Evaluation of potential actinomycetes Bioformulation on stem rot of groundnut caused by sclerotium rolfsii

Introduction

Groundnut is a versatile food crop which provides food for human, feed for livestock and poultry, manufacturing of artificial fibres and also used as fuel for manufacturing coarse boards and cork substitutes. Groundnut, being a legume crop with root nodules, it improves the soil fertility by the fixation of atmospheric nitrogen. It is also a good organic manure because of its high nitrogen content (7.0 -8.0%) and other nutrients. Globally, groundnut is cultivated throughout the tropical, sub-tropical and warm temperate regions of the world (Nwokolo, 1996) [9] . The major production constraints are unreliable and erratic distribution of rainfall and appearance of unpredictable diseases and pests accounting low productivity. Diseases are one of the major constraints responsible for the low productivity. Crop production losses due to plant diseases by pathogenic fungi and bacteria amounts to 13% worldwide which greatly reduces the production, quality and safety of food. Ground nut stem rot caused by Sclerotium rolfsii Sacc. damages the crop with pod loss of 30-40 per cent especially near harvest time (Johnson and Subramanyam, 2000) [6] . Due to its wide host range, soilborne nature and lack of disease resistance in existing commercial cultivars management of the stem rot is difficult with chemicals. Consequently, these situations in agriculture need a serious search to identify alternative methods for plant protection, which are environmentally safer and are of natural origin. Biological control methods involving use of natural antagonists of plant pathogens have been suggested as a safe alternative to chemical methods by several workers (Gumede, 2008 and Sharma, 2011) are capable of protecting plants against many pathogens. Soil actinomycetes have revealed their wide antifungal activity (Tinatin and Nuzrat, 2006) [14] . Actinomycetes are one of the important promising group of antagonists and root-colonizing microbes which survive in soils.

Hence the present research focussed on isolation, identification and invitro evaluation of actinomycetes and study the efficicacy of bioformulation on Sclerotium rolfsii in vivo under glasshouse conditions.

Methodology

Research was carried out in the Department of Plant Pathology at Agricultural College, Mahanandi, Acharya N.G. Ranga Agricultural University, Andhra Pradesh. Rhizospheric soil samples were collected the soils of major groundnut growing areas (10 districts and 20 mandals) of Andhra Pradesh. Isolation of Actinomycetes was done from the soil suspension of 10 -3 to 10 -6 dilutions by using Ken Knight and Munaiers Agar medium (KA) (Allen, 1953). Isolates of actinomycetes were tested for their ability to inhibit mycelial growth of S.rolfsii in vitro following the dual culture technique (Toumatia et al., 2014) [15] . Based on the results of various morphological and biochemical tests, the organisms were identified upto generic level by referring to the Bergy's manual (Lechevalier, 1989) [8] . For development of formulation of actinomycetes, the isolates were cultivated in Ken Knight's broth. Talc formulation of potential isolates were assessed for its efficacy in controlling stem rot of groundnut under glasshouse conditions. Surface sterilisation of groundnut seeds was performed by sequential dipping in ethanol solution (70% v/v, 3 min), NaClO solution (0.9% w/v, 4 min) followed by washing three times in sterile distilled water (Goudjal et al., 2013) [4] . Autoclaved soils were infested with the S. rolfsii using a modified method of Sadeghi et al. (2012) [11] . Earthen pots (30 cm in diameter) filled with 5 kg soil were infested with 50 g of the pathogen inoculum previously prepared by growing on sorghum grains (50 g autoclaved sorghum grains for uninoculated soil). Pots were then watered with 100 ml sterile distilled water and incubated at room temperature for 7 days to enhance the growth of the pathogen. The Streptomyces sp. were used in infested pots by adding 20 ml of talc powder suspension formulation (10 g talc powder watered in 100 ml sterile distilled water) before sowing and after 45 DAS. Just before sowing, groundnut variety Kadiri -6 seeds were coated with 0.2% CMC as an adhesive and treated with powder formulation of potential Streptomyces isolates at the rate of 5 g kg -1 and 10 g kg -1 seeds. Five seeds were sown per pot with 3 replicates per each treatment. Nineteen treatments were imposed in this experiment. To know the population of the Streptomyces cfu were counted at 30, 75 and 105 DAS. Replications: 3 Treatments: 19 • T1: Seed treatment with Streptomyces sp. Ggd @ 5 g kg -1 seed • T2: Seed treatment with Streptomyces sp. Kdr @ 5 g kg - 1 seed • T3: Seed treatment with Streptomyces sp. Kyd @ 5 g kg -1 seed • T4: Seed treatment with Streptomyces sp. Ggd @ 10 g kg -1 seed • T5: Seed treatment with Streptomyces sp. Kdr @ 10 g kg -1 seed

