DISCUSSION

Field-collected and existing B. lactucae isolates have been identified using the IBEB

system. The virulence factors of the field isolates from Lincolnshire have also been investigated by observing the results of the interaction between virulence genes in

the samples and the Dm gene in the IBEB lettuces varieties. During the last decade,

over 2000 new isolates have been found around Europe. In California isolates have been assigned up to Pathotype VIII (pathotype assignment, by University of California, Davis) (Michelmore and Wong, 2008). However, until 2011 only 28 isolates have been nominated by IBEB as Bl: 1 to Bl: 28 which is due to the decision of IBEB board based on whether the new isolates are important enough to be added to the existing Bl-set of races (Plantum NL, 2011). These new isolates were found in many different production areas and it is predicted that these new isolates will spread over Europe (Plantum NL, 2011). However, assignment of IBEB nomination is for a

pragmatic utility rather than comprehensive categorization of isolates (Michelmore et

al., 2009) and might miss important variation.

The results show some of the sextet codes of current isolate collections have the same or similar sextet code as the IBEB denominated isolates, which indicates the possibility that the collection contains the existing European Bl-set races and some of the collected isolates are new races. The IBEB sextet code results will be used to compare with the molecular marker differential results which will be described in the following chapters.

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The results of v-factor frequency comparison between 2009 and 2010 might only

show part of the variations of B. lactucae virulence in fields in Lincolnshire as the

collection were not from exactly the same lines of lettuce varieties in these two years. But as an investigation it might be useful to keep records for the presence and absent of v-factors, as it is very possible that these field collections are heterogeneous which means an isolate is a mixture of races. Therefore single spore races in this sample could not be identified by the given IBEB sextet code. Therefore single spore isolation is necessary in future work. Furthermore, the identifications were based on the EU-A system, while the new EU-B system has been introduced recently, therefore it would be helpful to re-identify and re-code the collections using the EU-B set in future work.

The results of IBEB characterization of field samples show that there are 29 different isolates in the collection. However these field samples had not been single spore isolated so it is very possible that these samples are mixed spore isolates. This means they are likely to be virulent on more cultivars than the single spore line by which the IBEB set was defined (Michelmore and Crute, 1982). The problem is that the IBEB differential set is designed for samples that are single spore isolates and shows the interaction of avirulence and resistance as the presence or absence of symptom. It cannot identify how many isolates are in the sample using the IBEB differential set. If the field sample is comprised of three different isolates, for example isolates BL801, BL806 and NL5 as Figure 3.4 shows below, then the IBEB sextet code for NL5 is EU-A 05-27-01-00 (v-factors:1, 3, 7, 10, 12, 13 and 15) BL801 is EU-A 63-31-02-00 (v-factors: 1, 2, 3, 4, 5/8, 6, 7, 10, 11, 12, 13, and 16) and BL806 is EU-A 55-62-00-01 (v-factors: 1, 2, 3, 5/8, 6, 10, 11, 12, 13 and 19). But the result of

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IBEB classification of this field sample would be EU-A 63-63-06-01 which comprised the v-factors of BL801 BL806 and NL5. This result could neither tell the actual race within the sample nor which race is dominant.

Figure 3. 4 An example of a field collected sample that consists of three races.

The results of IBEB differentiation only show the + for compatible and - for incompatible but ignore the degree of compatibility/incompatibility. For two different races, the cultivars would show a higher degree of compatibility for the more aggressive one, but as + and - IBEB scoring, they might share the same IBEB sextet

code and be considered as one race. Although there are twenty four different Dm

genes in IBEB EU-B set, two isolates sharing one sextet code might be differentiated

if more lettuce varieties carrying different Dm genes are introduced in the differential

set. Therefore more varieties used in the differential set would make the results more accurate. However that would require more time and labour to use.

Genotypic identification using molecular markers is a promising method to identify

the races. Some researchers found that avirulance to specific Dm genes were

inherited as single dominant unlinked Loci (Norwood et al., 1983; Norwood and

BL801 64% BL806 25% NL5 11%

Field sample

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Crute, 1984; Michelmore et al., 1984; Ilott et al., 1987). Avirulence is determined by

dominant alleles at the unlinked loci and might be modified depending on the genetics of the host and the pathogen (Michelmore and Wong, 2008). As the avirulence would vary among races, the avirulence related marker could be

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