Fusariumoxysporum and Ralstoniasolanacearum were isolated from the infected stem of eggplant, and Meloidogyne spp. was isolated from the knots of the infected root. F. oxysporum was purified by hyphal tip culture method from the diseased stem by blotter method and was identified by the key characters (CMI) (Figure 1). The bacterial wilt of an infected plant was confirmed by bacterial ooze test by making the water turbid. From the serial dilution of ooze suspension, 100 µL of each concentration was poured followed by spreading with a sterilized spreader onto the surface of the solidified TTC media. The inoculated plates were incubated at 32˚C for 48 hours. The isolated bacterium was streaked on CPG and NA media and was identified as R. solanacearum by morphological and colony characters [24] and gram staining [25] (Figure 2). The roots of nemic wilted plant were washed gently under running tap water. A branch of root bearing knots was examined under a stereomicroscope, and a number of egg masses were noticed. A single egg mass of the root knot was picked up in semi-permanent slide and crushed for studying under the compound microscope. An adult pear-shaped female nematode was pulled from the hole which was seen after removing an egg mass and identified as Meloidogyne spp. By

observing under stereo and compound microscope (Figure 3).

The fungicide Autostin 50WP (Carbendazim) (Supplementary Table 1) was assayed against Fusariumoxysporum by Food Poisoning Technique, cup or groove method [26]. Five mm disc of PDA media in each petri plate was scooped from 3 places maintaining an equal distance from the centre using a sterilized disc cutter. After putting Autostin 50WP (0.1%) solution into each hole, the plates were kept overnight for the diffusion of fungicide into the medium. 5 mm mycelial discs of 7 days old culture of Fusariumoxysporum were taken and placed at the centre of the diffused PDA plates. The plates with holes filled with sterile water were served as control. All the plates were incubated at 25˚C ± 2˚C for seven days, and mycelial growth of Fusariumoxysporum was recorded. The percent inhibition of mycelial growth was calculated using the formula used by Islam [26] and [27]. Bioassay of antibacterial Krosin 10SP (Streptomycin sulphate 9% and Tetracycline hydrochloride 1%) (Supplementary Table 1) against Ralstoniasolanacearum was done by zone inhibition method. 10 ml nutrient broth in a test tube was inoculated with 48 hours old pure culture of bacteria grown on NA media plate. After shaking in a shaker incubator at 30˚C and 150 rpm for 24 hours, the broth culture was spread uniformly on TTC media plate using the sterile cotton swabs. 5 mm disc was scooped and filled with Krosin 10SP suspension (0.05%) while inoculated plates filled with sterile water was treated as control. The plates were incubated at 30˚C in incubation chamber for 48 hours, and the diameter of the inhibition zone was measured.

The root-dip assay modified from Ramaiah and Garampalli [19] was followed for pathogenicity test of Fusariumoxysporum. Seedlings with wounded roots were submerged for 10 min in the conidial suspension (1 × 10⁶ ml⁻¹) obtained from 7 days old culture of Fusariumoxysporum on. In the case of Ralstoniasolanacearum, 5 ml spore suspension (10⁵ CFU ml⁻¹) was injected into the vascular system of the 25 days old seedlings (Figure 4). The plants expressing wilt symptoms were selected. The fungus and bacterium were re-isolated from the symptom and compared with the previous culture of Fusariumoxysporumon and

Ralstoniasolanacearum, respectively to satisfy the Koch’s postulates. Egg masses were picked up from the roots, kept for hatching in water for 24 to 36 hours. The hatched second stage (J2) juveniles were allowed to inoculate 21 days old seedlings of eggplant. After 100 to 150 days when the nematodes complete 2 - 3 generations, the root galls were explored for confirmation of the causal nematode. In each case, uninoculated plants served as a positive control.

