DIFFERENTIATION OF RHIZOCTONIA SPP . BASED ON THEIR ANTIGENIC PROPERTIES

Antigenic properties and serological relationship was investigated in binucleate and multinucleate Rhizoctonia spp. isolates from strawberries, soybean, alfalfa and potato plants from Serbia, from Spain, anastomosis group testers and in strawberry roots inoculated with binucleate Rhizoctonia AG A and AG I. Two polyclonal antisera, unabsorbed and cross absorbed, were used in dotimmunobinding assay for these investigations. Antisera were produced against mycelial antigens of two isolates, which belong to different anastomosis groups (AG) of binucleate Rhizoctonia AG A and AG I. Both unabsorbed antisera reacted positively with all tested Rhizoctonia spp. isolates, and the reaction was absent with control isolates (Pythium sp., Agaricus sp. and Fusarium sp.). The results prove a close serological relationship among Rhizoctonia spp. isolates, and diversity between Rhizoctonia spp. and isolates from different taxonomic groups. Also, both unabsorbed antisera reacted with higher intensity with closely related antigens (belonging to the same AG) than with ones from another AG of binucleate Rhizoctonia or R. solani (multinucleate Rhizoctonia). After cross absorption specificity of the antisera was enhanced, especially with the antiserum raised against mycelial proteins of binucleate Rhizoctonia AG I. This antiserum reacted positively only with antigens from the same AG, after cross absorption with antigens from AG A of binucleate Rhizoctonia and from R. solani AG 2-2. It proved to be specific to AG I of binucleate Rhizoctonia, and able to differentiate isolates of this AG from others. In this way the serological homology among isolates of one AG was proven, and also the diversity among isolates which belong to different AGs of binucleate Rhizoctonia as well as isolates of R. solani.


I n t r o d u c t i o n
Species of Rhizoctonia are significant soilborne plant pathogens which cause worldwide important diseases on most of world`s important crop plants, such as: cereals, cotton, sugarbeet, potato, vegetables, field crops, turfgrasses, ornamentals, fruit trees and forest trees.Economic importance, as well as the knowledge of Rhizoctonia spp. is growing rapidly but in spite of that there is still some lack of understanding in systematic relationships and genetic variations within and among the complex.The lack of fossil record, absence of the teleomorph, phenotypic variation and pathological diversity are the reasons why Rhizoctonia taxonomy is progressing slowly.Identification and differentiation of the species belonging to Rhizoctonia complex is difficult if one is using only conventional criteria.The present system of the identification of Rhizoctonia spp., 1996).The concept of AGs and subgroups represents a very successful method for the classification of Rhizoctonia spp.but it provides little insight into the genetic diversity of these fungi (J a b a j i -H a r e , 1996).This is the reason why, in recent years, a lot of attention has been given to new approaches among which serology takes an important place.This paper examines the possibilities of differentiating antigenic properties as well as serological relationship within the Rhizoctonia complex using dot-immunobinding assay.
Rhizoctonia spp.isolates designated as Rh-L and Rh-R, Rhizoctonia solani AG 3 (V i c o , 1997), originate from Spain and were supplied by dr Maria Muntanola -Cvetković (Faculty of Biology, University of Belgrade ), and also tester isolates of binucleate Rhizoctonia AG I and AG A (O g o s h i et al., 1983) were included in the study.
Antigen preparation.-Antigensused for the production of antisera were prepared according to A d a m s and B u t l e r (1979) from the mycelia of isolates MLE (binucleate Rhizoctonia AG A) and 4 (binucleate Rhizoctonia AG I).Protein concentration was determined according to L o w r y et al. (1951).
Antigens used for serological studies and those used for cross absorption of the antisera were prepared in Tris-HCl (A d a m s and B u t l e r , 1979), and in PBS (1g of frozen mycelium homogenized in 4 ml of PBS, centrifuged 1 hour at 1500 g).
