Identification of novel genetic regions associated with resistance to European canker in apple.

BACKGROUND: European canker, caused by the fungal pathogen Neonectria ditissima, is an economically damaging disease in apple producing regions of the world - especially in areas with moderate temperatures and high rainfall. The pathogen has a wide host range of hardwood perennial species, causing trunk cankers, dieback and branch lesions in its hosts. Although apple scion germplasm carrying partial resistance to the disease has been described, little is still known of the genetic basis for this quantitative resistance. RESULTS: Resistance to Neonectria ditissima was studied in a multiparental population of apple scions using several phenotyping methods. The studied population consists of individuals from multiple families connected through a common pedigree. The degree of disease of each individual in the population was assessed in three experiments: artificial inoculations of detached dormant shoots, potted trees in a glasshouse and in a replicated field experiment. The genetic basis of the differences in disease was studied using a pedigree-based analysis (PBA). Three quantitative trait loci (QTL), on linkage groups (LG) 6, 8 and 10 were identified in more than one of the phenotyping strategies. An additional four QTL, on LG 2, 5, 15 and 16 were only identified in the field experiment. The QTL on LG2 and 16 were further validated in a biparental population. QTL effect sizes were small to moderate with 4.3 to 19% of variance explained by a single QTL. A subsequent analysis of QTL haplotypes revealed a dynamic response to this disease, in which the estimated effect of a haplotype varied over the field time-points. CONCLUSIONS: This study describes the first identified QTL associated with resistance to N. ditissima in apple scion germplasm. The results from this study show that QTL present in germplasm commonly used in apple breeding have a low to medium effect on resistance to N. ditissima. Hence, multiple QTL will need to be considered to improve resistance through breeding.

Transformation and gene-disruption in the apple-pathogen, Neonectria ditissima.

BACKGROUND: Apple production in Sweden and elsewhere is being threatened by the fungus, Neonectria ditissima, which causes a disease known as European canker. The disease can cause extensive damage and the removal of diseased wood and heavily infected trees can be laborious and expensive. Currently, there is no way to eradicate the fungus from infected trees and our knowledge of the infection process is limited. Thus, to target and modify genes efficiently, the genetic transformation technique developed for N. ditissima back in 2003 was modified. RESULTS: The original protocol from 2003 was upgraded to use enzymes currently available in the market for making protoplasts. The protoplasts were viable, able to uptake foreign DNA, and able to regenerate back into a mycelial colony, either as targeted gene-disruption mutants or as ectopic mutants expressing the green fluorescent protein (GFP). CONCLUSIONS: A new genetic transformation protocol has been established and the inclusion of hydroxyurea in the buffer during the protoplast-generation step greatly increased the creation of knockout mutants via homologous recombination. Pathogenicity assays using the GFP-mutants showed that the mutants were able to infect the host and cause disease.

Apple endophyte community is shaped by tissue type, cultivar and site and has members with biocontrol potential against Neonectria ditissima.

AIMS: This research aimed to identify factors influencing endophyte community structure in apple shoots and the bioactivity of cultured representatives against the fungal pathogen Neonectria ditissima. METHODS AND RESULTS: The endophyte community in leaves and stems of the apple cultivars 'Royal Gala' and 'Braeburn' were analysed by a cultivation-independent method (PCR-DGGE) which showed that tissue type, cultivar and site were determinant factors, with the endophyte taxa in 'Royal Gala' more variable than that in 'Braeburn', with leaf endophyte communities typically differing from stems in both cultivars. Seasonal (spring vs autumn) and regional (Nelson vs Hawke's Bay) variations were not obvious in woody stems. A collection of 783 bacterial and 87 fungal endophytes were recovered from leaves and stems of 'Royal Gala', 'Braeburn', 'Scilate' and/or 'Scifresh' from Nelson (nine sites) and Hawke's Bay (five sites) in spring and from Nelson (three sites) in autumn. A dual culture plating assay was used to test their ability to inhibit the mycelial growth of N. ditissima. Thirteen bacterial (mean of percent inhibition ≥20%) and 17 fungal isolates were antagonistic towards N. ditissima. These isolates belonged to the bacterial genera Bacillus and Pseudomonas, and fungal genera Chaetomium, Epicoccum, Biscogniauxia, Penicillium, Diaporthe, Phlyctema and two unidentified fungal isolates. CONCLUSIONS: Endophyte communities in apple shoots were determined by tissue type, cultivar and site. Endophytic bacterial and fungal isolates inhibiting N. ditissima growth in vitro were found. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provided new evidence of factors influencing apple endophyte community in New Zealand. Endophytes with potential to reduce N. ditissima infection were identified, with the potential to be developed into a biocontrol strategy for European canker.

