AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: , ISSN Online: , ScienceHuβ,

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1 AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: , ISSN Online: , ScienceHuβ, Isolation and identification of some fungi from certain solanaceous seeds in Sulaimania and Germian regions and their exudate effects on germination rate Jalal H.S. Ismael College of Agriculture,Sulaimani University, Sulaimania, Kurdistan, Iraq ABSTRACT This study was conducted in order to determine the fungi associated with solanaceous seeds at two regions Sulaimania and Germian / Iraq. The fungi which have been isolated and detected from tomato and eggplant seeds at Sulaimania region were: Aspergillus clavatus, A.flavus, A.niger, Cladosporium sp., Penecillium digitatum, Pythium sp., Rhizoctonia sp., Rhizopus arrhizus, Rh.stolonifer and Sclerotinia sp. The highest % of repetition of the fungi A.flavus and Pythium sp. were 0.42 and 0.4 respectively in tomato seeds, while the lowest % of repetition were for each of the fungi A.clavatus and Cladosporium sp. in eggplant seeds and for the fungus Rhizoctonia sp. in tomato seeds. The fungi which were detected and diagnosed from tomato, pepper and eggplant seeds at Germian region were Alternaria alternata, Aspergillus niger A.versicolor, Fusarium oxysporum, Penicillium digitatum, Phytopthora sp., Rhizoctonia sp., Rhizopus arrhizus and Rh.stolonifer. The highest % of repetition was 0.77 for A.niger, while the the lowest % of repetition was 0.05 for Fusarium oxysporum An experiment was conducted to find the negative influences of some fungal exudates on the germination rate (gr) of the seeds. There were obvious and significant effects of the fungal exudates on the (gr) on the tested solanaceous seeds, the % of (gr) decreased significantly Keywords: Fungi, germination rate, seeds, exudates effect, tomato, egg plant, pepper INTRODUCTION Crop seeds will be contaminated with different fungi, some of them attack the seeds in the field internally (contaminating the seed sheaths and within the embryo and endosperm ), and others as an external contamination (Ismael 1997, 2000, Michael 2000 and Maudi 2000 ). On the other hand the contamination occur as an air borne fungi as a result of environmental conditions (high relative humidity (RH) and suitable temperature (temp.) and also high level of seed moisture content ( AbdelHafiz et al 1993 and Ismail et al 2002 ).Seeds are important input for crop production. Pathogen free healthy seed is urgently needed for desire plant propagation. Many plant pathogens are seedborne, causing enormous crop losses ( Islam et al 2009 ). Seedborne diseases are able to spread across international borders very easily and are often difficult to identify as their typical symptoms being rare on seed surfaces except in some legumes as economic impact has increased in recent years their important has increased with regards to many kinds of crop worldwide ( Nishikawa et al 2006 ). Little information is available regarding seedborne fungi in Kurdistan in solanaceous seeds.the purpose of this study is to obtain information on the fungi at different locations both in Sulaimania and Germian regions and the influence of some fungal exudates on seed germination rate MATERIALS AND METHODS Seed collections: Solanaceous seeds were collected from different regions of Sulaimania and Germian regions as amount of gm of each species and from each region. a Sulaimania regions Sharazour: Nawgrdan, Aliawa, Grdenaze, Kedefery, Grdisadiq and Grdisharif, Penjwen : Arozer, Deremiane and Kelaty Germik : Tatan, Kalendrawa b Germian region : Zorkan and Badeen ` Fungi isolation: seeds from each species were taken randomly from each region and 2530 seeds were laid on each (90mm) sterilized disposable Petri dish for surfacecontaminated seed fungi used blotter test while agar test used for inner fungi infesting seeds, in the second method (agar test) the seeds were sterilized by sodium hypochlorite (%1 v/v for 5 mins. ) All the cultured petri dishes were incubated at 25 ± 2º C for 57 days (Michael 2000, Maudi 2000 and Agarwal 1987 ). Percentage of no. of

