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Prunus trees in Germany—a hideout of unknown fungi?

July 2020
Mycological Progress 19(7):667-690

July 2020
19(7):667-690

DOI:10.1007/s11557-020-01586-4

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CC BY 4.0

Authors:

Nordwestdeutsche Forstliche Versuchsanstalt

Senckenberg Museum of Natural History Görlitz, Germany

Prunus belongs to the economically most important genera of fruit crops in Germany. Although wood pathogens possess the capability to damage the host substantially, the knowledge of the fungal pathogenic community and the mycobiome of Prunus wood in general is low. During a survey in important fruit production areas in Germany, branches with symptoms of fungal infection were sampled in Prunus avium, P. cerasus and P. domestica orchards, and 1018 fungal isolates were obtained primarily from the transition zone of symptomatic to non-symptomatic wood. By a combination of blastn searches and phylogenetic analyses based on ITS and LSU sequences with a strong focus on reliable reference data, a diversity of 172 fungal taxa belonging to Ascomycota, Basidiomycota and Mucoromycota were differentiated. The majority of the strains belonged to three classes of Ascomycota, namely Sordariomycetes, Leotiomycetes and Dothideomycetes. The dominant species were Aposphaeria corallinolutea (Dothideomycetes) and Pallidophorina paarla (Leotiomycetes) that were isolated more than a hundred times each, while all other taxa were isolated ≤ 30 times. Only part of them could be identified to species level. Because of the high plasticity of species boundaries, the identification certainty was divided into categories based on nucleotide differences to reference sequences. In total, 82 species were identified with high and 20 species with low (cf.) certainty. Moreover, about 70 species could not be assigned to a known species, which reveals Prunus wood to represent a habitat harbouring high numbers of potentially new species, even in a well-explored region like Germany.

Phylogeny of dataset 3 obtained by Bayesian inference analysis of the combined LSU and ITS sequence alignment of Leotiomycetes. Colletotrichum godetiae strain CBS 133.44 is used as outgroup. BI posterior probability support values above 0.9 (bold) and ML bootstrap

Phylogeny of dataset 3 obtained by Bayesian inference analysis of the combined LSU and ITS sequence alignment of Leotiomycetes. Colletotrichum godetiae strain CBS 133.44 is used as outgroup. BI posterior probability support values above 0.9 (bold) and ML bootstrap

Phylogeny of dataset 4 obtained by Bayesian inference analysis of the combined LSU and ITS sequence alignment of Eurotiomycetes. Diplodia intermedia strain CBS 124462 is used as outgroup. BI posterior probability support values above 0.9 (bold) and ML bootstrap

Phylogeny of dataset 4 obtained by Bayesian inference analysis of the combined LSU and ITS sequence alignment of Eurotiomycetes. Diplodia intermedia strain CBS 124462 is used as outgroup. BI posterior probability support values above 0.9 (bold) and ML bootstrap

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ORIGINAL ARTICLE

Prunus trees in Germany—a hideout of unknown fungi?

Steffen Bien

1

&Ulrike Damm

1

Received: 9 March 2020 /Revised: 23 April 2020 / Accepted: 27 April 2020

#The Author(s) 2020

Abstract

Prunus belongs to the economically most important genera of fruit crops in Germany. Although wood pathogens possess the

capability to damage the host substantially, the knowledge of the fungal pathogenic community and the mycobiome of Prunus

wood in general is low. During a survey in important fruit production areas in Germany, branches with symptoms of fungal

infection were sampled in Prunus avium,P. cerasus and P. domestica orchards, and 1018 fungal isolates were obtained primarily

from the transition zone of symptomatic to non-symptomatic wood. By a combination of blastn searches and phylogenetic

analyses based on ITS and LSU sequences with a strong focus on reliable reference data, a diversity of 172 fungal taxa belonging

to Ascomycota,Basidiomycota and Mucoromycota were differentiated. The majority of the strains belonged to three classes of

Ascomycota, namely Sordariomycetes,Leotiomycetes and Dothideomycetes. The dominant species were Aposphaeria

corallinolutea (Dothideomycetes)andPallidophorina paarla (Leotiomycetes) that were isolated more than a hundred times

each, while all other taxa were isolated ≤30 times. Only part of them could be identified to species level. Because of the high

plasticity of species boundaries, the identification certainty was divided into categories based on nucleotide differences to

reference sequences. In total, 82 species were identified with high and 20 species with low (cf.) certainty. Moreover, about 70

species could not be assigned to a known species, which reveals Prunus wood to represent a habitat harbouring high numbers of

potentially new species, even in a well-explored region like Germany.

Keywords Cultivation .Fungal community .Stone fruit trees .Systematics .Wood inhabitants

Introduction

Fungal pathogens inhabiting the woody plant body can plug

vessels and necrotise tissue, which causes wilting, inhibition

of blossoming and dieback of branches and whole trees. The

resulting decrease in fruit or timber yield can ruin the produc-

tivity of orchards, vineyards and forests and can even require

replanting. Additionally, some of the pathogens can reduce

the quality of fruits, which causes further yield losses.

Moreover, trees in forests and orchards are usually grown in

monocultures and are therefore especially threatened by fun-

gal plant pathogens, both due to the increasing global plant

trade (Roy et al. 2014, Ghelardini et al. 2017) and effects of

climate change (Anderson et al. 2004, Gange et al. 2011, Luck

et al. 2011,Fisheretal.2012, Altizer et al. 2013). An example

for the threat an exotic pathogen can pose to native trees is

Hymenoscyphus fraxineus, the causal agent of ash dieback

that moved from eastern Asia to Europe, encountering ash tree

species being more susceptible (McMullan et al. 2018). Due

to extreme conditions like drought, trees become also more

susceptible to fungi that are already living as endophytes in-

side their wood, so-called weak parasites. They include spe-

cies of Botryosphaeriales that have frequently been isolated

from Prunus trees in South Africa (Damm et al. 2007a,b). In

Germany, one of these species, Diplodia pinea, has been re-

ported to cause serious damage to pine trees that suffered from

drought stress and had been attacked by bark beetles

(Heydeck and Dahms 2012, Petercord 2017). Furthermore,

trees can become more susceptible to pathogens or encounter

new potential pathogens if they are planted outside their typ-

ical growing region, for example by the northward expansion

Section Editor: Marc Stadler

Electronic supplementary material The online version of this article

(https://doi.org/10.1007/s11557-020-01586-4) contains supplementary

material, which is available to authorized users.

*Steffen Bien

steffenbien@hotmail.com

1

Senckenberg Museum of Natural History Görlitz, PF 300 154,

02806 Görlitz, Germany

https://doi.org/10.1007/s11557-020-01586-4

(2020) 19:667–690

Mycological Progress

of European crop production due to global warming

(Maracchi et al. 2005,Santosetal.2017). In order to allow

an early detection and control of known and new threats for

the fruit industry, knowledge of the wood mycobiome of fruit

trees is crucial.

Fungal communities inside wood have frequently been

studied using culture-independent high-throughput sequenc-

ing (HTS) (e.g. Kubartová et al. 2012, Hoppe et al. 2016,

Purahong et al. 2018) and isolation techniques (e.g.

Kowalski 1983, Butin and Kowalski 1986, Lygis et al.

2005, Santamaría and Diez 2005, Simeto et al. 2005,Cloete

et al. 2011,Markakisetal.2017, Fischer et al. 2016).

However, many studies focused on endophytic fungi (e.g.

Barengo et al. 2000, Fröhlich et al. 2000, Gonthier et al.

2006) or were restricted to grapevine wood (e.g. Hofstetter

et al. 2012, Pancher et al. 2012, Bruez et al. 2014,2016).

Sweet cherry (Prunus avium), sour cherry (P. cerasus)and

plum (P. domestica) are the most important stone fruit crops

in German fruit industry (Garming et al. 2018). In 2018, more

than 350,000 t of sweet cherry, sour cherry and plum fruit

were produced on an area of around 12,000 ha (FAO 2020).

In spite of this economic importance, there are only a few

studies on the fungal diversity of aboveground woody parts

of Prunus trees (e.g. Bernadovičová and Ivanová 2011,

Haddadderafshi et al. 2011, Hortová and Novotný 2011,

Gramaje et al. 2012, Abdollahi Aghdam and Fotouhifar

2016,2017). Most of these studies are limited by a small

sample size, by a narrow sampling area or by relying solely

on morphological features for species identification.

The most extensive work so far has been conducted in a

survey on the fungal diversity of Prunus species in South

Africa (Damm et al. 2007a,b,2008a,b,c,2010,Moyoetal.

2018, Bien and Damm 2020). More than 40 taxa were report-

ed, predominantly within Botryosphaeriales (nine species)

and Phaeoacremonium (14 species). During this survey, 24

species of Botryosphaeria-ceae,Calosphaeriaceae,

Togniniaceae,Montagnulaceae,Coniochaetaceae,

Celotheliaceae,Tympanidaceae and Ploettnerulaceae were

recognised as new to science. However, these publications

aimed only on selected, very abundant or specifically interest-

ing taxa of wood-inhabiting fungi from Prunus wood in South

Africa; the complete diversity collected was not evaluated.

Moreover, no comprehensive study has been done on the

mycobiome of Prunus trees in Germany. In a study on several

tree species in the vicinity of a vineyard in Germany, only a

selection of eight fungal species (belonging to

Botryosphaeriaceae,Stereaceae,Tympanidaceae and

Valsaceae) isolated from wood of six Prunus species (includ-

ing P. cerasus and P. domestica) was reported (Gierl and

Fischer 2017).

With an extensive study such as the evaluation of a

mycobiome, time is the most limiting factor. For the selection

of an appropriate approach for identification, quantity and

quality have to be balanced against each other. Uncertainties

in identifications of fungi can arise due to deficiencies of both

morphological and molecular approaches. Morphological

identification of fungal cultures is hindered or impossible, if

strains do not develop identification-relevant features (fruiting

structures) or show phenotypic plasticity (Slepecky and

Starmer 2009), belong to a complex of cryptic species that

cannot be differentiated by morphological features (e.g.

Damm et al. 2012) or species had been described based on

one morph only, usually the sexual morph, that does not de-

velop in culture (e.g. Bien and Damm 2020). Even if morpho-

logical identification is possible, each genus requires a certain

amount of expertise (Hofstetter et al. 2019), as well as time to

obtain necessary literature and reference/type material. If

many taxa extending over the entire fungal kingdom need to

be identified in a reasonable time frame, an overall

morphology-based approach is not appropriate; identification

based on sequence data is the method of choice.

Fungal identification solely based on blastn searches

with ITS sequences is common practice (Hughes et al.

2009, Hofstetter et al. 2019); however, it has a lot of

shortcomings as well. Although the ITS region is consid-

ered as the universal barcode region for fungi and the

most commonly sequenced locus in mycology, it is not

suitable for species delimitation in each genus (Schoch

et al. 2012). Species identification in surveys using HTS

is even less certain, because the sequences generated are

very short, and the high number of sequences generated

puts even more time pressure on identification, allowing

only unquestioned/unvalued blastn searches. Moreover,

identification results cannot be verified by morphology

as no cultures are available. Therefore, species can often

only be identified up to genus level (LoBuglio and Pfister

2010, Johnston et al. 2014, Ekanayaka et al. 2017,Pärtel

et al. 2017, Purahong et al. 2018) or result in doubtful

identifications like those of Collophorina species that

are discussed in Bien et al. (2020).

The purpose of this study was to reveal the mycobiome

of Prunus trees in a temperate climate focusing on poten-

tial pathogens associated with wood necroses of P. avium,

P. cerasus and P. domestica in three important fruit pro-

duction areas in Germany. Some of the genera isolated

within this study, belonging to the Leotiomycetes and

Eurotiomycetes, have previously been analysed in depth

and several new taxa were revealed (Bien et al. 2020,

Bien and Damm 2020). The aim of this study was to give

an overview of the complete fungal diversity based on

LSU and ITS sequences, to highlight the possible depth

of identification based on these loci as part of a

mycobiome study and to detect potential new taxa. A

culture-dependent approach allowed verifying results by

morphology, if necessary, and facilitates further taxonom-

ic studies.

Mycol Progress (2020) 19:667–690

668

Materials and methods

Sampling and fungal isolation

Branches with wood symptoms (e.g. canker, necroses,

wood streaking, gummosis) were collected from Prunus

domestica (61 branches), P. cerasus (64) and P. avium

(43) orchards in Saxony; from P. domestica (30) and

P. avium (60) orchards in Lower Saxony; and from

P. domestica (38) and P. avium (48) orchards in Baden-

Württemberg, Germany, in 2015 and 2016. Additionally,

a symptomatic wood sample from a P. cerasus tree locat-

ed in a private garden in Bavaria was included. From each

of these 345 branches, ten wood pieces (5 × 5 × 5 mm)

from the transition zone of symptomatic to non-

symptomatic wood tissue as well as each three pieces of

the same size from non-symptomatic wood of the same

branch were surface sterilised 30 s in 70% ethanol, 1 min

in 3.5% NaOCl and 30 s in 70% ethanol and washed for

1 min in sterilised water. Five pieces from symptomatic

tissue were placed on synthetic nutrient-poor agar (SNA,

Nirenberg 1976) medium, and the remaining five pieces

from symptomatic tissue as well as the three pieces from

non-symptomatic tissue on oatmeal agar (OA; Crous et al.

2019) medium both supplemented with 100 mg/L penicil-

lin, 50 mg/L streptomycin sulphate and 1 mg/L chloram-

phenicol. After incubation for several days at 25 °C, hy-

phal tips of developing fungi were transferred to SNA

medium with a sterilised pine needle. Single-spore or

single-hyphae isolates were obtained from the fungi for

further study.

The resulting strains are preserved in cryotubes contain-

ing sterile distilled water with 10% glycerol at −80 °C and

in sterile distilled water at + 4 °C in the culture collection of

the Senckenberg Museum of Natural History Görlitz,

Germany (GLMC). Specimens (dried cultures) were depos-

ited in the fungarium of the Senckenberg Museum of

Natural History Görlitz (GLM).

Phylogenetic analysis

Genomic DNA of the isolates was extracted using the meth-

od of Damm et al. (2008b). A partial sequence of the 28S

nrDNA (LSU) and the 5.8S nuclear ribosomal gene with the

two flanking internal transcribed spacers ITS-1 and ITS-2

(ITS) were amplified and sequenced using the primer pairs

LROR (Rehner and Samuels 1994) + LR5 (Vilgalys and

Hester 1990) and ITS-1F (Gardes and Bruns 1993)+ITS-

4(Whiteetal.1990), respectively.

ThePCRmixturecontained1μL of 1:10 DNA template,

2.5 μL 10X buffer (Peqlab, Erlangen, Germany), 1 μLof

each primer (10 mM), 2.5 μLMgCl

2

(25 mM), 0.1 μLTaq

polymerase (0.5 U, Peqlab, Erlangen, Germany) and 2.5 μL

of 2 mM dNTPs. Each reaction was made up to a final

volume of 20 μL with sterile water. DNA amplifications

were carried out in a Mastercycler® pro S (Eppendorf,

Hamburg, Germany). Conditions for the amplification of

LSU and ITS were set according to Paulin and Harrington

(2000) and Bien et al. (2020), respectively. The PCR prod-

ucts were visualised on a 1% agarose gel and sequenced by

the Senckenberg Biodiversity and Climate Research Centre

(BiK-F) laboratory (Frankfurt, Germany). The forward and

reverse sequences were assembled by using BioEdit

Sequence Alignment Editor (v. 7.2.5; Hall 1999).

All strains were grouped based on comparison of their

ITS sequences. One strain of each group with an identical

ITS sequence was selected for blastn searches and phylo-

genetic analysis. For generic determination of the isolates

and selection of reference strains, blastn searches were per-

formed on the NCBI GenBank (www.ncbi.nlm.nih.gov)

and EPPO-Q-Bank (qbank.eppo.int) databases. For each

genus, sequences of strains identified to species level,

preferably of ex-type strains and strains of the type species,

with at least 97% identity were included as reference strains

in the phylogenetic analyses. If no type strains were avail-

able, strains with a CBS (culture collection of the

Westerdijk Fungal Biodiversity Institute, Utrecht, the

Netherlands) number were favoured. Strains without spe-

cies determination were only used, if blastn searches did not

result in any close match with a strain identified to species

level.

For the phylogenetic analyses, the sequences

downloaded were added to the sequences generated in this

study and those of the appropriate outgroup sequences in

five LSU-ITS datasets depending on phylum and class.

Four datasets were assembled for species of the

Ascomycota classes Sordario-mycetes,Dothideomycetes,

Leotiomycetes and Eurotiomycetes, respectively. A fifth

dataset encompasses species of the classes

Agaricomycetes,Tremellomycetes and

Cystobasidiomycetes (Basidiomycota); Lecanoromycetes,

Pezizomycetes and Saccharomycetes (Ascomycota); as well

as the subdivision Mucoromycotina of the Mucoromycota.

The datasets of each locus were aligned automatically using

MAFFT v. 7.308 (Katoh et al. 2002, Katoh and Standley

2013), manually adjusted where necessary and subsequent-

ly concatenated using Geneious v. 10.2.2 (Kearse et al.

2012).

The phylogenetic analyses were conducted using

Bayesian inference (BI) and maximum likelihood (ML) as

described in Bien et al. (2020). The DNA sequences gener-

ated in this study were deposited in GenBank (Table 1)and

the alignments in TreeBASE (treebase.org/treebase-web/

home.html; TB2:S25316). The complete list of strains

included in the phylogenetic analyses is provided in the

supplementary material table (suppl. material tab.).

Mycol Progress (2020) 19:667–690 669

Table 1 List of taxa isolated from Prunus wood in Germany with novelties and potential new reports, numbers of strains per wood tissue, host species

and sampling region, representative strains and GenBank numbers

Taxon Nov. Strains sy. n-

sy.

P.d. P.c. P.a. Sa LSa BW Ba Rep. strain GenBank no.

