Influence of Temperature on the Pathogenesis and Gene Expression of Rhizoctonia solani Causing Web Blight/Wet Root Rot Disease in Mungbean

Mungbean [Vigna radiata (L.) Wilzeck] is one of the most important pulse crop and grown in almost all parts of the India. With increased irrigation  facilities, remunerative prices and availability of short duration cultivars, this crop now occupies considerable area during summer season also in several parts of India. Mungbean is inflicted by a number of diseases caused by fungi, bacteria and viruses.
       
Wet root-rot and web/aerial bight caused by Rhizoctonia solani Kühn (Thanatephorus cucumeris Donk) is a destructive disease of mungbean and it is considered one of the important causes for stagnated productivity of the crop in India. The disease was observed to reduce 33 to 40 per cent grain yield and 28.6 per cent in test weight at different level of disease severity and in different varieties of mungbean (Singh, 2006; Gupta et al., 2010). Web blight symptoms on mungbean appears on roots, stems, petioles, as well as on pods, but the disease is devastating on foliage (Bashyal et al., 2018). Symptoms initiate with the development of small water soaked lesion which enlarged giving blighted appearance with web like mycelium of the pathogen on upper surface (Singh, 2006). The seedling is killed when the lesions on hypocotyle; girdles the stem after they coalesce or enlarge in size. During the period of high humidity, the disease spreads very fast. In the beginning, the infection occurs only on the basal parts including petiole of the lower leaf from where it extends upwards. The infected branches shrivel at the point of infection, leaves on it loose their normal green colour and wither. Leaves and branches fall down leaving behind the main stem bare. Affected plant parts rot rapidly in warm and wet weather.
       
Rhizoctonia solani, teleomorph Thanatephorus cucumeris, is a polyphagous necrotrophic plant pathogen belongs to order the Basidiomycete (Rioux et al., 2011). To date, R. solani has been splited into 14 anastmosis groups (AGs) designated as AG 1 through 13 and bridging isolate (BI) group (Carling et al., 2002; Rioux et al., 2011). Several AGs are further subdivided into intraspecific groups (ISGs) based on morphology of cultural growth, nutritional requirements, temperature effect on growth, host specificity, frequency of hyphal anastomosis and pathogenicity (Sneh et al., 1991). Recent protein and DNA-based studies support the separation of R. solani into genetically distinct groupings, but has also revealed considerable genetic diversity within one anastomosis group. Rhizoctonia solani casual agent of web blight of mungbean belongs to anastomosis group 1, intraspecific group IB (Singh, 2006). Aerial blight pathogen (R. solani) of soybean belongs to AG-1IA group (Yang and Chen, 1989) while both IA and IB intraspecific groups are responsible for causing foliar blight of legume crops especially in soybean (Harvillae et al., 1996). Dubey et al., (2014) identified the seven anastomosis groups (AGs) of R. solani associated with pulse crops. Ganeshamoorthi and Dubey (2015) suggested AGs did not show host specificity in legumes. However, effect of these AGs on the pathogenicity of mungbean needs to be studied.
       
Environmental factors play a vital role in the development of web blight disease caused by R. solani. An understanding of the role of environmental factors on the infection and survival of any root pathogen is necessary to develop cultural disease management practices as well as assays that evaluate host resistance or efficacy of seed treatments. Documenting the independent and interactive effects of soil moisture and temperature on plant disease is essential for developing effective management strategies or screening for host resistance. Temperature is relatively easy to control experimentally, especially in climate-controlled chambers. There are very limited information available about the influence of environmental factors for example temperature and relative humidity on the development of root-rot/web blight/areal blight disease of mungbean and the expression of genes which having vital role in pathogenesis process of R. solani. Therefore, the objectives of this study were to investigate the effect of temperature on web blight disease development and to elucidate the effect of temperature on the expression of selected pathogenicity associated genes of R. solani during the pathogenesis process in mungbean.

Experiments were conducted in the year 2016-17 at Division of Plant Pathology and National Phytotron Facility of ICAR-Indian Agricultural Research Institute, New Delhi.
 
Source of R. solani isolates
 
The culture of R. solani belonging to different anastomosis groups (AGs) were obtained from Pulse Pathology Lab, Division of Plant Pathology, ICAR-IARI, New Delhi, which were originally isolated from diseased host plant (Table 1) with typical symptoms. The anastomosis grouping and pathogenicity test using Koch’s postulates of these cultures were also determined earlier (Dubey et al., 2014).
 

