SEEDS AND THEIR BIO-CONTROL IN VITRO

SEEDS AND THEIR BIO-CONTROL IN VITRO Salem Milad and Ali Ben Milad AL-Asmarya Islamic University, Faculty of Science, Department of Microbiology, Libya. E-mail: sbinmeelad@gmail.com ABSTRACT Various fungal species (Eleven species) belonging to different five genera have been. Malty and Blanka found in or on localized broad bean seeds cultivar. These fungi were purified and identified as Aspergillus flavipes, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, Aspergillus wentii, Penicillium brevicompactum, Penicillium expansum, Fusarium moniliforme, Rhizopus stolonifer and Mucor eircinellricles. Results also indicated that Aspergillus flavus isolate No. (1) was significantly more pathogenic as it totally inhibited seed germination (0%) and caused the highest percentage of seed infection (100%), followed by Aspergillus niger isolate No.(1) and Aspergillus flavus isolate No. (3). It was found that Aspergillus flavus isolate No. (2) and No (5) were the least pathogenic. However, the difference between any two tested fungi was significant. In this study showed that Trichoderma album and Trichoderma harzianum significantly inhibited the growth of Penicillium expansum by 77.7 and 72.2%, respectively, followed by Aspergillus flavus and Aspergillus flavipes. However, Aspergillus ustus was the least affected fungus in this concern (22.2%). 1. INTRODUCTION Broad bean (Vicia faba L.) is the major annual leguminous crop. It is cultivated in winter season mainly for human consumption as green or dried seeds. In some cases, it is used for animal feeding. It is an excellent protein source (20-25%), calcium (0.15%), phosphorous (0.50%), lycine (1.5%) and methionine-cysteine (0.5%). Also, it is an excellent source of complex carbohydrates, dietary fiber and minerals [10]. Many agricultural commodities are vulnerable to attack by a group of fungi. Fungi are the major causal of deterioration and spoilage in stored crops [6]. - 38 -

Broad bean seeds are attacked by several fungal pathogens that causing a tremendous yield losses of these diseases, seed-rots which lead to reduction in seed size and seed discoloration [11]. Isolated twenty six species of fungi belong to fourteen genera from ninety seed samples of six leguminous crops namely pigeon pea, chickpea. Lablab hyacinth, alfalfa, phaseolus vulgaris, kidney bean, and cowpea. From these isolates. six species are new records to the mycoflora of Sudan, where as different fungal species were reported for the first time in the seeds [2]. Chemical fungicides and biological control provides support to disease management. However, the use of fungicids is being discouraged due to economic reasons and growing concern for environment and safety issues. On the other hand, the bioagents give an advantage in the competition for space and nutrients with plant pathogenic fungi [22]. The purpose of the present investigation was to the study the fungi associated with broad bean stored seeds of two cultivars. The effect two Trichoderma species on the growth of isolated fungi was investigated in vitro. 2. MATERIALS AND METHODS Seeds sampling: Stored broad bean seeds of two cultivar named (Maity and Blanka) were obtained from the Agricultural Research Center at keam of Libya. The seeds of each cultivar were put in plastic bags and kept at 40c for subsequent studies. - 39 -

Isolation of seed associated fungi: Working seed samples (200 seeds each) were tested using two standard agar plate techniques [15] method namely. Blotter and Czapek a. Blotter method: Seeds were divided into two parts (200 seeds / each), the seeds in the first part were surface sterilized by dipping in 2% sodium hypochlorite solution for three mins, then washed twice in sterilized distilled water to get red of sodium hypochlorite and plated on three layers of blotter papers moistened with sterilized distilled water containing rose bengal stain (1% of 10% solution) in petri-dishes to avoid bacterial growth [8]. The second part was carried out from the rest seeds, but without surface sterilization. Ten seeds were transferred to each plate under aseptic conditions and 20 dishes were employed for each treatment. b. Czapek s agar plate method: In this method rose bengal stain was also used. the practice procedures were carried out as in the Blotter method. The sterilized or un-sterilized seeds were separately plated on Czapek s agar medium at rate of ten seeds /dish. All the plates were incubated at 25 ± 2 for 10 days. Isolation and purification : The developed fungi were isolated and purified using a single spore technique [14] and / or hyphal tip method [20]. The pure cultures were kept on Potato Dextrose Agar medium (PDA) and maintained at 5-8 in the refrigerator till use. The identifications were made on 10-days old cultures - 40 -