1

LSU ITS

Ascomycota

Dothideomycetes

Alternaria conjuncta G, a, c, d 3 3 1 1 1 1 2 GLMC 1338 MT156154 MT153704

Alternaria destruens G, P 24 24 8 7 9 13 7 4 GLMC 1234 MT156155 MT153705

Alternaria rosae G, P 1 1 1 1 GLMC 636 MT156156 MT153706

Angustimassarina cf. spp. 8 8 3 5 4 1 3 GLMC 891 MT156157 MT153707

Aposphaeria corallinolutea G, P 138 125 13 99 18 21 72 41 23 2 GLMC 1355 MT156159 MT153708

Aureobasidium pullulans d 15 15 11 2 2 10 1 4 GLMC 1460 MT156164 MT153709

Bipolaris cf. spp. 1 1 1 1 GLMC 248 MT156165 MT153710

Cladosporium cf. spp. 1 10 8 2 2 1 7 2 3 5 GLMC 1289 MT156192 MT153711

Cladosporium cf. spp. 2 2 1 1 2 2 GLMC 711 MT156193 MT153712

Coniothyrium ferrarisianum G, P 24 13 11 6 14 4 24 GLMC 380 MT156201 MT153713

Constantinomyces sp. 1 1 1 1 GLMC 1767 MT156202 MT153714

Devriesia pseudoamericana P 1 1 1 1 GLMC 819 MT156209 MT153715

Didymella macrostoma a, c, d 8 8 2 5 1 7 1 GLMC 1392 MT156215 MT153716

Diplodia mutila d 1 1 1 1 GLMC 1759 MT156216 MT153717

Diplodia seriata 1 1 1 1 GLMC 1527 MT156217 MT153718

Epicoccum cf. spp. 7 7 2 5 5 2 GLMC 369 MT156218 MT153719

Jeremyomyces cf. labinae 3 2 1 1 2 2 1 GLMC 327 MT156244 MT153720

Kalmusia cf. ebuli 4 4 3 1 4 GLMC 767 MT156245 MT153721

Kalmusia variispora G, P 4 4 4 1 3 GLMC 1347 MT156246 MT153722

Neocucurbitaria populi G, P 1 1 1 1 GLMC 348 MT156266 MT153723

Neoleptosphaeria rubefaciens G, P 1 1 1 1 GLMC 337 MT156269 MT153724

Nothophoma cf. quercina 18 17 1 7 11 11 1 6 GLMC 432 MT156271 MT153725

Paraphaeosphaeria neglecta P 2 2 1 1 GLMC 857 MT156275 MT153726

Parapyrenochaeta protearum G, P 2 1 1 1 1 1 1 GLMC 301 MT156276 MT153727

Phoma laundoniae G, a 1 1 1 1 GLMC 1459 MT156298 MT153728

Preussia persica G, P 2 2 2 1 1 GLMC 447 MT156301 MT153731

Preussia cf. spp. 1 1 1 1 GLMC 1754 MT156302 MT153732

Roussoella euonymi G, P 1 1 1 1 GLMC 1544 MT156304 MT153733

Lentitheciaceae sp. 1 1 1 1 GLMC 1563 MT156299 MT153729

Pleosporales sp. 1 1 1 1 GLMC 1316 MT156300 MT153730

Eurotiomycetes

Aspergillus chevalieri G, P 2 2 2 2 GLMC 899 MT156162 MT156109

Aspergillus glaucus G, d 1 1 1 1 GLMC 771 MT156163 MT156110

Capronia sp. 1 1 1 1 GLMC 1254 MT156189 MT156111

Exophiala sp. 3 1 2 3 3 GLMC 1670 MT156225 MT156112

Minutiella pruni-avium N 2 2 2 2 GLMC 1624 MN232925 MN232957

Minutiella sp. P 2 2 2 2 GLMC 1636 MN232927 MN232959

Penicillium angulare G, P 1 1 1 1 GLMC 1646 MT156277 MT156114

Penicillium brevicompactum G, P 6 6 6 3 3 GLMC 1661 MT156278 MT156115

Penicillium glabrum G, P 2 2 2 2 GLMC 1400 MT156279 MT156116

Penicillium cf. spp. 1 1 1 1 GLMC 1288 MT156280 MT156117

Rhinocladiella cf. quercus 3 3 2 1 2 1 GLMC 1752 MT156303 MT156118

Talaromyces sp. 2 2 2 2 GLMC 1678 MT156312 MT156119

Herpotrichiellaceae sp. 1 1 1 1 GLMC 914 MT156229 MT156113

Mycol Progress (2020) 19:667–690

670

Table 1 (continued)

Taxon Nov. Strains sy. n-

sy.

P.d. P.c. P.a. Sa LSa BW Ba Rep. strain GenBank no.

1

LSU ITS

Lecanoromycetes

Lecanoromycetes sp. 2 2 1 1 1 1 GLMC 1733 MT156247 MT156137

Leotiomycetes

Arboricolonus simplex N 1 1 1 1 GLMC 459 MN232924 MN232935

Botrytis cinerea 4 1 3 4 3 1 GLMC 635 MT156168 MT156090

Cadophora luteo-olivacea P 12 12 12 1 8 3 GLMC 1264 MT156172 MN232938

Cadophora novi-eboraci P 8 8 8 7 1 GLMC 1472 MT156181 MN232947

Cadophora prunicola N 8 8 5 3 5 3 GLMC 1633 MT156183 MN232955

Cadophora ramosa N 1 1 1 1 GLMC 377 MT156187 MN232956

Collophorina africana d 21 20 1 21 7 14 GLMC 1736 MK314581 MK314542

Collophorina badensis N 10 10 10 10 GLMC 1684 MK314594 MK314546

Collophorina germanica N 2 2 2 1 1 GLMC 1445 MK314595 MK314550

Collophorina neorubra N 7 7 7 3 2 2 GLMC 929 MK314604 MK314533

Dermea cerasi A G 4 4 4 4 GLMC 1760 MT156207 MT156093

Dermea cerasi B G 1 1 1 1 GLMC 862 MT156206 MT156092

Dermea sp. 2 2 2 2 GLMC 867 MT156208 MT156094

Monilinia laxa G 4 4 4 2 2 GLMC 1290 MT156255 MT156091

Neofabraea vagabunda G, P 1 1 1 1 GLMC 718 MT156268 MT156100

Neofabraea sp. 8 8 5 3 7 1 GLMC 1284 MT156267 MT156099

Oidiodendron cf. griseum 8 7 1 6 2 8 GLMC 602 MT156272 MT156101

Oidiodendron sp. 1 1 1 1 1 GLMC 469 –MT156102

Oidiodendron sp. 2 7 7 7 7 GLMC 485 MT156273 MT156103

Pallidophorina paarla a, c, d 112 110 2 17 12 83 33 64 15 GLMC 452 MK314608 MK314555

Pezicula cf. carpinea 4 4 3 1 4 GLMC 416 MT156283 MT156095

Pezicula eucrita P 2 2 2 2 GLMC 643 MT156284 MT156096

Pezicula sporulosa G, P 4 4 4 4 GLMC 1224 MT156286 MT156097

Pezicula sp. 14 14 14 5 9 GLMC 1726 MT156285 MT156098

Phialocephala piceae G, P 26 26 12 2 13 25 1 GLMC 331 MT156294 MT156105

Phialocephala sp. 1 1 1 1 1 GLMC 803 MT156295 MT156106

Phialocephala sp. 2 2 2 2 2 GLMC 385 MT156296 MT156107

Phialocephala sp. 3 6 6 5 1 6 GLMC 833 MT156297 MT156108

Proliferodiscus ingens N 1 1 1 1 GLMC 1751 MN232929 MN232961

Proliferodiscus sp. 7 7 6 1 3 3 1 GLMC 460 MN232930 MN232962

Leotiomycetes sp. 1 1 1 1 GLMC 792 MT156248 MT156104

Pezizomycetes

Trichophaeopsis bicuspis G, P 1 1 1 1 GLMC 1596 MT156319 MT156139

Saccharomycetes

Nakazawaea cf. holstii 1 1 1 1 GLMC 1309 MT156261 MT156138

Wickerhamomyces silvicola G, P 1 1 1 1 GLMC 1708 MT156324 MT156140

Sordariomycetes

Acremonium sp. 1 1 1 1 GLMC 1762 MT156152 MT153618

Akanthomyces muscarius G, P 5 5 5 5 GLMC 347 MT156153 MT153619

Anthostomella cf. pinea 2 1 1 1 1 2 GLMC 451 MT156158 MT153620

Arthrinium cf. arundinis 1 1 1 1 GLMC 230 MT156160 MT153621

Ascotricha chartarum G, P 1 1 1 GLMC 453 MT156161 MT153622

Biscogniauxia nummularia a, d 3 1 2 2 1 3 GLMC 829 MT156166 MT153623

Brunneomyces hominis G, P 1 1 1 1 GLMC 717 MT156169 MT153624

Mycol Progress (2020) 19:667–690 671

Table 1 (continued)

Taxon Nov. Strains sy. n-

sy.

P.d. P.c. P.a. Sa LSa BW Ba Rep. strain GenBank no.

1

LSU ITS

Calosphaeria pulchella G, d 30 30 3 27 29 1 GLMC 1629 MT156188 MT153625

Chaetomium sp. 2 1 1 2 1 1 GLMC 946 MT156190 MT153626

Chaetosphaeria cf. spp. 1 1 1 1 GLMC 641 MT156191 MT153627

Clypeosphaeria sp. 1 1 1 1 GLMC 463 MT156194 MT153628

Colletotrichum godetiae c 8 8 8 8 GLMC 224 MT156195 MT153629

Coniochaeta cf. cipronana 1 1 1 1 GLMC 1710 MT156196 MT153633

Coniochaeta sp. 1 1 1 1 1 GLMC 355 MT156197 MT153630

Coniochaeta sp. 2 1 1 1 1 GLMC 723 MT156198 MT153632

Coniochaeta sp. 3 3 3 3 3 GLMC 487 MT156199 MT153631

Cordyceps farinosa P 2 2 2 2 GLMC 886 MT156151 MT153634

Diaporthe cf. eres 6 6 6 6 GLMC 532 MT156210 MT153637

Diaporthe cf. mahothocarpus 3 3 3 3 GLMC 260 MT156211 MT153635

Diaporthe rudis P 7 7 4 3 3 4 GLMC 1427 MT156212 MT153638

Diaporthe sp. 16 16 13 3 11 3 2 GLMC 309 MT156213 MT153636

Dichotomopilus cf. spp. 4 4 4 4 GLMC 425 MT156214 MT153639

Eutypa lata c 13 13 6 7 11 2 GLMC 427 MT156219 MT153640

Eutypa petraki var. hederae 1 1 1 1 GLMC 631 MT156220 MT153641

Eutypa petraki var. petrakii 6 6 6 2 4 GLMC 1645 MT156221 MT153642

Eutypa sp. 2 2 2 2 GLMC 1758 MT156222 MT153643

Eutypella cf. spp. 1 1 1 1 GLMC 625 MT156223 MT153644

Fusarium culmorum c 2 2 2 2 GLMC 218 MT156226 MT153645

Fusarium cf. spp. 1 10 10 4 6 6 1 3 GLMC 1465 MT156227 MT153647

Fusarium cf. spp. 2 7 7 3 4 4 3 GLMC 1293 MT156228 MT153646

Hypoxylon cf. fragiforme 5 4 1 3 2 2 1 2 GLMC 1653 MT156234 MT153653

Hypoxylon fuscum d 1 1 1 1 GLMC 1823 MT156235 MT153656

Hypoxylon howeanum a, d 5 3 2 3 2 4 1 GLMC 394 MT156236 MT153651

Hypoxylon sp. 1 15 12 3 7 8 1 2 12 GLMC 1456 MT156237 MT153652

Hypoxylon sp. 2 2 2 2 2 GLMC 1657 MT156238 MT153654

Hypoxylon sp. 3 1 1 1 1 GLMC 1725 MT156239 MT153655

Jackrogersella cf. cohaerens 7 6 1 3 1 3 3 4 GLMC 652 MT156240 MT153657

Jackrogersella sp. 1 1 1 1 GLMC 1516 MT156241 MT153658

Jattaea sp. 1 1 1 1 1 GLMC 503 MT156242 MT153659

Jattaea sp. 2 4 4 4 3 1 GLMC 853 MT156243 MT153660

Lepteutypa sp. 1 18 18 18 12 6 GLMC 1319 MT156249 MT153661

Lepteutypa sp. 2 5 5 4 1 1 4 GLMC 1557 MT156250 MT153662

Leucostoma cf. spp. 28 25 3 17 2 9 7 21 GLMC 1521 MT156251 MT153663

Lopadostoma dryophilum G, P 9 9 3 5 1 7 2 GLMC 1682 MT156254 MT153665

Lopadostoma cf. turgidum A 4 4 3 1 4 GLMC 757 MT156252 MT153664

Lopadostoma cf. turgidum B 1 1 1 1 GLMC 1768 MT156253 MT153666

Monocillium cf. tenue 10 10 8 2 10 GLMC 563 MT156256 MT153667

Nemania sp. 1 4 4 2 1 1 4 GLMC 413 MT156262 MT153668

Nemania sp. 2 1 1 1 1 GLMC 1515 MT156263 MT153669

Nemania sp. 3 4 3 1 3 1 4 GLMC 1799 MT156264 MT153670

Neocosmospora cf. perseae 1 1 1 1 GLMC 300 MT156265 MT153671

Neurospora sp. 6 5 1 1 4 1 5 1 GLMC 658 MT156270 MT153672

Ophiostoma sp. 4 4 3 1 4 GLMC 619 MT156274 MT153673

Phaeoacremonium hungaricum G, P 3 3 1 2 1 2 GLMC 1236 MT156288 MT153677

Mycol Progress (2020) 19:667–690

672

Table 1 (continued)

Taxon Nov. Strains sy. n-

sy.

P.d. P.c. P.a. Sa LSa BW Ba Rep. strain GenBank no.

1

LSU ITS

Phaeoacremonium iranianum G, d 1 1 1 1 GLMC 490 MT156289 MT153674

Phaeoacremonium scolyti G 1 1 1 1 GLMC 570 MT156290 MT153676

Phaeoacremonium cf. viticola 9 9 2 7 9 GLMC 498 MT156287 MT153675

Phialemonium sp. 2 2 2 2 GLMC 576 MT156293 MT153678

Seimatosporium sp. 10 10 7 3 1 7 2 GLMC 1722 MT156305 MT153679

Simplicillium aogashimaense G, P 4 4 4 4 GLMC 349 MT156306 MT153681

Simplicillium minatense G, P 1 1 1 1 GLMC 520 MT156307 MT153680

Sporothrix variecibatus G, P 2 2 2 2 GLMC 353 MT156310 MT153683

Tolypocladium sp. 3 3 3 3 GLMC 1695 MT156313 MT153684

Trichoderma citrinoviride c 1 1 1 1 GLMC 235 MT156317 MT153685

Trichoderma cf. simmonisii 2 2 2 2 GLMC 350 MT156316 MT153686

Trichoderma cf. spp. 1 1 1 1 GLMC 512 MT156318 MT153687

Truncatella angustata P 3 3 1 2 2 1 GLMC 253 MT156320 MT153688

Xylaria longipes P 5 2 3 5 5 GLMC 1499 MT156328 MT153690

Valsaceae sp. 17 15 2 15 1 1 15 2 GLMC 412 MT156323 MT153689

Xylariaceae sp. 1 1 1 1 1 GLMC 1660 MT156325 MT153693

Xylariaceae sp. 2 1 1 1 1 GLMC 1594 MT156326 MT153692

Xylariaceae sp. 3 2 1 1 1 1 2 GLMC 848 MT156327 MT153691

Hypocreales sp. 1 2 1 1 2 2 GLMC 550 MT156231 MT153648

Hypocreales sp. 2 3 3 3 3 GLMC 686 MT156232 MT153650

Hypocreales sp. 3 2 1 1 2 2 GLMC 556 MT156233 MT153649

Sordariales sp. 1 1 1 1 GLMC 1232 MT156309 MT153682

Basidiomycota

Agaricomycetes

Bjerkandera cf. adusta 5 5 3 1 1 3 2 GLMC 431 MT156167 MT156120

Coniophora puteana G, d 1 1 1 1 GLMC 420 MT156200 MT156121

Coprinellus cf. spp. 1 1 1 1 GLMC 737 MT156203 MT156122

Coriolopsis gallica G, d 1 1 1 1 GLMC 1308 MT156204 MT156123

Exidia glandulosa c 1 1 1 1 GLMC 374 MT156224 MT156125

Heterobasidion annosum d 1 1 1 1 GLMC 1320 MT156230 MT156126

Mycoacia fuscoatra G, P 1 1 1 1 GLMC 1268 MT156260 MT156127

Peniophora cinerea a, c, d 13 13 1 11 1 12 1 GLMC 947 MT156281 MT156128

Peniophora quercina 2 2 2 2 GLMC 1640 MT156282 MT156129

Phellinus tuberculosus A d 4 4 4 3 1 GLMC 396 MT156291 MT156130

Phellinus tuberculosus B a, d 3 3 2 1 3 GLMC 1755 MT156292 MT156131

Sistotrema sp. 2 2 2 2 GLMC 1593 MT156308 MT156132

Stereum cf. spp. 3 3 3 3 GLMC 475 MT156311 MT156133

Trametes hirsuta 1 1 1 1 GLMC 467 MT156314 MT156134

Trametes versicolor 2 2 1 1 1 1 GLMC 1717 MT156315 MT156135

Cystobasidiomycetes

Cystobasidium pinicola G, P 3 3 2 1 3 GLMC 1603 MT156205 MT156124

Tremellomycetes

Udeniomyces sp. 1 1 1 1 GLMC 1365 MT156321 MT156136

Mucoromycota

Mucor circinelloides a 1 1 1 1 GLMC 1405 MT156257 MT156141

Mucor hiemalis P 1 1 1 1 GLMC 1395 MT156258 MT156142

Mucor sp. 1 1 1 1 GLMC 656 MT156259 MT156143

Mycol Progress (2020) 19:667–690 673

Identification

The strains were identified to species, genus or higher level,

depending on the affinity to the available reference sequences.

These identifications were assigned to a level of identification

certainty based on an evaluation of the respective clades in the

phylogenetic trees and nucleotide differences in the respective

ITS alignments. A species was assigned to “identified with high

certainty”, if the strain showed ≤4 nucleotide differences in the

ITS sequence to a named reference sequence. Letters at the

species name indicate a sequence variation within strains that

were identified as the same species. A low certainty was indi-

cated with “cf.”, if the ITS sequence of a strain differed in 5–10

nucleotides from the closest named reference sequence. The

strain was assigned to a genus, but not to a species, if the ITS

sequence differed in > 10 nucleotides from the closest named

reference sequence or matched with more than one named ref-

erence sequence and marked with “sp.”or “cf. spp.”, respective-

ly. If the strain belonged to a clade, for which no named refer-

ence sequence was available or with reference sequences be-

longing to more than one genus, the name of family, order or

class was applied. Identifications of part of the taxa to genus

level were verified based on microscopic examination of mor-

phological features formed on the used standard media.

Results

In total, 1018 fungal strains were isolated from Prunus wood,

which belonged to 172 species. The numbers of species isolated

per host species were as follows: 113 species from Prunus

domestica,70fromP. avium and 61 from P. cerasus

Table 1 (continued)

Taxon Nov. Strains sy. n-

sy.

P.d. P.c. P.a. Sa LSa BW Ba Rep. strain GenBank no.

