Influence of Weather and Soil Parameters on Development of Wet Root Rot in Pulse Crops and Virulence Analysis of Rhizoctonia solani Isolates

Wet root rot caused by Rhizoctonia solani Kühn in pulse crops was favored by wide range of soil parameters like temperature, humidity, pH, electric conductivity and soil texture. The areas surveyed for the collection of the isolates showing variable atmospheric temperature and relative humidity and low to medium levels of soil organic carbon and high level of available phosphorus influenced the development of the disease incidence from 2-48%. Seventy three cultivars of mungbean, twenty eight cultivars of urdbean and eight cultivars of cowpea were evaluated against virulent isolate of R. solani (RASC 30) to design a set of differential cultivars for virulence analysis. Two cultivars of urdbean, namely, NDU3-4 and IPU2-14, one cultivar of mungbean, namely, HUM 1 and three cultivars of cowpea, namely, V240, V585 and DCP7 showed resistant reactions. Four cultivars of urdbean, namely, TU94-2, KU323, KUG216 and B3-8-8, one cultivar of mungbean, namely, PDM54 and two cultivars of cowpea, namely, V578 and DCP13 were moderately resistant against the pathogen. The virulence analysis of 90 isolates of the pathogen representing 7 anastomosis groups (AGs) isolated from pulse crops of 16 agro-ecological regions of India on a set of differential cultivars, namely, HUM 1, PDM 54 and Pusa Vishal of mungbean, NDU 3-4, KU 323, Uttara of urdbean and V 240, V 578 and Pusa Sukomal of cowpea grouped the isolates into five pathotypes. The differential cultivar for each pathotype was identified. The pathotypes were not corresponding to the AG type of the isolates. Except one pathotype (isolate RMPG28 belonging to AG2-3), each pathotype had the isolates from different AGs.


Introduction
India is recognized globally as a major pulse producing country sharing 25% of the global pulse production.India grows a variety of pulse crops under a wide range of agro-ecological conditions in 23.4 mh with 14.6 mt production and 625 kg ha -1 productivity (Agricultural Statistics at a Glance, 2010).Biotic stresses are the major cause of low productivity of pulse crops in India.Amongst the fungal pathogens, Rhizoctonia solani Kühn [teleomorph -Thanatephorus cucumeris (Fr.)Donk] is a seed-and soil-borne plant pathogen causing web blight/wet root rot in pulse crops besides other agricultural and horticultural crops.Chickpea (Cicer arietinum L), cowpea [Vigna unguiculata (L.) Walp.], mungbean [Vigna radiata (L) Wilczek], urdbean [Vigna mungo (L.) Hepper] lentil (Lens culinaris Medikus) and french bean (Phaseolus vulgaris L.) are the major pulse crops cultivated world wide under different agro-climatic conditions are affected by R. solani.The pathogen causes considerable yield loss in mungbean and urdbean in India (Dubey, 2003).Yield loss up to 57% in mungbean was reported from Iran (Kaiser, 1970).The intensive crop cultivation and modified agro-practices have increased the populations of R. solani in soil and gradually built up new disease problems.R. solani is genetically diverse in respect of pathogenicity as well as cultural/morphological and physiological characters.Initially, Rhizoctonia spp.are classified in different species or groups on the basis of morphological and cultural characters (Parmeter & Whiteny, 1970).Isolates of R. solani have been traditionally classified into different anastomosis groups (AGs) (Ogoshi, 1987;Carling, 1996).
with pulse crops based on virulence analysis on host differentials.The host differentials for pulse crops are also not available worldwide.The pathotypes of the pathogen have not been correlated with agro-ecological regions, AGs and soil and weather parameters.Keeping these points in view, the present study was aimed to standardize the host differentials to determine the pathotypes/races among the populations of R. solani representing various AGs associated with various pulse crops in different agro-ecological regions of India having diverse cropping sequences and to determine the influence of weather and soil parameters on disease development in different areas.

Cultures of Rhizoctonia Solani and Cultivars of Mungbean, Urdbean and Cowpea
Ninety (representative of 470 isolates) isolates of R. solani isolated from different pulse crops from 16 agro-ecological regions covering 21 states of India and being maintained in Pulse Laboratory, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi were used in the present study.The cultivars/genotypes of mungbean, urdbean and cowpea included in the present study were collected from Division of Genetics, Indian Agricultural Research Institute, New Delhi.

Weather and Soil Parameters
The soil samples along with diseased specimens were collected from 9 states, namely, Delhi, Uttar Pradesh, Rajasthan, Jammu and Kashmir, Uttarakhand, Panjab, Gujarat, Maharashtra and Assam.The pooled samples of each location (48 soil samples) were analyzed for nitrogen, carbon, phosphorus, salinity, pH and soil texture.The weather parameters as soil and atmospheric temperature, soil and atmospheric relative humidity were also recorded in these areas by using portable digital humidity and temperature recorder.

