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Research Article
Citizen science project reveals high diversity in Didymellaceae (Pleosporales, Dothideomycetes)
expand article infoLingwei Hou§, Margarita Hernández-Restrepo|, Johannes Zacharias Groenewald|, Lei Cai§, Pedro W. Crous|
‡ University of Chinese Academy of Sciences, Beijing, China
§ Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
| Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands
¶ Utrecht University, Utrecht, Netherlands
Open Access

Abstract

Fungal communities play a crucial role in maintaining the health of managed and natural soil environments, which directly or indirectly affect the properties of plants and other soil inhabitants. As part of a Citizen Science Project initiated by the Westerdijk Fungal Biodiversity Institute and the Utrecht University Museum, which aimed to describe novel fungal species from Dutch garden soil, the diversity of Didymellaceae, which is one of the largest families in the Dothideomycetes was investigated. A preliminary analysis of the ITS and LSU sequences from the obtained isolates allowed the identification of 148 strains belonging to the family. Based on a multi-locus phylogeny of a combined ITS, LSU, rpb2 and tub2 alignment, and morphological characteristics, 20 different species were identified in nine genera, namely Ascochyta, Calophoma, Didymella, Juxtiphoma, Nothophoma, Paraboeremia, Phomatodes, Stagonosporopsis, and Xenodidymella. Several isolates confirmed to be ubiquitous plant pathogens or endophytes were for the first time identified from soil, such as Ascochyta syringae, Calophoma clematidis-rectae, and Paraboeremia litseae. Furthermore, one new genus and 12 novel species were described from soil: Ascochyta benningiorum sp. nov., Didymella degraaffiae sp. nov., D. kooimaniorum sp. nov., Juxtiphoma kolkmaniorum sp. nov., Nothophoma brennandiae sp. nov., Paraboeremia rekkeri sp. nov., P. truiniorum sp. nov., Stagonosporopsis stuijvenbergii sp. nov., S. weymaniae sp. nov., Vandijckomycella joseae gen. nov. et sp. nov., V. snoekiae sp. nov., and Xenodidymella weymaniae sp. nov. From the results of this study, soil was revealed to be a rich substrate for members of Didymellaceae, several of which were previously known only from diseased or apparently healthy plant hosts.

Keywords

biodiversity, new taxa, Phoma, phylogeny, soil-borne fungi

Introduction

Due to high plasticity and the capacity to adapt and survive in adverse or unfavourable conditions, fungi are exceedingly successful soil inhabitants (Frąc et al. 2018). The majority of the fungal species presently known can survive in, or directly adapt to, the soil environment (Bridge and Spooner 2001; Botha 2011). Soil-borne fungi play essential roles in nutrient cycling in terrestrial ecosystems and are able to break down all kinds of organic matter, decompose soil components or act as effective biosorbents of toxic metals, thereby helping to maintain soil health (Anderson and Domsch 1973; Bender et al. 2013; Rudgers et al. 2014; Tedersoo et al. 2014; Yang et al. 2017; Frąc et al. 2018). Soil fungal communities also form symbiotic associations with plants, thereby improving nutrient absorption (Voøíšková and Baldrian 2012). Most fungal taxa found in the soil are continuously present in the environment as harmless saprobic organisms, but some also play a negative role. For instance, plant pathogenic fungi in soil could infect seedlings or other plant tissues when conditions are suitable, resulting in significant damage (van Agtmaal et al. 2017). In addition, some fungi reside in soil in the form of propagules to survive in an unsuitable environment, posing a long-term threat to other inhabitants (Maryani et al. 2019).

Didymellaceae is a ubiquitous fungal family including saprobic, endophytic and pathogenic species (Aveskamp et al. 2008, 2010; Marin-Felix et al. 2017). More than 50% of the species in this family have been reported as plant pathogens, causing great losses to a wide range of economic crops (Aveskamp et al. 2008). Other species are found in different substrates, including soil, air, and water or cyst nematodes (Dorenbosch 1970; Chen et al. 1996; Boerema et al. 2004; Aveskamp et al. 2010; Porras-Alfaro et al. 2011; Chen et al. 2015, 2017; Grishkan 2018; Valenzuela-Lopez et al. 2018), and even in some extreme environments such as deep-sea sediments, or soils in Antarctica, deserts, and karst caves (Ruisi et al. 2007; Li et al. 2016; Zhang et al. 2016a, 2016b, 2017; Chen et al. 2017; Nagano et al. 2017; Grishkan 2018). Although recent research has suggested that the soil environment represents an important niche for the discovery of novel phoma-like species (Chen et al. 2017, van Agtmaal et al. 2017), very few studies have investigated the diversity of Didymellaceae in soil, which is a massive reservoir for plant, animal and human pathogens.

The first paper systematically investigating Didymellaceae species from soil was published by Dorenbosch (1970), who provided diagnostic characteristics and a usable identification method (keys) for nine ubiquitous phoma-like fungi from soil, including Pyrenochaeta acicola, Phoma chrysanthemicola, Ph. eupyrena, Ph. exigua, Ph. fimeti, Ph. glomerata, Ph. herbarum, Ph. medicaginis var. pinodella, and Ph. prunicola (names used at that time). Later, Boerema et al. (2004) and Domsch et al. (2007) illustrated several Didymellaceae species from soil and provided their ecological distributions. Since then, a few species have been reported sporadically, along with the research of root and seed diseases, but studies of Didymellaceae from soil are still rare, with even fewer describing new taxa from soil. Most species in previous studies have been reallocated to other genera in this family based on their DNA phylogeny (Chen et al. 2015, 2017; Valenzuela-Lopez et al. 2018). To date, only approximately 30 species from eight genera in Didymellaceae have been recorded from soil, namely Ascochyta, Phoma, Didymella, Neodidymelliopsis, Epicoccum, Cumuliphoma, Ectophoma and Juxtiphoma (Dorenbosch 1970; Boerema et al. 2004; Domsch et al. 2007; Chen et al. 2017; Valenzuela-Lopez et al. 2018). Although most of the species documented from soil are plant-associated (pathogens and endophytes), some species, such as Ph. herbarum and J. eupyrena, are characterised as soil-borne (Dorenbosch 1970; Boerema et al. 2004).

Didymellaceae species from soil always produce diverse metabolites, some of which can be cytotoxic, including cytochalasin A and B, deoxaphomin, proxiphomin and tenuazonic acid (Bennett et al. 2018). Currently, most Didymellaceae species thus far found in the soil environment were originally described from plant substrates, such as leaves, seedlings, wood, stem bases or roots, some of which are even capable of wood decay (Boerema et al. 2004; Aveskamp et al. 2008, 2010; Chen et al. 2015). On the contrary, crops that are grown in close proximity to infected soil appear to be more contaminated, given that soil is a known source of plant pathogenic fungi (Paterson and Lima 2017). Besides, some species have also been reported to be opportunistic pathogens in animals and humans, such as J. eupyrena (= Phoma eupyrena) and Phoma herbarum (Bakerspigel et al. 1981; Tullio et al. 2010). Considering the potential threat and great losses caused by soil-borne pathogens, and the application in the biotechnological or pharmaceutical industries, knowledge of the diversity of Didymellaceae in soil is urgently needed to better understand the functions, interactions and ecosystem feedback of fungi in the terrestrial environment.

The present Citizen Science Project was initiated by the Westerdijk Fungal Biodiversity Institute (WI) and the Utrecht University Museum, aiming to investigate the diversity of fungi in Dutch garden soil collected by children in their home gardens from different regions in the Netherlands (Groenewald et al. 2018). During the course of this project thousands of isolates were obtained from 293 soil samples. Of these, 148 isolates were found to belong to Didymellaceae, and subsequently selected for study. The aim of the present study was to investigate the diversity of Didymellaceae from Dutch garden soil, describe and illustrate novel species, and compare them with known and related species.

Materials and methods

Sampling and isolation

Protocols for the collection and processing of soil samples are described in Groenewald et al. (2018) and Giraldo et al. (2019). Isolates are maintained in the Johanna Westerdijk (JW) working collection housed at the WI in Utrecht, the Netherlands. New and interesting strains were also deposited in the CBS fungal collection and holotypes in the fungarium at the WI, respectively.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, USA) following the manufacturer’s protocols. Initially, the internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA gene (ITS) and partial large subunit nrDNA (LSU) were amplified using primer pairs ITS5/ITS4 (White et al. 1990) and LR0R/LR5 (Vilgalys and Hester 1990; Vilgalys and Sun 1994), respectively. For members of Didymellaceae two extra loci were amplified, the partial beta-tubulin (tub2) and the partial RNA polymerase II second largest subunit (rpb2), using the primer pairs Tub2Fd/Tub4Rd (Woudenberg et al. 2009) and Rpb2-5F2/Rpb2-7cR (Liu et al. 1999; Sung et al. 2007), respectively. The PCR amplifications were performed following Chen et al. (2015), except for rpb2, which was amplified in a total volume of 12.5 µL containing 1.25 µL of 10× EasyTaq Buffer (Bioline, Luckenwalde, Germany), 0.5 µL of dNTPs (40 μM), 0.5 µL of MgCl2 (2 mM), 0.5 µL of bovine berum albumin (BSA, 1 μg/μL), 0.5 µL of each primer (0.2 μM), 0.1 µL of Taq DNA polymerase (Bioline) and 1 µL of genomic DNA. PCR conditions for rpb2 were set as follows: an initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 95 °C for 45 s, annealing at 55 °C for 80 s and extension at 72 °C for 2 min; and a final extension step at 72 °C for 10 min. The PCR products were sequenced according to the methods of Crous et al. (2013). Consensus sequences were assembled from forward and reverse sequences using Seqman Pro v.12.1.0 (DNASTAR, Madison, WI, USA). All sequences generated in this study were deposited in GenBank (Table 1).

Table 1.

Taxa used in this study and their GenBank accession numbers.

