New Reports of Melampsora Rust (Pucciniomycetes) on the Salix Retusa Complex in Balkan Countries
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(2020) 44 (1): 89-93 DOI: https://doi.org/10.2298/BOTSERB2001089S journal homepage: botanicaserbica.bio.bg.ac.rs Original Scientific Report New reports of Melampsora rust (Pucciniomycetes) on the Salix retusa complex in Balkan countries Miloš Stupar✳, Milica Ljaljević Grbić, Jelena Vukojević and Dmitar Lakušić University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Takovska 43, 11000 Belgrade, Serbia ✳ correspondence: [email protected] Keywords: ABSTRACT: plant pathogen, distribution, Melampsora epitea, known to cause rust on the complex of Salix retusa prostrate Melampsora epitea, basidiomycete, willows distributed in the subalpine and alpine belt of the mountains of Central snow willows Europe, is here documented for the first time in Montenegro and North Macedonia growing at six localities. It is not new for Serbia, but the records come from a newly reported host, namely Salix serpyllifolia. The pathogen’s distribution presumably is UDC: 528.28:582.681.81(234.42) wider than initially believed, and further surveys need to be conducted. Received: 09 January 2020 Revision accepted: 04 February 2020 During field investigation of the complex of Salix retusa were collected, placed in bags and transported to a labo- (family Salicaceae) on the Balkan Peninsula, six popula- ratory in the Department of Algology, Mycology and Li- tions with many individuals infected with rust fungus chenology of Faculty of Biology, University of Belgrade for were documented (Fig. 1). proper screening of symptoms, documented and used for The complex of Salix retusa includes S. retusa L. (s.str), further analyses by light and scanning microscopy. Symp- S. kitaibeliana Willd. and S. serpillifolia Scop., prostrate toms were detected both in the field and on herbarium willows distributed in the subalpine and alpine vegeta- specimens. tion belts of the mountains of Europe: the Pyrenees, Alps, Apennines, Dinarides, Scardians, Carpathians and Rila. Chorological data: The systematic positions and geographical distribution Specimens examined and pathogen identified: of these taxa are not fully resolved and are still disputed (Kosiński 2017, 2019). Although he states that S. retusa is Salix retusa s. str. common on the Balkan Peninsula in the Dinarid, Scardian North Macedonia: Mt. Jakupica, Solunska glava and Rila Mts., a number of prostrate willows with smaller N41.715351, E21.398691, 2221 m a.s.l., alpine-subni- leaves than those of typical S. retusa are found commonly val snow-bed vegetation (Salicetea herbaceae), marble, and have been classified as S. serpyllifolia. Accordingly, for leg. Lakušić D., Kuzmanović N., Janković I., 22.07.2016, prostrate willows with small spathulate leaves, we here use BEOU-46336. the name “S. serpyllifolia”. According to Pei (2005), willow Montenegro: Prokletije Mts., Maja Karanfili, Skrožnje, and poplar (family Salicacea) around the world are infect- N42.501556, E19.79057, 1985 m a.s.l., alpine-subnival ed with rust fungus of the genus Melampsora. snow-bed vegetation (Salicetea herbaceae), limestone, The conducted field study took place in the summer leg. Lakušić D., Kuzmanović N., Janković I., 29.07.2016, periods from 2016 to 2018. Herbarium material of infected BEOU-46512. S. retusa collected in the field were deposited in the BEOU Montenegro: Durmitor, Gornja Ališnica, N43.143605, herbarium, while leaf samples with visible rust symptoms E19.032165, 2050.2 m a.s.l., alpine calcareous grasslands, © 2020 Institute of Botany and Botanical Garden Jevremovac, Belgrade 90 | vol. 44 (1) Fig. 2. Powdery pustules corresponding to uredinia documented on infected Salix retusa leaves collected in the Međukomlje Mts., Mon- tenegro (magnification, 51.2×). “Salix serpyllifolia” Montenegro: Komovi Mts., Kom Vasojevićki, N42.691946, E19.670435, 2163 m a.s.l., alpine-subnival snow-bed veg- etation (Salicetea herbaceae), limestone, leg. Lakušić D., Kuzmanović N., Janković I., 11.07.2016, BEOU-46257. Symptoms observed in the field: Salix retusa s. str. Montenegro: Durmitor Mts., Bobotov kuk, Velika Previja, [UTM: CN47] N43.126501, E19.037171, 2350 m a.s.l., al- pine limestone grassland Festuco-Seslerietea, limestone, leg. Lakušić D., 14.08.2017, BEOU-46656. Montenegro: Mt. Maganik, Kurozeb (Petrov vrh) 1800- 2100 m a.s.l., heaths above Pinus heldreichii H. Christ belt, on limestone, Niketić M., Tomović G. 19.08. 