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Ochraceocephala foeniculi gen. et sp. nov., a new pathogen causing crown rot of fennel in Italy
expand article infoDalia Aiello, Alessandro Vitale, Giancarlo Polizzi, Hermann Voglmayr§|
‡ University of Catania, Catania, Italy
§ University of Natural Resources and Life Sciences, Vienna, Austria
| University of Vienna, Vienna, Austria
Open Access

Abstract

A new disease of fennel is described from Sicily (southern Italy). Surveys of the disease and sampling were conducted during spring 2017 and 2018 in Adrano and Bronte municipalities (Catania province) where this crop is widely cultivated. Isolations from the margin of symptomatic tissues resulted in fungal colonies with the same morphology. Pathogenicity tests with one isolate of the fungus on 6-month-old plants of fennel reproduced similar symptoms to those observed in nature. Inoculation experiments to assess the susceptibility of six different fennel cultivars to infection by the pathogen showed that the cultivars ‘Narciso’, ‘Apollo’, and ‘Pompeo’ were more susceptible than ‘Aurelio’, ‘Archimede’, and ‘Pegaso’. Phylogenetic analyses based on a matrix of the internal transcribed spacer (ITS), the large subunit (LSU), and the small subunit (SSU) rDNA regions revealed that the isolates represent a new genus and species within the Leptosphaeriaceae, which is here described as Ochraceocephala foeniculi gen. et sp. nov. This study improves the understanding of this new fennel disease, but further studies are needed for planning effective disease management strategies. According to the results of the phylogenetic analyses, Subplenodomus iridicola is transferred to the genus Alloleptosphaeria and Acicuseptoria rumicis to Paraleptosphaeria.

Keywords

Fungal disease, Leptosphaeriaceae, pathogenicity, susceptibility

Introduction

Fennel (Foeniculum vulgare Mill.), native in arid and semi-arid regions of southern Europe and the Mediterranean area, is used as a vegetable, herb, and seed spice in the food, pharmaceutical, cosmetic, and healthcare industries. Italy is the leading world producer of fennel (around 85% of the world production), with 20,035 ha of area cultivated and a total production of 537,444 tons. Fennel represents an important crop widely cultivated in Sicily (southern Italy) with 1,620 ha harvested and a production of 35,930 tons (ISTAT 2018). Several diseases caused by fungi have been reported from this crop throughout the world (Table 1). Amongst soilborne diseases, brown rot and wilt caused by Phytophthora megasperma and crown rot caused by Didymella glomerata (syn. Phoma glomerata) were reported in Italy (Cacciola et al. 2006; Lahoz et al. 2007).

In 2017, a new disease was first observed on fennel in a farm of Adrano area (Catania province, eastern Sicily, Italy). The disease symptoms were necrotic lesions on the crown, root, and stem of fennel plants. Disease incidence initially was about 5% on ‘Apollo’ cultivar. However, in 2018 different surveys conducted in the same area showed a high increase of the incidence on three different cultivars with yield losses of about 20–30%. The aims of the present study were to identify the causal agent obtained from symptomatic fennel plants, using morphological characteristics and DNA sequence analyses, to evaluate the pathogenicity of one representative isolate and to evaluate the susceptibility of different cultivars of fennel to the newly described disease.

Main diseases caused by fungal pathogens on fennel.

Disease Fungal pathogen Reference
Collar rot Sclerotium rolfsii Khare et al. 2014
Damping off and Root rot Pythium spp. Khare et al. 2014; Koike et al. 2015
Vascular wilt Fusarium oxysporum Shaker and Alhamadany 2015
Vascular wilt Verticillium dahliae Ghoneem et al. 2009
Root and Foot rot Rhizoctonia solani Shaker and Alhamadany 2015
Brown rot and Wilt Phytophthora megasperma Cacciola et al. 2006
Stem rot Sclerotinia sclerotiorum Choi et al. 2016
Blight and Leaf spot Alternaria alternata D'Amico et al. 2008
Blight and Leaf spot Ascochyta foeniculina Khare et al. 2014
Blight and Leaf spot Fusoidiella anethi Taubenrauch et al. 2008
syn. Cercospora foeniculi
Cercosporidium punctum
Mycosphaerella anethi
M. foeniculi
Passalora kirchneri
P. puncta
Ramularia foeniculi
Umbel browning and Stem necrosis Diaporthe angelicae Rodeva and Gabler 2011
Downy mildew Plasmopara mei-foeniculi Khare et al. 2014
syn. P. nivea sensu lato
Powdery mildew Leveillula languinosa Khare et al. 2014
Powdery mildew Erysiphe heraclei Choi et al. 2015
Leaf spot Leptosphaeria purpurea Odstrčilová et al. 2002
Leaf spot Subplenodomus apiicola Odstrčilová et al. 2002
syn. Phoma apiicola
Leaf spot and blight Phoma herbarum Shaker and Alhamadany 2015
Crown rot Didymella glomerata Lahoz et al. 2007
syn. Phoma glomerata

Materials and methods

Collection of samples and fungal isolates

In order to identify the causal agent of the fennel disease, 30 samples were collected during several surveys in Adrano and Bronte area (Catania province, eastern Sicily). Pieces of tissue obtained from different parts of fennel plants (crown, root, and stem) were surface disinfected for 1 min in 1.5% sodium hypochlorite solution, rinsed in sterile water, placed on potato dextrose agar (3.9% PDA, Oxoid, Basingstoke, UK) amended with 100 mg/L of streptomycin sulfate (Sigma-Aldrich, USA) to prevent bacteria growth, and then incubated at 25 ± 1 °C for seven days. Fungal colonies consistently grown from symptomatic tissues were subcultured on new PDA plates. Subsequently, single-spore isolates were obtained from these pure cultures and stored at –20 °C in sterile 15% glycerol solution. The fungal isolates were provisionally identified by cultural and morphological characteristics, and they were deposited in the culture collection of the Department of Agriculture, Food and Environment, University of Catania. One representative isolate (Di3A-F1; ex holotype culture) was deposited at the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, the Netherlands. The holotype specimen of the new pathogen species was deposited in the fungarium of the Department of Botany and Biodiversity Research, University of Vienna (WU).

