This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Downy mildew (Hyaloperonospora sp.) is an obligate oomycete which infects wild rocket (Diplotaxis tenuifolia) and has been identified by UK growers and breeders as an increasing problem to control. After conidia have germinated and penetrated the leaf, conidiophores emerge through stomata, mostly on the underside but can be on both sides in severe cases. It causes necrotic patches and white spores on young leaves, resulting in significant reductions in crop quality and major yield losses. Growers and breeders are experiencing a breakdown of resistance since previously tolerant varieties are now showing symptoms, so there is interest in finding new sources of resistance and confirming if different races of the pathogen exist. The current literature indicates that isolates of downy mildew are mostly species specific, so there is very little cross-infection to other brassica species, but little is known about host resistance or diversity between different races of the pathogen.
Italian and Dutch varieties are currently most popular with UK growers due to mildew resistance traits, so there is a niche for UK breeders to produce seeds suited to the UK climate. This project was in collaboration with seed companies (Tozers Seeds Ltd, Elsoms Seeds Ltd, CN Seeds Ltd) and the British Leafy Salads Association (BLSA), a grower sub-group part of the British Growers Association.
A collaboration with the British Leafy Salad Association (BLSA) provided survey results which looked at identifying resistant phenotypes in a range of wild rocket varieties and seeing if this agreed with initial observations from growers and breeders. Full analysis of this survey indicated that out of the 5 varieties which had a resistance claim on the breeder website, downy mildew had been seen in 3 of them. Further results indicated that around 60% of the varieties surveyed had shown some infection symptoms. This suggests that resistance is breaking down since infection is now being seen in varieties which have previously been thought of as tolerant, confirming downy mildew as an increasing problem for growers and breeders.
The primary aim of my project was to establish an infection protocol for rocket downy mildew and bulk up an isolate of the pathogen for screening assays. This was particularly difficult since downy mildew is an obligate biotroph and there are a lack of long-term storage options since freezer storage can decrease quality of spores. The pathogen was bulked up on 7-day old seedlings – using a susceptible accession supplied by Tozers Seeds. This is because cotyledons are more susceptible than first and second leaves and resistance at seedling stage is good indication of resistance at adult stage. The isolate we were given was contaminated which made this difficult since cleaning up the inoculum took several weeks. A 7-day cycling infection protocol from Tozers Seeds was optimised, resulting in an increase in spore numbers towards the end of my project. This involved only selecting visibly sporulating cotyledons to get rid of the contaminant, as well as adapting the volume of water to ensure there was a large enough concentration of spores in the inoculum. I performed germination tests on 3 different susceptible accessions, finding that germination at 20˚ C resulted in the highest numbers of cotyledons, whereas incubating the inoculated seedlings at 15˚ C was ideal, since warmer temperatures can inhibit sporulation. I also grew up mature plants to put around the site in the hopes to obtain an isolate through natural infection, as well as contacting local rocket farmers, however the weather during this project was too warm, so natural infection was inhibited and no further isolates were available.
The gene DMR6 encodes 2-oxoglutarate-Fe(II) oxygenase-like protein and has been identified as a susceptibility factor to bacterial and oomycete pathogens. Once transcription is activated during infection, salicylic acid is converted to 2,3-DHBA which supresses the defence genes PR-1, 2 and 5. I carried out a Blastn with DMR6-like oxygenase 2 from B. napus and the D. tenuifolia genome to design 3 primer pairs. I extracted DNA from 4 rocket accessions: Voyager, Napoli, Monza and a resistant accession (Athena) and carried out several PCR reactions to test for presence of DMR6, to see if it concurred with resistance phenotypes. Unfortunately, none of them amplified and I ran out of time to further optimise.
Hopefully, this project acts as a good foundation for future experiments within the VeGIN project, allowing confirmation of Hyaloperonopora sp. species identity and diversity, as well as aiding the production of resistant UK wild rocket seeds. I’d like to thank Lauren Chappell at Warwick Crop Centre who supported me through my project, particularly when facing certain challenges, and helped me develop my lab skills which I am sure I will use lots in the future.
Alice Monksfield
University of Cambridge
Cover image: Wild rocket seedlings 7 days post inoculation with downy mildew, with visible conidiophores.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
