Ophiostomatales associated with wounds on hardwood trees in Poland

Wounds on trees provide entrance for many members of the Ophiostomatales (Ascomycota), including economically damaging tree pathogens. Because very little is known regarding wound-associated fungi on hardwoods in Europe or their potential nitidulid beetle vectors (Coleoptera), the aim of this survey was to explore the diversity of these fungi in Poland. Wound samples associated with 26 tree species were collected from 18 stands in Poland. Fungi belonging to the Ophiostomatales were isolated and identified based on morphology and DNA sequence comparisons of five gene regions, combined with phylogenetic analyses. In total, 32 taxa were identified, including 20 known and 12 currently undescribed species. In addition, Grosmannia cainii, Ophiostoma cf. ponderosae, O. sparsiannulatum and Sporothrix aurorae were reported for the first time outside of the USA. The wound-associated members of the Ophiostomatales recovered from hardwoods showed high diversity and specificity. Among the species recovered, 16 belonged to Sporothrix, 11 represented Ophiostoma s. l., five could be assigned to Leptographium s. l., and one Graphilbum species was detected. Among the studied tree species, wounds on Quercus robur appeared to be the most vulnerable to infection by members of the Ophiostomatales. All the nitidulid beetle species were found in association with species assignable to Ophiostoma and Sporothrix. This study detected a significant association between the occurrence of species belonging to the Ophiostomatales and the health status of the wounded trees. Some of the isolated species were host specific although Ophiostoma quercus, the most frequently encountered species, had a broad host range.     

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.     

PCR‐based identification of cacao black pod causal agents and identification of biological factors possibly contributing to Phytophthora megakarya's field dominance in West Africa

Among the Phytophthora species that cause black pod of cacao, P. megakarya is the most virulent, posing a serious threat to cacao production in Africa. Correct identification of the species causing the black pod and understanding the virulence factors involved are important for developing sustainable disease management strategies. A simple PCR‐based species identification method was developed using the species‐specific sequences in the ITS regions of the rRNA gene. A phylogenetic tree generated for 119 Phytophthora isolates, based on the 60S ribosomal protein L10 gene and rDNA sequence, verified the PCR‐based identification assay and showed high interspecific variation among the species causing black pod. Phytophthora megakarya isolates were uniformly virulent in an assay using susceptible cacao pod husks inoculated with zoospores, while the P. palmivora isolates showed greater divergence in virulence. The virulence of P. megakarya was associated with earlier production of sporangia and an accelerated induction of necrosis. While zoospore germ tubes of both species penetrated pods through stomata, only P. megakarya produced significant numbers of appressoria. A hypersensitive‐like response was observed when attached SCA‐6 pods were inoculated with P. palmivora. SCA‐6 pods became vulnerable to P. palmivora when wounded prior to zoospore inoculation. Phytophthora megakarya was more aggressive than P. palmivora on attached SCA‐6 pods, causing expanding necrotic lesions with or without wounding. Phytophthora megakarya is predominant in the Volta region of Ghana and it remains to be seen whether it can displace P. palmivora from cacao plantations of Ghana as it has in Nigeria and Cameroon.     

Death of endemic Virgilia oroboides trees in South Africa caused by Diaporthe virgiliae sp. nov.