Antigens from strawberry plants were prepared by homogenizing 0.1g of roots in 1ml PBS or TBS-T alone or with the addition of the following antioxidants: 0.01 M DIECA with 0.01 M EDTA and 0,1 M ascorbic acid; or 0.01 M DIECA with 0.01 M EDTA and 0.2% β-mercaptoethanol; or 1% Na 2 SO 3 ; or 10% Na 2 SO 3 ; or 1% tioglicolic acid; or 0.1 M ascorbic acid with 0.2 ml dihlormethane.The suitability of the antioxidants was checked in tubes (when homogenized samples, after 10 minutes centrifugation at 2000 g, were incubated 3 hours at room temperature and than the color changes were assessed) and after a test on nitrocellulose membrane was performed.Suspension of carpophores of isolate ŠAM and mycelium of isolate L-Fus were used as control.Color changes were assessed after the reaction was developed.
Production of antisera.-Polyclonalantisera raised against mycelial proteins of isolate 4 (binucleate Rhizoctonia AG I) and isolate MLE (binucleate Rhizoctonia AG A) were produced by immunizing two rabbits.For the first one, during 10 days, 5.8 mg/ml of protein (mycelium of isolate 4), and for the second one 20.9 mg/ml of protein (mycelium of isolate MLE) was injected intramuscularly.After 4 days the booster injection was given, and the blood was taken 10 days later.Antisera raised against the mycelial proteins of isolate 4 was designated as As 1, and antisera raised against the mycelial protein of isolate MLE was designated as As 2. The titer of antibody was determined using agglutination test, Quachterlony agar double -diffusion test and dotimmunobinding assay.
Preparation of antisera for serological tests.-Unabsorbedand cross absorbed antisera As 1 and As 2 were used in serological investigations.Cross absorption was done by mixing the antigen with antisera in 1:2 ratio, for every step of absorption, according to A d a m s and B u t l e r (1979).
Serological methods.-Agglutinationtest was used to determine the level of antibody in As 1 and As 2 according to T o š i ć and Š u t i ć (1977).The first check to determine agglutination was done after 30-50 minutes, but the results were more obvious after a period of 3-4 hours.During that time probes were kept in humid chambers at 4 o C.
Quachterlony agar double-diffusion test was used for the assessment of antibody titer in As1 and As 2. The test was done using Ion agar according to T o š i ć and Š u t i ć (1977) in 1% concentration.40 g/l of poly-ethilen glicol (PEG, 6000 mol wt.) was added according to A d a m s and B u t l e r (1979) to enhance the visibility of precipitation lines, and for the same reason the gels were stained in 0.05% Comassie blue in 7% acetic acid and 50% methanol.Destaining was done in 10% methanol and 7.5% acetic acid.Dot-immunobinding assay was used for determination of antibody level in As 1 and As 2 and for the investigation of the serological relationship of different Rhizoctonia spp.isolates.The assay was done according to the method shown in catalog No 170-6545 Bio-Rad Laboratories, Richmond CA, USA, as described by O 'D o n n e l l et al. (1982), modified by S h u k l a et al. (1989).Antisera dilution 1:100 and 1:1000 were used when titer of antibody was assessed, the dilution 1:500 was used for unabsorbed and dilution 1:50, or 1:25 was used for absorbed antisera during the studies of serological relationship.Goat-anti-rabbit antisera was used in 1:2500 dilution.The reaction was developed for about 3 minutes until lilac-blue color appeared on the paper which was a sign of a positive reaction.

Results and Discussion
Antibody titer.-Twoantisera were produced during the investigations: As 1 and As 2. The titer of antibody in As 1 determined by agglutination test is 1/8, and of the As 2 is 1/32.Using agar double diffusion test titer of both As 1 and As 2 was ½, while it was much higher when determined by dot-immunobinding assay when it was 1/1000 for both antisera.
Suitability of buffers and antioxidants for use in dot-immunobinding assay.-Buffersand antioxidants used for antigen preparation from mycelium and strawberry roots have shown different efficiency.Results of these investigations are shown in Table 1 and 2 Reaction intensity depended on the serological relationships of the isolates but also on the buffer used.Antigens prepared in PBS have shown better reactivity than the ones prepared in Tris-HCl buffer.
T a b .2. -Influence of the antioxidants on color changes of root extracts from strawberry and mushroom extract (Agaricus sp.) .