Heterologous Expression of Ilicicolin H Biosynthetic Gene Cluster and Production of a New Potent Antifungal Reagent, Ilicicolin J.

Ilicicolin H is a broad-spectrum antifungal agent targeting mitochondrial cytochrome bc1 reductase. Unfortunately, ilicicolin H shows reduced activities in vivo. Here, we report our effort on the identification of ilicicolin H biosynthetic gene cluster (BGC) by genomic sequencing a producing strain, Neonectria sp. DH2, and its heterologous production in Aspergillus nidulans. In addition, a shunt product with similar antifungal activities, ilicicolin J, was uncovered. This effort would provide a base for future combinatorial biosynthesis of ilicicolin H analogues. Bioinformatics analysis suggests that the backbone of ilicicolin H is assembled by a polyketide-nonribosomal peptide synthethase (IliA), and then offloaded with a tetramic acid moiety. Similar to tenellin biosynthesis, the tetramic acid is then converted to pyridone by a putative P450, IliC. The decalin portion is most possibly constructed by a S-adenosyl-l-methionine (SAM)-dependent Diels-Alderase (IliD).

Generic concepts in Nectriaceae.

The ascomycete family Nectriaceae (Hypocreales) includes numerous important plant and human pathogens, as well as several species used extensively in industrial and commercial applications as biodegraders and biocontrol agents. Members of the family are unified by phenotypic characters such as uniloculate ascomata that are yellow, orange-red to purple, and with phialidic asexual morphs. The generic concepts in Nectriaceae are poorly defined, since DNA sequence data have not been available for many of these genera. To address this issue we performed a multi-gene phylogenetic analysis using partial sequences for the 28S large subunit (LSU) nrDNA, the internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), the large subunit of the ATP citrate lyase (acl1), the RNA polymerase II largest subunit (rpb1), RNA polymerase II second largest subunit (rpb2), α-actin (act), ß-tubulin (tub2), calmodulin (cmdA), histone H3 (his3), and translation elongation factor 1-alpha (tef1) gene regions for available type and authentic strains representing known genera in Nectriaceae, including several genera for which no sequence data were previously available. Supported by morphological observations, the data resolved 47 genera in the Nectriaceae. We re-evaluated the status of several genera, which resulted in the introduction of six new genera to accommodate species that were initially classified based solely on morphological characters. Several generic names are proposed for synonymy based on the abolishment of dual nomenclature. Additionally, a new family is introduced for two genera that were previously accommodated in the Nectriaceae.

A high-quality draft genome sequence of Neonectria faginata, causative agent of beech bark disease of Fagus grandifolia.

The draft genome of Neonectria faginata was sequenced with Oxford Nanopore and Illumina 250 bp paired-end sequencing technologies. The assembled genome was 42.9 Mb distributed over 24 contigs, with N50 of 4.4 Mb and 98.6% BUSCO completeness. This genome sequence will aid in understanding N. faginata population structure and ecology.

Neonectria bordenii sp. nov., a potential symbiote of the alder bark beetle, and its detection by quantitative PCR.

