Genetic Diversity of Lecanosticta acicola in Pinus Ecosystems in Northern Spain.

Lecanosticta acicola is one of the most damaging species affecting Pinus radiata plantations in Spain. Favourable climatic conditions and unknown endogenous factors of the pathogen and host led to a situation of high incidence and severity of the disease in these ecosystems. With the main aim of understanding the factors intrinsic to this pathogenic species, a study of the population structure in new established plantations with respect to older plantations was implemented. The genetic diversity, population structure and the ability of the pathogen to spread was determined in Northern Spain (Basque Country), where two thirds of the total Pinus radiata plantations of Spain are located. From a total of 153 Lecanosticta acicola isolates analysed, two lineages were present; the southern lineage, which was prevalent, and the northern lineage, which was scarce. A total of 22 multilocus genotypes were detected with a balanced composition of both mating types and evidence for sexual reproduction. In addition to the changing environmental conditions enhancing disease expression, the complexity and diversity of the pathogen will make it difficult to control and to maintain the wood productive system fundamentally based on this forest species.

Temporal and Spatial Variation in the Population Structure of Spanish Fusarium circinatum Infecting Pine Stands.

Fusarium circinatum is an introduced fungal pathogen extended to the northern regions of Spain that causes Pine Pitch Canker (PPC) disease. In this work, we analyzed the pathogen's genetic diversity to study changes over time and space since the first outbreak occurred in Spain. Using six polymorphic SSR markers, 15 MLGs were identified in 66 isolates, and only three haplotypes were found with frequencies higher than one. In general, genotypic diversity was low and decreased shortly over time in the northwestern regions while maintained at País Vasco, where only one haplotype (MLG32) was detected 10 years. This population also included isolates of a single mating type (MAT-2) and VCGs identified in only two groups, while isolates from NW regions were of both mating types and VCGs represented in 11 groups. The existence of haplotype MLG32 maintained on time and widely distributed suggests its good adaptation to the environment and the host. Results showed that the pathogen in País Vasco remains clearly differentiated from other northwestern populations. This fact was supported with no evidence of migration among regions. Results are explained by the asexual reproduction, but also selfing at least to a lesser extent that leads to identification of two new haplotypes.

Weather Variables Associated with Spore Dispersal of Lecanosticta acicola Causing Pine Needle Blight in Northern Spain.

In the last decade, the impact of needle blight fungal pathogens on the health status of forests in northern Spain has marked a turning point in forest production systems based on Pinus radiata species. Dothistroma needle blight caused by Dothistroma septosporum and D. pini, and brown spot needle blight caused by Lecanosticta acicola, coexist in these ecosystems. There is a clear dominance of L. acicola with respect to the other two pathogens and evidence of sexual reproduction in the area. Understanding L. acicola spore dispersal dynamics within climatic determinants is necessary to establish more efficient management strategies to increase the sustainability of forest ecosystems. In this study, spore counts of 15 spore traps placed in Pinus ecosystems were recorded in 2019 and spore abundance dependency on weather data was analysed using generalised additive models. During the collection period, the model that best fit the number of trapped spores included the daily maximum temperature and daily cumulative precipitation, which was associated to higher spore counts. The presence of conidia was detected from January and maximum peaks of spore dispersal were generally observed from September to November.

Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems.

Diplodia tip blight is the most ubiquitous and abundant disease in Spanish Pinus radiata plantations. The economic losses in forest stands can be very severe because of its abundance in cones and seeds together with the low genetic diversity of the host. Pinus resinosa is not genetically diverse in North America either, and Diplodia shoot blight is a common disease. Disease control may require management designs to be adapted for each region. The genetic diversity of the pathogen could be an indicator of its virulence and spreading capacity. Our objective was to understand the diversity of Diplodia spp. in Spanish plantations and to compare it with the structure of American populations to collaborate in future management guidelines. Genotypic diversity was investigated using microsatellite markers. Eight loci (SS9-SS16) were polymorphic for the 322 isolates genotyped. The results indicate that Diplodia sapinea is the most frequent Diplodia species present in plantations of the north of Spain and has high genetic diversity. The higher genetic diversity recorded in Spain in comparison to previous studies could be influenced by the intensity of the sampling and the evidence about the remarkable influence of the sample type.

