Use of Coniothyrium minitans transformed with the hygromycin B resistance gene to study survival and infection of Sclerotinia sclerotiorum sclerotia in soil

Abstract

A Coniothyrium minitans strain (T3) co-transformed with the genes for β-glucuronidase (uidA) and hygromycin phosphotransferase (hph), the latter providing resistance to the antibiotic hygromycin B, was used to investigate the survival and infection of sclerotia of Sclerotinia sclerotiorum by C. minitans over time in four different soils. Infection of sclerotia was rapid in all cases, with the behaviour of transformant T3 and wild type parent A69 being similar. Differences were seen between the soils in the rate of infection of sclerotia by C. minitans and in their indigenous fungal populations. Amendment of agar with hygromycin B enabled the quantification of C. minitans in soil by dilution plating where there was a high background of other microorganisms. In Lincoln soil from New Zealand, which had a natural but low population of C. minitans, the hygromycin B resistance marker allowed the umambiguous discrimination of the applied transformed isolate from the indigenous hygromycin B sensitive one. In this soil, although the indigenous C. minitans population was detected from sclerotia, none were recovered on the dilution plates, indicating the increased sensitivity of C. minitans detection from soil using sclerotial baiting. C. minitans was a very efficient parasite, being able to infect a large proportion of sclerotia within a relatively short time from an initially low soil population. The addition of hygromycin B to agar also allowed the detection of C. minitans from decaying sclerotia by inhibiting secondary fungal colonisers. This is the first report to show that fungi colonising sclerotia already infected by C. minitans mask the detection of C. minitans from sclerotia rather than displacing the original parasite.