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lower fungi such as the parasitic Phycomycetes conidiobolus (Ishikawa et al., 1979) and the soil born plant pathogen Rhizoctonia solani (Kellens and Peumans, ...

Comm. Appl. Biol. Sci, Ghent University, 72/3, 2007

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ANALYSIS OF LECTIN CONCENTRATIONS IN DIFFERENT RHIZOCTONIA SOLANI STRAINS M. HAMSHOU¹‫׳‬², G. SMAGGHE¹ & E.J.M. VAN DAMME² ¹ Laboratory of Agrozoology, Department of Crop Protection, ² Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology Faculty of Bioscience Engineering, Ghent University Coupure links 653, BE-9000 Gent, Belgium

SUMMARY Lectins are carbohydrate-binding proteins that contain at least one carbohydrate binding domain which can bind to a specific mono- or oligosaccharide. These proteins are widely distributed in plants. However, over the last decade evidence is accumulating that lectins occur also in numerous fungi belonging to both the Ascomycota and Basiodiomycota. Rhizoctonia solani is known to be an important pathogen to a wide range of host plants. In this study, isolates of R. solani from different anastomosis groups have been screened for the presence of lectin using agglutination assays to detect and quantitate lectin activity. The evaluation included determination of the lectin content in mycelium as well as in sclerotia. The amount of lectin in the sclerotia was higher than in the mycelium of the same strains. The R. solani strains with the highest amounts of lectin have been selected for cultivation, extraction and purification of the lectin. Key words: Rhizoctonia solani lectin, anastomosis group, mycelium, sclerotium, agglutination.

INTRODUCTION Lectins constitute a group of (glyco) proteins of non-immune origin, which bind reversibly to specific carbohydrates or more complex glycans. Plant lectins were defined by Peumans and Van Damme (1995) as ‘all proteins possessing at least one non-catalytic domain, which binds reversibly to a specific mono or oligosaccharide’. Because of their interaction with carbohydrate structures many lectins are able to agglutinate cells or precipitate polysaccharides and glycoconjugates. As a consequence of their biochemical properties, they have become a useful tool in several fields of biological research such as immunology, cell biology and cancer research (Van Damme et al., 1998). Living organisms of almost every taxonomic classification ranging from bacteria to higher animals contain carbohydrate binding proteins known as agglutinins or lectins (Van Damme et al., 1998). In recent years evidence has also accumulated that many fungi contain agglutinating substances. Fungal lectins have been isolated and characterized from fruiting bodies of several higher fungi such as commercial mushroom Agaricus bisporus (Presant and Kornfeld, 1972) and from mycelium of lower fungi such as the parasitic Phycomycetes conidiobolus (Ishikawa et al., 1979) and the soil born plant pathogen Rhizoctonia solani (Kellens and Peumans, 1990b). R. solani is the asexual stage (anamorph) of the fungus, whereas the sexual stage (teleomorph) is named Thanatephorus cucumeris. In nature R. solani primarily survives asexually and exists as vegetative mycelium and/or sclerotia (Meyer, 2002). It is a destructive plant pathogen and can cause damage worldwide to more than 142 plant species, including many agricultural and horticultural crops (Guillemaut et al., 2003). In addition, it is a selective species which is considered to survive in soil in the form of sclerotia associated with organic matter (Kumar et al.,

