Egypt. J. Exp. Biol. (Bot.), 7(2): 285 – 291 (2011) © The Egyptian Society of Experimental Biology

RESEARCH ARTICLE

El-Kassas H. Yassin Abdel Aziz N. El-Said

CONTROL OF FISH FUNGAL PATHOGEN PARASITICA USING BRACHIONUS PLICATILIS AND SODIUM CHLORIDE

ABSTRACT: This investigation was designed to compare INTRODUCTION: between the inhibiting effect of sodium There is a steady increase in chloride and the rotifer Brachionus plicatilis constructing Fish Farms in Egypt especially on the growth of a fish pathogen, Saprolegnia during the last fifty years as a source of parasitica as safe remediation techniques in alternative protein due to the rapid increase aquacultures. The results showed that the in population and also the high prices of addition of 100 cells of B. plicatilis, caused protein. Outbreaks of waterborne decrease in spores count of S. parasitica, by fungal infections (saprolegniasis) on fish, 34% after 2 hours, and 79% after 6 hours, fish eggs and water continue to cause respectively. Increasing the number of B. problems among cultured fish (Ali, 2009; plicatilis to 150 cells decreased the spores Refai et al., 2010). count of S. parasitica by 80.00%. The two way Members of the genus Saprolegnia analysis of variance showed that the tested cause saprolegniasis; a devastating disease, rotifer species exhibited a great efficiency in with symptoms of visible white or grey grazing fungal zoospores (P<0.001). On the patches of filamentous mycelium on the body other hand, NaCl solution reported stronger or fins of freshwater fish. Great losses in effect on the growth of the tested fish weight are sometimes observed due to the pathogen as compared to that occurred by the infection by this genus, as well as water rotifer species. After 14 days of incubation, S. mould (Lartseva, 1986) and loss of total parasitica was markedly suppressed incubated eggs (Dudkai et al., 1989; Sati and (P<0.001) at different concentrations of NaCl Khulbe, 1981). Kitancharoen and Hatai solution. (1997) stated that surrounding hyphal growth could reduce the water circulation around the KEY WORDS: eggs and block oxygen transportation, thus Saprolegnia parasitica, Sodium chloride, contributing to the death of the eggs. Brachionus plicatilis, Pathogenic inhibitors, Disease also reduces hatchery efficiency and Lake Edku. production. Several aquatic fungi, including chytrids and soil Hyphomycetes have been reported from two lakes on Soignée Island (Ellis Evans, 1985).

CORRESPONDENCE: Prevention and control of saprolegniasis are especially difficult, even Hala Yassin El-Kassas under fish-farming conditions, owing to the Hydrobiology Laboratory, Marine Environment ubiquitous nature and the rapid spreading of Division, National Institute of Oceanography and such fungi (Alderman, 1994). Fisheries, Alexandria, Egypt. E-mail: [email protected] Up till 2002, Saprolegnia infections in aquaculture were kept under control with malachite green (an organic dye) that is very efficient at killing the pathogen. However, the Nagwa El-Said Abdel Aziz use of malachite green has been banned worldwide due to its carcinogenic and Hydrobiology Laboratory, Marine Environment toxicological effects and this had resulted in Division, National Institute of Oceanography and a dramatic re-emergence of Saprolegnia Fisheries, Alexandria, Egypt. infections in aquaculture. As a consequence Saprolegnia parasitica is now, economically, a very important fish pathogen, especially on catfish, salmon and trout species, and warrants further investigation to develop new ARTICLE CODE: 35.02.11 alternative control strategies (West, 2006).

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org 286 Egypt. J. Exp. Biol. (Bot.), 7(2): 285 – 291 (2011)

