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Screening of Different Bioagents and Fungicides against Dry Root Rot of Chickpea Incited by Macrophomina phaseolina (Tassi) Goid

Nagaraj C. Malagi1, Data Ram Kumhar1, A.L. Yadav1, Vikash Kumar1,*, Rakesh Kumar1, Anand Choudhary1, Mahesh Kumar Mimrot1
1Department of Plant Pathology, College of Agriculture, Swami Keshwanand Rajasthan Agricultural University, Bikaner-334 006, Rajasthan, India.
Background: Dry root rot of chickpea caused by Macrophomina phaseolina (Tassi) Goid is a serious biotic constraint for chickpea production in Rajasthan. For the management of soil borne disease like dry root rot of chickpea, by using fungicides alone is not feasible due to environmental and health hazards. Hence integrated management of the disease by using bio-agents and fungicides is the best alternative. Therefore, in the present investigation, our main emphasis was to identify best fungicide and bio-agent for management of dry root rot in chickpea.

Methods: Eight fungicides and four bio-agents were evaluated against dry root rot pathogen (M. phaseolina) in lab as well as in field condition. The experiment was conducted at instructional farm, COA, SKRAU, Bikaner during Rabi-2019 on most popular cv. GNG-1581 in RBD design with the application of seed treatment and soil application of different bio-agents and fungicides at different concentrations against dry root rot disease and compared with an untreated control. 

Result: Among all the fungicides used in the present investigation, tebuconazole 50% + trifloxystrobin 25% was found most effective in inhibiting the mycelial growth of the pathogen. Among all the bio-agents, T. harzianum was found most effective in inhibiting the mycelial growth of the pathogen. Under field condition, tebuconazole 50%+ trifloxystrobin 25% WG as seed treatment @ 1.5 g/kg along with T. harzianum @ 10 kg/ha as soil application gave maximum (83.76 %) disease control with highest pod yield (19.5 q/ha) and net return (Rs 39,826/ha). These treatments can provide an effective and economical management of dry root rot disease for chickpea cultivators.
Chickpea (Cicer arietinum) is an annual legume crop in temperate and sub-tropical regions. It belongs to family Fabaceae. Just after the bean, chickpea is the second most grown legume in the world. Southwest Asia (Turkey) is the originating centre and it is cultivated from ancient times both in Asia and European countries. India is the largest producer and consumer of chickpea in the world. India accounts approximately 75 per cent of world’s chickpea production (Anonymous, 2016).

In world, chickpea is cultivated in 139 lakh ha with a production of 137.31 lakh tonnes, having average productivity of 982 kg ha-1. Asia accounts for 89.2 per cent of chickpea area and comprises of 84.47 per cent of production. The countries growing chickpea which contribute about 90 per cent of total global production are India (71%) followed by Australia (3.63%), Pakistan (6.79%), Turkey (2.78%), Myanmar (2.75%) and Iran (4.25%) (FAOSTAT. 2014). Gram recorded highest production of 11.23 MT with a record productivity of 1,063 kg/ha in area of 10.56 Mha. Major 7 states contributing more than 90 per cent gram production are Madhya Pradesh (4.60 MT), Maharashtra (1.78 MT), Rajasthan (1.67 MT), Karnataka (0.72 MT) andhra Pradesh (0.59 MT), Uttar Pradesh (0.58 MT) and Gujarat (0.37MT) (Anonymous, 2016-17). One of the major crops grown in Rajasthan is chickpea that is grown in 14,86,163 ha with total production of 13,82,207 tonnes (Anonymous, 2016). If we look at the district-wise contribution to over all chickpea production in the state, Bikaner leads with a share of 13%, followed by Churu (9%), Jhunjhunu (9%), Hanumangarh (8%), Sri Ganganagar (8%), Jaipur (8%), Jaisalmer (5%), Sikar (5%), Ajmer (5%) and other remaining districts (30%) (Anonymous, 2015-16).

