Combined Application of Bio-agents and Novel Fungicides for Management of Collar Rot of Chickpea 

The chickpea, scientifically known as Cicer arietinum L., is the third most significant pulse crop globally, following beans and peas. India is responsible for nearly 75% of the world’s chickpea production. The total land area occupied by this entity is 9.70 million hectares. Its production output is 11.08 million tonnes, with an average productivity of 1142 kg per hectare, according to the Directorate of Economics and Statistics in 2019-20. According to the Directorate of Economics and Statistics 2019-20, Uttar Pradesh is the fourth highest producer of chickpea in India, with a production of 0.85 million tonnes. The states of Madhya Pradesh, Rajasthan and Maharashtra hold the first, second and third places, with the production of 2.73, 2.66 and 2.24 million tonnes, respectively. Bundelkhand, located in southern part of Uttar Pradesh, is the primary region for cultivating pulses in the state. It is renowned as the main hub for pulse production in Uttar Pradesh. The chickpea is the predominant pulse crop in the Bundelkhand region of Uttar Pradesh. Overall, the crop productivity in this region is significantly limited by various biotic and abiotic causes Overall, the crop productivity in this region is significantly limited by various biotic and abiotic restrictions (Narayan and Kumar, 2015; Jha et al., 2018).                  

Chickpea cultivation is significantly impacted by a variety of diseases and insect pests. One of the major diseases affecting chickpea is collar rot, caused by Sclerotium rolfsii. This disease has become more prevalent due to climate change. The study conducted by Gurha and Dubey in 1982 found that it results in a much greater rate of seedling death, ranging from 55% to 95%. Due to its soil-borne origin, polyphagous behaviour and extended persistence in soil, controlling the pathogen on its own appears to be ineffectual and economically unviable. Various fungicides can be used to effectively manage soil borne plant diseases, such as Sclerotium rolfsii. In addition to their detrimental effects on the biotic and abiotic environments, fungicides pose a significant hazard for human and farm animals. Controlling collar rot of chickpea (Sclerotium rolfsii) cannot be achieved by the use of a single approach of plant disease control. The application of bioagents (Trichoderma harzianum, T. virens and Pseudo-monas fluorescens) were successfully decreases the collar rot incidence in chickpea (Singh et al., 2022; Bagul et al., 2024). The management can be achieved by the integration of all existing control mechanisms. Consequently, considering the aforementioned facts, tests were carried out to investigate the integration of Trichoderma, FYM, vermicompost and fungicides for the purpose of managing Collar rot disease in chickpea.

Isolation and purification of causal pathogen
 
The pathogenic fungus (Sclerotium rolfsii) was isolated and purified from infected plants exhibiting characteristic collar rot symptoms, which were taken from chickpea fields of Crop Research Farm of BUAT, Banda. The collar region, exhibiting characteristic signs of decomposition, was dissected into minute fragments. These fragments were washed in sterilized water. Then the parts underwent surface sterilization using the 1% sodium hypochlorite solution for a duration of 60 seconds. The pieces were rinsed extensively in sterile distilled water two to three times to eliminate any remaining sodium hypochlorite and then transferred to sterilized potato dextrose agar (PDA) plates under aseptic conditions. The plates were placed in an incubator set at a temperature of 27±1^oC for a duration of four days. After this period, the plates were inspected to determine if there was any growth of the pathogen. After a duration of four days, the newly growing and expanding hyphal fragments were transferred to newly prepared PDA plates using the hyphal tip procedure as described by Karr and Albersheim (1970).
 
Collection and maintenance of cultures of bio-agents (Trichoderma spp.)
 
Trichoderma isolates i.e. one isolate of Trichoderma viride-1 (TV-1) and 5 isolates of Trichoderma harzianum-1 (TH-1), Trichoderma harzianum-2 (TH-2), Trichoderma harzia-num-3 (TH-3), Trichoderma harzianum-4 (TH-4) and Tricho-derma harzianum-5 (TH-5) were collected from Plant Pathology Laboratory, Department of Plant Pathology, B.U.A.T., Banda.

