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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. |