Morphology

For culture characteristics, cultures were grown on 2% (w/v) malt extract agar (MEA, VWR) and on corn meal agar (CMA, Sigma-Aldrich) supplemented with 2% w/v dextrose (CMD). Colony diameters and morphologies were determined after seven days of incubation at room temperature (22 ± 1 °C) and daylight.

Microscopic observations were made in tap water. Methods of microscopy included stereomicroscopy using a Nikon SMZ 1500 equipped with a Nikon DS-U2 digital camera, and Nomarski differential interference contrast (DIC) using a Zeiss Axio Imager.A1 compound microscope equipped with a Zeiss Axiocam 506 colour digital camera. Images and data were gathered using the NIS-Elements D v. 3.22.15 or Zeiss ZEN Blue Edition software packages. Measurements are reported as maxima and minima in parentheses and the range representing the mean plus and minus the standard deviation of a number of measurements given in parentheses.

DNA extraction and PCR amplification

The extraction of genomic DNA from pure cultures was performed by using the Wizard Genomic DNA Purification Kit (Promega Corporation, WI, USA). Partial regions of six loci (ITS, LSU, and SSU rDNA, RPB2, TEF1, TUB2) were amplified; for details on the primers and annealing temperatures used for PCR and sequencing, see Table 2. The PCR products were sequenced in both directions by Macrogen Inc. (South Korea) or at the Department of Botany and Biodiversity Research, University of Vienna using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit v. 3.1 (Applied Biosystems, Warrington, UK) and an automated DNA sequencer (3730xl Genetic Analyser, Applied Biosystems). The DNA sequences generated were assembled with Lasergene SeqMan Pro (DNASTAR, Madison, USA). Sequences generated during the present study were uploaded to Genbank (Table 3).

Primers used to amplify and sequence the nuclear internal transcribed spacer (ITS), large subunit (LSU) and small subunit (SSU) rDNA regions, the RNA polymerase II second largest subunit (RPB2) gene, the translation elongation factor 1-α (TEF1) gene and the β-tubulin (TUB2) gene.

Gene Primer Sequence (5'–3') Direction Annealing t (°C) Reference
ITS ITS5 GGAAGTAAAAGTCGTAACAAGG forward 48 White et al. 1990
ITS4 TCCTCCGCTTATTGATATGC reverse White et al. 1990
LSU LR0R GTACCCGCTGAACTTAAGC forward 48 Vilgalys and Hester 1990
LR5 TCCTGAGGGAAACTTCG reverse Vilgalys and Hester 1990
ITS-LSU V9G TTAAGTCCCTGCCCTTTGTA forward 55 Hoog and Gerrits van den Ende 1998
LR5 TACTTGAAGGAACCCTTACC reverse Vilgalys and Hester 1990
LR2R-Az CAGAGACCGATAGCGCAC forward Voglmayr et al. 2012
LR3z CCGTGTTTCAAGACGGG reverse Vilgalys and Hester 1990
ITS4z TCCTCCGCTTATTGATATGC reverse White et al. 1990
SSU NS1 GTAGTCATATGCTTGTCTC forward 48 White et al. 1990
NS4 CTTCCGTCAATTCCTTTAAG reverse White et al. 1990
RPB2 RPB2-5F2 GGGGWGAYCAGAAGAAGGC forward 52 Sung et al. 2007
RPB2-7cR CCCATRGCTTGYTTRCCCAT reverse Liu et al. 1999
TEF1 EF1-728F CATCGAGAAGTTCGAGAAGG forward 52 Carbone and Kohn 1999
EF1-986R TACTTGAAGGAACCCTTACC reverse Carbone and Kohn 1999
EF1-728F CATCGAGAAGTTCGAGAAGG forward 55 Carbone and Kohn 1999
TEF1-LLErev AACTTGCAGGCAATGTGG reverse Jaklitsch et al. 2005
TEF1_INTFz CCGTGAYTTCATCAAGAACATG forward Jaklitsch 2009
TEF1_INT2z CCACTTNGTNGTGTCCATCTTRTT reverse Voglmayr and Jaklitsch 2017
TUB2 T1 AACATGCGTGAGATTGTAAGT forward 52 O'Donnell and Cigelnik 1997
bt2b ACCCTCAGTGTAGTGACCCTTGGC reverse Glass and Donaldson 1995

Characteristics and accession numbers of isolates collected from fennel plants in Sicily.

