A monoclonal antibody immunoassay for the detection of Nectria galligena in apple fruit and woody tissues ¹

Abstract

Relatively rapid, highly specific monoclonal antibody‐based enzyme‐linked immunosorbent and immunofluorescent assays have been developed to detect and visualize Nectria galligena in woody tissue. Hybridoma cell lines were raised by co‐immunization of mice with extracts from freeze‐dried cultures of N. galligena and murine polyclonal antiserum to a related pathogen, N. haematococca. This method appeared to reduce the number of hybridoma cell lines that secreted cross‐reactive antibodies. From two fusions, ten cell lines that recognized N. galligena and not N. cinnabarina were raised. Hybridoma supernatants were screened by ELISA using antigen‐coated wells. Monoclonal antibodies from one cell line, NG‐IE4, proved useful in the detection of the pathogen in woody tissue by ELISA and antibodies from another cell line, NG‐DF11, have been used to immunolocalize the fungus in woody tissue by immunofluorescence. Further applications are ongoing.

Limited asymptomatic colonization of apple tree shoots by Neonectria ditissima following infection of leaf scars and pruning wounds

“…We confirmed, for the first time with a DNA-based approach, that N. ditissima can asymptomatically colonize plant tissues adjacent to cankers. Dewey et al (1995) and Weber and Hahn (2013), using immunolocalization with fluorescent antibodies and isolation techniques, respectively, showed that N. ditissima may spread in the xylem beyond the limits of visible cankers and in absence of visual symptoms on the external bark surface. However, Dewey et al (1995) did not specify the distance from the canker at which the pathogen was detected, whereas Weber and Hahn (2013) tracked the fungus up to 20 cm away from lesions on the trunk of young trees of cv.…”

“…However, although fungal hyphae were observed in association with xylem staining at sites remote from the lesion (Dewey et al, 1995), more systematic studies (Weber & Hahn, 2013;Weber & Zabel, 2010) failed to consistently isolate N. ditissima from stained wood. Despite the limited evidence, some authors currently believe that fungal colonization can cause wood staining (Børve et al, 2019;Weber, 2014).…”

“…At the end of the 19th century, it was proposed, but not verified experimentally, that the occurrence of multiple cankers on the same trunk could be a consequence of the internal colonization of plant tissues by the pathogen (Hartig, 1889). More recently, following severe canker outbreaks in newly established orchards in the UK, N. ditissima was detected immunologically in the wood tissues of stem sections collected from young trees with multiple cankers on the rootstock or the mainstem, at an unspecified distance from the canker lesion (Dewey et al, 1995). This led to the hypothesis that N. ditissima might systemically colonize trees following infection during the propagation stage (McCracken et al, 2003) and thus cause multiple cankers.…”

Relative significance of nursery infections and orchard inoculum in the development and spread of apple canker (Nectria galligena) in young orchards

“…These individual trees could have multiple cankers, while neighbouring trees were disease‐free. When stem sections of infected trees were examined there was extensive staining, and hyphae of a type common to Nectria were detected in the xylem using immunofluorescent techniques (Dewey et al ., 1995). This combined evidence supported the idea that trees could become infected, without symptom development, during the propagation stages.…”

Specific In Situ Visualization of the Pathogenic Endophytic FungusAciculosporium take, the Cause of Witches’ Broom in Bamboo

“…In addition, this method cannot be applied to natural samples because it is necessary to inoculate host plants with fungal transformants. Although immunostaining techniques are also an effective approach (7,26), they require complicated preparations for generating individual species-specific antibodies.…”