Biochemical and serological comparison of lectins from different anastomosis groups of Rhizoctonia solani

Mycol. Res. 95 (10): 1235-1241 (1991)

1235

Printed in Great Britain

Biochemical and serological comparison of lectins from different anastomosis groups of Rhizoctonia solani

JAN T. C. KELLENS AND WILL Y J. PEUMANS Katholieke Universiteit Leuven, Lahoratorium voor Fytopathologie en Plantenhescherming, Fakulteit der Landhouwwetenschappen, Willem de Croylaan 42, 8-3001 Leuven, Belgium

Twenty five isolates of different anastomosis groups of Rhizoctonia solani and a few related species were checked for the presence of lectins. All isolates of Rhizoctonia solani as well as Aquathanatephorus pendulus, Rhizoctonia crocornm and Athelia rolfsii contained lectins which comprised between 0'2 and 35 % of the total protein. The lectins were isolated by affinity chromatography and analyzed by ion-exchange chromatography which revealed that some isolates contained different isolectins. SDS-PAGE and gel filtration indicated that the molecular weights of the monomer polypeptides varied between 11 and 20 kDa whereas that of the native lectins ranged from 28 to 44 kDa. Although the lectins are most likely dimers, one R. solani lectin behaves as a monomer. Serological relationships between the lectins were investigated by double immunodiffusion assays and quantified by ELiSAs. Thereby it became evident that lectins from R. solani AG-I isolates and those from A. pendulus are closely related serologically by reaction with an antiserum against a lectin of an isolate of AG-l. Similarly all AG-4 isolates of R. solani appear to be closely related to each other; they are all recognized by the antiserum to a lectin from R. solani AG-4 but not by antiserum to a lectin from R. solani AG-l. The use of the lectins in ELiSAs to determine the AG of R. solani isolates is discussed.

Rhizoctonia solani Kiihn, the mycelial state of Thanatephorus cucumeris (Frank) Dank is a complex species with many biotypes differing in pathogenicity, host range, distribution in nature, physiological characteristics, and morphology. Although many attempts have been made to divide the isolates into intraspecific groups according to these characteristics, most of them were not satisfactory. The first natural subdivision of R. solani was made by Schultz (1937) who introduced the anastomosis group (AG) concept. Hyphal anastomosis occurred only between isolates from the same group and hence anastomosis groups are genetically distinct (parmeter, Sherwood & Platt, 1969). Until now, nine anastomosis groups have been proposed (Carling, Liener & Kebler, 1987) along with a number of subgroups (Ogoshi, 1987). Since there is some correlation between AG and host range, morphology, infection, and ecological behaviour (Parmeter et a!', 1969), determination of the AG of isolates is important. Although direct observation of hyphal anastomosis is most Widely used, it is difficult and time-consuming. Therefore other criteria have been applied to differentiate between AGs. Ogoshi & Ui (1979) were able to correlate vitamin requirement of some isolates with their AG, whereas separation of isolates of R. solani AG-3 from other AGs was possible on 'Stewart's medium' (Castro, Davis & Wiere, 1983). Jones & Pettit (1987) demonstrated differences in sensitivity to gliotoxin, an antibiotic produced by Gliocladium virens Miller, Giddens & Foster, among anastomosis groups of R. solani. Others

compared soluble protein patterns of R. solani by polyacrylamide gel electrophoresis (Reynolds, Weinhold & Morris 1983) or used antisera raised against total protein extracts to separate serologically the different anastomosis groups (Adams & Butler, 1979). Genetic relationships between and within anastomosis groups in R. solani were also investigated by heterologous DNA/DNA hybridization and ribosomal DNA restriction fragment length polymorphisms (Vilgalys, 1988; Vilgalys & Gonzalez, 1990). These studies demonstrated a genetical basis for the anastomosis grouping concept but also an extensive variation within many AGs. The recent discovery of lectins in Rhizoctonia solani (AGgroup not known), Rhizoctonia crocorum (Pers.) DC. ex Fr. and Athelia rolisii (Curzi) Tu & Kimbrough (Vranken, Van Damme, Allen & Peumans, 1987; Kellens, Allen & Peumans, 1989) raised two questions; firstly whether isolates of different AGs of Rhizoctonia solani contained lectins and secondly whether lectins produced by R. solani were related to each other? The present study reports the isolation and comparison of lectins from isolates of different AGs of R. solani and a few related species, and discusses the possible use of these proteins to distinguish between anastomosis groups of R. solani.

