A Sensitive ELISA for Pythium ultimum Using Polyclonal and Species-Specific Monoclonal Antibodies

Abstract

A double-antibody sandwich indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantification of Pythium ultimum. A polyclonal antibody produced to cell walls of P. ultimum was used as the capture antibody, while a P. ultimum-specific mono-clonal antibody (MAb E5) was used for recognition of the fungus. In the ELISA, culture extracts of 7 isolates of P. ultimum exhibited strong positive reactions, whereas none of the 37 isolates of other Pythium spp. and fungal genera had positive reactions. P. ultimum was detected by ELISA in roots of bean, cabbage, and sugar beet seedlings grown in pathogen-infested soil. ELISA optical density readings for infected bean and sugar beet root samples were highly correlated (r > 0.9) with infection levels determined by culturing the samples on water agar. The correlation between the two methods of testing cabbage roots was low, but all cabbage roots in which P. ultimum was detected by culturing were strongly positive in the ELISA. Samples of roots infected with P. irregulare and those with no Pythium infection did not react in the ELISA. The ELISA was highly sensitive; the fungus was detected in culture extracts diluted 1:5,000,000 and in roots with less than 1 infection per 100 cm root.

A clp Gene Homologue Belonging to the Crp Gene Family Globally Regulates Lytic Enzyme Production, Antimicrobial Activity, and Biological Control Activity Expressed by Lysobacter enzymogenes Strain C3

“…The in vitro antimicrobial activities of L. enzymogenes strains were determined by coculturing bacterial strains with the fungus B. sorokiniana isolate Bs1 (42) and the oomycete P. ultimum isolate P201 (39). The bacterial strains were spot inoculated on the periphery of 10% TSA plates, and we included a mockinoculated plate as a control.…”

Plant Pathogen Forensics: Capabilities, Needs, and Recommendations

“…ELISA and indirect fluorescent antibody staining are serological assays commonly used for identification of plant pathogens, particularly viruses and bacteria. The sensitivity and specificity of serological assays vary with the titer and specificity of the antibody and whether the antibody is monoclonal or polyclonal (102,180,197). Recent adaptations by diagnostic industries for dipstick convenience and portability have enhanced the usefulness of these immunology-based technologies in the field.…”

Characterization of species associated with Pythium soft rot of ginger and evaluation of Pythium Oligandrum as a biocontrol

“…that have been designed (Schroeder et al, 2013). Besides qPCR, Yuen et al (1998) and Kyuchukova et al (2006) have successfully employed indirect enzyme-linked immunosorbent assay (ELISA) in order to enumerate the number of propagules of P. ultimum and P. aphanidermatum in a sample. This technique has proven to be sensitive enough to detect P. ultimum at 1:5,000,000 culture dilution rate (Yuen et al, 1998) or at a level of 0.05 g mycelium of P. aphanidermatum per litre of nutrient solution (Kyuchukova et al, 2006).…”

“…Besides qPCR, Yuen et al (1998) and Kyuchukova et al (2006) have successfully employed indirect enzyme-linked immunosorbent assay (ELISA) in order to enumerate the number of propagules of P. ultimum and P. aphanidermatum in a sample. This technique has proven to be sensitive enough to detect P. ultimum at 1:5,000,000 culture dilution rate (Yuen et al, 1998) or at a level of 0.05 g mycelium of P. aphanidermatum per litre of nutrient solution (Kyuchukova et al, 2006). Recently, Fukuta et al (2013Fukuta et al ( , 2014 successfully developed a highly sensitive, quick and easy detection method, called LAMP (loop-mediated isothermal amplification) to monitor P. aphanidermatum and P. myriotylum from hydroponic solutions (this will be discussed in a next section).…”

“…Kageyama et al (2002) were successful in using monoclonal antibodies (MAbs) in order to differentiate between P. sulcatum and P. myriotylum or P. zingiberis from infected tissues or soil. MAb E5 was developed to recognise P. ultimum in diseased roots of bean, cabbage and sugar beet by double-antibody sandwich ELISA (Yuen et al, 1998).…”