Biologia 63/1: 44—49, 2008 Section Botany DOI: 10.2478/s11756-008-0003-5 Leaf spot disease on Tilia cordata caused by the fungus Cercospora microsora Slávka Bernadovičová & Helena Ivanová Institute of Forest Ecology SAS Zvolen, Branch of Woody Plants Biology, Akademická 2, SK-94901 Nitra, Slovakia; e-mail: slavka.bernadovicova@sav.savzv.sk Abstract: Increased incidence of leaf spots on many tree species, up to the presence of peripheral importance only, including linden trees was noticed recently. First massive and continuous occurence of the fungus Cercospora microsora Sacc. [teleomorph Mycosphaerella millegrana (Cook.) Schröet., Mycosphaerella microsora Syd.], causal agent of anthracnose on linden trees (Tilia cordata Mill.) grown in urban plantings in Slovakia was reported. Along with this, certain of the important growth characteristics of this fungus were studied under laboratory conditions. To specify Cercospora biology mycelial growth of C. microsora in pure hyphal cultures was observed in relation to medium and locality. One-way ANOVA has confirmed a statistically significant influence of both factors, culture medium and locality on growth rate values of C. microsora. The effect of these factors has not proved unambiguously in all cases. In the case of one locality (Nitra), the significant influence of used media has not been proved (P > 0.05). PDAg showed generally as the most suitable medium, inducing the most intensive growth in three localities (41.06 mm/week on average). Comparing three localities, the effect of this factor is not so unambiguous. Growth rate values from the localities Bratislava and Pribeta indicate unsuitability of medium A for the fast radial growth. A Tukey test separately conducted for the factors medium and the locality revealed the significant combinations of means (P ≤ 0.05). Key words: Mycosphaerella millegrana; Tilia sp.; growth rate; leaf blight Abbreviations: GR, growth rate (mm/week); PDAg, potato-dextrose agar enriched with bromcresol green; MA, malt agar; A, water agar Introduction Tilia is a genus of over 20 species of deciduous trees occurring in woodland in Europe, Asia and North America. Tilia species in Slovakia are frequently planted as ornamental trees in urban plantings along city streets. Then the trees are susceptible to disease caused by some fungi. Diseases which fall among these are leaf spots. Leaf spots are the most common diseases of shade and ornamental trees. Most of these diseases are favored by cool weather, light and frequent rains, fog or heavy dews, high humidity, and crowded or shady plantings (Heimann & Mahr 1997; Pataky 1998). Many leaf spot diseases are caused by fungi, which attack one species of tree or several species. The spreading of fungus basically depends on meteorological conditions. The humidity and active solar radiation influence the development of these plant pathogens more than temperature (Stakvilevièienë 1999). These spots usually become conspicuous from late June through August. Leaf spot infections that start early in the growing season can lead to premature defoliation. Leaf spots commonly increase in number and size in late summer and early autumn as the leaves be- c 2008 Institute of Botany, Slovak Academy of Sciences gin to senesce. If it occurs over two or more successive years, it can seriously weaken a tree, reduce its growth, and increase its susceptibility to bark borers, winter injury, and other diseases (Pataky 1998). Leaf spots on lindens caused by Cercospora microsora Sacc. give rise to circular brown spots with dark borders. When the spots are very numerous, the entire leaf may turn brown and fall off the tree (Broembsen 2005). Each species of anthracnose fungus attacks only a limited number of tree species (Berry 1998). Various anthracnose-causing fungi have similar life cycles, but require slightly different moisture and temperature conditions for infection. Kaneko & Kaneko (2004) and Nix (2005) have confirmed varying influence of temperature, nutrition, humidity and pH value as external factors of the environment and conditions of in vitro growth (medium, temperature, pH) which play an important role in the pathogen growth and its spread. The present work aims to specify growth characteristics of Cercospora microsora Sacc. [teleomorph Mycosphaerella millegrana (Cook.) Schröet., Mycosphaerella microsora Syd.] on Tilia cordata Mill. planted in urban settings and on the basis of laboratory study to extend knowledge