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Taxonomic circumscription of melanconis-like fungi causing canker disease in China
expand article infoXinlei Fan, Zhuo Du§, Jadson D.P. Bezerra|, Chengming Tian
‡ Beijing Forestry University, Beijing, China
§ Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
| Universidade Federal de Pernambuco, Recife, Brazil
Open Access

Abstract

Melanconis-like species comprise latent fungal pathogens with a wide range of woody hosts. Taxonomy of these pathogens is difficult due to their uninformative descriptions and similar asexual morphology. Based on molecular phylogenies, many species of this group were placed in various families of Diaporthales. In this study, eight species of melanconis-like fungi were isolated from Betula albosinensis, B. platyphylla (Betulaceae), Cornus controversa (Cornaceae), Corylus mandshurica (Betulaceae) and Juglans regia (Juglandaceae) in China. These species were phylogenetically placed in three families of Diaporhthales, i.e. Juglanconis juglandina, J. oblonga (Juglanconidaceae), Melanconiella betulicola sp. nov., M. corylina sp. nov. (Melanconiellaceae), Melanconis betulae, Ms. itoana, Ms. stilbostoma (Melanconidaceae) and one new genus, Sheathospora (Melanconiellaceae). Sheathospora is proposed to accommodate Melanconiella cornuta with conical and discrete pycnidia with aseptate, hyaline, cylindrical to ellipsoidal conidia with distinct hyaline sheath on branches of Cornus controversa. Combined analyses of ITS, LSU, CAL, RPB2 and TEF1-α sequence data were used to construct the molecular phylogeny. Additionally, we provided separate phylogenetic trees for three families (Juglanconidaceae, Melanconidaceae and Melanconiellaceae) to show the species distribution of melanconis-like fungi in China.

Keywords

Diaporthales, phylogeny, taxonomy, wood-inhabiting fungi

Introduction

Melanconium (Diaporthales) was introduced by Link (1809) from dead branches of Fagus with M. atrum Link as the generic type. Corda (1837) extended this genus to 28 species. Subsequently, the genera Melanconis Tul. & C. Tul. and Melanconiella Sacc. were described as sexual morphs of Melanconium (Wehmeyer 1937, 1941). Sutton (1980) summarised more than 200 binomials that have been described in Melanconium, whereas no generic revision is available due to the uninformative descriptions and illustrations, few morphological characteristics, misplacement or poor condition of original specimens and lacking of ex-type cultures. In the Index Fungorum (2018), there are more than 235 species epithets of Melanconium with an estimated 50 species epithets by Kirk et al. (2008). Thus Melanconium species has serious obstacles for appropriate interpretation and is phylogenetically distributed throughout the Diaporthales, especially in Juglanconidaceae, Melanconidaceae and Melanconiellaceae. Although the genus Melanconium may be synonymous with Melanconis and would therefore have priority, the true identity of the generic type, M. atrum, is obscure and it was recommended to protect Melanconis over Melanconium (Rossman et al. 2015).

Molecular phylogenetics have had a major impact in taxonomic rearrangements of fungi since the early 1990s (White et al. 1990, Hibbett et al. 2007, Choi and Kim 2017, Fan et al. 2018). Castlebury et al. (2002) re-evaluated Diaporthales based on LSU rDNA sequences, indicating the single genus Melanconis with asexual morph Melanconium in Melanconidaceae s. str. Rossman et al. (2007) followed this differentiation and believed that many additional species of Melanconis sensu Wehmeyer (1941) should be separated from Melanconidaceae. One example is Melanconiella spodiaea (Tul. & C. Tul.) Sacc., type of the genus Melanconiella, which segregated from Melanconis (Rossman et al. 2007). Voglmayr et al. (2012) published sequences and molecular phylogenies for species of Melanconiella firstly and proposed that Melanconiella represented a distinct clade from Melanconis. Subsequently, Norphanphoun et al. (2016) introduced Lamproconiaceae to accommodate Melanconium desmazieri (Berk. & Broome) Sacc., with its sexual morph Melanconis desmazieri Petr. (Grove 1937, Sutton 1980). Voglmayr et al. (2017) proposed Juglanconidaceae to accommodate Melanconium juglandinum Kunze. Senanayake et al. (2017) introduced Melanconiellaceae to accommodate the previous unresolved Melanconiella.

During trips to collect forest pathogens that cause canker or dieback diseases in China, several melanconis-like taxa associated with various disease symptoms were collected in Beijing, Gansu, Heilongjian, Jilin, Ningxia, Shaanxi and Tibet Provinces. As the higher-level phylogeny of many genera within the melanconis-like taxa remains largely unresolved in China, this project was initiated to address this issue. In this paper, we identified eight melanconis-like species residing in three families of Diaporthales; recognised three genera within Melanoconiellaceae; and described two new species in Melanconiella as well as one new genus to accommodate Melanconiella cornuta.

Materials and methods

Isolation

Fresh specimens of melanconis-like fungi were collected from infected branches of seven hosts during collection trips in China (Table 1). A total of 47 isolates were established by removing a mucoid spore mass from ascomata or conidiomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) and incubating at 25 °C for up to 24 h. Single germinating conidia/ascospores were removed and plated on to fresh PDA plates. Specimens and isolates were deposited in the Key Laboratory for Silviculture and Conservation of the Ministry of Education in the Beijing Forestry University (BJFU) and the working Collection of X.L. Fan (CF) housed at the BJFU. Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Details of the strains included for molecular study used in this study.