Taxon name1 Strain number2 Substrate Country GenBank Accession numbers3
rpb2 tub2 ITS LSU
Allophoma cylindrispora CBS 142453T; FMR 13723 Human superficial tissue USA LT593058 LT592989 LN907376 LT592920
Al. nicaraguensis CBS 506.91T; IMI 215229; PD 91/876 Coffea sp. Nicaragua KT389551 GU237596 GU238058 GU237876
Al. piperis CBS 268.93T; PD 88/720 Peperomia pereskifolia The Netherlands KT389554 GU237644 GU238129 GU237816
Al. tropica CBS 436.75T Saintpaulia ionantha Germany KT389556 GU237663 GU238149 GU237864
Ascochyta benningiorum CBS 144957T; JW 196005 Garden soil The Netherlands MN824606 MN824755 MN823432 MN823581
CBS 144958; JW 196023 Garden soil The Netherlands MN824607 MN824756 MN823433 MN823582
JW 196013 Garden soil The Netherlands MN824608 MN824757 MN823434 MN823583
A. boeremae CBS 372.84T; PD 80/1246 Pisum sativum Australia KT389774 KT389697 KT389480
CBS 373.84; PD 80/1247 Pisum sativum Australia KT389560 KT389775 KT389698 KT389481
A. fabae CBS 649.71 Vicia faba The Netherlands GU237527 GU237964 GU237902
CBS 524.77 Phaseolus vulgaris Belgium GU237526 GU237963 GU237880
PD 83/492 Phaseolus vulgaris The Netherlands GU237528 GU237965 GU237917
A. herbicola CBS 629.97R; PD 76/1017 Water USA KP330421 GU237614 GU238083 GU237898
A. lentis CBS 370.84; PD 81/783 Lens culinaris Unknown KT389768 KT389691 KT389474
A. medicaginicola var. macrospora CBS 112.53T Medicago sativa USA GU237628 GU238101 GU237749
CBS 404.65R; IMI 116999 Medicago sativa Canada KP330423 GU237629 GU238102 GU237859
A. nigripycnidia CBS 116.96T; PD 95/7930 Vicia cracca Russia GU237637 GU238118 GU237756
A. phacae CBS 184.55T Phaca alpina Switzerland KT389769 KT389692 KT389475
A. pisi CBS 126.54 Pisum sativum The Netherlands DQ677967 GU237531 EU754137 GU237772
CBS 122785T; PD 78/517 Pisum sativum The Netherlands GU237532 GU237969 GU237763
CBS 122751; ATCC 201620 Pisum sativum Canada EU874867 KP330388 KP330444 KP330432
A. rabiei CBS 534.65 Cicer arietinum India KP330405 GU237533 GU237970 GU237886
CBS 237.37T Cicer arietinum Bulgaria KT389773 KT389696 KT389479
A. syringae CBS 545.72 Syringa vulgaris The Netherlands KT389777 KT389700 KT389483
JW 1074 Garden soil The Netherlands MN824605 MN824754 MN823431 MN823580
A. versabilis CBS 876.97R Silene sp. The Netherlands KT389561 GU237664 GU238152 GU237909
A. viciae CBS 451.68 Vicia sepium The Netherlands KT389562 KT389778 KT389701 KT389484
A. viciae-pannonicae CBS 254.92 Vicia pannonica Czech Republic KT389779 KT389702 KT389485
Boeremia exigua var. heteromorpha CBS 443.94T Nerium oleander Italy KT389573 GU237497 GU237935 GU237866
B. exigua var. populi CBS 100167T; PD 93/217 Populus (×)euramericana The Netherlands GU237501 GU237939 GU237707
Briansuttonomyces eucalypti CBS 114879T Eucalyptus sp. South Africa KU728595 KU728519 KU728479
CBS 114887 Eucalyptus sp. South Africa KU728596 KU728520 KU728480
Calophoma clematidina CBS 102.66 Clematis sp. UK KT389587 FJ427099 FJ515630 FJ426988
CBS 108.79T; PD 78/522 Clematis sp. The Netherlands KT389588 FJ427100 FJ515632 FJ426989
C. clematidis-rectae JW 179007 Garden soil The Netherlands MN824612 MN824761 MN823438 MN823587
CBS 507.63 Clematis sp. The Netherlands KT389589 FJ515624 FJ515647 FJ515606
C. vodakii CBS 173.53T Hepatica triloba Switzerland KT389791 KT389714 KT389497
Coniothyrium palmarum CBS 400.71 Chamaerops humilis Italy KT389592 KT389792 EU754153 AY720708
Cumuliphoma indica CBS 654.77T; FMR 15341 Unknown India LT623261 FJ427153 GU238122 FJ427043
Cu. omnivirens CBS 341.86T; FMR 14915 Phaseolus vulgaris Belgium LT62326 FJ427152 LT623214 FJ427042
Cu. pneumoniae CBS 142454T; FMR13739 Human respiratory tract USA LT593063 LT592994 LN907392 LT592925
Didymella aeria CGMCC 3.18353T; LC 7441 Air China KY742137 KY742293 KY742205 KY742051
D. aliena LC 8121 Pyrus calleryana Italy KY742295 KY742207 KY742053
CBS 379.93; PD 82/945 Berberis sp. The Netherlands KP330416 GU237578 GU238037 GU237851
D. americana CBS 185.85R; PD 80/1191 Zea mays USA KT389594 FJ427088 GU237990 FJ426972
D. anserina CBS 360.84R Potato flour The Netherlands KT389596 GU237551 GU237993 GU237839
D. aquatica CGMCC 3.18349T; LC 5556 Water China KY742140 KY742297 KY742209 KY742055
D. arachidicola CBS 333.75T; ATCC 28333; IMI 386092 Arachis hypogaea South Africa KT389598 GU237554 GU237996 GU237833
D. aurea CBS 269.93T; PD 78/1087 Medicago polymorpha New Zealand KT389599 GU237557 GU237999 GU237818
D. bellidis CBS 714.85R; PD 74/265 Bellis perennis The Netherlands KP330417 GU237586 GU238046 GU237904
D. boeremae CBS 109942T; PD 84/402 Medicago littoralis cv. harbi Australia KT389600 FJ427097 GU238048 FJ426982
D. brunneospora CBS 115.58T; FMR 15745 Chrysanthemum roseum Germany KT389625 KT389802 KT389723 KT389505
D. calidophila CBS 448.83T Desert soil Egypt FJ427168 GU238052 FJ427059
D. chenopodii CBS 128.93R; PD 79/140 Chenopodium quinoa cv. sajana Peru KT389602 GU237591 GU238055 GU237775
D. chloroguttulata CGMCC 3.18351T; LC 7435 Air China KY742142 KY742299 KY742211 KY742057
D. coffeae-arabicae CBS 123380T; PD 84/1013 Coffea arabica Ethiopia KT389603 FJ427104 GU238005 FJ426993
D. dactylidis CBS 124513T; PD 73/1414 Dactylis glomerata USA GU237599 GU238061 GU237766
D. degraaffiae CBS 144956T; JW 195004 Garden soil The Netherlands MN824470 MN824618 MN823295 MN823444
D. dimorpha CBS 346.82T Opuntia phyllocladium Spain GU237606 GU238068 GU237835
D. ellipsoidea CGMCC 3.18350T; LC 7434 Air China KY742145 KY742302 KY742214 KY742060
D. eucalyptica CBS 377.91R; PD 79/210 Eucalyptus sp. Australia KT389605 GU237562 GU238007 GU237846
D. exigua CBS 183.55T Rumex arifolius France EU874850 GU237525 EU754155 GU237794
D. gardeniae CBS 626.68T; IMI 108771 Gardenia jasminoides India KT389606 FJ427114 GQ387595 FJ427003
D. glomerata CBS 528.66R; PD 63/590 Chrysanthemum sp. The Netherlands GU371781 FJ427124 EU754184 FJ427013
D. heteroderae CBS 109.92T; PD 73/1405 Undefined food material The Netherlands KT389601 FJ427098 GU238002 FJ426983
D. ilicicola CGMCC 3.18355T; LC 8126 Ilex chinensis Italy KY742150 KY742307 KY742219 KY742065
D. infuscatispora CGMCC 3.18356T; LC 8128 Chrysanthemum indicum China KY742152 KY742309 KY742221 KY742067
D. keratinophila CBS 143032T; FMR 13690 Human superficial tissue USA LT593039 LT592970 LN907343 LT592901
D. kooimaniorum CBS 144951T; JW 27006 Garden soil The Netherlands MN824474 MN824622 MN823299 MN823448
D. lethalis CBS 103.25 Unknown Unknown KT389607 GU237564 GU238010 GU237729
D. macrophylla CGMCC 3.18357T; LC 8131 Hydrangea macrophylla Italy KY742154 KY742312 KY742224 KY742070
D. macrostoma JW 57015 Garden soil The Netherlands MN824472 MN824620 MN823297 MN823446
CBS 223.69R Acer pseudoplatanus Switzerland KT389608 GU237623 GU238096 GU237801
JW 149014 Garden soil The Netherlands MN824473 MN824621 MN823298 MN823447
CBS 482.95 Larix decidua Germany KT389609 GU237626 GU238099 GU237869
D. maydis CBS 588.69T Zea mays USA GU371782 FJ427190 EU754192 FJ427086
D. microchlamydospora CBS 105.95T Eucalyptus sp. UK KP330424 FJ427138 GU238104 FJ427028
D. molleriana CBS 229.79R Digitalis purpurea New Zealand KP330418 GU237605 GU238067 GU237802
D. negriana CBS 358.71R Vitis vinifera Germany KT389610 GU237635 GU238116 GU237838
D. nigricans CBS 444.81T; PDDCC 6546 Actinidia chinensis New Zealand GU237558 GU238000 GU237867
PD 77/919 Actinidea chinensis Unknown GU237559 GU238001 GU237915
D. ocimicola CGMCC 3.18358T; LC 8137 Ocimum sp. China KY742320 KY742232 KY742078
D. pedeiae CBS 124517T; PD 92/612A Schefflera elegantissima The Netherlands KT389612 GU237642 GU238127 GU237770
D. pinodella LC 8139 Acer palmatum Japan KY742161 KY742322 KY742234 KY742080
CBS 531.66 Trifolium pratense USA KT389613 FJ427162 GU238017 FJ427052
D. pinodes CBS 525.77T Pisum sativum Belgium KT389614 GU237572 GU238023 GU237883
D. pomorum JW 196022 Garden soil The Netherlands MN824469 MN824617 MN823294 MN823443
CBS 539.66R; IMI 122266; PD 64/914 Polygonum tataricum The Netherlands KT389618 FJ427166 GU238028 FJ427056
D. protuberans CBS 381.96T; PD 71/706 Lycium halifolium The Netherlands KT389620 GU237574 GU238029 GU237853
D. pteridis CBS 379.96T Pteris sp. The Netherlands KT389624 KT389801 KT389722 KT389504
D. rhei CBS 109177R; PD 2000/9941 Rheum rhaponticum New Zealand KP330428 GU237653 GU238139 GU237743
D. rumicicola CBS 683.79T Rumex obtusifolius New Zealand KT389622 KT389800 KT389721 KT389503
CBS 179.97 Rumex hydrolapathum The Netherlands KP330415 GU237575 GU238034 GU237793
CBS 539.77 Rumex obtusifolius New Zealand MN824471 MN824619 MN823296 MN823445
D. sancta CBS 281.83T Ailanthus altissima South Africa KT389623 FJ427170 GU238030 FJ427063
D. segeticola CGMCC 3.17489T; LC 1636 Cirsium segetum China KP330414 KP330399 KP330455 KP330443
D. senecionicola CBS 160.78R Senecio jacobaea New Zealand GU237657 GU238143 GU237787
D. subglomerata CBS 110.92R; PD 76/1010 Triticum sp. USA KT389626 FJ427186 GU238032 FJ427080
D. subherbarum CBS 250.92T; PD 92/371 Zea mays Canada GU237659 GU238145 GU237809
D. suiyangensis CGMCC 3.18352T; LC 7439 Air China KY742169 KY742332 KY742244 KY742090
D. viburnicola CBS 523.73R; PD 69/800 Viburnum cassioides The Netherlands KP330430 GU237667 GU238155 GU237879
Ectophoma multirostrata CBS 274.60T; FMR 15335; IMI 081598 Soil India LT623265 FJ427141 GU238111 FJ427031
Ec. pomi CBS 267.92T; FMR 15346; PD 76/1014 Coffea arabica India LT623263 GU237643 GU238128 GU237814
Epicoccum nigrum CBS 173.73T; IMI 164070 Dactylis glomerata USA KT389632 FJ427107 GU237975 FJ426996
LC 8157 Ocimum sp. China KY742179 KY742352 KY742264 KY742110
LC 5180 Lonicera japonica China KY742178 KY742351 KY742263 KY742109
LC 8158 Poa annua USA KY742180 KY742353 KY742265 KY742111
Ep. pimprinum CBS 246.60T; IMI 081601 Soil India FJ427159 GU237976 FJ427049
PD 77/1028 Unknown Unknown KT389633 FJ427160 GU237977 FJ427050
Heterophoma sylvatica CBS 874.97T; PD 93/764 Melampyrum pratense The Netherlands GU237662 GU238148 GU237907
H. verbascicola CGMCC 3.18364T; LC 8163 Verbascum thapsus China KY742187 KY742361 KY742273 KY742119
Juxtiphoma eupyrena JW 164001 Garden soil The Netherlands MN824541 MN824689 MN823366 MN823515
JW 263011 Garden soil The Netherlands MN824542 MN824690 MN823367 MN823516
JW 158007 Garden soil The Netherlands MN824543 MN824691 MN823368 MN823517
JW 201014 Garden soil The Netherlands MN824544 MN824692 MN823369 MN823518
JW 213001 Garden soil The Netherlands MN824545 MN824693 MN823370 MN823519
JW 201009 Garden soil The Netherlands MN824546 MN824694 MN823371 MN823520
JW 4005 Garden soil The Netherlands MN824547 MN824695 MN823372 MN823521
JW 4017 Garden soil The Netherlands MN824548 MN824696 MN823373 MN823522
JW 3015 Garden soil The Netherlands MN824549 MN824697 MN823374 MN823523
JW 224006 Garden soil The Netherlands MN824550 MN824698 MN823375 MN823524
JW 132015 Garden soil The Netherlands MN824551 MN824699 MN823376 MN823525
Juxtiphoma eupyrena JW 146002 Garden soil The Netherlands MN824700 MN823377 MN823526
JW 160021 Garden soil The Netherlands MN824552 MN824701 MN823378 MN823527
JW 18016 Garden soil The Netherlands MN824553 MN824702 MN823379 MN823528
JW 40009 Garden soil The Netherlands MN824554 MN824703 MN823380 MN823529
JW 40019 Garden soil The Netherlands MN824555 MN824704 MN823381 MN823530
JW 97009 Garden soil The Netherlands MN824556 MN824705 MN823382 MN823531
JW 96020 Garden soil The Netherlands MN824557 MN824706 MN823383 MN823532
JW 57007 Garden soil The Netherlands MN824558 MN824707 MN823384 MN823533
JW 149010 Garden soil The Netherlands MN824559 MN824708 MN823385 MN823534
JW 74008 Garden soil The Netherlands MN824560 MN824709 MN823386 MN823535
JW 247003 Garden soil The Netherlands MN824561 MN824710 MN823387 MN823536
JW 267005 Garden soil The Netherlands MN824562 MN824711 MN823388 MN823537
JW 261008 Garden soil The Netherlands MN824563 MN824712 MN823389 MN823538
JW 30012 Garden soil The Netherlands MN824564 MN824713 MN823390 MN823539
JW 167015 Garden soil The Netherlands MN824565 MN824714 MN823391 MN823540
JW 221022B Garden soil The Netherlands MN824566 MN824715 MN823392 MN823541
JW 259004 Garden soil The Netherlands MN824567 MN824716 MN823393 MN823542
JW 73004 Garden soil The Netherlands MN824568 MN824717 MN823394 MN823543
JW 170018 Garden soil The Netherlands MN824569 MN824718 MN823395 MN823544
JW 141018 Garden soil The Netherlands MN824570 MN824719 MN823396 MN823545
JW 181003 Garden soil The Netherlands MN824571 MN824720 MN823397 MN823546
JW 289013 Garden soil The Netherlands MN824572 MN824721 MN823398 MN823547
JW 127004 Garden soil The Netherlands MN824573 MN824722 MN823399 MN823548
JW 81007 Garden soil The Netherlands MN824574 MN824723 MN823400 MN823549
JW 182002 Garden soil The Netherlands MN824575 MN824724 MN823401 MN823550
JW 212001 Garden soil The Netherlands MN824576 MN824725 MN823402 MN823551
JW 191036 Garden soil The Netherlands MN824577 MN824726 MN823403 MN823552
JW 221020 Garden soil The Netherlands MN824578 MN824727 MN823404 MN823553
JW 96002 Garden soil The Netherlands MN824579 MN824728 MN823405 MN823554
JW 52011 Garden soil The Netherlands MN824580 MN824729 MN823406 MN823555
JW 38012 Garden soil The Netherlands MN824581 MN824730 MN823407 MN823556
JW 40007 Garden soil The Netherlands MN824582 MN824731 MN823408 MN823557
JW 43007 Garden soil The Netherlands MN824583 MN824732 MN823409 MN823558
Juxtiphoma eupyrena JW 75002 Garden soil The Netherlands MN824584 MN824733 MN823410 MN823559
JW 116017 Garden soil The Netherlands MN824585 MN824734 MN823411 MN823560
JW 170013 Garden soil The Netherlands MN824586 MN824735 MN823412 MN823561
JW 79016 Garden soil The Netherlands MN824587 MN824736 MN823413 MN823562
CBS 374.91; FMR 15329 Solanum tuberosum The Netherlands LT623268 FJ427110 GU238072 FJ426999
JW 125024 Garden soil The Netherlands MN824588 MN824737 MN823414 MN823563
