Potentiality of Formulated Bioagents from Lab to Field: A Sustainable Alternative for Minimizing the Use of Chemical Fungicide in Controlling Potato Late Blight

: Late blight of potato caused by an oomycete, Phytophthora infestans (Mont.) De Bary limits the production of potato worldwide. Late blight management has been based on chemical fungicide application, and the repeated use of these fungicides introduces new and more aggressive genotypes, which can rapidly overcome host resistance. Therefore, innovative and effective control measures are needed if fungicide use is to be reduced or eliminated. Some potential formulated bacterial bioagents viz. Pseudomonas putida (BDISO64RanP) and Bacillus subtilis (BDISO36ThaR), and fungal bioagents viz. Trichoderma paraviridicens (BDISOF67R) and T. erinaceum (BDISOF91R), were evaluated for their performance in controlling late blight of potato under growth chamber and ﬁeld conditions. Both artiﬁcial inoculation and ﬁeld experiments revealed that eight sprays of these bacterial ( P. putida and B. subtilis ) and fungal ( T. erinaceum ) bioagents were found to be most effective at reducing late blight severity by 99% up until 60 days after planting (DAP), whereas these bioagents were found to be partially effective until 70 DAP, reducing late blight severity by 46 to 60% and 58 to 60% in the ﬁeld and growth chamber conditions, respectively. However, these bioagents can reduce the spray frequencies of Curzate M8 by 50% (four sprays instead of eight) when applied together with this fungicide. Economic analysis revealed that T 6 (eight sprays of formulated P. putida + B. subtilis + four sprays of Curzate M8) and T 16 (eight sprays of formulated P. putida , B. subtilis, and T. erinaceum + four sprays of Curzate M8) performed better in consecutive two years, applying less fungicidal spray compared to T 1 (eight sprays of Curzate M8 (Positive control)), which indicated that the return ranged, by Bangladeshi Currency (Taka), from 0.85 to 0.90 over the investment of Bangladeshi Currency (Taka) 1.00 in these treatments, and these results together highlight the possibility of using bioagents in reducing late blight of potato under a proper warning system to reduce the application frequency of chemical fungicide.


Introduction
Late blight caused by P. infestans (Mont.) De Bary restricts the yield of potato notably in cool temperature regions globally. P. infestans (Mont.) De Bary is an oomycete that is well recognized for its explosive expansion when environmental circumstances are adequate and host plants are vulnerable to infection [1]. In 2019, Bangladesh produced 9.7 million tonnes of potato on 0.5 million ha, which represents 2.6% of the world production [2]. The yield of potatoes in Bangladesh in 2019 as calculated by the FAO [2] was 20.6 t/ha, which was dosporic and enzymatic components of B. subtulis have been found to be very potent against numerous fungal infections. Potato associated cyanogenic Pseudomonas spp. displays a volatile-mediated high potential against P. infestans [48,49]. In addition to supplying biofungicides as effective alternatives to synthetic fungicides, bacteria have an enormous potential for agricultural advantages such as secreting plant growth regulating hormones, fixing atmospheric nitrogen, and enhancing phosphorus nutrition [34]. Unlike synthetic fungicides, numerous microorganisms may also have the capacity to increase their hostile activity against plant pathogens over time by effectively colonizing plant surfaces [50]. Plant growth reduction is caused by drought stress [51], heavy metals [52], weed infestation [53], salt stress [54], and several adverse environmental states. PGPM may alter plant performance directly by producing chemicals that enhance plant growth, boost nutrient availability, and absorption under biotic stress, and trigger plant defense responses, or indirectly by suppressing plant infections [55]. Surprisingly, biocontrol agents (BCAs), including microorganisms and their secondary metabolites, were shown to be promising as efficient and environmentally friendly alternatives to chemicals [19,56]. Because disease symptoms occur early in the growth stage, chemical control programs should use prediction models and eco-friendly plant protection methods to minimize the fungicide dose and lengthen the treatment intervals [57].
Two new native fungal isolates identified from the rice rhizosphere and bacterial isolates identified from potato phylloplane and rhizosphere have been used in this study. We assessed their efficacy for controlling the late blight of potato, but P. infestans is a polycyclic pathogen that can hardly be completely controlled with bioagents only. The effects of fungicide application have numerous hazards to mankind and the environment, and apart from that, many fungicides are banned in developed countries due to their toxic effects to human beings and animals. Thus, in this study, we focused on the use of both fungicides and native formulated bioagents, considered as a novel approach in reducing the application frequency of chemical fungicide, to minimize the impact of late blight severity on potato yield.

