Fungal species associated with grapevine trunk diseases in Washington wine grapes and California table grapes, with novelties in the genera Cadophora, Cytospora, and Sporocadus

Abstract

Grapevine trunk diseases cause serious economic losses to grape growers worldwide. The identification of the causal fungi is critical to implementing appropriate management strategies. Through a culture-based approach, we identified the fungal species composition associated with symptomatic grapevines from wine grapes in southeastern Washington and table grapes in the southern San Joaquin Valley of California, two regions with contrasting winter climates. Species were confirmed through molecular identification, sequencing two to six gene regions per isolate. Multilocus phylogenetic analyses were used to identify novel species. We identified 36 species from 112 isolates, with a combination of species that are new to science, are known causal fungi of grapevine trunk diseases, or are known causal fungi of diseases of other woody plants. The novel species Cadophora columbiana , Cytospora macropycnidia , Cytospora yakimana , and Sporocadus incarnatus are formally described and introduced, six species are newly reported from North America, and grape is reported as a new host for three species. Six species were shared between the two regions: Cytospora viticola , Diatrype stigma , Diplodia seriata , Kalmusia variispora , Phaeoacremonium minimum , and Phaeomoniella chlamydospora . Dominating the fungal community in Washington wine grape vineyards were species in the fungal families Diatrypaceae, Cytosporaceae and Sporocadaceae, whereas in California table grape vineyards, the dominant species were in the families Diatrypaceae, Togniniaceae, Phaeomoniellaceae and Hymenochaetaceae. Pathogenicity tests demonstrated that 10 isolates caused wood discoloration similar to symptomatic wood from which they were originally isolated. Growth rates at temperatures from 5 to 35°C of 10 isolates per region, suggest that adaptation to local climate might explain their distribution.

Full text

Fungal species associated with
grapevine trunk diseases in
Washington wine grapes and
California table grapes, with
novelties in the genera
Cadophora,Cytospora,
and Sporocadus
Renaud Travadon
1
*, Daniel P. Lawrence
1
, Michelle M. Moyer
2
,
Phillip T. Fujiyoshi
3
and Kendra Baumgartner
3
*
1
Department of Plant Pathology, University of California, Davis, Davis, CA, United States,
2
Department of Horticulture, Irrigated Agriculture Research and Extension Center, Washington
State University, Prosser, WA, United States,
3
Crops Pathology and Genetics Research Unit, United
States Department of Agriculture –Agricultural Research Service, Davis, CA, United States
Grapevine trunk diseases cause serious economic losses to grape growers
worldwide. The identification of the causal fungi is critical to implementing
appropriate management strategies. Through a culture-based approach, we
identified the fungal species composition associated with symptomatic
grapevines from wine grapes in southeastern Washington and table grapes in
the southern San Joaquin Valley of California, two regions with contrasting
winter climates. Species were confirmed through molecular identification,
sequencing two to six gene regions per isolate. Multilocus phylogenetic
analyses were used to identify novel species. We identified 36 species from
112 isolates, with a combination of species that are new to science, are known
causal fungi of grapevine trunk diseases, or are known causal fungi of diseases
of other woody plants. The novel species Cadophora columbiana,Cytospora
macropycnidia,Cytospora yakimana, and Sporocadus incarnatus are formally
described and introduced, six species are newly reported from North America,
and grape is reported as a new host for three species. Six species were shared
between the two regions: Cytospora viticola,Diatrype stigma,Diplodia seriata,
Kalmusia variispora,Phaeoacremonium minimum,andPhaeomoniella
chlamydospora. Dominating the fungal community in Washington wine
grape vineyards were species in the fungal families Diatrypaceae,
Cytosporaceae and Sporocadaceae, whereas in California table grape
vineyards, the dominant species were in the families Diatrypaceae,
Togniniaceae, Phaeomoniellaceae and Hymenochaetaceae. Pathogenicity
tests demonstrated that 10 isolates caused wood discoloration similar to
symptomatic wood from which they were originally isolated. Growth rates at
Frontiers in Fungal Biology frontiersin.org01
OPEN ACCESS
EDITED BY
Gonzalo A. Diaz,
Universidad de Talca, Chile
REVIEWED BY
Christian Kraus,
Julius Kühn-Institut, Germany
Josep Armengol,
Universitat Politècnica de València,
Spain
*CORRESPONDENCE
Kendra Baumgartner
kendra.baumgartner@usda.gov
Renaud Travadon
rtravadon@ucdavis.edu
SPECIALTY SECTION
This article was submitted to
Fungi-Plant Interactions,
a section of the journal
Frontiers in Fungal Biology
RECEIVED 12 August 2022
ACCEPTED 09 September 2022
PUBLISHED 07 October 2022
CITATION
Travadon R, Lawrence DP, Moyer MM,
Fujiyoshi PT and Baumgartner K (2022)
Fungal species associated with
grapevine trunk diseases in
Washington wine grapes and
California table grapes, with
novelties in the genera Cadophora,
Cytospora, and Sporocadus.
Front. Fungal Bio. 3:1018140.
doi: 10.3389/ffunb.2022.1018140
COPYRIGHT
© 2022 Travadon, Lawrence, Moyer,
Fujiyoshi and Baumgartner. This is an
open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the
original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
cited, in accordance with accepted
academic practice. No use,
distribution or reproduction is
permitted which does not comply with
these terms.
TYPE Original Research
PUBLISHED 07 October 2022
DOI 10.3389/ffunb.2022.1018140

temperatures from 5 to 35°C of 10 isolates per region, suggest that
adaptation to local climate might explain their distribution.
KEYWORDS
fungal ecology, fungal diversity, taxonomy, Sordariomycetes, Leotiomycetes,
Diatrypaceae, Kalmusia, Thyrostroma
Introduction
The numerous microbes associated with grapevine (Vitis
sp.) play important roles in the plant’s nutrition and health
status (Deyett and Rolshausen, 2020;Bettenfeld et al., 2022).
Recent research efforts have characterized the grapevine
microbiota in the rhizosphere,phyllosphereand endosphere
(Bettenfeld et al., 2022;Cobos et al., 2022). The composition
and structure of microbial communities have been found to be
associated with vineyard management practices, vineyard
location, and cultivars, suggesting that, for example, fruit and
wine characteristics may be affected by the “microbial terroir”
(Bokulich et al., 2014;Bokulich et al., 2016;Burns et al., 2016).
The grapevine microbiota has also been investigated in relation
to plant health (Bettenfeld et al., 2022), including its role
relative to grapevine trunk diseases (Del Frari et al., 2019;
Bruez et al., 2020;Pacetti et al., 2021;Patanita et al., 2022).
These studies used culture-independent, DNA-metabarcoding
approaches to reveal a considerable diversity of microbes
associated with healthy and diseased grapevines, but these
approaches have their limitations. First, studies of the fungal
microbiota often rely on the sequencing of the ribosomal
internal transcribed spacer (ITS) (Schoch et al., 2012), which
may influencetheresultsduetothespecific PCR conditions
and primers used (Jayawardena et al., 2018;Morales-Cruz
et al., 2018b). Further, the high intra- and interspecific
variability of the ITS region often limit the characterization
of fungal communities to the genus level (Dissanayake et al.,
2018). Second, the relationship between gene abundance and
organismal abundance is not straightforward (Amend et al.,
2010). While there are a combination of methods to confirm
the viability of the taxa detected, such as metatranscriptomic
analyses and/or complementary isolations on culture media
(Eichmeier et al., 2018,Morales‐Cruz et al., 2018a,Niem et al.,
2020;Haidar et al., 2021;Nerva et al., 2022;Paolinelli et al.,
2022;Vanga et al., 2022), fungi detected strictly through DNA
sequencing may not be metabolically active (Baldrian et al.,
2012). Most importantly, although extensive datasets are
generated with these approaches, very little may be known
about the biology and ecology of some taxa; the understanding
of fungal communities cannot be achieved without
understanding the ecology of individual species (Peay, 2014).
Lastly, putatively novel species identified from metabarcoding
studies, in the absence of physical specimens or cultures,
cannot be properly nor formally characterized.
A traditional, field-based inventory of fungal biodiversity
gathers the crucial details of taxonomy and life-history traits,
which are required to test hypotheses on the roles of microbes
in plant health, and importantly provides a tangible
fungarium collection for sharing scientificknowledgethat
can last over time (Cazabonne et al., 2022). To describe fungal
communities in grapevine, such culture-dependent
approaches draw on mycological methods and rely on
direct observations and microscopic examinations of
diseased tissues and fungal structures, microbiological
isolations, morphological characterizations of cultures and
DNA-based phylogenetic analyses. The community
composition is undoubtedly underestimated because
artificial media and incubation conditions typically favor a
subset of taxa (Jayawardena et al., 2018). Nonetheless, these
approaches have enriched our understanding of fungal
communities inhabiting grapevine as it relates to trunk
diseases, either by comparing fungal community profiles
over time (Kraus et al., 2019), among vines from different
production areas (U

rbez-Torres et al., 2012), between
symptomatic or asymptomatic plants (Hofstetter et al.,
2012), or grown under different viticultural practices
(Travadon et al., 2016). With physical culture collections,
population genetics and genomics analyses can be conducted
(Peros and Berger, 1999;Smetham et al., 2010;Travadon
et al., 2012;Gramaje et al., 2013;Onetto et al., 2022), the
pathogenicity of fungal isolates can be assessed
experimentally (Cloete et al., 2015;Lawrence et al., 2015;
Travadon and Baumgartner, 2015;Baloyi et al., 2018),
interactions between species can be evaluated under
controlled conditions (Lawrence et al., 2018b;Silva-
Valderrama et al., 2021), and molecular databases including
the ecological guild of species can be built (Lawrence et al.,
2017b). Further, new pathogenic species can be described and
linked with type specimens deposited in public repositories
(Travadon et al., 2015;Lawrence et al., 2017a;Moyo et al.,
2018;Kraus et al., 2020).
Grapevine trunk diseases (GTDs) are widespread throughout
the world. The chronic wood infections cause a dieback and/or
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org02

decline of vines, affecting fruit quality, reducing the productivity and
longevity of vineyards, and causing serious economic losses to the
viticulture industry (Kaplan et al., 2016;Sosnowski and Mccarthy,
2017;Baumgartner et al., 2019). GTDs encompass a set of diseases
according to their etiology and fungal causal agents. The most
common diseases are black foot disease (causal agents in the order
Hypocreales and family Nectriaceae), Botryosphaeria dieback
(Botryosphaeriales, Botryosphaeriaceae), Eutypa dieback
(Xylariales, Diatrypaceae), esca and Petri disease (Helotiales,
Ploettnerulaceae; Phaeomoniellales, Phaeomoniellaceae;
Togniniales,Togniniaceae; Hymenochaetales, Hymenochaetaceae),
and Phomopsis dieback (Diaporthales, Diaporthaceae). The causal
agents are estimated to represent more than 130 fungal species
(Gramaje et al., 2018),andtheyspanfourclassesinthe
Pezizomycotina (Ascomycota) and 10 genera in the
Hymenochaetales (Basidiomycota) (Lawrence et al., 2017b). Taxa
belonging to the order Xylariales have recently been associated with
dieback symptoms of grapevines (Lawrence et al., 2018b;Bahmani
et al., 2021;Moghadam et al., 2022).
Among western North American viticulture regions, the fungal
species causing grapevine trunk diseases have been examined
primarily in California. Eleven species within the Diatrypaceae
(Trouillas et al., 2010) and 15 species within the Botryosphaeriaceae
(U

rbez-Torres et al., 2006;U

rbez-Torres, 2011;Rolshausen et al.,
2013;Nouri et al., 2018) have been isolated from grapevine cankers,
and their pathogenicity has been examined (U

rbez-Torres and
Gubler, 2009;Trouillas and Gubler, 2010). Regarding the fungi
associated with Petri disease and esca, Cadophora luteo-olivacea,
Cadophora melinii,Fomitiporia polymorpha,Inonotus vitis,
Phaeoacremonium fraxinopennsylvanicum,Phaeoacremonium
inflatipes,Phaeoacremonium minimum,Phaeoacremonium viticola,
and Phaeomoniella chlamydospora have all been isolated from
symptomatic grapevines and are confirmed pathogenic (Scheck
et al., 1998;Eskalen et al., 2005a;Eskalen et al., 2005b;Travadon
et al., 2015;Brown et al., 2020). Additional esca and Petri disease
pathogens identified in vineyards from British Columbia, Canada,
include Phaeoacremonium canadense,Phaeoacremonium iranianum,
and Phaeoacremonium roseum (U

rbez-Torres et al., 2014a). In
California and British Columbia, Black foot pathogens include six
and five species from the Nectriaceae, respectively, in the genera
Campylocarpon,Dactylonectria,Ilyonectria,andThelonectria (U

rbez-
Torres et al., 2014b;Lawrence et al., 2019). In addition, seven species
in the genus Diaporthe have been recovered from grapevines with
dieback symptoms in California, and most species were confirmed
pathogenic (Lawrence et al., 2015). Surveys of trunk diseases in
vineyards in dry grape-growing regions of southern California
(Coachella) and Mexico (Baja California and Sonora) report on
species causing Botryosphaeria dieback, Eutypa dieback, and esca
(Gispertetal.,2020;U

rbez-Torres et al., 2020;Rangel Montoya
et al., 2021).
Important differences in environmental conditions and
cultivar susceptibility may affect the species composition of
grapevine trunk pathogens. The state of Washington is the
second largest US producer of wine grapes and the largest
producer of juice grapes in the US (National Agricultural
Statistics Service, 2018). Wine grapes are grown principally in
southeastern Washington, where the climate is a semi-arid
steppe, characterized by very warm, dry summers, large
diurnal temperature changes, and cold winters (USDA Cold
Hardiness Zones 6-7), where injuries due to winter frost and
freezing conditions are common. Most Vitis vinifera cultivars
grown in Washington belong to the eco-geographic group
occidentalis, which includes the small-berried wine grapes
originating from western Europe (Aradhya et al., 2003). In
Washington wine grape vineyards, Eutypa dieback can result
in drastic yield reductions (Johnson and Lunden, 1987);
however, the characterization of the pathogenic species is
limited. To our knowledge, a single report listed the presence
of Cryptosphaeria pullmanensis,Diatrype whitmanensis,
Eutypa laevata,andEutypa lata within the Diatrypaceae,
Diplodia (Dip.) seriata and Dip.mutila within the
Botryosphaeriaceae, Cytospora chrysosperma and Cytospora
rhodophila within the Cytosporaceae, Diaporthe eres within
the Diaporthaceae, and Discostroma fuscellum within the
Amphisphaeriales [synonymized with Xylariales; (Holland
et al., 2015)].
In California, table and raisin grapes are grown extensively
in the southern San Joaquin Valley, which is characterized as a
semi-arid climate with hot, dry summers and cool winters. The
table grape cultivars belong to the eco-geographic group
orientalis (large-berried table grapes of West Asia; e.g.,
‘Thompson Seedless’, synonym ‘Sultanina’), pontica
(intermediate type from the basin of the Black Sea and Eastern
Europe; e.g., ‘Muscat of Alexandria’), and cultivars with mixed
pedigree from these two groups (e.g., ‘Scarlet Royal’). Differences
in susceptibility to vascular diseases among these three groups
have been postulated (Travadon et al., 2013;Deyett et al., 2019).
Reports of pathogenic species associated with trunk diseases in
the southern San Joaquin Valley include members of the
Botryosphaeriaceae, Diaporthe ampelina and Eutypa lata
(U

