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Abstract
Summary
The brown rot disease of potato is important plant disease because it leads to great decrease in potato yield in Egypt and different locations in the world. Therefore, this study aimed at isolate the causal agent of this disease (Ralstonia solanacearum) from different locations in El-Dakhlia and El-Sharkia Governorates and isolation of phages (from the rhizosphere of infected plants) active against this bacteria. Also, the biological and morphological characters of the isolated phages were studied. Moreover this study aimed to recognize the molecular biology of this bacteria and their phages. Furthermore, some physical factors affecting the infectivity of phages were studied. Finally, storage of the isolated phages in different media and different temperatures has been studied.
Different isolates of Ralstonia solancearum were been obtained from infected potato tubers collected from different locations in El-Dakhlia (6 locations) and El-Sharkia (2 locations) Governorates. Most of these isolates (13 isolates) contained both virulent strains, while few isolates (7 isolates) contained only the virulent strain.
From only the clay soil of El-Dakhlia Govenorate, the isolated phages (active against R. solanacearum) were obtained. On the other hand, no-phage was obtained from the sandy soil of El-Dakhlia Govenorate and from the soil of El-Sharkia Govenorate.
The isolated phages could be distinguished into three types according the plaque morphology and electron microscopy that were designated by RSP1, RSP2 and RSP3. Furthermore, all the isolated phages were active only against the avirulent strains.
The plaques of isolated phages could be distinguished into clear plaques (plaques of phages RSP1& RSP3) and turbid plaques (plaques of phage RSP2) Also, plaques of phage RSP1 were the largest ones.
It was observed that there were no lysogenic bacteria found for the three isolated phages after 7 days of incubations.
Plaque diameters of the isolated phages were increased with further incubation (growing plaques) and this increase continued for 6 days. After this period, the plaque diameters of phage RSP1 were the largest (20 mm), while those of phage RSP2 were the smallest (14 mm). Also, the plaque diameters of phages RSP3 were 16 mm. Moreover, this increase in diameter caused interfering and confusion of plaques, which might lead to complete lysis of bacterial lown. It is observed that plaques of phage RSP2 were clear with turbid center when they grow.
According to the electron microscopy, these phages belong to group B (phages RSP1 & RSP3) and group C (phage RSP2) of classification of Bradley (1967) and Family Styloviridae (phages RSP1 & RSP3) and family Podoviridae (phage RSP2) of classification of Mathews (1982). Where phages RSP1 and RSP3 had long tails with base plates and tail fibers but phage RSP1 had hexagonal head and phage RSP3 had pentagonal head (the measurements of heads, tails and base plates varied in both phages. Furthermore, phage RSP2 had hexagonal head with very short tail (like-knob).
Studying the rate of adsorption experiment clarified that phage RSP3 had the highest value of adsorption rate constant (9.75X10-4 PFU/ CFU /ml) and the lowest time of highest adsorption (6 minutes). While, phages RSP1 and RSP2 had the same time of maximum adsorption (8 minutes). Moreover phage RSP1 had the lowest value of adsorption rate constant (5.76X10-4 PFU/ CFU /ml). Furthermore, phage RSP2 had the highest value of maximum phage adsorption (78.4%), while phage RSP1 had the lowest corresponding value (65.38%).
Studying the One-step growth curve experiment revealed that the isolated phages had the same latent period (2 minutes) and the same rise period (6 minutes). However, the burst size values were different for the three phages, where phage RSP1 had the highest burst value (46.2 PFU/ cell) and phage RSP2 had the lowest corresponding value (25.4 PFU/ cell).
Molecular studies of R. solanacearum strains and their phages revealed that the chromosomal DNA of the two strains (virulent and avirulent strains) of the plant pathogen had a similarity of 92.7% and there was a positive relationship between the two strains in their proteins and DNA of plasmid. Moreover, isolated phages had similarities in their DNA composition, where the highest similarity was between two phages RSP1 and RSP3 (94.7%) and the lowest one was between two phages RSP1 and RSP2 (85.7%). Also, there were positive relationships between the protein composition of the three isolated phages.
The three isolated phages were resistant to high temperatures, where the complete inhibition of the three isolated phages was observed at 100 ºC after 15 minutes (phage RSP1) or 20 minutes (phages RSP2 and RSP3).
Furthermore, these phages were resistant to exposure UV-irradiation, where complete inhibition was observed after exposure to this irradiation at a distance 30 cm for 65 minutes (phage RSP3) or 70 minutes (phages RSP1 and RSP2).
Studying the effect of pH on the infectivity of the tested phages indicated that the optimum pH value for the growth of these phages was almost pH 8.0 Furthermore, these three phages were resistant to pH values. Where the complete inactivation was occurred below pH 3.0 in acidic media and at pH 13.0 (phage RSP1) or PH14 (phages RSP2 and RSP3)
Study of the effect of some organic solvents (Petroleum ether, acetone, ethyl alcohol and chloroform) on the infectivity of phages showed that the infectivity of these phages decreased with further incubation with these solvents. However, these phages were resistant to these solvents where, after 30 minutes of incubation the infectivity of these phages was about or more than 50%.
Study of the effect of fumigation by some oils on the infectivity of tested phages indicated that the infectivity approximately did not affected by fumigation of these oils.
Study of the effect of some diluents on the infectivity of isolated phages revealed that some of them were stimulant and others were inhibitors. Phage RSP1 was stimulated by the highest number of used diluents, where it was stimulated by distilled water, NaCl (1 & 0.1 M), NaNO3 (0.1 &0.01 M), KCl (0.01 M) and CaCl2 (1, 0.1 & 0.01 M). On the other hand, phage RSP3 stimulated only by distilled water, while phage RSP2 stimulated by distilled water, KCl (0.1 & 0.01 M) and MgSO4 (0.01M). Therefore, phage RSP1 was inhibited by the lowest number of used diluents, where it inhibited by NaCl (0.01 M), NaNO3 (1 M), KCl (1, 0.1 M), MgSO4 (1, 0.1 & 0.01 M) and ZnSO4 (1, 0.1 & 0.01 M). On the contrast, phage RSP3 was inhibited by the highest number of used diluents, where it inhibited with all used diluents except distilled water, while phage RSP2 inhibited by all used diluents except distilled water, KCl (0.1 & 0.01 M) and MgSO4 (0.01M).
Storage of the tested phages in different solutions SPB, distilled water, saline solution and soil extract solution) at different temperatures (4ºC, room temperature 30ºC) for 6 weeks revealed that all phages were maintained viability in all cases. Furthermore, results suggested that the best conditions for preserving these phages were at 4 ºC and SPB or distilled water. On the other direction, preservation of isolated phages in saline solution had the lowest corresponding values of infectivity.