Research Article |
Corresponding author: Samad Ashrafi ( ashrafi.samad@gmail.com ) Academic editor: Cecile Gueidan
© 2017 Samad Ashrafi, Marc Stadler, Abdelfattah A. Dababat, Katja R. Richert-Pöggeler, Maria R. Finckh, Wolfgang Maier .
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ashrafi S, Stadler M, Dababat AA, Richert-Pöggeler KR, Finckh MR, Maier W (2017) Monocillium gamsii sp. nov. and Monocillium bulbillosum: two nematode-associated fungi parasitising the eggs of Heterodera filipjevi. MycoKeys 27: 21-38. https://doi.org/10.3897/mycokeys.27.21254
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Monocillium gamsii sp. nov. (Ascomycota, Hypocreales, Niessliaceae) isolated from eggs of the cereal cyst nematode Heterodera filipjevi is described and illustrated based on morphological and molecular phylogenetic evidence. The new taxon discovered in wheat fields in Turkey destructively parasitises nematode eggs. The infected eggs were readily colonised by the fungus, which produced microsclerotia. The fungus could be grown on artificial media and the parasitism of M. gamsii towards H. filipjevi was reproducible in vitro. Hyphae penetrating the nematode eggs entirely colonised the embryo, developed into multicellular chlamydospore and dictyochlamydospore-like structures eventually forming microsclerotia. Molecular and morphological differences and similarities between M. gamsii and its phylogenetically related species are discussed. Monocillium bulbillosum was found to be closely related to the new species. The pathogenicity of M. bulbillosum against H. filipjevi was also assayed in vitro because of its sister group relationship to M. gamsii revealing that this species was also capable of colonising eggs of H. filipjevi.
Egg-parasitic fungi, Niessliaceae , new species, plant parasitic nematodes, taxonomy, molecular phylogeny, ITS, LSU, rpb1 , tef
Various fungi have been reported as natural enemies of plant parasitic nematodes (PPN) (
Egg-parasitic fungi attacking cyst nematodes have repeatedly been isolated from all agricultural soils in various geographic regions (
Experimental wheat fields of the International Maize and Wheat Improvement Centre (CIMMYT) in Turkey, where a significant reduction in population size of the cereal cyst nematode Heterodera filipjevi had been observed between two consecutive years (unpublished data), were sampled to isolate and study fungal candidates that could be causally involved in this drop of the nematode population size.
Here we report a so-far undescribed hypocrealean species which destructively parasitised the eggs of H. filipjevi. The antagonistic interaction of this fungus with the nematode eggs was studied based on in vitro tests. We also report the antagonistic potential of M. bulbillosum as the most closely related species to the herein described fungus, towards the eggs of H. filipjevi.
Cysts of H. filipjevi were collected from experimental wheat fields of CIMMYT in the Central Anatolian Plateau of Turkey in 2013. The fields located in Yozgat (39°08'N, 34°10'E; altitude 985 m.a.s.l) and Haymana (39°26'N, 39°29'E, altitude 1260 m.a.s.l) were naturally nematode infested. The samples including soil and roots were collected at random from the rhizosphere of wheat plants at the end of the growing season. Cysts were extracted from the collected samples using the modified flotation decanting method (
The field-collected cysts of H. filipjevi were scrutinised by using a dissecting microscope to separate symptomatic cysts showing defined discolourations or bearing discernible hyphae, from healthy-looking (i.e. homogeneously brown) or empty cysts. Symptomatic cysts were selected, surface-sterilised in 5% sodium hypochlorite (NaOCl), and dissected to collect their egg contents. Only the nematode eggs showing symptoms of fungal infection were processed for fungal isolation and culture-dependent species identification. A portion of the fungal infected eggs were additionally used for culture-independent identification. The methods applied here, have been described in greater detail in
