Indian Journal of Biotechnology Vol I, October 2002, pp 363-366

Dac-Elisa Technique for Early Detection of Red Rot Pathogen in Sugarcane var. CoC 671

Lingayya Hiremath and G R Naik*

Department of Biotechnology. Gulbarga University, Gulbarga 585106, India

Received 19 September 2001 : accepted 10 JUlle 2002

The sugarcane variety CoC 671 is an early maturing, high sugar content variety in India. Recently, it has become very susceptible to red rot caused by Colletolrichumfalcalllm Went. To detect the pathogen at an early stage, a new serological technique, enzyme-linked immunosorbent assay was developed based on antiserum developed against the protein of host pathogen. Direct antigen coating enzyme-linked immunosorbent assay (Dac-Elisa) for the early detection of red rot infection has been standardized. Further, this technique was found reliable to screened planting material of sugarcane variety CoC 671 for red rot infection at an earlier stage. Besides, ill vitro red rot treated as well as healthy calli were also screened for red rot infection.

Keywords: Collelolrichum falcal1l11l, Dac-Elisa, red rot pathogen, sugarcane

Introduction The enzyme-linked immunosorbent assay (ELISA)

has proved valuable tool in detection of the plant viruses (Clark & Adams, 1977; Clark & Bar-Joseph, 1984). The use of ELISA has been also extended to detect fungal plant pathogens (Casper & Mendgen, 1979; Johnson et al, 1982). The methods to quantify resistance of plants to fungal pathogens differ widely. Very often lesion size or sporulations are used to quantify the external manifestation of disease several days after incubation (Ferris, 1995).

Resistance to red rot disease of sugarcane caused by Colletotrichum falcatum Went. is assessed based on internodal symptoms such as lesion width, nodal transgression, presence of white spots and drying of tops (Srinivasan & Bhat, 1961). Field screening takes more time to assess the resistance by plug and nodal methods evaluation (Viswanathan & Mohanraj, 1998). Resistances assessed by the above methods are grossly influenced by environmental factors such as temperature and relative humidity. Since these methods are time-consuming and inconsistent, a controlled condition evaluation was devised to assess the host reaction under optimum environmental conditions (Mohanraj et al, 1997). Another ideal measure to evaluate resistance during early phase of host and pathogen interaction is the ability of the host

* Author for correspondence: Tel : 445446; Fax: 08472-45927 E-mail : grnaik@hotmail.com

to restrict fungal growth. By measuring the amount of fungal colorization in the infected host the resistance can be assessed. A direct microscopic method was standardized to quantify red rot resistance in the inoculated sugarcane tissues (Mohanraj et al, 1994). Estimation of fungal biomass by serological methods such ELISA has been attempted (Viswanathan et aI, 2000) in other host patho-systems (Aguelon & Dunez, 1984; Mullen & Hagan, 1989). During the present investigation, the ELISA technique was attempted to detect the pathogen in the host using polyclonal antiserum developed against the fungi during earlier stages of disease development in sugarcane, and in vitro red rot treated calli as well as healthy calli were also screened for red rot infection.

Materials and Methods Diseased stalks of var. coe 671 were collected

from Aland sugar factory area in Karnataka from 7-8 months old sugarcane field. They were cut into small pieces i.e. 0 .2-0.3 cm and washed thoroughly with the sterile distilled water. The stem pieces were disinfected with 70% alcohol for 30 sec followed by washing with sterile distilled water to remove the traces of alcohol. The diseased materials were again transferred to 0.1 % HgCIz for 3 min and washed thrice with sterile distilled water. After surface sterilization, the diseased materials were gently placed on potato dextrose agar (PDA) in petri plates. The petri plates were kept at room temperature (25 ± 3°C)

364 INDIAN J BIOTECHNOL, OCTOBER 2002

until the fungal growth almost covered the surface of the medium.

The identity of this pathogen was confirmed using cultural, morphological and pathogenicity tests. The morphological characters of pure pathogen were observed daily after incubation. The fungus showed the cottony growth of mycelium, which turned grey wi th the age. The conidial masses were dark grey at first and then changed into pink. Bulk of C. falcatunl mycelia were harvested from liquid Czepek's Oox liquid media and sugarcane host extract media (Naik & Vedamurthy, ] 997), containing sugarcane juice, 250 g; NaN03. 2 g, KH4P04, I g; MgS04, 7H20, 0.5 g; FeS04, 7H20, 0.01 g; and distilled water 1000 ml (pH 7.0) .

