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Efficacy of different combinations of microbial biocontrol agents against sheath blight of rice caused by Rhizoctonia solani

Abstract

Background

Sheath blight of rice caused by Rhizoctonia solani is one of the most important diseases worldwide, causing considerable yield losses. The estimation of losses due to sheath blight of rice in India has been reported to be up to 54.30%. As a consequence of this fact, eco-friendly approaches were explored in this investigation.

Results

The pathogen R. solani was isolated from the infected sheath of rice plant, which was identified and characterized on the basis of morphology and through molecular Sequencing. The sequences of ITS were submitted to NCBI GenBank and the accession number allotted is SUB11543577. In dual culture best two potential isolates of biocontrol agents were selected Pseudomonas fluorescens (Pf27) and Trichoderma harzianum (Th47). Their different combinations with Herbal Kunapajala (HKJ) were tested in glasshouse, experimental field and farmers’ fields against sheath blight pathogen R. solani. The maximum plant vigor index was found in treatment combination P. fluorescens (27) + T. harzanium (47) + Herbal Kunapajala, seed treatment, soil drenching and three foliar applications. Minimum disease severity and incidence at 30 days after sowing (DAS) and 60DAS, maximum Phenylalanine Ammonia Lyase Activity (PAL), Polyphenol Oxidase Activity (PPO) and Peroxidase Activity (PO) were also recorded in the same treatment. In field experiment the maximum population of biocontrol agents after 60 days of transplanting cfu/g soil for Trichoderma spp. (32.33 × 104) and Pseudomonas spp. (36.33 × 104) in rhizosphere and in rhizoplane. Maximum cfu/g soil for Trichoderma spp. (24.67 × 104) and Pseudomonas spp. (24.0 × 104) was observed in treatment Th + Pf + HKJ [seed treatment + soil drenching + foliar spray]. At experimental and farmers field minimum disease severity, disease incidence at 60 DAT was recorded in treatment Th + Pf + HKJ [seed treatment + soil drenching + foliar spray] and was at par with the chemical treatment Carbendazim, but the maximum yield was obtained in the treatment Th + PF + HKJ [seed treatment + soil drenching + foliar spray] due to the maximum growth promotion activity.

Conclusions

Among various treatment seed treatment, soil drenching and three foliar sprays with combination T. harzianum (47), P. fluorescens (27) and Herbal Kunapajala was found very effective in reducing the disease incidence, disease severity and increasing growth promotion activity at all conditions. Therefore, the recommendation of this investigation could be exploited under bio-intensive disease management program for sustainable cultivation of rice.

Background

Rice (Oryza sativa L.) is one of the most important cereal crops in the world that serves as a staple food source for more than 50 percent of the world’s population (Grossa and Zhao 2014). Over 90% of the world’s rice is produced and consumed in China, India, Indonesia, Bangladesh, Vietnam and Japan (Abdullah et al. 2015). Plant diseases are the major constraints for rice production. In India, average yield loss of 25–30% per annum due to plant diseases such as blast (Pyricularia grisea), brown spot (Helminthosporium oryzae), sheath blight (Rhizoctonia solani), false smut (Ustilaginoidea virens), bacterial leaf blight (Xanthomonas oryzae pv. oryzae), bacterial leaf streak (Xanthomonas oryzae pv. oryzicola) (Jha et al. 2012). Sheath blight of rice caused by Rhizoctonia solani is one of the most important diseases worldwide, causing considerable yield losses (Prasad and Reddi 2011). The disease of R. solani has been reported as being one of serious and destructive diseases limiting rice productivity all over the world, particularly where rice production is intensive, being second only to rice blast disease (Singh et al. 2016). The yield losses ranging from 4 to 50% have been reported depending on the crop stage at the time of infection, severity of the disease and environmental conditions (Singh et al. 2003). (Richa et al. 2016) also reported that under favorable environmental conditions, the sheath blight fungus can reduce yield by up to 50%. Chemical control of sheath blight is expensive and intensive use of fungicide places huge selection pressure on pathogen and thereby possibly develops fungicide resistance and is harmful for the environment and human health (Kabdwal et al. 2022). For the management of diseases in the ancient agriculture the most significant innovation, apparently a first in world Agri-history was the development of fermented liquid manures from organic wastes Kunapajala and now antagonists like Trichoderma viride, T. harzianum, Aspergillus terreus, A. niger and botanicals are the need of today (Kandhari 2007). In the present study, Herbal Kunapajala, two microbial bio control agents namely, T. harzianum and P. fluorescens known as potential biocontrol agents for effective management of the plant diseases were tested in vitro and field conditions as seed treatment, drenching and foliar spray alone and in combination to manage sheath blight disease in Kumoun region of Uttarakhand in India.

Methods

Laboratory experiments were carried out in Biological Control Laboratory, Department of Plant Pathology, College of Agriculture, G. B. Pant University of Agriculture & Technology Pantnagar, Uttarakhand, India. Field experiment was carried out at Norman Ernest Borlaug Crop Research Centre (CRC) of G.B. Pant University of Agriculture and Technology, Pantnagar during 2020. Farmers’ field trials were conducted in Sheetpuri and Vijayrampura villages situated in district Udam Singh Nagar and Devellamalla and Dumkabanga villages situated in District Nainital of Uttarakhand, India. Pure culture of T. harzianum (Th) 02, Th13, Th47, Th82, Th05 and P. fluorescens (Pf) 01, Pf24, Pf25, Pf27, Pf31 were obtained from the culture repository of Biocontrol Laboratory, Department of Plant Pathology, G. B. Pant University of Agriculture and Technology, Pantnagar. Meanwhile, the Herbal Kunapajala prepared by Asian Agri History Foundation Pantnagar center.

Isolation, identification and morphological characterizations of the fungal pathogen causing sheath blight of rice and determining the pathogenicity of tested isolates

Rhizoctonia solani was isolated from leaf sheaths of infected rice plant, which were collected from farmer’s field. The plant diseased parts were rinsed thoroughly under running tap water to remove soil present on the surface after that the washed sheaths were air dried. The sclerotial bodies were removed from diseased rice sheaths, using a sterilized blade, then the sclerotia were surface sterilized with 0.1% sodium hypochlorite solution for 1 min., then washed 3 times through sterilized distilled water, dry using sterilized blotting paper, after that placed on plates poured with potato dextrose agar (PDA). The plates were then incubated at 28 ± 2 °C in BOD incubator for 3 days. After three days, of incubation from young mycelia slide was prepared and observed under the microscope (Guttierrez et al. 1997).

Pathogenicity test

In 2019, to ensure the identity of the disease and its pathogen, pathogenicity test was conducted in glasshouse of Department of Plant Pathology, College of Agriculture, G. B. Pant University of Agriculture and Technology, Pantnagar (Uttarakhand). The soil was autoclaved twice with an interval of 24 h. at 15 p.s.i pressure for 20 min. Seeds of rice variety Pant dhan-4 were sown in plastic pots. Four seeds per pot. After germination of seeds, rice plants were inoculated during three leaf stage. Sclerotia were placed in between the base of the two tillers with cotton. This cotton was sprinkled with water, where one pot was kept as control and no pathogen was inoculated. The disease severity was assessed through daily examination of inoculated plants, 7 days after inoculation. All the inoculated plants showed symptoms, i.e., water soaked, greenish grey center with reddish brown margins. The symptoms expressed in glass house were matched with the symptoms as in the infected plants in the farmer’s field. After that the pathogen was re-isolated from these infected plants and was compared with previously isolated pathogen.