A taxonomically comprehensive perspective on the fungal associates of bark beetles (Coleoptera: Curculionidae: Scolytinae), and powerful molecular tools for detection of these fungi, are imperative to understanding bark beetle impacts on forest ecosystems. The most common filamentous fungi living alongside bark beetles in infested trees are ophiostomatoids (Ascomycota: Ophiostomatales and Microascales), yet an undescribed species of Neonectria (Neonectria sp. nov.; Ascomycota: Hypocreales) was recently identified cohabitating with the alder bark beetle, Alniphagus aspericollis, in red alder, Alnus rubra. The hardwood-infesting alder bark beetle is found throughout the range of its red alder host in the Pacific Coast region of North America and is associated with Neonectria sp. nov. in southwestern British Columbia, Canada. The aim of this study was to describe and name Neonectria sp. nov. and to develop a quantitative PCR (qPCR) assay to enable rapid detection of Neonectria sp. nov. from individual adult alder bark beetles and to define the distribution of the fungus. Neonectria sp. nov. was phylogenetically and morphologically determined to represent a distinct species closely related to N. ditissima and is described herein as Neonectria bordenii sp. nov. Neonectria bordenii was reliably detected from individual whole-beetle DNA extractions using a probe-based qPCR assay targeting multi-copy internal transcribed spacers (ITS) of nuclear ribosomal DNA. The qPCR assay amplified the fungus from 87.8 % (36/41) of individual alder bark beetle samples and was highly sensitive to N. bordenii, with a lower limit of detection of 1 × 10-6 ng/µL of culture DNA (or ~262 genome copies). Application of the qPCR assay developed in this study will expedite future research evaluating N. bordenii as a potential symbiote of the alder bark beetle. Citation: Wertman DL, Tanney JB, Hamelin RC, Carroll AL (2024). Neonectria bordenii sp. nov., a potential symbiote of the alder bark beetle, and its detection by quantitative PCR. Fungal Systematics and Evolution 13: 15-28. doi: 10.3114/fuse.2024.13.02.

Quantitative trait loci associated with apple endophytes during pathogen infection.

The plant phyllosphere is colonized by microbial communities that can influence the fitness and growth of their host, including the host's resilience to plant pathogens.There are multiple factors involved in shaping the assemblages of bacterial and fungal endophytes within the phyllosphere, including host genetics and environment. In this work, the role of host genetics in plant-microbiome assembly was studied in a full-sibling family of apple (Malus x domestica) trees infected with the fungal pathogen Neonectria ditissima. A Quantitative Trait Loci (QTL) analysis showed that there are multiple loci which influence the abundance of individual endophytic taxa, with the majority of QTL having a moderate to large effect (20-40%) on endophyte abundance. QTL regions on LG 1, 3, 4, 5, 10, 12, 13, 14 and 15 were shown to affect multiple taxa. Only a small proportion of the variation in overall taxonomic composition was affected by host genotype, with significant QTL hits for principal components explaining <8% and <7.4% of the total variance in bacterial and fungal composition, respectively. Four of the identified QTL colocalised with previously identified regions associated with tolerance to Neonectria ditissima. These results suggest that there is a genetic basis shaping apple endophyte composition and that microbe-host associations in apple could be tailored through breeding.

Susceptibility of Cider Apple Accessions to European Canker-Comparison between Evaluations in Field Planted Trees and Rapid Screening Tests.

European canker, caused by Neonectria ditissima Bres., is an economically damaging fungal disease of apple. Breeding new cultivars with a high level of resistance to European canker is the main aim of apple breeding programs. Observations of symptoms in naturally infected trees were carried out in 400 apple accessions in Asturias (north-western Spain). Young and mature field planted trees were assessed under conditions highly conducive for N. ditissima development. The results demonstrated that juvenile trees (4-year-old) barely showed noticeable symptoms whereas a wide variability in the levels of resistance among accession was observed in mature trees (14-year-old). Around 28% of the locally maintained collection resulted to be highly resistant to this disease in the region. Field observations on mature trees were also compared to four rapid screening tests based on artificially induced lesions. Spearman correlation analysis using two resistance parameters revealed that none of the methods resulted in similar rankings of cultivar susceptibility as some accessions that were ranked as resistant for a given test turned out to be susceptible in the field. This study might suggest that whilst conventional resistance phenotyping techniques are time-consuming, the outcomes of this approach still seem the preferred option to assess the response to N. ditissima of apple accessions.

Molecular and Pathogenic Characterization of Cylindrocarpon-like Anamorphs Causing Root and Basal Rot of Almonds.

