Bull 31 Goner A. Shaker & Hany S. Alhamadany Bull. Iraq nat. Hist. Mus. (2015) 13 (3): 31-38 ISOLATION AND IDENTIFICATION OF FUNGI WHICH INFECT FENNEL Foeniculum vulgare Mill. AND ITS IMPACT AS ANTIFUNGAL AGENT Goner A. Shaker and Hany S. Alhamadany Iraq Natural History Museum, University of Baghdad, Baghdad, Iraq ABSTRACT The study included isolation and diagnosis of fungi that infect Foeniculum vulgare Mill planted in the Department of Drugs and Medicinal Plants, Pharmacy College - University of Baghdad, different symptoms such as wilting and yellowing, stunting on the plants were observed fungi: Alternaria alternata, Rhizoctonia solani, Phoma herbarum and Fusarium oxysporum, The disease incidence ranging between 5-10%. Studied the effect of Foeniculum vulgare plant seeds extract against Alternaria alternata, Rhizoctonia solani, Phoma herbarum and Fusarium oxysporum,where tested the concentrations 0,2.5 and 5% of alcoholic extract of fennel seeds showed effectiveness noticeable trend all fungal isolates and showed significant differences between different levels of concentrations, at P = 0.01 and P = 0.05. INTRODUCTION The studies are looking for new products with little residues in order to comply with food safety standards, and Various industries are now looking to alternative sources for a number of reasons, including natural and environmentally friendly antimicrobials, antibiotics, antioxidants and Crop protection agents (1), the use of fungicides is more harmful in the post- harvest period because of the short time between treatment and consumption, However, these chemicals will be terminates in the near future due to their potential adverse impact on the environment. Some fungus have shown resistance against Fungicides, such as Benzimidazoles, Imazalil and Prochloraz due to repeated usage and some of them such as Mucor and Rhizopus are not sensitive and need especial fungicides to be controlled. (2). Foeniculum vulgare Mill, commonly known as Fennel an aromatic and medicinal Plant, it is refer to family Umbellifarae (Apiaceae). It is an indigenous herb Mediterranean sea, is a very popular spice as well as an important traditional Chinese medicine. (3,4). Is an annual, biennial or perennial herbs (5). The F.vulgare is believed to be one the oldest medicinal plant, because it has been known from the time the first prophet, Adam (6). Fennel is chiefly known as culinary herb but it is a commonly used household remedy for various medicinal purposes (7,8). An analysis of fennel shows it to consist of moist 6.3%, protein 9.5%, fat 10 %, minerals 13.4%, fiber18.5% and carbohydrates 42.3%. it is mineral and vitamin contents are calcium, phosphorous, iron, sodium, potassium, thiamine riboflavin, niacin, and vitamin C. (9). Recently, essential oils of the fruits of Egyptian fennel showed high antimicrobial and antioxidant activities,18 major monoterpenoids in essential oils as trans-Anethole,estragole, fenchone and limonene were highly abundant in all examind oils.(10) It is a well-known medicinal plants within a broad stimulant, diuretic, carminative and sedative and galactagogic, emmenagogic, expectorant and antispasmodic (4). Essential oil of fennel has been reported to possess antifungal activity (11). Fennel essential oils could also be used as possible bio fungicides alternative to synthetic fungicides against phytopathogenic fungi as it has been reported to reduce the mycelial growth and germination of Sclerotinia sclerotiorum (12). 32 Isolation and Identification of Fungi Fennel crop suffer from many diseases causing by fungi pathogens, such as Rhizoctonia solani, Pythium aphanidermatum, Cercospera sp, Sclerotinia sclerotiorum, Alternaria alternata and Fusarium oxysporum (13). Our study aimed to evaluate the effectiveness of inhibitory alcoholic extract against fungi that infect the plant because of the available benefits to Foeniculum vulgare and its effectiveness. MATERIALS AND METHODS 1-Sampling: The samples of Foeniculum vulgare were collected from the Department of Drugs and Medicinal Plants, Pharmacy College - University of Baghdad, which showed symptoms of yellowing and wilting and stunting and death of the seedling, collected the samples of F. vulgare seeds harvested in the previous season and brought to the laboratory and kept in the refrigerator degree1 25 C°. 