الفهرس 
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Abstract
The present investigation was undertaken during the period from2014-18 at the greenhouse and laboratories of Department of Plant Pathology, Faculty of Agriculture, University of Assiut, Assiut, Egypt. The specific objectives were to isolate and identify of the causal pathogen of bacterial ring rot of potato (Clavibacter michiganensis subsp. sepedonicus). Control of the disease under greenhouse conditions using bacterial bioagents, essential oils and porpolis. Enhancement of plant quarantine efforts by improvement of identification and detection methods of potato ring rot pathogen like design and evaluation of new target- specific PCR primers and characterization of the causal pathogen isolates using FT-IR spectroscopy and MALDI-TOF mass spectrometry.The salient features of the findings have summarized below:1. Out of collected samples of potato tubers (cv. Diamant and cv. Spunta) showing typical symptoms of bacterial ring rot disease caused by Clavibacter michiganensis subsp. sepedonicus (Cms), only 10 confirmed Cms isolates were obtained.>2. Pathogenicity tests of all ten isolates showed that all isolates were pathogenic on eggplant and produced typical symptoms of ring rot disease. Isolates No. 4 exhibited the highest disease severity percentage (100%), followed by isolate No. 5 which caused 83.5% disease severity. The rest of isolates caused moderate percentage of disease severity (78-66.5%), while isolate No. 8 caused the lowest percentage of the disease severity (60%).3. Potato tuber lesions assay has been used for more confirmation of the pathogenicity of Cms isolates and the results of this test confirmed that all Cms isolates were severely manifested aggressive lesions. Also, isolate No. 4 showed the highest aggressive lesions percentage (91.67%), followed by isolate No. 5 (83.33%). The rest of isolates recorded moderate percentage of aggressive lesions (75-50%), while isolate No. 8 recorded the lowest percentage (42%).4. Pure cultures of ten isolates were identified as Clavibacter michiganensis subsp. sepedonicus using biochemical and physiological studies and confirmed by PCR.5. Morphology of Cms isolates cells were observed by scanning electron microscope (SEM). Scanning electron microscopy of Cms isolates cells showed that they exist as non‐motile, non‐spore‐forming and colonies of rod-shape bacteria with an average cell length of 1.35 µm and cell width of 0.59 µm, which appeared straight to slightly curved or wedge shaped. Also, coccoid forms were observed. 6. Nineteen biocontrol agents were evaluated for their antagonistic activity against Cms. Bacillus aerius PST13, Bacillus subtilis MIRCEN and Pseudomonas fluorescens MIRCEN inhibited the growth of the bacterial ring rot pathogen. The other tested bacterial bioagents were had no effective. 7. Out of thirteen isolates of bacterial bioagents obtained from potato rhizosphere, surface of potato tubers and tuber sap, isolate PST13 showing antagonistic activity against Cms was molecularly characterized by means of 16S rRNA sequencing as Bacillus aerius.
8. Under greenhouse conditions, all three tested biocontrol agents were reduced significantly the disease: a- In trail 1 where potato tubers were treated with different biocontrol agents, Pseudomonas fluorescens MIRCEN exhibited the highest disease reduction percentage of potato ring potato disease (18%) followed by Bacillus subtilis MIRCEN and Bacillus aerius PST13 which recorded 15.78 and 9.13%, respectively.b- In trail 2 where treatments were done on potato eyeballs, Pseudomonas fluorescens MIRCEN exhibited the highest disease reduction percentage of potato ring potato disease (26.16%) followed by Bacillus subtilis MIRCEN and Bacillus aerius PST13 which recorded 24.05 and 19.83%, respectively c- In trail 3 where eggplant seedlings were treated with different biocontrol agents, Pseudomonas fluorescens MIRCEN recorded30.64% of disease reduction, Bacillus subtilis MIRCEN recorded
24.86 and Bacillus aerius PST13 recorded 19.08%.9. A screening for lytic exoenzymes in the effective bacterial bioagents under greenhouse conditions was investigated in vitro. The enzyme activities of Bacillus subtilis MIRCEN was found to be the highest for amylase, pectinase and protease. While Pseudomonas fluorescens MIRCEN found to be the highest for cellulase. Bacillus aerius PST13 record the minimum enzyme activities for all investigated enzymes.10. In vitro, twelve plant essential oils namely Lavender, Thyme, Peppermint, Marjoram, Mint, Lemon, Dill, Basil, Camphor, Common