Species Culture/strain/specimen Location Host GenBank accession numbers
ITS LSU CAL RPB2 TEF1-α
Apiosporopsis carpinea CBS 771.79 Switzerland Carpinus betulus NA AF277130 NA NA NA
Apiosporopsis sp. 11Af2-1 Japan Alnus firma NA AB669034 NA NA NA
Apoharknessia insueta CBS 111377 Brazil Eucalyptus pellita JQ706083 AY720814 NA NA NA
CBS 114575 Colombia Eucalyptus sp. NA AY720813 NA NA NA
Asterosporium asterospermum MFLU 15-3555 Italy Fagus sylvatica NA MF190062 NA MF377615 NA
CBS 112404 Italy Fagus sylvatica NA AB553745 NA NA NA
KT2138 Japan Fagus crenata NA AB553744 NA NA NA
Auratiopycnidiella tristaniopsidis CBS 132180 = CPC 16371 Australia Tristaniopsis laurina JQ685516 JQ685522 NA NA NA
Cainiella johansonii Kruys 731 Sweden Dryas octopetala NA JF701920 NA NA NA
Chapeckia nigrospora AR 3809 USA Betula sp. JF681957 EU683068 NA NA NA
Chiangraiomyces bauhiniae MFLUCC 17-1669 Thailand Bauhinia sp. MF190118 MF190064 NA MF377604 NA
MFLUCC 17-1670 Thailand Bauhinia sp. MF190119 MF190065 NA MF377603 NA
Chrysocrypta corymbiae CBS 132528 Australia Corymbia sp. JX069867 JX069851 NA NA NA
Coniella diplodiella CBS 111858 = CPC 3708 France Vitis vinifera AY339323 AY339284 NA KX833423 KX833603
Coniella koreana CBS 143.97 Korea NA KX833584 AF408378 NA KX833490 KX833684
Coniella musaiensis var. hibisci AR 3534 = CBS 109757 South Africa Hibiscus sp. KX833589 AF408337 NA NA KX833689
Coniella straminea CBS 149.22 = CPC 3932 USA Fragaria sp. AY339348 AF362569 NA KX833506 KX833704
Coniella wangiensis CBS 132530 = CPC 19397 Australia Eucalyptus sp. JX069873 JX069857 NA KX833509 KX833705
Coryneum depressum AR 3897 Austria Quercus cerris NA EU683074 NA NA NA
Coryneum modonium AR 3558 Austria Castanea sativa NA EU683073 NA NA NA
Coryneum umbonatum AR 3541 Austria Quercus cerris NA EU683072 NA NA NA
MFLUCC 15-1110 Italy Quercus sp. MF190121 MF190067 NA MF377610 NA
MFLUCC 13-0658 Italy Quercus sp. MF190120 MF190066 NA MF377609 NA
Cryphonectria macrospora AR 3444 = CBS 109764 Russia Quercus mongolica EU199182 AF408340 NA EU220029 NA
Cryphonectria nitschkei AR 3433 = CBS109776 Russia Quercus mongolica DQ120761 AF408341 NA NA NA
Cryphonectria parasitica ATCC 38755 USA Castanea dentata AY141856 EU199123 NA DQ862017 EU222014
Cryptodiaporthe aesculi AFTOL-ID 1238 = CBS 109765 Austria Aesculus hippocastanum DQ323530 AF408342 NA EU199138 GU354004
AR3640 = CBS 121905 USA Aesculus hippocastanum EU254994 EU255164 NA EU219269 DQ313558
LCM 447.01 Germany Aesculus hippocastanum GU367076 NA NA GU367110 GU354002
Cryptosporella betulae AR 3524 = CBS 109763 Austria Betula pendula EU199180 AF408375 NA EU199139 EU221884
Cryptosporella hypodermia AR 3552 Austria Ulmus minor EU199181 AF408346 NA EU199140 NA
Cryptosporella suffusa AR 3496 = CBS 109750 Austria Alnus incana EU199207 AF408376 NA EU199163 EU221945
Cytospora cenisia AR 3522 = CBS 109752 Austria Juniperus communis NA AF408385 NA NA NA
Cytospora chrysosperma CFCC 89600 China Sophora japonica KR045623 KR045623 NA KU710951 KU710915
Cytospora elaeagni CFCC 89633 China Elaeagnus angustifolia KF765677 KF765693 NA KU710956 KU710919
Cytospora leucostoma CFCC 50468 China Betula platyphylla KT732949 KT732968 NA NA NA
Cytospora nivea AR 3512 Austria Salix purpurea NA AF408367 NA NA NA
Cytospora sacculus AR 3416 = CBS 109756 Russia Quercus mongolica NA AF408386 NA NA NA
AR 3426 = CBS 109777 Austria Quercus robur NA AF408387 NA NA NA
Dendrostoma mali CFCC 52102 China Malus spectabilis MG682072 MG682012 NA MG682032 MG682052
Dendrostoma osmanthi CFCC 52106 China Osmanthus fragrans MG682073 MG682013 NA MG682033 MG682053
CFCC 52107 China Osmanthus fragrans MG682074 MG682014 NA MG682034 MG682054
CFCC 52108 China Osmanthus fragrans MG682075 MG682015 NA MG682035 MG682055
CFCC 52109 China Osmanthus fragrans MG682076 MG682016 NA MG682036 MG682056
Dendrostoma quercinum CFCC 52103 China Quercus acutissima MG682077 MG682017 NA MG682037 MG682057
CFCC 52104 China Quercus acutissima MG682078 MG682018 NA MG682038 MG682058
CFCC 52105 China Quercus acutissima MG682079 MG682019 NA MG682039 MG682059
Diaporthe decedens AR 3459 = CBS 109772 Austria Corylus avellana KC343059 AF408348 NA NA NA
Diaporthe detrusa AR 3424 = CBS 109770 Austria Berberis vulgaris KC343061 AF408349 NA NA KC343787
Diaporthe eres AR 3538 = CBS 109767 Austria Acer campestre KC343075 AF408350 NA NA KC343801
Diaporthella corylina CBS 121124 China Corylus sp. KC343004 NA NA NA NA
Diaporthella sp. CN5 Italy Corylus avellana KP205483 NA NA NA NA
CN13 Italy Corylus avellana KP205484 NA NA NA NA
Diaporthosporella cercidicola CFCC 51994 China Cercis chinensis KY852492 KY852515 NA NA NA
CFCC 51995 China Cercis chinensis KY852493 KY852516 NA NA NA
CFCC 51996 China Cercis chinensis KY852494 KY852517 NA NA NA
Diaporthostoma machili CFCC 52100 China Machilus leptophylla MG682080 MG682020 NA MG682040 MG682060
CFCC 52101 China Machilus leptophylla MG682081 MG682021 NA MG682041 MG682061
Disculoides eucalypti CPC 17650 Australia Eucalyptus sp. JQ685517 JQ685523 NA NA NA
Disculoides eucalyptorum CBS 132184 = CPC 17648 Australia Eucalyptus viminalis NR120090 JQ685524 NA NA NA
Ditopella ditopa AR 3423 = CBS 109748 Austria Alnus glutinosa EU199187 EU199126 NA EU199145 NA
Erythrogloeum hymenaeae CPC 18819 Brazil Hymenaea courbaril JQ685519 JQ685525 NA NA NA
Gnomonia gnomon CBS 199.53 Italy Corylus avellana AY818956 AF408361 NA EU219295 EU221885
Harknessia eucalypti CBS 342.97 Australia Eucalyptus regnans AY720745 AF408363 NA NA NA
Harknessia leucospermi CBS 775.97 South Africa Leucospermum sp. NR137147 AY720824 NA NA NA
Harknessia molokaiensis AR 3578 = CBS 109779 USA Eucalyptus robusta NA AF408390 NA NA NA
Harknessia syzygii CBS 111124 = CPC184 South Africa Syzygium cordatum AY720738 AY720834 NA NA NA
Hercospora tiliae AR 3526 Austria Tilia tomentosa NA AF408365 NA NA NA
Hyaliappendispora galii MFLUCC 16-1208 Italy Galium sp. MF190149 MF190095 NA NA NA
Involutscutellula rubra CBS 192.71 Japan Quercus phillyraeoides MG591899 MG591993 NA MG976476 MG592086
Juglanconis appendiculata D140 Greece Juglans nigra KY427138 KY427138 NA KY427188 KY427207
D96 Austria Juglans nigra KY427139 KY427139 NA KY427189 KY427208
D96A Austria Juglans nigra KY427140 KY427140 NA KY427190 KY427209
MC Greece Juglans nigra KY427141 KY427141 KY427242 KY427191 KY427210
MC2 Spain Juglans nigra KY427142 KY427142 KY427243 KY427192 KY427211
MC4 Spain Juglans nigra KY427143 KY427143 KY427244 KY427193 KY427212
ME17 Austria Juglans nigra KY427144 KY427144 KY427245 KY427194 KY427213
Juglanconis juglandina D142 Austria Juglans nigra KY427145 KY427145 NA KY427195 KY427214
CFCC 51727* China Juglans nigra KY363854 KY363859 MK096394 MK096439 NA
CFCC 51728* China Juglans nigra KY363855 KY363860 MK096395 MK096440 NA
CFCC 51729* China Juglans nigra KY363856 KY363861 MK096396 MK096441 NA
MC1 Austria Juglans nigra KY427146 NA KY427246 KY427196 KY427215
MC3 Spain Juglans nigra KY427147 KY427146 KY427247 KY427197 KY427216
ME16 Austria Juglans nigra KY427148 KY427147 KY427248 KY427198 KY427217
ME22 Austria Juglans nigra KY427149 KY427148 KY427249 KY427199 KY427218
ME23 Austria Juglans nigra KY427150 KY427150 KY427250 KY427200 KY427219
Juglanconis oblonga CFCC 51725* China Juglans nigra KY363852 KY363857 MK096392 MK096437 NA
CFCC 51726* China Juglans nigra KY363853 KY363858 MK096393 MK096438 NA
ME14 USA Juglans cinerea KY427151 KY427151 KY427251 KY427201 KY427220
ME15 USA Juglans cinerea KY427152 KY427152 KY427252 KY427202 KY427221
ME18 Japan Juglans ailanthifolia KY427153 KY427153 KY427253 KY427203 KY427222
ME19 Japan Juglans ailanthifolia KY427154 KY427154 KY427254 KY427204 KY427223
Juglanconis pterocaryae ME20 Japan Pterocarya rhoifolia KY427155 KY427155 KY427255 KY427205 KY427224
Lamproconium desmazieri MFLUCC 14-1047 Russia Tilia cordata KX430132 KX430133 NA NA MF377592
MFLUCC 15-0870 Russia Tilia tomentosa KX430134 KX430135 NA MF377605 MF377591
Lasmenia sp. CBS 124123 Puerto Rico Nephelium lappaceum GU797406 JF838338 NA NA NA
CBS 124124 Puerto Rico Nephelium lappaceum JF838336 JF838341 NA NA NA
Luteocirrhus shearii CBS 130776 Australia Banksia baxteri NR120254 NG042770 NA NA NA
Macrohilum eucalypti CPC 10945 New Zealand Eucalyptus sp. DQ195781 DQ195793 NA NA NA
CPC 19421 Australia Eucalyptus piperita KR873244 KR873275 NA NA NA
Melanconiella betulicola CFCC 52482* China Betula albosinensis MK096312 MK096352 NA MK096397 MK096272
CFCC 52483* China Betula albosinensis MK096313 MK096353 NA MK096398 MK096273
Melanconiella carpinicola MNM Austria Carpinus betulus JQ926232 JQ926232 NA JQ926304 JQ926370
MNUK UK Carpinus betulus JQ926234 JQ926234 NA JQ926306 JQ926372
MSMI Austria Carpinus betulus JQ926235 JQ926235 NA JQ926307 JQ926373
Melanconiella chrysodiscosporina MCH Austria Carpinus betulus JQ926238 JQ926238 NA JQ926310 JQ926376
MEE Austria Carpinus betulus JQ926240 JQ926240 NA JQ926312 JQ926378
MGG Austria Carpinus betulus JQ926242 JQ926242 NA JQ926314 JQ926380
Melanconiella chrysomelanconium MCM Austria Carpinus betulus JQ926247 JQ926247 NA JQ926319 JQ926385
MEUK UK Carpinus betulus JQ926249 JQ926249 NA JQ926321 JQ926387
MGUK UK Carpinus betulus JQ926255 JQ926255 NA JQ926327 JQ926393
Melanconiella chrysorientalis MGB Croatia Carpinus orientalis JQ926256 JQ926256 NA JQ926328 JQ926394
MGP Croatia Carpinus orientalis JQ926257 JQ926257 NA JQ926329 JQ926395
MVH Croatia Carpinus orientalis JQ926259 JQ926259 NA JQ926331 JQ926397
Melanconiella corylina CFCC 52484* China Corylus mandshurica MK096314 MK096354 NA MK096399 MK096274
CFCC 52485* China Corylus mandshurica MK096315 MK096355 NA MK096400 MK096275
Melanconiella decorahensis CBS 159.26 USA Betula sp. JQ926260 JQ926260 NA JQ926332 JQ926398
MD France Betula pendula JQ926261 JQ926261 NA JQ926333 JQ926399
MED France Betula pendula JQ926262 JQ926262 NA JQ926334 JQ926400
Melanconiella echinata DAOM 121196 USA Carpinus caroliniana JQ926263 JQ926263 NA N/A N/A
Melanconiella elegans AR 3830 USA Carpinus caroliniana JQ926264 JQ926264 NA JQ926335 JQ926401
BPI 843574 USA Carpinus caroliniana JQ926266 JQ926266 NA JQ926337 JQ926403
BPI 872067 USA Carpinus caroliniana JQ926267 JQ926267 NA JQ926338 JQ926404
Melanconiella ellisii BPI 843491 USA Carpinus caroliniana JQ926268 JQ926268 NA N/A JQ926405
BPI 878343 USA Carpinus caroliniana JQ926271 JQ926271 NA JQ926339 JQ926406
BPI 883227 USA Carpinus caroliniana JQ926269 JQ926269 NA N/A N/A
Melanconiella flavovirens MFV1 Austria Corylus avellana JQ926274 JQ926274 NA JQ926342 JQ926409
MFV2 Austria Corylus avellana JQ926275 JQ926275 NA JQ926343 JQ926410