Numerous dead and dying individuals of the Western Cape endemic tree Virgilia oroboides (Fabaceae) were recently observed within a South African national botanical garden. Root‐rot fungi and fungi symbiotic with bark beetles (Curculionidae; Scolytinae) from diseased trees were assessed for their respective roles in V. oroboides mortality. Disease progression was also monitored over 1 year. Fungi were isolated from surface sterilized bark and root samples from diseased trees and provisionally identified using data from the internal transcribed spacer regions (ITS1, ITS2), including the 5·8S rRNA gene (ITS). Pathogenicity of selected fungi towards V. oroboides was tested under field conditions. The pathogenicity of various bark beetle‐associated Geosmithia (Hypocreales: Hypocreomycetidae) spp. from V. oroboides were similarly assessed. The only fungus consistently isolated from lesions on the roots and bark of declining V. oroboides, and never from healthy individuals, represented an undescribed Diaporthe (Diaporthales, Diaporthaceae) species that was characterized using molecular (using data from the ITS marker and part of the β‐tubulin gene, TUB), cultural and morphological characters. It is an aggressive pathogen of V. oroboides, newly described here as Diaporthe virgiliae sp. nov. Trees of all ages are susceptible to this pathogen with subsequent bark beetle attack of mature trees only. All Geosmithia spp. from beetles and/or infected trees were nonpathogenic towards V. oroboides. Diaporthe virgiliae caused a severe decline in the health of the monitored V. oroboides population over a period of only 1 year and should be considered as a significant threat to these trees.     

Reassessment of rust fungi on weeping willows in the Americas and description of Melampsora ferrinii sp. nov.

Rust fungi in the genus Melampsora usually cause disease on hosts in the Salicaceae. Identification of Melampsora species is often complicated due to few differences in spore morphology and little publicly available comparative sequence data. Weeping willow trees (primarily Salix babylonica and its hybrids) have been reported to be infected by 11 Melampsora species; however, most of these records are based on morphological characterization. New collections of rust fungi on weeping willows from the central USA were analysed using a combination of morphology, ITS and LSU rDNA sequencing, and host data to determine that they represent an undescribed rust fungus, Melampsora ferrinii sp. nov. Additional studies of herbarium material revealed that M. ferrinii has occasionally been collected but identified as M. epitea. In addition to North America, M. ferrinii is also present in South America and has been infecting weeping willows there since at least the 1990s.     

Pathogenicity of fungi associated with ash dieback towards Fraxinus excelsior

A large part of the area in Europe in which Fraxinus excelsior is native is currently affected by ash dieback, a threatening disease caused by the ascomycetous fungus Hymenoscyphus fraxineus. Fungi other than H. fraxineus also occur in large numbers on stems of the dying ash trees. To clarify their possible role in the dieback process, six fungal species common on dying stems and twigs of ash in Poland, i.e. Cytospora pruinosa, Diaporthe eres, Diplodia mutila, Fusarium avenaceum, F. lateritium and F. solani, were tested for pathogenicity using a test based on artificial wound inoculations of 6‐year‐old F. excelsior plants under field conditions, with H. fraxineus included for comparison. There were significant differences in index of pathogenicity among the fungi tested. Hymenoscyphus fraxineus (mean index 5.78) was the most pathogenic. Diplodia mutila (4.23) and C. pruinosa (4.02) were significantly less pathogenic than H. fraxineus, but significantly more than the other fungi. Diaporthe eres (2.43), F. avenaceum (1.92), F. solani (1.86) and F. lateritium (1.08) were the least pathogenic (P < 0.0001). The extent of disease symptoms caused by F. solani and F. lateritium was statistically similar to the control (P = 0.05). All tested fungi were successfully reisolated from inoculated stems. The contribution of the results to understanding the possible role of these fungi in the ash dieback process in F. excelsior, particularly in trees weakened after primary infection by H. fraxineus, is discussed.     

Characterization of Cytospora isolates from wood cankers of declining grapevine in North America,with the descriptions of two new Cytospora species

