J Gen Plant Pathol (2009) 75:437–439 DOI 10.1007/s10327-009-0200-2 DISEASE NOTE Leaf spot disease of Hyoscyamus muticus (Egyptian henbane) caused by Cladosporium herbarum Fatma F. Abdel-Motaal • Magdi A. El-Sayed Soad A. El-Zayat • Mortada S. M. Nassar • Shin-ichi Ito • Received: 19 May 2009 / Accepted: 18 August 2009 / Published online: 15 October 2009 Ó The Phytopathological Society of Japan and Springer 2009 Abstract Severe brown spots caused by Cladosporium herbarum appeared on the leaves of Hyoscyamus muticus (Egyptian henbane) grown in a greenhouse at Yamaguchi city, Japan, in the summer of 2008. Nucleotide sequence analysis of rDNA-ITS and 28S rDNA supported the morphological identification of the isolate, which caused the same disease symptoms after reinoculation of the host. This new disease was named ‘‘Cladosporium leaf spot of Egyptian henbane’’. (Oksman-Caldentey et al. 1987). Brown spots were observed on the leaves of potted H. muticus plants in the greenhouse at Yamaguchi University, Yamaguchi city, Japan, during the summer of 2008. We isolated and identified the pathogen, and reinoculated the host with the isolate to clarify the causal agent of the disease. Keywords Leaf spot
Cladosporium herbarum
Egyptian henbane
Hyoscyamus muticus Disease symptoms on H. muticus plants appeared in the summer, and spores were produced in the leaf lesions under relatively humid conditions ([70%) and at slightly high temperatures (20–30°C). The disease was characterized by circular distinct patches of white spots that turned brown upon sporulation of the fungus. The spots increased in size over time, which led to leaf curling and defoliation (Fig. 1a, b). Germinated conidia settled on the leaves, and the fungus penetrated the leaf tissue through open or damaged stomata and continued to grow, and eventually the leaf tissue died. Later, conidiophores emerged from the stomatal openings (Fig. 1c). Introduction Hyoscyamus muticus L. (Solanaceae) is a tropical herb containing an array of pharmaceutically valuable tropane alkaloids. It is a source of hyoscyamine, particularly, the epoxide form of hyoscyamine, namely, scopolamine. Both alkaloids are widely used anticholinergic and antispasmodic medicinal agents. H. muticus is a valuable species because its total alkaloid content is higher than other Hyoscyamus species. The total alkaloid content of H. muticus obtained from Egypt and the East Indies often exceeds 1.25%, most of it being pure hyoscyamine F. F. Abdel-Motaal
M. A. El-Sayed
S. Ito (&) Department of Biological and Environmental Sciences, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan e-mail: shinsan@yamaguchi-u.ac.jp S. A. El-Zayat
M. S. M. Nassar Department of Botany, Faculty of Science, South Valley University, Aswan 81528, Egypt Symptoms Isolation and identification of the pathogen Diseased leaf spots were removed using a cork borer and placed on potato dextrose agar (PDA) in petri dishes and incubated at 25°C. Mycelia were visible growing from the discs within 2 days. Single hyphae were transferred and cultured on fresh PDA plates. The abundant aerial mycelia conferred a dark greenish to black color to the fungal colonies, which were brown to olivaceous brown mainly at the colony centre and had narrow, white margins (Fig. 1d). The mycelia of the isolate were well developed, loosely 123 438 J Gen Plant Pathol (2009) 75:437–439 Fig. 1 Symptoms of Cladosporium leaf spot disease in Egyptian henbane (Hyoscyamus muticus) and morphology of the causative agent Cladosporium herbarum. a Natural disease symptoms on H. muticus in the greenhouse. b Gradual increase in severity of disease by the pathogen in the leaves of H. muticus. c Leaf spots stained with 0.05% (w/v) trypan blue in lactoglycerol (1:1:1 mixture of lactic acid, glycerol, and water) and examined under a light microscope, showing conidiophores emerging from the leaf stomata. d–l Morphology of the pathogen (C. herbarum) cultured on PDA. d Colony, front (right) and back (left) views. e Conidiophores near the agar surface. f Conidiophore bearing conidia in chains. g Conidiophore arising near to the hyphal septa. h Proliferation of conidiophores (black arrow). i Macronematous conidiophore. j Micronematous conidiophore. k Ramoconidia with conidia. l Conidia. m Disease symptoms on the leaves of H. muticus inoculated with a conidial suspension prepared from a single-spore isolate of C. herbarum from a diseased H. muticus plant branched (0.5–3.5 lm wide), and septate with small swellings, and the mycelial walls were subhyaline, pale brown to olivaceous brown, smooth or slightly rough, and unthickened to somewhat thickened. Conidiophores formed on the septate hyphae near the agar surface, and branched chains of conidia were clearly visible (Fig. 1e, f). Macro- and micronematous conidiophores emerged near the hyphal septum laterally or terminally (Fig. 1g). The macronematous conidiophores were erect, straight to flexuous, 15–360 9 3.0–4.5 lm in size, and septate with smooth and thickened walls; they were unbranched or had 1–3 branches with single to multilateral swellings or lateral branch-like proliferations at the apex; these conidiophores turned darker brown with age (Fig. 1h, i). The micronematous conidiophores were short lateral outgrowths, narrow and cylindrical, aseptate, unbranched, subhyaline, pale brown to brown, verruculose, and formed clavate to ovate conidia (Fig. 1j). The ramoconidia were cylindrical, greenish