Rezumatul tezei de doctorat - USAMV Cluj-Napoca

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4,60%. The springtime end and the begining of the summer are overlapping with the cyprinid reproduction period, when the manipulations, as well as the stress caused by corticosteroid hormones are the factors of saprolegniasis incidence, at the weakened individuals. The average losses on the 69 samples studied is 4,30%. A lower percentage, of 4,10%, is remarked in the autumn period, when generally all the fish populations have a very good immune status, being prepared for wintering. CHAPTER III CONCLUSIONS AND RECOMMENDATIONS 1. The water presence of Saprolegnia, prooved by the spawns infestation, is observed irrespective of the water collecting month. 2. The higher infestation degree of the cyprinid spawns with Saprolegnia was remarked at the water temperature of 22ºC, when the medium and strong infestation percentage reaches 85,41%, irrespective of sample collecting months. 3. Irrespective of the solid culture media used (PDA and SGA), and of the reading interval (24, 48 and 72 hours), there are differences between the analyzed Saprolegnia colonies, from one fishpond and location to another. 4. The growing of Saprolegnia colonies on Potato Dextrose Broth (PDB) liquid culture medium is superior to the one realized on Sabouraud Dextrose Broth (SDB) liquid medium. 5. The morphologic characterization of Saprolegnia strains, from the obtained colonies, reveals unsegmented hyphae specific to the inferior fungi, and differences regarding the presence of sexual reproduction (at 59,42%) and its absence (at 40,58%). 6. At all Saprolegnia isolates it was identified the presence of flagella on the primary zoospores. 7. DNA purities illustrated by Nanodrop ND 1000 spectrophotometer, after the kit extraction (Qiagen), varies between 1,02 - 1,51, with a sample purity average of 1,250, at 260/280 nm ripple length, and the DNA quantities between 2,54 – 14,00 ng/µl, with an average of 5,543 ng/µl, and the DNA purities after PBS solution 72
extraction have values between 1,10 – 1,51, with an average of 1,394, at 260/280 nm ripple length, and the DNA quantities obtained by the same method, varied between 5,23 – 86,56 ng/µl, with an average of 30,653 ng/µl. 8. In the case of DNA samples extracted with kit QIAGEN, in all the situations the amplification produced with both primer pairs used, with the visualization of 743 bp fragments at Saprolegnia ferax, and of 761 bp at Achlya bisexualis. 9. In the case of the DNA samples extracted with PBS solutions, in spite of the normal DNA purities and quantities, the amplification of Saprolegnia DNA samples, with the two primer pairs, produced differently, because of the presence of possible fungal inhibitors, which do not allow the attachment of ITS1 and ITS4 primer pair, and in the case of using a PCR purification kit, the amplification performed. 10. In the case of the samples extracted with NE solutions, in spite of the normal purities and quantities, the amplification of Saprolegnia DNA samples, with the two primer pairs didn’t produced. 11. RsaI enzyme has a very good specificity for Saprolegnia and Achlya restriction sites, so we opted for its usage in all the fragment restriction tests. 12. AluI and HindIII enzymes, tested by us in the experiment, do not have any specificity for Saprolegnia and Achlya restriction sites, failing to produce any restricion of the DNA fragments. 13. In the case of the 10 fisheries and 69 samples analyzed, there are visualized two DNA length polymorphisms, in the gels. 14. The study of phylogenetic tree, using linkage and Euclidean distances, allows us to conclude the fact that in the studied fisheries and fishponds, there are different genera of fungi from Saprolegniaceae family, which have different pathogenicity upon cyprinid species. 15. After the DNA sequencing, there were identified two species of aquatic fungi, in the studied fishponds, represented by Saprolegnia ferax (743 bp, a identity of 99% and has two point mutations compared to the matrix species from GeneBank) and Achlya 73
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UNIVERSITATEA DE ŞTIINłE AGRICOLE
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II.6. REZULTATELE RESTRICłIEI ENZI
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diverse specii piscicole. De aici s
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Fig.1. LocaŃiile în care s-au efe
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A fost constituit un număr de 207
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Sabouraud Dextrose Broth (SDB-bulio
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Echipament, materiale şi reactivi
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Primerii folosiŃi de către noi î
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CAPITOLUL II REZULTATELE CERCETĂRI
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Mediile diametrului coloniilor de S
Page 21 and 22: laborator specifice metodologiei de
Page 23 and 24: Deoarece am utilizat în experiment
Page 25 and 26: fragmente: primul la aproximativ 45
Page 27 and 28: II.7. REZULTATE PRIVIND STUDIUL ÎN
Page 29 and 30: mai mare genetică, indivizii despr
Page 31 and 32: MenŃionăm faptul că în toate ba
Page 33 and 34: CAPITOLUL III CONCLUZII ŞI RECOMAN
Page 35 and 36: isexualis (761 pb, o identitate de
Page 37 and 38: BIBLIOGRAFIE SELECTIVĂ 1. Colao Ma
Page 39 and 40: 19. Oroian, T.E., 2006, SelecŃia a
Page 41 and 42: UNIVERSITY OF AGRICULTURAL SCIENCES
Page 43 and 44: SPECIES FROM SAPROLEGNIACEAE FAMILY
Page 45 and 46: The specific literature presents up
Page 47 and 48: Fig.1. The locations where the rese
Page 49 and 50: estimation of the spawns infestatio
Page 51 and 52: pellets were filtred, washed with d
Page 53 and 54: The DNA rapid extraction using PBS
Page 55 and 56: We used a comparatively mixture of
Page 57 and 58: II.1. THE RESULTS OF SAPROLEGNIA CU
Page 59 and 60: Reading time 24 48 72 n Culture med
Page 61 and 62: asexual one. We can precisely concl
Page 63 and 64: 1 2 3 4 5 6 7 8 9 700pb- Fig.3. Ele
Page 65 and 66: pairs length. The sequencing result
Page 67 and 68: II.7. RESULTS REGARDING THE GENETIC
Page 69 and 70: higher genetic distance, the indivi
Page 71: We mention the fact that in al the
Page 75 and 76: 13. In researches on the aquatic fu
Page 77 and 78: 40. Frisvad, J.C., Bridge, P.D., Ar
Page 79: 61. *** http://www.sigmaaldrich.com
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