Cryphonectria Species Research Articles

Life after isolation: Reconstructing the phylogenetic identity and a centennial chronology of historical Cryphonectria isolates

AbstractFungi of the genus Cryphonectria are of special interest to tree pathologists because they cause bark disease in numerous species of the Fagaceae and Betulaceae. Despite this special attention, several Cryphonectria species were not recognized as such for a long time. This also affected the identity of eight isolates with outdated names that have been deposited in our laboratory since 1954. The present study reconstructs the history of the Cryphonectria cultures M282–M289 using primary bibliographic sources. To verify the species identity, all isolates were DNA barcoded and taxonomic affiliation was assessed using a phylogenetic approach. The taxonomic identity combined with the history of the isolates confirms that the chestnut blight epidemic (Cryphonectria parasitica) in Spain was caused by the planting of Asian chestnuts and that the first documented case of bark canker in 1940 was caused by Cryphonectria radicalis on Japanese chestnuts imported from France. The history of isolate M289 could be traced back to CBS 165.32, which proved to be the oldest preserved isolate of the rare Cryphonectria naterciae, confirming the presence of this fungus in Europe for at least 100 years. Based on the ITS barcoding of the 32‐year‐old type specimen, Cryphonectria decipiens must be reclassified as a later synonym of Cryphonectria radicalis. The reconstruction of the history of isolates M282–M289 takes us on a scientific journey from Italy to France, Spain and Portugal in the early twentieth century and offers new insights into the historical context of the invasion of Cryphonectria parasitica in Europe.

Cryphonectria carpinicola discovered in Japan: first report of the sexual state on Carpinus tree.

Cryphonectria carpinicola is an ascomycetous fungus that has been regularly found in its asexual form on European hornbeam (Carpinus betulus) in Europe over the past two decades. Here we describe the discovery of C. carpinicola in Japan and report for the first time its sexual state on Carpinus species. No symptomatic trees were observed, but stromata were found saprotrophically on broken branches of Carpinus species on the forest floor. The sexual structures of C. carpinicola resembled that of other Cryphonectria species and strongly resembled those of the closely related species C. radicalis. A phylogenetic tree based on the internal transcribed spacer sequences showed monophyly for the Japanese and European isolates of C. carpinicola. Further studies on the distribution and host range of C. carpinicola in Japan and on the life history strategies of this fungus are needed.

Cryphonectria carpinicola sp. nov. Associated with hornbeam decline in Europe

Since the early 2000s, reports on declining hornbeam trees (Carpinus betulus) are spreading in Europe. Two fungi are involved in the decline phenomenon: One is Anthostoma decipiens, but the other etiological agent has not been identified yet. We examined the morphology, phylogenetic position, and pathogenicity of yellow fungal isolates obtained from hornbeam trees from Austria, Georgia and Switzerland, and compared data with disease reports from northern Italy documented since the early 2000s. Results demonstrate distinctive morphology and monophyletic status of Cryphonectria carpinicola sp. nov. as etiological agent of the European hornbeam decline. Interestingly, the genus Cryphonectria splits into two major clades. One includes Cry. carpinicola together with Cry. radicalis, Cry. decipiens and Cry. naterciae from Europe, while the other comprises species known from Asia-suggesting that the genus Cryphonectria has developed at two evolutionary centres, one in Europe and Asia Minor, the other in East Asia. Pathogenicity studies confirm that Car. betulus is a major host species of Cry. carpinicola. This clearly distinguished Cry. carpinicola from other Cryphonectria species, which mainly occur on Castanea and Quercus.

Testing the Pathogenic Potential of Cryphonectria parasitica and Related Species on Three Common European Fagaceae