Culture and Growth Condition for Bacterial and Fungal Bioagents
The cultures of bacterial bioagents were maintained in Luria−Bartani (LB) medium [58] and fungal bioagents were maintained in potato dextrose agar (PDA) medium. Two bacterial isolates viz. P. putida (BDISO64RanP) and B. subtilis (BDISO36ThaR) were isolated from potato phylloplane and rhizosphere identified previously by sequencing 16SrDNA [59] and were grown on the LB agar medium during the experimental period. Two fungal strains viz. T. paraviridicens (BDISOF67R) and T. erinaceum (BDISOF91R) (Islam et al., unpublished data) isolated from the rice rhizosphere were identified with ITS primer and were cultured on the PDA medium.

In Vitro Antagonist Test in the Laboratory
In order to test the efficacy of different bioagents against P. infestans in vitro, the growth inhibition of P. infestans by different bio-agents was compared with the controls (positive and negative) (Figure 1). For the bacteria, the bioagents were sub cultured for one week after being removed from −80 • C and then, overnight, the culture of B. subtilis/P. putida was inoculated in a triangle on pea agar plates. Then, 5 mm disc of P. infestans (9 days old) were placed at the center of the triangularly inoculated bacterial plates. In the control plates, only a 5 mm disc of P. infestans (9 days old) was inoculated. The radial growth inhibition of P. infestans was assessed at two to three weeks after inoculation by measuring the radial growth of P. infestans in the dual and control plates. The percent radial mycelial growth inhibition was calculated as follows: where R1 = radial growth of P. infestans in the control plates and R2 = radial growth of P. infestans in the dual culture plates.
measuring the radial growth of P. infestans in the dual and control plates. The percent radial mycelial growth inhibition was calculated as follows: % Radial growth inhibition = (R1 − R2) × 100 R1 (1) where R1 = radial growth of P. infestans in the control plates and R2 = radial growth of P. infestans in the dual culture plates. For fungal bioagents, a dual culture method was used to analyze whether T. paraviridescens/T. erinaceum inhibits the growth of P. infestans [60]. Trichoderma isolates were maintained in PDA strains at 4-8 °C for a short period of time. Briefly, a 5 mm diameter mycelial plug of P. infestans (9 days old) was placed on one side of a petri dish (9 cm diameter) containing pea agar and was pre-incubated at 18 °C for 2 days to initiate growth. Later, a 5 mm diameter disc of T. paraviridescens/T. erinaceum (7 days old) was placed 6 cm away from the pathogen on the dual plates, whereas a sterile PDA disc was placed in the control plates. The assay was done twice with five replications and the radial growth of the pathogen was measured 4 days after incubation at 18 °C . The percent radial mycelial growth inhibition (I) was calculated as follows [59]: where C is the radial growth measurement of the pathogen in the control plates and T is radial growth of the pathogen in the dual plates.

Experimental Location and Design
The efficacy of some selected formulated bio-agents was evaluated in both the plant growth chamber (18 °C and RH 90%) and field conditions. Plant growth chamber experiments were conducted at the Professor Golam Ali Fakir Seed Pathology Centre, Bangladesh Agricultural University, Mymensingh. The growth chamber was equipped with an For fungal bioagents, a dual culture method was used to analyze whether T. paraviridescens/T. erinaceum inhibits the growth of P. infestans [60]. Trichoderma isolates were maintained in PDA strains at 4-8 • C for a short period of time. Briefly, a 5 mm diameter mycelial plug of P. infestans (9 days old) was placed on one side of a petri dish (9 cm diameter) containing pea agar and was pre-incubated at 18 • C for 2 days to initiate growth. Later, a 5 mm diameter disc of T. paraviridescens/T. erinaceum (7 days old) was placed 6 cm away from the pathogen on the dual plates, whereas a sterile PDA disc was placed in the control plates. The assay was done twice with five replications and the radial growth of the pathogen was measured 4 days after incubation at 18 • C. The percent radial mycelial growth inhibition (I) was calculated as follows [59]: where C is the radial growth measurement of the pathogen in the control plates and T is radial growth of the pathogen in the dual plates.

Experimental Location and Design
The efficacy of some selected formulated bio-agents was evaluated in both the plant growth chamber (18 • C and RH 90%) and field conditions. Plant growth chamber experiments were conducted at the Professor Golam Ali Fakir Seed Pathology Centre, Bangladesh Agricultural University, Mymensingh. The growth chamber was equipped with an air cooler and sprinkling watering system, and sensors to maintain temperature (18-20 • C and adjust humidity (85-90%) two to three times in a day. Field experiments were conducted in the same farmer's field, Sutia Khali, Mymensingh Sadar, Mymensingh, from 2018-2021. Pot experiments were conducted in a plant growth chamber with completely randomized design (CRD) and field experiments were with randomized complete block design (RCBD) by maintaining three replications. The plot size for field experiments was 3 × 2 m 2 . The row to row distance was 60 cm, while the plant to plant distance was 20 cm.

