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

[EN] 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.This work was supported by COST action FP1406 Pinestrength . The work of the Estonian team was supported by the Estonian Science Foundation grants PSG136 and IUT21-04. The work of Portuguese team from INIAV was financed by INIAV I.P. Institute. The work at U. Aveiro (Portugal) was financed by European Funds through COMPETE and National Funds through the Portuguese Foundation for Science and Technology (FCT) to CESAM (UID/AMB/50017/2013 POCI-01- 0145-FEDER-007638). The work of Slovenian team was financed through Slovenian Research Agency (P4-0107) and by the Slovenian Ministry of Agriculture, Forestry and Food (Public Forestry Service). 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Global Geographic Distribution and Host Range of Fusarium circinatum, the Causal Agent of Pine Pitch Canker

Fusarium circinatum, the causal agent of pine pitch canker (PPC), is currently one of the most important threats of Pinus spp. globally. This pathogen is known in many pine-growing regions, including natural and planted forests, and can affect all life stages of trees, from emerging seedlings to mature trees. Despite the importance of PPC, the global distribution of F. circinatum is poorly documented, and this problem is also true of the hosts within countries that are affected. The aim of this study was to review the global distribution of F. circinatum, with a particular focus on Europe. We considered (1) the current and historical pathogen records, both positive and negative, based on confirmed reports from Europe and globally; (2) the genetic diversity and population structure of the pathogen; (3) the current distribution of PPC in Europe, comparing published models of predicted disease distribution; and (4) host susceptibility by reviewing literature and generating a comprehensive list of known hosts for the fungus. These data were collated from 41 countries and used to compile a specially constructed geo-database. A review of 6297 observation records showed that F. circinatum and the symptoms it causes on conifers occurred in 14 countries, including four in Europe, and is absent in 28 countries. Field observations and experimental data from 138 host species revealed 106 susceptible host species including 85 Pinus species, 6 non-pine tree species and 15 grass and herb species. Our data confirm that susceptibility to F. circinatum varies between different host species, tree ages and environmental characteristics. Knowledge on the geographic distribution, host range and the relative susceptibility of different hosts is essential for disease management, mitigation and containment strategies. The findings reported in this review will support countries that are currently free of F. circinatum in implementing effective procedures and restrictions and prevent further spread of the pathogen

Gene-expression in forest-tree species assessed with microarrays as a tool

DNA microarray technology is a powerful tool in functional genomics study of many organisms, the forest−trees included. This method allow to examine simultaneously the changes in expression of thousands of genes and it is based on specific hybridization of cDNA probes from an organism with the DNA library immobilized in an array. The power of this method consists in miniaturization, automation and parallel study of large−scale genome from multiple samples. In forest science, the microarray technology has already been applied in some study of gene−expression in Populus, Pinus and Picea species and the number of new reports is still increasing every year. Since far, some gene−expression have been studied among woody plants in regard of development and growth processes (xylem, adventious−root and zygotic embryo formation, flowering, ripening, shooting of leaves), resistance mechanisms against biotic (fungi, viruses) and abiotic factors (drought, NaCl, elevated CO2 and O3 concentrations). Many practical applications of the microarray technique may concern the early selection in nursery of trees for morphologically valuable traits, the sustainable forest regeneration and the production of genetically transformed species for the chosen trait

Application of DNA markers against illegal logging as a new tool for the Forest Guard Service