Invasions by non-native pathogens represent a major threat to managed and natural ecosystems worldwide. Although necessary for adopting preventive strategies, the identification of invasive species before they are introduced is particularly difficult. Indeed, most pathogenic species that have become established in the last decades were first described only after they became invasive. To prevent further biological invasions, not only the early identification of potential new invasive plant pathogens is crucial, but also the assessment of their potential host range. In this study, we determined the pathogenicity and the saprotrophic ability of three Cryphonectria species towards three potential hosts in the family Fagaceae. For this, seedlings and dormant stems of European chestnut (Castanea sativa), pedunculate oak (Quercus robur) and European beech (Fagus sylvatica) were inoculated with different genotypes of C. parasitica (Asian species, invasive in Europe), C. naterciae (European species), and C. japonica (Asian species, not present in Europe). Lesion growth was measured and mortality assessed for four months. The highest damage was caused by C. parasitica on European chestnut, while C. japonica and C. naterciae induced significantly smaller lesions on this host species. All three Cryphonectria species did not grow saprophytically on F. sylvatica and Q. robur, but successfully colonized dormant stems of C. sativa. In the context of biological invasions, our study shows that the Asian C. japonica most likely represents a much less severe threat than C. parasitica for the tested European host species. Nonetheless, the ability of C. naterciae and C. japonica to saprotrophically colonize fresh chestnut wood may suggest that they could become established in chestnut forests and eventually infect weakened chestnut trees or other hosts not tested in this study.

Identification and pathogenicity of Cryphonectriaceae species associated with chestnut canker in China

Cryphonectriaceae species cause serious canker diseases on chestnut, oak and eucalypt trees. Recently, canker symptoms with typical orange fruiting bodies were observed on Chinese chestnut and oak trees in Hebei, Hubei, Shaanxi and Shandong Provinces in China. In the present study, isolates of these fungi were identified based on phylogenetic and morphological evidence, and their pathogenicities were tested on detached chestnut (Castanea mollissima) branches. DNA sequence comparisons of the internal transcribed spacer (ITS) regions and two regions of β‐tubulin (TUB1/TUB2) indicate that these isolates represent five species in the Cryphonectriaceae, viz. Cryphonectria japonica, Cryphonectria parasitica, Aurantiosacculus castaneae sp. nov., Cryphonectria neoparasitica sp. nov. and Endothia chinensis sp. nov. The sexual morph of Aurantiosacculus is discovered for the first time and can be distinguished from the other genera in Cryphonectriaceae by dark brown ascospores and tubiform appendages at both ends. Cryphonectria neoparasitica sp. nov. is different from the other Cryphonectria species by its aseptate ascospores. Endothia chinensis sp. nov. is the sole species in Endothia infecting the host genus Castanea. Additionally, it is much smaller than E. gyrosa and narrower than E. singularis in ascospores. The inoculation results showed that these five Cryphonectriaceae fungi isolated from chestnut or oak are all pathogenic to tested chestnut branches. Cryphonectria parasitica appears to be the most aggressive fungus, followed by C. neoparasitica sp. nov., C. japonica, E. chinensis sp. nov. and A. castaneae sp. nov.

A multiplexed genotyping assay to determine vegetative incompatibility and mating type in Cryphonectria parasitica

The highly destructive chestnut blight disease can be successfully controlled by infecting the virulent strain of C. parasitica with the hyperparasitic mycovirus CHV-1. The artificial application of the virus-induced hypovirulence, however, requires that the vegetative compatibility (vc) diversity in the target C. parasitica population be determined beforehand. Conventional vc type determination by pairing the unknown isolate with tester strains of known vc types is time consuming and not always reliable. A genotyping assay was developed that uses DNA from C. parasitica isolates and discriminates the six known di-allelic vic loci and the two mating idiomorphs in two fluorescence-labeled multiplex PCRs. Validation was conducted by successfully genotyping the 74 known European vc tester strains and a conventionally characterized natural population of C. parasitica. Cross-species amplification tests with three Cryphonectria species (C. japonica, C. naterciae, and C. radicalis) revealed a high amplification specificity for C. parasitica, while the analytical sensitivity of the assay was established at 20 pg per reaction of genomic DNA. In conclusion, this fluorescent multiplex genotyping assay offers a simple and high-throughput tool for the characterization of the vegetative compatibility and mating type of C. parasitica at population level, which is particularly important for the application of mycovirus-mediated biological control of chestnut blight.