Morphological characterization of Rhizoctonia solani

Morphological characters of the pathogen were studied. Petri plates were seeded with PDA media. After getting full growth on culture plate, the permanent slide were made and examined under the compound microscope. The test pathogen was morphologically identified, according to Branching of hyphae, Color of mycelium, Formation of sclerotia (central, scattered, and peripheral), Texture of sclerotia (smooth/ rough), Location of sclerotia (Aerial and surface), color and size of sclerotia.

Molecular characterization of Rhizoctonia solani

Fungal DNA of R. solani was extracted from the pure culture by using standard CTAB protocol and genomic DNA of purified R. solani were amplified in PCR fungal universal primers (ITS1/4) and sequencing was done. Which further sequence was blast. Sequence were deposited at NCBI GenBank, and got accession numbers. The genomic DNA was extracted from following the protocol developed by Murray and Thompson (1980).

Amplification of genomic DNA with its primers

Sequencing was done in Biologia Research India Pvt. Ltd New Delhi. Using Universal primers, we amplified about 650 bp amplicon, the expected sized amplicon was seen in the positive control. ITS1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCCGCTTATTGATATGC. The test amplicon of 650 bp was purified by Gel elution/SAP. The purified product sequenced by Sanger’s method of DNA sequencing using protocol. These quenching results were assembled and compared with NCBI database.

Evaluation of efficacy of Herbal Kunapajala, Trichoderma harzianum and Pseudomonas fluorescens against Rhizoctonia solani

Under in vitro conditions

Dual culture technique

In vitro evaluations of different isolates of T. harzianum viz., Th 02, Th 05, Th 13, Th47, Th 82 and P. fluorescens viz; Pf 01,Pf 24,Pf 25, Pf 27, Pf 31, against R. solani were studied by dual culture technique described by (Rahman et al. 2009). Inhibition of mycelial growth of R. solani by the antagonist was calculated on the basis of radial growth in dual culture and control (having only R. solani) with the following formula given by (Dev et al. 2016).

$$I = \frac{C - T}{C} \times 100$$

where I Percent inhibition, C Radial growth of R. solani in check, T Radial growth of R. solani in treatment

Poisoned food technique

The present investigation was carried out using Herbal Kunapajala to test antifungal potential on the growth of R. solani through Poisoned Food Technique suggested by Nene and Thapliyal (1993). The effect of Herbal Kunapajala was tested at three different concentrations (5, 10 and 15%). Herbal Kunapajala of desired concentration was poised thoroughly in melted PDA just before pouring in sterilized Petri plates and was allowed to solidify for 03 h in Petri plates. Each plate was inoculated at the center of Petri plate with 5 mm disc of mycelia bit taken with the help of sterilized cork borer from the periphery of 7 days old culture of R. solani growing on PDA. The inoculated Petri plates were incubated at 28 ºC. Three Petri plates were used for each treatment serving as three replications. A control was also maintained where medium was not supplemented with Herbal Kunapajala. Colony diameter was observed at seventh day of incubation.

Under glasshouse conditions

Three glasshouse experiments were conducted for growth promotion, disease incidence & severity and ISR activity. First glasshouse experiment was conducted to test the plant growth promotion effect of T. harzianum (Th 47), P. fluorescens (27) and Herbal Kunapajala in Complete Randomized Design (CRD) with three replications. Each plastic pot (10 kg capacity) was filled with sterilized well pulverized sandy-loam soil. According to treatments, seeds were sown in plastic pots filled with sterilized soil and plastic pots without any treatment were kept as control. Ten numbers of seeds were sown in each pot on 15 May 2019. Observation on seed germination was recorded after 15 DAS; then 5 plants per pot were maintained for 45 days to assess growth promotion effect. Water was poured in pots, when required to maintain optimum level of moisture through visual observation.

Treatments
  1. 1.

    Trichoderma harzianum (Th) [seed treatment]

  2. 2.

    Pseudomonas fluorescens (Pf) [ST]

  3. 3.

    Th- + Pf [ST]

  4. 4.

    HKJ (herbal Kunapajal) [Foliar Spray (FS)]

  5. 5.

    HKJ (herbal Kunapajal) [Drenching]

  6. 6.

    Th + HKJ [ST + FS]

  7. 7.

    Pf + HKJ [ST + FS]

  8. 8.

    Th + Pf + HKJ [ST + FS]

  9. 9.

    Th + HKJ [ST + Drenching]

  10. 10.

    Pf + HKJ [ST + Drenching]

  11. 11.

    Th + Pf + HKJ [ST + Drenching]

  12. 12.

    Th + HKJ [ST + Drenching + FS]

  13. 13.

    Pf + HKJ [ST + Drenching + FS]

  14. 14.

    Th + Pf + HKJ [ST + Drenching + FS]

  15. 15.

    Carbendazim[ST + FS]

  16. 16.

    Control.

where (Th = Trichoderma harzianum, Pf = Pseudomonas fluorescens, HKJ = Herbal Kunapajala, ST = seed treatment, FS = foliar spray).

Evaluation of antagonistic potential of biocontrol agent and herbal Kunapajala under glasshouse conditions

Second glasshouse experiment was conducted to test antagonistic potential of alone and different combinations of sixteen treatments of T. harzianum (Th47), P. fluorescens (27) and herbal Kunapajala in Complete Randomized Design (CRD) with three replications. Each plastic pot (10 kg capacity) was filled with sterilized well pulverized sandy-loam soil. After sterilization, soil was inoculated with R. solani before one week of sowing. R. solani was inoculated in soil @25 gm/10 kg pot, followed by sowing individually seeds with treatments alone and with different combinations of Trichoderma, Pseudomonas and herbal Kunapajala and soil without any treatment inoculated with R. solani was kept as control. Five numbers of seeds were sown in each pot on 15 May 2019.

Evaluation of Induced Systemic Resistance (ISR) activity of bioagents and Herbal Kunapajala against the R. solani

Third glasshouse experiment was conducted after the screening of different treatments in above two glasshouse experiments and eleven potential different treatments were selected. This experiment was conducted to test Induced biochemical changes due to the different combinations treatments of T. harzianum (Th47), P. fluorescens (Pf27) and herbal Kunapajala. The experiment was conducted in Complete Randomized Design (CRD) with three replications. Each plastic pot (10 kg capacity) was filled with sterilized well pulverized sandy-loam soil. The seeds were sown according to the treatments in combinations of Trichoderma, Pseudomonas, herbal Kunapajala and soil without any treatment were kept as control. Five numbers of seeds were sown in each pot on 20th June 2020. After 30 days all the plants were challenged through the pathogen R. solani mycelium bits with sclerotia were placed in between the base of the two tillers with cotton, cotton was sprinkled with water.

Treatments

  1. 1.

    Th + HKJ [ST + FS].

  2. 2.

    Pf + HKJ [ST + FS].

  3. 3.

    Th + Pf + HKJ [ST + FS].

  4. 4.

    Th + HKJ [ST + Drenching].

  5. 5.

    Pf + HKJ [ST + Drenching].

  6. 6.

    Th + Pf + HKJ [ST + Drenching].

  7. 7.

    Th + HKJ [ST + Drenching + FS].

  8. 8.

    Pf + HKJ [ST + Drenching + FS].

  9. 9.

    Th + Pf + HKJ [ST + Drenching + FS].

  10. 10.

    Carbendazim[ST + FS].

  11. 11.

    Control.

where (Th = Trichoderma harzianum, Pf = Pseudomonas fluorescens, HKJ = Herbal Kunapajala, ST = seed treatment, FS = foliar spray).

Samples collection

These inoculated sheath samples were collected 0, 2, 4, 6, 8 days after the challenged inoculation. These sheath samples were kept in ice bags just after collecting them and then in deep fridge (− 80 °C) and were used for preparation of crude enzyme. Phenylalanine Ammonia Lyase Activity (PAL Activity) was determined using the method explained by (Whetten and Sederoff 1992). Peroxidase Activity (PO) activity was determined as per procedure given by Hammerschmidt et al. (1982). Polyphenol Oxidase Activity (PPO) was determined as per procedure suggested by (Mayer et al.1965).

