3.5.1. Application of FIGS for the identification of best-bet AB resistance sources in the global lentil collection
The predictions of finding useful resistance to AB through a FIGS approach revealed 17.2% accessions within the subset of lentil as resistant or moderately resistant at 21 dpi following expected responses of control cultivars ILL 7537 and ILL 6002 to inoculations with isolate FT13037 as previously established (Dadu et al., 2017). Further, evaluation of physiological defence responses of the highly resistant accession IG 207 indicated that the approach captured novel, highly effective, and potentially useful A. lentis resistance mechanisms. Thus, a possible conclusion would be that the FIGS approach for mining gene banks is an effective alternative compared to random selection or arduous and costly large collection screening. Certainly, this has been the conclusion of other authors who found multiple sources for rare resistance to Russian wheat aphid (Diuraphis noxia) and Sunn pest (Eurygaster integriceps) in relatively small FIGS subsets after unsuccessful screening of thousands of wheat landraces conserved in the ICARDA gene bank (El Bouhssini et al., 2009; El Bouhssini et al., 2011; El Bouhssini et al., 2013).
However, a cautionary note must be added here. While this study, like the others cited above, does show that FIGS was successful at identifying useful trait variation, it did not directly compare the FIGS set to a subset constructed using different methods, such as random selection or a core collection. In fact, it could be the case that the frequency of resistant accessions in the FIGS set used is merely a reflection of the native frequency of resistance present in the genepool at large. For example, 17% of total wild lentil accessions screened have shown resistant reaction against the virulent A. lentis isolate FT13038 (Dadu et al., 2017). Likewise, Bayaa et al. (1994) reported that 27% of L. orientalis genotypes were resistant to the tested A. lentis isolates. By contrast an evaluation of 188 Indian lentil genotypes revealed only 5% of the accessions were resistant to the tested isolates (Singh et al., 1982). Meanwhile in Australia, of 488 lentil accessions only 5% were resistant to the three tested isolates, while 29% showed variable reactions (Nasir and Bretag, 1998). Although it is difficult to draw firm conclusions by comparing such studies due to different lentil genotypes and A. lentis isolates
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used, the cited studies do demonstrate considerable variation in resistance reactions to A. lentis. Thus, to confirm the utility of the FIGS approach for gene bank mining would require a comparative study where the approach is compared to at least multiple random selections.
This study, however, did demonstrate that the incidence of resistance to A. lentis does not seem to be randomly distributed geographically. Rather, the bulk of the resistant and moderately resistant accessions came from relatively small pockets that included northern Morocco and southern Spain, Ethiopia and south west Russia (trans-Caucuses) (Figure 2). This link to geographic locations has been noted in previous studies. Bayaa et al. (1994) observed that the majority of resistant wild lentil genotypes came from Syria and Turkey as did Tullu et al. (2010a) and Dadu et al. (2017), who studied wild lentil resistance to Canadian and Australian A. lentis isolates. This is not surprising given that wild lentils are native to west Asia. However, geographic specificity has also been observed in cultivated lentils. Nasir and Bretag (1998), who evaluated 488 lentil accessions from 25 different countries for resistance to three Australian isolates of A. lentis, found that almost all the resistant genotypes came from Pakistan. Further, Iqbal et al. (2010), who studied resistance of Pakistani lentils to A. lentis, found that most of the resistant genotypes were from the Punjab province. The prevalence of resistance found in Punjab material was proposed to be due to a strong positive selection pressure imposed by the wide distribution and occurrence of A. lentis in the province (Singh et al., 1982).
In the above context, the reader is reminded that the FIGS selection strategy used for this study was designed to capture collection sites that have climatic profiles most likely to favour A. lentis development and thus impose a selection pressure for resistance. However, because there are multiple factors that influence seasonal population dynamics of the pathogen, it is likely that the selection process could be improved. For example, it is suggested that the selection algorithm could include measures of seasonal variation for each variable. The rationale is that, environments that have a high degree of inter-seasonal stability and popular cultivars with widespread planting, populations of the pathogen are likely to evolve and produce aggressive isolates, that can infect even the best of the resistant cultivars. Thus, the selection process would be designed to favour environments with higher inter-seasonal stability for climatic variables critical to disease development. Further improvements could be made by
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combining the results of this study with previous studies to refine the environmental profiles (including geographic locations) most likely to predict resistant genotypes.
3.5.2. Evidence of better resistance to A. lentis in IG 207 than ILL 7537 and Indianhead
The evolution of A. lentis pathogenicity is important to consider when developing breeding strategies. In Australia, the presence of both mating types (MAT1-1 and MAT1-2) of A. lentis (Skiba and Pang, 2003) leads to sexual ascospores, which is assumed to quickly increase the genetic diversity of the fungal populations (Martin et al., 2013). Indeed, an assessment of genetic diversity within the Australian A. lentis populations showed that there were large variations among isolates collected from different parts of Australia (Ford et al., 2000). Likely, this variation in aggressiveness within the pathogen genepool has helped it to rapidly overcome simply inherited resistances. Therefore, it is important that new resistances be stable and quantitative to withstand the pressure created by the aggressive and evolving pathogen population.