Strain1 Year Cultivar Farm ITS2 LSU2 SSU2 RPB2 2 TEF1 2 TUB2 2
Di3AF1 = CBS 145654* 2017 Apollo Farm 1 MN516753 MN516774 MN516743 MN520145 MN520149 MN520147
Di3AF2 2017 Apollo Farm 1 MN516754 MN516775 MN516744
Di3AF3 2018 Apollo Farm 1 MN516755 MN516776 MN516745
Di3AF4 2018 Apollo Farm 1
Di3AF5 2018 Apollo Farm 1 MN516756 MN516777 MN516746
Di3AF6 2018 Apollo Farm 1 MN516757 MN516778 MN516747
Di3AF7 2018 Apollo Farm 1 MN516758
Di3AF8 2018 Apollo Farm 1 MN516759
Di3AF9 2018 Apollo Farm 1 MN516760 MN516779 MN516748
Di3AF10 2018 Apollo Farm 1 MN516761 MN516780 MN516749 MN520146 MN520150 MN520148
Di3AF11 2018 Apollo Farm 1 MN516762
Di3AF12 2018 Apollo Farm 1 MN516763
Di3AF13 2018 Apollo Farm 1 MN516764 MN516781 MN516750
Di3AF14 2018 Apollo Farm 1 MN516765 MN516782 MN516751
Di3AF15 2018 Apollo Farm 1 MN516766 MN516783 MN516752
Di3AF16 2018 Apollo Farm 1 MN516767
Di3AF17 2018 Apollo Farm 1 MN516768
Di3AF18 2018 Narciso Farm 2
Di3AF19 2018 Narciso Farm 2 MN516769
Di3AF20 2018 Narciso Farm 2 MN516770
Di3AF21 2018 Narciso Farm 2 MN516771
Di3AF22 2018 Narciso Farm 2
Di3AF23 2018 Narciso Farm 2
Di3AF24 2018 Narciso Farm 2
Di3AF25 2018 Narciso Farm 2
Di3AF26 2018 Narciso Farm 3
Di3AF27 2018 Narciso Farm 3
Di3AF28 2018 Narciso Farm 3
Di3AF29 2018 Narciso Farm 4
Di3AF30 2018 Narciso Farm 4 MN516772
Di3AF31 2018 Narciso Farm 4
Di3AF32 2018 Aurelio Farm 5 MN516773

Phylogenetic analysis

According to the results of BLAST searches in GenBank, the newly generated ITS, LSU, and SSU rDNA sequences of the fennel pathogen were aligned with selected sequences of Leptosphaeriaceae from Gruyter et al. (2013) and complemented with a few recent additions from GenBank. The familial and generic concept of Leptosphaeriaceae implemented here follows the molecular phylogenetic studies of Gruyter et al. (2013), Ariyawansa et al. (2015), and Phookamsak et al. (2019). Due to insufficient RPB2, TEF1, and TUB2 sequence data available in Genbank for the study group, the sequences of these markers could not be included in phylogenetic analyses, but they were deposited in GenBank (Table 3). A combined SSU-ITS-LSU rDNA matrix was produced for phylogenetic analyses, with six species of Coniothyrium (C. carteri, C. dolichi, C. glycines, C. multiporum, C. telephii, C. palmarum) from Coniothyriaceae added as the outgroup according to the results of the phylogenetic analyses of Gruyter et al. (2013). As the rDNA sequences of the fennel pathogen isolates were (almost) identical (see Results section below), only a single isolate (CBS 145654 = Di3A-F1; ex holotype strain) was included in the final matrix. The GenBank accession numbers of sequences used in the analyses are given in Table 4. Sequence alignments were produced with the server version of MAFFT (http://mafft.cbrc.jp/alignment/server), checked and refined using BioEdit v. 7.2.6 (Hall 1999). The combined data matrix contained 3312 characters; i.e. 607 nucleotides of the ITS, 1333 nucleotides of the LSU and 1372 nucleotides of the SSU).

Isolates and accession numbers used in the phylogenetic analyses. Isolate/sequences in bold were isolated/sequenced in the present study.