rbez-Torres et al., 2006).
We investigated the fungal species cultured from the
wood of grapevines with external symptoms of trunk
diseases. To compare two western US regions with different
climates and different cultivars, we surveyed 10 wine grape
vineyards in Washington and 10 table grape vineyards in
California, with an emphasis on vineyards with leaf
symptoms of esca. We determined the pathogenicity to
grapevine of a sub-sample of undescribed and under-
studied species, and compared the thermophilic profiles of
isolates from each region under controlled conditions, in
order to assess if temperature could have an influence on
the fungal community compositionineachregion.Finally,we
described four species new to science.
Travadon et al. 10.3389/ffunb.2022.1018140
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Material and methods
Field sites and fungal isolations
In August 2018 and October 2018, we surveyed 10 wine
grape vineyards in southeastern Washington State and 10 table
grape vineyards in the southern San Joaquin Valley of California,
respectively. The cooperation of local grape-growers and
extension agents targeted vineyards with a history of trunk
disease symptoms. Symptoms included general dieback
symptoms, namely dead cordons, dead fruiting positions, and
dead canes, usually present on vines affected by Botryosphaeria,
Cytospora, Eutypa or Phomopsis diebacks. The selection of the
20 vineyards was also based on the presence of typical esca
symptoms (interveinal leaf chlorosis and vine apoplexy observed
in Summer) and typical Eutypa symptoms (stunted shoots,
dwarfing and cupping of leaves with tattered margins observed
in Spring). Sampled material consisted of diseased wood pieces
from trunks and cordons. Internal symptoms revealed after
cutting through the wood of symptomatic vines included
vascular streaking, light brown to black discolorations, and
white-rotted wood. Fifty-one wood samples were collected in
Washington and 53 wood samples were collected in California.
From each wood sample, 18 to 24 wood chips (4 × 4 × 2 mm)
were cut from the margins of necrotic wood with flame-sterilized
pruning shears, surface disinfected in 0.6% sodium hypochlorite
(pH 7.2) for 30 s, rinsed in two serial baths of sterile deionized
water for 30 s, dried on sterile filter paper and plated (6 to 8
wood chips per plate) either on potato dextrose agar (PDA,
Difco, Detroit, MI) amended with tetracycline (1 mg/L) or malt
extract agar (MEA, Difco) amended with streptomycin sulphate
(100 mg/L) or water agar (Difco) amended with benomyl 50WP
(4 mg/L) and streptomycin sulphate (100 mg/L). Incubation of
Petri dishes occurred at 22°C in the dark for up to 28 days.
Emerging colonies were hyphal-tip purified to PDA for
further analyses.
Fungal identification
Isolates were categorized into morphological groups based on
colony morphology on PDA and limited microscopic
observations. A preliminary species-level identification relied on
the sequencing of the ITS gene region. Mycelium collection and
DNA extraction followed the protocols outlined in Lawrence et al.
(2018b). Fungi that are generally not considered as causal
pathogens of trunk diseases were not examined further (62
isolates in the genera Acremonium,Alternaria,Aureobasidium,
Camarosporium,Collariella,Coniothyrium,Epicoccum,
Fusarium,Peyronellea,Pseudocamarosporium,Trichoderma,
and Tamaricicola). For the remaining 112 isolates (Table 1),
sequencing of the translation elongation factor 1 alpha (TEF-1a)
regionwasperformedforisolatesinthefamilies
Botryosphaeriaceae, Cytosporaceae, Diaporthaceae,
Didymosphaeriaceae, Dothidotthiaceae, Graphostromataceae,
Hymenochaetaceae, Ploettnerulaceae, Physalacriaceae,
Polyporaceae, Quambalariaceae, and Sporocadaceae.
Sequencing of beta-tubulin (TUB2) was performed for isolates
in the families Cytosporaceae, Diaporthaceae, Diatrypaceae,
Phaeomoniellaceae, Ploettnerulaceae, Sporocadaceae, and
Togniniaceae. The actin (ACT) gene region was sequenced for
isolates in the families Cytosporaceae, Diaporthaceae,
Dothidotthiaceae, and Togniniaceae. Sequencing of the second
largest subunit of RNA polymerase II (RPB2) was performed for
isolates in the families Cytosporaceae, Diaporthaceae,
Dothidotthiaceae, Graphostromataceae, Hymenochaetaceae,
Physalacriaceae, Polyporaceae, Quambalariaceae, and
Sporocadaceae. Sequencing of the 28S ribosomal RNA gene
(LSU) was performed for isolates in the families Cytosporaceae,
Diaporthaceae, Physalacriaceae, Pleosporineae, Polyporaceae,
and Sporocadaceae.
DNA samples were used undiluted as template in PCR, or
diluted 1:10 when the undiluted template yielded no product.
Amplifications were performed in 25 mL reactions containing 1×
GoTaq®Flexi colorless buffer (Promega, Madison, Wisconsin,
USA), 1 mM of each primer and 1 mL DNA (Supplementary
Table 1). Magnesium concentrations were titrated to 3 mM for
Actin for greater primer specificity. All extension steps were at
72°C. Conditions for some primer pairs were made more
stringent for difficult templates or were altered slightly for
efficient thermocycler use. Successful amplifications were
verified by gel electrophoresis and bidirectionally sequenced by
the UC Davis College of Biological Sciences DNA
Sequencing Facility.
Forward and reverse nucleotide sequences were assembled,
proofread, and edited in Sequencher 5.4.6 (Gene Codes
Corporation, Ann Arbor, Michigan, USA), and deposited in
GenBank (Table 1). Preliminary species identities were obtained
by BLASTn searches of sequences against the nucleotide
database of GenBank from the National Center for
Biotechnology Information (NCBI) and the curated molecular
repository of grapevine trunk pathogens TrunkDiseaseID.org
(Lawrence et al., 2017b). The sequences of some of the isolates
did not match any known molecular data in either database. In
these cases, sequences with high similarity from ex-type and
non-type isolates were included for phylogenetic reference
utilizing the BLASTn function in NCBI and extensive
literature review. Accordingly, relevant reference sequences
were retrieved from recent and comprehensive phylogenetic
studies for isolates in the genus Cadophora [(Travadon et al.,
2015;Linnakoski et al., 2018;Walsh et al., 2018;Bien and
Damm, 2020;Macia-Vicente et al., 2020;Aigoun-Mouhous
et al., 2021;Chen et al., 2022;Koukol and Macia-Vicente,
2022); Supplementary Table 2], Cytospora [(Lawrence et al.,
2018a;Pan et al., 2020;Shang et al., 2020;Pan et al., 2021);
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org04

TABLE 1 List of isolates obtained from grapevine (Vitis vinifera) with wood symptoms of trunk diseases in California table grapes and Washington wine grapes, and associated GenBank accession numbers.
Family Genus Specific epithet Isolate
1
State
2
County Site Cultivar ITS LSU ACT RPB2 TEF-1aTUB
Botryosphaeriaceae Diplodia seriata Kern802 CA Kern 6 Autumn King OP038084 –––OP106944 OP079902
Botryosphaeriaceae Lasiodiplodia exigua Kern801 CA Kern 2 Holiday OP038083 –––OP106943 –
Botryosphaeriaceae Lasiodiplodia gilanensis Kern803 CA Kern 9 Autumn King OP038085 –––OP106945 –
Botryosphaeriaceae Lasiodiplodia gilanensis Kern804 CA Kern 9 Autumn King OP038086 –––OP106946 –
Didymosphaeriaceae Kalmusia variispora Kern308 CA Kern 5 Muscat OP038056 –––OP106931 –
Didymosphaeriaceae Kalmusia variispora Kern309 CA Kern 5 Muscat OP038057 –––OP106932 –
Didymosphaeriaceae Kalmusia variispora Kern607 CA Kern 7 Flame Seedless OP038065 –––OP106939 –
Phaeomoniellaceae Phaeomoniella chlamydospora Kern703 CA Kern 3 Princess OP038067 –––OP106940 OP079888
Phaeomoniellaceae Phaeomoniella chlamydospora Kern707 CA Kern 5 Muscat OP038071 ––––OP079892
Phaeomoniellaceae Phaeomoniella chlamydospora Kern710 CA Kern 7 Flame Seedless OP038074 ––––OP079895
Phaeomoniellaceae Phaeomoniella chlamydospora Kern717 CA Kern 7 Flame Seedless OP038080 ––––OP079899
Phaeomoniellaceae Phaeomoniella chlamydospora Kern711 CA Kern 7 Flame Seedless OP038075 ––––OP079896
Diaporthaceae Diaporthe ampelina Kern903 CA Kern 3 Princess OP038090 OP076931 OP003973 OP095261 OP106950 OP079905
Diaporthaceae Diaporthe ampelina Kern904 CA Kern 3 Princess OP038091 OP076932 OP003974 OP095262 OP106951 OP079906
Diaporthaceae Diaporthe ampelina Kern902 CA Kern 3 Princess OP038089 OP076930 OP003972 OP095260 OP106949 OP079904
Diaporthaceae Diaporthe ampelina Kern906 CA Kern 7 Flame Seedless OP038093 OP076934 OP003976 OP095264 OP106953 OP079908
Cytosporaceae Cytospora macropycnidia Kern907 / CBS 149338 CA Kern 10 Scarlet Royal OP038094 OP076935 OP003977 OP095265 OP106954 OP079909
Cytosporaceae Cytospora viticola Kern504 CA Kern 5 Muscat OM976604 ON059352 ON012557 ON045095 ON012571 ON086752
Cytosporaceae Cytospora viticola Kern901 CA Kern 3 Princess OP038088 OP076929 OP003971 OP095259 OP106948 OP079903
Cytosporaceae Cytospora viticola Kern905 CA Kern 4 Crimson Seedless OP038092 OP076933 OP003975 OP095263 OP106952 OP079907
Togniniaceae Phaeoacremonium minimum Kern704 CA Kern 5 Muscat OP038068 –OP003962 ––OP079889
Togniniaceae Phaeoacremonium minimum Kern725 CA Kern 5 Muscat OP038082 –OP003970 ––OP079901
Togniniaceae Phaeoacremonium minimum Kern705 CA Kern 5 Muscat OP038069 –OP003963 ––OP079890
Togniniaceae Phaeoacremonium minimum Kern708 CA Kern 5 Muscat OP038072 –OP003965 ––OP079893
Togniniaceae Phaeoacremonium minimum Kern722 CA Kern 7 Flame Seedless OP038081 –OP003969 ––OP079900
Togniniaceae Phaeoacremonium minimum Kern709 CA Kern 7 Flame Seedless OP038073 –OP003966 ––OP079894
Togniniaceae Phaeoacremonium minimum Kern712 CA Kern 7 Flame Seedless OP038076 –OP003967 ––OP079897
Togniniaceae Phaeoacremonium minimum Kern714 CA Kern 7 Flame Seedless OP038077 –OP003968 ––OP079898
Togniniaceae Phaeoacremonium parasiticum Kern706 CA Kern 5 Muscat OP038070 –OP003964 ––OP079891
Togniniaceae Phaeoacremonium scolyti Kern701 CA Kern 4 Crimson Seedless OP038066 –OP003961 ––OP079887
Diatrypaceae Cryptovalsa ampelina Kern009 CA Kern 10 Scarlet Royal OP038052 ––––OP079881
Diatrypaceae Diatrype stigma Kern010 CA Kern 10 Scarlet Royal OP038053 ––––OP079882
Diatrypaceae Diatrype stigma Kern005 CA Kern 1 Princess OP038048 ––––OP079878
Diatrypaceae Diatrype stigma Kern306 CA Kern 1 Princess OP038054 ––––OP079883
(Continued)
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TABLE 1 Continued
Family Genus Specific epithet Isolate
1
State
2
County Site Cultivar ITS LSU ACT RPB2 TEF-1aTUB
Diatrypaceae Diatrype stigma Kern001 CA Kern 2 Holiday OP038044 ––––OP079874
Diatrypaceae Diatrype stigma Kern307 CA Kern 2 Holiday OP038055 ––––OP079884
Diatrypaceae Diatrypella verruciformis Kern002 CA Kern 2 Holiday OP038045 ––––OP079875
Diatrypaceae Diatrypella verruciformis Kern006 CA Kern 2 Holiday OP038049 ––––OP079879
Diatrypaceae Eutypella citricola Kern003 CA Kern 4 Crimson Seedless OP038046 ––––OP079876
Diatrypaceae Eutypella citricola Kern401 CA Kern 4 Crimson Seedless OP038063 ––––OP079885
Diatrypaceae Eutypella citricola Kern004 CA Kern 4 Crimson Seedless OP038047 ––––OP079877
Diatrypaceae Eutypella microtheca Kern008 CA Kern 10 Scarlet Royal OP038051 ––––OP079880
Sporocadaceae Hyalotiella transvalensis Kern605 / CBS 149294 CA Kern 4 Crimson Seedless OP038064 OP076926 OP003960 OP095255 OP106938 OP079886
Graphostromataceae Biscogniauxia mediterranea Kern007 CA Kern 8 Scarlet Royal OP038050 ––OP095249 OP106930 –
Graphostromataceae Biscogniauxia mediterranea Kern805 CA Kern 9 Autumn King OP038087 ––OP095258 OP106947 –
Hymenochaetaceae Fomitiporia polymorpha Kern311 CA Kern 7 Flame Seedless OP038058 OP076921 –OP095250 OP106933 –
Hymenochaetaceae Fomitiporia polymorpha Kern312 CA Kern 7 Flame Seedless OP038059 OP076922 –OP095251 OP106934 –
Hymenochaetaceae Fomitiporia polymorpha Kern313 CA Kern 7 Flame Seedless OP038060 OP076923 –OP095252 OP106935 –
Hymenochaetaceae Fomitiporia polymorpha Kern314 CA Kern 7 Flame Seedless OP038061 OP076924 –OP095253 OP106936 –
Hymenochaetaceae Fomitiporia polymorpha Kern315 CA Kern 7 Flame Seedless OP038062 OP076925 –OP095254 OP106937 –
Quambalariaceae Quambalaria cyanescens Kern715 CA Kern 6 Autumn King OP038078 OP076927 –OP095256 OP106941 –
Quambalariaceae Quambalaria cyanescens Kern716 CA Kern 6 Autumn King OP038079 OP076928 –OP095257 OP106942 –
Botryosphaeriaceae Diplodia seriata Bent802 WA Benton 1 Grenache / Sangiovese OP038033 –––
OP106920 OP079865
Botryosphaeriaceae Diplodia seriata Bent803 WA Benton 1 Grenache / Sangiovese OP038034 –––OP106921 OP079866
Botryosphaeriaceae Diplodia mutila Bent805 WA Benton 3 Cabernet Sauvignon OP038036 –––OP106923 –
Botryosphaeriaceae Diplodia seriata Bent801 WA Benton 1 Grenache / Sangiovese OP038032 –––OP106919 –
Botryosphaeriaceae Dothiorella iberica Bent804 WA Benton 2 Chardonnay OP038035 –––OP106922 –
Didymosphaeriaceae Kalmusia variispora Bent603 WA Yakima 10 Marsanne OP038019 –––OP106913 –
Pleosporineae Thyrostroma sp. Bent904 WA Benton 2 Chardonnay OP038037 OP076914 OP003954 OP095242 OP106924 OP079867
Phaeomoniellaceae Phaeomoniella chlamydospora Bent708 WA Benton 3 Cabernet Sauvignon OP038021 ––––OP079854
Phaeomoniellaceae Phaeomoniella chlamydospora Bent710 WA Benton 8 Chardonnay OP038023 ––––OP079856
Ploettnerulaceae Cadophora columbiana Bent717 / CBS 149300 WA Skamania 9 Pinot Noir OP038026 –––OP106915 OP079859
Ploettnerulaceae Cadophora columbiana Bent718 / CBS 149299 WA Skamania 9 Pinot Noir OP038027 –––OP106916 OP079860
Ploettnerulaceae Cadophora ferruginea Bent721 / CBS 149295 WA Grant 11 Grenache OP038030 –––OP106917 OP079863
Ploettnerulaceae Cadophora ferruginea Bent722 WA Grant 11 Grenache OP038031 –––OP106918 OP079864
Diaporthaceae Diaporthe eres Bent909 WA Skamania 9 Pinot Noir OP038042 OP076919 OP003959 OP095247 OP106929 OP079872
Cytosporaceae Cytospora yakimana Bent902 / CBS 149297 WA Benton 5 Tinto Cao OM976602 ON059350 ON012555 ON045093 ON012569 ON086750
Cytosporaceae Cytospora yakimana Bent903 / CBS 149298 WA Benton 5 Tinto Cao OM976603 ON059351 ON012556 ON045094 ON012570 ON086751
Cytosporaceae Cytospora viticola Bent503 WA Benton 5 Tinto Cao OP038011 OP076904 OP003949 OP095232 OP106912 OP079846
(Continued)
Travadon et al. 10.3389/ffunb.2022.1018140
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TABLE 1 Continued
Family Genus Specific epithet Isolate
1
State
2
County Site Cultivar ITS LSU ACT RPB2 TEF-1aTUB
Cytosporaceae Cytospora viticola Bent901 / CBS 149296 WA Benton 5 Souzao OM976601 ON059349 ON012554 ON045092 ON012568 ON086749
Cytosporaceae Cytospora viticola Bent907 WA Benton 8 Chardonnay OP038040 OP076917 OP003957 OP095245 OP106927 OP079870
Cytosporaceae Cytospora viticola Bent908 WA Benton 8 Chardonnay OP038041 OP076918 OP003958 OP095246 OP106928 OP079871
Cytosporaceae Cytospora viticola Bent411 WA Benton 8 Chardonnay OP038010 OP076903 OP003948 OP095231 OP106911 OP079845
Cytosporaceae Cytospora viticola Bent401 WA Benton 8 Chardonnay OM976600 ON059348 ON012553 ON045091 ON012567 ON086748
Cytosporaceae Cytospora viticola Bent905 WA Benton 8 Riesling OP038038 OP076915 OP003955 OP095243 OP106925 OP079868
Cytosporaceae Cytospora viticola Bent906 WA Benton 8 Riesling OP038039 OP076916 OP003956 OP095244 OP106926 OP079869
Togniniaceae Phaeoacremonium minimum Bent719 WA Grant 11 Grenache OP038028 –OP003952 ––OP079861
Togniniaceae Phaeoacremonium minimum Bent720 WA Grant 11 Grenache OP038029 –OP003953 ––OP079862
Togniniaceae Phaeoacremonium minimum Bent709 WA Benton 8 Chardonnay OP038022 –OP003950 ––OP079855
Togniniaceae Phaeoacremonium minimum Bent712 WA Benton 8 Chardonnay OP038024 –OP003951 ––OP079857
Diatrypaceae Diatrype stigma Bent015 WA Benton 1 Grenache / Sangiovese OP038001 ––––OP079837
Diatrypaceae Diatrype whitmanensis Bent019 WA Grant 11 Grenache OP038005 ––––OP079840
Diatrypaceae Diatrype whitmanensis Bent020 WA Grant 11 Grenache OP038006 ––––OP079841
Diatrypaceae Diatrype whitmanensis Bent023 WA Grant 11 Grenache OP038009 ––––OP079844
Diatrypaceae Diatrype whitmanensis Bent002 WA Benton 1 Grenache / Sangiovese OP037989 ––––OP079828
Diatrypaceae Diatrype whitmanensis Bent004 WA Benton 3 Cabernet Sauvignon OP037991 ––––OP079830
Diatrypaceae Eutypa cerasi Bent001 WA Benton 2 Chardonnay OP037988 ––––OP079827
Diatrypaceae Eutypa lata Bent021 WA Grant 11 Grenache OP038007 ––––OP079842
Diatrypaceae Eutypa lata Bent022 WA Grant 11 Grenache OP038008 ––––OP079843
Diatrypaceae Eutypa lata Bent003 WA Benton 1 Grenache / Sangiovese OP037990 ––––OP079829
Diatrypaceae Eutypa lata Bent009 WA Benton 6 Cabernet Sauvignon OP037995 ––––OP079831
Diatrypaceae Eutypa lata Bent010 WA Benton 6 Cabernet Sauvignon OP037996 ––––OP079832
Diatrypaceae Eutypa lata Bent011 WA Benton 6 Cabernet Sauvignon OP037997 ––––OP079833
Diatrypaceae Eutypa lata Bent012 WA Benton 6 Cabernet Sauvignon OP037998 ––––OP079834
Diatrypaceae Eutypa petrakii var hederae Bent014 WA Benton 8 Chardonnay OP038000 OP076901 –––OP079836
Diatrypaceae Eutypa petrakii var hederae Bent013 WA Benton 8 Chardonnay OP037999 OP076900 –––OP079835
Diatrypaceae Eutypella virescens Bent017 WA Grant 11 Grenache OP038003 ––––OP079838
Diatrypaceae Eutypella virescens Bent018 WA Grant 11 Grenache OP038004 ––––OP079839
Sporocadaceae Sporocadus kurdistanicus Bent506 / CBS 149336 WA Benton 2 Chardonnay OP038014 OP076907 –OP095235 –OP079849
Sporocadaceae Sporocadus kurdistanicus Bent504 WA Benton 3 Cabernet Sauvignon OP038012 OP076905 –OP095233 –OP079847
Sporocadaceae Sporocadus kurdistanicus Bent507 WA Benton 3 Cabernet Sauvignon OP038015 OP076908 –OP095236 –OP079850
Sporocadaceae Sporocadus kurdistanicus Bent505 WA Benton 3 Cabernet Sauvignon OP038013 OP076906 –OP095234 –OP079848
Sporocadaceae Sporocadus kurdistanicus Bent509 WA Benton 8 Chardonnay OP038016 OP076909 –OP095237 –OP079851
Sporocadaceae Sporocadus kurdistanicus Bent510 WA Benton 8 Riesling OP038017 OP076910 –OP095238 –OP079852
(Continued)
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org07