In tubes Extract from
Color change after the use of antioxidants Root samples prepared in 1% tioglicol acid and mushroom extracts prepared in 1% tioglicol acid and 0.1M ascorbic acid with 0.2 ml dihlormethane have shown no color changes after 3 hours of incubation in tubes (Table 2.).Also, after the test on nitrocellulose membrane there was no false reactions with antisera used.This means that the most suitable way of sample preparation is with 1% tioglicol acid.Mushroom extracts can also be prepared in 0.01M DIECA, 0.01M EDTA and 0.1M ascorbic acid in TBS-T (Table 2

.).
Serological reactions.-AntiseraAs 1 and As 2, which were produced in the course of investigations when used in dot-immunobinding assay, reacted with homologus but also with other antigens.Enhancement of the antisera and the clasification of serological relationships, that is specific antigenic properties were examined after cross absorbing the antisera.Summary of the results of the serological investigations is presented in Table 3.
Both antisera As 1 and As 2 have shown positive reaction with all Rhizoctonia spp.tested, and also with the samples of strawberry roots infected with binucleate Rhizoctonia AG A and AG I.The reaction was absent with antigens from other genera (Agaricus sp., Pythium sp. and Fusarium sp.), and with healthy root samples (Table 3. column 1 and 6).Among antigens from the same AG and the homologous antisera the reaction was of the highest intensity.As 1 (raised against antigen 4, binucleate Rhizoctonia AG I) had the most intensive reaction with isolates 4 and P6, which both belong to the same AG, as well as with the tester strain for AG I.The reaction was less intensive with isolates members of AG A of binucleate Rhizoctonia and isolates of R. solani.Antisera As 2 (raised against antigen MLE, binucleate Rhizoctonia AG A) behaved similarly as As 1 with his homologous and closely related antigens.The reaction was more intensive with isolates belonging to AG A (4a, 4c, MČA, M2, SS1, SS2 and tester isolate AG A), and less intensive with isolates from binucleate Rhizoctonia AG I, as well as R. solani isolates.As 1 reacted positively with root extract from strawberry cv.Senga sengana inoculated with isolate MLE and strawberry hybrid 3/2/86/88/R infected with both isolates MLE and 4.There was no reaction of this antiserum with strawberry roots of cv.Senga sengana inoculated with isolate 4. On the contrary, As 2 reacted with extracts from roots of cv.Senga sengana inoculated with both isolates, and the reaction was absent with strawberry hybrid 3/2/86/88/R.
The results obtained in the reactions with unabsorbed antisera show that Rhizoctonia spp.isolates are serologically related, and they differ from Agaricus sp. with whom they are taxonomically close (all belong to division Basidiomycota), from Fusarium sp. and Pythium spp. with whom they are taxonomically distant.Using dot-immunobinding assay both antisera As 1 and As 2 recognize all Rhizoctonia spp.isolates tested, and the way to differentiate AG is the intensity of the reaction.With homologus AG both antisera show the most intensive reaction, while the reaction is weaker with isolates of another AG of binucleate Rhizoctonia and isolates of R. solani (multinucleate Rhizoctonia).
Also both antisera can recognise infected strawberry roots-the reaction is positive.This means that both antisera can be used to detect Rhizoctonia spp. in strawberry roots.After cross absorption the specificity of the antisera was enhanced.After absorption of the antisera As 1 with antigen MLE, in one and two steps ( As 1 +MLE and As 1 + MLE + MLE) (Table 3, column 2 and 3) the reaction with antigens 4 and P6 (AG I) was very strong, but it was also present with members of the other AG (AG A) of binucleate Rhizoctonia and isolates of R. solani.After absorption with isolate RhKsp (R. solani) antisera As 1 reacted positively with isolates of binucleate Rhizoctonia while the reaction was absent with isolates of R. solani (Table 3, column 4).The result proves serological similarity between Rhizoctonia solani isolates, and among the isolates of binucleate Rhizoctonia spp.Also serological similarity inside the group of either binucleate or multinucleate Rhizoctonia spp. is greater than between the groups.Antiserum cross absorbed in the second step with antigen MLE (As 1+RhKsp+MLE) showed high specificity in reacting only with isolates of AG I to which it was raised against (Table 3, column 5).The results obtained show that it is possible to serologically differentiate isolates from different AGs of binucleate Rhizoctonia.Specificity of the antisera was enhanced with every step of cross-absorption and every step eliminated the positive reaction of the whole corresponding AG.