Taxon Culture, specimen Host, substrate Country GenBank accession no
ITS LSU SSU
Alloleptosphaeria iridicola CBS 143395 Iris sp. (Iridaceae) United Kingdom MH107919 MH107965
Alloleptosphaeria italica MFLUCC 14-934 Clematis vitalba (Ranunculaceae) Italy KT454722 KT454714
Alternariaster bidentis CBS 134021 Bidens sulphurea (Asteraceae) Brazil KC609333 KC609341
Alternariaster centaureae-diffusae MFLUCC 14-0992 Centaurea diffusa (Asteraceae) Russia KT454723 KT454715 KT454730
Alternariaster helianthi CBS 119672 Helianthus sp. (Asteraceae) USA KC609337 KC584368 KC584626
Alternariaster trigonosporus MFLU 15-2237 Cirsium sp. (Asteraceae) Russia KY674857 KY674858
Coniothyrium carteri CBS 105.91 Quercus robur (Fagaceae) Germany JF740181 GQ387594 GQ387533
Coniothyrium dolichi CBS 124140 Dolichos biforus (Fabaceae) India JF740183 GQ387611 GQ387550
Coniothyrium glycines CBS 124455 Glycine max (Fabaceae) Zambia JF740184 GQ387597 GQ387536
Coniothyrium multiporum CBS 501.91 Unknown Egypt JF740186 GU238109
Coniothyrium palmarum CBS 400.71 Chamaerops humilis (Arecaceae) Italy AY720708 EU754153 EU754054
Coniothyrium telephii CBS 188.71 Air Finland JF740188 GQ387599 GQ387538
Heterosporicola chenopodii CBS 448.68 Chenopodium album (Chenopodiaceae) Netherlands FJ427023 EU754187 EU754088
Heterosporicola dimorphospora CBS 165.78 Chenopodium quinoa (Chenopodiaceae) Peru JF740204 JF740281 JF740098
Leptosphaeria conoidea CBS 616.75 Lunaria annua (Brassicaceae) Netherlands JF740201 JF740279 JF740099
Leptosphaeria doliolum CBS 505.75 Urtica dioica (Urticaceae) Netherlands JF740205 GQ387576 GQ387515
Leptosphaeria errabunda CBS 617.75 Solidago sp. (hybrid) (Asteraceae) Netherlands JF740216 JF740289
Leptosphaeria macrocapsa CBS 640.93 Mercurialis perennis (Euphorbiaceae) Netherlands JF740237 JF740304
Leptosphaeria pedicularis CBS 126582 Gentiana punctata (Gentianaceae) Switzerland JF740223 JF740293
Leptosphaeria sclerotioides CBS 144.84 Medicago sativa (Fabaceae) Canada JF740192 JF740269
Leptosphaeria slovacica CBS 389.80 Balota nigra (Lamiaceae) Netherlands JF740247 JF740315 JF740101
Leptosphaeria sydowii CBS 385.80 Senecio jacobaea (Asteraceae) UK JF740244 JF740313
Leptosphaeria veronicae CBS 145.84 Veronica chamaedryoides (Scrophulariaceae) Netherlands JF740254 JF740320
Neoleptosphaeria rubefaciens CBS 387.80 Tilia (×) europea (Malvaceae) Netherlands JF740242 JF740311
Ochraceocephala foeniculi Di3AF1 = CBS 145654 Foeniculum vulgare (Apiaceae) Italy MN516753 MN516774 MN516743
Paraleptosphaeria dryadis CBS 643.86 Dryas octopetala (Rosaceae) Switzerland JF740213 GU301828
Paraleptosphaeria macrospora CBS 114198 Rumex domesticus (Chenopodiaceae) Norway JF740238 JF740305
Paraleptosphaeria nitschkei CBS 306.51 Cirsium spinosissimum (Asteraceae) Switzerland JF740239 JF740308
Paraleptosphaeria orobanches CBS 101638 Epifagus virginiana (Orobanchaceae) USA JF400230 JF740299
Paraleptosphaeria padi MFLU 15-2756 Prunus padus (Rosaceae) Russia KY554203 KY554198 KY554201
Paraleptosphaeria praetermissa CBS 114591 Rubus idaeus (Rosaceae) Sweden JF740241 JF740310
Paraleptosphaeria rubi MFLUCC 14-0211 Rubus sp. (Rosaceae) Italy KT454726 KT454718 KT454733
Paraleptosphaeria rumicis CBS 522.78 Rumex alpinus (Polygonaceae) France KF251144 KF251648
Plenodomus agnitus CBS 121.89 Eupatorium cannabinum (Asteraceae) Netherlands JF740194 JF740271
Plenodomus agnitus CBS 126584 Eupatorium cannabinum (Asteraceae) Netherlands JF740195 JF740272
Plenodomus artemisiae KUMCC 18-0151 Artemisia sp. (Asteraceae) China MK387920 MK387958 MK387928
Plenodomus biglobosus CBS 119951 Brassica rapa (Brassicaceae) Netherlands JF740198 JF740274 JF740102
Plenodomus biglobosus CBS 127249 Brassica juncea (Brassicaceae) France JF740199 JF740275
Plenodomus chrysanthemi CBS 539.63 Chrysanthemum sp. (Asteraceae) Greece JF740253 GU238151 GU238230
Plenodomus collinsoniae CBS 120227 Vitis coignetiae (Vitaceae) Japan JF740200 JF740276
Plenodomus confertus CBS 375.64 Anacyclus radiatus (Asteraceae) Spain AF439459 JF740277
Plenodomus congestus CBS 244.64 Erigeron canadensis (Asteraceae) Spain AF439460 JF740278
Plenodomus deqinensis CGMCC 3.18221 soil China KY064027 KY064031
Plenodomus enteroleucus CBS 142.84 Catalpa bignonioides (Bignoniaceae) Netherlands JF740214 JF740287
Plenodomus enteroleucus CBS 831.84 Triticum aestivum (Poaceae) Germany JF740215 JF740288
Plenodomus fallaciosus CBS 414.62 Satureia montana (Lamiaceae) France JF740222 JF740292
Plenodomus guttulatus MFLU 15-1876 unidentified dead stem Germany KT454721 KT454713 KT454729
Plenodomus hendersoniae CBS 113702 Salix cinerea (Salicaceae) Sweden JF740225 JF740295
Plenodomus hendersoniae CBS 139.78 Pyrus malus (Rosaceae) Netherlands JF740226 JF740296
Plenodomus hendersoniae LTO Salix appendiculata (Salicaceae) Austria MF795790 MF795790
Plenodomus influorescens CBS 143.84 Fraxinus excelsior (Oleaceae) Netherlands JF740228 JF740297
Plenodomus influorescens PD 73/1382 Lilium sp. (Liliaceae) Netherlands JF740229 JF740298
Plenodomus libanotidis CBS 113795 Seseli libanotis (Apiaceae) Sweden JF740231 JF740300
Plenodomus lijiangensis KUMCC 18-0186 dead fern fronds China MK387921 MK387959 MK387929
Plenodomus lindquistii CBS 386.80 Helianthus annuus (Asteraceae) former Yugoslavia JF740232 JF740301
Plenodomus lindquistii CBS 381.67 Helianthus annuus (Asteraceae) Canada JF740233 JF740302
Plenodomus lingam CBS 275.63 Brassica sp. (Brassicaceae) UK JF740234 JF740306 JF740103
Plenodomus lingam CBS 260.94 Brassica oleracea (Brassicaceae) Netherlands JF740235 JF740307
Plenodomus lupini CBS 248.92 Lupinus mutabilis (Fabaceae) Peru JF740236 JF740303
Plenodomus pimpinellae CBS 101637 Pimpinella anisum (Apiaceae) Israel JF740240 JF740309
Plenodomus salviae MFLUCC 13-0219 Salvia glutinosa (Lamiaceae) Italy KT454725 KT454717 KT454732
Plenodomus sinensis MFLU 17-0757 Plukenetia volubilis (Euphorbiaceae) China MF072722 MF072718 MF072720
Plenodomus tracheiphilus CBS 551.93 Citrus limonium (Rutaceae) Israel JF740249 JF740317 JF740104
Plenodomus tracheiphilus CBS 127250 Citrus sp. (Rutaceae) Italy JF740250 JF740318
Plenodomus visci CBS 122783 Viscum album (Viscaceae) France JF740256 EU754195 EU754096
Plenodomus wasabiae CBS 120119 Wasabia japonica (Brassicaceae) Taiwan JF740257 JF740323
Plenodomus wasabiae CBS 120120 Wasabia japonica (Brassicaceae) Taiwan JF740258 JF740324
Pseudoleptosphaeria etheridgei CBS 125980 Populus tremuloides (Salicaceae) Canada JF740221 JF740291
Sphaerellopsis filum CBS 317.68 Puccinia deschampsiae uredinium, on Deschampsia caespitosa Germany KP170657 KP170725
Sphaerellopsis hakeae CPC 29566 Hakea sp. (Proteaceae) Australia KY173466 KY173555
Sphaerellopsis isthmospora KUN-HKAS 102225 Unidentified twig China MK387925 MK387963 MK387934
Sphaerellopsis macroconidialis CBS 233.51 Uromyces caryophylli on Dianthus caryophyllus Italy KP170658 KP170726
Sphaerellopsis paraphysata CPC 21841 Pennisetum sp. (Poaceae) Brazil KP170662 KP170729
Subplenodomus apiicola CBS 285.72 Apium graveolens var. rapaceum (Apiaceae) Germany JF740196 GU238040
Subplenodomus drobnjacensis CBS 269.92 Eustoma exaltatum (Gentianaceae) Netherlands JF740211 JF740285 JF740100
Subplenodomus valerianae CBS 630.68 Valeriana phu (Valerianaceae) Netherlands JF740251 GU238150
Subplenodomus violicola CBS 306.68 Viola tricolor (Violaceae) Netherlands FJ427083 GU238156 GU238231

