroeste-Litoral and Leiria, displayed the largest susceptibility to pinewood nematode. Changes in susceptibility to B. xylophilus between experiments were influenced by temperature and seasonality. Autumn precipitation and mean maximum temperature during summer at the original provenance sites could be related to provenance susceptibility. Noroeste-Litoral and Leiria were the most disease-affected provenances. This study emphasizes the need for further research on how tree growth stage influences susceptibility and on the possibility of cross-breeding among provenances.

/pdf/variation-in-pinewood-nematode-susceptibility-among-pinus-1nnju25yxb.pdf

Variation in pinewood nematode susceptibility among Pinus pinaster Ait. provenances from the Iberian Peninsula and France

In the last decade, the worldwide production of kiwi fruit has been highly affected by Pseudomonas syringae pv. actinidiae (Psa), a phytopathogenic bacterium; this has led to severe economic losses that are seriously affecting the kiwi fruit trade. The available treatments for this disease are still scarce, with the most common involving frequently spraying the orchards with copper derivatives, in particular cuprous oxide (Cu2O). However, these copper formulations should be avoided due to their high toxicity; therefore, it is essential to search for new approaches for controlling Psa. Antimicrobial photodynamic therapy (aPDT) may be an alternative approach to inactivate Psa. aPDT consists in the use of a photosensitizer molecule (PS) that absorbs light and by transference of the excess of energy or electrons to molecular oxygen forms highly reactive oxygen species (ROS) that can affect different molecular targets, thus being very unlikely to lead to the development of microbe resistance. The aim of the present study was to evaluate the effectiveness of aPDT to photoinactivate Psa, using the porphyrin Tetra-Py+-Me and different light intensities. The degree of inactivation of Psa was assessed using the PS at 5.0 μM under low irradiance (4.0 mW cm−2). Afterward, ex vivo experiments, using artificially contaminated kiwi leaves, were conducted with a PS at 50 μM under 150 mW cm−2 and sunlight irradiation. A reduction of 6 log in the in vitro assays after 90 min of irradiation was observed. In the ex vivo tests, the decrease was lower, approximately 1.8 log reduction at an irradiance of 150 mW cm−2, 1.2 log at 4.0 mW cm−2, and 1.5 log under solar radiation. However, after three successive cycles of treatment under 150 mW cm−2, a 4 log inactivation was achieved. No negative effects were observed on leaves after treatment. Assays using Cu2O were also performed at the recommended concentration by law (50 g h L−1) and at concentrations 10 times lower, in which at both concentrations, Psa was efficiently inactivated (5 log inactivation) after a few minutes of treatment, but negative effects were observed on the leaves after treatment.

/pdf/an-insight-into-the-photodynamic-approach-versus-copper-ydr9nfugov.pdf

An insight into the photodynamic approach versus copper formulations in the control of Pseudomonas syringae pv. actinidiae in kiwi plants.

Huanglongbing (HLB) is one of the most catastrophic citrus diseases. HLB pathogens Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf), and Ca. L. americanus (Lam) and their insect vectors Asian citrus psyllids (ACP, Diaphorina citri) and African citrus psyllids (AfCP, Trioza erytreae) are invading citrus producing regions where HLB was absent previously, including the Mediterranean basin. Importantly, the Mediterranean region is one of the two major citrus producing areas without HLB. Here, I provide a short perspective regarding a) information related to the distribution of the HLB pathogens and psyllid vectors in this region and neighboring countries, b) predicted distribution of the HLB for this region, c) the possible evolution of Liberibacters and how they could have established their relationship with different hosts, and d) approaches to fend off HLB. Specifically, I emphasized the following measurements: quarantine measures against AfCP, ACP, Las, Laf, and Lam, early detection and diagnosis of HLB and removal of inoculum, surveillance of AfCP and ACP and eradication of citrus psyllids once identified.

/pdf/a-perspective-of-citrus-huanglongbing-in-the-context-of-the-3suz9qbbcg.pdf

A perspective of citrus Huanglongbing in the context of the Mediterranean Basin

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.

/pdf/transferability-of-pcr-based-diagnostic-protocols-an-47j865319u.pdf

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

The disease huanglongbing (HLB), caused by the phloem-limited and psyllid-vectored ‘ Candidatus Liberibacter’ spp., is threatening the Mediterranean citrus industry. The African psyllid ( Trioza erytreae ) vector of the pathogen was detected in Madeira (Portugal) in 1994 and in the Canary Islands (Spain) in 2002, and its arrival in 2014 in northwest Spain and Portugal along the Atlantic coast instigated a biological alert, and a contingency management plan was developed. Extensive surveys were conducted in Canary Islands from 2009 to 2015 and in the northwest mainland Spain (Galicia) since the first detection of T. erytreae . Symptoms of the psyllid were observed in most sweet orange orchards of five islands in Canary Islands (93% of the inspected plots). In northwest mainland Spain, 65% of the inspected plots up to 2016 showed T. erytreae symptoms. During the surveys, ten leaves/tree from trees showing suspicious symptoms and from symptomless trees, as well as adult psyllids, were collected and analysed by real-time PCR using a universal ‘ Ca . Liberibacter’ spp. kit, according to the EPPO standard. Suspected samples from other surveyed Spanish regions free of the vector were also analysed. The few samples that were positive in the screening test were tested by species-specific real-time PCR protocols, and they did not show amplification. These data confirm that the Spanish citrus industry is currently free of the ‘ Ca . Liberibacter’ spp., but strict measures to prevent the introduction of this pathogen are required as the presence of T. erytreae increases the risk of its dissemination.