The consistent resistance reaction demonstrated by IG 207 when challenged with four aggressive isolates (FT13038, FT15160, FT16299-2 and FT16112) indicates that its resistance to A. lentis is likely to be stable. These findings are in broad agreement with the hypothesis that resistance to A. lentis is probably far more polygenic than originally proposed (Gupta et al., 2012a), although further research is required to understand the genetics of resistance displayed by IG 207.
The literature suggests that most resistance sources to AB used in advanced lentil cultivars are derivatives of a single, or at most a few major resistance loci, and thus are likely to be vulnerable to the pathogen population at large (Ford et al., 1999; Nguyen et al., 2001; Sudheesh et al., 2016a). To sustain the lentil industry, it is suggested that multiple resistance genes need to be pyramided into elite cultivars (Sari, 2014). It is worth noting that the resistance genes derived from sources including CDC-Robin, ILL 7537, 964a-46 and ILL 1704 are proposed as non-allelic and therefore provide a unique opportunity to stack the different genes shared among them (Sari, 2014). In this study, clear discrimination of disease reactions to A. lentis (isolates FT13038, FT15160 and FT16299-2) between highly resistant IG 207 and currently employed resistance sources (Indianhead, ILL 7537 and Nipper) provided an initial
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indication that IG 207 carries resistance that is putatively different. The number of resistance genes and the nature of the resistance within the accession IG 207 and current resistance sources (Indianhead and ILL 7537) could be revealed through genetic analysis and allelism tests (Ye et al., 2001; Sari, 2014). Following confirmation of the allelic nature of these resistances and mapping of the genes, it would be desirable to pyramid these genes together in an effort to breed for durable resistance and benefit Australian lentil industry.
3.5.3. Evidence of better defence through early physical containment A. lentis in IG 207
The efficacy of a resistance source is dependent on the spatial and temporal distribution of defence responses to the invading pathogen, which are largely genotype-dependent (Khorramdelazad et al., 2018; Sari et al., 2018). Characterising such defence responses could help understand the resistance mechanism adopted by the newly identified resistance sources as well as indicate its novelty. This can be achieved by undertaking time-course histopathological assays and gene-expression studies. Such a study was undertaken by Sambasivam et al. (2016) and Dadu et al. (2018a) who histopathologically quantified and characterised A. lentis resistance of genotypes IG 7537 and ILWL 180. A more recent RNA- sequencing study revealed differential expression (DE) of defence related genes within lentil cultivars ILL 7537 and ILL 6002 and concluded that faster and stronger amplitudes in expression of these genes were critical for resistance to A. lentis (Khorramdelazad et al., 2018).
In this study, pre-penetration behavior including spore germination percentage, germ tube length, timing and formation percentage of appressoria of aggressive isolate FT13038 revealed significant differences between the resistance mechanisms deployed by accession IG 207 and Nipper. This again is in acceptance with the results of disease symptomology at all time intervals as mentioned above. Upon inoculation, spores of isolate FT13038 produced maximum germination within 24 hpi as previously observed on the leaflets of accession ILL 6002 (Sambasivam et al., 2016; Dadu et al., 2018a). It is worth noting that the spore germination percentage recorded for accession IG 207 was lower than well-known resistant accession ILL 7537 (Sambasivam et al., 2016; Sari et al., 2017) and though different isolates were tried, they were known to have similar aggressiveness (Dadu et al., 2017). The lower spore germination percentage for accession IG 207 might be due to early recognition of the pathogen associated molecular patterns (PAMPs) released by A. lentis assisted by pattern
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recognition receptors (PRRs) including receptor-like kinase proteins and receptor-like proteins (Dangl et al., 2013). By contrast, the delayed recognition by PRRs in cultivar Nipper might be responsible for the increased germination percentage.
Germinated isolate FT13038 produced significantly shorter germ tubes on IG 207 leaflets than those of cultivar Nipper at each time point except at 6 hpi. This hindrance of germ tube growth has also been observed on the leaflets of other resistant accessions including ILL 7537 (Sambasivam et al., 2016), CDC Robin, L-01-827A (Sari et al., 2017) and ILWL 180 (Dadu et al., 2018a). By contrast, the germ tubes grew extensively on the leaflets of cultivar Nipper, which indicates a delayed activation of defence mechanisms. On the other hand, it is proposed that the ability of IG 207 to recognize the presence of the fungus earlier than Nipper may result in the activation of the downstream defence responses in the host and provide an extended resistance against the pathogen infection.
Reduced germination and restricted growth of isolate FT13038 on the leaflets of accession IG 207 failed to produce adequate appressoria for tissue penetration. Conversely, spores of isolate FT13038 produced appressoria within 6 hpi on the leaflets of cultivar Nipper, which led to active penetration of tissue and colonization within 36 hpi. These findings are in agreement with previous studies Sambasivam et al. (2016) and Dadu et al. (2018), who reported similar differences in the timing and percent of appressoria produced between resistant and susceptible accessions (ILL 7537 and ILL 6002).
In conclusion, this study indicates that FIGS is an efficient sampling strategy to mine large germplasm collections for A. lentis resistance. However, studies that compare FIGS to alternate sampling strategies are required to support this assertion with more authority. Accession IG 207 was identified with higher and stable resistance to AB infection than resistant controls. A histopathological study suggested that relatively faster recognition of A. lentis presence on the leaflets is likely to contribute to the resistance displayed by IG 207. However, differential molecular studies would provide further evidence and a more complete understanding of the defence mechanisms within accession IG 207 to A. lentis infection.
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