Identification and Molecular Characterization of Little Leaf Disease Associated with Phytoplasma on Sugar Beet (Beta Vulgaris L.

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1 Middle East Journal of Applied Sciences Volume : 06 Issue :04 Oct.-Dec Pages: Identification and Molecular Characterization of Little Leaf Disease Associated with Phytoplasma on Sugar Beet (Beta Vulgaris L.) Plants in Egypt Manal A. El-Shazly, Eman M. EL-Abagy, Amira M.E.Aly and Sahar A.Youssef Virus and Phytoplasma Research Department, Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt. Received: 20 November 2016 / Accepted: 28 December 2016 / Publication date: 31 December 2016 ABSTRACT Little leaf disease is one of the most important economic diseases caused by phytoplasma in many crops such as tomato, eggplant, sweet potato and sugar beet. Symptoms of infection with little leaf disease on sugar beet plants were observed in the farm of Agricultural Research Center, Giza, Egypt for the first time during November Affected plants showed characteristic symptoms including a 'pineapple-shaped' crown, stunted, chlorotic and necrotic leaves and petioles, with recent re-growth being small and deformed. Phytoplasma was transmitted from diseased to healthy sugar beet plants by the dodder (Cuscuta campestris). Light microscopy was used for detecting the causal agent and Dienes stain showed blue areas in the phloem tissues region of infected plants but not in apparently normal one which confirmed the presence of phytoplasma. Transmission electron microscopy (TEM) of infected plants showed phytoplasma units inside phloem tissues appearing as spherical to oval structures of variable sizes but not in healthy plants. The molecular detection was done through PCR and it produced an amplification of~1.8 kb with universal primer P1/P7 and ~900- bp with specific primer fu5/ru3. Changes in chemical composition of infected leaves and roots of sugar beet plants showed increased in α- amino nitrogen, protein, sodium, potassium and total free amino acids contents. On the other hand, infected plants showed decreasing in total sugars and reducing sugar contents. Key words: Sugar beet, Phytoplasma, Dienes stain, detection, PCR, Dodder transmission, Transmission electron microscopy Introduction Sugar beet (Beta vulgaris L.) is considered the second sugar crop for sugar production after sugar cane in the world and especially in Egypt. It is an important crop that helps in establishing integrated agricultural-industrial societies, especially in the new reclaimed areas and contributes in many industries such as sugar industry, highly-value animal feed resulting from processing waste (Arzanlou et al., 2016 and Moliszewska et al., 2016). Phytoplasmas (formerly mycoplasma - like organism, MLO) have diverged from gram-positive bacteria, and belong to the Candidatus Phytoplasma genus within the Class Mollicutes (Bertaccini, 2007). Through evolution the genomes of phytoplasmas became greatly reduced in size and they also lack several biosynthetic pathways for the synthesis of compounds necessary for their survival, and they must obtain those substances from plants and insects in which they are parasites (Bai et al., 2006), thus they can't be cultured in vitro in cell-free media. Phytoplasmas plant pathogenic mollicutes have been associated with several hundred diseases affecting economically important crops, such as ornamentals, vegetables, fruit trees and grapevine (Lee et al., 2000). In host plants, phytoplasmas are restricted to the phloem sieve tubes and are transmitted between plants by phloem- sap- feeding leafhopper in a persistent manner (Gatineau et al., 2001, 2002; Ahmed et al., 2014). Phytoplasmas are obligate parasites inducing characteristic symptoms in host plants, such as low growth rate, stunting, yellowing or reddening of the leaves, reduced leaf size, shortening of internodes and loss of apical dominance. These lead to reduced yield, proliferation of shoots or roots, witches- Corresponding Author: Eman M. EL-Abagy, Virus and Phytoplasma Research Department, Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt. eman_abagy@hotmail.com 1054

