DIAGNOSTIC TOOLS FOR DISEASE DIAGNOSIS IN SUGARCANE M. L. Chhabra, B. Parameswari and M. R.

Meena Sugarcane Breeding Institute, Regional Centre, Karnal-132001, India In India, sugarcane is one of the most important commercial crops occupying an area of 4.5 M ha. Propagation of sugarcane through vegetative cuttings favours spread of diseases through planting materials. About 55 diseases of sugarcane caused by fungi, bacteria, viruses, phytoplasmas and nematodes have been reported from India (Rao et al., 2002). Among them red rot, smut, wilt, sett rot, leaf scald, ratoon stunting, mosaic, yellow leaf and grassy shoot are the major diseases seriously affecting sugarcane production in India (Viswanathan et al., 2011). Many of the sugarcane diseases do not cause diagnosable symptoms on the seed cane and different factors influence disease expression in the field hence we need to follow certain diagnostic techniques based on serology or molecular biology to detect them in the seed cane before planting in the field. Infected planting materials are responsible for the primary spread of the disease in the field. Hence, going for the disease-free setts would reduce the risk of disease introduction to disease free areas. Effort to detect and diagnose sugarcane pathogens using more advanced laboratory techniques have been developed in India during the past two decades. Development of molecular diagnostic techniques has also facilitated precise identification and characterization of different fungal, bacterial and viral pathogens of sugarcane (Viswanathan et al., 2009; Ramesh sundar et al., 2011; Singh et al., 2011). Also this technique has the potential to detect more than one pathogen in one reaction and diagnosis also amenable to automation. Increased sensitivity has generally made the assays for pathogens more reliable and frequently the increased sensitivity contributes in making the assays more rapid and economical for routine detection and identification. Diagnostic tools The first and the foremost important step in management of a plant disease is the correct identification of causal organism that causes it. Pathogen or disease diagnosis is therefore fundamental to all aspects of Plant Pathology. Fastidious microorganisms, which do not grow on artificial media, are very difficult to identify and detect by conventional methods. Again, the conventional methods of diagnosis are not always conclusive. In recent years, immuno assays

and nucleic-acid based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of sugarcane pathogens. In addition to detecting sugarcane pathogens in seed canes, the recent approaches in the disease diagnosis using serological and molecular approaches have applications to develop disease-free seedlings, disease surveillance and integrated disease management in sugarcane. Serological techniques ELISA (Enzyme-Linked immunosorbent assay), DIBA (Dot-immunobinding Assay), FIA (Fluorescent Immunoassay), ISEM (Immunosorbent electron microscopy) and TIBA (Tissue blot Immunoassay) are some of the commonly used serological techniques for the detection of plant pathogens. Among them, ELISA and tissue-blot technique have become very popular and largely used for routine detection of bacterial, phytoplasmal and viral pathogens in sugarcane (Viswanathan and Rao, 2011). ELISA: The amount of pathogen present is proportional to the amount of enzyme-labeled specific antibody forms the basis of the test. The process involves conjugating antibody to an enzyme that can be used to generate a colour change when specific substrate is added. ELISA is usually performed in a microtitre plate where the degree of colour change usually measured in a computer controlled plate reader, can be used to determine the amount of pathogen present. There are many variations in ELISA: the most common form is the double-antibody sandwich (DAS) method. In this case the antigen gets sandwiched between two immunoglobulins and thus is referred to as the double antibody-sandwich form of ELISA. Other widely used variations are direct antigen coating (DAC)-ELISA, wherein the immunoprobe (specific/ primary antibody) is not enzyme-labelled but is itself identified by another enzyme labeled probe (anti immuneglobulin moleculaes, secondary antibody). DIBA: Dot-immunobinding assay (DIBA), in principle, similar to ELISA; the polystyrene plates are replaced by nitrocellulose - or nylon-based membranes on which the antigen is mobilized. The unconjugated virus-specific antibody is then allowed to react with the immobilized antigen. The trapped antibody is then probed with alkaline-phosphatase, horse radish peroxidase-labeled protein A, anti Fc or anti-IgG. Appropriate substrate is provided to allow visual detection of a colored product.