Early detection of Cryphonectria parasitica by real-time PCR

The development and validation of a real-time PCR method for the detection of Cryphonectria parasitica in bark tissues is described. The selected region in the genome was a fragment of the internal transcribed spacer region. The DNA extraction and PCR conditions were optimized to be routinely applicable to fresh and dried bark. The sensitivity of the assay allowed the detection of 2 fg of genomic DNA, equivalent to one spore of the pathogen. There was no cross-reaction with closely related Cryphonectria species. The use of droplet digital PCR (ddPCR) confirmed the high sensitivity of the real-time PCR method, and its capacity to be used as an early detection method. A survey was conducted in Belgium on chestnut trees displaying cankers using isolation and the real-time PCR method. Both methods provided the same results for 83% of the samples (either negative or positive results). For the remaining samples, C. parasitica was not isolated, while amplifications close to the end of the PCR run (resulting in late Cycle threshold (Ct) values) were observed with the real-time PCR. Some of these samples were collected in stands where C. parasitica had already been detected on other chestnut trees, or in sites close to the infected stands. The application of this method may help plant protection services to detect new introductions of the pathogen within areas still free of chestnut blight and to prevent its establishment. It may also be useful for scientists involved in the epidemiology of the disease.

Two novel species of Cryphonectria from Quercus in China

Cryphonectria species associated with canker disease are presently difficult to identify, because of lack of ex-type cultures with molecular data and few distinguishable characters. During our survey about chestnut blight in western China, Cryphonectria parasitica was confirmed as the most important canker pathogen based on ten morphologically atypical isolates. Two novel species with orange stromata are introduced based on multigene sequence (ITS, TUB1, TUB2) analyses. This is the first study that has established the Cryphonectria species associated with oak canker in China using a multiphasic approach.

First report of Diplodia seriata causing canker on Castanea sativa in India

In India, sweet chestnut ( Castanea sativa ) trees are affected by pathogenic fungi including species of Cryphonectria and Gnomoniopsis (Dar & Rai, 2014; Dar & Rai, 2015). During a survey of fungal pathogens associated with cankers…

Distribution and severity of damage by Cryphonectria parasitica in the chestnut stands in Guilan province, Iran

SummaryChestnut blight caused by Cryphonectria parasitica has recently been reported from Guilan province, the only region with natural chestnut (Castanea sativa) stands in Iran. During the past few years, chestnut stands in Iran have been seriously threatened by the fungus, incidence of the disease is increasing and vast canopies are reduced to sprouts. As yet, there is no report on the disease distribution and severity in this region. Six sites from three main growing regions of chestnut in Guilan province were selected for investigation. We report occurrence and evaluation of the damage of the disease caused by C. parasitica. To evaluate the scale of damage, the investigated trees were classified into six categories based on the degree of crown damage, the number of canker wounds and the presence of the fungus. Index of health condition was calculated for all sites. During this study, a total of 250 isolates of Cryphonectria species were obtained, of which 232 isolates were C. parasitica and 18 were Cryphonectria radicalis. Castanea parasitica was observed in all regions. Index of health condition was scored from 0 to 6 with 0 being disease free and six being the most severe infection. Index results in the investigated sites varied between 0.69–5.45 and 0.93–5.55 for years 2006 and 2007, respectively. The highest damage was found in Doran (IH = 5.55), which is located some 100 km away from Shahbalutmahalleh, the site with lowest damage (IH = 0.93). This is the first extended report on aspects of chestnut blight in Iran.

Molecular diversity of chrysoviruses in Korean isolates of a new fungal species, Cryphonectria nitschkei

Genetic diversity of the chrysovirus within the four fungal strains was analyzed by comparing the full-length sequences of cloned chrysoviral genes encoding the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP). Because the morphological characteristics of four chrysovirus-infected Cryphonectria spp. strains were different, strain identification was conducted via sequence comparison of the internal transcribed spacers (ITSs) of the fungal rRNA gene. Phylogenic analysis of the ITS regions revealed that the four strains were closely clustered with the reference strain of Cryphonectria nitschkei, while they were more distantly related to other common Cryphonectria species, indicating that they were likely C. nitschkei. Sequence comparison among chrysoviruses from Korean C. nitschkei strains revealed that similarities of the RdRp and CP genes ranged from 98% to 100% and from 95% to 100%, respectively, at the protein level. Their corresponding nucleotide sequences showed 97% to 100% and 84% to 100% identities, respectively. Compared to RdRp, the CP gene had more divergence, suggesting the presence of genes possessing different evolutionary rates within the chrysovirus genome. Sequence comparisons with other known chrysoviruses showed that the four Korean chrysoviruses were clustered together at the next lineage level. Discovering why two strains (bsl31 and bsl32) containing identical ITS sequences and chrysoviruses display different phenotypes should prove interesting.

