Evaluation of fungicides and bioagents for the management of chickpea root rot

Introduction

Chickpea (Cicer arietinum L.) is the most important pulse crops grown, particularly in semi-arid and tropical regions worldwide. It contains high levels of protein, carbohydrates, fats, vitamins and minerals. Cicer arietinum L. plays very important role in sustainable agriculture through biological nitrogen fixation. In India, chickpea occupies a significant share of pulse production; however, its productivity is severely constrained by several biotic stresses, among which root rot is one of the most destructive soil-borne diseases. Root rot of chickpea, primarily caused by Rhizoctonia bataticola (Taub.) Butler (syn. Macrophomina phaseolina), leads to considerable yield losses, especially under high soil temperature and moisture stress conditions. The disease is characterized by yellowing, wilting, sudden drying of plants, and dark lesions on roots, ultimately resulting in plant death. The pathogen survives in soil and crop residues as sclerotia, making its management difficult and allowing it to persist across cropping seasons. Management of chickpea root rot has traditionally relied on chemical fungicides however, sole dependence on chemicals poses several challenges, including environmental pollution, development of pathogen resistance, high input costs, and adverse effects on beneficial soil microflora. In recent years, there has been increasing emphasis on eco-friendly and sustainable disease management strategies. Biological control using antagonistic microorganisms such as Trichoderma spp., Pseudomonas fluorescens and Bacillus subtilis has shown promising results in suppressing soil-borne pathogens through mechanisms like mycoparasitism, antibiosis, and induced systemic resistance. Integrated disease management approaches combining chemical fungicides and bioagents are gaining importance as they offer effective disease suppression while reducing chemical load and improving soil health. Evaluating the comparative and combined efficacy of chemical fungicides and bioagents against chickpea root rot is therefore essential for developing sustainable and practical disease management strategies. In view of the economic importance of chickpea and the destructive nature of root rot disease, the present investigation was undertaken to study the effectiveness of selected chemical fungicides and biological control agents in managing chickpea root rot under controlled and/or field conditions.

Materials and Methods 1) Experimental Location

The present investigation was conducted during the rabi season of 2011-2013 at the Department of Plant Pathology, Post graduate institute, Dr. PDKV, Akola. The experiment was carried out under pot culture. The soil of the experimental site was sandy loam with good drainage.

2) Pathogen Isolation and Identification

Root rot-infected chickpea plants showing typical symptoms such as sudden wilting and root discoloration were collected from the field. The infected root portions were washed thoroughly with tap water and cut into small pieces (5-10 mm). These pieces were surface sterilized with 0.1% HgCl₂ (mercuric chloride) for thirty second, followed by washings (three time) with sterile distilled water. The sterilized pieces were placed on Potato Dextrose Agar (PDA) medium and incubated at 27 ± 2 °C. The pathogen was identified as Rhizoctonia bataticola based on cultural and morphological characteristics.

3) Maintenance of Pathogen Culture

The pure culture of Rhizoctonia bataticola was maintained on PDA slants and subcultured periodically for further studies.

4) Mass Multiplication culture

The grains of sorghum were partially immersed in warm water (40 0 C to 45 0 C) for one hour. For air drying, grains spread on the clean blotting paper. Add an average 300 g air dried grains in a conical flask (1 liter) with 10 ml of water for autoclaved (30 minutes at 15 lbs psi pressure). After autoclave, in this flask add mycelial bits of Rhizoctonia bataticola and keep this flask under aseptic condition for incubation at 28 ± 2 0 C for 10 days. During this period, the flasks were shaken daily to prevent clumping of grains and to promote early fungal growth. Due to mycelial growth of fungus the grains turn whitish in colour. Well grown mass multiplied inoculum mix with an experimental sick plot before 15 days of sowing.