• T6: Seed treatment with Streptomyces sp. Kyd @ 10 g kg -1 seed • T7: Seed treatment with Streptomyces sp. Ggd 5 g kg -1 seed + soil application with Streptomyces sp. Ggd @ 1 g / 5 kg soil • T8: Seed treatment with Streptomyces sp. Kdr 5 g kg -1 seed + soil application with Streptomyces sp. Kdr @ 1 g / 5 kg soil • T9: Seed treatment with Streptomyces sp. Kyd 5 g kg -1 seed + soil application with Streptomyces sp. Kyd @ 1 g / 5 kg soil • T10:

Seed treatment with Streptomyces sp. Ggd 5 g kg -1 seed + soil application with Streptomyces sp. Ggd @ 2 g / 5 kg soil • T11:

Seed treatment with Streptomyces sp. Kdr 5 g kg -1 seed + soil application with Streptomyces sp. Kdr @ 2 g / 5 kg soil • T12:

Seed treatment with Streptomyces sp. Kyd 5 g kg -1 seed + soil application with Streptomyces sp. Kyd @ 2 g / 5 kg soil • T13:

Seed treatment with Streptomyces sp. Ggd @ 10 g kg -1 seed + soil application with Streptomyces sp. Ggd 5 g / 5 kg soil • T14:

Seed treatment with Streptomyces sp. Kdr @ 10 g kg -1 seed + soil application with Streptomyces sp. Kdr 5 g / 5 kg soil • T15:

Seed treatment with Streptomyces sp. Kyd @ 10 g kg -1 seed + soil application with Streptomyces sp. Kyd 5 g / 5 kg soil • T16:

Seed treatment with Carbendazim @ 2 g kg -1 seed • T17:

Commercial formulation of Trichoderma spp.@ 10 g kg -1 seed • T18:

Inoculated control (pathogen inoculated) • T19:

Un-inoculated Control (without seed & soil treatment) Per cent stem rot incidence were recorded at 30, 60, 90 and 105 DAS. The talc-based formulation of potential actinomycete isolate was assessed for its efficacy in controlling stem rot of groundnut under glasshouse conditions. The susceptible groundnut cultivar, K-6 (Bunch type; duration 105-110 days) was obtained from the Agricultural College Farm, Mahanandi. The fungus, S. rolfsii was mass multiplied on sorghum seeds (Elad et al., 1980) [2] for 15 days and the inoculum was mixed with the sterilized soil in the ratio of 1:19 in polyethylene bags. The polyethylene bags were shaken vigorously to ensure uniform distribution of the inoculum. Earthen pots (30 cm diameter) were filled up with 5 kg of infested soil and arranged on the glasshouse benches. The pots were incubated for three days before planting. Seeds were treated with the powder formulation of Streptomyces sp. at the rate of 5 g and 10 g of powder formulation per kg seed and sown in infested soil. In another set of pots, seed treatment was followed by soil application of talc-based powder formulation at 1 g and 2 g per pot.

Seeds were also treated with Carbendazim @ 2 g / kg seed and commercial formulation of Trichoderma spp. @ 10 g / kg seed. Soil mixed with pathogen inoculum and seed treated without actinomycetes formulation served as inoculated control. Soil without pathogen inoculum and seed mock-treated with plain talc powder formulation was kept as un-inoculated control. Five groundnut seeds were planted in each pot. Each pot served as a replicate, and each treatment was replicated three times. Pots were maintained in the glasshouse under controlled environment conditions. The percentage of stem rot incidence was recorded at 30, 60, 90 and 105 days after sowing by counting all plants per pot showing signs and symptoms, dividing by the total plant count per pot and multiplied by 100. Experiments were conducted in Completely Randomized Design (CRD). Arc sine transformation of data on percentage of stem rot incidence was done and Duncan's multiple range test (DMRT) was first applied to the transformed values and then transferred to the original means. The data were subjected to analysis of variance (ANOVA) using SPSS 13.0 software for windows (SPSS Inc., Chicago, USA). Mean comparisons were performed by Fisher's Protected Least Significant Difference (LSD) test (P<0.05).