Treatment Design and Combination
We assessed the efficacy of two bacterial (viz; P. putida and B. subtilis) and two fungal (T. paraviridescens and T. erinaceum) bioagents compared to the chemical fungicide (Curzate M8) in a different combination. Treatment combinations were T 0 (water (negative control), T 1 (foliar spray of formulation of T. paraviridescens), T 2 (foliar spray of formulation of T. erinaceum), T 3 (foliar spray of formulation of P. putida), T 4 (foliar spray of formulation of B. subtilis), T 5 (foliar spray of formulation of T. paraviridescens and P. putida), T 6 (foliar spray of formulation of T. erinaceum and P. putida), T 7 (foliar spray of formulation of T. paraviridescens and B. subtilis), T 8 (foliar spray of formulation of T. erinaceum and B. subtilis), T 9 (foliar spray of formulation of T. paraviridescens, T. erinaceum, P. putida, and B. subtilis), T 10 (foliar spray of Curzate M8 (Cymoxanil + Mancozeb), and T 11 (foliar spray of formulation of T. paraviridescens, T. erinaceum, P. putida, and B. subtilis with T 10 ).
According to the results of the previous experiments on the efficacy of the two formulated bacterial and two fungal bioagents in reducing late blight severity of potato under growth chamber conditions and field conditions during 2018-2019, we selected two bacterial (viz; P. putida and B. subtilis) and one fungal (viz. T. erinaceum) bioagents that were found to be effective for the total growth inhibition of late blight pathogen. The next step was to compare treatments (A) exclusively based on the current number of sprays of chemical fungicide; (B) based on the same number of sprays, but applying single or mixed bioagents; and (C) the same as (B), but reinforced by one to four additional sprays with chemical fungicide. Thus, the efficacy of these bioagents in reducing the application frequency of chemical fungicides for controlling late blight of potato was evaluated in the following treatments: T 0 = water (negative control), T 1 = eight sprays of Curzate M8 (positive control), T 2 = eight sprays of formulated P. putida + B. subtilis, T 3 = T 2 + one spray of Curzate M8, T 4 = T 2 + two sprays of Curzate M8, T 5 = T 2 + three sprays of Curzate M8, T 6 = T 2 + four sprays of Curzate M8, T 7 = Eight sprays of formulated T. erinaceum, T 8 = T 7 + one spray of Curzate M8, T 9 = T 7 + two sprays of Curzate M8, T 10 = T 7 + three sprays of Curzate M8, T 11 = T 7 + four sprays of Curzate M8, T 12 = Eight sprays of formulated P. putida, B. subtilis and T. erinaceum, T 13 = T 12 + one spray of Curzate M8, T 14 = T 12 + two sprays of Curzate M8, T 15 = T 12 + three sprays of Curzate M8, and T 16 = T 12 + four sprays of Curzate M8.

Growing Potato for Field Experiments
Land was fertilized with cow dung (7.5 t/ha), DAP (260 kg/ha), MOP (260 kg/ha), Gypsum (120 kg/ha), zinc (7.5 kg/ha), boron (7.5 kg/ha), magnesium (45 kg/ha), furadan (7.5 kg/ha), and urea (120 kg/ha) just before the final land preparation. Apparently disease free and uniform tubers of a popular potato cultivar (Diamant, a variety showing susceptibility under severe outbreak) were cut into pieces with at least one bud and were left for 24 h for suberization. Then, the suberized tuber pieces were treated by drenching with the formulated bioagents (0.4% w/v) and the treated tubers were left for at least 1 h for adherence. Treated and non-treated tuber pieces were planted in the pots filled with prepared soils, which were then kept in the net house until two days before the inoculation. For field experiments, the treated and non-treated tuber pieces were planted in respective experimental plots. Two top dressings of urea (120 kg/ha) were applied at 33 and 60 DAP along with two irrigations at 27 and 60 DAP. Weeding was performed at 25 DAP followed by earthen up at 33 and 43 DAP.

