Cryphonectria Cubensis Research Articles

Molecular characterisation of novel mitoviruses associated with Sclerotinia sclerotiorum

Seven putative mitoviral genomes, representing four species from three Sclerotinia sclerotiorum isolates, were fully sequenced. The genome lengths ranged from 2438 to 2815 nucleotides. The RNA-dependent RNA polymerase (RdRp) of one genome shared high amino acid (aa) sequence identity (98.5 %) with the previously described Sclerotinia sclerotiorum mitovirus 2 (SsMV2/NZ1) and was provisionally assigned the name SsMV2/14563. The RdRps of three of the genomes with closest aa sequence identity of 78.8-79.3 % to Sclerotinia sclerotiorum mitovirus 1 (SsMV1/KL1) were provisionally considered to represent a new species, and the corresponding virus was named Sclerotinia sclerotiorum mitovirus 5 (SsMV5/11691, SsMV5/14563 and SsMV5/Lu471). The remaining two novel genomes, for which the viruses were provisionally named Sclerotinia sclerotiorum mitovirus 6 (SsMV6/14563 and SsMV6/Lu471) and Sclerotinia sclerotiorum mitovirus 7 (SsMV7/Lu471), showed closest aa sequence identities to Sclerotinia sclerotiorum mitovirus 3 (SsMV3/NZ1; 57.5-57.8 %) and Cryphonectria cubensis mitovirus 1a (CcMV1a; 32 %), respectively. The RdRp proteins of all seven genomes contained the conserved aa sequence motifs (I-IV) previously reported for mitoviruses, and their 5' and 3' untranslated regions (UTRs) have the potential to fold into stem-loop secondary structures.

Cryphonectria cubensis on an indigenous host in Brazil: a possible origin for eucalyptus canker disease?

Cryphonectria cubensis on an indigenous host in Brazil: a possible origin for eucalyptus canker disease?

First record of natural infection of Marlierea edulis by the eucalyptus canker fungus Chrysoporthe cubensis

Eucalyptus is the most important plant genus for forest plantations worldwide, including Brazil where approximately 3 million ha are cultivated. Chrysoporthe cubensis (formerly known as Cryphonectria cubensis) is the aetiological agent of eucalyptus canker, one of the most serious diseases of eucalypt. Although in the past this fungus was only known to attack eucalyptus and cloves, it is now known to attack other hosts in the Myrtaceae and Melastomataceae families (Wingfield, 2003; Seixas et al., 2004; Rodas et al., 2005). A host-range study involving wound inoculations with an isolate from eucalyptus indicated a potentially wide host range for this fungus, particularly within the order Myrtales (Seixas et al., 2004). Among the species found susceptible in this test was Marlierea edulis, popularly known in Brazil as ‘cambucá’. This is a large, native tree of the Brazilian tropical rainforest and a minor fruit tree occasionally grown as a curiosity in orchards. Recently, M. edulis was found in the field (Saquarema, State of Rio de Janeiro, Brazil) showing typical canker symptoms: necrotic wounds with cracking and flaking of the bark that led to branch death. Diseased tissue showed abundant, black, spine-like perithecial ostioles. The fungus had the typical morphology of C. cubensis: ascomata perithecial, subglobose 205–445 × 220–440 µm, ostioles rostrate 300–750 × 65–125 µm; asci unitunicate, clavate 15–28 × 3–5 µm; ascospores ellipsoid, 5–7 × 1–3 µm, one-septate and hyaline. A sample was deposited in the local herbarium (VIC 27828). Wound inoculations of an isolate obtained from this host using the bark-replacement method (Ferreira, 1989) on adult, healthy specimens of both M. edulis and Corymbia citriodora (=Eucalyptus citriodora) resulted in typical canker symptoms and abundant formation of sporulating C. cubensis perithecia after 3 weeks. This is the first record of natural infection by this fungus on M. edulis, and the third native host for this fungus to be found in the field in Brazil. This finding confirms the susceptibility under natural conditions of plants included in the host-range study performed by Seixas et al. (2004). The demonstration of pathogenicity of an isolate obtained from M. edulis to eucalyptus represents additional evidence for the hypothesis that native plants serve as reservoirs for eucalyptus canker outbreaks in Brazil.