Results and Discussion

Roving survey was conducted to collect the isolates of actinomycetes in major groundnut growing areas of Andhra Pradesh. During the survey 180 rhizospheric soil samples were collected from the rhizosphere of healthy groundnut plants in stem rot infected fields. Isolated a total number of fifty morphologically different actinomycetes by soil dilution plate technique using Ken Knight's Agar medium (KA). Basic identification of actinomycetes was done by visual observation, general morphology, spore colony morphology including Gram stain and biochemical tests and an earthy odour. Among other inhabitants of soil samples, the isolates were identified by formation of clearing zones as the major evidence of antibiotics production. Among the fifty, twenty isolates were selected based on the growth rate of isolate on culture medium and clearing zones in dilution plates. All the twenty isolates were further identified as actinomycetes by general biochemical and morphological characters and proved that all were acid-fast negative, Gram stain positive and aerobic with aerial and substrate mycelia of different colors with varying spore chains. The phylogeny of antagonistic actinomycetes was analysed based on the nucleotide sequences of 16S rRNA gene amplified through F243 and A3R. The phylogenetic analysis revealed that the potential actinomycete isolates clustered with respective species of Streptomyces belonged to various countries (Sailaja Rani et al., 2023) [12] . These twenty isolates were selected for in vitro antagonistic study and were maintained as pure culture by routine microbiological methods on Ken Knight's agar slants. Among twenty isolates of actinomycetes, five isolates namely Ggd (73.72%), Kdr (71.44%), Kyd (70.61%), Lrp (61.33%) and Mkc (52.06%) showed maximum inhibition of S. rolfsii. Among the five actinomycete isolates (Ggd, Kdr, Kyd, Lrp and Mkc), three potential isolates were selected for preparation of bioformulations Glasshouse experiments were conducted for the purpose of further evaluation of the Streptomyces spp. as biocontrol agents. The efficacy of talc based formulations of Ggd, Kdr and Kyd were evaluated against S. rolfsii for their ability to reduce the stem rot incidence of groundnut under glasshouse conditions.Talc formulation of Streptomyces spp. Ggd, Kdr and Kyd were taken for glasshouse study. The data presented in the Table 1. Nineteen treatments were imposed to assess the combined effect of seed and soil application of Streptomyces spp. against stem rot disease of groundnut under pot culture conditions. Per cent disease incidence of stem rot was recorded at 30, 60, 90 and 105 DAS. At 30 DAS stem rot incidence was ranged from 7.2 to 18.6 per cent. Among the treatments, T13 (seed treatment with Streptomyces sp. Ggd @ 10 g kg -1 seed + soil application with Streptomyces sp. Ggd 5 g / 5 kg soil) was recorded least percent disease incidence of 7.2 and found to be highly effective in controlling stem rot disease and showed a reduction of 61.1 per cent stem rot disease over control which is on par with seed treatment with Streptomyces sp. Kyd @ 10 g kg -1 seed + soil application with Streptomyces sp. Kyd 5 g / 5 kg soil (T 15 ) (7.8), seed treatment with Streptomyces sp. Kdr @ 10 g kg -1 seed + soil application with Streptomyces sp. Kdr 5 g / 5 kg soil (T 14 ) (7.9), seed treatment with Streptomyces sp. Ggd 5 g kg -1 seed + soil application with Streptomyces sp. Ggd @ 2 g / 5 kg soil (T 10 ) (8.1) whereas seed treatment with Streptomyces sp. Kyd 5 g kg -1 seed + soil application with Streptomyces sp. Kyd @ 2 g / 5 kg soil (T 12 ) (8.2), seed treatment with Streptomyces sp. Kdr @ 5 g kg -1 seed + soil application with Streptomyces sp. Kdr 2 g / 5 kg soil (T 11 ) (8.3) and seed treatment with Streptomyces sp. Ggd 5 g kg -1 seed + soil with Streptomyces sp. Ggd @ 1 g / 5 kg soil (T 7 ) (8.3). Highest stem rot incidence of 18.6 per cent was recorded in pathogen inoculated control (T 18 ). There was an increase in incidence of stem rot at 60 DAS compared to 30 DAS in all the treatments. At 60 DAS stem rot incidence was ranged from 9.6 to 24.1%. It is evident from the Table 1 that lowest per cent disease incidence of stem rot (9.6) was recorded in the seed treatment with Streptomyces sp. Ggd @ 10 g kg -1 seed + soil application with Streptomyces sp. Ggd 5 g / 5 kg soil (T 13 ) by showing a reduction of 60.0 per cent disease over control which is on par with T 15 , (seed treatment with Streptomyces sp. Kyd @ 10 g kg -1 seed + soil application with Streptomyces sp. Kyd 5 g / 5 kg soil) (11.1), T 14 (seed treatment with Streptomyces