Phoma-Like Fungi in the Mycological Herbarium of All-Russian Instituteof Plant Protection (LEP)
Mycological herbaria constitute the basic and essential source of fungal biodiversity information. Mycological Herbarium of All-Russian Institute of Plant Protection (LEP) is the major collection of phytopathogenic fungi in Russia. A long-term storage of herbarium specimens results in fatal distruction of fungal morphological structures. Thus phytopathogenic fungi in such specimens could not be used for morphological studies. But these specimens content several amounts of fungal DNA and thus could be used for molecular phylogenetic studies, especially for PCR. Moreover herbarium specimens are also a finite resource and molecular methods require small quantity of herbarium material. Phoma-like fungi is a huge taxonomically complex and controversial group of anamorph ascomycetes, that are widely distributed in different ecological niches. Significant part of these fungi are phytopathogens, that causes diseases of important crops. Sum total in Mycological Herbarium LEP there are 1328 specimens of plants, infected by fungi, that were identified as Phoma-like species. We have studied 28 specimens of phomoid fungi from plants of family Asteraceae. These specimens were collected from Russia and the territory of former USSR countries and ranged in collection date from 1895 to 1960. The age of the oldest specimen LEP 129311 is 124 years. Morphological features of sporebearing structures survived for examination (pycnidia, conidia, fruiting bodies, ascospores, chlamydospores etc) were estimate for all 28 specimens. Whereas fungal DNA for subsequent sequencing were extracted from 18 specimens. Taxonomically significant internal transcribed spacer (ITS loci) was amplified and sequenced with primers ITS1F/ITS4. Additionally the specific primers (Did2F/Did2R) were constructed to amplify ITS loci of Phoma-like fungi in the family Didymellaceae. It was shown, that in majority of cases long-term storage of plants, infected by phomoid fungi results in destruction of sporebearing structures. Thus we had not enough data on morphological features to correct identifying of these fungi. Molecular features were more explicit and allowed us to determine Phoma-like fungi in 9 herbarium specimens to genera level as well in another 9 – to species. Among them nucleotide sequences of ITS loci of four specimens were obtained by PCR with primers ITS1F/ITS4, whereas fourteen sequences were derived by PCR with new primers Did2F/Did2R, which were designed by authors. As a result in Mycological Herbarium LEP on infected plants of family Asteraceae are stored five genera of Phoma-like fungi: Ascochyta, Boeremia, Didymella, Epicoccum, Stagonosporopsis and four species: Ascochyta rabiei, Boeremia exigua, Didymella pomorum (Epicoccum nigrum. Primers Did2F/Did2R could be recommended to amplify ITS loci in DNA of phomoid fungi from Didymellaceae and to phylogenetic studies in this group of fungi, especially in herbarium specimens of Phoma-like species. Molecular features were in corcondance with morphological characters only for 124 years old specimen Boeremia exigua. To date this specimen is the oldest herbarium sample of Phoma-like fungi, which DNA was sequenced. Previously the oldest sequenced herbarium specimen of phomoid fungi was 97 years old Didymella pinodes.