الفهرس 
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Abstract
Date palm (Phoenix dactylifera L.) is one of the most important
tropical and subtropical fruit tree in Egypt and in the Arabian region. Date
palm belongs to genus phonix in the family Arecaceae that
monocotyledon, dioecious plant. The Arabic terms of maturation stages of
dates are Hababouk, kimri, khalal, rutab and tamar respectively.
In the present work, different studies on bacterial diseases that
infected date-palm and their specific bacteriophages were carried out. The
results can be summarized as follows:
Bacillus subtilis that considered pathogenic bacteria on date palm
that caused systemic wilt and external malformation on date palm in
tissue culture. Bacteria was obtained from central laboratory of date palm,
Agricultural research center (ARC) Giza, Egypt
The other source of pathogenic bacteria was isolated from infected
date palm exhibited dark pink and black spots on trunk, the flowers were
colored with pink and in later stages flowers were colored black. Bacteria
was isolated from date palm trunk that obtained from Agricultural
research center (ARC) Giza, Egypt. The bacteria was isolated on NA
medium and streaked on EMB and XLD media.
The pathogenicity test of B.subtilis and the isolated bacteria was
carried out and same symptoms were appeared on plants.
The isolated bacterium was identified using light microscope and
was found to be Gram negative, rod shape, single, non-spore forming.
TEM showed that bacterium is short rod with length 1.8 micron and
peritrichous flagella. Physiological method using bergey’s Manual
referred to the isolated bacterium is Serratia marcescens. Molecular
method using 16SrDNA sequencing showed that the isolated bacterium is
Serratia marcescens.
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SUMMERY
Loay L. Abd Allah, (2018),Ph.D., Fac. Agric., Ain Shams Univ.
Pollen grain viability of infected flowers with S.marcescens
decreased to 73.2% comparing with the control that was 98%.
Bacteriophages were isolated from infected trunk and free soil. The
isolated phages for S.marcescens named Serratia plant 1 (SP1) and
serratia plant 2 (SP2) from infected trunk and serratia soil 1 (SS1) from
soil. The isolated phages for B.subtilis named bacillus soil 1 (BS1) and
bacillus soil 2 (BS2) and bacillus soil 3 (BS3) from soil. The phages were
isolated according to the shape of plaques of each plaque.
Detection of temperate phage in S.marcescens and B.subtilis was
assayed using UV lamp as induction tool. S.marcescens and B.subtilis
were found to be free from prophages.
Morphology of isolated bacteriophages was examined using TEM.
Three isolated phages of S.marcescens were 98x90nm in head dimensions
and 213 nm in tail length in SP1, 86x104 nm in head dimensions and 128
nm in the tail length in SP2 and 111x112nm in head dimensions and 215
nm in tail length in SS1. Three isolated phages of B.subtilis were
110x85nm in head dimensions and 191nm in tail length in phage BS1,
85x79nm in head dimensions and 245nm in tail length in BS2 and
109x110nm in head dimensions and 149nm of the tail length in BS3.
Taxonomy of isolated phages showed SP1 belongs to Siphoviridae
and SP2 and SS1 belong to Myoviridae in Serratia phages. BS1 and BS3
belong to Myoviridae and BS2 belong to Siphoviridae in Bacillus phages.
The ultraviolet spectrum was used to measure the absorbance of
purified phages using differential centrifugation. The A260 / A 280 ratio
were 1.2, 1.2, 1.6, 1.1, 1.3 and 1.2 for SP1, SP2, SS1, BS1, BS2 and BS3
respectively. A 280 /A260 ratio were 0.83, 0.84, 0.62, 0.82, 0.77 and 0.82
for SP1, SP2, SS1, BS1, BS2 and BS3 respectively.
Thermal inactivation point (TIP) of phages was 54±1, 78±1, 52±1,
58±1, 46±1 and 54±1 for phages SP1, SP2, SS1, BS1, BS2 and BS3,
respectively.
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Loay L. Abd Allah, (2018),Ph.D., Fac. Agric., Ain Shams Univ.
pH range of phages were 7-9, 6-9, 5-9, 6-8, 6-9 and 5-8 for phages
SP1, SP2, SS1, BS1, BS2 and BS3 respectively. Concentration of phages
in pH range was 3.5x104, 6x107and 5x105 for SP1 from pH 7-9, 1x103,
2.7x106, 5x109 and 9.8x106 for SP2 from pH 6-9, 8x102, 7x104, 2.4x107,
4x1010 and 4x105 for SS1 from pH 5-9, 1x102, 4x105 and 3.1x102 for BS1
from pH 6-8, 3x103, 3.7x105, 6x103 and 2.1x102 for BS2 from pH 6-9,
2x103, 3.3x104, 2.3x105 and 1x104 for BS3 from pH 5-8.
