JOURNAL OF FOREST SCIENCE, 60, 2014 (8): 330–335

Biotechnical control of tar spot (Rhytisma acerinum) disease on velvet maple (Acer velutinum Boiss) in vitro

S.M. Karami1, M.R. Kavosi1, G. Hajizadeh2, H. Jalilvand2

1Department of Forest Science, Faculty of Forest Ecology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran 2Department of Forestry, Faculty of Natural Resources, Sari University of Agricultural Sciences and Natural Resources, Sari, Mazandaran, Iran

ABSTRACT: Several different fungi can cause tar leaf spot diseases in maple trees, including three fungi of the ge- nus Rhytisma. Rhytisma acerinum (Pers.) Fries is an ascomycete that forms black stromata known as tar spot on the adaxial surface of the leaves of Acer species. The tar spot (R. acerinum) disease has been increasing in incidence and severity in maples of Hyrcanian forests, northern Iran, in recent years. One of the best ways to manage infestations by R. acerinum is through adequate biotechnical techniques. The isolation of fungal spore colonies was evaluated us- ing different dosages of Oxywet 10% (50, 100, 200, 500 μl), Gentamicin 5% (100, 200, 400; 1,000 μl), and Amoxicillin antibiotics 20% (25, 50, 100, 250 μl) in 100 ml of distilled water in each treatment. All possible combinations of single doses were applied using light and dark treatments. In light conditions, it appears that the Oxywet (200 μl) had the significant effect on controlling R. acerinum. Reduced fungal growth, coefficient and inhibition of fungal growth were observed in the light treatment. The other antibiotics (Gentamicin, Amoxicillin) were not so effective in controlling this pathogen. Results of spore germination showed a significant difference between all treatments. All treatments were tested in pure cultures in the laboratory only. The results obtained cannot be expected of the same effectiveness in open field trials.

Keywords: antibiotic; fungus

Th e genus Acer (commonly known as maple) is deforested or degraded lands. However, seed dor- comprised of 150 widely distributed species in the mancy has hampered attempts to raise seedlings in temperate zone, ranging from North America, Eu- tree nurseries for large-scale planting (Difazio et rope, and Asia to North Africa. Maples are highly al. 1988; Yousef-Zadeh et al. 2007). valuable for a variety of purposes, such as lumber Rhytisma acerinum is a plant pathogen that com- and veneer, ornamental uses, food and shelter for monly aff ects maples in late summer and autumn, wildlife and watershed protection (Farhadi et al. causing a problem known as tar spot (Hudler 2013). Acer velutinum Boiss (velvet maple) is one of 1998). Tar spot infection remains localized to the the most valuable native species in northern Iran, chlorotic areas on the leaves creating mostly a which is widely distributed in the Alborz mountain cosmetic issue, rather than an economically det- range from 200 to 2,000 m in elevation. Under fa- rimental disease. Rhytisma acerinum, a biotrophic vourable conditions, velvet maple reaches over 40 m ascomycete parasite, locally infects the leaves of in height and diameters exceeding 150 cm. It com- maple trees (Weber, Webster 2002). Symptoms prises nearly 8% of the growing stock in this area fi rst appear as inconspicuous, pale green to yellow and provides nearly 2% of domestic wood demand areas on the leaves. As the fungus grows within the (Farhadi et al. 2007). Th e fast growth of this spe- leaf, these areas develop into distinctive, slightly cies has drawn the attention of forest managers raised, shiny, tar-like, black spots on the leaves. Th e as one of the key species used in reforestation of size of the spot depends upon the fungal species;