Harvested mycelia were homogenized in ice cold Phosphate Buffer Saline (PBS) 0 . 1 M (pH 7.2) and total soluble proteins were extracted by centrifugation at 10,000 g for ]() min at 4 DC; and the total soluble proteins were precipitated with ammonium sulphate (60%). The partially purified protein samples were dialyzed and lyophilized. The protein concentration was assessed by using brilliant blue dye R-250 (Sigma) (Bradford, 1976). Later the protein sample was diluted with carbonate buffer (PH 9.6) to a concentration of 25-35 mg/ml. The protein adjusted ex tracts were used as antigen in the first set of experiment.

calli were maintained ill vitro on MS medium (Murashige & Skoog, 1962) containing3 mg/I 2,4-0.

The preliminary experiments wherein different dilutions of antigen, antiserum and secondary antibody + enzyme conjugate were tested. Antigen dilution I: 100, antiserum dilution I :500 and secondary antibody + enzyme conjugate dilution at I: 1000. These dilutions were used for standardization of Oac-Elisa technique. In the present investigation , the antigens used are the partially purified protein extracts of the OM (A g) and the antiserum raised represent the polyclonal antibodies. The antigen dilutions in carbonate buffer of 200 ).11 were incubated overnight at 4DC in micro-titer plates. The wells were then emptied, washed thrice with PBS (PH 7.2) containing 0.05% (w/v) Tween-20. The emptied well s were loaded with 200 ).11 antiserum raised against toxic pathogen diluted to I: 1000 or I :2000 in PBS (PH 7.2) containing 0.05 (v/v) Tween-20, 2% (w/v) polyvinyl pyrolidone (PVP) and 0.2% (w/v) ovalbumin (PBSTPO) and incubated at 37DC for 3 hrs. After rewashing the plates were incubated for 3

hrs at 37DC with 200 ).11 per well of anti-rabbit goat IgG + alkaline phosphatase (8 Genei, India) diluted to I: 1000 in PBSTPO. After final washing, the wells were loaded with 200 ).11 of solution containing I mg/ml para-nitrophenyl phosphate (Hi-media, India, phosphatase reagent) substrate in 10% diethanolamine buffer (PH 9.8). Colour development was monitored periodically at 405 nm by using ELlSA reader (Biotech Inc., USA). For terminating the reaction, I M sulphuric acid in distilled water was used.

Results and Discussion Immunodiffusion Technique

Ouchterlonie's double diffusion technique was carried out. Precipitin line formed on the plate showed antigen and antibody reaction , which shows polyclonal antibody production (Fig. I) .

The partially purified dikaryotic · mycelia] antigen and Freund's complete adjuvant (B Gene', India) mixture was used for raising the antiserum in New Zealand Albino Rabbit. The specificity of antiserum was confirmed by the Ouchterlony double diffusion method. The immunization schedu le was prepared as shown in the Table I. The polyclonal antiserum was raised agai nst the pathogen as followed by Alexander and 10thi (1995). One gram of each, test sample was homogenized in carbonate buffer (PH 9.6) . Extractions of antigen from various test samples of sugarcane were prepared from healthy plants as well as red rot infected plants. Healthy and red rot infected

Detection of C. falcatum by Dac-Elisa The sensitivity of Oac-Elisa tests showed that, the

Tnble I- Schedule of immunizntion

Date Ro ute o f inoculntion Protein Adjuvant (ILl) Total vol ume concentration injected (IL I)

20-04-01 r ntmdermal 1.5 1 mg/0.5ml 250 500

2S-04-0 I Intravenal 1.5 1 mg/0.5ml 250 500

06-05-01 Intradermal 1.51 mg/0.5ml 250 500

14-05-01 r ntradcrmnl 3. r 1 mg/O.Sml 400 800

LINGA YY A AND NAIK : DAC- ELISA DETECTION FOR SUGARCANE RED ROT 365

Fig .-I Ouchterlonic' s Double Diffusion. The I well was loaded with healthy cane ex tract. The II well was loaded with control bl ood serum (CI3S) of rabbit. Ill, IV and V well s were loaded with antigen of host pathogen. And center . A' well was loaded with antibody rai sed against host pathogen .

anti serum dilutions of 1: 1000 and 1 :2000 were compared against the antigen extract (from naturally infected stalks). The dilutions were made from 1: 10 to I :640; there was a decrease in the absorbance upon dilution (Fig. 2) . The same assay showed fast colour development in wells after substrate addition in extracts diluted to concentration of I: 10-1 :40. The purpose of the study was to determine whether or not EUSA based detection of red rot pathogen using polyclonal antiserum during the earliest stages of fungal invasion were feasible. The concentration of antigen (I: 100), antiserum (l :5000) and enzyme conjugate (1 :1000) used in the study were found optimum to detect C. fatcalUIIl in the infected host.

The absorbance at 405 nm for the six samples tested (I-VI) are given in the Table 2. Each value

1.4 E 1.2 c:

I() 0 ..,. - 0.8 ca 41 (.) 0.6 c: ca -e 0.4 0 tJ)

0.2 ~ 0 1)(20 1X40 1X80 1X160 1X320 1X640

Antigen dilution

Fig. 2-Detection of C. FilcatUlII infect ion in Sugal'cane Ti ssues by DAC-ELISA

represents the mean of three replicates (OD of 3 wel ls). The threshold baseline absorbance was calculated for healthy plant as well as for healthy callus in order to compare with the infected plant and infected callus. Formula for calculation of threshold baseline absorbance (TBA) value as , TBA = 3 X standard deviation of healthy sample + mean value for the healthy sample.

The threshold baseline absorbance for the healthy plant was 0.128 and for that of healthy callus was 0.112. The absorbance value of the buffer control was 0.006. The absorbance value for the antigen representing red rot infected plant (Ag II) was 0.912. Thus, it was about six and half times more than the threshold absorbance value for the healthy plant. Similarly, the absorbance value of the antiserum treated with ill vitro red rot treated callus (Ag IV) was also about 4-5 times more than the threshold absorbance value for the healthy callus . The absorbance value for the (Ag IV) ill vitro red rot treated callus was 0.475, while for ill vitro healthy callus (Ag V) was 0.112.

From these observations it can be inferred ~hat the EUSA method can be used as an important serological tool in the detection of the red rot fungu s

Table 2- Early detection of sugarcane red rot infection by DAC-ELISA

No. Sample Anti gen type Absorbance at 405 nm Threshold absorbance value"''''

Reel rot OM antigen (DM=Dikaryotic Mycelia) 1.641 (± 0.139)

2 Rcd rot infected cane. 11 0.912 (±0.021)

3 Health y cane. III 0. 128 (± 0.005) 0.143

4 III vitm red rot treated callu s IV 0.475 (± 00(8)

5 Heallhy ('{{IIIIS V 0.112 (± 0.(02) 0.118

6 IJIII!i>,. ('ulllrul VI 0.006 (± 0.0(3)

"'''' Mean of three replicates;values in parenthesis indicate the standard deviation.

366 INDIAN J 13IOTECHNOL, OCTOBER 2002

either in the planting material or in the callus. The absorbance for red rot dikaryotic mycelial antigen was quite high (1.641), which is because of the presence of the antigen only from the fungus. For the interpretation of this serological test and for the calculation of its value the procedure by Falk and Purcifull (1983) was adopted. Further standardization of serological test is necessary especially with reference to the standardization and dilutions and adopted the methodology for various field samples (both healthy & infected ones). The investigation proves the utility of ELlSA serological test in the early detection of the diseases like red rot pathogen . The experiments are also designed for the detection of other sugarcane di seases like smut (Naik et al. 2002), eye spot, sugarcane mosaic virus and grassy shoot di sease using ELlSA technique.

In this context, ELlSA technique is found very useful in indexing and certifying that canes are free from pathogen .

Of late, many sugar factories are recommending the use of micropropagated canes and somaclones derived from the callus tissues for plantation purposes.

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