Taxonomy and Geographic Distribution of
Vascular Pathogens in Perennial Crops
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-a 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.
Cadophora species associated with wood-decay of grapevine in North America
Cadophora species are reported from grapevine (Vitis vinifera L.) in California, South Africa, Spain, Uruguay, and Canada. Frequent isolation from vines co-infected with the Esca pathogens (Togninia minima and Phaeomoniella chlamydospora), and confirmation of its ability to cause wood lesions/discoloration in pathogenicity tests, suggest that C. luteo-olivacea is part of the trunk pathogen complex. In North America, little is known regarding the diversity, geographic distribution, and roles of Cadophora species as trunk pathogens. Accordingly, we characterized 37 Cadophora isolates from ten US states and two Canadian provinces, based on molecular and morphological comparisons, and pathogenicity. Phylogenetic analysis of three loci (ITS, translation elongation factor 1-alpha (TEF1-a) and beta-tubulin (BT)) distinguished two known species (C. luteo-olivacea and Cadophora melinii) and three newly-described species (Cadophora orientoamericana, Cadophora novi-eboraci, and Cadophora spadicis). C. orientoamericana, C. novi-eboraci, and C. spadicis were restricted to the northeastern US, whereas C. luteo-olivacea was only recovered from California. C. melinii was present in California and Ontario, Canada. Morphological characterization was less informative, due to significant overlap in dimensions of conidia, hyphae, conidiophores, and conidiogenous cells. Pathogenicity tests confirmed the presence of wood lesions after 24 m, suggesting that Cadophora species may have a role as grapevine trunk pathogens
Identification of Eutypa spp. Causing Eutypa Dieback of Grapevine in Eastern North America
Eutypa dieback of grapevine is caused by Eutypa lata in production areas with Mediterranean climates in California, Australasia, Europe, and South Africa. Eutypa dieback has also been described in the colder, eastern North American vineyards where cultivars adapted from native Vitis spp. (e.g., Vitis × labruscana ‘Concord’) are primarily grown. However, the causal agents associated with the diseases in this region have not been conclusively identified. Examination of 48 vineyards showing symptoms of dieback in the northeastern United States (Connecticut, Massachusetts, Michigan, New York, Ohio, and Rhode Island) and Ontario, Canada revealed that vineyards were mainly infected by Eutypa spp. other than E. lata. Multigene phylogenies (internal transcribed spacer ribosomal DNA, β-tubulin, and RNA polymerase II) of isolates recovered from these vineyards indicated that Eutypa dieback is caused primarily by an undescribed Eutypa sp. and E. laevata. Eutypa sp. was recovered from 56% of the vineyards examined, whereas E. laevata and E. lata were less far common (17 and 6%, respectively). Fruiting body morphology and spore dimensions supported phylogenetic separation of the three taxa. Pathogenicity tests conducted on Vitis vinifera ‘Chardonnay’ in the greenhouse and in the field verified that all three species were able to cause wood canker and to infect pruning wounds, respectively.
Characterization of Species of Diaporthe from Wood Cankers of Grape in Eastern North American Vineyards
In eastern North America, Phomopsis cane and leaf spot, caused by Phomopsis viticola, is a foliar disease of grape but, in the Mediterranean climate of western North America, P. viticola is primarily associated with wood cankers, along with other Diaporthe spp. To determine the identity of wood-infecting Diaporthe spp. in eastern North America, 65 isolates were cultured from 190 wood-canker samples from 23 vineyards with a history of Phomopsis cane and leaf spot. Identification of 29 representative isolates was based initially on morphology, followed by phylogenetic analyses of DNA sequences of the ribosomal DNA internal transcribed spacer region, elongation factor subunit 1-α, and actin in comparison with those of type specimens. Three species were identified: P. viticola, P. fukushii, and Diaporthe eres. Inoculations onto woody stems of potted Vitis labruscana ‘Concord’ and V. vinifera ‘Chardonnay’ showed that D. eres and P. fukushii were pathogenic (mean lesion lengths of 7.4 and 7.1 mm, respectively, compared with 3.5 mm for noninoculated controls) but significantly less so than wood-canker and leaf-spot isolates of P. viticola (13.5 mm). All three species infected pruning wounds of Concord and Chardonnay in the field. Our finding of pathogenic, wood-infecting Diaporthe spp. in all 23 vineyards suggests a frequent co-occurrence of the foliar symptoms of Phomopsis cane and leaf spot and wood cankers, although the latter are not always due to P. viticola.