Talc-Based Formulation of Selected Bacterial and Fungal Bioagents
First, 500 g talc powder, 5 g CMC (Carboxy methyl cellulose), and 7.5 g CaCO 3 were mixed at 121 • C for 30 min. To formulate the bioagents, the bacteria were cultured for 24 h on LB media. The bacteria were then cultured in LB broth for 6 h. They were then centrifuged and resuspended in 200 mL peptone broth with bactopeptone. This broth Sustainability 2022, 14, 4383 6 of 22 culture was shaken for 2 h more. Then, 5 mL of sterile 100% glycerol was added in a 200 mL culture. These cultures (5 × 10 8 CFU/mL) were added to 500 g powdered talc in the tray. The formulations were then air dried overnight in a laminar flow hood and later the formulations were powdered with hand wearing gloves and mask. The formulated bacterial antagonists were packed in plastic bags. For fungal bioagents, a mycelial disc (5 mm diameter) for each isolate was inoculated in 100 mL PDB broth. Conidia production was counted after 7 days and the mycelial mat along with conidia from PDB was mixed thoroughly with previously autoclaved talcum powder pretreated with 0.5% CMC (5 g CMC dissolved in 100 mL water mixed with 1 kg talcum powder). The mixture was then air-dried in a laminar flow hood and was kept in plastic bags, accordingly.

Artificial Inoculation of P. infestans
Inoculum was prepared from Petri plate cultures of the P. infestans isolates on pea agar with β-sitosterol (50 mg/L) grown until the maximum vegetative growth stage; on the day before inoculation, the mycelia were smashed with a sterile test tube and the plates were left at 18 • C in an incubator (VELP SCIENTIFICA) overnight for the production of sporangia. The sporangia were harvested by washing them off the plates with Sato's solution [61] and the concentration was determined by counting with a hemocytometer and was adjusted to 10 4 sporangia/mL of Sato's solution. The viability of the formulated bioagents was more than four months.

Application of Formulated Bacterial and Fungal Bioagents
In case of net house experiments in the growth chamber, formulated biagents and Curzate M8 were sprayed four times on plants before inoculation at 34, 41, 48, and 53 DAP and 2, 4, 7, and 9 days after inoculation, i.e., 57, 59, 62, and 64 DAP, whereas the inoculation was done at 55 DAP. In the case of field experiments, the chemical fungicide (s) and formulated bioagents were sprayed at 34, 41, 48, 53, 57, 62, 69, and 75 DAP over the potato plant surface when applied alone. However, in case of combined application with chemical fungicides, one chemical spray at 53 DAP; two chemical sprays at 53 and 57 DAP; three chemical sprays at 53, 57, and 62 DAP; and finally, four chemical sprays at 48, 53, 57, and 62 DAP were applied together. The formulated bacterial and fungal bioagents were sprayed (0.4% w/v) two days after fungicide application to avoid the interactions effects with chemical fungicide. The application concentration of each bioagent was reduced to half in case of the combined application of two bioagents, and one third when three bioagents were applied together.

Assessment of Late Blight Incidence and Severity
Ten potato plants were randomly selected and tagged for data collection.

Economic Analyses of Formulated Bioagents
The benefit−cost ratio (BCR) was calculated for each treatment according to the method of Mondal et al. [63]. The cost−benefit analysis compared the profitability of each treatment based on the gross returns and costs. Each treatment's gross and net returns were computed as follows. Gross return (TK/ha) = tuber Yield (kg/ha) × price (TK/kg); net return (TK/ha) = gross return (TK/ha) − cost of production plus treatment cost (TK/ha); the BCR was calculated as shown below: where A = selling price (Tk./kg), B = cost of cultivation + treatment cost (Tk./ha), and C = yield (kg/ha).

Statistical Analysis
Data were analyzed using the MStatC statistical program. Means were compared using Duncan's multiple range test (DMRT).
Experimental procedures are presented in Chart 1.
every leaflet infected, but plants retain normal form; plants may smell of blight; field looks green although every plant is affected), 6 = 50% blight (every plant affected and about 50% of leaf area destroyed), 7 = 75% blight (about 75% of leaf area destroyed; field appears neither predominantly brown or green), 8 = 95% blight (only a few leaves on plants, but stems green), and 9 = 100% blight (all leaves dead, stems dead or dying).

Economic Analyses of Formulated Bioagents
The benefit−cost ratio (BCR) was calculated for each treatment according to the method of Mondal et al. [63]. The cost−benefit analysis compared the profitability of each treatment based on the gross returns and costs. Each treatment's gross and net returns were computed as follows. Gross return (TK/ha) = tuber Yield (kg/ha) × price (TK/kg); net return (TK/ha) = gross return (TK/ha) − cost of production plus treatment cost (TK/ha); the BCR was calculated as shown below: where A = selling price (Tk./kg), B = cost of cultivation + treatment cost (Tk./ha), and C = yield (kg/ha).

