First report of cucumber mosaic virus infecting Azorina vidalii in Azores Islands.

Virus diseases that occur in crops pose a major threat not only to global food security but also to wild plant communities growing in natural ecosystems (Jones, 2020, and references therein). In Azores (Portugal) little is known about viruses present on native flora and therefore, they have not been taken into consideration in conservation programs. Considering this, we selected Azorina vidalii (Campanulaceae), an endangered (IUCN) plant, endemic to Azores (Bilz, 2011), to survey for plant viruses. A. vidalii, the sole species of its genus, is often found in crevices with no soil accumulation on coastal cliffs, exposed to storms and sea spray, and is used as an ornamental. Leaves from 53 plants of A. vidalii from three populations from Terceira Island and three populations from Flores Island were randomly collected without obvious symptoms of virus infection, between the summer of 2021 and fall of 2022. RNA extraction was performed using the Plant/Fungi Total RNA Purification Kit (Norgen Biotek, Canada). RNA extracts from each population were pooled into six distinct composite samples (AvT1, AvT2, AvT3, AvF1, AvF4 and AvF5) and sent to Lexogen (Austria) for small RNA library preparation and High-Throughput Sequencing. Single-end RNA sequencing using Illumina NextSeq2000 system yielded between 10.1 M and 33.8 M raw reads. Adaptors and low-quality reads were removed with Trim Galore! and PRINSEQ. Trimmed reads were mapped to the genome phylogenetically nearest to A. vidalii available at the NCBI database (Adenophora triphylla). The resulting 2.5 M - 13.5 M unmapped reads were analysed with VirusDetect online version (database v248) (Zheng et al., 2017) for virus detection and identification. Sequences of cucumber mosaic virus (CMV) (contigs of up to 3045 nt for RNA1, 2917 nt for RNA2 and 2086 nt for RNA3) were identified in five (AvT1, AvT2, AvT3, AvF1 and AvF5) of the six composite samples and CMV satellite sequences (two contigs with 145 and 197 nt) were identified in only one (AvT1) of the composite samples. To confirm the presence of CMV, all samples were tested by two-step RT-PCR using primers targeting CMV-specific RdRp gene (513 bp) (Grieco et al., 2000), which retrieved 18 positive samples (34%). Nine samples were selected for Sanger sequencing (six out of 13 from Terceira and three out of five from Flores) based on digestion profile obtained with AluI and MboI. The resulting sequences (OQ176229-OQ176233, OQ732757-OQ732760) share an identity of 97.2-100% and BLASTn showed them to have 98.3-99.6% identity to CMV strain TN (AB176848). A Neighbour-Joining tree (Supplementary material) inferred in MEGA11 (Tamura et al., 2021) with 237 additional CMV-RdRp sequences, showed that A. vidalii CMV-derived isolates clustered together with reference strains of subgroup II, as those used by Roossinck (2002) for phylogenetic analysis of the 2a ORF. Besides CMV, tomato spotted wilt virus and polerovirus-associated RNAs sequences were found in one of the A. vidalii populations, but with lower coverage, and need to be further investigated. To the best of our knowledge, this is the first report of CMV infecting A. vidalli. CMV, genus Cucumovirus, is an agriculturally important virus and one of the most successful viruses known, infecting over 1,200 species of plants (Palukaitis & García-Arenal, 2003). In addition to A. vidalii being a CMV reservoir, which may have implications on adjacent crop fields, further research is needed to investigate the impact of CMV on A. vidalli fitness.

Morphological and genetic characterization of an emerging Azorean horse breed: the Terceira Pony.

The Terceira Pony is a horse indigenous to Terceira Island in the Azores. These horses were very important during the colonization of the island. Due to their very balanced proportions and correct gaits, and with an average withers height of 1.28 m, the Terceira Pony is often confused with a miniature pure-bred Lusitano. This population was officially recognized as the fourth Portuguese equine breed by the national authorities in January, 2014. The aim of this study was to analyze the morphology and the genetic diversity by means of microsatellite markers of this emerging horse breed. The biometric data consisted of 28 body measurements and nine angles from 30 animals (11 sires, 19 dams). The Terceira Pony is now a recognized horse breed and is gaining in popularity amongst breeders and the younger riding classes. The information obtained from this study will be very useful for conservation and management purposes, including maximizing the breed's genetic diversity, and solidifying the desirable phenotypic traits.

Genetic diversity of an Azorean endemic and endangered plant species inferred from inter-simple sequence repeat markers.