Supplementary Table 3], and Sporocadus [(Liu et al., 2019;
Bundhun et al., 2021;Moghadam et al., 2022);
Supplementary Table 4].
Multiple sequence alignments were performed in MEGA v. 6
(Tamura et al. 2013) and manually adjusted where necessary in
Mesquite v. 3.10 (Maddison and Maddison 2016). Phylogenetic
analyses were performed for concatenated datasets. Each dataset
was analyzed using two different optimality search criteria,
maximum parsimony (MP) and maximum likelihood (ML), in
PAUP* v. 4.0a169 and GARLI v. 0.951, respectively (Swofford,
2002;Zwickl, 2006). For MP analyses, heuristic searches with
1000 random sequence additions were implemented with the
Tree-Bisection-Reconnection algorithm; gaps were treated as
missing data. Bootstrap analyses with 1000 pseudoreplicates
were used to estimate branch support. For ML analyses,
MEGA was used to infer a model of nucleotide substitution
for each dataset, using the Akaike Information Criterion (AIC).
All ML analyses were conducted using the best-fit model of
nucleotide substitution using the default parameters in GARLI.
Branch support was determined by 1000 bootstrap
pseudoreplicates. Sequences of Hyaloscypha finlandica,
Diaporthe ampelina,andSeimatosporium luteosporum and
Seimatosporium pistaciae served as the outgroup taxa in
phylogenetic analyses of the genus Cadophora,Cytospora, and
Sporocadus, respectively.
Morphological characterization of
putative new species
For all the morphological descriptions, colony colors were
assessed following Rayner (1970) and photographs were
obtained using either a Leica DM500B (Leica microsystems
CMS, Wetzlar, Germany) light microscope equipped with a
Leica color video camera (model DFC 310 FX) or a Olympus
CX31 (Olympus Corporation, Tokyo, Japan) light microscope
equipped with a ToupTek Photonics camera (model E3ISPM
Series C-mount USB3.0 CMOS; ToupTek Photonics Co., Ltd.,
Hangzhou, China). For macroscopic features, a binocular Wild
Heerbrugg M8 (Leica microsystems CMS) stereomicroscope
equipped with an identical ToupTek Photonics camera was
used. All measurements were made with the software
ToupView (ToupTek Photonics Co., Ltd.). Thirty
measurements were made for all the observed micro-
morphological structures and results are presented as min-
(average)-max.
Isolates of putative new species in the genus Cadophora were
characterized morphologically according to Travadon et al.
(2015). Colony growth and characters were obtained on 2%
MEA and 3.9% PDA media at 22°C in the dark. Microscopic
slides were produced by mounting vegetative hyphae, conidia,
conidiogenous cells, collarettes and conidiophores in water
or in 50% glycerol and measurements were obtained at
TABLE 1 Continued
Family Genus Specific epithet Isolate
1
State
2
County Site Cultivar ITS LSU ACT RPB2 TEF-1aTUB
Sporocadaceae Sporocadus kurdistanicus Bent910 WA Benton 8 Riesling OP038043 OP076920 –OP095248 –OP079873
Sporocadaceae Sporocadus kurdistanicus Bent511 WA Benton 8 Riesling OP038018 OP076911 –OP095239 –OP079853
Sporocadaceae Sporocadus incarnatus Bent716 / CBS 149301 WA Benton 8 Riesling OP038025 OP076913 –OP095241 –OP079858
Physalacriaceae Flammulina filiformis Bent016 WA Benton 1 Grenache / Sangiovese OP038002 OP076902 –OP095230 OP106910 –
Physalacriaceae Flammulina filiformis Bent006 WA Benton 6 Syrah OP037992 OP076897 –OP095227 OP106907 –
Physalacriaceae Flammulina filiformis Bent007 WA Benton 6 Syrah OP037993 OP076898 –OP095228 OP106908 –
Physalacriaceae Flammulina filiformis Bent008 WA Benton 6 Syrah OP037994 OP076899 –OP095229 OP106909 –
Polyporaceae Trametes versicolor Bent701 WA Benton 5 Tinto Cao OP038020 OP076912 –OP095240 OP106914 –
1
Kern isolates originate from California and Bent isolates originate from Washington state. Kern and Bent isolates are maintained in the culture collection of Dr. Kendra Baumgartner, USDA-ARS laboratory, UC Davis. CBS: Culture collection of the
Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands.
2
CA: California, isolates collected in October 2018; WA: Washington, isolates collected in August 2018.
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org08

1000× magnification. Isolates of putative new species in the
genus Cytospora were characterized morphologically according
to Lawrence et al. (2018a). Colony growth and characters were
obtained on 2% PDA medium after 4 to 21 days at 22°C in the
dark. Pycnidia were induced on grapevine wood embedded in
PDA as detailed by Lawrence et al. (2017a). Pycnidial squash
mounts were obtained in 50% glycerol and measurements of
vegetative hyphae, conidia, and conidiogenous cells were
obtained at 1000× magnification. Pycnidia characterization
(diameter, presence/absence of a conceptacle, and color) was
achieved with a binocular dissecting microscope. For isolates of
putative new species in the genus Sporocadus,colony
descriptions and microscopic structures were obtained
from cultures on 3.9% PDA and 2% MEA. Conidiomata,
mycelium, conidiophores, conidiogenous cells and conidia
were measured.
Pathogenicity tests
In a replicated greenhouse trial conducted in 2019, ten
fungal isolates (Bent708, Bent901, Bent718, Bent603, Bent008,
Bent904, Bent721, Kern004, Kern007, Bent902), representative
of ten distinct species identified by molecular analyses (Table 1),
were selected for inoculations to the woody stems of potted Vitis
vinifera ‘Chardonnay’. For four isolates that did not produce
spores readily in culture (Bent008, Bent904, Kern004, and
Kern007), mycelial suspensions were prepared as inoculum [5-
day-old cultures grown in Potato Dextrose Broth (PDB) and
homogenized with a hand-held disperser], following the
protocol outlined in Travadon et al. (2013).Conidial
suspensions served as inoculum for the six remaining isolates,
with all inoculum adjusted to 1 × 10
5
spores or mycelial
fragments/mL using a hemacytometer.
In the pathogenicity test, two replicate experiments were
performed, starting two weeks apart, on two sets of plants
propagated in two separate greenhouses. In each experiment,
32 plants inoculated with water served as control plants and 32
plants inoculated with conidia of Phaeomoniella chlamydospora
isolate Bent708 served as positive controls (64 plants × two
experiments = 128 plants). For the other nine isolates, 16 plants
were inoculated with each isolate per experiment (nine
inoculation treatments × 16 plants × two experiments = 288
plants). The plants were arranged in a completely randomized
design in each greenhouse. Plants were propagated from
dormant cuttings according to Travadon et al. (2013). Briefly,
starting in May 2019, cuttings were callused at 30°C and 100%
humidity in a mixture of perlite and vermiculite (1:1, vol/vol) for
21 days. Once shoot and root initials emerged from the callus
tissue, cuttings were coated with melted paraffin wax (Gulf Wax;
Royal Oak Enterprises, L.L.C, Roswell, GA, USA) to prevent
moisture loss and transplanted into pots containing sterile
potting mix amended with slow-release fertilizer (Osmocote®
Pro 24-4-9, Scotts, Marysville, Ohio, USA). Plants were grown in
thegreenhouseattheUniversityofCaliforniaExperiment
Station in Davis [natural sunlight photoperiod, 25 ± 1°C (day),
18 ± 3°C (night)], with some modifications to the temperature
conditions [10 ± 2°C (day), 4 ± 2°C (night)] during dormancy
(from December to February). During the growing season,
plants were watered four times per week for 10 min using a
drip-irrigation system (0.5 L h-1). Approximately 2 months after
being transplanted in September 2019, the woody stem of each
grapevine was wounded (2 mm-width × 3 mm-depth) with a
power drill to produce a wound approximately 2 cm below the
uppermost node. Inoculum (20 μl) was pipetted into the wound,
which was then sealed with Vaseline (Unilever, Rotterdam,
London, UK) and Parafilm (Bemis Co., Neenah, Wisconsin,
USA) to prevent inoculum desiccation. Non-inoculated controls
were wounded and ‘mock-inoculated’with sterile water.
As inoculated plants did not develop foliar symptoms of
trunk diseases during the 12-month incubation period, we used
as a measure of pathogenicity the length of wood discoloration
(LWD) extending from the inoculation site. To reveal these
wood lesions, the green shoots, roots, and bark of each plant
were removed with a flame sterilized knife or pruning shears.
The woody stems were surface disinfected in 1% sodium
hypochlorite for 2 min and rinsed with deionized water. The
length of each stem was recorded and cut longitudinally through
the inoculation site to expose wood discoloration, the length of
which was measured with a digital caliper. To confirm that the
pathogen was responsible for wood discoloration in inoculated
plants, recovery was attempted by cutting 10 pieces (2 × 5 × 5
mm) of wood from the distal margin of the lesion, followed by
surface disinfestation in 0.6% sodium hypochlorite (pH 7.2) for
30 s, two 30-s rinses in sterile deionized water, plating on PDA
amended with tetracycline (1 mg L-1), and incubation in the
dark at approximately 22°C for 14 to 21 days.
Normality and homogeneity of variances were evaluated
before an analysis of variance (ANOVA) was used to
determine the effect of each isolate on LWD. The ANOVA
was performed using the MIXED procedure in SAS, with
experiment considered as a random effect. Means were
calculated using the LS-Means procedure. Pairwise mean
differences in LWD between inoculated and mock-inoculated
plants were analyzed using Dunnett’stests(P< 0.05). The
recovery of isolates from inoculated plants served to complete
Koch’s postulates and, as such, was considered a second measure
of pathogenicity. Recovery rates were estimated as the number of
plants from which the isolate was recovered out of the total
number of plants inoculated.
Effect of temperature on mycelial growth
Ten isolates from each geographical region were assessed in
vitro for their optimal growth temperature. The ten isolates from
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org09