Evaluation of Herbal Kunapajala, T. harzianum and P. fluorescens for the management of sheath blight of rice at experimental field

Seed sowing in nursery

Rice seeds of cultivar (Pant Dhan 4) were sown in 11 nursery beds for rising rice seedlings keeping the consideration of elevation selected treatments. Seed treatments were given according to plan. Light irrigations were given at frequent intervals. Standard agronomical practices were followed for raising the seedlings.

Transplanting

Seedlings of rice were uprooted from nursery beds 20 DAS. The plot size was (2.5 × 3 m2). A spacing of 50 cm between rows and plants was maintained. As per treatments decided desired three foliar spray of bioagents/HKJ/Carbendazim given i.e. 1st 30, 2nd 60 and 3rd 75 days after transplanting (DAT). The untreated seedlings served as control. To keep the experimental field free from insect pest, 3 sprays of imidacloprid (0.1%) were given i.e. 25, 40, 60 and 80 (DAT). The experiment was laid out in a randomized block design with 11 selected treatments each with three replications.

Observations

Data on plant growth promotion, disease incidence, disease severity and total grain yield were recorded.

Disease incidence

Disease incidence was recorded for sheath blight of rice

$${\text{Percent}}\;{\text{Disease}}\;{\text{Incidence}} = \frac{{{\text{Number}}\;{\text{of}}\;{\text{diseased}}\;{\text{plants}}}}{{{\text{Total}}\;{\text{number}}\;{\text{of}}\;{\text{plants}}}} \times 100$$

Disease severity

Disease severity was recorded by observing the leaves lesions which were usually irregular, initially water soaked, ellipsoid and greenish grey spots are present and later lesions becomes greyish white centers with brown margin as they grow old. The rating was done on scale of 0–9 as developed by International Rice Research Institute (IRRI) in 2002.

Rating

Description

0

No infection

1

Vertical spread of lesions up to 20% of plant height

3

Vertical spread of lesions up to 21 to 30% of plant height

5

Vertical spread of lesions up to 31 to 45% of plant height

7

Vertical spread of lesions up to 46 to 65% of plant height

9

Vertical spread of lesions more than 65% of plant height

Percent disease index (PDI) was calculated by using the following formula:

$${\text{Disease}}\;{\text{index(\% ) }} = \, \frac{{{\text{Sum}}\;{\text{of}}\;{\text{all}}\;{\text{numerical}}\;{\text{ratings}}}}{{{\text{No}}{.}\;{\text{of}}\;{\text{leaves}}\;{\text{examined}} \times {\text{maximum}}\;{\text{grade}}}} \, \times { 100}$$

Population dynamics of the biocontrol agents in rhizosphere and rhizoplane soil

The dilution plate count technique was applied (Hirte 1969) to evaluate the biocontrol agents (Trichoderma spp., P. fluorescens) population in soil. Rhizosphere and rhizoplane soil samples were randomly taken from treated experimental rice nursery and field. One soil was weighted, transferred to 10 ml (W/V) of sterile distilled water; the soil suspension was diluted from 101 to 108, and stirred well. For checking the population of Trichoderma in rhizosphere soil, the dilution factors 104, 105, 106 were used. One ml of each dilution was poured into the Petriplates with the help of pipette. Then fresh Trichoderma selective media (TSM) medium was seeded on the same Petriplates, and mixed well. Three replications of each dilution were kept. Plates were wrapped by parafilm and incubated at 28 ± 2 °C in Biological Oxygen Demand (BOD) incubator. After 3 days colony were observed in Petriplates.

Same technique was used to evaluate P. fluorescens, but the King’s B medium was used for the growth of bacteria using dilution plate technique of rhizospheric and rhizoplane soil at the dilutions of 102 to 104. After 24 h. Colony was observed in Petriplates.

Evaluation and demonstration of effective selected treatments for the management of sheath blight of rice at farmers field

Evaluation of effective selected four treatments after glasshouse and research field experimental was carried out at farmers’ fields during Kharif season of 2021 in four villages Davlamalla and Dumkabangar in District Nainital and Seethpuri and Vijayrampura of district Udam Singh Nagar. The villages were included in important rice growing belt of Uttarakhand. Fields were prepared by ploughing three times and NPK was applied in the form of (140 kg) urea, (50 kg) single super phosphate, (60 kg) muriate of potash as basal application.

Treatments

  1. 1.

    Th + Pf + HKJ [ST + Drenching], Th + Pf + HKJ

  2. 2.

    Th + Pf + HKJ [ST + Drenching + FS],

  3. 3.

    Carbendazim[ST + FS],

  4. 4.

    Control

Data on disease incidence, disease severity and total grain yield were recorded.

Statistical analysis

Experimental data was analyzed, using standard procedure for ANOVA with the help of computer having analysis program STPR-3, programmed by the Department of Mathematics and Statistics, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar -263 145. The conclusions were drawn based on critical difference at 5% level of significance.

Results

Isolation, identification and morphological characterization of the fungal pathogen causing sheath blight of rice and determining the pathogenicity of tested isolates

The pathogen R. solani was isolated from the infected sheath of rice plant collected from farmer’s field of U.S.Nagar District of Uttarakhand. Morphological characteristics of the fungus studied on potato dextrose agar media, at the beginning the mycelium was white and then turned creamy white and dark brown in later stage of maturity (Fig. 1). From obtained fresh culture, the slides were prepared and observed under compound microscope. The fungal hyphae often branch at 90° angle having anastomosis reaction (Fig. 2). All the inoculated plants showed typical sheath blight symptoms i.e. water soaked, greenish grey center with reddish brown margins. In later stage of plant, the lesions coalesce and became greyish white center with reddish brown margin on the leaf sheath. The symptoms expressed were matched with the symptoms as in the infected plants in the field then the pathogen was re-isolation from this sample on PDA slants and was compared with previously isolated fungus. The pathogenicity test reveals that R. solani, isolated from infected rice plant tissues, caused sheath blight disease symptoms on the rice plants sown in glasshouse. Its pure culture was found pathogenic and produced typical sheath blight symptoms. Its isolate was characterized based on morphological and conidial characteristics for further confirmation at molecular level; molecular studies were conducted by using universal ITS primers.

Fig. 1
figure 1

Pure culture of Rhizoctonia solani on PDA; (A 5 Days old, B 10 Days old culture)

Fig. 2
figure 2

Microscopic images showing typical hyphal characters of Rhizoctonia solani (Tended to branch at right angle and constriction at base) under 40x

PCR primer pairs of ITS1/ITS4 yielded specific PCR products of approximately 651 bp (ITS). PCR product was gel eluted by using Medauxin® gel extraction kit and then sanger sequenced the genes. DNA sequences for ITS1/ ITS4 gene obtained were compared by using NCBI BLAST programme. Based on ITS sequences the results of BLAST showed that the. R. solani was showed 100 per cent homology with the reference GenBank accession number is [MT587794.1] and other strains showing homology with R. solani infecting rice are [MT587795.1], [MT587796.1] and [MT587791.1]. Sequences of ITS were submitted to NCBI GenBank and the accession numbers SUB11543577 Rhizoctonia solani ITS1/4 [ON627716] was allotted.

Evaluation of efficacy of Herbal Kunapajala, T. harzanium and P. fluorescens against R. solani:

In vitro and in vivo conditions

The observations of the dual culture reveal that all isolates were able to inhibit growth of R. solani significantly. Maximum % growth inhibition (90.74%) was recorded by P. fluorescens (27) and T. harzanium (47) (89.63%), the inhibition % ranged from 44.08 to 90.74%at 6 DAI (days after incubation) were significantly higher than other isolates (Table 1).