2 brooming general decline and sometimes, death of the plant. At the same time, infected plants normally show symptoms such as green coloration of flower tissues (virescence), retrograde metamorphosis of floral organs into leafy structures (phyllody), growth of elongated stalks (bolting) and are associated typically with little leaf diseases of several hundred plant species such as eggplant (Mitra, 1988), cowpea, tomato (Saqib et al., 2006), sugar beet (Thilagavathi et al., 2011) and sweet potato (Tran-Nguyen et al., 2012). Sugar beet little leaf disease causes a loss of root sugar content which can have dramatic economic consequences for growers and the local sugar beet industry (Gatineau et al., 2002). Early symptoms including a 'pineapple-shaped', stunted, chlorotic and necrotic leaves and petioles, with recent re-growth being small and deformed. Affected plants produce new shoots with small, narrow leaves and chlorotic laminae. Old leaves become yellow, necrotic and incurvation. (Gatineau et al., 2002; Sémétey et al., 2007 and Thilagavathi et al., 2011). Transmission Electron Microscopy (TEM) allows the observation of characteristic phytoplasma morphology in sieve tubes of plant host (Doi et al., 1967 and Sertkaya et al., 2005). The ability to detect and identify phytoplasmas is necessary for accurate disease diagnosis. However, detection has been hampered by the inability to culture phytoplasmas in vitro. Recently, considerable progress has been made by the use of DNA-based methods in detecting, identifying and classifying phytoplasmas. In particular, by restriction site and sequence analysis of 16S rdna, many phytoplasmas have been distinguished and phylogenetically classified (Lee et al., 1998; Seemueller et al., 1998). The introduction of the polymerase chain reaction (PCR) to amplify conserved genes has greatly improved the detection and identification of a broad range of phytoplasmas (Pollini et al., 2001; Abou-Jawdah et al., 2002; Castro and Romero, 2002 ; Harrison, 2002). The phytoplasma associated with little leaf disease of sugar beet has never been detected in Egypt, so the objectives of this study was to determine association of phytoplasma with little leaf disease in sugar beet plant (Beta vulgaris L.) based on symptomatology and dodder transmission. The combined applications of light, transmission electron microscopy and molecular technique were used to verify and identify phytoplasma under study. On the other hand, the effect of infection on sugar content and quality parameters of artificially infected sugar beet plants compared with healthy one were studied. Materials and Methods Source of the disease: Sugar beet (Beta vulgaris L.) plants with little leaf symptoms were collected from the farm of Agricultural Research Center, Giza, Egypt during November Infected plants were transferred and grown in 30 cm plastic pots filled with natural soil under the greenhouse conditions. Upon recovery of plants, they were detected firstly using light microscope and verified by PCR assay. Sugar beet seedling of the same age and not exposed to insects were used as healthy controls. Detection and identification of phytoplasma: Samples from leaves and tap roots of naturally infected and healthy sugar beet plants were transferred to a small Petri dish containing 70% ethanol for 3 min and then immersed three times in distilled water. The midribs of the leaves and tap root were removed and grinded to release the juice contents. Three microliters from the juice were transferred to a glass slide covered with a cover slip. Motility and shape of phytoplasma under study were examined at high contrast phase, 100x oil immersion objective on a light microscope (Olympus, optoscient) according to Davis & Worley (1973). Naturally infected sugar beet plants showing symptoms and checked for presence of phytoplasma were used as source of phytoplasma and dodder transmission. Dodder transmission: Thirty seeds of dodder (Cuscuta campestris) were surface sterilized with 1% sodium hypochlorite solution, washed with distilled water and dried. Seeds were in vitro germinated in Petri- 1055