Magaraggia et al. (2006) studied the treatment of microbiologically polluted aquaculture waters by a novel photochemical technique of potentially low environmental impact. Antifungal agents are essential for the maintenance of healthy stocks of fish and their eggs in intensive aquaculture operations. Ali (2009) reported the antifungal activity of sodium chloride on sp. and Saprolegnia diclina. The rotifer Brachionus plicatilis is a euryhaline species that has been most widely used as essential food source for various types of small larvae, in rearing fish and shrimp, as well as crab larvae due to its tolerance to the marine environment (Walkes, 1981; Watanabe et al., 1983; Fig.1. Map showing Lake Edku and the study Lubzens, 1987; Dhert and Sorgeloos, 1994). Farms, Alexandria, Egypt Kagami et al. (2004) reported that the Daphnia galeata hyalina can Isolation of Saprolegnia parasitica: protect from fungal parasitism through reducing the abundance of fungal Previously prepared glass bottles each containing eight germinated and sterilized zoospores. seeds was used to capture and Accordingly, this investigation was recovery of the resident zoosporic fungi designed, for studying the occurrence of a (Khallil, 1984). Water samples in these bottles zoosporic fungus in some fish farms and fish were poured under aseptic conditions into in Lake Edku, Egypt. A comparison between equivalent numbers of sterilized Petri-dishes, the Pathogenic inhibitors of sodium chloride and then kept at 22°C for 24 hours for the and Brachionus plicatilis as safe remediation colonization of sesame seeds by fungal techniques in aquacultures were carried out. propagules. Colonized sesame seeds were then transferred into other clean and sterile Salinity in Lake Edku and in Boughaz Petri-dishes containing sterile distilled water El-Maadiya fluctuated between a minimum of to which crystalline penicillin (2000 units per 0.48 ppt and a maximum of 35.69 ppt (Abdel- liter of water) was added (Roberts, 1963). Aziz and Dorgham, 1999). Therefore, These dishes were incubated at 22°C for suppression tests using sodium chloride on twenty days during which the fungal growth the two tested pathogens were performed was daily examined. The water surrounding using sodium chloride concentrations ranged the seeds contained numerous motile from 5 ppt to 30 ppt. secondary zoospores. Zoospore suspensions As far as the authors knowledge, this were then obtained by filtering this water could be considered as the first trial to follow through double layers of Whatman 541 filter up the grazing effect of Brachionus plicatilis paper (Carballo and Munoz, 1991). on Saprolegnia parasitica. Identification of the recovered fungus: The isolated fungus was identified to the MATERIAL AND METHODS: species level at the Regional Center for Mycology and Biotechnology (RCMB), Al- Sampling and collection: Azhar University, Cairo, Egypt (Khulbe, 2001; Surface water samples and about 10 random Watanabe, 2002). The most dominating fungal fishes (Tillapia spp. and Mugil spp.) were species identified was Saprolegnia parasitica. collected. Each water sample (three Media: replicates) was collected during March 2011 Glucose peptone (GP) agar medium was from eight basins from fish farms at Lake used for purification of S. parasitica and in a Edku (Fig. 1) and put in brown and sterilized broth form for estimation of the zoospores glass bottles (400 ml capacity each). Some counts as affected by the different treatments fungal isolates were collected from skin, gills of the rotifer: Brachionus plicatilis. The fungal and stomachs of the collected fish samples. isolate namely S. parasitica which dominated The rotifer Brachionus plicatilis was kindly in fish farms and fish hatcheries were used for provided by Dr. Heba Saad, Aquaculture further studies. S. parasitica was preserved in division, National Institute of Oceanography pure culture (water sesame seeds culture). and Fisheries, Alexandria.

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org Yassin & El-Said, Control of Fish Fungal Pathogen Saprolegnia parasitica Using Rotifer Brachionus plicatilis and NaCl 287

Growth of Saprolegnia parasitica in and the result was expressed as the mean of presence of Brachionus plicatilis: two perpendicular radii. Grazing experiments: b. Dry weight (DW) method: Each experiment was triplicates and the Glucose peptone broth (100 ml) amended average result was recorded. At the end of with different NaCl concentrations (0, 5, 10, 15, each experiment the counts were 20, 25, & 30 ppt), in 250 ml Erlenmeyer flasks recorded to detect if there were mortality then inoculated with 5 mm discs of each fungus among the rotifers during the experiments. separately. The flasks were incubated at 25 ± Experiment 1: 2°C for 7 days. The fungal mats and the spores were removed by filtration and dried at 60 ± 3°C For estimating the grazing efficiency of to constant weight. Fungal dry weights were B. plicatilis on S. parasitica, a set of six 100- expressed as g/l. ml flasks were inoculated with 20 ml of fungal spores suspension (known initial count) c. Germinating spores count: and100 individuals of B. plicatilis were added The tested fungus was grown on glucose to each flask. Flasks were incubated for 2, 4 peptone broth amended with different & 6 hrs under florescent light 40 µmol quanta concentrations of NaCl (0, 5, 10, 15, 20, 25, & m-2 s-1 then zoospore's counts were recorded 30 ppt) and incubated at 25 ± 2°C for 10 days. after each time interval (Fig. 2). The germinating spores were counted at 800x magnification with a compound microscope (CARLZEISS) according to (Guzman and Axtell, 1986). Statistical analyses: Each single treatment was triplicates and the mean value ± standard deviation was considered. The data were statistically analyzed by applying ESPSS program, using the simple linear correlation Tuckey’s-b test and analysis of variance (ANOVA) test.