Maximum likelihood (ML) analyses were performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 1.3 (Silvestro and Michalak 2012), using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates.

Maximum parsimony (MP) bootstrap analyses were performed with PAUP v. 4.0a165 (Swofford 2002). All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data; the COLLAPSE command was set to MINBRLEN. MP bootstrap analyses were performed with 1000 replicates, using 5 rounds of random sequence addition and subsequent TBR branch swapping (MULTREES option in effect, steepest descent option not in effect) during each bootstrap replicate. In the Results and Discussion, bootstrap values below 70 % are considered low, between 70–90 % medium and above 90 % high.

Pathogenicity test

To determine the ability of the representative isolate Di3A-F1 (CBS 145654) to cause disease symptoms, pathogenicity tests were conducted on 6-month-old plants of fennel grown in a growth chamber. Five plants for each of the three replicates were used. The inoculum, which consisted of a 6-mm-diameter mycelial plug from a 10-day-old culture on PDA, was inserted in four points for each crown and the wounds wrapped with Parafilm to prevent desiccation. Fennel plants inoculated with sterile PDA plugs served as a control. After inoculation, plants were covered with a plastic bag for 48 h and maintained at 25 ± 1 °C and 95% relative humidity (RH) under a 12 h fluorescent light/dark regime. Five days after inoculation the presence of a lesion was evaluated in each inoculation point. To fulfill Koch’s postulates, symptomatic tissues taken from the crown of each inoculated plant were plated on PDA and the identity of the fungal isolates was confirmed as described above.

Cultivar susceptibility

To evaluate the susceptibility of six different cultivars of fennel to infection by the pathogen, one experiment was conducted on 1 to 2-month-old seedlings of fennel in a growth chamber. Eight plants for each of three replicates were used. The inoculum, which consisted of a 6-mm-diameter mycelial plug from a 10-day-old culture on PDA, was inserted at the crown of each plant and wrapped with Parafilm to prevent desiccation. Fennel plants inoculated with sterile PDA plugs served as a control. All the replicates were enclosed in plastic bags and maintained at 25 ± 1 °C and 95% relative humidity (RH) under a 12 h fluorescent light/dark regime in a growth chamber until the symptoms were observed. Plant mortality (PM), disease incidence (DI) and symptom severity (SS) were evaluated. Symptom severity was rated using a category scale from 0 to 5, where 0 = healthy plant; 1 = necrotic lesion on crown from 0.1 to 0.2 cm; 2 = from 0.3 to 1 cm; 3 = from 1.1 to 2 cm; 4 = from 2.1 to 3.5 cm; 5 = dead plant. The experiment was performed twice.

Statistical analysis

Data about disease susceptibility of examined fennel cultivars from the repeated experiments were analysed by using the Statistica package software (v. 10; Statsoft Inc., Tulsa, OK, USA). The arithmetic means of PM, DI, and SS were calculated, averaging the values determined for the single replicates of each treatment. Percentage data concerning PM and DI were transformed into the arcsine (sin–1 square rootx) prior to analysis of variance (ANOVA), whereas SS values were not transformed. Initial analyses of PM and DI were performed by calculating F and P values associated to evaluate whether the effects of single factor (cultivar) and cultivar × trial interactions are significant. In the post hoc analyses, the corresponding mean values of PM and DI were subsequently separated by the Fisher’s least significant difference test (P = 0.05). Because ordinal scales were adopted for SS data calculation, different nonparametric approaches were used. Kendall’s coefficient of concordance (W) was calculated to assess whether the rankings of the SS scores among fennel cultivars are similar within each trial (cultivar × trial interactions). Since in the susceptibility experiment W was higher than 0.9, the SS scores were at first analysed by using Friedman’s nonparametric rank test, and subsequently followed by the all possible pairwise performed with the Wilcoxon signed-rank at P < 0.05. On the other hand, when only the cultivar effects were examined, the Kruskal-Wallis non parametric one-way test was preliminarily applied, calculating χ2 and P value associated.