3 dishes 12 cm diameter with moist filter paper for four days at room temperature (27-30 C) and transferred to 20 healthy sugar beet plants cv. Baraka (2 per pot) were used as recipient. After three weeks, connection was established between dodder strands from healthy sugar beet and a symptomatic sugar beets were used as donor plants. Four weeks later, newly developed dodder strands from symptomatic sugar beets were connected to healthy sugar beet plants. Connections were maintained for four weeks for transmission before discarded. Controls were exposed to dodder grown on healthy sugar beet. Infection of dodder-inoculated plants were examined for symptoms expression by visual inspection, light and electron microscope, and verified by PCR assay.on the other hand,the inoculated plants showing symptoms and checked for phytoplasma presence by light microscopy were further tested for chemical-composition analysis. Light microscopy of Dienes stain: Dienes stain was used to detect little leaf disease caused by phytoplasma in sugar beet plants. Plant samples are taken from stems and tap root of naturally, artificially infected and healthy plants. Free hand cross sections were prepared by hand and transferred into distilled water, using razor blade edge. The sections were transferred to Dienes stain for 10 min. The stain was prepared by dissolving 2.5 g methylene blue, 1.25 g azure 11, 10.0 g maltose and 0.25 g sodium carbonate (Na 2CO 3) in 100 ml distilled water filter through filter paper and dilute to 0.2% (v/v) in distilled water according to, Hibben et al. (1986) and Musetti (2013). The stained sections were later washed in distilled water and examined by light microscope. Transmission electron microscopy (TEM): Examination of ultra thin section from symptomatic tap root tissues of infected and healthy sugar beet were cut into small pieces about 1-2 mm, fixed in 2% (v/v) glutraldhyde dissolved in 0.1 M sodium cacodylate buffer, ph 7.2 and subjected to a vacuum for 1-4 min every 15 min for 2 hours on ice. Post fixation was done with 1% osmium tetroxide dissolved in 0.1 M sodium cacodylate buffer for 1.5 hours at room temperature. The samples were then washed with distilled water, treated with 5% uranyl acetate for 1.5 hours, washed again with distilled water and then dehydrated with ascending concentrations of ethanol for 15 min for each concentration. After dehydration, ultra-thin sections were cut using ultra microtome Leica model EM-UC6 at thickness 90 nm, mounted on copper grids (400 mish). Sections were stained with double stain (Uranyl acetate 2% for 10 min followed by 0.4% Lead citrate for 5 min and examined by transmission electron microscope (JOEL- JM-100-C). Images were captured by CCD camera model AMT, optronics camera with 1632x1632 pixel format as side mount configuration. DNA extraction: The total DNA was extracted from leaves of artificially infected and healthy control sugar beet plants according to Dellaporta et al., (1983) with minor modification as follows. Dellaporta extraction method (About 1g of small leaves were immersed in liquid nitrogen and ground to a very fine powder in a cooled mortar. Powder sample was collected in 1.5 ml microfuge tube placed in 500 l of Dellaporta extraction buffer (100 mm Tris ph 8.0, 50 mm EDTA, 500 mm sodium chloride and 10 mm -mercaptoethanol), and then ground with knots pestle (Vineland, NJ). 33 l of 20% sodium dodecyl sulphate (SDS) (w/v) was added, vortexed and incubated at 65 C for 15 min. Then, 160 l of 5 M potassium acetate was added, vortexed and followed by centrifugation for 15min at 10,000 rpm. The supernatant (450 l) was removed, carefully avoiding any debris and the potassium acetate extraction repeated. The supernatant (450 l) was removed to a new eppendorf tube, 0.5 volume of isopropanol was added and incubated at -80 for 1hr, then centrifuged at l4,000 rpm for 20 min. The supernatant discarded carefully and the pellet washed twice with 70% ethanol, spins 5 min at 10,000 rpm, and then carefully removed the supernatant. The pellet was dried and resuspended in MilliQ water, DNA concentration was determined using NanoDrop (Thermo scientific) spectrophotometer at O.D. 280; the DNA was diluted to approximately 10 ng /µl and stored at 20 C. 1056

4 PCR detection of phytolasma: The DNA extracted from symptomatic sugar beet plants were used as a template for PCR and DNA extracted from healthy sugar beet plants was used as negative control. The universal phytoplasma primer pair P1(5'-AAGAGTTTGATCCTGGCTCAGGATT-3) and P7 (5- 'CGTCCTTCATCG GCTCTT-3 ) Sinclair et al., (2000) was used in direct PCR to amplify a 1.8-kb product of 16SrRNA gene. For nested PCR another specific forward primer pair fu5 (5 - CGGCAATGGAGGAAACT-3 ) and reverse primer ru3 (5 -TTCAGCTACTCTTTG TAA CA-3 ) was used to detect little leaf phytoplasma as described by Lorenz et al., (1995). The PCR reaction mixture of 25 l contained (3 l) extracted DNA, 0.5 l (0.5mM) of each primer and 15.8 l ddh2o, 5 l 5x MyTaq Reaction Buffer and 0.2 l Paq5000tmDNA polymerase, (STRATAGENE). The amplification