 
Effect of temperature on mycelial growth of R. solani
 
The influence of different temperature (4^oC, 20^oC, 25^oC, 30^oC and 35^oC) on mycelial growth of seven different R. solani isolates belonging to different anastomosis group were investigated on Potato Dextrose Agar (PDA) medium (200 g potatoes; 20 g agar; 20 g dextrose in 1 L of water). Each Petri plate was centrally inoculated with 5 mm mycelial disc from the 48 hours old culture of test fungus. Five replications were maintained for each treatment and colony diameter was measured after 48 hrs of inoculation.
 
Effect of temperature on web blight disease development of mungbean under phytotron conditions
 
Four sets of pot experiments were conducted under controlled environment (plant growth chamber) in phytotron at ICAR-IARI, New Delhi to determine the disease development on susceptible variety of mungbean Pusa Vishal. The condition for these sets were temperature of 20^oC, 25^oC, 30^oC and 35^oC, relative humidity (85%) and illumination (alternate dark and light period) of 12 hrs. To study the virulence of different isolates on susceptible genotype Pusa Vishal, isolates were multiplied on sorghum grains and 15 days old inoculums was mixed on soil at the rate of 10 g/kg of soil (Bashyal et al., 2018). Disease incidence was observed at the interval of 15 days and final data was calculated as mean value of five observations. Experiment was conducted on randomized block design (RBD) with five replications per treatment and analysis of variance was calculated using two factor (isolates and temperature) analysis.
 
Effect of temperature on pathogenicity related gene expression
 
To study the effect of temperature on pathogenicity related gene expression most virulent isolate RUPU-18 was selected. The above mentioned temperatures (20^oC, 25^oC, 30^oC and 35^oC) and humidity conditions were selected for the experiments under phytotron conditions. For each temperature twenty seeds of susceptible variety of mungbean Pusa Vishal were sown in 4 inch pots filled with sterilized soil. After 15 days of sowing of mungbean, in all sets of experiment, the soil in pots was inoculated with the mycelial suspension (10 g in 50 ml water) of 15 days old sorghum grain multiplied inoculums of R. solani isolate RUPU-18. After 24 hrs, 48 hrs, 72 hrs and 96 hrs of inoculations, plants were uprooted carefully, preserved in liquid nitrogen and stored at -80^oC. The experiment was conducted in completely randomized design with 5 replications per treatments.
 
Primer designing for pathogenicity associated genes of R. solani
 
Putative pathogenesis related genes as described by Rioux et al., (2011) were selected for the gene expression studies. Sequences of these genes were retrieved from National Center for Biotechnology Information (NCBI). Primers sequences were designed by the IDT oligo analyser (https://www.idtdna.com/Primerquest/Home/Index) at default settings. Primer specificity test was conducted through in silico analysis using Basic Local Alignment Search Tool (BLAST) of NCBI non redundant data with the identification of short and nearly exact match’s parameters.
 
Real time PCR based expression analysis for the selected genes
 
Seedlings were kept in pre-chilled mortar and homogenized in liquid nitrogen with the help of pestle. Total RNA was isolated from the powdered samples (100 mg each) using Tri-reagent (Invitrogen) following the manufacturer protocol. Further, 2 mg of RNA was treated with RNase- free DNase (Genetix) and kept at 37°C for 30 minutes. For the inactivation of DNase, DNase stop solution was given for 10 minutes at 65°C. Oligo (dT) 12-18 primers were added to  the treated RNA (2 μg)  and the sample (20 μl) was briefly denatured at 65°C for 10 min and chilled on ice for 2 min. Protocol of MuMLv (Fermentas) was followed for the reverse transcription. Gene expression was quantified through real time PCR detection system (MJ miniopticon-48 wells; Bio-Rad Labs, Inc). The final volume of 20 µl PCR mixture prepared with 2X dynamo colour flash SYBR green mix dye (Thermo Scientific, USA): 10 µl;  forward and reverse primers:  100 nM and cDNA template: 100 ng, no template controls (NTC) and a reference gene (Actin) was used for each treatment. Thermal cycling conditions were as follows: 95°C for 10 mins followed by 39 cycles of 95°C for 15 secs and 53°C for 30 secs and a melt curve from 65°C to 95°C. Expression of target gene was normalized by reference gene and fold expression of target gene was calculated using 2^-ΔΔCT method of Livak and Schmittgen (2001). Three biological replicates with 3 technical replicates for each biological replicate was used for the entire experiment.

Effect of temperatures on growth of R. solani isolates
 
Mycelial growth of seven R. solani isolates was investigated under in vitro conditions at different temperatures. Maximum radial growth in R. solani isolates was observed at 30^oC followed by 25^oC and 20^oC after 48 hrs of incubation (Fig 1). However, growth of R. solani at 25^oC was also at par with 30^oC. Significant difference in growth among seven isolates was observed at 20^oC. R. solani isolates could not grow at 4^oC. Radial growth of one isolate RDLM-1 was found statistically at par at three temperatures 20^oC, 25^oC and 30^oC. Kumar et al., (2014), in similar studies, have also recorded that the optimum hyphal growth of Rhizoctonia occurred at temperature range of 20 to 30^oC with an optimum at 25^oC.
 

