Romanian Biotechnological Letters Vol. 2, No. 1, 215 Copyright 215 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Research on micoflora present in the caryopses of wheat (Triticum aestivum) in the S-E of Romania, in terms of 214 Received for publication, December 16, 214 Accepted, January 2, 215 CRISTEA (MANOLE) M.S. 1 *, CRISTEA S. 1, ZALĂ C. 1 1 University of Agronomic Sciences and Veterinary Medicine, Bd. Mărăsti nr 59, sect. 1, 1146, Bucharest, România * Corresponding author: E-mail: malismanole@yahoo.com Abstract Romania is a traditional grower and producer of wheat; therefore it is necessary to obtain a high quality of seed material. Seeds are considered an extremely effective means for pathogens spreading both from year to year also during the same year from harvest to harvest. The micromycetes incidence on caryopses (kernels) of wheat (Triticum aestivum) was monitored in surrounding area of Bucharest using standard methods. For determinations were taken representative samples of wheat varieties for this area. Micoflora spectrum determined from samples that were analyzed consists of a variety of parasitic and saprophytic fungi. Skin fungal genres identified in wheat caryopses were the following: Fusarium sp. present in 9, Alternaria sp.: 9, Stemphylium radicinum: 5, Oedocephalum sp.:3, Rhizopus sp.: 2, Penicillium sp.: 1, Epicoccum purpurascens: 6. Keywords: pathogens, caryopses, wheat, varieties 1. Introduction Wheat, a plant that grows in all favorable regions, is one of the main cereals and basic food for humans. Good seedlings material offers a good start for obtaining high yields and superior quality indicators. Seedlings material should be considered an important means of production because its contamination can decrease or eliminate the germination capacity, a uniform emergence. It can also transmit diseases inside the cultures in the later stages of plant growth, in which case they would be showing a systemic or local infection with negative consequences on production (RAICU [12]; KHANZADA et al. [8]; BATEMAN & KWASNA [2]; KUBIAK & KORBAS [1]; WEBER et al. [14]). For some pathogens, the seeds have an exclusive role in the transmission and spreading. In the literature, there are summarized data on the spread of plant diseases due to the import of grains which have been infected or contaminated with pathogens (AGARWAL & SINCLAIR [1]). To achieve good agronomic results, it is required an examination in order to determine the spectrum of plant pathogens found on seeds (VARGA & MATEI [13]). The knowledge of seed pathology enables both the prevention of the introduction of new pathogens in wheat and also avoiding the emergence of massive infection in the field (CRISTEA & BERCA [4]). In addition, micoflora associated to the caryopses of wheat can produce mycotoxins that can be dangerous to humans and animals (HALT [6], MATEI et al. [11]). Romanian Biotechnological Letters, Vol. 2, No. 1, 215 1183
CRISTEA (MANOLE) M.S., CRISTEA S., ZALĂ C. 2. Materials and methods Samples of wheat (Triticum aestivum) that were examined were collected from different batches of grain naturally infected with pathogens during the growing season, in the climatic conditions of 214, in South-Eastern of Romania. In this work 1 varieties of wheat have been examined. The research aimed to identify the spectrum of pathogens present on wheat caryopses in order to determine their health status and recommend an effective treatment. Biological material was represented by caryopses belonging to the wheat varieties grown in this area:,,,,,,,, and. In order to study the micoflora, two (standard) methods were used: the method culture medium in Petri dishes and germinating seeds on filter papers. Wheat caryopses were placed in Petri dishes ø 8cm on PDA culture environment (potato- dextrose- agar), sterilized at 121ºC / 2 min (HULEA [7], CONSTANTINESCU [3]); incubation was performed at a temperature of 22ºC for 1 days. The seeds were placed in groups of 4 at equal distances. The results are the mean of two separate experiments with three repetitions for each sample. For identifications, pathogens were isolated and transplanted successively on PDA culture medium and incubated under the same conditions (CONSTANTINESCU [3]). To determine the micomycetes placed on the caryopses and also to determine the germination, 4 seeds were used. These were placed in groups of 1seeds / rehearsal on filter paper / strip-pp, incubated at a temperature of 2 C. To determine the germination rate, as the seeds were from samples of 214, it was required a prior pre-cooling to 4 C for 4 days. Observations were made at 8th day. The micromycetes identification was performed with Zeiss Primo Star microscope. 