Nearly 172 pathogens (67 fungi, 22 viruses, 3 bacteria, 80 nematodes and phytoplasma) will cause diseases in chickpea (Nene et al.1996). These pathogens may be of  bacteria, mycoplasma, fungi, nematodes and viruses which contain high genotypic variation. Commonly seen pathogens causing diseases in chickpea crop are Ascochyta rabiei found in 35 countries, Fusarium oxysporum f.sp. ciceris (32 countries), Uromyces ciceris-arietini (25 countries), bean leafroll virus (23 countries) and Macrophomina phaseolina (21 countries). Dry root rot is a major disease causing significant losses in chickpea that is 10-25 per cent losses (Pal, 1998). But it became severe in most of the chickpea cultivating areas and become reason for more than 50 per cent losses (Massoud and Kumar, 2001).

The fungicides are the most common tools for controlling plant disease. But they are not feasible for environment, health and it leads to environment hazard. Hence integrated management of the disease by using bio-agents and fungicides is the best alternative.  Therefore, it is an urgent need to use some bio-agents and fungicides which are effective against dry root rot disease. Hence, keeping in view the importance of chickpea crop and potential threat of Macrophomina phaseolina in all the chickpea growing areas in the Rajasthan, the present investigation was undertaken to manage of this important disease through bio-agents and fungicides.
Efficacy of bio-agents and fungicides were tested against Macrophomina phaseolina both in lab as well as in field condition.
Evaluation of the efficacy of bioagents against Macrophomina phaseolina in vitro
The fungal and bacterial bioagents (Table 1) were evaluated in vitro for testing their efficacy against M. phaseolina by dual culture method (Morton and Strouble, 1955) and paper disc inoculation method, respectively on PDA medium.

Table 1: Efficacy of bioagents against M. phaseolina in vitro.

Per cent growth inhibition was calculated by following formula as; (Skidmore and Dickinson, 1976).
C = Mycelial growth of M. phaseolina in control (mm).
T = Mycelial growth of M. phaseolina in presence of antagonist (mm).
Evaluation of efficacy of fungicides against M. phaseolina in vitro
Eight fungicides (Table 2) were tested at a concentration of 100, 200, 300 and 500 ppm against M. phaseolina using poison food technique (Nene and Thapliyal, 1973) in vitro.

Table 2: Efficacy of fungicides against M. phaseolina in vitro.

Colony diameter (two diagonals) of pathogen was measured after 5 days of incubation. Medium without fungicide served as control. Per cent growth inhibition was calculated by formula (Skidmore and Dickinson, 1976).
Management of dry root rot of chickpea by bioagents and fungicides in vivo
A field trial was conducted for the management of dry root rot of chickpea using bioagents and fungicides during Rabi season 2019. A most popular chickpea cultivar GNG-1581 was used in this experiment. Seed treatment of fungicides and seed and soil treatment of the talc based bioagent formulations were done. In case of control, seeds were sown in pathogen inoculated soil without any bioagents and fungicides. Observations were taken periodically for the disease incidence.

12 treatments were conducted in the field using randomised block design and a plot size of 3  x 3 m2. Each treatment was replicated thrice. The experiment was conducted under artificial soil infested conditions. For this purpose, sand maize meal inoculum of M. phaseolina was applied at 50 g per plot and mixed properly on top surface soil using a hand rack. Standard agronomic practices recommended for cultivation of chickpea crop in this region were followed. In case of control, the untreated seeds were sown. Observations on Dry Root Rot incidence were recorded periodically as well as the grain yield was recorded after harvesting the crop.
Field trial
In the view to study the efficacy of these biocontrol agents and fungicides as soil application, seed treatment alone and in combination against dry root rot incidence, field experiments were carried out during rabi 2019 at experimental farm, College of Agriculture, Swami Keshwanand Rajasthan Agricultural University, Bikaner. The chickpea crop was sown on 9th November 2019 with 30 ´ 10 cm row to row and plant to plant spacing.
Calculation and statistical analysis
The data of per cent disease incidence in all the experiments were transformed to their Arc sin values (Fisher and Yates, 1963). The statistical analysis of the data of all the laboratory experiments was done following completely randomized design. The data of field experiments were analyzed following Randomized Block Design (Cochran and Cox, 1957). Economics of each treatment was also computed.
Efficacy of bioagents and fungicides against M. phaseolina in vitro
Efficacy of bioagents
The results presented in the Table 1 and Plate 1 shows that all the bioagents were significantly superior in controlling the test fungus over the control.