Reaction of Different Cultivars of Mungbean, Urdbean and Cowpea against R. solani
In order to find out resistant sources to constitute a set of differential cultivars for analysis of virulence, 28 cultivars of urdbean, 8 cultivars of cowpea and 73 cultivars of mungbean were evaluated against R. solani (RASC 30) under sick pot soil condition.Surface sterilized (0.1% formalin) plastic pots (20 cm dia) were filled (2 kg pot -1 ) with sterilized soil (1% formalin).The soil was inoculated 2-days prior sowing with 10-day-old inoculum (10 g kg -1 soil) of R. solani multiplied on sorghum grains (Dubey et al., 2009).Ten seeds of urdbean, mungbean and cowpea in each pot were sown on August 22, 2009 seperately in three replications.The incidence of wet root rot was recorded at 15 days interval up to maturity of the crop plants.

Virulence Analysis
Virulence analysis of 90 representative isolates including international testers of 7 AGs of the pathogen was carried out on a set of 10 differential cultivars, namely, HUM 1, PDM 54 and Pusa Vishal of mungbean, NDU 3-4, KU 323, Uttara and KUG 216 of urdbean, and V 240, V 578 and Pusa Sukomal of cowpea in net house.Twelve seeds of each cultivar were sown on August 3, 2010 and July 13, 2011 in 20 cm diameter surface sterilized plastic pots (0.1 % mercuric chloride) filled with 2 kg sterilized soil (1 % formalin for 15 days) and inoculated with 10-day old culture of R. solani multiplied on sorghum grains (10g kg -1 soil) 2 days before sowing.Pots with un-inoculated soils were also maintained as control for comparison.The incidence of wet root rot was recorded at 15 days interval up to maturity of the crop plants.

Virulence Analysis of AG Groups and Their Combinations
A set of pot experiment was conducted to test the virulence of the isolates representing two AG groups for AG1isolate RUPM83 and RUPU82 and for AG4-isolate RUPM66 and RUPU69) individually and in combinations along with hybrid culture of the same group in three replications.Surface sterilized (0.1% formalin) plastic pots (10 cm diameter) were filled (500g pot -1 ) with sterilized soil (1% formalin).The soil was inoculated 2 day prior sowing with 10-day-old inoculum as mentioned earlier.Ten seeds of mungbean (cv Ratna) were sown on August 1, 2009 in each pot.The incidence of wet root rot was recorded at 15 days interval up to maturity of the crop plants.

Observations Recorded and Data Analysis
The wet root rot incidence was recorded on the basis of number of plants showing wet root rot symptoms out of the total plants and the cultivars/genotypes were categorized into resistant (0-10% root rot incidence), moderate resistant (>10-20% root rot incidence), susceptible (>20-50% wet root rot incidence) and highly susceptible (>50% root rot incidence).Finally, the reactions were presented as resistant (0-20%) and susceptible (>20%) for pathotype grouping (Dubey, 2003).

Influence of Weather and Soil Parameters
The soil and atmospheric temperature and relative humidity recorded in different parts of the country were variable.The results (Table 1) indicated that the disease favoured by wide range of soil (18.1-41.0°C)and atmospheric (8.1-46.3°C)temperature and soil (28-68%) and atmospheric (20-87%) relative humidity.The disease development was also favoured by variable soil parameters.The soil pH range from 5.56-8.71and electric conductivities from 0.03 -0.51 ds m -1 favoured the disease development.The majority of areas surveyed showed low to medium level of soil organic carbon and total nitrogen ranging from 62.7-492.8kg ha -1 .Available phosphorus was from 1.2-98.3kg ha -1 and the soil samples collected from most of the areas showed high phosphorus status.Soil texture was also variable in areas surveyed from loam to sandy loam and silty to clay loam (Table 2)