Washington represented six species and the ten isolates from
California represented eight species. All isolates were cultured on
3.9% PDA, except for isolates of the genus Cadophora, which
were cultured on 2% MEA. Optimal growth temperature was
tested by culturing each isolate in triplicate in the dark at
temperatures ranging from 5°C to 35°C at 5°C increments.
Mycelial plugs (5mm diam.) were taken from the margin of an
actively growing culture and transferred to the center of 90-mm
diam. dishes. Radial growth was measured from two to 14 days
after inoculations, depending on how fast each isolate’s
mycelium grew in culture (e.g., 2 days for Biscogniauxia
mediterranea and 14 days for Phaeoacremonium scolyti). Two
perpendicular measurements were taken of the colony diameter.
Each experiment was conducted twice. For each isolate, the
average colony diameters at each temperature were adjusted to a
non-linear regression curve to estimate their optimal growth
temperatureusingtheprogramSigmaPlotv.14.0(Systat
Software Inc., San Jose, California). For this purpose, data
were assumed to follow a Gaussian function of the form:
Y=ae½−0:5(x–x0
b)2
With athe height of the curve’s peak, x
0
the position of the
center of the peak (i.e., optimal temperature) and bthe Gaussian
root mean square controlling the width of the curve. For each
isolate, normality of data distribution was tested with the
Shapiro-Wilk test and homoscedasticity was tested by
computing the Spearman rank correlation between the
absolute values of the residuals and the observed value of the
dependent variable (a= 0.05). Goodness of fit was evaluated
through the computation of the coefficient of determination R
2
.
Results
Vineyard sampling and symptom
observations
Of the 10 wine grape vineyards in Washington, five sites
(sites 1, 2, 5, 9 and 11; see Table 1 for locations and cultivars) had
typical leaf symptoms of esca (interveinal discolorations and
scorching), with apoplectic vines (severe form of esca) also
present at sites 5 and 9. Among the five sites with esca
symptoms, cross-sections through trunks and cordons revealed
black spots (sometimes present in concentric rings in the wood)
at all five sites, with white-rotted wood also present at sites 1, 5
and 11. Among the Washington sites with general dieback
symptoms (dead spur positions and/or entire dead cordons at
sites 6, 8 and 10) cross-sections through trunks and cordons
revealed dark brown to black wood cankers at all three sites.
White-rotted wood was observed at sites 1, 5 and 11. Leaf
symptoms of grapevine leaf roll viruses were present at site 8.
Leaf symptoms of Eutypa dieback were present at site 3.
Pathogenic species were isolated from the nine Washington
sites with trunk disease symptoms, with up to six species
isolated from site 1 (Table 1). In contrast, one Washington site
(site 7) had peculiar canopy symptoms with uncommon leaf
scorching, black spots on shoots, petioles swollen at the base, and
vines with wilted and dead cordons; isolation attempts from
wood samples did not yield any fungal pathogens.
From the 10 table grape vineyards in California, seven sites
(sites 1, 2, 3, 5, 6, 7, and 8) had typical leaf symptoms of esca,
with apoplectic vines also present at site 7. Wood symptoms in
these seven sites included both black spotting and wood
discolorations, varying in color from black to brown to
pinkish. General dieback symptoms were present at sites 1, 2,
3, 4, 7, 9 and 10. Wood symptoms at site 10 were extensive
brown lesions present in young cordons, whereas wood
symptoms at site 7 were exclusively black spots (sometimes
present in concentric rings). There were four sites (sites 1, 2, 3
and 7) with symptoms of both esca and dieback. Pathogenic
species were isolated from all 10 vineyards.
Identification of isolates
Molecular identification based on the sequencing of two to six
loci per isolate, and subsequent sequence comparisons with type/
voucher specimens, identified 112 isolates of 36 fungal species either
frequently associated with grapevine trunk diseases or known causal
agents of dieback in other woody plants. Sixty isolates from
Washington represented 22 species whereas 52 isolates from
California represented 20 species (Table 1). The following six
species were shared between Washington and California: Cytospora
viticola,Diatrype stigma,Diplodia seriata,Kalmusia variispora,
Phaeoacremonium minimum,andPhaeomoniella chlamydospora.
Dominating the community of pathogenic species in
California table grape vineyards (61.5% of California isolates)
were species in the families Diatrypaceae (12 isolates of five
species: Cryptovalsa ampelina,Diatrype stigma,Diatrypella
verruciformis,Eutypella citricola and Eutypella microtheca),
Togniniaceae (10 isolates of three species: Phaeoacremonium
minimum,Phaeoacremonium parasiticum and
Phaeoacremonium scolyti), Phaeomoniellaceae (five isolates of
Phaeomoniella chlamydospora) and Hymenochaetaceae (five
isolates of Fomitiporia polymorpha). Other known pathogenic
speciesincludedfourisolatesfromthefamily
Botryosphaeriaceae (Diplodia seriata,Lasiodiplodia exigua and
Lasiodiplodia gilanensis) and eight isolates from the order
Diaporthales (Diaporthe ampelina,Cytospora viticola and a
second Cytospora species whose sequences did not match any
known species in molecular databases). From the 112 total
isolates recovered from the two regions, two isolates from the
genus Cadophora, three isolates from the genus Cytospora, and
one isolate from the genus Sporocadus did not have any
affiliations with type or non-type specimens in GenBank;
Travadon et al. 10.3389/ffunb.2022.1018140
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hence, multilocus phylogenetic analyses were conducted to
circumscribe these undescribed taxa.
Dominating the community of pathogenic species in
Washington wine grape vineyards (61.7% of Washington
isolates) were species in the fungal families Diatrypaceae (18
isolates of six species), Cytosporaceae (10 isolates of two
Cytospora species) and Sporocadaceae (nine isolates of two
species). Fungal species in the Diatrypaceae included Diatrype
stigma,Diatrype whitmanensis,Eutypa cerasi,Eutypa lata,
Eutypa petrakii var hederae,andEutypella virescens.
Pathogenic species often associated with esca included
Phaeomoniella chlamydospora and Phaeoacremonium
minimum in addition to Basidiomycetes Flammulina filiformis
and Trametes versicolor. Two species of Cadophora were also
recovered. Only five isolates of the family Botryosphaeriaceae
(Diplodia seriata,Diplodia mutila and Dothiorella iberica) were
isolated from Washington sites.
Phylogenetic analyses of the genus
Cadophora
The concatenated sequences (ITS, TUB and TEF-1a) of the
four Cadophora isolates recovered in this study along with those
of 57 representative isolates (including two isolates of the
outgroup species Hyaloscypha finlandica;Supplementary
Table 2) were subjected to ML and MP analyses. For ML
analyses, the best-fit model of nucleotide substitution was
deduced based on the AIC criterion (ITS: K2+G+I; TEF-1a:
HKY+G; TUB: HKY+G+I). The concatenated sequence
alignment resulted in a 1,689 character dataset (834 characters
were constant, 682 characters were parsimony-informative, and
173 characters were parsimony-uninformative). MP analysis
produced 100 equally most parsimonious trees of 2,286 steps
and a consistency index (CI), retention index (RI), and rescaled
consistency index (RC) of 0.599, 0.88 and 0.527, respectively.
The phylogeny consisted of two main clades supported by MP
and ML analyses (98/99% and 100/71% MP and ML bootstrap
values, respectively). Within the first main clade (98/99%
support), including Cadophora sensu lato following Koukol
and Macia-Vicente (2022), all known species were well-
supported as independent phylogenetic lineages, except that
three isolates of the recently described Cadophora sabaouae
species did cluster with strong support (100/100%) with eight
isolates of the well-known species Cadophora luteo-olivacea, and
these three isolates were placed on a branch internal to the one of
the type specimen for this species (CBS 141.41). Within the
second main clade (Cadophora sensu stricto) that includes the
type species for the genus (Cadophora fastigiata), MP and ML
analyses of the dataset revealed that two isolates (Bent721 and
Bent722) clustered strongly (98/99%) with the type specimen of
Cadophora ferruginea (CBS 146363) but also with the type
specimen (CBS 146263) associated with the recently described
species Cadophora vinaceae (Figure 1). The isolate of Cadophora
vinaceae CBS 146263 was placed on a short branch within this
clade, indicating very short phylogenetic distance between this
isolate and the type specimen of Cadophora ferruginea. Two
isolates (Bent717 and Bent718) formed a well-supported clade
(100/100%) that does not include any previously described
species. This clade was strongly supported as sister clade (95/
97%) to the type specimen of Cadophora margaritata (CBS
144083), this latter isolate being placed on a moderately long
branch in this clade grouping the two species hence supporting
important phylogenetic distance between them.
Phylogenetic analyses of the genus
Cytospora
The concatenated sequences (ITS, LSU, TEF-1a,RPB2,TUBand
ACT) of the 14 Cytospora isolates recovered in this study, along with
those of 43 representative isolates (including two isolates of the
outgroup species Diaporthe ampelina;Supplementary Table 3), and
four isolates of Diaporthe ampelina from this study were subjected to
ML and MP analyses. For ML analyses, the best-fit model of nucleotide
substitution was deduced based on the AIC criterion (ITS: GTR+G+I;
LSU: TrN+G+I; TEF-1a: K2+G+I; RPB2: TrN+G+I; TUB: HKY+G;
ACT: K2+G+I). The concatenated sequence alignment resulted in a
4,510 character dataset (3,130 characters were constant, 1,100
characters were parsimony-informative, and 280 characters were
parsimony-uninformative). MP analysis produced 100 equally most
parsimonious trees of 2,201 steps and a CI, RI, and RC of 0.794, 0.948
and 0.753, respectively. One early-divergent, well-supported clade (100/
75%) included 11 isolates from the current study and was identified as
Cytospora viticola, based on their clustering with the type specimen for
this species (CBS 141586). Two other well-supported, main clades (100/
100% and 100/97% respectively) were revealed by MP and ML analyses
(Figure 2), with one clade including the type species for the genus,
Cytospora chrysosperma, and one clade including species such as
Cytospora ribis and Cytospora cotini. Within the Cytospora
chrysosperma clade, MP and ML analyses revealed that two isolates
(Bent902 and Bent903) formed a well-supported clade (100/100%) that
does not include any previously described species. This clade was
supported as sister clade (87/88%) to the one including the type
specimen for Cytospora joaquinensis (CBS 144235). Within the
Cytospora ribis main clade, MP and ML analyses revealed that one
isolate (Kern907) represented a unique phylogenetic lineage moderately
supported as sister taxon (77/80%) to Cytospora cotini and
Cytospora ampulliformis.
Phylogenetic analyses of the
genus Sporocadus
The concatenated sequences (ITS, LSU, TEF-1a,RPB2,and
TUB) of the nine Sporocadus isolates recovered in this study, along
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withthoseof36representativeisolates (including two isolates of the
outgroup species Seimatosporium luteosporum,andoneisolateof
the outgroup species Seimatosporium pistaciae;Supplementary
Table 4) were subjected to ML and MP analyses. For ML
analyses, the best-fit model of nucleotide substitution was
deduced based on the AIC criterion (ITS: T92+G; LSU: K2+G+I;
TEF-1a:HKY+G+I;RPB2:T92+G;TUB:HKY+G+I).The
concatenated sequence alignment resulted in a 3,908 character
dataset (2,940 characters were constant, 639 characters were
parsimony-informative, and 329 characters were parsimony-
FIGURE 1
Phylogram generated from maximum parsimony analysis of the genus Cadophora based on a combined ITS, TEF1-aand TUB sequence dataset.
Bootstrap support values for MP and ML equal to or greater than 70% are presented. The tree is rooted to Hyaloscypha finlandica (CBS 444.86
and IFM 50530). The new isolates are in bold and isolates of new species are in blue. Ex-type strains are noted with superscript T.
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FIGURE 2
Phylogram generated from maximum parsimony analysis of the genus Cytospora based on a combined ITS, LSU, TEF-1a, RPB2, TUB and ACT
sequence dataset. Bootstrap support values for MP and ML equal to or greater than 70% are presented. The tree is rooted to Diaporthe
ampelina (CBS 114016 and Wolf912). The new isolates are in bold and isolates of new species are in blue. Ex-type strains are noted with
superscript T. .
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uninformative). MP analysis produced 100 equally most
parsimonious trees of 2,004 steps and a CI, RI, RC of 0.658, 0.784
and 0.516, respectively. Eight isolates were placed in a well-
supported clade (88/94%), including the type specimen for the
recently described species Sporocadus kurdistanicus (CBS 143778).
The ninth isolate (Bent716) was placed on a relatively long
branch outside this clade (74/87% support) and did not
cluster with a type or non-type isolate with DNA sequence
data available (Figure 3); isolate Bent716 represented a novel
phylogenetic species.
Taxonomy
Based on DNA sequence data and morphological
examinations, two isolates represent an undescribed
Cadophora species, three isolates represent two
undescribed Cytospora species, and one isolate represents an
undescribed Sporocadus species. These newly discovered species
are described below.
Cadophora columbiana Travadon & D.P. Lawr., sp. nov.
MycoBank No: MB844778
FIGURE 3
Phylogram generated from maximum parsimony analysis of the genus Sporocadus based on a combined ITS, LSU, TEF-1a, RPB2, and TUB
sequence dataset. Bootstrap support values for MP and ML equal to or greater than 70% are presented. The tree is rooted to Seimatosporium
luteosporum (CBS 142599 and Napa754) and Seimatosporium pistaciae (CBS 138865). The new isolates are in bold and isolates of new species
are in blue. Ex-type strains are noted with superscript T.
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Figure 4
Typification: USA, Washington: Skamania County, 45°
44'13.05"N, 121°34'32.40"W, 557m asl, isolated from necrotic
wood of Vitis vinifera ‘Pinot noir’, August 2018, R. Travadon
number Bent718 (holotype BPI 911228, dried culture; ex-type
CBS 149299). ITS sequence GenBank OP038027; TEF-1a
sequence GenBank OP106917; TUB sequence
GenBank OP079860.
Etymology: The epithet refers to the location from which the
fungus originates, in a vineyard near the Columbia River in
Washington State, USA.
Description: Sexual morph not observed. Asexual morph
observed in culture on 2% malt extract agar (MEA). Vegetative
mycelium hyaline, smooth-walled, septate, branched, 1.4-(2.3)-
3.2 μm wide, becoming brown with age, chlamydospores not
observed. Conidiophores arising from aerial hyphae, hyaline,
smooth-walled, septate, short, 5.2-(11.6)-23.2 μm long.
Conidiogenous cells hyaline, smooth-walled, terminal or lateral,
monophialidic, obclavate, with a flask-shaped appearance, 6.7-
(12.7)-20.1 μm long × 2.7-(3.5)-4.2 μm wide at the widest part,
tapering towards collarette; collarettes flaring, cup-shaped, 1.4-
(1.8)-2.3 μm wide at upper edge. Conidia hyaline, smooth-
walled, aseptate, ovoid to ellipsoidal, enteroblastic, aggregated
in heads, 2.9-(4.8)-6.7 × 1.2-(1.8)-2.5 μm.
Culture characteristics: Colonies on MEA reaching 28 mm
diam after 13 d at 20°C in the dark. Flat colony with undulate
margin, color ranging from isabelline to honey to off-white
from center to edge. Colonies on 3.9% potato dextrose agar
(PDA) displaying aerial, fasciculate hyphae with aerial
mycelium tufts at the center, margin undulate, colony color
ranging from isabelline to dark isabelline to off-white from
center to edge.
Notes: Based on the phylogenetic inference obtained in this
study, Cadophora columbiana is the closest relative to
Cadophora margaritata, with 95% Maximum Parsimony
bootstrap support. Both species are lignicolous and
characterized by phialidic conidiogenesis with non-septate
conidia attached in heads. Colony colors on MEA are very
distinct between the two species, with Cadophora margaritata
being olivaceous black and Cadophora columbiana ranging from
isabelline to white. Cadophora margaritata CBS 144083
produces longer phialides (up to 29 μm) than Cadophora
columbiana Bent718 (up to 13.6 μm), and conidia of the
former species are truncated at the base, a characteristic not
observed for those of Cadophora columbiana.
The ITS sequence of Cadophora columbiana Bent718 differs
at four nucleotide positions (99% identity) from that of the ex-
holotype of Cadophora margaritata CBS 144083, whereas the
TUB sequences differ at 72 nucleotide positions (84% identity).
Additional specimens examined: USA, Washington: Benton
County, 45°44'13.05"N, 121°34'32.40"W, 557 m asl, isolated from
necrotic wood of Vitis vinifera ‘Pinot noir’, August 2018, R.
Travadon number Bent717 (CBS 149300).
Cytospora macropycnidia Travadon & D.P. Lawr., sp. nov.
MycoBank No: MB844777
Figure 5
FIGURE 4
Cadophora columbiana (CBS 149299/Bent718). (A, B) 14-day-old colonies on MEA and PDA media, respectively. (C, D). 55-day-old colonies on
MEA and PDA media, respectively. (E). Conidiophores, conidiogenous cells and conidia aggregated in heads. (F) Phialides and colarettes. (G, H).
Conidia. Scale bars: 20 µm (E–G);10µm(H).
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Typification: USA, California: Kern County, 35°42'13.8"N,
119°23'16.1"W, 77m asl, isolated from necrotic wood of Vitis
vinifera ‘Scarlet Royal’, October 2018, R. Travadon number
Kern907 (holotype BPI 911229; dried culture; ex-type CBS
149338). ITS sequence GenBank OP038094; LSU sequence
GenBank OP076935; ACT sequence GenBank OP003977;
RPB2 sequence GenBank OP095265; TEF-1asequence
GenBank OP106954; TUB sequence GenBank OP079909.
Etymology: The epithet refers to the large pycnidia, which
form on grapevine wood in the laboratory.
Description: Sexual morph not observed. Asexual morph
observed in culture on 2% potato dextrose agar (PDA).
Conidiomata pycnidial, ostiolated, black to mouse grey with
white to off-white surface hyphae, 2.3-(3.2)-4.3 mm diameter,
erumpent on bark surface, solitary and gregarious, with multiple
internal locules sharing invaginated walls, conceptacle absent.
Mycelium hyaline, smooth-walled, septate, branched, 2.8-(4.4)-
5.6 μm wide. Conidiophores reduced to conidiogenous cells.
Conidiogenous cells hyaline, smooth-walled, with a flask-
shaped appearance, 13.3-(17)-23.6 μm long × 2-(2.5)-2.9 μm
wide. Conidia hyaline, allantoid, smooth, aseptate, 4.9-(6)-7.6
μm long × 1.7-(2.1)-2.4 μm wide.
Culture characteristics: Colonies on PDA reaching 85 mm
diam after 5 d at 25°C in the dark, fast-growing with relatively
even but slightly dentate margin, white to off-white with short
aerial tufts giving a cottony appearance, becoming straw-colored
with age.
Notes: Based on the phylogenetic inference obtained in this
study, Cytospora macropycnidia forms a moderately supported
clade (77% Maximum Parsimony and 80% Maximum
Likelihood bootstrap support) with Cytospora ampulliformis
(MFLUCC 16-0583 isolated from Sorbus intermedia in Russia)
and Cytospora cotini (MFLUC 14-1050 isolated from Cotinus
coggygria in Russia). Cytospora macropycnidia has larger
pycnidia (average 3.2 mm in diameter) than Cytospora
ampulliformis and Cytospora cotini (< 1 mm).
The ITS sequence of Cytospora macropycnidia Kern907
differs at three nucleotide positions (99% identity) from those
of both Cytospora ampulliformis MFLUCC 16-0583 and of
Cytospora cotini MFLUC 14-1050. The LSU sequence of
Cytospora macropycnidia Kern907 differs at three nucleotide
positions (99% identity) from that of Cytospora ampulliformis
MFLUCC 16-0583 and has 100% identity with that of Cytospora
cotini MFLUC 14-1050. The RPB2 sequence of Cytospora
macropycnidia Kern907 differs at 12 nucleotide positions (98%
identity) from that of Cytospora ampulliformis MFLUCC 16-
0583 and at 22 nucleotide positions (98% identity) from that of
Cytospora cotini MFLUC 14-1050. The ACT sequence of
Cytospora macropycnidia Kern907 differs at six nucleotide
position (98% identity) from that of Cytospora ampulliformis
MFLUCC 16-0583.
Cytospora yakimana Travadon & D.P. Lawr., sp. nov.
MycoBank No: MB844775
Figure 6
Typification: USA, Washington: Benton County, 46°
12'53.7"N, 119°44'47.9"W, 208m asl, isolated from necrotic
wood of Vitis vinifera ‘Tinto Cão’, October 2018, R. Travadon
number Bent902 (holotype BPI 911230; dried culture; ex-type
FIGURE 5
Cytospora macropycnidia (CBS 149338/Kern907). (A) 4-day-old culture on 2% PDA. (B) 7-day-old culture on 2% PDA. (C) 32-day-old culture on
3.5% PDA. (D) Gregarious pycnidia produced in vitro on sterile grapevine wood. (E) Solitary pycnidium exudating pale yellow cirrhus (mucilage).
(F) Longitudinal section of a pycnidium. (G) Conidiogenous cells and conidia. (H) Conidia. Scale bars: 1mm (D–F); 10µm (G, H).
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CBS 149297). ITS sequence GenBank OM976602; LSU sequence
GenBank ON059350; ACT sequence GenBank ON012555;
RPB2 sequence GenBank ON045093; TEF-1asequence
GenBank ON012569; TUB sequence GenBank ON086750.
Etymology: The epithet refers to the Yakima River in
Washington State, USA, bordering the vineyard where the
specimen was originally isolated.
Description: Sexual morph not observed. Asexual morph
observed in culture on sterile grapevine wood embedded in
3.9% potato dextrose agar (PDA). Conidiomata pycnidial,
ostiolated, mouse grey with white to off-white surface hyphae,
1.1-(2)-2.4 mm diameter, erumpent or semi-immersed on
bark surface, mostly solitary, rarely aggregated, without
conceptacle, with multiple internal locules with shared
invaginated walls. Mycelium hyaline, smooth-walled,
septate, branched, 2.3-(3.9)-5 μm wide. Conidiophores
hyaline, smooth-walled, reduced to 2—3 monoblastic,
branching filamentous conidiogenous cells. Conidiogenous
cells hyaline, smooth-walled, tapering towards the apex, 11-
(14.9)-18.6 μm long × 0.7-(0.9)-1.4 μm wide. Conidia hyaline,
allantoid, smooth, aseptate, 3.9-(4.3)-4.9 μm long × 1.4-(1.8)-
2μmwide.
Culture characteristics: Colonies on 2% PDA reaching 70
mm diam after 7 d at 25°C in the dark, moderately fast-growing
with an uneven margin, white to off-white with hints of pale rose
with age.
Notes: Based on the phylogenetic inference obtained in this
study, Cytospora yakimana is the closest relative to Cytospora
joaquinensis, with 87% Maximum Parsimony and 88%
Maximum Likelihood bootstrap support. Cytospora yakimana
originates from Vitis vinifera in Washington state, whereas
Cytospora joaquinensis is reported from Juglans regia,Pistacia
vera and Populus deltoides in California. Cytospora yakimana
produces conidiogenous cells that may be branched, which is not
a culture characteristic of Cytospora joaquinensis. Conidia of
Cytospora yakimana (3.9 to 4.9 μm long) are slightly shorter
than those of Cytospora joaquinensis (5 to 6 μm long).
The ITS and ACT sequences of Cytospora yakimana
Bent902 both differ at only one nucleotide position (99%
identity) from those of the ex-holotype of Cytospora
joaquinensis CBS 144235. However, the TEF-1asequence of
Cytospora yakimana Bent902 differs at 22 nucleotide positions
(92% identity) from that of Cytospora joaquinensis CBS 144235,
whereas the TUB sequences of these two isolates differ at 17
nucleotide positions (96% identity).
Additional specimens examined: USA, Washington: Benton
County, 46°12'53.7"N, 119°44'47.9"W, 208m asl, isolated from
necrotic wood of Vitis vinifera ‘Tinto Cão’, October 2018, R.
Travadon number Bent903 (CBS 149298).
Sporocadus incarnatus Travadon & D.P. Lawr., sp. nov.
MycoBank No: MB844774
Figure 7
FIGURE 6
Cytospora yakimana (CBS 149297/Bent902). (A) 4-day-old culture on PDA. (B) 14-day-old culture on PDA. (C) Pycnidia produced in vitro on sterile
grapevine wood. (D) Pycnidium exudating pale luteoustosaffroncoloredmucilage.(E) Locular chamber lined with conidiogenous cells. (F) Straight and
branched conidiogenous cells. (G) Conidiogenous cells and conidia. (H) Conidia. Scale bars: 1 mm (C, D);50µm(E);10µm(F–H).
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Typification: USA, Washington: Benton County, 46°
17'56.8"N, 119°46'53.1"W, 370m asl, isolated from necrotic
wood of Vitis vinifera ‘Riesling’, August 2018, R. Travadon
number Bent716 (holotype BPI 911231; dried culture; ex-type
CBS 149301). ITS sequence GenBank OP038025; LSU sequence
GenBank OP076913; RPB2 sequence GenBank OP095241; TUB
sequence GenBank OP079858.
Etymology: The epithet refers to the flesh to salmon color of
the colony when grown in culture.
Description: Sexual morph not observed. Asexual morph
observed in culture on 3.9% potato dextrose agar (PDA).
Conidiomata black, erumpent, semi-immersed or immersed,
mainly gregarious, sometimes scattered. Mycelium hyaline,
smooth-walled, septate, branched, 2.9-(3.9)-4.8 μm wide.
Conidiophores hyaline, smooth-walled, septate, up to 60 μm.
Conidiogenous cells hyaline, smooth-walled, mainly cylindrical
but occasionally ampulliform, 8.6-(17.7)-28.9 μm long × 2.5-
(3.4)-4.2 μm wide. Conidia ovoid to subcylindrical, straight, 2—
7-septate, wall smooth, often slightly constricted at the septa, 20-
(28)-46.5 × 8.2-(9.2)-10.8 μm, lacking appendages; basal cell
obconic, occasionally with a truncate base, hyaline to pale
brown, thin-walled, 3.6-(6.3)-9.7 μm long; median cells 2—5
with fairly thick walls, pale to mid-brown, dolioform to
subcylindrical, 4-(6)-8.7 μm long; apical cell obtuse,
concolorous with median cells, 5.3-(6.6)-9.2 μm long.
Culture characteristics: Colonies on PDA reaching 63 mm
diam after 14 d at 25°C in the dark. Colony displaying aerial
mycelium tufts at the center, margin irregular, colony color
ranging from cinnamon to fleshish salmon to off-white from
center to edge. Colonies on 2% malt extract agar (MEA) flat with
irregular margin, color ranging from saffron to fleshish salmon
to saffron to off-white from center to edge.
Notes: Based on the phylogenetic inference obtained in this
study, Sporocadus incarnatus is the closest relative to Sporocadus
kurdistanicus, with 74% Maximum Parsimony and 87% Maximum
Likelihood bootstrap support. Both species have been isolated from
necrotic wood of Vitis vinifera. Colony colors on PDA are distinct
between the two species, with Sporocadus kurdistanicus described as
buff to sepia whereas Sporocadus incarnatus colorrangesfromflesh
to salmon. Growth rate for Sporocadus kurdistanicus is higher (48 to
56 mm after 14 d at 20°C, compared to 33 to 45 mm after 14 d at
21°C for Sporocadus incarnatus). Conidia of Sporocadus incarnatus
have 2 to 7 septa, whereas those of Sporocadus kurdistanicus (like
most Sporocadus species), have three septa.
The ITS and LSU sequences of Sporocadus incarnatus
Bent716 both differ at three nucleotide positions (99%
identity) from those of the ex-holotype of S. kurdistanicus CBS
143778, whereas the TUB sequences differ at 34 nucleotide
positions (95% identity).
Pathogenicity tests
At 12 months after inoculation, the wood surrounding the
inoculation sites of water-inoculated control plants was
discolored, but the discoloration was restricted (LWD = 6.8
mm; n= 53 plants, averaged across experiments) and no
pathogenic fungi were isolated from these lesions (Table 2).
The woody stems of plants inoculated with Phaeomoniella
chlamydospora isolate Bent708 (positive-control plants) had
FIGURE 7
Sporocadus incarnatus (CBS 149301/Bent716). (A, B) 14-day-old colonies on MEA and PDA media, respectively. (C, D). Conidiomata on PDA. (E)
Conidiophores, conidiogenous cells and conidia. (F) Germinating conidia. (G, H) Conidia. Scale bars: 20µm (E); 10µm (F–H).
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black, vascular discolorations spreading above and below the
inoculation site, with an average LWD of 19.1 mm (n=62
plants, averaged across experiments). ANOVA revealed a
significant effect of inoculation treatment on lesion length (P<
0.0001). All inoculated plants had significantly larger lesions
than the water-inoculated controls (Dunnett's test; P< 0.0001, a
= 0.05). These lesions varied in color from light orange to brown
to black, depending on the isolate. Plants inoculated with
Flammulina filiformis Bent008 had significantly smaller lesions
(average of 12.7 mm), compared to those inoculated with
Phaeomoniella chlamydospora Bent708, Thyrostroma sp.
Bent904, Biscogniauxia mediterranea Kern007, Cadophora
columbiana Bent718, and Kalmusia variispora Bent603
(Table 2). Plants inoculated with Cadophora ferruginea,
Cytospora viticola,Cytospora yakimana, and Eutypella citricola
had lesions intermediate in length. From the wood lesions of the
inoculated plants, we recovered isolates matching
morphologically the inoculated isolates, with recovery rates
ranging from 43% for Flammulina filiformis Bent008 to 67%
for Biscogniauxia mediterranea Kern007. Both LWD and
recovery rates suggest that all ten isolates are indeed pathogenic.
Effect of temperature on mycelial growth
All 10 isolates from Washington grew at 5°C, whereas only
three of the 10 isolates from California grew at 5°C (Eutypella
citricola Kern004, Diaporthe ampelina Kern904 and Cytospora
macropycnidia Kern907; Figure 8). In contrast, at the highest
temperature of 35°C, nine of 10 isolates from California grew (all
species except Cytospora macropycnidia Kern907), whereas only
three of 10 isolates from Washington grew at 35°C (Diatrype
stigma Bent015, Cytospora yakimana Bent902 and Bent903). All
isolates from Washington had an optimal growth temperature
below 24°C, except for the two Cytospora yakimana isolates
(optimal temperatures of 27.1 and 27.4°C, respectively;
Supplementary Table 5). All isolates from California had an
optimal growth temperature above 24°C, except for two isolates
(Cytospora macropycnidia Kern907 at 20.3°C and Diatrypella
verruciformis Kern006 at 21.5°C; Supplementary Table 5). The
lowest optimal growth temperature was 16.2°C for Thyrostroma
sp. Bent904 and the highest optimal growth temperature was
29.8C for Biscogniauxia mediterranea Kern004.
Discussion
We examined the communities of pathogenic fungi
associated with esca-like symptomatic vines from two grape
production systems and two regions with distinct climates and
environmental conditions: southeastern Washington wine
grapes and southern San Joaquin Valley of California table
grapes. Although our fieldsurveywaslimitedtoonly10
vineyards in each region, we established a fungal collection of
more than 110 isolates representing 36 species, either
consistently associated with grapevine trunk diseases or known
causal agents of dieback in other woody plants. For the first time,
we report esca in vineyards from Washington, which was
expected, as this disease is present in virtually all grape-
growing regions of western North America, including
neighboring British Columbia (U

