Extraction, chemical composition, use in induced protection and cross-reactive antigens between exopolisaccharides from Tremella fuciformis Berk and Xanthomonas campestris pv. citri (Hasse) Dye

Exopolysaccharides (PS) are the major components on the surface of bacteria and also produced by fungi. These molecules are important in human health, in order to control diabetes as well as protect plants against attacks of foliage diseases. The objective of the present work was to study the partial chemical structure of the carbohydrate, use in control disease in plants and cross-serological relationship (cross-reactive antigens between isolates from fungi (Tremella fuciformis (Tf) and bacteria (Xanthomonas campestris pv. citri (Xcc)). Tf was developed in culture medium containing sorghum seeds during 20 days, and Xcc in the PDA (potato dextrose agar) medium for an 8 days period. The polysaccharide was removed from the culture medium, precipitated with ethanol, and quantified total sugar. By TLC was observed that 2 isolates presented galactose, glucose, mannose, arabinose and xylose in different proportions. Fucose and ribose was not found in the PS from Xcc but present in Tf. In ELISA, antiserum to Xcc revealed an antigenic homologous reaction with the same bacteria and heterologous with Tf. Barley plants pretreated with PS from Tf and later challenged with conidia from B.sorokiniana, demonstrated protection against the pathogen. Results suggested that PS from Tf presented induction of protection. Both PS (antigens) present an identical epitope demonstrated by reaction in Elisa test. The antibody against Xcc was specific for an epitope and bounded to another antigen due to having similar chemical properties.

by reducing cholesterol, triglyceride, GPT, urea and increasing HDL cholesterol [6,7]. Another important application from PS was with promoted induction of protection against disease in plants. Bach et al. [8] demonstrated that PS from Tf in three concentrations, stimulated the ability of plants to compensate the damaging effects of the pathogens (Bipolaris sorokiniana) on barley plant (Variety BRS Brau) metabolism. The same protection was observed with xantham gum from Xanthomonas campestris pv. citri (Xcc), Xanthomonas campestris pv campestris and commercial xantham gum against the same disease on barley and wheat plants [9,10]. About chemical structure of the PS obtained from dried Tf, commercially avaliable in China, and extracted by boiling water, determined that it consists of a linear backbone of (1→3) α-D-mannan with side chains composed of glucuronic acid, xylose and fucose [11,12]. Xanthomonas produced xanthan gum and is an important industrial biopolymer as example food. According to several authors, the PS are antigenic macromolecules and may be related to pathogenicity [13 -15]. The objective from the present work was to study the partial chemical structure of the carbohydrate and investigate the use under greenhouse conditions as an inducer of protection in barley plants (cultivar Embrapa BRS Elis) against pathogen. Another method for identification PS was study the cross-serological relationship (cross-reactive antigens between isolates from fungi (Tremella fuciformis Tf) and bacterial (Xanthomonas campestris pv. citri Xcc). This could aid in the possible explanation for its use in control of diseases both for the use in plants such as for humans beings.

II. MATERIALS AND METHODS Preparation of polysaccharide from Tremella fuciformis
Tremella fuciformis received from Brasmicel, were cultured in potato dextrose agar (PDA) for 8 days, and then transferred to plastic bag containing sorghum seeds. The bags were incubated during 45 days for micelial growth in chamber with controlled temperature (27±1 0 C) and dark. For production of polysaccharides (PS), a solid medium was made and added 100g of sorghum seeds (Embrapa seed variety 308) that was first cooked in water and after crushed in a blender in 400mL of water and boiled again. The mixture was filtered through sieve, gauze, cotton cloth, completely to 500mL water and supplemented with 0.5g of agar. After boiled, the solution was transferred to bottles and sterilized [8]. Sorghum seeds with mycelium were inoculated to bottle with solid medium and incubated for 10, 20, 30 days in chamber with controlled temperature (25 ± 1 0 C) and dark.
After the period, PS was removed and solution was reduced to half-volume by vacuum evaporation. This reduced volume was, then, treated with cold ethanol (70%) to polysaccharide precipitation. To facilitate the precipitation (ppt), the solution was kept at 4 o C for an additional 24 h and then centrifuged at 4000g for 10 min at 18 o C, and the precipitates were collected, washed twice with alcohol and then solubilized in water. After, the gum was submitted to dialysis in bags with 10.000 daltons against buffer phosphate pH=7 (0.05mol/L) for retired phenols. Tests were performed to quantify beta glucan and total sugar. Beta-glucan was determined by Lever method [16] involving the beta-glucanase enzyme (Sigma). For standard in test, was used laminarin that said one unit of enzyme can be liberated 1 M of glucose/min at 37C [17]. For total sugar the method used was Anthrone [17][18][19]. The PS was loaded onto a Sepharose CL_4B column (2.4 cm x 100 cm, Pharmacia) and eluted with the same buffer at a flow rate of 2ml/min. The carbohydrate moiety in the PS was monitored by absorbance at 480 nm. A pool fractions from 10, 12 and 14mL was quantify by Anthrone.