profile initiated with 3 min at 94, and then 35 cycles of the following steps: denaturation for 1 min at 94 C, annealing for 2 min at 53 C (P1/P7) or 1 min at 57 C (fu5/ru3), extension for 2 min at 72 C and final extension for 5 min at 72 C. PCR-amplified DNA fragments were separated by gel electrophoreses in 1% agarose in 0.5x TBE buffer (Tris borate-edta, 90 mm tris-acetate, 90 mm boric acid, 2 mm EDTA) and visualized with UV trans-eliminator after staining for 15 min with ethidium bromide (Sambrook et al., 1989) and photographed using a Bio-Rad Gel documentation system. Effect of phytoplasma infection on chemical composition of sugar beet leaves and roots: The effect of phytoplasma infection on sugar content and chemical components of sugar beet leaves and roots cv. Baraka including total free amino acid, sodium, potassium, nitrogen, total soluble sugars, reducing sugars and protein content analysis were done according to the following methods. Determination of total nitrogen and crude protein: Total nitrogen was determined by the modified micro-kjeldahl method as described by Pregl (1945) and The crude protein content was calculated by multiplying the total nitrogen by 6.25 according to the method of A.O.A.C (1990). Sugar beet impurities: Impurities of sugar beet including potassium and sodium percentage were determined by using a flame photometer apparatus as described by Brown and Lilleland (1946). Determination of total free amino acids: Total free amino acids were determined according to the method of Jayaraman (1985). The developed pink color was measuring using a spectrophotometer at 750 nm. The concentration of free amino acids was calculated using the stander curve of lysine. Determination of total soluble and reducing sugars: Extraction of total soluble and reducing sugars was conducted according to the method described by Daniel and Geoorge (1972) and the total soluble sugars was determined by the phenol sulphuric acid method as described by (Dubois et al., 1956) and reducing sugars were determined using dinitrosalicylic acid (DNS) method (Miller,1959). Statistical analysis: The obtained data were statistically analysed according to the method described by Snedecor and Cochran (1980). 1057

5 Results Source of plant and Symptomatology: Characteristic symptoms of sugar beet plants which collected from the farm of Agricultural Research Center included 'pineapple-shaped', stunted growth with numerous, small, narrow leaves, reduced tuber size, chlorotic and necrotic leaves and petioles, with recent re-growth being small and deformed compared to the healthy one (Fig.1and 2). A B C Fig. 1: Symptoms of phytoplasma on naturally infected sugar beet plants. A, 'pineapple-shaped', stunted growth with numerous, little, narrow leaves. B, 'pineapple-shaped and necrotic local lesions on infected leaf compared with healthy one (left). C, reduced tuber size (left) compared with healthy plant (right). A B C D Fig. 2: Symptoms development of phytoplasma infection on naturally sugar beet plants under the greenhouse conditions. A, 'pineapple-shaped', stunted growth with numerous, little, narrow leaves with chlorotic, development to yellowing in B, and then to red -brown with deformation in C, compared with healthy plant in D. Dodder transmission: The transmission experiments by the dodder were carried out to transmit phytoplasma that cause little leaf disease from infected to healthy sugar beet plants. The causative agent was successfully transmitted to healthy plants. Fourteen out of 20 sugar beet plants parasitized by the dodder from naturally infected sugar beet developed disease symptoms within days post inoculation. The major symptoms shown by experimentally infected sugar beet plants were small leaves, yellowing and stunting (Fig.3). Infection of symptomatic dodder-inoculated plants was verified by light transmission electron microscopy and PCR. 1058