Three almond nurseries were prospected in the South of Spain (Sevilla) to evaluate the sanitary status of the nursery plant material. Samples consisted of main roots, secondary roots and six-month-old basal stems 'GxN-15', 'Nemaguard', 'Cadaman', 'Rootpac-40' and 'Rootpac-20' rootstocks planted in the soil, and twigs of mother plants from 'Lauranne', 'Guara', 'Marcona', 'Marta' and 'Ferragnes' almond cultivars. Endophytic and potential pathogenic fungi were identified in mother plants and 70 Cylindrocarpon-like anamorph isolates were detected in the root system and basal stems of analyzed rootstocks. Based on partial sequencing of the his3 gene and multilocus phylogenetic analysis of the concatenated ITS, tub2, his3 and tef1-α partial sequences, seven Cylindrocarpon-like anamorph species were identified as Dactylonectria torresensis, D. novozelandica, D. macrodidyma, Ilyonectria liriodendri, Neonectria sp. 1, N. quercicola and Cylindrocladiella variabilis. Pathogenicity was assessed on young healthy detached twigs of 'Guara' almond cultivar and one-year-old 'Lauranne' potted almonds grafted onto 'GxN-15' rootstocks. Among the seven Cylindrocarpon-like anamorph species, I. liriodendri, Neonectria sp. 1 and N. quercicola were the most aggressive. Inoculated detached shoots developed necrotic lesions 15 days after inoculation. Inoculated trees showed sectorized necrosis in the main and secondary roots and the basal stem of the rootstock 5 months after inoculation. The most aggressive species were able to cause necrosis also in the grafted cultivar, and I. liriodendri, and N. quercicola also reduced the root biomass. This is the first report of Cylindrocarpon-like anamorph species causing root and basal rot of almonds.

Investigating Host Preference of Root Endophytes of Three European Tree Species, with a Focus on Members of the Phialocephala fortinii-Acephala applanata Species Complex (PAC).

Host preference of root endophytes of the three European tree species of Norway spruce (Picea abies), common ash (Fraxinus excelsior), and sycamore maple (Acer pseudoplatanus) were investigated in two forest stands near Zurich, Switzerland. The focus was placed on members of the Phialocephala fortinii s.l. (sensu lato)-Acephala applanata species complex (PAC), as well as other dark septate endopyhtes (DSE). PAC species were identified based on 13 microsatellite loci. Eleven PAC species were found, with Phialocephala helvetica, P. europaea being the most frequent. All but cryptic species 12 (CSP12) preferred Norway spruce as a host. Though very rare in general, CSP12 was most frequently isolated from maple roots. Regarding the abundant PAC species, P. helvetica and P. europaea, the preference of spruce as a host was least pronounced in P. europaea, as it was also often isolated from ash and maple. It is the first record of PAC found on common ash (Fraxinus excelsior). Cadophora orchidicola, a close relative of PAC, has frequently been isolated from ash. Various species of the Nectriaceae (Cylindrocarpon spp.) have often been isolated, particularly from maple roots. By comparison, Pezicula spp. (Cryptosporiopsis spp.) was found to be abundant on all three hosts. Phomopsis phaseoli exhibits a clear preference for spruce.

Apple endophyte community in relation to location, scion and rootstock genotypes and susceptibility to European canker.

European apple canker, caused by Neonectria ditissima, is a severe disease of apple. Achieving effective control is difficult with the currently available pesticides. Specific apple endophytes associated with cultivars may partially contribute to the cultivar response to the pathogen and thus could be used for disease management. We sought to determine whether the overall endophyte community differed among cultivars differing in their susceptibility to N. ditissima and to identify specific microbial groups associated with the susceptibility. Using Illumina MiSeq meta-barcoding, we profiled apple tree endophytes in 16 scion-rootstock combinations at two locations and quantified the relative contribution of scion, rootstock and location to the observed variability in the endophyte communities. Endophyte diversity was primarily affected by the orchard location (accounting for 29.4% and 85.9% of the total variation in the PC1 for bacteria and fungi, respectively), followed by the scion genotype (24.3% and 19.5% of PC2), whereas rootstock effects were small (<3% of PC1 and PC2). There were significant differences in the endophyte community between canker-resistant and -susceptible cultivars. Several bacterial and fungal endophyte groups had different relative abundance between susceptible and resistant cultivars. These endophyte groups included putative pathogen antagonists as well as plant pathogens. Their possible ecological roles in the N. ditissima pathosystem are discussed.