Preparation of Suspension of pathogen
The pathogen used was B.sorokiniana obtained from infected barley leaves (Fundação Guarapuava-Agraria, Paraná) and grown on plates using potato dextrose agar (PDA). After 10 days of incubation, conidia, removed by brushing the surface of the agar, were suspended in 10 ml of sterile water and filtered through gauze. The concentration of conidia was adjusted to 10 5 conidia per ml and Tween 20 (poly-oxyethylene sorbitan monolauret, Sigma Chemical Co) was added to give a final concentration of 0.05% [20,21].

Preparation of barley and treatments
Barley plants (Embrapa Elisfrom Foundation Agraria, state of Paraná), were grown in clay pots (15cm diameter, ten seeds) contained red soil fertilized (NPK 10:10:10 and micronutrients) in a greenhouse under a 12h photoperiod (approximately 190IE/m 2 /s) for approximately 3 weeks when plants reached the tillering stage (stage 5) [22]. Groups of 10 plants was used in each treatment and replicated three times. The data were submitted to variance analysis. Plants were arranged in a complete randomized block design and the combination of challenger and protector in each treatment. Plants were maintained at room temperature and a 12h photoperiod (7.35Wm-2 of fluorescent light) throughout each treatment unless indicate otherwise [20]. Approximately 10ml of the conidia suspension, PS of Tf, or water were used in each treatment. Treatments were: (a) healthy: plants were sprayed with water; (b) Inducer: plants were sprayed with PS of Tf (conc 2mmol of sugar); (c) pathogen inoculated: plants were pulverized with the conidial suspension of the pathogen; (d) 24 hour Inducerpathogen: first treated with PS and 24 hours later inoculated with the conidial suspension; (e) 48 hour Inducer-pathogen: as in group d but inoculated with the conidial suspension 48 hours after inducer; (f) 72 hour Inducer-pathogen: as in group d but inoculated with the conidial suspension 72 hours after inducer. During the first 24 hours after inoculation with the pathogen, all plants were kept in the dark at room temperature in a humid chamber (80% relative humidity). After that, plants were transferred to the greenhouse. Protection level was evaluated 7 days after inoculation with the pathogen, based on the number of infected leaves in ten plants [20,21].

Preparation of polysaccharide and antisera for Xanthomonas campestris pv. citri
Bacterial cells were grown for 8 days on potato dextrose agar (PDA) in Petri disches at 27 o C in the dark. The PS was removed from surface with water. One hundred mL of solution were centrifuged (7,000g-20min), supernatants were reduced to half-volume by vacuum evaporation at 40 o C and precipitated with 75% ethanol. The precipitates were dried under a N2 stream and dissolved in distillate water. Beta glucan and total sugar was measured as described for Tremella. Antisera for Xanthomonas campestris pv.citri pathotype A were produced in New Zealand White rabbits (approx. 2kg) in Biological Institute, São Paulo, Brazil in year 2000. Rabbits were immunized with one single 1.0 mL intra-lymph node injection of 900ug of glucose equivalents emulsified with an equal volume of Freund complete adjuvant (Difco). Sera were collected from the marginal ear veins 20 days after the injection [23]. Immunoglobulin (Ig) was precipitated from rabbit sera with 100% sat. ammonium sulfate, repeated twice, and dissolved in 0.85% NaCl solution followed by dialysis against the same solution. Enzyme conjugation was performed by adding 0.5mL of Ig (1 mg/mL) to 0.09 mL of alkaline phosphatase (Boehringer) followed by dyalisis against 2mM sodium phosphate buffer pH 7.4 plus 0.85% NaCl and 0.8mL of 0.25% glutaraldehyde for 1h at 4 o C. Ig and conjugates were diluted 1:2 and antigens (PS) were used in the concentration of 1mmol of glucose equivalents/mL. Normal sera were prepared with the same method as negative control.

Treatment of antigens with sodium periodate
The method was based in Bach and Guzzo [13] when 1mmol of glucose/mL of each PS, as measured by the anthrone test [19] and treated with 0.05mol sodium periodate. Another group of equally treated samples were then reduced with sodium borohydride (Sigma) and subjected to hydrolysis with 0.03mol acetic acid for 1h (Hydrolyzed sample). Control samples were treated with ethylene glycol prior to the periodate treatment. Periodate-treated, hydrolyzed and control samples were used as antigen for serological tests by ELISA test.