by studying the cultural and microscopic characteristics of each isolate [4,19]. The identification was verified by Mycology Research and survey of Plant Diseases Section, Plant Pathology Research Institute, Giza Egypt. The number of fungi and their frequency were determined. Pathogenicity test: The pathogenic capability of nine different fungal isolated which were previously isolated in high frequency from broad bean seeds was carried out in vitro. These isolates consist of five isolates of Aspergillus flavus, two isolates of Aspergillus niger and isolate of penicilium expansum and Rhizopus stolonifer. Seeds of Blanka cultivar were more susceptible than the other cultivar and thus they were used in this study. Apparently free seeds of any mechanical injury were employed. They were surface sterilized and separately inoculated with the previous test fungal isolates as follows: The spores of each tested fungal isolate were liberated using sterilized needle by scrapping the surface of 10- days old cultures grown on PDA medium. The spores were then suspended into 20ml of sterilized water. The inoculum density was determined by a Haemocytometer slide and adjusted to 10 5 spores /ml. The moisture content of broad been seeds cv. Blanka was determined in three replicates according to [12] using the following formula: X-Y X 100 Where : X = Initial weight of the sample. - 41 -

72 hrs. Y = Final weight of the dried sample after in an oven at 104 for The moisture content of the intact seeds was raised to 20% according to [12] using the following formula : B-A / 100-B 100 Were : B = The desired level of moisture A = The original level of moisture The adjusted spores suspersion (2000 spores /1 gm seeds ) was employed to inoculate the seeds as the proposed method of [17]. The control seeds were only treated with sterilized distilled water, four replicates 50 seeds / replicate ) were used for each treatment. All the artificially inoculated and the control seeds were incubated at 25± 20C for 15 days. The percentage of infection and seed viability were calculated according to [3,9]. Determination of infected seeds percentage: Two hundred Czapek s agar medium (10 seeds / dish) and incubated at 28±2. The plates were daily examined up to 10 days. The seeds which exhibited fungal growth were counted and the percentage of seed invasion was calculated. Determination of Seed Viability: Two hundred seeds were maintained on sterile moist filter papers in petridishes kept at 28± 2 for 10 days and the germination was determined. - 42 -

Biological Control of Seed Associated Fungi: Two species of Trichoerma namely; Trichoderma album obtained from Egypt and Trichoderma harzianum obtained from Libya were used to estimate their antagonistic effect against the growth of fungi in vitro [16]. In dual culture plante assay, a 5mm diameter of inoculum discs of each Trichoderma, (10 days old cultures ) were placed at one cm away of plates edge containing PDA medium. Similar inoculums discs of each tested fungi (10 days old culture ) were placed at one cm to the opposite side of the plate. Control treatment were inoculated only with each tested fungus without a bioagent.tree replication dishes were used for each treatment. All plates were incubated at 25± 2 till the fungi growth in the control treatment reached to the plate edge. The growth inhibition pereentage (GIP) was calculated according to [27] as follows. GIP = Average growth in the control Average growth in the treatment Average growth in the control Statistical Analysis: The obtained data were statistically analyzed using the standard procedure of split design as mention by [23]. The averages were compared at 5% level of probability using least significant differences (L. S. D.) according to [13]. - 43 -