Knowledge of the levels and distribution of genetic diversity is important for designing conservation strategies for threatened and endangered species so as to guarantee sustainable survival of populations and to preserve their evolutionary potential. Picconia azorica is a valuable Azorean endemic species recently classified as endangered. To contribute with information useful for the establishment of conservation programmes, the genetic variability and differentiation among 230 samples from 11 populations collected in three Azorean islands was accessed with eight inter-simple sequence repeat markers. A total of 64 polymorphic loci were detected. The majority of genetic variability was found within populations and no genetic structure was detected between populations and between islands. Also the coefficient of genetic differentiation and the level of gene flow indicate that geographical distances do not act as barriers for gene flow. In order to ensure the survival of populations in situ and ex situ management practices should be considered, including artificial propagation through the use of plant tissue culture techniques, not only for the restoration of habitat but also for the sustainable use of its valuable wood.

Lignification of developing maize (Zea mays L.) endosperm transfer cells and starchy endosperm cells.

Endosperm transfer cells in maize have extensive cell wall ingrowths that play a key role in kernel development. Although the incorporation of lignin would support this process, its presence in these structures has not been reported in previous studies. We used potassium permanganate staining combined with transmission electron microscopy - energy dispersive X-ray spectrometry as well as acriflavine staining combined with confocal laser scanning microscopy to determine whether the most basal endosperm transfer cells (MBETCs) contain lignified cell walls, using starchy endosperm cells for comparison. We investigated the lignin content of ultrathin sections of MBETCs treated with hydrogen peroxide. The lignin content of transfer and starchy cell walls was also determined by the acetyl bromide method. Finally, the relationship between cell wall lignification and MBETC growth/flange ingrowth orientation was evaluated. MBETC walls and ingrowths contained lignin throughout the period of cell growth we monitored. The same was true of the starchy cells, but those underwent an even more extensive growth period than the transfer cells. Both the reticulate and flange ingrowths were also lignified early in development. The significance of the lignification of maize endosperm cell walls is discussed in terms of its impact on cell growth and flange ingrowth orientation.

Diagnosis of Theileria equi infections in horses in the Azores using cELISA and nested PCR.

Equine piroplasmosis is a tick-borne disease of equids that is often caused by the parasite Theileria equi. We applied competitive ELISA (cELISA) and nested PCR diagnostic methods to detect this parasite in horses by screening 162 samples from mainland Portugal where the parasite is endemic, and 143 from the Azores representing both native and imported horse populations. We found that 2.8% of the Azorean samples tested positive exclusively by cELISA, 1.4% tested positive only by nested PCR, and 9.1% tested positive using both tests. Samples from the native Terceira Pony population were negative for both tests. The parasite was more prevalent in samples from mainland Portugal when both test methods were considered (9.3% positive exclusively by cELISA, 1.9% positive exclusively by nested PCR, and 16.7% positive for both tests). To our knowledge, this is the first time that molecular techniques have been used to detect T. equi in the Azores and the first report of this parasite in the archipelago. Based on this study, it is clear that the import of horses into the Azores and the movement of horses between the islands must be controlled to reduce the risk of new infections, contributing to the protection of native horse populations such as the Terceira Pony population.

Biotechnology of temperate fruit trees and grapevines.

Challenges concerning fruit trees and grapevines as long lived woody perennial crops require adapted biotechnological approaches, if solutions are to be found within a reasonable time frame. These challenges are represented by the need for correct identification of genetic resources, with the foreseen use either in conservation or in breeding programmes. Molecular markers provide most accurate information and will be the major solution for questions about plant breeders rights. Providing healthy planting material and rapid detection of newly introduced pathogens by reliable methods involving serological and molecular biological tools will be a future challenge of increases importance, given the fact that plant material travels freely in the entire European Union. But also new breeding goals and transgenic solutions are part of the biotechnological benefits, e.g. resistance against biotic and abiotic stress factors, modified growth habits, modified nutritional properties and altered processing and storage qualities. The successful characterization of transgenic grapevines and stone fruit trees carrying genes of viral origin in different vectors constructed under ecological consideration, will be presented. Beyond technical feasibility, efficiency of resistance, environmental safety and Intellectual Property Rights, also public acceptance needs consideration and has been addressed in a specific project. The molecular determination of internal quality parameters of food can also be addressed by the use of biotechnological tools. Patient independent detection tools for apple allergens have been developed and should allow to compare fruits from different production systems, sites, and genotypes for their content of health threatening compounds.