3. Results and discussion Microscopic examinations revealed a spectrum of pathogenic fungi belonging to framed Ascomycetes and Deuteromycetes (GHEORGHIES & CRISTEA [5], RAICU [12], KIFFER & MORELET [9], VARGA & MATEI[13]). During the study period, there were identified seven pathogens (Table 1, Figure 1), which have inhabited differently the caryopses varieties analyzed. Tabel 1. Identified micoflora in wheat caryopses Pathogen Variety Fusarium sp.alternaria sp. Stemphylium Rhizopus Penicillium Epicoccum Oedocephalum radicinum sp. sp. purpurascens sp x x - - - x - x x x - - x x x x x - - - - x x - - - x x - x - x x - - x x x - - x - Flamura85 x x x - - - - x - x - - - x x x - x - - - x x - - - x - Therefore, the frequency of 9 was recorded in pathogens belonging to Fusarium and Alternaria, 5 for Stemphylium, 3 for Oedocephalum species, 2 from Rhizopus, 1 for Penicillium, and Epicoccum of 6 (Figure 2). Fusarium fungus was not identified in 1184 Romanian Biotechnological Letters, Vol. 2, No. 1, 215
Research on micoflora present in the caryopses of wheat (Triticum aestivum) in the S-E of Romania, in terms of 214 variety, but it can be seen that on the wheat caryopses were developed species of the Penicillium and Rhizopus genres. Rhizopus sp. fungal developed only on and caryopses varieties. Species of the Alternaria genus were identified in most varieties analyzed except variety. Epicoccum purpurascens fungal contaminated 4 of the tested varieties (,, and ) and Oedocephalum sp. fungus, often seen on seeds of cereals used in bread industry (RAICU [12]) has been identified at, and varieties. Average number of pathogens present in the caryopses was 3.4 and for variety there were determined five pathogens. Apach Solehi Figure 1. Colonization caryopses (kernels) of wheat varieties studied fungi detected Figure 2. Incidence of pathogens spectrum in the caryopses of wheat analyzed The percentage values of the micoflora incidence detected in caryopses are shown in figure 2, 3, 4, and 5. It was found that the highest incidence values were determined in the case of Fusarium (Figure 3) and Alternaria (Figure 4) micromycetes. The values were between 3 and 8 to the genus of Fusarium species and 14 and 6 in genus of Alternaria species. Fusarium sp. recorded the highest value of the frequency of the variety, 8 and for Alternaria sp., the highest incidence was noted for the variety, 6. Romanian Biotechnological Letters, Vol. 2, No. 1, 215 1185
CRISTEA (MANOLE) M.S., CRISTEA S., ZALĂ C. 9 8 7 6 5 4 3 2 1 Fusarium sp. 7 6 5 4 3 2 1 Alternaria sp. Figure 3. The incidence of Fusarium species on caryopses Figure 4. The incidence of Alternaria species on caryopses From exposed presented data, it can be noticed the presence of Stemphylium radicinum fungal who often accompanies Alternaria species with a level of frequency between 6 ( variety) and 2 (, ), (Figure 5). Rhizopus species were found only in two varieties ( and ) with incidence values of 35 and 45 respectively. Penicillium fungus was detected only in variety in which the incidence was 25. Epicoccum purpurascens was reflected in a relatively low frequency not exceeding 1 in five studied varieties (,,, and ). The presence of Oedocephalum species was noted at varieties (19), (12) and 45 (Figure 6). 25 2 15 1 5 Stemphylium sp. 5 4 3 2 1 Rhizopus sp. Epicoccum sp. Penicillium sp. Oedocephalum sp. Figure 5. The incidence of Stemphylium sp. on caryopses Figure 6. The incidence of Rhizopus sp, Penicillium sp, Epicoccum purpurascens and Oedocephalum sp. on caryopses The percentage of seeds germination rate was between 87 and 99 (Figure 7). The germination rate for the variety was of 87, which can be accounted for by the attack of the following pathogens: Fusarium sp, Alternaria sp, Stemphylium radicinum, Epicoccum purpurascens and Oedocephalum sp. For the and varieties for which the germination represented 9 and 92 respectively, we consider that the reduction in germination was due to the presence of the Fusarium sp, Alternaria sp and Stemphylium radicinum pathogens.the presence of Fusarium sp, Alternaria sp and Epicoccum purpurascens pathogens reduced the germination for the and varieties to 94. 1186 Romanian Biotechnological Letters, Vol. 2, No. 1, 215