rbez-Torres et al., 2014a). A
core set of six species was shared between the two regions:
Cytospora viticola,Diatrype stigma,Diplodia seriata,Kalmusia
variispora,Phaeoacremonium minimum,andPhaeomoniella
TABLE 2 Mean lesion lengths and recovery rates of ten fungal isolates from the woody stems of Vitis vinifera ‘Chardonnay’at 12 months post-
inoculations in duplicated greenhouse experiments.
Inoculation treatment Lesion length
(mm)
1
95% Confidence limits of lesion length (mm) Recovery rate
(%)
2
Sample
size
Non-inoculated control 6.81a 5.41 - 8.59 0 53
Flammulina filiformis Bent008 12.72b 10.18 - 15.90 43.33 30
Cadophora ferruginea Bent721 16.95bc 13.56 - 21.19 53.33 30
Eutypella citricola Kern004 17.92bc 14.32 - 22.43 57.14 28
Cytospora viticola Bent901 18.40bc 14.72 - 22.99 48.48 33
Cytospora yakimana Bent902 18.77bc 15.02 - 23.47 60 30
Phaeomoniella chlamydospora
Bent708
19.12bc 15.02 - 24.35 46.77 62
Thyrostroma sp. Bent904 19.75bc 15.80 - 24.68 43.75 32
Biscogniauxia mediterranea Kern007 20.38c 16.28 - 25.50 66.67 27
Cadophora columbiana Bent718 21.19c 16.92 - 26.54 53.57 28
Kalmusia variispora Bent603 21.73c 17.38 - 27.16 46.88 32
1
Mean lesion lengths with the same letters have overlapping 95% confidence intervals.
2
Recovery rates were estimated as the number of plants from which the isolate was recovered out of the total number of plants inoculated.
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chlamydospora, These species may thus be adapted to a wide
array of environmental and cultural conditions. These species
are widely reported as trunk pathogens, except for Cytospora
viticola and Kalmusia variispora, which were more recently
characterized as pathogenic in vineyards from the northeastern
US and northwestern Iran, respectively (Lawrence et al., 2017a;
Abed-Ashtiani et al., 2019).
Members of the genus Cytospora include species that cause
cankers on a range of horticultural crops, and Cytospora canker
is recognized as a common disease in many fruit and nut crops
(Adams et al., 2002;Fan et al., 2015;Lawrence et al., 2018a).
However, Cytospora species are not frequently reported causal
fungi of grapevine trunk diseases [see Fotouhifar et al. (2010);
Lawrence et al. (2017a) and Dekrey et al. (2022). In Washington
vineyards, Cytospora viticola was found at two sites on four
distinct cultivars, and in California, it was found at three sites
and three cultivars. This is a first report of Cytospora viticola in
these two states; this species was previously reported causing
Cytospora canker in vineyards from the northeastern US (US
states of Connecticut, Michigan, Minnesota, New York, Ohio,
Vermont, Virginia, and Wisconsin), and southeastern Canada
(Canadian provinces of Ontario and Quebec) (Lawrence et al.,
2017a;Dekrey et al., 2022). Our results of controlled
inoculations confirmed Cytospora viticola as pathogenic to
grapevine wood and support Cytospora canker as a grapevine
trunk disease. Like Botryosphaeria and Phomospsis dieback of
grapevines, Cytospora canker is not associated with peculiar
foliar symptoms, but instead causes reduced vine vigor and the
gradual death of spurs and cordons. It appears that several
species of Cytospora can be considered pathogenic to grapevine,
including Cytospora vinaceae (Lawrence et al., 2017a), but also
the presently described species Cytospora yakimana, which was
as virulent as Cytospora viticola. In addition, we discovered and
described a new species from table grape ‘Scarlet Royal’in
California, Cytospora macropycnidia, but further studies are
required to evaluate its pathogenicity. Cytospora species are
among the most prevalent species reported in some grape-
growing regions (Dekrey et al., 2022), multiple Cytospora
species have been shown to naturally infect grapevine pruning
wounds in Washington (Baumgartner et al., 2022), and many
Cytospora species are damaging pathogens of woody hosts
(Spielman, 1985;Lawrence et al., 2018a;Pan et al., 2020). The
contribution of Cytospora species to grapevine dieback may have
been overlooked in the past.
A
B
FIGURE 8
Average colony diameter of ten isolates from Washington (A) and ten isolates from California (B) assessed at temperatures ranging from 5 to 35°C in 5°C
increments. At each temperature, three media dishes were used per isolate in each of two experiments.
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org20