Distribution of Chrysoporthe Canker Pathogens on Eucalyptus and Syzygium spp. in Eastern and Southern Africa

Chrysoporthe cubensis and C. austroafricana, collectively known as Cryphonectria cubensis in the past, are important canker pathogens of Eucalyptus spp. worldwide. Previous studies have suggested that Chrysoporthe austroafricana occurs only in South Africa, whereas C. cubensis occurs in Australia, Cameroon, Tanzania, Democratic Republic of Congo, Republic of Congo, Southeast Asia, and South, Central, and North America. In South Africa, C. austroafricana is a pathogen on nonnative Eucalyptus (Myrtaceae) and Tibouchina (Melastomataceae) spp., both residing in the order Myrtales. Recently, the fungus also has been found on native Syzygium cordatum trees in the country, leading to the hypothesis that it is native to Africa. In contrast, C. cubensis is thought to have been introduced into Africa and is known only on non-native Eucalyptus spp. and S. aromaticum (clove) in four countries. The aim of this study was to consider the distribution of Chrysoporthe spp. on non-native Eucalyptus spp. as well as on native Myrtales in southern and eastern Africa. Isolates were collected from as many trees as possible and characterized based on their morphology and DNA sequence data for two gene regions. Results show, for the first time, that C. cubensis occurs in Kenya, Malawi, and Mozambique on nonnative Eucalyptus spp. C. austroafricana was found for the first time in Mozambique, Malawi, and Zambia on non-native Eucalyptus spp. and native S. cordatum. The known distribution range of C. austroafricana within South Africa also was extended during these surveys.

Discovery of the Canker Pathogen Chrysoporthe austroafricana on Native Syzygium spp. in South Africa.

Chrysoporthe canker is one of the most important diseases of plantation-grown Eucalyptus spp. in tropical and subtropical regions worldwide. For many years, the disease was reported to be caused by the fungal pathogen Cryphonectria cubensis. Recent DNA-based studies have shown that the fungus in South Africa is not conspecific with Chr. cubensis and it was recently described in the new genus Chrysoporthe as Chrysoporthe austroafricana. Chr. austroafricana is known only from South Africa, where it causes severe cankers on Eucalyptus spp. and on ornamental Tibouchina trees, both of which have been introduced into South Africa. The origin of Chr. austroafricana is unknown, but it is possible that it expanded its host range from native trees related to Eucalyptus and Tibouchina spp. to these exotic hosts. Subsequent surveys of some indigenous South African Myrtales led to the discovery of fruiting structures resembling those of Chr. austroafricana on native Syzygium cordatum and S. guineense. The fungus from these Syzygium spp. was identified as Chr. austroafricana based on morphological characteristics and β-tubulin gene sequences. Pathogenicity trials showed that Chr. austroafricana is more virulent on exotic Eucalyptus spp. than on native S. cordatum. This study represents the first report of Chr. austroafricana from native hosts in South Africa and adds credence to the view that the fungus could be native to this country.

Relative Pathogenicity of Cryphonectria cubensis on Eucalyptus Clones Differing in Their Resistance to C. cubensis.

Cryphonectria cubensis causes a destructive canker disease of Eucalyptus species. Management of this disease is primarily through breeding and selection of disease resistant trees. One means of selecting such trees is by artificial inoculation with the pathogen. In routine screening trials in South Africa, an isolate of C. cubensis, considered to be highly pathogenic, has been used for such inoculations. Although the most resistant clones under natural conditions are the same as those detected in inoculation trials, a question has arisen whether all clones respond similarly to different C. cubensis isolates. Thus, a trial consisting of five clones, known to differ in susceptibility to infection by C. cubensis, was established. These trees were inoculated with nine South African C. cubensis isolates previously shown to differ in pathogenicity. Inoculations showed a significant isolate × clone interaction as well as an "apparent immunity" for one clone × isolate interaction, providing evidence highly suggestive of a vertical resistance component in the pathosystem. Disease screening in this pathosystem has traditionally relied on a single pathogen isolate; however, considering data presented here, future reliance on a single isolate may be inadequate.

Cryphonectria cubensis on an indigenous host in Brazil: a possible origin for eucalyptus canker disease?

