**3. Genetics of Resistance to Fusarium Wilt**

The *Fusarium oxysporum* f. sp. ciceris (*FOC*) pathogenies known to possess great pathogenic diversity that is classified into different pathogenic races, including races 0 and 1A, 1B/C, 2, 3, 4, 5, and 6. Additionally, two categories of FW symptoms have been identified: early yellowing and late wilting [55,56]. Additionally, researchers have also looked at the genetics of races 1A, 2, 3, 4, and 5 [57]. The symptomatic wilting pathotype induces quick and severe chlorosis, flaccidity, vascular discoloration, and early plant death, mostly in races 1A, 2, 3, 4, 5, and 6 [55], whereas the symptomatic yellowing pathotype instigates slow foliar yellowing, vascular discoloration, and late plant death in races 0 and1B/C [56,57].

It has been documented that chickpea resistance to Fusarium wilt can be either monogenic or oligogenic (Table 1) depending on the source or race of the resistance [57]. Three distinct genes (h1, h2, and H3) independently govern resistance to race 1A, according to early investigations on *FOC* [58]. Late wilting resistance can be conferred by any one of these three genes, but total resistance can be conferred by any two of these genes (h1h2, h1H3, or h2H3) [59]. While resistance to race 3 has been proven to be monogenic, resistance to race 2 is controlled by a single recessive gene [60,61]. As stated in earlier studies, race 4 resistance is recessive and digenic, but race 5 resistance is governed by a single gene [62].

Geographical classifications of the pathogenic races of *FOC* have been made. Indian, Mediterranean, and American populations of race 1A have been documented [63]. In addition, race 4 has been documented in Ethiopia, India, and Iraq [64,65]. Races 0,1B/C,5, and 6 are most common in the Mediterranean Basin and California (USA) [66], while races 2 and 3 have been observed in Ethiopia, India, and Turkey [50].


**Table 1.** Genetics of resistance to races of the chickpea wilt *Fusarium oxysporum* f. sp. ciceris.

#### **4. Breeding Methods Employed for Fusarium Wilt Resistance in Chickpea**

Higher and more consistent yields are the main objectives of chickpea breeding programs [15]. According to an investigation conducted by Srivastava et al. [68], chickpea resistance to Fusarium wilt may be either monogenic or oligogenic, depending on the resistance source or race. The selection of plants for characteristics and disease resistance is the second most important step in a breeding program involving evaluation of the plant for commercial production.

Breeding programs are dependent upon the magnitude of genetic variation present in the population. The type and degree of diversity influence a breeding strategy's efficacy. Even though the disease is soil-borne, chemical control is ineffective and impractical to use [69]. Utilizing host plant resistance is the most reliable strategy for solving the problem. Several sources of chickpea resistance to Fusarium wilt have been found in the past. These resistance sources have been identified using different methods, including a wilt-diseased plot in the field and hot spot location screening, as well as greenhouse and laboratory procedures [70–72]. The majority of these methods were employed in resistance breeding programs at the National Agricultural Research System (NARS) and International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), which significantly increased chickpea productivity in semi-arid parts of Africa and Asia [73,74]. However, in these areas, substantial genetic diversity in the pathogen and GxE interaction have an impact on resistance durability. A variety of strategies, including the GGE billet technique, have been utilized in different studies to investigate the GxE interaction [75]. Utilizing biplot analysis of GxE data, it is now possible to graphically address many important aspects to develop a better understanding, including genotype stability, mean performance, discriminating ability, mega-environmental investigation, representativeness of the environment, and who-resistant-where pattern [76–78].

The process of using plants as a strategy involves gathering and analyzing genotypes from different sources in order to find suitable genotypes that are adapted to the local environment and have high productivity or any other desired specialized attribute [79]. As a result, the type of material introduced determines whether plant introductions are successful. Genes must be fixed in breeding lines in order to create pure-line cultivars. The

initial selection process that uses landraces is the simplest and is known as mass or pure-line selection. Crossover programs and several iterations of pedigree and bulk approaches were employed to manage segregating generations [79,80]. Through pure-line selection, the JG315 chickpea cultivar evolved resistance to Fusarium wilt in Madhya Pradesh, India. The JG 62 cultivar, in addition to race 0, is a variety that is very vulnerable to FW, whereas ICCV 05530 is a cultivar that is highly resistant to FW [81].

