**7.6. Modern breeding approach**

study suggests that the application of Zn and Mn salts at 80 ppm concentration on presoaked

The initial step in managing the disease is to develop a reliable and reproducible disease screening techniques, so that a large number of germplasm (cultivated and wild relatives) can be evaluated in wilt sick plot and in greenhouse. The varietal resistance is a major goal of lentil improvement programme currently running at the International Centre for Agricultural Research in the Dry Areas (ICARDA). In order to identify the resistant variety of Fusarium wilt, screening under field and controlled conditions (green house and laboratory conditions) has been suggested [93, 94]. The systematic utilization of resistant source for wilt from cultivated accessions such as 'ILL 5883', 'ILL 5588', 'ILL 4400' and 'ILL 590' has resulted in the development of a wide spectrum of Fusarium wilt resistant varieties at ICARDA. Some of the prominent wilt resistant varieties in Syria ('Idleb 2', 'Idleb 3', 'Idleb 4' and 'Ebla 1'), Lebanon ('Talya 2', 'Rachayya' and 'Hala'), Turkey ('Firat 87' and 'Syran 96'), Ethiopia ('Adaa', 'Alemaya', 'Assano', 'Alemtena' and 'Teshale'), Iran ('Kimiya') and Iraq ('IPA 98') [95]. In India, several wilt resistant varieties are released such as 'L 4147', 'Pant L 406', 'Pant L 4', 'Pant L 639', 'Priya', 'Seri', 'JL 3',

The lentil germplasm can be screened under natural condition with natural inoculum of *Fol* in field. Wilt sick plot (WSP) is the most common method used to screen disease resistant plants under natural conditions. The WSPs have been developed by ICARDA, and NARS. The advantage of this method is that, large number of genotypes can be screened. Bayaa et al. (1997) has screened a core collection of 577 lentil germplasm accessions from 33 countries. The result reveals that the most resistant accessions came from Chile, Egypt, India, Iran and Romania and also emphasize the relative uniformity of disease pressure in WSPs [19].

Different inoculation methods have been used to for the infectivity of wilt in chickpea but in lentil very limited work has been conducted [12, 98, 99]. The inoculum density of about

The most economical means to control the Fusarium wilt of lentil is through the development of resistant varieties [12]. Due to the evolution of new races and co-existence of more than one

 conidia ml−1 is generally been used to establish the pathogen [100]. Wild species are an invaluable source for disease resistance. The wild germplasm of lentil was evaluated for resistance against biotic and abiotic stresses was done at ICARDA [101]. The crosses were made between the wild lentil (*L. culinaris* ssp. *orientalis*) and the cultigen has resulted in highyielding selections under dryland conditions. Similarly, another study was done to screen the 221 accessions representing five species/subspecies, showed resistance in ILWL 113 (*L. culinaris* ssp. *orientalis*) from Turkey and ILWL 138 (*L. ervoides*) from Syria [102]. In India, seventy accessions representing four wild species/sub-species were evaluated and the donors for *Fol* resistance were identified in all species. The wild accessions of lentil (one of *L. culinaris* ssp. *orientalis* (ILWL76), five of *L. odemensis* (ILWLs 35, 39, 153, 237, 300), eleven of *L. ervoides* (ILWLs 40, 41, 42, 133, 204, 251, 258, 261, 271, 280 and 299) and six of *L. nigricans* (ILWLs 22, 26, 31, 37, 38, 430) can provide an important source of alien genes for disease resistance [103].

seeds of lentil has shown promising results on the control of wilt disease [92].

126 Fusarium - Plant Diseases, Pathogen Diversity, Genetic Diversity, Resistance and Molecular Markers

**7.4. Fusarium wilt resistant cultivars**

106

'Noori', and 'VL 507' under national program [65, 96, 97].

**7.5. Genetic of Fusarium wilt resistance**

The classical plant breeding is based on recombination breeding approach by selecting the desirable plants on the basis of their phenotypic characters. However, this approach is less precise and time consuming when dealing with quantitative traits which are highly influenced by environment and genotype-environment (GE) interaction [112]. Therefore, it is important to integrate modern biotechnological tools such as genetic engineering and marker assisted selection (MAS) in lentil breeding program to mainstream new genetic variability in the cultivated gene pool.

In early 1980s, the first genetic linkage map of lentil was constructed using morphological and isozyme markers [113, 114]. Later, Eujayl et al. (1998) has reported first comprehensive linkage map with 177 RAPD, AFLP, RFLP, and morphological markers was developed using inter specific recombinant inbred lines (RIL) population of a single cross of *L. culinaris* × *L. orientalis* [104, 106]. Hamwieh et al. (2005) added 39 SSR and 50 AFLP markers to the comprehensive Lens map constructed by Eujayl et al. (1998), comprising 283 genetic markers covering 715 cM. They have constructed first genomic library from a cultivated accession, ILL5588 using the restriction enzyme Sau3AI (*Staphylococcus aureus* 3A) and screened with (GT)10, (GA)10, (GC)10, (GAA)8, (TA)10, and (TAA) probes. This study reveals that only SSR59-2B was closely linked with Fw at 19.7 cM [115]. In an another study, a set of 122 functional SSR markers have been developed using a genomic library enriched for GA/CT motifs for utilization in the lentil breeding program [116].