2 Agric. Biol. J. N. Am., 2010, 1(4): each fungus appeared on each plant species and region was taken using the following formula (Akkasha et.al. 1985). fpr = no. of the repeatability of the fungi on the tested seeds no. of the whole seeds fpr = frequency percentage rate of the fungus species Fungi purification and identification: Each fungus isolated and and cultured on PDA and kept in a refrigerator at 8 ºC, the fungi identified according to external morphological characters using { Barnett and Hunter ( 1972 ) for the genera and Raper and Fennel ( 1965 ), Booth (1971), Ellis (1971, 1976 ), Pitt 1979, Domsch et. al. ( 1980 ), Kozakiewiez (1989), and Pitt and Hocking (1997 ) for the species }. Preparation of fungal exudates and their effects on seed germination rate : After the purification of the fungi, Czapik Dox broth medium was prepared and obtained 150 ml in to the 250 ml sterilized flasks, after sterilization of the medium, cultured with the fungi : Aspergillus niger, A clavatus and Selertinia sp., the flasks incubated at 25±2 C after 21 days of incubation, the flasks component were filtered through two layered filter papers (80mm whatman 1) for 4 to 5 times to ensure complete filtration without fungal mycelia growth, each filtered exudates (50ml) laid into Petri dishes and the seeds soaked into them for each species and varieties (tomato ; Lycopersicum esculentum L.var.Sun,Karina and King Rock ; and pepper ; Capsicum annum L.var, California Wonder ) seeds of each species and varieties laid on the filtered fungus exudates for periods 6,12,18 and 24 hours (lab. temp, was 25± 2ºC), several no. of seeds for each sp. and var. were maintained as control irrigated with Czapic Dox broth only, The factorial CRBD experimental design was applied, the treatments were replicated 3 times. the percentage of germinated seed were calculated according to Warham et. al The means were compared by using LSD test at 5% level. RESULTS AND DISCUSSION Table 1. shows the fungal species isolated from solanaceous seeds in Sulaimania region from tomato seeds the following fungi were detected :Aspergillus clavatus, A. flavus, A.niger, Penicillium digitatum, Pythium sp., Rhizopus arrhizus and Rh. stolonifer, the frequency percentage rate ( fpr ) were : 0.42, 0.08, 0.4, 0.04, 0.025, 0.02, and respectively.whereas the fungi detected were : A.clavatus,A.niger, Cladosporium sp., P.digitatum, Pythium sp. Rh.stolonifer and Sclerotium sp. from eggplant seeds, the ( fpr ) of the above fungi were : 0.025, 0.025, 0.025, 0.1, 0.05, 0.2 and 0.05 respectively. While the average frequency percentage rate ( fpr ) of the fungi as a general for each crop seeds was in tomato seeds and in eggplant seeds. The fungi A.flavus and Rh.stolonifer isolated from tomato seeds had highest repetition at Sharezoor locations with a rate 0.42, while the fungus Rhizoctonia sp. had the lowest repetition with a rate at the region. Regarding Penjween location, the fungus Rh.stolonifer was ranked the highest with a rate 0.2, while Rh.arrhizus possessed the least rate of The only fungus which isolated from tomato seeds at Germik location was Rh.stolonifer with a rate No fungi isolated from seeds of eggplant at Penjween and Germik locations. Table 2. shows fungal species isolated from solanaceous seeds in Germian