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

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

Introduction of Ophiobolus graminis into new polders and its decline

After a short introductory chapter on the occurrence of Ophiobolus graminis (take-all disease) in the polders, in chapter 2 the course of the disease and the biology of the fungus are described. The third chapter deals with materials and methods. The following chapters deal with investigations on two aspects of the disease: chapter 4 on the introduction of Ophiobolus into the polders; chapters 5 to 8 on decline of Ophiobolus with continuous cereal growing and the backgrounds of this phenomenon.The reclamation of the Ysselmeer Polders after drainage follows a fixed pattern. In East Flevoland it was:reed as a soil cover,gradual reclamation,rape as a first and wheat as a second crop.In many fields Ophiobolus already patchily occurs in this wheat crop, mostly at the border of a field. But in field trials with wheat directly after reed, Ophiobolus was more widespread and evenly distributed over the field. Shortly after reclamation Ophiobolus was already present on grasses. The Ophiobolus infection of wheat grown directly after reed was suggested to originate from the reed roots, whereas on wheat grown after rape the grasses at the border of the field were primarily responsible.The occurrence of Ophiobolus on grasses on an artificial island, 1½ year after its construction, suggested infection by air-borne ascospores. In the greenhouse, where perithecia-bearing wheat plants of other experiments were present, spontaneous infection of wheat by ascospores occurred regularly, if plants were grown on subsoil clay from East Flevoland. In experiments with wheat and grasses sterility or semisterility of the soil proved to be of paramount importance for successful infection by ascospores. Thus grasses, originating from seed and establishing themselves in the polder directly after drainage, are rapidly infected by Ophiobolus. Reed replacing the grasses picks up the infection, which in turn is passed to wheat after reclamation, and to grasses which establish themselves along roads and ditches.In the second or third year of continuous cereal growing there is a peak attack of Ophiobolus. Then the incidence of disease declines to a very low level. This could be clearly demonstrated on field trials in the North-East Polder and East Flevoland. In greenhouse trials decline was induced only by soil application of virulent Ophiobolus (1 % w/w; fresh weight). Wheat growing itself and application of avirulent Ophiobolus or some other fungi were ineffective. Ophiobolus-induced decline is governed by a specific antagonism, as will be shown; by contrast, a non-specific action of the soil microflora is responsible for a less effective antagonism present in every soil. Hereafter antagonism refers to specific antagonism.By applying Ophiobolus in greenhouse trials once in three months antagonism reaches a maximum within 3-6 months. It is preserved by growing wheat or barley continuously (in the field) or by continued application of Ophiobolus (in the greenhouse). Interruption of continuous cereal growing with another crop for one season, or for two cycles of three months in the greenhouse, considerably decreases the antagonistic capacity of the soil.The antagonistic capacities of a soil are lost by heating (50°C or higher for 30 min.) or application of chemical disinfectants. A mixture of antagonistic and nonantagonistic soil shows an antagonistic capacity in proportion to logarithm concentration of antagonistic soil. Growth of Ophiobolus in vivo is reduced by a sterilized water-extract of antagonistic soil. These three results show that antagonism is governed by antibiotics from soil micro-organisms. (The reduction of growth by the waterextract was measured by the length of runner hyphae on roots of wheat grown in sand. In vitro no effect could be demonstrated. Therefore isolation and identification of the active material had to be suspended).No differences could be found in the microflora of antagonistic and non-antagonistic soil by isolating fungi by the dilution method and similar techniques, and by using precolonized or clean baits, followed by testing the antagonistic properties of the isolates in vitro and in vivo. None of the isolates could be demonstrated to be of paramount importance in decline.This failure can be explained as follows. Since antibiotic properties depend on the environment, the actual capacities of an organism cannot be measured in vitro. Conditions of trial even in vivo differ so much from natural conditions that results are not conclusive.Wastie's method did not demonstrate any correlation between the antagonism of a series of soils and their effect on growth of Ophiobolus in vitro. But there was a clear effect on survival and colonization in the seedling test and the Cambridge test, in which the wheat plant indicates the presence of OphiobolusThe trials described show that antagonistic soil reduces the growth of Ophiobolus in its parasitic stage as well as its survival in the saprophytic stage

Biologische bestrijding sclerotiënrot in witlof

Results of experiments with the fungus Coniothyrium minitans for biological control of Sclerotinia sclerotiorum in lettuce. Method and time of application were investigate