Evaluation of different fungicides and antagonists In vitro and In vivo condition against Alternaria blight of pigeonpea 

Pigeonpea [Cajanus cajan (L.) Millsp.] is one of the important food legume crops and rank second after chickpea in area, production and productivity of the tropics and sub-tropics. India has largest acreage under pigeonpea (4.65 mh) with a total production and productivity of 3.02 mt and 650 kg/ha, respectively (Pulses status, 2016). The shift of pigeonpea cultivation from the traditional kharif season to pre rabi September sowing in north Bihar has not only shown an increased production potential of this important pulse crop but has also opened altogether new possibilities in land use pattern of the rainfed areas of Bihar and adjoining states (Gupta et al., 2015). Among the several factors responsible for reduction yield and quality deterioration of pigeonpea in India, diseases occupy a vital place. Among the diseases, Alternaria blight (A. tenuissima (Kunze ex pers.) Wiltshire) was reported for the first time from Varanasi, India by Pavgi and Singh (1971). Alternaria blight has become one of the serious fungal diseases of pigeonpea, especially in September sown crop. This disease has been reported to cause yield losses up to the tune of 40-50 percent (Kushwaha et al., 2010a) and 20-80 percent (Sharma et al., 2012) reduction in yield, in pigeonpea. Fungicide application can minimize disease and thus increase the genetic potential and ultimately yield. However, there are reports of resistances development against fungicides (Chattopadhyay and Bagchi, 1994). So this situation compels to focus on disease management by utilizing bio agents and fungicides in lowest concentration. Application of bio agents was a sustainable approach apart from being a promising alternative to fungicide application. Thus the present study was aimed to evaluate In vitro two bio agents and six fungicides were studies after combination of fungicides and antagonistic activity under field conditions and to determine optimal timing of their application for the control of Alternaria blight on pigeonpea.

In vitro evaluation of bio-agents by dual culture plate method
 
In vitro efficacy of two bio-agents i.e., Trichoderma harzianum and T. viride an anamorph of Hypocrea rufa, is a known bio- control agent against Alternaria tenuissima was tested by using dual culture plate method on PDA medium (Singh et al., 2005). Culture of both bio agents was taken from Biocontrol laboratory, Department of Mycology and Plant Pathology, BHU, Varanasi. PDA was used as the basal medium twenty ml of sterilized melted PDA was poured in each 90 mm diameter glass Petri plates and allowed to solidify. After 12 hours of pouring, these Petri plates were inoculated with 5 mm discs with the help of a sterilized cork borer from the edge taken from eight days old culture of test pathogen and antagonistic bio agents. Both were placed separately at equal distance on the periphery of the Petri dishes. Inoculated Petri dishes were incubated at 25+2°C in BOD incubator. The test fungus and bio-agents were recorded at an interval of 24 h and continued till untreated control plate was fully covered with mycelial growth of the test fungus. Inhibition zone was recorded after seven days of inoculation (Singh et al., 2005). PDA Petri plates inoculated with pathogen alone served as control. Four replications were maintained for each treatment.  
 
In vitro evaluation of fungicides by poisoned food technique
 
Efficacy of six systemic and non-systemic fungicides (Carbendazim, Chlorothalonil, Copper Oxychloride, Iprodione, Mancozeb and Propineb) against mycelial growth of A. tenuissima was tested by poisoned food technique (Schmitzs, 1930) and using PDA as basal medium. Five different concentrations viz., 50, 100, 200, 250 and 500 ppm of each fungicide were evaluated. Required concentration of each fungicides were added separately to sterilized PDA medium mixed thoroughly and poured in sterilized (90 mm) glass petri plates and allowed to solidify. All the test fungicides and their concentrations were replicated thrice. A suitable untreated control was also maintained where, medium was not supplemented with any fungicides. Each Petri plates were inoculated with active growing 5 mm mycelial of discs pathogen with the help of sterilized cork borer from the edge of the fungal culture and incubated at 25+2°C temperature for 8 days. The experiment was planned with completely randomized design (CRD). The data on the per cent infection of the disease was also converted into angular values and analyzed statistically. Colony diameter (two diagonals) was measured at 24 h after interval and continued till the untreated control plate was fully covered with mycelial growth of the test fungus. Per cent growth inhibition over control was calculated by vincent’s (1947) formula follows

 
 

 
Where,
C = diameter of the colony in check (average of bothdiagonals)
T = diameter of colony in treatment (average of both diagonals)
 
Effect of seed treatment, foliar spray, seed treatment + foliar spray with fungicides, bio- agents and their treatment combinations on Alternaria blight of pigeonpea in field condition
 
Three different experiments were carried out for the management of the disease by seed treatment, foliar spray and seed treatment + foliar spray of the fungicides, bio agents and their treatment combinations which were found most effective in the bioassay test in a field trial was conducted on research trials field during the years of September 2010-11 and 2011-12 at B.H.U., Varanasi in randomized block design (RBD) with three replications using pigeonpea Bahar susceptible cultivar. Six fungicides which completely inhibited mycelial growth were employed for seed treatment. The highly susceptible cultivar seeds were inoculated by rolling with ten days old culture of test pathogen in Petri plates. After inoculation with the pathogen, the inoculated seeds were soaked for 30 minutes in concentration 2.5 percent of six fungicides and two bio agents. The required amounts of fungicides and bio agents were added to seed in 100 ml Erlenmeyer flask and shaken thoroughly to give a uniform coating of respective fungicides before sowing (Agarwal and Sinclair, 1987). The treated seeds were sown in plot size of 10.80 × 3.0 m and row to row and plant to plant distance was 45 and 20 cm respectively with three replications per treatment.
        
In second experiment was sowing cultivar seeds in twenty five treatment combination growing in fields. After attaining the age of sixty days old plants were inoculated by spray with spore suspension of test pathogen. The spore cum mycelial suspension was prepared in sterilized distilled water a desired spore concentration (1.5 × 10⁴ spores/ml) as viewed under light microscope/haemocytometer was obtained. Pigeonpea plants were inoculated by spraying the spore cum mycelial suspension with the help of an atomizer. Similarly control plants were sprayed with the sterilized distilled water for comparison. Before inoculation of the plants to maintain relative humidity to about 90 percent by spraying the sterilized distilled water on pigeonpea plants fields for 24 hr. High humidity was maintained. The plants were irrigated from time to time to maintain proper moisture 48 hours to ensure successful penetration of the pathogen into the tissue. Spraying six fungicides and two bio agents were done forty eight hours after inoculation and repeated at ten days of interval with two subsequent sprays. Similarly control plants were sprayed with the sterilized distilled water for comparison.
            
In third experiment seed treatment + foliar spray was conducted with above procedure in seed treatments experiment, cultivar seed inoculated with test pathogen after fungicides, bio agents and their combination treated. The treated seeds were sown in twenty four plots and one plot has only pathogen inoculated seed as a controls. After attending sixty days olds cultivar plants same procedure was adopted in foliar spray and were inoculated with test pathogen after fungicides and bio-agents and their combination. Observations were made regularly for the appearance and development of symptoms. First spray of fungicides was given as soon as the blight symptoms were observed in the experimental plots followed by 2 more applications at an interval of 15 days. The recorded of disease intensity was after 10 days of last fungicides and bio-agents based on per cent leaf area covered by disease symptoms (leaf spot). Per cent disease intensity (PDI) was calculated and categorized them as highly resistant (PDI < 0), resistant (PDI >0.1-5.0%), moderately resistant (PDI >5.1-10%), moderately susceptible (PDI >10.1-25.0%), susceptible (PDI >25.1-50.0%) and highly susceptible (PDI >50%) differentials against the disease rating key using a 0-5 scale (Mayee and Datar, 1986). The data on the per cent infection of the disease were also converted into angular values and analyzed statistically. Data were recorded at per cent disease intensity and per cent disease control recorded. Total treatment combination used in fungicides and bio-agents are twenty five which denotes following i.e., T₀, T₁,  T₂, T₃, T₄, T₅, T₆, T₇, T₈, T₁×T₇, T₂×T₇, T₃×T₇, T₄×T₇, T₅×T₇, T₆×T₇, T₇×T₇, T₈×T₇, T₁×T₈, T₂×T₈, T₃×T₈, T₄,×T₈, T₅×T₈, T₆×T₈, T₇×T₈ and T₈×T₈.
 
Whereas,
 
T₀= Control,  
T₁=  carbendazim (Bavistin),
T₂= copper oxychloride (Blitox-50),
T₃=  chlorothalonil (Kavach),
T₄= mancozeb (Indofil M-45),
T₅= iprodione (Rovral -50),
T₆=  propineb (Proximain),
T₇= Hypocrea rufa,
T₈= Trichoderma harzianum,

Efficacy of Bio-agents on radial growth of Alternaria tenuissima in dual culture technique
 
Results (Table 1) revealed that all the bio agents evaluated exhibited fungistatic/antifungal activity against A. tenuissima and significantly inhibited its mycelial growth over untreated control. Hypocrea rufa was found most effective and recorded least linear mycelial growth (32.72 mm) with highest mycelial inhibition (63.64%) of the test pathogen over untreated control (90.00 mm and 00.00%). The second best antagonists were found Trichoderma harzianum recorded mycelial growth of 33.90 mm and 33.95 mm respectively and inhibition of 62.33 and 62.27 per cent, respectively both of which was at par. The antagonistic effect of the fungal bio agents against A. tenuissima found in present study may be attributed to the mechanisms viz., competition, lysis, myco-parasitism, antibiosis and production of volatile and non volatile compounds by the bio agents.
 