Table 1 In vitro screening through dual culture technique of potential biocontrol agents against Rhizoctonia solani at 6 days after inoculation

After in vitro screening of ten (five each) Trichoderma and Pseudomonas isolates, the best T. harzianum (47) and P. fluorescens (27) were selected on the basis of antagonistic potential and further their sixteen different treatments alone and in combinations with Herbal Kunapajala, were tested under glasshouse conditions. As presented in Table 2, the maximum germination percentage was recorded in treatments Th + Pf + HKJ [ST + Drenching + FS] (88.33%), followed by Th + Pf + HKJ [ST + Drenching] (84.33%), Th + Pf + HKJ [ST + FS] (83.12%), Th- + Pf [ST] (82.66%), Carbendazim [ST + FS] (81.33%) and minimum germination was recorded in treatment control (68.33%). Shoot length, root length, fresh weight of shoot, dry weight of shoot and fresh weight root and dry weight of root were higher in all the treatments than in the control. Maximum shoot & root length (72.70 cm), (24.08 cm), fresh & dry shoot weight (4.15 g), (0.79 g), fresh & dry root weight (0.93 g), (0.68 g), were recorded in Th + Pf + HKJ [ST + Drenching + FS]. Maximum plant vigor index (8548.58) was observed in Th + Pf + HKJ [ST + Drenching + FS] and was significantly higher than other treatments and control (4215.96).

Table 2 Effect of different treatments of Trichoderma harzianum- 47, Pseudomonas fluorescens -27 and herbal Kunapajala on the growth promotion activity

Under experimental field conditions, all the treatments were found exceeded the control in plant height, panicle length and number of tiller (Table 3). Maximum plant height (82.66 cm) was observed in Th + Pf + HKJ [ST + Drenching + FS], followed by Th + Pf + HKJ [ST + Drenching] (81.33%), Carbendazim [ST + FS](81.0%), Th + Pf + HKJ [ST + FS](80.34%), all the other treatments were also higher than control (63.0%). Maximum percent plant height increase was recorded in the treatments Th + Pf + HKJ [ST + Drenching + FS] (31.21%) followed by Th + Pf + HKJ [ST + Drenching] (29.10%), Carbendazim [ST + FS](28.57%), Th + Pf + HKJ [ST + FS] (27.52%) over control (Table 4).

Table 3 Effect of different treatments of Trichoderma harzianum (47), PF = Pseudomonas fluorescens (27) and Herbal Kunapajala on Plant vigor at experimental field
Table 4 Phenylalanine Ammonia Lyase (PAL), Polyphenol Oxidase (PPO) and Peroxidase (PO) activity in rice plants against the sheath blight pathogen Rhizoctonia solani through the different combination of biocontrol agents and Herbal Kunapajala

In glasshouse conditions

The data recorded 30 days after sowing (Table 5) revealed that the minimum disease severity (14%) was recorded in treatment Carbendazim [ST + FS], followed by Th + Pf + HKJ [ST + Drenching + FS] (15%), Th + Pf + HKJ [ST + Drenching] (17%), Th + Pf + HKJ [ST + FS](17.67%), Th + Pf [ST](17.67%) there treatments were at par with each other but significantly different from the other treatments and control (38%). The maximum reduction in disease severity at 30DAS was observed in Carbendazim [ST + FS] (63.16%), followed by Th + Pf + HKJ [ST + Drenching + FS] (60.52%), Th + Pf + HKJ [ST + Drenching] (55.26%), Th + Pf + HKJ [ST + FS](53.50%), Th- + Pf [ST](53.50%) over control. After 60 days of sowing, minimum disease severity (25.33%) was recorded in treatment Carbendazim [ST + FS] and was at par with Th + Pf + HKJ [ST + Drenching + FS] (28%) but significantly different from the other treatments and control (76.33%). The maximum reduction in disease severity at 60 DAS was recorded in Carbendazim [ST + FS] (66.81%), followed by Th + Pf + HKJ [ST + Drenching + FS] (63.32%), Th + Pf + HKJ [ST + FS](57.64%), Th + Pf + HKJ [ST + Drenching](52.84%), Th- + Pf [ST](50.65) over control (Table 5). Minimum disease incidence 60 DAS was observed in Carbendazim [ST + FS] (25%) and was at par with Th + Pf + HKJ [ST + Drenching + FS] (28.33%) but significantly different from the other treatments and control (73.33%). The maximum reduction in disease incidence at 60 DAS was recorded in Carbendazim [ST + FS] 65.91%), followed by Th + Pf + HKJ [ST + Drenching + FS] (61.37%), Th + Pf + HKJ [ST + Drenching](40.91%), Th [ST] (40.45%), TH + HKJ [ST + Drenching + FS](39.99%) over control.

Table 5 Screening of different treatments of Trichoderma harzianum (47), Pseudomonas fluorescens (27) and herbal Kunapajala against Rhizoctonia solani under glasshouse conditions

The maximum yield was observed in Th + Pf + HKJ [ST + Drenching + FS] (27 g/plant), followed by Carbendazim [ST + FS] (24.33 g/plant), Pf + HKJ [ST + Drenching] (22.67 g/plant) respectively, TH + HKJ [ST + Drenching] (20 g/plant) and significantly different from control (14 g/plant). Maximum percent increase in grain yield was Th + Pf + HKJ [ST + Drenching + FS] (92.86%), followed by Carbendazim [ST + FS](73.79%), Pf + HKJ [ST + Drenching] (61.93%), Th + HKJ [ST + Drenching] (50%) over control. The maximum test weight was recorded in Th + Pf + HKJ [ST + Drenching + FS] (26 g), followed by Carbendazim [ST + FS](22.33 g), Pf + HKJ [ST + Drenching] (21 g), Th- + Pf [ST] (20.33 g/plant), Th [ST] (20.33 g/plant), Th + HKJ [ST + Drenching] (20.33 g/plant) and significantly different from control (14 g/plant). Maximum percent in increase in test weight was observed Th + Pf + HKJ [ST + Drenching + FS] (62.50%), followed by Carbendazim [ST + FS](39.56%), Pf + HKJ [ST + Drenching](31.25%), Th- + Pf [ST] (27.06%), Th [ST] (27.06%),TH + HKJ [ST + Drenching] (27.06%) over control.

Induction of the activity in rice plants through the different combination of biocontrol agents and Herbal Kunapajala

PAL activity

The data recorded in Table 4 revealed that the PAL activity was increased in every treatment up to 6 days after challenged by the R. solani, thereafter, 6 days the activity was decreased. The maximum PAL activity (0.98, 1.12, 1.26 and 1.33 ∆OD/mm/g of fresh tissue) on 0 DAI, 2 DAI, 4 DAI and 6 DAI was recorded in treatment combination Th + Pf + HKJ [ST + Drenching + FS], followed by Pf + HKJ [ST + Drenching + FS] (0.92, 0.98, 1.03 and 1.16 ∆OD/mm/g of fresh tissue), Th + Pf + HKJ [ST + FS] (0.92, 0.96, 1.03 and 1.14 ∆OD/mm/g of fresh tissue) and Pf + HKJ [ST + Drenching] (0.91, 0.98, 1.02 and 1.12 ∆OD/mm/g of fresh tissue). The minimum PAL activity (0.11, 0.18, 0.20, 0.23 ∆OD/mm/g of fresh tissue) was recorded in control. On eighth day after inoculation the PAL activity was decreased. On 8 DAI the maximum PAL activity (1.11 ∆OD/mm/g of fresh tissue) was recorded in treatment combination Th + Pf + HKJ [ST + Drenching + FS],followed by Pf + HKJ [ST + Drenching + FS] (1.03 ∆OD/mm/g of fresh tissue), Pf + HKJ [ST + Drenching] (0.99 ∆OD/mm/g of fresh tissue) and Th + Pf + HKJ [ST + FS] (0.96 ∆OD/mm/g of fresh tissue). The minimum PAL activity (0.14 ∆OD/mm/g of fresh tissue) was recorded in control.