Plate 1: Efficacy of bioagents against M. phaseolina in vitro.

Among the tested fungal bioagents, maximum pathogen inhibition was resulted from T. harzianum (74.49%) followed by T. viride (64.25%), whereas in case of bacterial bioagents P. fluorescens (51.65%) and then B. subtilis (38.15). The observed results were very similar to the report of Meena et al., (2017) who observed antagonistic character of Trichoderma and other species isolated from infested soil by growing them on Czaper’s dox agar medium in Petri dishes. According to them, Four fungi Aspergillus nigerTrichoderma atrovirideT. harzianumT. viride showed different antagonistic characters inhibiting the pathogen.

The same trends were observed by Cherkupally et al., (2016). Who found that radial growth of M. phaseolina was inhibited to the maximum by T. harzianum under in vitro conditions. Similarly efficacy of Trichoderma spp. against pathogen causing brinjal root rot by dual culture method under in vitro conditions. Among the seven Trichoderma spp, T. harzianum inhibited maximum of 77.77%. Similar results were published by Sangappa and mallesh (2016). Thombre B. B. and Kohire O.D. (2018) tested seven fungal antagonists viz., Trichoderma viride, T. harzianum, T. hamatum, T. longibrachiatum, T. koningii, Gliocladiumvirens, Aspergillus niger and two bacterial antagonists Pseudomonas fluorescens and Bacillus subtilis under in vitro conditions against M. phaseolina. In these T. harzianum, T. viride and P. fluorescens showing 77.59%, 65.46%, 51.37% disease inhibition, respectively. Swamy et al., (2018) evaluated two isolates of the Trichoderma spp., four Pseudomonas fluorescens isolates, one Pseudomonas putida and one Bacillus subtilis for their efficacy. Among them maximum disease inhibition was resulted by T. harzianum (41.86%) followed by T. viride (39.07%), P. fluorescens (18.77%) and B. subtilis (14.85%). Our results are somewhat similar to these findings in efficacy of bioagents against M. phaseolina and these findings support our findings.
Efficacy of fungicides
Eight fungicides were evaluated at four concentrations, viz., 100, 200, 300 and 500 ppm against M. phaseolina using poison food technique in vitro. The data presented in Table 2 and Plate 2 showed that all the eight fungicides caused significant reduction in mycelial growth as compared to control.

Plate 2: Efficacy of fungicides against M. phaseolina in vitro.

Cent per cent growth inhibition was observed in treatment with Tebuconazole 50%+ Trifloxystrobin 25% WG at all the concentrations. Carbendazim 12%+ Mancozeb 63% inhibited cent per cent growth at three concentrations viz., 200, 300 and 500 ppm, then Thiophanate methyl inhibited cent per cent growth at two concentrations (300 and 500 ppm). Most fungicides in this study were effective @ 500 ppm like Tebuconazole 50%+ Trifloxystrobin 25% WG, Carbendazim 12%+ Mancozeb 63%, Thiophanate methyl, Carboxin 37.5%+ Thiram 37.5% and Carbendazim 50% WP. But we have to recommend the lowest possible quantity of the effective fungicide to the farmers. So Tebuconazole 50%+ Trifloxystrobin 25% WG is effective (100% control) even at 100 ppm viz. is lowest concentration in the present study.

These results were similar with the report given by Meena et al. (2018) who tested tebuconazole 50%+ trifloxystrobin 25%WG and carbendazim 12%+ Mancozeb 63% under in vitro conditions against R. solani. The results stated that tebuconazole 50%+ trifloxystrobin 25% WG fungicide reported maximum inhibition of mycelial growth at 100 ppm followed by carbendazim 12%+ mancozeb 63% WP.  Sangappa and Mallesh (2016) revealed that cent per cent mycelial growth inhibition was seen in treatment with carbendazim at 0.05, 0.1 and 0.2%.concentrations. Kumar et al., (2021) found that tebuconazole 25.9% EC was most effective in inhibiting the mycelial growth of the pathogen followed by trifloxystrobin 25% + tebuconazole 50% WG.
Management of dry root rot of chickpea by bioagents and fungicides in vivo
The obtained results were displayed in the Table 3.