MFV3 Italy Corylus avellana JQ926276 JQ926276 NA JQ926344 JQ926411
Melanconiella hyperopta MCHBV Austria Carpinus betulus JQ926280 JQ926280 NA JQ926346 JQ926413
MCR Austria Carpinus betulus JQ926283 JQ926283 NA JQ926349 JQ926416
MHG Switzerland Carpinus betulus JQ926285 JQ926285 NA JQ926351 JQ926418
Melanconiella hyperopta var. orientalis MHP Croatia Carpinus orientalis JQ926288 JQ926288 NA JQ926352 JQ926420
MHVA Croatia Carpinus orientalis JQ926287 JQ926287 NA JQ926353 JQ926419
MSK Croatia Carpinus orientalis JQ926286 JQ926286 NA JQ926354 JQ926421
Melanconiella meridionalis MOA Austria Ostrya carpinifolia JQ926289 JQ926289 NA JQ926355 JQ926422
MOK Croatia Ostrya carpinifolia JQ926290 JQ926290 NA JQ926356 JQ926423
MOM Austria Ostrya carpinifolia JQ926291 JQ926291 NA JQ926357 JQ926424
Melanconiella ostryae CBS 208.38 USA Ostrya virginiana JQ926297 JQ926297 NA JQ926363 JQ926430
Melanconiella spodiaea MVS Croatia Carpinus orientalis JQ926299 JQ926299 NA JQ926365 JQ926432
MSH Austria Carpinus betulus JQ926298 JQ926298 NA JQ926364 JQ926431
SPOD Croatia Carpinus betulus JQ926300 JQ926300 NA JQ926366 JQ926433
Melanconis alni AR 3529 Russia Duschekia maximowiczii NA AF362566 NA NA NA
AR 3748 Austria Alnus viridis EU199195 EU199130 NA EU199153 NA
AR 4016 = CBS 121480 Austria Alnus alnobetula EU254863 NA NA EU219298 EU221894
CBS 109773 Austria Alnus viridis DQ323523 AF408371 NA EU219300 EU221896
Melanconis betulae CFCC 50471* China Betula albosinensis KT732952 KT732971 NA KT732984 KT733001
CFCC 50472* China Betula albosinensis KT732953 KT732972 NA KT732985 KT733002
CFCC 50473* China Betula albosinensis KT732954 KT732973 NA KT732986 KT733003
Melanconis italica MFLUCC 16-1199 Italy Alnus cordata MF190151 MF190096 NA NA NA
MFLUCC 17-1659 Italy Alnus cordata MF190151 MF190097 NA MF377602 NA
Melanconis itoana CFCC 50474* China Betula albosinensis KT732955 KT732974 NA KT732987 KT733004
CFCC 52876* China Betula albosinensis MK096324 MK096364 NA MK096409 MK096284
CFCC 52877* China Betula albosinensis MK096326 MK096366 NA MK096411 MK096286
CFCC 52878* China Betula albosinensis MK096327 MK096367 NA MK096412 MK096287
MAFF 410080 Japan Betula ermanii JX522738 NA NA NA NA
Melanconis marginalis AR 3442 = CBS 109744 Canada Alnus rubra EU199197 AF408373 NA EU219301 EU221991
MAFF 410218 Japan Alnus maximowiczii JX522742 NA NA NA NA
Melanconis stilbostoma CBS 109778 = AR 3501 Austria Betula pendula DQ323524 AF408374 NA EU219299 EU221886
CBS 121894 = MS NA Betula pendula JQ926229 JQ926229 NA JQ926302 JQ926368
CFCC 50475* China Betula platyphylla KT732956 KT732975 NA KT732988 KT733005
CFCC 50476* China Betula platyphylla KT732957 KT732976 NA KT732989 KT733006
CFCC 50477* China Betula platyphylla KT732958 KT732977 NA KT732990 KT733007
CFCC 50478* China Betula platyphylla KT732959 KT732978 NA KT732991 KT733008
CFCC 50479* China Betula platyphylla KT732960 KT732979 NA KT732992 KT733009
CFCC 50480* China Betula platyphylla KT732961 KT732980 NA KT732993 KT733010
Melanconis stilbostoma CFCC 50481* China Betula platyphylla KT732962 KT732981 NA KT732994 KT733011
CFCC 50482* China Betula platyphylla KT732963 KT732982 NA KT732995 KT733012
CFCC 52843* China Betula platyphylla MK096338 MK096378 NA MK096423 MK096298
CFCC 52844* China Betula platyphylla MK096341 MK096381 NA MK096426 MK096301
CFCC 52845* China Betula platyphylla MK096343 MK096383 NA MK096428 MK096303
CFCC 52846* China Betula platyphylla MK096347 MK096387 NA MK096432 MK096307
CFCC 52847* China Betula platyphylla MK096348 MK096388 NA MK096433 MK096308
CFCC 52848* China Betula platyphylla MK096349 MK096389 NA MK096434 MK096309
CFCC 52849* China Betula platyphylla MK096328 MK096368 NA MK096413 MK096288
CFCC 52850* China Betula platyphylla MK096329 MK096369 NA MK096414 MK096289
CFCC 52851* China Betula platyphylla MK096330 MK096370 NA MK096415 MK096290
CFCC 52852* China Betula platyphylla MK096331 MK096371 NA MK096416 MK096291
CFCC 52853* China Betula platyphylla MK096332 MK096372 NA MK096417 MK096292
CFCC 52854* China Betula platyphylla MK096333 MK096373 NA MK096418 MK096293
CFCC 52855* China Betula platyphylla MK096334 MK096374 NA MK096419 MK096294
CFCC 52856* China Betula platyphylla MK096335 MK096375 NA MK096420 MK096295
CFCC 52857* China Betula platyphylla MK096336 MK096376 NA MK096421 MK096296
CFCC 52858* China Betula platyphylla MK096337 MK096377 NA MK096422 MK096297
CFCC 52859* China Betula platyphylla MK096339 MK096379 NA MK096424 MK096299
CFCC 52860* China Betula platyphylla MK096340 MK096380 NA MK096425 MK096300
CFCC 52861* China Betula platyphylla MK096342 MK096382 NA MK096427 MK096302
CFCC 52862* China Betula platyphylla MK096344 MK096384 NA MK096429 MK096304
CFCC 52863* China Betula platyphylla MK096345 MK096385 NA MK096430 MK096305
CFCC 52864* China Betula platyphylla MK096346 MK096386 NA MK096431 MK096306
CFCC 52865* China Betula platyphylla MK096316 MK096356 NA MK096401 MK096276
CFCC 52866* China Betula platyphylla MK096317 MK096357 NA MK096402 MK096277
CFCC 52867* China Betula platyphylla MK096318 MK096358 NA MK096403 MK096278
CFCC 52868* China Betula platyphylla MK096319 MK096359 NA MK096404 MK096279
CFCC 52869* China Betula platyphylla MK096320 MK096360 NA MK096405 MK096280
CFCC 52870* China Betula platyphylla MK096321 MK096361 NA MK096406 MK096281
CFCC 52871* China Betula platyphylla MK096322 MK096362 NA MK096407 MK096282
CFCC 52872* China Betula platyphylla MK096323 MK096363 NA MK096408 MK096283
CFCC 52873* China Betula platyphylla MK096350 MK096390 NA MK096435 MK096310
CFCC 52874* China Betula platyphylla MK096351 MK096391 NA MK096436 MK096311
CFCC 52875* China Betula platyphylla MK096325 MK096365 NA MK096410 MK096285
Microascospora fragariae CBS 118.16 USA Fragaria sp. NR156500 NA NA NA NA
CBS 128350 USA Rubus sp. JF514854 NA NA NA NA
1-1 China Fragaria ananassa HM854850 NA NA NA NA
1-2 China Fragaria ananassa HM854849 NA NA NA NA
1-3 China Fragaria ananassa HM854852 NA NA NA NA
Microascospora rubi MFLU 15-1112 Italy Rubus ulmifolia MF190154 MF190098 NA MF377581 MF377611
MFLU 17-0883 Italy Rubus ulmifolia MF190153 MF190099 NA MF377582 MF377612
Nakataea oryzae CBS 243.76 NA NA KM484861 DQ341498 NA NA NA
Oblongisporothyrium castanopsidis ATCC 22470 Japan Castanopsis cuspidata MG591850 MG591943 NA MG592038 MG976454
Ophiodiaporthe cyatheae YMJ1364 China Cyathea lepifera JX570889 JX570891 NA JX570893 NA
Pachytrype princeps Rogers S USA NA NA FJ532382 NA NA NA
Pachytrype rimosa FF1066 Costa Rica NA NA FJ532381 NA NA NA
Paradiaporthe artemisiae MFLUCC 14-0850 Italy Artemisia sp. MF190155 MF190100 NA NA NA
MFLUCC 17-1663 Italy Artemisia sp. MF190156 MF190101 NA NA NA
Phaeoappendispora thailandensis MFLUCC 13-0161 Thailand Quercus sp. MF190157 MF190102 NA MF377613 NA
Phaeodiaporthe appendiculata CBS 123821 = D77 Austria Acer campestre KF570156 KF570156 NA NA NA
CBS 123809 = D76 Austria Acer campestre KF570155 KF570155 NA NA NA
Phragmoporthe conformis AR 3632 = CBS 109783 Canada Alnus rubra DQ323527 AF408377 NA NA NA
Plagiostoma euphorbiae CBS 340.78 Netherlands Euphorbia palustris EU199198 AF408382 NA DQ368643 NA
Plagiostoma salicellum AR 3455 = CBS 109775 Austria Salix sp. DQ323529 AF408345 NA EU199141 EU221916
Prosopidicola mexicana CBS 113530 USA Prosopis glandulosa AY720710 NA NA NA NA
CBS 113529 USA Prosopis glandulosa AY720709 KX228354 NA NA NA
Pseudomelanconis caryae CFCC 52110 China Carya cathayensis MG682082 MG682022 NA MG682042 MG682062
CFCC 52111 China Carya cathayensis MG682083 MG682023 NA MG682043 MG682063
CFCC 52112 China Carya cathayensis MG682084 MG682024 NA MG682044 MG682064
CFCC 52113 China Carya cathayensis MG682085 MG682025 NA MG682045 MG682065
Pseudoplagiostoma eucalypti CBS 124807 Venezuela Eucalyptus urophylla GU973512 GU973606 NA NA NA
CBS 116382 Thailand Eucalyptus camaldulensis GU973514 GU973608 NA NA NA
Pseudoplagiostoma oldii CBS 115722 Australia Eucalyptus camaldulensis GU973535 GU973610 NA NA NA
Pseudoplagiostoma variabile CBS 113067 Uruguay Eucalyptus globulus GU973536 GU973611 NA NA NA
Pyricularia grisea Ina168 NA NA AB026819 AB026819 NA NA NA
Racheliella saprophytica NTCL052-1 Thailand Syzygium cumini KJ021933 KJ021935 NA NA NA
Racheliella wingfieldiana CBS 143669 South Africa Syzigium guineense MG591911 MG592006 NA MG592100 MG976487
Rossmania ukurunduensis AR 3484 Russia Acer ukurunduense NA EU683075 NA NA NA
Saprothyrium thailandense MFLU 13-0260 Thailand Decaying leaf MF190163 MF190110 NA NA NA
Sheathospora cornuta CFCC 51990* China Cornus controversa MF360006 MF360008 NA MF360002 MF360004
CFCC 51991* China Juglans regia MF360007 MF360009 NA MF360003 MF360005
Sillia ferruginea AR 3440 = CBS 126567 Austria Corylus avellana JF681959 EU683076 NA NA NA
Sphaerosporithyrium mexicanum CFNL 2945 Mexico Quercus eduardi MG591896 MG591990 NA MG592083 MG976473
Stegonsporium protopyriforme CBS 117041 Austria Acer pseudoplatanus NR126119 EU039992 NA NA NA
Stegonsporium pyriforme CBS 124487 UK Acer heldreichii KF570160 KF570160 NA KF570190 NA
Stilbospora macrosperma CBS 121883 Austria Carpinus betulus JX517290 JX517299 NA KF570196 NA
CBS 121695 Netherlands Carpinus betulus JX517288 JX517297 NA NA NA
Sydowiella depressula CBS 813.79 Switzerland Rubus sp. NA EU683077 NA NA NA
Sydowiella fenestrans AR 3777 = CBS 125530 Russia Chamerion angustifolium JF681956 EU683078 NA NA NA
Synnemasporella aculeans AR 3878 = CBS 126566 USA Rhus glabra NA EU255134 NA NA NA
CFCC 52094 China Rhus chinensis MG682086 MG682026 NA MG682046 MG682066
CFCC 52095 China Rhus chinensis MG682087 MG682027 NA MG682047 MG682067
CFCC 52096 China Rhus chinensis MG682088 MG682028 NA MG682048 MG682068
Synnemasporella toxicodendri CFCC 52097 China Toxicodendron sylvestre MG682089 MG682029 NA MG682049 MG682069
CFCC 52098 China Toxicodendron sylvestre MG682090 MG682030 NA MG682050 MG682070
CFCC 52099 China Toxicodendron sylvestre MG682091 MG682031 NA MG682051 MG682071
Tubakia japonica ATCC 22472 Japan Castanea crenata MG591886 MG591978 NA MG592071 MG976465
CBS 191.71 Japan Castanea crenata MG591885 MG591977 NA MG592070 MG976464
MUCC 2297 Japan Castanea crenata NA MG591979 NA MG592072 MG976466
MUCC 2298 Japan Castanea crenata NA MG591980 NA MG592073 MG976467
MUCC 2300 Japan Castanea crenata NA MG591981 NA MG592074 MG976468
MUCC 2301 Japan Castanea crenata NA MG591982 NA MG592075 MG976469
Tubakia seoraksanensis CBS 127490 South Korea Quercus mongolica MG591907 KP260499 NA MG592094 NA
Tubakia sutoniana ICMP 14042 New Zealand Quercus sp. KC145909 NA NA NA KC145954
ICMP 14043 New Zealand Quercus ilex KC145858 NA NA NA KC145955

