Optimization of Sprout Seed Abrasion Method for Efficient Inoculation of Leaf Crinkle Disease in Urdbean [Vigna mungo (L.) Hepper]

Urdbean (Vigna mungo (L.) Hepper) or Blackgram, mungo bean or black matpe bean is one of the important pulse crops cultivated in the South Asia, widely adopted to both subtropical and semi-arid areas (Ganguly and Bhat, 2012). The area under cultivation of urdbean in Tamil Nadu during Kharif 2019-20 is 0.36 lakh ha with 0.33 lakh tonnes production (Department of Economics and Statistics, 3^rd Advance estimates 2019-20). Nonetheless, India has largest acreage and is the highest consumer of urdbean, however production is constrained due to prevalence of biotic stresses.
       
Among the biotic stresses, Urdbean leaf crinkle disease (ULCD) is a destructive viral disease that infects urdbean and has become a potential danger to the crop in recent years since most of the cultivars are susceptible to the disease (Gautam et al., 2016). Symptom expression is highly influenced by temperature (Baranwal et al., 2019; Binyamin et al., 2011) and causes high yield reductions (Baranwal et al., 2021; Priyanga, 2021; Sravika, 2018; Sharma et al., 2015; Kanimozhi et al., 2009; Mandhare, 2007). Mechanical inoculation of Leaf crinkle disease onto different cultivars by sprout seed abrasive method was followed by Biswas et al., (2012). Dubey et al., (2020) conducted mechanical inoculation of leaf crinkle disease onto different stages of urdbean plant and observed 100% disease incidence when inoculated onto germinated seeds. In accordance with various investigations on inoculation of leaf crinkle disease carried by various workers, the present work was outlined to standardize the sprout seed abrasion method in order to increase the transmission efficiency.  In an attempt to identify viruses associated with leaf crinkle disease in mungbean and urdbean by Next Generation Sequencing (NGS) of small RNAs from field samples, presence of Cowpea mild mottle virus (CpMMV) was detected but its exact role in causing leaf crinkle symptom and host-pathogen interaction was not established (Baranwal et al., 2015).
               
In recent years, a variety of methods have been employed to study novel plant virus which include Next generation deep sequencing of infected samples followed by bioinformatics analysis. In the process of identifying the etiology of uncharacterized disease through NGS, some of the asymptomatic viruses present in the field sample are always interfering which suggests the need for the maintenance of pure inoculum under glasshouse conditions. These viral inoculation investigations are influenced by a variety of factors such as plant age, inoculum source, buffer with antioxidants and abrasives, anti-inhibitors of sap transmission and so on. As a result, most attention has been placed on the standardization of effective high throughput sap inoculation procedures for viruses (Mandal et al., 2002, 2008; Laidlaw, 1987). In order to identify the putative virus associated with ULCV in urdbean by NGS, it is essential to maintain the virus inoculum under insect proof conditions. Therefore, present investigation was carried out to optimize the inoculation procedure by sprout seed abrasive method and its validation with different genotypes.

Collection and maintenance of the infected plants
 
As the leaf crinkle disease is highly seed transmissible, symptomatic plants in the experimental farms at Tamil Nadu Agricultural University were identified, tagged during summer 2020 and seeds from those infected plants were collected at the time of harvest and were sown in the glasshouse during kharif 2020. Symptomatic leaves developed were used as inoculum for further use.
 
Optimization of mechanical inoculation protocol
 
The present protocol was optimized by modifying the method described by Biswas et al., (2012) in the inoculation procedure by adding abrasives and additives to obtain high symptomatic transmissibility and to attain the infection on urdbean plants at the earliest. The present study was performed during rabi 2020-21 using VBN 8 cultivar of urdbean where in, two methods of incubating sprouted seeds in the leaf crinkle infected leaf sap were followed (i) with pin prick of the sprouted seed and (ii) without pin pricking, where in the virus suspension was grinded with 0.05 M PPB (pH 7.0) with different combinations of additives and abrasives. In pin prick method of sap inoculation, 2-3 gentle pricks were made to the emerging meristem by wounding with a fine needle (Fig 1A).
 

       
To evaluate the effect of antioxidants, four different combinations of inoculum was prepared viz., 0.05M PPB (pH 7.0) containing 0.2% Sodium Sulphite (SS), PPB containing 0.01M 2-Mercaptoethanol (2-ME) and PPB with 0.15% SS + 0.01M 2-ME. Similarly, to determine the role of abrasives on percent transmission of virus, the sap was divided into three parts i.e., only 2% carborundum (320 grit), only 1% celite (545) and both 2% carborundum + 1% celite. Seeds soaked in infected leaf suspension were sown in pots (20 x 20 cm) filled with potting mixture consisting of red soil, farm yard manure, vermiculite and sand in the ratio of 3:2:2:1 (W/W). Total 30 plants for each parameter (6 pots: 5 plants/pot) for three replications along with respective healthy control were maintained under 28±2°C and percent transmission was recorded from 14 to 55 Days post inoculation (DPI).
 
