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Pathogen fitness in fungal systems without major gene resistance using Venturia inaequalis as a model system - HH2302STF

Through a study of apple scab (Venturia inaequalis) and apple the main objective of this project is give greater understanding of the parameters affecting the optimum management of weak to moderately strong polygenic resistances. This information will be directly relevant to scab but will also inform our understanding of all systems using resistances of this type and will therefore underpin many plant breeding programmes. Improved use of all available resistances is relevant both to improved quality but also to environmental concerns through the reduced use of pesticides.
(i) Variation in fitness will be assessed by collecting field isolates and inoculating tests plants to be scored for disease. Three major UK varieties which do not have 'major gene' resistance have been chosen (Bramley, Cox, Worcester) and orchards which are single variety (except for the inevitable pollinators) or mixed will be identified. The mixed orchard will be either all three varieties in one place or two orchards with pairs of varieties. These mixed orchards may also contain other varieties. 50 lesions will be collected from each variety in both the pure and mixed orchards to give 6 isolate 'types' (i.e. single orchard Cox isolates (SCox), mixed orchards Cox isolates (MCox) etc.). V. inaequalis will be isolated from each of these lesions via single sporing to give pure in vitro cultures which will be assessed for conidiation. 20 of these (the first 20 in numerical order which conidiate reasonably) will be grown in vitro and conidia harvested. (Unlike HH1753STF conidia will be harvested from in vitro cultures rather than being bulked up on seedlings to avoid unintentional selection occurring.) For each of the 120 isolates, 2 trees each of Bramley, Cox and Worcester will be inoculated and the disease induced scored for symptom type and approximate level of conidial production. Variety preferences will be apparent as lower scores on the heterologous host than on the homologous host but if no variety preference is present there would be no predictable ranking of virulence. The need to grow indicator trees and do the isolations means that testing on plants will not start until at least the middle of the first season.(ii) Competitive fitness will be measured by serially inoculating mixtures of the above isolates to two varieties and looking to see which isolates 'win'.The isolates from (i) will be tested using microsatellittes to try to identify all isolates individually but at least differentiate the types. (Any isolates not 'fit' i.e. not conidiating on the homologous variety, will be discarded but otherwise isolates will be taken in numerical order to avoid bias.) Groups of five isolates from each type will then be used to create mixtures used to inoculate two trees each of Cox and Bramley . The inoculum will be such that the 'test' isolates will be mixed with a four-fold greater amount of the competitor isolates. Hence SCox would be mixed with MCox, MBramley, MWorcester, SBramley and SWorcester or nothing (the last as a control for the general fitness of the isolates). Disease developed would be scored and conidia collected to inoculate a further set of trees and this repeated again. After these three passages, lesions would be collected and tested by microsatellites. At least 20 and up to 40 would be tested. Competitive fitness would be seen as the propensity for various isolate types to 'win' by becoming more frequent than the initial 20%. The precise numbers of trees might have to be adjusted after an initial experiment to ensure that sufficient inoculum were produced to infect the next round. The experiment would then be repeated using SBramley as the test isolates, again inoculating Bramley and Cox. Lack of resources means that this could not also be done for the Worcester isolates. This experiment would be done in the second season (2002 now 2003) but might stretch into the third (now 2004).(iii) Genetics of specificity. As soon as isolates with variety specificty had been identified in vitro crosses would be set up to look at the inheritance of those preferences. For each cross (SCox x SWorcester; SCox x SBramley; SBramley x SWorcester) at least 25 repeats with different isolates of each type would be set up so that at least some should be of opposite mating types and 'competent'. From the ascospores produced at least 35 and up to 60 monoconidial isolates would be produced and used to inoculate two trees each of the three varieties and scored for disease. The results would be analysed for pattern of inheritance. Polygenic resistance would be apparent as continuous variation in virulence for the two homologous hosts and no variation on the heterologous host. (iv) Small scale diversity. A large orchard of a single variety will be identified and three areas delineated within it with two areas c. 100metres apart and the third c. 40 and 70 from the first two. Approx. 150 isolates will be collected from each area (as soon after primary infection occurs as possible) and each tested with five microsatellites as has occurred in HH1753STF. Collection will take place in the first season in which large scale infection occurs. 'Gene' frequencies for the three areas will be compared. This information is relevant to understanding how closely alternative hosts should be planted to give improved control of Venturia by polygenic resistance.(v) Changes in fitness correlated with orchard practice. Traditional cider orchards are generally low intensity mixed orchards but these are increasingly being replaced by intensive orchards of single varieties. If polygenic resistance occurs in these varieties it is to be predicted that isolates from the older orchards are less virulent for the homologous varieties than isolates from modern orchards. Two sets of paired orchards (one mixed, the other single variety) of two different cider varieties will be identified and 10 lesions collected. As before collection will be as soon as possible and more than 10 isolates collected to ensure that at least 10 conidiating isolates will be obtained. These isolates will be tested on their homologous varieties (two trees per isolate) and average virulences compared. The testing for this objective will largely take place in the third year and will be subject to constraints on time. If possible more than 10 isolates will be tested but if necessary this part may be dropped without major impact on the project as it is largely confirmatory of Objective (i).(vi) The evidence for major gene resistances occurring in apple will be examined by a standard literature search, by discussion with apple breeders at HRI-EM and where possible by examining breeding records.(viii) (Additional funding 2003/4) Initially rRNA ITS based primers will be assessed for specificity using a range of isolates from the HRI culture collection and standard PCR. Simultaneously a fluorescent (“real-time”) PCR will developed for P. violae using isolates in culture. Then rapid procedures for the extraction of P. violae from soil and subsequent DNA extraction will be developed. these will be based on sucrose flotation/wet sieving (already known to work for initial isolation of Pythium spp. from soil) and commercially available kits (Qiagen DNeasy which work well for other soil-borne fungi e.g. Verticillium spp.)

Time-Scale and Cost
From: 2001

To: 2004

Cost: £259,539
Contractor / Funded Organisations
Warwick - HRI, Horticulture Research International
Disease Control              
Organic Farming              
Fields of Study