Research on micoflora present in the caryopses of wheat (Triticum aestivum) in the S-E of Romania, in terms of 214 For the and varieties, the seed germination was of 95, although the caryopses of the seeds were populated with different pathogens, the presence of the Alternaria sp pathogen can be noticed in both varieties. Higher values of germination were registered in 97, 98 and varieties, where the germination rate reached 99, although the seeds of these varieties hosted a lot of pathogens (Fusarium sp, Alternaria sp., Stemphylium radicinum, Rhizopus sp., Penicillium sp.) the germination percentage has not been significantly altered. 1 95 9 85 8 germination Figure 7. The varieties germination (after 8 th day) In figures 8-13 are presented aspects captured in the visual field of the Zeiss Primo Star microscope during determinations. Figure 8. Fusarium sp macroconidia Figure 9. Alternaria sp.-conidia Figure 1. Stemphylium radicinum- conidia Romanian Biotechnological Letters, Vol. 2, No. 1, 215 1187
CRISTEA (MANOLE) M.S., CRISTEA S., ZALĂ C. Figure 11. Oedocephalum sp, the apex of conidiophores the conidia Figure 12. Penicillium sp conidiophores the conidia Figure 13. Rhizopus sp a. sporangium spore b. thizoizi 4. Conclusion In the wheat caryopsis detected micoflora was numerous, pathogens belonging to the Fusarium, Alternaria, Stemphylium, Epicoccum purpurascens, Oedocephalum, Rhizopus and Penicillium genera. Fusarium and Alternaria genera were present in 9 of the investigated wheat varieties. Pathogen association reduced the percentage of the germination rate of seeds such as for variety at 87 and for variety at 9. 1188 Romanian Biotechnological Letters, Vol. 2, No. 1, 215
Research on micoflora present in the caryopses of wheat (Triticum aestivum) in the S-E of Romania, in terms of 214 Germination rate of the seeds was not influenced by this pathogen presence on the kernels for example in the variety, for which the germination rate was 99. Pathogens detected in wheat caryopsis can be associated to black-point phenomenon. 5. Acknowledgments This paper was published under the frame of European Social Fund, Human Resources Development Operational Programme 27-213, project no. POSDRU/159/1.5/S/132765. References 1. V.K. AGARWAL, J.B. SINCLAIR. 1996. Principles of Pathology. 2nd ed, CRC Press, Inc., Boca Raton, Fl. 539 pp. 2. G.L. BATEMAN, H. KWASNA. 1999. Effects of number of winter wheat crops grown successively on fungal communities on wheat roots. Applied Soil Ecology, 13: 271-282. 3. O.CONSTANTINESCU, 1974, Metode şi tehnici în micologie. Ed. Ceres, Bucureşti. 4. C. M. CRISTEA, M.BERCA, 213, Researches concerning the caryopses mycoflora of wheat to varieties grown in Modelu Location, Calarasi country, Research Journal of Agricultural Science (RJAS), vol 43, Agroprint Timisoara. 5. GHEORGHIES, C., CRISTEA S. 21- Fitopatologie, Vol.1, Ed. Ceres, Bucuresti. 6. M. HALT, 1994. Aspergillus flavus and aflatoxin B1 in flour production. Eur. J. Epidermiol., 1(5): 555-8. 7. A.HULEA, 1969. Ghid pentru laboratoarele de micologie si bacteriologie, Ed. Agrosilvica, Bucuresti. 8. K.A. KHANZADA, M.A. RAJPUT, G.S. SHAH, A.M. LODHI AND F. MEHBOOB. 22. Effect of seed dressing fungicides for the control of seed borne mycoflora of Wheat. Asian Journal of Plant Sciences, 1(4): 441-444. 9. E. KIFFER, MORELET, M., 2: The Deuteromycetes. Mitosporic fungi, classification and genera keys. Science Publishers Inc., Enfield, New Hampshire. 1. K. KUBIAK, M. KORBAS.1999. Occurrence of fungal diseases on selected winter wheat cultivars. Postepy Ochronie Roslin 39 (2), 81-84. 11. F. MATEI, F. ISRAEL-ROMING, I. SMEU, A. RADU, S. CRISTEA, 211: Quantitative study of Deoxynivalenol and Ochratoxin accumulation in sintetic media. Romanian Biotechnol. Lett., Vol 16, No.1, Supplement, Bucharest, p.33-39. 12. C. RAICU, BACIU D., 1978. Patologia semintei. Editura Ceres, Bucuresti. 13. M.VARGA, F. MATEI, 29: The application of statistical modeling tools the growth evolution of food born moulds. Romanian Biotechnol. Lett., Vol 15, No.2, Bucharest, p.4249-4259. 14. R. WEBER, B. HRYNCZUK, B. RUNOWSKA-HRYNCZUK, W. KITA. 21. Influence of the mode of tillage on diseases of culm base in some winter wheat varieties, oats, and spring wheat. J. Phytopathol., 149: 185-188. Romanian Biotechnological Letters, Vol. 2, No. 1, 215 1189