In vitro efficacy of fungal antagonists (Trichoderma spp.) against pathogen (Sclerotium rolfsii)
 
The effectiveness of fungal antagonists (Trichoderma spp) against the pathogen (Sclerotium rolfsii) was evaluated using dual culture and culture filtrate procedures. The antagonistic potential of Trichoderma harzianum (TH-2) against S. rolfsii was tested using a dual culture approach developed by Morton and Stroube (1955). The antagonists and pathogens were grown on PDA medium. “Aseptically, 20 ml of sterilized PDA media was poured into a sterilized Petri plate with a diameter of 90 mm. A 5 mm mycelial disc obtained from the five-day-old vigorously growing Trichoderma spp culture was moved to one side of a Petri dish. Similarly, the opposite side of the Petri plate was inoculated with five days-old culture of S. rolfsii. The test pathogen was inoculated at centre of the Petri plate for the purpose of comparison. The plates that were treated with an inoculum were placed in a BOD incubator at the temperature of 27±1^oC. This process was repeated three times. The percentage of inhibition in the growth of test pathogens in the presence of Trichoderma spp was determined by using the formula provided by Bliss (1934) to compare it with the control.
 
Per cent inhibition
 

 

 
Where,
I = Per cent inhibition.
C = Growth of test pathogen in control.
T = Growth of test pathogen.
 
Efficacy of culture filtrates of the Trichoderma spp. against  S. rolfsii
 
The Trichoderma spp. and S. rolfsii isolates were cultivated on PDA medium in Petri plates at a temperature of 27±s1^oC. for a duration of 4 days. Two blocks of Trichoderma species, each measuring 5 mm in size, were taken from the actively growing edges of 4-day-old cultures. These blocks were then separately placed into 250 ml conical flasks, with each flask holding 100 ml of sterilised potato dextrose broth. This process was repeated three times to ensure triplicate samples. Following a 10-day period of incubation at a temperature of 27±1^oC, the static cultures were passed through Whatman filter paper number 42 into sterilized flasks and subsequently through a cellulose Millipore membrane filter. To get the desired concentration of culture filtrate, 5 ml of the filtrate was added to 95 ml of melted PDA media before pouring. The modified medium was carefully poured into sterilized Petri plates and reproduced three times for each treatment. The solid plates were inoculated at the centre with a 5 mm diameter disc of mycelium from the pathogen S. rolfsii. The plates were then incubated at a temperature of 27±1^oC for a period of 5-6 days. The PDA media without the addition of culture filtrate of Trichoderma and infected with the pathogen was used as the control. The expansion of mycelium in the test pathogens was quantified and the percentage of inhibition in mycelial growth was computed.
 
Determination of tolerance in pathogen (Sclerotium rolfsii) and fungal bio-agent agent (Trichoderma harzianum) to novel fungicides
 
Two set of experiments were conducted to evaluate novel fungicides against S. rolfsii and T. harzianum-2 (TH-2) through Poisoned food technique (Schmitz 1930). Nine different fungicides viz; carbendazim 12% + mancozeb 63% WP, captan 70% + hexaconazole 5% WP, Propineb 70% WP, carbendazim 50% WP, mancozeb 75% WP, Penflufen 13.28% w/w + Trifloxystrobin 13.28% w/w FS, propiconazole 25% EC., hexaconazole 5% SC and tebuconazole 25.9% w/w EC were tested at four concentrations (50 ppm, 100 ppm, 500 ppm and 1000 ppm) in vitro. Hundred ml of PDA medium was sterilized in conical flask, requisite quantity of fungicide was incorporated in laminar air flow in molten media to make 50, 100, 500 and 1000 ppm concen-tration. Media was then poured aseptically in sterilized Petri plates. After solidification of media five mm discs of the S. rolfsii was cut with the help of sterilized cork borer from 5-6 days old culture and placed centrally in each Petri plates. Three replications of each treatment were maintained and control treatment maintained without the added of fungicide. The Inoculated plates were incubated at 27±1^oC in BOD incubator. Observation of linear growth of fungus was recorded after 24 hours of incubation.
       
Another set of experiment was conducted for evaluation of fungicides against T. harzianum in which nine fungicides, viz., carbendazim 12% + mancozeb 63% WP), captan 70% + hexaconazole 5% WP, propineb 70% W, carbendazim 50% WP, mancozeb 75% WP, Penflufen 13.28% w/w + Trifloxystrobin 13.28% w/w FS, propiconazole 25% E.C., hexaconazole 5% SC and tebuconazole 25.9% w/w EC were tested at four concentrations i.e. 50 ppm, 100 ppm, 500 ppm and 1000 ppm, in vitro using poisoned food technique (Table 4). Three replications were maintained by inoculating 5 mm disc of 3 days old cultures of T. harzianum (TH-2) control plates without any fungicide were also inoculated simultaneously for comparison. Inoculated plates were incubated at 25±1oC for seven days. Colony diameter in each plate was measured to find the fungicidal and fungistatic behaviour.
 