PPO activity

Maximum PPO activity i.e. 1.12 ∆OD/min/g of fresh tissue were recorded on 6 DAI for treatment combination Th + Pf + HKJ [ST + Drenching + FS], followed by Th + Pf + HKJ [ST + Drenching](1.02 ∆OD/mm/g of fresh tissue), Th + Pf + HKJ [ST + FS]](1.01 ∆OD/mm/g of fresh tissue), Pf + HKJ [ST + Drenching] (0.89 ∆OD/mm/g of fresh tissue), Pf + HKJ [ST + Drenching + FS] (0.89 ∆OD/mm/g of fresh tissue). The minimum PPO activity (0.21 ∆OD/mm/g of fresh tissue) was recorded in control. On eighth day PPO activity decreased. Maximum PPO activity i.e. 0.99 ∆OD/mm/g of fresh tissue were recorded on 8 DAI for treatment combination Th + Pf + HKJ [ST + Drenching + FS], followed by Th + Pf + HKJ [ST + Drenching] (0.89 ∆OD /mm/g of fresh tissue), Th + Pf + HKJ [ST + FS](0.85 ∆O.D. /min/g of fresh tissue), Pf + HKJ [ST + Drenching + FS] (0.79 ∆OD/mm/g of fresh tissue), Pf + HKJ [ST + Drenching] (0.78 ∆OD/mm/g of fresh tissue). The minimum PPO activity (0.14 ∆OD/mm/g of fresh tissue) was recorded in control (Table 4).

PO activity

PO activity (Table 4) was found different in all the treatments at all the days’ interval and was observed higher than the control. At 8DAI fall in the PO activity was recorded. At 0 DAI maximum PO activity (0.96∆OD /mm/g of fresh tissue), recorded in treatment Th + Pf + HKJ [ST + Drenching + FS] and Th + Pf + HKJ [ST + Drenching], followed by 0.90 ∆OD/mm/g of fresh tissue in treatment Pf + HKJ [ST + Drenching] and Th + Pf + HKJ [ST + FS], Pf + HKJ [ST + Drenching + FS] (0.85∆OD/mm/g of fresh tissue) and minimum PO activity was found in control (0.21∆OD/mm/g of fresh tissue). On 2DAI maximum PO activity was in Th + Pf + HKJ [ST + Drenching + FS] (1.05∆OD/mm/g of fresh tissue) and minimum PO activity was in control (0.23∆OD/mm/g of fresh tissue). On 4 DAI maximum PO activity was in Th + Pf + HKJ [ST + Drenching + FS] (1.23 ∆OD/mm/g of fresh tissue) and minimum PO activity was in control (0.25∆OD/mm/g of fresh tissue). It was recorded that on 6 DAI maximum PO activity was in Th + Pf + HKJ [ST + Drenching + FS] (1.30 ∆OD/mm/g of fresh tissue) and minimum PO activity was in control (0.29 ∆OD/mm/g of fresh tissue). On eighth day the PO activity decreased in all the treatments. Maximum PO activity on 8DAI was in Th + Pf + HKJ [ST + Drenching + FS] (1.02 ∆OD/mm/g of fresh tissue) and minimum PO activity was found in control (0.13 ∆OD/mm/g of fresh tissue).

Effect of different treatments on population dynamics of the Trichoderma spp. and P. fluorescens in rhizosphere and rhizoplane soil

Population of Trichoderma spp. and P. fluorescens was observed in colony forming unit (cfu) from rhizosphere and rhizoplane soil experimental field in CRC at 0, 30, 60 and 90 DAT. Results recorded from rhizosphere soil of experimental field plots and presented in Fig. 3 revealed that population of both the biocontrol agents Trichoderma spp., P. fluorescens increased up to 60 DAT, there after their populations declined in all the treatments.

Fig. 3
figure 3

Rhizosphere Population of Trichoderma spp. and Pseudomonas fluorescens in different combinations of treatments

On zero DAT population of Trichoderma spp. was recorded in the range of 1.0 × 104 to 9.00 × 104 cfu and population of P. fluorescens was recorded in the range of 2.0 × 104 to 11.33 × 104 cfu. Maximum cfu/g soil (9.0 × 104) for Trichoderma spp. was observed in treatment Th + PF + HKJ [ST + Drenching] which was seed treatment with T. harzianum (47), P. fluorescens (27) and Herbal Kunapajala, the population of Pseudomonas spp. was in this treatment was 8.33 × 104 cfu. But in case of P. fluorescens maximum cfu/g soil (11.33 × 104) was recorded in treatment Th + Pf + HKJ [ST + Drenching + FS] and was higher than the P. fluorescens population in control (6.33 × 104) and carbendazim (2.00 × 104), respectively. While Trichoderma spp. population in this treatment was 7.33 × 104 cfu/g soil, which was higher than the Trichoderma spp. population in control and carbendazim, which was (4.33 × 104) and (1.0 × 104), respectively. The population of Trichoderma spp. was recorded on 30 DAT and found in the range of 5.0 × 104 to 23.66 × 104 cfu and population of P. fluorescens was recorded in the range of 6.0 × 104 to 27.66 × 104 cfu. Maximum cfu/g soil for Trichoderma spp. (23.66 × 104) and P. fluorescens (27.66 × 104) was observed in treatment Th + Pf + HKJ [ST + Drenching + FS] and was significantly higher than the population of Trichoderma spp. and P. fluorescens population in control (5.0 × 104), (6.0 × 104) and carbendazim (5.0 × 104), (6.0 × 104) respectively.

The population of Trichoderma spp. was recorded on 60 DAT and found in the range of 5.0 × 104 to 32.33 × 104 cfu and population of P. fluorescens was recorded in the range of 5.0 × 104 to 36.33 × 104 cfu. Maximum cfu/g soil for Trichoderma spp. (32.33 × 104) and P. fluorescens (36.33 × 104) was observed in treatment Th + Pf + HKJ [ST + Drenching + FS] and was significantly higher than the population of Trichoderma spp. and P. fluorescens population in control (5.0 × 104), (7.0 × 104) and carbendazim (6.0 × 104), (5.0 × 104), respectively.

After 90 days of transplanting population of Trichoderma spp. and P.fluorescens was declined in all the treatments. Maximum cfu/g soil for Trichoderma spp. (26.0 × 104) and P. fluorescens (30.66 × 104) was recorded in treatment Th + Pf + HKJ [ST + Drenching + FS] and was significantly higher than the population of Trichoderma spp. and P. fluorescens in control (3.33 × 104) and (5.0 × 104) and carbendazim (3.0 × 104) and (3.66 × 104), respectively.

Results recorded from rhizoplane soil of experimental field plots and presented in (Fig. 4) revealed that population of both the biocontrol agents Trichoderma spp., P. fluorescens increased up to 60 DAT, thereafter their populations declined in all the treatments. On zero DAT population in rhizoplane soil of Trichoderma spp. was recorded in the range of 0.66 × 104 to 5.33 × 104 cfu and population of P. fluorescens was recorded in the range of 1.33 × 104 to 8.0 × 104 cfu. Maximum cfu/g soil (5.33 × 104) for Trichoderma spp. was observed in treatment Th + Pf + HKJ [ST + Drenching], the population of P. fluorescens was in this treatment was also 5.33 × 104 cfu. The maximum population of Pseudomonas spp. (8.00 × 104) cfu/g soil was recorded in treatment Th + Pf + HKJ [ST + Drenching + FS] and was higher than the Pseudomonas fluorescens population in control (3.0 × 104) and carbendazim (1.33 × 104), respectively, while Trichoderma spp. population in this treatment was 5.0 × 104 cfu/g soil, which was higher than the Trichoderma spp. population in control (2.0 × 104) and carbendazim (0.66 × 104), respectively.