Growth rates were determined at various temperatures from 15 to 35 °C at 5 °C intervals in the dark or in ambient conditions by placing agar disks (5 mm diam.), excised from the margin of a young potato dextrose agar (PDA) culture onto five replicate plates of PDA, cornmeal agar (CMA), oatmeal agar (OA; 30 g oatmeal, 18 g agar-agar, 1L deionised water), synthetic nutrient-poor agar (SNA;
The antagonistic potential of the below described species and M. bulbillosum, respectively, was assessed towards H. filipjevi in vitro as previously described (
Nematode eggs and fungal structures were examined and photographed by a Zeiss Axioskop 2 plus compound microscope and an Olympus SZX 12 stereo microscope equipped with a Jenoptik ProgRes® digital camera. Images were recorded using CapturePro 2.8 software (Jenoptic, Jena, Germany). Nematode eggs colonised by fungi, and fungal structures were mounted in water or lactic acid and photographed. Cysts were photographed in water in a square cavity dish (40×40×16 mm). To illustrate different stages of fungal development and fungal colonisation of nematode eggs, slide cultures were prepared (
Scanning electron microscopy was performed on a Quanta 250 scanning electron microscope (FEI Deutschland GmbH, Frankfurt, Germany). Fungal structures of interest were obtained from a one-month-old OA culture grown at 23 °C in the dark and directly analysed using environmental scanning electron microscopy (ESEM). For the experiment, pressures between 410 and 490 Pa at 4 °C were employed. For cooling the sample chamber was equipped with a Peltier stage. Fungal mycelia with abundant conidia were placed on non-conductive double-sided adhesive discs on a flat specimen stub and positioned on the Peltier stage for cooling. Images were taken at acceleration voltage of 12.5 kV. Scanning speed was 60 µsec. For imaging of beam sensitive fungal structures, the scanning modus was changed to 3 µsec with 20-fold line integration. Images were adjusted in brightness and contrast using Adobe Photoshop software CS 5.1.
Fungal genomic DNA was isolated from mycelia grown on PDA using a modified CTAB method, and from individual nematode eggs infected by fungi using the Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) as reported in
For each specimen, four nuclear loci were amplified: The internal transcribed spacers including the 5.8S rDNA gene (ITS) using the primers ITS1F (
The newly generated sequences together with closely related sequences selected as revealed by BLASTn searches were used for phylogenetic analyses (Table
Species | Isolate number | Host / substrate | Locality | GenBank accession numbers | Reference | |||
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ITS | LSU | rpb1 | tef | |||||
Bionectria byssicola | CBS 914.97–GML2665 | Alchornea branches- leaf litter | Uganda, Brazil | AF358252 | GQ506011 | GQ506040 | KX184977 | ( |
Hyaloseta nolinae | CBS109837 | Nolina micrantha, leaf litter | USA, New Mexico | KM231846 | KM231726 | KM232279 | – | ( |
Ijuhya vitellina | DSM104494 | Heterodera filipjevi, egg | Turkey | KY607535 | KY607549 | KY607576 | – | ( |
Monocillium bulbillosum | CBS344.70 | mouldy wallpaper | Germany | MF681488 | MF681501 | MF681513 | MF681507 | This study |
Monocillium gamsii | DSM105458 | Heterodera filipjevi, egg | Turkey | MF681485 | MF681496 | MF681512 | MF681506 | This study |
Monocillium gamsii | DSM105459 | Heterodera filipjevi, egg | Turkey | MF681483 | MF681493 | MF681511 | MF681505 | This study |
Monocillium gamsii | DSM105460 | Heterodera filipjevi, egg | Turkey | MF681482 | MF681492 | MF681510 | MF681504 | This study |
Monocillium gamsii | DSM105461 | Heterodera filipjevi, egg | Turkey | MF681481 | MF681490 | MF681509 | MF681503 | This study |
Monocillium ligusticum | CBS684.95 | ectomycorrhizae of Pinus halapensis | Italy | MF681489 | MF681502 | MF681514 | MF681508 | This study |
Nisslia exilis | CBS357.70 | Picea abies, bark | Germany | – | AY489718 | AY489645 | AY489613 | ( |
Nisslia exilis | CBS560.74 | Pinus sylvestris, decayed needle | England | – | AY489720 | AY489647 | AY489614 | ( |
Among the field-collected samples, a high proportion of cysts was found containing blackish bodies resembling microsclerotia-like structures upon microscopy (Fig.