6 A B C Fig. 3: Symptoms of development infection with phytoplasma in healthy plant through dodder transmission showing A, little leaf symptoms. B, little leaf symptoms, deformation and incurvation compared with the healthy plant in C. Detection and identification of phytoplasma: The presence of phytoplasma in infected leaves and/or tap roots of sugar beet plants under field and greenhouse conditions after inoculation were verified through examination with the light microscopy. Micrograph observation illustrated the presence of rounded or elongated shape of phytoplasma compared with healthy one Fig. 4.. A B Fig. 4: Detection of phytoplasma in tap roots 'sap of sugar beet. (A), presence of rounded or elongated shape of phytoplasma in infected plant compared with healthy one (B). The light microscopy of Dienes stain: The previously stained sections of stems and tap roots of infected and healthy sugar beet plants were examined by light microscope. Infected samples which stained with Dienes stain, showed some phloem cells were stained with blue color which indicating the presence of phytoplasma Fig. 5A. The phloem of sections prepared from healthy plants remained unstained Fig. 5B. Transmission electron microscopy (TEM): Ultra thin section from symptomatic tap root tissues of infected and healthy sugar beet plants showed phytoplasma-like organisms present in the phloem cell. The majority of particles were oval spherical or elongated bounded by a unit membrane (figs. 6A and B). However, phytoplasma have not detected in the phloem tissues of symptomless plants. 1059

7 A B Fig. 5: Micrograph of transverse section of infected sugar beet plant stained with Dienes stain showing blue areas in phloem cells (A), section of healthy plant with unstained phloem cells (B) A B B Fig. 6. (A, B): Transmission electron micrograph of ultrathin sections of the tap roots showing completely units were oval, spherical or elongated bounded by a unit membrane in A, B, magnification (x ). PCR detection of phytolasma: DNA fragment of approximately 1800 bp was amplified with universal phytoplasma primer pair P1/P7 from total nucleic acid extracted from samples of infected sugar beet plants. The same primer pair failed to generate the amplicon in a PCR mixture containing DNA from the healthy plants (Fig.7A) which confirm the phytoplasma infection. An amplification product of the expected size (about 900 bp) was consistently obtained in PCR using specific primer fu5/ru3 when used the same DNA extracted from infected sugar beet leaves (Fig. 7B), which indicate and confirm the presence of phytoplasma associated with little leaf disease in tested samples. Effect of phytoplasma infection on chemical composition of sugar beet leaves and roots: Chemical components of leaves and roots of sugar beet such as α- amino nitrogen, protein, sodium, potassium, total free amino acids and total, solube and reducing sugars were determined and the data presented in Fig. (8) indicated that all tested sugar beet plants infected with phytoplasma showed increased in α- amino nitrogen, protein, sodium, potassium and total free amino acids contents in leaves and roots. On the other hand, infected plants showed decreasing in total soluble and reducing sugars contents compared with the healthy plants. 1060

8 A B Fig. 7A, B: Gel electrophoresis analysis for the detection of the phytoplasma in sugar beet symptomatic leaves using A, universal primers pair P1/ P7 (lane 1: 1800 bp) and B, specific primer fu5/ru3(lane 1: ~900) base pair. lane M:1 kb DNA ladder, lane1: symptomatic sugar beet sample and lane H, healthy sugar beet sample used as negative control. Fig. 8: Effect of infection with phytoplasma on α- amino nitrogen, protein, total free amino- acids, sodium, potassium, total sugars and reducing sugars contents in leaves and roots of beet plants. Discussion Phytoplama is considered as one of the most important plant pathogens reducing the productivity of several economic crops including sugar beet. In the current study, the little leaf disease of sugar beet associated with phytoplasma was identified showing unusual symptoms on sugar beet plants grown at Agricultural Research Center farm, Giza, Egypt, including a 'pineapple-shaped', stunted growth with numerous, small, narrow leaves, reduced tuber size, chlorotic and necrotic leaves and 1061

