Optimising the use of biocontrol agents to improve the control of Botrytis cinerea in key vegetable and fruit crops (Studentship)

This project aims to obtain ecological knowledge on BCAs that are currently registered and being registered in the UK and then use the knowledge to develop and evaluate strategies of applying BCAs to improve efficacy against Botrytis development on strawberry and lettuce. Specifically, we shall (1) develop molecular methods to quantify the viable population of two commercial BCAs (one fungus and one bacterium), and two new candidate BCAs (identified at EMR and are being formulated by a commercial company); (2) use the method to study BCA population dynamics under different conditions; (3) investigate BCA dispersal under different rainfall intensities in both glasshouse and field conditions; (4) use the new knowledge to optimise BCA applications and evaluate the strategies on strawberry and lettuce; (5) conduct field studies to assess whether combined use of BCAs as well as with reduced fungicide input would lead to synergy and reduced variability in biocontrol efficacy.

Although quantification methods based on real-time PCR of DNA have been developed for the two BCAs, these methods are not what is required for the purposes of the present project. This project focuses on dynamics of viable BCA population (propagules); DNA-based methods cannot, however, distinguish viable propagules from non-viable propagules. RT-PCR can be used to quantify viable propagules but it will be too expensive to develop for the present project. Instead, this project will develop quantification methods based on the following approach:
Cytological quantification. For B. subtilus, we shall try to genetically transform the isolates with the GFP gene to facilitate direct quantification of this BCA under the microscope. This technique has been used in many pathological studies to understand infection and subsequent invasion of pathogens inside host tissues by visualising the organisms in question. An important question is to ensure that the transformed isolate has the same or similar ecological requirements.
Plating technique. For all three BCAs, plating techniques will be used to directly estimate the amount of viable colony forming units (CFU) on agar media. This approach depends on whether there are sufficient unique colony characteristics for each BCA strain to distinguish them from other resident microflora.
Indirect estimation. If neither the cytological nor the plating method has worked for one or more BCA, an indirect method will be used to estimate CFU based on Botrytis severity on treated leaves. This method assumes that BCA population size is monotonically related to disease control efficacy.

 

Objective 2:  Determining the relationship of each BCA with external conditions (by month 24)
Temperature and humidity will be studied for their effects on BCA dynamics. Experimental protocols will depend on how BCA propagule density is quantified (WP1). Experiments will be conducted initially in controlled conditions where temperature and humidity (including rainfall) are controlled and later in semi-commercial conditions (glasshouse and/or open field). These experiments are conducted to answer the following questions:

 

• Can we predict the dynamics of BCA propagules based on temperature and humidity under constant and fluctuating conditions? If so, this predictive model can be used in conjunction with disease models to time BCA applications.
• To what extent are BCA propagules dispersed to newly emerged plant tissues? This will determine whether (and if so, at what frequency) repeated application of BCAs is necessary.

Objective 3:  Determining whether combined use of BCAs leads to synergy and reduced variability in efficacy (by month 28)
Initial experiments will be conducted under controlled conditions to study combined use of BCAs in relation to synergy and the variability in biocontrol efficacy. Each product will be applied either alone or in combination with another. Disease development will be assessed at several time points. Later, similar experiments will be conducted on commercial crops with multiple applications of BCAs. Results from these experiments will answer the following questions:
• What combinations of BCAs can result in synergy or independent interaction? This will enable selections of BCAs to improve efficacy.
• Could combined use of BCAs lead to reduced variability in biocontrol efficacy? This is important for assessing biocontrol potentials of individual products. Inconsistency in biocontrol among different studies is one factor limiting the use of BCAs.

Objective 4:  Developing and evaluating strategies for BCA application (by month 36).
Recently we developed a model to evaluate biocontrol with one or two BCAs under fluctuating conditions. We shall use the data obtained in WP2 to estimate BCA-related model parameters. We also attempt to incorporate fungicide effects into the model in order to evaluate the efficacy of joint use of fungicides and BCAs. Strategies will be compared theoretically using models and experimentally in large field experiments, including use of individual BCAs, combined use of BCAs at the same time, and use of alternative BCAs over time. Data from field and modelling studies will answer the following questions:
• How often does combined use of BCAs outperform individual BCAs based on modelling studies using historical weather data recorded over a period of 90 years at East Malling?
• Does one strategy consistently outperform another one in terms of disease suppression and variability in disease suppression?
• Does the performance of a particular strategy depend on particular crops (strawberry and lettuce) or BCAs?
Answers to these questions form the foundation of optimising strategies of applying BCAs in practice.