5) Preparation of sick soil & Pathogenicity test

Take a gunny bag and put sick plot soil for sterilization in autoclave 15 lbs psi pressure for 1 hour, continue for 3 days. After 3 days in that sterilized soil thoroughly mix mass multiplied inoculum in 1:10 proportion. Thus soil become a sick. Take 15 cm diameter size plastic pots and surface sterilized with 0.1% HgCl 2 (mercuric chloride). In sterilized plastic pots filled sick soil 1/4 th of its capacity and watered lightly. Keep these plastic pots 4 days for incubation. Sown 10 chickpea seeds of variety JG-62 and keep these pots under observation for seedling mortality. After sowing, an interval of 10 days record symptoms of root rot. Four pots served as control without inoculation. From roots of root rot seedling re-isolate fungus on PDA media.

6) Preparation of Bioagents

The bioagents used in the experiment included Trichoderma viride, Bacillus subtilis and Pseudomonas fluorescens. The cultures were obtained from the Department of Plant Pathology. Talc-based formulations of bioagents were prepared and used at recommended concentrations.

7) Fungicides Used

The chemical fungicides selected for the study were Carbendazim, Benomyl and Fosetyl AL. These were used as seed treatment at recommended doses.

8) Evaluation of fungicides, insecticides and weedicide

against root rot under in vitro condition. To estimate the effect of fungicides, insecticides, and weedicides against Rhizoctonia bataticola, the compatibility was employed. The required amount of each fungicide, calculated on the basis of its active ingredient, was added to autoclaved potato dextrose agar (PDA) to achieve the desired concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 Percent. PDA medium without any chemical served as the control. The amended medium was poured into 90 mm Petri plates with three replications for each treatment. After solidification, each plate was inoculated aseptically at the center with a 6 mm mycelial disc cut from the actively growing margin of the test fungus. For seven days inoculated petri plates were incubated at 27 ± 2 °C. After the incubation period, radial growth of the fungal colony was measured. After control plates were fully covered by the mycelial growth of pathogen, the percent inhibition of mycelial growth was calculated based on the mean colony diameter, using the following formula: Percent inhibition = C -T/C x 100 Where C = radial growth of the test fungus in the control (mm) T = radial growth of the test fungus in the treatment (mm)

9) Antagonistic test against chickpea root rot under in vitro condition

The fungal and bacterial biocontrol agent viz, Trichoderma viride, Bacillus subtilis and Pseudomonas fluorescens were tested for their biocontrol activity against Rhizoctonia bataticola by dual culture method. From the margin of 5 days old cultures cut mycelial disc of 6 mm diameter of both test pathogen and antagonists were placed opposite to each other on PDA in petriplates (90mm). The distance between two inoculum was 6 cm. The petriplates along with disc of Rhizoctonia bataticola served as the control. The inoculated petriplates were incubated at 27± 2 0 C for 7 days. The growth of Rhizoctonia bataticola was measured and the Percent growth inhibition of intersecting colonies was calculated by following formula.

10) Management of root rot of chickpea under in vivo condition

The experiment of pot culture was conducted in green house condition with above 17 treatments of 3 replication by using chickpea variety JG-62 additional with control pots without treatment. The mass multiplied inoculum of test fungus Rhizoctonia bataticola was added to the soil @ 100 g per kg soil. The earthen pots were sterilized using 0.1% HgCl₂ solution. The chickpea seeds were treated and sown 5 seeds per sterlilized earthen pots and water lightly. The seedling mortality was recorded at 30 and 60 days after sowing. The details of the treatments used in the pot experiment are given below. The details of layout and plan for pot experiment

11) Observations recorded

Germination of seeds, shoot and root length, plant dry weight, shoot and root dry weight, measurement of disease incidence, statistical analysis was recorded.

Results and Discussion 1. Isolation of Rhizoctonia bataticola

Rhizoctonia bataticola was isolated from infected roots of chickpea Plate 1, showing typical root rot symptoms. On the basis of morphological features as described by cultural and vegetative structure, Rhizoctonia bataticola was identified.

Patel and Anahosur (2000) [13] found association frequency of soil borne fungi with wilted chickpea plants and correlation coefficient between their frequency and other factors. Isolations made at 10 days interval from 10th day of sowing upto 90th day revealed preferential association of Fusarium spp.