JW 158014 Garden soil The Netherlands MN824589 MN824738 MN823415 MN823564
JW 4010 Garden soil The Netherlands MN824590 MN824739 MN823416 MN823565
JW 202020 Garden soil The Netherlands MN824591 MN824740 MN823417 MN823566
J. kolkmaniorum JW 125028 Garden soil The Netherlands MN824592 MN824741 MN823418 MN823567
CBS 146005T; JW 185006 Garden soil The Netherlands MN824593 MN824742 MN823419 MN823568
JW 191004 Garden soil The Netherlands MN824594 MN824743 MN823420 MN823569
JW 23021 Garden soil The Netherlands MN824595 MN824744 MN823421 MN823570
JW 167004 Garden soil The Netherlands MN824596 MN824745 MN823422 MN823571
JW 221010 Garden soil The Netherlands MN824597 MN824746 MN823423 MN823572
JW 220011 Garden soil The Netherlands MN824598 MN824747 MN823424 MN823573
JW 241011 Garden soil The Netherlands MN824599 MN824748 MN823425 MN823574
JW 94009 Garden soil The Netherlands MN824600 MN824749 MN823426 MN823575
CBS 527.66; FMR 15337 Wheat field soil Germany LT623269 FJ427111 GU238073 FJ427000
JW 63001 Garden soil The Netherlands MN824601 MN824750 MN823427 MN823576
JW 168007 Garden soil The Netherlands MN824602 MN824751 MN823428 MN823577
Leptosphaeria doliolum CBS 505.75T Urtica dioica The Netherlands KT389640 JF740144 GQ387576 JF740205
Leptosphaerulina australis CBS 311.51 Lawn Switzerland FJ795508
L. saccharicola CBS 939.69 Soil The Netherlands GU237541 JX681098 GU237911
L. trifolii CBS 235.58 Trifolium sp. The Netherlands GU237542 GU237982 GU237806
Macroventuria anomochaeta CBS 525.71T Decayed canvas South Africa GU456346 GU237544 GU237984 GU237881
Ma. wentii CBS 526.71T Plant litter USA KT389642 GU237546 GU237986 GU237884
Microsphaeropsis olivacea CBS 233.77 Pinus laricio France KT389643 GU237549 GU237988 GU237803
CBS 442.83 Taxus baccata The Netherlands GU237547 EU754171 GU237865
Mi. proteae CBS 111319T; CPC 1425 Protea nitida Unknown JN712650 JN712563 JN712497
Neoascochyta argentina CBS 112524T Triticum aestivum Argentina KT389822 KT389742 KT389524
Neoa. desmazieri CBS 297.69T Lolium perenne Germany KT389644 KT389806 KT389726 KT389508
Neoa. paspali CBS 560.81T; PDDCC 6614 Paspalum dilatatum New Zealand KP330426 FJ427158 GU238124 FJ427048
Neoa. tardicrebrescens CBS 689.97T Hay Norway KT389654 KT389824 KT389744 KT389526
Neoa. triticicola CBS 544.74T Triticum aestivum South Africa KT389652 GU237488 EU754134 GU237887
Neodidymelliopsis cannabis CBS 234.37 Cannabis sativa Unknown KP330403 GU237523 GU237961 GU237804
CBS 121.75T; IMI 194767; PD 73/584 Urtica dioica The Netherlands GU237535 GU237972 GU237761
Neod. polemonii CBS 109181T; PD 83/757 Polemonium caeruleum The Netherlands KP330427 GU237648 GU238133 GU237746
Neod. xanthina CBS 383.68T Delphinium sp. The Netherlands KP330431 GU237668 GU238157 GU237855
Neomicrosphaeropsis italica MFLUCC 16-0284 Tamarix sp. Italy KU714604 KU900296 KU900321
MFLUCC 15-0484 Tamarix sp. Italy KU695539 KU729853 KU900319
MFLUCC 15-0485T Tamarix sp. Italy KU674820 KU729854 KU900318
Nothophoma anigozanthi CBS 381.91T; FMR 14914 Anigozanthus maugleisii The Netherlands KT389655 GU237580 GU238039 GU237852
N. arachidis-hypogaeae CBS 125.93R; PD 77/1029 Arachis hypogaea India KT389656 GU237583 GU238043 GU237771
N. brennandiae JW 1066 Garden soil The Netherlands MN824603 MN824752 MN823429 MN823578
CBS 145912T; JW 53011 Garden soil The Netherlands MN824604 MN824753 MN823430 MN823579
MFLUCC 16-1392 Ulmus (×) hollandica Italy KY053898 KY053899 KY053897 KY053896
N. gossypiicola CBS 377.67; FMR 14912 Gossypium sp. USA KT389658 GU237611 GU238079 GU237845
UTHSC:DI16-294 Human deep tissue/ fluids USA LT593082 LT593012 LN907437 LT592943
N. infossa CBS 123395T Fraxinus pennsylvanica Argentina KT389659 FJ427135 GU238089 FJ427025
CBS 123394 Fraxinus pennsylvanica Argentina FJ427134 GU238088 FJ427024
N. macrospora CBS 140674T; FMR 13767 Human respiratory tract USA LT593073 LN880539 LN880537 LN880536
N. pruni MFLUCC 18-1600T Prunus avium China MH853664 MH853671 MH827028 MH827007
N. quercina MFLUCC 18-1588 Prunus avium China MH853665 MH853672 MH827029 MH827008
CBS 633.92R; ATCC 36786 Microsphaera alphitoides from Quercus sp. Ukraine KT389657 GU237609 EU754127 GU237900
UTHSC:DI16-270; FMR 13761 Human superficial tissue USA LT593067 LT592998 LN907413 LT592929
N. variabilis CBS 142457T; FMR 13777 Human respiratory tract USA LT593078 LT593008 LN907428 LT592939
Paraboeremia adianticola CBS 260.92; PD 86/1103 Pteris ensiformis Unknown KT389832 KT389752 KT389534
P. adianticola CBS 187.83; PD 82/128; FMR 15344 Polystichum adiantiforme USA KP330401 GU237576 GU238035 GU237796
P. camelliae CGMCC 3.18108 Camellia sp. China KX829052 KX829060 KX829044 KX829036
CGMCC 3.18106T Camellia sp. China KX829050 KX829058 KX829042 KX829034
CGMCC 3.18107 Camellia sp. China KX829051 KX829059 KX829043 KX829035
P. litseae CGMCC 3.18110; LC 5030 Litsea sp. China KX829046 KX829054 KX829038 KX829030
JW 157001 Garden soil The Netherlands MN824519 MN824667 MN823344 MN823493
CGMCC 3.18109T; LC 5028 Litsea sp. China KX829045 KX829053 KX829037 KX829029
P. putaminum JW 110005 Garden soil The Netherlands MN824480 MN824628 MN823305 MN823454
JW 126003 Garden soil The Netherlands MN824481 MN824629 MN823306 MN823455
JW 265009 Garden soil The Netherlands MN824482 MN824630 MN823307 MN823456
JW 221011 Garden soil The Netherlands MN824483 MN824631 MN823308 MN823457
JW 165006 Garden soil The Netherlands MN824484 MN824632 MN823309 MN823458
JW 232004 Garden soil The Netherlands MN824485 MN824633 MN823310 MN823459
JW 192007 Garden soil The Netherlands MN824486 MN824634 MN823311 MN823460
JW 125011 Garden soil The Netherlands MN824487 MN824635 MN823312 MN823461
JW 18014 Garden soil The Netherlands MN824488 MN824636 MN823313 MN823462
JW 142002 Garden soil The Netherlands MN824489 MN824637 MN823314 MN823463
JW 221018 Garden soil The Netherlands MN824490 MN824638 MN823315 MN823464
JW 238003 Garden soil The Netherlands MN824491 MN824639 MN823316 MN823465
JW 192019 Garden soil The Netherlands MN824492 MN824640 MN823317 MN823466
JW 213009 Garden soil The Netherlands MN824493 MN824641 MN823318 MN823467
JW 226017 Garden soil The Netherlands MN824494 MN824642 MN823319 MN823468
JW 109022 Garden soil The Netherlands MN824495 MN824643 MN823320 MN823469
JW 4002 Garden soil The Netherlands MN824496 MN824644 MN823321 MN823470
CBS 130.69R; IMI 331916 Malus sylvestris Denmark GU237652 GU238138 GU237777
JW 16015 Garden soil The Netherlands MN824497 MN824645 MN823322 MN823471
JW 16001 Garden soil The Netherlands MN824498 MN824646 MN823323 MN823472
JW 25002 Garden soil The Netherlands MN824499 MN824647 MN823324 MN823473
JW 276009 Garden soil The Netherlands MN824500 MN824648 MN823325 MN823474
JW 48011 Garden soil The Netherlands MN824501 MN824649 MN823326 MN823475
JW 4011 Garden soil The Netherlands MN824502 MN824650 MN823327 MN823476
JW 276008 Garden soil The Netherlands MN824503 MN824651 MN823328 MN823477
JW 65008 Garden soil The Netherlands MN824505 MN824653 MN823330 MN823479
JW 132016 Garden soil The Netherlands MN824506 MN824654 MN823331 MN823480
JW 226014 Garden soil The Netherlands MN824507 MN824655 MN823332 MN823481
JW 226015 Garden soil The Netherlands MN824508 MN824656 MN823333 MN823482
JW 25012 Garden soil The Netherlands MN824509 MN824657 MN823334 MN823483
P. putaminum JW 11007 Garden soil The Netherlands MN824510 MN824658 MN823335 MN823484
JW 129005 Garden soil The Netherlands MN824511 MN824659 MN823336 MN823485
CBS 372.91R; PD 75/690 Ceratocystis ulmi The Netherlands GU237651 GU238137 GU237843
JW 145026 Garden soil The Netherlands MN824504 MN824652 MN823329 MN823478
JW 4006 Garden soil The Netherlands MN824512 MN824660 MN823337 MN823486
JW 191017 Garden soil The Netherlands MN824513 MN824661 MN823338 MN823487
JW 161002 Garden soil The Netherlands MN824514 MN824662 MN823339 MN823488
JW 116031 Garden soil The Netherlands MN824515 MN824663 MN823340 MN823489
JW 1008 Garden soil The Netherlands MN824516 MN824664 MN823341 MN823490
JW 1020 Garden soil The Netherlands MN824517 MN824665 MN823342 MN823491
JW 1046 Garden soil The Netherlands MN824518 MN824666 MN823343 MN823492
P. rekkeri JW 13016 Garden soil The Netherlands MN824526 MN824674 MN823351 MN823500
JW 13030 Garden soil The Netherlands MN824527 MN824675 MN823352 MN823501
JW 79024 Garden soil The Netherlands MN824528 MN824676 MN823353 MN823502
JW 25013 Garden soil The Netherlands MN824529 MN824677 MN823354 MN823503
JW 167006 Garden soil The Netherlands MN824530 MN824678 MN823355 MN823504
JW 132004 Garden soil The Netherlands MN824531 MN824679 MN823356 MN823505
CBS 144949; JW 4024 Garden soil The Netherlands MN824532 MN824680 MN823357 MN823506
JW 13017 Garden soil The Netherlands MN824533 MN824681 MN823358 MN823507
JW 91008 Garden soil The Netherlands MN824534 MN824682 MN823359 MN823508
JW 226002 Garden soil The Netherlands MN824535 MN824683 MN823360 MN823509
JW 3018 Garden soil The Netherlands MN824536 MN824684 MN823361 MN823510
CBS 144955T; JW 172002 Garden soil The Netherlands MN824537 MN824685 MN823362 MN823511
JW 51014 Garden soil The Netherlands MN824538 MN824686 MN823363 MN823512
JW 196020 Garden soil The Netherlands MN824539 MN824687 MN823364 MN823513
CBS 144950; JW 6005 Garden soil The Netherlands MN824540 MN824688 MN823365 MN823514
P. selaginellae CBS 122.93T; PD 77/1049 Selaginella sp. The Netherlands GU237656 GU238142 GU237762
P. truiniorum JW 270002 Garden soil The Netherlands MN824520 MN824668 MN823345 MN823494
CBS 144952T; JW 47002 Garden soil The Netherlands MN824521 MN824669 MN823346 MN823495
JW 147025 Garden soil The Netherlands MN824522 MN824670 MN823347 MN823496
JW 182014 Garden soil The Netherlands MN824523 MN824671 MN823348 MN823497
JW 192003 Garden soil The Netherlands MN824524 MN824672 MN823349 MN823498
CBS 144961; JW 203021 Garden soil The Netherlands MN824525 MN824673 MN823350 MN823499
Phoma herbarum CBS 274.37 Picea excelsa UK KT389662 KT389835 KT389754 KT389537
CBS 615.75R; IMI 199779; PD 73/655 Rosa multiflora cv. cathayensis The Netherlands KP330420 FJ427133 EU754186 FJ427022
Phomatodes aubrietiae CBS 627.97T; PD 70/714 Aubrietia sp. The Netherlands KT389665 GU237585 GU238045 GU237895
Phomat. nebulosa JW 166004 Garden soil The Netherlands MN824609 MN824758 MN823435 MN823584
JW 166006 Garden soil The Netherlands MN824610 MN824759 MN823436 MN823585
JW 166013 Garden soil The Netherlands MN824611 MN824760 MN823437 MN823586
CBS 100191 Thlapsi arvense Poland KT389666 KP330390 KP330446 KP330434
CBS 117.93; PD 83/90 Mercurialis perennis The Netherlands KP330425 GU237633 GU238114 GU237757
Pseudoascochyta novae-zelandiae CBS 141689T; FMR 15110 Cordyline australis New Zealand LT592895 LT592894 LT592893 LT592892
Pse. pratensis CBS 141688T; FMR 14524 Soil Spain LT223133 LT223132 LT223131 LT223130
Remotididymella anthropophylica CBS 142462T; FMR 13770 Human respiratory tract USA LT593075 LT593005 LN907421 LT592936
R. destructiva CBS 378.73T; FMR 15328 Lycopersicon esculentum Tonga LT623258 GU237601 GU238063 GU237849
Stagonosporopsis andigena CBS 269.80; PD 75/914 Solanum sp. Peru GU237675 GU238170 GU237817
S. astragali CBS 178.25R; MUCL 9915 Astragalus sp. Unknown GU237677 GU238172 GU237792
S. bomiensis LC 8168 Boraginaceae China KY742190 KY742366 KY742278 KY742124
CGMCC 3.18366T; LC 8167 Boraginaceae China KY742189 KY742365 KY742277 KY742123
S. crystalliniformis CBS 713.85T; ATCC 76027; PD 83/826 Lycopersicon esculentum Colombia KT389675 GU237683 GU238178 GU237903
S. dorenboschii CBS 426.90T; IMI 386093; PD 86/551 Physostegia virginiana The Netherlands KT389678 GU237690 GU238185 GU237862
S. hortensis CBS 104.42R The Netherlands KT389680 GU237703 GU238198 GU237730
CBS 572.85; PD 79/269 Phaseolus vulgaris The Netherlands KT389681 GU237704 GU238199 GU237893
S. loticola CBS 562.81T; PDDCC 6884 Lotus pedunculatus New Zealand KT389684 GU237697 GU238192 GU237890
S. papillata LC 8170 Rumex nepalensis China KY742192 KY742368 KY742280 KY742126
CGMCC 3.18367T; LC 8169 Rumex nepalensis China KY742191 KY742367 KY742279 KY742125
S. stuijvenbergii CBS 144953T; JW 132011 Garden soil The Netherlands MN824475 MN824623 MN823300 MN823449
JW 33021 Garden soil The Netherlands MN824476 MN824624 MN823301 MN823450
JW 14003 Garden soil The Netherlands MN824477 MN824625 MN823302 MN823451
JW 44014 Garden soil The Netherlands MN824478 MN824626 MN823303 MN823452
S. weymaniae CBS 144959T; JW 201003 Garden soil The Netherlands MN824479 MN824627 MN823304 MN823453
Vacuiphoma bulgarica CBS 357.84T Trachystemon orientale Bulgaria LT623256 GU237589 GU238050 GU237837
Vac. oculihominis UTHSC:DI16-308T; FMR 13801 Human superficial tissue USA LT593093 LT593023 LN907451 LT592954
Vandijckomycella joseae CBS 144948; JW 1068 Garden soil The Netherlands MN824614 MN824763 MN823440 MN823589
Van. joseae CBS 143011T; JW 1073 Garden soil The Netherlands MN824615 MN824764 MN823441 MN823590
Van. snoekiae CBS 144954T; JW 149017 Garden soil The Netherlands MN824616 MN824765 MN823442 MN823591
Xenodidymella applanata CBS 115577 Rubus idaeus Sweden KT389688 KT389850 KT389762 KT389546
CBS 195.36T Rubus idaeus The Netherlands KT389852 KT389764 KT389548
CBS 205.63 Rubus idaeus The Netherlands KP330402 GU237556 GU237998 GU237798
CBS 115578 Rubus arcticus nothossp. stellarcticus Sweden KT389851 KT389763 KT389547
X. asphodeli CBS 375.62T Asphodelus albus France KT389689 KT389853 KT389765 KT389549
CBS 499.72 Asphodelus ramosus Italy KT389853 KT389766 KT389550
X. catariae CBS 102635; PD 77/1131 Nepeta catenaria The Netherlands KP330404 GU237524 GU237962 GU237727
X. humicola CBS 220.85R; PD 71/1030 Franseria sp. USA KP330422 GU237617 GU238086 GU237800
X. weymaniae CBS 144960T; JW 201005 Garden soil The Netherlands MN824613 MN824762 MN823439 MN823588

