Abstract: Spiroplasmas are helical motile filamentous, wall-less and culturable mollicutes. Thirty six spiroplasma species have been identified. Only S. citri, S. kunkelii and S. phoeniceum have been identified as plant pathogens. Spiroplasma citri, the causal agent of citrus stubborn disease, have a wide host range. S. citri infects most citrus species and cultivars and a wide range of non-rutaceous plant species. Citrus srubborn disease widely distributed in the southwestern united states of America, northern Africa and Mediterranean countries. It is naturally transmitted by phloem-feeding leafhopper vectors. S. citri can be detected by grafting to citrus indicators, culturing on artificial media, serological, DNA probes, dot-immunobinding assay, Immunocapture Polymerase Chain Reaction (I C- PCR), Polymerase Chain Reaction (PCR) and real-time PCR. There is genetic variability among isolates of S. citri.
INTRODUCTION
Spiroplasmas (spira: a coil, spiral; plasma: something formed or molded) are motile, filamentous, wall-less and culturable mollicutes (Whitcomb and Tully, 1982; Bove, 1997). Spiroplasmas have helical, tubular and pleomorphic morphology. Extracellular spiroplasmas are more helical and tubular whilst round or flask forms are more dominant in intracellular spiroplasmas (Ammar et al., 2004). They were discovered from infected corn (Zea mays L.) plants with the corn stunt agent by dark-field microscopy (Davis et al., 1972). In 1971, Spiroplasma citri was cultured in artificial media (Saglio et al., 1971). The genus Spiroplasma was stablished in 1973 through studies on motile and helical microorganism associated with corn stunt disease (Davis and Worley, 1973). In 1973, the first spiroplasma species has been named as Spiroplasma citri as well (Saglio et al., 1973). S. citri is the type species of the type genus Spiroplasma of the family Spiroplasmataceae. Taxonomically, Spiroplasmas are in the kingdom Bacteria, Phylum Tenericutes, Class Mollicutes, Order Entomoplasmatales, Family Spiroplasmataceae and Genus Spiroplasma (Gasparich, 2002, 2010; Tully et al., 1987; Williamson et al., 1998). Based on the spiroplasma species concept as determined by the International Research Programme on Comparative Mycoplasmology and the International Subcommittee on the Taxonomy of Mollicutes, 36 spiroplasma species have been identified (Whitcomb et al., 1987; Whitcomb, 1977; Wang et al., 2010) (Table 1). Only S. citri, S. kunkelii and S. phoeniceum have been identified as plant pathogens.
The genome size of spiroplasmas ranges from 780 to 2220 kbp (Carle et al., 1992, 1995; Williamson et al., 2010) and the S. citri genome is one of the largest among Mollicutes with a high adenosine-thymidine content (up to 75%) (Melcher and Fletcher, 1999). G+C base composition of spiroplasmas ranges from 24-31% (Carle et al., 1983; Gasparich et al., 2004). They are helical filaments with 3-15 μm length range and 200-250 nm in width and an amplitude of 0.4 μm (Fletcher et al., 2006). Spiroplasmas require sterol for growth. They are resistance to penicillin. In spiroplasmas, UGA is not used as a stop codon. They use both UGA and UGG as tryptophan codons (Citti et al., 1992; Renaudin et al., 1986; Stamburski et al., 1992). In most mollicutes, comprising spiroplasmas, UAA and UAG are possible termination codons (Melcher and Fletcher, 1999).
Spiroplasma citri: Spiroplasma citri, the causal agent of citrus stubborn disease, is restricted to the phloem sieve tubes in plants. It is an obligate parasite, surviving in citrus or in a variety of other host plants, with no saprophytic phase. Citrus Stubborn Disease (CSD) was first observed in Washington navel orange trees in California about 1915 (Fawcett et al., 1944).
Spiroplasma citri infects most citrus species and cultivars and a wide range of non-rutaceous plant species (Gumpf and Calavan, 1981; Oldfield and Calavan, 1980; Whiteside et al., 1988).
| Table 1: | Spiroplasmas classification and characterization |
Citrus is the main economic host of Spiroplasma citri. The name "stubborn" should be restricted to the disease in citrus.
Genome characteristics: G+C content of S. citri GII3-3X chromosome is 26.1%. It encodes one single 16S-23S-5S rRNA operon (Carle et al., 2010). Several pathways have been identified in S. citri, including phosphoenolpyruvate Phosphotransferase System (PTS) to import sugars to synthesize ATP using F0F1-ATP synthase, purine and pyrimidine metabolism pathways, pathway for the biosynthesis of a C55 terpenoid, 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate (MEP/DOXP) pathway to the synthesis of isopentenyl pyrophosphate, glycolytic and lactate dehydrogenase pathways to enhance fermentation. S. citri chromosome also encodes essential subunits for ATP synthase and ATP Binding Cassette (ABC) transporters. Whilst 21% of truncated CDS in S. citri GII-3X chromosome compared to their bacterial orthologs, reveals an important gene decay. It indicate that the reductive evolution of the spiroplasma genome to smaller genomes may be is still ongoing on the way (Carle et al., 2010).