Fig. 4
figure 4

Rhizoplane Population of Trichoderma spp. and Pseudomonas fluorescens in different combinations of treatments

The population of Trichoderma spp. was recorded on 30DAT and found in the range of 3.33 × 104 to 19.33 × 104 cfu and population of P. flurencens was recorded in the range of 4.0 × 104 to 20.33 × 104 cfu. Maximum cfu/g soil for Trichoderma spp. (19.33 × 104) and P. fluorescens (20.33 × 104) was observed in treatment Th + Pf + HKJ [ST + Drenching + FS] and was significantly higher than the population of Trichoderma spp. and P. fluorescens population in control (4.0 × 104), (4.0 × 104) and carbendazim (3.33 × 104), (4.0 × 104), respectively.

The population of Trichoderma spp. was recorded on 60 DAT and found in the range of 4.67 × 104 to 24.67 × 104 cfu and population of Pseudomonas spp. was recorded in the range of 4.3 × 104 to 24.0 × 104 cfu. Maximum cfu/g soil for Trichoderma spp. (24.67 × 104) and P. fluorescens (24.0 × 104) was observed in treatment Th + Pf + HKJ [ST + Drenching + FS] and was significantly higher than the population of Trichoderma spp. and P. fluorescens population in control (4.67 × 104), (5.0 × 104) and carbendazim (5.0 × 104), (4.33 × 104), respectively.

After 90 days of transplanting population of Trichoderma spp. and P. fluorescens was fall down in all the treatments. Maximum cfu/g soil for Trichoderma spp. (22.0 × 104) and P. fluorescens (22.66 × 104) was recorded in treatment Th + Pf + HKJ [ST + Drenching + FS] and was significantly higher than the population of Trichoderma spp. and P. fluorescens in control (3.33 × 104) and (3.33 × 104) and carbendazim (2.66 × 104) and (2.66 × 104), respectively.

Evaluation of the potential selected combinations of Herbal Kunapajala, T. harzianium and P. fluorescens against Sheath blight of rice disease

Under experimental field conditions

To compare the effect of selected different combinations of T. harzianum, P. fluorescens and Herbal Kunapajala against R. solani causes sheath blight of rice, a field experiment was conducted during the Kharif season of year 2020. The observations on percent disease severity, disease incidence, grain yield and test weight were recorded in (Table 6). The maximum reduction in disease severity at 30 DAT was observed in Carbendazim [ST + FS](73.62%), followed by Th + Pf + HKJ [ST + Drenching + FS] (67.03%), Th + HKJ [ST + Drenching + FS] (50.54%), Th + Pf + HKJ [ST + FS] (47.25%) over control. Maximum reduction in disease severity at 60 DAT was observed in Carbendazim [ST + FS](72.42%), followed by Th + Pf + HKJ [ST + Drenching + FS] (68.96%), Pf + HKJ [ST + Drenching + FS] (61.37%), Th + HKJ [ST + Drenching + FS] (54.48%), over control. Maximum reduction in disease incidence at 60 DAT was observed in Carbendazim [ST + FS] (69.07%), followed by Th + Pf + HKJ [ST + Drenching + FS] (65.78%), Th + HKJ [ST + Drenching + FS] (53.30%), Th + Pf + HKJ [ST + Drenching] (52.58%), Th + Pf + HKJ [ST + FS] (50.51%), over control. Maximum percent increase in grain yield was Th + Pf + HKJ [ST + Drenching + FS] (32.01%), followed by Carbendazim [ST + FS](29.67%), Th + Pf + HKJ [ST + Drenching] (23.43%),over control. Maximum percent in increase in test weight was observed Th + Pf + HKJ [ST + Drenching + FS] (69.78%), followed by Carbendazim [ST + FS](48.85%), Pf + HKJ [ST + Drenching + FS] (28.68%), Th + Pf + HKJ [ST + Drenching] (27.22%), Th + Pf + HKJ [ST + FS] (25.61%),over control.

Table 6 Effect of different treatments on disease severity, disease incidence, grain yield and test weight at experimental field

At farmers’ field

Four selected effective treatments from experimental field were evaluated and demonstrated at four farmers’ fields in U.S.Nagar and Nainital District during the Kharif season of 2021. The average of results of four farmers were calculated and presented in (Table 7). Maximum reduction in disease severity at 30 DAT was observed in Carbendazim [ST + FS](66.19%) and Th + Pf + HKJ [ST + Drenching + FS](65.65%), TH + Pf + HKJ [ST + Drenching] (46.31%), over control. Maximum reduction in disease severity at 60 DAT was observed in Carbendazim [ST + FS] (55.05%) and Th + Pf + HKJ [ST + Drenching + FS] (53.39%), TH + Pf + HKJ [ST + Drenching] (21.39%), over control. Maximum disease incidence reduction 60 days after transplanting was recorded in Carbendazim [ST + FS] (63.11%), followed by Th + Pf + HKJ [ST + Drenching + FS] (61.65%), and Th + Pf + HKJ [ST + Drenching] (40.15%), over control. Maximum increase in grain yield was Carbendazim [ST + FS] (19.57%), followed by Th + Pf + HKJ [ST + Drenching + FS] (17.56%), and Th + Pf + HKJ [ST + Drenching] (7.80%), over control. Maximum increase in test weight was observed Carbendazim [ST + FS] (34.15%), followed by Th + Pf + HKJ [ST + Drenching + FS] (30.00%), and Th + Pf + HKJ [ST + Drenching] (16.34%), over control.

Table 7 Effect of selected treatments at farmers’ fields of four villages in District Nainital and U.S.Nagar of Uttarakhand