Thirty-five days old seedlings of BARI hybrid begoon 3 (oblong) were transplanted in the research field of Plant Pathology Division, Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur, Bangladesh during September, 2017. Sixteen treatments were arranged with 3 replications in Randomized Complete Block Design (RCBD) where each plot size was 3 m² (3.0 m × 1.0 m) containing 5 plants per plot with 0.6 m × 0.6 m planting distance. Every treatment was placed once at each block. Treatments were T₁ = Autostin 50 WP (seedling treatment + foliar spray @ 0.2%); T₂ = Furadan 3 G (Supplementary Table 1) (soil treatment@ 10 g pit⁻¹); T3 = Krosin 10SP (seedling treatment + drenching of rhizosphere soil @ 0.1%); T₄ = Trichoderma formulation (Soil application @ 20 g·m⁻²: 1 × 108 cfu g⁻¹ of soil); T₅ = Poultry manure (soil amendment @ 2 kg 3 m⁻²; T₆ = Autostin 50 WP + Trichoderma formulation; T₇ = Furadan 3 G + Trichoderma formulation; T₈ = Krosin 10SP + Trichoderma formulation; T₉ = Autostin 50 WP + Poultry manure; T₁₀ = Furadan 3 G + Poultry manure; T₁₁ = Krosin 10SP + Poultry manure; T₁₂ =Trichoderma formulation + Poultry manure; T₁₃ = Autostin 50 WP + Trichoderma formulation + Poultry manure; T₁₄ = Furadan 3 G + Trichoderma formulation + Krosin 10SP; T₁₅ = Krosin 10SP + Furadan 3 G + Trichoderma formulation + Poultry manure; T₁₆ = Control.

Autostin (0.2%) and Krosin (0.1%) solutions were applied for seedling-root treatment before transplanting, followed by foliar spraying. Spraying of treatments were done 3 times with 10 days’ interval after transplanting. Furadan 3G was applied in pit soil (5 gm pit⁻¹) during transplanting of seedlings. Poultry manure was applied @ 2 kg plot⁻¹ as a soil amendment before 21 days of transplanting to the allotted plots. Spore suspension of Trichodermaharzianum. was prepared by scraping the 10 - 12 days old culture, maintaining the concentration 1 × 10⁸ conidia ml⁻¹ solution. The roots of seedlings were soaked with Trichoderma suspension. The specific pit soil was treated with black gram based Trichodermaformulation (50 gm pit⁻¹) where Black gram: peat soil: Cowdung = 1:2:1.

The Disease Incidence or severity was measured, and costing for estimation of Benefit Cost Ratio (BCR) was done as exercised by Islam et al. [28] (Supplementary Table 2 and Table 3).

%   Disease   Incidence / Severity = Number of infected plant ( s ) Numaber of total plant ( s ) × 100

BCR = Gross return ( Tk . / ha ) ÷ Total cost of production ( Tk . / ha )

Initially, F. oxysporum produced white cottony mycelium on PDA media. The fungus grew slowly and with time made pink pigmentation (Figure 5). Several-celled and slightly curved, hyaline macro conidia were observed in PDA culture. Single-celled, hyaline, ovoid to globose micro conidia were also found from 21 days old pure culture of Fusariumoxysporum. In case of bacteria, colonies were appeared as the red coloured centre with whitish margins (virulent) within 24 - 48 hours culturing on semi selective media Tetrazolium chloride (TZC) medium. While culturing on NA media, circular, mucoid, convex, lucid coloured colonies were found (virulent). The bacterium was rod-shaped with rounded ends and gram-negative (red colour) under the compound microscope at 1000X magnification with oil immersion objective. These characteristics were matched

with Ralstonia solanacearum (Figure 6). In the case of nematode, pear-shaped female surrounded by egg mass were found under stereomicroscope which was prepared from root knot and identified as Meloidogyne spp. Eggs were found ellipsoid, lack of uniformity. Larvae and adults were also observed (Figure 7).

