الفهرس 
Aim of the WorkOnly 14 pages are availabe for public view
 |  | from | 163 |  |  |
| | | from | 163 | | |
Abstract
Summary
Pythium is a large and important genus in the class Oomycetes. It is cosmopolitan and can be found as saprophytes, parasites, facultative parasites or mutualistic; aquatic, amphibious or terrestrial fungi. They occur most abundantly in cultivated soils near the rhizosphere of cultivated and wild plant region and less commonly in non-cultivated soil. In Egypt, little attention has been paid to the isolation and identification of Pythium species from rhizosphere soils of wild plants. This work was undertaken to obtain information about the occurrence, identification, physiology of reproduction, pathogenicity and biological control efficiency of Pythium species in different localities in Egyptian soils particularly in the rhizosphere zone of different wild plants growing in Egypt.
Three hundred and seven isolates belonging to 6 species of Pythium were isolated from the rhizosphere soil of 37 different plant species growing in different localities in Egypt. The (6) isolated different species of Pythium, which were identified on both morphological and molecular basis are: P. aphanidermatum (Edson) Fitzp, P. diclinum Tokunaga, P. irregulare (Buisman), P. longisporangium (Paul), P. oligandrum Drechsler, P. ultimum Trow var. ultimum.
P. aphanidermatum was the most predominant species in the rhizosphere soil of the different wild plants studied. Ninety isolates of P. aphanidermatum, representing 29.3% of the total Pythium isolates and were obtained from the rhizosphere soil of Avena fatua, Emex spinosa, Sensio desfontanei, Conyza dioscoridis, Sonchus oleraceus, Amaranthus sp., Cynodon dactylon and Salicornia fruticosa plants growing in Botanical garden of Department of Botany and Microbiology, Minia University in the spring of (2008); Minia city, in front of Alragaa farm, a reclaimed farm in the western desert (2006) and (2008); Bank of the River Nile at Assiut city (2008); At the end of the road side from Assiut to the New Valley (Kharga) (2006); Gezeret Sharona a Nile island near Maghagha, Minia (2007) and (2008); Ekhsas island in the River Nile in Minia city (2006) and (2008) ; Fayoum at Shalshuk closed to Qaroon lake (2005).
On the other hand, moderate isolations were recorded for P. ultimum var. ultimum and seventy-five isolates representing 24.4% occurrence, were isolated from the rhizosphere soil of Avena fatua, Cynodon dactylon, Conyza dioscoridis, Sonchus oleraceus, Zygophyllum coccenum, Imperata cylindrica, Sensio desfontainei and Alhagi maurorum growing in Botanical garden of Department of Botany and Microbiology, Minia University in the spring of (2006); Bank of the River Nile at Assiut city (2005, 2007); Bank of Ibrahimia canal at Assiut city (2007), Hagool valley , suez (2006), Ekhsas island in the River Nile in Minia city (2007); Fayoum at Shalshuk closed to Qaroon lake (2008).
Sixty isolates of P. diclinum, representing 19.5% occurrence, were isolated from the rhizosphere soil of Chenopodium album, Malva parviflora, Zygophyllum coccinium, Sonchus oleraceus, Beta vulgaris, Mesembriyanthemum nodiflorum, Sensio vulgaris, Melilotus indica, Sensio desfontainei, Amaranthus ascendens, Phragmites communis and Zygophyllum album growing in the Botanical garden of Department of Botany and Microbiology, Minia University in the spring of (2005, 2008); Estern desert opposite to Mallawy city, Minia governorate (2006); Bank of the River Nile at Assiut city (2006, 2008); Bank of Ibrahimia canal at Assuit city (2006); At the end of the road side from Assiut to the New Valley (Kharga) (2005); Gezeret Sharona a Nile island near Maghagha, Minia (2006, 2008); Ekhsas island in the River Nile in Minia city (2006, 2008).
Fourty isolates of P. oligandrum representing 13% occurrence, were isolated from the rhizosphere soil of Chenopodium album, Sonchus oleraceus, Amaranthus sp., Melilotus indica, Imperata cylindrica , Phragmites communis plants growing in Botanical garden of Department of Botany and Microbiology, Minia University in the spring of (2007); Bank of the River Nile at Assiut city (2006); Bank of Ibrahimia canal at Assiut city (2005); Gezeret Sharona, a Nile island near Maghagha, Minia (2006, 2007); Ekhsas island in the River Nile in Minia city (2005) ), Fayoum at Shalshuk closed to Qaroon lake (2007) and Fayoum, Shalalat, Wadi Elrayan (2008). This fungus is a special interest since it is used as a biological control agent of some fungal diseases.
Isolations were also recorded for P. irregulare and twenty-six isolates representing 9.8% occurrence was recorded for isolates from rhizosphere soil of four plants only which are; Beta vulgaris, Melilotus indica, Zygophyllum album, cynodon dactylon, which were growing at the Bank of Ibrahiimia canal at Assiut city (2005); Gezeret Sharona, a Nile Island near Maghagha, Minia (2006); Ekhsas Island in the River Nile in Minia city (2005).
Low occurrence was recorded for P. longisporangium and sixteen isolates representing 5.2% occurrence, were isolated from the rhizosphere soil of Mesembryanthemum nodiflorum growing at the end of the side road from Assiut to the New Valley (Kharga).