Draft genome sequence of Neonectria sp. DH2 isolated from Meconopsis grandis Prain in Tibet.

In the current study, we report the high-quality draft genome sequence of Neonectria sp. DH2, an endophytic fungus isolated from Meconopsis grandis Prain in Tibet. The whole genome is about 45.8 Mbp, with a GC content of 53%. A total of 14,163 genes are predicted to encode proteins, and 557 of them are considered as unique, as no matches are found in five gene databases. A neighbor-joining phylogenetic tree based on internal transcribed spacer (ITS) region sequences shows that Neonectria sp. DH2 was most closely related to Neonectria ramulariae. 47 biosynthetic gene clusters (BGC) were identified in Neonectria sp. DH2 genome, and only 5 BGCs shows significant similarities to previously reported BGCs. The presence of 42 unique BGCs in Neonectria sp. DH2 suggests that it has great potential to produce novel secondary metabolites.

Multi-Locus Phylogeny and Taxonomy of the Fungal Complex Associated With Rusty Root Rot of Panax ginseng in China.

Panax ginseng rusty root rot caused by the Ilyonectria species complex is a devastating disease, and it is one of the main factors contributing to the difficulty in continual cropping. Rusty root rot occurs in all ginseng fields, but little is known about the taxonomy of the fungal pathogen complex, especially Ilyonectria and Ilyonectria-like species. Rusty root rot samples were collected from commercial ginseng cultivation areas of China, and the pathogens were isolated and purified as single spores. Based on the combination analysis of multiple loci (rDNA-ITS, TUB, HIS3, TEF, ACT, LSU, RPB1, RPB2, and SSU) and morphological characteristics, the pathogens causing ginseng rusty root rot were determined. Fungal isolates were obtained from infected roots in 56 locations within main cultivation areas in China. A total of 766 strains were identified as Ilyonectria, Ilyonectria-like and Rhexocercosporidium species, including I. robusta (55.0%), I. communis (21.7%), I. mors-panacis (10.9%), I. pseudodestructans (2.0%), I. changbaiensis (1.3%), I. qitaiheensis (1.3%), Neonectria obtusispora (2.0%), Dactylonectria torresensis (0.5%), D. sp. (0.5%), and R. panacis (1.5%), and four novel species, Thelonectria ginsengicola (1.0%), T. jixiensis (1.0%), T. mulanensis (0.8%) and T. fusongensis (0.5%), with a total of 14 species. As the pathogen present in the highest proportion, I. robusta was the most prevalent and damaging species, unlike the pathogens reported previously. All of the examined strains were proven to cause ginseng rusty root rot. Our results indicate that the taxonomy of the fungal complex associated with ginseng rusty root rot includes Ilyonectria, Ilyonectria-like genera (Dactylonectria, Neonectria, and Thelonectria) and Rhexocercosporidium.

Ilicicolinic acids and ilicicolinal derivatives from the fungus Neonectria discophora SNB-CN63 isolated from the nest of the termite Nasutitermes corniger found in French Guiana show antimicrobial activity.

The fungus Neonectria discophora SNB-CN63 has been isolated from the nest of the termite Nasutitermes corniger found in French Guiana. From the ethyl acetate extract of fungal culture, bioassay guided fractionation led to the isolation of fourteen ilicicolinic acids and ilicicolinal derivatives. Their structures were elucidated by analyses of 1D and 2D NMR and MS spectroscopic data. All metabolites were tested against several microbial pathogens and six displayed antimicrobial activities with MIC <16 µg/mL on T. rubrum, Staphylococcus aureus and methicillin-resistant S. aureus. Based on their structural similarities, a common biosynthetic pathway is proposed for all isolated metabolites.

Variation in Host and Pathogen in the Neonectria/Malus Interaction; toward an Understanding of the Genetic Basis of Resistance to European Canker.