There are many Coding Sequences (CDS) of plectovirus in the S. citri chromosome (Carle et al., 2010). Spiroplasma citri use fructose for pathogenicity and growth in plants (Andre et al., 2005; Gaurivaud et al., 2000). Spiralin, the most abundant and major membrane lipoprotein of 26 kDa, is essential for transmission of S. citri by the leafhopper vector Circulifer haematoceps (Duret et al., 2003).
Host range: Stubborn is an important disease of citrus. Sweet orange (C. sinensis (L.) Osbeck), sour orange (C. aurantium L.), mandarin (C. reticulata Blanco), grapefruit (C. paradisi Macfad.), lemon (C. limon (L.) Burm.f.), pomelo (C. maxima Merr.), sweet lime (C. limettioides Tan.), Rangpur (C. limonia Osbeck), Calamondin (C. madurensis Lour.), rough lemon (C. jambhiri Lush.), satsuma mandarin (C. unshiu Marcow), tangelo (C. paradisi x C. reticulata), kumquat (Fortunella spp.) and citrange (C. sinensis x Poncirus trifoliata) are susceptible to infection. Acid limes, trifoliate orange and trifoliate orange hybrids are tolerant (Calavan, 1980; Whiteside et al., 1988).
Many varieties of sweet orange comprising Washington navel, Valencia, Thomson navel, Frost Navel, Frost Valencia, Washington sanguine, Hamlin, Cadenera, Portugaise, Surprise navel, Beni Selman, Petit Jaffa, local cultivar of Fars and Ramsar Number 4 are infected to S. citri with different range of suscebtibility (Childs and Carpenter, 1960; Nejat et al., 2007).
Citrus is not the only host plant of S. citri. Many non-citrus plant species throughout the world have been also found to naturally and experimentally infected with S. citri (Table 2, 3).
Periwinkles (Catharanthus roseus (L.) G. Don) were the first non-rutaceous plants to have been found naturally infected by the Spiroplasma citri in California and Arizona (Allen, 1975; Granett et al., 1976) and then has been found in Mediterranean countries including Syria, United Arab Emirates, Oman, Cyprus and Turkey (Bove, 1986) and Malaysia (Nejat et al., 2011).
Symptoms: Stubborn disease can much reduces the quality and quantity of yields. Affected trees by stubborn disease usually are stunted and have a dense or bunchy type and upright position of growth with shortened stem internodes and multiple axillary buds. Typical leaves symptoms of stubborn are small, to call the disease little leaf, cupped shaped with rounded tip and leathery appearance. Infected leaves sometimes indicate a variety of chlorotic or mottled resembling zinc, iron and manganese deficiencies.
Fruits symptoms on stubborn-affected citrus trees is variable and include small, lopsided (curved columella), acorn-shaped with stem-end peel of normal thickness and thin rind at stylar end, stylar-end greening (retention of green color of the stylar end after ripening and become orange color of fruit) and small fruits drop. The taste of the diseased-fruits sometimes is insipid or bitter and show seed abortion (Fig. 1). (Bove, 1995; Calavan, 1968, 1979; Calavan and Carpenter, 1965; Calavan and Oldfield, 1979; Fawcett et al., 1944; Gumpf and Calavan, 1981).
Naturally infected periwinkle showing the following symptoms: Rapid decline in the number and size of the flowers were observed until flowering ceased. The buds and flowers were abscised prematurely with the reduction in leaf size and yellowing of the leaves that starts from the margin and tip, progressing to the center part. General chlorosis starts from down part, proliferation of auxillary buds, stunting and death.
Geographical distribution: Citrus srubborn disease widely distributed in the southwestern united states of America, northern Africa, Mediterranean countries and Southeast Asia including the Arizona, California, Illinois and Maryland in the United States of America, France, Greece, Italy, Spain, Libya, Algeria, Cyprus, Egypt, Iran, Iraq, Jordan, Lebanon, Palestine, Morocco, Oman, Saudi Arabia, , Syria, Tunisia, Turkey, Pakistan, Yemen and the United Arab Emirates in Mediterranean and Middle East areas, Mexico and Malaysia (Bove, 1995; Nejat et al., 2011).
| Table 2: | Natural non-rutaceous hosts of Spiroplasma citri |
| Table 3: | Experimental non-rutaceous hosts of Spiroplasma citri |
The disease does not appear to be a problem in cool areas or areas with warm, humid climates (Whiteside et al., 1988). While there is Spiroplasma citri in equatorial areas and can cause severe symptoms and lethal disease on periwinkle (Nejat et al., 2011).