Microthia, Holocryphia and Ursicollum, three new genera on Eucalyptus and Coccoloba for fungi previously known as Cryphonectria

Cryphonectria havanensis is a fungus associated with Eucalyptus species in Cuba and Florida (U.S.A.). Until recently, there have been no living cultures of C. havanensis and it has thus not been possible to assess its taxonomic status. Isolates thought to represent this fungus have, however, emerged from surveys of Eucalyptus in Mexico and Hawaii (U.S.A.). Results of this study showed that these isolates represent C. havanensis but reside in a genus distinct from Cryphonectria sensu stricto, which is described here as Microthia. Isolates of an unidentified fungus occurring on Myrica faya in the Azores and Madeira also grouped in Microthia and were identical to other M. havanensis isolates. Cryphonectria coccolobae, a fungus occurring on sea grape (Coccoloba uvifera) in Bermuda and Florida, was found to be morphologically identical to Microthia and is transferred to this genus, but as a distinct species. Surveys for M. coccolobae on sea grape in Florida, yielded a second diaporthalean fungus from this host. This fungus is morphologically and phylogenetically distinct from M. coccolobae and other closely related taxa and is described as Ursicollum fallax gen. et sp. nov. Phylogenetic analyses in this study have also shown that isolates of C. eucalypti, a pathogen of Eucalyptus in South Africa and Australia, group in a clade separate from all other groups including that representing Cryphonectria sensu stricto. This difference is supported by the fact that Cryphonectria eucalypti has ascospore septation different to that of all other Cryphonectria species. A new genus, Holocryphia, is thus erected for C. eucalypti.

New taxonomic concepts for the important forest pathogenCryphonectria parasiticaand related fungi

Species of Cryphonectria include some of the world's most important and devastating tree pathogens. Largely through the application of DNA sequence phylogenies, the taxonomy of these fungi has undergone major changes in recent years. Cryphonectria, including the chestnut blight pathogen Cryphonectria parasitica, has been restricted to species that have semi-immersed stromata, orange and pulvinate conidiomata, and one-septate ascospores. Other species of Cryphonectria with different morphological characteristics have been transferred to new genera that are strongly supported by phylogenetic data. This review represents a summary of the taxonomic changes to species of Cryphonectria sensu lato, and we discuss the impact that these changes might have on the understanding of their ecology, pathology and worldwide distribution.

Amphilogia gen. nov. for Cryphonectria‐like fungi from Elaeocarpus spp. in New Zealand and Sri Lanka

AbstractThe ascomycete genera Cryphonectria and Endothia are closely related members of Diaporthales. Recent DNA sequence comparisons have shown that isolates from Elaeocarpus spp. in New Zealand, previously identified as Cryphonectria radicalis and Cryphonectria gyrosa, represent a phylogenetic group distinct from those including other species of Cryphonectria and Endothia. Cryphonectria gyrosa applies to a species that occurs on Elaeocarpus glandulifer in Sri Lanka, the same host genus but a different species from which the New Zealand collections were made. The aim of this study was to provide a name for the fungi residing in the phylogenetic group from New Zealand. Morphological characters that define these fungi include superficial conical conidiomata, conidia of variable size, and ascospores with one to three septa. These characteristics are not found in other species of Cryphonectria. We also recognise a second species in the group from New Zealand that has distinctly larger ascospores. Herbarium specimens of C. gyrosa exhibit the same primary characteristics as the specimens from New Zealand and C. gyrosa is regarded as conspecific with one of the species in the New Zealand collections. A new genus, Amphilogia, is described for the collections of C. gyrosa from Sri Lanka and New Zealand, which also contains the second species from New Zealand, Amphilogia major sp. nov.