Selection of Didymellaceae strains

A preliminary species identification of the strains was carried-out by a BLASTn search performed with each ITS and/or LSU sequence against the NCBI (http://blast.ncbi.nlm.nih.gov) and WI (http://www.westerdijkinstitute.nl/Collections) databases. The ITS and/or LSU sequences generated in this study with more than 98 % similarity with reference sequences for Didymellaceae were selected for further study (Table 1).

Sequence alignment and molecular phylogenetic analysis

To further study the phylogenetic relationships, reference sequences of Didymellaceae were downloaded from GenBank (Table 1). Sequences of single loci were aligned with MAFFT v.7 using default settings (Katoh et al. 2017), and manually edited in MEGA v.6.0 when necessary (Tamura et al. 2013). MrModeltest v.2.3 (Nylander 2004) was used to select the best-fit models of evolution for the four data partitions according to the Akaike information criterion. Bayesian inference (BI), maximum-likelihood (ML) and maximum parsimony (MP) methods were implemented for phylogenetic analysis of individual gene regions and the combined dataset. The multi-locus gene dataset was generated using SequenceMatrix v.1.8 (Vaidya et al. 2011).

Bayesian analyses were performed using MrBayes v.3.2.6 (Ronquist et al. 2012) as described by Chen et al. (2015). The burn-in fraction was set to 0.25, after which the 50 % majority rule consensus trees and posterior probability (PP) values were calculated. The ML analyses including 1000 bootstrap replicates were conducted using RAxML v.7.2.6 (Stamatakis and Alachiotis et al. 2010) as described by Chen et al. (2015). Statistical support for the branches was evaluated using a bootstrap analysis (BS) of 1000 replicates. MP analyses were carried out using PAUP v.4.0b10 (Swofford 2003) as described by Braun et al. (2018). Statistical support for the branches was evaluated using a bootstrap analysis (PBS) of 1000 replicates. Trees were visualised in FigTree v.1.4.0 (Rambaut 2014) and the layout was created in Adobe Illustrator. Alignments and phylogenetic trees derived from this study were uploaded to TreeBASE (www.treebase.org) and sequences deposited in GenBank (Table 1).

Morphological characterisation

Isolates of Didymellaceae were transferred to fresh oatmeal agar (OA), 2 % malt extract agar (MEA) and potato dextrose agar (PDA) (Crous et al. 2019) plates and incubated at 25 °C under near-ultraviolet (UV) light (12 h light/12 h dark) to induce sporulation. Colony diameters were measured after 7 d of incubation (Boerema et al. 2004), and macroscopic characters and colony colours were described after 14 days of incubation and rated according to the colour charts of Rayner (1970). Preparations were mounted in distilled water to study the micro-morphological structures of mature conidiomata, conidiogenous cells and conidia from OA cultures (Aveskamp et al. 2010; Chen et al. 2015). Morphological observations included the general characteristics of the conidiomata, shape, presence of mycelium/setae on conidiomata, number of ostioles, thickness and texture of the pycnidial wall, length and width of the conidiogenous cells and conidia. To study the pycnidial wall, sections of mature conidiomata were generated using a Leica CM 1900 freezing microtome (Aveskamp et al. 2010; Chen et al. 2015). Observations of micro-morphological characteristics were processed with a Nikon Eclipse 80i compound microscope with differential interference contrast (DIC) optics and a Nikon AZ100 dissecting microscope, both equipped with a Nikon DS-Ri2 high-definition colour digital camera (Nikon, Tokyo, Japan) using NIS-elements imaging software v.4.3. The NaOH spot test was carried out using a drop of concentrated NaOH to determine the secretion of metabolite E on OA cultures (Boerema et al. 2004). Morphological descriptions and taxonomic information for the new taxa were deposited in MycoBank (Crous et al. 2004).

