BSPP News Spring 2002 - Online Edition

The Newsletter of the British Society for Plant Pathology
Number 41, Spring 2002 

Powdery mildew: coping with the hostile environment of the plant

This Summer I was lucky enough to work under the supervision of Dr Ziguo Zhang, in the Gurr Lab of the Department of Plant Sciences, Oxford, on barley powdery mildew.  Doing this studentship has given me a real taste of what research is all about.  During my ten weeks I learned a huge amount and hopefully made some worthwhile contributions as well as really enjoying it all.

 My project was to investigate some aspects of how the obligate pathogen Blumeria graminis f. sp. hordei copes with the oxidative environment that it is faced with when it attempts to attack and colonise barley plants.  This is set in context with the aim of eventually breeding for resistance in plants, as some plants show an oxidative burst as an early response associated with disease resistance.  My work started with getting to know my fungus in its normal lifecycle, which I knew only a little about, and then set about fine tuning experimental design and getting to grips with the various new techniques that I would have to use.  In order to see the effect of oxidative stress on the germination and differentiation of spores I applied the pro-oxidants H2O2 and Paraquat to barley epidermal peels at a variety of concentrations.  Armed with my newly acquired knowledge of what normal development should look like, I did a series of scoring experiments for spores inoculated onto the different treatments, looking at germination and differentiation to see if the treatments had any effect.  With practice I eventually got over the seasickness of looking down a microscope all day and gathered a lot of data.  The analysis carried out seems to show that germination of spores is unaffected by the oxidative stress applied but the subsequent differentiation of spores is increasingly inhibited with increasing stress levels.  I was then able to extend my investigation further by looking at the effects of a catalase inhibitor, 3-amino triazole, in the same way, and again found that differentiation of spores was inhibited while germination remained unaffected.  The inhibition of the endogenous fungal catalase may reduce its pathogenicity, rendering the pathogen less able to cope with an oxidative environment and so give the results seen.  Keen to see where else I could go with my investigation, I learned a whole new range of protocols that allowed me to look at the activity levels of known antioxidant enzymes in the developing spores when subjected to the pro-oxidants and inhibitor.  In this way I was able to look more specifically at the fungal scavenging activity and see the relative importance of the enzymes playing major roles in coping with the oxidative stress.  Unfortunately it was at this point that I came to the end of my ten weeks and so was unable to carry my project on further, even though it quickly became apparent that there were endless directions that I could have gone in, given more time.  Hopefully, however, the work that I was able to carry out will have provided some useful ground work for further studies into separating the host and parasite oxidative responses and a lead to a better understanding of how the fungus deals with the hostile environment of the plant. 

I am very grateful and would like to thank the BSPP for giving me this opportunity to get a closer look at an area that I enjoy and find interesting, as well as teaching me a lot about what it is like to control your own lab work with all the trouble shooting, adapting and learning along the way that goes with it.  Thanks must also go to all the members of the Gurr lab who made my time with them so enjoyable and successful. 

Catherine Eyre
University of Oxford

Molecular markers for studying the population biology of late blight (Phytophthora infestans)

Late Blight, caused by Phytophthora infestans is one of the most devastating diseases of potato crops worldwide. The P.infestans population in Europe has been displaced since the introduction of new A1 and A2 mating types from Mexico during the 1980s. This introduction changed the population into a dynamic one, as the occurrence of sexual recombination between the two mating types gave rise to an increase in the genetic variation.

During my time in the Scottish Crop Research Institute I learnt many novel procedures involved in the study of P.infestans. I observed the development and scoring of a single nucleotide polymorphism (SNP) PCR detection system that would allow a fast effective way of mapping P.infestans populations.

SNPs are good molecular markers as they have a high frequency; they are stable, and have a lower mutation rate than repeat sequences. The SNP was detected by sequencing the 2-phospho-glyceratedehydratase gene. After detection the next procedure was to design SNP primers, which I helped to design using the web software SNAPER. Producing an SNP primer involves mismatching the terminal 3' base pair and one other in the terminal 4 bases. The final stage was to test the primers on different isolates around Scotland, which resulted in a clear discrimination of the different base pairs at this SNP locus. 

The second experiment I was involved with was the development of a PCR diagnostic method for P.infestans detection in potato tubers. Seven seed tubers samples were tested. The tubers were washed and peeled; the peeled strips were immediately passed through a sap press that consisted of two revolving and interlocking rollers. DNA was extracted from the peeled extract and diluted down into a 1/20 solution. PCR amplification of the samples for the first round was with the outer primer sets DC6/ITS4 or ITS6/ITS4. The nested PCR primers (INF2fwd and INFrev) were P.infestans-specific. The method worked well and showed a number of samples to be positive despite a lack of blight symptoms. 

The majority of my time was spent on the third experiment, investigating the competitive ability of blight isolates on different potato cultivars. The intention was to be able to rapidly discriminate isolates recovered from co-infected leaves on the basis of their growth on metalaxyl ammended media. Metalaxyl resistant and sensitive P. infestans isolates were therefore selected for testing. The two isolates were used to infect a range of potato cultivars in 0:100, 25:75, 50:50, 75:25 and 100:0 ratios. Lesion type and sporulation were scored and differential responses observed.  Distinguishing isolates recovered from these leaves on the basis of growth on metalaxyl proved a challenge! First I tried washing the spores onto metalaxyl (100 mg/ml) amended pea agar and count the germinated and ungerminated spores under the binocular microscope. 