Cytospora species are ubiquitous pathogens of numerous woody plants, causing dieback and wood cankers in agronomic crops, timber trees and wildland trees (e.g. Prunus, Eucalyptus and Salix, respectively). Cytospora chrysosperma, C. cincta and C. leucostoma have been reported from grapevines in Iran showing symptoms of one or more recognized trunk diseases (esca, botryosphaeria‐, eutypa‐ and phomopsis diebacks); however, only C. chrysosperma was shown to be pathogenic to grapevine. To understand the potential role of Cytospora species in the grapevine trunk‐disease complex, 21 Cytospora isolates were examined that were recovered from dieback and wood cankers of Vitis vinifera and Vitis interspecific hybrids in seven northeastern U.S. states and two Canadian provinces. Phylogenetic analyses of ITS and translation elongation factor 1‐α identified two novel species: Cytospora vinacea sp. nov. and Cytospora viticola sp. nov. Differences in culture morphology and conidial dimensions also distinguished the species. When inoculated to the woody stems of potted V. vinifera ‘Thompson Seedless’ in the greenhouse, both species were pathogenic, based on development of wood lesions and fulfilment of Koch's postulates. Cytospora viticola was the most virulent based on lesion length at 12 months post‐inoculation. As cytospora canker shares some of the same general dieback‐type symptoms as botryosphaeria‐, eutypa‐ and phomopsis diebacks, it may be considered part of the grapevine trunk‐disease complex in eastern North America.     

A disease and production index (DPI) for selection of cacao (Theobroma cacao) clones highly productive and tolerant to pod rot diseases

Frosty pod rot (FPR) (Moniliophthora roreri), along with black pod rot (Phytophthora species) and witches’ broom disease (Moniliophthora perniciosa) constitute the main phytosanitary problems limiting cacao (Theobroma cacao) production causing severe yield losses. One of the main sought after methods of pod rot management is the selection of tolerant cacao genotypes. Typically, the selection is carried out through the quantification of the percentage of diseased pods (PDP). However, PDP does not consider the relative productivity, or production potential (PT) of the genotype. Production potential can vary among cacao genotypes. Consequently, genotypes with similar PT can have similar or vastly different disease tolerance levels as measured by PDP. The disease and production index (DPI) was developed to integrate a genotype's tolerance to M. roreri and other diseases as measured by PDP, with its PT. Here, we evaluated the number of healthy pods, number of diseased pods, and weight of fresh seed for 29 clones grown in replicated five-tree plots over 4 years. The data obtained was used to calculate PDP and DPI for each clone for three different disease combinations: frosty pod rot alone, pod rots other than frosty pod rot, and the combination of all pod rots. Multivariate analysis verified that DPI discriminated between clones based on productivity and disease tolerance. Surprisingly, there was a close ranking of clones between resistance to FPR and resistance to all other pod rots. The DPI can be used in breeding programmes focused on the selection of high yielding disease-tolerant cacao genotypes.     

Hymenoscyphus fraxineus is a leaf pathogen of local Fraxinus species in the Russian Far East

Dieback of European ash was first observed in Europe in the early 1990s. The disease is caused by the invasive ascomycete Hymenoscyphus fraxineus, proposed to originate from Far East Asia, where it has been considered a harmless saprotroph. This study investigates the occurrence of H. fraxineus in tissues of local ash species in the Russian Far East, and assesses its population‐specific genetic variation by ITS sequencing. Shoot dieback symptoms, characteristic of H. fraxineus infection on European ash, were common, but not abundant, on Fraxinus mandshurica and Fraxinus rhynchophylla trees in Far East Russia. High levels of pathogen DNA were associated with necrotic leaf tissues of these ash species, indicating that the local H. fraxineus population is pathogenic to their leaves. However, the low levels of H. fraxineus DNA detected in shoots with symptoms, the failure to isolate this fungus from such tissues, and the presence of other fungi with pathogenic potential in shoots with symptoms indicate that local H. fraxineus strains may not be responsible (or their role is negligible) for the observed ash shoot dieback symptoms in the region. Conspicuous differences in ITS rDNA sequences detected between H. fraxineus isolates from Russian Far East and European populations suggest that the current ash dieback epidemic in Europe might not directly originate from the Russian Far East. Revision of the herbarium material shows that the earliest specimen of H. fraxineus was collected in 1962 from the Russian Far East and the oldest H. fraxineus specimen of China was collected in 2004.     