Macrophomina phaseolina (Tassi) Goid and Sclerotium rolfsii Sacc. In relation to plant age, soil temperature and moisture and found that the Fusarium spp. was associated consistently throughout the crop period. However, it showed increasing association upto 50 days. Low temperature was more congenial for Fusarium spp. Andrabi (2011) [1] isolated Fusarium oxysporum f.sp. ciceri, F. solani and Rhizoctonia solani from the wilted chickpea (Cicer arietinum) plants.

Morphology of Rhizoctonia bataticola

In Plate 1, mycellium of Rhizoctonia were superficial or immersed, hyaline to brown, branched, septate, often tree like in form. The basic characters of the genus is formation of sclerotia. Sclerotia was seen dark brown to black, smooth, hard, and was formed of thick walled cells of irregular size but of uniform texture. The hyphae cells was barrel shaped, branching more or less at right angles and faint brown to brown colour.

Pathogenicity test of Rhizoctonia bataticola

The data presented in Table 1 revealed that in sick pots Rhizoctonia bataticola caused (100%) root rot on JG-62 in 30 DAS. In control no symptoms of root rot was observed as compared to the typical root rot symptoms was observed within 30 Days after sowing in remaining pots. The pathogen was reisolated from the infected plants, compared with the original culture, and found to be identical. Singh and Nene (1990) [15] reported that pure cultures of Rhizoctonia bataticola (Macrophomina phaseolina) were isolated from infected chickpea, pigeon pea, groundnut and sorghum plants from ICRISAT fields.

In vitro effect of herbicides on growth of Rhizoctonia bataticola

The herbicides viz., Pendimethalin 30% EC, Imazathapyr 10% SL, Targa Super, Whip Super, Oxadiargyl, and Oxyfluorfen were tested at three different concentrations.

In Table 4, Plate 4, showed that highest Percent inhibition growth of Rhizoctonia bataticola was observed in Pendimethalin 0.1, 0.2, 0.3g (100%), followed by Oxyflorfen 0.1ml, 0.2ml, 0.3ml (100%) and Targa super 0.3 ml(100%). The lowest growth inhibition was observed in Imazathapyr 0.1ml,0.2ml,0.3ml (0.00%). The results recorded in present studies are found in consences with (Khan (2010) [7] , (Kathresan (2004), Chen HouDe et al. (2005) [3] reported that Fenoxaprop-P-ethyl [16] reported that Trichoderma viride, the next best antagonist caused 62.3 and 87.0% inhibition in mycelial growth and sclerotia production followed by Trichoderma harzianum, Aapergillus ochraceous, Bacillus subtilis and Pseudomonas citrinum. Aapergillus flavus and Aapergillus niger proved to be least effective in comparison to other antagonists but were superior to control. Kumar et al.

(2011) [8] reported the efficacy of Trichoderma harzianum and In in vitro conditions Trichoderma viride inhibiting mycelial growth of Rhizoctonia bataticola compared with Pseudomonas maltophila, Pseudomonas fluorescens and Bacillus subtilis. [2] who found that seed treatment with Trichoderma viride, Trichoderma harzianum and Trichoderma aureoviride were as effective as Bavistin seed treatment. It resulted germination highest and mortality reduced. Murugesan et al. (2009) [10] reported that Imidacloprid, Monocrotophos and Pseudomonas fluorescens improved germination and increased shoot length. Whereas adverse effect of neem oil on shoot length. Thomas Moly and Vyas (1984) [18] reported that seed treatment with Carbendazim resulted in significantly higher emergence in comparison with other fungicide treatments.