1

LSU ITS

Umbelopsis isabellina G, P 5 3 2 5 5 GLMC 521 MT156322 MT156144

# branches sampled 129 64 151 168 90 86

Nov., novelties and potential first reports during this survey; sy., from symptomatic wood tissue; n-sy., from non-symptomatic wood tissue; P.d., from

Prunus domestica;P.c., from P. cerasus;P.a., from P. avium;Sa, from Saxony; LSa, from Lower Saxony; BW, from Baden-Württemberg; Ba, from

Bavaria; N, newly described in Bien et al. 2020 or in Bien and Damm 2020;G,P,a,c,d, potential first report from Germany, Prunus,P. avium,

P. cerasus or P. domestica,respectively;rep. strain, representative strain for the taxon

1

LSU, 28S nrDNA; ITS, internal transcribed spacers and intervening 5.8S nrDNA

Saxony

(n=168)

Baden-

Württemberg

(n=86)

Lower

Saxony

(n=90)

73

13

22

34

14

610

Prunus

avium

(n=151)

66

Prunus

domestica

(n=129)

31 20

17

16

14

8

Prunus

cerasus

(n=64)

ab

Fig. 1 Number of species isolated from Prunus wood in Germany aper host species and bper sampling region. n, number of sampled branches

Mycol Progress (2020) 19:667–690

674

(Fig. 1a). While 66, 31 and 20 species, respectively, were ex-

clusively isolated from one of these hosts, 17 species occurred in

all of them. Regarding the main sampling regions, 122, 75 and

43 species were isolated from Prunus wood collected in Saxony,

Baden-Württemberg and Lower Saxony, respectively. While

73, 34 and 10 species, respectively, were exclusively isolated

from wood collected from one of these regions, 13 species oc-

curred in all of them (Fig. 1b). Five species were isolated from

all three Prunus species and in all collection areas, namely

Alternaria destruens,Aposphaeria corallinolutea,

Aureobasidium pullulans,Pallidophorina paarla and

Cladosporium cf. spp. 1 (Table 1). Aposphaeria corallinolutea

and Pa. were isolated 138 and 112 times, respectively, all other

taxa ≤30 times. Most of the taxa with 15–30 strains were iso-

latedfromatleasttwohostspeciesandinatleasttwocollection

regions, except for Collophorina africana and Lepteutypa sp. 1

that were collected only from P. domestica,andConiothyrium

ferrarisianum that was collected only from Saxony.

The majority of the species (166 species) was isolated from

the transition zone between symptomatic and non-symptomatic

tissue, 138 species exclusively from this tissue, while 34 spe-

cies were isolated from asymptomatic tissue, six species (each

one isolate) exclusively from asymptomatic tissue.

Of the 172 species, 152 species belonged to the

Ascomycota (965 strains), 16 to the Basidiomycota (45 strains)

and four to the Mucoromycota (eight strains). Within the

Ascomycota, 75 species belonged to the Sordariomycetes

(356 strains), 30 to the Leotiomycetes (290 strains), 30 to the

Dothideomycetes (287 strains) and 13 to the Eurotiomycetes

(27 strains), representing 43.6%, 17.4%, 17.4% and 7.6%,

respectively, of the total diversity and 35%, 28.5%, 28.2%

and 2.7%, respectively, of the abundance of the complete

mycobiome of Prunus wood isolated in this study

(Fig. 2a, b). The sequences of the four most abundant classes

of Ascomycota were analysed in separate alignments, while

the remaining classes of the Ascomycota were analysed to-

gether with Basidiomycota and Mucoromycota.

Phylogenetic analyses

The combined sequence dataset 1 of the Sordariomycetes

consisted of 246 strains including the reference strains and

the outgroup Cadophora luteo-olivacea strain CBS 141.41

(Leotiomycetes) and comprised 1884 characters (gene bound-

aries: LSU: 1–902, ITS: 903–1884, including gaps). The final

ML optimisation likelihood of ML analysis was lnL = -

39,461.875859. In total, 356 isolates from Prunus wood

belonged to 75 taxa (Fig. 3). Thirty-one species (136 isolates)

were placed in the order Xylariales, of which 15 taxa were

determined to species, 13 to genus and three to family level.

Six species (77 isolates) were placed in the Diaporthales;the

generic determination of one of them was unclear. Three taxa

(35 isolates) were placed in the Calosphaeriales, four (14

isolates) in the genus Phaeoacremonium,Togniniales, and

two (six isolates) in the Ophiostomatales. Seventeen species

(57 isolates) were placed in the order Hypocreales; the generic

placement of three of them was unclear. Two taxa (nine iso-

lates) were placed in the order Glomerellales and determined

to species level. Four species (13 isolates) were placed in the

order Sordariales, one of which not determined to genus level.

One isolate was placed in the genus Chaetosphaeria,

Chaetosphaeriales. Four species (six isolates) were placed in

the genus Coniochaeta,Coniochaetales; one of them was

identified to species level. One species (two isolates) was

placed in a clade formed by strains of Phialemonium sp., sister

to the single-strain clade of the ex-type strain of Ph.

dimorphosporum (incertae sedis). With 30 strains,

BM

A

a

A

B

M

BM

A

b

Sordariomycetes

Leotiomycetes

Dothideomycetes

Eurotiomycetes

Saccharomycetes

Lecanoromycetes

Pezizomycetes

Agaricomycetes

Tremellomycetes

Cystobasidiomycete

s

Mucoromycota

Fig. 2 Percentage aof taxa per class and phylum and bof strains per class and phylum isolated from Prunus wood in Germany. A, Ascomycota;B,

Basidiomycota;M,Mucoromycota

Mycol Progress (2020) 19:667–690 675

Calosphaeria pulchella (Calosphaeriales) was the most fre-

quently isolated species in the Sordariomycetes.

The combined sequence dataset 2 of the Dothideomycetes

consisted of 113 strains including the outgroup Penicillium

resticulosum strain CBS 609.94 (Eurotiomycetes) and com-

prised 1585 characters (gene boundaries: LSU: 1–870, ITS:

871–1585, including gaps). The final ML optimisation likeli-

hood of ML analysis was lnL =- 17,660.376634. In total, 287

isolates belonged to 30 taxa (Fig. 4). Twenty-three taxa (258

isolates) were placed in the Pleosporales, of which 17 were

determined to species, four to genus and each one to family

and order level. Four taxa (14 isolates) were placed in the

Capnodiales and determined to species or genus level. One

taxon (15 isolates) of the Dothideales was identified as

Aureobasidium pullulans. Each one isolate was identified as

Diplodia mutila and D. seriata (Botryosphaeriales). With 138

strains, Aposphaeria corallinolutea (Pleosporales) was the

most frequently isolated species in the Dothideomycetes.

The combined sequence dataset 3 of the Leotiomycetes

consisted of 84 strains including the outgroup Colletotrichum

godetiae strain CBS 133.44 (Sordariomycetes)andcomprised

1557 characters (gene boundaries: LSU: 1–912, ITS: 913–

1557, including gaps). The final ML optimisation likelihood of

ML analysis was lnL = - 11,950.782384. In total, 290 isolates

belonged to 30 taxa (Fig. 5). Twenty-four taxa (137 isolates)

were placed in the Helotiales, of which 15 were determined to

species and nine to genus level. Five taxa (152 isolates) were

placed in Phacidiales and determined to species level. One strain

remained in an uncertain taxonomic position on order level. With

112 strains, Pallidophorina paarla (Phacidiales)wasthemost

frequently isolated species in the Leotiomycetes.

The combined sequence dataset 4 of the Eurotiomycetes

consisted of 38 strains including the outgroup Diplodia

intermedia strain CBS 124462 (Dothideomycetes)andcom-

prised 1573 characters (gene boundaries: LSU: 1–908, ITS:

909–1573, including gaps). The final ML optimisation likeli-

hood of ML analysis was lnL =- 9837.561743. In total, 27 iso-

lates belonged to 13 taxa (Fig. 6). Seven taxa (15 isolates) were

placed within Eurotiales, of which five were determined to spe-

cies and two to genus level. Four taxa (eight isolates) were

placed in Chaetothyriales; one was determined to species, two

to genus and one to family level. Two taxa (four isolates) were

placed in Phaeomoniellales, of which one was determined to

species and one to genus level. All species of the Eurotiomycetes

were isolated with low frequencies (≤6strains).

The combined sequence dataset 5 of the remaining classes

of the Ascomycota,aswellasallBasidiomycota and

Mucoromycota consisted of 105 strains including the

outgroup Entomophthora sphaerosperma strain CBS 530.75

(Entomophthoromycotina,Zoopagomycota) and comprised

2058 characters (gene boundaries: LSU: 1–1115, ITS:

1116–2058, including gaps). The final ML optimisation like-

lihood of ML analysis was lnL =- 33,834.310764. Within the

16 taxa (45 strains) of Basidiomycota, 14 taxa (41 strains)

belonged to the in Agaricomycetes, of which 11 taxa were

identified to species and three to genus level (Fig. 7). One

isolate of the Tremellomycetes and one taxon (three isolates)

of the Cystobasidiomycetes were determined to genus and

species level, respectively. With 13 strains, Peniophora

cinerea was the most frequently isolated species in the

Basidiomycota. Species of the other phyla were isolated with

low frequencies (< 10 strains). One strain of the class

Pezizomycetes (Ascomycota) was determined as

Trichophaeopsis bicuspis. One taxon of the

Lecanoromycetes (two strains) could not be further deter-

mined. Two strains of the Saccharomycetes were determined

as Nakazawaea cf. holstii and Wickerhamomyces silvicola,

respectively. Within the 8 strains of Mucoromycota,five

strains were identified as Umbelopsis isabellina, two strains

as Mucor circinelloides and M. hiemalis, respectively, while

one further Mucor strain could not be assigned to a species.

Identification certainty

In total, 102 taxa were assigned to a particular species with

high (82 taxa) or low (20 taxa) certainty. A further 57 species

were determined to genus level. Thirteen species could not be

assigned to any genus and were identified to family (six),

order (five) or class (two), level, respectively. Almost all of

the 70 taxa that were not identified to species level belonged to

the Ascomycota, with the largest number of taxa belonging to

the Sordariomycetes (39), followed by Leotiomycetes (ten),

Dothideomycetes (nine) and Eurotiomycetes (five) (Fig. 8).

Only few undetermined species belonged to Basidiomycota,

Mucoromycota and to the remaining classes of Ascomycota.

Discussion

Fungal diversity of necrotic Prunus wood in Germany

In total, 172 fungal species were detected in the wood samples

of Prunus trees studied. The diversity detected in this study far

exceeds the number of taxa usually reported from isolation

studies of woody plants. In many cases, not more than 30 taxa

were reported (e.g. Barengo et al. 2000,Gonthieretal.2006,

Hortová and Novotný 2011,Markakisetal.2017). Only in

few studies up to or more than a hundred taxa were isolated



Fig. 3 Phylogeny of dataset 1 obtained by Bayesian inference analysis of

the combined LSU and ITS sequence alignment of Sordariomycetes.

Cadophora luteo-olivacea strain CBS 141.41 is used as outgroup. BI

posterior probability support values above 0.9 (bold) and ML bootstrap

support values above 70% are shown at the nodes. The strains isolated in

this study are emphasised in bold. Numbers in parentheses indicate the

number of isolated strains per taxon. Branches that are crossed by diag-

onal lines are shortened by 50%. T, ex-type strain; #, type species

Mycol Progress (2020) 19:667–690

676

Xylariales

Diaporthales

Calosphaeriales

Xylariaceae sp. 1 GLMC 1660 (1)

Rosellinia sp. SO1_T24 L1A

Rosellinia sp. SO1_T34_L4A

Fungal sp. V-I7

Xylariaceae sp. 2 GLMC 1594 (1)

Fungal sp. V-E9

Rosellinia aquila MUCL 51703 #

Rosellinia australiensis CBS 142160 T

Rosellinia mearnsii MFLU 16-1382 T

Nemania diffusa GZ AT-F006

Nemania sp. 3 GLMC 1799 (4)

Nemania diffusa FR AT-113

Nemania serpens BHI-F650a #

Nemania sp. 1 GLMC 413 (4)

Nemania serpens CBS 679.86 #

Nemania sp. 2 GLMC 1515 (1)

Xylaria longipes GLMC 1499 (5)

Xylaria longipes CBS 347.37

Xylaria hypoxylon CBS 120.16 #

Xylaria hypoxylon CBS 126417 #

Anthostomella proteae CBS 110127 T

Clypeosphaeria sp. GLMC 463 (1)

Clypeosphaeria mamillana CBS 140735 T #

Sordariomycetes sp. 11262

Sordariomycetes sp. 11280

Xylariaceae sp. 3 GLMC 848 (2)

Anthostomella pinea CBS 128205 T

Anthostomella cf. pinea GLMC 451 (2)

Barrmaelia moravica CBS 142769 T

Barrmaelia rhamnicola CBS 142772 T #

Entosordaria perfidiosa CBS 142773 T #

Entosordaria quercina CBS 142774 T

Biscogniauxia marginata MFLUCC 12.0740

Biscogniauxia repanda ATCC 62606

Biscogniauxia nummularia GLMC 829 (3)

Biscogniauxia nummularia MUCL 51395 T #

Jackrogersella cf. cohaerens GLMC 652 (7)

Jackrogersella cohaerens CBS 119126

Jackrogersella minutella CBS 336.70

Jackrogersella multiformis CBS 119016 T #

Jackrogersella sp. GLMC 1516 (1)

Annulohypoxylon truncatum CBS 140778 T #

Hypoxylon fuscum CBS 113049 T

Hypoxylon fuscum GLMC 1823 (1)

Hypoxylon rubiginosum MUCL 52887 T

Pyrenomyxa morganii CBS 118186 T

Hypoxylon sp. 1 GLMC 1456 (15)

Hypoxylon perforatum CBS 115281

Hypoxylon sp. 2 GLMC 1657 (2)

Hypoxylon sp. 3 GLMC 1725 (1)

Hypoxylon fragiforme MUCL 51264 T #

Hypoxylon cf. fragiforme GLMC 1653 (5)

Hypoxylon howeanum MUCL 47599

Hypoxylon howeanum GLMC 394 (5)

Seimatosporium cornii MFLUCC 14-0467 T

Seimatosporium sp. GLMC 1722 (10)

Seimatosporium physocarpi CBS 139968 T

Seimatosporium pistaciae CBS 138865 T

Seimatosporium rosae CBS 139823 T

Truncatella angustata CBS 144025 T #

Truncatella angustata GLMC 253 (3)

Truncatella hartigii CBS 118148

Lepteutypa sp. 1 GLMC 1319 (18)

Lepteutypa sp. 2 GLMC 1557 (5)

Lepteutypa uniseptata HKUCC 6349

Lepteutypa fuckelii CBS 140409 T

Lepteutypa sambuci CBS 131707 T

Arthrinium arundinis CBS 114316

Arthrinium arundinis CBS 124788

Arthrinium cf. arundinis GLMC 230 (1)

Arthrinium malaysianum CBS 102053 T

Arthrinium thailandicum MFLUCC 15-0202 T

Eutypa lata CBS 208.87 T #

Eutypa lata GLMC 427 (13)

Eutypa petrakii var. hederae CBS 285.87 T

Eutypa petrakii var. hederae GLMC 631 (1)

Eutypa sp. GLMC 1758 (2)

Eutypa petrakii var. petrakii CBS 244.87

Eutypa petrakii var. petrakii GLMC 1645 (6)

Xylaria cubensis CBS 116.85

Eutypella virescens CBS 205.36 T

Eutypella cf. spp. GLMC 625 (1)

Eutypa tetragona CBS 284.87

Lopadostoma cf. turgidum A GLMC 757 (4)

Lopadostoma cf. turgidum B GLMC 1768 (1)

Lopadostoma turgidum CBS 133207 T #

Lopadostoma dryophilum GLMC 1682 (9)

Lopadostoma dryophilum CBS 133213 T

Lopadostoma lechatii CBS 133694 T

Ascotricha chartarum GLMC 453 (1)

Ascotricha chartarum CBS 104.25 #

Ascotricha lusitanica CBS 462.70 T

Ascotricha pusilla CBS 132.60

Diaporthe cotoneastri CBS 439.82 T

Diaporthe eres CBS 138594 T #

Diaporthe cf. eres GLMC 532 (6)

Diaporthe celeris CBS 143349 T

Diaporthe sp. GLMC 309 (16)

Diaporthe celastrina CBS 139.27

Diaporthe rudis CBS 113201 T

Diaporthe rudis GLMC 1427 (7)

Diaporthe mahothocarpus CGMCC 3.15181 T

Diaporthe cf. mahothocarpus GLMC 260 (3)

Leucostoma persoonii CBS 129.22

Leucostoma persoonii CBS 260.34

Leucostoma cf. spp. GLMC 1521 (28)

Leucostoma cinctum CBS 254.34

Valseutypella multicollis CBS 105.89 T

Valsaceae sp. GLMC 412 (17)

Valsa sordida CBS 197.50

Valsa ambiens CBS 423.52 #

Jattaea sp. 1 GLMC 503 (1)

Jattaea taediosa MR 3669 T

Jattaea discreta CBS 127681 T

Jattaea sp. 2 GLMC 853 (4)

Jattaea algeriensis STE-U 6201 #

Calosphaeria pulchella CBS 115999

Calosphaeria pulchella GLMC 1629 (30)

Calosphaeria africana CBS 120870 T

1/100

1/100

1/99

0.99/–

1/84

1/100

1/92

1/100

0.99/71

1/96

1/100

1/74

1/100

1/100

–/75

1/95

1/100

1/100

0.99/78

1/100

1/100

1/92

1/100

1/100

1/100

1/100

1/100

1/100

0.99/87

1/100

1/100

1/100

1/100

1/100

0.99/86

1/100

1/100

0.91/–

1/97

1/95

0.95/81

1/100

0.98/100

1/100

1/100

1/100

1/91

1/97

0.99/72

1/75

0.97/94

1/100

1/100

1/97

1/86

1/97

1/91

1/100

1/96

1/100

1/100

0.94/–

1/74

0.98/98 1/100

1/100

1/100

1/100

1/100

1/100

1/90

0.99/84

1/99

1/99

1/100 1/100

1/100

1/100

1/100

1/99

0.95/95

1/100

1/100 1/100

1/100

0.98/–

1/100

1/100

Mycol Progress (2020) 19:667–690 677

Ophiostomatales

Togniniales

Glomerellales

Hypocreales

incertae sedis

Trichoderma citrinoviride CBS 258.85 T

Phaeoacremonium cf. viticola GLMC 498 (9)

Phaeoacremonium viticola CBS 101738 T

Phaeoacremonium roseum DAOM 242365 T

Phaeoacremonium hungaricum CBS 123036 T

Phaeoacremonium hungaricum GLMC 1236 (3)

Phaeoacremonium iranianum CBS 101357 T

Phaeoacremonium iranianum GLMC 490 (1)

Phaeoacremonium angustius CBS 249.95 T

Phaeoacremonium scolyti GLMC 570 (1)