brown to brown verruculose, and slightly tapering 123 towards the apex and base with somewhat thickened walls (Fig. 1k). Conidia were present in unbranched or loosely branched chains; they were elliptical to oblong in shape with slightly to distinctly thickened walls, verruculose to verrucose, subhyaline to olivaceous brown; they had 0–1 septum and were 3–15 9 2–7.5 lm in size with prominent scars at one or both ends, and emerged from pale brown or brown pigmented conidiophores (Fig. 1l). The morphological characteristics of the isolate were consistent with previously published descriptions of Cladosporium herbarum (Pers.:Fr.) Link (teleomorph: Davidiella tassiana (De Not.) Crous & U. Braun) (Moubasher 1993; Schubert et al. 2007). C. herbarum causes diseases in several plant species. In Japan, C. herbarum has been reported to cause various diseases in plants (Phytopathological Society of Japan 2000); one of these diseases is brown spot disease of grapes (Ouchi et al. 1976). It invades tea farms in Korea causing scab in plants (Kwon et al. 2001). Perelló et al. (2003) reported the infection of wheat leaves by J Gen Plant Pathol (2009) 75:437–439 C. herbarum in Argentina. The fungus also causes leaf spot disease of Centaurea solstitialis in Greece (Berner et al. 2007) and Caltha leptosepala (marsh marigold) in North America (Johnson et al. 2008). In Egypt, C. herbarum causes leaf spot disease in Cleome amblyocarpa, Haplophyllum tuberculatum, and Pluchea dioscorides, which grow naturally in the Mediterranean coastal area (El Naggar and Abdel-Hafez 2003). However, there is no report of disease of H. muticus caused by C. herbarum. Nuclear DNA from C. herbarum was extracted with the Dr. GenTLE kit (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. Nucleotide sequences of the internal transcribed spacer region of ribosomal DNA (rDNA-ITS) and the D1/D2 region of 28S rDNA of the isolate were determined directly from the polymerase chain reaction (PCR) products (White et al. 1990) and deposited into GenBank as accessions, AB500699 and AB500698, respectively. Sequence analysis revealed that the isolate shared 98–100% (rDNA-ITS) and 96–97% (28S rDNA) nucleotide sequence identities with those of C. herbarum found in sequence databases, supporting the morphological identification of the isolate. Pathogenicity Pathogenicity was tested by the inoculation method described by Berner et al. (2007). The isolate was grown on PDA. The conidia were harvested and suspended in sterile distilled water at 106 conidia/ml, and the conidial suspension was sprayed on the leaves of H. muticus plants. The plants were covered with polyethylene bags and incubated at 28°C under moist conditions. Disease symptoms first appeared after 2 weeks in the form of small white spots, which enlarged and turned brown after 3 weeks (Fig. 1m). Control plants, grown under the same conditions and sprayed with water instead of the fungal conidial suspension, did not develop any symptoms. C. herbarum was reisolated from the diseased plants but not from the control plants. Name of the disease 439 Cladosporium leaf spot disease caused by the fungus in H. muticus. Hence, we proposed the name Cladosporium leaf spot (Hanten-byo in Japanese) of Egyptian henbane for this new disease. Acknowledgments The authors thank the Department of Missions (Ministry of Higher Education and Scientific Research, Egypt) for providing financial support through a channel system scholarship to F. F. Abdel-Motaal. References Berner DK, Smallwood EL, McMahon MB, Luster DG (2007) First report of leaf spot caused by Cladosporium herbarum on Centaurea solstitialis in Greece. Plant Dis 91:463 El Naggar SM, Abdel-Hafez SII (2003) Leaf surface, pollen morphology, fungal biodiversity and leaf spots of some wild plants in Sinai, Egypt. Feddes Repert 114:74–90 Johnson DA, Pimentel G, Dugan FM (2008) Cladosporium herbarum causes a leaf spot on Caltha leptosepala (marsh marigold) in Western North America. Online Plant Health Prog. doi: 10.1094/PHP-2008-1121-01-RS Kwon JH, Kang SW, Kim JS, Park CS (2001) Scab of tea (Thea sinensis) caused by Cladosporium herbarum in Korea. Plant Pathol J 17:350–353 Moubasher AH (1993) Soil fungi in Qatar and other Arab countries. Scientific and Applied Research Center, University of Qatar, Doha Oksman-Caldentey KM, Vuorela H, Isenegger M, Strauss A, Hiltunen R (1987) Selection for high tropane alkaloid content in Hyoscysamus muticus plants. Plant Breed 99:318–326 Ouchi S, Hatamoto M, Oku H, Shiraishi T, Yokoyama T, Tateishi M, Fujii S (1976) Brown spot of grapes caused by Cladosporium cladosporioides and Cladosporium herbarum. Sci Rep Fac Agr Okayama Univ 48:17–22 Perelló AE, Sisterna MN, Moreno MV (2003) Occurrence of Cladosporium herbarum on wheat leaves (Triticum aestivum) in Argentina. Australas Plant Pathol 32:327–328 Phytopathological Society of Japan (2000) Common names of plant diseases in Japan. Plant Protection Association, Tokyo, Japan Schubert K, Groenewald JZ, Braun U, Dijksterhuis J, Starink M, Hill CF, Zalar P, de Hoog GS, Crous PW (2007) Biodiversity in the Cladosporium herbarum complex (Davidiellaceae, Capnodiales), with standardisation of methods for Cladosporium taxonomy and diagnostics. Stud Mycol 58:105–156 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, San Diego, pp 315–322 We established that the present disease in H. muticus was caused by C. herbarum. This is the first report of 123