Naturally infested cysts and eggs of Heterodera filipjevi with Monocillium gamsii, and pure cultures obtained from infected eggs. A Field collected symptomatic cysts bearing parasitised eggs. B–E Nematode eggs infected by M. gamsii B, D Nematode eggs containing microsclerotia of M. gamsii E An embryonated egg containing a second stage juvenile (J2) parasitised by M. gamsii (arrow points at nematode’s stylet) F A nematode egg containing microsclerotia, and hyphae growing out of it G–H colony of M. gamsii grown on PDAG colonies developing from three individually plated infected eggs H A 25-d-old culture grown at 25 °C in the dark I The surface of a five-month-old culture detailing the sclerotioid masses covering the colony surface. Single microsclerotia can be seen as little black dots at the margin of the culture. Scale bars: 800 µm (A); 30 µm (B–E); 50 µm (F); 2 cm (H); 5 mm (I).
The DNA sequences of four different gene regions obtained from the examined specimens of the here described nematode-parasitic fungus were either identical (in TEF and RPB1), or nearly identical (1 base pair (bp) substitution in LSU, and up to 2 bp substitutions in ITS). The most similar DNA sequences found in GenBank using BLASTn searches belonged to Hyaloseta nolinae, the sexual morph of Monocillium nolinae, and shared similarities of 96% in the ITS region, 99% in the LSU, and 89% in rpb1. A similar BLASTn search in MycoBank showed identities of the ITS sequence of 96.6% with M. bulbillosum, 93.9% with H. nolinae and 92.7% with Niesslia exosporioides, and of the LSU sequence of 99.5% with H. nolinae, and 99% with both M. bulbillosum and Niesslia exosporioides suggesting a close relationship with the representatives of the Niessliaceae. Fungal DNA could also be directly isolated and sequenced from individual eggs displaying the typical symptoms of fungal infection. These DNA sequences were identical to the sequences retrieved from pure cultures supporting the conspecificity of the symptom-causing structures within the egg with the isolated pure cultures derived from the eggs.
The final combined ITS, LSU, rpb1 and tef dataset comprised 11 strains representing 7 species with a total alignment length of 2949 bp (603 ITS, 797 LSU, 649 rpb1, 900 tef). The topologies of the phylogenetic trees were identical using Bayesian inference (Fig.
Bayesian inference of phylogenetic relationships using four strains of the here described nematode parasite and all Niessliaceae present in GenBank based on an alignment of ITS, LSU, rpb1, and tef sequences using GTR+I+G as nucleotide substitution model. Depicted is a 50% majority rule consensus tree derived from 3500 trees from the stationary phase of a Monte Carlo Markov Chain. A posteriori probability (BIpp) values greater than 0.95, and bootstrap values of neighbor-joining (NJBT) and maximum likelihood (MLBT) analyses greater than 0.7 are given above branches (BIpp/NJBT/MLBT). Two representatives of the Bionectriaceae, Bionectria byssicola and Ijuhya vitellina, were used to root the tree.
Turkey, Yozgat, experimental wheat field: dried culture on PDA, originating from an individual egg from a cyst of Heterodera filipjevi, isolated by Samad Ashrafi, August 2013, dried culture on PDA, deposited at the herbarium of the Botanic Garden and Botanical Museum Berlin-Dahlem: B700016491.
Ex-holotype strain: DSM 105458, deposited in the open collection of the Leibniz-Institut
From the same location: DSM 105459 (dried culture on PDA, B700016492), GenBank accession number: MF681483 (ITS), MF681493 (LSU), MF681511 (rpb1), MF681505 (tef); DSM 105460 (dried culture on PDA, B700016493), GenBank accession number: MF681482 (ITS), MF681492 (LSU), MF681510 (rpb1), MF681504 (tef); DSM 105461, GenBank accession number: MF681481 (ITS), MF681490 (LSU), MF681509 (rpb1), MF681503 (tef); and CBS 141176.
In honour and memory of Prof Walter Gams for his outstanding works on the genera Monocillium and Niesslia.
Naturally occurring infected eggs often accommodating one subglobose, strongly pigmented, dark brownish microsclerotium.