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2002). The relative importance of sclerotia in the life cycle of the fungus varies with different anastomosis groups. Sclerotia play an important source of inoculum for some anastomosis groups. However, the mycelium infests plant debris, and forms the main component of soil-borne inoculum (Kumar et al., 2002). The sexual fruiting structures and basidiospores were firstly observed and described in detail by Prillieux and Delacroiz in 1891 (Meyer, 2002). The variability in disease symptoms, host range, and geographical location of R. solani isolates suggest that there are several strains of the species. At present, 13 different anastomosis groups of the fungus (AG-1 through AG-13) have been recognized (Mghalu et al., 2004) based on affinities for hyphal fusion (anastomosis), a genetic feature that results in exchange of nuclei and the combining of different genotypes (Blazier and Conway, 2004). The presence of lectins on the hyphal surface and tissues of R. solani has been reported (Mghalu et al., 2004). It was shown that the amount of lectin in R. solani is dependent of the stage of the life cycle. Whereas in young mycelium the lectin concentration is very low, the amount of lectin increases gradually until formation of sclerotia and reaches its maximum in adult sclerotia. At the start of the mycelioginic germination, the lectin content in sclerotia rapidly decreases. The high concentration of R. solani lectin in the sclerotia, and its developmental regulation, indicate that the lectin probably serves as a storage protein in the resting structures of this fungus (Kellens and Peumans, 1990a). Until now there have been a few reports that document the occurrence, purification and characterization of the R. solani lectin or agglutinin (abbreviated as RSA). The lectin is a dimeric protein composed of two identical subunits of 13 kDa, and exhibits specificity towards N-acetylgalactosamine (GalNAc) and several other simple sugars (Vranken et al., 1987; Kellens and Peumans, 1991). The lectin is present in R. solani strains of different anatomosis groups. RSA agglutinates both human and rabbit erythrocytes (Kellens and Peumans, 1990b). However, the lectin exhibits a much higher activity with rabbit red blood cells. With respect to human erythrocytes RSA prefers blood type A over type B and type O erythrocytes. The concentrations of lectin ranged from 0.05 to 30 mg/g lyophilized mycelium (Kellens and Peumans, 1990b). At present the physiological role of Rhizoctonia lectin remains unclear. Elad et al. (1983) suggested that the lectin may play a role in specific recognition of the fungus by mycoparasites. Later Kellens and Peumans (1990a) suggested that the lectin could play a role as a storage protein and potentially as defence protein against herbivorous insects. The objectives of this study were to screen for a R. solani strain that expresses a high amount of lectin. In future experiments the lectin of this strain can be purified in sufficient amounts to test the insecticidal activity of the lectin towards biting-chewing insects (e.g. cotton leaf worm, Spodoptera littoralis) as well as piercing-sucking insects (e.g. pea aphid, Acyrthosiphon pisum).

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MATERIALS AND METHODS Isolates and growth conditions Ten Rhizoctonia solani strains of different anastomosis groups (AG) were obtained from the Laboratory of Plant Pathology and Virology at Ghent University (Table 1) and grown on two different media, being potato dextrose agar (for use in petri dishes) and potato dextrose broth (for liquid cultures in erlenmeyer flasks). The liquid cultures were started by inoculating 200 ml of liquid medium with a few pieces of approximately 1 cm² agar covered with mycelium from a 5-day old Rhizoctonia culture grown on potato dextrose agar. All fungal cultures were incubated in a growth chamber at a temperature of 25-27°C. Table 1. Different strains of Rhizoctonia solani tested Strain No. 1 2 3 4 5 6 7 8 9 10

Anastomosis group 3 1-1C 4 HGI 10 1-1A 6 GV 1-1B 1-1C 2-2LP 8 (ZG-1-2)

Host Potato Endive Peanut Lettuce Rice Soil Lettuce Endive Saint Augustine grass Barley

Origin Japan Belgium Japan Belgium Japan Japan Belgium Belgium Japan West-Australia

Protein extraction Mycelium from cultures of about 70-days old was collected and strongly squeezed by hand before determining the wet weight. Subsequently samples were lyophilized and dry weight was determined. Protein extracts were made in phosphate buffered saline (PBS; 170 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) using mortar and pestle. Approximately 0.1 g of dry mycelium was extracted in 3 ml of PBS. The liquid was transferred to eppendorf tubes and centrifuged for 2 min at 12,000 g. Similarly, sclerotia were harvested from the petri dishes and homogenized using a mortar and pestle (0.1 g in 3 ml PBS). After centrifugation, the supernatants were transferred to new eppendorf tubes and stored in the freezer at -20 °C until use. Determination of total protein content Total protein content was determined using the method of Bradford (Bradford, 1976). Therefore 10 μl of each sample was mixed with 250 µl of the Coomassie Reagent in the wells of a microtiter plate. Each sample was analysed in triplicate. The absorbance of the samples was measured in a microtiter plate reader (BioTek Instruments, Inc., Winooski, USA). Analysis of lectin activity in different Rhizoctonia strains Lectin activity in extracts from mycelium and sclerotia was analyzed using agglutination assays with trypsin-treated rabbit erythrocytes (BioMérieux, Marcy L'Etoile,