Discussions

Among the all rice diseases, most concern that has the potential to disrupt productivity and production is sheath blight disease of rice caused by R. solani, which is dynamic pathogen with a broad host range that enables it to survive and overwinter in crop residues. The most widely utilized management strategy is chemical control, which is not only hazardous for the environment but also promotes the emergence of new, aggressive strains of the virus (Senapati et al. 2022). On the other hand microbial biological control agents provide excellent option to reduce the use of hazardous chemical pesticides. To achieve this goal the present investigation was carried out to test the potential of existing fungal and bacterial biological control agents. The pathogen R. solani was isolated from the infected sheath of rice plant was further identified and characterized on the basis of morphology. The sheath blight symptoms produced in this study were accordance with that symptom described by (Turaidar et al. 2018). The sequences of ITS1/4 were submitted to NCBI GenBank and the accession number allotted is SUB11543577. Molecular results were in accordance with studies conducted by (Pralhad et al. 2019). Different combinations with Herbal Kunapajala (HKJ) were tested in glasshouse, experimental field and farmers’ fields against R. solani. The maximum plant vigor index was found in treatment combination P. fluorescens (27) + T. harzanium (47) + Herbal Kunapajala, seed treatment (ST), soil drenching and three foliar applications (FS). The findings of the study on increase in growth parameters are accordance with the findings of (Elad et al. 2007). (Mwangi et al. 2008) proved that treatment with T. harzianum, significantly enhanced the plant heights; shoot and root dry weight of tomato plant. (Tanwar et al. 2013) observed greater plant growth promotion activity in tomato in the combination of T. harzianum and Pseudomonas. In Trichoderma, induced systemic resistance is one of the most important mechanisms of bio-control (Harman 2006). Minimum disease severity and disease incidence at 30 and 60 DAS, maximum PAL, PPO and PO activity, was also recorded in the same treatment. Numerous strains of T. virens, T. asperellum, T. atroviride, and T. harzianum alter plant metabolism, increasing the plants' resistance to a variety of plant diseases. Induced systemic resistance is the result of Trichoderma colonizing and penetrating into plant root tissues, which results in a number of morphological and biochemical changes in the plant. These changes are thought to be a plant defense response (ISR). Elicitation of resistance in plants by bio-control agents has received little study attention but is growing in popularity among scientists (Bailey and Lumsden 1998). Some Trichoderma strains clearly showed induced resistance like responses. It was reported that xylanases from Trichoderma spp. is responsible for induction of systemic resistance in cotton, tobacco, grapevine etc. (Djonovic et al. 2006) demonstrated that the hydrophobin like elicitor of T. virens induces defense like responses in maize. The bio-control agents, Trichoderma viride, T. harzianum, T. hamatum, T. koningii have been found to enhance the germination and seedling vigor of sorghum seeds infected with mold fungus (Indira et al. 2006). (Elsharkawy et al. 2022) conducted an experiment to evaluate different isolates of Pseudomonas spp. against R. solani. Pseudomonas isolates enhanced the production of peroxidase and polyphenol oxidase enzymes and the expression of the phenylalanine ammonia lyase (PAL) and NPR1 genes, which could be involved in disease incidence reduction and also has been demonstrated to improve rice growth and resistance to R. solani. In field experiment, the maximum population of biocontrol agents after 60 days of transplanting cfu/g soil for Trichoderma spp. (32.33 × 104) and P. fluorescens (36.33 × 104) in rhizosphere and in rhizoplane. Maximum cfu/g soil for Trichoderma spp. (24.67 × 104) and P. fluorescens (24.0 × 104) was observed in treatment Th + Pf + HKJ [ST + Drenching + FS]. These results are supported by the findings of (Odunfa and Oso 1979). They reported an initial increase of antagonist population in rhizosphere and rhizoplane, which could be due to the abundance of sugar and amino acids exuded from roots in earlier stages, while the subsequent decrease could be due to reduction or change in composition of root exudates with the plant age. At experimental and farmers fields, minimum disease severity, disease incidence at 60DAT was recorded in treatment Th + Pf + HKJ [ST + Drenching + FS] and was at par with the chemical treatment Carbendazim but, the maximum yield was obtained in the treatment Th + Pf + HKJ [ST + Drenching + FS] due to the maximum growth promotion activity shown in the treatment. Jash and Pan (2007) found that there is a rapid increase in the antagonist population of the rhizosphere and rhizoplane soil up to 20 to 30 days thereafter declined which might be due to germination of different spore forms and there subsequent proliferation with or without food base. (Sharma et al. 2012) studied the field performance of bio-formulation of T. harzianum (Th3), in different crops and reported seed treatment with powdered bio formulation of T. harzianum, followed by spray of liquid bio formulation were conducted at 20 different locations of two districts of Rajasthan (viz., Jaipur and Kota) showed colonizing behavior to rhizosphere. Survivability of T. harzianum was found maximum with R.C. Index value: 0.31 at flowering stage and cfu. Value: 5.16 × 107/gm at pre-harvesting stage, while it was minimum with R.C. Index value: 0.16 and cfu: 0.72 × 107/gm at seedling stage of most of the crops. Rhizosphere population densities of T. harzianum (Th3) were consistently higher than in untreated rhizosphere soils. In the present research, the application of combination of T. harzianum (47), P. fluorescens (27), and Herbal Kunapjala as seed treatment; soil drenching and three foliar spray to control the R. solani was the most effective treatment due to their multiple mechanism such as competition, hyperparasitism, secretion of volatile compounds, plant growth promotion activity and induction of defense mechanism, which plays an important role as potential biocontrol agents to controlling the R. solani and promoting the growth of the rice plant. The result agreed with the work of the (Kazempour et al. 2003) observed that, P. fluorescens were found to inhibit the rice sheath blight pathogen under in vitro conditions. All the strains of the bioagent (Biovar 2) produced siderophores on King’s B media. The volatile metabolites, extra cellular secretions and antibiotics of these isolates were inhibitory to R. solani. All the antagonists could reduce germination and caused lysis of sclerotial bodies. (Reddy et al. 2010) reported that P. fluorescens (P.f. 003 strain) inhibited the growth of R. solani by 77.78% and P.f. 008 by 20%. (Khan and Sinha 2007) observed that the radial growth of different isolates of Trichoderma spp. against R. solani. Volatile compounds produced by T. harzianum resulted in maximum inhibition (59.7%) of mycelial growth of R. solani after 48 h. (Rani et al. 2011) recorded that the per cent growth inhibition was maximum (67.02%) in case of T. harzianum, followed by T. viride against R. solani causing sheath blight of rice. (Rajput and Zacharia 2017) reported that the maximum reduction in colony growth of R. solani of paddy was recorded in T. harzianum (63.37%), followed by

Conclusion

Through the study, it was concluded that mono cropping, use of susceptible verities, lack of proper scientific knowledge and indiscriminate, use of chemical pesticides making rice crop of farmers more vulnerable to the attack with sheath blight disease. Trichoderma harzianum (47) and Pseudomonas fluorescence (27) and Herbal Kunapjala in various combinations were tested at laboratory, glasshouse, experimental field and farmers’ fields. Among various treatments, seed treatment, soil drenching and three foliar spray with the combination of T. harzianum (47), P. fluorescens (27), and Herbal Kunapjala was found very effective in reducing the disease incidence, disease severity and increasing growth promotion activity including yield in glasshouse, experimental field and farmer’s field. Therefore, the recommendation of present investigation could be exploited under bio-intensive disease management program for sustainable cultivation of rice.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the Library of G.B. Pant University Pantnagar.

References

  • Abdullah A, Kobayashi H, Matsumura I, Shoichi, ITO (2015) World rice demand towards 2050: impact of decreasing demand of per capita rice consumption for China and India. Research Gate, 1–18

  • Abbas A, Jiang D, Fu Y (2017) Trichoderma spp. as antagonist of Rhizoctonia solani. J Plant Pathol Microbiol 8:402. https://doi.org/10.4172/2157-7471.1000402

    Article  CAS  Google Scholar 

  • Bailey BA, Lumsden RD (1998) Gliocladium on plant growth and resistance to pathogens. In: Trichoderma and gliocladium: enzymes, biological control and commercial applications. Taylor and Francis, London, pp 185–204

  • Djonovic S, Pozo MJ, Dangott LJ, Howell CR, Kenerley CM (2006) Sm1, a proteinaceous elicitor secreted by the biocontrol fungus Trichoderma virens induces plant defense responses and systemic resistance. Mol Plant Microbe Interact 19:838–853

    Article  CAS  PubMed  Google Scholar 

  • Dev D, Konda S, Puneeth ME, TanujaN SinghP, Narendrappa T (2016) In vitro evaluation of bioagents and botanicals against Colletotrichum gloeosporioides (Penz.) Penz&Sacc. causing anthracnose of pomegranate. Int J Ecol Environ Conserv 22(3):1229–1232

    Google Scholar 

  • Elad Y, Zaqs ZY, Zuriel S, Chet I (2007) Use of Trichodermaharzianumor alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions. Can J Bot 42(3):324–332

    Google Scholar 

  • Elsharkawy MM, Sakran RM, Ahmad AA, Behiry SI, Abdelkhalek A, Hassan MM, Khedr AA (2022) Induction of systemic resistance against sheath blight in rice by different Pseudomonas isolates. Life 12:349. https://doi.org/10.3390/life12030349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grossa BL, Zhao Z (2014) Archaeological and genetic insights into the origins of domesticated rice. https://doi.org/10.1073/pnas.1308942110