Most breeding operations for chickpeas use single-cross hybridization. Hybridization almost occurs within the same species of the genetically distinct Desi and Kabuli varieties [82]. To promote genetic diversity and introduce beneficial genes from wild *Cicer* spp. into cultivated species, interspecific crosses have been attempted. *FOC* race resistance has largely been found in the Desi germplasm and in wild *Cicer* spp. In fact, accessions of *C. bijigum*, *C. cuneatum*, and *C. judaicum* showed combined resistance against races 0 and 5, but accessions *C. canariense* and *C. chorassanicum* were found to be resistant to race 0 whenever vulnerable to race 5. Additionally, the *C. pinnatifidum* accessions evaluated were found to be vulnerable to race 5, whereas some were resistant to race 0 [83].

Various chickpea breeders have used traditional methodologies and breeding techniques, and the population has improved in terms of increased output, different resistance, and desired plant types. Regarding FW response, genetic heterogeneity in chickpea genotypes has been recorded [84]. In accordance with the earlier findings, resistant sources were identified against FW in both Kabuli (ICCV 2 and UC 15) and Desi types (FLIP 85-20C, FLIP 85-29C, and FLIP 85-30C). Numerous chickpea Fusarium wilt-resistant genotypes, including ICCV 98505, ICCV 07105, ICCV 07111, and ICCV 07305, were identified by Sharma et al [85] using GGE biplot analysis. Four Kabuli chickpea genotypes resistant to FW, including ICCV 2, ICCV 3, ICCV 4, and ICCV 5 (Table 2), were previously generated using the pedigree method. Crop breeders now have a range of more effective tools for resistance breeding owing to recent developments in legume genomic technologies. As a result, legume crops can now be improved using genomics to better withstand different biotic and abiotic challenges [86,87].


**Table 2.** Important cultivars/donors (genetic resource) contributing to Fusarium wilt resistance.

Pande et al. [70] found twenty-one accessions free from FW disease and twenty-five that were resistant during their study on the screening of chickpea genotypes against FW. In a separate study, genotypes JG 315, Avrodhi, DCP 92-3, JG 74, BG 372, and KWR 108 were found to be resistant to Fusarium wilt [87], while ICCV 05530 maintained its resistance against two FW races, viz., 1 and 3. Among these genotypes, JG 62 showed 89–100% wilt incidence against both FW races.

The use of nested association mapping (NAM) and multi-parent advanced generation intercross (MAGIC) populations is being developed in chickpea to make inter-crosses between multiple (4, 8, or 16) parental lines that originate from diverse regions. The creation of these crosses is possible through the balanced funnel crossing method, which recombines mosaics of founder parents, resulting in novel genotype and haplotype combinations [89]. At ICRISAT, a MAGIC population was created by mating cultivars and elite breeding lines, including ICC 4958, ICCV 10, JAKI 9218, JG 11, JG 130, JG 16, ICCV 97105, and ICCV 00108, with eight varied founder parents [73,85,88].

#### **5. Screening Strategies to Identify Wilt-Resistant Genotypes**

The utilization of host plant resistance (HPR) begins with the development of trustworthy and reproducible disease screening techniques to assess many germplasm accessions and breeding materials. It has been claimed that screening in the field and under controlled conditions (such as in greenhouse and lab settings) may help to identify resistant genotypes against FW [94]. However, there are some problems associated with maintaining uniform conditions for each plant during the screening of genotypes. So, it is important to develop a simple and efficient technique to screen chickpea genotypes for the identification of FW-resistant cultivars for future breeding programs. Generally, the following methods are applied for the screening of Fusarium wilt-resistant chickpea genotypes.