          
Similar results were also observed by Lal and Upadhyay (2002) and Khare (2006) against A. tenuissima of pigeonpea In vitro, Abdul et al., (2001) in T. harzianum in A. solani of tomato, Ambuse et al., (2012) against of leaf spot (A. tenuissima) of Sorrel, Gholve et al., (2012) and Gholve et al., (2014) in A. macrospora.
 
Efficacy of fungicide on radial growth of Alternaria tenuissima in poisoned food technique
 
Efficacy of each fungicide at five different concentrations was tested In vitro for radial growth of A. tenuissima. The data recorded as per cent inhibition of radial growth were presented in Table 2. A perusal of data revels that all the fungicides were superior in inhibiting the radial growth of the pathogen over control.
 

        
Irrespective of concentration Mancozeb was proved to be most effective fungicides inhibiting the radial growth of pathogen (75.26%) followed by Chlorothalonil (66.34%) and Iprodione (63.85%). Propineb (23.29%) was least effective followed by Carbendazim (27.24%) and Copper oxychloride (41.96%). Higher concentration (500 ppm) of fungicides significantly inhibit the radial growth over low concentrations (50, 100, 200 and 250 ppm) and the maximum inhibition was observed at 500 ppm and minimum at 50 ppm concentration in all the fungicides. Maximum inhibition of radial growth was recorded by Mancozeb (90.16%) followed by Chlorothalonil (83.60%) both at 500 ppm concentration and minimum was recorded by Propineb (8.19%) followed by Carbendazim (13.11%) at 50 ppm. Data further reveals that significant into interaction was observed between fungicides and their concentration. Application of Mancozeb followed by Chlorothalonil and Iprodione all at 500 ppm concentration significantly inhibited the radial growth A. tenuissima over control Propineb, Carbendazim and Copper oxychloride as compared to other concentrations least effective.
        
Present findings are collaborated with Hegde (1988) Mancozeb against A. tenuissima, Iprodione, Mancozeb, Copper oxychloride, Chlorothalonil and Carbendazim against A. tenuissima in pigeonpea other worker agree like Lal et al., (2000), Amaresh and Nargund (2002) against A. helianthi of sunflower, Gholve et al., (2012) and Gholve et al., (2014), Khan et al., (2007) against A. macrospora.
 
Effect of seed treatment with fungicides, antagonist and their treatment combination on Alternaria blight of pigeonpea in field condition
 
All the six fungicides and two bio agents and their combination tested in seed treatment field condition found significantly or partially effective, reduced the disease intensity as compared to control (Table 3 and Table 4). Twenty five treatment amongst them combination of Mancozeb with H. rufa was found most effective in reducing the disease intensity two consecutive years (6.33%) followed Mancozeb with T. harzianum (6.40%) and alone Mancozeb (6.53%), respectively. While, T. harzianum alone was least effective and maximum disease intensity (14.87%) two consecutive years were observed as compared to control followed by T. harzianum double dose (14.65%) and T. harzianum and H. rufa (15.70%) combination treatment, respectively. The combination of Mancozeb with H. rufa, Mancozeb with T. harzianum and Mancozeb single were significantly at par in reducing the disease intensity of A. tenuissima over control. Data further reveals that no significant interaction was observed between fungicides and bio agents in both the years. Application of fungicide and bio-agents combination treatment Mancozeb with H. rufa followed by Mancozeb with T. harzianum and Mancozeb alone at recommended dose significantly reduced disease intensity of A. tenuissima over control. While, T. harzianum alone, H. rufa and T. harzianum treatment combination and H. rufa alone as compared to other concentration were least effective.
 

 

        
Our results with chemical confirmed with similar findings Khare and Kumar (2006) Mancozeb and Carbendazim of pigeonpea against the Alternaria blight, Girish et al., (2007) A. alternate and A. brassicae of pigeonpea against Iprodione and Carbendazim, Ansari et al., (1990) against A. brassicae infected rapeseed mustard Mancozeb and Copper oxychloride. Our results with bio-agents confirmed with similar findings of Kumar et al., (2000) with H. rufa pathogens (A. alternata) of pigeonpea. Use of antagonists has been reported quite effective against different pathogens (Chattopadhyay et al., 2002) particularly as seed treatment material followed by fungicidal spray in managing many fungal diseases (Rohila et al., 2001). 
 