Phaeoacremonium scolyti CBS 113597 T

Phaeoacremonium parasiticum CBS 127262 #

Ophiostoma tasmaniense CBS 127212 T

Ophiostoma tsotsi CBS 122287 T

Ophiostoma sp. GLMC 619 (4)

Ophiostoma denticiliatum CBS 124498 T

Ophiostoma piliferum CBS 129.32 #

Ophiostoma piliferum CBS 138.33 #

Sporothrix variecibatus GLMC 353 (2)

Sporothrix variecibatus CMW23051 T

Sporothrix rossii CBS 116.78 T

Sporothrix schenckii CBS 359.36 T #

Cordyceps farinosa CBS 156.65

Cordyceps farinosa CBS 264.35

Cordyceps farinosa GLMC 886 (2)

Cordyceps militaris CBS 128.25 #

Cordyceps tenuipes CBS 226.60

Akanthomyces muscarius CBS 143.62 T

Akanthomyces muscarius GLMC 347 (5)

Akanthomyces attenuatus CBS 402.78

Akanthomyces aculeatus TS772 #

Simplicillium minatense GLMC 520 (1)

Simplicillium minatense JCM 18176 T

Simplicillium lanosoniveum CBS 123.42 T #

Simplicillium aogashimaense JCM 18167 T

Simplicillium aogashimaense GLMC 349 (4)

Hypocreales sp. 1 GLMC 550 (2)

Sarocladium implicatum MUT ITA 2365

Hypocreales sp. 2 GLMC 686 (3)

Hypocreales sp. ICMP 16980

Annulohypoxylon multiforme BHI-F464a

Hypocreales sp. 3 GLMC 556 (2)

Tolypocladium inflatum CBS 824.70 T #

Tolypocladium ovalisporum CBS 700.92 T

Tolypocladium sp. GLMC 1695 (3)

Tolypocladium album CBS 830.73

Tolypocladium album CBS 869.73 T

Fusarium reticulatum var. reticulatum CBS 190.31

Fusarium cf. spp. 1 GLMC 1465 (10)

Fusarium avenaceum CBS 128538

Fusarium sinensis CBS 122710 T

Fusarium cf. spp. 2 GLMC 1293 (7)

Fusarium culmorum CBS 128537

Fusarium culmorum GLMC 218 (2)

Neocosmospora perseae CBS 144142 T

Neocosmospora cf. perseae GLMC 300 (1)

Neocosmospora pseudensiformis CBS 125729 T

Trichoderma reesei CBS 383.78 T

Trichoderma citrinoviride GLMC 235 (1)

Trichoderma simmonsii CBS 130431 T

Trichoderma cf. simmonsii GLMC 350 (2)

Trichoderma neotropicale CBS 130633 T

Trichoderma atroviride CBS 142.95 T

Trichoderma scalesiae CBS 120069 T

Trichoderma cf. spp. GLMC 512 (1)

Trichoderma viride CBS 127113 #

Sarocladium oryzae CBS 399.73 #

Sarocladium zeae CBS 801.69 T

Sarocladium kiliense CBS 122.29 T

Sarocladium bacillisporum CBS 425.67 T

Sarocladium implicatum CBS 397.70A

Acremonium alternatum CBS 407.66 T #

Acremonium sp. GLMC 1762 (1)

Acremonium sclerotigenum CBS 124.42 T

Acremonium sordidulum CBS 385.73 T

Monocillium cf. tenue GLMC 563 (10)

Monocillium tenue CBS 432.66 T

Monocillium indicum CBS 313.61 T #

Brunneomyces hominis GLMC 717 (1)

Brunneomyces hominis CBS 139053 T

Brunneomyces europaeus CBS 652.96 T

Brunneomyces brunnescens CBS 559.73 T #

Colletotrichum godetiae GLMC 224 (8)

Colletotrichum godetiae CBS 133.44 T

Colletotrichum johnstonii CBS 128532 T

Colletotrichum pyricola CBS 128531 T

Chaetomium angustispirale CBS 137.58 T

Chaetomium coarctatum CBS 162.62 T

Chaetomium sp. GLMC 946 (2)

Chaetomium globosum CBS 160.62 T #

Dichotomopilus cf. spp. GLMC 425 (4)

Dichotomopilus funicola CBS 159.52 T

Dichotomopilus pseudofunicola CBS 142033 T

Dichotomopilus indicus CGMCC 3.14184 T #

Neurospora crassa CBS 367.70

Neurospora sitophila CBS 112.19 #

Neurospora sp. GLMC 658 (6)

Neurospora tetraspora CBS 178.33 T

Zopfiella tabulata CBS 230.78 #

Cercophora sp. CIM1_17

Sordariales sp. GLMC 1232 (1)

Uncultured Ascomycota dfmo0690_036

Zopfiella tardifaciens CBS 670.82 T

Clypeosphaeria perfidiosa CBS 407.68

Chaetosphaeria myriocarpa CBS 878.70

Chaetosphaeria pygmaea MR 1365

Chaetosphaeria cf. spp. GLMC 641 (1)

Chaetosphaeria myriocarpa CBS 141.53

Chaetosphaeria innumera MR 1175 #

Coniochaeta lignicola CBS 267.33 T

Coniochaeta luteoviridis CBS 206.38 T

Coniochaeta sp. 3 GLMC 487 (3)

Coniochaeta ligniaria CBS 424.65 #

Coniochaeta sp. 1 GLMC 355 (1)

Coniochaeta prunicola CBS 120875 T

Coniochaeta sp. 2 GLMC 723 (1)

Coniochaeta cipronana CBS 144016 T

Coniochaeta cf. cipronana GLMC 1710 (1)

Coniochaeta navarrae CBS 141016 T

Phialemonium atrogriseum CBS 604.67 T

Phialemonium obovatum CBS 279.76 T #

Phialemonium inflatum CBS 263.58

Phialemonium sp. GLMC 576 (2)

Phialemonium sp. 507-C

Phialemonium sp. Gall_27

Phialemonium dimorphosporum CBS 491.82 T

Cadophora luteo-olivacea CBS 141.41 T

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Chaetosphaeriales

Coniochaetales

Fig. 3 (continued)

Mycol Progress (2020) 19:667–690

678

from wood (Lygis et al. 2005, Simeto et al. 2005, Hofstetter

et al. 2012). The high number of detected taxa in our study

presumably results from the high sample number of three

different target host species over a wider geographical area,

in contrast to most of the studies that display less diversity.

However, the isolated taxa only encompass those fungi pres-

ent at the time of sampling and accessible by isolation; a

multitude of fungi cannot be cultured in general or on standard

media (Allen et al. 2003,O’Brien et al. 2005,Tsuietal.2011,

Muggia et al. 2017). Therefore, studies using culture-

independent high-throughput sequencing (HTS) techniques

usually report much higher species numbers from the fungal

diversity inside living or dead plant parts (up to 2000 opera-

tional taxonomic units, OTU) than studies using isolation

techniques (e.g. Kubartová et al. 2012, Hoppe et al. 2016,

Dissanayake et al. 2018, Jayawardena et al. 2018, Purahong

et al. 2018). As most of the taxa were isolated in this study

only once or few times, we would expect the number of taxa to

increase tremendously, if the number of wood samples would

be increased. The mycobiome of the woodof the three Prunus

species in Germany is far from being complete.

The two most abundant species, Aposphaeria

corallinolutea (Dothideomycetes,Pleosporales)and

Pallidophorina paarla (Leotiomycetes,Phacidiales), were

isolated >100 times each and from all three host species and

in all three collection areas. Aposphaeria corallinolutea was

revealed as the most dominant inhabitant of Prunus wood in

Germany in our study, while there are only five reports from

previous studies: from Fraxinus excelsior and Kerria japonica

in the Netherlands (de Gruyter et al. 2013), from decaying

wood in Thailand (Li et al. 2016), from dead branches of

Prunus padus in Russia (Tibpromma et al. 2017) and from

needles of Pseudotsuga menziesii in the USA (Daniels 2017).

The only ITS sequence of this species in GenBank originates

from the study in Thailand. Thus, A. corallinolutea is known

from several hosts, including Prunus, and from different coun-

tries, however, has not previously been reported from

Germany or from any of the Prunus species studied here.

The low number of reports could be explained by the lack of

studies on its main host plants/substrates that, based on this

study, includes necrotic wood of Prunus in Germany, but also

by the facts that A. corallinolutea was described only 2013 (de

Gruyter et al. 2013) and that the first and so far only ITS

sequence of a strain identified as this species was submitted

to GenBank only 2017 (Li et al. 2016). A blastn search with

the ITS sequence of strain GLMC 1355 revealed a 100%

match with an unidentified Ascomycota strain from leaves of

Fagus sylvatica in Germany (Unterseher and Schnittler 2009)

indicating the occurrence of this fungus on a further host as

well as in Germany. In contrast, the second most abundant

species, Pa. paarla, has previously frequently been reported

from a number of Prunus species in several countries includ-

ing Germany (Gierl and Fischer 2017,Bienetal.2020).

Part of the taxa isolated in this study probably represent

first reports for the genus Prunus, for specific Prunus species

or for Germany. We conducted a search of the 82 taxa iden-

tified to species level with high certainty on the USDA data-

base (Farr and Rossman 2019). For 41 of these taxa, no pre-

vious report from Germany and for 40 taxa, no previous report

from the host genus Prunus was listed (Table 1). Of further 25

taxa, there was no previous report from one or more of the

Prunus hosts, on which they were collected from in our study.

However, as some of the latest publications are missing, this

database is apparently not complete. Therefore, and due to the

unreliable identification results of many species, we consider

these reports as potential first reports. They need to be con-

firmed by in-depth studies of the respective species, which

was beyond the scope of this study.

The aim of this study was to reveal the mycobiome asso-

ciated with necroses of Prunus wood in Germany as complete

as possible in a reasonable time frame using a cultivation

approach. As the study was based on commercial orchards,

it was not possible to collect the exact amount of samples from

each host species with the same age, same cultivars etc. at the

same collection area. For some of the orchards, data like tree

age and cultivar were not even available. Therefore, a direct

comparison of the three collection areas and host species re-

garding strain or species numbers cannot be made as it is most

probably biased by other factors.

Comparison with other studies from Prunus

The results obtained in this study could only be compared to a

few other studies that used similar methods (culturing,

sequence-based identification). However, most of them were

conducted on different Prunus species and in different cli-

mates. The extensive survey of fungi in Prunus wood

(P. armeniaca,P. dulcis,P. persica,P. persica var.

nucipersica,P. salicina) in South Africa resulted in reports

of 47 species in several publications by Damm et al. (2007a,b,

2008a,b,c,2010), Moyo et al. (2018) and Bien and Damm

(2020) focusing on specific genera. Gramaje et al. (2012)iso-

lated nine fungal species from Prunus dulcis in Spain (Island

of Mallorca) including five species belonging to the

Botryosphaeriales as well as Collophorina hispanica,

Diaporthe amygdali,Eutypa lata and Phaeoacremonium

amygdalinum. The study of Inderbitzin et al. (2010) was re-

stricted to Botryosphaeriaceae from Prunus dulcis in CA,

USA, and that of Tian et al. (2018)toDiaporthe amygdali

and Botryosphaeria dothidea of P. persica in Yangshan,

China. The only study from Germany was that by Gierl and

Fischer (2017), who reported only eight fungal species from

symptomatic wood of six Prunus species, two of which were

also sampled by us, namely P. cerasus and P. domestica.

Botryosphaeriales are known as pathogens and endophytes

of various woody hosts (Slippers et al. 2007, Cloete et al.

Mycol Progress (2020) 19:667–690 679

Pleosporales

Capnodiales

Dothideales

Botryosphaeriales

Epicoccum layuense CGMCC 3.18362 T

Epicoccum cf. spp. GLMC 369 (7)

Epicoccum nigrum CBS 173.73 T #

Epicoccum dendrobii CGMCC 3.18359 T

Nothophoma cf. quercina GLMC 432 (18)

Nothophoma quercina CBS 633.92

Nothophoma anigozanthi CBS 381.91 T

Nothophoma infossa CBS 123395 T #

Didymella macrostoma GLMC 1392 (8)

Didymella macrostoma CBS 839.84

Didymella ru micicola CBS 683.79 T

Didymella exigua CBS 183.55 T #

Didymella heteroderae CBS 630.97 T

Phoma laundoniae ICMP 10843

Phoma laundoniae GLMC 1459 (1)

Coniothyrium ferrarisianum GLMC 380 (24)

Coniothyrium ferrarisianum CBS 285.74

Sclerostagonospora cycadis CBS 123538 T

Sclerostagonospora ericae CBS 141318 T

Jeremyomyces labinae CBS 144617 T

Jeremyomyces cf. labinae GLMC 327 (3)

Alternaria conjuncta CBS 196.86 T

Alternaria conjuncta GLMC 1338 (3)

Alternaria arbusti CBS 596.93 T

Alternaria rosae CBS 121341 T

Alternaria rosae GLMC 636 (1)

Alternaria angustiovoidea CBS 195.86 T

Alternaria destruens ATCC 204363 T

Alternaria destruens GLMC 1234 (24)

Bipolaris drechsleri CBS 136207 T

Bipolaris cf. spp. GLMC 248 (1)

Bipolaris variabilis CBS 127716 T

Bipolaris maydis CBS 136.29 T #

Leptosphaeria sp. LQ122417

Leptosphaeria sp. LCC1-2

Pleosporales sp. GLMC 1316 (1)

Leptosphaeria cichorium MFLUCC 14-1063

Leptosphaeria doliolum MFLU 15-1875

Leptosphaeria slovacica CBS 389.80

Neoleptosphaeria rubefaciens GLMC 337 (1)

Neoleptosphaeria rubefaciens CBS 387.80

Neoleptosphaeria jonesii MFLUCC 16-1442 T

Coniothyrium glycines CBS 124455

Coniothyrium dolichi CBS 124140

Coniothyrium telephii CBS 188.71

Parapyrenochaeta protearum GLMC 301 (2)

Parapyrenochaeta protearum CBS 131315 T #

Parapyrenochaeta acaciae CBS 141291 T

Neocucurbitaria populi GLMC 348 (1)

Neocucurbitaria populi CBS 142393 T

Neocucurbitaria cava CBS 257.68 T

Neocucurbitaria unguis-hominis CBS 378.92 #

Kalmusia cf. ebuli GLMC 767 (4)

Kalmusia ebuli CBS 123120 T #

Paraconiothyrium fuscomaculans CBS 116.16 T

Kalmusia longispora CBS 582.83 T

Kalmusia variispora GLMC 1347 (4)

Kalmusia variispora CBS 121517 T

Paraphaeosphaeria neglecta GLMC 857 (2)

Paraphaeosphaeria neglecta CBS 124078 T

Paraphaeosphaeria michotii MFLUCC-13-0349 T #

Paraconiothyrium brasiliense CBS 100299 T

Paraconiothyrium archidendri CBS 168.77 T

Paraconiothyrium estuarinum CBS 109850 T #

Lentitheciaceae sp. GLMC 1563 (1)

Sclerostagonospora cycadis CBS 291.76

Murilentithecium clematidis MFLUCC 14-0562 T #

Murilentithecium rosae MFLUCC 15-0044 T

Preussia flanaganii CBS 112.73 T

Preussia funiculata CBS 659.74 #

Preussia cf. spp. GLMC 1754 (1)

Preussia persica CBS 117680 T

Preussia persica GLMC 447 (2)

Angustimassarina italica MFLUCC 15-0082 T

Angustimassarina lonicerae MFLUCC 15-0087 T

Angustimassarina populi MFLUCC 13-0034 T #

Angustimassarina cf. spp. GLMC 891 (8)

Aposphaeria corallinolutea CBS 131287 T

Aposphaeria corallinolutea GLMC 1355 (138)

Aposphaeria populina CBS 350.82

Roussoella euonymi GLMC 1544 (1)

Roussoella euonymi CBS 143426 T

Roussoella mukdahanensis MFLUCC 11-0201 T

Roussoella nitidula MFLUCC 11-0634 #

Constantinomyces sp. GLMC 1767 (1)

Constantinomyces patonensis CBS 117950 T

Constantinomyces oldenburgensis CBS 144642 T

Constantinomyces virgultus CBS 117930 T #

Devriesia shelburniensis (Devriesia s. str.) CBS 115876

Devriesia staurophora (Devriesia s. str.) CBS 375.81 #

Devriesia pseudoamericana GLMC 819 (1)

Devriesia pseudoamericana (Devriesia s. lat.) CBS 126270 T

Devriesia americana (Devriesia s. lat.) CBS 117726

Cladosporium acalyphae CBS 125982 T

Cladosporium needhamense CBS 143359 T

Cladosporium cf. spp. 1 GLMC 1289 (10)

Cladosporium xylophilum CBS 125997 T

Cladosporium herbarum CBS 121621 T #

Cladosporium macrocarpum CBS 121623 T

Cladosporium cf. spp. 2 GLMC 711 (2)

Cladosporium variabile CBS 121636 T

Aureobasidium pullulans GLMC 1460 (15)

Aureobasidium pullulans CBS 584.75 T #

Aureobasidium melanogenum CBS 105.22 T

Aureobasidium namibiae CBS 147.97 T

Diplodia mutila GLMC 1759 (1)

Diplodia mutila CBS 136014 T #

Diplodia neojuniperi CPC 22753 T

Diplodia seriata CBS 112555 T

Diplodia seriata GLMC 1527 (1)

Diplodia intermedia CBS 124462 T

Penicillium resticulosum CBS 609.94 T

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Mycol Progress (2020) 19:667–690

680

2011). In previous studies, species of this order were reported

to be very abundant in wood of Prunus trees in South Africa,

the USA, Spain and China (Damm et al. 2007a,b, Inderbitzin

et al. 2010, Gramaje et al. 2012,Tianetal.2018). The dom-

inating species in the studies from South Africa and Spain

were D. seriata and Neofusicoccum parvum, respectively,

while only Botryosphaeria dothidea was reported in that from

China.Moreover, D. pinea, a pathogen of several Pinus spe-

cies in many countries (Farr and Rossman 2019), that also

cause serious damage to pine trees suffering from drought

stress and bark beetle attacks in Germany (Heydeck and

Dahms 2012,Petercord2017), had frequently been isolated

from P. persica in South Africa and tested positive for its

pathogenicity on this host (Damm et al. 2007a). Therefore,

host jumps from infected Pinus plantations to Prunus orchards

in close vicinity are possible. However, Botryosphaeriales

were surprisingly rare in this study. Only one strain each of

D. seriata and D. mutila was detected in wood of P. domestica

in the most southern sampling region in Germany; D. pinea

was not isolated at all. Brodde et al. (2019)documentedan

outbreak of Diplodia tip blight on Pinus sylvestris stands in

Sweden in 2016 caused by D. pinea and attributed it to the

increased summer temperatures. An influence of different cli-

matic conditions on distribution patterns of Botryosphaeriales

species has also been observed in the USA and Australia

(Taylor et al. 2005, Úrbez-Torres et al. 2006,Pittetal.