Discussion
Rhizoctonia solani produced typical web blight /wet root rot symptoms on different pulse crops under diverse environmental conditions.The development of disease was favoured by wide range of atmospheric and soil temperature and relative humidity.Wide range of soil properties as pH, EC and texture along with nutritional conditions as organic carbon, nitrogen and phosphorus also favoured disease development in the areas surveyed.Most of the areas surveyed showed low to medium level of soil organic carbon.Tewoldemedhin et al. (2006) reported variation in temperature requirement for the growth of R. solani and observed that the isolates of R. solani belonging to AG 2-2 and AG 4 HG-II had significantly higher optimum growth temperatures than those from other AGs.In the present study, the type of AGs were not correlated with either temperature or relative humidity requirement in the field conditions The present findings clearly indicated that the disease is favoured by wide rage of soil ranging from silt, loam, sandy and clay.In general all type of soils was found suitable for the development of the disease in various crops.
The pathogenicity of the combined as well as hybrid culture of different AGs showed that the first generation of hybrid culture of two AGs was more or less similar in the aggressiveness on susceptible cultivars of mungbean of their parental AGs.
First time through the present study, an attempts was made to standardize the differential cultivars for virulence analysis of the populations of R. solani originating from different pulse crops.Availability of resistant cultivars against the pathogen in pulse crops is major limitation for differentials.Therefore, a large number of cultivars of mungbean, urdbean and cowpea were evaluated against the pathogen with an aim to find out resistant cultivars to constitute an effective set of differentials.Out of 109 cultivars evaluated, ten cultivars having different degree of resistance and susceptibility against the pathogen (3 of mungbean, 4 of urdbean and 3 of cowpea) were selected to constitute a set of differentials for virulence analysis.The results confirmed that there were differences in virulence among the isolates tested.The results of virulence analysis of 90 isolates including 7 international isolates of AGs tester of R. solani indicated that most of the isolates caused susceptible reaction on the set of cultivars evaluated.The isolates were grouped into the five pathotypes and differential cultivars for each pathotype were determined.Attempts have not been made earlier to determine the pathotypes of R. solani.Scanty information is available on the pathogenic grouping of the limited number of the isolates on a few cultivars and the isolates were found variable in respect of pathogenicity.Similar to the present study Engelkes and Windels (1996) reported that the isolates of R. solani AG2-2IIIB and AG2-2 IV were pathogenic to sugarbeet and bean crops and the severity of the isolates on these crops differed.Bohlooli et al. (2006) observed that 81 isolates of R. solani isolated from roots and hypocotyls of bean showed variability in respect of virulence on the cultivars of beans.Tewoldemedhin et al. (2006) also observed differences among the virulence of R. solani isolates representing different AGs on different crops.The isolates of AG 2-2 and AG 4 HG-II were the most virulent on all crops evaluated.AG 2-1 was highly virulent on canola, moderately virulent on medic and lupin, weakly virulent on lucerne and barley, and nonpathogenic on wheat.AG-11 isolates were moderate to weakly virulent on all crops, with the exception of barley and wheat.AG-3 was weakly virulent on canola, lupin, and medic.In the present study, the isolates representing 7 AGs were highly variable on mungbean, urdbean and cowpea cultivars included in the present study, but there was no definite correlation in between AGs and cultivar or crops.Godoy-Lutz et al. (2008) observed at least six different subgroups of R solani causing web blight symptoms in common bean.Khandaker et al. (2008) tested pathogenicity of two potato isolates of R. solani on 33 hosts including pulse crops and observed differences in the degree of pathogenicity of both the isolates in respect of hosts.Seventy eight isolates of R. solani were isolates from diseased lupin plants and various other crops in Alberta, Canada.Isolates belonging to AG-4 produced typical symptoms of stem rot and root rot on lupin seedlings and showed greater virulence compared with AG-2-1 and AG-2-2 isolates (Zhou et al., 2009).Caesar et al. (2010) observed significant differences among the isolates in respect of their virulence.Pathogenic behaviour of 368 isolates of R. solani representing 7 AGs collected from different leguminous crops from 16 agro-ecological regions of India were highly variable in pathogenicity and caused 10% to100% wet root rot incidence in mungbean, urdbean and cowpea and 11% to100% in chickpea (Dubey et al., 2011).The pathogenicity tests on Beta vulgaris revealed that R. solani AG2-2 IIIB and AG2-2 IV isolates were more virulent than R. cerealis (Taheri & Tarighi, 2012).

Conclusion
Wet root rot/web blight caused by R. solani is an important disease of pulse crops.Pathotypes based on virulence on a set of differential cultivars is not known for the populations of R. solani.No attempt has been made in the past to develop a set of differential cultivars for pathotyping of the pathogen.Considering the wide host range of pulses, a set of differential cultivars consisting of mungbean, urdbean and cowpea was developed in the present study.Based on the virulence on the a set of differential cultivars, the populations of R. solani representing 7 AGs associated with pulse crops were grouped into 5 pathotypes and differential cultivar for each pathotype was identified.The weather and soil parameters like temperature, humidity, pH, electric conductivity, soil texture, organic carbon, nitrogen and phosphorus influenced the disease development ranging from 2-48% in the areas surveyed for collection of the isolates.The pathotype groups generated in the present study were not corresponding to AGs/crop of origin/area of origin of the isolates.The information generated on pathotypes can be used for resistant breeding against the pathogen.

Table 1 .
. Soil and atmospheric temperature and humidity recorded during survey in different parts of the country

Table 5 .
Pathogenic groups of R. solani and their differential cultivars