Screening Chickpea Genotypes for Resistance to Rhizoctonia bataticola in Controlled Conditions

Chickpea (Cicer arietinum L.) is one of the oldest (earlier than 9500 BC) and widely cultivated pulse crops in over 50 countries of the world. Southwest Asia and the Mediterranean are the two primary centers of origin and Ethiopia, the secondary centre of diversity (Vavilov, 1926; 1951). Wild annual Cicer originated mainly in the Mediterranean regions having a wide eco-geographic range, differing in habitat, topographic and climatic conditions (Abbo et al., 2003; Berger et al., 2003). Globally, chickpea is grown in an area of 11.08 million hectares with the production of  9.77 millon tonnes and an average productivity of  881.9 kg ha^-1. It  is the second most important  pulse crop in the world in terms of area under cultivation after dry bean but ranks third in production following dry bean and peas. India is the largest producer of chickpea in the world sharing 65.25 and 65.49 per cent (FAOSTAT, 2010) of the total area (11.97 million ha) and production (10.89 million tonnes) with productivity of 920 kg/ha.
       
The realized productivity of this pulse crop is not appreciable due to various production constraints. The major being biotic stresses like diseases, insect pests and abiotic stresses like drought, extreme temperatures and salinity. Among the biotic stresses, chickpea is severely affected by Fusarium wilt, dry root rot, Ascochyta blight, collar rot, bacterial blight, filiform virus and root nematode causing economic yield loss (Nene and Sheila, 1996). Of late, dry root rot is becoming severe in most chickpea growing regions of India (Masood Ali and Shivakumar, 2001). Dry root rot of chickpea caused by Rhizoctonia bataticola is a soil borne fungus that survives in the soil in the form of sclerotia for long time. The fungus lacks fruiting bodies and spores. The mycelium is light brown, thick in which black sclerotia are formed.
       
Although dry root rot disease is found in all chickpea growing areas, it is most severe in Central and South India, where the crop is grown under rain fed conditions causing 30-40 per cent yield loss. High day temperature (>30°C) and dry soil conditions at flowering and podding stage rapidly increase the severity of the disease (Gurha et al., 2003). The disease results in sudden drying of the plants in the field at flowering and poding, where as the leaves and stems of affected plants remain straw-colored. Roots dry, becomes brittle exhibiting shredding of bark and break easily. The disease appears suddenly when the ambient temperatures are between 25°C and 30°C (Haware, 1990).
       
Among the different approaches of prevention and management of diseases, the introgression of genetic resistance into cultivars is as an important and successful method as that is relatively inexpensive, biologically safe and sustainable for long term. The screening and evaluation of available germplasm is the first step in successful disease resistance breeding programme to identify the sources of resistance which could be utilized further for development of resistant varieties. However, the research work carried out with respect to the identification of resistance sources to dry root rot of chickpea is meager. In this context, an effort was made to identify the resistant sources for dry root rot in chickpea.

The present investigation started during rabi 2011, continued up to summer 2012-13 at the experimental plots of AICRP on Chickpea, Zonal Agriculture Research Station, Gandhi Krishi Vignana Kendra Campus, University of Agricultural Sciences, Bangalore.
 
Collection and isolation of the fungus
 
The chickpea plants showing typical symptoms of dry root rot (Isolate Rb1, Aghakhani and Dubey 2009) were collected from experimental plots of AICRP on Chickpea at GKVK, Bangalore (Fig 1A and 1B) and the samples were brought to the laboratory and washed thoroughly in running water. The plants were dried in shade for 3-4 days and preserved for further studies. Fungus was isolated following standard tissue isolation method under aseptic condition. Infected roots were cut into small bits of size 1-2 mm, surface sterilized in 1:1000 mercuric chloride solution for a minute and then washed repeatedly thrice in sterile distilled water to remove traces of mercuric chloride before transferring them to sterile potato dextrose agar [PDA] slants under aseptic conditions and incubated at  27 ±1°C.
 

