Mycol. Res. 99 (10): 1191 1194 (1995) 1191 Prilllcd ill Creal Brilaill Leaf blotch of lime associated with Asteromella tiliae comb. nov. and the latter's connection to Didymosphaeria petrakiana H. BUTIN AND R. KEHR Institut fur P{/anzenschutz im Forst der Biologischen Bundesanslalt fur Land- und Forstwirtschaft, Messeweg 11/12, D-38104 Braunschweig, Germany Asteromella tiUae is described and its connection to the ascomycete Didymosphaeria petrakiana demonstrated. The fungus is associated with a leaf blotch symptom of lime trees to which the questionable name' Asteroma tiliae' was applied previously. In the past, a leaf blotch disease of several lime species (Tilia spp.) was identified as being caused by 'Asteroma tiliae F. Rudolphi'. Typically, symptoms consist of blotches with black branch-like veining which result in premature yellowing of the affected leaves. Taxonomically, the name' Asteroma tiliae' is an unusual combination, since Rudolphi (1829), who first established it, neither characterized fruit bodies nor spores. He limited his description solely to the symptoms and deduced from similar diseases that the causal agent must belong to Asteroma. Such a fonn, however, was subsequently never found. Diedicke (1911) remarked 'Fruchtkorper und Sporen unbekannt' (fruit bodies and spores not observed). Nevertheless, the name Asteroma tiliae became established in phytopathological and floristic literature (Diedicke, 1915; Viennot-Bourgin, 1949; Lanier et aI., 1968; Hepting, 1971). Because of difficulties in characterizing the fungus, Asteroma tiliae was eventually treated together with the more recently described Asteroma vagans Desm. as a collective species (Saccardo, 1884). Grove (1935) also treated A. tiliae and A. vagans collectively, but called the latter' a useless collective name'. He went on to state: 'A. tiliae Rud. differs in its dendroid, not round, spots and its fibrils covered with greyish arachnoid threads'. The most precise assessment of the problem was given recently by Sinclair, Lyon & Johnson (1989), who stated: 'Until the disease cycle is studied, the reason for the late-season appearance of symptoms will be unknown'. This study attempted to find the casual agent of the conspicuous symptoms on lime leaves and detennine its life cycle in order to substantiate or reject the 'Asteroma ti/iae'hypothesis. immediately, the rest were placed in small cloth sacks and left on the ground under deciduous trees in the vicinity of Braunschweig, Gennany. Leaf parts affected at this time were marked prior to storage using a waterproof pen (Edding paint marker 751) so that they could be found after several months. At intervals of 14 d, several leaves were taken into the laboratory and examined using a dissecting microscope in order to study the development of fungal fruit bodies. These were removed from the leaf tissue and either hand-sectioned with a razor blade and examined in water or fixed in FAA (Gerlach, 1969) and subsequently embedded in glycolmethacrylate (Kulzer, Gennany). Sections from embedded material were prepared on a rotation microtome (Microm HM 350) and in some cases stained with a I % aqueous solution of thionine. The sections were examined and photographed using a Leitz Aristoplan microscope with a Wild MPS 48 photomicrographic system. Tissue was also removed from dried material with fruit bodies, stored in a moist chamber until fully hydrated and then treated as above. In addition to the leaf material gathered by the authors, dried samples of' Asteroma tiliae' from the Museum of Natural History in Vienna were examined. Unfortunately, exsiccata gathered by Rudolphi could not be inspected since they were destroyed during the Second World War. For cultural studies, ascospores or complete anamorphic fruit bodies were placed on 2 % malt agar and incubated at 20°C. Fruit bodies produced in culture were also studied using the above methods. RESULTS Disease symptoms (Fig. I) MATERIALS AND METHODS Fresh leaf material of Tilia platyphyllos Scop. showing the typical symptoms of 'Asteroma tiliae' was gathered on 10 Sep. 1993 in Amlach, Austria. Some of the leaves were dried The first symptoms of leaf blotch disease appeared in late summer on lime (Tilia spp.) and were comprised of black irregularly shaped patterns several mm across on the upper side of otherwise green leaves. Subsequently, the black veinlike patterns ramified and enlarged, leading to increasingly 1192 H. Butin and R. Kehr 4 \\°0 \J (J Figs 1-9. Didymosphaeria pefrakiana. Fig. 1. Infected leaf of Tilia platyphy/los with various stages of symptoms (bar, 3 cm). Fig. 2. Conidioma of the anamorph-stage Asteromella tiliae, cross-section (bar, 30 11m). Fig. 3. Conidiophores and conidiogenous cells with conidia (bar, 10 1Jffi). Fig. 4. Conidia (bar, 5 1Jffi). Fig. 5. Pycnidium of Asterome/la tiliae in leaf tissue of Tilia platyphyllos, cross-section (bar, 30 11m). Fig. 6. Ascoma of Didymosphaeria petrakiana, cross-section (bar, 60 11m). Fig. 