Monocillium gamsii Ashrafi & W. Maier

Ashrafi, Samad, Stadler, Marc, Dababat, Abdelfattah A., Richert-Poeggeler, Katja R., Finckh, Maria R. & Maier, Wolfgang, 2017, Monocilliumgamsii sp. nov. and Monocilliumbulbillosum: two nematode-associated fungi parasitising the eggs of Heteroderafilipjevi, MycoKeys 27, pp. 21-38 : 28-30

Monocillium gamsii Ashrafi & W. Maier sp. nov. Figs 1H, I, 3

Holotype.

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 DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, GenBank accession numbers: ITS: MF681485; LSU: MF681496; rpb1: MF681512; tef: MF681506.

Additional material examined.

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.

Etymology.

In honour and memory of Prof Walter Gams for his outstanding works on the genera Monocillium and Niesslia .

Diagnosis.

Naturally occurring infected eggs often accommodating one subglobose, strongly pigmented, dark brownish microsclerotium.

Description.

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.

Development of M. gamsii in nematode eggs in vitro

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. 4A, B). The fungus rendered the homogenously brown healthy looking cysts black-dotted, bearing a strong resemblance to the naturally infected cysts collected from fields. By dissecting the symptomatic cysts, nematode eggs were found to be colonised with darkly pigmented spherical to subglobous microsclerotia formed inside the body cavity of the developing juveniles (Fig. 4C). Similar to some naturally infected eggs, sclerotioid masses were also found in some artificially infected samples colonising almost the entire egg (Fig. 4D).

In the slide cultures, fungal infection of eggs was initiated by individual hyphae directly penetrating the eggshell and body cuticle of developing juveniles (Fig. 4E, F). Following penetration, filamentous hyphae entirely colonised the unembryonated eggs (Fig. 4G, H) or the body cavity of the developing juveniles (Fig. 4I, J), enlarged (Fig. 4 H–J), occasionally inflated, forming thick-walled, finely pigmented, and guttules-filled cells (Fig. 3J), which eventually coalesced to form discrete microsclerotia with a textura angularis appearance (Fig. 4 K–M). Infection studies revealed that such microsclerotia could be formed 7-10 d after the incubation of nematode eggs with the fungus.

Pigmentation of microsclerotia occurred during fungal development from hyaline to olivaceous brown and later strongly brownish melanised cells (Fig. 4 N–P). Microsclerotia developing inside the artificially infected eggs displayed a textura angularis and were indistinguishable from those found in the naturally infected samples. At the early stages of development, microsclerotial cells were often filled with guttules (oil-like bodies), which were not observed in the mature microsclerotia. At the early stages of fungal infection (up to 10 d after inoculation), some vacuole-like structures were observed inside the eggs along the body cavity of developing juveniles (cf. Fig. 4E, F) with a glistening reflexive appearance, which were not observed at later stages of development, or in the field collected samples containing mature sclerotia.

Parasitism of M. bulbillosum towards H. filipjevi

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. 5 A–F).