The 6 isolated species of Pythium were described morphology and detailed plates for each species were provided. P. aphanidermatum showed temperature maxima higher than other Pythia studied (43°C). The optimum temperature for all the fungi investigated ranged between 25°C and 30°C. The minimum temperature supporting mycelial growth of Pythium spp. was around 5°C whereas P. aphanidermatum started to grow at 10°C. Sequencing of rDNA-ITS including the 5.8SrDNA were performed for the Pythium species isolated to confirm their identification. The sequence of (HS24) was closely related with that of P. aphanidermatum (Genbank accession number, AB274404.1) with 100% similarity. The sequence of (HS 1) was closely related with that of P. diclinum ((Genbank accession number, AY598689.1) with 99% similarity. The sequence of (HS13) was closely related with that of P. irregulare (Genbank accession number, AF452142.1) with 100% similarity. The sequence of (HS2) was closely related with that of P. longisporangium (Genbank accession number, AY455693.1) with 100% similarity. The sequence of (HS10) was closely related with that of P. oligandrum (Genbank accession number, AY986954.1) with 100% similarity. The sequence of (HS21) was closely related with that of P. ultimum var. ultimum (Genbank accession number, AY598657.1) with 100% similarity.
Pathogenicity (pre-emergence damping-off) of Pythia was tested on cucumber seeds. P. aphanidermatum and P. diclinum were highly pathogenic to cucumber seeds causing 100% damping-off, where as P. ultimum was moderately pathogenic (67%) damping off, P. longisporangium and P. irregulare were weakly pathogenic producing not more than 33% damping-off in case of cucumber germinating seeds. P. oligandrum, on the other hand was non-pathogenic to cucumber seeds as tested in agar bottles. Pre-emergence damping-off pathogenicity tests were performed also in pots using 2.5% inoculum concentration of the tested Pythia added to soil and incubated for 2-3 weeks before cucumber seeds was cultivated. P. aphanidermatum proved to be the highly pathogenic to cucumber seeds causing 100% damping-off. P. diclinum was similarly highly pathogenic to cucumber germinating seeds causing 80%, whereas P. ultimum was moderately pathogenic causing 60% and P. irregulare and P. longisporangium were weakly pathogenic causing 40% and 20% respectively, only P. oligandrum was non-pathogenic to cucumber seed.
Three selected species ( P. aphanidermatum, P. irregulare and P. oligandrum) were chosen to study some factors affecting oospore production and germination. Oospores were produced over a temperature ranging of 5-35 º C for P. irregulare, 10-35 ºC for P. longisporangium and 15-35 ºC for P. oligandrum. The optimum temperatures were 30, 28 and 25°C for oospores production by P. irregulare, P. longisporangium and P. oligandrum, respectively. oospores production happened over a range of pH 5-9. Optimum pH values were noticed between 6-7.5 for P.irregulare, P. longisporangium and P.oligandrum respectively. The effect of osmotic potential on oospores production at 28°C is studied. Tested species showed similar responses. Oospores produced at –0.13 to –1.65 MPa with the optimum production rate at –0.27 to –0.47 MPa. Regarding oospore germination, oospores of P. irregulare showed good germination between 25ºC and 35 ºC, with optimum at 30°C but not below 20 ºC and very low at 40 ºC; whereas those of P. longisporangium and P. oligandrum showed good germination between 20 ºC and 30 ºC with optimum also at 30°C, but did not germinate below 15°C or above 35ºC. All the fungi tested germinated over a range of pH 5 to pH 9 with an optimum between 6.5~8. The effect of osmotic potential on oospores germination at 28ºC was studied. All of the species tested showed similar responses. At –3.40 MPa, non could germinate. They germinated at the other osmotic potentials tested, with good germination between –0.13 and –0.27 MPa and the optimum at –0.47MPa.
Antagonism between P. oligandrum and pathogenic Pythium species was done in each of agar bottles, pots and the field. In agar bottles, controlling damping-off of cucumber caused by P. aphanidermatum, P. diclinum, and controlled by P. oligandrum grown on 2% water agar were done. Application of P. oligandrum improved emergence of seedling in case of seeds subjected to infection by the pathogenic pythia as: P. aphanidermatum from 0% to100%; P. diclinum from 0% to 100%. In pot experiment, controlling damping-off of cucumber seeds and seedling caused by P. aphanidermatum, P. diclinum, and controlled by P. oligandrium were performed. Soil treatment by P. oligandrum antagonised the effect of the pathogenic pythia improved seed emergence subjected infection by: P. aphanidermatum from 0% to 80% and P. diclinum from 20 % to 80%. In order to expand the pot experiments to the field to verify the results obtained in these experiments and test the applicability of the biocontrol to the field using cucumber seeds, seeds were sown in four lines. The first line contains soil infested with P. aphanidermatum and a second line infested with both P. aphanidermatum and P. oligandrum and a third line infested with P. oligandrum only as well as a line with no added Pythium. Results indicated very poor emergence in case of line infested with P. aphanidermatum only while the line infested with P. oligandrum alone gave very good emergence and healthy seedlings. In case of the line in which the soil infested with P. aphanidermatum and treated with P. oligandrum before planting cucumber seeds, the treatment increased emergence of seedlings which was almost similar to that of absence of the pathogen. Therefore, verifying the suitability of P. oligandrum as a biocontrol agent to damping-off disease of cucumber caused by P. aphanidermatum under field conditions was confirmed.