The mango sudden decline pathogen, Ceratocystis manginecans, is vectored by Hypocryphalus mangiferae (Coleoptera: Scolytinae) in Oman

In Oman, the bark beetle Hypocryphalus mangiferae is closely associated with trees affected by mango sudden decline disease caused by Ceratocystis manginecans. Although it has previously been assumed that this beetle plays a role in the dispersal of the pathogen, this has not been established experimentally. The aim of this study was to determine whether H. mangiferae vectors C. manginecans from infected to healthy mango trees. A survey conducted in northern Al Batinah region of Oman revealed that H. mangiferae was closely associated with mango sudden decline disease symptoms and it was found on trees in the early stages of the disease. Healthy, 2-year-old mango seedlings were exposed to H. mangiferae collected from diseased mango trees. Seedlings were infested by the bark beetles and after 6 weeks, typical mango sudden decline disease symptoms were observed. Ceratocystis manginecans was isolated from the wilted mango seedlings while uncolonized control seedlings remained healthy. The results show that H. mangiferae vectors C. manginecans in Oman and is, therefore, an important factor in the epidemiology of this disease.     

Rapid Screening of Cacao Genotypes for Field Resistance to Phytophthora palmivora Using Leaves, Twigs and Roots

Black pod, caused by Phytophthora spp. is one of the most important diseases of cacao occurring worldwide. Losses due to black pod caused by P. palmivora are still moderate in Côte d'Ivoire but P. megakarya causes high losses in Ghana and other Central African countries. Variation in field attack has been observed between cacao genotypes, but evaluation of pod losses is unsuitable for obtaining rapid progress in breeding. Results of inoculation tests using young detached leaves, twigs and roots, obtained from field and nursery plants, are presented here and compared to field resistance of similar genotypes observed over a 10-year period. Nine different Upper Amazon Forastero genotypes were tested together with progenies obtained by crossing these with the susceptible check IFC5 (Amelonado genotype). Rank correlations between the early screening tests and the level of field attack were positive and mostly significant (r=0.58–0.95). The coefficient of correlation was slightly higher for leaves (r=0.88) and roots (r=0.89) than for twigs (r=0.76). Also, resistance of the different plant organs was correlated (r=0.6–0.9). Resistance of the Upper Amazon parents was well correlated with the resistance of their cross progenies (r=0.7–0.9), suggesting that resistance is highly heritable. Resistance of leaves and twigs from the nursery was better correlated with field resistance than resistance of leaves and twigs from the field, which might result from more uniform growing conditions in the nursery. Inoculation of leaves appears the most suitable early screening method for black pod resistance. Application of this test in breeding more resistant cacao cultivars is discussed.     

Isolation of actinomycetes and endospore-forming bacteria from the cacao pod surface and their antagonistic activity against the witches’ broom and black pod pathogens

In this study, actinomycetes and endospore-forming bacteria were isolated from the surface of cacao pods. The activity of these microorganisms againstCrinipellis perniciosa andPhytophthora palmivora, causal agents of witches’ broom and black pod diseases of cacao, respectively, was investigated. A total of 336 isolates of actinomycetes and endosporeforming bacteria were tested on a detached pod assay againstC. perniciosa. The screening procedure used proved to be fast and inexpensive, allowing the selection of five actinomycetes as the most promising isolates for the biocontrol ofC. perniciosa. Under laboratory conditions the actinomycetes were able to inhibit 100% ofC. perniciosa basidiospore germination. However, under field conditions the selected actinomycetes were unable to protect cacao pods against both pathogens. In these experiments, inhibition ofC. perniciosa ranged from 6% to 21% in relation to the control, whereas there was no inhibition of black pod caused byP. palmivora. Formulations need to be improved in order to enhance the activity of the actinomycetes against cacao pathogens in the field. Molecular identification of the selected isolates showed that they are species of the genusStreptomyces.     