2010). However, a climatical or geographical explanation in

general can be ruled out, since species of this order have been

detected from fruit trees and grapevine in Central Europe be-

fore, even in different parts of Germany, including a report of

the same two species from P. armeniaca (Trapman et al. 2008,

Quaglia et al. 2014, Fischer et al. 2016, Gierl and Fischer

2017). Based on the results in this study, species of

Botryosphaeriaceae are currently not regarded as a threat for

German Prunus orchards.

With 14 species, Phaeoacremonium was the genus with the

highest diversity in the study on Prunus wood in South Africa

(Damm et al. 2008b), while only four Phaeoacremonium spe-

cies were isolated in Germany (this study). Three of them

were isolated in both studies, namely Pm. iranianum,Pm.

scolyti and Pm. viticola, provided the identification of the

latter, which was with low certainty (cf.), is correct.

Although the genera were found in Prunus wood in both

countries, completely different species of Coniochaeta

(Coniochaetales,Sordariomycetes), Calosphaeria,Jattaea

(Calosphaeriales,Sordariomycetes), Paraconiothyrium/

Paraphaeosphaeria (Pleosporales,Dothideomycetes)and

Phaeomoniellales (Eurotiomycetes) were collected in

Germany and in South Africa (Damm et al. 2008a,c,2010,

Bien and Damm 2020, this study). The latter order was much

more diverse and frequent in Prunus wood in South Africa; in

Germany, only two Minutiella species were collected. In con-

trast, Cadophora species were more frequently detected in

wood of different Prunus species in Germany, but only rarely

detected in South Africa; only Ca. prunicola was collected in

Prunus wood in both countries (Bien and Damm 2020).

Collophorina (syn. Collophora)andPallidophorina spe-

cies were isolated frequently in Prunus wood both in South

Africa and in Germany (Damm et al. 2010,Bienetal.2020,

this study). The dominating Collophorina species isolated

from several Prunus species in South Africa was C. rubra,a

species not reported from Germany, while the dominating one

in Germany was C. africana (syn. Collophora capensis). The

latter was originally found exclusively on wood of P. salicina

in South Africa, while in our study, it was exclusively present

on P. domestica. In the study by Damm et al. (2010), Pa.

paarla (syn. C. paarla,Collophora pallida) was mostly iso-

lated from P. salicina in South Africa, while this species was

one of the two dominating species in this survey occurring in

all Prunus species studied (Bien et al. 2020, this study). The

Collophorina species isolated from P. dulcis wood in Spain

(Gramaje et al. 2012), C. hispanica, was not found in our study.

Gierl and Fischer (2017) isolated Pa. paarla from symptomatic

wood of P. cerasus and P. persica,aswellasC. hispanica and

C. africana from P. armeniaca and P. dulcis, respectively.

Although five species of Diatrypaceae were collected in

the surveys in Germany and South Africa, Eutypa lata was the

only species found in both of them, in wood of P. cerasus and

P. domestica in Germany, as well as in P. armeniaca,

P. avium,P. dulcis and P. salicina in South Africa (Moyo

et al. 2018, this study). It was also found in wood of

P. dulcis in Mallorca (Gramaje et al. 2012). Furthermore,

Diaporthe species have been isolated in all three studies as

well. Based on preliminary studies, none of the species is

overlapping with those found in this study (Gramaje et al.

2012, U. Damm, unpubl. data). The remaining taxa cannot

be compared as no data were published from the survey in

South Africa.

Function of the fungal species inside wood

Only for part of the species/genera isolated in this study infor-

mation on lifestyle, like pathogenicity on Prunus species, is

available. In the survey on Prunus wood in South Africa,

preliminary pathogenicity tests on detached shoots revealed

the majority of tested species belonging to

Botryosphaeriaceae,Celotheliaceae,Coniochaetaceae,

Togniniaceae and Tympanidaceae to be potentially

Fig. 4 Phylogeny of dataset 2 obtained by Bayesian inferenceanalysis of

the combined LSU and ITS sequence alignment of Dothideomycetes.

Penicillium resticulosum strain CBS 609.94 is used as outgroup. BI

posterior probability support values above 0.9 (bold) and ML bootstrap

support values above 70% are shown at the nodes. The strains isolated in

this study are emphasised in bold. Numbers in parentheses indicate the

number of isolated strains per taxon. Branches that are crossed by diag-

onal lines are shortened by 50%. T, ex-type strain; #, type species

Mycol Progress (2020) 19:667–690 681

pathogenic to P. persica var. nucipersica and/or P. salicina

(Damm et al. 2007a,2008b,2010). Species of all these fam-

ilies have been isolated in this study as well. However, apart

from the fact that these pathogenicity tests were preliminary

and not followed up by field tests, these results cannot be

transferred to this study, because most of the fungal species

isolated were different, and even the few species isolated in

both studies, for example Pa. paarla and C. africana,werenot

isolated from the same Prunus species. Therefore, the patho-

genicity of each fungal species isolated in this study would

need to be tested on its host species in Germany.

As we aimed at isolating pathogens causing necroses inside

Prunus wood, the majority of wood pieces we isolated from

were from the transition zone of symptomatic to non-

Helotiales

Phacidiales

1/100

0.98/100

1/100

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0.93/–

1/94

1/99

1/94

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1/86

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1/100

1/99

1/83

1/100

1/100

1/96

1/98

1/93

–/85

1/100

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incertae sedis

Cadophora luteo-olivacea CBS 141.41 T

Cadophora luteo-olivacea GLMC 1264 (12)

Cadophora malorum CBS 165.42

Cadophora obscura CBS 269.33

Cadophora bubakii CBS 198.30

Cadophora novi-eboraci GLMC 1472 (8)

Cadophora novi-eboraci NYC14 T

Cadophora africana CBS 120890 T

Cadophora prunicola CBS 120891 T

Cadophora prunicola GLMC 1633 (8)

Cadophora fastigiata CBS 307.49 #

Cadophora melinii CBS 268.33

Cadophora ramosa GLMC 377 T (1)

Cadophora orientoamericana MYA-4972 T

Phialocephala piceae GLMC 331 (26)

Phialocephala piceae UAMH 10851 T

Phialocephala sp. 1 GLMC 803 (1)

Phialocephala compacta CBS 506.94

Phialocephala sp. 2 GLMC 385 (2)

Phialocephala catenospora DAOM C 250108 T

Phialocephala nodosa NB475 T

Phialocephala sp. 3 GLMC 833 (6)

Phialocephala dimorphospora CBS 300.62 T #

Proliferodiscus chiangraiensis MFLU 16-0588 T

Proliferodiscus sp. GLMC 460 (7)

Proliferodiscus inspersus var. magniascus KUS-F52660

Proliferodiscus ingens GLMC 1751 T (1)

Proliferodiscus tricolor CBS 122000

Pezicula eucrita GLMC 643 (2)

Pezicula eucrita CBS 662.96

Pezicula sporulosa GLMC 1224 (4)

Pezicula sporulosa CBS 224.96 T

Pezicula cf. carpinea GLMC 416 (4)

Pezicula carpinea CBS 283.39 #

Pezicula cinnamomea CBS 290.39

Pezicula sp. GLMC 1726 (14)

Pezicula sp. 3 CC-2015 CBS 268.78

Dermea cerasi A GLMC 1760 (4)

Dermea cerasi CBS 141.26 #

Dermea cerasi CBS 432.67 #

Dermea cerasi B GLMC 862 (1)

Dermea pruni CFCC 53007

Dermea sp. GLMC 867 (2)

Dermea persica MFLU 16-0259 T

Dermea pinicola CBS 142.46 T

Neofabraea sp. GLMC 1284 (8)

Neofabraea kienholzii CBS 126461 T

Neofabraea malicorticis AFTOL-ID 149 T #

Neofabraea malicorticis CBS 141.22 #

Neofabraea vagabunda GLMC 718 (1)

Neofabraea vagabunda CBS 261.32

Neofabraea vagabunda CBS 109875

Monilinia fructicola CBS 203.25 #

Monilinia fructicola CBS 329.35 #

Monilinia laxa GLMC 1290 (4)

Monilinia laxa AFTOL-ID 169

Monilinia laxa CBS 132.21

Botrytis cinerea GLMC 635 (4)

Botrytis cinerea CBS 261.71 T #

Botrytis fabae CBS 120.29 T

Botrytis porri CBS 190.26 T

Arboricolonus simplex GLMC 459 T # (1)

Oidiodendron cf. griseum GLMC 602 (8)

Oidiodendron griseum CBS 249.33

Oidiodendron sp. 1 GLMC 469 (1)

Oidiodendron griseum CBS 128807

Oidiodendron eucalypti CPC 32659 T

Oidiodendron tenuissimum CBS 238.31 T #

Oidiodendron maius CBS 126948

Oidiodendron sp. 2 GLMC 485 (7)

Collophorina africana GLMC 1736 (21)

Collophorina africana CBS 120872 T

Collophorina badensis GLMC 1684 T (10)

Collophorina germanica GLMC 1445 T (2)

Collophorina neorubra GLMC 929 T (7)

Collophorina rubra CBS 120873 T #

Pallidophorina paarla GLMC 452 (112)

Pallidophorina paarla CBS 120877 T #

Pseudeurotium bakeri CBS 878.71 T

Pseudeurotium zonatum CBS 329.36 T #

Pseudeurotium desertorum CBS 986.72 T

Leotiomycetes sp. GLMC 792 (1)

Pseudeurotium sp. 30404-E

Colletotrichum godetiae CBS 133.44 T

0.09

0.98/71

1/93

–/77 1/88

1/91

1/100

Thelebolales

Fig. 5 Phylogeny of dataset 3 obtained by Bayesian inference analysis of

the combined LSU and ITS sequence alignment of Leotiomycetes.

Colletotrichum godetiae strain CBS 133.44 is used as outgroup. BI

posterior probability support values above 0.9 (bold) and ML bootstrap

support values above 70% are shown at the nodes. The strains isolated in

this study are emphasised in bold. Numbers in parentheses indicate the

number of isolated strains per taxon. Branches that are crossed by diag-

onal lines are shortened by 50%. T, ex-type strain; #, type species

Mycol Progress (2020) 19:667–690

682

symptomatic wood tissue. From most of the wood samples,

we isolated several fungi. Wood diseases are caused by a

complex of fungal pathogens, which is known from grapevine

trunk diseases like esca and Botryosphaeria dieback (Larignon

and Dubos 1997, Bertsch et al. 2013). Therefore, more than

one of the isolated fungi could be responsible for the symp-

toms on the respective branch. Moreover, transitions between

different lifestyles have been shown in a high number of fungi

(Promputtha et al. 2010, Álvarez-Loayza et al. 2011, Eaton

et al. 2011,O’Connell et al. 2012, Kuo et al. 2014). As an

example, many of the wood-inhabiting fungi, including

Botryosphaeriaceae, are known as weak pathogens: they do

not cause symptoms and live inside their host endophytically

and become pathogenic, if the host plant is exposed to stress,

e.g. drought (Desprez-Loustau et al. 2006,Slippersand

Wingfield 2007). However, not only the presence of one or

more pathogens decides, if a disease develops, but also the

absence of other fungi or other organisms that prevent the

disease and keep the tree healthy. Thus, in a study of

Gennaro et al. (2003), the endophytic communities on declin-

ing oaks were less diverse than those on healthy trees, and

endophyte communities of needles of Norway spruce have

been proposed as indicators of tree health (Rajala et al.

2014). It is therefore hardly possible to draw conclusions

concerning the particular role of the individual species within

the temporal-spatial succession of fungal communities associ-

ated with wood necroses of Prunus trees in Germany.

We isolated fungi both from the transition zone of symp-

tomatic to non-symptomatic tissue and from non-symptomatic

tissue of the same branch providing that the sole isolation of a

certain species from one of the two zones would indicate a

certain life style, e.g. the sole isolation from non-symptomatic

tissue would indicate an endophytic life style. However, the

resulting data are not directly comparable, because the number

of wood pieces of the non-symptomatic tissue of a branch with

wood symptoms studied was lower than the number of wood

pieces from the transition zone of symptomatic to non-

symptomatic wood tissue. Moreover, in some branches, little

Eurotiales

Chaetothyriales

Phaeomoniellales

0.99/97

1/86

1/98

1/100

1/100

1/97

0.97/75

1/93

1/100

1/79

0.99/85

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1/94

1/100

1/100

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1/95

1/100

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Penicillium expansum CBS 325.48 T

Penicillium resticulosum CBS 609.94 T

Penicillium cf. spp. GLMC 1288 (1)

Penicillium roqueforti CBS 221.30 T

Penicillium brevicompactum GLMC 1661 (6)

Penicillium brevicompactum CBS 257.29 T

Penicillium glabrum CBS 125543 T

Penicillium glabrum GLMC 1400 (2)

Penicillium angulare GLMC 1646 (1)

Penicillium angulare NRRL 28157 T

Penicillium dierckxii CBS 304.48

Aspergillus chevalieri GLMC 899 (2)

Aspergillus chevalieri CBS 522.65 T

Aspergillus chevalieri CBS 129311

Aspergillus glaucus GLMC 771 (1)

Aspergillus glaucus CBS 297.71 #

Aspergillus leucocarpus CBS 353.68 T

Talaromyces sp. GLMC 1678 (2)

Talaromyces dendriticus CBS 660.80 T

Talaromyces aculeatus CBS 289.48 T

Talaromyces islandicus CBS 394.50

Exophiala sp. GLMC 1670 (3)

Exophiala sideris CBS 127096

Exophiala capensis CBS 128771 T

Capronia kleinmondensis CBS 122671 T

Rhinocladiella atrovirens CBS 317.33 aut #

Herpotrichiellaceae sp. GLMC 914 (1)

Capronia sp. GLMC 1254 (1)

Capronia moravica CBS 603.96

Capronia fungicola CBS 614.96 T

Capronia mansonii CBS 101.67 T

Rhinocladiella quercus CPC 26621 T

Rhinocladiella cf. quercus GLMC 1752 (3)

Minutiella sp. GLMC 1636 (2)

Minutiella sp. CBS 145008

Minutiella pruni-avium GLMC 1624 T (2)

Minutiella tardicola CBS 121757 T

Diplodia intermedia CBS 124462 T

0.2

1/100

0.97/82

1/100

Fig. 6 Phylogeny of dataset 4 obtained by Bayesian inference analysis of

the combined LSU and ITS sequence alignment of Eurotiomycetes.

Diplodia intermedia strain CBS 124462 is used as outgroup. BI

posterior probability support values above 0.9 (bold) and ML bootstrap

support values above 70% are shown at the nodes. The strains isolated in

this study are emphasised in bold. Numbers in parentheses indicate the

number of isolated strains per taxon. Branches that are crossed by diag-

onal lines are shortened by 50%. T, ex-type strain; #, type species

Mycol Progress (2020) 19:667–690 683

Polyporales

Russulales

Agaricales

Boletales

Hymenochaetales

Cantharellales

Auriculariales

Tremellomycetes

Cystobasidiomycetes

Pezizomycetes

Lecanoromycetes

Saccharomycetes

Mucoromycota Ascomycota

Agaricomycetes

Basidiomycota

Trametes versicolor GLMC 1717 (2)

Trametes versicolor CBS 296.33

Trametes suaveolens CBS 279.28 #

Trametes hirsuta GLMC 467 (1)

Trametes hirsuta CBS 282.73

Coriolopsis gallica GLMC 1308 (1)

Coriolopsis gallica CBS 429.34

Coriolopsis gallica CBS 428.34

Coriolopsis trogii RLG4286sp

Mycoacia nothofagi CBS 125847

Mycoacia fuscoatra CBS 125883 #

Mycoacia fuscoatra KHL13275 #

Mycoacia fuscoatra GLMC 1268 (1)

Mycoacia uda CBS 224.56

Geotrichopsis mycoparasitica CBS 687.93 T #

Bjerkandera adusta CBS 371.52 #

Bjerkandera cf. adusta GLMC 431 (5)

Bjerkandera fumosa CBS 152.79

Peniophora cinerea CBS 261.37

Peniophora cinerea GLMC 947 (13)

Peniophora pilatiana CBS 269.56

Peniophora quercina GLMC 1640 (2)

Peniophora quercina CBS 409.50 #

Peniophora simulans CBS 875.84

Heterobasidion annosum GLMC 1320 (1)

Heterobasidion annosum CBS 567.67 #

Heterobasidion annosum CBS 169.28 #

Heterobasidion araucariae CBS 743.94 T

Stereum armeniacum CBS 944.96

Stereum hirsutum CBS 930.70 #

Stereum cf. spp. GLMC 475 (3)

Stereum ostrea CBS 361.36

Coprinellus radians SZMC-NL-3986

Coprinellus xanthothrix SZMC-NL-3417

Coprinellus cf. spp. GLMC 737 (1)

Coprinellus curtus SZMC-NL-1490

Coprinellus heptemerus SZMC-NL-2144

Coprinellus deliquescens Wat27209 #

Coniophora puteana GLMC 420 (1)

Coniophora puteana CBS 148.32 #

Coniophora arida CBS 109.40

Coniophora merulioides CBS 152.35 aut

Phellinus tuberculosus A GLMC 396 (4)

Phellinus tuberculosus CBS 383.72

Phellinus tuberculosus B GLMC 1755 (3)

Phellinus igniarius CBS 380.72 #

Phellinus laevigatus CBS 256.30

Sistotrema eximum CBS 531.91

Sistotrema octosporum CBS 126038

Sistotrema hypogaeum CBS 393.63

Sistotrema brinkmannii CBS 402.54

Sistotrema sp. GLMC 1593 (2)

Exidia glandulosa GLMC 374 (1)

Exidia glandulosa CBS 126.24 #

Exidia nigricans MW 313

Exidia thuretiana CBS 215.63

Udeniomyces sp. GLMC 1365 (1)

Udeniomyces pyricola CBS 6754 T #

Udeniomyces pseudopyricola CBS 10076 T

Udeniomyces megalosporus CBS 7236 T

Cystobasidium pinicola CBS 9130 T

Cystobasidium pinicola GLMC 1603 (3)

Cystobasidium laryngis CBS 2221 T

Cystobasidium benthicum CBS 9124 T

Trichophaeopsis bicuspis GLMC 1596 (1)

Trichophaeopsis bicuspis NSW 8316 #

Trichophaeopsis tetraspora C F-47525

Trichophaeopsis sp. DHP-AR-19

Trichophaeopsis sp. DHP-CH-58

Melastiza contorta KH 01.06

Pseudaleuria quinaultiana p712L

Chaetothiersia vernalis BAP 492 T #

Melastiza cornubiensis KH 03.43

Orbicula parietina CBS 238.32

Lecanoromycetes sp. GLMC 1733 (2)

Uncultured Ascomycota agrFF142

Uncultured Ascomycota SMOTU80

Anthopsis catenata CBS 492.81 T

Anthopsis deltoidea CBS 263.77 T #

Cyanodermella oleoligni CBS 140345 T

Umbilicaria esculenta A6

Nakazawaea ernobii CBS 1737 T

Nakazawaea holstii CBS 4140 T #

Nakazawaea cf. holstii GLMC 1309 (1)

Nakazawaea populi CBS 7351 T

Nakazawaea pomicola CBS 4242 T

Wickerhamomyces silvicola CBS 1705 T

Wickerhamomyces silvicola GLMC 1708 (1)

Candida peoriensis CBS 8800 T

Wickerhamomyces rabaulensis CBS 6797 T

Wickerhamomyces canadensis CBS 5676 #

Umbelopsis isabellina CBS 560.63

Umbelopsis isabellina GLMC 521 (5)

Umbelopsis ovata CBS 499.82 T

Umbelopsis versiformis CBS 150.81 T #

Mucor bainieri CBS 293.63 T

Mucor circinelloides f. lusitanicus CBS 108.17 T

Ellisomyces anomalus CBS 243.57 T #

Mucor sp. GLMC 656 (1)

Mucor circinelloides GLMC 1405 (1)

Mucor circinelloides f. circinelloides CBS 195.68 T

Mucor hiemalis GLMC 1395 (1)

Mucor hiemalis f. hiemalis CBS 201.65

Mucor mucedo CBS 640.67 T #

Entomophthora sphaerosperma CBS 530.75

0.8

1/98

0.97/72

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1/99

1/100

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0.94/70

0.94/72

1/92

1/97

1/91

1/100

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1/92

1/96

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1/100

1/99

0.94/81

1/100

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1/100

1/93

0.99/–

1/100

1/100

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1/100

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1/85

1/100

1/100

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0.96/88

1/100

1/100

1/100

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1/100

1/97

–/100

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–/86

1/99

1/100

1/96

1/96

1/99

1/100

1/100

1/97

0.90/79

0.96/82

1/100

1/100 1/100

1/92

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Mycol Progress (2020) 19:667–690

684

non-symptomatic tissue was available due to the large expan-

sion of the necroses and the “symptomless tissue”placed on

OA for isolation was very closely located to the necrotic tis-

sue. Biggs et al. (1983) detected hyphae of Cytospora

chrysosperma up to 2 cm away from xylem tissue of

Populus with visible necroses caused by this fungus.