Influence of Temperature and Moisture Duration on Pathogenic Life History Traits of Predominant Haplotypes of Fusarium circinatum on Pinus spp. in Spain.

Pathogen life history traits influence epidemic development and pathogen adaptive ability to interact with their hosts in different environments. Reduced traits variation may compromise pathogen evolutionary potential, which is particularly important for introduced pathogens. Fusarium circinatum (cause of pine pitch canker) is an invasive fungal pathogen in Europe, with current distribution restricted to forest stands of Pinus radiata and Pinus pinaster in northern Spain and Portugal. This study aimed to quantify pathogenic traits of Spanish isolates of F. circinatum, with two of the strains representing the two dominant haplotypes in the Spanish population. Disease severity was measured on P. radiata, analyzing the influence of temperature and moisture duration on infection as well as the influence of temperature on spore germination, sporulation, and mycelial growth. Results indicated that the isolate representing the most common haplotype caused more severe disease on P. radiata at 25 and 30°C compared with the second most common haplotype but caused less severe disease at 15°C. Spore germination was higher for the most common haplotype, which produced more spores at 20 and 25°C. The isolate showed hyphal melanization at 5°C, which has been associated with survival and may be important because no resting structures have been described for F. circinatum. Our study determined that longer moisture periods during infection result in more severe disease from 7 to 24 h, regardless of the isolate virulence. This is the first study on virulence of the most abundant haplotypes of F. circinatum in Spain as affected by temperatures and moisture.

The transcriptome of Pinus pinaster under Fusarium circinatum challenge.

BACKGROUND: Fusarium circinatum, the causal agent of pitch canker disease, poses a serious threat to several Pinus species affecting plantations and nurseries. Although Pinus pinaster has shown moderate resistance to F. circinatum, the molecular mechanisms of defense in this host are still unknown. Phytohormones produced by the plant and by the pathogen are known to play a crucial role in determining the outcome of plant-pathogen interactions. Therefore, the aim of this study was to determine the role of phytohormones in F. circinatum virulence, that compromise host resistance. RESULTS: A high quality P. pinaster de novo transcriptome assembly was generated, represented by 24,375 sequences from which 17,593 were full length genes, and utilized to determine the expression profiles of both organisms during the infection process at 3, 5 and 10 days post-inoculation using a dual RNA-sequencing approach. The moderate resistance shown by Pinus pinaster at the early time points may be explained by the expression profiles pertaining to early recognition of the pathogen, the induction of pathogenesis-related proteins and the activation of complex phytohormone signaling pathways that involves crosstalk between salicylic acid, jasmonic acid, ethylene and possibly auxins. Moreover, the expression of F. circinatum genes related to hormone biosynthesis suggests manipulation of the host phytohormone balance to its own benefit. CONCLUSIONS: We hypothesize three key steps of host manipulation: perturbing ethylene homeostasis by fungal expression of genes related to ethylene biosynthesis, blocking jasmonic acid signaling by coronatine insensitive 1 (COI1) suppression, and preventing salicylic acid biosynthesis from the chorismate pathway by the synthesis of isochorismatase family hydrolase (ICSH) genes. These results warrant further testing in F. circinatum mutants to confirm the mechanism behind perturbing host phytohormone homeostasis.

Synergistic abiotic and biotic stressors explain widespread decline of Pinus pinaster in a mixed forest.

