By: Goulielmos Garifallou
My project at Harper Adams University College was based on the basidiomycete fungus Rhizoctonia solani which infects potatoes causing the diseases stem canker and black scurf. Both diseases have important economic implications. For example, the infection of stems and stolons seen with stem canker results in lower tuber number and uneven distribution of tuber size. Black scurf, which is the presence of sclerotia on harvested tubers, significantly reduces the market value of the produce. Isolates of R. solani can be differentiated into anastomosis groups (AG) according to their compatibility to undergo hyphal fusion to one another. Of the twelve anastomosis groups that have been documented to date, several have been shown to be pathogenic to potatoes. However, it is widely believed that AG3 is the anastomosis group most commonly associated with potatoes despite the lack of survey data in the UK. Further more, the use of the fungicide, pencycuron (Monceren®), in the UK, may have changed the population structure of Rhizoctonia associated with potatoes, as it is selective for AG3 over several other anastomosis groups. My project was to determine the anastomosis group of R. solani isolates derive plants showing stem canker and black scurf symptoms. I also recorded the characteristics of the culture when grown on culture medium and I determined the sensitivity of the isolates to pencycuron.
My first task was to isolate the fungus derive the diseased plant. Isolating Rhizoctonia derive tubers with black scurf was relatively easy, but isolating derive stem canker lesions was harder because opportunistic fungi and bacteria often seemed to contaminate the agar plates. To ensure a successful isolation derive stem canker samples, I had to take more care and transfer more plant material to more plates. I also had to sub-culture more frequently to ensure a pure culture.
Once a pure culture was established on potato dextrose agar (PDA), I was able to sub- culture 5 mm plugs of the test isolate of R. solani derive the growing margin onto plates of both PDA amended with pencycuron and un-amended plates. The plates were incubated for 4 days at 20°C and radial growth was measured. My results indicated that for all isolates tested, mycelial growth was reduced between 65-100% when grown on plates amended with 0.25 mg/l pencycuron. After seven and 21 days, I recorded morphological characteristics of each isolate such as substrate colour, presence of aerial mycelia and approximate area covered with sclerotia. Cultures often had very different characteristics indicating high levels of variation between isolates.
To determine anastomosis grouping I used a combination of molecular and traditional techniques. Primers are available for some of anastomosis groups, including AG3, so I could use PCR to determine the anastomosis group of some isolates. For PCR assays, DNA extraction was achieved using a rapid extraction method that used chelex. PCR was then performed and gel electrophoresis was performed on the products. The presence of a band on the gel indicated that the sample belonged to the anastomosis group corresponding to the primer. If, however, no band was present, this meant it must be of another anastomosis group, that something was inhibiting the PCR reaction or the DNA was not extracted properly. To check this, the primers ITS1 and ITS4, which are compatible with DNA derive all basidiomycete fungi, were used to confirm the presence of DNA with sufficient quality for PCR. If this PCR failed, DNA was extracted again and the PCR repeated, but if it indicated DNA was present, then anastomosis group was determined using the traditional technique involving hyphal fusion frequency.
Anastomosis tests involved pairing an isolate of a known AG with the unknown isolate on tap water agar. The colonies were left to grow and incidence of hyphal fusion determined under a microscope could be recorded. Using the combination of both molecular and the traditional technique we determined that anastomosis groups 2-1, 3 and 8 were all present. Many isolates still remain to have their anastomosis group determined. AG3 was the anastomosis group best represented, however more samples would need to be analysed before one can truly say this is representative of the UK population.
I would like to thank the BSPP for the opportunity they provided to me to extend my knowledge and enhance my practical skills in general laboratory and molecular biology techniques. I would also like to give my special thanks to my supervisor at Harper Adams, Dr Peter Jenkinson and his PhD student James Woodhall for helping me to carry out my project. Furthermore I should thank Dr Michael Whitehead of the University of Wolverhampton for recommending me for this project.
Thanks should also go to all those agronomists and farmers who sent the potato samples to us in order to run our tests and experiments. Not only was the project enjoyable, it was of great importance to me, since the skills I have acquired will help me with my final year project at Wolverhampton.