Therefore, isolation of a pathogenic fungus from nearby

symptomless tissue cannot be excluded and a similar fungal

diversity and abundance was expected. However, the number

of fungi isolated from non-symptomatic tissue was exception-

ally low compared to that from the transition zone of symp-

tomatic to non-symptomatic tissue. The fungi isolated solely

from symptomless tissue were isolated only once. And none

of the few species isolated more often from symptomless than

from symptomatic tissue was found more than five times in

total. This cannot be explained by the lower subsample num-

ber of non-symptomatic wood pieces. We attribute this to the

larger number of ecological niches of the wood pieces from

the transition zone resulting in a temporal-spatial succession

of fungal communities including endophytes, pathogens and

saprobionts.

Uncertainties in identifications

Of the 172 species isolated from Prunus wood in this study, 102

could be assigned to a particular species with different levels of

certainty. The ITS region of many species is highly variable,

which decreases the similarity values and results in unjustified

uncertainty (Nilsson et al. 2008, Simon and Weiß 2008, Hughes

et al. 2009). In contrast, ITS sequences of closely related species

can be identical or nearly so, which results in similarity values of

up to 100% and therefore unjustified certainty (e.g. Houbraken

et al. 2011,Dammetal.2019). This means, on the one hand,

some of the 70 taxa not assigned to a species could possibly be

identified to species level by including the whole variability of

the ITS sequences of the respective genus. On the other hand,

some of the 102 species that were assigned to a particular species

with high or low (cf.) certainty, even of those with identical ITS

sequences, could represent new species or species with no se-

quence data in GenBank. This demonstrates how imprecise an

identification based on solely ITS data is, even by availability of

full-length sequences, careful selecting the reference data and

inclusion of the nucleotide differences/identities.

The different inter- and intraspecific variability of ITS se-

quences is a dilemma of species identification in fungal diversity

studies dealing with big and diverse sampling datasets. It is sim-

ply not possible to study the variability of each taxon and con-

sider DNA variability of each species while defining a uniform

threshold for species differentiation. On the one hand, a rigorous

application of strict criteria for species delimitation ignores the

variability of different fungal taxa. On the other hand, if no clear

criteria are applied, species delimitation is to a certain degree

subjective and the different reasons for a specific decision hard

to compare.

0

10

20

30

40

50

60

70

80

Sordariomycetes

Dothideomycetes

Leotiomycetes

Eurotiomycetes

Saccharomycetes

Lecanoromycetes

Pezizomycetes

Agaricomycetes

Cystobasidiomycetes

Tremellomycetes

Mucoromycota

genus unknown6

species unknown5

species uncertain (cf. spp.)4

species uncertain (cf.)3

species identified2

species studied1

Number of species

Ascom

y

cota Basidiomycota

Fig. 8 Number of species in

different classes of Ascomycota,

Basidiomycota and

Mucoromycota detected inside

Prunus wood in Germany and

level of certainty of identification

based on ITS/LSU sequence

comparisons.

1

Species treated in

Bien et al. 2020 or Bien and

Damm 2020,

2

ITS 0–4 nucleotide

differences to a named reference

sequence,

3

ITS 5–10 nucleotide

differences to a named reference

sequence,

4

ITS 0–4 nucleotide

difference to reference sequences

of ≥2 different species,

5

ITS > 10

nucleotide differences to a named

reference sequence,

6

no reference

sequence in the same clade

Fig. 7 Phylogeny of dataset 5 obtained by Bayesian inferenceanalysis of

the combined LSU and ITS sequence alignment of miscellaneous

Ascomycota (Lecanoromycetes,Pezizomycetes,Saccharomycetes),

Basidiomycota and Mucoromycota.Entomophthora sphaerosperma

strain CBS 530.75 is used as outgroup. BI posterior probability support

values above 0.9 (bold) and ML bootstrap support values above 70% are

shown at the nodes. The strains isolated in this study are emphasised in

bold. Numbers in parentheses indicate the number of isolated strains per

taxon. Branches that are crossed by diagonal lines are shortened by 50%.

T, ex-type strain; #, type species

Mycol Progress (2020) 19:667–690 685

Moreover, blastn searches often not only result in uncertain

but more importantly in wrong identifications due to the se-

quence data in NCBI GenBank, of which many are incom-

plete, include artefacts, are mixed up or derived from wrongly

identified samples and therefore not suitable as reference data

(Vilgalys 2003, Nilsson et al. 2006, Bidartondo 2008, Hyde

et al. 2010,Koetal.2011). Therefore, only sequences of ex-

type strains can be reliable references. However, even se-

quences of ex-type strains can be unreliable, if they are based

on sequences withlow quality or mixed up with other species,

as revealed for example in Colletotrichum hymenocallidicola

(Damm et al. 2019). Nonetheless, the main drawback of iden-

tification based on sequence data (in GenBank) is the limited

part of the overall known fungal diversity with available se-

quence data, especially those from type material. It is possible

that the number of species that were not identified to species

level and regarded as new species is lower. That means, some

of these 70 potentially new species as well as some of those

with uncertainty identified species could represent species that

had previously been described based on morphology, howev-

er, lacking sequence data in GenBank.

Species identified with high certainty

In total, 82 taxa (630 strains) were identified to species level

with certainty. All species belonging to the genera

Arboricolonus,Cadophora,Collophorina,Pallidophorina

and Proliferodiscus in the Leotiomycetes and Minutiella in

the Eurotiomycetes have previously been studied in detail

morphologically as well as by multi-locus phylogenetic anal-

yses (Bien et al. 2020,BienandDamm2020). Therefore, their

identifications are reliable. Three and two species of

Cadophora and Collophorina, respectively, and one species

each of Proliferodiscus and Minutiella, as well as the genus

Arboricolonus have been described in these two previous

studies based on strains isolated from Prunus wood in

Germany collected within our survey.

Strain GLMC 380 belonging to the Dothideomycetes

shows that also strains assigned to a species with certainty

can actually be of uncertain systematic position. The sequence

of this strain (representing further 23 strains) was identical

with that of a strain referred to as Coniothyrium ferrarisianum

(CBS 285.74). Both strains form a clade sister to a clade

formed by two ex-type strains of Sclerostagonospora.Other

strains of Coniothyrium,Co. dolichi,Co. glycines and Co.

telephi, formed a distant clade also within the Pleosporales.

There is no DNA sequence of the type species Co. palmarum

available; the genus Coniothyrium is currently regarded as

polyphyletic (Verkley et al. 2004,2014). Therefore, the sys-

tematic placement of the genus Coniothyrium as well as of the

individual species, including Co. ferrarisianum, still needs to

be clarified.

Species identified with low certainty

In total, 20 taxa (98 strains) were assigned to a species with

low certainty, because the ITS sequences differed in 5–10

nucleotides from the closest named reference sequences.

These are taxa that need to be studied in depth; species bound-

aries need to be evaluated, etc. It is possible that part of these

taxa represent new species. Even the affiliation of some of the

taxa to genus level still needs to be clarified, for example

Anthostomella cf. pinea strain GLMC 451 (see below).

Taxa identified to genus level

In total, 57 taxa (255 strains) were assigned to a genus, but not

to a species. Of these, 16 taxa (86 strains) matched with more

than one named reference sequence (cf. spp.); these fungi are

unlikely to represent new species and can probably be identi-

fied to species level based on secondary barcodes. It is also

possible that one of the species in the respective clade repre-

sents a synonym that has previously not been revealed yet.

The ITS sequences of the other 41 taxa (169 strains) dif-

fered in > 10 nucleotides from the closest named reference

sequence. Most of these taxa represent new species, unless

the species was described only based on morphology and no

ITS sequence is available.

Species not identified to genus level

Thirteen species could not be identified to genus level (35

strains), because they did not match with named reference se-

quences in blastn searches and were placed isolated within the

phylogenies (e.g. Leotiomycetes sp. GLMC 792,

Lecanoromycetes sp. GLMC 1733) or because the respective

genus is polyphyletic and sequences of the type species are either

not available or belong to a different clade within the phylogeny.

The 13 taxa were therefore identified to family (six taxa), order

(five taxa) or class (two taxa) level only; most of them belong to

the Sordariomycetes.

Although some of the closest matches in blastn searches with

the ITS sequences of strains GLMC 1660 (Xylariaceae sp. 1) and

GLMC 1594 (Xylariaceae sp. 2) were strains previously identi-

fied as Rosellinia sp., we doubt these taxa belong to this genus,

because sequences of ex-type strains of two species and of a

strain of the type species, R. aquila (Wendt et al. 2018), belong

to different clades. Affiliation of the strains isolated in this study

to the genus Rosellinia cannot be clarified with the data at hand.

Strain GLMC 848 (Xylariaceae sp. 3) is placed together

with two strains from Juniperus deppeana in the USA referred

to as Sordariomycetes sp. (Hoffman and Arnold 2010). The

clade formed by these strains is sister to a clade formed by

strain GLMC 451 (Anthostomella cf. pinea, this study) and the

ex-type strain of Anthostomella pinea (CBS 128205).

However, the genus Anthostomella is polyphyletic

Mycol Progress (2020) 19:667–690

686

(Daranagama et al. 2015), which is confirmed here as the ex-type

strain of another species, An. proteae (CBS 110127), belongs to

a different clade. None of these clades was confirmed to repre-

sent the genus Anthostomella, because there is no sequence of

the type species of the genus, An. limitata, available. Therefore,

the affinity of both strains, GLMC 848 and GLMC 451, to

Anthostomella is unclear.

Strain GLMC 1232 (Sordariales sp.) groups with a

strain referred to as Cercophora sp. (CIM1_17,

Mapperson and Dearnaley, unpubl. data), an uncultured

Ascomycota (dfmo0690_036) from soil in the USA

(O’Brien et al. 2005) and the ex-type strain of Zopfiella

tardifaciens (CBS 670.82). A strain of the type species of

Zopfiella,Z. tabulata (CBS 230.78), is placed in a single-

strain clade sister to this group. The intergeneric relation-

ships of Lasiosphaeriaceae genera including Zopfiella

and Cercophora were described as inconclusive due to

the uncertainty about the phylogenetic value of different

morphological characters (Cai et al. 2005).

Strain GLMC 1316 (Pleosporales sp.) clustered with two

strains referred to as Leptosphaeria sp. (LCC1-2, Li et al.

unpubl.; LQ122417, Qiong et al., unpubl. data) that are distant

from a clade formed by strains of three further Leptosphaeria

species, none of which are ex-type strains. The affiliation of the

isolated strain to this genus is therefore doubtful.

The ITS sequence of strain GLMC 1563 (Lentitheciaceae

sp.) is identical with that of a strain previously identified as

Sclerostagonospora cycadis (CBS 291.76). Both strains form

a clade sister to a clade formed by two ex-type strains of

Murilentithecium species, including the type species of the

genus. As the ex-type strain of S. cycadis (CBS 123538) be-

longs to a different clade within the Pleosporales, sister to the

ex-type strain of S. ericae, strain CBS 291.76 must have been

wrongly identified. Both strains are likely to be a

Murilentithecium species, which needs to be confirmed.

Strain GLMC 792 (Leotiomycetes sp.), belonging to the

Leotiomycetes, grouped with strain 30404-E that had been iso-

lated from wood in Greenland and identified as Pseudeurotium

sp. (Pedersen et al., unpubl. data). However, the placement in

this genus is doubtful, because this clade is distant from the

Pseudeurotium clade formed by three ex-type strains including

the type species of the genus.

This study highlights that a common substrate like wood of

fruit trees in Germany actually represents an underexplored hab-

itat and houses a widely unknown mycobiome with widely un-

known host spectrum/specificity, distribution, conservation sta-

tus, life cycle and function and probably large potentials for

applications. We expect most of the taxa not assigned to a spe-

cies and part of the species identified with more or less certainty

to represent new species or even new genera. In order to clarify

their identity, these species should be treated in depth in further

follow-up studies by a polyphasic approach consisting of multi-

locus sequence analyses and sound morphological examinations.

Availability of data and material The DNA sequences generated in this

study were deposited in GenBank (Table 1, suppl. material tab.). The

datasets generated and analysed during the current study are available

from the TreeBASE website, http://purl.org/phylo/treebase/phylows/

study/TB2: S25316.

Authors’contributions Both authors have contributed equally. Both au-

thors read and approved the final manuscript.

Funding information Open Access funding provided by Projekt DEAL.

This study contributes to the German Barcode of Life project, funded by

the Federal Ministry of Education and Research of Germany (www.

bolgermany.de).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflicts of

interest.

Ethics approval Not applicable

Consent to participate Not applicable

Consent for publication Not applicable

Open Access This article is licensed under a Creative Commons

Attribution 4.0 International License, which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as

long as you give appropriate credit to the original author(s) and the

source, provide a link to the Creative Commons licence, and indicate if

changes were made. The images or other third party material in this article

are included in the article's Creative Commons licence, unless indicated

otherwise in a credit line to the material. If material is not included in the

article's Creative Commons licence and your intended use is not

permitted by statutory regulation or exceeds the permitted use, you will

need to obtain permission directly from the copyright holder. To view a

copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Endophytic community in juvenile Acer pseudoplatanus and pathogenicity of Cryptostroma corticale and other associated fungi under controlled conditions

Article

Full-text available

Feb 2024
J PLANT PATHOL

The causative agent of sooty bark disease, Cryptostroma corticale , has, for some years, caused increased damage to maples ( Acer spp.) in Germany and throughout Europe. It has been stated that this pathogen can occur latently in Acer . In this study, the composition of endophytic fungi of woody stem tissues of sycamore ( Acer pseudoplatanus ) saplings were analysed in order to investigate whether C. corticale is present in young, visually healthy maple trees from natural regeneration. The most abundant taxa of the 30 isolated endophytes were Diaporthe cf. rudis and Petrakia irregularis . An association of five fungal species ( Biscogniauxia mediterranea , Coniochaeta velutina , Gibellulopsis catenata , Neocucurbitaria quercina , Tangerinosporium thalitricola ) with A. pseudoplatanus was reported for the first time. Cryptostroma corticale was not present in the juvenile sycamore stems. Furthermore, its pathogenicity was studied in comparison to other pathogens associated with A. pseudoplatanus , namely Diplodia mutila , Dothiorella sp., Nectria cinnabarina , Neonectria coccinea , Neonectria punicea and Stegonsporium pyriforme . The longest necroses were induced by C. corticale followed by N. coccinea . In a trial with a C. corticale spore suspension sprayed directly on stem bark, the fungus could be re-isolated from woody tissue, however necroses were evoked only when the bark was wounded prior to infection. The results from the experiments presented here function as additional evidence for the ability of C. corticale to establish endophytically within the host and, in particular, for the ability of the fungus to establish a successful infection/entrance through intact bark.

Diversity of caulicolous species of the genus Diaporthe on Prunus sensu lato in Japan

Article

Full-text available

Jun 2024

The genus Diaporthe Fuckel consists of a large number of species recorded as pathogens, saprobes, and endophytes. While more than 1200 species of Diaporthe have been recorded to date, only pathogenic strains of Diaporthe were reported from Japan, notably affecting economically important plants. This study successfully elucidated the diversity of endophytic Diaporthe spp. from Prunus sensu lato (s.l.), commonly known as the Sakura tree, along with the addition of two novel species of Diaporthe to the Japanese mycobiota. In this study, a polyphasic approach was employed, using multi-locus analysis of 5 loci, including internal transcribed spacer (ITS) region, partial sequence of translation elongation factor 1–alpha (TEF), beta-tubulin (TUB), histone H3 (HIS) and calmodulin (CAL) genes. Additionally, morphological observation was also conducted by inducing sporulation of the isolates in artificial media. As a result, two novel species were introduced: Diaporthe endoprunicola A.H. Ujat & Y. Hattori and Diaporthe pseudoamygdali A.H. Ujat & Y. Hattori, along with confirmation of previously documented Diaporthe species in Prunus s.l.