Colonies slow-growing, at 20 °C on PDA reaching 10–12 mm diam. (7d), 19–22 mm (14 d), 25–32 (21 d); optimum temperature for growth 25 °C, 14–16 mm (7 d), 22–25 mm (14 d), 31–34 mm (21d); at 30 °C 10–11 mm (7d), 15–17 (14 d), 22–25 mm (21 d), no growth observed at 35 °C; optimum temperature for growth in other examined cultural media 25 °C, after 21 d reaching 31–32 mm diam. (CMA), 36–40 mm (MEA), 40–50 mm (OA), 32–40 mm (SNA); colonies on PDA finely wrinkled, slightly elevated centrally, first pale creamy, later centrally becoming dotted, greyish-brown to fuscous black due to formation of darkly pigmented microsclerotia, margins and reverse pale creamy. Vegetative hyphae hyaline, thin-walled, forming strands or coils, often with dictyochlamydospore-like structures, occasionally bearing setae with elongate, ellipsoid tips, variable in size. Chlamydospores or dictyochlamydospores mostly developing intercalary, filled with small guttules, gradually pigmented, turning brownish firstly at cell walls, interweaving to form microsclerotia. Cells of microsclerotia angular, pigmented, first pale olivaceous brown filled with guttules, later dark brown, forming a textura angularis in surface view. Guttules often absent in mature and strongly melanised sclerotial cells. Microsclerotia later covering the entire colony, developing sclerotioid masses, not changing colour in KOH. Phialides often separated from hyphae by a basal septum, thick-walled in the lower part, the wall thickening distinct at about 1/3 to 1/2 of the total length from the base, thin-walled from ca. midpoint extending to the tip, occasionally slightly inflated in the middle part, gradually tapering to the tip, 21–39 µm (28.7 ± 4.4) in length, 1.0–2.1 µm (1.4 ± 0.2) wide at the base (n = 90), solitary, arising directly from hyphae or hyphal rope, occasionally arising from hyphal coils surrounding several conidia. Conidiogenesis abundant, conidia hydrophilic, adhering in watery droplets, oblong, rarely clavate or ampulliform, one-celled, smooth-walled, 4.1–7.4 × 1.4–2.9 µm (4.9 ± 0.6 × 2.1 ± 0.3) (n = 250). Sexual morph not observed.
Monocillium gamsii infected cysts and eggs of H. filipjevi in vitro. Initial indications of infection were observed in healthy nematode cysts placed on the fungal colonies within 2–3 weeks (Fig.
In the slide cultures, fungal infection of eggs was initiated by individual hyphae directly penetrating the eggshell and body cuticle of developing juveniles (Fig.
Micrographs of Monocillium gamsii. A–C Hyphal growth by intercalary development of chlamydospore and dityochlamydospore-like structures filled with guttules D–H initiation of microsclerotia by interweaving or coiling of dictyochlamydospores, and growth to full size I Highly pigmented sclerotium at maturity displaying a textura angularis on surface view J–N Setae, phialides and conidia M–O Formation of phialides on coiling hyphae P Conidial heads, conidia cohering in wet heads Q, R SEM: Q Phialides from mycelium with conidial heads, arising from hyphae R coiling hyphae (arrows), and detail of phialides bearing conidia A–I from PDA 1/3 strength J–P from PDA; Q, R from OA. Scale bars: 30 µm (A, H, I, K, M, O, R); 20 µm (B–G, J, L, N); 200 µm (P), 50 µm (Q).
Cysts and eggs of Heterodera filipjevi infected by Monocillium gamsii exhibiting colonisation in vitro. A, B infected cysts rendered black-dotted due to fungal-colonised eggs containing microsclerotia C, D Eggs with mature sclerotia, extracted from symptomatic cysts E, F Individual hyphae penetrating eggshell (arrows indicate the individual hyphae; V indicates vacuole-like structures) G–M Fungal development inside the eggs G, H Earlier stages of infection in unembryonated eggs I, J Fungal development in the body cavity of developing juveniles where enlarged, thick-walled cells are formed and coalesce to initiate microsclerotia formation K–M Microsclerotia developing to full size and pigmentation N–P Pigmentation in microsclerotia from pale-olivaceous to darkly brown. Scale bars: 600 µm (A); 300 µm (B); 30 µm (C–O); 50 µm (P).