Apple canker caused by the phytopathogenic fungus Neonectria ditissima is an economically important disease, which has spread in recent years to almost all pome-producing regions of the world. N. ditissima is able to cross-infect a wide range of apple varieties and causes branch and trunk lesions, known as cankers. Most modern apple varieties are susceptible and in extreme cases suffer from high mortality (up to 50%) in the early phase of orchard establishment. There is no known race structure of the pathogen and the global level of genetic diversity of the pathogen population is unknown. Resistance breeding is underway in many global breeding programmes, but nevertheless, a total resistance to canker has not yet been demonstrated. Here we present preliminary data from a survey of the phylogenetic relationships between global isolates of N. ditissima which reveals only slight evidence for population structure. In addition we report the results of four rapid screening tests to assess the response to N. ditissima in different apple scion and rootstock varieties, which reveals abundant variation in resistance responses in both cultivar and rootstock material. Further seedling tests show that the segregation patterns of resistance and susceptibility vary widely between crosses. We discuss inconsistencies in test performance with field observations and discuss future research opportunities in this area.

Relación in vitro entre cylindrocarpon cylindroides y neonectria fuckeliana asociados a cancros fustales de pinus radiata en Chile / In vitro relationship between cylindrocarpon cylindroides and neonectria fuckeliana associated with stem cankers on pinus radiata in Chile

Desde el punto de vista estrictamente micológico, se ha discutido por décadas la relación de Cylindrocarpon cylindroides como anamorfo de Neonectria fuckeliana, lo que entre otras consecuencias, ha incidido en la controversial ubicación taxonómica de este teleomorfo. Se añade a esto que en patología forestal se ha brindado poca atención a la presencia de C. cylindroides dentro del ciclo de la enfermedad causada por Neonectria fuckeliana en coníferas, razón por la cual los estudios específicos respecto de este anamorfo han sido también muy escasos. El presente trabajo tuvo como objetivo verificar la relación entre C. cylindroides y Neonectria fuckeliana, asociados a cancros en troncos de Pinus radiata en Chile, mediante el análisis de las características macro y micromorfológicas que ellos manifiestan en cultivos in vitro, además de análisis de tipo molecular. Se pudo constatar que ambos agentes dieron origen a colonias con idénticas características macro y micromorfológicas, destacando entre éstas la producción de abundantes conodióforos y microconidios del tipo Acremonium, el cual ya ha sido señalado en anteriores estudios como estado anamorfo de Neonectria fuckeliana. El que este mismo estado asexual del tipo Acremonium haya sido la manifestación in vitro tanto de C. cylindroides como de Neonectria fuckeliana, permitió confirmar la relación existente entre estos dos agentes en estudio, la que también fue ratificada mediante los análisis moleculares realizados.

From the point of view strictly mycological has been discussed for decades the relationship of Cylindrocarpon cylindroides as anamorph of Neonectria fuckeliana, which among other consequences, has influenced the controversial taxonomic status of this teleomorph. Add to this that in forest pathology has given little attention to the presence of C. cylindroides within the cycle of the disease caused by Neonectria fuckeliana on conifers, why specific studies regarding this anamorph have been very few. This study aimed to verify the relationship between C. cylindroides and Neonectria fuckeliana associated with cankers on trunks of Pinus radiata in Chile, by analyzing the macro and micromorphological characteristics they manifest in cultures in vitro, further analysis type molecular. It was found that both agents gave rise to colonies with identical macro and micromorphological characteristics, highlighting among them the production of abundant microconidia and conidiophores of Acremonium type, which has already been noted in previous studies as anamorph state of Neonectria fuckeliana. The asexual state that this same type of Acremonium has been the in vitro demons-tration of both C. cylindroides as Neonectria fuckeliana, allowed us to confirm the relationship between these two agents in the study, which was also ratified by the molecular analyzes performed.