Results

A total of 293 soil samples were analysed, and nearly 3000 fungal strains were obtained. Among them, 148 Didymellaceae isolates were identified from 89 different garden soil samples, representing several locations in the Netherlands (Table 1).

Phylogenetic identification

A multi-locus phylogeny comprising 325 strains, including the JW soil isolates and reference strains from GenBank, was used to infer the relationships among species in Didymellaceae (Figure 1, Table 1). Coniothyrium palmarum (CBS 400.71) and Leptosphaeria doliolum (CBS 505.75) were used as outgroups. The final combined ITS, LSU, rpb2 and tub2 alignment comprised 2317 characters including gaps (500 for ITS; 859 for LSU; 602 for rpb2; 356 for tub2), of which 1563 characters were constant, 106 parsimony-uninformative, and 618 were parsimony-informative. For the Bayesian analysis, SYM+I+G was selected as the best-fit model for the ITS dataset, and GTR+I+G was selected as the best model for the LSU, tub2 and rpb2 datasets. The phylogenetic trees obtained with three analyses showed a similar topology and were congruent with each other, and only the ML tree is presented herein with BS, PP, and PBS values plotted on the branches (Figure 1).

In the phylogenetic analysis, the 148 isolates from Dutch soil were distributed in 10 clades (Figure 1). The majority of the isolates clustered in Juxtiphoma (n=63) which were recovered from 48 soil samples and 28 cities, followed by Paraboeremia (n=61) from 29 soil samples and 19 cities. Other isolates belonged to Didymella spp. (n=5), Stagonosporopsis spp. (n=5), Ascochyta spp. (n=4), Phomatodes nebulosa (n=3), Nothophoma spp. (n=2), Calophoma clematidis (n=1), and Xenodidymella applanata (n=1), and three isolates clustered in an unknown clade (Figure 1, Table 1).

Figure 1. 

Phylogenetic tree generated from the maximum-likelihood analysis based on the combined ITS, LSU, tub2 and rpb2 sequence alignment of Didymellaceae members. The RAxML bootstrap support values (BS), Bayesian posterior probabilities (PP), and parsimony bootstrap support values (PBS) are given at the nodes (BS/PP/PBS). BS and PBS values represent parsimony bootstrap support values >50 %. Full supported branches are indicated in bold. The scale bar represents the expected number of changes per site. Ex-type strains are represented in bold. Strains obtained in the current study are printed in green; among them, whilst strains that represent new taxa are printed in red. Some of the basal branches were shortened to facilitate layout (the fraction in round parentheses refers to the presented length compared to the actual length of the branch). The tree was rooted to Coniothyrium palmarum CBS 400.71 and Leptosphaeria doliolum CBS 505.75.

In the Juxtiphoma clade species clustered in two lineages, one corresponding to J. eupyrena (77/1/-) and the other representing a potentially new species (100/1/99). In the Paraboeremia clade, the soil isolates clustered in P. putaminum (86/0.99/67) and P. litseae (98/1/97). However, 21 isolates were distributed in two different lineages (with 6 and 15 isolates, respectively) that were phylogenetically distant from other species, representing two potentially new taxa. The soil isolates belonging to Stagonosporopsis clustered in a clade (100/1/99) that was phylogenetically distant from the other species, representing two potentially new species. In Didymella, the species were distributed in D. macrostoma (100/1/100) and D. pomorum (100/1/100), while isolates JW 195004 and JW 27006 were placed in two different branches, representing two putative new species. In Ascochyta one isolate grouped with A. syringae (93/1/86), whereas three isolates grouped in a different clade distant from previously known species, representing a potentially new species (100/1/100). The other three isolates grouped together at the bottom of the tree in a distant unknown lineage, which is introduced herein as a new genus with two species (100/1/90). All the new taxa are introduced in the taxonomy section based on the phylogenetic analysis and supported by morphological data. Descriptions and illustrations of the new taxa are provided in the taxonomy section below.

Loci resolution

The single locus phylogenies of rpb2 and tub2 performed quite well at both generic and species levels. The rpb2 phylogeny was able to discriminate all 27 generic clades included in the phylogeny (Figure 1), with good resolution of species among these genera (140 of 143 species). The tub2 phylogeny was able to distinguish 26 of 27 generic clades recognising 134 of 143 species, but proved unsuccessful for Vacuiphoma and Ascochyta, mainly because species of these genera did not cluster into monophyletic lineages, but were sometimes intermixed or formed separate lineages. However, the LSU phylogeny displayed a low resolution at both generic and species levels, being able to distinguish only 12 of 27 genera and 50 of 143 species. The ITS phylogeny was able to distinguish 17 of the 27 generic clades and 44 of the 143 species.

Taxonomy

Ascochyta benningiorum Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833194
Figure 2

Etymology

benningiorum refers to Eva, Bas & Anne Benning who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Gelderland province, Wijchen, isolated from garden soil, Mar. 2017, E. Benning, B. Benning & A. Benning (holotype designated here CBS H-24104, living ex-type culture CBS 144957 = JW 196005).

Conidiomata pycnidial, mostly solitary, sometimes confluent, globose or subglobose, irregularly-shaped with age, brown to dark brown, glabrous, mostly produced on the agar surface and some immersed, 140–480(–580) × 100–370(–440) μm; with 1–6(–10) slightly papillate ostioles; pycnidial wall pseudoparenchymatous, 4–8 layers, 14.5–65 μm thick, outer layers composed of brown, flattened polygonal cells of 11–28 μm diam. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform to lageniform, 5.5–9 × 4–6.5 μm. Conidia cylindrical, hyaline, smooth- and thin-walled, mostly straight, occasionally curved, aseptate, (3.5–)4.5–7 × 1.5–2.5 μm, 2-guttulate, small. Conidia matrix whitish.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 50–55 mm diam, aerial mycelium floccose, olivaceous to olivaceous black, buff towards the periphery, abundant production of pycnidia, margin irregular; reverse concolorous with the surface. On MEA reaching 40–45 mm diam, aerial mycelium floccose, concentric circles, centre pink, grey olivaceous, mouse grey, rosy buff toward periphery, moderate production of pycnidia, margin irregular; reverse orange, olivaceous black toward periphery. On PDA reaching 45–50 mm diam, aerial mycelium floccose, dark brick to olivaceous grey, buff towards periphery, abundant production of pycnidia, margin irregular; reverse concolorous with the surface. NaOH spot test negative on OA.

Additional specimens examined

The Netherlands. Gelderland province, Wijchen, isolated from garden soil, Mar. 2017, E. Benning, B. Benning & A. Benning, JW 196023 = CBS 144958; ibid. JW 196013.

Notes

Ascochyta benningiorum is represented in the phylogenetic tree by three isolates (CBS 144957, CBS 144958 and JW 196013) from the same soil sample collected in Wijchen (Gelderland province). Ascochyta benningiorum grouped in a distinct clade close to A. phacae (Figure 1). However, it morphologically differs from A. phacae by having smaller (3.5–7 × 1.5–2.5 μm) and aseptate conidia. In A. phacae the conidia are 7–10 × 2–4 μm and 0–1-septate (Corbaz 1955).

Species in Ascochyta are commonly regarded as plant pathogens, especially of cereal crops and legumes (Davidson and Kimber 2007; Tivoli and Banniza 2007), and only a few species were reported from soil, namely A. fabae, A. lentis, A. pisi, A. rabiei (Gossen and Morrall 1986; Tivoli and Banniza 2007) and A. syringae in the current study. Nevertheless, A. benningiorum is phylogenetically and morphologically distinct from these species (Figure 1; Chen et al. 2015).

Figure 2. 

Ascochyta benningiorum (CBS 144957). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G pycnidia forming on OA H pycnidium I section of pycnidium J section of pycnidial wall K–M conidiogenous cells N conidia. Scale bars: 100 μm (H, I); 10 μm (J); 5 μm (K–N).

Didymella degraaffiae Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833195
Figure 3

Etymology

degraaffiae refers to Janne de Graaff who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Limburg province, Weert, isolated from garden soil, Mar. 2017, J. de Graaff (holotype designated here CBS H-24105, living ex-type culture CBS 144956 = JW 195004).

Conidiomata pycnidial, superficial on the agar or semi-immersed in the agar, scattered or aggregated, mostly confluent, globose, subglobose, lageniform to irregularly-shaped with age, brown to dark brown, ostiolate, covered by hyphal outgrowths, especially near the ostiole, 150–485 × 120–330 μm; non-papillate or with up to two papillate ostioles; pycnidial wall pseudoparenchymatous, 3–6 layers, 10–55 μm thick, outer layers composed of brown, isodiametric cells, 16–33 μm diam. Conidiogenous cells phialidic, hyaline, smooth, ampulliform, lageniform, pyriform or globose, 5.5–8.5 × 5–8 μm. Conidia ellipsoidal, oblong or oval, thin- and smooth-walled, hyaline, aseptate, 4.5–9(–11) × 3–4.5 μm, 2–6-guttulate, small. Conidial matrix milky white.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 65–70 mm diam, aerial mycelium floccose, orange to olivaceous, margin regular; reverse black near the centre, pale grey towards the periphery. On MEA reaching 55–60 mm diam, aerial mycelium floccose, buff to pale olivaceous, with white mycelium pellet and radially furrowed zones near the centre, margin regular; reverse buff near the centre, olivaceous to yellow towards the periphery. On PDA reaching 50–55 mm diam, aerial mycelium floccose, concentric circles pale brown, pale olivaceous grey, dark olivaceous, honey, margin irregular; reverse black with a pale olivaceous edge. NaOH spot test negative on OA.

Notes

In our phylogenetic analysis, D. degraaffiae grouped with D. americana and D. maydis (Figure 1). However, morphologically, D. americana differs by its smaller conidiogenous cells (3–5 × 3–4 μm) and conidia (5–7 × 2–2.5 μm) (Boerema 1993); while D. maydis differs in having larger conidia (15–17 × 3.5–5 μm) (de Gruyter 2002). Furthermore, D. americana and D. maydis occasionally produced 1-septate conidia, while septate conidia were not observed in D. degraaffiae.

Figure 3. 

Didymella degraaffiae (CBS 144956). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G, H pycnidia on OA I section of pycnidium J section of pycnidial wall K, L conidiogenous cells M chlamydospores N conidia. Scale bars: 50 μm (H, I); 10 μm (J); 5 μm (K–N).

Didymella kooimaniorum Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833196
Figure 4

Etymology

kooimaniorum refers to Noud & Robin Kooiman who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Utrecht province, Vleuten, isolated from garden soil, Mar. 2017, N. Kooiman & R. Kooiman (holotype designated here CBS H-24106, living ex-type culture CBS 144951 = JW 27006).

Conidiomata pycnidial, superficial or semi-immersed, scattered or solitary, sometimes confluent, globose to subglobose, irregularly-shaped with age, pale brown to brown, covered by hyphal outgrowths, especially near the ostioles, 200–375 × 195–280 μm; with 1–3(–6) papillate ostioles; pycnidial wall pseudoparenchymatous, 3–5 layers, 10–35 μm thick, outer layers composed of pale brown, flattened polygonal cells of 16–32 μm diam. Conidiogenous cells phialidic, hyaline, smooth, ampulliform, lageniform or somewhat isodiametric, (4.5–)5.5–10 × 3.5–9 μm. Conidia ellipsoidal to oblong, straight, thin- and smooth-walled, hyaline, aseptate, 3.5–7 × 2–3 μm, 2-guttulate, big. Conidial matrix buff.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 55–60 mm diam, aerial mycelium floccose, pale smoke grey, pale brown towards periphery, abundant production of confluent pycnidia, margin regular; reverse pale olivaceous, with some olivaceous black zones. On MEA reaching 50–55 mm diam, aerial mycelium woolly, pale olivaceous grey, margin irregular; reverse buff near the centre, dark brown with orange edge. On PDA reaching 50–55 mm diam, aerial mycelium floccose, pale mouse grey with olivaceous edge, margin irregular; reverse dark brown with pale brown edge. NaOH spot test negative on OA.