on Mycosphaerella biology in ecological conditions of Slovakia. Leaf spot disease on Tilia cordata 45 b a Bratislava Current effect: F(2, 57)=112.61, p=0.0000 52 52 50 50 48 48 46 46 44 44 42 42 40 40 38 38 36 36 GR GR Pribeta Current effect: F(2, 57)=189.77, p=0.0000 34 34 32 32 30 30 28 28 26 26 24 24 22 22 20 20 18 18 PDAg MA PDAg A MA A Medium Medium c Nitra Current effect: F(2, 57)=1.4217, p=0.24972 52 50 48 46 44 42 40 GR 38 36 34 32 30 28 26 24 22 20 18 PDAg MA A Medium Fig. 1. Influence of medium on growth rate (mm/week) of Cercospora microsora isolated from Tilia cordata at different localities (one-way ANOVA). PDAg – potato-dextrose agar enriched with bromcresol green; MA – malt agar; A – water agar. Material and methods To determine growth rates of Cercospora microsora on Tilia cordata leaf samples were taken from affected host trees grown in urban environment during growing season at selected localities (Bratislava, Nitra, Pribeta). Altogether 50 leaf samples were used for isolation from one location. The age of evaluated trees varied from 20 to 60 years. Samples were surface-sterilized in 70% ethanol and for 15–20 minutes in sodium hypochlorite (1% available chlorine), rinsed in sterile distilled water (2–3 times) and dried carefully between filter paper. After surface sterilization, tissue samples were cut in small pieces (2–3 mm) and placed on 1% malt extract agar (MA) (10 g L−1 Difco agar, 7 g L−1 malt extract) and subsequently incubated in Petri dishes at 24–25 ◦C, in the dark. Pure fungal cultures (from 15 to 25) were isolated from attacked leaves after multiple purification from each investigated locality. Mycelial growth was assessed on 10-day-old pure cultures grown in Petri dishes at 24–25 ◦C, in darkness. Three different artificial media, 3% malt agar (MA) (30 g L−1 Difco agar, 21 g L−1 malt extract), 3% potato-dextrose agar (Difco) (PDAg) enriched with bromcresol green (50 mg L−1 ) and 3% water agar (A) were used in cultivation (30 g L−1 agar). The pH values of the cultivation media were adjusted on 6 with KOH. Growth rates of pure cultures were performed by recording of daily growths of mycelium (mm/day; with precision 0.5 mm) during a week. Agar columns about 0.5 × 0.5 mm of “maternal” mycelia were used for inoculation of plates. Altogether 25 repetitions were made for each locality and medium and growth extremes (atypical mycelial growth, minimal/maximal dimensions) were excluded from the subsequent analysis for more effectual results representativeness. The one-way analysis of variance (ANOVA) was used to assess the influence of two factors – locality and medium on growth rate of Cercospora microsora. Overall growth rate after one week was taken (differences in individual time intervals were not compared). Influence of used medium was tested separately for each locality, as well as influence of locality for each medium. A Tukey test (ANOVA) was performed to determine the combinations of growth rate means that are significantly different. All localities and media were compared with respect to growth rate including all combinations. The statistical package STATISTICA-7 (StatSoft) was used for all analysis. Results When using one-way ANOVA, a significant influence of both two factors, culture medium (Figs 1a–c) and locality (Figs 2a–c) on growth rate of investigated fungus was confirmed. The effect of these factors has not been S. Bernadovičová & H. Ivanová 46 b a MA Current effect: F(2, 57)=22.784, p=0.00000 52 50 50 48 48 46 46 44 44 42 42 40 40 38 38 36 36 GR GR PDAg Current effect: F(2, 57)=71.056, p=0.00000 52 34 34 32 32 30 30 28 28 26 26 24 24 22 22 20 20 18 18 PRIBETA BRATISLAVA NITRA PRIBETA BRATISLAVA Locality NITRA Locality c A Current effect: F(2, 57)=45.409, p=0.00000 52 50 48 46 44 42 40 GR 38 36 34 32 30 28 26 24 22 20 18 PRIBETA BRATISLAVA NITRA Locality Fig. 2. Influence of locality on growth rate (mm/week) of Cercospora microsora isolated from Tilia cordata on different media (one-way ANOVA). PDAg – potato-dextrose agar enriched with bromcresol green; MA – malt agar; A – water agar. proved unambiguously in all cases. In the case of the locality Nitra, the significant influence of used media has not been proved (P > 0.05). PDAg showed to be generally the most suitable medium, inducing the most intensive growth in three localities (41.06 mm/week on average), although a significant difference was not proved in every case. On the contrary, samples from all localities showed