Weak virulence has limited the development of Colletorichum coccodes (Wallr.) Hughes (DAOM 183088) as a bioherbicide for Abutilon theophrasti Medik. (velvetleaf) control. This study examines the role of chemical synergy to suppress host defense mechanisms. Dry weight of A. theophrasti was reduced by treatment with C. coccodes in a tank-mix with 0.25 kg a.i. ha )1 bentazon more than by the treatment with C. coccodes or bentazon alone, while the effect of a sub-lethal rate of glyphosate was not significant. C. coccodes did not affect phenylalanine ammonia lyase (PAL) activity in infected leaves of A. theophrasti, whereas PAL activity was significantly inhibited by the presence of bentazon 5 days after treatment. The inhibition was stronger when bentazon was applied alone than when bentazon was applied with C. coccodes. Treatment with glyphosate or a mixture of glyphosate and C. coccodes did not affect PAL activity. Peroxidase activity was strongly induced by C. coccodes treatment and increased over time. Peroxidase activity was not induced by 0.25 kg a.i. ha )1 bentazon alone. However, when bentazon was applied in combination with C. coccodes, it prevented the activation of peroxidase by C. coccodes infection. Treatment with 0.2 kg a.i. ha )1 glyphosate did not affect peroxidase activity. These results suggest that these enzymes are involved in the resistance mechanism of A. theophrasti, and the synergistic effect of bentazon on C. coccodes efficacy as a bioherbicide may result from suppressing the activation of these defense-related enzymes.
The plant pathogenic fungus, Sclerotinia minor IMI 344141, has been developed as a bioherbicide for broadleaf weed control in turfgrass and a means to differentiate this biocontrol agent from like organisms is required. A strain specific molecular marker was developed to detect and monitor the Sclerotinia minor IMI 344141 bioherbicide strain. The method was based on polymerase chain reaction (PCR) amplification of two sequence-characterized amplified regions (SCAR) primer pairs for a first round PCR, and another two sets of nested primers was used for a second round PCR if higher sensitivity was needed. Sclerotinia minor IMI 344141 was successfully traced from both pure cultures and environmental samples originating from bioherbicide-released field trials. DNA of the S. minor bioherbicide isolate IMI 344141 was detected in the soil 2 months after application, but was not detected in the 3-and 9-month samples after application. When applied as a bioherbicide, S. minor (IMI 344141) did not persist into the following spring season in turf environments. This molecular detection method provides a mechanism to distinguish this isolate from related organisms and a tool to monitor behavior of the biocontrol agent S. minor IMI 344141 in nature, particularly in soil.
Two strains of Colletotrichum coccodes, the wild type (DAOM 183088) and T-20a, engineered with the necrosis- and ethylene-inducing peptide (NEP1) gene for hypervirulence on velvetleaf (Abutilon theophrasti, Medik.), were monitored in planta for the first 2 weeks after infection. Real-time quantitative polymerase chain reaction (QPCR) was used to assess the extent of colonization of both strains on velvetleaf using SYBR Green chemistry. Quantification of both strains was successful as soon as the conidia were sprayed on the leaves and up to 14 days after infection. The increase in fungal DNA amounts corroborated with the appearance of necrotic lesions on velvetleaf leaves infected with the wild-type strain. The wild-type C. coccodes was more efficient at infecting velvetleaf than the transgenic T-20a strain. In addition, detection of host DNA allowed us to quantitatively monitor the decrease in plant DNA amounts in response to wild-type strain infection. Expression of the NEP1 transgene by conventional retro-transcription (RT)-PCR was absent from T-20a growing on either V8 agar or in planta, suggesting that the gene may be silenced. The application of QPCR to monitor fungal growth was proven to detect the target organisms in planta prior to the appearance of symptoms.
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