 
Development of root rot/web blight disease
 
Development of root rot/ web blight disease in mungbean was evaluated under phytotron conditions as described above (Fig 2). Effect of isolates and temperatures was significant for the disease development (Table 2). Maximum disease incidence (73.11%) was produced by R. solani isolate (RUPU-18) followed by R-17 (68.75%), RDLM-1 (63.45%) at 25^oC (Fig 3). Only RASC-27 isolate produced disease (36.75%) on mungbean at 20^oC. When plants were grown at 30^oC, maximum disease incidence (35.95%) was recorded with RUPU-18 isolate. At temperature, 35^oC, only two isolates (RDLM-1 and RUPU-18) were able to produce disease on Pusa Vishal. Sharma and Tripathi (2001) have noted that the higher aerial temperature (26 to 32^oC), relative humidity near 100% and soil temperature 30-33^oC favors the development of high disease  severity of web blight of urdbean caused by Rhizoctonia solani Kuhn. They also noted that the rainfall (91-97 mm) has a significant role in severe development of web blight during early stage of crop in urdbean. Dubey (1997) have shown that the high temperature (26-28^oC) and high relative humidity (90-100%) favor severe web blight disease development on groundnut. The pathogenic ability of R. solani at different temperatures enhances the fitness in nature and might be responsible for making it most successful pathogen with wide range under diverse climatic conditions.
 

 

 

 
Effect of temperature on expression of pathogenesis related genes
 
List of primers designed for the real time PCR based studies were presented in Table 3. Primers were screened for the constitutive expression and those present constitutively were further selected for the study. Real Time-PCR analysis of expression of pathogenesis associated genes showed that the expression of these genes is dependent on the temperature and time interval post pathogen inoculation (ppi) of isolate RUPU-18 (Fig 4).  For gene β-1, 3 glucan hydrolase significant expression was observed after 24, 48 and 72 hrs post pathogen inoculation (ppi) at 25^oC. At 92 hrs post inoculation maximum expression of gene was observed at 30^oC. Similarly, gene 7 i.e. Polysaccharide synthtase expression was maximum at 25^oC after 24, 48 and 72hrs post inoculation. At 96 hrs significant expression of the gene was observed at 20 and 25^oC. Gene 8 i.e. Potassium hydrogen antiporter was expressed maximum at 20^oC after 24 and 48 hrs of inoculation. It was maximum at 25^oC at 72 hrs ppi and expression was maximum at 35^oC after 96 hrs of inoculation. Gene 13 i.e. ABC transporter was observed to be expressed maximum at 30^oC after 24, 48, 72 and 96 hrs post inoculation (Fig 3).
 

 

       
Cell wall polysaccharides, such as β-1, 3-glucan and chitin, are recognized by host cells as pathogen-associated molecular patterns (PAMPs) and the fungal cells are attacked and removed by host immune systems. Glycoside hydrolases degrade β-glucans, mainly to regulate the plasticity of the cell wall for cell expansion, cell division, cell separation and sporulation. Potassium hydrogen antiporter regulates the pH and stress response. Polysaccharide synthase is responsible for the appressorium formation (Rioux et al., 2011). Three major events involved in pathogenesis process of R. solani in mungbean are early host contact or attachment and penetration, adjustment to the host environment and proliferation of pathogen through necrotic tissues (Bashyal et al., 2018). Higher expression of β-1, 3 glucan hydrolase and Polysaccharide synthase at early hrs of inoculation i.e. 24 and 48 hrs at 25^oC suggests the early colonization and pathogenicity at this temperature. The ABC transporter family is a group of membrane proteins that use the hydrolysis of ATP to power the translocation of a wide variety of substrates across cellular membranes. The ABC transporter of Pseudomonas fluorescens was optimally functional at 20 and 25^oC. Transporter proteins are one of an organism’s primary interfaces with the environment. The expressed set of transporters mediates cellular metabolic capabilities and influences signal transduction pathways and regulatory networks.

Our study showed that there is direct link between the temperature and the induction level of key genes that likely to affect the efficiency in the disease development in mungbean and fitness of R. solani in nature. Real Time-PCR analysis of expression of pathogenesis associated genes showed that the expression of these genes is dependent on the temperature and time interval post pathogen inoculation.

Authors are thankful to the ICAR-NASF (project code BSP- 4011/2013-14) for financial support and Head, Division of Plant Pathology, for providing facilities to carry out the research work.