Tuberculina-Helicobasidium: Host Specificity of the Tuberculina-Stage Reveals Unexpected Diversity Within the Group1
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Mycologia, 96(6), 2004, pp. 1316±1329. q 2004 by The Mycological Society of America, Lawrence, KS 66044-8897 Tuberculina-Helicobasidium: Host speci®city of the Tuberculina-stage reveals unexpected diversity within the group1 Matthias Lutz2 erculina species become visible during sporulation as Robert Bauer hemispherical, lilac to violet mycelia that burst Dominik Begerow through the plant surfaces generally close to rust Franz Oberwinkler sori, releasing a powdery mass of conidia. Tuberculina UniversitaÈt TuÈbingen, Botanisches Institut, Lehrstuhl recently was demonstrated phylogenetically to be Spezielle Botanik und Mykologie, Auf der Morgenstelle closely related to its associates, the rusts, some species 1, D-72076 TuÈbingen, Germany representing the asexual life stage of species of the plant-parasitic genus Helicobasidium (Lutz et al 2004a, b). In contrast to the mycoparasitic genus Tub- Abstract: Tuberculina species are mitosporic para- erculina, Helicobasidium species are serious plant sites of rust fungi. It was demonstrated recently that pathogens causing the economically important violet Tuberculina represents the asexual life stage of the root rot. They are unspeci®c in their choice of hosts plant-parasitic genus Helicobasidium. Here we reveal (Buddin and Wake®eld 1924, Hering 1962) and re- the host speci®cities of Tuberculina and Helicobasi- ported as parasites of more than 120 plant species dium species on rust fungal hosts by means of infec- tion experiments and molecular analyses. We inocu- representing more than 50 families of the spermato- lated species of the rust genera Chrysomyxa, Coleos- phytes and ferns (Itoà 1949, Viennot-Bourgin 1949). porium, Cronartium, Gymnosporangium, Puccinia, Extensive research has been conducted on the biol- Tranzschelia and Uromyces with conidia and with ba- ogy of and the combat against Helicobasidium, but sidiospores of Helicobasidium longisporum and H. pur- the diversity of the genus remains poorly understood pureum and with conidia of Tuberculina maxima, T. (Aimi et al 2003a, b; Uetake et al 2002) and hypoth- persicina and T. sbrozzii. In addition we analyzed base eses on the life cycle con¯ict (Aimi et al 2003b, Lutz sequences from the nuclear ITS region of 51 Tuber- et al 2004a). culina and Helicobasidium specimens collected in the Although the strong inhibitory effect of Tuberculi- ®eld together with the sequences from the Tubercu- na on rust spore production has resulted in extensive lina infections obtained by infection experiments. research dealing with Tuberculina as an agent in bi- The resulting data show that at least six monophyletic ological rust control (for review see Wicker 1981), lineages are within the Tuberculina/Helicobasidium- the relationship among Tuberculina, rusts and plants group that can be unambiguously distinguished by was the subject of controversy. Tuberculina species combining molecular and morphological characters have been interpreted as mycoparasites speci®c to and by speci®c host spectra of the Tuberculina-stage. rusts (Kirulis 1940, Tubeuf 1901, Tulasne 1854, Zam- This diversity opens up new vistas on the evolution bettakis et al 1985), as nonspeci®c parasites on sev- of this exceptional mycoparasitic-phytoparasitic fun- eral substrates (Petrak 1956, Schroeter 1889) or even gal group. as specialized parasites on rust-infected plant tissues Key words: Helicobasidium, host speci®city, infec- (Biraghi 1940; Hulea 1939; Marchal 1902; Wicker tion experiments, ITS, Tuberculina, molecular phy- and Woo 1969, 1973). Thus plant parasites and par- logeny, mycoparasitism, Urediniomycetes asites of non-rust fungi were included in the genus adopting a concept based on morphological charac- ters (Ellis 1893, Patouillard and Gaillard 1888). Oth- INTRODUCTION er authors used a species concept based on host spec- i®cities, distinguishing Tuberculina species on differ- Tuberculina species occur all over the world, living in ent rust hosts (Spegazzini 1880, 1884) or even plant association with more than 150 rust species from at hosts (Gobi 1885). However, Barkai-Golan (1959) least 15 genera. Growing between the hyphae of their demonstrated that Tuberculina could not infect rust- rust fungal hosts in leaves and stems of plants, Tub- free plants and we recently substantiated the myco- parasitic nature of Tuberculina persicina on rust fun- 1 Part 215 in the series Studies in Heterobasidiomycetes from the Bo- tanical Institute, University of TuÈbingen. gal hosts by ultrastructural observations that reveal a 2 Corresponding author. E-mail: [email protected] speci®c and morphologically uncommon cellular in- 1316 LUTZ ET AL:HOST SPECIFICITY OF TUBERCULINA SPECIES 