Morphological studies

Species identification was based on morphological features of the ascomata or conidiomata produced on infected plant tissues and micromorphology, supplemented by cultural characteristics. Cross-sections were prepared by hand using a double-edge blade under a dissecting microscope. More than 10 conidiomata/ascomata, 10 asci and/or 50 conidia/ascospores were measured to calculate the mean size and standard deviation (SD). Microscopic photographs were captured with a Nikon Eclipse 80i microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast (DIC) illumination and the Nikon software NIS-Elements D Package v. 3.00. Adobe Bridge CS v. 6 and Adobe Photoshop CS v. 5 were used for the manual editing. Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004). Colony diameters were measured and the colony colours described after 3 weeks according to the colour charts of Rayner (1970).

DNA extraction and sequencing

Genomic DNA was extracted using a modified CTAB method, with fungal mycelium harvested from PDA plates with cellophane (Doyle and Doyle 1990). The DNA was estimated by electrophoresis in 1% agarose gel and the quality was measured by NanoDrop 2000 (Thermo, USA) according to the user’s manual (Desjardins et al. 2009). The PCR amplifications were performed in DNA Engine (PTC-200) Peltier Thermal Cycler (Bio-Rad Laboratories, CA, USA). The ITS region was amplified with the primers ITS1 and ITS4 (White et al. 1990), the LSU region with the primers LR0R and LR5 (Vilgalys and Hester 1990), the CAL gene (for Juglanconidaceae) with primers CAL-228F and CAL-737R (Carbone and Kohn 1999), the RPB2 region with primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999), the TEF1-α gene with the primers EF1-728F and EF1-LLErev for Melanconiellaceae (Carbone and Kohn 1999, Jaklitsch et al. 2005) and the primers EF1-983F and EF1-1567R for Melanconidaceae (Carbone and Kohn 1999, Rehner and Buckley 2005). The PCR mixture for all the regions consisted of 1 μl genomic DNA, 3 mM MgCl2, 20 μM of each dNTP, 0.2 μM of each primer and 0.25 U BIOTAQ DNA polymerase (Bioline). Conditions for PCR of ITS and LSU regions constituted an initial denaturation step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 45 s at 51 °C and 1 min at 72 °C and a final extension step of 8 min at 72 °C, while the TEF1-α gene was performed using an initial denaturation step of 2 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 45 s at 56 °C and 1 min at 72 °C and a final extension step of 8 min at 72 °C. For the RPB2 amplification, conditions consisted of five cycles of 45 s at 95 °C, 45 s at 56 °C and 2 min at 72 °C, then five cycles with a 53 °C annealing temperature and 30 cycles with a 50 °C annealing temperature. The DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with BigDye Terminater Kit v. 3.1 (Invitrogen) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses

DNA sequences generated by each primer combination were used to obtain consensus sequences using SeqMan v. 7.1.0 in the DNASTAR Lasergene Core Suite software package (DNASTAR Inc., Madison, WI, USA). Reference sequences were selected based on ex-type or ex-epitype sequences available from relevant published literature (Voglmayr et al. 2012, 2017, Fan et al. 2016, 2018, Du et al. 2017, Senanayake et al. 2017) (Table 1). All sequences were aligned using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html) and edited manually using MEGA v. 6 (Tamura et al. 2013). Phylogenetic analyses were performed using PAUP v. 4.0b10 for maximum parsimony (MP) analysis (Swofford 2003), MrBayes v. 3.1.2 for Bayesian Inference (BI) analysis (Ronquist and Huelsenbeck 2003) and PhyML v. 7.2.8 for Maximum Likelihood (ML) analysis (Guindon et al. 2010). The first analyses were performed on the combined multi-gene dataset (ITS, LSU, RPB2, TEF1-α) to compare isolates of Diaporthales species to ex-type sequence data from recent studies (Table 1).