Endothiella sp. was found associated with witches' broom symptoms of Tibouchina granulosa growing in a stretch of Atlantic rain forest in Rio de Janeiro, Brazil. The fungus was the anamorph of Cryphonectria cubensis , the eucalypt canker fungus. Host-range studies were performed involving 40 plant species belonging to 19 families and showed that C. cubensis has a wide potential host-range, concentrated on the Myrtales but including Persea americana (avocado) and Pouteria caimito . These are members respectively of the subclasses Magnoliidae and Dilleniidae, distantly related to plants of the subclass to which both eucalypt and T. granulosa belong (Rosidae). The finding of C. cubensis in balance with a population of a native species at a site isolated and distant from both eucalypt and clove plantations might be regarded as reinforcing the hypothesis of a Neotropical origin for the fungus as opposed to the hypothesis of the fungus being from Oceania and having cloves as its original host. The new alternative hypothesis (of separate origins from endemic hosts in the Neotropics, Africa and Oceania) should nevertheless be also considered.

Biological and Phylogenetic Analyses Suggest that Two Cryphonectria spp. Cause Cankers of Eucalyptus in Africa.

Cryphonectria cubensis is an economically important pathogen of commercial Eucalyptus spp. Differences have been reported for disease symptoms associated with Cryphonectria canker in South Africa and other parts of the world, and recent DNA-based comparisons have confirmed that the fungus in South Africa is different from that in South America and Australasia. During a disease survey in the Republic of Congo, Cryphonectria canker was identified as an important disease on Eucalyptus grandis and E. urophylla. In this study, we compared Congolese and South African isolates of C. cubensis using DNA sequence data and pathogenicity under greenhouse conditions. The β-tubulin and internal transcribed spacer (ITS) region sequences show that C. cubensis in Congo is different from the fungus in South Africa and that Congolese isolates group most closely with South American isolates. Furthermore, pathogenicity tests showed that a South African isolate was more aggressive than two Congolese isolates. We conclude that two distinct Cryphonectria spp. occur in Africa and hypothesize that the fungus in the Congo probably was introduced into Africa from South America. Both fungi are important pathogens causing disease and death of economically important plantation trees. However, they apparently have different origins and must be treated separately in terms of disease management and quarantine considerations.

Modelling the spatial distribution of two important South African plantation forestry pathogens

Pathogens, pests and diseases impact heavily on commercial plantation forestry in South Africa, and must thus be considered in any diversified and adaptive management approach. Two important fungal pathogens of Pinus and Eucalyptus species, respectively, are Sphaeropsis sapinea and Cryphonectria cubensis. The aim of this study was to explore the use of bioclimatic modelling to predict the habitat distribution for these pathogens, and to consider potential distribution patterns under conditions of climate change. High-risk areas identified for Sphaeropsis dieback coincide with the summer rainfall hail belt, emphasising the need for planting resistant Pinus spp. in these regions. A much smaller area of South Africa is predicted to be suitable for the occurrence of C. cubensis than for S. sapinea, but a range shift westward in suitable habitat for C. cubensis is predicted under a climate change scenario. Of concern is that many of these areas are currently being planted with disease susceptible Eucalyptus clones. These preliminary results, and further refinement of the model, will lay a valuable foundation for future risk assessment and strategic management planning in the South African forestry industry.

Primers for the amplification of sequence‐characterized loci in Cryphonectria cubensis populations

AbstractWe describe the development of DNA markers for the fungal pathogen of Eucalyptus, Cryphonectria cubensis. These markers originated from cloned intershort sequence repeat polymerase chain reactions, which enrich for medium to highly repetitive DNA sequences. In total, 10 markers were isolated, eight of which were polymorphic, and these can subsequently be applied to study populations of C. cubensis.

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.

2003 Daniel McAlpine Memorial Lecture Increasing threat of diseases to exotic plantation forests in the Southern Hemisphere: lessons from Cryphonectria canker

Plantation forestry in the Southern Hemisphere has grown dramatically in the last hundred years. This has largely been due to the exceptional growth and performance of exotic species, particularly Pinus, Eucalyptus and Acacia. It is generally recognised that the success of these plantations has, at least in part, been due to the separation of the trees from their natural enemies. In this regard, the trees have performed similarly to weeds. Indeed, in some situations, species regarded as highly desirable for forestry are also recognised as noxious weeds. The artificial barrier between exotic plantation species and their pathogens is, however, crumbling. Despite intensive efforts to exclude pests and pathogens from countries now dependent on plantations of exotic trees, new and seriously damaging incursions are occurring with increasing frequency. Cryphonectria canker of Eucalyptus caused by Cryphonectria cubensis provides an important and interesting example and is used in this paper to illustrate emerging trends. These include evidence to suggest that native pathogens, previously thought to be relatively host specific and non-threatening, are adapting to infect exotic plantation trees. Other than the damage that these pathogens are causing to exotics, they now pose a serious threat to the same or related tree species in their areas of origin. This tremendous threat is only just being recognised and it is little understood. This is, at least in part, due to a poor understanding of the taxonomy and ecology of even some of the better known tree pathogens. Intensive efforts will be required to protect the sustainability of exotic plantation forestry. They will also be needed to ensure that ‘new pathogens’ do not lead to destruction of the same or related tree species in their areas of origin.