#### *5.1. Field Screening*

The most frequent and recurrently applied technique for identifying FW-resistant genotypes is the wilt-diseased plot (WDP) strategy. The primary advantage of the WDP technique is that it makes it possible to screen a vast array of genetic materials under field conditions [95]. Effective wilt-diseased plots for field and hot spot location screening, as well as greenhouse and laboratory methodologies and successful breeding programs, have all been created [96]. Assessing inoculum homogeneity in a plot involves planting test genotypes next to susceptible cultivars as an indicator line or checking susceptibility after every 2–4 test entries. The widely applied susceptibility checks for races 1 to 4 in India include "JG 62", a twin-podded chickpea type that is extremely susceptible to all *FOC* races except race 0. The cultivar "JG 74" and the germplasm line "WR 315" (ICC 11322) of chickpeas are the two main sources of resistance. While the latter is resistant to all races but race 2, the prior is resistant to all *FOC* races except for race 3. The stepwise identification of host plant resistance to diseases has recently been revised by Pande et al. [89]. In order to screen many germplasm lines against FW, WDPs have been created at the International Center for Agricultural Research in the Dry Areas (ICARDA), ICRISAT, and NARS of countries that cultivate these crops.

Chickpea wilt has been investigated globally since the last decade of the 20th century using several methods. These efforts have involved the creation of multiple disease grading scales to calculate disease incidence and prevalence when evaluating new chickpea germplasm lines. Disease reactions are categorized based on the proportion of dead plants, whereas physiological maturity represents the reaction score of each genotype. To determine phenotypic resistance and susceptibility for race identification, different disease scoring scales are applied.

The six-point scale makes scoring simple (Table 3). Interpretation of the scale is as follows:


**Table 3.** Details of scoring scale to calculate Fusarium wilt disease incidence in chickpea.

#### *5.2. Screening under Controlled Conditions*

#### 5.2.1. Greenhouse Screening

Conducting screening under controlled conditions using a greenhouse can be a useful technique to verify the outcomes of evaluating wilt-diseased plots (WDP). This is crucial for researching the molecular mapping and tagging of a specific disease race, as well as the inheritance of pathogens [85]. Furthermore, pathogenic diversity studies can be carried out under controlled circumstances to learn the disease's genotypic information [89]. To screen the chickpea germplasm in greenhouses, the pot culture method has been standardized [97]. Another method that is frequently used for growing chickpea is root dip inoculation under greenhouse screens [94]. The identification of ninety percent of wilt in susceptible lines is guaranteed using the pot screening technique, although soil compaction from repeated irrigation may impair the association between pot and field performance. The chickpea seedlings are raised in autoclaved soil, dipped in inoculum at the roots, and then, transplanted into pots containing autoclaved soil, and the disease incidence is then measured [97]. There are some limitations to the greenhouse screening method, as well. It is very difficult to maintain uniform density of the inoculums in each diseased plot. So, it is not possible to differentiate the wilted plants in to early, late, and resistant categories.

#### 5.2.2. Laboratory Screening

Laboratory screening methods include various technologies, such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), quantitative PCR (qPCR), etc., for the accurate detection of *FOC*. In chickpea, artificial screening methods have been created by ensuring uniform inoculum load at the same vegetative stage of each test plant. This method guarantees that all inoculated plants have a roughly equal chance of infection by injuring the roots prior to inoculation [98]. Using this method, 25 resistant genotypes and 21 asymptomatic genotypes were identified. The method was applied to 211 genotypes from a core collection that included more than 16,000 unique chickpea germplasm accessions [70]. It has been suggested that pollen bioassays be employed as a quick and effective screening method to distinguish between resistant, late wilting, and susceptible genotypes [99]. One of the poisons produced by the fungus, fusaric acid (FA), is used as a selection agent to examine the genotypes of chickpeas.

#### **6. Management of Fusarium Wilt in Chickpea**

Management techniques to treat the disease are always adopted after a thorough disease evaluation. The management of Fusarium wilt in chickpea cannot be fully accomplished using a single control measure [100]. Elimination of the pathogen, as well as a reduction in the quantity and/or effectiveness of the main inoculums, are necessary for disease management [101]. The ideal control measure for such a goal should include the efficient application of one or a combination of the following management strategies:
