Análisis integral de los resultados

In investigations of pea root rot diseases worldwide, many Fusarium species have been associated with symptoms of root rot comprising black discolouration of the roots and stem base, yellowing of lower leaves, and stunted growth (Persson et al., 1997). A range

of Fusarium species are also commonly isolated from roots and the surrounding soil

(Oyarzun et al., 1993; Skovgaard et al., 2002). For many years, F. solani was thought to be the major pea root rot pathogen (Chittem et al., 2015), but several surveys have revealed that it is not always the most frequently isolated species. For instance, a study of root rot pathogens of pea in Canada found that Fusarium species were the most frequently isolated, with the highest proportion belonging to F. oxysporum (Hwang & Chang, 1989). However, subsequent studies in Canada identified F. avenaceum as the most prevalent species isolated from diseased pea roots (Fernandez, 2007). Similarly, findings from North Dakota in 2004/5 and 2008/9 also report F. avenaceum and F.

oxysporum as the most frequently isolated species, along with other species such as F.

solani and F. redolens (Chittem et al., 2015). Research on root rot pathogens carried out

in Denmark, Sweden and the Netherlands, found that F. oxysporum, F. solani and F.

avenaceum were the most prevalent species, supporting results seen in other countries

(Oyarzun et al., 1993; Persson et al., 1997). In the UK, F. solani and F. oxysporum were the most common species isolated from plants displaying root rot symptoms collected from 20 sites across Nottinghamshire, Lincolnshire and Worcestershire (Clarkson, 1978). This is similar to previous findings where F. solani, F. oxysporum and F. redolens were isolated from diseased peas across the major pea growing areas of the UK (Buxton, 1955). In most studies, pathogenicity tests were carried out with varying results, suggesting that not all species isolated are highly pathogenic (Chittem et al., 2015; Persson et al., 1997; Skovgaard et al., 2002). Additionally, in most studies isolates were identified using morphological techniques only, reducing the accuracy and reliability of the results.

Fusarium oxysporum f. sp. pisi (FOP) was first described and distinguished from Fusarium root rot in 1925, by Jones and Linford, after it was found in around 50 fields in Wisconsin (Jones & Linford, 1925; Kraft, 1994). Symptoms differ from those caused by root rot pathogens in that leaves become dry and wilted, progressing from the lower leaves to the apex, until the whole plant was wilted and dried (Kraft & Pfleger, 2001). It was later assigned the name of FOP race 1 and has since been controlled using resistant varieties. Race 2 was described in 1933 as it was capable of causing disease in pea plants resistant to race 1, and was found to be as widespread as race 1 (Kraft, 1994). Race 5 was not identified until the late 1960’s, where commercial cultivars resistant to race 1 and 2 were found to be susceptible in north-western Washington (Haglund & Kraft, 1970). The final race to be identified was race 6, found in western Washington, and was pathogenic on cultivars resistant to the other identified races (Haglund & Kraft, 1979). Race 6 was identified in 27% of the 640 fields sampled, with 40% containing race 5 isolates, and race 6 was distinguished from race 5 using two differential cultivars (Haglund & Kraft, 1979). The development of resistant varieties has enabled the successful control of all these four races of FOP infections on pea (Kraft, 1994). Early studies on FOP in the UK showed that some isolates of F. oxysporum collected from affected pea fields caused wilting similar to that observed previously for an isolate of race 1, while others showed root rot symptoms similar to those seen by F. solani (Buxton, 1955).

As with the pea root rot surveys, the majority of these studies with FOP have relied on morphological identification and race testing using a core set of pea differential cultivars (Kraft & Pfleger, 2001). Molecular techniques for identification are now common practice, using sequencing and phylogeny of housekeeping genes, which greatly improves the accuracy of species classification. In fungi, the internal transcribed spacer (ITS) is universally used for identification, but it has been shown to be inadequate for distinguishing different Fusarium species due to non-orthologous sequence types found within Fusarium species and within isolates (O’Donnell & Cigelnik, 1997; Summerell & Leslie, 2011). However, the translation elongation factor 1a (TEF) gene, which encodes an essential part of the protein translation machinery, resolves different Fusarium spp. and is therefore the marker of choice for identification. The gene has been shown to be consistently single copy in Fusarium and shows a high level of sequence polymorphism between closely related species (Geiser et al., 2004). TEF sequencing was also used to investigate lineages within the F. oxysporum complex, but failed to reliably distinguish

between different formae speciales due to isolates being as genetically similar to other

formae speciales as they are to isolates in their own formae speciales (O'Donnell et al.,

1998). Other housekeeping genes such as b-tubulin (TUB2) and RNA polymerase II

second largest subunit (RPB2) have also been used to provide a more robust phylogeny

within species (O’Donnell & Cigelnik, 1997; O'Donnell et al., 2010).

Although sequencing of these genes is a useful tool for identification and understanding the phylogeny of the genus, they still do not allow different F. oxysporum formae

speciales, races or pathogenic/non-pathogenic isolates to be distinguished. Molecular

discrimination between F. oxysporum isolates is very difficult due to the polyphyletic nature of the many formae speciales, meaning that isolates from different formae

speciales may be more closely related to each other than to isolates of the same forma

specialis (Lievens et al., 2009). More recently however, progress has been made through

the use of effector gene presence / absence or sequence for distinguishing between certain

formae speciales, between pathogenic and non-pathogenic isolates, and even between

races of F. oxysporum f. sp. lycopersici (FOL) (Lievens et al., 2009). However, in many cases, traditional pathogenicity tests are still required to confirm the formae speciales, race and pathogenicity of F. oxysporum. In FOP, there is currently no molecular way of distinguishing the four races, which still relies on pathogenicity tests using the pea differential cultivars.

There are a number of ways of assessing pathogenicity of root rot and wilt pathogens in pea. A widely used pathogenicity test is the root trim and dip method, used to distinguish pathogenic races of FOP (Haglund, 1989; Kraft, 1994). Although this assay has also been used to test the pathogenicity of Fusarium root rot isolates (Persson et al., 1997), it is predominantly used for testing isolates causing Fusarium wilt. Pathogenicity tests with

Fusarium isolates affecting pea have included tube tests with liquid inoculum (Dyer &

Ingram, 1990), a sand-cornmeal inoculum layer between layers of vermiculite (Chittem

et al., 2015), sterilised soil inoculated with liquid inoculum (Clarkson, 1978) and soaking

germinated seeds in a conidial suspension (Feng et al., 2010). Each test appears successful in that the pathogenicity of different isolates can be assessed on the pea plants, allowing visible symptoms to be scored. Other tests used to evaluate the pathogenicity of other F.

could also be applied to assess root rotting effects of F.oxysporum in pea (Taylor et al., 2013).

The main aim of this chapter was to identify and characterise Fusarium species affecting peas in the UK and develop methods for investigating pathogenicity.

The specific objectives were:

1. To obtain Fusarium isolates from pea plants displaying symptoms of root rot/wilt from UK pea fields

2. To identify and characterise Fusarium species using common fungal housekeeping genes (TEF, TUB2, RBP2)

3. To assess methods for testing the pathogenicity of different isolates of F.