MATERIALS AND METHODS Isolates and growth conditions The fungal isolates listed in Table I were obtained from the Centraalbureau voor Schimmelcultures (Baarn, the Nether78-2

Lectins in Rhizoctonia solani

1236

Table 1. List of isolates used' No. I

2 3 4 5 6 7 8 9 10 II 12

13 14 15 16 17 18 19 20 21 22 23 24 25

Source

Isolate

(AG)

CBSt CBS CBS Sherwood, R. T. Sumner D. CBS Sumner, D. CBS Sumner, D. Herr, L. J. CBS CBS Herr, L. J. Sumner, D. Sumner, D. Sumner, D. Sherwood, R. T. Sherwood, R. T. Herr, L. j. CBS CBS CBS CBS CBS CBS

205'84 206.84 207.84 Sc 192 RHS8 208.84 145-405 ALDF 209.84 264-405 LDF 207 211.84 336.84 AG4 RHS 109 279 223-5£-2 5-283 5-287 AG5 214.84 700.82 324.47 719.83 316.84 810.85

AG-1A AG-1B AG-1C AG-1 AG-1 AG-2-1 AG-2-1 AG-2-2 AG-2-2 AG-2 AG-3 AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-5 AG-7

, Species: nos 1-20, Rhizoctonia solan;; no. 21, AquathaMtephoros pendulus; no. 22, Rhizoctonia crocorum; no. 23, Athelia rolfsii; no. 24, Wailea circi'lala; no. 25, Phanerochaele salmonicolor. t CBS, Centraalbureau voor Schimmelcultures (Baam, the Netherlands).

lands) or were kindly provided by Prof. Sumner (Michigan State University, East Lansing, Michigan, USA.), Prof. Sherwood (North Carolina State University, Raleigh, North Carolina, U.S.A.) and Prof. Herr (Ohio Agricultural Research and Development Center, Wooster, Ohio, USA.). All the strains were grown at room temperature (approx. 18-20°C) in 2-1 Erlenmeyer flasks containing 750 ml of the medium of Okon, Chet & Henis. (1973). Agar discs (5 mm diameter) covered with mycelium were cut from 5-day old cultures grown at 27° on potato dextrose agar (PDA) and were used as inocula. Mycelium from approximately 50-day old cultures was collected, extensively washed with distilled water, frozen and lyophilized. After lyophilization the dried mycelium was ground in liquid nitrogen.

Preparation of immobilized V-galactose D-Galactose was coupled to Epoxy-activated Sepharose-6B (Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions using a 10% (w/v) solution of D-galactose.

Ledin extraction and purification Powdered mycelium (10-20 g) was suspended in 25 volumes (v/w) of phosphate buffered saline (PBS; 1'5 mM KH 2 P0 4 1