Effect of environment on the response of Eucalyptus clones to inoculation with Cryphonectria cubensis

SummaryThe ascomycete Cryphonectria cubensis causes severe losses in Eucalyptus plantations in South Africa and selection programmes for disease tolerance are necessary. The aim of this study was to use two C. cubensis inoculation trials, planted at different locations to assess the disease susceptibility of the clones and the effect of the environment on disease development. These two trials consisted each of 21 Eucalyptus clones (E. grandis, E. grandis × camaldulensis and E. grandis × urophylla). All trees were inoculated with a single virulent strain of C. cubensis and lesion widths measured 6 and 12 months after inoculation. Clones differed significantly in their tolerance to C. cubensis. Further, disease severity differed depending on the geographical location of the trial. A significant clone × locality (genotype × environment) interaction was observed. Therefore, screening for disease resistance should take place only in areas where clones will be commercially grown.

Cryphonectria canker on Tibouchina in South Africa

Cryphonectria cubensis is an important canker pathogen of plantation Eucalyptus spp. in tropical and sub-tropical areas of the world, including South Africa. It is best known on Eucalyptus spp., but it also occurs on Syzygium aromaticum (clove). In 1998, C. cubensis was found to cause cankers on the non-myrtaceous hosts Tibouchina urvilleana and T. lepidota in Colombia. In this study, we report on a similar canker disease that has recently been found in South Africa on T. granulosa commonly grown as an ornamental tree. The identity of the pathogen was determined through morphological comparisons and phylogenetic analyses of ITS and β-tubulin gene sequences. The pathogenicity of the fungus was also tested on T. granulosa and E. grandis. Morphological as well as DNA sequence comparisons showed that the fungus on T. granulosa is the same as C. cubensis occurring on Eucalyptus spp. in South Africa. Pathogenicity tests on T. granulosa and E. grandis clones showed that the fungus from T. granulosa is able to cause cankers on both hosts.

β-Tubulin and histoneH3gene sequences distinguishCryphonectria cubensisfrom South Africa, Asia, and South America

Cryphonectria cubensis (Bruner) Hodges is the causal agent of an important stem canker disease of Eucalyptus. Previous phylogenetic studies based on sequence data have shown that C. cubensis is distinct from other species of Cryphonectria but that C. cubensis isolates reside in two distinct groups, consistent with geographical origin. Thus, isolates of C. cubensis from South America and South Africa grouped together but apart from those originating from Southeast Asia and Australia. These results were in contrast with the symptoms of Cryphonectria canker in South Africa, which are different from those observed elsewhere in the world. The aim of this study was to use more variable regions of the fungal genome to test whether South African isolates of C. cubensis are genetically distinct from those from other parts of the world. For this comparison, β-tubulin and histone H3 gene sequences were used. Specimens from South America, Southeast Asia, Australia, and South Africa were also compared morphologically. The phylogram emerging from the analysis indicated that South American and Southeast Asian – Australian isolates resided in two well-resolved but closely related clades. However, isolates from South Africa were distinct from other groups. This is consistent with ecological aspects of the South African fungus, although no obvious morphological differences between the fungi from the various regions could be found. Our results suggest that the South African fungus represents a species distinct from C. cubensis occurring elsewhere in the world.Key words: β-tubulin, histone H3, molecular phylogeny, Cryphonectria cubensis.

Molecular analysis of an endopolygalacturonase gene from a Eucalyptus canker pathogen, Cryphonectria cubensis.