Pigmentation of microsclerotia occurred during fungal development from hyaline to olivaceous brown and later strongly brownish melanised cells (Fig.
The antagonistic potential of M. bulbillosum was also examined against H. filipjevi in vitro. Eggs of H. filipjevi were infected by M. bulbillosum in the course of 2–4 weeks. The infection symptoms were similar to the symptoms described for M. gamsii. Monocillium bulbillosum rendered cysts black dotted, containing eggs colonised with microsclerotia. In early stages of infection, eggs were entirely colonised with filamentous hyphae which later developed into microsclerotia with a textura angularis on the surface (Fig.
Cysts and eggs of Heterodera filipjevi infected by Monocillium bulbillosum in vitro. A Symptomatic cysts infected by M. bulbillosum B Infected egg showing early stage of fungal colonisation C–F Formation of microsclerotia in nematode eggs (arrow points at nematode’s stylet). Scale bars: 300 µm (A); 30 µm (B–F).
The results obtained from comparative morphological characteristics and molecular phylogenetic inference using four gene regions, suggested M. gamsii as a new species. Within the genus Monocillium, only M. bulbillosum, M. curvisetosum, M. indicum and M. ligusticum have been reported to form (micro-) sclerotia in culture, as is also the case in M. gamsii. In the (micro-) sclerotia forming group, M. gamsii can be separated from M. indicum by producing conidia cohering in watery heads instead of dry conidia. Monocillium curvisetosum produces dry and globose conidia, while M. gamsii produces oblong and watery conidia. The new taxon differs from M. ligusticum by having much shorter phialides: 21–39 µm vs (35–) 40–70 (–140) µm (
Hyaloseta nolinae (asexual morph: Monocillium nolinae) was included in this study according to a BLASTn search in GenBank, showing a high sequence similarity with the sequences of M. gamsii as query. In the phylogenetic analyses presented here it forms a highly supported monophyletic group with M. gamsii and M. bulbillosum (Fig.
It is intriguing that microsclerotia, which represent the main symptoms of fungal infection of nematode cysts and eggs in both M. bulbillosum and M. gamsii were readily reproduced in fungal pure cultures and were also formed in artificially infected nematode eggs. Apart from the essential role of conidia in fungal reproduction and dispersal, it seems that microsclerotia also play an important part in the developmental cycle of these fungi, at least with respect to those parts of their life cycle that could be assayed in vitro here and during which it interacts with nematodes. Monocillium gamsii was found in field-collected dried cysts in the semiarid Central Anatolian Plateau. In nature, fungal sclerotia are generally considered as resting structures by which the fungus may tolerate abiotic stresses like dessication, and can thus survive until favourable conditions return. Support for this hypothesis comes from the observation that we were able to isolate M. gamsii from field-collected cysts obtained by culturing the microslerotium-containing infected eggs that had been kept for more than one year in dry conditions either at 4 °C or at room temperature. Furthermore, nematode cysts are protective structures in which nematode eggs can survive for many years in soil in the absence of host plants or in adverse environments. By colonising the cyst contents, i.e. the mucilaginous matrix and the eggs, the egg-colonising fungi for example M. gamsii and M. bulbillosum, may thus benefit from this “specific” niche where they may have equivalent prolonged-survival conditions.
Our microscopic observations of the in vitro tests revealed that M. gamsii is capable of destructively and quickly parasiting the nematode eggs within the cysts first by penetrating the eggshell, followed by profilic formation of microsclerotia. We did not observe formation of any specific infecting structure like in the case of the recently described cyst and egg-parasitic fungus Ijuhya vitellina which developed appressoria (
This work was finacially supported by the Federal Ministery for Economic Cooporation and Development, Germany, Grant ID: W0267 GIZ/BMZ-Endophyte as Biocontrol. Additional support of the International Maize and Wheat Improvement Centre (CIMMYT) and the Julius Kühn-Institut are gratefully acknowledged. Special thanks go to the Fiat Panis Foundation, Ulm, Germany, for a “PhD completion stipend” to SA. We thank Dr Thomas Kühne for carefully reading the manuscript, and Anke Brisske-Rode and Katrin Balke for technical support in the molecular studies and culture maintaining.