10 mM Na 2 HP0 4 (pH 7'4); 3 mM KCl; 140 mM NaCl) and extracted for I h at room temperature on a magnetic stirrer. The homogenate was frozen at - 20°, After thawing the

homogenate was centrifuged (10 min; 3°; 6,000 g) using a Sorvall RC-5B centrifuge and a GS-34 rotor, the supernatant taken off and the pellet re-extracted in 25 volumes (v Iw) of PBS. After centrifugation (10 min; 3°; 6,000 g) of the second extract, both supernatant fractions were pooled and the pH adjusted to 4'0 with I M acetic acid. Since a new precipitate was formed at this pH. the extract was recentrifuged (10 min; 3°; 6,000 g) and the supernatant taken off and brought to pH 7'0 with I mM NaOH and applied to a column (bed size: 6 em x 2' 5 cm; bed volume: 30 ml) of immobilized D-galactose preequilibrated with PBS. The volume of the supernatans was 500-1000 ml containing a protein concentration of 0'4-1 mg ml- I. The flow rate was 10 ml min-I. Unbound proteins were eluted with PBS until the A 280 of the eluate was below 0'01 and the lectin was desorbed with 0'1 M D-galactose in PBS. The affinity-purified fractions were collected, pooled and dialyzed overnight against formate buffer (25 mM Na-formate, pH 4'0) at 3° and further analyzed by ion-exchange chromatography.

Ion-exchange chromatography The affinity-purified lectins were chromatographed on a Pharmacia Mono-S (type HR 515: bed size 5 mm x 50 mm; bed volume: I ml) cation-exchange column using a Pharmacia Fast Protein Liquid Chromatography (FPLC) system (type GP250). Lectin samples of approXimately 5 mg were loaded on the column equilibrated with 25 mM Na-formate, pH 4'0. After washing the column with the same buffer (4 ml), the lectins were eluted using a linear gradient (30 ml) from 0 to 0'6 M NaCl in formate buffer. The flow rate was 2 ml min-I. The elution position of the isolectins was determined by measuring the A 280 using the UV monitor of the FPLC system connected to an integrator (ENICA 21 Delsi Instruments, Suresnes, France). Peak fractions were collected and further purified by repeated chromatography on the Mono-S column. The purified isolectins were stored at - 20° and used for further analyses or were dialyzed against H 2 0 (three times for 3 h, once overnight and once for 6 h), frozen and lyophilized.

Assays and analytical methods Agglutination assays were done in small glass tubes in a final volume of 0'1 ml containing 80 J,J.l of a I % (v Iv) suspension in PBS of trypsin-treated rabbit erythrocytes and 20 J.ll of crude extract or lectin solution. After the mixtures had been incubated for I h at 20° the agglutination was monitored visually. Total protein was estimated by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Munich, FRG) which is based on the principle of Bradford (1976) using bovine serum albumin as a standard. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was done on a 12'5-25 % polyacrylamide gradient gel using a discontinuous system (Laemmli, 1970). ElectrophoreSiS was performed under constant voltage (180 V). Gel filtration was carried out on a Superose 12 column (type HR 10/30 from Pharmacia; bed size: I cm x 30 cm; bed volume: 24 ml) using a Pharmacia FPLC system as described by Vranken ef al. (1987).

J. T. C. Kellens and W. J. Peumans Immunization procedure One milligram of highly purified lectin dissolved in 1 ml of PBS was emulsified in 1 ml of complete Freund's adjuvant and injected subcutaneously in a rabbit. Five booster injections were given at 10-day intervals. Ten days after the last injection blood was taken from an ear marginal vein and allowed to clot overnight at room temperature. Crude antiserum was obtained by centrifugation at 3,000 g for 5 min.

Partial purification of crude antiserum Crude antiserum was partially purified by repeated precipitation (6 times) with (NH4)2S04 (40% relative saturation). The final precipitate was dissolved in 25 mM 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris)-HCl (pH 8'7) containing 0'1 M NaCl and dialyzed for 3 h and then overnight at room temperature (approx. 18°) against the same buffer. This fraction of the serum was then applied to an anionexchange column (bed size: 3 cm x 25 cm; bed volume; 175 ml) of Q-Fast Flow from Pharmacia (Uppsala, Sweden), equilibrated with the same Tris-buffer. Under these conditions, IgG immunoglobulins did not bind to the column and eluted in a large peak just behind the void volume. The IgG fraction was collected and used in all further experiments. The antibody titre as determined in a double immunodiffusion assay was 4 in both cases.

