Analysis of populations of Rhizoctonia solani  on potatoes

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 from 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 from the diseased plant. Isolating Rhizoctonia from tubers with black scurf was relatively easy, but isolating from stem canker lesions was harder because opportunistic fungi and bacteria often seemed to contaminate the agar plates.  To ensure a successful isolation from 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 from 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 from 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.

Goulielmos Garifallou
University of Wolverhampton

Genetic characterisation of resistance in Arabidopsis to important Brassica isolates of Xanthomonas campestris pv.campestris

tThe yellow-pigmented, gram-negative bacteria Xanthomonas campestris pv.campestris, the causal agent of black rot (primarily a seed borne disease) is one of the major world-wide diseases of Crucifer. Studies of recent Xcc outbreaks on oilseeds have suggested that the spread of new highly aggressive variants of this pathogen has been involved in localised epidemics. Indeed, within the UK, Xcc has caused such a problem to growers in recent years that it has been identified as a priority disease by the International Seed Health Initiative. The aim of my bursary project was to screen for differential responses in UK and common laboratory accessions of Arabidopsis, using six races of Xcc identified by Vincente and colleagues on the basis of their reactions on a series of differential Brassica genotypes (Vincente et al., 2001).

We developed a method of infecting Arabidopsis involving introducing the Xcc by clipping the mid vein of 5-week-old plants and scoring symptoms over a period of 9 days.  Five leaves of each plant (5 weeks old) of the lines are inoculated in the mid-vein with Xcc by making 4-5 incisions using ‘rabbit ear’ forceps. Susceptible accessions of Arabidopsis thaliana express yellow, V-shaped lesions. If severe disease is present, these lead to rapid chlorosis and finally collapse of the leaf (Figure 1). We could also distinguish clearly resistant and susceptible interactions 7 days after infection by counting bacteria isolated from challenged leaves. Susceptible accessions wth the typical yellow, V-shaped lesions supported at least 3 orders of magnitude more bacterial growth than the symptomless leaves.

Figure 1. Typical symptoms for resistant (left) and  susceptible (right) interactions on Arabidopsis with different Xcc races.

 To further characterise this interaction at the cellular level we tested whether a molecular marker of hypersensitive cell death developed in Murray Grant’s laboratory was also induced in an avirulent Xcc interaction. Leaves of Col-5 Arabidopsis plants were inoculated with race 3 (resistant), race 6 (susceptible) or a control (10mM MgCl₂) by both clipping and with a needle-less syringe. RNA blots showed much larger and more rapid induction of a cell death marker after inoculation with race 3 than with race 6 (Figure 2). This indicates that race 3 can elicit a marker of hypersensitive cell death in a manner similar to that seen in gene-for-gene interactions involving Pseudomonas (avrRpm1/RPM1). 

Figure 2. A marker of cell death induced after challenge with avirulent Pseudomonas isolates
is also induced in Arabidopsis leaves during an incompatible interaction with Xcc. 
Lanes:1, race 3, 24 hpi; 2, race 6, 24 hpi; 3, mock infiltration at 24 h; 4, race 3, 32 hpi; 5, race 6, 32 hpi; 6, mock infilatration at 32 h. 

Previous trials of Xcc races on several core and UK accessions of Arabidopsis thaliana indicated that the Landsberg erecta and Cape Verde Island accessions were suitable for mapping resistance to races 5 and 6, both of which were virulent on Cvi and avirulent on La-er. F8 single seed decent lines of La-er x Cvi were used to map resistance to Xcc races  5 and 6. Results were scored as disease presence or absence after 6 days. There was no clear segregation of resonses to race 6, but race 5 was predicted to lie at the bottom of chromosome III, and this was indeed found to be the case in the La-er x Cvi population. 

It is of interest that three typical NBS-LRR resistance genes are also located in this region in the Col-0 accession (www.niblrrs.ucdavis.edu/At_RGenes). Given that Col-0 is also resistant to race 5, these represent potential resistance genes and work is in progress to transform these sequences into the Cvi background. 

