Atlas Journal of Biology 1 (3): 47-51, 2011 doi: 10.5147/ajb.2011.0047 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 5. P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Punica granatum: A New Host of Bipolaris spicifera in Morocco Oumama Kadri*, Rachid Benkirane, Amina Ouazzani Touhami, and Allal Douira* Laboratoire de Botanique et de Protection des Plantes, Université Ibn Tofail, Faculté des Sciences, Kénitra, Morocco Received: February 12, 2011 / Accepted: May 8, 2011 __________________________________________________ *Corresponding authors: douiraallal@hotmail.com & ok_ouma@ hotmail.com. Introduction Pomegranate (Punica granatum L.) is a fruitful ornamental tree in the tropical and subtropical areas of the world, including the Mediterranean Basin and Asia. Several varieties are gener- ally associated with this species. Punica granatum L. ‘Nana’ is a dwarf variety more valuable for its numerous small flowers and long flowering period (Jianzhu et al., 2003). In March 2009, the survey in the public and the University of Sciences gardens of Kenitra city (Morocco) showed that 60% of leaves of 100 plants of Punica granatum L. ’Nana’ presented foliar lesions affecting the aesthetic appearance of this orna- mental plant. At the time of attacking, temperatures varied be- tween 10 and 25°C (average of 17.5°C) and the rainfall for the month was 19 mm. The necrotic lesions developed on the periphery and in the middle of leaves were sometimes numer- ous, often circular with dark brown color and measuring 1 to 4 mm in diameter. Later, the midst of these lesions becomes more clear and surrounded by a border of dark purple (Fig. 1). This was the first time that these symptoms were observed on Punica granatum L. ’Nana’. 47 SHORT COMMUNICATION Abstract During March 2009 in the public gardens and the Univer- sity of Sciences of Kenitra city (Morocco), 60% of leaves of 100 plants of pomegranate (Punica granatum L. ’Nana’) presented foliar lesions that affecting the aesthetic appear- ance of this ornamental plant. Bipolaris spicifera was isolated from ten necrotic lesions of 25 leaves. The artificial inocula- tion of the healthy leaves of three plants of Punica granatum L. ’Nana’ by conidial suspension of the pathogen induced the same lesions to that observed in nature. The diseased fo- liar surface and the total number of diseased leaves 30 days after inoculation with B. spicifera conidial suspension were 37.5% and 78. Conidia production of B. spicifera on inocu- lated leaves was 0.81 x 105 spore cm-2 and the fungus was re-isolated from lesions on inoculated plants. This is the first report of B. spicifera on pomegranate in Morocco. Keywords: Punica granatum L.’Nana’, Bipolaris spicifera, Lesions, Inoculation. 48 Material and Methods Twenty five leaves showing necrotic lesions were collected from 5 plants of Punica granatum L. ‘Nana’. Leaves with lesions were cut in small fragments, washed with water, disinfected in alcohol and placed in 90 mm Petri dishes on three filter paper discs moistened with sterile distilled water with 5 lesions per Pe- tri dish. The lesions were incubated at 22°C under continuous fluorescent lighting. Lesions were examined using an optical mi- croscope after 4 days and conidia were taken with a capillary tube and placed on PSA media (Potato Sucrose Agar: 200 g potato, 20 g sucrose, 15 g Agar-agar, and 1000 ml distilled water) and incubated at 28°C to determine the pathogen. Three plants of Punica granatum L. ’Nana’ (plant I: 252 total healthy leaves; plant II: 327 total healthy leaves; plant III: 349 total healthy leaves) were maintained in a greenhouse at 17- 25°C (April 2009). These plants were inoculated by spraying with 60 ml of a conidial suspension containing 105 conidia ml-1 of fungus. The conidia concentration was adjusted with distilled water, containing 0.02% of Tween 20 and 1% gelatin. The con- trol plants were inoculated with distilled water containing Tween 20 and gelatin. The inoculated plants were kept covered for 48h with black bags and then moved to a greenhouse. The test was repeated twice and the same fungus was consistently re-isolated from these lesions. The notation of results was made seven days af- ter inoculation and during one month. The diseased foliar sur- face (%) was scored according to the scale of Notteghem et al. (1980). The conidia production (conidia cm-2) of fungus on the in- fected pomegranate leaves was estimated according to the technique of Hill and Nelson (1983). One month after inocula- tion, the leaves with lesions were harvested from the inoculated plants, cut into pieces of 1cm2 and placed in 90 mm Petri dishes on three filter paper discs moistened with sterile distilled water. The dishes were incubated from 48 to 72 hours at 22°C un- A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 5. P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 5. P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Fig. 1. Foliar symptoms of Bipolaris spicifera infection on Pomegranate plants (Punica graminum L. ‘Nana’) in nature. (A): dark brown circular lesions; (B) lesion surrounded by a border of dark purple color; (C): lesions are sometimes numerous. A B C 49 der continuous fluorescent lighting. Then, each fragment was placed in a test tube containing 1 ml of sterile distilled water and agitated by a vortex mixer for 2 min. The conidia of the pathogen were counted using a Malassez slide under an optical microscope at magnification ×100 with three counting of each sample. Results and Discussion On PSA, the monospore culture of the fungus isolated was black olive, dark inside, becoming clearer toward the periphery. The conidia were brown, 3 septates, elliptical often cylindrical (16.75±4) μm long, (8.25±2) μm wide, rounded at the ends, each with an evident hilum (Figure 2). The conidiophores were erected, unbranched, partitioned, 4-8 μm wide and geniculat- ed. Based on the morphological description (Ellis, 1971), the fun- gus was identified as Bipolaris spicifera (Bainier) Subram (1971). Symptoms on leaves of Punica granatum L. ‘Nana’ appeared seven days after inoculation by B. spicifera and the necrotic le- sions were identical to that observed in nature (Figure 3). The tested plants of P. granatum L. ‘Nana’, all showed sensitivity relative to B. spicifera. Between the 7th and 30th day after inocu- lation, the amount of disease increased with time. The diseased foliar surface of inoculated plants was 0.5% on the 7th day and increased to 37.5% on the 30th day pot inoculation (DPI). The total number of leaves with symptoms increased from 10 on the 7th day to 78 on the 30th. Bipolaris spicifera was also able to produce conidia on the lesions of P. granatum L. ‘Nana‘plants with an average of 0.81×105 spore cm-2. Bipolaris spicifera isolated from 77 host plants, from air and soil (Ellis, 1971). In Morocco, this pathogen was isolated for the first time from foliar lesions of Oryza sativa (Ennaffah et al., 1997), foliar lesions of Hibiscus rosa-sinensis (Meddah et al., 2007) and from watermelon (El Mhadri et al., 2009). This fungus was also isolated from Chloris verticillata, Eragrostis cilianensis and Paspalum leave (Roane and Roane, 1996, 1997); Cynodon A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 5. P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Fig. 2. Colonies (A), Conidiophore (B) and conidia (C) of Bipolaris spicifera isolated from Punica granatum L. ‘Nana’ and cultivated on PSA medium. A B C 50 dactylon and Deschampsia flexuosa (Roane, 2004, 2009); Av- ena sativa, Hordeum vulgare, Secale cerea, and Triticum aestivum (Cook and Dubé, 1989); Oryza sativa, Phaseolus vulgaris, Sor- ghum bicolor, and from Zea mays (Mendes et al., 1998). 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