region ; from tomato seeds the following fungi were isolated and detected : A.versicolor, P.digitatum, Rh.arrhizus and Rh.stolonifer, with ( fpr ) 0.10, 0.10, and 0.25 respectively. While the fungi isolated and detected from eggplant seeds were : Alternaria alternata, A.niger, Fusarium oxysporum, Phytophthora sp. and Rhizoctonia sp. with ( fpr ) : 0.075, 0.77, 0.05, 0.07 and respectively. The only fungus isolated and detected from pepper seeds was A.niger, the ( fpr ) was 0.6. The average fugal ( fpr )s were as a general 0.214, 0.09 and 0.6 for tomato, eggplant and pepper seeds There are similar studies on the fungi associated with solanaceous seeds and others ( Basak et. al. 1989, Fiume 1994, Essien et. al. 1999, Nishikawa et. al and Dutta 2007 ). Table 3. shows significant ( P = 0.05 ) effects of the fungal exudates on the % germination rate ( gr ) of tomato and pepper seeds in different cultivars and for different regions. There were also significant effect of the fungal exudates ( among them ) on the % of ( gr ). The results can be arranged as following : 1. Fungal exudates of Sclerotinia sp. 2. Fugal exudates of A.clavatus 3. Fungal exudates of A.niger 616

3 Agric. Biol. J. N. Am., 2010, 1(4): There were significant effects of different fungal exudates ( The toxins which were produced by the fungi : A.niger, A.clavatus and Sclerotinia sp.) at the different periods seed soaking within the fungal exudates on the % of ( gr ) of tomato and pepper seeds ( Ismael 1997, 2000 ). It appeared that the influence of fungal exudates were less at the first periods especially at 6 hrs, while the effects increased by raising the periods to 24 hrs.. On the other hand Sclerotinia fungus exudates at the period after 12 hrs affect the % of ( gr ) and reached to zero ( 0.00 ), while the % of ( gr ) of the control was in pepper seeds ( California wonder cultivar ) ( Table 3 ) Table 1. The percentage of frequency of fungi isolated from tomato and eggplant seeds produced by farmers in different regions of Sulaimania regions / Iraq. Sharezoor Penjween Germik Regions Fungi Nawgrdan Aliawa Grdenaze Kedefery Grdisadiq Grdisherif Arozer Deremiane Kelaty Tatan Kelendrawa General Aspergillus clavatus A.flavus A.niger Cladosporium sp Penicillium digitatum Pythium sp Rhizoctonia sp Rhizopus arrhizus Rhizopus stolonifer Sclerotinia sp General seeds were tested for each plant species and region ( ) : No fungus isolated ( ) : The percentage of the fungi isolated from tomato seeds and ( ) : the percentage of the fungi isolated from eggplant seeds. 617

4 Agric. Biol. J. N. Am., 2010, 1(4): Table 2 : The percentage of the frequency of fungi isolated from tomato, eggplant and pepper seeds produced by farmers in Germian regions / Iraq. Gwezila Badeen Germian Brdbrd Zorkan Sedelan Regions Fungi Alternaria alternata Aspergillus niger A. versicolor Fusarium oxysporum Penicillium digitatum Phytophthora sp Rhizoctonia sp Rhizopus arrhizus Rhizopus stolonifer seeds were tested for each plant species and region ( ) No fungus isolated. ( ) : The percentage of the fungi isolated from tomato seeds, ( ) : the percentage of the fungi isolated from eggplant seeds and ( ) : the percentage of the fungi isolated from pepper seeds. Table 3 : The effect of Aspergillus niger, A.clavatus and Sclerotinia sp. fungal exudates on the percentage of seed germination rate of three tomato varieties and one pepper var. within different soaking periods. Pepper Tomato California Wonder a b b bc bcd cde def efg fgh gh King Rock a b bc bc c c d d d d e e e Karina a a bc bc bc cd de de e e f f f Sun a ab b bc bc cd de def ef fg gh h h Each figure is the mean of three replications. There are significant differences among variable letters in the columns. *P = Periods of soaking the seeds within the fungal exudates ( P1,P2,P3,P4 : 6,12,18,24 hrs respectively ) An = Aspergllus niger, Ac = A. clavatus and S = Sclertinia sp. Treatments * Control AnP1 AnP2 AnP3 AnP4 AcP1 AcP2 AcP3 AcP4 SP1 SP2 SP3 SP4 LSD ( P = 0.05 ) Varieties