Results

Collection of samples and isolates

Symptoms referable to infection (Fig. 1a, b) were detected in five commercial farms surveyed in eastern Sicily, Italy. The disease was observed on 3 different cultivars of fennel (4 to 6-month-old) in open fields. The symptoms consisted of depressed necrotic lesions formed near the soil line and affected crown, root, and stem. The lesion was first light brown with wet appearance, becoming dark brown to black with age and sometimes appearing dry. Under favourable conditions (high humidity), the lesion extended and the infection resulted in a crown and root rot. Fungal colonies representing the new fennel pathogen were consistently obtained from symptomatic tissues. A total of 32 single-spore isolates were collected (Table 3). Preliminary identity of the fungal isolates was based on cultural and morphological characteristics. Among these, 17 isolates were obtained from ‘Apollo’, 14 from ‘Narciso’, and one from ‘Aurelio’ cultivars.

Figure 1. 

Symptoms caused by Ochraceocephala foeniculi on fennel plants. a, b Necrotic lesions and crown rot on ‘Narciso’ cultivar. c, d Necrotic lesions and crown rot on ‘Apollo’ cultivar. e Symptoms on artificially inoculated seedlings of ‘Pompeo’ cultivar.

Sequencing

All strains of the new fennel pathogen sequenced had identical LSU, SSU, RPB2, TEF1, and TUB2 sequences. Also all ITS sequences were identical, except for a single nucleotide polymorphism (A/G) towards the end of the ITS2 region. All sequences generated during this study were deposited at GenBank; for GenBank accession numbers, see Table 3.

Phylogenetic analyses

Of the 3312 characters included in the phylogenetic analyses, 294 were parsimony informative (222 from the ITS, 62 from the LSU, 10 from the SSU). The best ML tree (lnL = –14211.5558) revealed by RAxML is shown in Figure 2. In the phylogenetic tree, the Leptosphaeriaceae received high (96% ML and MP) support. Within Leptosphaeriaceae, most of the deeper nodes of the tree backbone received low to insignificant support. Highly supported genera include Alloleptosphaeria, Heterosporicola, Leptosphaeria (all three with maximum support) and Alternariaster (99% ML and 100% MP), while Sphaerellopsis received low (53%) and Paraleptosphaeria medium (75%) support only in the ML analyses, and Plenodomus and Subplenodomus were unsupported. Subplenodomus iridicola was not contained within the Subplenodomus clade, but sister species to Alloleptosphaeria italica with maximum support, and Acicuseptoria rumicis was embedded within the Paraleptosphaeria clade, indicating that they are generically misplaced. The new fennel pathogen was placed basal to the Plenodomus clade, however, without significant support. Although the new fennel pathogen is closely related to the genus Plenodomus, it is morphologically highly distinct. As no suitable described genus is available, a new genus is therefore established here.

Figure 2. 

Phylogram of the best ML tree (–lnL = 14211.5558) revealed by RAxML from an analysis of the combined SSU-ITS-LSU matrix of selected Leptosphaeriaceae, showing the phylogenetic position of Ochraceocephala foeniculi (bold red). Taxa in bold black denote new combinations proposed here. ML and MP bootstrap support above 50% are given above or below the branches.

Taxonomy

Ochraceocephala Voglmayr & Aiello, gen. nov.

MycoBank No: 833933

Etymology

referring to the ochraceous conidial capitula of the type species.

Conidiophores erect, variable in shape and branching, from unbranched, loosely to densely branched up to several times; branching commonly irregularly verticillate. Phialides arising singly or in irregular whorls, cylindrical, lageniform or ampulliform, producing basipetal conidial chains. Conidia in chains, unicellular, thick-walled.

Type species

Ochraceocephala foeniculi Voglmayr & Aiello.

Notes

Ochraceocephala is phylogenetically closely related to Plenodomus, from which it deviates substantially in morphology. Plenodomus species are characterised by pycnidial phoma-like asexual morphs, and while in two Plenodomus species (P. chrysanthemi, P. tracheiphilus) simple hyphomycetous, phialophora-like synanamorphs have been recorded (Boerema et al. 1994), these are very different from the complex conidiophores of the present fennel pathogen. These morphological differences, the lack of a suitable genus within Leptosphaeriaceae and its phylogenetic position therefore warrants the establishment of a new genus.

Ochraceocephala foeniculi Voglmayr & Aiello, sp. nov.

MycoBank No: 833934
Figure 3

Etymology

referring to its host genus, Foeniculum (Apiaceae).

Colonies fast-growing, at room temperature (22 ± 1 °C) on CMD reaching 80 mm after 7 d; on MEA 38 mm after 7 d; with dull white to cream surface, upon conidiation becoming beige to olive yellow from the centre, reverse cream with greyish to dark brown centre; cottony, with abundant surface mycelium; sporulation abundant on aerial hyphae. Aerial hyphae hyaline, 2–6 µm wide. Conidiophores hyaline, produced terminally or laterally on aerial hyphae, variable in shape and branching, unbranched, loosely or densely branched up to two times; branching commonly irregularly verticillate. Phialides arising singly or in whorls of 2–5, (3.8–)5.8–13.5(–21.0) × (2.5–)3.0–4.3(–5.5) µm (n = 100), cylindrical, lageniform or ampulliform, often with a distinct collarette, producing basipetal conidial chains; polyphialides rarely present. Conidia (3.2–)3.5–6.0(–8.5) × (2.5–)3.0–4.2(–6.0) µm, l/w (1.0–)1.1–1.5(–2.1) (n = 155), hyaline to yellowish, in masses sand to olive yellow, smooth, mostly globose to subglobose, rarely broadly ellipsoid to pip-shaped, thick-walled.

