This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Botrytis cinerea is a necrotrophic fungal pathogen responsible for grey mould of strawberries. Strawberries produce volatile organic compounds (VOCs) which could be altered by the presence of B. cinerea. Previous work has shown Drosophila melanogaster, a vector for B. cinerea, is attracted to VOCs produced by healthy strawberries, although the role of B. cinerea inoculation on strawberry VOC production, and insect behaviour, has not yet been established. Here, we determined whether B. cinerea inoculation alters the VOC profile of strawberries, and whether these changes can influence the behaviour of D. melanogaster. We showed that B. cinerea inoculation causes changes in strawberry VOC production, and that D. melanogaster show a preference towards B. cinerea inoculated fruit. Findings from this work could be used to develop B. cinerea based semiochemical attractants to reduce pathogen transmission by D. melanogaster, reducing post-harvest losses.
Botrytis cinerea is a necrotrophic fungal pathogen which causes grey mould of strawberries, resulting in substantial global losses per year. B. cinerea produces spores that are spread by wind, water, or insect vectors such as D. melanogaster. Insects can be attracted to food through various cues such as VOCs, which are detected by their antennae. Whilst the attraction of D. melanogaster towards healthy fruit VOCs is well-established, the influence of B. cinerea inoculation of fruits on their VOC profiles, and D. melanogaster behaviour, is unknown.
First, we determined how long after strawberry inoculation with B. cinerea visual symptoms would appear, and established that symptoms can be observed at 72 h. After 72 h inoculation, dynamic headspace collections were performed to collect VOCs from either mock-inoculated or B. cinerea-inoculated strawberries. The strawberries were enclosed in glass chambers, and purified air was blown over the headspace of the strawberry (pictured). A glass tube containing an adsorbent polymer (Porapak) was placed within the vessel, which the VOCs adsorb to. After 24 h, a solvent was then used to elute the Porapak tubing to collect the VOCs. These samples collected using dynamic headspace collections were then analysed using gas chromatography (GC), which separates the compounds and enables us to quantify them, and our data show that B. cinerea inoculation causes quantitative and qualitative changes in strawberry VOC production.
To determine whether these differences in strawberry VOC production post inoculation cause changes to D. melanogaster behaviour, a two-choice bioassay was established. Within a large glass chamber, a falcon tube containing a strawberry inoculated with B. cinerea, and another falcon tube containing a mock inoculated strawberry (control), was placed. Thirty D. melanogaster flies were then released into each chamber, and could choose whether to move towards the B. cinerea fruit or the mock-inoculated control strawberry. After 24 h, the number of flies in each falcon tube was counted, and we found that there were significantly more flies captured in the B. cinerea-inoculated strawberry treatment compared to the mock-inoculated controls. This suggests D. melanogaster prefer the smell of B. cinerea-inoculated strawberries.
To locate which of the VOCs produced by the B. cinerea-inoculated strawberry was detected by the flies’ antennae, GC-electroantennography (GC-EAG) was used. The headspace sample containing the VOCs was injected into a GC, which separates the compounds. Simultaneously, the compounds were blown over a fly antenna, which is attached to two electrodes. When the antenna responds to a specific compound, there is an electrical change. From the headspace collection sample of the B. cinerea-inoculated strawberry, 24 of the compounds were found to elicit a response in D. melanogaster antennae. Future work will involve identifying these EAG-active compounds, which involves analysing the samples using coupled GC-mass spectrometry to obtain tentative identifications of the compounds, and peak enhancement using GC and authentic standards of the compounds. From this, a synthetic blend of the compounds, which mimic the strawberry VOCs, can be made. This blend would then be tested in a bioassay to determine if the blend is as attractive to the flies as the B. cinerea-inoculated strawberry.
I learned so much over my 8-week studentship at Rothamsted and I would like to thank everyone who has made this such an enjoyable and insightful experience, specifically my supervisors Gareth Thomas and József Vuts. Thank you as well to the BSPP for allowing me to have this amazing opportunity.
Arwen Gean
Dundee University
IMAGE: Dynamic headspace collection set-up of Botrytis cinerea-inoculated strawberry (left) and mock inoculated strawberry (right)
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