Double immunodiffusion assay Double immunodiffusion was done in small Petri dishes (9 cm in diameter) filled with 10 ml of 1 % (w Iv) agarose in PBS containing 4% (w/v) polyethylene glycol 6000 and 0'1 M Dgalactose (to prevent aspecific binding of the lectin to immunoglobulins). A central well and six peripheral wells, all of 3 mm diameter were made on a distance of 8 mm from each other. Five I-li of lectin samples (approx. 1 mg ml- 1 in PBS) (peripheral wells) were challenged with 5 I-li of antiserum (central well). After 24 h, unprecipitated proteins were eluted with PBS during 3 days. The precipitation lines were visualized as described by Peumans, Stinissen & Carlier (1982).

Preparation of the enzyme-labelled antibodies Alkaline-phosphatase conjugated rabbit anti-lectin antibodies were prepared following the procedure of Voller, Bidwell & Bartlett (1980) using glutaraldehyde to couple the enzyme to the antibody. The alkaline phosphatase (type VII-N from bovine intestinal mucosa) was purchased from Sigma.

Quantitation of the serological relationships between the lectins by an enzyme-linked immunosorbent assay (ELISA) To measure the relative serological relationships between the lectins, a sensitive double sandwich ELISA was developed. Flat-bottomed polyvinylchloride microtiter plates (96 wells, from Costar, Cambridge, Mass., U.s.A.) were coated with 50 I-li

1237 per well of a 0'002 % (v Iv) solution of partially purified rabbit antibodies in Borate-saline buffer (6'19 g 1-1 H 3 B0 3 ; 8'50 g 1-1 Na 2B40 7 • 10H 20; 4'39 g 1-1 NaCl; pH 8'4) for 2 h at 37°C. Then the plates were washed twice with PBS containing 0'02 % (w Iv) sodium azide and 0'05 % Tween-20 (PBST) and twice with PBS containing 0'02 % (w Iv) sodium azide alone (PBSN). To block the remaining non-specific protein-binding sites the wells were filled for 30 min at room temperature with 60 I-li of borate-saline buffer containing 1 % (w Iv) bovine serum albumin and 0'02 % (w Iv) sodium azide. After washing twice with PBSN, different dilutions of the purified (iso)lectins which were dissolved in dilution buffer (PBST containing 1 % (w Iv) bovine serum albumin and 100 mM D-galactose) at a concentration of 1 mg ml- 1 and a standard series (8 wells) of the lectin from the tester isolate at concentrations from 0 to 20 ng ml- 1 in dilution buffer, were incubated for 2 h at 37° (50 I-li per well). Thereafter, the plates were washed twice with PBST and twice with PBSN. Then 50 I-li of 1 % (v Iv) alkalinephosphatase-labelled antibodies in dilution buffer was added to each well and incubated at 37° for 2 h. Unbound conjugate was washed away by rinsing the plates twice with PBST and twice with PBSN. The enzymic reaction was performed in 50 I-li per well of diethanolamine buffer (9'6% (v/v) diethanolamine: pH 9'8; 0'005% (w/v) MgCl 2 and 0'1 % (w Iv) p-nitrophenyl phosphate (Sigma, St Louis, Mo., USA)). After approx. 30 min at room temperature the reaction was stopped by the addition of 4 N KOH (50 I-li per well) and quantified by measuring the absorbance of the yellow reaction product (produced by the reaction of the enzyme with pnitrophenyl phosphate) at 405 nm using an SLT Easy Reader EAR 340 AT (SLT Labinstruments, Salzburg, Austria). The specificity of the antibodies was confirmed by SDS-PAGE and Western blotting (Kellens & Peumans, 1990).