Genetic Structure of the Fungal Grapevine Pathogen Eutypa lata from Four Continents
The generalist ascomycete fungus Eutypa lata causes Eutypa dieback of grapevine (Vitis vinifera) worldwide. To decipher the cosmopolitan distribution of this fungus, the population genetic structure of 17 geographic samples was investigated from four continental regions (Australia, California, Europe and South Africa), based on analysis of 293 isolates genotyped with nine microsatellite markers. High levels of haplotypic richness (R = 0.91–1) and absence of multilocus linkage disequilibrium among loci supported the preponderance of sexual reproduction in all regions examined. Nonetheless, the identification of identical multilocus haplotypes with identical vegetative compatibility groups, in some vineyards in California and South Africa, suggests that asexual dispersal of the fungus among neighbouring plants could be a rare means of disease spread. The greatest levels of allelic richness (A = 4.89–4.97) and gene diversity (H = 0.66–0.69) were found in Europe among geographic samples from coastal areas surrounding the Mediterranean Sea, whereas the lowest genetic diversity was found in South Africa and Australia (A = 2.78–3.74; H = 0.49–0.57). Samples from California, Australia and South Africa, which had lower genetic diversity than those of Europe, were also characterized by demographic disequilibrium and, thus, may represent founding populations of the pathogen. Low but significant levels of genetic differentiation among all samples (DEST = 0.12, P = 0.001; FST = 0.03, P = 0.001) are consistent with historical gene flow preventing differentiation at continental scales. These findings suggest that global, human-mediated spread of the fungus may have resulted in its current global distribution.
Cylindrocarpon Species Associated with Black-Foot of Grapevine in Northeastern United States and Southeastern Canada
Black-foot disease of grapevine is caused by a complex of soilborne fungi. The most common and virulent species, which are found across all major grapegrowing regions of the world, are Cylindrocarpon liriodendri (C. liriodendri) and C. macrodidymum (teleomorph = Neonectria). Other species with a more limited distribution and uncertainty regarding their pathogenicity include C. destructans, C. obtusisporum, C. pauciseptatum, Campylocarpon fasciculare (C. fasciculare), and C. pseudofasciculare. The goal was to identify the species associated with black-foot disease in vineyards of the northeastern United States (U.S.) and southeastern Canada as such regions have not previously been surveyed. Recent expansion of winegrape acreage in these regions necessitates a clear understanding of the disease risks. Eleven U.S. states and two Canadian provinces were surveyed. Genus-level identification was based preliminarily on colony morphology. Species-level identity was based on phylogenetic analysis of two nuclear loci, 5.8S rDNA and ß-tubulin, using voucher specimens and sequences with high sequence identity. We report for the first time from Canada recovery of C. liriodendri, C. macrodidymum, and C. destructans from symptomatic grapevines. Also reported are species not previously identified from black-foot symptomatic grapes anywhere in the world, including C. didymum and a Neonectria mammoidea-like species. Results suggest that local viticultural practices, primarily burying the vine underground during winter, may create injuries, and thus exacerbate infection by wound pathogens such as Cylindrocarpon. Overall this work improves the knowledge of black-foot disease in these nascent grapegrowing regions and will be helpful to growers in their decisions regarding viticultural practices, planting, and disease management.
