الفهرس 
CitrusOnly 14 pages are availabe for public view
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Abstract
Citrus is considered the main fruit crop in Egypt, which
represents about 15% of the total citrus production in the
Mediterranean Basin and is considered the ninth largest citrus
producer in the world (FAO, 2013). Citrus is belonging to the
genus citrus L., Family: Rutaceae, Sub family: Aurantioideae.
Citrus trees are subjected to invasion by several bacterial, fungal,
virus and virus –like diseases. In recent years, trees of citrus have
been seriously affected by stubborn disease caused by a
prokaryotic pathogen Spiroplasma citri (Gumpf et al., 1981).
Spiroplasma citri are a Pleomorphic, phloem-limited, cellwall-less bacterium in class Mollicutes. Members of the class
Mollicutes, phylogentically related to Gram-positive bacteria are
the smallest free-living organisms known. They are characterized
by a small genome size (580 to 2200 kbp) and low G+C contents
(Barre et al., 2004). In the Mollicutes, the genus Spiroplasma
consists of a group of motile helical prokaryotes that are
associated primarily with arthropods, mainly insects (Gasparich
et al., 2004). Only three species, Spiroplasma citri, S. kunkelii,
and S. phoeniceum are pathogenic to plants (Saglio et al., 1973;
Whitcomb et al., 1986; Saillard et al., 1987). S. citri, the first
plant pathogenic mollicute to be cultured and characterized
(Saglio, et al., 1971). It multiplies and moves slowly through the tree, cholesterol and fructose from the requirement to growth. S.
Citri absolute resistance to penicillin, low guanine and cytosine
content of cellular deoxyribonucleic acid (DNA), has a small
genome size, and several cultural properties (Gaurivaud et al.
2000a; Bové et al. 2003).
S. citri cannot be transmitted mechanically but transmitted
in a circulative-propagative manner by several phloem-feeding
homopteran insects including the leafhoppers (Circulifer tenellus)
in the American basin and C. haematoceps (syn. Neoaliturus
haematoceps) in the Mediterranean region (Calavan and Bové,
1989). S. citri are also transmitted by grafting and dodder
(Gaurivaud et al., 2000a; Lee et al., 2000).
Among the major constraints, Stubborn disease of citrus
(CSD) is a very serious disease in most citrus growing regions
and dramatically decreases citrus yields (Bové, 1986). Symptoms
vary in intensity with variety, and are not limited to, localized to
generalized bunchy growth of foliage induced by a shortening of
nodes on branches; and include dense, small and abnormally
upright leaves showing variable chlorotic patterns resembling
nutritional deficiencies; and off-season blooming that results in
variable size and maturity of fruit.
Although the disease is rarely lethal, affected trees can be
severely stunted, produce lopsided fruit that remain green at the
stem (acropetal) end and have aborted seeds fruits (Bové and Garnier, 2002; Yokomi et al, 2010; Shi et al., 2014). Field
diagnosis of CSD, however, is difficult and often inaccurate as
symptoms can be confused with those of other citrus pathogens or
nutritional problems (Polek et al., 2007).
In addition, detection in field samples is erratic due to low
titer and uneven distribution of the pathogen. Since S. citri grows
well at warm temperatures, stubborn disease diagnosis may be
most reliable in the summer months. Further genetic studies have
led to the identification of Spiroplasmal genes associated with
biological functions such as motility, insect transmission, and
pathogenicity (Bové et al., 2003).
S. citri can be reliably detected by culturing in cell-free
liquid medium and observing the organism by dark field
microscopy to confirm its typical helical morphology and motility
(Yokomi et al., 2008). Detection of S. citri by polymerase chain
reaction (PCR) has been described by others, who employed
primers designed from several sources: sequences of S. citri virus
SpV1 strain R8A2B (Saillard et al, 1996), another spiroplasma
virus, 16S rDNA (Lee et al, 2006), spiralin sequences (Rangel et
al, 2005), and two adhesin-associated S. citri proteins, P89 and
P58 (Yokomi et al., 2008). Real-time PCR is useful method for
Spiroplasma detection in infected plant phloem or insect vectors
with 100-1000 times of sensitivity greater than convential PCR
(Yokomi et al., 2008). Our results can be summarized in the following points:
1. Different citrus samples showed symptoms suspected to
stubborn disease were collected from different
Governorates in Egypt including Ismailia, Al-Qalyubia and
Kafr El- Shakh.
2. The causal agent (S. citri) was isolated from infected plants
using:
C-3G liquid media to obtain the change of the normal
red color of the C-3G media to the yellow color which
indicate the presence of the S. citri growth.
C-3G solid media by Inoculating it with liquid growth
S.citri media to obtain the fried-egg shape which
confirm the presence of S.citri.
3- Several identification methods were used including:
Dienes’ stain was carried out to confirm the S. citri
infection through an investigation on the anatomy of
diseased plants. The phloem of infected stem
sections showed many irregularly areas that stained
in a distinct blue. However, the phloem tissue of
healthy stem sections remained unstained. This
might be due to the sieve tubes of the phloem tissue
without nucleus. Hence, the distinct blue color of the phloem tissue of the infected plant might be a result
of the staining of S citri nucleic acids.
Fresh leaves of infected citrus were used for
extraction of DNA extracted using three different
extraction methods, the extracted DNA were
amplified by PCR. The obtained results
demonstrated that by using different methods we are
able to detect the S. citri in infected tissue in most
experiments.
Primers for isolating different genes were used to
identify S. cirti including P89, P58, and spiralin
gens,
The PCR assays results prove the efficiency of
primers based on gene sequences for putative P89
adhesin and putative P58 adhesin-like for the
molecular detection of the Egyptian isolates of S.
citri than spirallin gene
Cloning and sequencing for P89 and spiralin gene
were prepared, and sequences were analyzed with
the other sequences available in the Gene Bank. The
highest degree of similarity with P89 gene of
Egyptian isolate was found with P89 gene of GizaMan isolate (HE617172) from Egypt. The highest
similarity degree of spiralin gene Egypt isolate was found with spiralin gene of Qualubia isolate
(AM157771).
4. Repetitive extra palindromic-polymerase chain reaction
(rep-PCR) analysis, based on the occurrence and
distribution of repetitive elements within the genome, has
been used to assess genetic diversity in S. citri. The results
of rep- PCR revealed the presence of repetitive elements in
Spiroplasma genomes. The elements varied in number and
distribution in different locations in the genomes of samples
which suggested that the different isolates of Spiroplasma
citri were present.
5. The different concentrations of Tetracycline-HCl antibiotic
were used to control the growth of S. citri.
0.2 µg/ ml was a lowest concentration of tetracycline
– HCl that prevented growth of S. citri in broth
media so the color of C-3G media wasn’t change.
2 mg/L was the most effective concentration of
tetracycline-HCl antibiotic which gave the highest
control degree as symptoms reduction in infected S.
citri seedling.
Reverse transcriptase quantitative real-time
polymerase chain reaction (RT-qPCR) has become a
very powerful technique for confirm the reduction S.
citri growth in infected seedling. These results will help to reveal the true image of the real
distribution and spread of CSD, and will also facilitate and
improve the citrus certification programs as well as the
management of this disease through the early and precise
detection even if the pathogen titer is low in the collected
samples. This will help for the enhancement of the quality
of the produced citrus fruits in Egypt in the future.