the lowest growth on the A (28.79 mm/week on average). The MA induced 38.45 mm/week, on average. Comparing three localities (Figs 2a–c), the effect of this factor is not so unambiguous. The samples from the locality Bratislava averagely showed the highest values of the growth rate in the cases of PDAg (49.8 mm/week) and MA (43.05 mm/week). Only in the case of medium A it was a lower value. The samples from Nitra grew evenly regardless of the medium (35.21 mm/week on average). Samples from this locality growing on two media types (PDAg, MA) generally showed the slowest growth. Hovewer, in case of the medium A, samples from Bratislava and Pribeta demonstrated slower growth than isolates from Nitra. Thus, growth rate values from the localities Bratislava and Pribeta indicate unsuitability of medium A for the fast radial growth. When using PDAg and MA, the isolates grew faster than samples from Nitra. A Tukey test separately conducted for the factors medium and the locality revealed significant combinations (P ≤ 0.05) of means (those picked out in the Tables 1, 2). The test has revealed significant differences between average growth rate values of the samples from Bratislava cultivated on PDAg, MA and A. On the contrary, in the case of isolates from the locality Nitra, the differences between three media are statistically insignificant. In the locality Pribeta, only differences between the medium A and other two media are significant. Discussion The fungus Mycosphaerella millegrana (Cook.) Schröet., syn. M. microsora Syd. [anamorph Cercospora microsora Sacc., syn. C. exitiosa Syd. & P. Syd., syn. Cercospora tiliae Peck., syn. Passalora microsora (Sacc.)], the causative agent of the leaf spot disease, currently belongs to the most widespread fungi on linden trees in Slovakia. In 2002, only range of leaf parasitic fungi that could be found on various linden species in our conditions was showed in general (Juhásová 2002). First report Leaf spot disease on Tilia cordata 47 Table 1. Influence of medium on growth rate of Cercospora microsora isolated from Tilia cordata and combinations of growth rate means using a Tukey test. Significant combinations (P ≤ 0.05) are picked out. Locality Pribeta Medium PDAg MA A PDAg MA A Average x 0.877307 0.000119 0.877307 x 0.000119 0.000119 0.000119 x 37.10 36.65 21.32 Locality Bratislava Medium PDAg MA A PDAg MA A Average x 0.000121 0.000119 0.000121 x 0.000119 0.000119 0.000119 x 49.80 43.05 31.35 Locality Nitra Medium PDAg MA A PDAg MA A Average x 0.918852 0.247149 0.918852 x 0.444100 0.247149 0.444100 x 36.27 35.65 33.70 PDAg – potato-dextrose agar enriched with bromcresol green; MA – malt agar; A – water agar Table 2. Influence of locality on growth rate of Cercospora microsora isolated from Tilia cordata and combinations of growth rate means using a Tukey test. Significant combinations (P ≤ 0.05) are picked out. Medium PDAg Locality Pribeta Bratislava Nitra Pribeta Bratislava Nitra Average x 0.000119 0.793998 0.000119 x 0.000119 0.793998 0.000119 x 37.10 49.80 36.27 Medium MA Locality Pribeta Bratislava Nitra Pribeta Bratislava Nitra Average x 0.000122 0.679569 0.000122 x 0.000119 0.679569 0.000119 x 36.65 43.05000 35.65000 Medium A Locality Pribeta Bratislava Nitra Pribeta Bratislava Nitra Average x 0.000119 0.000119 0.000119 x 0.212677 0.000119 0.212677 x 21.32500 31.35000 33.70000 PDAg – potato-dextrose agar enriched with bromcresol green; MA – malt agar; A – water agar about Cercospora microsora Sacc. in connection with its increased and massive occurence on Tilia cordata was reported during growing season in 2006. There are more than 3 000 named species in the genus Cercospora (Pollack 1987). Most of them have no known sexual stage, although a Mycosphaerella teleomorph has been identified for a few (Crous & Brau 2003). According to Pollack (1987) there is some disagreement over the identity of the teleomorph. In our health state evaluation, circular or oval light-brown spots about 3–4 mm in diameter were visible to the naked eye on the upper and lower side of leaves. Dark areas and speck-sized, fungus-fruiting bodies commonly form in the dead tissues of many older spots. Ascospores cause the primary infection of leaves. During the growing season, the fungus is spread by conidia, which are formed in great quantity as cinnamonbrown figure in the middle of spots. Conidia, formed in the pycnidia under epidermis on the lower side of leaves are filamentous, 35–100 µm long, light brown, septate (3–8). The spores are spread primarily by air currents, splashing rains, and insects, to newly emerging leaves of susceptible trees where in the presence of free water they germinate and penetrate and the infection begins. 