sp. Kdr @ 10 g kg -1 seed + soil application with Streptomyces sp. Kdr 5 g / 5 kg soil) (11.8), T 10 (seed with Streptomyces sp. Ggd 5 g kg -1 seed + soil with Streptomyces sp. Ggd @ 2 g / 5 kg soil) (11.9) and T 11 (seed treatment with Streptomyces sp. Kdr 5 g kg -1 seed + soil application with Streptomyces sp. Kdr @ 2 g / 5 kg soil) (12.1), seed treatment with Streptomyces sp. Kyd 5 g kg -1 seed + soil application with Streptomyces sp. Kyd @ 2 g / 5 kg soil (T 12 ) (12.3), seed treatment with Streptomyces sp. Ggd 5 g kg -1 seed + soil application with Streptomyces sp. Ggd @ 1 g / 5 kg soil (T 7 ) (13.0) and seed treatment with Streptomyces sp. Kyd 5 g kg -1 seed + soil with Streptomyces sp. Kyd @ 1 g / 5 kg soil (T 9 ) (13.3) which were showed a reduction of 53.8, 50.9, 50.4, 49.8, 49.0, 46.0 and 45.0 per cent over control respectively. Highest stem rot incidence of 24.1 per cent was recorded in pathogen inoculated control (T 18 ) (inoculated). While in other treatments the stem rot disease incidence recorded was minimum. There was an increase in incidence of stem rot at 90 DAS compared to 30 and 60 DAS in all the treatments. At 90 DAS stem rot incidence ranged from 17.4 to 56.1 per cent. Lowest per cent disease incidence of stem rot (17.4) was recorded in the seed treatment with Streptomyces sp. Ggd @ 10 g kg -1 seed + soil application with Streptomyces sp. Ggd 5 g / 5 kg soil (T 13 ) which is on par with (T 15 ) seed treatment with Streptomyces sp. Kyd @ 10 g kg -1 seed + soil application with Streptomyces sp. Kyd 5 g / 5 kg soil (18.5) and T 14 (seed treatment with Streptomyces sp. Kdr @ 10 g kg -1 seed + soil application with Streptomyces sp. Kdr 5 g / 5 kg soil) (20.9) showed a reduction of 69.0, 67.0 and 62.7 per cent disease over control respectively. Highest stem rot incidence of 56.1 per cent was recorded in pathogen inoculated control (T 18 ). In other treatments the stem rot disease incidence recorded was moderate in (T 10 ) seed treatment with Streptomyces sp. Ggd 5 g kg -1 seed + soil application with Streptomyces sp. Ggd @ 2 g / 5 kg soil (24.4%) and seed treatment with Streptomyces sp. Kyd 5 g kg -1 seed + soil application with Streptomyces sp. Kyd @ 2 g / 5 kg soil (T 12 ) (25.4%) which were showed a reduction of 56.5 and 54.7 per cent over control respectively. Hence, in the present study more number of Streptomyces colonies were observed in red soils which may be due to more leaf drop that served as food base for colonization of Streptomyces spp. Kanti (2005) [7] stated that actinomycetes has a vital role as a saprophytic organism which decompose organic material and is essential to increase soil fertility. Several workers have also reported the potential of actinomycetes to control plant diseases (Pattanapipitpaisal and Kamlandharn, 2012) [10] . Exposure of the host plant to the heavy inoculum load of the pathogen along with the antagonist will provide ecological data on the performance of the antagonist under glasshouse conditions. Promising antagonists from controlled environment may lead to testing its efficacy under field conditions. The decomposed product of the defoliated leaves might have served as carbon sources and improved microbial activity in the rhizosphere. This results in balanced plant nutrition leading to resistance against the pathogens. Wu et al. (2019) [16] reported that a novel antifungalmycin was found to be responsible for the inhibitory effects of Streptomyces isolate N2, and when directly applied, was also able to reduce the symptoms of sheath blight on rice leaves in pot culture experiments.

Conclusion

Nineteen treatments were imposed to assess the combined effect of seed and soil application of Streptomyces spp. against stem rot disease of groundnut in pot culture under glasshouse conditions. Lowest per cent disease incidence of stem rot was recorded in the T13 (seed treatment with Streptomyces sp. Ggd @ 10 g kg -1 seed + soil application with Streptomyces sp. Ggd 5 g / 5 kg soil) and found significantly effective. Per cent disease incidence of stem rot recorded at 30, 60, 90 and 105 DAS was 7.2, 9.6, 17.4 and 26.6 per cent respectively.

Disclaimer (Artificial Intelligence)

Author(s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc.) and text-to-image generators have been used during the writing or editing of this manuscript.

Acknowledgement

AcknowledgementI take this opportunity to sincerely acknowledge Acharya N.G.Ranga Agricultural University for giving me financial assistance and a chance to explore and carry out the wide aspect of research in Plant Pathology.

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