2-Isolation: washed the plant samples collected which are leaves, stems and roots, with water, cut off to parts of a length of 2-5 mm, and sterilized with sodium hypochlorite 1.2% and then washed with sterilized distilled water and dried by sterilized filter papers and then transferred to the Potato Dextrose Ager plate and incubated in the incubator at 1 25 C° until the pathogen growth emergence. 3- Morphological Diagnosis: purified fungal isolates by transfer from the end of the isolated fungal culture by sterile needle the fungi mounted on slide, stained with lacto phenol-cotton blue and examined under microscope diagnosis based on morphological characteristics of the colonies and spores depending on taxonomic keys cited by (14,15) and preserved in Slants at 4-5 C ° . 4-Estimation the disease incidence:: the survey have been done, in order to determine the infection percentage and follow from the first stages of the growing of the beginning of October until the harvest in June, F. vulgare was planted in two boards and the percentage of wilting infection and seedling damping - off calculated depending on the equation: The percentage of disease infection No. of diseased plants / No. of all plants 100 5-Assays the efficiency of F. vulgare alcoholic extract as antifungal: Adopted a F. vulgare seeds collected from the previous planting season after cleaned, washed and dried and milled by electric mill and preserved in a sterile polyethylene bags. Preparation alcoholic extract: attended the alcoholic extract 5 gm of F. vulgare seeds powder was soaked in 50 ml of alcohol ethanol 95% overnight at room temperature and then the extract was centrifuged at (7000 rpm) for 20 minutes and the extract was filtered by using Whatman No 0.1 filter papers and the alcoholic extract of fennel seeds was stored at 4 C ° till further use (16) To increase the required size of the alcoholic extract of the seeds of F. vulgare will be on the basis of (V/V) ratio of the same material. Testing the effectiveness of alcoholic extract: alcoholic extract was added to the media PDA before solidification, three concentrations of extract were prepared, Zero, 2.5, 5%, shake the media before it pour (in order to homogenize) in a sterile Petri dish 9 cm in diameter and 33 Goner A. Shaker & Hany S. Alhamadany leave at room temperature to next day, three replicates of each concentration were inoculated by placing 5 mm diameter discs of Alternaria alternata, Fusarium oxysporum, Rhizoctonia solani and Phoma herbarum and incubated at temperature1 25 C° until the mycelia growth appears and measuring the linear growth. The percentage inhibition of mycelia growth was calculated by using the formula (17): % inhibition of mycelia growth Dc. Dt. / Dc. 100 Dc. diameter of control, Dt. diameter of test (treatment) RESULTS AND DISCUSSION 1-Isolation and morphological diagnosis: the fungi were purified, identify and diagnosis as Alternaria alternata, Rhizoctonia solani, Phoma herbarum and Fusarium oxysporum. The study (13) indicated that Rhizoctonia solani, Fusarium oxysporum and Fusarium moniliforme are pathogens attack the fennel plant causing diseases such as Root rot, wilting and blight. 2-Estimation the fungal disease incidence: It is clear in the Fig. 1. That the percentages of disease incidence in the months January, March and May (5,7,10%), respectively, the diseases more common in nurseries and be clear and severely when the condition is favorable, high moisture in soil and a little are exposed to sunlight. Figure (1): The Percentage of disease incidence of Foeniculum vulgare. 3-Testing the efficiency of F.vulgare alcoholic extract: The antifungal activities of the tested plant extract (Foeniculum vulgare) were investigated at different concentrations Table (1) and Fig.2. The results clearly revealed that fennel extract could cause growth inhibition on the four tested fungi, although the rate of inhibition of tested fungi shows that two concentrations 2.5 and 5% were found to be inhibitory to mycelia growth and the rate of inhibition increased generally by increasing the concentration, and 5% concentration was the most effective on the mycelia growth of Alternaria alternata, Rhizoctonia solani, and Phoma herbarum and Fusarium oxysporum, were 1.31, 1.56, 1.13 and 1.65 cm, respectively Fig.1 compared to the treatment of control where the concentration was 0% the rates of fungal growth diameter was 2.4, 9, 2.5 and 2.63 cm, and shown the existence significant differences between mycelia growth rates in the dishes and between concentrations of alcoholic extract of