  • Guttierez WA, Shew HD, Melton TA (1997) Sources of inoculum and management for Rhizoctonia solani damping­off on tobacco transplants under greenhouse conditions. Plant Dis 81:604–606

    Article  Google Scholar 

  • Hammerschmidt R, Nuckles EM, Kuc J (1982) Association of enhanced peroxidase activity with induced systemic resistance of cucumber of Colletotrichum lagenarium. Physiol Plant Pathol 20:73–82

    Article  CAS  Google Scholar 

  • Harman GE (2006) Overview of mechanisms and uses of Trichoderma spp. Phytopathol 96(2):190–194

    Article  CAS  Google Scholar 

  • Hirte WF (1969) The use of dilution plate method for the determination of soil microflora: the qualitative demonstration of bacteria and actinomycetes. Zen- Trall Bakteriol Parasitenkd Infektionskr Hyg 123(2):167–178

    CAS  Google Scholar 

  • Indira S, Muthusubramanian V, Tonapi VA, Varanavasiappan S, Seetharama N (2006) Utility of biocontrol agents in the suppression of seed borne pathogenic mycoflora and their effect on seed quality in sorghum (Sorghum bicolor (L.) Moench). Arch. Phytopathol. Plant Prot. 39(5):379–387

    Article  Google Scholar 

  • Jash S, Pan S (2007) Variability in antagonistic activity and root colonizing behaviour of Trichoderma isolates. J Trop Agric 45(1–2):29–35

    Google Scholar 

  • Jha A, Singh KM, Meena M, Singh R (2012) Constraints of rainfed rice production in Eastern India: an overview. Available at SSRN 2061953

  • Kabdwal BC, Sharma R, Kumar S, Singh KP, Srivastava RM (2022) Occurrence and status of sheath blight of rice in Kumaun region of Uttarakhand. Pl Dis Res 36(2):209–214

    Article  Google Scholar 

  • Kandhari J (2007) Management of sheath blight of rice through fungicides and botanicals. Indian Phytopathol 60(2):214–217

    CAS  Google Scholar 

  • Kazempour MN, Pedramfar H, Elahinia SA (2003) Effect of certain fungicides and isolates of antagonistic fungi on Rhizoctoniasolani, the causal agent of rice sheath blight. J Sci Technol Agri Natural Resour 6:151–158

    Google Scholar 

  • Khan AA, Sinha AP (2007) Screening of Trichoderma spp. against Rhizoctonia solani the causal agent of rice sheath blight. Indian Phytopathol 60(4):450–456

  • Mayer AM, Harel E, Shaul RB (1965) Assay of catechol oxidase a critical comparison of methods. Phytochemistry 5:783–789

    Article  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8(19):4321–4325

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mwangi MW, Monda EO, Sheila A, Okot J, Joyce M (2008) Inoculation of tomato seedlings with Trichodermaharzianumand arbuscularmycorrhizal fungi and their effect on growth and control of wilt in tomato seedlings. Braz J Microbiol 42:508–513

    Article  Google Scholar 

  • Nene YL, Thapliyal PN (1993) Fungicides in plant disease control. Oxford and IBH Publication Company, New Delhi, p 507

    Google Scholar 

  • Odunfa VS, Ayo O (1979) Fungal populations in the rhizosphere and rhizoplane of cowpea. Trans Br Mycol Soc 73:21–26

    Article  Google Scholar 

  • Prasad BN, Reddi MK (2011) Effect of Non-Volatile Compounds Produced byTrichoderma spp. on Growth and Sclerotial Viability of Rhizoctonia Solani, incitant of Sheath Blight of rice. Indian J Fundam Appl Life Sci 2:2231–6345

    Google Scholar 

  • Pralhad SP, Krishnaraj PU, Prashanthi SK (2019) Morphological and molecular characterization of Rhizoctonia solani causing sheath blight in rice. Int J Curr Microbiol App Sci 8(1):1714–1721

    Article  CAS  Google Scholar 

  • Rajput DK, Zacharia S (2017) Efficacy of plant extracts and Trichodermaspp in the management of sheath blight of paddy (Oryzaesativa L.). J Pharmacogn Phytochem 6(4):1950–1952

  • Rani VD, Reddy PN, Devi RG, Reddy SS (2011) Evaluation of biological agents against Rhizoctonia solani f. sp. Sasakii causing Banded leaf and Sheath blight of maize. Int J Pl Prot 39(3):208–211

  • Rahman MA, Begum MF, Alam MF (2009) Screening of Trichoderma isolates as a biological control agent against Ceratocystis paradoxa causing pineapple disease of sugarcane. Mycobiology 37(4):277–285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reddy PB, Rani J, Reddy MS, Kumar KVK (2010 In vitro antagonistic potential of Pseudomonas fluorescens isolates and there metabolites against rice sheath blight pathogen Rhizoctonia solani. Int J Appl Biol Pharma Technol I. ISSN: 0976-4550

  • Richa K, Tiwari IM, Kumari M, Devanna BN, Sonah H, Kumar A, Nagar R, Sharma V, Botel JR, Sharma TR (2016) Functional characterization of novel Chitinase genes present in the sheath blight resistance QTL: qSBR11-1in Rice Line Tetep. Front Plant Sci 7:1–10

    Article  Google Scholar 

  • Sharma P, Patel AN, Deep S, Saini MK, Jambulkar PP, Gangwar OP, Prakasham V (2012) Field performance of Trichoderma harzianum (TH3) for rhizosphere competence and survival in different agriculturally important rabi crops. e-planet 9(2):8–13

  • Singh A, Rashmi R, Savary S, Willocquet L, Singh US (2003) Infection process in sheath blight of rice caused by Rhizoctonia solani. Indian Phytopathol 56(4):434–438

  • Singh R, Sunder S, Kumar P (2016) Sheath blight of rice: current status and perspectives. Indian Phytopath 69(4):340–351

    Google Scholar 

  • Senapati M, Tiwari A, Sharma N, Chandra P, Bashyal BM, Ellur RK, Krishnan SG (2022) Rhizoctonia solani Kühn pathophysiology: status and prospects of sheath blight disease management in rice. Front Plant Sci 13:881116–881116

    Article  PubMed  PubMed Central  Google Scholar 

  • Tanwar A, Aggarwal A, Kadian AN, Gupta A (2013) Arbuscular mycorrhizal inoculation and super phosphate application influence plant growth and yield of Capsicum annuum. J Plant Nutr Soil Sci 13(1):55–66

    Google Scholar 

  • Turaidar V, Reddy M, Anantapur R, Krupa KN, Dalawai N, Deepak CA, Harini Kumar KM (2018) Rice sheath blight: major disease in rice. Int J Curr Microbiol App Sci 7:976–988

    Google Scholar 

  • Whetten RW, Sederoff RR (1992) Phenylalanine ammonia-lyase from loblolly pine: purification of the enzyme and isolation of complementary DNA clones. Plant Physiol 98(1):380–386

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Authors are thankful to Joint Director, CRC, Pantnagar and farmers of District Nainital and U.S.Nagar in Uttarakhand for providing land and other resources during the experiment and their kind cooperation, the valuable support from all the members of bio control lab of Plant Pathology department, GBPUAT Pantnagar is highly appreciated.

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Kabdwal, B.C., Sharma, R., Kumar, A. et al. Efficacy of different combinations of microbial biocontrol agents against sheath blight of rice caused by Rhizoctonia solani. Egypt J Biol Pest Control 33, 29 (2023). https://doi.org/10.1186/s41938-023-00671-6

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