3. RESULTS AND DISSCUSSION Fungal isolation from broad bean malty cultivar localized seeds using both blotter and Czapek s agar plate techniques : a. Isolation from sterilized seeds The isolation from surface sterilized seeds trials yielded11species of fungi belonging to 5 genera (Table 1). They were purified and indentified as Asperiglls flavipes, A. flavus, A. niger, A. terrus, Fusarium moniliforme, Mucor circinelloids, Penicillium brevicompactum, P.expansum and Rhizopus stolonifer. The most frequent fungi were Aspergillus flavus and Aspergillus niger. Their frequency percentages were 39.80 and 15.79%, respectively. On the other hand, each of Aspergillus flavies, Aspergillus ustus and Penicillium brevicomactum were less frequency (3.75% for each). The blotter method yielded a great number of fungi around (83) compared to that of Czapek s agar plate method (50). This means the majority of fungi need a high level of relative humidity (RH) that present in the plotter technique and thus encourage the fungal growth. These results are in agreement with those recorded by [11] who reported that the Plotter method - 44 -

(TABLE 1): THE NUMBER AND FREQUENCY OF FUNGI ASSOCIATED WITH BROAD BEAN MALTY CULTIVAR SEEDS* USING BLOTTER AND CZAPEK S AGAR PLATE TECHNIQUES *The infection percentage of broad bean Malty cultivar seeds = 42.5% - 45 -

yielded a great number of fungi compared to the deep-freezing method for both sterilized and unsterilized surface seeds. Also, [21] stated that Aspergillus and Penicillium were the most common genera in 10 different broad bean cultivars. On the contrary, present data are, somewhat, in agreement with those recorded by [26] who found that the most frequent species were Aspergillus niger, od Aspergillus colummaries, Penicillium citrinum, Penicillium funiculosum, Rhizopus stolonifer and Fusarium moniliforme. The difference between these results could be due to the cultivar tested, seeds situation (sterilized or unsterilized) and / or the storage period. b. Isolation from surface unsterilized seeds: The total number of 207 fungal isolates were isolated from broad bean unsterilized surface sterilized seeds compared to 133 fungal isolates which isolated from surface sterilized seeds (Table 1). Several fungl species belonging to 4 genera were isolated. Aspergillus niger was of the highest frequency (29.95%), followed by Aspergillus flavus (25.12%), Penicillium expansum, (12.07%) and Rhizopus stolonifer (12.07%). On the other contrary, Aspergillus flavipes was less frequency (1.45%). The Results reveal that Fusarium moniliforme was not isolated from unsterilized surface seeds using either Blotter or Czapek s agar plating technique. This might be attributed to the presence of numerous microorganisms on the unsterilized surface seeds which are competitive to Fusarium. Meanwhile, Rizopus stolonifer was not isolated using Czapek s - 46 -

agar method and this might be due to its high requirement form RH which is not present using Czapek s agar method. The obtained data are in agreement with those recorded by [5] who stated that 81% of the seeds which were not disinfected produced mold fungi, where as mold fungi were detected in 73% of the seeds that were surface disinfected. This is a true finding since the sterilization could eliminate the weaked saprophyte pathogens. Fungal isolation from broad bean Blanka cultivar localized seeds using both Blotter and Czapek s agar methods. a. Isolation from surface sterilized seeds: The Blotter method (Table 2) yielded high number of fungal isolates (109) compared to Cazpek s agar plate method (45). The isolates fungi were 11 species belonging to 5 genera. Aspergillus flavus was the most dominant fungus (27.27%), followed by A.niger (25.90%). Aspergillus wentii showed the least frequency (1.29%). It was found that Aspergillus niger and Rhizopus stolonifer could not be isolated using plating technique. b. Isolation from surface unsterilized seeds: The total number of 209 fungal isolates were isolated compared to those recovered from the surface sterilized seeds, being 154 isolates (Table 2). Aspergillus niger and Aspergillus flavus recorded more frequent, being 36.36 and 20.50%, respectively. On the other hand, Aspergillus ustus showed low frequency, being 0.96%. - 47 -

(TABLE 2): THE NUMBER AND FREQUENCY OF FUNGI ASSOCIATED WITH BROAD BEAN BLANKA CULTIVAR SEEDS* USING BLOTTER AND CZAPEK S AGAR PLATE TECHNIQUES *The infection percentage of broad bean Blanka cultivar seeds = 45.4%. - 48 -