330 J. FOR. SCI., 60, 2014 (8): 330–335 spots can be irregular and up to 1.3 cm in diam- MATERIAL AND METHODS eter (R. acerinum) or can appear as tiny, pinpoint dots (Rhytisma punctatum). Signifi cant premature Sterilization and isolation of R. acerinum. For autumn coloration and defoliation can occur, espe- isolation and sterilization of R. acerinum colonies, cially when the infection is heavy, as is often the infected leaves from tar spot samples were collected situation in Norway maple (Hudler, Banik 1987; from the Shast Kalate forest, Gorgan, northern Iran Hsiang et al. 2008). (36°43'–36°48'N and 54°21'–54°24'E) (Etminan et Rhytisma acerinum can occur in many tree spe- al. 2011). Th e surfaces of the leaves were sterilized cies, although it most commonly infects species before separation of the fungal spots. Samples of of the genus Acer. Th e large tar spot (R. acerinum) spots on leaves were isolated, cut into approxi- most commonly infects Norway maple (Acer plata- mately 5 × 5 mm sections, and the surfaces were noides), silver maple (Acer saccharinum), sycamore sterilized with 0.05% NaOCl and EtOH for 2 min maple (Acer pseudoplatanus) and recently velvet (Hudler, Banik 1987), rinsed with 96% alcohol maple (A. velutinum). Th e small tar spot (R. punc- for one min and then washed with distilled water tatum) occurs in madrone (Arbutus menziesii), under laboratory conditions. Leaf cultures on 4% yellow poplar (Liriodendron tulipifera) and wil- (w/v) Potato-Dextrose-Agar (PDA) )Pinkerton, low (Salix) (Sinclair et al. 1987; Tainter, Baker Strobel 1976 (media were incubated at 24 ± 1°C 1996; Hsiang, Tian 2007). and, after 21 days, the isolation of R. acerinum was Tar spots contain the teleomorph of R. acerinum. performed. Cultures of R. acerinum were exposed In the spring, needle-shaped ascospores are released to three antibiotics: Oxywet, Gentamicin and from overwintering apothecia in fallen leaf debris Amoxicillin. Th e selected treatments were Oxywet (Weber, Webster 2002; Hsiang et al. 2009). Th e (50, 100, 200, 500 μl), Gentamicin 5% (100, 200, 400; wind disseminates the spores, which have a sticky 1,000 μl) and Amoxicillin 20% (25, 50, 100, 250 μl) coating allowing them to attach to new healthy in 100 ml distilled water. Each was prepared in con- leaves. Once on the leaves, the spores germinate ditions of both light and darkness. Th e growth rates and penetrate through the stoma. Th e subsequent of the R. acerinum colony and mycelium were stud- infection causes chlorosis of the leaves in localized ied to test the eff ect of each antibiotic and radial yellow spots. As the season continues into summer, growth was measured for a week. apothecia begin to form, giving rise to brown-black In the next step for the preparation of a spore leaf lesions that resemble spots of tar. Leaves retain suspension, a liquid potato-dextrose agar medium their yellow border during the initial chlorosis. Apo- was used. Two circles of the fungus from fi ve my- thecia overwinter in the fallen plant debris, releas- celia at the edge of fungi colonies were transferred ing spores when the temperature warms again. Th e to media in laminar air fl ow. Th e media were placed use of adequate sanitation techniques provides one on shakers set at 70 rpm. After 14 days, the media of the best methods of managing the pathogen. Be- were transferred from sterilized Whatman fi lter pa- cause the fungus overwinters in diseased leaf debris, per onto a vacuum hopper for separating the myce- removing the debris in autumn can help reduce the lium. Samples were centrifuged about three times, occurrence of the disease. In certain severe cases, for 10 min at 10,000 rpm. Spores in suspension fungicides can also assist with control. Copper- were counted with a haemocytometer for achiev- based fungicides sprayed in early spring when leaves ing 2 × 102 spores per ml. To evaluate the eff ects are budding and twice more throughout the season on spore germination, the three antibiotics test- also helps to reduce the incidence of the disease ed here were integrated with PDA media. One μl (Hsiang et al. 2009). of the suspension was distributed at the media Th e objectives of this study were to determine level. Rates of inhibition of spore germination were (i) appropriate biotechnical method that can be determined by counting germinated spores after used to control R. acerinum, (ii) suitable concen- 7 days for each treatment. Th e spores obtained were trations of selected antibiotics, and (iii) to analyze exposed under conditions of light and darkness in individual and combined eff ects of biotechnical an- media containing the three antibiotics: Oxywet, tibiotic on fungal growth, area under fungi growth Gentamicin and Amoxicillin. curve, called the AUFGC coeffi cient, and inhibition Treatments used were 10% Oxywet at four con- of fungal growth. Here it is essential to state that all centrations (50, 100, 200, 500 μl), 20% Amoxicillin this was tested in pure cultures in the lab only. Th e at four concentrations (25, 50, 100, 250 μl) and 5% results obtained cannot be expected of the same ef- Gentamicin at four concentrations (100, 200, 400; fectiveness in open fi eld trials. 1,000 μl) in four replications, with all tests com-