Taxonomic analysis reveals host preference of rare fungi in endophytes of Vitis vinifera from the Czech Republic

Article

Full-text available

Jun 2023
FOLIA MICROBIOL

This paper represents the results of screening a diversity of fungal endophytes associated with Vitis vinifera leaves and canes in the Czech Republic. The characterization of strains is based on morphological and phylogenetic analyses of ITS, EF1α and TUB2 sequence data. Our strain selection covers 16 species and seven orders belonging to Ascomycota and Basidiomycota. Together with ubiquitous fungi, we report on several poorly known plant-associated fungi, Angustimassarina quercicola (= A. coryli, a synonym proposed in this study) and Pleurophoma pleurospora. Other species, such as Didymella negriana, D. variabilis, Neosetophoma sp. (species identical or sister to N. rosae), Phragmocamarosporium qujingensis and Sporocadus rosigena, have so far been little known and rarely found, but are frequent on V. vinifera in different parts of the world and obviously belong to a microbiota with a strong preference for this plant. Detailed taxonomical identification allowed us to identify species with apparent stable associations with V. vinifera, for which further interactions with V. vinifera can be expected. Our study is the first to focus on V. vinifera endophytes in Central Europe and expands the knowledge about their taxonomy, ecology and geography.

Rustøen et al. 2023 Substrate affinities of wood decay fungi are foremost structured by wood properties not climate

Article

Jun 2023
FUNGAL ECOL

Wood decomposing fungi differ in their substrate affinities, but to what extent factors like wood properties influence host specialization, compared to climate, is largely unknown. In this study, we analysed British field observations of 61 common wood decay species associated with 41 tree and shrub genera. While white rot fungi ranged from low-to high-substrate affinity, brown rot fungi were exclusively mid-to high-affinity. White rot fungi associated with dead fallen wood demonstrated the least substrate affinity. The composition of wood decomposer fungi was mostly structured by substrate properties, sorted between angiosperms and conifers. Any relationships with temporal and regional climate variability were of far less significance, but did predict community-based and substrate-usage host shifts, especially for fungi on fallen deadwood. Our results demonstrate that substrate shifts by wood-decay fungi will depend primarily upon their degree of affinity to, and the distribution of, related woody genera, followed less at regional levels by climate impacts.

Identification and Pathogenicity of Biscogniauxia and Sordaria Species Isolated from Olive Trees

Article

Full-text available

Mar 2024

A field investigation of olive trees in Istria, Croatia, revealed branch dieback and cracked bark. Samples of diseased branches were collected from eight different locations and analysed. Additionally, meteorological data from two locations were analysed to determine if there was a connection between climatic changes and the appearance of pathogens in the region. Pathogenicity tests were conducted on olive seedlings. This study provides a description of Biscogniauxia and Sordaria species' morphology and elucidates their phylogeny based on the internal transcribed spacer (ITS), beta-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1-α) regions. This research represents the first documented occurrence of Biscogniauxia mediterranea causing charcoal disease in olive trees in Croatia. Additionally, it is the first report of Biscogniauxia nummularia (Bull.) Kuntze and Sordaria fimicola causing diseases in olive trees anywhere in the world. Furthermore, this study marks one of the initial forays into molecular investigations of these species isolated from olive trees. Considering the potential threat posed by the inherent aggressiveness of Biscogniauxia species, further research is deemed necessary to curb the development of charcoal disease.

Fungal Trunk Diseases Causing Decline of Apricot and Plum Trees in the Czech Republic

Article

Full-text available

Dec 2023
PLANT DIS

Fungal trunk diseases (FTDs) have been a significant threat to the global stone fruit industry. FTDs are caused by a consortium of wood-decaying fungi. These fungi colonize woody tissues, causing cankers, dieback, and other decline-related symptoms in host plants. In this study, a detailed screening of the fungal microbiota associated with the decline of stone fruit trees in the Czech Republic was performed. The wood fragments of plum and apricot trees showing symptoms of FTDs were subjected to fungal isolation. The partial internal transcribed spacer (ITS) region, partial beta-tubulin (tub2) and translation elongation factor 1-α (tef) genes were amplified from genomic DNA extracted from fungal cultures. All isolates were classified, and the taxonomic placement of pathogenic strains was illustrated in phylogenetic trees. The most abundant pathogenic genus was Dactylonectria (31 %), followed by Biscogniauxia (13 %), Thelonectria (10 %), Eutypa (9 %), Dothiorella (7 %), Diplodia (6 %), and Diaporthe (6 %). The most frequent endophytic genus was Aposphaeria (17 %). The pathogenicity of six fungal spp. (Cadophora daguensis, Collophorina africana, Cytospora sorbicola, Dothiorella sarmentorum, Eutypa lata, and Eutypa petrakii var. petrakii to four Prunus spp. was evaluated and the Koch’s postulates were fulfilled. All tested isolates caused lesions on at least one Prunus sp. The most aggressive species was E. lata, which caused the largest lesions on all four tested Prunus spp., followed by E. petrakii var. petrakii, and D. sarmentorum. Japanese plum (Prunus salicina) and almond (P. amygdalus) were the most susceptible hosts while apricot (P. armeniaca) was the least susceptible host in the pathogenicity trial.

Fungal Community Taxa Differ in Diversity and Number between Live and Dead Prunus serotina Ehrh. Wood in a Protected Forest within Its Secondary Range of Distribution

Article

Full-text available

Jun 2023

Prunus serotina is an important invasive plant species in Poland. It was introduced to European forests, even in places currently protected by law, such as national parks. One major factor contributing to the spread of this species is the lack of infecting pathogens and incomplete knowledge about the relationship between the plant and microorganisms. This study aimed to identify fungal communities collected from freshly cut stumps of live and dead black cherry tree specimens growing in a protected forest. The main working hypotheses were as follows: (i) fungal communities inhabiting the wood of dead and living trees will differ in diversity and the number of taxa; and (ii) saprotrophic fungi will dominate in the wood of dead tree stumps. This study applied Illumina sequencing based on the amplification of the fungal ribosomal ITSI region. The average number of sequences (OTU) obtained from the analysis of dead tree wood was 101,758, while that of living trees was 94,150. These sequences belonged to 312 taxa, among which 254 were isolated from the wood of dead trees and 171 from that of living trees. Among the saprotrophs on dead trees, the following species were identified: Stereum rugosum, Ganoderma adspersum, G. applanatum, Peniophora cinerea, and Ascocoryne cylichnium. On the other hand, in the wood of living trees, Cytospora leucostoma and Botrytis cinerea were the most abundant saprotrophic species. The fungal communities inhabiting the wood of dead and living trees differed in the diversity and abundance of taxa, thus confirming our hypothesis. The results of our research conducted in a protected area indicate that black cherry wood can be naturally colonized by many pathogen species that can further limit its expansion.

Fungi associated with stem collar necroses of Fraxinus excelsior affected by ash dieback

Article

Full-text available

Jun 2023

In recent decades the vitality and physical stability of European ash trees in Germany have been reduced by European ash dieback, especially when associated with stem collar necroses and rots. This study was carried out to investigate the composition of the fungal communities associated with stem collar necroses. Filamentous fungi were isolated from 58 ash trees out of nine forest stands in northern, eastern, and central Germany. Obtained isolates were identified to a genus or species level by means of morphological and molecular analyses. In total 162 morphotypes including endophytic, saprotrophic, and pathogenic fungi were isolated. For 33 species found no prior reports from Fraxinus excelsior were recognised, including Cryptostroma corticale and Diplodia sapinea . None of the identified species were found at all studied sites, though Diplodia fraxini was the most common fungus with regard to frequency within all isolates , occurring at seven sample sites. This species is followed by Hymenoscyphus fraxineus , Armillaria spp., Neonectria punicea , Diaporthe cf. eres , Fusarium cf. lateritium , and Paracucurbitaria sp. in order of frequency within all isolates. The aforementioned species are characterised and analysed in respect to their occurrence in stem collar necroses and at sample sites. The influence of site conditions on the fungal composition was described for five intensively sampled sites with a minimum of five studied trees (Schwansee, Rhüden, Berggießhübel, Satrup, and Schlangen). The sampling site of Schlangen was further subdivided into four subplots with different positions in the terrain. In the remaining four extensive sample sites, either one or two trees, respectively, were sampled and analysed (Oranienbaumer Heide, Woltershausen, Wolfenbüttel, and Neuhege). Over all sample sites, fungal communities of symptomatic stem tissue are similar concerning the most frequent fungi, but vary greatly according to singularly isolated fungi.

Habitat and tree species identity shape aboveground and belowground fungal communities in central European forests

Article

Full-text available

Mar 2023

IntroductionTrees interact with fungi in mutualistic, saprotrophic, and pathogenic relationships. With their extensive aboveground and belowground structures, trees provide diverse habitats for fungi. Thus, tree species identity is an important driver of fungal community composition in forests.Methods Here we investigate how forest habitat (bark surface vs. soil) and tree species identity (deciduous vs. coniferous) affect fungal communities in two Central European forests. We assess differences and interactions between fungal communities associated with bark surfaces and soil, in forest plots dominated either by Fagus sylvatica, Picea abies, or Pinus sylvestris in two study regions in southwestern and northeastern Germany.ResultsITS metabarcoding yielded 3,357 fungal amplicon sequence variants (ASVs) in the northern and 6,088 in the southern region. Overall, soil communities were 4.7 times more diverse than bark communities. Habitat type explained 48–69% of the variation in alpha diversity, while tree species identity explained >1–3%. NMDS ordinations showed that habitat type and host tree species structured the fungal communities. Overall, few fungal taxa were shared between habitats, or between tree species, but the shared taxa were highly abundant. Network analyses, based on co-occurrence patterns, indicate that aboveground and belowground communities form distinct subnetworks.DiscussionOur study suggests that habitat (bark versus soil) and tree species identity are important factors structuring fungal communities in temperate European forests. The aboveground (bark-associated) fungal community is currently poorly known, including a high proportion of reads assigned to “unknown Ascomycota” or “unknown Dothideomycetes.” The role of bark as a habitat and reservoir of unique fungal diversity in forests has been underestimated.

Fungi associated with stem collar necroses of Fraxinus excelsior affected by ash dieback

Preprint

Full-text available

Jan 2023

In recent decades the vitality and physical stability of European ash trees in Germany have been reduced by European ash dieback, especially when associated with stem collar necroses and rots. This study was carried out to investigate the composition of the fungal communities associated with stem collar necroses. Filamentous fungi were isolated from 58 ash trees out of nine forest stands in northern, eastern, and central Germany. Obtained isolates were identified to a genus or species level by means of morphological and molecular analyses. In total 162 morphotypes including endophytic, saprotrophic and pathogenic fungi were isolated. For 33 species found no prior reports from Fraxinus excelsior were recognised, including Cryptostroma corticale and Diplodia sapinea . None of the identified species were found at all studied sites, though Diplodia fraxini was the most common fungi with regard to frequency within all isolates, occurring at seven sample sites. This species is followed by Hymenoscyphus fraxineus , Armillaria spp., Neonectria punicea , Diaporthe cf. eres , Fusarium cf. lateritium , and Paracucurbitaria sp. in order of frequency within all isolates. The aforementioned species are characterised and analysed in respect to their occurrence in stem collar necroses and at sample sites. The influence of site conditions on the fungal composition was described for five intensively sampled sites with a minimum of five studied trees (Schwansee, Rhüden, Berggießhübel, Satrup, and Schlangen). The sampling site of Schlangen was further subdivided into four subplots with different positions in the terrain. In the remaining four extensive sample sites either one or two trees, respectively, were sampled and analysed (Oranienbaumer Heide, Woltershausen, Wolfenbüttel, and Neuhege). Over all sample sites fungal communities of symptomatic stem tissue are similar concerning the most frequent fungi, but vary greatly according to singularly isolated fungi.

Arboricolonus simplex gen. et sp. nov. and novelties in Cadophora, Minutiella and Proliferodiscus from Prunus wood in Germany

Article

Full-text available

Mar 2020

During a survey on fungi associated with wood necroses of Prunus trees in Germany, strains belonging to the Leotiomycetes and Eurotiomycetes were detected by preliminary analyses of ITS sequences. Multi-locus phylogenetic analyses (LSU, ITS, TUB, EF-1α, depending on genus) of 31 of the 45 strains from Prunus and reference strains revealed several new taxa, including Arboricolonus gen. nov., a new genus in the Helotiales (Leotiomycetes) with a collophorina-like asexual morph. Seven Cadophora species (Heloti-ales, Leotiomycetes) were treated. The 29 strains from Prunus belonged to five species, of which C. luteo-olivacea and C. novi-eboraci were dominating; C. africana sp. nov., C. prunicola sp. nov. and C. ramosa sp. nov. were revealed as new species. The genus Cadophora was reported from Prunus for the first time. Phialophora bubakii was combined in Cadophora and differentiated from C. obscura, which was resurrected. Asexual morphs of two Proliferodiscus species (Helotiales, Leotiomycetes) were described, including one new species, Pr. ingens sp. nov. Two Minutiella species (Phaeomoniellales, Eurotiomycetes) were detected, including the new species M. pruni-avium sp. nov. Prunus avium and P. domestica are reported as host plants of Minutiella.

Novel collophorina-like genera and species from Prunus trees and vineyards in Germany

Article

Full-text available

Sep 2019

Strains with a yeast-like appearance were frequently collected in two surveys on the biodiversity of fungi in Germany, either associated with necroses in wood of Prunus trees in orchards in Saxony, Lower Saxony and Baden-Württemberg or captured in spore traps mounted on grapevine shoots in a vineyard in Rhineland-Palatinate. The morphology of the strains was reminiscent of the genus Collophorina : all strains produced aseptate conidia on integrated conidiogenous cells directly on hyphae, on discrete phialides, adelophialides and by microcyclic conidiation, while in some strains additionally endoconidia or conidia in conidiomata were observed. Blastn searches with the ITS region placed the strains in the Leotiomycetes close to Collophorina spp. Analyses based on morphological and multi-locus sequence data (LSU, ITS, EF-1α , GAPDH ) revealed that the 152 isolates from wood of Prunus spp. belong to five species including C. paarla , C. africana and three new species. A further ten isolates from spore traps belonged to seven new species, of which one was isolated from Prunus wood as well. However, a comparison with both LSU and ITS sequence data of these collophorina-like species with reference sequences from further Leotiomycetes revealed the genus Collophorina to be polyphyletic and the strains to pertain to several genera within the Phacidiales . Collophorina paarla and C. euphorbiae are transferred to the newly erected genera Pallidophorina and Ramoconidiophora , respectively. The new genera Capturomyces , Variabilispora and Vexillomyces are erected to accommodate five new species isolated from spore traps. In total nine species were recognised as new to science and described as Collophorina badensis , C. germanica , C. neorubra , Capturomyces funiculosus , Ca. luteus , Tympanis inflata , Variabilispora flava , Vexillomyces palatinus and V. verruculosus .

The unbearable lightness of sequence-based identification

Article

Full-text available

May 2019

Using the basic GenBank local alignment search tool program (BLAST) to identify fungi collected in a recently protected beech forest at Montricher (Switzerland), the number of ITS sequences associated to the wrong taxon name appears to be around 30%, even higher than previously estimated. Such results rely on the in-depth re-examination of BLAST results for the most interesting species that were collected, viz. first records for Switzerland, rare or patrimonial species and problematic species (when BLAST top scores were equally high for different species), all belonging to Agaricomycotina. This paper dissects for the first time a number of sequence-based identifications, thereby showing in every detail-particularly to the user community of taxonomic information-why sequence-based identification in the context of a fungal inventory can easily go wrong. Our first conclusion is that in-depth examination of BLAST results is too time consuming to be considered as a routine approach for future inventories: we spent two months on verification of approx. 20 identifications. Apart from the fact that poor taxon coverage in public depositories remains the principal impediment for successful species identification, it can be deplored that even very recent fungal sequence deposits in GenBank involve an uncomfortably high number of misidentifications or errors with associated metadata. While checking the original publications associated with top score sequences for the few examples that were here reexamined , a positive consequence is that we uncovered over 80 type sequences that were not annotated as types in GenBank. Advantages and pitfalls of sequence-based identification are discussed, particularly in the light of undertaking fungal inventories. Recommendations are made to avoid or reduce some of the major problems with sequence-based identification. Nevertheless, the prospects for a more reliable sequence-based identification of fungi remain quite dim, unless authors are ready to check and update the metadata associated with previously deposited sequences in their publications.

Entwicklungen des Obstbaus in Deutschland von 2005 bis 2017: Obstarten, Anbauregionen, Betriebsstrukturen und Handel

