Isolation and characterisation of novel nematophagous fungi from eggs of the cereal cyst nematode Heterodera filipjevi and identifying their secondary metabolites with nematicidal activity

Parasitism caused by cereal cyst nematodes (CCNs) is a major limiting biotic factor in cereal cropping systems. CCNs attack cereal crops and can lead to significant yield reductions. Field observations in experimental wheat fields of the International Maize and Wheat Improvement Centre (CIMMYT) in Turkey had revealed a sharp decline in nematode populations of the cereal cyst nematode Heterodera filipjevi. Microscopic studies of the cyst samples obtained from these fields regularly demonstrated the presence of fungi colonising nematode cysts and eggs. It was hypothesised that these or other fungi could be involved in the reduction of the population size of H. filipjevi. The present study therefore aimed at (i) isolating fungi from cysts of H. filipjevi; (ii) specifically isolating symptom-causing fungi from single eggs by establishing a specific isolation technique; (iii) classifying the fungal isolates of interest by morphological studies and multi-locus molecular phylogenetic analyses; (iv) studying the nematode-fungus interaction microscopically; (v) fulfilling Koch’s postulates for the fungi isolated from the nematodes; (vi) isolating and identifying secondary metabolites produced by the fungal isolates of interest. Two screening approaches were applied to isolate a maximum number of fungi associated with nematode eggs: In a general screening, field-collected cyst samples and their contents were cultured without prior selection. In a focused screening approach, only those nematode eggs exhibiting symptoms of fungal infection were individually cultured. This approach resulted in finding six new fungal species, three of which are described here: Ijuhya vitellina sp. nov., Monocillium gamsii sp. nov., and Polyphilus sieberi nom. prov. Ijuhya vitellina, M. gamsii belong to the families of Bionectriaceae and Niessliaceae, respectively and are the first reports of nematophagous fungi within the genera Ijuhya and Monocillium. The fungal genus Polyphilus nom. prov. was proposed to accommodate two species (P. sieberi nom. prov. and P. frankenii nom. prov.), of which only P. sieberi was isolated from nematode eggs. This is the first case where a dark septate endophyte (DSE) was shown to parasitise nematode eggs. In addition to the species described here, three hitherto undescribed pleosporalean species were isolated, two of which were also DSEs. All the new fungal species could be successfully re-isolated from the eggs of artificially infected nematode cysts in vitro, and Koch’s postulates were thus fulfilled. Additionally, their antagonistic interactions with nematode eggs were documented in detail using light microscopy: After penetrating the eggshell and the body cuticle of developing juveniles inside the eggs, hyphae of I. vitellina and M. gamsii developed and formed microsclerotia. These microsclerotia were rich in oil-like droplets and germinated on artificial media. They are interpreted as long-term survival structures. Parasitism of nematode eggs caused by P. sieberi as a DSE suggests a multifunctional lifestyle that could potentially benefit the plant host by killing nematodes as well as by transferring nutrients from colonised nematodes. The isolated fungi were screened for secondary metabolites, and their chemical structures were elucidated. Chaetoglobosin A and its derivative 19-O-acetylchaetoglobosin A were isolated from I. vitellina, which is a new source for this compound with broad bioactivity that also showed inhibiting activity against the second-stage juvenile of H. filipjevi. These compounds were not present in the plant-associated species of the genus Ijuhya investigated here. From a so-far undescribed nematode-egg parasitic pleosporalean species several novel compounds were identified. Two of these are cyclodepsipeptides, a class of compounds known for their anthelmintic effects. Taken together, these results suggest that nematode-antagonistic fungi represent a promising source for finding new natural compounds with nematicidal activity. The discovery of several novel fungal species is intriguing given that cyst nematodes have been intensively screened for nematophagous fungi since the pioneering studies of Julius Kühn for over 150 years. The new findings became possible by applying a new isolation technique that can be used in future to discover even more fungi from other plant parasitic nematodes or in other geographic regions.