J. FOR. SCI., 60, 2014 (8): 330–335 331 pleted under conditions of both light and darkness. Table 1. Analysis of variance Equation (1) was used to calculate the AUFGC on Treatment df P various days: AUFGC coeffi cient Σn (X + X ) AUFGC = i+1 i+1 i (t – t) (1) Light 1 0.22ns 2 i+1 Antibiotic 12 0.015* where: Light × antibiotic 12 0.20ns

Xi – observed colony size, Growth of fungus mycelium t – time (days), Light 1 0.96ns n – number of observations. Antibiotic 12 0.001** Statistical analysis. Th is study was conducted as Light × antibiotic 12 0.98ns a two-factor factorial experiment with a completely Spore germination of Rhytisma acerinum randomized design. Means were compared using Light 1 0.001** Duncan’s new multiple range tests with the results Antibiotic 12 0.002** considered signifi cant at P < 0.01. Light × antibiotic 12 0.005**

*P < 0.05, **P < 0.01 – signifi cant diff erences between the RESULTS treatments; ns not signifi cant

Results showed that 200 μl of Oxywet antibi- pact on the AUFGC coefficient and the 200 μl Oxy- otic in 100 ml distilled water prevented the fungal wet concentrations had the minimum impact on colony growth of the maple tar spot disease patho- the AUFGC coefficient (Fig. 3a) in light conditions, gen (R. acerinum) and caused the degradation and Oxywet in concentrations of 50, 100 and 500 μl growth of fewer fungal mycelium fi laments of Rhyti- and control treatment had a significant impact on sma acerinum (Fig. 1). the AUFGC coefficient and Oxywet with concen- Th e eff ect of light and antibiotic on the AUFGC trations of 200 μl had the smallest impact on the factor did not show any signifi cant diff erences AUFGC coefficient (Fig. 3b). (P < 0.01) but the type of antibiotic used resulted in a The effect of the antibiotic in light and darkness signifi cant diff erence in AUFGC (P < 0.05) (Table 1). on the mycelial growth of the fungus was not sig- Th e mean comparison of diff erent antibiotics on nificantly different between treatments. The ef- AUFGC showed that among used antibiotics, 200 μl fect of the antibiotic on the mycelial growth of the of Oxywet had a minimal eff ect on AUFGC (Fig. 2). fungus was significantly different (Table 1). Based In dark conditions, Oxywet antibiotic with con- on Fig. 4, the mean comparison of various treat- centrations of 50, 100, 500 μl had the maximum im- ments on the mycelium growth of maple tar spot fungus shows that Oxywet with 50, 100 and 500 (a) (b) μl and amoxicillin with 250 μl had the maximum effect.

300 a O –a Oxywet, Ga – Gentamicin, A – Amoxicillin, C – Control 250 a c b 200 c c c c c c 150 d

AUFGC 100 (c) (d) 50 0

Treatment

Fig. 2. Mean (± SE) comparison of AUFGC coeffi cient in diff erent treatments. Th e same letters indicate mean values are not signifi cantly diff erent

Fig. 1. Th e fungus Rhytisma acerinum in diff erent treatments: (a) Oxywet 200 μl, (b) Gentamicin, (c) Amoxicillin and (d) control treatment

332 J. FOR. SCI., 60, 2014 (8): 330–335 (a) (a) 300 O – Oxywet, G – Gentamicin, A – Amoxicillin, C – Control 140 Number of days a c b 7 5 3 1 250 AUFGC STD 120 O 50 248.5533 a 7.183156 f d O 100 245.93 c 7.800635 g e 200 100 O 200 99.32i ml 3.6149 j h O 500 246.5458 b 4.343962k 80 150 G 100 161.295 i 8.495366 G 200m 151.34 L 21.28733 60 AUFGC 100 G 400 148.9542 k 1.737472 G 1000 160.8075 j 1.608858 40 Mycelium growth 50 A 25 171.57 h 5.74352 A 50 195.9167 f 30.9 20 0 A 100 180.25 g 13.89019 (b) A 250 197 1667 e 2 020726 0 300 (b) a 160 d AUFGCc STD b O – Oxywet, G – Gentamicin, A – Amoxicillin, C – Control 250 O 50 248.5 a 3.5 140 O 100 238 d 4.40407g 120 200 O 200 93.91667 L 5.625463 e f hh i O 500 246.25j c 2.537223 k 100 150 G 100 151.6667 j 7.285831 G 200 165.0833 h 7.285831 80