Effect of combinations on plant dry weight

Table 6 showed that the lowest plant dry weight was recorded in T 14 , i.e., Pendimethalin + Bacillus subtilis, with values of 3.06 g and 4.5 g at 30 and 60 DAS, respectively, whereas the control recorded 2.33 g and 4.06 g at 30 and 60 DAS, respectively. The highest plant dry weight of 6.9 g and 8.13 g at 30 and 60 DAS, respectively, was observed in T 11 , i.e., Carbendazim + Trichoderma viride + Bacillus subtilis, followed by 6.2 g and 8.13 g at 30 and 60 DAS, respectively, in T 9 , i.e., Carbendazim + Trichoderma viride. The above findings are in conformity with, Sindhan et al. (2002) [16] Carbendazim 0.2% was most effective with 79.2% disease control and also significantly increased root and shoot length and dry weight in comparison to control. The above findings are in conformity with, (Pahwa and Prakash (1996) [14] . Sindhan et al. (2002) [16] Carbendazim 0.2% was most effective with 79.2% disease control and also significantly increased root and shoot length and dry weight in comparison to control. Nagamani et al. (2011) [12] , Field experiments were conducted during 2009-2010 and 2010-11 rabi seasons at Andhra Pradesh, India to devise an effective management strategy for dry root rot of chickpea. The treatments were T ). It also recorded highest shoot length and root length and also the highest yield.

Table 6, showed that highest root dry weight at 30 DAS and 60 DAS was observed in T 11 i.e. Carbendazim + Trichoderma viride + Bacillus subtilis i.e. 1.37g and 2.04g respectively. The lowest plant dry weight was 0.34 g and 0.54 g at 30 and 60 DAS respectively observed in T 14 i.e. Pendimethylene + Bacillus subtilis followed by T 13 i.e, Pendimethylene + Trichoderma viride whereas in control it was 0.32 g and 0.52 g at 30 and 60 DAS respectively. The results were in confirmation with, (Yadav et al.

(2006) [19] , Sindhan et al. (2002) [16] Carbendazim 0.2% was most effective with 79.2% disease control and also significantly increased root and shoot length and dry weight in comparison to control.

The effect of the treatments was found to be significant at 30 and 60 DAS. Table 6 showed that the highest shoot length at both 30 DAS and 60 DAS was observed in T 11 , i.e., Carbendazim + Trichoderma viride + Bacillus subtilis 22.88 cm and 27.03cm respectively followed by T 9 i.e. Carbendazim + Trichoderma viride i.e. 21.30 cm and 25.82 cm. The lowest Shoot length was observed in T 14 Pendimethalene + Bacillus subtilis 14.03cm, 20.7 cm at 30 and 60 DAS whereas in control it was 14.93cm and 17.36 cm at 30 and 60 DAS respectively. The findings of the present study are in agreement with, (Pahwa and Prakash (1996) [14] , (Sindhan et al. (2002) [16] .

The effect of treatments found significant at 30 and 60 DAS. The data presented in Table 6, showed that the lowest shoot length was observed in T 14 Pendimethalene + Bacillus subtilis 2.39 cm, 3.03 cm at 30 and 60 DAS whereas in control it was 2.87cm and 3.87cm at 30 and 60 DAS respectively. The highest shoot dry weight at 30 DAS and 60 DAS was 6.88 cm and 9.88 cm respectively observed in T 11 i.e. Carbendazim + Trichoderma viride + Bacillus subtilis next to 6.63cm and 9.63 cm in T 9 i.e. Carbendazim + Trichoderma viride. The findings of the present study are in agreement with, Sindhan et al. (2002) [16] . Sharma et al. (2004) [17] seed treatment with Carbendazim gave the highest grain yields during both the years. Increase of phenolic compounds has also been observed earlier in mustard and chickpea plants with application of Bavistin (Carbendazim) and Carboxin In the present study, the effectiveness of Carbendazim against Rhizoctonia bataticola may probably be attributed to the increasing levels of phenols, which provided resistance to chickpea plants against the pathogen.