Integrated management of collar rot of chickpea in pots and field condition
 
Pot culture experiment
 
The pot culture experiments were conducted during Rabi 2019-20 and 2020-21 for evaluating the effect of biocontrol agents, fungicides alone or various integrated treatments in complete randomized design (CRD). Plastic pots were filled with sterilized soil. The formulation of T. harzianum-3 (TH-3) was multiplied on FYM and vermicompost was mixed separately in pots @ 100 g/pot. After 5-7 days mass culture of S. rolfsii was mixed in pots @ 25 g/kg soil. Seeds of chickpea were surface sterilized with sodium hypochlorite solution for 3 minutes, rinsed thoroughly in sterilized distilled water. Seeds of collar rot susceptible chickpea cultivar L550 were sown with different treatments seed treatment. Data on seedling emergence and mortality per cent were recorded 10-15 days after sowing and final plant stand was counted after 60 days of sowing.
 
Field experiment
 
Field experiments were carried out during the Rabi season of 2019-20 and 2020-21 at the Crop Research Centre, BUAT, Banda. The trials followed a randomised block design (RBD) with ten treatments and three replications. The chickpea cultivar L550, which is prone to susceptibility to collar rot, was planted in a plot measuring 6.0 m². The spacing between each plant was 30 cm x 10 cm. This setup was replicated for each treatment. Chickpea seeds, which had been treated with bio-agents and fungicide according to the specified treatments, were planted in six rows in each plot, with a total of 180 seeds. The fungicide Captan 70% + Hexaconazole 5% EC and a mixture of Bavistin+Thiram (1:1) were applied at a rate of 2 grams per kilogram of seed, while a talc formulation of T. harzianum was applied at a rate of 5 grammes per kilograms of seed. T. harzianum, with a concentration of 2 x 10⁸ colony forming units per gramme, was applied to the soil together with enriched farmyard manure at a rate of 5 kilograms of bioagent per tonne of manure. The application was done in furrows at a rate of 1 ton per hectare or 100 grams per square meter, according to the specified treatments. The occurrence of collar rot was monitored at 20-days intervals until the crop reached maturity and the total number of diseased plants was tallied. The grain yield observations were recorded post-harvest.
 
Treatment number
 
Treatments detail                
 
T1   Seed treatment with Thiram + carbendazim (1:1) @ 2 g/ kg of seed.
T2   Seed treatment with Captan 70% + Hexaconazole 5%EC (Taquat) @ 2 g/kg of seed.
T3   Seed treatment with Trichoderma harzianum @ 5 g/kg seed.    
T4   Soil application with Trichoderma harzianum enriched FYM@ 100 g/m2.
T5    Soil application with Trichoderma harzianum enriched FYM vermicompost @ 100 g/m².
T6   Seed treatment with Captan 70% + Hexaconazole 5% EC (Taquat) @ 2 g/kg of seed + soil application Trichoderma harzianum enriched FYM @ 100 g/m².
T7   Seed treatment with Captan 70% + Hexaconazole 5%EC (Taquat)  @2g/kg of seed+ soil application Trichoderma harzianum enriched vermicompost@100 g/m².
T8  Seed treatment with Trichoderma harzianum @ 5 g/kg seed + soil application with Trichoderma harzianum enriched FYM @100 g/m².
T9   Seed Treatment with Trichoderma harzianum @ 5g/kg seed + soil application with Trichoderma harzianum enriched vermicompost @100 g/m².
T10   Control.