Table 3: Management of Dry root rot of chickpea by bioagents and fungicides in vivo.

These observations indicate that the dry root rot of chickpea incidence was greatly reduced by combined effect of chemical and biocontrol agents than the chemicals alone or biocontrol agents alone. Incidence of disease being reduced 83.76% in combined effect of bioagent and chemical that is tebuconazole 50%+ trifloxystrobin 25% WG as seed treatment @ 1.5 g/kg along with T. harzianum@ 10 kg/ha as soil application. This treatment was followed by combined application of carbendazim 12%+ mancozeb 63% WP @ 2g/kg as seed treatment along with T. harzianum as soil application @ 10 kg/ha (79.19%). It was closely followed by tebuconazole 50%+ trifloxystrobin 25% WG as seed treatment with soil application of P. fluorescens @ 10kg/ha. Similar results were obtained by Meena et al., (2018), Veena and Reddy (2016) and Lakhran and Ahir (2020).

Biological control of diseases through antagonists is helpful for inducing the long term disease resistance in the plants. Even though the biocontrol is somewhat slow in effect, long term disease resistance can be achieved by these. Chemicals will give immediate effect but they exhaust the nutritional capacity of the soil as well as cause pollution hazards. But presently a number of scientists are working on reduction of plant diseases with combined effect of chemicals as well as biocontrol agents. Antibiotics secreted by biocontrol agents can be utilized for inhibiting the pathogen in soil, so as to reduce the soil borne diseases caused by phytopathogens as stated by Harman et al., (2010) and Nawar (2008).
The yield results given in Table 3 revealed that grain yield was enhanced considerably with combined effect of fungicide as seed treatment and bioagents as soil application than the treatments containing fungicide or bioagent alone. The grain yield was highest (19.50 q ha-1) in tebuconazole 50%+ trifloxystrobin 25%  as seed treatment @ 1.5 g/kg seed plus T. harzianum as soil application @ 10kg/ha and minimum 5.80 per cent disease incidence followed by carbendazim 12%+ mancozeb 63 %  as seed treatment @ 2 g/kg seed and T. harzianum as soil application @ 10kg/ha (18.72 qha1 and 7.83 % disease incidence). The control of soil borne plant pathogens and increase in yield of different crops after treatment with fungicides and bioagents have been reported by Elaigwu et al., (2017).

The economics computed on various treatments (Table 4) reveals that highest net gain (Rs 39,826 /ha) was obtained in the treatment T6 followed by T4 (Rs. 36,820 /ha).

Table 4: Economics of different treatments of chickpea.

Similarly Nagamani et al., (2011) conducted experiment on dry root rot of chickpea and reported that seed treatment with carbendazim @ 2 g/kg of seed+ seed treatment with T. viride @ 4 g/kg of seed + soil application of FYM fortified with T. viride recorded least disease incidence with highest yield and BC ratio. Manjunatha and Saifulla (2021) observed that fungicides and bioagents are effective for the the management of dry root rot of chickpea.

So by this present study, we can conclude that combined effect of chemicals and bioagents would be effective against the dry root rot disease in chickpea than the application of chemicals or bioagents alone.
It can be concluded that among the four bioagents, T. harzianum was found most effective in controlling the pathogen followed by T. viride. Their growth was superior than the causal organism and restricted the growth of the pathogen. Among the tested fungicides, tebuconazole 50% + trifloxystrobin 25% WG was found most effective against the mycelial growth of pathogen. Combined effect of fungicide as seed treatment and bioagent as soil application was most effective in controlling dry root rot of chickpea in field conditions. Among the twelve treatments, tebuconazole 50%+ trifloxystrobin 25% WG @ 1.5 g/kg seed and T. harzianum @ 10 kg/ha soil application yielded maximum grain yield (19.50 qha-1) and minimum disease incidence of 5.80 per cent. This treatment is better option of chickpea cultivators for enhancing the yield by managing dry root rot disease with maximum net return under field conditions.
The authors are very much thankful to Department of Plant Pathology, College of Agriculture, Swami Keshwanand  Rajasthan Agricultural University, Bikaner for providing  chickpea seeds, land to conduct the experiment and lab facilities.

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