As mentioned before, Fusarium moniliforme was not isolated from unsterilized surface seeds. Rhizopus stoloifer was not isolated using Czapec s agar method. Aspergillus ustus was not isolated using Blotter method and this might be due to that this fungus needs very low level of RH that not present using lotter technique. This study could be concluded that the percentage of Blanka cultivar seed infection was relatively more than that recorded for Malty cultivar, being 45.4% and 42.5%, respectively. (Tables 1, 2) The obtained data are in agreement with those recorded by [1,11] who isolated Aspergillus, Penicillium Rhizopus, Mucor and Fusarium genera from broad bean seeds. Also, [25] stated that the seeds of legumes were found to be hesvily infected with numerous mycoflora. Pathogenicity test: ( Table 3) reveal that the used fungi were significantly differed in their ability to infect broad bean seeds compared to the control treatment. It was found that Aspergillus flavus isolate No. (1) was more deleterious as it profoundly affected significantly seed germination and caused the highest seed invasion percentage, followed by Aspergillus niger isolate No. (1) and Aspergilllus flavus isolate No. (3) if compared with control. On the other hand, Aspergillus flavus isolate No. (2) and (5) were the least pathogenic. The difference among the used isolates might be, partially, attributed to the parasitic pattern of each fungus and the amount of enzymes calborated. The reduction in germinability might be principally attributed to the interior - 49 -

(TABLE 3): PATHOGENICITY OF NINE DIFFERENT FUNGAL ISOLATES USING BROAD BEAN BLANKA CLTIVAR SEEDS, 15 DAYS AFTER INCUBATION AT 25±2 L.S.D. at 5% 3.93 2.84 * Each figure represent average of four replicates (50 seed /replicate). - 50 -

changes in the seed constituents during fungal invasion. The oxidation of some components such as oil and pigments. The obtained data are in agreement with those recorded by [18] who stated that the percentage of seed germination was inversely proportional to RH of the storage vessel Trichoderma antagonistic effect on some isolated fungi growth in vitro: The inhibition effect of each bioagent Trichoderma harzianum and Trichoderma album equals (Table 4) on Aspergillus ustus growth, being 22.2% and thus this fungus was resistant to the action of Trichoderma spp. Compared to other tested fungi. Results also show that Trichoderma penicillium expansum and Aspergillus flavus isolate No. (1). The respective averages of growth inhibition were 77.7 and 52.7% respectively. On the contrary, Trichoderma harzianum caused a moderate effect in this respect against Aspergillus flavipes growth compared to other Trichoderma isolate, being 38.8 and 33.3%, respectively. This might be attributed to antifungal compounds production that compete fungal growth as stated by [22]. The obtained results are in agreement with those recorded by [7] who reported that Trichoderma strain is able to secrete some lytic enzymes such as chitinases acting upon fungal cell walls causing growth inhibition. - 51 -

(TABLE 4): EFFECT OF ANTAGONISTIC TRICHODERMA SPP. ON GROWTH OF FOUR BROAD BEAN SEEDS ASSOCIATED FUNGI IN VITRO. L.S.D. at 5% for : Anatagonistic (A) = not significant Growth inhibition (G) = 2.8 AXG = 3.5-52 -