Eutypa dieback was responsible for a loss in net income for California wine grapes estimated to be over $260 million in 1999. Many growers consider Eutypa to be the most significant disease of grapevines. Typical symptoms of E. lata include formation of a wedge-shaped canker and stunted shoots with cupped, tattered, chlorotic, and necrotic leaves that are best seen in spring time. Foliar symptoms are due to toxins produced by E. lata. Differences in susceptibility of grapevine cultivars to infection have been reported, although no cultivars are immune. Cankers develop downward at a faster rate than toward the end of cordons and also increase in diameter over time. Extended infection of grapevines by E. lata leads to vine death.
Neofusicoccum parvum, a member of the Botryosphaeriaceae family, is a vascular pathogen that causes severe decline and dieback symptoms in grapevines worldwide. The draft genome of the grapevine isolate N. parvum UCR-NP2 provides a first glimpse into the complex set of putative virulence factors that this pathogen may use to rapidly colonize plants.
Eutypa dieback is a vascular disease of several cultivated crops and trees worldwide. The attribution of the name to the agent responsible for branch dieback is ambiguous. Pathogenicity of Eutypa sp. first was reported on apricot and the causal agent was named E. armeniacae. However, no morphological differences were reported with the previously described E. lata, and some authors considered both species synonymous. Others regarded them as distinct species on the basis of pathogenesis and molecular analysis. We further investigated the relatedness of both species by phylogenetic analyses of the internal transcribed spacer region and β-tubulin gene. These analyses included several other taxa placed in the same family (Diatrypaceae), and yielded three groups. The isolates referred to as E. lata in previous work clustered with Diatrype stigma in one group. Isolates of E. armeniacae and E. lata clustered in a second group, supporting the synonymy of these species. The third group included other Eutypa spp. supporting the polyphyletic origin of this genus. Measurements of conidia length and secondary metabolite production of isolates supported the phylogenetic analyses. Secondary metabolites appeared to be a synapomorphic character shared by several taxa including E. lata, E. armeniacae, E. laevata, and E. petrakii var. petrakii.
The vascular pathogen Eutypa lata, which causes Eutypa dieback in grapevines, is a major threat to grape production worldwide. Here, we present the first draft genome sequence of E. lata (UCR-EL1). The computational prediction and annotation of the protein-coding genes of UCR-EL1 provide an initial inventory of its potential virulence factors.
Eutypa lata is a vascular canker pathogen of woody plants commonly diagnosed by isolating the pathogen from infected tissue. Related fungi from the same family, the Diatrypaceae, also have been found in association with grapevine in Californian vineyards. An in situ polymerase chain reaction (PCR) method has been developed for detection of E. lata in infected wood tissue. However, our results indicate that this method also would amplify other Diatrypaceous fungi, which could potentially lead to an incorrect diagnosis. Therefore, we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The internal transcribed spacer (ITS)1/5.8S/ITS2 ribosomal DNA region was amplified by PCR using universal primers, and RFLP patterns were determined after digestion with AluI. The restriction profiles obtained served to distinguish E. lata from wood trunk pathogens of grapevine (Phomopsis viticola, Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora), Diatrypaceous fungi (Diatrype sp., Diatrypella sp., Eutypella vitis, and Eutypa leptoplaca), and Cryptovalsa sp. found on dead wood of grapevine, and other Eutypa spp. (E.petrakii var. hederae, E. astroidea, E. crustata, and E. lejoplaca), with the exception of E. armeniacae, which we regard as a synonym for E. lata, and E. laevata, which has not been found on grapevine.
Grapevine infections by Phaeoacremonium aleophilum in association with other pathogenic fungi cause complex and economically important vascular diseases. Here we present the first draft of the P. aleophilum genome sequence, which comprises 624 scaffolds with a total length of 47.5 Mb (L50, 45; N50, 336 kb) and 8,926 predicted protein-coding genes.