Future work will involve complementation of Cvi with NBS-LRR resistance gene homologues located within this region of the resistant Col-0 accession (www.niblrrs.ucdavis.edu/At_RGenes). 

References: J.G. Vicente, J. Conway, S.J. Roberts and J.D. Taylor (2001). Phytopathology 91: 492-499.

Susanna Atwell
Imperial College at Wye

Molecular characterisation of a putative novel double stranded RNA virus in Agaricus bisporus 

Mushroom Virus X (MVX) is an emerging infection of the cultivated mushroom Agaricus bisporus.  The present diagnostic for MVX is a reliable test involving the total dsRNA extraction and electrophoretic visualization of the virus.  Although a satisfactory test, it is both uneconomically expensive and highly time consuming.  The PCR primer test currently being developed at HRI to detect MVX would significantly reduce the amount of time required for disease diagnosis from about three days to approximately three hours.  In addition, the PCR test would enable the testing of mushroom mycelium for diagnostic purposes - at present only relatively large quantities of fully developed mushrooms can be tested.  The PCR test has greater specificity, is more sensitive than the electrophoretic test and the costs involved for disease detection are significantly reduced.  Mushroom growers worldwide are very anxious to see the introduction of the PCR primer diagnostic test as soon as possible.

MVX has been described as the ‘horticultural equivalent to foot and mouth’ (Peter Mills, personal correspondence) and at present it seems that its consequences could be equally as significant.  The main and most damaging symptom associated with MVX is a large loss of yield resulting in bare patches in otherwise uniformly growing mushroom patches.  Other symptoms include browning of white mushrooms and the premature opening of the mushroom cap.  In the past twelve months alone, the mushroom industry has experienced a £50 million reduction in profits.  Three United Kingdom mushroom farms have been declared bankrupt and total UK mushroom production has fallen by 10 – 15%.  Recently, farms in South Africa, Italy and New Zealand amongst others have experienced cropping problems characteristic of MVX.  The electrophoretic test at HRI has confirmed that these international farms are also infected with MVX. 

Twenty three dsRNA bands (B1-B23) and three wild type hybrid dsRNA bands (BH1-BH3) have been visualised in infected A. bisporus samples.  They differ in their intensity and frequency and recently it has been found that certain viral bands are associated with particular symptoms. 

My 10 week vacation bursary project at HRI involved developing PCR primers to two of the MVX viral bands, B23 and BH3, for the purpose of developing a reliable diagnostic based on the amplification of the nucleotide sequences between these flanking regions (the primers).  This was achieved by extracting the entire nucleic acid content of spawn cultivated mushrooms and then separating and precipitating the ds viral RNA.  The ds RNA was cloned and sequenced and primers were designed to these sequences. 
B23 is the smallest band of the viral complex.  There have been suggestions that it may be linked to B3 and shows homology to the cryphonectria virus.  B23 was linked to the browning symptom of the disease and found to be very rare in occurrence, normally in association with Bands 18, 19 and 22.

BH3 is a hybrid wild type band, which does not appear to be connected to any disease symptoms, and is present in the vast majority of A. bisporus specimens.  For the purpose of the diagnostic test, BH3 was used as an internal positive for ensuring that the PCR test is working effectively. 

Homology between each virus band and other bands in the complex as well as other genetic sequences worldwide are also being studied.  Further research will provide information on whether there are sub-groups within the complex, which work as units, or whether each band within the complex is a single independent viral entity. 

I would like to thank the BSPP for the vacation bursary and HRI Wellesbourne for the use of their facilities.  My supervisors Dr Bruce Adie and Dr Mike Challen provided invaluable advice and support throughout my placement - I thank them for their time and patience.  The skills and knowledge I obtained during my 10 weeks at HRI have already proved useful for my final year studies and I’m sure will continue to do so during my scientific career.  I enjoyed my placement immensely and look forward to returning to HRI and monitoring the progress of the MVX project.

Kulvinder Kaur
University of Oxford

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