RESULTS Occurrence and isolation of ledins from different isolates Twenty isolates of different AGs of Rhizoetonia solani and strains of Aquathanatephorus pendulus Tu & Kimbrough, Rhizoetonia crocorum, Athelia rolfsii, Waitea circinata Warcup & Talbot, and Phanerochaete salmonicolor Karst. em. Donk were checked for the presence of lectins. Crude extracts were made from each isolate and their protein content and agglutination titre (defined as the highest dilution that still gave a visible agglutination of red blood cells) determined. The agglutination titre as well as the protein content of the different isolates varied strongly (Table 2). Extracts of all the R. solani isolates and of A. pendulus, R. crocorum and A. rolfsii exhibited high titers in agglutination assays with trypsin-treated rabbit erythrocytes, whereas no agglutination activity could be detected in extracts of W. circinata and P. salmonicolor (Table 2). Extracts from the latter two species did not agglutinate trypsin-treated pigeon, porcine or human erythrocytes (results n()t shown). Since the agglutinating activity of the extracts of most of the strains could be inhibited by D-galactose, lectins were isolated by affinity chromatography on immobilized Dgalactose. However, the lectins from R. crocorum and A. rolfsii

Ledins in Rhizoctonia solani

1238

Table 2. Agglutination titre. protein content and relative ledin content from different isolates of Rhiwctonia'

1 2 3 4 5 6 7 8 9 10 11

12 13

14 15 16 17 18 19 20 21 22 23 24 25

Agglutination titre

AG

No.

AG-1A AG-1B AG-IC AG-1 AG-1 AG-2-1 AG-2-1 AG-2-2 AG-2-2 AG-2 AG-3 AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-5 AG-7

Relative lectin content (%)

Protein content of PBS extrad (mg (g dry wt)-l)

400 200

1600 400

3200 700

100 800

20 400 800 1200 600

52 80 66 49 57 63 48 52 100

0'1

25 52 68

4 0 0

72

600 800 800 800 1200

1600 800 1200 700 0 0

20·1 14-4

R 21 21 21 ABC

13

A

19

4

c:

1·0

.2 ~

A

B

0,5

,

0·0

~

C

C

\.

10 20 30

,

,,

~\

10 20 30

,

/

,

,

,

B

/

~

10 20 30

,

/ /

~

10 20 30

0·6 C 1:)

0,4 g t)

0·2

I I 1 19 3 ABC

10

20202011 ABC

22 23

fig. 2. 50S-PAGE of some isolated (iso)lectins. About 20 ~g of each (iso)lectin was loaded on a 12'5-25% acrylamide gradient gel in the presence of 2 % l3-mercaptoethanol. Numbers correspond to those used in Table 1 and characters refer to the order of elution position of the isolectins in ion-exchange chromatography. Mol. wt reference proteins are shown in lane R. They are: lysozyme (14'4 kOa), soybean trypsin inhibitor (20'1 kDa), carbonic anhydrase (30 kOa), ovalbumin (43 kOa), bovine serum albumin (67 kOa) and phosporylase b (94 kOa).

, Fungal cultures were extraded in 50 volumes (v/w) of PBS and the agglutination titres of those extrads were determined with trypsin-treated rabbit erythrocytes. Protein content was determined by the Bio-Rad protein assay. The relative ledin content is the percentage of the total protein content represented by the ledin content (lectin content = agglutinating adivity (mg ml- 1) x agglutination titre x 50 (ml(g dry wt)-l)) (agglutinating adivity = minimal ledin concentration of the purified ledins required for agglutination of trypsin-treated rabbit erythrocytes). The numbers of the isolates correspond to the numbers in Table 1 and to the species listed in the footnote.