Although mycelial growth of sensitive isolates is severely restricted by metalaxyl, unfortunately its zoospore cyst germination rates were unaffected and I therefore couldn't distinguish the two isolates. I then tested other methods of distinguishing the isolates, for example using an ELISA plate reader to rapidly estimate the extent of mycelial growth of mixtures of the two isolates in pea broth amended with metalaxyl and antibiotics. The results were not conclusive, probably as a result of unpredictable levels of bacterial contamination afftecting the optical density readings of the ELISA. There was no time left to continue the development of a method to distinguish the isolates.

I would like to thank the BSPP for providing me with the opportunity to work in a research environment were I acquired a number of new skills, particularly lab techniques such as PCR, gel electrophoresis, and culturing, as well as experimental design and data analysis.  Also thanks to David Cooke, Naomi Williams and Shaukat Hussain for their help and guidance over the 10 weeks I spent at SCRI in Dundee.

Dewi S. Davies
University of Wales, Bangor

The fitness cost of fungicide resistance in Rhynchosporium secalis, causal agent of leaf blotch of barley 

Leaf blotch, caused by Rhynchosporium secalis (Oudem) J.J. Davis is the most important disease of barley in the UK, mainly due to an increase in the acreage of barley sown and a change in agricultural practices over the last 40 years. Yield losses of 35-40% have been reported in some barley varieties, but more commonly losses are less than 10%. The disease is spread mainly by splash-dispersed conidia, and is thus more severe in the wetter areas of the UK. Using resistant varieties is one means of controlling R. secalis, but resistance has been prone to break down. The use of fungicides is an alternative means of control, but fungicide tolerance in R. secalis has become common, particularly to the MBC (benzimadozole carbamantes) and DMI (sterol demethylation inhibitors) classes of fungicide.

The effect of fungicide tolerance on the fitness of R. secalis has not been addressed, and such information would be useful in the management of pathogen populations when using fungicides with contrasting modes of action. This project, with Dr C. H. Bock at IACR-Rothamsted, investigates the effect of carbendazim tolerance in R. secalis.

R. secalis isolates were obtained from barley leaves from four locations: a field at IACR-Rothamsted, two different fields from Trentholme Farm in Nottinghamshire and from Northern Ireland (kindly provided by Dr P. Mercer). The isolates from Northern Ireland were known to be resistant to carbendazim. Leaves were surface-sterilised and then incubated in petri dishes lined with moist filter paper at 16°C for 48 hours. After this period mycelial tufts of R. secalis could be seen under a dissecting microscope. These tufts of mycelia were then transferred to Potato Dextrose Agar (PDA) plates and the plates placed in an incubator at 16°C for 2-3 weeks. Spores from each colony were saved on silica and stored at -80°C. A total of seventy-eight isolates were processed in this way.

Eleven isolates were included in an experiment to investigate the effect of fungicide resistance on growth of the fungus. Five of the eleven isolates used in the fitness experiment were those previously characterised as resistant to carbendazim. Agar plugs were taken of the eleven isolates from the PDA plates and a spore suspension prepared, of which 250 ul was added to Erlenmeyer flasks containing 70 ml Butter Bean Broth  (BBB) either amended with carbendazim (10 ug ml-1) to test for fungicide tolerance, or left as a control to determine the fitness of the different strains. There were three replicates of each treatment. 

The broth was harvested and filtered off, the mycelia were oven-dried at 80°C for 12 h, and the dry weight of the mycelia was calculated. Two of the isolates previously found to be resistant were not found to be resistant in this study, and two of the previously uncharacterised isolates from Nottinghamshire were resistant. In the presence of fungicide the tolerant strains produced a similar mass, while the sensitive strains were greatly reduced in growth. The results show that fungicide resistance is important when considering the efficacy of carbendazim (Table 1), and that the tolerant isolates did not grow as rapidly as the sensitive isolates. Thus fungicide tolerance in R. secalis appears to carry a cost by reducing the ability of the fungus to grow, which may be a useful tool in managing fungicide resistance.

Table 1 The effect of carbendazim on fungal mass of tolerant and sensitive strains grown in butter bean broth (standard errors in parentheses).
  TreatmentTolerant isolatesSensitive isolatesMeanAmmended with fungicide0.55 (0.04)0.22 (0.03)0.38No fungicide0.46 (0.03)0.96 (0.03)0.71Mean0.510.59

Five isolates were chosen (three tolerant to MBCs) and grown in Potato Dextrose Broth (PDB) at 16 °C. The DNA was extracted from these, and PCR-RAPDs was performed with five different primers to determine genetic variability of these selected isolates. This gave me the opportunity to use a technique that I had previously only learnt in theory. The resulting banding pattern in the gel showed minor differences between the two carbendazim tolerant isolates. There were also minor differences among the other three isolates, but most noticeable was the large difference in the DNA fragment sizes between the two groups. Clearly this is a very small sample size, and further work using tolerant and sensitive isolates from a wider geographic range is required to compare these strains.

Whilst at Rothamsted I helped with other projects. I worked on an investigation of the progress of Tapesia yallundae and T. acuformis (eyespot) in winter wheat, the incidence of Septoria tritici and Stagonosporanodorum (septoria) in winter wheat, and the incidence of Leptosphaeria maculans (stem canker) in oil seed rape. I also helped with the laboratory isolation of Verticillium dahliae from spring linseed stems, and with PCR analysis of A and B types of L. maculans. In addition I prepared meteorological data sets from the Rothamsted archives, and performed a literature search on the epidemiology of R. secalis.

From the project, I gained an enjoyable insight into laboratory work in a research institute. I was able to contribute to current knowledge of plant pathology, which will help in the understanding of disease control and subsequent benefit to agriculture. I would like to thank the BSPP for awarding me the undergraduate vacation bursary, Dr C.H. Bock for his help, and all of the people at IACR-Rothamsted who have made me feel welcome. 

Martin Croft
University of Birmingham