Species from the Diatrypaceae family were well represented
in our survey, with five species from California table grapes
(Cryptovalsa ampelina,Diatrype stigma,Diatrypella
verruciformis,Eutypella citricola and Eutypella microtheca) and
six species from Washington wine grapes (Diatrype stigma,
Diatrype whitmanensis,Eutypa cerasi,Eutypa lata,Eutypa
petrakii var hederae,andEutypella virescens). All five
California species are reported there (Trouillas et al., 2010)
and most species have been proven vascular pathogens of
grapevines, except Diatrypella verruciformis,whichis
considered saprophytic (Trouillas and Gubler, 2010).
Inoculation assays confirmed Eutypella citricola pathogenic to
grapevine and these findings support the role of this species as a
trunk pathogen, as previously demonstrated for this species and
Eutypella microtheca (Trouillas et al., 2011;Luque et al., 2012;
U

rbez-Torres et al., 2020). Eutypella citricola is best known as a
canker pathogen of Citrus sp. (Mayorquin et al., 2016).
Diatrypaceae species frequently infect a large diversity of
cultivated and wild, perennial hosts in the same landscape
(Moyo et al., 2019), and have sometimes been shown to be
able to cross-infect these distinct hosts (Travadon and
Baumgartner, 2015). Because the southern San Joaquin Valley
includes both vineyards and citrus orchards, it is possible there
are infection routes between these crops. The most common
Diatrypaceae species in Washington wine grapes was Eutypa
lata, the only species that has been proven responsible for
causing Eutypa dieback foliar symptoms. Such foliar
symptoms were observed during the present survey and
previously from another study in the same region (Johnson
and Lunden, 1987), hence the presence of Eutypa lata is
confirmed (Glawe et al., 1982). A comprehensive taxonomic
study of Diatrypaceous fungi from Washington, Oregon and
Idaho described 28 species, collected from dead or declining
angiosperms, with all species belonging to the genera
Cryptosphaeria,Cryptovalsa,Diatrype,Diatrypella,Eutypa, and
Eutypella, with only Eutypa lata associated with Vitis vinifera
and V. labruscana (Glawe and Rogers, 1984). Among the less
common species found in Washington, our study constitutes the
first report of Eutypa cerasi on V. vinifera; this is a first report of
Eutypa cerasi in North America, as it was only known from
Prunus cerasus in China (Long et al., 2021). We also report for
the first time Eutypa petrakii var hederae on Vitis vinifera, as this
species was only known from Hedera helix in France and
Switzerland (Rappaz, 1987). The pathogenicity to grapevine of
these two latter Eutypa species requires further evaluation.
Multiple isolates of the genus Sporocadus were isolated from
four sites in Washington and they represented the third most
abundant group of fungi in this region. Members of the family
Sporocadaceae (order Xylariales) are ceolomycetous fungi also
referred to as ‘pestalotioid fungi’as they produce multiseptate
conidia that often bear apical and/or basal appendages. Among
the 30 genera in this family (Liu et al., 2019), some have been
characterized as pathogenic to grapevine, such as
Seimatosporium (Dı
az et al., 2012;Lawrence et al., 2018b),
Truncatella,Pestalotiopsis and Neopestalotiospis (U

rbez-Torres
et al., 2009;Arzanlou et al., 2013;Jayawardena et al., 2015;
Maharachchikumbura et al., 2017). More recently, three newly
described species of pestalotioid fungi were isolated from
diseased vines from the Kurdistan Province of Iran,
Seimatosporium marivanicum,Sporocadus kurdistanicus, and
Xenoseimatosporium kurdistanicum; however, their
pathogenicity could not be demonstrated in short-term field
pathogenicity trials and they were thus considered saprophytic
(Moghadam et al., 2022). Phylogenetic analyses of a dataset
combining sequences of five loci grouped eight Washington
isolates with the type specimen of Sporocadus kurdistanicus,
which is here reported for the first time in North America. Two
Washington isolates (Bent506 and Bent507) formed a sister
clade to this species, but morphological observations
supported their affiliation with Sporocadus kurdistanicus (data
not shown). In contrast, Washington isolate Bent716 was
strongly supported as a new species, sister to Sporocadus
kurdistanicus, but placed on a long branch outside that clade
in the phylogeny. Moreover, morphological observations
confirmed this distinction with isolate Bent716 producing
conidia with up to seven septa, whereas those of Sporocadus
kurdistanicus have three septa, like most Sporocadus species (Liu
et al., 2019). The species Sporocadus incarnatus is newly
described and typified to accommodate isolate Bent716, which
was deposited in two public repositories for future investigations
of its ecology. Future research should determine the pathogenic
status of isolates from this genus in long-term experiments, as
they are reported from diseased grapevines around the world
(Mundy et al., 2020;Moghadam et al., 2022).
Since our surveys principally targeted vineyards with a history
of esca symptoms, it was anticipated to isolate the widely
recognized causal agents of this disease, namely Phaeomoniella
chlamydospora and Phaeoacremonium minimum. The frequent
white-rot symptoms in the trunks of affected vines are usually
associated with species from the phylum Basidiomycota, with
Fomitiporia mediterranea the main causal agent in European
vineyards (Moretti et al., 2021), but multiple basidiomycetes
species have been associated with esca worldwide (Fischer,
2006). In California, the predominant species is Fomitiporia
polymorpha (Brown et al., 2020) and our isolations confirmed
the presence of this species in California table grape vineyards. In
addition, we recovered two isolates of Quambalaria cyanescens
(Microstromatales, Quambalariaceae), reporting this species for
the first time in North America. This species has recently been
categorized as a grapevine trunk pathogen in northwest Iran
(Narmani and Arzanlou, 2019) and we hypothesize it may also
be part of the complex of fungi causing trunk diseases in
California. Basidiomycetes recovered from Washington wine
grapes included Flammulina filiformis and Trametes versicolor,
the former species being reported from Vitis vinifera for the first
time worldwide. As generalist wood-rotting Basidiomycetes, their
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association with perennial crops such as grapevines is not
surprising. Flammulina filiformis was the least virulent species
in our pathogenicity test. Among the species associated with esca
and young vine decline, we identified Phaeoacremonium
parasiticum and Phaeoacremonium scolyti in California, two
species that have been isolated from diseased grapevines in
various grape-growing regions (Spies et al., 2018).
Phaeoacremonium scolyti is now reported for the first time in
North America.
In addition, two Cadophora species were recovered from
Washington wine grapes, including the recently described species
Cadophora ferruginea (Macia-Vicente et al., 2020), and a new
phylogenetic lineage described here as Cadophora columbiana.
Pathogenicity testing revealed they are both pathogenic to Vitis
vinifera. In the multilocus phylogenetic analyses of a comprehensive
dataset, including all currently known Cadophora species, all species
were well-supported as independent phylogenetic lineages, except
for two taxa: Cadophora vinaceae CBS 146263 (Chen et al., 2022),
which clustered with strong support with the type isolate of
Cadophora ferruginea CBS 146363, and Cadophora sabaouae
(Aigoun-Mouhous et al., 2021), which was nested with strong
support with the type isolate of Cadophora luteo-olivacea CBS
141.41. Cadophora vinaceae CBS 146263 was described as
phylogenetically related to Cadophora ferruginea CBS 146363
with slight differences in culture growth rate and color, and 17
nucleotide differences in their TEF-1asequences. The TEF-1a
sequences of Cadophora vinaceae CBS 146263 are identical to the
ones of our isolates Bent721 and Bent722, and morphological
features of Bent721 matched the description of Cadophora
ferruginea CBS 146363. Accordingly, we considered isolates
Bent721 and Bent722 to be Cadophora ferruginea based on the
present phylogeny of the genus, which further suggests that
Cadophora vinaceae is invalid and should be synonymized with
Cadophora ferruginea. Furthermore, three isolates of Cadophora
sabaouae identified from grapevines in Algeria clustered with strong
support (100/100%) with eight isolates of the well-known species
Cadophora luteo-olivacea, and these three isolates were placed on a
branch internal to the one of the type specimen for this species (CBS
141.41). Cadophora sabaouae CBS 147192 was described as
phylogenetically related to Cadophora luteo-olivacea with
differences in culture growth rate and 14 nucleotide differences in
an alignment of more than 1,400 characters. Given the
morphological variability of this species in culture (Harrington
and Mcnew, 2003 Gramaje et al., 2011) and the minimal amount of
nucleotide differences between sequences of the two species, our
phylogenetic results strongly suggest that ‘Cadophora sabaouae’is
an internally nested clade that is actually Cadophora luteo-olivacea.
If not, the clade containing Cadophora luteo-olivacea is
polyphyletic, which does not conform to the principles of
cladistics and monophyly. Therefore, the name Cadophora
sabaouae is reduced to synonymy with Cadophora luteo-olivacea.
Kalmusia variispora was isolated from diseased grapevines
in Washington and California. The pathogenicity of this species
to grape is reported by Abed-Ashtiani et al. (2019), who fulfilled
Koch’s postulates and reproduced wood discolorations and
wilting symptoms in controlled inoculations of grapevines. In
our pathogenicity test, this species was the most virulent among
10 species evaluated. This is the first report of Kalmusia
variispora in North America. Members of the family
Didymosphaeriaceae include plant endophytes, saprobes and
pathogens (Ariyawansa et al., 2014), and Kalmusia variispora is
known as a causal agent of apple fruit core rot (Ntasiou et al.,
2021;Gutierrez et al., 2022).ItssisterspeciesKalmusia
longispora has been shown to cause vascular necrosis in
grapevines (Karacsony et al., 2021). Considering Kalmusia
variispora produces a wide array of phytotoxic metabolites
(Cimmino et al., 2021), this species may be associated with
other overlooked symptoms.
The charcoal oak decline fungus Biscogniauxia mediterranea
is a well-known wood pathogen around the world, but has not
been reported as a grapevine trunk pathogen, although it is
reported from Vitis sp. in the US (Jayawardena et al., 2018). The
closely related species Biscogniauxia rosacearum has been shown
pathogenic to grapevine in Iran (Bahmani et al., 2021), and is
also known to cause charcoal cankers of pear, plum and quince
trees in Italy (Raimondo et al., 2016). Biscogniauxia
mediterranea isolate Kern007 proved pathogenic in our
pathogenicity test. Our findings support the role of
Biscogniauxia mediterranea as a grapevine trunk pathogen
in California.
Members of the genus Thyrostroma (Pleosporales,
Pleosporineae) frequently are plant pathogens, causing wood
cankers, dieback and leaf spots on numerous hosts, with, for
example, Thyrostroma celtidis and Thyrostroma lycii associated
with twig cankers of Celtidis occidentalis and Lycium barbarum,
respectively (Senwanna et al., 2019). There are only 14 species in
this genus that are currently supported with molecular data, with
these species known only from Korea, Russia, Ukraine, USA and
Uzbekistan (Hyde et al., 2020). One isolate of Thyrostroma
(Bent904) was recovered from a wood canker of a Vitis
vinifera ‘Chardonnay’vine in Washington. We generated and
deposited in GenBank sequences for six loci for this isolate and
multilocus phylogenetic analyses revealed this isolate constituted
a new phylogenetic species (data not shown) whose closest
relative was Thyrostroma ephedricola (Pem et al., 2019). We
did not formally described our isolate as a new species as we
could not obtain, despite multiple attempts, fruiting bodies in
culture. Our pathogenicity test revealed Thyrostroma sp.
Bent904 as pathogenic to grapevine.
In assessing the optimal mycelium growth temperature of 10
isolates from each region, an important finding was the sharp
contrast in their growth ability at the extremes of the
temperature ranges tested. Most isolates from Washington
grew at 5°C, but not at 35°C, whereas most isolates from
California did not grow at 5°C, but grew at 35°C. In addition,
differences in optimal growth temperatures were observed for
Travadon et al. 10.3389/ffunb.2022.1018140
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isolates from each region. Our findings suggest that the
differences in species composition in each region (with only
six species shared between regions), may be influenced by local
environmental conditions. Although these findings are based on
a subset of isolates from each region, they suggest that
Washington isolates seem, on average, better adapted to the
cooler winter / spring climate found in this region, compared to
Californian isolates adaptations to warmer winter / spring
climates. Considering that some evidence points to a
latitudinal, geographical range shift of plant pests and
pathogens influenced by climate change (Bebber, 2015), and
that some trunk pathogens are often found in grapevine planting
material (Carbone et al., 2022) that is commonly transported
across viticulture regions, such future shift in geographical
ranges of grapevine trunk pathogens might be expected.
In conclusion, this study illustrates the differences in
community composition that can be observed in grapevines
expressing similar symptoms of trunk diseases from distinct
grape-growing regions and production systems, with climate
adaptation a plausible driver of pathogen distributions. By
establishing a physical fungal collection, the characterization of
the thermophilic profiles and pathogenic status of some isolates
could be achieved, expanding the list of fungal pathogens
associated with grapevine trunk diseases. Moreover, this study
introduced four new fungal species in important genera of plant
pathogens. Accurate taxonomic identification coupled with the
evaluation of life history traits are essential to understanding the
basic ecology and management of individual species, which will
undeniably assist into the interpretation of fungal community
sequencing datasets.
Data availability statement
The molecular data presented in this study can be found at the
National Institute of Health, genetic sequence database GenBank
(https://www.ncbi.nlm.nih.gov/genbank/), with accession numbers
provided in Table 1. Dried specimens of newly described taxa were
deposited at the U.S. National Fungus Collections (BPI) under the
following accession numbers: BPI 911228 - BPI 911231 and can be
accessed at the herbarium specimen database (https://nt.ars-grin.
gov/fungaldatabases/specimens/specimens.cfm). Living cultures of
newly described taxa and additional strains presented in Table 1
have been deposited at the Westerdijk Fungal Biodiversity Institute
(https://wi.knaw.nl/) with accession numbers CBS 149294-CBS
149301, CBS 149336 and CBS 149338. Further queries can be
directed to the corresponding author.
Author contributions
RT, MM and KB obtained the funding and designed the
study. MM and KB directed the liaison with industry for
vineyard selection. DL, PTF and RT contributed to the
analyses and interpretation of genetic data. DL conducted
phylogenetic analyses. PF and RT guided the acquisition of
molecular data. RT and KB conducted the acquisition and
interpretation of the pathogenicity data. RT conducted the
acquisition and interpretation of the morphological and
temperature data. RT wrote the manuscript with contributions
from DL, MM and KB. All authors reviewed the manuscript.
Funding
Funding was provided by the USDA Specialty Crop Multi-
State Program, grant 17-0728-001-SF. Additional funding was
provided by USDA National Institute of Food and Agriculture,
Hatch project 1016563.
Acknowledgments
The authors would like to express their gratitude to Gabriel
A. Torres (University of California Cooperative Extension) for
his help in identifying table grape vineyards. The assistance of
local grape growers was very much appreciated. Authors thank
Maria Mireles (Washington State University) for her help with
plant sample acquisition. We also thank Paula J. Eschen and
Alejandro I. Hernandez (UC Davis) for assistance with
generating molecular sequences and microscopy. Mention of
trade names or commercial products is solely for the purpose of
providing specific information and does not imply
recommendation or endorsement by the USDA. USDA is an
equal opportunity provider and employer.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/
ffunb.2022.1018140/full#supplementary-material
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org23

References
Abed-Ashtiani, F., Narmani, A., and Arzanlou, M. (2019). Analysis of Kalmusia
variispora associated with grapevine decline in Iran. Eur. J. Plant Pathol. 154, 787–
799. doi: 10.1007/s10658-019-01702-5
Adams, G. C., Surve-Iyer, R. S., and Iezzoni, A. F. (2002). Ribosomal DNA
sequence divergence and group I introns within the Leucostoma species L. cinctum,
L. persoonii, and l. parapersoonii sp. nov., ascomycetes that cause cytospora canker
of fruit trees. Mycologia 94, 947–967. doi: 10.1080/15572536.2003.11833153
Aigoun-Mouhous, W., Mahamedi, A. E., Leon, M., Chaouia, C., Zitouni, A.,
Barankova, K., et al. (2021). Cadophora sabaouae sp. nov. and Phaeoacremonium
species associated with Petri disease on grapevine propagation material and young
grapevines in Algeria. Plant Dis 105, 3657–68. doi: 10.1094/pdis-11-20-2380-re
Amend, A. S., Seifert, K. A., and Bruns, T. D. (2010). Quantifying microbial
communities with 454 pyrosequencing: does read abundance count? Mol. Ecol. 19,
5555–5565. doi: 10.1111/j.1365-294X.2010.04898.x
Aradhya, M. K., Dangl, G. S., Prins, B. H., Boursiquot, J. M., Walker, M. A.,
Meredith, C. P., et al. (2003). Genetic structure and differentiation in cultivated
grape, Vitis vinifera l. Genetical Res. 81, 179–192. doi: 10.1017/S0016672303006177
Ariyawansa, H. A., Tanaka, K., Thambugala, K. M., Phookamsak, R., Tian, Q.,
Camporesi, E., et al. (2014). A molecular phylogenetic reappraisal of the
didymosphaeriaceae (= montagnulaceae). Fungal Diversity 68, 69–104. doi:
10.1007/s13225-014-0305-6
Arzanlou, M., Narmani, A., Moshari, S., Khodaei, S., and Babai-Ahari, A. (2013).
Truncatella angustata associated with grapevine trunk disease in northern Iran.
Arch. Phytopathol. Plant Prot. 46, 1168–1181. doi: 10.1080/03235408.2012.761417
Bahmani, Z., Abdollahzadeh, J., Amini, J., and Evidente, A. (2021).
Biscogniauxia rosacearum the charcoal canker agent as a pathogen associated
with grapevine trunk diseases in zagros region of Iran. Sci. Rep. 11, 1–7. doi:
10.1038/s41598-021-93630-w
Baldrian, P., Kolar
ı