23

67

30

0'3

5'2 9'2 16'9 11'2 7'9 12'8 16'9 14 19 30 15'4

94

43

II'5 5'8 3'8 22 7'5 35 9'2 1'8 15'6

67 69 55 55 51 84 73 63 51 67 73 83

800

kDa

<0

of the affinity-purified ledins was determined. (After affinity chromatography on Gal-Sepharose and elution with 0'1 M Dgalactose the lectins were virtually pure. the ledins from R. crocorum and A. rolfsii were further purified by ion-exchange chromatography on a Mono-S column before use.) The relative lectin content of all the isolates was calculated as the percentage of the total soluble protein content (mg (g dry wtrl) represented by the ledin content (mg (g dry wt)-l; (lectin content = agglutinating activity (mg ml- l ) X agglutination titer x 50 (ml(g dry wt)-l)). These values range from 0'2 to 35 % of total protein (Table 2). The affinity-purified ledins were analysed by ion-exchange chromatography on a Mono-S cation-exchange column using an FPLC system. The ledins from different isolates eluted at different NaCi concentration (Fig. 1). Whereas some isolates contained only one ledin, others contained different isoledins which were purified by repeated ion-exchange chromatography. The isoledins are indicated with charaders (A, B, C) in order of elution position.

Z

0·0

Elution volume (ml) Fig. 1. Ion-exchange chromatography of some lectins isolated from different R. solani strains. Affinity-purified lectins were chromatographed on a Mono-S column as described in the Materials and Methods section. The numbers correspond to the numbers of the isolates listed in Table I, Isolectins are labelled A, B, C in order of elution position.

were isolated on immobilized mucin since they were not inhibited by D-galadose (Kellens et al., 1989), Thereafter the agglutinating adivity (= minimal lectin concentration required for agglutination of trypsin-treated rabbit erythrocytes)

Characterization of the lectins The molecular weight and subunit stoichiometry of the lectins was determined by SDS-PAGE and gel filtration. Upon SDSPAGE the lectins of all strains yielded one or more polypeptides ranging in size from 1 I kDa to 20 kDa except that of one isolate of R. solani AG-3 (no. 11) which yielded a single polypeptide band of 34 kDa (Fig, 2). Since reduced (with 13-mercaptoethanol) and unreduced samples yielded the same pattern (results not shown), the subunits are not held together by intramolecular bridges (Laemmli, 1970), The native lectins eluted from a Superose 12 gel filtration column with an apparent mol. wt ranging from 26 to 44 kDa (Table 3). Therefore all the lectins are most probably dimeric proteins

]. T. C. Kellens and W.

J. Peumans

1239

Table 3. Molecular weight and subunit stoichiometry of isolated (iso)lectins'

(Iso)lectin

lA B, C 2 A, B, C 3 4A

B C 5 A, B 6 A, B, C 7A

B C 8 9A

B C 10 11 12 13A

B, C 14 15 A, B, C 16 17 A B, C 18 19 20 A, B, C

21 A B C

22 23

Mol. wt of subunits

Mol. wt of native lectin

Molecular stoichiometry

18,20 16, 18 16 15 17 IS, 17 15 16 15 15 17 IS, 17 15 15 17 IS, 17 20 34 16 15 16 15 16 15 15 16 15 18, 19, 21, 22 20 18 16, 18 16

34 34 28 27 32 31 30 27 26 30 32 31 30 30 32 31 34 32 30 30 30 30 28 28 30 30 28 38 38 36 34 32 44 37

dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer

11

17

monomer

dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer dimer tetramer dimer

The molecular weight of the subunits was detennined by SDS-PAGE whereas the molecular weight of the native lectins was estimated by gel filtration on a Superose 12 column. The most probable subunit stoichiometry is given. The numbers in the first column refer to those used in Table I, and the characters (A, B, C) correspond to the isolated isolectins in order of elution from the Mono-S column.

composed of either identical (or at least identically sized) or different subunits except that from R. solani isolate CBS 211.84 (no. 11) which is a monomer of 34 kDa and that from Rhizoetonia crocorum which is a tetramer composed of 4 identical subunits (Kellens et aI., 1989).