Of mushrooms and chocolate trees: aetiology and phylogeny of witches' broom and frosty pod diseases of cacao

The troubled history of the two major diseases of the chocolate tree (Theobroma cacao) in South America, witches' broom and frosty pod, is reviewed, concentrating on critical aspects of the aetiology as well as the phylogeny of the causal agents. Both diseases are caused by sister species within the genus Moniliophthora, belonging to the Marasmiaceae family of mushrooms. The witches' broom pathogen, Moniliophthora perniciosa, evolved on the Amazonian side of the Andes and induces brooms not only in cacao and its relatives in the genera Theobroma and Herrania (Malvaceae), but also in species in the plant families Bignoniaceae, Malpighiaceae and Solanaceae, on which the mushrooms (basidiomata) are produced. Moniliophthora roreri, the type species of the genus, evolved as a pod pathogen on endemic Theobroma species on the western side of the northern Andean Cordillera. Because Moniliophthora was described originally as the asexual form of an unknown basidiomycete, the generic diagnosis is amended here to accommodate species with agaricoid basidiomata. In addition, the new variety M. roreri var. gileri is designated for the morphotype occurring on Theobroma gileri, in northwest Ecuador. Cytology studies indicate that the supposed conidia of M. roreri are, in fact, sexual spores (meiospores) and it is posited that the fruiting structure represents a much‐modified mushroom. Finally, based on preliminary data from pathogenicity testing, it is hypothesized that the true causal agent of both diseases is an as yet unidentified infectious agent vectored into the host by the fungus.     

Trichoderma harzianum produces nonanoic acid, an inhibitor of spore germination and mycelial growth of two cacao pathogens

An isolate of Trichoderma harzianum Rifai from an infected cacao pod produces and secretes nonanoic (pelargonic) acid into a liquid culture medium. Nonanoic acid (NA) was very inhibitory to spore germination and mycelial growth of two cacao pathogens, Crinipellis perniciosa Stahel and Moniliophthora roreri Cif. H.C. Evans. It was highly active causing 75% inhibition of spore germination in an in vitro assay at a rate as low as 0.09 μM for M. roreri and 0.92 μM for C. perniciosa. Mycelial growth was comparatively less sensitive to inhibition, but still there was a 75% reduction in growth with 0.62 μM in M. roreri and 151 μM NA in C. perniciosa. In contrast, NA did not affect Trichoderma mycelial growth or spore germination at concentrations that were inhibitory to the pathogens. 6-pentyl-α-pyrone was also produced and secreted into the medium by T. harzianum, however; it was not antagonistic to the cacao pathogens. Although a number of metabolites produced by Trichoderma spp. have been identified in the past, this is the first report of NA production and secretion by any Trichoderma. The results suggest that NA may play a role in the successful use of some Trichoderma spp. isolates in the biocontrol of fungal diseases of plants.     

Transmission of ‘Candidatus Phytoplasma pyri’ by naturally infected Cacopsylla pyri to peach,an approach to the epidemiology of peach yellow leaf roll (PYLR) in Spain

Peach orchards in the northeast of Spain were severely affected in 2012 by a previously unreported disease in this area. The symptoms included early reddening, leaf curling, decline, abnormal fruits, and in some cases death of the peach trees. All the infected peach samples were positive for ‘Candidatus Phytoplasma pyri’, but none were infected by the ‘Ca. Phytoplasma prunorum’. In this work, potential vectors able to transmit ‘Ca. Phytoplasma pyri’ from pear to peach and between peach trees were studied and their infective potential was analysed at different times of the year. Transmission trials of the phytoplasma with potential vectors to an artificial feeding medium for insects and to healthy peach trees were conducted. Additionally, isolated phytoplasmas were genetically characterized to determine which isolates were able to infect peach trees. Results showed that the only insect species captured inside peach plots that was a carrier of the ‘Ca. Phytoplasma pyri’ phytoplasma was Cacopsylla pyri. Other insect species captured and known to be phytoplasma transmitters were present in very low numbers, and were not infected with ‘Ca. Phytoplasma pyri’ phytoplasma. A total of 1928 individuals of C. pyri were captured in the peach orchards, of which around 49% were phytoplasma carriers. All the peach trees exposed to C. pyri in 2014, and 65% in 2015, were infected by ‘Ca. Phytoplasma pyri’ 1 year after exposure, showing that this species is able to transmit the phytoplasma to peach. Molecular characterization showed that some genotypes are preferentially determined in peach.     