The Botryosphaeriaceae is a fungal family that includes many destructive vascular pathogens of woody plants (e.g., Botryosphaeria dieback of grape, Panicle blight of pistachio). Species in the genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum, and Neoscytalidium attack a range of horticultural crops, but they vary in virulence and their abilities to infect their hosts via different infection courts (flowers, green shoots, woody twigs). Isolates of seventeen species, originating from symptomatic apricot, grape, pistachio, and walnut were tested for pathogenicity on grapevine wood after 4 months of incubation in potted plants in the greenhouse. Results revealed significant variation in virulence in terms of the length of the internal wood lesions caused by these seventeen species. Phylogenomic comparisons of the seventeen species of wood-colonizing fungi revealed clade-specific expansion of gene families representing putative virulence factors involved in toxin production and mobilization, wood degradation, and nutrient uptake. Statistical analyses of the evolution of the size of gene families revealed expansions of secondary metabolism and transporter gene families in Lasiodiplodia and of secreted cell wall degrading enzymes (CAZymes) in Botryosphaeria and Neofusicoccum genomes. In contrast, Diplodia,
Dothiorella, and Neoscytalidium generally showed a contraction in the number of members of these gene families. Overall, species with expansions of gene families, such as secreted CAZymes, secondary metabolism, and transporters, were the most virulent (i.e., were associated with the largest lesions), based on our pathogenicity tests and published reports. This study represents the first comparative phylogenomic investigation into the evolution of possible virulence factors from diverse, cosmopolitan members of the Botryosphaeriaceae.
Characterization of Lasiodiplodia species associated with grapevines in Mexico
Botryosphaeria dieback is one of the most prevalent grapevine trunk diseases (GTDs), and is caused by fungi in the Botryosphaeriaceae. Fungi invade grapevine vascular systems mainly through pruning wounds, and cause cankers and necrotic lesions, which lead to grapevine decline and death. Lasiodiplodia theobromae has been reported as a highly virulent pathogen of grapevine, and was previously reported in Mexican vineyards. The taxonomy of Lasiodiplodia was recently revised, adding new species, and some were reduced to synonymy. This study aimed to characterize Lasio- diplodia producing grapevine dieback symptoms in Sonora and Baja California, Mexico. Using the phylogenetic markers tef1-α and ITS regions, Lasiodiplodia brasiliensis, L. crassispora, L. exigua, and L. gilanensis were identified. Lasidiplodia exigua was the most prevalent species. Lasiodiplodia brasiliensis and L. gilanensis were very virulent to ‘Cabernet Sauvignon’ plants, while L. exigua and L. gilanensis were less virulent, and L. crassispora did not produce lesions at 2 months post-inoculation. The optimum temperature of the Lasiodiplodia spp. was 28°C, but all four species grew up to 37°C, and the isolates of L. exigua grew slowly at 40°C. This is the first report of the four of Lasiodiplodia species in vineyards of Mexico.
Identity and pathogenicity of Botryosphaeriaceae and Diaporthaceae from Juglans
regia in Chile
English walnut (Juglans regia) has become an important crop in Chile, representing 11.5% of the total area of fruit trees, surpassed only by grapevine. As the Chilean walnut industry rapidly expands,
young orchards are at risk from the emergence of new fungal diseases. Botryosphaeriaceae and
Diaporthaceae fungi have been recognized as main causes of wood diseases in walnut, with symptoms of dieback, canker, and blight. In winter 2017, samples were collected from different orchards in ValparaÃso and Maule regions. Fungal isolates recovered were cultured, characterized morphologically, and identified using DNA sequence analyses. Three species (Neofusicoccum parvum, Diplodia mutila, Diplodia seriata) were characterized in Botryosphaeriaceae and two (Diaporthe cynaroidis, Diaporthe australafricana) in Diaporthaceae. Pathogenicity tests showed that N. parvum was the most aggressive species to walnut. This study confirmed the presence of pathogenic Botryosphaeriaceae and Diaporthaceae in J. regia that should be considered an increasing risk for the growing Chilean walnut industry.