Statistical Analysis
Data were analyzed using the MStatC statistical program. Means were compared using Duncan's multiple range test (DMRT).
A flow chart depicting the entire experimental procedures.

Development of an Eco-Friendly Sustainable Management Alternative against Late Blight of Potato Using Potential Formulated Bio-Agents under Field Conditions
The present study was designed to develop an eco-friendly sustainable management alternative against potato late blight using some potential formulated bio-agents under both growth chamber and field condition. Experiments were conducted in both a net house with artificial inoculation and in the field with natural infection conditions to compare the efficacy of the selected formulated bacterial and fungal bio-agents for controlling Chart 1. A flow chart depicting the entire experimental procedures.

Development of an Eco-Friendly Sustainable Management Alternative against Late Blight of Potato Using Potential Formulated Bio-Agents under Field Conditions
The present study was designed to develop an eco-friendly sustainable management alternative against potato late blight using some potential formulated bio-agents under both growth chamber and field condition. Experiments were conducted in both a net house with artificial inoculation and in the field with natural infection conditions to compare the efficacy of the selected formulated bacterial and fungal bio-agents for controlling late blight of potato. Before using those with chemical fungicide (Curzate M8), the interactions effect of different bacterial and fungal bioagents were studied. The results showed that no interactions effect was observed among the bioagents. However, the growth of both bacterial bioagents (P. putida and B. subtilis) and fungal bioagent (T. paraviridescens and T. erinaceum) were slightly delayed due to CurzateM8 (Supplementary Figure S1). Thus, the bioagents were applied after two days of Curzate M8 application.

In Vitro Growth Inhibition and Morphological Changes of P. infestans by Bacterial and Fungal Bioagents
The in vitro antagonistic assay of B. subtilis and P. putida with P. infestans revealed that the growth of P. infestans was inhibited by 93.99% over the control (Figure 2). On the other hand, T. paraviridicens and T. erinaceum inhibited the growth of P. infestans by 46 and 51.5%, respectively, over the control (Figure 3). Considering the morphological changes, we observed the deformation of mycelial structures when bioagents were applied against P. infestans in a duel culture method in the laboratory (Figure 1). late blight of potato. Before using those with chemical fungicide (Curzate M8), the interactions effect of different bacterial and fungal bioagents were studied. The results showed that no interactions effect was observed among the bioagents. However, the growth of both bacterial bioagents (P. putida and B. subtilis) and fungal bioagent (T. paraviridescens and T. erinaceum) were slightly delayed due to CurzateM8 (Supplementary Figure S1). Thus, the bioagents were applied after two days of Curzate M8 application.

In Vitro Growth Inhibition And Morphological Changes of P. infestans by Bacterial and Fungal Bioagents
The in vitro antagonistic assay of B. subtilis and P. putida with P. infestans revealed that the growth of P. infestans was inhibited by 93.99% over the control (Figure 2). On the other hand, T. paraviridicens and T. erinaceum inhibited the growth of P. infestans by 46 and 51.5%, respectively, over the control (Figure 3). Considering the morphological changes, we observed the deformation of mycelial structures when bioagents were applied against P. infestans in a duel culture method in the laboratory (Figure 1).

Figure 2.
In vitro growth inhibition (mm) and percent reduction of mycelia growth of P. infestans by two antagonistic fungal isolates (BDISOF67R and BDISOF91R). actions effect of different bacterial and fungal bioagents were studied. The results showed that no interactions effect was observed among the bioagents. However, the growth of both bacterial bioagents (P. putida and B. subtilis) and fungal bioagent (T. paraviridescens and T. erinaceum) were slightly delayed due to CurzateM8 (Supplementary Figure S1). Thus, the bioagents were applied after two days of Curzate M8 application.

In Vitro Growth Inhibition And Morphological Changes of P. infestans by Bacterial and Fungal Bioagents
The in vitro antagonistic assay of B. subtilis and P. putida with P. infestans revealed that the growth of P. infestans was inhibited by 93.99% over the control (Figure 2). On the other hand, T. paraviridicens and T. erinaceum inhibited the growth of P. infestans by 46 and 51.5%, respectively, over the control (Figure 3). Considering the morphological changes, we observed the deformation of mycelial structures when bioagents were applied against P. infestans in a duel culture method in the laboratory (Figure 1).

Figure 2.
In vitro growth inhibition (mm) and percent reduction of mycelia growth of P. infestans by two antagonistic fungal isolates (BDISOF67R and BDISOF91R).