Lateral transfer of a phytopathogenic symbiont among native and exotic ambrosia beetles

Different ambrosia beetle species can coexist in tree trunks, where their immature stages feed upon symbiotic fungi. Although most ambrosia beetles are not primary pests and their fungal symbionts are not pathogenic to the host tree, exceptional situations exist. Notably, Xyleborus glabratus carries a phytopathogenic symbiont, Raffaelea lauricola, which causes laurel wilt, a lethal disease of some Lauraceae species. Both X. glabratus and R. lauricola are natives of Asia that recently invaded much of the coastal plain of the southeastern USA. This study examined ambrosia beetles that breed in susceptible trees in Florida (USA), including avocado (Persea americana), redbay (P. borbonia) and swampbay (P. palustris). Raffaelea lauricola was recovered from six of eight ambrosia beetle species that emerged from laurel wilt‐affected swampbay trees, in addition to X. glabratus. Controlled infestations with cohorts of the six species other than X. glabratus revealed that each could transmit the pathogen to healthy redbay trees and two could transmit the pathogen to healthy avocado trees; laurel wilt developed in five and one of the respective beetle × host interactions. These results indicate flexibility in the lateral transfer of a non‐native ambrosial fungus to other ambrosia beetles, and for the first time documents the transmission of a laterally transferred phytopathogenic symbiont by new ambrosia beetle species. Additional work is needed to determine whether, or to what extent, the new beetle × R. lauricola combinations play a role in spreading laurel wilt.     

Bark beetle (Coleoptera, Scolytidae) outbreak and system of IPM measures in an area affected by intensive forest decline connected with honey fungus (Armillaria sp.)

The character of a bark beetle outbreak, planning system, and implementation of IPM measures in a forest affected by intensive decline connected with honey fungus (Armillaria sp.) is described. It is possible to distinguish two levels of outbreak in the study area. The first level is characterized by spruce mortality connected with yellowing of spruce and presence of plant pathogens, mostly Armillaria sp. Forest stands have disperse infestations of bark beetles. Identification of infested trees and salvage cutting in time are problematic. In case of late processing of infested trees, the second level of outbreak could become manifest. The populations of bark beetle increase. Beetles could attack relatively healthy trees. The classical outbreak with forest edges and spot infestations starts. The identification of infested trees is easier. The application of pheromone trap barriers is effective. Thus, the second level of outbreak could be easily managed. The planning of IPM measures is concerned with localization of various pheromone trap systems in particular forest stands. It is based on field survey, data acquisition, estimation of swarming bark beetle population, and information of pheromone system effectiveness.     

Characterization of <Emphasis Type="Italic">Trichoderma</Emphasis> species isolated in Ecuador and their antagonistic activities against phytopathogenic fungi from Ecuador and Japan

Native Trichoderma spp. were isolated from agricultural fields in several regions of Ecuador. These isolates were characterized via morphological observation as well as molecular phylogenetic analysis based on DNA sequences of the rDNA internal transcribed spacer region, elongation factor-1α gene and RNA polymerase subunit II gene. Fifteen native Trichoderma spp. were identified as T. harzianum, T. asperellum, T. virens and T. reesei. Some of these strains showed strong antagonistic activities against several important pathogens in Ecuador, such as Fusarium oxysporum f. sp. cubense (Panama disease) and Mycosphaerella fijiensis (black Sigatoka) on banana, as well as Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom disease) on cacao. The isolates also showed inhibitory effects on in vitro colony growth tests against Japanese isolates of Fusarium oxysporum f. sp. lycopersici, Alternaria alternata and Rosellinia necatrix. The native Trichoderma strains characterized here are potential biocontrol agents against important pathogens of banana and cacao in Ecuador.