9 petioles, with recent re-growth being small and deformed. These symptoms are similar to those described by Gatineau et al. (2001 ; 2002), Bressan et al. (2008) and Thilagavathi et al. (2011). Transmission of phytoplasma from naturally infected to healthy sugar beet plants using the parasitic plant Cuscuta spp. (dodder) is an effective way to maintain and propagate the pytoplasma under study. At the same time, the healthy dodder is one of the main ways by which phytoplasma infection is achieved under artificial conditions (Marcone et al., 1997). The same symptoms that are typical for the little leaf disease of sugar beet appeared within days post inoculation. This result agreed with similar to previous studies on phytoplasma transmission by the dodder (Gatineau et al., 2001, Salehi et al., 2009, Akhtar et al., 2009, Ahmed et al., 2014 and Hamed et al., 2014) Light microscopy techniques have been used successfully as preliminary methods for diagnosis to verity the presence of phytopalsma in symptomatic plants so they constitute the first steps towards understanding the possible association between phytoplasma and the disease symptoms in the plants. Infected samples stained with Dienes, showed some phloem cells are stained with blue color which indicating the presence of phytoplasma compared with healthy plants these results is similar to those described by Deeley et al. (1979), Ahmed et al. (2014) and Hamed et al. (2014) Transmission Electron Microscopy (TEM) has traditionally been used to demonstrate the presence of phytoplasmas in phloem tissues (Chang et al., 1996; Hwang et al., 1997). The morphological characteristics of the structures are similar to organisms found in plants infected with little leaf disease (Gibb et al., 1995; Gatineau et al and Ajayakumar et al., 2007). Also Maust et al (2003) reported that, the main effect of phytoplasmal infections apparently is the impairment of the sieve tube function and causes inhibition of phloem transport in phytoplasma- infected plants, which in turn leads to an accumulation of abnormal amounts of carbohydrates in source leaves and a marked reduction of these essential energy storage compounds in sink organs and roots. The presence of phytoplasmas in ultrathin sections of diseased sugar beet and the absence of other pathogens in the symptomless ones support a phytoplasma etiology of the disease. Molecular diagnosis confirmed the infection of sugar beet with phytoplasma associated with little leaf disease for the first time in Egypt. The DNA extracted from sugar beet plants, those showed little leaf symptoms, was used as a template for phytoplasma- universal and specific PCR primers, respectively. The results of using the universal phytoplasma primers P1/P7 showed a clear band at the specific size 1.8-kb (Saqib et al., 2006, Tran-Nguyen et al., 2012, Ahmed et al., 2014 and EL-Banna et al., 2015) while the PCR using -specific PCR primers fu5/ru3 showed a clear band at ~900-bp (Gatineau et al. 2001, 2002 and Thilagavathi et al., 2011). Those PCR results clearly demonstrated the natural infection of sugar beet plants with phytoplasma associated with little leaf disease and confirmed the successful transmission of the phytoplasma associated with little leaf disease into the healthy sugar beet plants using dodders. Biochemical changes of infected leaves and roots of sugar beet plants showed the increase of protein content, these changes may be due to the fact that the polyphenol-oxidase plays a role in the catalase synthesis of the phytoalexins and other compounds as indicators of the infection process. Also, phytoplasma infection can lead to the production of defense proteins including peroxidases, chitinases and β- 1,3 glucanases increase in phenolic compounds and involvement of important signal molecules such as H 2O 2, and salicylic acid (Trandafirescu et al 2011). Regarding to, the free amino- acids content, our results have evinced that the infected leaves and roots have higher content than healthy plants, these may be due to the reduction in the protein synthesis and increase the protein decomposition due to the disturbance induced by the pathogenic agent and phytoplasma infection causes limitation of transport in the phloem which led to amino acid accumulation. Also, infection increased impurities i.e., nitrogen, sodium and potassium in storage roots due to infection is a manifestation of the inhibition of photosynthetic enzymes as stated by Goodman et al. (1965). On the other hand, infected plants showed decreasing in total sugars and reducing sugar contents it may be due to the phytoplasma infection causes the reduction in chlorophyll contents by interfere in photosynthesis and perhaps speedy senescence in the leaf tissue (Junquira et al., 2004 and Giorno et al., 2013). Similarly, results indicated by Colver et al. (1999) on sugar beet infected by some pathogens (viruses, phytoplasma, bacteria or fungi) led to an increase in storage- root impurities in the roots at the harvest, which causes decreasing in sugar yield by up to 47%. 1062

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