2010, BEOU- 31409. “Salix serpyllifolia” Serbia (Kosovo): Šara Mts., Piribeg, N42.177782, E21.048534, alpine-subnival snow-bed vegetation (Salicetea herbaceae), limestone, leg. Lazarević M., Lazarević P. 13.08.2018 (field observation, photo documented Fig. 1). Fig. 1. Melampsora epitea rust symptoms. a) infected Salix retusa On both sides of the leaves, formation of yellow to orange from Montenegro, Durmitor, Ališnica (photo: D. Lakušić, 6.8.2018); indistinguishable spots corresponding to orange powdery b) infected “Salix serpyllifolia” from Montenegro, Komovi (photo: pustules (uredinia), scattered or rarely grouped and cover- D. Lakušić, 11.07.2016); c) infected “Salix serpyllifolia” from Serbia ing approximately 10 to 50% of the leaf surface, was docu- (Kosovo: Šara Mts., Piribeg) (photo: M. Lazarević, 13.08.2018). mented (Fig. 2). Pustules originating from infected leaf surfaces sam- Scabioso silenifoliae-Caricetum laevis, limestone, leg. pled at all investigated localities were gently removed Lakušić, D., 06.08.2018, (field observation, photo docu- with the aid of adhesive tape and by scraping, mounted mented - Fig. 1). with Lactophenol Cotton Blue and observed under a light M. Stupar et al.: Melampsora on the Salix retusa complex | 91 Fig. 3. Melampsora epitea s. str. - morphological characteristics of pathogen uredial stage documented on Salix retusa. a, c) uredinium; b) urediniospore with capitate paraphyses dyed with Lactophenol Cotton Blue; d) urediniospore with dense spines and thick cell walls docu- mented. a, b – light microscopy; c, d – SEM. microscope (Nikon Eclipse E200 equipped with a Bresser mum symmetry, such as a perfect circle. Urediniospores MikroCam PROHDMI camera). To observe and analyse with documented CIRC values in the case presented here fungal structures present on S. retusa leaves using SEM, ranged from 0.7 to 0.8, indicating more ovoid to ellipsoidal sample fragments were taken from leaf surface with vis- shape of the pathogen spore (14–24×11–20 μm Ø). The ible symptoms by means of adhesive carbon tape on alu- cell wall of urediniospores was approximately 1 μm thick, minum cylinders. SEM images were obtained at the Fac- uniformly echinulate with dense spines. Mean SD ranged ulty of Mining and Geology, University of Belgrade, using from 1.5 to 2 μm. Paraphyses were capitate, with a thick- a JEOL JSM–6610 LVSEM instrument (Tokyo, Japan). ened wall, placed intermixed or peripherally in the uredin- Samples (d=15nm, ρ=19.2g/cm3) were gold-coated with ia (30–61×9–26 μm Ø) (Fig. 3). Documented structures a Leica EMS CD005 sputter coater (Wetzlar, Germany). of rust fungus, urediniospores and paraphyses, and their Both types of microscopy, light and SEM, revealed in all morphological characters (shape, dimensions, CIRC, SD) tested samples the presence of numerous urediniospores from all leaf samples analysed microscopically showed and paraphyses emerging from amphigenous uredinia. no significant variation in morphology and size, thereby Further morphological analyses of documented structures suggesting that S. retusa specimens from all investigated of the uredial stage were conducted using ImageJ soft- populations suffer from the same pathogen. ware and included: characters of urediniospores (shape, Documented micromorphological features, as well as dimensions, cell wall thickness, circularity-CIRC, spine documented symptoms present on host plants identified distance-SD) and paraphyses (shape and dimensions). as S. retusa correnspond to the genus Melampsora (family The CIRC value was normalised and ranged from 0.0 to Melampsoraceae, order Pucciniales, class Pucciniomycetes, 1.0, where the value 1.0 represents an ideal case of maxi- division Basidiomycota). Species of the genus Melampsora 92 | vol. 44 (1) are biotrophic fungal pathogens of willows (Salix spp.) in REFERENCES the northern temperate zone (Bennet et al. 2011). Apart from M. amygdalinae Kleb, all Melampsora species that in- Bennett CM, Aime MC & Newcombe G. 2011. Molecular fect willows are heteroecious. Corresponding to the ploidy and pathogenic variation within Melampsora on Salix level of the fungus, Salix spp. are hosts for the dikaryotic in western North America reveals numerous cryptic stages of the fungus [uredinia (III) and telia (IV)], while species. Mycologia 103: 1004‒1018. the stages of spermogonia (0) and aecia (I) form on hosts Braun U. 1982. Die Rostpilze (Uredinales) der Deutschen Demokratischen Republik. Feddes Repertorium 93: in different lineages of seed plants, e.g., Abies, Euonymus, 213‒334. Larix, Ribes, Saxifraga, Tsuga, Viola and several genera of Damadi SM, Pei MH, Smith JA & Abbasi M. 2011. A new Orchidaceae (Pei 2005). 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