Elisa Test
Antigens (PS) from Xcc and Tf were tested by double antibody sandwich-ELISA [24] and the experimental conditions for the serological reactions and immunoglobulins was based in Bach and Alba [23] and, Bach and Guzzo [13]. ELISA values were obtained by measuring absorbance at 405nm. The concentration of PS from Tf and Xcc used in work was equivalent to 1mmol of glucose.

Chromatography Thin layer (TLC)
The sugar was analyzed by thin layer chromatography (TLC) carried out on Merck silica gel 60 F254 plates (20 cm x 20 cm). Aliquots of standards solutions of glucose, galactose, mannose, xylose, arabinose, ribose and fucose (10mg/mL of each from Sigma) were applied as spots at the origin on a plate and developed with butanol: acetic acid: water (4:1:1 by vol.) in a pre-saturated chromatography chamber. The thin layer chromatography plates were dried at 60°C and the sugars were visualized at 254nm UV light and after detected by spraying chromatograms with 5% ethanolic sulphuric and heating in the oven at 100°C until clear sugars spots appeared [25,26]. The CPATLAS program was used to determine the area of spots and relative front from each standard of developed chromatoplate.
10mmol from PS samples were treated with 100uL of beta glucanase (10UI) and 200uL of alfa glucanase (10UI) from Sigma in presence of citrate buffer (50mM, pH=5) and after 10min at 37C, was submitted to boiling for one hour at 100C. Those was separated in TLC.

Extraction of polysaccharide and concentration of beta glucan
When isolates of Tf and Xcc grown in medium, was observed as loose slime secreted by the microorganisms. After precipitation and solubilized in water we have a gum solution. Table 1 compared concentration of sugars present in PS from Xcc and from Tf. Tf polysaccharide increased until day 20, being after it decreased, most likely due to the increased oxidation, or enzyme presence. Present sugars were Alfa-glucose (millimol) and Betaglucan.  Concentration of beta-glucan linked-(1,3)(1,6)

and total sugars from preparations of samples from Tremella fuciformis (Tf) and Xanthomonas campestris pv. citri (Xcc).
Polysaccharide from mg Beta-glucan/mL mmol total sugar In parallel studies with suspension of Tf was observed presence of spores. The appearance in 10 days was ovoid like yeasts. In 20 and 30 days the appearance was type yeast elongated. In work we used for studies Tf from 20 days because PS presented white color and from 30 days PS was transformed a brown color.

Gel Filtration of PS
The PS from Tf 20 days and from Xcc was monitored by a gel filtration in a Sepharose CL_4B column, by which one polysaccharide was eluted as shown in Fig. 1. Peaks from tubes 0-4 included external volum. Pool fractions (tubes 5,6,7) from Tf presented 5.12mmol of glucose and from Xcc only 2.04mmol of glucose.

Thin Layer Chromatography
By TLC was possible to observe that isolates from Xcc and Tf contain galactose, glucose, mannose, arabinose and xylose in different proportions but similar. Fucose and ribose was not found in the PS from Xcc (Table 2).

Elisa test
Results obtained through ELISA from the combination of PS from Xcc and the same antisera have specific reaction. In contrast, negative reactions occurred with normal serum when used in the test. Similarly, antigen from Tf reacted specifically in heterologous antigen-antibody combinations and give reaction in Elisa at 0.82. The results can indicated that reaction was similar as observed with antigen from Xcc or that was equivalent to 100% of interaction. To confirm that the serological reaction depends from the polysaccharides antigen, samples could be treated with sodium periodate. Homologous and