Notes

Based on the multi-gene phylogenetic analyses, D. kooimaniorum forms an independent branch, clearly separated from other species in Didymella (Figure 1). Morphologically, D. kooimaniorum is characterised by pale brown pycnidia densely covered by long hairs, and ostioles with up to six papillae with a darker neck.

Figure 4. 

Didymella kooimaniorum (CBS 144951). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G pycnidia forming on OA H pycnidia I section of pycnidium J section of pycnidial wall K–M conidiogenous cells N conidia. Scale bars: 100 μm (H); 50 μm (I); 10 μm (J); 5 μm (K–N).

Juxtiphoma kolkmaniorum Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833197
Figure 5

Etymology

kolkmaniorum refers to Linde & Mette Kolkman who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Ophemert, isolated from garden soil, Mar. 2017, L. & M. Kolkman (holotype designated here CBS H-24214, living ex-type culture CBS 146005 = JW 185006).

Conidiomata pycnidial, superficial, solitary or confluent, globose to subglobose, brown to dark brown, glabrous, covered by dark hyphae and chlamydospores, 100–350 μm; uniostiolate papillate; pycnidial wall pseudoparenchymatous, 2–4 layers, 7.5–12.5 μm thick, outer layer composed of brown, flattened polygonal cells. Conidiogenous cells mono- or polyphialidic, hyaline, smooth, subcylindrical, ampulliform or somewhat isodiametric, 5.5–11.5 × 2.5–5.5 μm. Conidia ellipsoidal to oblong, straight or curved, thin- and smooth-walled, hyaline, aseptate, 3.5–7.5 × 2–3 μm, 1–3-guttulate, medium. Conidial matrix white to buff. Chlamydospores terminal or intercalary, solitary, or in simple or branched chains, barrel-shaped, subglobose or ellipsoidal, pale brown to brown, guttulate, 5.5–12 × 4–8 μm.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 45–60 mm diam, aerial mycelium cottony to floccose, isabelline to olivaceous, margin irregular; reverse concolorous. On MEA reaching 45–55 mm diam, aerial mycelium cottony to floccose, smoke grey to pale olivaceous grey with white edge, margin entire; reverse buff to smoke grey near the centre, olivaceous black with buff edge. On PDA reaching 45–50 mm diam, aerial mycelium cottony to floccose, olivaceous buff, dull green to buff, margin irregular; reverse smoke grey near the centre, olivaceous black with buff edge. NaOH spot test negative on OA.

Additional specimens examined

Germany. Kiel-Kitzeberg, from wheat field soil, 1966, W. Gams, living cultures CBS 527.66 = FMR 15337 = ATCC 22238; The Netherlands. North Brabant province, Breda, isolated from garden soil, Mar. 2017, F. Versantvoort, JW 167004; ibid. JW 168007; Rijen, isolated from garden soil, Mar. 2017, G. & L. Schijvenaars, JW 94009. North Holland province, Hilversum, isolated from garden soil, Mar. 2017, S. Nieuwenhuijsen, JW 23021. Utrecht province, Amersfoort, isolated from garden soil, Mar. 2017, M. Kerssen, JW 125028; Amersfoort, isolated from garden soil, Mar. 2017, E., K. & O. de Jong Verpaalen, JW 241011; Amersfoort, isolated from garden soil, Mar. 2017, F. Wiegerinck, specimen CBS H-24102, culture CBS 145911 = JW 4017; Amersfoort, isolated from garden soil, Mar. 2017, T. & K. Wesselink, JW 191004; Bilthoven, isolated from garden soil, Mar. 2017, Y. El Ghazi, JW 220011; Utrecht, isolated from garden soil, Mar. 2017, J. Kooijmans, JW 63001.

Figure 5. 

Juxtiphoma kolkmaniorum (CBS 146005). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G, H pycnidium forming on OA I chlamydospores J–L conidiogenous cells M conidia. Scale bars: 100 μm (G, H); 10 μm (I–M).

Notes

Juxtiphoma kolkmaniorum is very similar and phylogenetically close to J. eupyrena. However, based on the multi-gene phylogenetic analyses, J. kolkmaniorum forms a separate clade (Figure 1). Morphologically, J. kolkmaniorum has conidia slightly larger and with more guttules than those of J. eupyrena (3.5–7.5 × 2–3 μm, 1–3-guttulate vs. 4.2–5.6 × 1.8–2.4 μm, 2-guttulate, de Gruyter and Noordeloos 1992) and smaller chlamydospores (5.5–12 × 4–8 μm vs. 8–20 × 6–15 μm, de Gruyter and Noordeloos 1992).

Nothophoma brennandiae Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833198
Figure 6

Etymology

brennandiae refers to Kristel Brennand who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Limburg province, Ell, isolated from garden soil, Mar. 2017, K. Brennand (holotype designated here CBS H-24103, living ex-type culture CBS 145912 = JW 53011).

Conidiomata pycnidial, superficial to semi-immersed, solitary to confluent, globose to subglobose, irregularly-shaped with age, brown, setose, especially near the ostioles, 155–350 × 100–300 μm; with 1–4 papillate ostioles; pycnidial wall pseudoparenchymatous, 3–6 layers, 13.5–21.5 μm thick, outer layers composed of brown, flattened polygonal cells. Conidiogenous cells phialidic, hyaline, smooth, ampulliform or somewhat isodiametric, 3–5 × 5–8 μm. Conidia ellipsoidal, broadly ellipsoidal to oblong, straight, thick- and smooth-walled, hyaline becoming brown, aseptate, 3–8.5 × 1.5–3 μm, 1–6-guttulate, minute. Conidial matrix sepia to brown vinaceous.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 50–55 mm diam, aerial mycelium scarce, spore mass with grease-like appearance, dark brick to sepia, cinnamon to the edge, abundant production of confluent pycnidia, margin entire; reverse concentric rings umber to cinnamon. On MEA reaching 47–50 mm diam, aerial mycelium scarce, spore mass with grease-like appearance, dark brick to sepia, cinnamon to the edge, abundant production of confluent pycnidia, margin entire; reverse concentric rings umber to cinnamon. On PDA reaching 50–55 mm diam, aerial mycelium moderate to scarce, cottony, buff, spore mass with grease-like appearance, dark brick, ochreous to the edge, margin entire; reverse concentric rings dark brick to cinnamon. NaOH spot test negative on OA.

Additional specimen examined

The Netherlands. North Holland province, Amsterdam, isolated from garden soil, Mar. 2017, J. van Dijk, JW 1066.

Notes

In the phylogenetic tree N. brennandiae was close to N. quercina and N. pruni (Figure 1). Morphologically, N. brennandiae can be distinguished from N. quercina by having setose conidiomata with up to 4 ostioles, while in N. quercina conidiomata are glabrous with a single ostiole (Sydow and Sydow 1915; Aveskamp et al. 2010). Furthermore, conidia in N. quercina are larger and have less guttules (5.5–9 × 2.5–5 μm, 0–2(–3) guttules) (Sydow and Sydow 1915; Aveskamp et al. 2010). On the other hand, N. pruni is characterised by hyaline conidia (Chethana et al. 2019), while N. brennandiae produces conidia that turn brown with age.

Figure 6. 

Nothophoma brennandiae (CBS 145912). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G pycnidia forming on OA. H, I pycnidia J section of pycnidial wall K–M conidiogenous cells N conidia. Scale bars: 50 μm (H, I); 10 μm (J); 5 μm (K–N).

Paraboeremia rekkeri Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833199
Figure 7

Etymology

rekkeri refers to Daan Rekker who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Gelderland province, Geldermalsen, isolated from garden soil, Mar. 2017, D. Rekker (holotype designated here CBS H-24107, living ex-type culture CBS 144955 = JW 172002).

Conidiomata pycnidial, superficial, scattered or aggregated, solitary or confluent, globose or subglobose, irregularly-shaped with age, buff to brown, covered with abundant mycelial outgrowths especially when young, 150–390 × 120–320 μm; 1–2 papillate or non-papillate ostioles; pycnidial wall pseudoparenchymatous, 3–7 layers, 17.5–37 μm thick, outer layers composed of brown, flattened polygonal cells, 10–21 μm diam. Conidiogenous cells phialidic, hyaline, smooth, globose, subglobose or ampulliform, 5–10 × 4.5–7.5 μm. Conidia ellipsoidal to oblong, thin- and smooth-walled, hyaline, aseptate, 3.5–5 × 2.5–3 μm, with 2(–3) large guttules. Conidial matrix pink.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 75–80 mm diam, aerial mycelium floccose, saffron, vinaceous buff, pale olivaceous, margin regular; reverse concentric circles saffron, grey, olivaceous grey. On MEA reaching 55–60 mm diam, aerial mycelium floccose, margin irregular, pale olivaceous grey to whitish, orange near edge; reverse brown to dark brown, orange towards the periphery. On PDA reaching 70–75 mm diam, margin irregular, covered by felty aerial mycelium, buff, olivaceous grey towards periphery; reverse mouse, olivaceous towards periphery. NaOH spot test negative on OA.

Additional specimens examined

Gelderland province, Culemborg, isolated from garden soil, Mar. 2017, H. van de Warenburg, JW 3018; Kapel-Avezaath, isolated from garden soil, Mar. 2017, A. Panneman, JW 79024; Meteren, isolated from garden soil, S. van Stuijvenberg, JW 132004; North Brabant province, Breda, isolated from garden soil, Mar. 2017, F. Wiegerinck, CBS 144949 = JW 4024; Breda, isolated from garden soil, Mar. 2017, F. Versantvoort, JW 167006; Zwanenburg, isolated from garden soil, Mar. 2017, J. Rebergen, JW 91008; North Holland province, Alkmaar, Mar. 2017, B. Verschoor, JW 13016, ibid. JW 13017 and JW13030; Utrecht province, Bilthoven, isolated from garden soil, Mar. 2017, H. Vos & S. Vos, JW 51014; Eemnes, isolated from garden soil, Mar. 2017, H.W. Vos, CBS 144950 = JW 6005; Hooglanderveen, isolated from garden soil, Mar. 2017, F. Rijpma, JW 25013; Utrecht, isolated from garden soil, R. van Zijl, JW 226002.

Notes

Paraboeremia rekkeri formed a well-supported (1.0/100/96) distinct lineage in Paraboeremia (Figure 1). It is most closely related with P. truiniorum, another novel species collected from Dutch soil and described in the present study. However, P. rekkeri is distinguished by producing larger pycnidia (150–390 × 120–320 μm), with a thinner pycnidial wall (3–7 layers and 17.5–37 μm thick). Pycnidia in P. truiniorum are 160–420 × 135–430 μm, and have a wall of 7–11 layers and 40–70 μm thick.

Figure 7. 

Paraboeremia rekkeri (CBS 144955). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G pycnidia forming on OA H pycnidium I section of pycnidium J section of pycnidial wall K–N conidiogenous cells O conidia. Scale bars: 100 μm (H); 20 μm (I); 10 μm (J); 5 μm (K–O).

Paraboeremia truiniorum Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833201
Figure 8

Etymology

truiniorum refers to Cuno & Tygo Truin who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Gelderland province, Barneveld, Voorthuizen, isolated from garden soil, Mar. 2017, C. Truin & T. Truin (holotype designated here CBS H-24108, living ex-type culture CBS 144952 = JW 47002).

Conidiomata pycnidial, superficial, scattered or aggregated, most solitary, globose or subglobose, confluent and irregularly-shaped with age, pale brown, thick-walled, covered with abundant mycelial outgrowths, 160–420 × 135–430 μm; 1-papillate or non-papillate ostioles, sometimes elongated to a short neck; pycnidial wall pseudoparenchymatous, 7–11 layers, 40–70 μm thick, outer layers composed of brown, flattened polygonal cells of 22–45.5 μm diam. Conidiogenous cells phialidic, hyaline, smooth, globose, subglobose, ampulliform or doliiform, 4.5–8.5 × 4–7 μm. Conidia ellipsoidal to oblong, thin- and smooth-walled, hyaline, aseptate, 3.5–5 × 2–3 μm, with (1–)2 large guttules. Conidial matrix whitish.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 70–75 mm diam, aerial mycelium floccose, vinaceous buff to hazel, margin regular; reverse buff to olivaceous. On MEA reaching 65–70 mm diam, aerial mycelium felty, whitish, pale mouse grey toward periphery, margin regular; reverse dark brick to dark brown, with pale brown edge. On PDA reaching 75–80 mm diam, aerial mycelium felty, olivaceous buff to pale mouse grey, olivaceous toward periphery, margin irregular; reverse mouse grey, olivaceous toward periphery. NaOH spot test negative on OA.