7. Upper side of a moist Tilia platyphyllos leaf with subcuticular hyphae on discolored fibrils (bar,S mm). Fig. 8. Colony on malt agar, 4-wk-old (bar, 20 mm). Fig. 9. Segment of 4-wk-old colony on malt agar with pycnidia of the anamorph stage (bar, 1 mm). Leaf blotch of lime finer branching. Eventually, they extended over the whole leaf surface. Rudolphi (1829) described the branching as follows: , ... fibrillis ramosissimis, divaricatis, longe radiatis, catenulatim constrictis ... '. Ultimately, the blotches became dark brown from the centre outward; on the underside of the leaf, they appeared brown to olive. At this stage, the remaining leaf area became yellow and the affected leaves were shed prematurely. The black, tree-like fibrils were caused by brown discoloration of the palisade and sponge parenchyma preceded by subcuticular growth of hyphaI bundles with a whitish appearance on the upper surface of the leaves. These appeared to determine the shape of the patterns. In addition to these hyphal bundles, which could easily be observed through a dissecting microscope (Fig. 7), hyphae growing mainly intercellularly were also found in discoloured leaf tissue. Our observations of lime trees and exsiccata show that in Europe Tilia cordata and T. pIatyphylIos are both affected by this disease, but that the frequency varies geographically. For North America, Sinclair et aI. (1989) name indigenous Tilia species as hosts. We found two different types of fungal fruit bodies at different times of year: from the end of August till midNovember, the anamorph was observed, while the teleomorph was found from March to May. During the periodical examination of leaves stored in the open, the fruit bodies of other fungi were seldom observed on parts on which either one of these forms sporulated. An exception was the ascomycete (Sphaeriales) Phaeosphaeria vagans (Niessl) O. E. Erikss., which could in some cases be found on older, decomposing leaf material from May onward; it is known almost exclusively from grasses (Eriksson, 1967). The frequent absence of other fungi on leaf tissue colonized by Asteromella tiIiae suggests that it has a high degree of antibiotic or antagonistic activity, a view reinforced by observation of its cultural behaviour, when, for instance, isolates were contaminated by other fungi. In addition, the leaf parts colonized by the fungus showed a much lesser degree of decomposition compared to neighbouring leaf tissues, on which other fungi such as Discosia artocreas (Tode) Fr., Mycosphaerella punctiformis (Pers.) Starback and Ophiognomonia meIanostyIa (OC.:Fr.) SacCo were present. Asteromella tiliae (Rud.) Butin & Kehr comb. nov. (Figs. 2-5) Status anamorphosus Conidiomata pycnidia, hypophylla, subglobosa vel plus minusque depressa, pallido-brunneola, intraepidermalia, 60-120 ｾ diam., irregulariler dispersa, in conlextu foliorum innata. Parte superiore semiorbiculata recava extrinseca, apicaliler ostiolo papilliformi praedita. Paries 10-20 ｾ crassus; pars inferior ceUulis pseudoparenchymaticis, fere achromaticis, tenuilunicatis, 4-6 !Jm composita; pars superior exterior cellulis pallido-brunneis, ovalibus, crassitunicatis; pars interior cellulis hyalinis. applanatis et tenuibus formata. Paries interior conidiophoris unicellularibus, brevibus et cellulis conidiogenis, ampulliformibus, 4-6 x 3'5 Ilm tunicatus. Conidia hyalina, unicellulata, bacilliformia vel ellipsoidea, rede vel plus minusque curvata, 4-5 x 1'5-2 !Jm. In foliis decoloribus Ti/iae piatyphyllos Scop., Amlach-OberdrautaL Austria, 10 Sep. 1993, leg. H. Butin, neotypus IMI 362854, cultura viva CBS 265.94. 1193 Conidiomata pycnidiaL semisphericaL depressed, 60 to 120 ｾ ｭ across, irregularly distributed intraepidermally on the underside of leaves. Upper, light brown part of the fruit body curved outwards semispherically when hydrated, covered only by the epidermis, through which an ostiolum protrudes slightly (Figs 2, 5). Pycnidial wall 10-20 ｾ thick lower part composed of almost hyaline, pseudoparenchymatous, thinwalled cells 4--6 ｾ across. Upper part of wall composed of light brown, thick-walled oval outer cells and thinner-walled hyaline, compressed inner cells. Inner wall of the pycnidial cavity almost completely lined by short, I-celled hyaline conidiophores and ampulliform, 4-6 x 3-5 ｾ ｭ large conidiogenous cells which at their tips enteroblastically produce conidia (Fig. 3). Conidia non-germinating, hyaline, one-celled, rod shaped to ellipsoidal, straight or slightly curved, 4-5 x 1'5-2 ｾｭＬ apparently serving as spermatia (Fig. 4). Neotype on fading leaves of Tilia pIatyphyIIos Scop., Amlach/Oberdrautal, Austria, 10 Sep. 1993, leg. H. Butin, Neotype IMI 362854, Culture CBS 265.94. Specimen examined: Herb. W No. 120 (sub Asteroma tiliae Rud.), on Ti/ia cordata, 12 Sept. 1935, Riga/Latvia, leg. J. Smarods. Herb. W No. 4989 (sub Asteroma tiliae Rud.), on Tilia platyphyllos, Aug. 1940, WeiBkirchen (presently Czech Republic), leg. F. Petrak. Herb. W