Freezing and thawing of phages showed that phages still active for
3, 3, 2, 4, 2 and 1 times of freezing and thawing for phages SP1, SP2,
SS1, BS1, BS2 and BS3 respectively. The concentration was 6.2x105,
5x104 and 2.2x103 for SP1, 3x107, 2.7x104 and 5.2x103 for SP2, 1x105
and 3.7x103 for SS1, 2x106, 2x105, 1x105 and 3x103 for BS1, 4.6x104 and
2.6x103 for BS2 and 4.1x103 for BS3
DNA of the phages was extracted and the concentration of the DNA
was 10.13, 9.76, 11.48, 12.14, 10.75 and 7.26 μg/ml for SP1, SP2, SS1,
BS1, BS2 and BS3 phages respectively. RAPD-PCR was carried out for
each phage’s DNA. SP1 amplified to 4 fragments with 2000, 1500, 1000 and
600 bp, phage SP2 amplified to 4 fragments with 1500, 1000, 700 and 600
bp, SS1 phage amplified to 5 fragments with 1500, 1000, 800, 700 and 600
bp, Monomorphic bands among Serratia phages. BS1 amplified to 3
fragments with 1000, 600 and 500 bp, phage BS2 amplified to 2 fragments
with 1000 and 600 bp, BS3 phage amplified to 2 fragments with 1000 and
500 bp.
Purified phages were used to measure the quantity of viral protein
using Bradford method. The obtained results showed that the phages
contained 0.29, 0.35, 0.28, 0.23, 0.14 and 0.28 mg/ml of protein for SP1,
SP2, SS1, BS1, BS2 and BS3, respectively.
Stability of the phages for exposure to UV radiation was estimated.
Sensitivity of S. marcescens and B.subtilis phages to UV radiation for 25,
20, 25, 20, 15 and 20 min for phages SP1, SP2, SS1, BS1, BS2 and BS3
respectively. the concentration of phages was2x1010, 5.7x109, 4.3x107,
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Loay L. Abd Allah, (2018),Ph.D., Fac. Agric., Ain Shams Univ.
1.3x106 and 3.1x103 for SP1 from 5-25min, 4.1x108, 2x106 and 5.4x103
for SP2 from 5-15min, 2.6x109, 3x108, 1x107, 2.2x105 and 1.6x102 for
SS1 from 5-25min, 2.1x105, 1.3x104, 1x103 and 1.1x102 for BS1 from 5-
20min, 1x106, 1.3x104 and 1.1x102 for BS2 from 5-15min, 2x106,
2.5x105, 1x103 and 4.2x102 for BS3 from 5-20min
Structural protein of the isolated phages was determined using SDSPAGE
method. SP1 phage contain of 3 proteins with molecular weight
∼66, 35 and 29 KDa. SP2 phage contains of 4 proteins with molecular
weight∼ 38, 35, 30 and 29 KDa. SS1 phage results showed that contain of
5 with ∼97, 38, 35, 30 and 29 KDa. Four proteins in BS1 with molecular
weight ∼97, 70, 37 and 35KDa. BS2 phage that contains of 5 proteins
with molecular weight ∼120, 97, 70, 35 and 25 KDa. BS3 phage contains
of 4 proteins with molecular weight ∼97, 70, 35 and 27 KDa.
LIV of isolated phages showed that all phages were stable at room
temperature for 90 days.
Host range for the isolated phages for S. marcescens and B.subtilis
were assayed with the identified bacteria. SP1 and SP2 infected S.
marcescens A, B and C while SP1 and SS1 infected S. marcescens D. All
B.subtilis phages couldn’t infect any identified bacteria that used as hosts.
Five formulas were added to isolated bacteriophages to protect from
UV irradiation. The formulas are skim milk +sugar, Beet-root juice,
Carrot juice, Sodium alginate and Casein. The best used formulas for that
used with all phages from the results and statical analysis were Beet-root
juice and Carrot juice for S. marcescens phages. The best used formulas
for that used with all phages were Beet-root juice and Casein formulas
B.subtilis phages.
Three different methods were used to inoculate bacteria in dateplants.
The plants inoculated with 1x1012 cfu of bacteria with syringe,
needle and blade. Blade method considered the most method to kill the
treated plants with S.marcescens.
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Loay L. Abd Allah, (2018),Ph.D., Fac. Agric., Ain Shams Univ.
Antimicrobial sensitivity using ampicillin antibiotic with S.
marcescens and B.subtilis was tested and the results showed that
concentration 400mg/L of ampicillin stopped growth S. marcescens and
100 mg/L of ampicillin stopped growth B.subtilis.
Detection of bacteria and phages on treated bacteria showed
bacteria were isolated at zero time and phages were detected at 1hour in
preservation and prevention in In vivo and In vitro experiments.
Total phenolic compounds in treated plants decreased in
preservation and prevention In vivo and In vitro experiments comparing
with control. The activity of polyphenol oxidase in preservation treatment
increased comparing with control. The total indols in treated plants
showed that preservation experiment In vivo and In vitro experiments
comparing with contro