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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).
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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.
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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).
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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).
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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).