sage, Ginger and Clove oils were evaluated for their antagonistic activity against Cms. Thyme oil exhibited the highest index of antimicrobial effectivity percentage (17.97%), followed by clove oil recoded 16.27 then peppermint and mint oils with 9.6 and 8.36%, respectively. While, lavender and ginger showed the lowest index of antimicrobial effectivity percentages against Cms with 6.93 and 6.31%, respectively 11. Under greenhouse conditions, six plant essential oils were evaluated for their control efficacy against potato ring potato disease. All tested plant essential oils reduced significantly the disease in all trails. Disease reduction percentages were varied from 13.04 to 46.59%.12. The most effective essential oils under greenhouse were chemically analyzed using Gas chromatography Mass Spectrum (GC-MS) system. Gas chromatography mass spectrum analysis on thyme oil identified and quantified nineteen constituents and the main constituents were identified as 2, 6, 10, 14-tetramethyl-7- (3-methylpent-4-enylidene) pentadecane (23.63%), hexadecanoic acid, methyl ester (21.69), 1- Nonadecene (8.13%), benzene (5.81%) and (3, 4- dimethoxyphenyl) (3H- imidazol-4-yl) methanone (4.94%), while others were below 4%. Also, Gas chromatography mass spectrum analysis on clove oil identified and quantified eighteen constituents and the main constituents were identified as tetracosane (20.23%), 1-nonadecene (17.59%), 2-piperidinone, N- [4- bromo-n-butyl]- (13.26%), 1-eicosene (11.77%), tetrapentacontane, 1, 54- dibromo- (7.97%) and tetratriacontyl trifluoroacetate (6.06%), while others constituents were below 6%. Finally, Gas chromatography mass spectrum analysis on peppermint oil identified and quantified eighteen constituents and the main constituents were identified as cyclohexanol, 5- methyl-2-(1-methylethyl)- (28.47%), cyclohexanone, 5-methyl-2-(1-
methylethyl)- (19.91%), 1-menthone (14.53%) and 4-cyanobenzophenone
(6.95%), while others constituents were below 6%.
13. In vitro, the antibacterial activities of propolis water extract were tested at four different concentrations (1, 5, 10 and 20%) against Cms. All concentrations of water extract of propolis inhibited significantly the growth of Cms. Concentration of 20% recorded the highest inhabitation growth percentage of Cms by 30.48%, followed by 10 and 5% concentrations which inhibited the growth of Cms by 21.53 18.73 and %, respectively. Whereas, 1% concentration recorded the lowest growth inhibition percentage (7.33%).
14. The in vivo trails results revealed that all applied concentrations of propolis water extract reduced significantly the disease severity. In all trails, applied contractions reduced the disease and the reduction percentages were varied from 12.5 to 52.52%. 15. Gas chromatography mass spectrum analysis on propolis water extract identified and quantified ten constituents and the main constituents were identified as benzoic acid, p-chloroaniline, 3-decen-5-one, phthalic anhydride, benzenepentanoic acid, 2-propenoic acid, 3-phenyl-, ethyl and –norephedrine.16. Among ten target-specific PCR primers designed using Primer- BLAST based on Cms plasmid pCSL1 (NCBI reference NC_010408.1) with the range was 60000 – 75000 base pairs (bp), primer pair 6 which is based on range of 63808- 64165 with a predicted PCR product of 358 bp was selected. Primer pair 6 was tested in vitro and PCR amplification of all ten Cms isolates produced the expected DNA products of 358 bp.
17. Ten FT-IR spectroscopy spectra were recorded from all Cms isolates. The fingerprint bands varied from 1 to 12 according to isolate. Isolate Cms5 recorded the highest fingerprint bands (12) followed by isolate Cms6 with 11 fingerprint bands then isolate Cms4 (10). While, the lowest fingerprint bands recorded by isolates Cms1, Cms3 and Cms10 with 1, 2 and 2 fingerprint bands, respectively.18. All Cms isolates were subjected to MALDI-TOF MS analysis. The obtained spectral profiles were screened for the presence of recurring peaks or biomarker ions specific for Cms. Forty six m/z values were detected in about all isolates, making them characteristic for Cms. The most frequency peaks were assigned 50S ribosomal protein L35 followed by 50S ribosomal protein L30 then 50S ribosomal protein L33. While the lowest frequency peaks were assigned 50S ribosomal protein L36 1, 50S ribosomal protein L36 2, 30S ribosomal protein S20, 30S ribosomal protein S18, 50S ribosomal protein L31 type B and 50S ribosomal protein L21.