Validation of the optimized protocol
 
Following the optimization of the mechanical inoculation to urdbean by sprout seed abrasion method, the experiment was extended for protocol validation. Nine cultivated urdbean genotypes viz., VBN4, VBN5, VBN6, VBN9, VBN10, VBN11, MDU-1, CO 6 including the susceptible check variety CO5 were obtained from National Pulse Research Centre, Vamban, Tamil Nadu and Department of Pulses, Tamil Nadu Agricultural University (TNAU). All the sprouted seeds of different genotypes were mechanically inoculated with standardised method and buffer control (without any inoculation) was maintained for all the genotypes separately in the glasshouse.
 
Statistical analysis
 
Analysis of variance was performed on the data (ANOVA). For analysis, the per cent data was arcsine transformed. A completely randomised design was used to calculate the standard error of mean (SEm), standard error of deviations (SEd) and critical difference (C.D.) values (Gomez and Gomez, 1984).

Effect of inoculation methods
 
Among the inoculation procedures tested, maximum transmission efficiency (83-100%) was obtained with pin pricking method when infected leaf suspension was prepared with PPB supplemented with SS+2-ME and celite and carborundum as abrasive (Table 1), while symptom expression started at the earliest at 2^nd trifoliate stage and reached maximum (100%) at 20 DPI followed by PPB supplemented with only 2-ME (60-100%) expressing symptoms at 2nd trifoliate stage. Comparing various combinations of additives employed with the buffer, PPB+ SS+2-ME showed 40-95% transmissibility at 25^th DPI followed by SS alone and 2-ME alone (21-92%) at 30^th DPI, which indicate that both SS and 2-ME show same efficacy when supplemented alone with the buffer. Similarly, while comparing the effect of abrasives used carborundum + celite showed a maximum of 40-95% transmissibility at 25^th DPI followed by carborundum (35-92%) and celite (16-53%) alone at 30 DPI which shows that carborundum when used alone is more effective than celite. There was a significant difference between the buffer, abrasives, additives and inoculation method and minor variations were observed in the severity of symptoms across various treatments.
 

       
In a study to increase the effectiveness of mechanical transmission in watermelon genotypes for Watermelon bud necrosis orthotospovirus, protocol was standardized by studying the effect of different inoculation methods and buffer formulations on virus transmission and validated in 17 watermelon genotypes (Jain et al., 2018). Sreevathsa et al., (2012) developed efficient sap inoculation technique method for tobacco streak virus (TSV) by injuring 2-day old seedling at the meristem and immersing in the serially diluted sap and attained 80% transmission at 10-5 dilution. Pensuk et al., (2002) adopted mechanical inoculation to screen peanut genotypes for resistance to peanut bud necrosis virus (PBNV) in both field and glasshouse environments and reported 60-100 per cent transmissibility in susceptible genotypes and consequently, in 2010 he studied the relationship of temperature and relative humidity on the efficacy of PBNV mechanical inoculation on peanuts. From the earlier studies it was evidenced that the optimization of mechanical transmission protocol will improve the transmission efficiency of virus. Therefore, in the present investigation for maintenance of Urdbean leaf crinkle virus (ULCV) isolate, we have followed the buffer composition as described by Biswas et al., (2012) with modifications in inoculation method with addition of additives and abrasives.
 
Symptom observation
 
The urdbean leaf crinkle virus inoculated plants maintained in the glasshouse were observed up to 55 DPI. The inoculated plants showed similar symptoms as recorded earlier in the field conditions. Typical symptoms like crinkling of lamina, malformation of floral organs, stunting of the plant was observed (Fig 1B, D). Enlargement and rugosity of lamina was more prominent in the glasshouse developed symptoms when compared to field conditions (Fig 1C). Most of the inoculated plants did not show flowering up to 45DPI.
 
Validation of inoculation method
 
The optimized protocol was further evaluated on 9 urdbean genotypes which recorded per cent transmission ranging from 92-100% (Table 2). Amongst all the genotypes tested, CO5 cultivar showed higher (42-100%) transmissibility 20 DPI i.e., at 2^nd trifoliate stage followed by VBN11, VBN 10 and CO6 (38-92%; 53-82%; 32-82%) respectively. Besides symptomatology, substantial changes in ULCD transmission percent were observed among the cultivars, demonstrating the pathogen preference and efficacy of the methodology. The authenticity and reproducibility of the methodology was further confirmed and it can be used for maintenance of virus inoculum as well as for screening of resistant genotypes against ULCD both under field and glasshouse conditions.
 

Evaluation of several factors affecting the sap transmission of ULCD resulted in development of a high throughput methodology for inoculation of ULCV which allows rapid transmission of large number of seedlings by immersing the sprouted seed in the virus suspension and expression of symptoms at the earliest. The standardized protocol will be highly useful for the maintenance of virus isolate under vector free conditions as well as for rapid evaluation of large number of urdbean cultivars for the source of ULCD resistance both under glasshouse and miniplot conditions. Further research is being conducted to determine the complete etiology of ULCD employing NGS techniques which will help to develop diagnostic techniques and to devise better disease management strategy.