Rostraureum tropicale gen. sp. nov. ( Diaporthales) associated with dying Terminalia ivorensis in Ecuador

Terminalia ivorensis, a tree of central African origin, is planted in several tropical countries for timber and veneer production. During the course of a recent disease survey, an unknown fungus was found associated with basal cankers on dying T. ivorensis in Ecuador. The fungus has orange fruiting structures and septate, fusoid ascospores, similar to those of Cryphonectria, a well-known genus of canker pathogens. The aim of this study was to identify the fungus and to assess its pathogenicity. Identification was based on morphological characteristics as well as DNA sequence data. DNA sequence data from the ITS regions of the rDNA operon and two regions of the beta-tubulin gene, were compared with published sequences of Cryphonectria species and the closely related genera Endothia and Chrysoporthe. Pathogenicity tests were conducted on T. superba saplings. Morphological characterisations revealed that the conidiomata of the fungus from T. ivorensis, differed from those typical of Cryphonectria in being superficial and rostrate. Only Cryphonectria longirostris was similar to the fungus from T. ivorensis, but could be distinguished from it based on conidial size. Phylogenetic analyses showed that the fungus from T. ivorensis grouped closely with species of Cryphonectria, Chrysoporthe and Endothia, yet formed a distinct clade. Pathogenicity tests on T. superba provided evidence that the fungus is able to cause distinct stem cankers. We conclude that the pathogenic fungus from T. ivorensis represents a new genus and new species in the Diaporthales and we provide the name Rostraureum tropicale for it. The genus is typified by R. tropicale. Furthermore, C. longirostris is transferred to Rostraureum.

DNA sequence data and morphology defineCryphonectriaspecies in Europe, China, and Japan

Cryphonectria includes important tree pathogens as well as species believed to be saprophytes. Recent phylogenetic studies have concentrated on North American and southern hemisphere Cryphonectria spp., but little is known about Asian and European taxa. In this study we identify and differentiate among the species occurring in Europe, China, and Japan using morphological and phylogenetic comparisons among the Cryphonectria species. Phylogenetic comparisons were based on sequence data from the ribosomal ITS operon and two regions in the β-tubulin gene. Unknown Japanese and Chinese isolates showing different cultural features than those of Cryphonectria parasitica (Murrill) M.E. Barr from Japan and the USA, grouped with isolates of Cryphonectria nitschkei (G.H. Otth) M.E. Barr from Quercus spp. and Rhus javanica L. Isolates of Cryphonectria havanensis (Bruner) M.E. Barr from Quercus grosseserrata Blume, Castanopsis cuspidata Schottky, Pyrus sinensis Lindl., and Eucalyptus globulus Labill. also grouped in this phylogenetic clade. We propose that Cryphonectria nitschkei and the fungus that has been referred to as Cryphonectria havanensis in Japan should be treated as a single taxon. Phylogenetic and morphological data also suggest that there are two species currently representing Cryphonectria radicalis (Schwein.: Fr.) M.E. Barr in Europe. One of these species is similar to the type specimen of Cryphonectria radicalis, while the other species probably is new.Key words: Cryphonectria parasitica, Cryphonectria radicalis, Cryphonectria havanensis, Cryphonectria macrospora, Cryphonectria nitschkei, Diaporthales.

Phylogenetic Relationships of Cryphonectria and Endothia Species, Based on DNA Sequence Data and Morphology

The fungal genera Endothia and Cryphonectria include some of the most important pathogens of forest trees. Despite available new technology, no comprehensive comparative study based on DNA sequence data and morphology has been done on the available isolates representing these two genera. The main objectives of this study were to assess the phylogenetic relationships among species of Cryphonectria and Endothia, for which cultures are available, and to establish a taxonomic framework based on DNA sequence and morphological data, which will aid future studies and identification of species in these and related genera. Comparisons were based on sequence variation found in the ITS region of the ribosomal RNA operon and two regions of the β-tu-bulin gene. In addition, the morphology of these species was examined. The phylogenetic data indicated that Endothia and Cryphonectria reside in two distinct phylogenetic clades. Cryphonectria parasitica, C. macrospora, C. nitschkei, C. eucalypti and C. radicalis represented the Cryphonectria clade. Endothia gyrosa and E. singularis were included in the Endothia clade. An isolate representing E. viridistroma grouped outside the Endothia clade and separately from other groups. Other clades outside the one encompassing Cryphonectria were those represented by the C. cubensis isolates and fungi isolated from Elaeocarpus dentatus originating from New Zealand. These clades could be distinguished from Endothia and Cryphonectria, based on anamorph morphology, stromatal structure and ascospore septation. Cryphonectria and Endothia, therefore, appear to be paraphyletic and taxonomic relationships for these fungi need to be revised.