Efficacy of Formulated Two Bacterial and Two Fungal Bioagents in Reducing Late Blight Severity of Potato under Artificial Inoculation Conditions
The minimum severity (3.67% and 5.00%) was recorded in T 11 at 61 and 65 DAP, respectively, in 2018-2019 compared to the control and treatments, as T 0 showed maximum severity at both 61 and 65 DAP. However, for T 1 to T 10 , all exhibited statistically similar data in both 61 and 65 DAP, except T 5 in the 65 DAP. Considering the percent reduction of severity at 65 DAP, T 11 showed the best (92.69%) result, followed by T 9 (72.44%), T 10 (71.87%), and T 2 (70.47%) compared to the other treatments (Table 1).

Assessment of Field Potential of Formulated Two Bacterial and Two Fungal Bioagents in Reducing Late Blight Infection and Severity under Field Conditions
The performance of the treatments on the percent of infected plants and late blight severity was recorded at three different time point viz. 48, 59, and 71 DAP in 2018-2019. Maximum (74.36%) and no plant infection were found in T 8 and T 11, respectively, while at 59 and 71 DAP, 100% infection was calculated, with almost all treatments possessing statistically identical data except T 9 (95.00), T 10 (64.96%), and T 11 (29.91%). Regarding the percentage of late blight severity at 48 DAP, no infected plant was found in T 10 and T 11 and maximum severity was recorded in T 5 (2.84%), and the others were calculated as a moderate rate of severity. At 59 DAP, minimal severity was recorded in T 11 (2.43%), followed by T 10 (3.37%) showing statistically identical data. These treatments performed better compared to all other treatments. In the case of 71 DAP, the minimum (10.33%) severity was recorded in T 11 followed by T 10 (21.30%), which was statistically similar and performed better among all of the other treatments. Considering the percent reduction of late blight severity over the control, the highest reduction was found when applied with T 11 (89.16%) followed by T 10 (77.50%), T 9 (27.07%), and T 2 (16.71%) ( Table 2).

Economic Analysis of Formulated Two Bacterial and Two Fungal Bioagents Used for Reducing Late Blight Infection and Severity under Field Conditions
The benefit−cost ratio (BCR) was calculated based on the data obtained from formulated bacterial and fungal bioagents during 2018-2019 for each of the treatments, and is tabulated in Table 3. The results from the table of the cost−benefit analysis revealed that all treatments provided BCR lower than 1, except T 10 (0.45) and T 11 (0.50), which previously recorded significant results in the reduction of severity over the control. The maximum gross return (Tk. 321,760.00/ha) and the net return (106,660.00 Tk./ha) were obtained from the treatment T 11 . Thus, the highest BCR was calculated from treatment T 11 (0.50) followed by T 10 (0.45). The results indicated that a return of Tk. of 0.45 and 0.50 was obtained over the investment of Tk. 1.00 in case of T 10 (0.45) and T 11 (0.50) (Table 3), respectively.

Economic Analysis of Formulated Two Bacterial and One Fungal Bioagents Used for Reducing the Application Frequency of Fungicide for Controlling Late Blight of Potato
During 2019-2020 and 2020-2021, the average cost−benefit analysis revealed that the highest (Tk. 395,111.11/ha) gross return was obtained from treatment T 16 , followed by T 6 (Tk. 390,222.23/ha), T 1 (379,200.00/ha), and T 11 (Tk. 374,888.89/ha). Thus, the highest BCR (0.90) was calculated from treatments T 16 and T 6 (0.88), which performed better than T 1 (0.85). BCR results indicated a return ranging from Taka 0.85 to 0.90 over the investment of Taka 1.00 in these treatments in those two years. In both years, treatments (T 16, T 6, T 11 , and T 1 ) performed better in the field conditions, reducing the fungicide application frequency to mitigate late blight severity, as those treatments also performed better in the cost−benefit analysis (Table 8).

Detailed Economic Analysis of the Improved Management of Late Blight Using Bioagents during 2019-2020 and 2020-2021
Bangladesh has been producing 9.7 million tonnes potato on 0.5 million hectares of land, as mentioned earlier. Farmers are spending 6500 million Tk of their total expenditures on fungicides per year with conventional approaches (eight sprays of Curzate M8 (positive control)). Conversely, if we could apply two improved management approaches with bioagents 1 ((T 2 ) + four sprays of Curzate M8) and 2 ((T 12 ) + four sprays of Curzate M8), then the total expenditures for fungicides could be drastically reduced to 3250 million Tk. Among the three approaches, improved management with bioagents 1 and 2 showed better economic returns compared to the farmers' approach. Cultivation of potato with improved management approaches with bioagents 1 and 2 were satisfactory, because farmers benefited from a 7.19% and 10.98% increase in their income for one hectare of land, respectively. With regards to the country's economic impact within two years, 9361.5 million Tk was the total increase of the country's return when applying improved management with bioagents 2 and 6135 million dollars from improved management with bioagents 1. Approximately 0.3 million farm families are closely engaged with potato production. In our detailed analysis, we observed that the income of an individual farm family was raised 31.21 thousand Tk when we applied improved management with bioagents 2, which indicated that the use of bioagents with chemical fungicide to minimize the late blight severity had a tremendous economic and social impact on our country. Thus, farmers will likely be willing to accept this technology, as several factors are closely associated with their income return from one hectare of potato land (Table 9).