Figure 3. 

Ochraceocephala foeniculi, holotype a culture on CMD (7d, 22 °C) b culture on MEA (21d, 22 °C) c conidiophores on aerial hyphae producing yellowish brown conidial masses in chains d–j, l, m unbranched (g–i) and verticillately branched (d–f, j, l, m) conidiophores (MEA, 21d, 22 °C) with phialides; in f with polyphialide (arrow) k, n, o phialides with collarettes (arrows) and young conidia p conidia. All microscopic preparations from MEA (21d, 22 °C) and mounted in water. Scale bars: 200 µm (c); 10 µm (d–j, l, m, p); 5 µm (k, n, o).

Distribution

Italy (Sicily).

Host and substrate

Pathogenic on crown, roots and stems of living Foeniculum vulgare.

Holotype

Italy, Sicily, Catania province, Adrano, May 2017 (WU 40034); ex-holotype culture CBS 145654; ex holotype sequences MN516753 (ITS), MN516774 (LSU), MN516743 (SSU), MN520145 (RPB2), MN520149 (TEF1), MN520147 (TUB2).

Alloleptosphaeria iridicola (Crous & Denman) Voglmayr, comb. nov.

MycoBank No: 833935

Basionym

Subplenodomus iridicola Crous & Denman, in Crous, Schumacher, Wingfield, Akulov, Denman, Roux, Braun, Burgess, Carnegie, Váczy, Guatimosim, Schwartsburd, Barreto, Hernández-Restrepo, Lombard & Groenewald, Fungal Systematics and Evolution 1: 207. 2018.

Notes

In the phylogenetic analyses (Fig. 2) Subplenodomus iridicola is placed remote from the other species of Subplenodomus, but is sister species to Alloleptosphaeria italica with maximum support; S. iridicola is therefore transferred to the genus Alloleptosphaeria.

Paraleptosphaeria rumicis (Quaedvl., Verkley & Crous) Voglmayr, comb. nov.

MycoBank No: 833936

Basionym

Acicuseptoria rumicis Quaedvl., Verkley & Crous, Stud. Mycol. 75: 376 (2013).

Notes

The monotypic genus Acicuseptoria was described by Quaedvlieg et al. (2013) as a segregate of the polyphyletic genus Septoria, and it was characterised by brown, globose pycnidia with conidiophores reduced to ampulliform conidiogenous cells bearing acicular, hyaline, euseptate conidia. However, its position within the Leptosphaeriaceae remained undetermined as no other representatives of the family were included in their phylogenetic tree (Quaedvlieg et al. 2013: fig. 2). In our phylogenetic analyses (Fig. 2), Acicuseptoria rumicis is embedded within the genus Paraleptosphaeria and placed in a highly supported subclade that also contains the generic type, P. nitschkei. Acicuseptoria rumicis is therefore transferred to the genus Paraleptosphaeria.

Pathogenicity test

The representative isolate (CBS 145654) was pathogenic to fennel plants, and produced symptoms similar to those observed in open field after five days (Fig. 1e). The pathogen was re-isolated from the artificially inoculated plants, and identified as previously described. No symptoms were observed on control plants.

Cultivar susceptibility

In the experiments on fennel susceptibility there was always a significant effect of the cultivar on all disease parameters (PM, DI and SS) of pathogen infections (p < 0.0001). Otherwise, a not significant cultivar × trial effect (p > 0.56) was observed for parametric variables (PM and DI) in this repeated experiment (Table 5). Besides, Kendall’s coefficient of concordance was 0.96 for SS data, thus indicating very high concordance between the two trials (Table 5). Therefore, the two trials were combined.

ANOVA effects of cultivar and cultivar × trial interactions on plant mortality, disease incidence and severity of symptoms caused by Ochraceocephala foeniculi on inoculated young fennel plants.

Model effect Parameter
Factor(s) Plant mortality (PM) 1 Disease incidence (DI) 1 Symptom severity (SS) 2
df F P value df F P value χ2 W P value
Cultivar 5 70.6286 < 0.0001 5 33.659 < 0.0001 89.2051 < 0.0001
Cultivar × trial 5 0.1273 0.98475ns 5 0.789ns 0.56797ns 0.95873 0.0003

Regarding susceptibility of fennel to this phytopathogenic fungus, a great variability was detected among the tested cultivars eight days after inoculation. Comprehensively, cultivar ‘Narciso’ was the most susceptible since all disease parameters and its PM value were significantly the highest among the tested cultivars. ‘Apollo’ was also highly susceptible to infection by the new fennel pathogen, significantly differing only in a slightly lower PM value. ‘Pompeo’ displayed PM and DI values similar to those recorded for ‘Apollo’, but its SS score was significantly lower than in the former (Table 6). In decreasing order of susceptibility, ‘Aurelio’ did not significantly differ from ‘Pompeo’ for DI and SS values, but its PM caused by the fennel pathogen was strongly reduced. No dead seedlings (PM = 0) were recorded for both ‘Archimede’ and ‘Pegaso’, that significantly differed for DI and SS from the other remaining cultivars. Altogether, ‘Pegaso’ was the least susceptible cultivar to fungal infection since it showed the lowest values of disease severity.

Compared susceptibility to crown and root rot infections of six commercial fennel cultivars.

