Biochemical Basis of Resistance to Sterility Mosaic Disease in Pigeon Pea Genotypes

Pigeon pea [Cajanus cajan (L.) Millsp.] is an important pulse crop of India with >75 per cent contribution to the world’s pigeon pea production. It is a substantial source of protein for the human diet. It is a perennial shrub with the chromosomal number 2n = 2x = 22 grown annually for food, feed, fodder or fuel throughout Asia, Africa, Latin America and the Caribbean. Globally, pigeon pea is cultivated in over 6 million hectares, with a production of 5.47 million tonnes (FAO, 2022). India is leading in pigeon pea production with an area of 4.54 million hectares and annual production of 3.83 million tonnes (Anonymous, 2021).
       
Sterility mosaic disease which is caused by Pigeon pea Sterility Mosaic Virus (PPSMV), is considered as an economically important disease of pigeon pea in India and Aceria cajani Channabasavanna transmits the virus in a semi-persistent manner (Manjunatha et al., 2012). Based on their molecular characterization, PPSMV - I has five negative sense dsRNA segments whereas, PPSMV - II has more than six negative sense dsRNA segments. Based on the morphological and molecular characteristics, both PPSMV - I and PPSMV - II viruses are placed in the family Fimoviridae and under the genus Emaravirus (Kumar et al., 2017).
       
The field symptoms of SMD (Sterility Mosaic Disease) include bushy plants, mild mosaic, severe mosaic, smaller leaves, excessive vegetative growth, stunted development, chlorotic ring patches, partial sterility and complete sterility (Siril et al., 2022). Early infection can cause 95 to 100 per cent yield loss, but late infection loses vary depending on the level of infection and ranges from 26 to 100 per cent (Dharmaraj et al., 2004). This is a major area of concern as pigeon pea demand in India is constantly increasing. It is worthwhile to study pigeon pea genotypes that are resistant to sterility mosaic disease and the mechanisms underlying their resistance, since cultivating resistant genotypes is an efficient and affordable alternative to chemically controlling plant diseases. Host plant resistance may be genetic or ecological. A possible defense mechanism against mite vectors and viruses is morphological and phenological formation of secondary metabolites, anatomical features, nutritional status and biochemical and microbiological characteristics (Manjunatha et al., 2021).
       
It is generally known that the disease resistance mechanism is a complicated process and that plants create a variety of biochemical responses in response to the invasion of a disease-causing organism. The current study focuses on identifying SMD-resistant pigeon pea genotypes by screening under field conditions and biochemical components which aids in defining the features that play a major role in SMD resistance in pigeon pea.

Screening of pigeon pea genotypes for resistance to sterility mosaic disease
 
A large number of genotypes were screened at AICRP on Pigeon pea, Voluntary Center, RARS, Vijayapur during Kharif 2020. A total of 41 pigeon pea genotypes were collected from AICRP on Pigeon pea, Voluntary Center, RARS, Vijayapur and ICAR - IIPR, RRC, Dharwad. Further, the genotypes were subjected to field screening at Indian Institute of Pulse Research, Regional Research Center, Dharwad during Kharif 2021 and 2022 against Sterility Mosaic Disease (SMD). These were sown in rows of 4 m in length with a spacing of 90 × 20 cm with ICP 8863 and Gulyal Local as susceptible checks (infector row) and Bahar as a resistant check in a non-replicated augmented design. All the recommended agronomic practices were followed, except disease management practices. All the test seedlings of pigeon pea genotypes were artificially inoculated by leaf stapling technique as per Nene et al., (1981) at 15-20 days after sowing and infector hedge row technique was also employed to develop high disease pressure. Per cent disease incidence (PDI) was calculated by using the following formula and the genotypes were categorized into different disease reactions based on average PDI by adopting the criterion followed in All India Co-ordinated Research Project (AICRP) on pigeon pea.