Comparative Profiling of Wood Canker Pathogens from Spore Traps and Symptomatic Plant Samples Within California Almond and Walnut Orchards
Fungi causing wood canker diseases are major factors limiting productivity and longevity of almond and walnut orchards. The goal of this study was to compare pathogen profiles from spore traps with those of plant samples collected from symptomatic almond and walnut trees and assess if profiles could be influenced by orchard type and age, rainfall amount and frequency, and/or neighboring trees. Three almond orchards and one walnut orchard with different characteristics were selected for this study. Fungal inoculum was captured weekly from nine trees per orchard using a passive spore-trapping device, during a 30-week period in the rainy season (October to April) and for two consecutive years. Fungal taxa identified from spore traps were compared with a collection of fungal isolates obtained from 61 symptomatic wood samples collected from the orchards. Using a culture-dependent approach coupled with molecular identification, we identified 18 known pathogenic species from 10 fungal genera (Ceratocystis destructans, Collophorina hispanica, Cytospora eucalypti, Diaporthe ampelina, Diaporthe chamaeropis/rhusicola, Diaporthe eres, Diaporthe novem, Diplodia corticola, Diplodia mutila, Diplodia seriata, Dothiorella iberica, Dothiorella sarmentorum, Dothiorella viticola, Eutypa lata, Neofusicoccum mediterraneum, Neofusicoccum parvum, Neoscytalidium dimidiatum, and Pleurostoma richardsiae), plus two unidentified Cytospora and Diaporthe species. However, only four species were identified with both methods (Diplodia mutila, Diplodia seriata, Dothiorella Iberica, and E. lata), albeit not consistently across orchards. Our results demonstrate a clear disparity between the two diagnostic methods and caution against using passive spore traps to predict disease risks. In particular, the spore trap approach failed to capture: insect-vectored pathogens such as Ceratocystis destructans that were often recovered from almond trunk and scaffold; Diaporthe chamaeropis/rhusicola commonly isolated from wood samples likely because Diaporthe species have a spatially restricted dispersal mechanism, as spores are exuded in a cirrus; and pathogenic species with low incidence in wood samples such as P. richardsiae and Collophorina hispanica. We propose that orchard inoculum is composed of both endemic taxa that are characterized by frequent and repeated trapping events from the same trees and isolated from plant samples, as well as immigrant taxa characterized by rare trapping events. We hypothesize that host type, orchard age, precipitation, and alternative hosts at the periphery of orchards are factors that could affect pathogen profile. We discuss the limitations and benefits of our methodology and experimental design to develop guidelines and prediction tools for fungal wood canker diseases in California orchards.
First Report of Diaporthe ambigua Associated with Dead Arm Disease on Grapevine in Chile
Grapevine (Vitis vinifera L.) is one of the most important fruit crops in Chile based on economic value. Phaeomoniella chlamydospora and Botryosphaeriaceae species have been reported as the major causal agents associated with dieback symptoms in Chile commercial vineyards (Besoain 2018; Díaz and Latorre 2014; Larach et al. 2020). Recently, Eutypa lata has been reported attacking Chilean vineyards with dieback symptoms (Lolas et al. 2020). In this study, two commercial cultivar Cabernet Sauvignon vineyards in the O’Higgins Region of Chile showing dead cordons, dead spur with a grayish color, canker, and vascular necrosis were sampled in fall 2018, and a high incidence of symptoms was observed. Four symptomatic wood samples were analyzed from these vineyards...
First Report of Diaporthe cynaroidis and D. australafricana Associated with Walnut Branch Canker in Chile
In 2017, with more than 40,000 planted hectares, Chile became the second exporter of English walnut (Juglans regia) worldwide with a production of about 100,000 tons (Muñoz 2017). In 2017, low incidence of branch canker from old pruning wounds was observed in young walnut orchards of cultivar ‘Chandler’ in the Maule region of Chile. Fifty symptomatic wood samples were collected from 10 trees in two orchards. Isolates were recovered from the wood necrotic lesions, displaying a light to dark brown discoloration in the cortical and vascular tissue, as previously described (Chen et al. 2014). The isolates were identified as Diaporthe cynaroidis and D. australafricana based on morphological characteristics and DNA sequencing...