AUFGC G 400l 165.0833 h 10.25406 100 60 G 1000 174.4167 g 15.70743 A 25 181.4667 e 2.380301 40 50 Mycelium growth A 50 170.3333 i 7.891187 20 A 100 183 f 1.732051 0 A 250 150.5 k 1.064483 0 C 247.0833 b 4.474465

Treatment Treatment

Fig. 3. AUFGC mean (±1 SE) in diff erent treatments in Fig. 5. Th e fungal mycelium growth of Rhytisma acerinum terms of dark (a) and light (b). Th e same letters indicate in dark (a) and light (b) conditions at diff erent times after mean values that are not signifi cantly diff erent culture

Fig. 5a shows that Oxywet 200 μl was the least ef- tion rate in the control and in the treatment of germi- fective in preventing the fungus mycelial growth in nation spores were released at roughly the same level the absence of light. Of the other media containing and other antibiotics the spore germination could diff erent antibiotics, Amoxicillin (25 and 250 μl) have been used to control (Fig. 6). and Oxywet (50 and 250 μl) in 100 ml of distilled 60 water had the greatest impact on the mycelium O – Oxywet, G – Gentamicin, A – Amoxicillin, C – Control a growth in darkness. Fig. 5b indicates that Oxywet 50 40 of 200 μl in light conditions had the smallest eff ect b 30 d c on the fungal mycelium growth. Th e other Oxywet ffg e gggf f concentrations (50, 100 and 500 μl) can stop the 20 fungal mycelium growth in light conditions and it Spore germination 10 was the most eff ective antibiotic. 0 Th e analysis of antibiotic and light eff ects on spore germination showed that the eff ects of antibiotic and Treatment light during spore germination were signifi cantly dif- ferent (Table 1). Th e results of the mean comparison Fig. 6. Mean (±1 SE) comparison of diff erent treatments on on spore germination showed the highest germina- the spore germination of Rhytisma acerinum O – Oxywet, G – Gentamicin, A – Amoxicillin, C – Control Among the antibiotics used here, Oxywet 200 μl 50 a a a b a a 45 d b c had the greatest impact on deterrence in dark con- e f f 40 ditions and Oxywet antibiotic concentrations of 50, 35 30 100 and 500 μl showed a minimum eff ect on the 25 inhibition of the fungal mycelium growth (Fig. 7a). 20 g 15 As Fig. 7b shows, the Oxywet antibiotic had the ef- 10 Mycelium growth Mycelium 5 fective impact on growth inhibition in light condi- 0 tions at 200 μl and Oxywet had the smallest eff ect with concentrations of 50, 100 and 500 μl. Th e oth- Treatment er antibiotics initially inhibited the growth of the Fig. 4. Mean (±1 SE) comparison of the fungal mycelium mycelium but over time, the ability to inhibit the growth in diff erent treatments. Th e same letters indicate mycelium growth was lost. Overall, 200 μl of Oxy- mean values that are not signifi cantly diff erent wet inhibited the uniform mycelium growth.