IV.
DISCUSSION Polysaccharides have the highest capacity for carrying biological information and differ greatly in their chemical composition, molecular weight, conformation, glycosidic linkage, and degree of branching, etc [27]. Weintraub [28] working with immunology said that the surface polysaccharide confers protection against the disease. The immunological properties of PS from bacterial can be used in vaccines, for study cross-reactive antigens, serogrouping or serotyping systems and others. PS from Xcc and Tf were used and demonstrated protection in barley plants against infection by Bipolaris sorokiniana [8][9][10]13]. So the objective was to evaluate the presence of crossed antigens. The chemical composition and efficiency of extraction processes of polysaccharides in fruiting bodies from T. fuciformis were not completely clear because there are different methods. In Brazil, it is difficult to find fruiting bodies, and work in labor is possible when grown in medium. Many solid medium were used, but the best was made with sorghum seeds (variety 308-Embrapa). Polysaccharides from Tf in this work, after precipitation with alcohol, presented white color that can be transformed in brown color in some days because enzyme peroxidase was presented and it acted in compound. After washing, precipitate and dialysis, the compound became white colored that can be kept in freeze for several years [7,8]. With PS from Xcc, the color after precipitation, was white. The concentration of beta glucan from Tf demonstrated that increased until day 20, being after it decreased, most likely due to the increased oxidation and correlated with the color from precipitated after 20 days. About the content of sugars Tf present more beta glucan and total sugars when compared with Xcc ( Table 1). The results with production of polysaccharides with spores its important and the appearance are according with those observed by Chen & Hou [29]; Cho et al. [30] and Seviour et al. [31]. Chen & Hou [29] said that spores were broadly ellipsoid with 7-9 x 6-7um, smooth and hyaline. According to Cho et al. [30] and Seviour et al. [31], exopolysaccharide production from Tf revealed that the morphological form grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. Cho et al. [29] said that it is noteworthy to mention that the increased population of elongated yeast probably contributed to an increased PS production. So, in results (Table 1) we have formation of elongated form in Tf in 20 days that coincide with a higher glucose concentration. Gel permeation chromatography of the polysaccharides on a Sepharose CL-4B column yielded two major peaks: a narrower peak in fractions (tubes 0-4) and another broad peak present in tubes 5-6 corresponded from 10 to 12mL. In this separation can be see that have the same peaks for Xcc and Tf that correspond to 5.12mmol of glucose for Tf and 2.04mmol for Xcc. By TLC was observed that PS from Xcc and Tf presented in polymers the sugars as: glactose, glucose, mannose, arabinose and xylose. The results with Tf in part are in accord with several authors that estimated ratio of mannose, fucose, xylose, and glucuronic acid is 9:1:4:3 making glucoronic acid accounted for 17.6% of the polysaccharides [32][33][34]. Khondkar [34] was isolated PS from liquid cultures of nine Tremella species grown in Malt-yeast extract media for 6 days at 27 o C, and the results demonstrated that the polysaccharides in aqueous solution consisted of the following monomeric sugars: fucose, ribose, xylose, arabinose, mannose, galactose, glucose and glucuronic acid. The backbones of the polysaccharide structures consisted of α-(1→3)-links while the side chains were β-linked. In results ( Table 2) from this work have not observed glucuronic acid. Bach and Guzzo [13] worked with PS from Xanthomonas campestris pv. citri and Xanthomonas campestris pv. manihotis and observed the sugar compositions in isolates that suggesting that antigenic determinants may depend on the sequence of sugars, linkages, branching and stereochemistry of PS. Lozano and Sequeira [35] said that PS coats the outer membrane and seems to play a role in the specific recognition mechanism between the bacterial or fungal cell and the host cell walls. The PS can also prevent the interaction to host cell wall. For determinated serological relationships among the preparations of PS, preliminary assays showed that Ouchterlony double-diffusion technique detect a weak cross-reactive antigens between Xcc and Tf. This indicates that these substances occur in low concentration, both in bacterial and fungi. For more contributions was made by sensitive serological techniques like ELISA. Table 3 demonstrated that have a clear indication that cross-reactive antigens must be involved in this case. This assumption is corroborated by the evidence that antiserum to Xcc reacted with PS from Xcc (homologous reaction) and also with PS from Tf (heterologous reaction). To confirm that serological reaction depends from the polysaccharides antigen, samples was treated with sodium periodate. Sodium periodate oxidation eliminated all serological reactions suggesting that, perhaps, periodatesusceptible 1,2; 1,4; 1,3; 1,6 linked non-terminal residues or non-reducing terminal units that could be present in antigenic sites. It can be assumed that all antigenic determinants did not recover serological activity after reduction with sodium borohydride and mild acidic hydrolysis, suggesting that perhaps the periodate degradation led to splitting of the polysaccharide chain. Apart from this, it can be concluded that PS have antigenic determinants of Xanthomonas and Tremella and with an identical epitope. The antibody against Xcc was specific for an epitope and bounded to another antigen due to having similar chemical properties. The results presented in Table 4 indicate that protection was conferred to barley plants when the inducer (PS) was applied in the plant, and after 72h from the treatments the protection was higher. This same effect was observed by Bach et al [20], Castro and Bach [21] in work with other elicitors.

V.
CONCLUSION The use of polysaccharides in agriculture aimed at disease control on plants has been important as a alternative control in biotechnology. The results suggest that PS from Tremella can be induce protection against disease in barley plants. In study from PS in the Xanthomonas-Tremella, interaction there is a "key" cross-reactive antigens. The hypothesis is that the molecular structures of polysaccharides should be similar and have the same common properties and both can be used for the same aim: the plant protection against diseases or as a medicinal usage.