Effect of foliar spray + seed treatment with fungicides, bio agents and their treatment combination on Alternaria blight of pigeonpea
 
All the fungicides and bio agents and their treatment combinations were tested in seed treatment + foliar spray of field condition were significantly or partially effective in reduced the disease intensity of Alternaria blight as compared to control (Table 3 and Table 4). Out of twenty five treatments, combination of Mancozeb with H. rufa treatment was found most effective in reducing the disease intensity two consecutive years (11.59%) followed by Mancozeb with T. harzianum (11.60%) and Mancozeb (11.77%) single treatment, respectively. While, least effective and maximum disease intensity in two consecutive years were observed T. harzianum (35.12%) single dose followed by T. harzianum (34.68%) double dose and combination of T. harzianum and H. rufa treatment (33.83%), respectively as compared control. A combination of Mancozeb with H. rufa, Mancozeb with T. harzianum and Mancozeb alone were significantly at par with each other in reducing the disease intensity of test pathogen over control. Data further reveals that not significantly difference in various interactions was observed between fungicides and bio agents as both the years. Application of combination of Mancozeb with H. rufa followed by Mancozeb with T. harzianum and Mancozeb alone at recommended dose significantly reduced disease intensity of A. tenuissima over control. While, T. harzianum alone, combination of H. rufa and T. harzianum and H. rufa alone as compared to other concentrations were least effective.
        
In our results with chemical fungicides Mancozeb and Copper oxychloride against blight Ansari et al., (1990) of A. brassicae, Amaresh and Nargund (2002) of Mancozeb, Chlorothalonil, Copper oxychloride, Iprodione and Carbendazim on sunflower percent disease intensity of leaf blight. Rathi and Singh (2009) bio agents and fungicides as seed treatment with T. harzianum seed followed by foliar spray, Karthikeyan et al., (2008) against onion blight disease (A. palandui) seed treatment+foliar spray Mancozeb was most effective followed by H. rufa.
 
Effect of foliar spray with fungicides, bio-agents and their treatment combination on Alternaria blight of pigeonpea
 
In the present investigation of all the fungicides and bio agents and their treatment combination tested in foliar spray under field conditions were significantly or partially reduced the disease intensity of Alternaria blight in pigeonpea as compared to control (Table 3 and Table 4). Out of twenty five treatments, combination of Mancozeb with H. rufa treatment was found most effective in reducing the disease intensity two consecutive years (12.07%) followed Mancozeb with T. harzianum (12.52%) and Mancozeb (12.77%) alone treatments, respectively. Whereas, least effective and maximum disease intensity two for consecutive years were observed with T. harzianum (36.10%) at a single dose followed by T. harzianum (35.28%) at a double dose and combination of T. harzianum and H. rufa (34.72%) respectively as a compared control. A combination of Mancozeb with H. rufa, Mancozeb with T. harzianum and Mancozeb alone were significantly at par with each other in reducing the disease intensity over control. Data further reveals that no significant difference in various interactions was observed between fungicides and bio agents in both years. Application of fungicide and bio agents’ combination treatment Mancozeb with H. rufa followed by Mancozeb with T. harzianum and Mancozeb alone at recommended dose significantly reduced disease intensity of A. tenuissima over control. While, T. harzianum alone, H. rufa and T. harzianum treatment combination and H. rufa alone as compared to other concentration were least effective.
        
Our results with chemicals (Mancozeb and Chlorothalonil) is confirmed with Kushwaha et al., (2010b) in pigeonpea infected by A. tenuissima, Lal et al., (2000); Kushwaha and Narain (2001) Iprodione, Mancozeb, Copper oxychloride, Chlorothalonil and Carbendazim of A. tenuissima in pigeonpea,  Khan et al., (2007) Carbendazim and Mancozeb in Alternaria blight (A. brassicae) of rapeseed mustard, Alternaria blight in Mancozeb (Gondal et al., 2012), Kumar et al., (2005) findings antagonistic against Alternaria leaf spot of Vicia faba in H. rufa and T. harzianum, Kumar et al., (2000) of pigeonpea against H. rufa, Lal and Upadhyay (2002) H. rufa and T. harzianum against A. tenuissima (pre-rabi pigeonpea), sprays of H. rufa. Varma et al., (2008) against A. solani of tomato, Foliar spray of H. rufa, Mishra (2011) of H. rufa application Alternaria leaf spot in pigeonpea, Reshu and Khan (2012) against A. brassicae in Mustard H. rufa. Hiremath and Sundaresh (1985) the fungal (A. tenuissima) growth in vitro and Mancozeb In vivo Katiyar et al., (2001) Mancozeb and Chlorothalonil was effective.

Chemical management is most favorable and widely used method against disease. It also has hazardous effect in our environment. Fungicides with antagonistic combination approach to integrated disease management in this area may be effective and eco friendly environment disease management.

The authors are grateful to UGC; for providing fellowship during investigation and Department of Mycology and Plant Pathology, Agricultural Farm In charge, Banaras Hindu University, Varanasi for providing necessary facilities during the course of investigation.