1317 teraction via large fusion pores with a direct cyto- The rusts for inoculation were obtained in four different plasm-cytoplasm connection and interspeci®c trans- ways. Accordingly, four experimental settings were estab- fer of Tuberculina-nuclei to rust hyphae (Bauer et al lished. (i) Rust-infected plants growing in nature were used 2004, Lutz et al 2004b). (Chrysomyxa rhododendri on Picea abies, Coleosporium tussi- Even though several authors described Tuberculina laginis on Pinus sylvestris, Cronartium ribicola on Pinus ar- istata, Gymnosporangium cornutum on Sorbus aucuparia, species based on the occurrence on different hosts Puccinia sessilis on Allium ursinum, and Tranzschelia pruni- (e.g., Spegazzini 1880, 1884) and the vast majority of spinosae on Anemone ranunculoides). We chose places where Tuberculina maxima specimens are described from we were not able to detect any Tuberculina infection in the Cronartium- and Gymnosporangium- hosts, no exper- previous growth season. We divided the places in isolated imental proof for host speci®city of any Tuberculina areas where we inoculated the rust infected plants and areas species was provided. On the contrary, Barkai-Golan without inoculation for control. (ii) We inoculated pear (1959) demonstrated that Tuberculina persicina spec- trees growing in nature with Gymnosporangium sabinae to imens from one rust host are capable of infecting obtain heavy rust infections. To this end we attached freshly several other rust species. collected branches of Juniperus sp., which showed intensive In this report, we present both results of infection infections with germinating and sporulating teliospores of experiments and molecular data that reveal the host Gymnosporangium sabinae to several distant trees where we were not able to detect any Tuberculina infection in the speci®cities of several Tuberculina and Helicobasidium previous vegetation period. One rust-inoculated, isolated lineages, respectively, on their rust fungal hosts. tree at each location served as control and was not inocu- These ®ndings yield new insights in the Tuberculina± lated with the hyperparasite. (iii) We transferred Euphorbia Helicobasidium life cycle and in the diversity of the cyparissias plants that systemically were infected by Uromyces group. pisi s.l., young shoots of Vinca major infected with Puccinia vincae and little Pinus sylvestris trees infected with Coleos- porium tussilaginis from nature to our greenhouse. The MATERIALS AND METHODS plants inoculated with the hyperparasite and control plants Infection experiments.ÐThe aim of our infection experi- were cultivated in different houses. After inoculation the ments was to test whether differences among Tuberculina single plants were incubated 3 d under transparent plastic maxima, T. persicina, T. sbrozzii, Helicobasidium longisporum bags to provide high relative humidity. (iv) We cultivated and H. purpureum exist in the choice of their rust hosts. In Taraxacum of®cinale agg. in our greenhouse and infected addition we wanted to test whether both basidiospores of the plants with Puccinia silvatica using the wire net method Helicobasidium and conidia produced in Tuberculina and (Kakishima et al 1999). Freshly collected leaves of Carex Helicobasidium cultures and from Tuberculina sporodochia, brizoides L. harboring sori of teliospores of the rust, which respectively, are capable of causing Tuberculina infections were incubated at room temperature 3 d in plastic bags at of rust hosts. high humidity, served as inoculum. Inoculation with the hy- We have gathered data on inoculated rust species, the perparasite and control were handled as in (iii). species used as inocula, the results of the different inocu- Inoculated plants and the control then were observed lations, the respective kind of inoculum and the respective until the respective rusts completed their life cycles on the experimental approach of the different inoculations (TABLE respective host plant. If sporodochia were detected, the I). Tuberculina infection was veri®ed microscopically and a The experimental approach depended on the kind of part of the material was conserved for further examina- inoculum applied and on the way the rusts for inoculation tions. were obtained. Field observations.ÐWe checked several rust species growing We used two kinds of inocula: (i) We collected intensively in an area of 200 square m around several sporulating bas- sporulating basidiocarps of Helicobasidium longisporum and idiocarps of Helicobasidium purpureum for Tuberculina in- H. purpureum in the ®eld, tore the fresh basidiocarps into fections during 2 y, and we introduced some additional pieces of about 4 square cm and tagged the pieces to pear plant species harboring young spermogonia of different trees at the same places where we had ®xed the rust inocula rust species at that