 

 
Hyphal tip culture
 
Dilute mycelial suspensions (10^-6 ml^-1) of the fungus were prepared in sterile distilled water. One ml of such (10^-6 ml^-1) suspension was spread uniformly on two per cent water agar plates and observed for mycelial growth initiation under microscope. Such growth initiation was marked and mycelial bit transferred on PDA plates under aseptic conditions. The PDA plates were incubated at a temperature of 27±1°C and observed for fungal growth. No sectoring was observed in any of the plates after isolating them by hyphal tip culture technique. This method was followed for maintaining pure culture as the fungus is known to be highly heterozygous.

Maintenance of culture
 
The fungus was sub cultured on Petri plates containing PDA (Fig 2) and allowed to grow at 27±1°C for ten days. Such plates were stored in refrigerator at 4°C and maintained by sub culturing at 20 to 25 days interval and used throughout the study.
 

 
Screening of germplasm lines for dry root rot resistance in laboratory condition
 
Five hundred and twenty chickpea germplasm lines procured from different national and international research institutes were screened in the laboratory condition using Blotter Paper Technique (Nene et al., 1981; Fig 3) to study the reaction of lines to Rhizoctonia bataticola infection and  to identify the resistance sources for the disease. Potato Dextrose Broth (PDB) was used for culturing the fungus, Rhizoctonia bataticola. 250 ml of PDB was poured into 500 ml conical flasks and steam sterilized in autoclave at 15 PSI for 15 minutes. The flasks were then inoculated with the fungus and incubated at 35°C for seven days. The mycelial mat formed in the flask was removed and macerated in a waring blender along with distilled water for a minute. The inoculum was later collected in a beaker. In the mean time, chickpea seedlings were raised in polythene bags containing sterilized sand. One week old ten seedlings were uprooted and the roots were immersed in sterile water in order to remove the adhered soil particles. The seedlings were then immersed completely in the inoculum in a beaker for a minute. The seedlings particularly the root portion were then placed side by side on a blotter paper (45 cm x 25 cm with one fold) in such a way that only the cotyledons and roots are covered and the green tops of seedlings remained outside and then blotter paper was folded. One folder blotter paper contained the seedlings of one test line.
 

       
The folded blotter papers were then placed in trays and trays were placed in an incubator at 35°C for eight days. During the incubation period, twelve hour artificial light was provided and the blotters were moistened with sterile water every day. At the end of the incubation period, seedlings were examined for the extent of root damage and scored for the disease severity on 1- 9 rating scale (Nene et al., 1981, Table 1).
 

       
Four resistant genotypes thus identified were again screened under green house condition in the pots containing the soil with Rhizoctonia fungus for reconfirmation of resistance along with the highly susceptible genotype L-550. Ten seeds per genotype were planted in each pot with two replications.

Before initiating crop improvement programme in any crop, breeder should thoroughly evaluate, screen and understand the genetic architecture of the germplasm he is handling. Allard and Bradshaw (1964) indicated the need for thorough evaluation and utilization of germplasm for improvement of of genotypes with moderate resistance reaction was reported by many workers (Pande et al., 2004, Muhammad Saifulla et al., (2011a and 2011b), Khan et al., 2013 and Nagamma et al., 2015).  However, very few investigations have identified the resistant genotypes. Om Gupta and Anita (2006) observed the resistance reaction of H 99-264, PG 9425-5, PG 9425-9, HK 00297 and PG 97-313 genotypes for dry root rot out of 271 genotypes that were screened at in disease sick plot at JNKVV, Jabalpur, India. The genotypes viz., WCG 97-29, WCG 97-37, IPC 97-72 and H 00-256 (desi) showed less than 10% mortality for dry root rot. Jayalakshmi et al., (2008) reported two resistant genotypes from a two year screening of 12 chickpea genotypes in pot cultures. Further, several other workers viz., Mishra et al., (2005), Khan et al., (2012), Om Gupta et al., (2012), Khan et al., (2013), Nagamma et al., (2015) reported the resistance reaction of genotypes for dry root rot through blotter paper technique in the laboratory condition, pot culture method and sick plot screening. The resistant genotypes identified in the present investigation could be utilized as potential donors for future resistance breeding programme for dry root rot in chickpea.