RESULTS: Relationship between spores abundances and Brachionus Plicatilis:

Fig. 2. The rotifer Brachionus plicatilis Brachionus plicatilis demonstrated remarkable inhibitory effect on the growth of Experiment 2: the zoosporic fungus, S. parasitica (Fig. 3 & To test the combined effect of rotifers Table1). Up on using 100 cells of B. plicatilis, number and time on the grazing efficiency of the results showed that the zoospores' counts B. plicatilis, experiment 1 was carried out of S. parasitica were reduced by about 37% using 50, 100, and 150 individuals of B. after only 2 hours of attack. Increasing the plicatilis, and incubated. Results were attack time (6 hrs), led to an increase in the recorded every 2 hours up to 6 hours of grazing efficiency of B. plicatilis to be about incubation. During this period any decrease in 77%. This reduction was highly significant (P spore density would be mainly due to grazing <0.001). by B. plicatilis, because zoospores do not die within 6 hrs without host cells (Bruning, 1991), and they do not make any cysts without host cells (Doggett and Porter, 1996) . Growth of Saprolegnia parasitica under the stress caused by NaCl: Experiment 3: a. Radial fungal growth method: To determine the effect of NaCl on the growth rate of the tested fungal species, a cork borers of 5 mm diameter were cut from the circular blocks of actively growing edges of the fungal colony and centrally placed onto glucose peptone agar plates amended with different concentrations of NaCl (0, 5, 10, 15, 20, 25, & 30 ppt), and incubated at 25 ± 2°C. Colony diameters were measured every 48 hours starting from the 2nd up to the 14th day, Fig. 3. Time course effect on grazing spores of Saprolegnia parasitica by individuals of Brachionus plicatilis

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Table 1. One way ANOVA with Tuckey’s-b test for the time course analysis of Brachionus plicatilis to graze the germinating spores of Saprolegnia parasitica (spore numbers x104). (a>b>c>d)

Time (Hours) S. parasitica (Mean ± SD)

0 19.8 ± 22546.25a

2 12.0 ± 12055.43b

4 6. 7 ± 11590.23 c

6 3.9 ± 6658.33 d

F 61.79 ANOVA P <0.001 Fig. 4. Effect of Individual numbers of Brachionus P <0.001 <0.001 plicatilis on grazing of spores of Saprolegnia Parasitica during different incubation periods. When the individual number of B. plicatilis increased (150 cells), the Radial growth reduction of Saprolegnia parasitica zooplankton displayed higher grazing activity treated with various concentrations of sodium against the spores of the fungus (S. chloride during different incubation periods: parasitica) by 80.00% (Fig. 4). Even at low Sodium chloride showed a good potency dose (50 individuals), B. plicatilis able to as an antifungal against the growth of S. graze 22% of the zoospores of S. parasitica. parasitica (Table 2). All concentrations reported The results of this experiment indicated the good inhibitory effect on the fungal growth combined effect of rotifers number and time through reduction of its colony diameter. The on the grazing efficiency of B. plicatilis. Also, reduction increased with increasing the results revealed that there were no concentration of sodium chloride, since the changes in B. plicatilis counts during the highest concentration (30 ppt) revealed experiments. The results of two way ANOVA significant effect (P<0.001) and caused reduction test, indicated that the increase in incubation of 76.67 ± 1.53% in colony diameter. At low dose time of B. plicatilis enhance its grazing (5 ppt), the efficiency of sodium chloride to activity on the fungus (P<0.001). suppress the growth of the pathogen was comparatively high, reducing 45.33 ± 0.58% of colony diameter of S. parasitica. Table 2. One way ANOVA for the percent reduction of the colony diameters per time for Saprolegnia parasitica