Effect of combination on Percent root rot

The effect of treatments was found to be significant. The data presented in Table 7, showed that highest% wilt over control was 75.6% observed in T 8 i.e. Carbendazim next to 75.6% in T 1 i.e. Trichoderma viride. The lowest% wilt over control was observed in T 14 i.e, Pendimethylene + Bacillus subtilis 35.8% whereas in control it was 29.00%. The above results are in conformity with findings Mukhopadhyay and Mukherjee (1991) [11] and Lokesha and Benagi (2007) [9] studied that causes of dry root rot of pigeonpea the efficacy of biocontrol agents on Macrophomina phaseolina. In dual culture technique both Trichoderma virens (PDBC TVS-2) and Pseudomonas fluorescens (PDBC Pf1) significantly inhibited the mycelial growth of Macrophomina phaseolina by 78.22 Percent and 76.66 Percent respectively. However, it was found to be on par with soil drenching using Carbendazim (0.1%). Seed treatment combined with soil application of Trichoderma virens recorded the lowest root rot incidence (2.89%) among the four bioagents tested. Among the three methods of applying talc formulations of bioagents, seed treatment (4 g/kg seed) along with soil application (2 g talc powder mixed with FYM/pot) supported maximum plant growth and reduced root rot incidence compared to the other treatments, and was found significantly superior to seed treatment alone (2 g/kg seed The pathogenicity was proved by soil inoculation method. Based on the culture, morphological and pathogenic characteristics, the isolated cultures were identified as, Rhizoctonia bataticola.

The findings observed during the experimentation are summarized below.

Evaluation of Fungicides, insecticides, herbicides, and bioagents against Rhizoctonia bataticola

The effect of three fungicides was evaluated in vitro against Rhizoctonia bataticola by employing poison food technique. Inhibiting mycelial growth of Rhizoctonia bataticola among the fungicides tested, Carbendazim, Benomyl and Fosetyl Al were found effective. Antagonist effect of Trichoderma viride among herbicides Pendimethalin and Oxyfluorfen was more effective. Among the fungicide tested, Carbendazim and Benomyl were found effective in inhibiting mycelial growth of Trichoderma viride.

Percent germination

An interaction effect of treatments found significant. In treatment T 8 i.e., Carbendazim (80%) followed by treatment T 11 Carbendazim + Trichoderma viride + Bacillus subtilis i.e. (73.2%) the highest Percent germination was observed whereas in inoculated control T 1 6 (26.6%).

Plant dry weight

The maximum effect was observed in treatment T 11 , i.e., Carbendazim + Trichoderma viride + Bacillus subtilis, at every growth stage, showing significant differences over other treatments. The plant dry weight recorded in this treatment was 6.9 g and 8.53 g at 30 and 60 DAS, respectively. The minimum plant dry weight was observed in treatment T 14 , i.e., Pendimethalin + Bacillus subtilis, with values of 3.06 g and 4.5 g at 30 and 60 DAS, respectively, whereas the control recorded 2.33 g and 4.06 g at 30 and 60 DAS, respectively

Root length

The effect of treatments was found significant at 30 and 60 DAS. In T 11 i.e. Carbendazim + Trichoderma viride + Bacillus subtilis observed the highest root length recorded at 30 DAS and 60 DAS was 15.31 cm and 22.01 cm, respectively, whereas the control showed root lengths of 8.88 cm and 14.44 cm at 30 and 60 DAS, respectively.

Root dry weight

In Rhizoctonia bataticola, the more root dry weight at 30 DAS and 60 DAS was 1.37g & 2.04g respectively observed in T 11 i.e. Carbendazim + Trichoderma viride + Bacillus subtilis whereas in control at 30 and 60 DAS it was 0.32 g and 0.52 g respectively.

Shoot length.

Rhizoctonia bataticola was highest shoot length is 22.88 cm and 27.03cm observed in T 11 i.e. Carbendazim + Trichoderma viride + Bacillus subtilis respectively at 30 DAS and 60 DAS. Whereas in control it was 14.93cm and 17.36cm at 30 and 60 DAS respectively.

Shoot dry weight

The more shoot dry weight was 6.88 cm and 9.88 cm observed in T 11 i.e. Carbendazim + Trichoderma viride + Bacillus subtilis respectively at 30 DAS and 60 DAS next by 6.63cm and 9.63 cm in T 9 i.e. Carbendazim + Trichoderma viride. whereas in control it was 2.87cm and 3.87cm at 30 and 60 DAS respectively.

Percent dry root rot incidence

It was less in treatment T 8 Carbendazim i.e 24.4% and the Percent dry root rot over control is i.e. 75.6% followed by in treatment T 1 Trichoderma viride 4g/kg seed i.e.24.4% and the Percent root rot over control is i.e. 75.6% whereas in inoculated control% root rot was 71.00% and% root rot over control was 29.00%.

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