Transmission: It is naturally transmitted by phloem-feeding leafhopper vectors in propagative manner: Circulifer tenellus Baker, Scaphytopius nitridus Delong and S. acutus delongi (Order Hemiptera, suborder Homoptera, family Cicadellidae) in California (USA) (Kaloostian et al., 1979; Mello et al., 2009; Oldfield, 1988; Oldfield et al., 1976, 1977), Neoaliturus haematoceps Mulsant and Rey (Bove et al., 1986) and C. tenellus (Klein et al., 1988) in the Mediterranean area. S. citri has been also transmitted experimentally by Euscelis plejebus Fallen (Markham and Towsend, 1974; Towsend et al., 1977) and Macrosteles fascifrons (O’Hayer et al., 1983). It is graft-transmissible through infected budwood but is neither seed nor mechanically transmissible (Whiteside et al., 1988; Rangel et al., 2005). It can be also transmitted via the parasitic plant, dodder (Cuscuta compestris or C. subinclusa) (Lee et al., 2001).
S. citri detection: S. citri can be detected by grafting to citrus indicators, culturing on artificial media, serological, DNA probes, dot-immunobinding assay, Immunocapture Polymerase Chain Reaction (I C- PCR), Polymerase Chain Reaction (PCR) and real-time PCR.
S. citri can be detected by graft inoculation of indicator plants of which the most suitables are sweet orange cv. Madame Vinous, grapefruit cv. Marsh and tangelo cv. Sexton, respectively (Bove, 1988). Side grafting is more successful than bud grafting to transmit S. citri (Rangel et al., 2005).
Phytopathogenic spiroplasmas is often detected by cultivation in artificial media. Several media have been used for the cultivation of S. citi such as: SMC (Saglio et al., 1971, 1973), C-3 (Chen and Liao, 1975), M-1 (Williamson and Whitcomb, 1975); LD8 (Lee and Davis, 1978, 1984); SP4 (Whitcomb, 1983).
| Fig. 1: | Citrus stubborn disease symptoms |
S. citri culturing is time-consuming and 2-3 weeks need to growth S. citri in culture media and contamination can cause by non-target microorganisms (Rangel et al., 2005).
The Enzyme-linked Immunosorbent Assay (ELISA) with polyclonal and monoclonal antibodies as a sensitive serological method has been applied for identifying spiroplasmas directly in plant or insect material (Archer and Best, 1980; Archer et al., 1982; Clark et al., 1978; Lin and Chen, 1985; Saillard et al., 1980, 1993; Tully et al., 1973).
The DNA probes and dot-immunobinding assay (DIMA) were applied to detect the Spiroplasma citri in medium, infected plants and insects. DIMA is rapid assay but less sensitive than ELISA while DNA probe has a high level of sensitivity (Fletcher, 1987; Nur et al., 1986; Saillard et al., 1993).
Immuno-capture (IC)-PCR method has been applied for detection of stubborn (El-Banna et al., 2005; Saillard et al., 1993, 1996).
Polymerase Chain Reaction (PCR) is useful method for spiroplasma detection in infected plant phloem or insect vectors with 100 -1000 times of sensitivity greater than ELISA and it are also more rapid than serological techniques and culturing (Fletcher et al., 2006; Rangel et al., 2005). PCR detection of S. citri has been used with primers based on gene sequences for spiralin (Foissac et al., 1996), 16S rRNA gene in particular spiroplasma infection of carrot in the United States (Lee et al., 2006), Putative P89 adhesin and Putative P58 adhesin-like genes (Yokomi et al., 2008) (Table 4).
There is genetic diversity among isolates of S. citri based on the Crossed Immunoelectrophoresis (CIE) with intermediate gel and polyacrylamide gel electrophoresis, three repetitive extragenic palindromic elements (BOX, ERIC and REP), random amplified polymorphic DNA (RAPD) and spiroplasma bacteriophage insertions as discriminative techniques (Mello et al., 2006, 2008; Omar et al., 2006).
Control: Stablish S. citri –free mother trees and citrus nurseries in locations where very low or no spread of S. citri occur. Elimination of brassicaceous weed hosts within and around citrus groves.
| Table 4: | Several oligonucleotide primers used for spiroplasma detection by PCR and real-time PCR |
Budwood should be taken from clean propagative materials and healthy trees. Trees that appear diseased and showing symptoms or abnormally stunted should be removed and replaced with healthy replants or more tolerant varieties. Use of trap plants such as sugarbeet which is leafhopper attractive but not host of S. citri can be reduced the incidence of stubborn disease in the citrus orchards. Although S. citri is highly sensitive to tetracylines in vitro but is not practical (Bowyer and Calavan, 1974; Fletcher et al., 2006; Gumpf and Calavan, 1981; Saglio et al., 1973; Whiteside et al., 1988).