**5. Diagnosis of pathogen**

Earlier detection of plant pathogens is very important for plant health certification and to conduct the disease management appropriately [58]. The detection and enumeration of disease causing pathogen have always been challenging issues over the years. The environment form which they are originated, pose difficulties in identification, isolation and quantification of pathogen. Developing the accurate and effective detection methods and assay is very challenging for the pathogen like *Fusarium* spp. as they exists as multiple species complex, different pathogenic profiles and virulence levels to the host [59]. Over the years, several techniques and methods have been developed in order to detect and identify disease causing pathogen in crop plants. Some of the techniques widely used in identification of pathogen are morphological based identification; Immunological assay and PCR base methods. The morphology based identification of any plant pathogen is the first and one of most difficult steps in the direction of proper identification of disease causing pathogen in plants. The identification of fungal pathogen isolates based on morphological characteristics of culture is still in use for many laboratories. Since many *Fusarium* spp. offer species complex instead of single species and look similar in many aspects, it is always challenging for proper identification of the pathogen and hence mere morphology based identification may not work good for complete identification and may lead to wrong identification for sure. It is very difficult, time consuming method and requires experts [60]. Although morphological identification is not sufficient for proper identification of the pathogen, the observable morphological characters may give us great amount of information from the culture they grow [61]. Immunoassays technique for early detection and precise identification of pathogen has been significantly increased following the development of enzyme-linked immunosorbent assay (ELISA) and monoclonal antibodies with greater sensitivity and specificity compared to morphological based methods [62]. Serology-based technique can be used to detect fungi present even in low quantities on plant tissues at an earlier stage of disease development. The earliest serological techniques used, were polyclonal antisera from immunized animals by centrifugation of clotted blood [63]. This method is further refined to a serum fraction in classical enzyme-linked immunosorbent assay (ELISA) with IgG as dominant which is obtained by ammonium sulphate precipitation, then passage over an ion exchange cellulose column is followed [64]. Now monoclonal antibodies in plant pathology are more routinely used but polyclonal antisera are also in use in this kind of techniques. Detection or quantification of binding of the diagnostic antibody with the target antigen is the principal aim of all the serological techniques used in plant pathology and double immunodiffusion techniques, indirect immunofluorescence assays and ELISA are most common used techniques. Techniques using antibodies are now suitable for both laboratory and field conditions and can identify pathogenic strains within species in very short span of time. In recent years, the PCR based identification techniques is most preferred and widely used technique among others due to its greater speed, specificity, sensitivity and reliable reproducibility. Polymerase chain reaction (PCR) is one of the greatest achievements of molecular biology. PCR synthesizes DNA and nucleic acid fragments can be specifically replicated in a semiconservative way. Currently, with the help of PCR-based technique, the taxonomic status of any fungal isolates can easily be determined. This technique has the ability to differentiate fungi species which are very closely related and morphologically similar in nature e.g. *F. proliferatum*, *F. temperatum*, and *F. verticillioides* belonged to *Fusarium fujikuroi* species complex (FFSC) [61]. PCR-based molecular methods along with sequencing of ribosomal DNA is now being successfully used to detect and identify the richness of the species from

*Fusarium Disease of Maize and Its Management through Sustainable Approach DOI: http://dx.doi.org/10.5772/intechopen.100575*

different environments [65]. The ribosomal internal transcribed spacer (ITS) region of nuclear rDNA from gDNA of the *Fusarium* isolates is amplified through PCR by using ITS1 and ITS4 primers followed by analysis of ITS sequences based on a ClustalW Multiple alignment using suitable BioEdit software [66, 67]. The search for homologous sequences is then conducted using Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI) for exact identification of fungal pathogen [68].