Serological relationships Antisera were prepared against ledins from R. solani isolate CBS 207.84 (AG-IC) (no, 3) and R. solani isolate 223-SE-2 (AG-4) (no. 16), and challenged with purified ledins in double immunodiffusion assays (Fig. 3). When readed with antiserum to isolate no. 3, the ledins from isolates of R. solani AG-1 and A. pendulus were identical with each other (readion type I: the precipitin lines are continuous) (Fig. 3, wells I to 6), when tested by Ouchterlony double immunodiffusion assays, whereas all the other ledins were serologically related to them (reaction type 2: the precipitin lines form traces) (Fig. 3, wells 7 to 12). When challenged with antiserum against isolate no.

7

10

Fig. 3. Double immunodiffusion of purified ledins hom Rhizoctonia so/ani. Five Ilg of purified ledins hom R. so/ani isolate no. 3 (wells I, 2, 3, 4, 7, and 10), no. 5 (well 5), no, 6 (well 8), no. 11 (well 9), no. 12 (well 11), no. 20 (well 12) and Aquathanatephorus pendu/us no. 21 (well 6) were challenged with 5 III antiserum to the ledin of isolate no. 3 (AG-Ie) (central wells).

Table 4. Serological relationship of purified (iso)lectins' Serological reaction of antisera to isolate No.

AG

Isolectin

3 (AG- IC)

16 (AG-4)

1 2 3 4 5 6 7 8 9 10

AG-IA AG-IB AG-IC AG-1 AG-1 AG-2-1 AG-2-1 AG-2-2 AG-2-2 AG-2 AG-3 AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-5 AG-7

A, B, C A, B, C

I'

2 2 2 2 2 2 2 2

11

12 13 14 15 16 17 18 19 20 21 22 23

1 1

A, B, C A,B A, B, C A, B, C

2 2

A, B, C

2

2

2

2

2

2

2

1

1 1

2

A, B, C

2

1

2 2 2 2 2

1

2

2

A, B, C

2

A, B, C

1

2 2 2 2

A, B, C A, B, C

2

2

1

1 1 1

Double immunodiffusion assays. Antisera (5 I-lI) against Rhizoctonia solani isolate no. 3 (AG-IC) and isolate no. 16 (AG-4) challenged with 5 I-lg of purified (iso)lectins (numbers and species correspond to those in Table 1 and characters refer to the isolectins in order of elution by ion exchange chromatography). Fonnation of precipitation lines was identical (reaction type 1) or related (reaction type 2) to the lectins of the tester isolates.

16 (AG-4) on the other hand, all the ledins from isolates of R. solani AG-4 readed identically with each other (readion type I) while all other ledins were serologically related to them (readion type 2) (Table 4). To quantify the serological relationships between the ledins, double sandwich ELISAs were developed with both

1240

Lectins in Rhizoctonia so/ani 1·5 r - - - - - - - - - - : : - - - - - - : : - , - - - - - - - - - - , 12 1·0

0·5

3 (AG-le) when tested against antiserum to AG-l (Fig. SA), whereas the lectins from isolates of other AGs reacted less strongly (about three orders of magnitude). Similarly all the lectins of isolates belonging to AG-4 when tested against antiserum to AG-4 reacted as well as the lectin of the tester isolate no. 16 (AG-4) whereas the others reacted much less strongly (Fig. 5B).

DISCUSSION 0·0 4-~~~~::_l:_:_----=~-ll::lt:t::~-____L_-_'_-1 0·001 0·01 0·1 I 10 100 1000

Lectin concentration (~g ml- I )

Fig. 4. ELISA dose response curve of purified lectins to antiserum against the lectin of isolate no. 3 (AG-IC). The numbers refer to the numbers of the isolates listed in Table 1 and the charaders to the order of elution position in ion-exchange chromatography.

