Biochemical Basis of Resistance in Chickpea (Cicer arietinum L.) against Wilt Complex

Chickpea (Cicer arietinum L.) is an important pulse crop of India. It is grown in more than 50 countries and India is the largest producer of chickpea in the world. India occupied about 10.56 million hectares area and total production of 11.23 million tonnes with average productivity of 1063 kg/ha during 2017-18 (Anonymous, 2018).
       
Nearly 172 pathogens (67 fungi, 3 bacteria, 22 viruses and 80 nematodes) have been reported infecting chickpea world-wide (Nene et al., 1996). Chickpea wilt complex caused by several soil-borne pathogens is highly destructive disease that causes severe yield losses. In these soil-borne pathogens, Fusarium wilt [Fusarium oxysporum Schlech tend. Fr. f. sp. ciceri (Padwick) T. Matuo and K. Satõ], black root rot [Fusarium solani (Mart.) Sacc.], dry root rot [Macrophomina phaseolina (Tassi) Goidanich], wet root rot [Rhizoctonia solani Kühn] and collar rot [Sclerotium rolfsii Sacc.] are of considerable importance (Nene et al., 1981).
       
 
Management of wilt complex of chickpea is difficult to achieve as the pathogens are soil-borne, surviving through resistant structure i.e. chlamydospores and sclerotia in the soil for years even in the absence of host and the crop remains susceptible throughout all the growth stages (Haware et al., 1986; Kaiser et al., 1994).
               
The development of resistant cultivars is the most viable, environmentally safe and economically sound technique for the management of the disease. Further, screening of disease resistant plants can be effective if the biochemical parameters of disease resistance in plants are well understood as the host-pathogen interaction is very complex and diverse in nature. During the survey in major chickpea growing regions of Gujarat, Fusarium oxysporum f. sp. ciceri, Fusarium solani and Macrophomina phaseolina were found predominantly associated with wilt complex infected chickpea plants. Hence, the present investigation was therefore undertaken to find out the resistant sources of chickpea varieties/germplasm against these soil-borne pathogens during rabi 2018-19 and rabi 2019-20 and then to identify biochemical parameters associated with the resistance.

A total of thirty-seven varieties/germplasm of chickpea including national check JG 62 were screened under pot conditions at the Department of Plant Pathology, B. A. College of Agriculture, AAU, Anand during rabi 2018-19 and 2019-20.
 
Screening of chickpea varieties/germplasm
 
The sick pot technique developed by Nene et al., (1981) was followed for the experiment. Sterile soil was placed in 30x30 cm earthen pots. Pure cultures of F. oxysporum f. sp. ciceri, F. solani and M. phaseolina were grown on Potato Dextrose Agar (PDA) medium and multiplied on 100 g of 9:1 sand maize meal medium in 250 ml conical flasks for 15 days at 25±1^oC. The fungus grown on sand maize meal medium in flasks was added at 100 g/2 kg of soil. Twenty seeds of respective varieties/germplasm of chickpea were sown in the pot. Observations on seedling mortality were recorded up to 30 days after sowing. The final observation on seedling mortality was considered to categorize the chickpea varieties/germplasm (Table 1) into different reactions (Nene et al., 1981).
 

       
Seedling mortality was calculated (Pande et al., 2012) as follow:
   
Biochemical analysis
 
The effect of biochemical characters was estimated from two of each promising varieties/germplasm representing resistant and susceptible disease reaction from diseased and healthy plant roots. The sampling was done during the morning hours. The samples were then transferred to polythene bags separately and carried to the laboratory in an icebox containing ice cubes to prevent any denaturation of enzymes.
       
Moisture content was estimated by using the following formula:
   
The protein content (%) was estimated by Lowry et al., (1951), phenols (mg/g of fresh weight) were estimated in acetone extract followed by addition of Folin-Ciocalteau reagent using catechol as a standard method (Bray and Thorpe, 1954) and total soluble sugars (mg/g of fresh weight) were determined by following the method suggested by Bhatnagar et al., (2006) with some modifications.
               
All these experiments were done in triplicate in completely randomized design (CRD) and the results were statistically analyzed. The biochemical analyses were carried out at the Department of Biochemistry, BACA, AAU, Anand.

Screening of chickpea varieties/germplasm
 
The varieties/germplasm were grouped under different degrees of resistance based on per cent disease incidence. The result presented in Table 2 showed that there were considerable differences among the varieties/germplasm for the level of resistance against chickpea wilt complex during two years of experimentation.
 

       
Based on two years of data, the final disease reaction has been worked out. The final reaction indicated that only two varieties/germplasm viz., Gujarat Junagadh Gram 6 and GAG0624 showed a resistant reaction while three varieties/germplasm viz., Gujarat Junagadh Gram 3, Dayadara and Khedbrahma showed a moderately resistant reaction.
       
The 20 varieties/germplasm viz., Gujarat Gram 4, Gujarat Gram 5, Arva, Puhri, Varantha, Godhara nazik, Saliya, Potiya zol, Kheroj, Pavi,  Ghelvat, Khakhariya, ACP-21, GAG 1107,  GAG 1423, CSJ 740, H 14-22,  ACP 1070, ACP 1075 and ACP 1076 showed as moderately susceptible and twelve varieties/germplasm viz., Gujarat Gram 1, Gujarat Gram 2, JG 62, Bavsar, Bagodara, Rayka, Baspa, Kheralu, Vitthalgadh, Mulava, CSJ 882 and ACP 1071 were as susceptible to the disease.
       