Per cent disease incidence
 

 

 
Per cent disease control
 

 

 
Per cent increase yield
 

In vitro efficacy of antagonists (Trichoderma spp.) against Sclerotium rolfsii
 
The antagonistic actions of five indigenous isolates of Trichoderma harzianum, namely TH-1, TH-2, TH-3, TH-4 and TH-5, were demonstrated by assessing the growth of the pathogen using the dual culture technique. Additionally, one isolate of Trichoderma viride (TV-1) was also included in the study. The radial expansion of S. rolfsii was limited by the presence of antagonistic strains of Trichoderma spp., resulting in considerable suppression. Among the six Trichoderma isolates, Trichoderma viride-1 exhibited the highest level of growth inhibition against S. rolfsii, with a rate of 75.44%. Trichoderma harzianum-2 also showed a similar level of inhibition at 75.18%, making it comparable to T. viride-1. The subsequent potent antagonist was T. harzianum-4, which effectively suppressed 71.43% of the pathogen’s mycelial growth. This was followed by T. harzianum-3 (70.70%) and T. harzianum-1 (68.81%), demonstrating their relative superiority. There was no substantial difference between them in terms of their ability to prevent the growth of the infection. Nevertheless, the antagonist T. harzianum-5 exhibited the lowest level of inhibition (64.25%) on the growth of the pathogen’s mycelium, as seen in Table 1. Ali and Javaid (2016) and Darvin et al., (2013) have verified that T. harzianum and T. viride exhibit antagonistic effects in dual culture against S. rolfsii.


 
Effect of non-volatile (cultural filtrate) metabolites of potential Trichoderma spp on mycelial growth of Sclerotium rolfsii
 
The impact of Trichoderma species’ non-volatile compounds was assessed using the procedures outlined by Dennis and Webster (1971) as described in the materials and methods section. The data in Table 2 shows that among the six isolates of Trichoderma spp, T. harzianum- 2 exhibited the highest inhibition of mycelial growth of S. rolfsii through the production of non-volatile compounds, with a rate of 61.85%. This was followed by T. harzianum-1 with a rate of 57.77% and T. harzianum-4 with a rate of 52.40%. These differences in inhibition rates between the isolates were statistically significant. T. viride -1, the subsequent potent antagonist, hindered 42.22% of the pathogen’s mycelial growth by producing an inhibitory metabolite. Nevertheless, the lowest level of inhibition (22.95%) in the growth of S. rolfsii was seen when exposed to T. harzianum-5. A study conducted by several workers focused on the development of antibiotics, both volatile and non-volatile. The study found that T. harzianum and T. viride were particularly successful in inhibiting the radial growth of S. rolfsii through the production of these compounds. This conclusion was reached by Rao and Kulkarni (2003). Trichoderma spp. isolates have the ability to create both volatile and non-volatile compounds that exhibit activity against a broad spectrum of fungi (Dennis and Webster, 1971). In their study, Kucuk and Kivanc (2003) found that the culture filtrate of T. harzianum exhibited inhibitory properties against various plant pathogenic fungi.


 
In vitro evaluation of the efficacy novel fungicides against Sclerotium rolfsii
 
The data from Table 3 showed that out of the nine fungicides tested, namely captan 70% + hexaconazole 5% WP (Taquat), penflufen 13.28% w/w + trifloxystrobin (Evergold), hexaconazole 5%SC (Contaf) and tebuconazole 25.9% w/w EC (Folicur), all of them were highly effective at all concentrations. They achieved a 100% inhibition of mycelial growth of S. rolfsii. Propiconazole 25% EC (Arihant) demonstrated a 70.92% inhibition of mycelia growth of the pathogen at a concentration of 50 ppm and an 82.96% inhibition at a concentration of 100 ppm. At higher concen-trations of 500 and 1000 ppm, it completely inhibited the mycelial growth of the pathogen. Propineb 70 WP (Antracol) showed a lower inhibition rate of 8.51% and 61.66% at concentrations of 50 and 100 ppm respectively, but achieved 100% inhibition at concentrations of 500 and 1000 ppm in the mycelia growth of S. rolfsii. The effectiveness of Mancozeb 75% WP (Indofil M-45) in inhibiting the growth of the pathogen’s mycelia was found to be the least. It demonstrated inhibition rates of 24.03%, 26.29%, 54.07% and 70.92% at doses of 50, 100, 500 and 1000 ppm, respectively. Nevertheless, the fungicide carbendazim 50% WP (Bavistin) proved to be ineffective in inhibiting the growth of the pathogen’s mycelium at all dosages tested. These findings were corroborated by other prior researchers, specifically Arunasri et al., (2011), who documented that Triazoles (Hexaconazole, Propiconazole, Difenconazole) had significant efficacy in suppressing the growth of S. rolfsii. According to Manu et al., (2012), Hexaconazole, Tebuconazole and Propiconazole were found to strongly suppress the growth of S.rolfsii isolated from finger millet, even at lower concentrations. According to Das et al., (2014), Hexaconazole and Tebuconazole had high efficacy at all concentrations against S. rolfsii. Propiconazole showed moderate inhibition, while Thiophanate methyl and Bavistin exhibited the least inhibition.


