Inheritance of Urdbean Leaf Crinkle Virus Disease in Blackgram [Vigna mungo (L.)  Hepper]

Blackgram or Urdbean [Vigna mungo (L.) Hepper] is an important food legume crop of Indian sub-continent. It is a self-pollinated crop rich in protein (24%) in addition to lysine and phosphoric acid but are deficient in sulphur containing amino acids (Gautam et al., 2016). The center of origin is India where blackgram has been cultivated from ancient times. Both biotic and abiotic constraints limit the grain legume production. Across southern Asia, viral diseases are one of the major biotic causes of loss to production. Urdbean leaf crinkle virus (ULCV) disease is emerging as one of the major limiting factors for the low productivity of the pulse crop. This disease is economically important, destructive, widespread and inflicts heavy losses annually. 
       
ULCV disease is characterized by the appearance of extreme crinkling, curling, puckering, rugosity of leaves, stunting of plants and malformation of floral organs. Pollen fertility and pod formation is severely reduced on infected plants (Nene, 1968). The yield loss due to ULCV disease ranges from 62-100%, which depends upon type of genotype, location and infection time (Nene, 1972). This disease was first reported by Williams et al., (1968) from India. The virus infection at an early stage of the plants is known to cause heavy losses (Bashir et al., 1991). The disease was recognized as a serious disease in Tamil Nadu as early in 1973 (Narayanaswamy and Jaganathan, 1973). The affected crops are blackgram, green gram, moth bean, pigeon pea, cowpea and soybean. ULCV is transmitted through sap inoculation, grafting and seeds (Kolte and Nene, 1972). Leaf feeding beetle (Henosepilachna dodecastigma (Beniwal and Bharathan, 1980), whitefly (Narayanaswamy and Jaganathan, 1973) and two aphid species (Dhingra, 1975) have been reported as insect vectors of ULCV. A few resistant germplasms against ULCV had been reported by various researchers (Bashir et al., 2005). However, no information is available on the genetics of this disease. Hence, the present investigation was carried out to study the nature of inheritance of ULCV disease in urdbean.

The present investigation was conducted at the National Pulses Research Center, Vamban, Tamil Nadu, India (Latitude: 11^o36' N; Longitude: 78^o90' E; Altitude 93 MSL).  Crosses were made between ULCV susceptible [VBN (Bg) 4, VBN 6 and VBN 8] and resistant (Mash 114, Mash 1008 and VBG 11053) genotypes during Nov-Dec 2016 season (Table 1). The crosses are viz., VBN (Bg) 4 × Mash 114, VBN (Bg) 4 × Mash 1008, VBN 6 × Mash 114, VBN 6 × Mash 1008, VBN 8 × Mash 114, VBN 8 × Mash 1008, VBN 8 × VBG 11053. True F₁ plants were identified during Mar-April, 2017 to build up F₂ population. All F₂ populations were artificially screened during July-Aug, 2017 for their resistance against ULCV disease through sap transmission technique.  Individual plants in F₂ generation were scored as resistant and susceptible based on the symptom appearance. Disease reaction was recorded on 60 days after sap transmission. To confirm goodness of fit, Mendelian segregation ratio for ULCV (resistance: susceptible) in the segregating population was tested through Chi-square test.
 

 
Artificial sap transmission protocol
 
Buffer preparation
 
Solution A and B were used in buffer preparation in which solution A contain 1.74 g of K₂HPO₄ in 10 ml and solution B contain 1.36g of KH₂PO₄ in 10 ml. 6.15 ml of solution A and 3.85 ml solution B mixed and volume was made up to 10 ml using distilled water.
 
Sap transmission procedure
 
The ULCV infected fresh and young leaves were collected in the morning hours and kept in refrigerator. Then by evening samples were grounded by using pestle and mortar with 0.1M phosphate buffer. Ten ml of buffer solution was used to ground the 1g of leaf sample. While grinding, 10µl of mercapto ethanol was added along with buffer. Then carborundum powder was dusted over the leaf surface of the plant to be inoculated to cause injury. Inoculation was done at two leaf stage i.e., 7 days old plant. The grounded inoculation sap was applied on leaf surface by giving a gentle pressure using pestle. After 5-10 minutes of inoculation, the leaf surface was washed with distilled water using hand sprayer. The viral infection on leaves was clearly observed between 15-30 days after inoculation. Disease reaction was recorded on 60 days after sap transmission (Bashir et al., 2005).

ULCV disease is the one of the major yield limiting factor in blackgram. The severity symptoms of ULCV depend on cultivated varieties and their cropping seasons. The per cent transmission of disease was gradually reduced with increase in plant age (Reddy et al., 2005). The disease caused stunting of plants, crinkling, curling, puckering and rugosity on leaves (Fig 1, 2 and 3).  Some branches show crinkling symptoms, whereas other branches of the same plant appear normal in growth (Brar and Rataul, 1986). Identification of resistant genotypes and incorporation of this resistance into other promising varieties are important aspects in the resistance breeding programme. As there is no information available on the genetic nature of inheritance of this disease, the present study was made in that direction. 
 

 

 

       
Six genotypes of blackgram were selected for the present study. The details of these genotypes were presented in Table 1. Hybridization was made between ULCV resistant (Mash 114, Mash 1008 and VBG 11053) and susceptible (VBN (Bg) 4, VBN 6 and VBN 8) to study the inheritance pattern of ULCV disease. The chi-square results of F₂ generation (Table 2) indicated that the segregation ratios fit into inhibitory gene action ratio (13R:3S or 49R:15S) for all the crosses except VBN (Bg) 4 × Mash 114. Cross VBN (Bg) 4 × Mash 1008 had trigenic inhibitory gene action while other crosses had digenic inhibitory gene action. The difference in the number of genes might be due to similar allelic form of one gene for both male and female parents. Based on the gene action following gene symbols were proposed for each parent. S₁S₁S₂S₂ii [VBN (Bg) 4], S₁S₁s₂s₂ii (VBN 6 and VBN 8) and s₁s₁s₂s₂ii (Mash 114, Mash 1008 and VBG 11053) (Table 3). The parent VBN (Bg) 4 had the second gene in dominant allelic form. Hence the cross VBN (Bg) 4 × Mash 1008 fit into the trigenic inhibitory gene action. The second gene is in homozygous recessive allelic form for remaining parents. Which explains the digenic nature of inheritance in the crosses. Hence delayed selection in recombination breeding is suggested for the development of ULCV disease resistant blackgram variety.




 

Conflict of interest
 
The authors declare that they have no conflict of interest.
 
Data availability
 
The datasets generated during and/or analyzed during the current study are available from the authors upon request.
 
Contribution of authors
 
PS performed the field experiments, measurements, data analysis and drafted the manuscript, NM and AM supervised the work, worked on the manuscript and aided in interpreting the results, NM, AM, VKS and CP were involved in planning. All authors provided critical feedback on research, analysis and manuscript.

Authors are acknowledging the help rendered by Mr. Arul Doss, Agricultural Supervisor, NPRC, Vamban in the trial.