Global change potentially increases forest vulnerability. Different abiotic and biotic factors may interact to cause forest decline and accelerated tree mortality. We studied a mixed Mediterranean continental forest where Pinus pinaster Ait. (maritime pine) shows widespread decline to analyse the role of different abiotic and biotic factors on health status and growth dynamics both at the individual and plot levels. We also analysed stand composition and regeneration of tree species to check whether there is a change in species dominance. Fungal pathogens were seldom present and we detected no pervasive fungi or insect infestation and no presence of pathogens like Heterobasidion or Phytophthora. Infection of hemiparasite plants like Viscum album L. (mistletoe) can reduce leaf area and its abundance is generally considered an expression of host decline. Yet, the existence among declining trees of high defoliation levels without mistletoe, but not vice versa, suggests that defoliation in response to some abiotic stressor could be a predisposing factor preceding mistletoe infection. Compared to healthy trees, declining and dead trees exhibited higher defoliation rates, smaller needles and lower recent growth with steeper negative trends. Dead and declining trees showed similar negative growth trends since the early 1990s droughts, which we interpreted as early warning signals anticipating mortality of currently declining trees in the near future. Mortality of maritime pine extending across all size classes, the lower presence of this species in the smallest size classes and its lack of regeneration suggest it is potentially losing its current dominance and being replaced by other co-occurring, more drought-tolerant species. Our results unravelled that maritime pine decline seems to be mainly driven by a combination of predisposing and inciting abiotic factors (microenvironment and drought stress) and biotic factors (mistletoe). The absence of widespread fungal pathogens suggests that they may have a minor role on pine decline acting only eventually as contributing factors. Although there could be other interrelations among factors or other biotic agents at play, our results strongly suggest that water stress plays a major role in the decline process of the dominant species on an ecosystem with strong land-use legacies.

Incorporating exposure to pitch canker disease to support management decisions of Pinus pinaster Ait. in the face of climate change.

Climate change is gravely affecting forest ecosystems, resulting in large distribution shifts as well as in increasing infection diseases and biological invasions. Accordingly, forest management requires an evaluation of exposure to climate change that should integrate both its abiotic and biotic components. Here we address the implications of climate change in an emerging disease by analysing both the host species (Pinus pinaster, Maritime pine) and the pathogen's (Fusarium circinatum, pitch canker) environmental suitability i.e. estimating the host's risk of habitat loss and the disease`s future environmental range. We constrained our study area to the Spanish Iberian Peninsula, where accurate climate and pitch canker occurrence databases were available. While P. pinaster is widely distributed across the study area, the disease has only been detected in its north-central and north-western edges. We fitted species distribution models for the current distribution of the conifer and the disease. Then, these models were projected into nine Global Climate Models and two different climatic scenarios which totalled to 18 different future climate predictions representative of 2050. Based on the level of agreement among them, we created future suitability maps for the pine and for the disease independently, which were then used to assess exposure of current populations of P. pinaster to abiotic and biotic effects of climate change. Almost the entire distribution of P. pinaster in the Spanish Iberian Peninsula will be subjected to abiotic exposure likely to be driven by the predicted increase in drought events in the future. Furthermore, we detected a reduction in exposure to pitch canker that will be concentrated along the north-western edge of the study area. Setting up breeding programs is recommended in highly exposed and productive populations, while silvicultural methods and monitoring should be applied in those less productive, but still exposed, populations.

Adaptive potential of maritime pine (Pinus pinaster) populations to the emerging pitch canker pathogen, Fusarium circinatum.

There is a concern on how emerging pests and diseases will affect the distribution range and adaptability of their host species, especially due to different conditions derived from climate change and growing globalization. Fusarium circinatum, which causes pitch canker disease in Pinus species, is an exotic pathogen of recent introduction in Spain that threatens its maritime pine (P. pinaster) stands. To predict the impact this disease will have on the species, we examine host resistance traits and their genetic architecture. Resistance phenotyping was done in a clonal provenance/progeny trial, using three-year-old cuttings artificially inoculated with the pathogen and maintained under controlled environmental conditions. A total number of 670 ramets were assessed, distributed in 10 populations, with a total of 47 families, 2 to 5 half-sibs per family, and 3-7 ramets per clone. High genetic variation was found at the three hierarchical levels studied: population, family and clone, being both additive and non-additive effects important. Narrow-sense and broad-sense heritability estimates were relatively high, with respective values of 0.43-0.58 and 0.51-0.8, depending on the resistance traits measured (lesion length, lesion length rate, time to wilting, and survival). These values suggest the species' high capacity of evolutionary response to the F. circinatum pathogen. A population originated in Northern Spain was the most resistant, while another from Morocco was the most susceptible. The total number of plants that did not show lesion development or presented a small lesion (length<30 mm) was 224 out of 670, indicating a high proportion of resistant trees in the offspring within the analyzed populations. We found large differences among populations and considerable genetic variation within populations, which should allow, through natural or artificial selection, the successful adaptation of maritime pine to pitch canker disease.

Role of motility and chemotaxis in the pathogenesis of Dickeya dadantii 3937 (ex Erwinia chrysanthemi 3937).

Dickeya dadantii 3937 (ex Erwinia chrysanthemi), a member of the Enterobacteriaceae, causes soft rot in many economically important crops. A successful pathogen has to reach the interior of the plant in order to cause disease. To study the role of motility and chemotaxis in the pathogenicity of D. dadantii 3937, genes involved in the chemotactic signal transduction system (cheW, cheB, cheY and cheZ) and in the structure of the flagellar motor (motA) were mutagenized. All the mutant strains grew like the wild-type in culture media, and the production and secretion of pectolytic enzymes was not affected. As expected, the swimming ability of the mutant strains was reduced with respect to the wild-type: motA (94%), cheY (80%), cheW (74%), cheB (54%) and cheZ (48%). The virulence of the mutant strains was analysed in chicory, Saintpaulia and potato. The mutant strains were also tested for their capability to enter into Arabidopsis leaves. All the mutants showed a significant decrease of virulence in certain hosts; however, the degree of virulence reduction varied depending on the virulence assay. The ability to penetrate Arabidopsis leaves was impaired in all the mutants, whereas the capacity to colonize potato tubers after artificial inoculation was affected in only two mutant strains. In general, the virulence of the mutants could be ranked as motA

A biomarker for the identification of four Phaeoacremonium species using the beta-tubulin gene as the target sequence.

A real-time polymerase chain reaction (PCR) was developed for the rapid detection and identification of Phaeoacremonium species, the fungi associated with severe diseases in grapevines. A degenerate primer pair (F2bt-R1bt) with homology to the beta-tubulin gene was designed to be used in the amplification of 11 species of Phaeoacremonium. Four species-specific probes labelled with three different fluorescent dyes were designed to be used with the degenerate primers in a real-time PCR for the identification of Phaeoacremonium aleophilum, P. parasiticum, P. viticola and P. mortoniae. Combinations of two probes in a duplex real-time PCR allowed to detect and identify a mixture of Phaeoacremonium species and cross-amplifications were not detected. This method was applied to detect Phaeoacremonium species in eight wood fragments from grapevine plants naturally infected, and results were compared with those obtained with nested PCR and culturing on growth media. Real-time PCR detected Phaeoacremonium in 100% of the analysed fragments, whereas nested PCR did only in the 62% of them and requiring subsequent restriction fragment-length polymorphism analysis to identify the species. This method is a sensitive tool to detect and identify Phaeoacremonium species in infected grapevine wood. Real-time PCR assay defined here can be used in a plant nursery program to identify pathogen-free plants in order to manage Petri disease of grapevines.

PCR-based strategy to detect and identify species of Phaeoacremonium causing grapevine diseases.

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial beta-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.

Hipertensão arterial sistêmica e diabetes mellitus em idosos: práticas e controle e autocuidado na área de abrangência da equipe de saúde da família Baixa Quente no município de Minas Novas - Minas Gerais

A Hipertensão Arterial Sistêmica (HAS) e o Diabetes Mellitus(DM) são doenças de elevada prevalência na população brasileira. O presente estudo tem como objetivo elaborar um plano de intervenção educativa que possibilite a melhoria do controle da hipertensão arterial sistêmica e do diabetes mellitus tipo 2 no PSF Baixa Quente- Minas Novas. Para tal, foi realizado um diagnóstico situacional, definindo-se a prevalência das respectivas doenças e o risco cardiovascular dos indivíduos em estudo, com posterior cadastro dos mesmos no sistema HIPERDIA do SUS para acompanhamento. Definidos os principais problemas quanto ao tratamento das morbidades, foram propostas medidas, que vão desde políticas públicas pela secretaria de saúde da cidade até anúncios informativos no rádio local, com intuito de conscientização quanto a doença e suas complicações em si e o estímulo de práticas de alimentação saudável e exercício físico regular, que constituem tratamento não medicamentoso da doença. Acredita-se que a proposta de intervenção contribuirá de forma importante ao aprimoramento das condições de vida e de saúde da população em estudo.