Book

Full-text available

Aug 2018

In Deutschland wurde im Jahr 2017 auf rund 76.000 ha in rund 11.000 Betrieben Obst angebaut. Der Obstbau ist gegliedert in den Anbau von Baumobst (49.934 ha), Strauchbeeren (6.838 ha) und Erdbeeren (18.649 ha). Der Bericht gibt einen umfassenden Überblick über die Entwicklung der Anbauflächen und Betriebsstrukturen in den Bundesländern im Zeitraum von 2005/06 bis 2016/17 sowie eine detaillierte Charakterisierung des Obstanbaus in den Bundesländern nach angebauten Obstkulturen in den Landkreisen. Darüber hinaus wird der nationale und internationale Handel mit Frischobst in diesem Working Paper beschrieben. Die Baumobstanbauflächen sind im betrachteten Zeitraum zunächst leicht zurückgegangen, um dann laut Baumobsterhebung 2017 wieder anzusteigen. Vor allem die Anbauflächen von Sauerkirschen haben deutlich abgenommen, während die Süßkirschenfläche nach einem Rückgang bis 2012 wieder leicht angestiegen ist. Die Anbauflächen von Birnen sowie Pflaumen und Zwetschen haben zwischen 2007 und 2012 abgenommen und sind seitdem auf fast gleichem Niveau stabil geblieben. Mit etwa 68 % der Baumobstfläche stehen die Äpfel an erster Stelle. Dabei ist die Apfelanbaufläche in den letzten 10 Jahren in etwa konstant geblieben. Der Strauchbeerenanbau ist im gleichen Zeitraum um rund 44 % angestiegen. Dies ist vor allem auf den Anstieg des Heidel-beeranbaus zurückzuführen, aber auch die Gesamtgröße der Anbaufläche von Sanddorn, Aroniabeeren, Stachelbeeren, Brombeeren und Holunder ist gestiegen. Die Größe der Anbaufläche von Himbeeren im Freiland ist zwar zurückgegangen, allerdings gab es starke Zuwächse im Anbau unter Glas. Etwa ein Viertel der Strauchbeerenfläche entfällt auf Betriebe mit vollständigem oder teilweisem ökologischen Anbau. Die Fläche für den Erdbeeranbau hat sich im Betrachtungszeitraum von 17.500 ha in 2006 zunächst auf rund 16.000 ha in 2009 reduziert, um dann bis 2013 auf einen Wert von rund 19.400 ha zu wachsen. Seitdem ist die Erdbeeranbaufläche besonders im Freiland rückläufig, während der geschützte Anbau stetig ansteigt. Die Betriebsstrukturen zeigen eine typische Verteilung der Betriebsgrößen zwischen den Bundesländern. In Süddeutschland sind die Obstbaubetriebe im Durchschnitt deutlich kleiner als in Nord- und Ostdeutschland. Die größten durchschnittlichen Anbauflächen je Betrieb sind in Thüringen, Sachsen und Brandenburg zu finden. Allerdings hat sich die durchschnittliche Anbaufläche von Baumobst je Betrieb in den neuen Bundesländern zwischen 2012 und 2017 verringert. Bezogen auf die Obstarten ist die durchschnittliche Anbaufläche je Betrieb für Erdbeeren mit 7,9 ha (Daten für 2016) am größten, gefolgt von den Strauchbeeren mit 6,8 ha (Daten für 2017) und dem Baumobst mit 7 ha (Daten für 2017). Dabei ist die Variation zwischen den Bundesländern enorm. Im Baumobstanbau liegt Sachsen mit durchschnittlich 58 ha je Betrieb vorn, im Strauchbeerenanbau Mecklenburg-Vorpommern mit rund 18 ha und bei den Erdbeeren Schleswig-Holstein mit rund 13 ha durchschnittlicher Anbaufläche je Betrieb. Die Verteilung der Anbauflächen nach Größenklassen zeigt, dass bei allen Obstkulturen ein großer Anteil der Anbaufläche in Betrieben mit über 10 ha liegt. Die Entwicklung der Betriebsstrukturen im Betrachtungszeitraum verdeutlicht, dass sich der Strukturwandel im Obstbau fortsetzt: Die Anzahl der Betriebe sinkt vor allem in den niedrigen Größenklassen, während die durchschnittliche Anbaufläche je Betrieb stetig ansteigt. Die Analyse der regionalen Verteilung des Obstbaus zeigt deutliche und jeweils typische Anbauschwerpunkte. Beispielhaft zu nennen sind die Bodenseeregion (v. a. der Bodenseekreis) und das Alte Land (v. a. der Landkreis Stade) mit dem Apfelanbau sowie die Rheinebene Baden-Württembergs (v. a. der Ortenaukreis) mit einem Schwerpunkt im Anbau von Süßkirschen, Pflaumen und Zwetschen. Im niedersächsischen Vechta werden mehr Erdbeeren angebaut als in jedem anderen Landkreis Deutschlands, wohingegen ganz in der Nähe, in den Landkreisen Diepholz und Nienburg, Deutschlands größtes Heidelbeeranbaugebiet liegt. Auf Großhandelsebene standen im Jahr 2014 aus deutscher Erzeugung knapp 1,5 Mio. t Frischobst zur Verfügung. Davon wurden rund 43 % über Erzeugerorganisationen vermarktet, während 17 % von meist größeren Erzeugern selbst an den Lebensmitteleinzelhandel vermarktet wurden. Etwa ein Viertel ging in die Verarbeitung. Ergänzt wird die deutsche Erzeugung auf Großhandelsebene durch Importe, die im Jahr 2014 etwa 6,3 Mio. t erreichten. Knapp zwei Drittel davon waren Südfrüchte. Demgegenüber sind die Exporte mit rund 0,7 Mio. t nur von geringer Bedeutung. Bei den Einkaufsstätten der Verbraucher für Frischobst nimmt der Lebensmitteleinzelhandel mit 83 % der Menge eine herausragende Bedeutung ein. Auch wenn der Einkauf auf Wochenmärkten und Hofläden an Bedeutung gewonnen hat, werden hier insgesamt nur 6,4 % der Frischobstmenge an die Verbraucher abgesetzt. Der Export von Obst aus Deutschland zeigt insgesamt keinen eindeutigen Trend auf. Die Apfelexportmengen schwanken stark im Betrachtungszeitraum von 2007 bis 2016, bedingt unter anderem durch die jährlichen Erntemengen, die Bedingungen auf den europäischen Märkten und auch durch den russischen Importstopp. Bei Birnen und Quitten scheinen sich die Ausfuhren leicht rückläufig zu entwickeln, während die Exporte von Heidelbeeren zwar auf einem insgesamt niedrigen Niveau liegen, aber deutlich angestiegen sind. Bei den Importen sind verschiedene Entwicklungen zu beobachten: Während die Einfuhren traditioneller Obstarten inklusive der Südfrüchte wie Bananen, Orangen, Ananas und Tafeltrauben annähernd gleich geblieben oder leicht zurück-gegangen sind, sind die Importe von Wasser- und Zuckermelonen, exotischen Früchten wie Mangos und Avocados sowie von Beerenfrüchten, insbesondere Heidelbeeren und Himbeeren, deutlich angestiegen. Eine gemeinsame Betrachtung der Veränderungen in der Obsterzeugung und im internationalen Handel zeigt eine allgemeine Entwicklungsrichtung des Obstsektors hin zu einer Diversifizierung. Der Anbau bzw. Import von Obstarten, die traditionell in großen Mengen nachgefragt werden (z. B. Äpfel, Birnen, Pflaumen und Zwetschen, Bananen und Orangen), stagniert oder ist sogar rückläufig. Parallel dazu steigt die Nachfrage nach verschiedenen Beerenfrüchten, insbesondere Heidelbeeren und Himbeeren, sowie nach exotischen Südfrüchten. Auf der Angebotsseite führen die Ausweitung des geschützten Anbaus von Erdbeeren und Himbeeren sowie eine zunehmende Überdachung von Süßkirschen zu einer höheren Qualität und einer Ausweitung der zeitlichen Verfügbarkeit dieser stark saisonalen Früchte. Den gleichen Effekt haben eine verbesserte Lager- und Transporttechnik sowie Innovationen im Bereich Verpackung für eine Verlängerung der Haltbarkeit. Dies betrifft sowohl die deutsche Erzeugung als auch die europäischen und außereuropäischen Wettbewerber, so dass die Verfügbarkeit an Obst in Deutschland insgesamt besser und vielfältiger geworden ist. JEL Codes: Q15, Q 17 Schlüsselwörter: Baumobstanbau, Strauchbeerenanbau, Erdbeeren, Strukturwandel, Obstan-baustatistik, Warenstromanalyse Frischobst, Importe und Exporte Obst

Phylogenetic Identification of Pathogenic and Endophytic Fungal Populations in West Coast Douglas-fir (Pseudotsuga menziesii) Foliage

Thesis

Full-text available

Sep 2017

Hazel Daniels

Douglas-fir provides social, economic, and ecological benefits in the Pacific Northwest (PNW). In addition to timber, forests support abundant plant and animal biodiversity and provide socioeconomic viability for many rural communities. Products derived from Douglas-fir account for approximately 17% of the U.S. lumber output with an estimated value of $1.9 billion dollars. Employment related to wood production accounts for approximately 61,000 jobs in Oregon. Timberland also supports water resources, recreation, and wildlife habitat. Minor defoliation has previously been linked to Swiss Needle Cast, associated with the fungus Phaeocryptopus gaeumannii, however, unprecedented large-scale defoliation began in the 1990s and has increased since, leading to decreased growth and yield. Areas affected areas by SNC exceed 500,000 acres in Oregon. Defoliation symptoms are inconsistent with predicted effects of P. gaeumannii, and targeted chemical control has had mixed results. While the microbiome community of conifer needles is poorly described to date, we hypothesize the full interstitial microbiome complex is involved in disease response in conifers.

Diplodia Tip Blight on Its Way to the North: Drivers of Disease Emergence in Northern Europe

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Jan 2019

Disease emergence in northern and boreal forests has been mostly due to tree-pathogen encounters lacking a co-evolutionary past. However, outbreaks involving novel interactions of the host or the pathogen with the environment have been less well documented. Following an increase of records in Northern Europe, the first large outbreak of Diplodia sapinea on Pinus sylvestris was discovered in Sweden in 2016. By reconstructing the development of the epidemic, we found that the attacks started approx. 10 years back from several isolated trees in the stand and ended up affecting almost 90% of the trees in 2016. Limited damage was observed in other plantations in the surroundings of the affected stand, pointing to a new introduced pathogen as the cause of the outbreak. Nevertheless, no genetic differences based on SSR markers were found between isolates of the outbreak area and other Swedish isolates predating the outbreak or from other populations in Europe and Asia Minor. On a temporal scale, we saw that warm May and June temperatures were associated with higher damage and low tree growth, while cold and rainy conditions seemed to favor growth and deter disease. At a spatial scale, we saw that spread occurred predominantly in the SW aspect-area of the stand. Within that area and based on tree-ring and isotope (δ¹³C) analyses, we saw that disease occurred on trees that over the years had shown a lower water-use efficiency (WUE). Spore traps showed that highly infected trees were those producing the largest amount of inoculum. D. sapinea impaired latewood growth and reduced C reserves in needles and branches. D. sapinea attacks can cause serious economic damage by killing new shoots, disrupting the crown, and affecting the quality of stems. Our results show that D. sapinea has no limitations in becoming a serious pathogen in Northern Europe. Management should focus on reducing inoculum, especially since climate change may bring more favorable conditions for this pathogen. Seedlings for planting should be carefully inspected as D. sapinea may be present in a latent stage in asymptomatic tissues.

Identification and Characterization of Phomopsis amygdali and Botryosphaeria dothidea Associated with Peach Shoot Blight in Yangshan, China

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Jun 2018
PLANT DIS

Peach (Prunus persica (L.) Batsch) is produced locally in Yangshan, Wuxi City, China. In recent years, a widespread shoot blight has been observed in many peach orchards of Yangshan that kills the twigs and results in high losses in fruit production. Disease incidences ranged from 10 to 20% in the affected orchards and, in extreme cases, 40% of the trees were affected. Shoot blight of peach is caused by a fungus, previously identified as Phomopsis amygdali. Between 2014 and 2015, samples were collected four times from three peach orchards located in Yangshan to understand the etiology of shoot blight. Interestingly, two types of shoot blight symptoms were observed: one characterized by necrotic lesions with rings and one without rings. Based on conidial morphology, cultural characteristics, and analysis of nucleotide sequences of three genomic regions (the internal transcribed spacer region, a partial sequence of the β-tubulin gene, and the translation elongation factor 1-α), isolates were identified as P. amygdali and Botryosphaeria dothidea. Remarkably, most of the P. amygdali isolates were recovered from twigs showing necrotic lesions without rings. In contrast, most of the B. dothidea isolates were recovered from twigs with rings in the necrotic lesions. Correlations among pathogens, sampling regions, and disease symptoms were noted, and growth rates of these pathogens were characterized. Pathogenicity tests showed that B. dothidea isolates could induce necrotic lesions with rings but P. amygdali isolates could only induce necrotic lesions. Moreover, the B. dothidea isolates exhibited higher levels of virulence than P. amygdali isolates on the peach twig. Additionally, high frequencies of detection of both P. amygdali and B. dothidea from buds indicated that buds may be the primary site of fungal invasion. Cankers and necrotic twigs may also serve as infection courts. Our results suggest that B. dothidea and P. amygdali are the common causal agents of peach shoot blight in Yangshan, China. This finding provides a basis for the development of effective management strategies.

The ash dieback invasion of Europe was founded by two genetically divergent individuals

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Jun 2018
Nat. Ecol. Evol.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

Direct comparison of culture-dependent and culture-independent molecular approaches reveal the diversity of fungal endophytic communities in stems of grapevine (Vitis vinifera)

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Apr 2018

Grapevines (Vitis vinifera) are colonized by ubiquitous microorganisms known as endophytes, which may have advantageous or neutral effects without causing disease symptoms. Certain endophytes are uncultivable, so culture-independent approaches such as next generation sequencing (NGS) can help for a better understanding of their ecology and distribution. To date, there are no studies which directly link NGS results with taxa derived from a culturing approach, integrating morphological and multi-gene phylogenetic analysis of endophytes. In this study, a culture-dependent and high-resolution culture-independent approach (next generation sequencing) were used to identify endophytes in grapevine stems. In the culture-dependent approach, a total of 94 isolates were recovered from 84 of 144 healthy grapevine stem fragments (colonization rate = 58.3%). The study is unique as we used subsets of combined multi-gene regions to identify the endophytes to species level. Based on each multi-gene phylogenetic analysis, 28 species belong to 19 genera (Acremonium, Alternaria, Arthrinium, Ascorhizoctonia, Aspergillus, Aureobasidium, Bipolaris, Botryosphaeria, Botrytis, Chaetomium, Cladosporium, Curvularia, Hypoxylon, Lasiodiplodia, Mycosphaerella, Nigrospora, Penicillium, Phoma, Scopulariopsis) were identified. A higher number of culturable fungi were obtained from 13 year-old vines, followed by eight and three year-old vines. In the culture-independent approach, a fungal richness of 59 operational taxonomic units (OTU) was detected, being highest in 13 year-old grapevines, followed by eight and three years. Even though the cultivation approach detected lower fungal richness, the results related to stem are consistent for fungal community composition and richness. Comparison of the fungal taxa identified by the two approaches resulted in an overlap of 53% of the fungal genera. Due to interspecific variability of the sequences from NGS, in many cases the OTUs (even with the highly abundant ones) were only assignable to order, family or genus level. Incorporating multi-gene phylogenies we successfully identified many of the NGS derived OTUs with poor taxonomic information in reference databases to the genus or species levels. Hence, this study signifies the importance of applying both culture-dependent and culture-independent approaches to study the fungal endophytic community composition in Vitis vinifera. This principle could also be applied to other host species and ecosystem level studies.

Endophytic fungi in branches of sour cherry trees: a preliminary study.

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Aug 2011

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Arboricolonus simplex gen. et sp. nov. and novelties in Cadophora, Minutiella and Proliferodiscus fr...

March 2020 · Mycokeys

During a survey on fungi associated with wood necroses of Prunus trees in Germany, strains belonging to the Leotiomycetes and Eurotiomycetes were detected by preliminary analyses of ITS sequences. Multi-locus phylogenetic analyses (LSU, ITS, TUB, EF-1α, depending on genus) of 31 of the 45 strains from Prunus and reference strains revealed several new taxa, including Arboricolonus gen. nov., a new ... [Show full abstract] genus in the Helotiales (Leotiomycetes) with a collophorina-like asexual morph. Seven Cadophora species (Heloti-ales, Leotiomycetes) were treated. The 29 strains from Prunus belonged to five species, of which C. luteo-olivacea and C. novi-eboraci were dominating; C. africana sp. nov., C. prunicola sp. nov. and C. ramosa sp. nov. were revealed as new species. The genus Cadophora was reported from Prunus for the first time. Phialophora bubakii was combined in Cadophora and differentiated from C. obscura, which was resurrected. Asexual morphs of two Proliferodiscus species (Helotiales, Leotiomycetes) were described, including one new species, Pr. ingens sp. nov. Two Minutiella species (Phaeomoniellales, Eurotiomycetes) were detected, including the new species M. pruni-avium sp. nov. Prunus avium and P. domestica are reported as host plants of Minutiella.

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Novel collophorina-like genera and species from Prunus trees and vineyards in Germany

September 2019 · Persoonia - Molecular Phylogeny and Evolution of Fungi

Strains with a yeast-like appearance were frequently collected in two surveys on the biodiversity of fungi in Germany, either associated with necroses in wood of Prunus trees in orchards in Saxony, Lower Saxony and Baden-Württemberg or captured in spore traps mounted on grapevine shoots in a vineyard in Rhineland-Palatinate. The morphology of the strains was reminiscent of the genus Collophorina ... [Show full abstract] : all strains produced aseptate conidia on integrated conidiogenous cells directly on hyphae, on discrete phialides, adelophialides and by microcyclic conidiation, while in some strains additionally endoconidia or conidia in conidiomata were observed. Blastn searches with the ITS region placed the strains in the Leotiomycetes close to Collophorina spp. Analyses based on morphological and multi-locus sequence data (LSU, ITS, EF-1α , GAPDH ) revealed that the 152 isolates from wood of Prunus spp. belong to five species including C. paarla , C. africana and three new species. A further ten isolates from spore traps belonged to seven new species, of which one was isolated from Prunus wood as well. However, a comparison with both LSU and ITS sequence data of these collophorina-like species with reference sequences from further Leotiomycetes revealed the genus Collophorina to be polyphyletic and the strains to pertain to several genera within the Phacidiales . Collophorina paarla and C. euphorbiae are transferred to the newly erected genera Pallidophorina and Ramoconidiophora , respectively. The new genera Capturomyces , Variabilispora and Vexillomyces are erected to accommodate five new species isolated from spore traps. In total nine species were recognised as new to science and described as Collophorina badensis , C. germanica , C. neorubra , Capturomyces funiculosus , Ca. luteus , Tympanis inflata , Variabilispora flava , Vexillomyces palatinus and V. verruculosus .

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Characterization of the Castanopsis carlesii Deadwood Mycobiome by Pacbio Sequencing of the Full-Len...

May 2019 · Frontiers in Microbiology

Short-read next generation sequencing (NGS) platforms can easily and quickly generate thousands to hundreds of thousands of sequences per sample. However, the limited length of these sequences can cause problems during fungal taxonomic identification. Here we validate the use of Pacbio sequencing, a long-read NGS method, for characterizing the fungal community (mycobiome) of Castanopsis carlesii ... [Show full abstract] deadwood. We report the successful use of Pacbio sequencing to generate long-read sequences of the full-length (500-780 bp) fungal ITS regions of the C. carlesii mycobiome. Our results show that the studied deadwood mycobiome is taxonomically and functionally diverse, with an average of 85 fungal OTUs representing five functional groups (animal endosymbionts, endophytes, mycoparasites, plant pathogens, and saprotrophs). Based on relative abundance data, Basidiomycota were the most frequently detected phyla (50% of total sequences), followed by unidentified phyla, and Ascomycota. However, based on presence/absence data, the most OTU-rich phyla were Ascomycota (58% of total OTUs, 72 OTUs) followed by Basidiomycota and unidentified phyla. The majority of fungal OTUs were identified as saprotrophs (70% of successfully function-assigned OTUs) followed by plant pathogens. Finally, we used phylogenetic analysis based on the full-length ITS sequences to confirm the species identification of 14/36 OTUs with high bootstrap support (99-100%). Based on the numbers of sequence reads obtained per sample, which ranged from 3,047 to 13,463, we conclude that Pacbio sequencing can be a powerful tool for characterizing moderate- and possibly high-complexity fungal communities.

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Grapevine pruning time affects natural wound colonization by wood- invading fungi

October 2020 · Fungal Ecology

Timing of pruning may affect wound susceptibility to wood-invading fungi, such as those associated with grapevine trunk diseases (GTDs). This study aimed to determine the effect of pruning time on natural fungal infection in six vineyards in Galicia, Spain, during two periods of three months each, from November to February and from February to May by ITS2 high-throughput amplicon sequencing. ... [Show full abstract] Fungal microbiome composition did not differ significantly with year of sampling. Pruned canes harboured a core community of fungal species, which appeared to be independent of the infection period. Accumulated rainfall over 8 and 11 weeks after pruning positively correlated with the total fungal microbiome. A seasonal effect on GTD fungal infection was detected for most genera, with a higher percent detected after pruning in February (winter) as compared with that of pruning in November (mid-autumn). Early pruning is recommended to reduce the infections caused by GTD fungi in Galicia.