A partition homogeneity test (PHT) with heuristic search and 1 000 search replicates was performed using PAUP to test for incongruence amongst the ITS, LSU, RPB2 and TEF1-α sequence datasets in reconstructing phylogenetic trees. Maximum parsimony (MP) analysis was run using 1 000 heuristic search replicates with random-additions of sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were set to 5 000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). Maximum likelihood (ML) analysis was performed with a GTR site substitution model, including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated with a bootstrapping (BS) method of 1 000 replicates.

MrModeltest v. 2.3 was used to estimate the best nucleotide substitution model settings for each gene (Posada and Crandall 1998). Bayesian inference (BI) was performed based on the DNA dataset from the results of the MrModeltest, using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). Two MCMC chains were run from random trees for 1 000 M generations and stopped when the average standard deviation of split frequencies fell below 0.01. Trees were saved each 1 000 generations. The first 25% of trees were discarded as the burn-in phase of each analysis and the posterior probabilities (BPP) were calculated from the remaining trees (Rannala and Yang 1996).

In addition to the above analyses, we provided separate phylogenetic trees for Juglanconidaceae, Melanconidaceae and Melanconiellaceae, based on various gene regions (see below) and the same analyses parameters as given above. Phylograms were edited using FigTree v. 1.3.1 (Rambaut and Drummond 2010). Novel sequences generated in the current study were deposited in GenBank (Table 1). The aligned matrices used for phylogenetic analyses and the resulting trees can be found in TreeBASE (www.treebase.org; accession number: S23477).

Results

Phylogenetic analyses

The combined matrix of ITS, LSU, RPB2 and TEF1-α of Diaporthales included 209 ingroup and two outgroup taxa, comprising 3 269 characters including gaps (776 characters for ITS, 517 for LSU, 1107 for RPB2 and 869 for TEF1-α) in the aligned matrix. Of these, 1 417 characters were constant, 192 variable characters were parsimony-uninformative and 1 660 characters were parsimony informative. The MP analysis resulted in 100 most parsimonious trees (TL = 10 370, CI = 0.341, RI = 0.806, RC = 0.275) and the first tree is shown as Fig. 1. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Branches with significant Bayesian posterior probability (≥ 0.95) in Bayesian analyses were thickened in the phylogenetic tree. The phylogram based on four genes resolved 28 known lineages, representing 26 known families and two incertae sedis genera Diaporthella and Phaeoappendispora due to lack of sequence data on their types. The current 47 melanconis-like isolates are herein placed within Juglanconidaceae, Melanconidaceae and Melanconiellaceae in Diaporthales (Fig. 1). A phylogenetic tree of each family or genus was constructed separately based on different DNA datasets. Tree topologies of all genera computed from the MP, ML and Bayesian analyses were similar for the individual gene region and in the combined dataset.

Figure 1. 

Phylogram of Diaporthales obtained from an MP analysis of a combined matrix of ITS, LSU, RPB2 and TEF1-α. MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.95 from BI. Scale bar = 200 changes. Type species are in bold. Strains obtained in the current study are in blue.

For the single genus Juglanconis (Juglanconidaceae), a combined ITS, LSU, CAL and RPB2 matrix of 23 ingroup accessions (five from this study and 18 retrieved from GenBank) was produced, which comprised 2 736 characters including gaps (2 427 constant, 216 variable and parsimony-uninformative, 93 parsimony-informative). A heuristic MP search generated nine equally most parsimonious trees (TL = 332, CI = 0.976, RI = 0.985, RC = 0.961), one of which is shown in Fig. 2. Isolates of Juglanconis clustered in four clades, corresponding to the four known species in this genus. The five Chinese strains sequenced in this study were revealed to belong to Juglanconis juglandina (3) and J. oblonga (2).

Figure 2. 

Phylogram of Juglanconis (Juglanconidaceae) obtained from an MP analysis of a combined matrix of ITS, LSU, CAL and RPB2. MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.95 from BI. Scale bar = 20 changes. Type species are in bold. Strains obtained in the current study are in blue.

For Melanconiellaceae, a combined ITS, LSU, RPB2 and TEF1-α matrix was produced from 53 ingroup accessions (six from this study and 47 retrieved from GenBank), which comprised 4 122 characters including gaps (2 829 constant, 87 variable and parsimony-uninformative, 1 206 parsimony-informative). A heuristic MP search generated 24 most parsimonious trees (TL = 2 716, CI = 0.652, RI = 0.880, RC = 0.573), one of which is shown in Fig. 5. Isolates of Melanconiellaceae clustered in three clades, corresponding to the type genus Melanconiella, Microascospora and a lineage described as the new genus Sheathospora below. Melanconiella betulicola and M. corylina formed two distinct strongly supported clades (MP/ML/BI = 100/100/1), which differ from the other species of the Melanconiella clade.

For the single genus Melanconis (Melanconidaceae), a combined ITS, LSU, RPB2 and TEF1-α matrix was produced for 57 ingroup accessions (49 from this study and eight retrieved from GenBank), which comprised 2 597 characters including gaps (2 238 constant, 219 variable and parsimony-uninformative, 140 parsimony-informative). A heuristic MP search generated 144 most parsimonious trees (TL = 459, CI = 0.861, RI = 0.919, RC = 0.791), one of which is shown in Fig. 6. Isolates of Melanconis clustered in six clades, corresponding to six known species in this genus. Melanconis betulae, Ms. stilbostoma and Ms. itoana were confirmed from China in this study.

Taxonomy

Juglanconidaceae Voglmayr & Jaklitsch, Persoonia 38: 142 (2017)

Type genus

Juglanconis Voglmayr & Jaklitsch, Persoonia 38: 142 (2017)

Notes

Juglanconidaceae, with the single genus Juglanconis, was newly introduced by Voglmayr et al. (2017) for Melanconium juglandinum, M. oblongum and M. pterocaryae. In this paper, we provide an updated tree including accessions of two Juglanconis species from China (Fig. 2).

Juglanconis Voglmayr & Jaklitsch, Persoonia 38: 142 (2017)

Type species

Juglanconis juglandina (Kunze) Voglmayr & Jaklitsch, Persoonia 38: 144 (2017).

Notes

Juglanconis was newly introduced by Voglmayr et al. (2017). The genus is characterised by having perithecial ascomata, octosporous asci with an apical ring, hyaline, bicellular ascospores with or without gelatinous appendages and acervular conidiomata with brown conidia with gelatinous sheaths and with verruculous inner surface of the conidal wall (Voglmayr et al. 2017). Juglanconis includes four species (J. appendiculata, J. juglandina, J. oblonga and J. pterocariae), which were restricted to host in Juglandaceae (Voglmayr et al. 2017).

Juglanconis juglandina (Kunze) Voglmayr & Jaklitsch, Persoonia 38: 144 (2017)

Fig. 3

Melanconium juglandinum Kunze, Fl. Dresd., 2. Aufl.: 260. 1823.

Descriptions

Conidiomata acervular, immersed in host bark, erumpent from surface of host branches, scattered or occasionally confluent, 1.5–2.5 mm, covered by black discharged conidial masses at maturity, usually conspicuous. Ectostromatic disc straw to honey, surrounded by bark or not. Central column beneath the disc more or less conical, straw to buff. Conidiophores cylindrical to lageniform, simple, rarely branched at the base, smooth, subhyaline to pale brown. Conidiogenous cells annellidic with distinct annellations, integrated. Conidia unicellular, initially hyaline, becoming brown to blackish when mature, broadly ellipsoid to broadly pip-shaped, truncate with distinct scar at the base, densely multiguttulate, thick-walled, (17–)19–22(–24.5) × (9–)11–14(–16.5) μm (av. = 20 × 13 μm, n = 50), with 0.8–1 µm wide gelatinous sheath. Sexual morph was not observed.

Figure 3. 

Morphology of Juglanconis juglandina from Juglans regia. A–B habit of acervuli on branches C transverse section through acervulus D longitudinal section through acervulus E–F conidiophores, conidiogenous cells and conidia. Scale bars: 1 mm (A–D), 20 μm (E–F).

Culture characteristics

On PDA, cultures are initially white, becoming straw after 3–5 d and grey olivaceous after 7–10 d. The colonies are felty with an irregular edge; sterile.

Materials examined

(all on twigs and branches of Juglans regia). CHINA, Gansu Province, Qingyang City, Shishe village, 35°38'17.08"N, 107°47'48.68"E, 14 July 2013, X.L. Fan (BJFC-S908; living culture, CFCC 51727); Gansu Province, Qingyang City, Zhongwan Forest Farm, 35°26'26.33"N, 108°34'09.38"E, 11 July 2013, X.L. Fan (BJFC-S947; living culture, CFCC 51728); Gansu Province, Qingyang City, Zhongwan Forest Farm, 35°26'25.52"N, 108°34'09.03"E, 11 July 2013, X.L. Fan (BJFC-S955; living culture, CFCC 51729).

Notes

Juglanconis juglandina is the type species of Juglanconis and is thus far only known to occur on Juglans regia distributed in Asia and Europe (Voglmayr et al. 2017). Juglanconis juglandina is described based on Melanconium juglandinum (= Melanconis carthusiana) (Voglmayr et al. 2017), which was regarded as the main causal agent of canker and dieback disease of Juglans regia in China (China Microbiology and Virology Databases, http://www.micro.csdb.cn/).