REFERENCES [1] Abdel-Hafes, A.I.I.(1988).Mycoflora of broad bean, chickpea and lentil seeds in Egypt. Cryptogamine Mycologie, 9:335-363. [2] Abdul Wehab S.A, S.A, El-Nagerabi and A. El-shafie, (2015). Leguminicolous fungi associated with some seeds of Sudanese legumes. Biodiversitas 16,No.2 269-280. [3] Anonymous(1959). International Rules For Seed Testing. Int. Seed Testing Assoc. Proc. 24:275-584. [4] Barnett, H.I. and B.B. Hunter (1972). Illustrated Genera of Imperfect Fungi. Burguess, Minneapolis. [5] Bean,G. and T. Fernando (1985). Winged bean (Psoohocapus tetragonolobus L.) as a substrate for growth and aflatoxin production by aflatoxigenic strains of Aspergillus spp. Mycopathologia 90:141-145. [6] Bhat, R.V.and S. Vasanthi(2003). Mycotoxin food safety risks in developing countries. Food Safety in food Trade. Vision 2020 for food, Agriculture and Environment, focus 10:1-2. [7] Chat, I. Y. Henis and R. Mitchell (1967). Chemical composition of hyphal and sclerotial walls of Sclerotium rolfsii Sacc. Can.J. Microbiol., 13:137-141. [8] Christensen, C.M. (1957). Deterioration of stored grains by fungi. Botanical Review,23:108-134. [9] Christensen, C. M. and R. F. Drechslera (1954). Grain storage studies. 14. Changes in moisture content, germination percentage and moldness of wheat samples stored in different portions of bulk wheat commercial bines. Cereal chem., 31:206-216. [10] El-Sayed, F. H. Nakoul and P. Williams (1982). Distribution of protein content in the world collection of faba bean (Vicia faba L.) FABIS, 5:37. [11] El-Wakil, M. A, I. M. El-Refai O.A. Awadallah, M. A. El-Metwally and M. S. Mohammed (2009). Seed- borne pathogens of faba bean in Egypt : Detection and Pathogenicity. Plant Pathol. J., 8:90-97. [12] Fahmy, T.A. (1960). Fumigation of rice against insect and its effect on the quality of the grain. M.Sc. Thesis, Cairo University. [13] Fisher, R. A. (1984). Statistical Methods 6th ed. Iowa State Univ. press, Ames, Iowa, USA. [14] Hansen, H. N. (1926). A simple method of obtaining single spore culture. Science, 64 :384, 1659. [15] ISTA (International Seed Testing Association) (1966). International Rules Health Testing Proc. International Seed Test Assoc. 31, 1-152. - 53 -

[16] Kucuk,C. and M.Kivane (2003). Isolation of Trichoderma spp. and their antifungal, biochemical and physiological features. Turk,J.Biol.,27:247-253. [17] Papavizas, G.C. and C.M. Christensen (1960). Grain storage studies: effect of invasion by individual species development of discolored germs in wheat. Cereal Chem., 37:197-203. [18] Rao, V. V. R., M. S. Reddy and K. C. Roa (1996). Effect of fungicidal treatment on the viability of groundnut seeds in storage. Seed Res., 24:66-68. [19] Raper, K. B. and D. I. Fennel (1965). The Genus Aspergillus. Williams and Wilkins Company, Baltimore, 686 pp. [20] Riker, A. J. and R. S. Riker (1936). International to Research on Plant Diseases. John. S. Swift Co. Inc. Sta. Iovis, Chicago, New York. [21] Saber, S. M. (1992). Fungal contamination, natural occurrence of mycotoxins and resistance for aflatoxin accumulation of some broad bean (Viba faba L.) cultivar. J. Basic Microbiol 32 (4) : 249-258. [22] Simon, C. and M. Sivasithamparam (1988). Interactions among G. graminis var. tritici, Trichoderma kooningii and soil bacteria. Can. J. microbial., 34:871-876. [23] Snedecor, G. W. and W.G. Cochran, (1967). Statistical Methods 8th ed. Iowa State Univ. Press, Ames, Iowa, USA. [24] Snedecor, G. W. and SK. Alane (2013). Efficacy of some botanical against seed borne Fungi or green gram (Phaseolus aureus Robb.) Biosci Discov., 4(1), 107-110. [25] Swami CS, Alane SK( 2013).Efficacy fo some botanical against seed-borne fungi of green gram (phaseolus aureus Robb.).Biosci Discov 4 (1):107-110. [26] Tseng, T. C. J. C. Tu and the L. C. Soo (1995). Comparison of the profiles of seeds borne fungi and the occurrence of aflatoxins in mould-damaged bean and soybeans. Microbiol., 84:105-116. [27] Zhou. T. and R. D. Releder (1990). Selection of Epicoccum purpurascens and improved biocontrol of Sclerotinia sclerotiorum. Can. J. Microbial., 36:754-759. - 54 -