Phylogenetic relationships of Cryphonectria and Endothia species, based on DNA sequence data and morphology

The fungal genera Endothia and Cryphonectria include some of the most important pathogens of forest trees. Despite available new technology, no comprehensive comparative study based on DNA sequence data and morphology has been done on the available isolates representing these two genera. The main objectives of this study were to assess the phylogenetic relationships among species of Cryphonectria and Endothia, for which cultures are available, and to establish a taxonomic framework based on DNA sequence and morphological data, which will aid future studies and identification of species in these and related genera. Comparisons were based on sequence variation found in the ITS region of the ribosomal RNA operon and two regions of the β-tu-bulin gene. In addition, the morphology of these species was examined. The phylogenetic data indicated that Endothia and Cryphonectria reside in two distinct phylogenetic clades. Cryphonectria parasitica, C. macrospora, C. nitschkei, C. eucalypti and C. radicalis represented the Cryphonectria clade. Endothia gyrosa and E. singularis were included in the Endothia clade. An isolate representing E. viridistroma grouped outside the Endothia clade and separately from other groups. Other clades outside the one encompassing Cryphonectria were those represented by the C. cubensis isolates and fungi isolated from Elaeocarpus dentatus originating from New Zealand. These clades could be distinguished from Endothia and Cryphonectria, based on anamorph morphology, stromatal structure and ascospore septation. Cryphonectria and Endothia, therefore, appear to be paraphyletic and taxonomic relationships for these fungi need to be revised.

Conspecificity of Endothia eugeniae and Cryphonectria cubensis: a re-evaluation based on morphology and DNA sequence data

Cryphonectria cubensis and Endothia eugeniae are fungal pathogens of Eucalyptus and clove that were reduced to synonymy on the basis of results of crossinoculation studies, isozyme analysis, cultural studies, and morphology. A previous phylogenetic study on Cryphonectria, based on sequence variation in the ITS region of the ribosomal RNA operon, also supported the conspecificity of C. cubensis and E. eugeniae, but was based on only one E. eugeniae isolate. New collections from clove in Brazil and Indonesia have become available, providing the opportunity to reconsider the conspecificity of C. cubensis and E. eugeniae. The occurrence of C. cubensis on clove was confirmed based on morphological comparisons and phylogenetic analyses of ribosomal DNA and β-tubulin gene sequence data. In addition to C. cubensis, other fungi morphologically similar to Cryphonectria species on the basis of their orange stromata were present on some clove specimens, but no isolates were available for these fungi. Furthermore, some isolates, for which no herbarium material exists, grouped separately from the C. cubensis clade and closer to the Cryphonectria clade. The presence of more than one closely related fungus on clove raises questions relating to the legitimacy of the synonymy of E. eugeniae and C. cubensis. Based on the presence of C. cubensis on the type specimen of E. eugeniae, we recognize the synonymy of the two fungi but provide evidence that other fungi, more closely related to Cryphonectria spp. than to C. cubensis, are present on clove.

Evidence for interspecies transmission of viruses in natural populations of filamentous fungi in the genus Cryphonectria.

Interspecies transmission is a significant evolutionary event that has allowed a variety of pathogens to invade new host species. We investigated interspecies transmission of viruses between the chestnut blight fungus, Cryphonectria parasitica, and a sympatric unidentified Cryphonectria species in Japan. Two isolates of Cryphonectria sp. were found to contain Cryphonectria hypovirus 1 (CHV-1), which has been typically found in C. parasitica. Three lines of evidence support the hypothesis of interspecies transmission of CHV-1. First, host species occur sympatrically and therefore have the opportunity to come into physical contact. Second, we transmitted CHV-1 between species experimentally in the laboratory. Third, phylogenetic analysis of 476 bp of the ORF B region of CHV-1 showed that sequences from Cryphonectria sp. were more closely related to those from C. parasitica than to each other. Local geographical subdivision of virus sequences from both host species argues against the alternative hypothesis of independent evolution of CHV-1 since speciation of their hosts. Based on these findings, we rule out the hypotheses that CHV-1 diverged from viruses in a common ancestor of the hosts, or that ancestral polymorphisms in CHV-1 persisted in the two host taxa. Estimating the direction and frequency of interspecies transmission in nature will require more extensive samples of CHV-1 from both host species.