Antisera As 1 +RhKsp can be used to differentiate binucleate Rhizoctonia from R. solani, and the antisera As 1 +RhKsp+MLE can be used to differentiate AG of binucleate Rhizoctonia.
Cross absorbed antisera As 2+4 and As 2+4+4 reacted with strong intensity with homologous antigens (AG A), while the reaction was less intensive with isolates from AG I (P6) of binucleate Rhizoctonia, as well as with R. solani isolates (9, L-Rh-II and RhKSp) (Table 3, column 7 and 8).Reactions with these antisera show that isolate P6 (binucleate Rhizoctonia AG I) is serologically more closely related to isolates of AG A, than isolate 4. On the other hand antisera As 2+RhKsp reacted with stronger intensity with antigens from AG A than from AG I (P6,4), AG 1 (9), AG 2-2 (L-Rh-II)(Table 3, column 9) which showed that isolates 9 and L-Rh-II (R. solani AG 1 and AG 2-2) are serologically closer related with isolates of AG A than AG I of binucleate Rhizoctonia.Isolate RhKSp ( R. solani AG 2-2) is serologically closer to AG I of binucleate Rhizoctonia spp.Serologically, the most distant from binucleate Rhizoctonia are isolates Rh-L and Rh-R (R. solani AG 3).This part of investigations shows that antisera As 2+4 and As 2+4+4 differentiate isolates from AG A and AG I of binucleate Rhizoctonia, as well as from R. solani, based on reaction intensity, and can be used for this purpose.
Serological investigations using both polyclonal and monoclonal antisera raised against mycelial proteins, secreted proteins or lecitins have been used to immunologically differentiate isolates of different AG of R. solani.First polyclonal antisera against mycelial proteins of isolates which belong to six AG R. solani (AG 1, 2-1, 2-2, 3, 4 and 5) were developed by A d a m s and B u t l e r (1979).These antisera distinguished all but the subgroups 2-1 and 2-2 when used in agar double diffusion test.More recently, M a t h i e w and B r o k e r (1991) serologically differentiated AG 8 from AG 2-1, 3 and 4 with an antisera prepared against secreted proteins of R. solani AG 8 isolates.Results of these authors have shown that there are also differences within AG.Although achieving these results polyclonal antisera are still of little use because of their cross-reactivity with several AGs (J a b a j i -H a r e , 1996).In the attempts to solve this problem monoclonal antisera have shown more specificity.T h o r t o n et al. (1994) produced an antisera specific for R. solani AG 4, but D u s u n c e l i and F o x (1992) produced a monoclonal antisera which had a positive reaction with a range of AGs of R. solani (AG 1, 2-1,2-2, 3, 4, 5, 6 and 8).
The results of our investigations have shown that cross absorbtion of polyclonal antisera is a reliable and less difficult way of distinguishing among isolates of Rhizoctonia spp.

C o n c l u s i o n
Based on the results obtained in our investigation with polyclonal antisera we can conclude that Rhizoctonia spp.differ in their antigenic properties from other taxonomically more or less related genera.When antisera raised against mycelial proteins of binucleate Rhizoctonia AG I and AG A are used in dotimmunobinding assay, these antisera react with homologous antigens and related Rhizoctonia spp.This means that Rhizoctonia spp.are serologically related, but they can easily be differentiated after cross absorption of the antisera which shows the antigenic diversity of these isolates.Also, cross absorption of the antiserum enhances the specificity of an antiserum and when cross absorbed antisera are used in dot-immunobinding assay AG of an unknown Rhizoctonia spp.isolate can be identified.
*b Strawberry root inoculated with isolate 4.
T a b .3. -Serological reactions of Rhizoctonia spp.isolates from different hosts, Rhizoctonia spp.tester isolates, root samples, healthy and inoculated with isolates of binucleate Rhizoctonia, and isolates Agaricus sp., Pythium sp.i Fusarium sp. with unabsorbed and cross absorbed antisera As 1 and As 2.
a *