5 Agric. Biol. J. N. Am., 2010, 1(4): REFERENCES AbdelHafiz,S.I.I.; Moubasher,A.H. and Barakat,A. (1993). Seasonal variations of Fungi of outdoor air and sediment dust at Assiut region, Upper Egypt, Grana 32: Agarwal, V.K. (1987). Principles of seed pathology 1 and 2. CRC press, Boca Rotan, Florida, USA. Akkasha, M.A.; AlHasan, K.K. and AbdAlhemid M.T., (1986). Isolation and Identification of some seed fungi of wheat and their effects on germination rate. Journal of Agricultural Researches and Water Resources, 24 (2):89102 ( In Arabic ). Barnett,H.L. and Hunter, B.B. (1972). Illustrated genera of imperfect fungi. 3 rd edition, Burgess Publishing Co., 273 pp. Basak,A.B.; Mridha M.A.U., and Shamima, S. (1989). Mycoflora associated with different cultivars of brinjal seeds collected from Chittagong district. Seed research,17(1):9395. Booth,C. (1971).Genus Fusarium. Commonwealth Mycological Institute ( CMI ), Kew,Surry,England. 273 pp. Domsch,K.H.; Games, W. and Anderson, T.H. (1980).Compendium of soil fungi. Academic press, London. Dutta,P.S. (2007). Seed borne fungi of mustard ( Brassica juncea Coss ). J.Mycopathol. Res., 45(2): Ellis,M.B. (1971). Dematiacous Hyphomycetes. CMI, Kew, Surry, England. 608 pp. Ellis,M.B. and Ellis, J.P. (1985). Microfungi on land plants, An Identification hand book, Croom Helm. Ltd, Kent, U.K. Essien,J.P.; Odoemena, C.S. and Essien, E.P. (1999). Effect of traditional preservation on seed borne pathogens and seed viability of capsicum okra and tomato. Trop. Sci., 39: Fiume, F. (1994). Seed disinfection of pepper ( Capsicum annum L. ) from Alternaria Nees. Acta Horticulturae 362, ( Seed research in Horticulture V. ). Islam, S.M.M. ; Masum, M.M.I. and Fakir, M.G.A. (2009). Prevalence of seedborne fungi in sorghum of different location of Bangladesh. Scientific Research and Essay, 4(3): Ismael, J.H.S. (1997). The effect of some isolated fungi from some wheat cultivars and their exudates on seed germination rate in Sulaimani Province, Zankoy Sulaimany Journal, 1(1):8795.( In Arabic ) Ismael, J.H.S. (2000). Isolation and identification of some pathogenic fungi from rice seeds. Kurdistan Academician Journal ( KAJ ), 1A(1):1721. Ismail, M.A. ; AbdelHafez, S.I.I. and Moharam, A.M.(2002). Aeromycobiota of western desert of Egypt. African Journal of Science and Technology ( AJST ) Science and Engineering Series, 3(1):19. Kozakieweiz, Z. (1989). Aspergillus species on stored products. Mycological papers No.161, Kew, Surry, England, 188 pp. Maudi, R.B. (2000). Seed borne diseases and their control 1 st edition, 2 nd print CAMI publishing, Wallingford, U.K., 188 pp. Michael, S. (2000). Seed pathology. 3 rd edition, AlMaarif Establishment, Alexandria, Egypt, 334 pp. ( In Arabic ) Nishikawa,J. ; Kobayashi, T. ; Shirata, K. ; Chibana, T. and Natsuaki, T. (2006). Seed borne fungi detected on stored solanaceous berry seeds and their biological activities. J. Gen. Plant Pathol.,72: Pitt,J.I. (1979). The genus Penicillium and its telomorphic states Eupenicillium and Talaromyces.Academic press, London. 634 pp. Pitt, J.I. and Hocking, A.D. (1997). Fungi and food spoilage. 2 nd edition, Blackie Academic and Professional Press, Cambridge, U.K. 593 pp. Raper, K. and Fennel, D.I. (1965). The genus Aspergillus. Williams and Wilkins, Baltimore, USA, 686 pp. Warham, E.J.; Bulter, L.D. and Sutton, B.C. (2003). Seed testing of maize and wheat ( A laboratory guide). 1 st edition. CIMMYT, Mexico 619