Xanthomonas arboricola pv. juglandis (Xaj) is the etiological agent of walnut (Juglans regia L.) bacterial blight (WBB), and has been associated to other walnut emerging diseases, namely brown apical necrosis (BAN) and vertical oozing canker (VOC), altogether severely affecting the walnut production worldwide. Despite the research efforts carried out to disclose Xaj genetic diversity, reliable molecular methods for rapid identification of Xaj isolates and culture-independent detection of Xaj in infected plant samples are still missing. In this work, we propose nine novel specific DNA markers (XAJ1 to XAJ9) selected by dedicated in silico approaches to identify Xaj isolates and detect these bacteria in infected plant material. To confirm the efficacy and specificity of these markers, dot blot hybridization was carried out across a large set of xanthomonads. This analysis, which confirmed the pathovar specificity of these markers, allowed to identify four broad-range markers (XAJ1, XAJ4, XAJ6, and XAJ8) and five narrow-range markers (XAJ2, XAJ3, XAJ5, XAJ7, and XAJ9), originating 12 hybridization patterns (HP1 to HP12). No evident relatedness was observed between these hybridization patterns and the geographic origin from which the isolates were obtained. Interestingly, four isolates that clustered together according the gyrB phylogenetic analysis (CPBF 1507, 1508, 1514, and 1522) presented the same hybridization pattern (HP11), suggesting that these nine markers might be informative to rapidly discriminate and identify different Xaj lineages. Taking into account that a culture-independent detection of Xaj in plant material has never been described, a multiplex PCR was optimized using markers XAJ1, XAJ6, and XAJ8. This triplex PCR, besides confirming the dot blot data for each of the 52 Xaj, was able to detect Xaj in field infected walnut leaves and fruits. Altogether, these nine Xaj-specific markers allow conciliating the specificity of DNA-detection assays with typing resolution, contributing to rapid detection and identification of potential emergent and acutely virulent Xaj genotypes, infer their distribution, disclose the presence of this phytopathogen on potential alternative host species and improve phytosanitary control.

Assessing a plant’s nutritional status and fertilizer rates and types that can optimize fruit quality and yield are critical in intensive apple orchards. The aim of this work was to identify correlations between nutrients in the different organs that allow the early diagnosis of the nutritional status and to assess the impact on the optimal nutrient content in apple leaves, as well as in the yield and quality of chemical and organic fertilization. Five orchards of ‘Gala’ were fertilized with different levels of NPK over a period of four years. Macro and micronutrients of buds, flowers, 45 and 90–110 days after full bloom (DAFB) leaves and 60 DAFB and 15 days before harvest (DBH) fruits were determined. Boron was the only element for which strong correlations, 0.7 < r < 0.9, were observed between all organ pairs. The fertilization treatments did not affect the nutrient concentrations in the leaves of 90–110 DAFB other than P, Ca and Mg and did not affect the macronutrients in the fruit. In one of the five orchards, the yield increased by 26% with double fertilization compared to standard fertilization and, for the other four orchards, the impact depended on the year. Fruit size was more related to crop load than to fertilization and TSS and firmness were not affected by the type or amount of fertilizers. Replacing part of the chemical fertilizer with organic materials did not affect productivity or fruit quality.

A rich heritage of traditional pear varieties is kept in national Portuguese collections. Out of these varieties, "Rocha" dominates national pear production. Although a noticeable phenotypic variation among clones of this variety has been reported, little is known about its genetic variability, as to date molecular studies have been performed on a single "Rocha" clone. Eleven Simple Sequence Repeats (SSR) markers were used to assess the genetic diversity of 130 local cultivars, 80 of them being "Rocha" clones. The results allowed the differentiation of 75 genotypes of which 29 are "Rocha". Three synonyms groups and four homonymous groups of other local varieties were confirmed. A Bayesian model-based clustering approach identified two distinct clusters. Using flow cytometry, six cultivars were found to be triploids. These results show high genetic variability among "Rocha" clones. In conclusion, there is a need for different "Rocha" clones to be preserved to enable the correct selection of the multiplication material.

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