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France) Therefore 10 µl of the protein extract was mixed with 10 µl of ammonium sulphate and 30 µl of red blood cells in small glass tubes. The agglutination reaction was assessed after incubation for 30 min at room temperature. For those samples that showed good agglutination activity a dilution series was made in order to quantify the amount of lectin. Gel electrophoresis Crude extracts and pure lectin were analyzed by SDS-PAGE in 15 % (w/v) acrylamide gels as described by Laemmli (1970). Approximately 22 µg of total protein from each sample and 12.5 µg of RSA were loaded on the gel. RESULTS Agglutination assays Using agglutination assays with trypsin-treated rabbit erythrocytes lectin activity was checked in extracts from mycelium as well as sclerotia (if available) of all Rhizoctonia strains under study. Only Rhizoctonia strains 5 (AG 1-1A), 7 (AG 1-1B) and 8 (AG 1-1C) gave a clear positive result with protein extracts from mycelium. The lectin in the extracts was quantified using a dilution series and comparison to a lectin preparation with known concentration. Strain 5 (AG 1-1A) yielded the highest lectin concentration, representing approximately 2% of the total protein. The minimal concentration of lectin that could be detected was 0.097 µg/ml. Only strains 2 (AG 1-1C), 6 (AG 6GV), 7 (AG 1-1B) and 8 (AG 1-1C) yielded sclerotia. Although extracts from all these strains showed agglutination activity, strains 7 (AG 1-1B) and 8 (AG 1-1C) clearly had the highest lectin content (Table 2). As shown in Table 2 the lectin concentration in the sclerotia is considerably higher than in the mycelium of the same strain. Sclerotia of strain 7 (AG 1-1B) contain about 8 times higher lectin content than in the mycelium, sclerotia of strain 8 (AG 1-1C) contain about 128 times the lectin concentration found in the mycelium. Table 2. Quantification of total protein and lectin content in Rhizoctonia strains

Mycelium Sclerotia

Strain No.

Lectin content (mg/g tissue)

5 7 8 7 8

3.750 0.9375 0.0585 7.500 7.500

Protein content (mg/g tissue) 181.62 68.31 66.24 282.45 246.42

% lectin 2.06 1.37 0.088 2.65 3.04

Protein analysis Total protein content was determined in extracts from mycelium and sclerotia of all Rhizoctonia strains which showed lectin activity. As shown in table 2, sclerotia of strains 7 (AG 1-1B) and 8 (AG 1-1C) show a high concentration of total protein. The concentration of protein in the sclerotia is considerably higher than in the mycelium of the same strains. R. solani strain 5 (AG 1-1A) reveals a high concentration of total protein in the mycelium. Crude protein extracts from different Rhizoctonia strains were analysed by SDSPAGE and compared to a sample of the pure R. solani lectin (RSA). All strains that

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yielded lectin activity also revealed a clear band in the protein extract at the same position as that of RSA. It is clear from the gel that the lectin represents an important fraction of the total protein. Furthermore it is evident that the lectin band in extracts from sclerotia represents a more prominent band than in extracts from mycelia, which is in agreement with the results presented in Table 2.

Figure 1. SDS-PAGE of total protein extracts from mycelium (M) and sclerotia (S) of different Rhizoctonia strains. RSA refers to the pure lectin of R. solani, whereas R shows a reference marker (ß-galactosidase, 116.0 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; lactate dehydrogenase, 35.0 kDa; restriction endonuclease Bsp981, 25.0 kDa; ß-lactoglobulin, 18.4 kDa; lysozyme, 14.4 kDa).

DISCUSSION A screening of different strains of Rhizoctonia demonstrated that although many strains reveal lectin activity, the lectin activity in the different strains/anastomosis groups is quite variable. The lectin concentration ranged from 0.058 to 7.5 mg/g lyophilized mycelium or sclerotia, which is in agreement with the data reported by Kellens and Peumans (1990b). The latter authors have shown that the R. solani lectin concentrations ranged from 0.05 to 30 mg/g lyophilized mycelium. The same authors also mentioned that the amount of lectin in sclerotia is higher than in mycelium. Our experiment revealed that the amount of lectin in sclerotia is 8-128 times higher than in the mycelium of the same strain. Mghalu et al. (2004) performed a large screening of 81 R. solani isolates for lectin activity in mycelium. However, they did not check the presence of lectin activity in sclerotia. It was shown that all R. solani strains of especially anastomosis groups 1 and 2 yielded high lectin activity. Within anastomosis group 1, AG 1-1A yielded the highest activity. Similarly, our screening revealed that mycelium of strain 5 of anastomosis group 1-1A yielded the highest lectin concentration. In addition, R. solani strains of anatomosis groups 1-1B (strain 7) and 1-1C (strain 8) also yielded high lectin activity both in the mycelium and in the sclerotia. At present we cannot