FIA: Fluorescent Immunoassay test involving the use of antibodies labeled with fluorescent dyes, ferritin and radioactive iodine have been employed to study the intracellular location and distribution of plant pathogens in infected plants and in insect vectors. The labeled antibodies present in the tissues are detected by fluorescent microscopy, electron microscopy and radio autography. ISEM: Immunosorbent electron micrsoscopy is a useful tool to visualize the immunological reactions on electron microscope grids and to reveal the localization and distribution of various pathogens in different tisssues. This test involves the attachement of the antigen to the electron microscopic grids coated with antibody raised against the pathogen. TBIA: Tissue blot immunoassay uses specific antibody as a detection tool. The infected plant parts or tissues are crushed on the surface of nitrocellulose or other suitable membranes and assayed for the presence of antigen by allowing it to react with antibodies by the protocol followed for western detection. These simple antibody based assays makes the detection and quantification of target pathogens relatively easier. Molecular techniques Molecualr methods using PCR and/or probes have increasingly been used in recent years to develop diagnostic assays for plant pathogens. These methods, particularly those based on PCR are precise, rapid and sensitive. Nucleic acid based assays mostly detect the pathogens by detecting their DNA or RNA. Probes are single stranded fragments of nucleic acid molecules labelled with reporter\ molecule such as radioactive isotopes, enzymes or fluorescent dyes. They bind to complementary DNA or RNA sequences of disease-causing pathogen, which in turn can be detected depending on the type of reporter molecule used to label the probes. Nucleic acid (DNA/RNA) extracted from the infected plants can be directly applied to nylon membranes (dot blot) or can be transferred from gels (Southern blot) and detected. Hybridization will occur when like complementary sequences are present. Molecular techniques like polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR and nested PCR assays are most sensitive than the serological techniques especially to detect sugarcane viruses and phytoplasmas which occur at low concentrations (Viswanathan and Rao, 2011)..

PCR: The Poymerase chain reaction (PCR) is an invitro method of nucleic acid synthesis by which a particular segment of DNA can be specifically replicated. The procedure essentially involves heat denaturation of the target dsRNA and hybridization of a pair of synthetic oligonucleotide primers to both strands of the target DNA, one to the 5 end of the sense and one to the 5 end of the antisense strand by an annealing step. Then by using thermostable Taq DNA polymerase enzyme, new DNA is synthesized on templates to produce twice the number of target DNAs. Newly synthesized DNA strands are used as targets for subsequent DNA synthesis, and the same steps repeated up to 50 times. RT-PCR: Reverse transcription polymerase chain reaction is used to reverse-transcribe and amplify RNA to cDNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions may be combined in a tube, with the initial heating step of PCR being used to inactivate the transcriptase. Nested PCR: Nested polymerase chain reaction is a modification of polymerase chain reaction intended to reduce the contamination in products due to the amplification of unexpected primer binding sites. It is used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first PCR, one pair of primers is used to generate DNA products, which may contain products amplified from non-target areas. The products from the first PCR are then used as template in a second PCR, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity. Nested PCR is often more successful in specifically amplifying long DNA products than conventional PCR, but it requires more detailed knowledge of the sequence of the target. m-PCR: Multiplex PCR uses several pairs of primers annealing to different target sequences. This permits the simultaneous analysis of multiple targets in a single sample. This PCR technique allows simultaneous amplification of several viruses using a mixture of specific primers. In this case choice of primers for amplification depends upon the desired type of detection i.e. when closely related viruses are tested a single polyvalent primer pair is often enough for simultaneous detection, but when a mixture of unrelated viruses are being detected a mixture virus specific primer pairs are used. However for the success of mPCR concentration of various reagents and primers need to be determined by manipulation. The mPCR thus offers the possibility of cutting down the cost of indexing by reducing the number of assays to perform.