No. 15431 (sub Asterama tiliae Rud.), on Tilia cordata 30 Aug. 1910, Tambov/Russia, leg. J. Schizagewski. Herb. W No. 16970 (sub Asteroma tiliae Rud.), on Tilia platyphylIos, Sonntagsberg/Austria, leg. F. Petrak. On the basis of the morphological charaderistics of the anamorph, the fungus must be placed in Asteromella Pass. & Thurn. (Sutton, 1980). Although it has conspicuously lightcoloured fruit bodies, the typical structure of the conidiogenous apparatus and the small, rod-shaped conidia, which possibly serve as spermatia, warrant its placement in the formgenus AsteromeIIa. Cultural studies (Figs 8, 9) Colonies on 2 % malt agar at 20° attaining 20 mm diam. after 8 d and 55 mm after 28 d; after ca 8 d, the first pycnidial strudures are formed as compact hyphaI bodies. Mature pycnidia present in 3-wk-old cultures, 150-220 ｾ ｭ diam. rounded, covered with numerous white hyphae (Fig. 9), with a more or less conspicuous ostiolum, especially following staining with cotton blue, sometimes in small agglomerations. Conidiomata later becoming light grey to light tan-coloured. large, pseudoConidiomal wall composed of hyaline, 5-8 ｾ ｭ parenchymatous cells, inner conidiomal wall lined by ampulliform conidiogenous cells. Conidia hyaline, one-celled, rodshaped, 5--6 x 1·5-2 ｾ Ｎ After ca 6 wk in diffuse daylight, some parts of the colonies turn light grey to light tan in colour. Didymosphaeria petrakiana Sacco (Fig. 6) Status teleomorphosus Beginning in January, an ascomycete developed in the same leaf areas that formerly yielded the conidiomata of the AsteromeIla-anamorph, and in March the first mature fruit bodies with ascospores capable of germination were present. H. Butin and R. Kehr The fungus could be determined according to Saccardo (1914) and Scheinpflug (1958) by the following characteristics: Ascoma irregularly immersed in leaf tissue, depressed to spherical, distending the upper and lower leaf surface, grey to brown, 110-160 I-lm diam., with a cylindrical, slightly protruding ostiolum. Asci club-shaped to cylindrical, containing eight ascospores, upper part biseriate, lower part uniseriate. Ascospores 13-16 x 5-6 I-lm, medianly or almost medianly one septate, strongly constricted at the septum, elongate-fusiform, olive-brown when mature. Paraphysoids numerous, septate but not branched, 3-5 I-lm diam. In the laboratory, ascospores were transferred onto malt agar and incubated. The resulting white colonies produced white, pseudoparenchymatous pycnidia after 14 d which were in complete agreement with Asteromella tiliae. Therefore, it can be safely stated that both isolates belong to the same fungus, Asteromella tiliae being the spermatial state of Didymosphaeria 1194 In respect to the teleomorph, earlier authors were more successful, although they were not able to establish firm proof. Petrak (1916) concluded from his studies that the symptom 'Asteroma tiliae Rud.' must be a sterile, premature form of Didymosphaeria petrakiana. Unfortunately, his remark was subsequently ignored. The present study should serve to assign conclusively the leaf blotch syndrome of lime to its true casual fungus Didymosphaeria petrakiana and its anamorph Asteromella tiliae comb. nov. In respect to the taxonomical placement of the teleomorph, we have been informed that there may be forthcoming changes (A. Aptroot, Baam, pers. comm.). We are grateful to Dr H. A. van der Aa, Baam, for critical review of the manuscript. Thanks also to Angelika Krischbin (Hann. Munden) for preparing the illustration outline and to the Director of the Museum of Natural History, Vienna. petrakiana. DISCUSSION The discovery of the anamorph and the elucidation of the disease cycle make it possible to correct the previously used name Asteroma tiliae Rud. for this leaf blotch disease of lime. The fibrillous symptoms observed on lime leaves by Rudolphi (1829) are constantly associated with the presence of fungal fruit bodies. On the basis of morphology, these conidiomata do not belong to Asteroma, as suspected by Rudolphi, but instead to the form-genus Asteromella (Sutton, 1980). In appreciation of the name introduced by Rudolphi, the new combination should, therefore, be Asteromella tiliae (Rud.) Butin & Kehr. It is astonishing that the mycologists who studied the 'Asteroma tiliae' -symptoms did not notice the presence of fungal fruit bodies. Instead of an acervular Asteroma, we found the pycnidial form presented here as Asteromella tiliae on several older exsiccata (Nos. 120, 4989, 15431, 16970 of the Museum of Natural History, Vienna). The failure to find the fruit bodies earlier may be due to the fact that they are very inconspicuous. When dry, they do not protrude outwards and, due to their light brown colour, they blend in with the necrotic leaf tissue. In addition, the majority of pycnidia are produced after September, when the leaves do not show clearly the symptoms or have already been shed. 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