k, M., S

tursova, M., Kopecky, J., Valaskova, V., Vetrovsky, T.,
et al. (2012). Active and total microbial communities in forest soil are largely
different and highly stratified during decomposition. ISME J. 6, 248–258. doi:
10.1038/ismej.2011.95
Baloyi, M. A., Mostert, L., and Halleen, F. (2018). Pathogenicity of ten
Phaeoacremonium species associated with esca and Petri disease of grapevine.
Phytopathol. Mediterr. 57, 538–546. doi: 10.14601/Phytopathol_Mediterr-23940
Baumgartner, K., Hillis, V., Lubell, M., Norton, M., and Kaplan, J. (2019).
Managing grapevine trunk diseases in california’s southern San Joaquin valley. Am.
J. Enol Viticult 70, 267–276. doi: 10.5344/ajev.2019.18075
Baumgartner, K., Travadon, R., Fujiyoshi, P., Mireles, M., and Moyer, M. (2022).
Preventing trunk diseases with fungicide applications to pruning wounds in
Washington wine grapes. Am. J. Viticult Enol.
Bebber, D. P. (2015). Range-expanding pests and pathogens in a warming world.
Annu. Rev. Phytopathol. 53, 335–356. doi: 10.1146/annurev-phyto-080614-120207
Bettenfeld, P., Cadena I Canals, J., Jacquens, L., Fernandez, O., Fontaine, F., Van
Schaik, E., et al. (2022). The microbiota of the grapevine holobio nt: a key
component of plant health. J. Advanced Res 40, 1–15. doi: 10.1016/j.jare.2021.
12.008
Bien, S., and Damm, U. (2020). Arboricolonus simplex gen. et sp. nov. and
novelties in Cadophora,Minutiella and Proliferodiscus from Prunus wood in
Germany. MycoKeys 63, 119. doi: 10.3897/mycokeys.63.46836
Bokulich, N. A., Collins, T. S., Masarweh, C., Allen, G., Heymann, H., Ebeler, S.
E., et al. (2016). Associations among wine grape microbiome, metabolome, and
fermentation behavior suggest microbial contribution to regional wine
characteristics. MBio 7, e00631-16. doi: 10.1128/mBio.00631-16
Bokulich, N. A., Thorngate, J. H., Richardson, P. M., and Mills, D. A. (2014).
Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and
climate. Proc. Natl. Acad. Sci. 111, E139-E148. doi: 10.1073/pnas.1317377110
Brown, A., Lawrence, D., and Baumgartner, K. (2020). Role of basidiomycete
fungi in the grapevine trunk disease esca. Plant Pathol. 69, 205–220. doi: 10.1111/
ppa.13116
Bruez, E., Vallance, J., Gautier, A., Laval, V., Compant, S., Maurer, W., et al.
(2020). Major changes in grapevine wood microbiota are associated with the onset
of esca, a devastating trunk disease. Environ. Microbiol. 22, 5189–5206. doi:
10.1111/1462-2920.15180
Bundhun, D., Jeewon, R., Senanayake, I. C., Camporesi, E., Aluthmuhandiram, J.
V., Tang, A. M., et al. (2021). Morpho-molecular characterization of discosia
ravennica sp. nov. and a new host record for Sporocadus rosigena.MycoKeys 79,
173. doi: 10.3897/mycokeys.79.60662
Burns, K. N., Bokulich, N. A., Cantu, D., Greenhut, R. F., Kluepfel, D. A., O'geen,
A. T., et al. (2016). Vineyard soil bacterial diversity and composition revealed by
16S rRNA genes: differentiation by vineyard management. Soil Biol. Biochem. 103,
337–348. doi: 10.1016/j.soilbio.2016.09.007
Carbone, M. J., Gelabert, M., Moreira, V., Mondino, P., and Alaniz, S. (2022).
Grapevine nursery propagation material as source of fungal trunk disease
pathogens in Uruguay. Front. Fungal Biol. 3. doi: 10.3389/ffunb.2022.958466
Cazabonne, J., Bartrop, L., Dierickx, G., Gafforov, Y., Hofmann, T. A., Martin, T.
E., et al. (2022). Molecular-based diversity studies and field surveys are not
mutually exclusive: On the importance of integrated methodologies in
mycological research. Front. Fungal Biol. 3, 860777. doi: 10.3389/
ffunb.2022.860777
Chen, Q., Bakhshi, M., Balci, Y., Broders, K., Cheewangkoon, R., Chen, S., et al.
(2022). Genera of phytopathogenic fungi: GOPHY 4. Stud. Mycol 101, 417–564.
doi: 10.3114/sim.2022.101.06
Cimmino, A., Bahmani, Z., Masi, M., Di Lecce, R., Amini, J., Abdollahzadeh, J.,
et al. (2021). Massarilactones d and h, phytotoxins produced by kalmusia
variispora, associated with grapevine trunk diseases (GTDs) in Iran. Natural
Product Res. 35, 5192–5198. doi: 10.1080/14786419.2020.1791116
Cloete, M., Mostert, L., Fischer, M., and Halleen, F. (2015). Pathogenicity of
south African hymenochaetales taxa isolated from esca-infected grapevines.
Phytopathol. Mediterr. 54, 368–379. doi: 10.14601/Phytopathol_Mediterr-16237
Cobos, R., Ibañez, A., Diez-Galan, A., Calvo-Peña, C., Ghoreshizadeh, S., and
Coque, J. J. R. (2022). The grapevine microbiome to the rescue: Implications for the
biocontrol of trunk diseases. Plants 11, 840. doi: 10.3390/plants11070840
Dekrey, D. H., Klodd, A. E., Clark, M. D., and Blanchette, R. A. (2022).
Grapevine trunk diseases of cold-hardy varieties grown in northern Midwest
vineyards coincide with canker fungi and winter injury. PloS One 17, e0269555.
doi: 10.1371/journal.pone.0269555
Del Frari, G., Gobbi, A., Aggerbeck, M. R., Oliveira, H., Hansen, L. H., and
Ferreira, R. B. (2019). Characterization of the wood mycobiome of Vitis vinifera in
a vineyard affected by esca. spatial distribution of fungal communities and their
putative relation with leaf symptoms. Front. Plant Sci. 910. doi: 10.3389/
fpls.2019.00910
Deyett, E., Pouzoulet, J., Yang, J. I., Ashworth, V., Castro, C., Roper, M., et al.
(2019). Assessment of pierce's disease susceptibility in vitis vinifera cultivars with
different pedigrees. Plant Pathol. 68, 1079–1087. doi: 10.1111/ppa.13027
Deyett, E., and Rolshausen, P. (2020). Endophytic microbial assemblage in
grapevine. FEMS Microbiol. Ecol. 96, fiaa053. doi: 10.1093/femsec/fiaa053
Dı

az, G., Elfar, K., and Latorre, B. (2012). First report of Seimatosporium botan
associated with trunk disease of grapevine (Vitis vinifera) in Chile. Plant Dis. 101,
253. doi: 10.1094/pdis-05-12-0478-pdn
Dissanayake, A. J., Purahong, W., Wubet, T., Hyde, K. D., Zhang, W., Xu, H.,
et al. (2018). Direct comparison of culture-dependent and culture-independent
molecular approaches reveal the diversity of fungal endophytic communities in
stems of grapevine (Vitis vinifera). Fungal Diversity 90, 85–107. doi: 10.1007/
s13225-018-0399-3
Eichmeier, A., Pecenka, J., Penazova, E., Baranek, M., Català-Garcı

a, S., Leon,
M., et al. (2018). High-throughput amplicon sequencing-based analysis of active
fungal communities inhabiting grapevine after hot-water treatments reveals
unexpectedly high fungal diversity. Fungal Ecol. 36, 26–38. doi: 10.1016/
j.funeco.2018.07.011
Eskalen, A., Rooney-Latham, S., and Gubler, W. (2005a). First report of
perithecia of Phaeoacremonium viticola on grapevine (Vitis vinifera) and ash tree
(Fraxinus latifolia) in California. Plant Dis. 89, 686–686. doi: 10.1094/PD-89-
0686B
Eskalen, A., Rooney-Latham, S., and Gubler, W. (2005b). Occurrence of
Togninia fraxinopennsylvanica on esca-diseased grapevines (Vitis vinifera) and
declining ash trees (Fraxinus latifolia) in California. Plant Dis. 89, 528–528. doi:
10.1094/PD-89-0528C
Fan, X., Hyde, K. D., Liu, M., Liang, Y., and Tian, C. (2015). Cytospora species
associated with walnut canker disease in China, with description of a new species C.
gigalocus.Fungal Biol. 119, 310–319. doi: 10.1016/j.funbio.2014.12.011
Fischer, M. (2006). Biodiversity and geographic distribution of basidiomycetes
causing esca-associates white rot in grapevine: a worldwide perspective.
Phytopathol. Mediterr. 45, 30–42. doi: 10.14601/Phytopathol_Mediterr-1846
Fotouhifar, K.-B., Hedjaroude, G.-A., and Leuchtmann, A. (2010). ITS rDNA
phylogeny of Iranian strains of Cytospora and associated teleomorphs. Mycologia
102, 1369–1382. doi: 10.3852/10-034
Gispert, C., Kaplan, J. D., Deyett, E., and Rolshausen, P. E. (2020). Long-term
benefits of protecting table grape vineyards against trunk diseases in the California
desert. Agronomy 101895. doi: 10.3390/agronomy10121895
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org24

Glawe, D. A., and Rogers, J. D. (1984). Diatrypaceae in the pacific Northwest.
Mycotaxon 20, 401–460.
Glawe, D., Skotland, C., and Moller, W. J. (1982). Isolation and identification of
Eutypa armeniacae from diseased grapevines in Washington state. Mycotaxon 16,
123–132.
Gramaje, D., Armengol, J., and Ridgway, H. J. (2013). Genetic and virulence
diversity, and mating type distribution of Togninia minima causing grapevine
trunk diseases in Spain. Eur. J. Plant Pathol. 135, 727–743. doi: 10.1007/s10658-
012-0110-6
Gramaje, D., Mostert, L., and Armengol, J. (2011). Characterization of
Cadophora luteo-olivacea and C. melinii isolates obtained from grapevines and
environmental samples from grapevine nurseries in Spain. Phytopathol. Mediterr.
50, 112–126. doi: 10.14601/Phytopathol_Me
Gramaje, D., Urbez-Torres, J. R., and Sosnowski, M. R. (2018). Managing
grapevine trunk diseases with respect to etiology and epidemiology: current
strategies and future prospects. Plant Dis. 102, 12–39. doi: 10.1094/PDIS-04-17-
0512-FE
Gutierrez, M., Pinheiro, C., Duarte, J., Sumonte, C., Ferrada, E., Elfar, K., et al.
(2022). Severe outbreak of dry core rot in apple fruits cv. Fuji caused by Kalmusia
variispora during pre-harvest in maule region, Chile. Plant Disease. doi: 10.1094/
PDIS-12-21-2776-PDN
Haidar, R., Yacoub, A., Vallance, J., Compant, S., Antonielli, L., Saad, A., et al.
(2021). Bacteria associated with wood tissues of esca-diseased grapevines:
functional diversity and synergy with Fomitiporia mediterranea to degrade
wood components. Environ. Microbiol. 23, 6104–6121. doi: 10.1111/1462-2920.
15676
Harrington, T. C., and Mcnew, D. L. (2003). Phylogenetic analysis places the
Phialophora-like anamorph genus Cadophora in the helotiales. Mycotaxon 87, 141–
151.
Hofstetter, V., Buyck, B., Croll, D., Viret, O., Couloux, A., and Gindro, K. (2012).
What if esca disease of grapevine were not a fungal disease? Fungal Diversity 54,
51–67. doi: 10.1007/s13225-012-0171-z
Holland, L., Grove, G. G., and Glawe, D. A. (2015). Washington Grapevine trunk
disease survey. Viticult Enol Extension News 2015, 4.
Hyde, K. D., Jeewon, R., Chen, Y.-J., Bhunjun, C. S., Calabon, M. S., Jiang, H.-B.,
et al. (2020). The numbers of fungi: is the descriptive curve flattening? Fungal
Diversity 103, 219–271. doi: 10.1007/s13225-020-00458-2
Jayawardena, R. S., Purahong, W., Zhang, W., Wubet, T., Li, X., Liu, M., et al.
(2018). Biodiversity of fungi on Vitis vinifera l. revealed by traditional and high-
resolution culture-independent approaches. Fungal Diversity 90, 1–84. doi:
10.1007/s13225-018-0398-4
Jayawardena, R. S., Zhang, W., Liu, M., Maharachchikumbura, S. S., Zhou, Y.,
Huang, J., et al. (2015). Identification and characterization of pestalotiopsis-like
fungi related to grapevine diseases in China. Fungal Biol. 119, 348–361. doi:
10.1016/j.funbio.2014.11.001
Johnson, D. A., and Lunden, J. D. (1987). Incidence and yield impact of eutypa
dieback of grapevine in Washington state USA. Washington State Univ. Agric. Res.
Center Res. Bull.,1–7.
Kaplan, J., Travadon, R., Cooper, M., Hillis, V., Lubell, M., and Baumgartner, K.
(2016). Identifying economic hurdles to early adoption of preventative practices:
The case of trunk diseases in California winegrape vineyards. Wine Economics
Policy 5, 127–141. doi: 10.1016/j.wep.2016.11.001
Karacsony, Z., Knapp, D. G., Lengyel, S., Kovacs, G. M., and Vaczy, K. Z. (2021).
The fungus Kalmusia longispora is able to cause vascular necrosis on Vitis vinifera.
PloS One 16, e0258043. doi: 10.1371/journal.pone.0258043
Koukol, O., and Macia-Vicente, J. G. (2022). Leptodophora gen. nov. (Helotiales,
leotiomycetes) proposed to accommodate selected root-associated members of the
genus Cadophora.Czech Mycol 74, 57–66. doi: 10.33585/cmy.74104
Kraus, C., Damm, U., Bien, S., Voegele, R., and Fischer, M. (2020). New species
of phaeomoniellales from a German vineyard and their potential threat to
grapevine (Vitis vinifera) health. Fungal Syst Evol. 6, 139–155. doi: 10.3114/
fuse.2020.06.08
Kraus, C., Voegele, R. T., and Fischer, M. (2019). Temporal development of the
culturable, endophytic fungal community in healthy grapevine branches and
occurrence of GTD-associated fungi. Microbial Ecol. 77, 866–876. doi: 10.1007/
s00248-018-1280-3
Lawrence, D. P., Holland, L. A., Nouri, M. T., Travadon, R., Abramians, A.,
Michailides, T. J., et al. (2018a). Molecular phylogeny of Cytospora species
associated with canker diseases of fruit and nut crops in California, with the
descriptions of ten new species and one new combination. IMA fungus 9, 333–369.
doi: 10.5598/imafungus.2018.09.02.07
Lawrence, D., Nouri, M., and Trouillas, F. (2019). Taxonomy and multi-locus
phylogeny of cylindrocarpon-like species associated with diseased roots of
grapevine and other fruit and nut crops in California. Fungal syst Evol. 4, 59–75.
doi: 10.3114/fuse.2019.04.06
Lawrence, D. P., Travadon, R., and Baumgartner, K. (2015). Diversity of
diaporthe species associated with wood cankers of fruit and nut crops in
northern California. Mycologia 107, 926–940. doi: 10.3852/14-353
Lawrence,D.P.,Travadon,R.,andBaumgartner,K.(2018b).Novel
Seimatosporium species from grapevine in northern California and their
interactions with fungal pathogens involved in the trunk-disease complex. Plant
Dis. 102, 1081–1092. doi: 10.1094/PDIS-08-17-1247-RE
Lawrence,D.P.,Travadon,R.,Nita,M.,andBaumgartner,K.(2017b).
TrunkDiseaseID. org: A molecular database for fast and accurate identification
of fungi commonly isolated from grapevine wood. Crop Prot. 102, 110–117. doi:
10.1016/j.cropro.2017.08.017
Lawrence, D., Travadon, R., Pouzoulet, J., Rolshausen, P., Wilcox, W., and
Baumgartner, K. (2017a). Ch aracterization of Cytospora isolates from wood
cankers of declining grapevine in north America, with the descriptions of two
new Cytospora species. Plant Pathol. 66, 713–725. doi: 10.1111/ppa.12621
Linnakoski, R., Kasanen, R., Lasarov, I., Marttinen, T., Oghenekaro, A. O., Sun,
H., et al. (2018). Cadophora margaritata sp. nov. and other fungi associated with
the longhorn beetles Anoplophora glabripennis and Saperda carcharias in Finland.
Antonie Van Leeuwenhoek 111, 2195–2211. doi: 10.1007/s10482-018-1112-y
Liu,F.,Bonthond,G.,Groenewald,J.,Cai,L.,andCrous,P.(2019).
Sporocadaceae, a family of coelomycetous fungi with appendage-bearing conidia.
Stud. Mycol 92, 287–415. doi: 10.1016/j.simyco.2018.11.001
Long,S.,Liu,L.,Pi,Y.,Wu,Y.,Lin,Y.,Zhang,X.,etal.(2021).Newcontributionsto
diatrypaceae from karst areas in China. MycoKeys 83,1.doi:10.3897/mycokeys.83.68926
Luque, J., Garcia-Figueres, F., Legorburu, F. J., Muruamendiaraz, A., Armengol,
J., and Trouillas, F. P. (2012). Species of diatrypaceae associated with grapevine
trunk diseases in Eastern Spain. Phytopathol. Mediterr. 51, 528–540.
Macia-Vicente, J. G., Piepenbring, M., and Koukol, O. (2020). Brassicaceous
roots as an unexpected diversity hot-spot of helotialean endophytes. IMA fungus
11, 1–23. doi: 10.1186/s43008-020-00036-w
Maddison, W. P., and Maddison, D. R. (2016). Mesquite: A modular system for
evolutionary analysis. Version 3.10. Available at: http://mesquiteproject.org.
Maharachchikumbura, S. S., Larignon, P., Al-Sadi, A. M., and Zuo-Yi, L. (2017).
Characterization of Neopestalotiopsis,Pestalotiopsis and Truncatella species
associated with grapevine trunk diseases in France. Phytopathol. Mediterr. 55,
380–390. doi: 10.14601/Phytopathol_Mediterr-18298
Mayorquin, J. S., Wang, D. H., Twizeyimana, M., and Eskalen, A. (2016).
Identification, distribution, and pathogenicity of diatrypaceae and
botryosphaeriaceae associated with citrus branch canker in the southern
California desert. Plant Dis. 100, 2402–2413. doi: 10.1094/PDIS-03-16-0362-RE
Moghadam,J.N.,Khaledi,E.,Abdollahzadeh,J.,andAmini,J.(2022).
Seimatosporium marivanicum,Sporocadus kurdistanicus,and
Xenoseimatosporium kurdistanicum: three new pestalotioid species associated
with grapevine trunk diseases from the Kurdistan province, Iran. Mycol Prog. 21,
427–446. doi: 10.1007/s11557-021-01764-y
Morales-Cruz, A., Allenbeck, G.,Figueroa-Balderas, R., Ashworth, V. E., Lawrence,
D. P., Travadon, R., et al. (2018a). Closed-reference metatranscriptomics enables in
planta profilingof putative virulence activities in the grapevine trunk disease complex.
Mol. Plant Pathol. 19, 490–503. doi: 10.1111/mpp.12 544
Morales-Cruz, A., Figueroa-Balderas, R., Garcı