Juglanconis oblonga (Berk.) Voglmayr & Jaklitsch, Persoonia 38: 147 (2017)

Fig. 4

Melanconium oblongum Berk., Grevillea 2 (no. 22): 153. 1874.

= Diaporthe juglandis Ellis & Everh., Proc. Acad. Nat. Sci. Philadelphia 45: 448. 1893.

Melanconis juglandis (Ellis & Everh.) A.H. Graves, Phytopathology 13: 311. 1923.

Descriptions

Pseudostromata immersed in host bark, distinctly erumpent from surface of host branches, 1.5–3 mm diam. Ectostromatic disc indistinct, usually circular, greyish to brownish. Perithecia often appearing as rounded bumps beneath the bark surface surrounding the ectostromatic disc, prolonged black neck from the top, (450–)525–700(–780) µm diam. (av. = 580 μm, n = 30). Asci hyaline, clavate to fusoid, (120–)122–135 × (12.5–)13–16.5 (–17) μm (av. = 126.5 × 15 μm, n = 20). Ascospores hyaline, ellipsoid, broadly ellipsoid or broadly fusoid, symmetric to slightly asymmetric, straight, rarely slightly curved, constricted at the septum, (17–)17.5–22(–23.5) × (7.5–)8–10.5(–11) μm (av. = 19.5 × 9.5 μm, n = 50). Conidiomata acervular, immersed in host bark, erumpent from surface of host branches, scattered or occasionally confluent, 1–2 mm, covered by black discharged conidial masses at maturity, usually conspicuous. Ectostromatic disc buff to honey, surrounded by bark or not. Central column beneath the disc more or less conical, isabelline to olivaceous grey. Conidiophores cylindrical to lageniform, simple, rarely branched at the base, smooth, subhyaline to pale brown. Conidiogenous cells annellidic with distinct annellations, integrated. Conidia unicellular, initially hyaline, becoming brown to blackish when mature, broadly ellipsoid to broadly pip-shaped, truncate with distinct scar at the base, densely multiguttulate, thick-walled, (14–)19–23.5(–28) × (6.5–)9–13(–15) μm (av. = 22 × 12.5 μm, n = 50), with 0.8–1 µm wide gelatinous sheath.

Figure 4. 

Morphology of Juglanconis oblonga from Juglans regia. A–B habit of acervuli on branches C transverse section through acervulus D longitudinal section through perithecia E longitudinal section through acervulus F conidiophores, conidiogenous cells G conidia H asci and ascospores I ascospores. Scale bars: 10 mm (A), 500 μm (B–E), 20 μm (F–I).

Culture characteristics

On PDA, cultures are initially white, becoming pale olivaceous grey after 10 d. The colonies are felty with an irregular edge; texture uniform; sterile.

Materials examined

(all on twigs and branches of Juglans regia). CHINA, Heilongjiang Province, Harbin City, Linan, Heilongjiang Botanical Garden, 45°42'21.10"N, 126°38'42.87"E, 2 August 2016, Q. Yang & Z. Du (BJFC-S1374; living culture, CFCC 51725; ibid. CFCC 51726).

Notes

Juglanconis oblonga is based on Melanconium oblongum (= Melanconis juglandis) (Voglmayr et al. 2017). This species can be distinguished from J. juglandina by on average longer length of conidia (22 × 12.5 vs.> 20 × 13 µm). However, there is a substantial size overlap between both species and sequence data are sometimes necessary for reliable species identification. It was also recorded to cause canker and dieback disease of Juglans regia in China (China Microbiology and Virology Databases, http://www.micro.csdb.cn/).

Melanconidaceae G. Winter, Rabenh. Krypt.-Fl., Edn 2 (Leipzig) 1.2: 764 (1886)

Type genus

Melanconis Tul. & C. Tul., Select. fung. carpol. (Paris) 2: 115 (1863)

Notes

Melanconidaceae was introduced by Winter (1886) and subsequently involved many genera with perithecia immersed in a well-developed stroma with ostioles (beaks) that emerge through an ectostromatic disc (Barr 1978). Castlebury et al. (2002) and Rossman et al. (2007) reduced this family to the type genus Melanconis based on LSU rDNA sequences. In this paper, we provide an updated tree with additional isolates of Melanconis (Melanconidaceae) from China (Fig. 5). All species have been described and illustrated by Fan et al. (2016).

Figure 5. 

Phylogram of Melanconis (Melanconidaceae) obtained from an MP analysis of a combined matrix of ITS, LSU, RPB2 and TEF1-α. MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.95 from BI. Scale bar = 20 changes. Type species are in bold. Strains obtained in the current study are in blue.

Melanconis Tul. & C. Tul., Select. fung. carpol. (Paris) 2: 115 (1863)

Type species

Melanconis stilbostoma (Fr.) Tul. & C. Tul., Select. fung. carpol. (Paris) 2: 115 (1863)

Notes

The type genus Melanconis was established by Tulasne and Tulasne (1863) based on Sphaeria stilbostoma Fr. This genus is characterised by circularly arranged perithecia immersed in well developed to reduced entostromata with a concolourous central column and ostioles erumpent through a light-coloured ectostromatic disc with hyaline, one-septate ascospores; acervuli with light-coloured central column producing brown to olive-brown, fusiform to pyriform alpha conidia and hyaline, cylindrical or allantoid beta conidia (Barr 1978; Castlebury et al. 2002; Voglmayr et al. 2012; Fan et al. 2016). Melanconis has approximately 105 species epithets recorded in Index Fungorum (2018), whereas Rossman et al. (2007) suggested that many of the species previously residing in Melanconis may belong somewhere else. Fan et al. (2016) provided an account on this genus including five species (Melanconis alni, Ms. betulae, Ms. marginalis, Ms. itoana and the type species Ms. stilbostoma), which were restricted to hosts in Betulaceae.

Melanconis betulae C.M. Tian & X.L. Fan, Mycol. Progr. 15(4/40): 4 (2016)

Materials examined

(all on twigs and branches of Betula albosinensis). CHINA, Gansu Province, Gannan Tibetan Autonomous Prefecture, Zhouqu County, Qiban Forestry Centre, 33°56'35.36"N, 104°07'13.03"E, 20 August 2014, Y.M. Liang (BJFC-S1319, holotype; living ex-type culture, CFCC 50471); Gansu Province, Gannan Tibetan Autonomous Prefecture, Zhouqu County, Qiban Forestry Centre, 33°56'37.05"N, 104°07'13.78"E, 20 August 2014, Y.M. Liang (BJFC-S13200; living culture, CFCC 50472); Gansu Province, Gannan Tibetan Autonomous Prefecture, Zhouqu County, Qiban Forestry Centre, 33°56'34.44"N, 104°07'15.59"E, 20 August 2014, Y.M. Liang (BJFC-S1321; living culture, CFCC 50473).

Notes

Melanconis betulae was described from Betula albosinensis (Fan et al. 2016). Morphologically, M. betulae is characterised by ovoid, olive-brown, aseptate alpha conidia, which are different from other Melanconis species but similar to the type species Ms. stilbostoma. However, it can be distinguished by the smaller length of its alpha conidia (10 vs.> 12 μm) and sequence data.

Melanconis itoana Tak. Kobay., Bull. Govt Forest Exp. Stn Meguro 226: 19 (1970)

Materials examined

(all on twigs and branches of Betula albosinensis). CHINA, Gansu Province, Gannan Tibetan Autonomous Prefecture, Zhouqu County, Qiban Forestry Centre, 33°56'34.49"N, 104°07'15.21"E, 20 August 2014, X.L. Fan (BJFC-S1322; living culture, CFCC 50474); Shaanxi Province, Ankang City, Ningshan County, Huoditang Forest Farm, 33°26'24.80"N, 108°26'45.10"E, 3 August 2015, Q. Yang (BJFC-S1349; living culture, CFCC 52877; ibid, CFCC 52878); Jilin Province, Jiaohe City, Haiqing Forest Farm, 43°79'88.71"N, 127°15'83.04"E, 26 June 2017, X.W. Wang (CF 20170668; living culture, CFCC 52876).

Notes

Melanconis itoana was described from Betula ermanii in Japan (Kobayashi 1970). Fan et al. (2016) isolated it from Betula albosinensis as a new record in China. Melanconis itoana is characterised by fusoid, green-brown alpha conidia with acute ends (13 × 4 μm) and hyaline, cylindrical or crescent beta conidia (9.5 × 1.5 μm).

Melanconis stilbostoma (Fr.) Tul. & C. Tul., Select. fung. carpol. (Paris) 2: 115 (1863)

Materials examined

(all on twigs and branches of Betula platyphylla). CHINA, Tibet Autonomous Region, Linzhi City, Juemu Valley, 29°39'50.13"N, 94°18'50.70"E, 22 July 2016, X.L. Fan (CF 20160703; living culture, CFCC 528433); Heilongjiang Province, Yichun City, Dailing District, Liangshui Natural Reserve, 47°11'05.26"N, 128°57'26.15"E, 29 July 2016, Q. Yang & Z. Du (CF 20161703; living culture, CFCC 52867); Heilongjiang Province, Harbin City, Heilongjiang Botanical Garden, 45°42'27.58"N, 126°38'36.72"E, 2 August 2016, Q. Yang & Z. Du (CF 20161709; living culture, CFCC 52868); Qinghai Province, Menyuan City, Xianmi Forest Farm, 37°16'35.27"N, 101°46'53.78"E, 3 September 2016, J.H. Zuo (CF 20160911; living culture, CFCC 52865); Ningxia Autonomous Region, Yinchuan City, Helan County, Taihedizhonghai, 38°31'50.40"N, 106°17'46.10"E, 5 August 2015, X.L. Fan & Z. Du (CF 20150802; living culture, CFCC 52873); Ningxia Autonomous Region, Jingyuan City, Jingguan Road, 35°29'50.32"N, 106°18'27.10"E, 13 August 2014, X.L. Fan & Z. Du (BJFC-S1324; living culture, CFCC 50476); Beijing City, Tongzhou District, Song Village, 35°59'49.50"N, 116°39'32.35"E, 20 May 2015, X.L. Fan (BJFC-S1325; living culture, CFCC 50477); other materials with similar locations and hosts are listed in Table 1.

