Evaluation of a polyclonal antibody immunoassay for detection and quantification of Macrophomina phaseolina

Abstract

Detection and quantification of Macrophomina phaseolina, causal agent of charcoal/dry root rot disease in many crop plants, was carried out using the ELISA serological technique. Polyclonal antisera raised to water soluble extracts of mycelium, the residual water insoluble mycelial materials or ribosomal proteins were evaluated for specificity and cross-reactivity with 16 common soil fungi by ODD and DAS-ELISA. Soluble and cell wall antisera exhibited strong cross-reactivity with most of the fungal isolates. Ribosomal antibodies were less reactive to common soil fungi except Fusarium oxysporum f.sp. ciceri. Mycelial antigens of M. phaseolina on chickpea roots were detectable with DAS-ELISA at a minimum concentration of 10 ng g ¹1 at 1 : 100 root : buffer dilution. Quantitative estimation of M. phaseolina on roots was evaluated by ELISA under different temperatures and moisture conditions, and in soil amended with a potential antagonist (Trichoderma harzianum 25-92). A significant reduction in ELISA values was observed in T. harzianumamended treatments. This method may be useful for detection and rapid screening of M. phaseolina under different environmental conditions.

Evaluation of arbuscular mycorrhiza and other biocontrol agents in managingFusarium oxysporumf. sp.Cubenseinfection in banana cv. Neypoovan

“…Under field condition also BCA treatment brought about a reduction in Fusarium population. Srivastava and Arora (1997) have studied the detection and quantification of Macrophomina phaseolina, causal agent of charcoal/dry root rot disease in many crop plants, using the ELISA. Skottrup, Nicolaisen, and Justesen (2007) have used ELISA for early disease detection of P. infestans causing, late blight disease in potato.…”

Immunodiagnostic potential of a 27 kDa protein of Fusarium xylarioides, the cause of coffee wilt disease in Robusta coffee in Uganda

“…The present study has produced a specific anti-27 kDa protein antiserum and provides a significant breakthrough in the development of immune-assay procedure for sensitive, specific and rapid diagno-sis of CWD. Species specificity of the anti-27 kDa protein antiserum, therefore, disagrees with earlier work done by some workers which indicated that polyclonal antibodies cannot differentiate between species and more so between Fusarium species (Srivastava and Arora, 1997); and that polyclonal antisera raised to crude mycelial extract of one species, have often been found to be generally genus-specific (Jamaux and Spire, 1994). Cloning and expressing the 27-kDa protein can undoubtedly improve the procedure of antigen preparation, thereby making it more costeffective.…”

“…Accurate and specific antigen to the pathogen has been identified. The potential of such immunodiagnostic tools to detect colonization of roots by several soil-borne fungal pathogens has been demonstrated (Priestley and Dever, 1993;Srivastava and Arora, 1997). This is the first report describing the use of a serological technique for the detection of F. xylarioides.…”

“…Six Fusarium species common to coffee as saprophytes or true pathogen (Hakiza and Webesa, 1997;Serani, 2000) namely F. xylarioides, F. solani, F. oxysporum, F. decemcellulare, Fusarium Palldoroseum, and F. Lateritium identified according to Nirenberg (1976) and Booth, (1971) were used to identify the target antigen in this study. Soluble proteins, crude cell component, fungal homogenate, culture fluids, and ribosomal proteins from fungal pathogens have been used to raise antibodies (Jamaux and Spire, 1994; Priestley and Dever, 1993; Srivastava and Arora, 1997). In this study, part of the soluble mycelial extract was used as the antigen for antibody production.…”

“…No attempt was made to further purify the protein by cross -absorption technique since some workers have reported that the procedure rarely improves the specificity of the antiserum raised to such protein (Srivastava and Arora, 1997) or even it specificity is improved, sensitivity is reduced (Jamaux and Spire, 1994). The specificity of the antiserum to the 27 kDa protein band was evaluated by dot blot and western blot analyses.…”