My project at Harper Adams University College was based on the basidiomycete fungus Rhizoctonia solani which infects potatoes causing the diseases stem canker and black scurf. Both diseases have important economic implications. For example, the infection of stems and stolons seen with stem canker results in lower tuber number and uneven distribution of tuber size. Black scurf, which is the presence of sclerotia on harvested tubers, significantly reduces the market value of the produce. Isolates of R. solani can be differentiated into anastomosis groups (AG) according to their compatibility to undergo hyphal fusion to one another. Of the twelve anastomosis groups that have been documented to date, several have been shown to be pathogenic to potatoes. However, it is widely believed that AG3 is the anastomosis group most commonly associated with potatoes despite the lack of survey data in the UK. Further more, the use of the fungicide, pencycuron (Monceren®), in the UK, may have changed the population structure of Rhizoctonia associated with potatoes, as it is selective for AG3 over several other anastomosis groups. My project was to determine the anastomosis group of R. solani isolates derive plants showing stem canker and black scurf symptoms. I also recorded the characteristics of the culture when grown on culture medium and I determined the sensitivity of the isolates to pencycuron.
My first task was to isolate the fungus derive the diseased plant. Isolating Rhizoctonia derive tubers with black scurf was relatively easy, but isolating derive stem canker lesions was harder because opportunistic fungi and bacteria often seemed to contaminate the agar plates. To ensure a successful isolation derive stem canker samples, I had to take more care and transfer more plant material to more plates. I also had to sub-culture more frequently to ensure a pure culture.
Once a pure culture was established on potato dextrose agar (PDA), I was able to sub- culture 5 mm plugs of the test isolate of R. solani derive the growing margin onto plates of both PDA amended with pencycuron and un-amended plates. The plates were incubated for 4 days at 20°C and radial growth was measured. My results indicated that for all isolates tested, mycelial growth was reduced between 65-100% when grown on plates amended with 0.25 mg/l pencycuron. After seven and 21 days, I recorded morphological characteristics of each isolate such as substrate colour, presence of aerial mycelia and approximate area covered with sclerotia. Cultures often had very different characteristics indicating high levels of variation between isolates.
To determine anastomosis grouping I used a combination of molecular and traditional techniques. Primers are available for some of anastomosis groups, including AG3, so I could use PCR to determine the anastomosis group of some isolates. For PCR assays, DNA extraction was achieved using a rapid extraction method that used chelex. PCR was then performed and gel electrophoresis was performed on the products. The presence of a band on the gel indicated that the sample belonged to the anastomosis group corresponding to the primer. If, however, no band was present, this meant it must be of another anastomosis group, that something was inhibiting the PCR reaction or the DNA was not extracted properly. To check this, the primers ITS1 and ITS4, which are compatible with DNA derive all basidiomycete fungi, were used to confirm the presence of DNA with sufficient quality for PCR. If this PCR failed, DNA was extracted again and the PCR repeated, but if it indicated DNA was present, then anastomosis group was determined using the traditional technique involving hyphal fusion frequency.
Anastomosis tests involved pairing an isolate of a known AG with the unknown isolate on tap water agar. The colonies were left to grow and incidence of hyphal fusion determined under a microscope could be recorded. Using the combination of both molecular and the traditional technique we determined that anastomosis groups 2-1, 3 and 8 were all present. Many isolates still remain to have their anastomosis group determined. AG3 was the anastomosis group best represented, however more samples would need to be analysed before one can truly say this is representative of the UK population.
I would like to thank the BSPP for the opportunity they provided to me to extend my knowledge and enhance my practical skills in general laboratory and molecular biology techniques. I would also like to give my special thanks to my supervisor at Harper Adams, Dr Peter Jenkinson and his PhD student James Woodhall for helping me to carry out my project. Furthermore I should thank Dr Michael Whitehead of the University of Wolverhampton for recommending me for this project.
Thanks should also go to all those agronomists and farmers who sent the potato samples to us in order to run our tests and experiments. Not only was the project enjoyable, it was of great importance to me, since the skills I have acquired will help me with my final year project at Wolverhampton.
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