48 Pataky (1998) describes disease on linden trees caused by species of Cercospora as formation of circular to angular dead areas in the leaves at first dark green and later yellow, tan or dark-brown spots with distinct dark margins. Some spots may drop out, leaving ragged shot-holes. The spots may be numerous and cause the leaf to turn brown and fall prematurely. Donaubauer (1999) ascertained the occurrence of the small necrosen on linden growing in west and middle Austria. The spots formed by C. microsora appeared from late July through August. According to Juhásová (2002) and Broembsen (2005) leaf spots on linden in urban settings are caused mainly by C. microsora. The spots are small, circular or circular to oval, brown with dark borders. When the spots are numerous, the entire leaf may turn brown and fall off the tree. Although infected leaves and other plant tissue harboring the fungus usually persist from one growing season to the next, the cool, rainy periods in early to mid-spring are often not long enough for the fungus to grow, multiply, and infect new leaves. Consequently, the presence and severity of leaf spot diseases are variable from year to year. Leaf spots are most likely to develop when there are extended periods of cool, moist weather during April, May, and June when the new leaf growth is expanding. Influence of the external factors of the environment such as temperature, nutrition, humidity and pH value as well as conditions of in vitro growth (medium, temperature, pH) play an important role in the pathogen growth and its spread (Caltrider 1961; Zimmermannová-Pastirčáková 2002; Kaneko & Kaneko 2004). Stakvilevièienë (1999) presents the results of the investigations on environment influence on the distribution of the cercosporoid fungi in Lithuania over the years 1992–1998. The fungus Passalora microsora Sacc. was ascertained on nine tree species of the Tilia genus. According to this author the humidity and active solar radiation influence the development of these plant pathogens more than the temperature. They occured only in the second half of summer when the amount of summed radiation decreased. During rainy and foggy summers they may appear earlier. Area pollution, industrial and health-resort cities and small towns have no influence on the distribution of brown leaf spots (Cercospora microsora, Passalora microsora) on trees. The spreading of fungus basically depends on meteorological conditions. Due to scant knowledge of the biology of this fungus in our conditions, this study was supplemented with initial data about some growth characteristics of the fungus on the basis of experiments under laboratory conditions. Of course, for a complete picture of the biology of this fungal pathogen and adequate conclusions it is needful to extend the results on the basis of numerous replications with an appropriate number of isolates from various localities in next study. Kaneko & Kaneko (2004) observed the little difference among three artificial media, 2% MA, 2% LCA (Miura medium) and 2% PDA, regarding fungal growth S. Bernadovičová & H. Ivanová rates, but growth rates differed among fungal species. For example Mycosphaerella buna (R. Kaneko & Kakish.) and Ascochyta fagi (Woron.) grew well between 15 and 30 ◦C. Insignificant to no growth was observed at 5 or 35 ◦C for all fungus. Zimmermannová-Pastirčáková (2002) summarised the effect of different pH values of the medium on growth of Phyllosticta sphaeropsoidea Ellis et Everh. Mycelium of the studied fungus grew on malt extract agar within the range of pH 3–12. Radial growth rate was greatest over the optimum pH range (6–8). The pH values of the cultivation media used for our experiments were adjusted on pH 6 with KOH. According to Sundari & Adholeya (2003) the substrate pH would not only determine the growth rate of the fungus but also limit further proliferation of the fungus in medium. In our experiments the fungus in initial stages forms aerated, light-yellow and later light-pink colonies, reddish purple with age. The fungus grows relatively fast on potato-dextrose agar and malt agar. Juhásová et al. (2006) have studied the biology of similar anthracnose fungi Gnomonia leptostyla (Fr.) Ces. Et de Not. and Marssonina