Time 4 days 6 days 8 days 10 days 12 days 14 days NaCl Conc.(ppt) (Mean ± SD) (Mean ± SD) (Mean ± SD) (Mean ± SD) (Mean ±SD) (Mean ±SD) 5 15.33 ± 1.53 19.67 ± 2.08 26.33 ± 1.53 34.00 ± 1.00 39.33 ± 1.50 45.33 ± 0.58 10 21.67 ± 2.50 29.67 ± 1.53 34.67 ± 2.08 38.00 ± 1.00 47.33 ± 1.50 51.67 ± 1.50 15 28.00 ± 3.00 37.33 ± 1.53 37.67 ± 1.53 46.00 ± 1.00 55.00 ± 2.00 65.33 ± 1.50 20 34.00 ± 2.65 43.00 ± 2.00 41.00 ± 1.00 47.33 ± 2.00 56.00 ± 2.00 68.00 ± 1.00 25 42.00 ± 2.00 46.00 ± 1.73 49.33 ± 2.52 54.00 ± 1.00 67.00 ± 2.00 71.67 ± 1.50 30 51.33 ± 1.50 53.33 ± 1.53 54.67 ± 1.53 58.67 ± 2.00 70.00 ± 1.00 76.67 ± 1.53 F 517.0510 145.6029 179.6123 222.6096 127.2815 60.4168 ANOVA P <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 One way ANOVA test was carried out to study the inhibitory effect of sodium chloride on the radial growth of the pathogen during different incubation periods. The results indicated the negative significant effect of sodium chloride concentrations during different incubation periods at (P <0.001). Reduction of Saprolegnia parasitica dry weight (g/L) after 7 days of using different NaCl concentrations: The different concentrations of sodium chloride greatly reduced the mat dry weight of the pathogenic fungus after 7 days of incubation (Fig. 5). The dry weight of S. parasitica was reduced by nearly 12% up on using 5 ppt of sodium chloride, reached 50% with increasing Fig. 5. Dry weight (g/ml) of Saprolegnia parasitica, as sodium chloride concentration to 30ppt. affected by NaCl concentrations, based on the results of one way ANOVA (p ratio <0.05)

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org Yassin & El-Said, Control of Fish Fungal Pathogen Saprolegnia parasitica Using Rotifer Brachionus plicatilis and NaCl 289

Reduction of Saprolegnia parasitica to the significant increase in the grazing germinating spores after 10 days using activity of B. plicatilis for the fungal isolate different NaCl concentrations: (P<0.001). This may be because the rotifer B. Similar to the fungal dry weight, the plicatilis preferred zoospores of S. parasitica fungal spores exhibited the same pattern of as food substances. In this respect, it was reduction under the stress of different reported that rotifers are filter feeders, concentrations of sodium chloride (Fig. 6). For accepting small particles up to 30 μm in size S. parasitica the spores number was 16.24 ± including bacteria, algae, yeast and protozoa 21.90 x104 \ 20 ml samples in the presence of (Arndt, 1993). 5ppt sodium chloride dropped to 6.4 ± 4.00 x The tested fish pathogenic fungus 104 at 30 ppt. couldn't tolerate NaCl solution. After 14 days of incubation, the colony diameters of the pathogen were markedly reduced 76.67 ± 1.53% for S. parasitica. The diameters of the 20 S . parasitica vegetative colonies of the tested fungus