Notes

Melanconis stilbostoma is the type species of Melanconis and is thus far only known to occur on Betula spp. with a worldwide distribution (Fan et al. 2016). Betula pendula, B. rotundifolia and B. tianschanica are recorded as hosts in China (Zhuang 2005). The current investigation suggested that this species is restricted to and widespread on Betula platyphylla in China.

Melanconiellaceae Senan., Maharachch. & K.D. Hyde, Stud. Mycol. 86: 275 (2017)

Type genus

Melanconiella Sacc., Syll. fung. (Abellini) 1: 740 (1882)

Notes

Melanconiellaceae was validated by Senanayake et al. (2017) for the invalid Melanconiellaceae of Locquin (1984). Senanayake et al. (2017) emended this family to accommodate Dicarpella, Greeneria, Melanconiella, Microascospora and Tubakia. Braun et al. (2018) recommended an exclusion of Dicarpella, Greeneria and Tubakia. In this paper, we introduce the new genus Sheathospora and two new species of Melanconiella in Melanconiellaceae (Fig. 6).

Figure 6. 

Phylogram of Melanconiellaceae obtained from an MP analysis from a combined matrix of ITS, LSU, RPB2 and TEF1-α. MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.95 from BI. Scale bar = 80 changes. Type species are in bold. Strains obtained in the current study are in blue.

Melanconiella Sacc., Syll. fung. (Abellini) 1: 740 (1882)

Type species

Melanconiella spodiaea (Tul. & C. Tul.) Sacc., Syll. fung. (Abellini) 1: 740 (1882)

Notes

The genus Melanconiella was established by Saccardo (1882) for two species, Melanconis spodiaea Tul. & C. Tul. and M. chrysostroma (Fr.) Tul. & C. Tul. The genus subsequently entered a long period of confusion with a broad concept of the melanconidaceous genera Melanconium and Melanconis Tul. & C. Tul. (Wehmeyer 1937, 1941; Barr 1987). Melanconiella has 37 species epithets recorded in Index Fungorum (2018). Voglmayr et al. (2012) revised the generic circumscriptions of Melanconiella with 13 accepted species, excluded numerous species and confirmed that it is genetically distinct from the genus Melanconis based on morphology and multi-gene phylogeny (ITS, LSU, RPB2 and TEF1-α). Melanconiella is characterised by forming circularly arranged perithecia immersed in the substrate with oblique or lateral ostioles convergent and erumpent through an ectostromatic disc with dark coloured or hyaline ascospores; acervuli with light-coloured central column, producing dark brown melanconium-like or hyaline discosporina-like conidia (not in the same species) (Barr 1978; Voglmayr et al. 2012). Melanconiella species were observed to be highly host-specific, as they were found to be confined to a single genus or sometimes even species within the host family Betulaceae from Europe and North America (Voglmayr et al. 2012).

Melanconiella betulicola Fan, sp. nov.

MycoBank No: 828427
Fig. 7

Etymology

betulicola (Lat.): referring to the host genus on which it was collected, Betula.

Diagnosis

This species is distinguished by hyaline ascospores, (16.5–)18–22(–24) × (3–)4–6 μm, with slightly constricted at the septum and with hyaline broad cap-like appendages at both ends.

Holotype

CHINA. Shaanxi Province: Ningshan County, Huoditang Forest Farm, Huodi Valley, 33°26'36.32"N, 108°26'46.48"E, 3 August 2015, on twigs and branches of Betula albosinensis, Q. Yang (BJFC-S1347 holotype; living culture, CFCC 52482).

Descriptions

Pseudostromata inconspicuous, immersed in host bark, slightly erumpent from surface of host branches, 1.5–3 mm diam. Ectostromatic disc indistinct, usually circular, buff to hazel. Central column circular, mouse grey to iron grey. Ostioles numerous, violaceous black to black, scarcely projecting, 70–150 μm diam. Perithecia flask-shaped to spherical, arranged circularly or irregularly, 7–12 per disc, often appearing as rounded bumps beneath the bark surface surrounding the ectostromatic disc, (320–)350–550(–610) µm diam. (av. = 480 μm, n = 30). Asci hyaline, clavate to fusoid, (50–)55–65(–70) × (7–)8.5–14(–16) μm (av. = 60 × 11 μm, n = 20). Ascospores hyaline, ellipsoid, broadly ellipsoid or broadly fusoid, 2–4 guttulate, symmetric to slightly asymmetric, straight, rarely slightly curved, slightly constricted at the septum, (16.5–)18–22(–24) × (3–)4–6 μm (av. = 20 × 4.5 μm, n = 50), with hyaline broad cap-like appendages at both ends. Conidiomata acervular, immersed in host bark, erumpent from surface of host branches, scattered or occasionally confluent, 1.3–2.5 mm, covered by fawn to dark brick discharged conidial masses at maturity, usually conspicuous. Ectostromatic disc inconspicuous. Central column beneath the disc more or less conical, olivaceous grey to iron grey. Conidiophores hyaline, smooth, cylindrical to lageniform, simple, rarely branched at the base. Conidiogenous cells hyaline, phialidic. Conidia unicellular, hyaline, narrowly ellipsoid, elongate to slightly allantoid, (9.5–)10–13.5(–15) × (2–)3–4.5(–5.5) μm (av. = 13 × 3.5 µm, n = 50), with 0.5 µm wide gelatinous sheath.

Figure 7. 

Morphology of Melanconiella betulicola from Betula albosinensis. A–B habit of pseudostromata on branches C transverse section through perithecia D longitudinal section through perithecia E–F habit of acervuli on branches G transverse section through acervulus H longitudinal section through acervulus I asci and ascospores J–K ascus and ascospores L–O ascospores P conidiophores, conidiogenous cells and conidia Q conidia. Scale bars: 2 mm (A, E), 500 μm (B–D, F–H), 10 μm (J–K, P–Q), 5 μm (L–O).

Culture characteristics

On PDA, cultures are initially white, becoming greyish-sepia after 3 d and distensible radially after 10 d. The colonies are felty with an irregular edge; texture uniform; sterile.

Additional material examined

CHINA. Shaanxi Province: Ningshan County, Huoditang Forest Farm, Huodi Valley, 33°26'37.53"N, 108°26'44.14"E, 3 August 2015, on twigs and branches of Betula albosinensis, Q. Yang (CF 20150847; living culture, CFCC 52483);

Notes

Melanconiella betulicola is associated with canker disease of Betula albosinensis in China. It is similar to M. ellisii but differs by larger ascospores (18–22 × 4–6 vs.> 12.5–16 × 4.0–5.5 μm) with hyaline, broad cap-like appendages at both ends (Voglmayr et al. 2012), distribution (China vs.> eastern North America) and a different host, Betula albosinensis vs.> Carpinus caroliniana. Melanconiella decorahensis also occurs on Betula (in Europe and North America) and it can be distinguished from M. betulicola based on dark brown ascospores without appendages and dark brown conidia (Voglmayr et al. 2012). The clear phylogenetic position confirmed a distinction from all other available strains included in this study and we therefore result in our decision to describe this species as new, based on DNA sequence data and morphology.

Melanconiella corylina Fan, sp. nov.

MycoBank No: 828428
Fig. 8

Etymology

corylina (Lat.): referring to the host genus on which it was collected, Corylus.

Diagnosis

This species is distinguished by acervuli erumpent through circularly cracked host bark and covered by olivaceous buff to honey discharged conidial masses at maturity; conidia unicellular, hyaline, with various shapes and 1–3 guttulate, (7–)8–13.5(–14.5) × (2–)2.5–4(–5) μm.

Figure 8. 

Morphology of Melanconiella corylina from Corylus mandshurica. A habit of acervuli on branches B–F process of development of acervulus G transverse section through acervulus H–I longitudinal section through acervulus J conidiophores K conidiogenous cells and conidia L–W conidia. Scale bars: 2 mm (A), 500 μm (B–I), 10 μm (J–K), 5 μm (L–W).

Holotype

CHINA. Shaanxi Province: Baoji County, Taibai Mountain, 34°15'43.32"N, 107°88'42.16"E, 13 July 2017, on twigs and branches of Corylus mandshurica, N. Jiang (BJFC-FB56 holotype; living culture, CFCC 52484).