 
 
 
Biochemical analysis of SMD resistant and susceptible pigeon pea genotypes
 
The biochemical parameters were assessed for healthy plants at 15 DAS and diseased plants at 45 DAS to know the biochemical activity before and after the infection of the virus. The leaf samples collected from the selected pigeon pea genotypes showing both resistant and susceptible reactions and were analysed for chlorophyll content using SPAD Meter (60 and 90 DAS), total phenol content, peroxidase and poly phenol oxidase activity. Selected genotypes viz., Bahar (Resistant), KA 17-2 (Resistant), BDN 2013-45 (Moderately resistant) and ICP 8863, Gulyal local and IPA 15-02 (Susceptible) genotypes were taken for phenol and enzyme activity assay. Analysis of variance (ANOVA) was carried out for different parameters by following a Completely Randomized Design with three replications using IBM SPSS statistics 21. Estimation of phenols was carried out with the Folin-Ciocalteu reagent by following the method of Bray and Thorpe (1954). The peroxidase activity from the leaves was assayed by the method of Kumar and Khan (1982) and the polyphenol oxidase activity was assayed spectrophotometrically by the procedure given by Mayer et al., (1965). 

The most practical and economically advantageous method of controlling SMD is the production and cultivation of resistant/tolerant cultivars of the crop. In the present study, seven genotypes among 41 showed resistant reactions with a mean SMD incidence ranging between 1.9 - 8.86 per cent. KA 17-2 (3.70%), IPA 19-26 (1.9%), BDN 711 (3.8%), MAL 52 (5.56%), BAUPP 17-14 (5.58%), IPA 14-4A (6.67%) and MAL 53 (8.86%) were the pigeon pea genotypes found to be resistant to SMD (Table 1). The study showed that 14 genotypes were moderately resistant and 20 genotypes were susceptible to SMD. Among susceptible genotypes, IPA 15-06 recorded the maximum SMD incidence (95.24%). Similarly, susceptible checks ICP 8863 and Gulyal local showed SMD incidence of 96.3 per cent and 100 per cent, respectively and resistant check Bahar recorded the least incidence of 1.8 per cent (Fig 1). Similar screening studies by Raghavendra (2003), concluded that ICP 8863, TTB 7, TS 3R, BRG 2, HY 3C and locally cultivated cultivars such a Gulyal local, Benur local  have a high incidence of sterility mosaic disease with mild to severe mosaic symptoms. In the multilocational screening trials, genotypes ICPL-16078, ICPL-16086 and ICPL-16087 displayed resistance reactions (Sayiprathap, 2022). SMD prevalence reached 100% in several cultivars, completely ruining the harvest and causing severe mosaic symptoms.
 

 
Biochemical analysis of SMD-resistant and susceptible pigeon pea genotypes
 
Plant pathogens induce changes in the normal metabolism of the host following infection. Similarly, some biochemical changes in the tissues are brought by viral infection in plants. As the infection progresses, the host develops several defense mechanisms. Initial defensive reactions include various antimicrobial substances in host plants, including phenols and defense enzymes (Osbourn, 1996). The present investigation focused on the study of some biochemical elements of SMD-resistant and susceptible pigeon pea genotypes to understand the effect of sterility mosaic disease and its role in resistance reactions.
 
Total phenol content
 
In the current investigation, resistant genotypes, Bahar (3.55 mg/g) and KA 17-2 (3.60 mg/g) recorded a higher amount of phenolic content, followed by moderately resistant genotypes BDN 2013-45 (2.75 mg/g) and IPA 15-06 (2.15 mg/g), ICP 8863 (1.95 mg/g) and Gulyal local (1.10 mg/g) genotypes at 45 DAS (Table 2 and Fig 1). A higher per cent increase of phenol content over healthy was recorded in the resistant lines than in the moderately or susceptible genotypes.  Resistant and moderately resistant genotypes showed a range of 32.00 - 42.50 per cent increase over the healthy, whereas it was 13.95-16.92 per cent increase over the healthy sample in the case of susceptible lines. Per cent increase of phenol content over healthy was found in KA 17-2 and Bahar with 42.50 and 40.85, respectively. The high phenol content in resistant genotypes might be explained by the presence of more sugar content, which serves as a precursor for the production of phenolic compounds. In susceptible varieties, phenol content and its increase during disease progression were the least. This agrees with the findings of Rathi (1986). Total phenolic content of healthy leaves of resistant/moderately resistant varieties, ICP 7035 (4.416 mg) and BRG 1 (2.981 mg), exhibited slightly higher phenolic content than the corresponding leaves of susceptible varieties, Maruti (2.241 mg) and TTB 7 (2.251 mg) in a study conducted by (Manjunatha et al., 2012).
 