4 decreased with increasing the concentration 15 of NaCl solution. Growth of the pathogen in presence of increasing concentrations of sodium chloride revealed a progressive 10 decrease in the fungal mat dry weight. In this respect, several authors found that NaCl Number of spores x 10 of spores Number 5 reduced growth and was the most effective in controlling the growth of fungus-like organisms Phytophthora cinnamomi (Sterne et 0 0 5 10 15 20 25 30 al., 1976), and ascomycete fungi such as NaCl Conc (ppt) Aspergillus niger and Paecilomyces lilacinum (Mert and Dizbay, 1977), fungus-like organisms such as Saprolegnia diclina (Taylor Fig. 6. Number of germinating spores (x104) of and Bailey, 1979), Saprolegnia spp. Saprolegnia parasitica as affected by NaCl concentrations, based on the results of one (Martinez-Palacios et al., 2004; Khodabandeh way ANOVA test (p ratio <0.001). and Abtahi, 2006) and Saprolegnia parasitica (Ali, 2005). However, Mert and Ekmekci (1987) reported that the vegetative growth of DISCUSSION: ascomycetous fungi Aspergillus flavus and The rotifers B. plicatilis constitute a Penicillium chrysogenum increased with an major and in most cases the only food source increase in the NaCl content of the nutrient for larval stages of several organisms reared medium. in aquatic aquaculture including fish and Similar to the fungal dry weight, the various invertebrates (Lubzens et al., 1990). fungal spores exhibit the same pattern of The widely applied batch culture procedure reduction up on using different concentrations appears to be simple, using micro algae of sodium chloride; where it reached about and/or bakers' yeast or other formulated 70% reduction for S. parasitica. In accordance products as food source. The development of with our results, some investigators found that the larval rearing industry is primarily due to relatively low concentrations of NaCl the advancement of mass culture technology suppressed or eliminated sporogenesis and of the rotifer B. plicatilis (Fulks and Main, reproductive growth in Trichophyton 1991). Since the rotifer is one of the main mentagrophytes (Kane and Fischer, 1973), food sources for larvae in aquacultures, it is Saprolegnia species (Harrison and Jones, important to follow up the grazing efficiency of 1975; Smith et al., 1990), Lagenidium them on pathogenic fungal spores. giganteum (Jeffrey and Roberts 1985), The presence of rotifer B. plicatilis with Aspergillus flavus (Mert and Ekmekci, 1987) the pathogenic fungus S. parasitica and Saprolegnia parasitica (Ali, 2005). significantly inhibit their germinating spores, P<0.001. The reduction of the abundance of CONCLUSION: fungi by the rotifers can be explained by grazing on free swimming fungal zoospores. Saprolegnia parasitica from the study Free-swimming zoospores would be digested Farms is an opportunistic fish pathogen. The in the gut of B. plicatilis, because they do not results of testing the effect of using B. have thick cell walls or sheaths (Beakes et plicatilis and NaCl solution as safe al., 1988). remediation techniques on S. parasitica indicated that presence of B. plicatilis was The results of two way analysis of effective for grazing of the zoospores. Also variance confirmed that the rotifer had great the high sodium chloride levels were efficiency in grazing fungal zoospores. successful in controlling and suppressing the Increasing the time needed for B. plicatilis activities of this fungus. The rotifer B. along with their suitable individual number led plicatilis is more effective in reducing fungal

I ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org 290 Egypt. J. Exp. Biol. (Bot.), 7(2): 285 – 291 (2011) zoospores and it is used as fish food in aquacultures; it is preferred to be used as ACKNOWLEDGEMENTS: antifungal remediation as compared to NaCl The authors wish to express their grateful since salinity may has undesirable effect on thanks to Dr: Hoda H. Yuesef, Faculty of some fish species. Thus, further Science, Alexandria University. The authors investigations are required to study the effect also would like to thank Dr. Hanan Khairy and of the presence of sodium chloride and B. Dr. Heba Saad, National Institute of plicatilis on the fungal and fish growth. Oceanography and Fisheries, Alexandria, for their kind help during this work. 389–394.

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دراﺳﺔ ﻣﻘﺎرﻧﺔ ﺑﺸﺄن اﻟﺘﺤﻜﻢ ﻓﻰاﻟﻔﻄﺮة اﻟﻤﻤﺮﺿﺔ ﻟﻸﺳﻤﺎك ﺳﺎﺑﺮوﻟﺠﻨﯿﺎ ﺑﺮاﺳﯿﺘﯿﻜﺎﺑﺎﺳﺘﺨﺪام اﻟﺮوﺗﯿﻔﺮ ﺑﺮاﻛﯿﻮﻧﺲ ﺑﻠﯿﻜﺎﺗﻠﺲوﻛﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم ھﺎﻟﻪ ﻳﺎﺳﯿﻦ اﻟﻘﺼﺎص، ﻧﺠﻮى اﻟﺴﯿﺪ ﻋﺒﺪ اﻟﻌﺰﻳﺰ اﻟﻤﻌﮫﺪ اﻟﻘﻮﻣﻰ ﻟﻌﻠﻮم اﻟﺒﺤﺎر و اﻟﻤﺼﺎﻳﺪ،ﻓﺮع اﻻﺳﻜﻨﺪرﻳﺔ ﻣــﻦ اﻟﻤﻌــﺮوف ﺟﯿــﺪا أناﻹﻣــﺮاضاﻟﻔﻄﺮﻳــﺔﻓــﻲﺑــﯿﺾ اﻟﺼﻮدﻳﻮم ﻓﻘﺪ وﺟﺪ انﺗﺄﺛﯿﺮه ﻣ ﮫ ﻢ إﺣﺼﺎﺋﯿﺎو انﻧﻤﻮ اﻟﻔﻄﺮة ﻗﺪ وﻳﺮﻗﺎت واﺳﻤﺎك اﻟﻤﯿﺎهاﻟﻌﺬﺑﺔﺗﺴﺒﺐ ﻣﺸﺎﻛﻞ اﻗﺘﺼﺎدﻳﻪﻣﮫﻤﺔ ﻗﻀﻰ ﻋﻠﯿﻪ ﺗﻤﺎﻣﺎ ﺑﻌﺪ ﻳﻮﻣﺎ ﻛﺬﻟﻚ ﻓﻰ وﺟﻮد ﺗﺮﻛﯿﺰات ﻟﺬا ﻛﺎن اﻟﮫﺪف ﻣﻦ ھﺬهاﻟﺪراﺳﺔﻋﺰل و ﺗﻌﺮﻳﻒ ﺑﻌﺾ اﻟﻔﻄﺮﻳﺎت ﻣﺨﺘﻠﻔﺔ ﻣﻦ ﻛﻠﻮرﻳﺪ اﻟﺼﻮدﻳﻮم ﻓﺎﻧـﻪ ﺗـﻢ ﺗﺨﻔـﯿﺾ اﻻوزان اﻟﺠﺎﻓـﻪ اﻟﻤﺴﺒﺒﻪ ﻟﻼﻣﺮاض ﻣﻦ اﻟﻤﯿﺎه اﻟﺘﻲﺗﺮﺑﻰ ﻓﯿﮫﺎ اﻷﺳﻤﺎكﺑﺎﻟﻤﺰارع ﻟﻠﻔﻄﺮة و ﻛﺬﻟﻚ ﻋﺪد اﻟﺠﺮاﺛﯿﻢﺑﻨﺴﺐﻋﺎﻟﯿﻪ و ﻟﻤﺎ ﻛﺎن اﻟﺮوﺗﯿﻔﺮ اﻟﻤﻮﺟﻮدة ﺑ ﻤ ﻨ ﻄ ﻘ ﺔ ﺑ ﺤ ﯿ ﺮ ة ا د ﻛ ﻮ ﻣ ﺼ ﺮ اﺟﺮﻳﺖ ھـﺬه اﻟﺪراﺳـﻪ ﺗﺴﺘﺨﺪم ﻛﻤﺎدهﻏﺬاﺋﯿﺔﻟﻠﯿﺮﻗﺎتﻓـﻲ ا ﻟ ﻤ ـ ﺰ ا ر ع اﻟـﺴﻤﻜﯿﺔ ﻓ ﻤ ـ ﻦ ﺧــﻼل ﻓــﺼﻞ اﻟﺮﺑﯿــﻊ و ﻛﺎﻧــﺖ اﻟﻔﻄــﺮة اﻟــﺴﺎﺑﺮوﻟﺠﻨﯿﺎ اﻟﻤﻔﻀﻞ اﺳﺘﺨﺪاﻣﮫﺎ ﻛﻤﻀﺎد ﻟﻠﻔﻄﺮﻳﺎت ﻋـﻦ ﻛﻠﻮرﻳـﺪ اﻟـﺼﻮدﻳﻮم ﺑﺮاﺳﯿﺘﯿﻜﺎھﻲاﻟﻔﻄﺮة اﻟﻤﮫﯿﻤﻨﻪﻓـﻲ اﻟﻤـﺰارع اﻟـﺴﻤﻜﯿﻪ وﻗـﺪ ﻻن اﺧﺘﻼفاﻟﻤﻠﻮﺣﺔﻗﺪ ﻳﻜﻮن ﻟﻪﺗﺄﺛﯿﺮﻏﯿﺮ ﻣﺮﻏـﻮب ﻓﯿـﻪ ﻋﻠـﻰ رﻛﺰتاﻟﺪراﺳﺔﻷولﻣﺮه ﻋﻠﻰاﻷﻗﻞﻓـﻲ ﻣـﺼﺮ ﻋﻠـﻰاﻟﻤﻘﺎرﻧـﺔ ﺑﻌﺾأﻧﻮاعاﻷﺳﻤﺎك و ﻳﻮﺻﻰ اﻟﺒﺤﺚ ﺑﺈﺟﺮاء ﻣﺰﻳﺪ ﻣﻦ اﻟﺪراﺳـﺔ ﺑـــﯿﻦ ﻛﻔـــﺎءة ﻛﻠﻮرﻳـــﺪ اﻟـــﺼﻮدﻳﻮم و اﻟﺮوﺗﯿﻔـــﺮ اﻟﺒﺮاﻛﯿـــﻮﻧﺲ ﻟﻤﻌﺮﻓــﺔ اﻟﺘــﺄﺛﯿﺮاﻟﻤــﺸﺘﺮك ﻟﻜﻠﻮرﻳــﺪ اﻟــﺼﻮدﻳﻮم واﻟﺒﺮاﻛﯿــﻮﻧﺲ ﺑﻠﯿﻜﺎﺗﻠﺲوأﻇﮫﺮت ﻧﺘﺎﺋﺞ اﻟﺘﺤﻠﯿـﻞ اﻻﺣـﺼﺎﺋﻰ اﻟﺘﺒـﺎﻳﻦﻓـﻲ ﺑﻠﯿﻜﺎﺗﻠﺲ ﻋﻠﻰاﻟﻔﻄﺮﻳﺎت اﻟﻤﻤﺮﺿﺔﻟﻸﺳﻤﺎك اﺗﺠﺎھﯿﻦ ان اﻟﺮوﺗﯿﻔﺮ ذو ﻛﻔﺎءه ﻋﺎﻟﯿـﻪ و ﻣﮫﻤـﺔ ﻟﻠﻘـﻀﺎء ﻋﻠـﻰ اﻟﺠﺮاﺛﯿﻢاﻟﻔﻄﺮﻳﺔأﻇﮫﺮت اﻟﻨﺘﺎﺋﺞ اﻧﻪ ﺑﺎﺳﺘﺨﺪامﻓﺮد ﻓﻘﻂ ﻣﻦاﻟﺒﺮاﻛﯿﻮﻧﺲ ﺑﻠﻜﺎﺗﻠﺲﻓﺎﻧﻪ ﺗﻢ اﻟﺘﺨﻠﺺ ﻣﻦ ﻣ ـ ﻦ ﻋ ـ ﺪ د ﺟـﺮاﺛﯿﻢ اﻟـﺴﺎﺑﺮوﻟﺠﻨﯿﺎ ﺑﺎراﺳـﯿﺘﯿﻜﺎوذﻟـﻚ ﺑﻌـﺪ ﺳـﺎﻋﺘﯿﻦ ﻓﻘـﻂ وﺑﺰﻳﺎد ةﻋﺪد اﻟﺮوﺗﯿﻔﺮ إﻟﻰﻓﺮد ﺗﻢ اﻟﺘﺨﻠﺺ ﻣﻦ و ﻣﻦ اﻟﻤﺤﻜﻤﻮن اﻟﺠــﺮاﺛﯿﻢاﻟﻔﻄﺮﻳــﺔ و ﺑﺰﻳــﺎده وﻗــﺖ اﻟﻨﻤــﻮ ﻟﻠﻔﻄــﺮةﻓــﻲوﺟــﻮد اﻟﺮ وﺗﯿﻔﺮ ﻟﻤـﺪة ﺳـﺎﻋﺎت اﺳـﺘﻄﺎعاﻟﺒﺮاﻛﯿـﻮﻧﺲ ﺑﻠﯿﻜـﺎﺗﻠﺲان أدﻣﺤﻤﺪ إﺑﺮاھﯿﻢ أﺣﻤﺪ ﻋﻠﻲﻗﺴﻢ اﻟﻨﺒﺎت، ﻋﻠﻮم اﻟﻘﺎھﺮة ﻳﺮﻋﻰ ﻋﻠﻰ ﺣﻮاﻟﻲﻣﻦ اﻟﺠﺮاﺛﯿﻢأﻣﺎ ﺑﺎﺳﺘﺨﺪام ﻛﻠﻮرﻳﺪ أ دﺳﻌﺎد ﻣﺤﻤﺪ أﺑﻮاﻟﺴﻌﻮدﻗﺴﻢ اﻟﻨﺒﺎت، ﻋﻠﻮم ﻃﻨﻄﺎ

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