Cultivar Plant mortality (PM) 1 Disease incidence (DI) 1 Symptom severity (SS) 2
Narciso 72.92 ± 2.08 a 100 a 4.15 ± 0.10 a
Apollo 58.33 ± 4.17 b 100 a 4.33 ± 0.17 a
Pompeo 45.83 ± 4.17 b 100 a 3.37 ± 0.13 b
Aurelio 10.42 ± 2.08 c 100 a 2.56 ± 0.06 b
Archimede 0.00 d 83.33 ± 4.17 b 1.94 ± 0.10 c
Pegaso 0.00 d 77.08 ± 2.08 b 1.48 ± 0.10 d

Discussion

In the present study, 32 fungal isolates were recovered from symptomatic fennel plants in Sicily over a 2-year period. Disease symptoms were observed in three farms, and included necrotic lesions and crown and root rot on three different cultivars. The fungal species obtained from symptomatic tissues was identified based on morphological characters and molecular phylogenetic analyses of an ITS-LSU-SSU rDNA matrix, resulting in the description of the fennel pathogen as a new genus and species, Ochraceocephala foeniculi.

In the phylogenetic analyses, O. foeniculi was revealed as sister group of Plenodomus; however, without significant support (Fig. 2). As commonly observed with ITS-LSU-SSU rDNA data, support of many backbone nodes is low or absent, and additional protein-coding markers like RPB2, TEF1 and TUB2 are necessary for an improved phylogenetic resolution of genera and families in Pleosporales (Voglmayr and Jaklitsch 2017; Jaklitsch et al. 2018). Although we sequenced RPB2, TEF1, and TUB2 for O. foeniculi, it was currently not feasible to perform multi-gene analyses due to insufficient sequence data for most species of Leptosphaeriaceae, in particular for Plenodomus. However, we consider the phylogenetic and morphological evidence conclusive for establishing the new genus Ochraceocephala. Also the generic transfer of Subplenodomus iridicola to Alloleptosphaeria is well substantiated, considering its highly supported phylogenetic position as sister species of Alloleptosphaeria italica, remote from the generic type (S. violicola) and other species of Subplenodomus (Fig. 2). In the phylogenetic analyses of the LSU rDNA matrix of Crous et al. (2018: fig. 1), only few taxa of Leptosphaeriaceae were included, and the phylogenetic position of S. iridicola remained inconclusive due to low resolution; however, also in their analyses it was placed remote from the generic type, S. violicola. In addition, they did not include its closest relative, Alloleptosphaeria italica, although it was mentioned as the closest match of an ITS BLAST search (Crous et al. 2018). No asexual morph is known for A. italica (Dayarathne et al. 2015), but the ascomata, asci and ascospores of A. iridicola and A. italica share many traits. Our phylogenetic analyses also showed that Acicuseptoria rumicis should be included within Paraleptosphaeria (Fig. 2). Although it was correctly placed within Leptosphaeriaceae by Quaedvlieg et al. (2013), its position within the family remained undetermined as no other representatives of the family were included in their phylogenetic analyses. As for most other species of Paraleptosphaeria no asexual morphs are known, no comprehensive morphological comparison can currently be made with P. rumicis.

Within Leptosphaeriaceae, O. foeniculi is remarkable and unique by its complex hyphomycetous asexual morph composed of branched conidiophores with phialidic conidiation and conidia produced in basipetal chains. Asexual morphs in Leptosphaeriaceae are typically coelomycetous and phoma-like, which is also the case in the closest relative of Ochraceocephala, Plenodomus (Gruyter et al. 2013). Another genus of Leptosphaeriaceae with a hyphomycetous asexual morph is Alternariaster, which, however, differs significantly by tretic condiogenous cells forming large, brown, septate conidia not produced in chains (Simmons 2007; Alves et al. 2013). Therefore, the unique morphology in combination with an isolated phylogenetic position within Leptosphaeriaceae warrant the establishment of a new genus.

Other fungal species belonging to Leptosphaeriaceae, as well as the closely related Didymellaceae (Odstrčilová et al. 2002; Shaker and Alhamadany 2015) have been reported worldwide in fennel crops. In Italy, crown rot of fennel caused by Didymella glomerata (syn. Phoma glomerata) was recorded from southern Italy (Lahoz et al. 2007). As confirmed in the pathogenicity tests, O. foeniculi caused symptoms on artificially inoculated plants of the same cultivar and, moreover, also on different fennel cultivars that showed some variability in disease susceptibility. To this regard, it is noteworthy that this study also represents a preliminary evaluation of fennel germplasm according to their susceptibility to this new disease. Although these data should be confirmed by additional investigations, this study might provide very useful information for local farmers and technicians. The determination of the extent of susceptibility to O. foeniculi is a starting point for evaluating the tolerance of commercial fennel cultivars to this disease under different agronomic and phytosanitary conditions.

On the basis of the disease incidence and severity observed in the field, we believe that this disease represents a serious threat to fennel crop in Sicily and may become a major problem also to other areas of fennel production if accidentally introduced. Moreover, infected soil could represent an inoculum source for this fungus. Further studies are needed to examine the life cycle of O. foeniculi and to ascertain the cardinal temperatures of the fungus for successful infection since this pathogen is well established in this representative fennel production area. This information is required for the setup and timing of sustainable approaches for soil disinfection, including solarization and/or fumigation at low rates, to reduce the level of the primary inoculum in the soil and hence the disease amount, like successfully applied for other soilborne plant pathogens (Vitale et al. 2013; Aiello et al. 2018).

Although not always conclusive, soil disinfestation and host resistance can be considered environmentally friendly means to be included within integrated pest management (IPM) strategies against crown rot caused by O. foeniculi in order to minimize the number and intensity of fungicide applications.

Acknowledgements

This research was supported by Research Project 2016–2018 “Monitoraggio, caratterizzazione e controllo sostenibile di microrganismi e artropodi di interesse agrario” WP2-5A722192134.

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