Development of a Sensitive Serological Method for Specific Detection of Latent Infection of Macrophomina phaseolina in Cowpea

Abstract

A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific detection and quantification of Macrophomina phaseolina in plant tissue. Both polyclonal antisera produced against immunogens from mycelium and culture filtrate of M. phaseolina detected the fungus in mycelial and plant extracts, although the antibodies raised against mycelium were more sensitive. No crossreaction occurred with Rhizopus stolonifer, Pythium ultimum, Mucor hiemalis, Fusarium oxysporum, Septoria nodorum, Rhizoctonia solani, Sclerotinia sclerotiorum, Phytophthora infestans and Aspergillus niger. In enzyme assays, activity of the endo-acting hydrolytic enzymes 1,3-b-glucanase and, less, cellulase, but not xylanase was detected in infected plants. DAS-ELISA was more sensitive than the 1,3-b-glucanase assay. In polyacrylamide gel electrophoresis (PAGE) up to 18 protein bands were observed, with four bands occurring in the 12 tested isolates deriving from various geographical origin in Niger and Nigeria. The enzyme assays and protein patterns were considered not suitable for specific M. phaseolina detection. Macrophomina phaseolina was essentially located in the roots and hypocotyls, and less in epicotyls and leaves of infected plants. The antibodies were also useful to detect latent infection and the infection of cowpea seeds.www.blackwell-synergy.com

Achievements and Challenges in Legume Breeding for Pest and Disease Resistance

“…Macrophomina phaseolina, responsible for charcoal rot disease, is another important root rot inducing pathogen. It has been reported in many legume growing areas from Northern European countries to New Zealand as an important pathogen of common bean, soybean, mungbean (Vigna radiata), peanut, cowpea, faba bean and chickpea (Abawi and Pastor-Corrales, 1989;Afouda et al, 2009;Devi et al, 2011;Gupta et al, 2012). Brown root rot of alfalfa and other perennial legumes, caused by Phoma sclerotioides (synonym Plenodomus meliloti), develops as plants emerge from winter dormancy.…”

“…By contrast, fusarium wilt and root rots caused by Fusarium spp. and Macrophomina phaseolina are generally favored by warm and dry conditions (Chaudhary et al, 2006;Afouda et al, 2009).…”

Diagnostic methods for detecting fungal pathogens on vegetable seeds

“…However, fungal reproductive structures are not always produced on these substrates. To stimulate fungal sporulation, a medium that is less rich in nutrients is more advisable, such as water agar or cornmeal agar (Roy et al, 2000;Afouda et al, 2009).…”

“…These assays are widely applied to detect seedborne viruses, but less so for fungal plant pathogens, due to the lack of species-specific antibodies. Furthermore, serologybased assays can also detect non-viable pathogens, which can result in erroneous interpretations (Walcott, 2003;Afouda et al, 2009).…”

Biology, Epidemiology and Management of the Pathogenic Fungus Macrophomina phaseolina (Tassi) Goid with Special Reference to Charcoal Rot of Soybean (Glycine max (L.) Merrill)

“…Average numbers of microsclerotia in naturally infested soils vary considerably from 0 to 1000/g soil. Recently, a double‐antibody sandwich enzyme‐linked immunosorbent assay was employed for specific detection and quantification of M. phaseolina in plant tissues (Fouda et al. 2009).…”