Additional specimens examined

The Netherlands, Gelderland province, Culemborg, isolated from garden soil, Mar. 2017, R. Fuld, JW 182014; The Netherlands. South Holland province, Alphen aan den Rijn, isolated from garden soil, Mar. 2017, K. Boutwell, CBS 144961 = JW 203021; The Netherlands. South Holland province, Gorinchem, isolated from garden soil, Mar. 2017, L. van Rosmalen, JW 270002; The Netherlands. Utrecht province, Utrecht, isolated from garden soil, Mar. 2017, L. van Rijnberk, JW 147025; The Netherlands. Utrecht province, Woerden, isolated from garden soil, Mar. 2017, L. Borsboom, JW 192003.

Notes

Based on the phylogenetic analyses, P. truiniorum is represented by six isolates, forming a distinct lineage (Figure 1). Paraboeremia truiniorum is characterised by the dense mycelial outgrowths on its pycnidia. Both P. truiniorum and P. rekkeri are phylogenetically close to the well-known soil-borne species, P. putaminum. However, P. putaminum is distinguished from these two new species by producing smaller conidia (3.2–4.2 × 2–2.6 μm) with greenish guttules (Boerema et al 2004).

Figure 8. 

Paraboeremia truiniorum (CBS 144952). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G pycnidia forming on OA H pycnidium I section of pycnidium J section of pycnidial wall K–N conidiogenous cells O conidia. Scale bars: 20 μm (H); 50 μm (I); 5 μm (J–O).

Stagonosporopsis stuijvenbergii Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833203
Figure 9

Etymology

stuijvenbergii refers to Simon van Stuijvenberg, who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Gelderland province, Meteren, from garden soil, Mar. 2017, S. van Stuijvenberg (holotype designated here CBS H-24109; living ex-type culture CBS 144953 = JW 132011).

Conidiomata pycnidial, produced on the agar surface, scattered or aggregated, solitary globose to subglobose, or 4–7(–10) confluent and irregularly-shaped, brownish, glabrous, ostiolate, 200–1000 × 195–930 μm; with 1–2 slightly papillate ostioles, sometimes elongated to a short neck; pycnidial wall pseudoparenchymatous, 4–5 layers, 6.5–35 μm thick, outer layers composed of brown, flattened polygonal cells, 9.5–33 μm diam. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform or lageniform, 4.5–9 × 4–8 μm. Conidia ellipsoidal to oblong, smooth- and thin-walled, hyaline, aseptate, 3.5–6.5 × 2–3 μm, 1–2-guttulate. Conidial matrix whitish.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 75–80 mm diam, floccose aerial mycelium, olivaceous to pale olivaceous, whitish to pink near the edge, margin regular; reverse iron grey. On MEA reaching 65–70 mm diam, margin regular, aerial mycelium floccose, vinaceous buff with olivaceous edge; reverse darker brown with olivaceous black edge, buff near the centre. On PDA reaching 70–75 mm diam, margin regular, covered by floccose aerial mycelium, olivaceous, olivaceous black towards periphery, with pinkish to pale brown edge; reverse iron-grey, buff towards periphery. NaOH spot test negative on OA.

Additional specimens examined

The Netherlands, Gelderland province, Arnhem, from garden soil, Mar. 2017, D. Peters, JW 14003; Utrecht province, Utrecht, from garden soil, Mar. 2017, N. Francisca, JW 44014; Utrecht, from garden soil, Mar. 2017, P. de Koff, JW 33021.

Notes

Phylogenetically, S. stuijvenbergii is most closely related to S. weymaniae, another novel species collected from Dutch soil in this study (Figure 1). However, S. stuijvenbergii is distinguishable from S. weymaniae by the colour and the size of its pycnidia, being brown and measuring 200–1000 × 195–930 μm in S. stuijvenbergii, whereas S. weymaniae produces whitish pycnidia, measuring 330–650 × 250–550 μm. Furthermore, S. weymaniae produces microconidia and chlamydospores, which were not observed in S. stuijvenbergii. Although there are several reports that Stagonosporopsis spp. could survive in soil for a short time (Vaghefi et al. 2016), this is the first record of a Stagonosporopsis species only known from soil (Domsch et al. 2007). Stagonosporopsis stuijvenbergii is represented by four strains isolated from different samples collected in Utrecht and Gelderland provinces.

Figure 9. 

Stagonosporopsis stuijvenbergii (CBS 144953). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G pycnidia forming on OA H pycnidia I ostiole J–L conidiogenous cells M stromatic hyphal aggregations N conidia. Scale bars: 50 μm (H); 10 μm (I, M); 5 μm (J–L, N).

Stagonosporopsis weymaniae Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833204
Figure 10

Etymology

weymaniae refers to Anna Weyman, who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Utrecht province, Baarn, isolated from garden soil, Mar. 2017, A. Weyman (holotype designated here CBS H-24110; living ex-type culture CBS 144959 = JW 201003).

Conidiomata pycnidial, semi-immersed or immersed, mostly solitary, scattered or aggregated, (sub-)globose, whitish to buff, glabrous, 330–650 × 250–550 μm; non-ostiolate or with a single, inconspicuous ostiole; pycnidial wall pseudoparenchymatous, 2–9 layers, 20–60 μm thick, outer layers composed of hyaline, flattened polygonal cells. Conidiogenous cells phialidic, hyaline, smooth, (sub-)globose to ampulliform, 4.5–7.5 × 4–7.5 μm. Macroconidia oblong, smooth- and thin-walled, hyaline, aseptate, 4–6.5(–8) × 2–3 μm, 1–3(–4)-guttulate, with one large central guttule or two large polar guttules. Microconidia produced in the same pycnidia with macroconidia, globose to subglobose, smooth, hyaline, aseptate, 3–4 × 2.5–3.5 μm, with a single, small guttule. Conidial matrix whitish. Chlamydospores unicellular, intercalary in chains, barrel-shaped, thick-walled, pale brown to green brown, guttulate, 9.5–14 × 11–16 μm diam.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 70–75 mm diam, sparse aerial mycelium, buff to pale olivaceous with sparse olivaceous zones, darker grey near the centre, abundant production of buff pycnidia, margin regular; reverse pale olivaceous, olivaceous black near the centre. On MEA reaching 80–85 mm diam, margin regular, aerial mycelium floccose, yellow to vinaceous buff; reverse orange to olivaceous. On PDA reaching 75–80 mm diam, margin regular, covered by floccose aerial mycelium, centre vinaceous buff, dark olivaceous towards the periphery with production of buff pycnidia; reverse olivaceous black, olivaceous towards the periphery. NaOH spot test: pale reddish discolouration on OA plate.

Notes

Stagonosporopsis weymaniae is phylogenetically closely related to S. stuijvenbergii (Figure 1). Morphological differences between S. weymaniae and S. stuijvenbergii are discussed under the latter species. Stagonosporopsis weymaniae together with S. stuijvenbergii formed a sister group with S. bomiensis and S. papillata, two plant pathogens from China (Chen et al. 2017). However, S. weymaniae differs from them by producing larger pycnidia [330–650 × 250–550 μm vs. 100–200 × 100–180 μm in S. bomiensis and (130–)200–280 × (100–)150–250 μm in S. papillata] and microconidia which are absent in S. papillata and S. bomiensis (Chen et al. 2017).

Figure 10. 

Stagonosporopsis weymaniae (CBS 144959). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G–I pycnidia forming on OA J, L conidiogenous cells K subglobose conidia M stromatic hyphal aggregations N chlamydospores O oblong conidia. Scale bars: 100 μm (I); 10 μm (J–N); 5 μm (O).

Vandijckomycella Hern.-Restr., L. W. Hou, L. Cai & Crous, gen. nov.

MycoBank No: 833205

Etymology

Named in honour of José F.T.M. van Dijck, who was elected as the first female President (2015–2018) of the Royal Dutch Academy of Arts and Sciences (KNAW).

Type species

Vandijckomycella joseae Hern.-Restr., L.W. Hou, L. Cai & Crous.

Conidiomata pycnidial, superficial on the surface of the agar, solitary or confluent, globose to lageniform, covered by hyphal outgrowths, ostiolate, pycnidial wall pseudoparenchymatous, with 3–9 layers. Conidiogenous cells phialidic, hyaline, smooth, globose or ampulliform. Conidia hyaline, smooth- and thin-walled, aseptate, ovoid, oblong or ellipsoidal, with 2–4 polar guttules.

Vandijckomycella joseae Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833208
Figure 11

Etymology

Named in honour of the first female President (2015–2018) of the Royal Dutch Academy of Arts and Sciences (KNAW), José F.T.M. van Dijck, who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. North Holland province, Amsterdam, isolated from garden soil, Mar. 2017, J.F.T.M. van Dijk (holotype designated here CBS H-24112; living ex-type culture CBS 143011 = JW 1073).

Conidiomata pycnidial, produced on the agar surface, scattered or aggregated, solitary, (sub-)globose, confluent and irregularly-shaped with age, pale brown, covered in abundant long and thin mycelium hair, 150–340 × 130–250 μm; with 1–2 slightly papillate or non-papillate ostioles, sometimes elongated to a short neck; pycnidial wall pseudoparenchymatous, 3–5 layers, 13–25 μm thick, outer layers composed of brown, flattened, polygonal cells of 10–23 μm diam. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform, lageniform or subglobose, 5–8(–9.5) × 4–8 μm. Conidia ellipsoidal to oblong, smooth- and thin-walled, hyaline, aseptate, 3.5–5.5 × 2–2.5 μm, (1–)2(–3)-guttulate. Conidial matrix whitish.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 75–80 mm diam after 7 d, covered by woolly aerial mycelium, concentric circles, pale olivaceous grey, pink, pale greenish grey, whitish near the edge, margin regular; reverse concentric circles dark brown, pale brown, orange, and pale olivaceous. On MEA reaching 75–80 mm diam, aerial mycelium woolly, margin regular, pale olivaceous grey; reverse dark brown, reddish towards the periphery. On PDA reaching 75–80 mm diam, margin regular, covered by felty aerial mycelium, pale olivaceous grey or olivaceous grey, with whitish parts near the centre or through the plate; reverse zonate, orange to reddish, brown and yellow. NaOH spot test: a coral discolouration on OA.

Additional specimen examined

The Netherlands. North Holland province, Amsterdam, isolated from garden soil, Mar. 2017, J.F.T.M. van Dijk, CBS 144948 = JW 1068.

Notes

The new genus Vandijckomycella is introduced to accommodate two new species isolated from soil samples which form an independent lineage in Didymellaceae, being clearly separated from other genera (Figure 1). Based on the phylogenetic analysis, V. joseae forms a distinct lineage which is distant from the nearest species V. snoekiae, and chiefly differs on tub2 and rpb2 sequences. Morphological differences between V. joseae and V. snoekiae are discussed under the latter species. Vandijckomycella joseae is characterised by producing pycnidia with longer whitish hyphal outgrowths, and with elongated necks.

Figure 11. 

Vandijckomycella joseae (CBS 143011). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G, H pycnidia forming on OA I, J section of pycnidial wall K–N conidiogenous cells O conidia. Scale bars: 100 μm (H); 20 μm (I); 10 μm (J); 5 μm (K–O).

Vandijckomycella snoekiae Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833207
Figure 12

Etymology

snoekiae refers to Rana Marit Ida Snoek who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Utrecht province, Utrecht, isolated from garden soil, Mar. 2017, R.M.I. Snoek (holotype designated here CBS H-24111, living ex-type culture CBS 144954 = JW 149017).