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explain why R. solani strain 2 which also belongs to anatomosis group 1-1C, did not show lectin activity. From our comparative analysis we can conclude that Rhizoctonia strain 7 (AG 1-1B; Origin: Belgium) has the highest amount of lectin in the sclerotia and also contains a high lectin concentration in the mycelium. Since this strain also shows very good growth, strain 7 (AG 1-1B) was selected for cultivation, extraction and purification of the lectin. In future experiments the insecticidal activity of the lectin will be tested towards biting-chewing insects (e.g. cotton leaf worm, Spodoptera littoralis) as well as piercing-sucking insects (e.g. pea aphid, Acyrthosiphon pisum). ACKNOWLEDGEMENTS M. Hamshou receives a scholarship from the Ministry of Higher Education in Syria. We are grateful to Mrs. S. Van Beneden and Prof. M. Höfte (Laboratory of Plant Pathology and Virology, Ghent University) for providing the Rhizoctonia strains.

REFERENCES BLAZIER S.R. & CONWAY K.E. (2004). Characterization of Rhizoctonia solani isolates associated with patch diseases on turfgrass. Proc. Oklah. Acad. Sci. 84:41-51. BRADFORD M. (1976). A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. ELAD Y., BARAK R. & CHET I. (1983). Possible role of lectins in mycoparasitism. J. Bacteriol. 154:1431-1435. GUILLEMAUT C., EDEL-HERMANN V., CAMPOROTA P., ALABOUVETTE C., RICHARD-MOLARD M. & STEINBERG C. (2003). Typing of anastomosis groups of Rhizoctonia solani by restriction analysis of ribosomal DNA. Can. J. Med. Technol. 49:556-568. ISHIKAWA F., OISHI K. & AIDA K. (1979) Chitin-binding hemagglutinin produced by Conidiobolus strains. Appl. Environ. Microbiol. 37:1110-1112. KELLENS J.T.C. & PEUMANS W.J. (1990a). Developmental accumulation of lectins in Rhizoctonia solani: potential role as a storage protein. J. Gen. Microbiol. 136:2489-2495. KELLENS J.T.C. & PEUMANS W.J. (1990b). Occurrence of lectins in different strains of Rhizoctonia solani. Clin. Biochem. 7:57-62. KELLENS J.T.C. & PEUMANS W.J. (1991). Biochemical and serological comparison of lectins from different anastomosis groups of Rhizoctonia solani. Mycol. Res. 95:1235-1241. KUMAR S., SIVASITHAMPARAM K. & SWEETINGHAM M.W. (2002). Prolific production of sclerotia in soil by Rhizoctonia solani anastomosis group (AG) 11 pathogenic on lupine. Ann. Appl. Biol. 141:11-18. LAEMMLI U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. MEYER K.M. (2002). Impact of nitrogen management strategies on yield, N-use efficiency, and Rhizoctonia diseases of Irish potato. M.Sc. Thesis, Graduate Faculty of North Carolina State University, USA, pp.100. MGHALU J.M., KOBAYASHI Y., KAWAGISHI H. & HYAKUMACHI M. (2004). Lectin variation in members of Rhizoctonia species. Microbes Environ. 19:227-235. PEUMANS W.J. & VAN DAMME E.J.M. (1995). Lectin as plant defense proteins. Plant Physiol. 109:347-352. PRESANT C.A. & KORNFELD S. (1972). Characterisation of the cell surface receptor for the Agaricus bisporus heamagglutinin. J. Biol. Chem. 247:6937-6945. VAN DAMME E.J.M., PEUMANS W.J., PUSZTAI A. & BARDOCZ S. (1998). Handbook of Plant Lectins: Properties and Biomedical Applications. John Wiley & Sons, Chichester, UK (ISBN 0-47196445-X). VRANKEN A.M., VAN DAMME E.J.M., ALLEN A.K. & PEUMANS W.J. (1987). Purification and properties of an N-acetylgalactosamine specific lectin from the plant pathogenic fungus Rhizoctonia solani. FEBS Lett. 216:67-72.

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