In the present studies, varieties/ germplasm viz., Gujarat Junagadh Gram 6 and GAG0624 were identified as resistant which can be further exploited in resistance breeding programme. A similar type of results was reported by earlier workers viz., Pande et al., (2007), Amule et al., (2014), Wagh et al., (2018), Sharma et al., (2019) and Talekar et al., (2021) against soil-borne pathogens of chickpea.
 
Biochemical analysis
 
The biochemical variation was estimated from diseased and healthy plant roots from two each of resistant (GJG 6and GAG 0624) and susceptible (JG 62 and Bagodara) varieties/germplasm (Table 3 to 6).
 

 

 

 

 
Moisture
 
Healthy roots of both germplasm contained a higher amount of moisture per cent than diseased roots (Table 3). The moisture content was higher (88.42 and 87.53%) in healthy roots of resistant germplasm i.e. GJG 6 and GAG 0624 and 85.90 and 86.21% in susceptible germplasm (JG 62 and Bagodara) followed by diseased roots (80.22 and 77.18%) of resistant germplasm and 74.71 and 75.52% of susceptible germplasm, respectively. So, the moisture content was higher in roots of healthy variety/germplasm than diseased roots of both groups of variety/germplasm.
 
Protein content
 
The result revealed that protein content was higher in healthy (GJG 6: 5.74; GAG 0624: 5.58%) as well as diseased (GJG 6: 3.92; GAG 0624: 3.76%) roots of resistant germplasm than healthy (JG 62: 3.60; Bagodara: 3.33%) and diseased (JG 62: 2.32; Bagodara: 1.72%) roots of susceptible germplasm. The least rate of decrease over healthy 32.14% was recorded in resistant germplasm, whereas it was highest 41.82% in susceptible germplasm (Table 4).
     
Protein in plant response to adverse conditions is very important as proteins are an indispensable component of innate immune responses in plants under biotic or abiotic stress conditions. Saxena and Karan (1991) and Ushamalini et al., (1998) also reported a decline in the protein content of sesame and sunflower seeds artificially inoculated with F. moniliforme, M. phaseolina, Alternaria alternata, Aspergillus flavus and A. niger.
 
Phenol content
 
The result presented in Table 5 revealed that healthy roots of resistant germplasm (GJG 6: 0.62; GAG 0624: 0.61 mg/g fresh weight) contained a higher amount of total phenol than susceptible one (JG 62: 0.51; Bagodara: 0.51 mg/g fresh weight).
       
The phenol amount was increased in resistant and susceptible diseased roots, but the higher increase of phenol was observed in resistant germplasm, while it was at low insusceptible germplasm. The result revealed that per cent increase in total phenol was higher (27.95%) in resistant germplasm, while it was low (25.14%) in susceptible germplasm.
       
Phenol is known to impart resistance against pathogens, because of their antimicrobial activity. It is often assumed that their main role in plants is to act as a protective compound against disease caused by fungi, bacteria and viruses. The higher amount of phenolic compounds in diseased roots of chickpea germplasm may be due to several factors including either enhancement of synthesis or translocation of phenolic to the site of infection. These findings are in harmony with the similar studies carried out on activity of phenol changes due to pathogen infection by Rathod and Vakharia (2011), Belkar et al., (2018) and Jyothi et al., (2018). They also found an increase in the activity of phenol in chickpea due to infection of F. oxysporum f. sp. ciceri and M. phaseolina.
 
Total soluble sugars
 
Higher amount of total soluble sugars content were presented in healthy roots of resistant germplasm (GJG 6: 5.09; GAG 0624: 4.94 mg/g fresh weight) than susceptible germplasm (JG 62: 3.19; Bagodara: 2.95 mg/g fresh weight). The least rate of decrease (32.14%) was recorded in resistant germplasm, whereas the highest rate of decrease (41.82%) was recorded in susceptible germplasm (Table 6).
               
Sugars play an important role in the inhibition of pectinolytic and cellulolytic enzymes, which are essential for the pathogen. Moreover, sugars are precursors of phenolics, which are highly toxic to germplasm. Results similar to the present investigations were observed by Ushamalini et al., (1998), Belkar et al., (2018) and Jyothi et al., (2018) as they reported high sugars content in resistant genotypes compared to susceptible genotypes.

Among the thirty-seven chickpea varieties/ germplasm screened, two lines viz., Gujarat Junagadh Gram 6 and GAG0624 can be used for wilt complex resistance breeding program based on their resistance reaction. Further, biochemical constituents revealed that the diseased plant roots had a lower percentage of moisture, protein and total soluble sugars over the healthy plant roots whereas, the phenol content was higher in the diseased plant roots.

Authors gratefully acknowledge Agricultural Research Station, Anand Agricultural University, Arnej and Pulse Research Station, Junagadh Agricultural University, Junagadh for providing screening material.