Sugarcane (a complex hybrid of Saccharum spp.) is the oldest energy source (sucrose) for human beings and more recently a source of fuel for motor vehicles. It is a high value cash crop and plays vital role in the enhancement of socio-economic conditions of farming community as well as in industry and trade. It’s by-products, molasses and bagasse, are the cheapest source of feed stock for animals and industries [1].

Pakistan ranks fifth in area of sugarcane production in the world with 1.00 million hectares of area. Average cane yield in Pakistan is 50 tons/ha and sugar recovery is 8.0-8.6%, which are lower than the world average of 63 tons/ha and 10.6%, respectively [2]. Per capita demand of sugar in Pakistan is the 25 kg per annum [3]. Low sugarcane yields are mainly attributed to different biotic and abiotic factors: insect pests, diseases caused by different pathogens (fungal, bacterial, viral and nematodes), poor nutrition and management, salinity, frost, unfavorable environmental conditions and low yielding varieties.

Pathological problems alone are responsible for colossal losses in production, 15-20% under ordinary and more than 50% under severe conditions. Losses of 15-20% due to diseases are common, sometimes complete crop failure occurs [4]. Red stripe disease has emerged as a serious bacterial disease in sugarcane, infecting commercial varieties and promising clones showing stalk rot symptoms [5]. Red stripe is particularly a problem when the pathogen invades the stalk where it can cause high yield losses. When the pathogen is only present in leaf tissue the disease is minor. The pathogen associated with red stripe of sugarcane also infects oats [6], corn [7] and rice [8].

Red stripe was first observed on sugarcane in Hawaii [9]. The bacterial pathogen was identified as Pseudomonas rubrilineans; now named Acidovorax avenae ssp. avenae (Aaa). Bacterium is gram negative, rod 0.4-0.6 μm×0.8-1.6 μm. The pathogen is now established and reported from 50 sugarcane growing countries of the world [10] and is present in several locations in Pakistan [11].

The disease is initially characterized by the appearance of green stripes near the midrib that later on turn reddish to dark red stripes. The stripes initially develop at the base of the leaves in June and July in Pakistan. Symptoms spread quickly to more leaves. Disease development is favored by high doses of nitrogen application, high temperature and humidity [12].

The pathogen was initially identified on the basis of symptomatology, growth behavior of the bacterium, grown out test and morphological characteristic under light microscope. These methods of identification were uneconomical, laborious, time consuming and non specific. The pathogen was specifically identified by serological methods mainly ELISA. Electron microscopy and PCR were also employed, which have been hardly attempted in Pakistan.

Because of the spread and potential yield losses of red stripe in Pakistan, an accurate and sensitive diagnostic method(s) are required to identify bacterial free planting material at the early stage. Thus, a stalk diagnostic assay procedure is required guarantee disease-free seed cane. The objective of the study to specifically confirm the identity of the pathogen through antigen-antibody reaction through tissue blot immunoassay (TBIA) and direct antibody coating enzyme linked immunoassay (DAC-ELISA) for the identification of resistance source to be used in Integrated Disease Management (IDM).

Antisera

Acidovorax avenae subsp. avenae was isolated from highly susceptible sugarcane variety CSSG-2402 on Yeast extract Dextrose Chalk-agar (YDC) medium. Bacterium was grown in 150 mL YDC broth in 350 ml flasks. Bacterial culture was centrifuged at 10,000 rpm for 30 minutes at 4°C. Supernatant was discarded and pellets were washed twice with 100 ml membrane filtered sterilized saline (0.85%).

Concentrated suspension was prepared using spectrophotometer to get 10⁹ cell/ml at 600 nm. The suspension was steamed for one hour to get somatic antigen in sonicator (yamato bran sonic-12 ultrasonic cleaner, US PATENT NO 3, 6.81, 6.26) and then cooled down. One ml specific antigen of Aaa was injected intravenously into albino rabbit 6-12 months old. Five such injections were given at weekly interval. Test bleed of rabbit was taken using bleeding rack and obtain approximately 1 ml blood from the ear after one week of the last injection. Blood was relocated into a sterile screw-cap test tube of 50-ml capacity and allowed to clot at room temperature for 2 hours. The clot was separated from the wall of tube using sterilized glass. The serum was kept overnight at 4s. Bacterial culture was centrifuged at 10,000 rpm for 30 minutes at 4°C. Purification was done by centrifugation at 5000 rpm for 10 min. Merthiolate was added in the antiserum and kept in refrigerator at 4s. Bacterial culture was centrifuged at 10,000 rpm for 30 minutes at 4°C.

The antiserum titer was determined by agglutinations in microtitre trays. The first bleed (approximately 10 ml) was taken at two weeks after the last injection and antiserum titer was determined [13]. Preparation of antigen and antiserum against Aaa was completed for the first time at National Institute for Biotechnology and Genetic Engineering (NIBGE) Faisalabad, Pakistan. The second antibody used in the TBIA and DACELISA tests were goat anti-rabbit IgG-alkaline phosphatase conjugate (Sigma-USA).

Plant materials

The sugarcane producing areas of Punjab Province, Pakistan including Jhang and Faisalabad regions were surveyed in 2010 and 2011. Samples (i.e., one stalk of each clone) were obtained from mature plants 9-10 month old of sugarcane varieties: HoSG-315, CPSG-437, NSG- 555, SPF-238, CP77-400, SPSG-79, CPF-237, HSF-240, SPF-213, CSSG- 668, CSSG-676, NSG-311, GT-11, CPSG-3481, NSG-59, CPSG-2923, CSSG-212, CPSG-104, HoSG-1257, CPSG-25, CSSG-239, CPSG-2713, NSG-49, CPSG-2453, US-114 and CP-NIA-82-223 showing naturally infected diseased symptoms of red stripe [11]. Sugarcane variety CSSG- 2402 was used as standard check because of its susceptibility to the red stripe of sugarcane. All samples were collected in polyethylene bags and stored in a refrigerator at 4s. Bacterial culture was centrifuged at 10,000 rpm for 30 minutes at 4°C until processed. All collected stalk samples were tested by two different serological assays TBIA and DAC-ELISA.

Detection of Acidovorax avenae subsp. avenae (Aaa) by direct antibody coating enzyme linked immunoassay (DAC-ELISA)

Vascular sap of infected samples was used as antigen. Stalk sections were centrifuged at 8500 rpm for 10 minutes to collect vascular sap. One hundred μl of vascular sap were loaded into wells of microtitter assay plate. Positive control well had 50 μl of cultured Aaa cells of unknown titer. Negative control well had uninfected xylem sap sample. ELISA plate was incubated overnight at 4°C in a refrigerator. Wash the sample wells with 150 μl PBST buffer 3 times for 3 minutes each. 100 μl of primary antibody at 1:1000 was loaded in each well of the ELISA plate [14] and incubated at 37°C for 2-3 hours. Then, the plates were washed with washing buffer 3 times with 3 minutes interval. Conjugate (Goat anti-rabbit immunogloblin IgG with alkaline phosphatase (ALP) enzyme) 100 μl was loaded in each well of the ELISA plate. The plates were incubated at 37°C for 2-3 hours. 5 mg p- nitrophenyl phosphate diluted, in 10 ml of substrate buffer. 150 μl of substrate buffer was loaded in each well of the ELISA plate. Results were observed visually for half an hour at room temperature. Later absorbance was also determined spectrophotometrically at 405 nm in an ELISA reader (ASYS Hitech GmbH. Model. Expert Plus. Type. G020150 [15]. A cut off/threshold OD₄₀₅ (Optical density) of 0.066 was calculated to indicate the presence of Aaa [16].

Detection of Acidovorax avenae subsp. avenae (Aaa) by tissue blot immunoassay (TBIA)

The tissue blot immunoassay (TBIA) procedures were performed for detecting the red stripe disease pathogen of sugarcane. 1cm core of tissue from each internode was cut in 1-cm lengths and the cut end was placed on a nitrocellulose membrane having 2-cm diameter and centrifuged at 1,700 rpm for 15 min. Membranes were blocked with blocking solution (Tris buffer saline 100 ml and dried milk powder 2 g) and incubated for one hour at room temperature. After washing for 1 min in 75 ml Tris buffered saline (TBS), each membrane was immersed in 10 ml primary antibody solution. Incubated and washed 3 times for 5 min in 75 ml TBS. Each membrane (single layer) was exposed with 10 ml anti-rabbit antibody diluted solution and Incubated for 1 hour. Washed 2 times for 15 min in 75 ml TBS. Immersed membranes in working substrate (Tris-24 g,Napthol-AS-phosphate-3 g, Dimethylformamide-5 ml and distilled water-1000 ml) with 0.075 g of fast blue and 0.375 ml of MgCl₂ (in 75 ml working substrate) in dark for 15-30 min. Membranes were washed and treated with 20% bleach solution for 20 minutes to remove brownish discoloration. After washing and drying, color of stained/processed nitrocellulose membranes were visually observed [15].

Evaluation by DAC-ELISA

A total of 27 samples of sugarcane varieties were collected from the field surveys during 2010-2011. A summary of the results of the samples tested with DAC-ELISA is shown in table 1. In DAC-ELISA, Aaa was detected from the infected or suspected to be infected samples showing positive reactions (yellow color) of the bacterium (Figure 1). Results showed that two representatives, US-114 and CSSG-2402 have the higher bacterial optical density values (1.379 and 1.390 respectively) were detected and showed highly positive reaction by DAC-ELISA. Five sugarcane varieties HoSG-315 (0.560), CPSG-437 (0.555), NSG- 49 (0.564), CPSG-2453 (0.556) and CP-NIA-82-223 (0.564) had higher level of absorbance values were positive in the ELISA plate wells [14]. From the representative stalks of 04 varieties like CP77-400, SPF-238, SPF-213 and GT-11 had moderate level of the values (0.101, 0.099, 0.098 and 0.104) respectively, showed the light yellow color (Positive reaction). All of the remaining 16 representative were showed negative reaction by the DAC-ELISA test [17,18].

Table 1: Visual and serological (DAC-ELISA) determination of Aaa.

Figure 1: ELISA plate showing positive reactions (Yellow color) against Aaa.

Detection of Acidovorax avenae subsp. avenae (Aaa) by TBIA

Tests were made by making blots of Aaa infected or suspected to be infected tissue on nitrocellulose membranes using standard procedures [15]. Of the twenty seven promising clones of sugarcane, only seven clones like HoSG-315, CPSG-437, NSG-49, CPSG-2453, CP-NIA-82-223, CSSG-2402 and US-114 were positive for Aaa using TBIA and showed dark blue stain colour where the vascular bundles were imprinted on the nitrocellulose membranes (Figure 2). Representative samples of twenty clones were negative by TBIA and no colour appearance was observed on the membranes [15,19].

Figure 2: Nitrocellulose membrane showing positive reactions (Dark blue color) against Aaa received from symptomatic and suspected to be infected stalks in TBIA Test.

Comparison between DAC-ELISA and TBIA

Both serological tests were performed on duplicate samples of the 27 sugarcane clones. According to the results of the tests, Aaa was not detected in 16 clones viz: NSG-555, SPSG- 79, CPF-237, HSF-240, CSSG-668, CSSG-676, NSG-311, CPSG-3481, NSG-59, CPSG-2923, CSSG-212, CPSG-104, HoSG-1257, CPSG-25, CSSG-239 and CPSG- 2713 using both DCA-ELISA and TBIA tests. DAC-ELISA showed false positive due to low optical density readings above background were inconsistent results in the 4 clones (SPF-238, CP77-400, SPF-213 and GT-11) based on slight yellow coloration in its test [20]. While, in sugarcane samples viz: HoSG-315, CPSG-437, NSG-49, CPSG- 2453, CP-NIA-82-223, CSSG-2402 and US-114 were showed positive detection of Acidovorax avenae subsp. Avenae, by both serological tests [15]. In DAC-ELISA test highly positive values were noted from the samples of CSSG-2402 and US-114 but in TBIA test positive reaction was observed of both clones (Table 2).

Table 2: Comparison of observation between TBIA and DAC-ELISA.

As per results, TBIA test was used to confirm results positive or negative. In DAC-ELISA test because of the vascular coloration corresponds to where Aaa is located. Since clones (SPF-238, CP77-400, SPF-213 & GT-11) did not test positive using TBIA the DAC-ELISA results are considered false [21].

Among 27 randomly collected sugarcane samples, TBIA showed 26% positive and 74% negative result. On the other hand DAC-ELISA was showed 41% positive (36% false+64% true) and 59% negative results, when compared with TBIA test. Assay time was also minimized in performing TBIA test as equivalence to DAC-ELISA. Related to the test performing time at least 25-30 hours were consumed for the completion of DAC-ELISA assay, while only 6-9 hours was required for TBIA assay with reliable results [22,23].

diagnostic tests have made a significant contribution in diseases control and eradication. Among the variety of immunological tests, TBIA proved to be extremely helpful for large scale testing at a very low cost without much compromise on sensitivity or specificity [14]. It is an essential test to be used in developing countries where hi-tech laboratories for diagnoses of the pathogens are not available and cost per sample is a limiting factor for any assay to be adopted for large scale testing [15,19].

The author is grateful to Dr. Shahid Mansoor, Head Plant Biotechnology Division, NIBGE Faisalabad, Pakistan for providing the facilities for the preparation of antisera. I owe my respectful thanks to Dr. Jack C. Comstock, Research Leader, ARS USDA, Canal Point Florida, USA for providing the nitrocellulose membranes and valuable guidance for TBIA test.