F. vulgare and at P 0.01 and P 0.05, the same results obtained from study (18) indicated the fennel (F. vulgare) seed is a potential source of natural antioxidant of the both water and ethanol seed extracts, and (19) showed that Foeniculum vulgare extract which was the one of the 49 medicinal plants extracts inhibited both fungal growth and production of AFs B1 and G1 producing of Aspergillus parasiticus and intentioned to use this plant as effective antimicrobial to protect foods and feeds from 0 5 10 Jan March May %disease incidence 34 Isolation and Identification of Fungi toxigenic fungus growth and subsequent (AF) Aflatoxin contamination. The reasearcher (20) the main constituents were E-anethol (92.9%), p-alylanisol (2.2%), Z-α-biosabolene (1.8%) for anise oil and E-anethol (71.2%), limonene (8.2%), Fenchone (8.53%), Methylchavicol (7.01%) for fennel oil and especial antifungal activities arise from interaction of components or complexity of the oils. The study (10) suggests that antioxidant activity is mostly related to the concentration of trans-anethole. The major component the volatile oil was trans-anethole (70.1%) and showed complete zone inhibition against Aspergillus niger, Aspergillus flavus, Fusarium graminearum and Fusarium moniliforme (21). Aqueous and ethanol extract of F.vulgare were used showed significant inhibition of growth of Fusarium solani, Rhizoctonia solani and Macrophomina phaseolina (22). The aqueous and alcoholic seed extract of F. vulgare were evaluated for their antifungal activity against A. alternata, Mucor rouxii and Aspergillus flavus (Neet et al. 2013). Concentrations of 10, 20 and 30% extracts of F. vulgare reduced the fungal biomass production in all tested pathogenic fungal species especially in initial growth stage (23). Figure (2): The activity of F.vulgare alcoholic extract on the mycelia growth of Alternaria alternata, Rhizoctonia solani, Phoma herbarum and Fusarium oxysporum (from right to left), at the concentration 0, 2.5, 5 %( from up to down). Table (1): Effect of extract of F. vulgare plant on the linear growth (cm) of Alternaria alternata, Rhizoctonia solani, and Phoma herbarum and Fusarium oxysporum. Ethanol extract concentrations Fungi species 0 % 2.5% 5% 2.4* 2 1.31 1-Alternaria alternata 9 4.8 1.56 2- Rhizoctonia solani 2.5 1.81 1.13 3-Phoma herbarum 2.63 2.13 1.65 4-Fusarium oxysporum * The three rate readings with significant differences at probability 0.05 and 0.01, and value of LSD fungi = 7.3, LSD conc. = 8.2 and LSD fungi x conc. = 5.5. 35 Goner A. Shaker & Hany S. Alhamadany Table (2): The percentage inhibition of mycelia growth by fennel seed extract of Alternaria alternate, Rhizoctonia solani, Phoma herbarum and Fusarium oxysporum. inhibition of mycelia growth % Fungi species Extract conc.5% Extract conc.2.5% 45.30 14.06 1-Alternaria alternata 70.55 25.55 2- Rhizoctonia solani 46.66 13.33 3-Phoma herbarum 32.85 21.41 4-Fusarium oxysporum LITERATURE CITED Abdelaaty, A. Shahat, Abeer Y. Ibrahim, Saber F. Hendawy, Elsayed A. Omer, Faiza M. Hammouda, Fawzia H. Abdel-Rahman and Mahmoud A. Saleh. (2011). Chemical Composition, Antimicrobial and Antioxidant Activities of Essential Oils from Organically Cultivated Fennel Cultivars. Molecules, Vol. 16, 1366-1377. Aboelsoud, H.N. (2010). Herbal medicine in ancient Egypt. J. Med. Plants Res. Vol.4 (2). pp. 82-86. Aghili MH, ed. Drug reservoir (Persian). Tehran, Iran: Tehran Univ. of Med. Sci. 405-406. Barnett, H.L. and Hunter, B. B. (1972). Illustrated genera of imperfect fungi. Burgess Pub. 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(2015) 13 (3): 31-38 وتأثيره بذوره Foeniculum vulgare Millعزل وتشخيص الفطريات التي تصيب نبات كمضاد فطري كونر عبدالوهاب شاكر و هاني صابر الحمداني جامعة بغداد/ مركز بحوث ومتحف التاريخ الطبيعي الخالصة ، املزروعة Foeniculum vulgare Millشملت الدراسة عزل وتشخيص الفطريات التي تصيب نبات حبة الحلوة ها حاالت من أعراض الاصفرار يجامعة بغداد، وشوهدت عل -الطبية، كلية الصيدلة في قسم العقاقير والنباتات :وتم تشخيص الفطريات ،والذبول والتقزم Alternaria alternata, Rhizoctonia solani, Phoma herbarum وFusarium oxysporum وكانت نسبة إلاصابة ,Alternariaضد Foeniculum vulgareكما وتم دراسة تأثير املستخلص الكحولي لبذور نبات .٪01-5تتراوح بين Rhizoctonia, Phoma وFusarium من املستخلص الكحولي لبذور نبات حبة 5و 5.5، 1، حيث اختبرت التراكيز ٪ معنوية ما بين املعامالت ما الحلوة وأظهر فعالية ملحوظة اتجاه جميع العزالت الفطرية من خالل ظهور فروقات .P 0.05و P 0.01بين الفطريات والتراكيز عنداحتمال