The findings of the present investigation coronated with the results of previous research with so much extent. Nelson et al. [29] and Joshi et al. [30] described that the colony appearances of F. oxysporum on PDA, which was highly variable. The pink colony colour and white cottony mycelium is formed on PDA media. Three types of asexual spores viz. macroconidia, microconidia and chlamydospore were produced. Macroconidia are slightly curved, sickle shaped and 4 - 5 celled where microconidia are round/globose and single-celled. Kumar & Sarma [31] and Dhital [32] narrated that Ralstonia solanacearum is a soil-borne aerobic, rod-shaped, non-spore-forming, gram-negative bacterium. Colonies of Ralstonia solanacearum appear as red coloured with whitish margins (virulent) on TTC medium which was highly fluidal. Circular, mucoid, convex, lucid coloured colonies were found on NA medium with respect to some samples. The pear-shaped female nematode of Meloidogyne sp. produced a huge number of knots or galls in the root system, and egg masses were attached with the knots [33].

The pathogenicity of Fusariumoxysporum, Ralstonia solanacearum and Meloidogynespp. was satisfied the Koch’s postulates (Figure 8). In Fusariumoxysporum, the inoculated plants showed wilt symptom after 20 - 30 days of post-inoculation. But in the case of bacteria, it was taken only one week for wilting. In contrast, the control plants were symptomless. On reisolation, the colonies were found to be similar to that fungus and bacteria inoculated previously. In the case of Meloidogyne spp., knots were found on the root system after 100 - 150 days of inoculation. Female nematode, egg masses and juveniles were similar to the previousMeloidogyne spp.

The antipathogenic activity of different chemicals were evaluated using the poison plate method. The chemicals tested in the present study showed a gradual decline in growth of associated pathogens even in low concentration (Figure 9). Autostin 50 WP (0.1%) and Krosin 10SP (0.05%) exhibited potent antifungal and antibacterial activity against Fusariumoxysporum and Ralstoniasolanacearum, respectively. Autostin 50WP (0.1%) and Krosin 10SP (0.05%) were found to be effective and gave 81% and 87% reduction of growth of Fusariumoxysporum and Ralstoniasolanacearum, respectively (Table 1 & Table 2).

Autostin 50 WP was found to be promising in the reduction of the growth of Fusariumoxysporum. Amini et al., [34] observed that Carbendazim (Autostin 50 WP) was effective in controlling Fusariumoxysporum in in vitro and fieldcondition. Murakoshi and Takahashi [35] and Svetlana Milijasevic et al. [36] stated that antibiotics like Streptocyclin found to be very effective in controlling Ralstoniasolanacearum, causing bacterial wilt. Krosin contains two types of active ingredient i.e. Streptomycin sulphate 9% and Tetracycline hydrochloride 1% that reduces the selection pressure and controls bacteria efficiently.

The incidence of fungal wilt with the application of different treatments singly or in combination varied sharply ranging from 0 to 13.33% against Fusariumoxysporum. Plants in control treatments showed the highest disease incidence (13.33%) while no wilt incidence was noticed in case of treatments T₁, T₄, T₅, T₆, T₇, T₈, T₉, T₁₂, T₁₃, T₁₄ and T₁₅ (Table 3). In case of incidence of bacterial wilt, treatment T₂, T₃, T₄, T₇, T₈, T₁₂, T₁₃, T₁₄ and T₁₅ exhibited complete reduction of Ralstoniasolanacearum compared to control (33.33%). Nemic wilt incidence was also greater in control plots (13.33%) than in treatment T₁, T₆, T₉ and T₁₂ (6.67%) whereas no nemic wilt incidence was observed in case of treatment T₂, T₄, T₇, T₈, T₁₃, T₁₄ and T₁₅. The cumulative incidence wilt complex also had the similar trend where the maximum incidence of wilt complex was recorded in control (60%). In contrast, no wilt complex was noticed in case of treatment T₁₅ where Krosin, Furadan, Trichoderma formulation and Poultry manure were applied combinedly. The minimum yield was obtained in control treatment T₁₆ (8.02 t·ha⁻¹) (Table 3).

*Values in a column with same letter (s) do not differ significantly (p = 0.05). *Figures in parenthesis are the mean of square root transformed values.