Cryphonectria cubensis causes a serious Eucalyptus canker disease. Fungal cell wall degrading enzymes (CWDEs) are important during the early stages of interaction of the fungus with Eucalyptus. To improve our understanding of the molecular regulation of the interaction of Eucalyptus and C. cubensis, the relevant genes involved in this interaction should be identified, cloned and studied. The aim of this study was, therefore, to clone the endopolygalacturonase (endoPG) gene of C. cubensis. C. cubensis was grown on a medium supplemented with Eucalyptus cell wall extracts. Degenerate primers were designed to amplify part of the endoPG gene from C. cubensis genomic DNA. The resulting sequence was used to design specific primers for use in inverse PCR to amplify the entire endoPG gene of C. cubensis (ccen-1). The endoPG sequence of C. cubensis has 93% amino acid sequence similarity to that of the chestnut blight pathogen, Cryphonectria parasitica.

Characterization of South African Cryphonectria cubensis Isolates Infected with a C. parasitica Hypovirus

ABSTRACT Cryphonectria cubensis is the causal agent of a serious canker disease of Eucalyptus spp. in tropical and subtropical parts of the world. In this study, a South African C. cubensis isolate was transfected by electroporation with a synthetic RNA transcript corresponding to the full-length coding strand of the C. parasitica hypovirus (CHV1-EP713). Hypovirus infection resulted in pronounced morphological changes that included a striking increase in bright yellow-orange pigment production, a reduction in mycelial growth rate, and reduced sporulation. Greenhouse studies revealed that the virus-containing strain was significantly less virulent than the original virulent C. cubensis isolate. Although the hypovirus was not transmitted through conidia produced by infected C. cubensis, the virus was readily transmitted via hyphal anastomosis to C. cubensis isolates representing a broad range of vegetative compatibility groups. These results suggest that vegetative incompatibility may not pose a strong barrier against virus transmission in South African isolates of C. cubensis and that hypovirus-mediated biological control could provide opportunities to reduce the impact of Cryphonectria canker in South Africa.

Genetic diversity of Cryphonectria cubensis isolates in South Africa

Cryphonectria canker caused by Cryphonectria cubensis is one of the most destructive diseases of Eucalyptus plantations in South Africa. To implement a meaningful management of plantation diseases, it is important to have an understanding of the population diversity of the pathogen. In this study, trees were surveyed to determine whether C. cubensis reproduces sexually in South Africa. The diversity of the South African C. cubensis population was assessed based on vegetative compatibility tests. Field inoculations were used to determine whether VC groups correlated with virulence. Only pycnidia were found on cankered trees, indicating that sexual reproduction does not occur. Only 23 VC groups were found amongst 100 isolates each collected from single diseased trees. A low degree of genetic diversity also indicated that sexual reproduction is absent or rare in the South African C. cubensis population. Inoculation studies revealed that isolates belonging to different VC groups differ significantly in their ability to cause lesions. The low level of genetic diversity enhances opportunities to capitalise on hypovirulence to reduce the impact of the pathogen in the future. It also supports the view that the fungus was recently introduced into South Africa.

Wound response of Eucalyptus clones after inoculation with Cryphonectria cubensis

SummaryTwenty‐five different Eucalyptus grandis clones were artificially wounded and inoculated with a virulent isolate of Cryphonectria cubensis. The capacity of wounds to close through callus production was correlated with the relative susceptibility of these clones to infection by C. cubensis. Clones with the greatest capacity to close wounds were those that were also most tolerant to C. cubensis infection. Those with a lower capacity to close wounds were most susceptible to Cryphonectria canker.

Hypovirulence detected in Brazilian isolates of Cryphonectria cubensis

A single 3 kb segment of double‐stranded (dsRNA) was present in three of 30 Brazilian isolates of Cryphonectria cubensis. These dsRNA‐containing isolates showed morphological characteristics suggestive of hypovirulence and were significantly less virulent than dsRNA‐free isolates. One isolate, however, with morphological characteristics suggestive of hypovirulence, showed reduced virulence, but was free from dsRNA. Conversion of virulent isolates with normal morphology to a morphology associated with hypovirulence was achieved by pairing hypovirulent and virulent isolates of the same vegetative compatibility group (VCG). This suggests that dsRNA can be transmitted to isolates of the same vegetative compatibility group by hyphal anastomosis. Converted isolates exhibited the same hypovirulence‐associated traits as those of the original dsRNA‐containing hypovirulent isolates. These studies suggest that a single 3 kb segment of dsRNA alters both morphology and virulence by conferring hypovirulence on the pathogen; the first such report for Brazilian isolates of C. cubensis.