a, J. F., Tran, E., Rolshausen, P. E.,
Baumgartner, K., et al. (2018b). Profiling grapevine trunk pathogens in planta: a
case for community-targeted DNA metabarcoding. BMC Microbiol. 18, 1–14. doi:
10.1186/s12866-018-1343-0
Moretti, S., Pacetti, A., Pierron, R., Kassemeyer, H.-H., Fischer, M., Peros, J.-P.,
et al. (2021). Fomitiporia mediterranea m. fisch., the historical esca agent: A
comprehensive review on the main grapevine wood rot agent in Europe.
Phytopathol. Mediterr. 60, 351–379. doi: 10.36253/phyto-13021
Moyo, P., Mostert, L., and Halleen, F. (2019). Diatrypaceae species overlap
between vineyards and natural ecosystems in south Africa. Fungal Ecol. 39, 142–
151. doi: 10.1016/j.funeco.2018.11.015
Moyo, P., Mostert, L., Spies, C. F., Damm, U., and Halleen, F. (2018). Diversity of
diatrypaceae species associated with dieback of grapevines in south Africa, with the
description of eutypa cremea sp. nov. Plant Dis. 102, 220–230. doi: 10.1094/PDIS-
05-17-0738-RE
Mundy, D. C., Brown, A., Jacobo, F., Tennakoon, K., Woolley, R. H., Vanga, B.,
et al. (2020). Pathogenic fungi isolated in association with grapevine trunk diseases
in new Zealand. NewZ.J.CropHortic.Sci.48, 84–96. doi: 10.1080/
01140671.2020.1716813
Narmani, A., and Arzanlou, M. (2019). Quambalaria cyanescens, a new fungal
trunk pathogen associated with grapevine decline in Iran. Crop Prot. 124, 104875.
doi: 10.1016/j.cropro.2019.104875
National Agricultural Statistics Service. (2018). Noncitrus fruits and nuts 2017
summary. Washington, D. C.: United States Department of Agriculture, National
Agricultural Statistics Service. Available at: https://www.nass.usda.gov/
Publications/Todays_Reports/reports/ncit0618.pdf.
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org25

Nerva, L., Garcia, J. F., Favaretto, F., Giudice, G., Moffa, L., Sandrini, M., et al.
(2022). The hidden world within plants: metatranscriptomics unveils the complexity
of wood microbiomes. J. Exp. Bot. 73, 2682–2697. doi: 10.1093/jxb/erac032
Niem, J. M., Billones-Baaijens, R., Stodart, B., and Savocchia, S. (2020). Diversity
profiling of grapevine microbial endosphere and antagonistic potential of
endophytic Pseudomonas against grapevine trunk diseases. Front. Microbiol. 11,
477. doi: 10.3389/fmicb.2020.00477
Nouri, M., Zhuang, G., Culumber, C., and Trouillas, F. (2018). First report of
Macrophomina phaseolina causing trunk and cordon canker disease of grapevine in
the united states. Plant Dis. 103, 579. doi: 10.1094/PDIS-07-18-1189-PDN
Ntasiou, P., Samaras, A., and Karaoglanidis, G. (2021). Apple fruit core rot
agents in Greece and control with succinate dehydrogenase inhibitor fungicides.
Plant Dis. 105, 3072–3081. doi: 10.1094/PDIS-11-20-2422-RE
Onetto,C.A.,Sosnowski,M.R.,VanDenHeuvel,S.,andBorneman,A.R.(2022).
Population genomics of the grapevine pathogen Eutypa lata reveals evidence for
population expansion and intraspecific differences in secondary metabolite gene
clusters. PloS Genet. 18, e1010153. doi: 10.1371/journal.pgen.1010153
Pacetti, A., Moretti, S., Pinto, C., Compant, S., Farine, S., Bertsch, C., et al. (2021).
Trunk surgery as a tool to reduce foliar symptoms in diseases of the esca complex and its
influence on vine wood microbiota. J. Fungi 7, 521. doi: 10.3390/jof7070521
Pan, M., Zhu, H., Bonthond, G., Tian, C., and Fan, X. (2020). High diversity of
Cytospora associated with canker and dieback of rosaceae in China, with 10 new
species described. Front. Plant Sci. 690. doi: 10.3389/fpls.2020.00690
Pan, M., Zhu, H., Tian, C., Huang, M., and Fan, X. (2021). Assessment of
Cytospora isolates from conifer cankers in China, with the descriptions of four new
Cytospora species. Front. Plant Sci. 12. doi: 10.3389/fpls.2021.636460
Paolinelli, M., Escoriaza, G., Cesari, C., Garcia-Lampasona, S., and Hernandez-
Martinez, R. (2022). Characterization of grapevine wood microbiome through a
metatranscriptomic approach. Microbial Ecol. 83, 658–668. doi: 10.1007/s00248-
021-01801-z
Patanita, M., Albuquerque, A., Campos, M. D., Materatski, P., Varanda, C. M.,
Ribeiro, J. A., et al. (2022). Metagenomic assessment unravels fungal microbiota
associated to grapevine trunk diseases. Horticulturae 8, 288. doi: 10.3390/
horticulturae8040288
Peay, K. G. (2014). Back to the future: natural history and the way forward in
modern fungal ecology. Fungal Ecol. 12, 4–9. doi: 10.1016/j.funeco.2014.06.001
Pem, D., Jeewon, R., Bulgakov, T., Gafforov, Y., Hongsanan, S., Phookamsak, R.,
et al. (2019). Taxonomy and molecular phylogeny of thyrostroma ephedricola sp.
nov.(Dothidotthiaceae) and proposal for Thyrostroma jaczewskii comb. nov.
Phytotaxa 416, 243–256. doi: 10.11646/phytotaxa.416.4.3
Peros, J. P., and Berger, G. (1999). Diversity within natural progenies of the grapevine
dieback fungus Eutypa lata.Curr. Genet. 36, 301–309. doi: 10.1007/s002940050504
Raimondo, M. L., Lops, F., and Carlucci, A. (2016). Charcoal canker of pear,
plum, and quince trees caused by biscogniauxia rosacearum sp. nov. in southern
Italy. Plant Dis. 100, 1813–1822. doi: 10.1094/PDIS-09-15-1037-RE
Rangel Montoya, E. A., Paolinelli, M., Rolshausen, P. E., Valenzuela Solano, C., and
Hernandez Martinez, R. (2021). Characterization of Lasiodiplodia species associated with
grapevines in Mexico. Phytopathol. Mediterr. 60, 237–251. doi: 10.36253/phyto-12576
Rappaz, F. (1987). Taxonomie et nomenclature de s diatrypacees à asques
octosporees. Mycologia Helv. 2, 285–648.
Rolshausen, P., Akgül, D., Perez, R., Eskalen, A., and Gispert, C. (2013). First
report of wood canker caused by Neoscytalidium dimidiatum on grapevine in
California. Plant Dis. 97, 1511–1511. doi: 10.1094/PDIS-04-13-0451-PDN
Scheck, H., Vasquez, S., Gubler, W., and Fogle, D. (1998). First report of three
phaeoacremonium spp. causing young grapevine decline in California. Plant Dis.
82, 590–590. doi: 10.1094/PDIS.1998.82.5.590C
Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C.
A., et al. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a
universal DNA barcode marker for fungi. Proc. Natl. Acad. Sci. 109, 6241–6246.
doi: 10.1073/pnas.1117018109
Senwanna, C., Wanasinghe, D., Bulgakov, T., Wang, Y., Bhat, D., Tang, A., et al.
(2019). Towards a natural classification of Dothidotthia and Thyrostroma in
dothidotthiaceae (Pleosporineae, pleosporales). Mycosphere 10, 701. doi: 10.5943/
mycosphere/10/1/15
Shang, Q., Hyde, K., Camporesi, E., Maharachchikumbura, S., Norphanphoun,
C., Brooks, S., et al. (2020). Additions to the genus Cytospora with sexual morph in
cytosporaceae. Mycosphere 11, 189–224. doi: 10.5943/mycosphere/11/1/2
Silva-Valderrama, I., Toapanta, D., Miccono, M. D. L. A., Lolas, M., Dı

az, G. A.,
Cantu, D., et al. (2021). Biocontrol potential of grapevine endophytic and
rhizospheric fungi against trunk pathogens. Front. Microbiol. 113311. doi:
10.3389/fmicb.2020.614620
Smetham, G. M., Ades, P. K., Peros, J. P., and Ford, R. (2010). Genetic structure of the
grapevine fungal pathogen Phaeomoniella chlamydospora in southeastern Australia and
southern France. Plant Pathol. 59, 736–744. doi: 10.1111/j.1365-3059.2010.02302.x
Sosnowski, M., and Mccarthy, G. (2017). Trunk disease: Economic impact of
grapevine trunk disease management in sauvignon blanc vineyards of new Zealand.
Wine Viticult J. 32, 42–48. doi: 10.3316/informit.409675897089506
Spielman, L. J. (1985). A monograph of valsa on hardwoods in north America.
Can. J. Bot. 63, 1355–1378. doi: 10.1139/b85-190
Spies, C., Moyo, P., Halleen, F., and Mostert, L. (2018). Phaeoacremonium
species diversity on woody hosts in the Western cape province of south Africa.
Persoonia-Molecular Phylogeny Evol. Fungi 40, 26–62. doi: 10.3767/
persoonia.2018.40.02
Swofford, D. L. (2002). PAUP: Phylogenetic analysis using parsimony (and other
methods), version 4.0a169. (MA, USA: Sunderland).
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013).
MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol.
30, 2775–29. doi: 10.1093/molbev/mst197
Travadon, R., and Baumgartner, K. (2015). Molecular polymorphism and
phenotypic diversity in the eutypa dieback pathogen Eutypa lata.Phytopathology
105, 255–264. doi: 10.1094/PHYTO-04-14-0117-R
Travadon, R., Baumgartner, K., Rolshausen, P. E., Gubler, W. D., Sosnowski, M.
R., Lecomte, P., et al. (2012). Genetic structure of the fungal grapevine pathogen
Eutypa lata from four continents. Plant Pathol. 61, 85–95. doi: 10.1111/j.1365-
3059.2011.02496.x
Travadon, R., Lawrence, D. P., Rooney-Latham, S., Gubler, W. D., Wilcox, W.
W., Rolshausen, P. E., et al. (2015). Cadophora species associated with wood decay
of grapevine in north America. Fungal Biol. 119, 53–66. doi: 10.1016/
j.funbio.2014.11.002
Travadon, R., Lecomte, P., Diarra, B., Lawrence, D. P., Renault, D., Ojeda, H.,
et al. (2016). Grapevine pruning systems and cultivars influence the diversity of
wood-colonizing fungi. Fungal Ecol. 24, 82–93. doi: 10.1016/
j.funeco.2016.09.003
Travadon, R., Rolshausen, P. E., Gubler, W. D., Cadle-Davidson, L., and
Baumgartner, K. (2013). Susceptibility of cultivated and wild vitis spp. to wood
infection by fungal trunk pathogens. Plant Dis. 97, 1529–1536. doi: 10.1094/PDIS-
05-13-0525-RE
Trouillas,F.P.,andGubler,W.D.(2010).Pathogenicityofdiatrypaceae
speciesingrapevinesinCalifornia.Plant Dis. 94, 867–872. doi: 10.1094/PDIS-
94-7-0867
Trouillas, F. P., Pitt, W. M., Sosnowski, M. R., Huang, R., Peduto, F., Loschiavo,
A., et al. (2011). Taxonomy and DNA phylogeny of diatrypaceae associated with
Vitis vinifera and other woody plants in Australia. Fungal Diversity 49, 203–223.
doi: 10.1007/s13225-011-0094-0
Trouillas, F. P., Urbez-Torres, J. R., and Gubler, W. D. (2010). Diversity of
diatrypaceous fungi associ ated with grapevine canker di seases in California.
Mycologia 102, 319–336. doi: 10.3852/08-185
U

rbez-Torres, J. R. (2011). The status of botryosphaeriaceae species infecting
grapevines. Phytopathol. Mediterr. 50, S5–S45. doi: 10.14601/Phytopathol_
Mediterr-9316
U

rbez-Torres,J.R.,Adams,P.,Kamas,J.,andGubler,W.D.(2009).
Identification, incidence, and pathogenicity of fungal species associated with
grapevine dieback in Texas. Am. J. Enol Viticult 60, 497–507.
U

rbez-Torres, J. R., Gispert, C., and Trouillas, F. P. (2020). Spore dispersal of
eutypella species under desert grape-growing conditions of southern California.
Phytopathol. Mediterr. 59, 485–501. doi: 10.14601/Phyto-11807
U

rbez-Torres, J. R., and Gubler, W. D. (2009). Pathogenicity of
botryosphaeriaceae species isolated from grapevine cankers in California. Plant
Dis. 93, 584–592. doi: 10.1094/PDIS-93-6-0584
U

rbez-Torres, J. R., Haag, P., Bowen, P., and O'gorman, D. (2014a). Grapevine
trunk diseases in British Columbia: incidence and characterization of the fungal
pathogens associated with esca and Petri diseases of grapevine. Plant Dis. 98, 469–
482. doi: 10.1094/PDIS-05-13-0523-RE
U

rbez-Torres,J.R.,Haag,P.,Bowen,P.,andO'gorman,D.T.(2014b).
Grapevine trunk diseases in British Columbia: incidence and characterization of
the fungal pathogens associated with black foot disease of grapevine. Plant Dis. 98,
456–468. doi: 10.1094/PDIS-05-13-0524-RE
U

rbez-Torres, J. R., Leavitt, G. M., Voegel, T. M., and Gubler, W. D. (2006).
Identification and distribution of botryosphaeria spp. associated with grapevine
cankers in California. Plant Dis. 90, 1490–1503. doi: 10.1094/PD-90-1490
U

rbez-Torres, J. R., Peduto, F., Striegler, R., Urrea-Romero, K., Rup e, J.,
Cartwright, R., et al. (2012). Characterization of fungal pathogens associated with
grapevine trunk diseases in Arkansas and Missouri. Fungal Diversity 52, 169–189.
doi: 10.1007/s13225-011-0110-4
Vanga, B. R., Panda, P., Shah, A. S., Thompson, S., Woolley, R. H., Ridgway, H.
J., et al. (2022). DNA Metabarcoding reveals high relative abundance of trunk
disease fungi in grapevines from Marlborough, new Zealand. BMC Microbiol. 22,
1–11. doi: 10.1186/s12866-022-02520-2
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org26

Walsh,E.,Duan,W.,Mehdi,M.,Naphri,K.,Khiste,S.,Scalera,A.,etal.(2018).Cadophora
meredithiae and C. interclivum, new species from roots of sedge and spruce in a western
Canada subalpine forest. Mycologia 110, 201–214. doi: 10.1080/00275514.2017.1406748
Zwickl, D. J. (2006). Genetic algorithm approaches for the phylogenetic analysis of
large biological sequence datasets under the maximum likelihood criterion (Austin,
Texas: The University of Texas at Austin).
Travadon et al. 10.3389/ffunb.2022.1018140
Frontiers in Fungal Biology frontiersin.org27