 
Determination of tolerance of Trichoderma harzianum to novel fungicides
 
The nine previously mentioned fungicides were assessed at four different concentrations: 50 ppm, 100 ppm, 500 ppm and 1000 ppm (Table 4). The results demonstrated that lower concentrations of fungicides exhibited a reduced inhibitory impact in comparison to larger concentrations. The combination of Carbendazim 12%+ Mancozeb 63% (Saff), Carbendazim (Bavistin), Propiconazole (Arihant), Hexaconazole 5% SC (Contaf plus) and Tebuconazole 25.9% EC (Folicur) completely prevented the growth of T. harzianum-2 at all tested concentrations of 50 ppm, 100 ppm, 500 ppm and 1000 ppm. The next fungicide in terms of effectiveness was a combination of Captan 70% + hexaconazole 5% WP (Taquat). This combination inhibited 35.36% and 52.77% of TH-2 growth at concentrations of 50 ppm and 100 ppm, respectively. It completely inhibited the growth of the bio-agent at concentrations of 500 ppm and 1000 ppm. Following this, the fungicide Penflufen 13.28% w/w + Trifloxystrobin (Evergold) inhibited 38.7%, 41.11%, 43.33% and 46.29% of pathogen mycelial growth at concentrations of 50 ppm, 100 ppm, 500 ppm and 1000 ppm, respectively. Propineb (Antracol) was determined to have the lowest effectiveness but was very compatible with Trichoderma harzianum isolates. It inhibited the growth of T. harzianum-2 by 2.4%, 5.55%, 12.59% and 21.44% at concentrations of 50 ppm, 100 ppm, 500 ppm and 1000 ppm, respectively. According to Bagwan’s research in 2010, it was found that thiram (0.2%), copper oxychloride (0.2%) and mancozeb (0.2%) are suitable to be used together with Trichoderma harzianum. Rai et al., (2016) reported that Trichoderma harzianum (Th-14) demonstrated compatibility with Mancozeb and Metalaxyl at low dosages. This study presents facts regarding the compatibility and incompatibility of Trichoderma harzianum with fungicides. Dubey et al., (2015) have also reported a similar study on the compatibility of T. harzianum with fungicides and other agrochemicals.


 
Evaluation of the effect of different integrated treatments for management of collar rot in pots under net house conditions
 
The findings from net-house studies (Table 5) carried out in pots with pathogen inoculation during the Rabi crop seasons of 2019-20 and 2020-21 shown that all the treatments effectively improved seed germination and decreased the occurrence of collar rot compared to the control group. The seeds treated with Captan 70% WP + hexaconazole 5% EC at a rate of 2 g/kg of seed, along with soil application of Trichoderma harzianum-2 enriched vermi-compost at 100 g/pot, resulted in the highest germination rate of 100% and the lowest disease incidence of 18.05%. This was followed by seed treatment with Captan 70% WP + Hexaconazole 5% EC at a rate of 2 g/kg of seed, along with soil application of T. harzianum-2 enriched FYM at 100 g/pot, which provided a germination rate of 96.33% and an incidence of collar rot of 19.68%. This trend was consistent across both years of experimentation and in the combined data. Nevertheless, the lowest germination rate of 74.33% and the highest disease incidence of 39.06% were observed after applying T. harzianum-2 enriched FYM at a rate of 100 g per pot. Veena and Reddy (2016) conducted a study to assess the impact of various organic amendments, such as FYM, vermicompost and neem cake, both individually and in combination with the fungal antagonist Trichoderma isolate, on the root rot of chickpea. They found that treating the seeds with the fungicide Copper oxychloride and applying the potential fungal antagonist Trichoderma harzianum to the soil, along with a bacterial biocontrol agent, resulted in the best outcomes. This treatment achieved a germination percentage of 100% and the lowest incidence of disease at 16%. A study conducted by Ahsan et al., (2020) found that the most effective method for controlling collar rot in chickpea was the combination of soil application of Trichoderma harzianum (10 g/pot) with Carboxin Vitavax seed treatment at a rate of 2 g/kg seed. Combining biocontrol agents (Trichoderma spp) with fungicides resulted in significantly improved disease control in various crops (Sugar beetroot, cauliflower and chickpea) compared to using either the biocontrol agent or fungicide alone (Upadhyay and Mukhopadhyay, 1986 and Dubey et al., 2015). The current findings are consistent with earlier findings.