Descriptions

Conidiomata acervular, immersed in host bark, erumpent from surface of host branches, scattered or occasionally confluent, 1–1.5 mm, erumpent through circularly cracked host bark and covered by olivaceous buff to honey discharged conidial masses at maturity, usually conspicuous. Ectostromatic disc inconspicuous and cracked circularly at maturity. Central column beneath the disc more or less oblate, iron grey to dark grey. Conidiophores hyaline, smooth, cylindrical, simple, rarely branched at the base. Conidiogenous cells hyaline, phialidic. Conidia unicellular, hyaline, narrowly ellipsoid to fusoid, elongate to slightly allantoid, 1–3 guttulate, (7–)8–13.5(–14.5) × (2–)2.5–4(–5) μm (av. = 10 × 3.5 µm, n = 50) μm (av. = 13 × 3.5 µm, n = 50). Sexual morph was not observed.

Culture characteristics

On PDA, cultures are initially white, becoming fuscous black in the centre and edge after 5 d. The colonies are felty with an irregular edge; texture uniform; sterile.

Additional material examined

CHINA. Shaanxi Province: Baoji County, Taibai Mountain, 34°15'40.05"N, 107°88'43.33"E, 13 July 2017, on twigs and branches of Corylus mandshurica, N. Jiang (CF 20170756 holotype; living culture, CFCC 52485).

Notes

Melanconiella corylina is associated with canker disease of Corylus mandshurica in China. It can be distinguished from its closest relative, the generic type M. spodiaea growing in Carpinus spp., by its hyaline, discosporina-like conidia, and the smaller size of conidia (8–13.5 × 2.5–4 vs.> 13.3–15.2 × 7.5–8.5 μm) as well as the hosts (Voglmayr et al. 2012). Melanconiella flavovirens also occurs on Corylus (in Europe and North America), and it can be distinguished from M. corylina based on larger conidia (12–15 × 5.0–5.5 vs.> 8–13.5 ×2.5–4 μm) (Voglmayr et al. 2012). The phylogenetic inferences indicated M. corylina as an individual well-supported clade (MP/ML/BI=100/99/1) within Melanconiella and we therefore describe it as new, based on sequence data and morphology.

Sheathospora Fan, gen. nov.

MycoBank No: 828429

Etymology

Sheathospora (Lat.): referring to the conidia with distinct hyaline sheath.

Diagnosis

This genus differs from other genera in Melanconiellaceae by conical and discrete pycnidia with aseptate, cylindrical to ellipsoidal conidia with distinct hyaline sheath.

Type species

Sheathospora cornuta (C.M. Tian & Z. Du) Fan.

Descriptions

Conidiomata pycnidial, immersed in host bark, erumpent through the surface of host branches. Ectostromatic disc inconspicuous and extended to form a beak at maturity. Central column absent. Conidiophores hyaline, smooth, cylindrical, simple, rarely branched at the base. Conidiogenous cells hyaline, phialidic. Conidia hyaline, aseptate, with distinct hyaline sheath. Sexual morph was not observed.

Notes

Sheathospora is established for Melanconiella cornuta, which was previously included in the Melanconiella clade (Voglmayr et al. 2012; Du et al. 2017). Morphologically, it differs from other genera in Melanconiellaceae by pycnidial conidiomata and conidia with distinct hyaline sheath. In our phylogenetic analyses, Melanconiella cornuta formed a distinct clade basal to Melanconiella within Melanconiellaceae. Based on morphology and different hosts (Cornus and Juglans vs. Betulaceae), it is here excluded from Melanconiella and transferred to the new genus Sheathospora. In our revised circumscription, Melanconiellaceae include three genera named Melanconiella, Microascospora and Sheathospora.

Sheathospora cornuta (C.M. Tian & Z. Du) Fan, comb. nov.

MycoBank No: 828430
Fig. 9

Basionym

Melanconiella cornuta C.M. Tian & Z. Du, Phytotaxa 327(3): 257 (2017)

Diagnosis

This species is distinguished by conical and discrete pycnidia without central column and aseptate, cylindrical to ellipsoidal, (19–)19.5–22.5(–23) × (8–)8.5–10.5(–11) μm conidia, with a distinct hyaline sheath 1–1.5 μm wide.

Holotype

CHINA. Shaanxi Province: Ankang City, Ningshan County, Huoditang Forest Farm, 33°26'04.46"N, 108°26'59.91"E, 3 July 2016, on twigs and branches of Cornus controversa, X.L. Fan (BJFC-S1375 holotype; living ex-type culture CFCC 51990).

Descriptions

Conidiomata pycnidial, immersed in host bark, conical, with single necks erumpent through the surface of host branches, scattered, (250–)270–330(–410) μm (av. = 300 μm, n = 20) diam. Ectostromatic disc inconspicuous and extended to form a beak at maturity, pale luteous to amber. Central column absent. Conidiophores hyaline, smooth, cylindrical, simple, rarely branched at the base, 17–24(–25) × 2.5–4(–4.5) μm (av. = 21.5 × 3.5 µm, n = 50). Conidiogenous cells hyaline, phialidic. Conidia hyaline, aseptate, cylindrical to ellipsoidal, (19–)19.5–22.5(–23) × (8–)8.5–10.5(–11) μm (av. = 21 × 10 µm, n = 50), with distinct hyaline sheath, 1–1.5 μm wide at maturity. Sexual morph was not observed.

Figure 9. 

Morphology of Sheathospora cornuta from Cornus controversa. A–B Habit of pycnidia on branches C–D transverse section through pycnidium E longitudinal section through pycnidium F conidiophores, conidiogenous cells G conidia. Scale bars: 5 mm (A), 1 mm (B), 500 μm (C–E), 20 μm (F–G).

Culture characteristics

Colony growth on PDA originally white, becoming pale yellowish after 7–10 days. Colony flat, felty-like, with a uniform texture and yellowish to dark brown conidiomata irregularly scattered on the colony surface.

Additional specimens examined (paratypes)

CHINA. Shaanxi Province: Ankang City, Ningshan County, Huoditang Forest Farm, 36°26'13.30"N, 108°26'48.32"E, 3 August 2015, on twigs and branches of Juglans regia, Q. Yang (BJFC-S1345 paratype; living ex-paratype culture CFCC 51991).

Notes

Sheathospora cornuta is proposed here as a new combination for Melanconiella cornuta. It is the type and currently only species of Sheathospora and so far known from Cornus controversa and Juglans regia in China. The sexual morph of this species is unknown and further collections are required to elucidate its life cycle.

Discussion

During the investigation of melanconis-like fungi in China, we identified eight species residing in three families (Juglanconidaceae, Melanconidaceae and Melanconiellaceae) of Diaporthales. It includes Juglanconis juglandina, J. oblonga, Melanconis betulae, Ms. itoana, Ms. stilbostoma, the two new species Melanconiella betulicola and M. corylina and the new combination Sheathospora cornuta in the new genus Sheathospora.

All specimens in the current study were collected from symptomatic branches and twigs associated with canker or dieback diseases, of which Juglanconis (Juglanconidaceae) species were isolated from Juglans regia (Juglandaceae), Melanconiella (Melanconiellaceae) species from Betula albosinensis and Corylus mandshurica (Betulaceae) and Melanconis (Melanconidaceae) species from Betula albosinensis and Betula platyphylla (Betulaceae). It may indicate that many melanconis-like species have obvious host specificity. The type species of the new genus Sheathospora (Melanconiellaceae) was isolated from Cornaceae (Cornus controversa) and Juglans regia (Juglandaceae), suggesting a low host specificity and that additional undiscovered hosts species of this taxon may exist in China.

As the morphological features in previous melanconis-like fungi are highly overlapping, phylogenetic studies using DNA sequences have been useful to elucidate the diversity and systematics in this group. The current results indicated that Juglanconis and Melanconis are still unique, the only genera in Juglanconidaceae and Melanconidaceae, respectively, due to the lacking of extensive fresh collections. The family Melanconiellaceae was recently proposed by Senanayake et al. (2017) to accommodate Dicarpella, Greeneria, Melanconiella, Microascospora and Tubakia based on morphological features and phylogenetic analyses. In this study, the phylogenetic affinity of Dicarpella, Greeneria and Tubakia was evaluated in Diaporthales (Fig. 1), which conformed to the recently described family Tubakiaceae (Diaporthales) (Braun et al. 2018). We here establish a new genus within Melanconiellaceae, Sheathospora, which is characterised by typical diaporthalean-like pycnidia and aseptate, cylindrical to ellipsoidal conidia with distinct hyaline sheath. Thus Melanconiellaceae is here restricted to the three genera Melanconiella, Microascospora and Sheathospora (Fig. 6).

As shown in this paper, future studies addressing the fungal diversity associated with canker or dieback diseases should routinely include sequence data for protein-coding genes to achieve stable, supported topologies in phylogenetic trees. It is hoped that the classification proposed here will also provide an updated phylogenetic framework that will facilitate further revision of the families with melanconis-like asexual morphs. Although the current study provides additional new data on melanconis-like genera, typification, species concept and taxonomic affiliation of many described Melanconium species are yet unclear, including the type species M. atrum, which currently represents a doubtful taxon (Rossman et al. 2015). In addition, sequence data are missing for most described Melanconium species. Thus, a thorough revision of the genus Melanconium based on robust sampling, reliable identification, cultures and DNA data is urgently needed. The fact that new records and species from three related families of Diaporthales were recorded in China further suggests that Asia may harbour many more species awaiting collections and descriptions.

Acknowledgements

This study is financed by National Natural Science Foundation of China (Project No.: 31670647) and National Key R&D Program of China (Project No.: 2017YFD0600105). All authors want to thank the Experimental Teaching Centre (College of Forestry, Beijing Forestry University) for providing installed scientific equipments during the whole process.

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