The yield contributing characters of eggplants significantly influenced by the treatments applied against wilt complex. The highest shoot length (76.40 cm) was observed in T₅, whereas the lowest length of the shoot was in the control plants (53.97 cm). The number of branches (17.03) as well as leaves (179.0) was maximum in T₁₅ where Trichoderma formulation, Krosin, Furadan and Poultry manure were applied combinedly. The plants treated with T₁₅ produced the maximum number of fruits (37.33) while the lowest number of fruits plant⁻¹ (23.37) was recorded in the control plots. The highest fruit length (23.00 cm) was observed in case of treatment T₁₅ followed by the treatments T₁₃, T₁₄ and T₁ (Table 4).

Treatment T₁₅ (Krosin 10SP + Furadan 5G + Trichoderma formulation + Poultry manure) was found to influence the number of branches, leaves, fruits, fruit

*Values in a column with same letter (s) do not differ significantly (p = 0.05).

length and fruit yield of eggplant by 32%, 29%, 60%, 53% and 300%, respectively. Poultry manure increased plant height by 42% over control where treatment T₁₅ increased 36% (Figure 10).

The benefit cost ratio (BCR) of treatment T₁₅ (Krosin + Furadan + Trichoderma formulation + Poultry manure) was the highest (5.68) followed by T₁₄ (5.56) and T₄ (5.48) (Table 5).

Integrated approach by employing three chemicals viz. Autostin 50WP (Carbendazim), Furadan 3G (Carbofuran), and Krosin 10SP (streptomycin sulphate) along with a bio-agent Trichoderma harzianum and a soil amendment by poultry manure singly and in combinations were explored for the management of wilt complex of eggplant. The treatment combinations showed a better result

*1 United States dollar = 84.83 Bangladeshi taka (Tk). *Costing was done on the basis of market price’ 2018.

than the individual application of each of the management option except Trichoderma formulation. The combination of bactericide (Krosin 10 SP), nematicide (Furadan 3G), Trichoderma formulation and poultry manure showed promising performances satisfying the Benefit Cost Ratio (BCR). Trichoderma formulation and Poultry manure not only suppress the soil-borne pathogens but also improved soil properties which resulted in higher yield.

It is worth mentioning here that the combination of treatments without Autostin 50 WP also showed superior results in reducing the fungal wilt incidence. The possible reason might be the Trichoderma formulation which acted against Fusariumoxysporum, consequently controlled the fungal incidence and some cases can cause 95% recovery of infection [37]. It is assumed that nematicide controlled not only nematode but also bacteria. The farmers of Philippine noticed no wilt symptom even in infectected sucker of banana caused by Ralstoniasolanacearum after treating with Furadan^® (a.i. carbofuran 48%) [38]. For this mechanism, Furadan 3G was enabled to combat the bacteria, even in absence of bactericide in the treatment. As the nemic infection facilitates the penetration of bacteria through wounds, Furadan indirectly control the bacteria by managing nematode. Combined application of Carbofuran 3G, Neem cake, Streptocyclin and Trichodermaharazianum was effective in controlling Meloidogyneincognita and Ralstoniasolanacearum causing wilt complex of eggplant under field condition [39]. The integration of Streptocylin @0.5 g/L + COC 50% WP (1 g/L) intercropped with Mustard cake was successful in reducing bacterial wilt incidence by 23.35% and formation of knots by 72.4% [40].

Trichoderma spp. enhance plant growth and productivity [41] [42]. The mechanisms of Trichoderma spp. were antagonism, mycoparasitism, competition with pathogens for nutrient and space and induction of systemic resistance in plants resulting in the reduction of disease and increase in yield. Carbofuran also showed a prominent influence on the growth parameters and suppression of Meloidogyne spp. and Ralstoniasolanacearum [38] [43]. The addition of poultry manure in the treatments ensued the increased yield by boosting up the soil fertility and the suppressing nature of the soil [44]. Krosin was responsible for inducing resistance in plants against Ralstoniasolanacearum. Thus the integrated use of Krosin, Furadan, Trichoderma formulation, and Poultry manure showed the maximum performances in yield and yield contributing characters. Though treatment T₁₃ comprised ofTrichoderma formulation and Poultry manure, it gave a lower yield than T₁₄. It might be due to the presence of component Autostin 50 WP which has antifungal properties on Trichoderma formulation.