Survey of huanglongbing associated with ‘Candidatus Liberibacter’ species in Spain: analyses of citrus plants and Trioza erytreae

Pseudomonas syringae pv. actinidiae (Psa) e o agente causal do cancro bacteriano da actinidea, doenca mais grave desta cultura na atualidade que provoca importantes perdas economicas nos principais paises produtores de kiwi. Os sintomas mais caracteristicos da doenca incluem pequenas necroses castanhas rodeadas de um halo amarelo nas folhas e exsudados avermelhados nos caules, podendo conduzir a morte da planta. Em Portugal, a doenca foi detetada pela primeira vez em 2010, na Regiao do Entre Douro e Minho. Com este estudo pretende-se identificar e caracterizar isolados de Psa da Regiao do Entre Douro e Minho. A partir de material vegetal com sintomas da doenca proveniente de diferentes pomares de Actinidia deliciosa, isolou-se o agente patogenico e realizou-se a sua identificacao e caracterizacao morfologica e fenotipica atraves de testes LOPAT, utilizacao de fontes de carbono (sistema Biolog), perfis de acidos gordos e atraves de tecnicas moleculares (PCR e BOX-PCR). Os resultados obtidos mostram que os isolados estudados apresentam caracteristicas morfologicas identicas, existindo variabilidade ao nivel das caracteristicas fenotipicas. A caracterizacao molecular mostra que os isolados de Psa da Regiao do Entre Douro e Minho sao semelhantes, sendo identicas a populacoes de Psa conhecidas como virulentas (Psa3) identificadas na Europa e na Nova Zelândia.

/pdf/identificacao-e-caracterizacao-de-pseudomonas-syringae-pv-1apye88sa9.pdf

Identificação e caracterização de Pseudomonas syringae pv. actinidiae (Psa) na Região do Entre Douro e Minho (Portugal)

Method for the detection of Pseudomonas syringae pv. actinidiae (Psa) in asymptomatic branches of Actinidia sp.

Pseudomonas syringae pv. actinidiae (Psa) es la responsable del chancro bacteriano del kiwi, una enfermedad que se ha extendido rapidamente en los principales paises productores causando importantes perdidas economicas. Para intentar frenar la dispersion del patogeno y controlar el desarrollo de esta enfermedad, es necesario utilizar tecnicas de diagnostico precoz. El objetivo de este estudio fue aplicar un metodo de deteccion temprana en muestras asintomaticas. Se seleccionaron 20 plantas de kiwi (15 de Actinidia deliciosa, 3 de A. arguta y 2 de A. kolomikta) en las que se habia confirmado previamente la presencia de Psa, localizadas en Pontevedra (Espana). La recogida de muestras se realizo durante el reposo vegetativo, tomando de cada planta cuatro ramas del ano, con diferente orientacion, que se subdividieron en tres submuestras y fueron procesadas mediante lavado interno con una solucion salina tamponada. Tres alicuotas de cada submuestra se analizaron por tecnicas moleculares y se sembraron en medio King B. Las colonias obtenidas con morfologia tipica de Psa tambien se identificaron por tecnicas moleculares. La bacteria fue detectada en el 55% de las plantas muestreadas, pero no en todas las ramas de una misma planta. En las ramas que resultaron positivas, Psa se aislo y detecto en las tres submuestras analizadas.

/pdf/metodo-de-deteccion-de-pseudomonas-syringae-pv-actinidiae-58q6eoj6dl.pdf

Método de detección de Pseudomonas syringae pv. actinidiae (Psa) en ramas asintomáticas de actinidia

The reproductive strategy of the pinewood nematode (PWN), Bursaphelenchus xylophilus , is sexual amphimictic and oviparous. The incidence of intrauterine egg development and hatching in plant-parasitic nematodes is not a very common phe- nomenon. During the process of maintaining and breeding a B. xylophilus population isolated in Spain under laboratory conditions, evidence of matricidal hatching was observed. This is the first described case of this phenomenon in this species.

/pdf/first-report-of-matricidal-hatching-in-bursaphelenchus-17sjf46tm9.pdf

First Report of Matricidal Hatching in Bursaphelenchus xylophilus.

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