juglandis (Lib.) Magnus on Juglans regia L. Statistically significant differences between three different media (2% water agar, 2% Czapek-Dox agar, 2% malt agar) and between the times of cultivation were denoted by multiple range analysis. The level of variability between the localities was statistically insignificant. The fungal genus Cercospora contains many species capable of infecting a large number of diverse and economically important plants. The success of this group of fungi as pathogens is attributed to their production of a toxin, cercosporin, first isolated in 1957 by Kuyama and Tamura, which is considered to be a primary pathogenicity factor (Daub & Ehrenshaft 2000). Many plant pathogenic Cercospora species produced light-activated non-host-selective perylenequinone toxins that are toxic to most organisms, including plants, animals and microorganisms via the generation of activated oxygen species, particularly singlet oxygen (Chung 2003). In our experiments cercosporin was produced by the fungus Cercospora microsora Sacc. and appeared in the cultures cultivated on light. Cercosporin production was observed three days after inoculation as a red or redpurple pigment in the agar. This knowledge is comparable with results of Daub & Ehrenshaft (2000) and Chung (2003). Studies on the cercosporin toxin produced by Cercospora species have documented an important role for this toxin in pathogenesis of host plants. Cercosporin-generated active oxygen species destroy the membranes of host plants, providing nutriens to support the growth of these intercellular pathogens. Further work is intended to specify pathogenicity and relevance of Cercospora microsora on Tilia cordata in relation to definite external factors and extend knowledge on Cercospora biology on the basis of morphological features and biological characteristics specification of this fungus. Leaf spot disease on Tilia cordata Acknowledgements This study was supported by Slovak grant agency VEGA project No. 2/7026/27. References Berry F.H. 1998. Anthracnose Diseases of Eastern Hardwoods. file://F:\Anthracnose%20Diseases%20of%20Eastern%20 Hardwoods20%FIDL.htm Broembsen S.V. 2005. Anthracnose and Other Common Leaf Diseases of Deciduous Shade Trees. pods.dasnr.okstate.edu/ docustare/dsweb/Get/Documnet-2831/F-7634web.pdf Caltrider P.G. 1961. Growth and sporulation of Guignardia bidwellii. Phytopathol. 51: 860–863. Chung K. R. 2003. Involvement of calcium/calmodulin signaling in cercosporin toxin biosynthesis by Cercospora nicotianae. Appl. Environm. Microbiol. 69: 1187–1196. Crous P.W. & Braun U. 2003. Mycosphaerella and its anamorphs: 1. Manes published in Cercospora and Passalora. CBS Biodiverzity Series 1, 571pp. Daub M.E. & Ehrenshaft M. 2000. The photoactivated Cercospora toxin cercosporin: Contributios to plant diesease and fundamental biology. Annu. Rev. Phytopathol. 38: 461–490. Donaubauer E. 1999. On the occurrence of some leaf diseases during summer and autum 1999. Forschutz Aktuell, Abstracts, p. 25. 49 Heimann M.F. & Mahr D.L. 1997. Common foliage diseases of shade trees in Wisconsin. University of Wisconsin-System Board of Regents and University of Wisconsin – Extention, 170 pp. Juhásová, G. 2002. Škodlivosť parazitických húb na lipách. Zahradnictví 6: 13. Juhásová G., Ivanová H. & Spišák J. 2006. Biology of fungus Gnomonia leptostyla in agro-ecological environments of Slovakia. Mikologija i Fitopatologija 40: 538–547. Kaneko R. & Kaneko S. 2004. The effect of bagging branches on levels of endophytic fungal infection in Japanese beech leaves. Forest Pathol. 34: 65–78. Nix S. 2005. Leaf spot tree disease – Prevention and control. http://forestry.about.com/od/forestdiseases/p/dis-com-1sp.htm Pataky N.R. 1998. Fungal leaf spot diseases of shade and ornamental trees in the midwest. Plant Disease 648: 1–8. Pollack F.G. 1987. An annotated compilation of Cercospora names. Mycol. Mem. 12: 1–212. Stakvilevièienë S. 1999. Influence of environmental conditions on the distribution of the cercosporoid fungi in Lithuania. Bot. Lithuanica Suppl. 3: 87–89. Sundari K.S. & Adholey A. 2003. Growth profile of ectomycorrhizal fungi mycelium: emphasis on substrate pH influence. J. Microbiol. 83: 209–214. Zimmermannová-Pastirčáková K. 2002. Occurrence of Horsechestnut leaf blotch and cultural characteristics of its causal agent-fungus Phyllosticta sphaeropsoidea, an anamorph of Guignardia aesculi. Folia oecologica 30: 245–250. Received June 9, 2006 Accepted Jan. 17, 2007