In Syria, the effect of different control options on disease parameters and yields were conducted as field experiment. The control options are changes in sowing dates, host plant resistance and fungicide seed treatment and the disease parameters like wilt onset, duration, per cent terminal wilt and areas under the disease progress curve were considered. The results revealed that the lentil genotypes had a greater effect on the onset and duration of Fusarium wilt than planting date or fungicide seed treatment. The percent terminal wilt and areas under the disease progress curve was observed lowest during November plantings for all lentil genotypes [78]. Therefore, different individual control options should be recommended to mitigate the effect of Fusarium wilt on lentil yield include manipulation of sowing date,

Fusarium Wilt: A Killer Disease of Lentil http://dx.doi.org/10.5772/intechopen.72508 129

Knowledge about the pathogen has improved since it was observed, but still few challenges remain. A region specific race of the pathogen is needed. Since there are potential differences in the reaction of lentil cultivars to different races of the pathogen, so information about the distribution of races will be of great importance for breeding programs and the development of resistant genotypes. Along with this, a standardized set of host differentials is required to correlate pathogenicity with DNA techniques. A robust screening techniques for resistant to the pathogen is also required. With the lack of host-pathogen interaction studies, management remains elusive and additional research is needed in this area. Marker assisted selection (MAS) offers great opportunity for improved efficiency and effectiveness in the selection of plant genotypes with a desired combination of traits. Through marker assisted selection, disease resistance can be evaluated in the absence of the disease and in early stages of plant development. Implementation of markers for routine use in lentil breeding programs is currently very limited, integration of the markers within the breeding program to ensure that cost effective utilization of the technology is achieved. Establishment of a tight linkage between a molecular marker and the chromosome allocation of the gene(s) governing the trait to be selected in a particular environment is required. The information from multiple populationspecific genetic maps can be integrated to produce high-density consensus structures utilizing the sequence-linked genetic markers which enables the identification of bridging loci between maps. This will further assist in the identification of more closely linked markers for Fusarium wilt resistance in lentil that can be effectively used in breeding and there is a need to develop and map more functional markers like EST-SSRs and SNPs on such maps to enhance their relevance in lentil genetics and breeding. The study based on SNP markers is still limited in lentil due to the lack of available sequence data. For effective variety development marker assisted selection is very imprint that requires much attention in lentil breeding program. Comparative genomics and synteny analyses with closely related legumes can play an important role in enhancing the knowledge of the lentil genome and can provide the genes and selectable markers for use in MAS. Transgenic and non-transgenic approaches including RNAi technology and virus-induced gene silencing (VIGS) can be explore to understand the molecular mechanisms of host resistance in lentil. Additional refined genetic materials are required in order to apply advanced genomic tools such as transcriptome profiling and

fungicide seed treatment, biological control agents and host resistance [52].

**8. Future directions**

As lentil has a narrow genetic base an inter-varietal linkage maps were developed by utilizing diverge parents from the wild and cultivated species but these maps have low recombination rate and the map size is also small. QTLs responsible for many traits can be identifying by intra specific mapping population and desirable gene of interest can be tagged. First intra specific lentil map was developed by Ford et al. (2003) through RAPD and ISSR markers [117]. Bi-parental mapping populations derived from the most divergent parents are always better for developing recombinant inbred line and through that a dense mapping or fine mapping can be done from the population developed through the cross of resistant and susceptible parents. These maps are useful to identify genes and major QTLs responsible for the variation of the trait of interest.

Gene cloning can help to characterize the function of the gene or QTLs responsible for the wilt and the knowledge of the genes cloned in lentil can facilitate the development of functional markers for the marker assisted selection. Resistant genes for different functions have cloned in lentil [118]. Using functional genomics approaches, genes expressing differentially in contrasting lentil genotypes can be identified.

Focusing towards the natural defense of host plant may reduce the impact of the pathogen on productivity. However, our poor knowledge about the molecular interaction between the crop and the pathogen limits support for breeding disease-resistant varieties. Due to the development of sequencing technologies, several genes coding transcription factors (TFs) and candidate defense genes (CDGs) from lentil are identified [112]. The full sequence of candidate defense genes like a β-1,3-glucanase (GLU1) (CV793598), a pathogenesis-relate (PR) protein from the Bet v I superfamily (AY792956), a disease resistance response protein 230 (DRR230-A) from pea (AJ308155), another disease resistance response protein (DRRG49-C) from pea (J03680), a pathogenesis-related 4 (PR4) type gene (DY396388) and a gene encoding an antimicrobial SNAKIN2 protein from tomato (HQ008860) are available in NCBI Genbank [112]. A partial sequence of translation elongation factor-1α (TEF-1α) (KR061303 and KR061304) from *Fusarium nygamai* infecting lentil were also deposited in Genbank [33]. These candidate genes and TFs should be further biologically characterized and can help us in decoding the defense pathways and pathogen recognition.

### **7.7. Integrated management of Fusarium wilt of lentil**

Integration of two or more disease management option can reduce the impact of any disease affecting crops. The expected benefit in opting this strategy is improved and sustainable control of disease. The use of biocontrol agents in combination with chemical control can act as one of the strategies in controlling some soil-borne diseases. Therefore, some researchers have used the combination of *Bacillus megaterium* with carbendazim, which provided an effective control of Fusarium crown and root rot of tomato [119]. Similarly, the combination of soil amendments and biological control agents such as *Trichoderma* spp., have been shown to increase disease control and horticultural productivity [120]. Nowadays the use of organic amendments to improve soil properties, plant health and yield has expanded [121].

In Syria, the effect of different control options on disease parameters and yields were conducted as field experiment. The control options are changes in sowing dates, host plant resistance and fungicide seed treatment and the disease parameters like wilt onset, duration, per cent terminal wilt and areas under the disease progress curve were considered. The results revealed that the lentil genotypes had a greater effect on the onset and duration of Fusarium wilt than planting date or fungicide seed treatment. The percent terminal wilt and areas under the disease progress curve was observed lowest during November plantings for all lentil genotypes [78]. Therefore, different individual control options should be recommended to mitigate the effect of Fusarium wilt on lentil yield include manipulation of sowing date, fungicide seed treatment, biological control agents and host resistance [52].