 

 
Peroxidase (PO) activity
 
Peroxidase (PO) is one of the first enzymes responding and providing fast defense against plant pathogens. An increase in the activity of peroxidase is observed in all the genotypes infected by sterility mosaic disease (Table 2 and Fig 1). But resistant and moderately resistant lines showed a high per cent increase of peroxidase activity over healthy (16.24 - 22.74) compared to the susceptible lines (4.78-6.56). The highest enzymatic activity was recorded in the resistant genotypes, KA 17-2 (4.92 DA min^-1g^-1) and Bahar (4.75 DA min^-1g^-1) at 45 days after sowing compared to the susceptible genotypes viz., PA 15-06 (2.44 DA min^-1g^-1), ICP 8863 (2.30 DA min^-1g^-1) and Gulyal local (2.60 DA min^-1g^-1).  Moderately resistant line, BDN 2013-45 recorded PO activity of 3.94 DA min^-1g^-1 fresh weight. The least PO activity was recorded in the healthy sample of ICP 8863 (2.19 DA min^-1g^-1) and IPA 15-06 (2.28 DA min^-1g^-1).  These results agree with the findings of Sudharani (2016). Less peroxidase activity in susceptible genotypes indicated that PO was suppressed leading to the weakening of defense mechanisms against Pigeon pea sterility mosaic virus.
 
Polyphenol oxidase (PPO) activity
 
Polyphenol oxidase (PPO) is an important in the initial stage of plant defense where membrane damage causes release of phenols such as chlorogenic acid. In the present study, it was found that infection of PPSMV resulted an increase in PPO activity in all the pigeon pea genotypes analysed, but susceptible lines showed small change only (Table 2 and Fig 1). At 45 days after sowing, resistant and moderately resistant varieties such as KA 17-2, Bahar and BDN 2013-45 exhibited higher PPO activity (1.1, 1.24 and 0.77 DA min^-1 g^-1 respectively) than that in the susceptible varieties, ICP 8863, Gulyal Local and IPA 15-06 (0.54, 0.37 and 0.50 DA min^-1 g^-1, respectively). The least PPO activity was recorded in the healthy sample of ICP 8863 (0.54 DA min^-1 g^-1) and IPA 15-06 (0.50 DA min^-1 g^-1). PPO activity was increased from 15 DAS to 45 DAS in all the genotypes, but resistant genotypes showed a higher per cent increase of PPO activity over healthy compared to susceptible genotypes. Resistant genotypes exhibited higher per cent increase of PPO activity over healthy sample which ranged from 42.86-60.91 compared to susceptible genotypes that showed 28.00- 32.43 per cent increase over healthy sample. These results are in conformity with reports of Anuradha et al., (2015). Upon infection with SMD, the only appreciable change recorded was the activity of PPO in susceptible variety where it increased considerably in the resistant genotypes.
 
SPAD chlorophyll meter reading (SCMR)
 
SPAD chlorophyll meter (SCMR) showed that chlorophyll content increased slightly in resistant genotypes after infection while it was decreased in all other genotypes (Table 2). SPAD chlorophyll meter readings (SCMR) of different pigeon pea genotypes recorded at 60 DAS and 90 DAS is presented in the Table 2. The highest SCMR was recorded in Bahar with 48.20, but the least was recorded in the susceptible genotype Gulyal local with 34.55. The highest drop SPAD readings was recorded in susceptible genotype compared to the moderately resistant genotype. Reduction trend was more in susceptible variety than moderately resistant varieties while it was increased in resistant genotypes. Similar conclusions on decline of chlorophyll concentration following pathogen infection were made by Arora et al., (2009) and Sudharani (2016).

One of the key control approaches for reducing viral disease losses is host plant resistance. This technique of management is reasonably affordable, easy to use and “ecologically sound” for farmers. Seven of the 41 pigeon pea genotypes screened were identified to be resistant to the sterility mosaic disease. Resistant genotypes include KA 17-2, IPA 19-26, BDN 711, MAL 52, BAUPP 17-14, IPA 14-4A and MAL 53. Chlorophyll content remained high in all resistant/moderately resistant genotypes as compared to susceptible genotypes. Resistant genotypes were shown to have higher phenol content, peroxidase and polyphenol oxidase activity. Although phenol content, PO and PPO activity increased after infection in all genotypes, but the per cent increase over healthy was observed to be higher in resistant and moderately resistant genotypes. High phenolic compounds and higher activity of peroxidase and polyphenol oxidase enzymes in leaf tissue of pigeon pea appeared to be important biochemical constituents in imparting resistance to SMD. These biochemical parameters can be used as markers for screening genotypes for PPSMV.