0·001 0·0001 3 2

5 4

7 6

9

8

I I 13 15 17 10 12 14 16 Number or isolate

19 2 I 23 20 22

Fig. 5. ELISA of different purified (iso)lectins with antisera against (A) Rhizoctonia so/ani isolate no. 3 (AG-IC) and (B) R. so/ani isolate no. 16 (AG-4). Relative reactivities to the tester ledin expressed as percentages are presented in a logarithmic scale. The numbers correspond to the numbers in Table 1. D, Isolectin A; i!ill, isoledin B; _, isoledin C.

antisera. Solutions of purified (iso)lectins were diluted and the highest dilution that still reacted in the assay determined. The dose response curves of some lectins in an ELISA with antiserum to isolate no. 3 lectin are presented in Fig. 4. The lectins of all the isolates of AG-l as well as those of A. pendu/us reacted almost equally well as the lectin of isolate no.

Besides morphological characteristics, biochemical analyses have become important for the identification and classification of fungi. Analyses of total protein and enzyme patterns are used since they reflect easily recognizable genetic differences. However, a major problem related to this type of comparative biochemical analysis is to find a well-defined protein that occurs in all strains to be tested, and that can easily be detected and isolated. In this report, we present evidence that the lectin occurring in different Rhizoctonia species and strains is a good taxonomical marker when used in serological assays. The results from the experiments described above indicate that all the R. so/ani isolates tested as well as R. crocorum, A. ro/fsii and A. pendu/us contain lectins, whereas W. circinafa and P. sa/monic%r did not agglutinate with different types of erythrocytes. The lectins from the R. so/ani isolates as well as those from A. pendu/us were specific for D-galactose whereas the lectins from R. crocorum and A. ro/fsii were only inhibited by fetuin and mucin. No correlation could be found between the anastomosis groups of R. solani isolates on the basis of their lectin content. molecular weight or stoichiometry of the lectins or isolectin patterns. On the other hand a clear distinction between the anastomosis groups of the isolates can be made based on the serological relationships of their lectins. In double immunodiffusion assays only lectins of isolates of the same AG as the tester isolate reacted identically with tester-lectin antiserum apart from A. pendu/us and antiserum to AG-l. In an ELISA the reaction of antiserum against a lectin with purified lectins from isolates of the same AG was nearly as strong as the reaction with the lectin itself whereas lectins from isolates of other AGs reacted about thousand times less strongly. Since there are great differences in reactivity of lectins from different AGs with antiserum against a lectin of a well-defined AG, a rapid and reliable method could be developed to determine the AG of Rhizoctonia so/ani isolates. When lectins are purified from tester isolates of all the different AGs of R. so/ani and antisera raised against them, ELISAs can be developed for each AG. Purified lectins or even total protein extracts of isolates of unknown AG can be tested in ELISAs for each AG. The lectin concentration of the same extract will be determined with antisera to different AGs. The isolate will belong to the AG to which the lectin in its extract has the highest relative reactivity in ELISA. These tests are not dependent on the relative lectin content in the extract since the reaction is highly specific for lectins (Kellens & Peumans, 1990). Since this method is very sensitive (it detects concentrations of less than 10 ng lectin ml- 1 ) only little material is required.

J. T. C. KeIIens and W. J. Peumans Our results emphasize the need to consider biochemical markers along with morphological features in fungal taxonomy. A. pendu/us, an aquatic Rhizoctonia species from water hyacinth (Eichhornia azurea (Swartz) Kunth.) with an imperfect state indistinguishable from Rhizoctonia so/ani was placed in the genus Aquathanatephorus because it produced a highly modified sexual state (Tu & Kimbrough, 1978). Nevertheless the lectins from A. pendu/us are identical in immunodiffusion tests to those of the isolates of R. so/ani AG-l.

This work was supported in part by grants from the 'Nationale Bank' and the National Fund for Scientific Research (Belgium) (FGWO project No. 2005989 N), of which W. J. Peumans is a Senior Research Director. J. T. C. KeIIens received a fellowship from the Belgian' Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw'.

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(Received for publication 14 January 1991 and in revised form 29 March 1991)

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