Conidiomata pycnidial, superficial on the agar or covered under a thick mycelial layer, scattered or aggregated, mostly solitary, globose to subglobose, sometimes confluent, ellipsoidal, dark brown, covered by abundant long hyphal outgrowths, 150–650(–850) × 145–600(–730) μm; ostioles inconspicuous; pycnidial wall pseudoparenchymatous, 5–9 layers, 37–58.5 μm thick, outer layers composed of brown, flattened polygonal cells, 10–23 μm diam. Conidiogenous cells phialidic, hyaline, smooth, globose, ampulliform or lageniform, 5–8.5 × 5–7.5 μm. Conidia oblong, smooth- and thin-walled, hyaline, aseptate, 4–6.5 × 2–2.5 μm, with two small polar guttules. Conidial matrix whitish.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 50–55 mm diam after 7 d, covered by floccose aerial mycelium, pink to grey, darker grey near the centre, margin regular; reverse black near the centre, yellow towards the periphery. On MEA reaching 50–55 mm diam, aerial mycelium floccose to cottony, buff with some mouse grey zones, margin regular; reverse orange with some radial yellow lines and some black zones. On PDA, reaching 45–50 mm diam, covered by floccose aerial mycelium, vinaceous grey to pale olivaceous, olivaceous grey near the centre, margin irregular; reverse buff to orange, black near the centre. NaOH spot test on OA: pale reddish discolouration.

Notes

Morphologically, V. snoekiae differs from its closest phylogenetic neighbour V. joseae in the size of its pycnidia and the number of ostioles. Vandijckomycella snoekiae produces larger pycnidia with inconspicuous ostioles, measuring 150–650(–850) × 145–600(–730) μm, while V. joseae produces pycnidia with 1–2 ostioles, measuring 150–340 × 130–250 μm. In addition, V. snoekiae produces conidia with less and smaller guttules than V. joseae (2 guttules, vs. 1–3 large guttules).

Figure 12. 

Vandijckomycella snoekiae (CBS 144954). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G, H pycnidia forming on OA I, J section of pycnidial wall K–N conidiogenous cells O conidia. Scale bars: 100 μm (H); 50 μm (I); 10 μm (J); 5 μm (K–O).

Xenodidymella weymaniae Hern.-Restr., L. W. Hou, L. Cai & Crous, sp. nov.

MycoBank No: 833209
Figure 13

Etymology

weymaniae refers to Anna Weyman who collected the soil sample from which the ex-type strain was isolated.

Typus

The Netherlands. Utrecht province, Baarn, isolated from garden soil, Mar. 2017, A. Weyman (holotype designated here CBS H-24113; living ex-type culture CBS 144960 = JW 201005).

Conidiomata pycnidial, semi-immersed on the agar, mostly confluent, sometimes solitary, scattered or aggregated, subglobose or ellipsoidal, irregularly-shaped when confluent, dark brown, ostiolate, glabrous or with long hyphal outgrowths around the ostiole, 100–700 × 100–400(–590) μm; with 1–2(–6) ostioles, papillate or elongated into a long neck, up to 113 μm in length; pycnidial wall pseudoparenchymatous, 3–5 layers, 17–45 μm thick, outer layers composed of pale brown to brown, flattened polygonal cells of 10–35 μm diam. Conidiogenous cells phialidic, hyaline, smooth, sub-globose, ampulliform or lageniform, 4.5–8 × 4–6.5 μm. Conidia oblong, smooth- and thin-walled, hyaline, aseptate, 4–6(–8) × 2–2.5 μm, with two small, polar guttules. Conidial matrix whitish.

Culture characteristics

Colonies after 7 d at 25 °C, on OA reaching 55–60 mm diam, aerial mycelium floccose near the centre, flat towards the periphery, pale olivaceous to whitish, black pycnidia visible near the centre, margin regular; reverse buff to salmon, pale olivaceous towards the periphery. On MEA reaching 40–45 mm diam, aerial mycelium felty, sectors with cottony mycelium, white, buff to pale olivaceous, margin regular; reverse yellow to orange, dark brown and pale grey near the centre. On PDA reaching 45–60 mm, aerial mycelium floccose, whitish in the centre, honey towards the periphery, margin regular; reverse concentric circles dark brown in centre, orange, yellow, buff towards the periphery. NaOH spot test negative on OA.

Notes

Xenodidymella weymaniae formed a distinct branch basal to X. applanata (Figure 1). Morphologically, X. weymaniae could be clearly differentiated from X. applanata in pycnidial and conidial characteristics. In X. weymaniae pycnidia are dark brown, ostioles have elongated necks, 100–700 × 100–400(–590) μm, and conidia are oblong, with 2 small polar guttules. In X. applanata pycnidia are pale brown, with single, slightly papillate ostioles, 85–175 × 60–145 μm, and ellipsoidal to ovoid conidia, with several guttules (Chen et al. 2015). Furthermore, the two species can also be distinguished from the NaOH spot test on OA medium (negative vs. pale reddish discoloration). This is the first record of a Xenodidymella species isolated from soil (Boerema et al. 2004; Chen et al. 2015, 2017).

Figure 13. 

Xenodidymella weymaniae (CBS 144960). A, B Colony on OA (front and reverse) C, D colony on MEA (front and reverse) E, F colony on PDA (front and reverse) G, H pycnidia forming on OA I, J section of pycnidial wall K–N conidiogenous cells O conidia. Scale bars: 50 μm (H); 20 μm (I); 10 μm (J); 5 μm (K–O).

Discussion

During the present Citizen Science project which focused on Dutch soil fungi, numerous unknown species of filamentous and yeast fungi were described (Crous et al. 2017, 2018; Groenewald et al. 2018; Giraldo et al. 2019). As part of the project, we focused on investigating species diversity of Didymellaceae from soil samples obtained in the Netherlands.

As one of the largest families in the fungal kingdom, at least 26 genera are accepted in Didymellaceae (Chen et al. 2015, 2017; Valenzuela-Lopez et al. 2018) and more than 5400 species names are recorded in MycoBank to date (Crous et al. 2004), representing 4.2 % of the 120000 accepted fungal species. However, only around 30 ubiquitous species have been found in the soil environment as saprophytes, mainly in Ascochyta, Boeremia, Didymella, Epicoccum and Phoma (Boerema et al. 2004; Aveskamp et al. 2008, 2010; Chen et al. 2015, 2017). In our set of samples, we found 20 species distributed in 10 genera i.e. Ascochyta, Calophoma, Didymella, Juxtiphoma, Nothophoma, Paraboeremia, Phomatodes, Stagonosporopsis, Vandijckomycella and Xenodidymella. However, we did not find any species of Phoma and Epicoccum, probably due to the media used for primary isolation, and also because of the taxonomical changes that have been suffered by many species of both genera in recent years (Aveskamp et al. 2010, Chen et al. 2015)

Paraboeremia and Juxtiphoma were the most dominant genera. Species of Paraboeremia are more common on plants than in soil, except for P. putaminum, which is regarded as a widespread soil-borne fungus isolated from the subterranean parts of various herbaceous and woody plants (de Gruyter and Noordeloos 1992; Boerema et al. 2004). In the present study this species was the most abundant species, being recovered from 29 soil samples from 19 cities. Besides P. putaminum, one isolate was identified as P. litseae, which was previously only known on diseased leaves of Litsea from China (Jiang et al. 2016). In addition, two new species were described, namely P. rekkeri found in Gelderland, North Brabant, North Holland and Utrecht provinces and P. truiniorum found in South Holland and Utrecht provinces.

The second most abundant species was Juxtiphoma eupyrena. The monotypic genus Juxtiphoma was recently introduced to accommodate Phoma eupyrena (Valenzuela-Lopez et al. 2018), a cosmopolitan soil-inhabiting fungus, which may cause damping-off of seedlings of herbaceous and woody plants (de Gruyter and Noordeloos 1992; Boerema et al. 2004; Morgan-Jones and Burch 1988), but was also reported as an opportunistic human pathogen (Bakerspigel et al. 1981). Furthermore, a new species was introduced in this genus as Juxtiphoma kolkmaniorum which includes 12 of our soil isolates (JW) and one strain (CBS 527.66) isolated from soil in a wheat field in Germany.

Among our isolates we found Phomatodes nebulosa, Didymella macrostoma and D. pomorum which are plurivorous and cosmopolitan species often isolated from soil (Boerema 1993; de Gruyter et al. 1993; Farr and Rossman 2019). Interestingly, we found two species identified as plant pathogens that had not been previously reported from soil, including Ascochyta syringae and Calophoma clematidis-rectae. Ascochyta syringae causes ascochyta blight of Lilac (Syringa vulgaris) in America, Australia and Europe (Farr and Rossman 2019), while Calophoma clematidis-rectae is known on Clematidis spp. in the Netherlands (Aveskamp et al. 2010). In addition, Stagonosporopsis is recognised mainly as a phytopathogenic genus on different plant hosts (Marin-Felix et al. 2019). However, we found two new species from soil, namely S. stuijvenbergii and S. weymaniae. Other new species described include A. benningiorum, D. degraaffiae, D. kooimaniorum, N. brennandiae, V. joseae, V. snoekiae, and X. weymaniae.

These findings suggest that species of Didymellaceae are also widely distributed in soil. Previous studies have revealed that many pathogens survive in soil by producing resting bodies (Dorenbosch 1970; Aveskamp et al. 2008), such as A. pinodes (currently: Didymella pinodes) and Phoma medicaginis var. pinodella (currently: Didymella pinodella) that produce chlamydospores or brown, thick-walled, swollen hyphae associated with sporocarps, which allow these species to survive in the soil for several years after the decay of their host tissues (Tivoli and Banniza 2007). On the other hand, some harmless saprobes in this family have also been observed to switch from an opportunistic to pathogenic lifestyle once in contact with the appropriate host (Aveskamp et al. 2008). Therefore, it is probable that the described new taxa are dormant in soil, remaining able to infect hosts under favourable conditions, especially species from phytopathogenic genera such as S. stuijvenbergii, S. weymaniae, N. brennandiae and X. weymaniae. However, considering that soil is a dynamic and multifunctional system and that the fungal community and its distribution are closely related to various living organisms such as plants, animals and insects, it was difficult to establish whether the species found in this study were true soil inhabitants or transferred to the soil via external vectors (such as worms, nematodes, etc.). Whether these new taxa originate from other habitats, or could change to pathogenic or endophytic lifestyles given the right conditions, remains to be determined. Furthermore, as the soil ecosystem is very complex and each type of soil and location may possess its own unique species diversity, the true diversity of Didymellaceae and their role in soil remains to be elucidated.

Recently, additional research based on cultivation-independent and cultivation-dependent methods has revealed that Didymellaceae species present in various soil environments are more diverse than one might have expected (Bell et al. 2014; Nallanchakravarthula et al. 2014; Li et al. 2016; Miao et al. 2016; Zhang et al. 2016a, 2016b; Chen et al. 2017; Nagano et al. 2017). Although recent high-throughput methods have detected a higher diversity of soil fungi compared with those based on culture-dependent methods, it is not possible to identify these taxa to species or even to genus level, as ITS sequence data alone are insufficient for species delimitation in most fungal families including Didymellaceae. Therefore, cultivation-dependent methods are still indispensable in the investigation of true species diversity of Didymellaceae based on additional loci such as rpb2 and tub2 obtained from cultivated isolates.

In summary, results of our study revealed the presence of a large number of unknown species and even a novel genus in soil, illustrating that this substrate is an important source for the discovery of novel taxa, and demonstrating that species diversity of Didymellaceae in soil is considerably greater than current estimates.

Acknowledgements

This study was financially supported by the Utrecht University Museum and the Royal Dutch Academy of Arts and Sciences for promoting the Citizen Science project, and for providing a platform to facilitate interaction with various Dutch primary schools. Lingwei Hou acknowledges CAS QYZDB-SSW-SMC044 for supporting her postgraduate studentship.

We are grateful to all the children and parents who participated in this project, collecting samples in their gardens and submitting them to the Westerdijk Institute for analyses; to José F.T.M. van Dijck, the first female President (2015–2018) of the Royal Dutch Academy of Arts and Sciences, who enthusiastically took part in this project and submitted the first soil sample in the Citizen Science Project. We are thankful to the staff from the Westerdijk Institute: Manon Verweij, Karin Schagen and Mariëtte Oosterwegel for promoting the project and establishing communication with the collectors and schools; to Trix Merkx and Arien van Iperen for depositing the isolates and specimens in the culture collection and fungarium.

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