Discussion
Managing late blight using eco-friendly methods is always challenging under high disease pressure in severe environments. Biological management in this country is more relevant due to the detrimental effect of chemicals on the environment and human health. In this study, it was observed that bacterial species belonging to the genera Pseudomonas and Bacillus are were able to inhibit the growth of P. infestans in vitro by 94% over the control. These results are in accordance with the findings of [42]. They observed the best antagonistic activity of Pseudomonas and Bacillus against P. infestans, as they produced a wide range of antibiotics, chemical surfactants, and biosurfactants. The antagonist B. subtilis B5 strain effectively inhibited P. infestans growth [43]. The route of action seems to be the ability of B subtilis strains to create mycotoxins that suppress P. infestans growth and stimulate peroxidase activity [44]. Elliott et al. [45] noted that Companion ® and Serenade ® are marketed B. subtilis biocontrol agents that reduce P. infestans. Bacillus strains might control P. infestans directly by reducing mycelial development, cyst germination, or motile zoospore swimming by creating antifungal chemicals that suppress the pathogen, or indirectly by stimulating active oxygen burst, nitrogen synthesis, callose accumulation, and lignification [64][65][66]. In our study, we also observed alteration of mycelia growth and morphological changes with the spore formation when formulated bioagents were applied in an in vitro condition. The metabolite of the biosurfactant producing bacterium, P. aeruginosa has shown high efficacy against P. infestans under in vitro conditions [67]. Pseudomonas and Bacillus isolates were antagonistic to P. infestans. Twenty-three effective microorganisms (spore-forming and non-spore-forming bacteria, yeasts, and fungi) isolated from potato phyllosphere on P. infestans growth were investigated in dual cultures, including their patterns of inhibition [68]. PCA (Phenazine-1-carboxylic) promotes biofilm development, allowing PCA-producing Pseudomonas spp. to bind to plant roots and act as biocontrol agents [69]. Pseudomonas biocontrol of P. infestans was previously shown to suppress sporangia and zoospore germination, implying the existence of several undiscovered antioomycete determinants. By up and down regulating the gene expression in P. infestans, Roquigny et al. [46] showed that Pseudomonas spp.-produced Phenazine-1-carboxylic PCA is involved in growth inhibition in P. infestans.
Bacterial (P. putida and B. subtilis) and fungal (T. erinaceum) bioagents were found to be effective at reducing late blight severity by 99% until 60 DAP, whereas these bioagents were found to be partially effective until 70 DAP, and reduced late blight severity by 46% when applied together under high disease pressure conditions. The use of these bacterial and fungal bioagents in combination with four sprays of chemical fungicide (Curzate M8) could reduce late blight severity up to 98% and could reduce the application frequencies of fungicide by 50% in both net house and field conditions; generally, all farmers of Bangladesh have been using at least eight sprays of chemical fungicides, which might be raised up to 16 sprays depending on the weather conditions, per hectare of land, whether late blight severity is present or not, thus, we have standardized it (8 sprays) based on the field surveys in our experiment to evaluate the reduction of spray frequency of chemical fungicide with bioagents. Furthermore, the cost−benefit analysis revealed that treatments T 10 and T 11 showed a better performance in terms of BCR in 2018-2019, as well as treatmentsT 6 and T 16 in 2019-2020 and 2020-2021, respectively, compared to other treatments applied. Yan et al. [70] observed that B. velezensis reduced late blight severity by 40.79% and 37.67% in a two-year field trial. They found that a low fungicide concentration and a high concentration of B. velezensis SDTB038 could reduce potato late blight. In addition, B. velezensis SDTB038 may successfully suppress the infection of potato leaves by P. infestans, making it a promising biological fungicide against potato late blight. Compared to untreated plants, the B. subtilis 26D strain reduced P. infestans mycelium growth and reduced late blight symptoms by 35%, respectively. Sorokan et al. [71] explained that B. strains induced systemic resistance to P. infestans through the activation of the transcription of PR genes in potato plants. The development of ectoenzymes and antifungal medicines like surfactin and iturinA gives B. subtilis strains a broad range of antifungal action. Antifungal metabolite-induced mycelium damage is thought to be mostly osmotic cell stress. In intimate contact with phytopathogenic fungus, the bacteria aggressively move towards fungal hyphae, kill them, and feed on them [72]. These observations are highly similar in accordance with our observations of the morphological deformation of P. infestans in a dual culture