Rezumatul tezei de doctorat - USAMV Cluj-Napoca
Rezumatul tezei de doctorat - USAMV Cluj-Napoca Rezumatul tezei de doctorat - USAMV Cluj-Napoca
- extension 90 seconds at 68°C - final extension: 7 minutes at 68°C (after Coalo Maria Chiara, 1999). The amplification performed on 69 samples, developed on Potato Dextrose Broth (PDB) medium, using an adapted method after Ristaino and al., 1998. I.3.8.2. PCR-RFLP techique - Restriction Fragment Length Polymorphism The amplified fragments were digested with 3 restriction enzymes from Fermentas: RsaI, HindIII and AluI. The digestion protocol was pursuant to the manufacturer (http://www.fermentas.com). The PCR products were overnight incubated, at 37ºC, and at the end at 65ºC for 10 minutes. The digestion products were then separated by electrophoresis, in a 3% agarose gel, in 1x TBE buffer, for 2 hours at 60V. The DNA was stained with Sybr Safe (Invitrogen), for the observation of amplified fragments polymorphism, under UV light (Biorad). There was used a DNA Ladder Low Range (700 bp), for the fragment comparison (Fermentas). I.3.9. Methods used in establishing the phylogenetic diversity and relationship of Saprolegniaceae family fungi species I.3.9.1. Automatic sequencing The DNA strand was sequenced with universal primers ITS1 (forward) and ITS4 (reverse), by Mycrosinth (Switzerland). The sequencing results were interpreted using a specific software called Chromas Lite. For the establishing of genetic diversity there were used specific genetical software. CHAPTER II PERSONAL RESEARCH RESULTS 56
II.1. THE RESULTS OF SAPROLEGNIA CULTURE IN LABORATORY CONDITIONS We expressed in percentage from the total number of analyzed samples, every infestational score from each temperature level, regardless the fishponds and fisheries (table 1). Spawns infestation degree with Saprolegnia based on temperature percentage estimated from the total number of samples (207) Temperature Infestational degree Table 1 0 1 2 3 4 10°C 4,34 38,64 54,58 2,41 0 15°C 0,96 11,11 32,36 51,20 4,34 22°C 0,96 0,96 12,56 45,41 40,09 Legend: 0 – uninfested; 1 - very weak infested; 2 - weak infested; 3 – medium infested; 4 – strong infested. From the table data results the fact that saprolegniasis development is different from one incubation temperature to another. At 10°C, 38,64% from the samples had the score of 1, which indicates a very weak infestation at 144 hours of incubation. A percentage of 54,58%, more than half of the analyzed samples, are weak infested, and only 2,41% are medium infested. At 15°C, only 0,96% from the samples do not manifest any infestation sign, comparative to 4,34% uninfested samples at 10°C. A weak infestation degree, of 32,36% from samples is reached at 15°C, and the infestation medium level at 51,20%. Compared to the temperature gradient of 10ºC, at 15°C there is a percentage of 4,34% with strong infestation. At 22ºC, 40,09% from the samples have a strong infestation degree, 45,41% have a medium infestation, and only 0,96% are uninfested. Those data indicate the fact that regardless the fishpond, the area and the time of the year, saprolegniasis as disease spreads with a high intensity and a shorter period at 20°C. This result confirms the literature data regarding the carp spawns and larvae losses, communicated by fishery owners and 57
- Page 5 and 6: diverse specii piscicole. De aici s
- Page 7 and 8: Fig.1. LocaŃiile în care s-au efe
- Page 9 and 10: A fost constituit un număr de 207
- Page 11 and 12: Sabouraud Dextrose Broth (SDB-bulio
- Page 13 and 14: Echipament, materiale şi reactivi
- Page 15 and 16: Primerii folosiŃi de către noi î
- Page 17 and 18: CAPITOLUL II REZULTATELE CERCETĂRI
- Page 19 and 20: 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
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- Page 59 and 60: Reading time 24 48 72 n Culture med
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- Page 67 and 68: II.7. RESULTS REGARDING THE GENETIC
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- 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
II.1. THE RESULTS OF SAPROLEGNIA CULTURE IN<br />
LABORATORY CONDITIONS<br />
We expressed in percentage from the total number of analyzed samples, every<br />
infestational score from each temperature level, regardless the fishponds and fisheries (table<br />
1).<br />
Spawns infestation <strong>de</strong>gree with Saprolegnia based on temperature<br />
percentage estimated from the total number of samples (207)<br />
Temperature<br />
Infestational <strong>de</strong>gree<br />
Table 1<br />
0 1 2 3 4<br />
10°C 4,34 38,64 54,58 2,41 0<br />
15°C 0,96 11,11 32,36 51,20 4,34<br />
22°C 0,96 0,96 12,56 45,41 40,09<br />
Legend: 0 – uninfested; 1 - very weak infested; 2 - weak infested; 3 – medium infested; 4 –<br />
strong infested.<br />
From the table data results the fact that saprolegniasis <strong>de</strong>velopment is different from<br />
one incubation temperature to another. At 10°C, 38,64% from the samples had the score of<br />
1, which indicates a very weak infestation at 144 hours of incubation. A percentage of<br />
54,58%, more than half of the analyzed samples, are weak infested, and only 2,41% are<br />
medium infested.<br />
At 15°C, only 0,96% from the samples do not manifest any infestation sign,<br />
comparative to 4,34% uninfested samples at 10°C. A weak infestation <strong>de</strong>gree, of 32,36%<br />
from samples is reached at 15°C, and the infestation medium level at 51,20%. Compared to<br />
the temperature gradient of 10ºC, at 15°C there is a percentage of 4,34% with strong<br />
infestation.<br />
At 22ºC, 40,09% from the samples have a strong infestation <strong>de</strong>gree, 45,41% have a<br />
medium infestation, and only 0,96% are uninfested. Those data indicate the fact that<br />
regardless the fishpond, the area and the time of the year, saprolegniasis as disease spreads<br />
with a high intensity and a shorter period at 20°C. This result confirms the literature data<br />
regarding the carp spawns and larvae losses, communicated by fishery owners and<br />
57
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