DNA markers are currently the most precise tool for forest tree species identification and can be used for comparative analyses of plant material. Molecular diagnosis of evidence and reference material is based on comparing the structure of DNA markers duplicated in the PCR reaction and estimation of the DNA profiles obtained in studied wood samples. For this purpose, the microsatellite DNA markers are the most suitable tool because of their high polymorphism and accurate detection of structural changes in the genome. The analysis of tree stump DNA profiles let avoid timely collection of data such as tree age, diameter, height and thickness, although such a piece of information may advantageous in wood identification process. For each examined tree species, i.e. Pinus sylvestris L., Picea abies (L.) Karst., Quercus robur L. and Q. petraea (Matt.) Liebl., Fagus sylvatica L., Betula pendula L., and Alnus glutinosa L., wood identification was possible via the DNA profiles established on a basis of minimum 4 microsatellite nuclear DNA loci, and at least one cytoplasmatic (mitochondrial or chloroplast) DNA marker. Determination of the DNA profiles provided fast and reliable comparison of genetic similarity between material of evidence (wood, needles, leaves, seeds) and material of reference (tree stumps) in the forest. This was done with high probability (approximately 98– 99%)

Health condition and genetic differentiation level of beech in the Siewierz Forest District assessed with cpDNA markers

Beech decline phenomenon has been observed in Poland since the 1980s. The reasons are still unclear but the discovery of pathogens genus Phytophthora shed a new light on it. Stems as well as roots of beech trees were severely affected by P. citricola and P. cambivora. In order to find out the genetic background of infection, three loci of chloroplast microsatellite DNA were investigated in the genetic differentiation study of beeches growing in the Siewierz Forest District in Poland. Parameters of genetic diversity (h Nei) and differentiation (GST) in the chloroplast genome were estimated and compared between healthy and damaged trees. Healthy beeches were more heterozygous (h=0.243) than the damaged ones (h=0.113), and the distribution of cpDNA alleles was different between these two groups. Healthy trees were nearly 50% more genetically diversed than declining ones

Phosphite fertilizers as a plant growth stimulators in forest nurseries

In the situation of limited number of pesticides being approved for use in forest nurseries it is necessary to examine new products available on European market that stimulate growth and improve resilience and vitality of seedlings as a part of intgrated plant protection program. Paper presents test of fertilizer Actifos used in seven Polish nurseries in order to stimulate the growth of seedlings. In 64% of cases, Actifos stimulated increament of shoots (significantly for oaks, beeches, pines, spruces and alders) as well as for roots (significantly only for alders and oaks)

Changes in genetic structure of sessile oak (Quercus petraea [Matt.] Liebl.) natural regeneration in relation to maternal trees

The genetic variability of sessile oak (Quercus petraea L.) mature stand and its natural progeny was investigated. Comparison between genetic structure of parental and progeny trees was based on frequencies of nuclear microsatellite (SSR) alleles occurring in three DNA loci. A slight (4%) increase of gene pool between oak mature and progeny trees was revealed by heterozygosity level estimation, maintaining 86.3% of genetic similarity between generations. Also allele richness, partition probability of basic clustering and inbreed coefficient proved the high genetic similarity between parental and progeny of investigated oak trees. The gene flow occurred within the stands as far as rare alleles were transmitted or new ones appeared in the progenies. The results highlight the necessity of such a study for silvicultural measures taken in order to proceed natural or artificial regeneration in forest tree stand

Monitoring of diseases caused by pathogenic oomycetes using DNA analysis

Phytophthora species are best known as pathogens of agricultural crops (e.g. P. infestans), but there are also invasive pathogens destroying forest atands (e.g. P. ramorum in the USA) or even whole forest ecosystems (e.g. P. cinnamomi in Australia). Still, little is known about indiginous species, especially in wild ecosystems. Rhododendrons are well known as a reservuar for oomycetes’ development. The main objectives of the present study were to develop the new tool for the identification of pathogenic Phytophthora and Phytium based on DNA sequence analysis of the ITS1, 5.8S gene and ITS2 region. Rhododendrons leaves served as specific plant baits. In order to reach the goal the real time PCR, the nested PCR and the DNA sequencing of the rDNA ITS region were carried out. The genomic DNA was extracted form the symptomatic rhododendron leaves. Three distinct Oomycetes species: Phytophthora cactorum, Pythium mercuriale and em>Pythium recalcitrans were detected in rhododendron leaves and registered in GenBank