Detección del sinanamorfo Cylindrocarpon cylindroides var. Tenue sobre cancros causados por Neonectria fuckeliana en Pinus radiata / Detection of sinanamorph Cylindrocarpon cylindroides var. tenue on Neonectria fuckeliana cankers in Pinus radiata

El presente trabajo tuvo por finalidad reportar y analizar la presencia de estructuras fungosas (esporodoquios) sobre cancros causados por Neonectria fuckeliana en árboles de Pinus radiata en Chile. Las muestras fueron colectadas en las Regiones de La Araucanía y de Los Ríos y fueron analizadas en el laboratorio. Los resultados de los análisis morfológicos y taxonómicos permitieron concluir que los esporodoquios hallados correspondían al sinanamorfo de Neonectria fuckeliana (forma cylindrocarpon cylindroides var tenue), siendo el primer reporte en el país. Estos antecedentes contribuyen al conocimiento y comprensión del ciclo patológico y epidemiológico de Neonectria fuckeliana en plantaciones de Pinus radiata.

The present work was aimed at reporting and analyzing the presence of fungal structures (sporodochia) present on cankers caused by Neonectria fuckeliana in Pinus radiata trees in Chile. The samples were collected in the regions of La Araucanía and Los Ríos and analyzed in the laboratory. The results of morphological and taxonomic analysis led to the conclusion that thesporodochia found corresponded to sinanamorph of Neonectria fuckeliana (cylindrocarpon cylindroides var. tenue morph), the first report in the country. This work contribute to understanding and comprehension of the disease cycle and epidemiology of Neonectria fuckeliana on plantations of Pinus radiata.

Caracterización morfológica de Acremonium sp. asociado a Neonectria fuckeliana en Pinus radiata en Chile / Morphological characterization of Acremonium-like asociated with Neonectria fuckeliana in Pinus radiata in Chile

En este estudio se realizaron los primeros aislamientos del estado asexual del patógeno Neonectria fuckeliana asociado a cancros o ®revirado del pino¼ en plantaciones de Pinus radiata. Se caracterizaron morfológicamente las cepas del sinanamorfo semejante a Verticillium (Acremonium), obtenidas en cultivo in vitro a partir de peritecios. El material para los aislamientos consistió en trozos de corteza de P. radiata con presencia de peritecios, colectados en Toltén, región de La Araucanía, lugar donde se realizó el primer reporte de N. fuckeliana en Chile. Se utilizaron diez cepas del semejante a Acremonium para la caracterización morfológica, mediciones de estructuras fúngicas, ritmo de crecimiento in vitro y morfología de las colonias. Las colonias presentaron un micelio flocoso y ralo de bordes blanquecinos e irregulares, destacándose tres tipos de colonias, blancas, naranjo oscura y naranjo claro. Taxonómicamente, las cepas coinciden con las estructuras mencionadas en la literatura, caracterizándose por la presencia de glioconidios. Las fialides midieron entre 7 – 78,4 x 1,4 - 4,9 ìm. Los conidios, de formas ovoides y algunas bicelulares, midieron entre 4,2 - 8,4 x 2,6 - 3,5 ìm. El ritmo de crecimiento in vitro fue lento, completando su desarrollo a los 19 días con un promedio de 71 +/- 0,3 mm de diámetro, a una tasa de crecimiento diario de 3,8 mm. Los resultados obtenidos hacen necesario futuros estudios de carácter molecular para analizar la variabilidad genética poblacional que puede presentarse en Chile.

First Neonectria fuckeliana isolates in Chile. Strains of Acremonium obtained from in vitro peritecia culture were morphologically characterized. The samples were collected in Toltén, La Araucanía region, were Neonectria fuckeliana was first reported in Chile. The material used for the isolation came from pieces of Pinus radiata bark with peritecia. Ten Acremonium-like strains were used for characterization of fungal structures, in vitro growth and strains morphology. The colonies were a floccose mycelium and thin edges whitish and irregular, varying color highlighting three types of strains, white, dark orange and pale orange. Taxonomically, the strains match the structures referred in the literature, characterized by the presence of gliospores. The phialides dimensions ranged from 7 to 7.8 ìm long and 1.4 to 4.9 ìm wide. The conidio of ovoid shapes and some bicelular measured between 4.2 to 8.4 ìm in length and width 2.6 and 3.5 ìm. In vitro growth rates were slow, the complete development take19 days with a daily growth average of 71 +/-3 mm in diameter, at a rate of 3.8 mm. It is necessary future molecular studies to analyze the population genetic variation that may occur in Chile.