 
Evaluation of the effect of various integrated treatments under field conditions on collar incidence and grain yield
 
Integrated disease management refers to a compre-hensive approach that incorporates many ways to promote the healthy growth of crop plants, resulting in good yields (Youdeowei, 2004). The utilization of fungicides, bioagents and organic amendments has the potential to decrease disease occurrence and enhance grain production. Utilizing a combination of seed treatment using fungicide and soil application with bioagent (T. harzianum-2), along with soil amendment, resulted in a decrease in disease occurrence and an increase in grain yield compared to the control group in both the consecutive years (2019-20 and 2020-21) as well as in the combined data. The most effective treatment among those tested was seed treatment with captan 70% WP + hexaconazole 5% EC (Taquat) combined with soil application of T. harzianum-2 enriched vermicompost. This treatment resulted in the lowest disease incidence (11.67%) and the highest grain yield (15.27 q/ha) compared to the control. The second most effective treatment was seed treatment with captan 70% WP + hexaconazole 5% EC combined with soil application of T. harzianum-2 enriched FYM, which had a disease incidence of 13.51% and a grain yield of 15.07 q/ha (Table 6). The results showed that the combined use of fungicides as seed treatment and bio-control agents as soil treatment was successful in reducing collar rot disease in chickpea and increasing grain production. This could be attributed to the immediate impact of fungicides and the long-term effectiveness of bio-control agents. In 2013-14 and 2014-15, Singh et al., (2017) conducted an experiment to investigate the effectiveness of integrating Trichoderma, Pseudomonas and fungicides in controlling collar rot disease in chickpea. The results showed that the treatment that had the greatest effectiveness was the application of Trichoderma harzianum enriched FYM at a rate of 8 q/ha-1 (Soil) combined with Hexaconazole at a rate of 3 ml/kg-1 seed”. This treatment resulted in the lowest mortality rate (4.30% and 2.25%) and the biggest increase in grain production (44.85%). Pandey et al., (2017) found that applying Trichoderma viride or T. harzianum (2 x 108 cfu/g) enriched FYM (10 kg bioagent/ tonne FYM) to the soil in furrows at a rate of 1 tonne/ ha, followed by soaking chickpea seeds in a suspension of talc-based formulation 1% WP (2 x 108cfu/g) of T. viride or T. harzianum for 10 hours at a rate of 50 g product/ 250 ml of water/ kg seed and shade drying, was effective in managing wilt and root rot complex. The conclusions of prior researchers strongly corroborate our findings.

The present study’s results indicated that it provided information on the effectiveness and compatibility of fungal antagonists (Trichoderma spp.) and new fungicides. The module comprises the formulation of a putative native bioagent, Trichoderma harzianum-2, along with organic amendments such as vermicompost, farmyard manure (FYM) and a fungicide. The formulation of Captan 70% WP + Hexaconazole 5% EC has been specifically designed to effectively control collar rot and maximize the grain yield of chickpea through integrated management techniques.

The authors acknowledge the assistance extended by Department of Plant Pathology, BUAT, Banda. The authors are thankful to the Centre of Excellence on Dryland Agriculture with special focus on pulses and oilseeds, B.U.A.T., Banda for extending the experimental facilities and providing materials for research work.
 
Author’s contribution
 
Arvind Kumar: Investigation, data curation, formal analysis and draft preparation. Vivek Singh: designed the research and experiments, supervision, original draft preparation, review and editing. Harshita: draft preparation, formal analysis, review and editing. Girijesh Jaisval:  data curation, draft preparation, formal analysis. All authors provided critical feedback and helped shape the research, analysis and manuscript.

On behalf of all the authors, I wish to confirm that there is no conflict of interest in the publication of this manuscript.