method. Wang et al. [73] highlighted that B. subtilis WL-2 and IturinA produced by B. subtilis WL-2 have great potential as candidates for inhibiting P. infestans mycelium growth and controlling potato late blight. B. subtilis 30B-B6 was shown to significantly decrease late blight severity [74]. As revealed by [48], P. infestans is very sensitive to bacterial volatiles such as 1-undecene generated by potato-associated Pseudomonas strains. It was shown that several potato-associated Pseudomonas strains could effectively suppress extremely pathogenic P. infestans isolates by inhibiting mycelial growth of all P. infestans isolates when co-cultured with the most active Pseudomonas strain (R47) [49]. Tomar et al. [67] in another study observed that five isolates of bacteria were found to be effective against P. infestans out of 95 tested as biocontrol agents. Both P. aeruginosa-1 and -3 had 62.22% and 46.42% inhibition after 72 h, respectively. P. aeruginosa-1 culture supernatant and bacterial cell suspension exhibited 10.42%, 9.94%, and 17.96% disease severity in potato plants, respectively, compared to 53.96% in the control. Zhang et al. [9] observed in greenhouse and field trials that the combined application of Rhodopseudomonas palustris GJ-22 and Curzate resulted in better disease control than the use of either agent alone. They highlighted the potentialities of the combined application of R. palustris strain GJ-22 and Curzate to control potato late blight in a more environment friendly way by a reduced level of harmful chemical fungicides application. In this study, we observed that T. paraviridescens and T. erinaceum reduced the late blight severity in both net house and field conditions. Kariukiet al. [28] observed the inhibitory action of T. asperellum and T. harzianum on the P. infestans mycelial growth and the suppression of late blight disease in the greenhouse experiment. Elsherbiny et al. [75] reported that Trichoderma VOCs suppressed the mycelial development of P. infestans cultured on laboratory media by 80% and on potato tubers by 93.1%. Electron microscopy demonstrated substantial morphological and ultrastructural malformations in T. atroviride VOC-treated hyphae, including cell deformation, collapse, and organelle disintegration. Purwantisari et al. [76] reported T. viride induced resistance in potato plants against late blight. Cwalina-Ambroziak et al. [77] found that using an integrated chemical and biological approach decreased the symptoms of P. infestans infections. Trichoderma's rhizosphere competence and competitive ability could be a factor in its biocontrol roles against P. infestans [66]. This is because Trichoderma uses many mycoparasitic strategies, which are direct methods for biological control that work by parasitizing, detecting, growing, and colonizing pathogens. These strategies include the detection of pathogens through chemotropism; the lysis of the pathogen's cell wall, the pathogen's hyphal penetration by appresorial formation; and the production of toxins [78]. Considering the detailed economic analysis, improved management with bioagents 1 and 2 performed better compared to the farmers' conventional approach in terms of economic return, and income of per farm family was raised up to 31.21 thousand Tk as well, which indicated that using these bioagents had a positive economic impact on farmer income and on the country. Farmers benefitted while using the improved management with bioagents, which significantly focused the acceptability of these bioagents among stakeholders, consumers, and farmers. These findings support our observation on the potentiality of the combined use of bacterial and fungal bioagents with Curzate M8 to reduce late blight severity almost at the same level as the conventional eight sprays of Curzate M8 did. This was observed with either single or combined use of bacterial (P. putida and B. subtilis) and fungal (T. erinaceum) bioagents. Therefore, the possibility of using formulated bacterial and fungal bioagents could be an alternative for reducing the application of chemical fungicides for controlling late blight of potato and producing export quality organic potato in the country.

Conclusions
Bacterial (P. putida and B. subtilis) and fungal (T. erinaceum) bioagents were found to be effective at reducing late blight severity by 99% until 60 DAP, whereas these bioagents were found to be partially effective until 70 DAP and reduced late blight severity by 46% when applied together under field conditions. The use of these bacterial and fungal bioagents in combination with four sprays of chemical fungicide (Curzate M8) could reduce late blight severity by up to 98% and could reduce the application frequencies of fungicide by 50% in both net house and field conditions. However, the possibility of the commercial formulation and application of these bioagents needs to be investigated with a proper late blight forecasting system. Proper warning systems shed light on when and how many times chemical fungicides need to be applied in the future.