J. FOR. SCI., 60, 2014 (8): 330–335 333 (a) 250 Number of days nies; the mycelium was damaged and the other con- 200 7 5 3 1 centrations of this antibiotic (50, 100 and 500 μl)

150 were not eff ective in controlling this disease. Based on the results, in terms of the presence and absence 100

Inhibition of light, Oxywet 200 μl showed the greatest impact 50 on the amount of the minimum inhibitory eff ect 0 on the fungus mycelium growth and Oxywet (50, (b) 300 100 and 500 μl). Amoxicillin (25 and 50 μl) in terms O – Oxywet, G – Gentamicin, A – Amoxicillin, C – Control 250 of light and darkness had the minimum inhibitory 1357 eff ect and the maximum eff ect on mycelium eff ect 200 O 50 9.62 0 0 0 O 100 15.84 5.12 0 0 as mycelium growth in addition to the control that 150 O 200 56.58 61 62 62.88 O 500 10 1.4 0 0 G 100 66.13 45.09 33.74 26.61 showed the highest growth in media containing 50, Inhibition 100 G 200 62.15 40.5 30.41 15.27 G 400 64.65 29.06 32.3 18.2 100 and 500 μl of the antibiotic. Th e presence or 50 G 1000 64.01 45.4 11.74 16.33 A 25 55.79 34.46 23.89 9.02 absence of light had a small eff ect on inhibiting the 0 A 50 56.55 39.46 26.79 13.04 A 100 59.01 35.59 20.56 6.9 mycelium growth. In this study, spore germination A 250 45.87 23 9.53 0 C 0000 in light, antibiotic and reciprocal eff ect of antibiotic Treatment and light resulted in signifi cant diff erences in spore germination. In controlling spore germination in Fig. 7. Inhibition growth curve of the Rhytisma acerinum contrast to mycelium growth and fungal colonies, fungus in dark (a) and light (b) conditions at diff erent time only Oxywet 200 μl was able to control the myce- after culture lium growth; the other antibiotics controlled the DISCUSSION disease according to comparison. Light or dark- ness conditions were eff ective in spore germination Several maple species are growing in the Hyrca- as in light conditions; spores of the tar maple spot nian region; of these the following velvet and Cap- fungus (R. acerinum) were more able to germinate padocian maples are widely distributed in diff erent rather than to look for light. parts of the Caspian forests (Sagheb-Talebi et al. 2014). Velvet maple is one of the fast-growing and productive tree species in the Caspian region that CONCLUSION accounts for 5.8% of the standing volume and 2.7% of the stem number in the Hyrcanian forests of Iran Acer velutinum Boiss (velvet maple) is one of the (Rasaneh et al. 2001). Some individuals of velvet most popular trees for plantations and restoration maple can reach large dimensions: 50 m in height of diff erent forest stands in the Caspian region, and and more than 2 m in diameter. It is a light demand- is widely produced in nurseries. Tar spot caused by ing species and can be found as individual trees or ascomycete fungi is problematic for maples in some in small groups from the plateau up to 2,000 m a.s.l. parts of the world and can even cause health disor- in diff erent forest communities. ders in some vertebrates. Th is paper is concerned Th e most important pathogen on this tree spe- with the biotechnical control of the pathogen in vitro. cies is R. acerinum. In general, tar spot diseases Th ere has been relatively little research done on tar appear on maple leaves in early autumn, causing a spot in northern Iran. reduction in wood and seed production. Finding an Th is was not a successful attempt; this was a basic appropriate way to control this disease is very im- experiment for checking the eff ects of certain antibi- portant. Th e results of this study demonstrate the otics against Rhytisma mycelia in the laboratory with eff ects of three antibiotics, Oxywet, Gentamicin quite promising results. And to conclude that they and amoxicillin in diff erent concentrations, on con- can be eff ective in controlling maple tar spot is too trolling the tar spot disease. Oxywet with a 200 μl early. For that, additional tests would be necessary! concentration was eff ective in that it had a minimal impact on the AUFGC coeffi cient and Oxywet at 50, 100 and 500 μl concentrations had the great- Acknowledgements est impact on the AUFGC coeffi cient. Th e eff ect of light and light antibiotic on the AUFGC coeffi cient We would like to thank the Gorgan University of and growth was not signifi cant but the eff ect of an- Agricultural Sciences and Natural Resources for tibiotic on the AUFGC coeffi cient and growth was supporting this research. We also thank anonymous signifi cant by using Oxywet 200 μl antibiotic colo- reviewers for helpful comments on the manuscript.

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Corresponding author: Dr. Goodarz Hajizadeh, Sari University of Agricultural Sciences and Natural Resources, Faculty of Natural Resources, Department of Forestry, P.O. Box 578, Sari, Mazandaran, Iran; email: [email protected]

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