Management of Wilt Complex of Eggplant ( Solanum melongena L.) Caused by Fusarium oxysporum , Ralstonia solanacearum and Meloidogyne spp.

Wilt complex disease of eggplant is a severe problem in Bangladesh as well as in the world. The pathogenic variability and their survival nature make the disease complex. The pathogen includes a fungus (Fusarium oxysporum), a bacterium (Ralstonia solanacearum) and a nematode (Meloidogyne spp.) that attack the plant individually or combinedly. The present investigation aimed to evaluate the integrated effects of different chemicals, poultry manure and Trichoderma harzianum to manage the disease. Carbendazim (Autostin 50WP), Streptomycin sulphate 9% and Tetracycline hydrochloride 1% (Krosin 10SP) and Carbofuran (Furadan 3G) were used as a fungicide, bactericide and nematicide, respectively which were integrated with poultry manure and Trichoderma harzianum resulting in sixteen treatments. The treatment efficacy varied significantly in comparison to control in respect of wilt incidence, yield and yield contributing characters. No wilt incidence was observed where Trichoderma harzianum was applied individually or in combination with Furadan 3G, Krosin 10SP, Autostin


Introduction
Eggplant (Solanum melongena L.) is one of the most important and highly productive vegetable crops in the tropical and sub-tropical region of the world. It is cultivated widely in Indian sub-continent for its year-round availability, food value, taste, cash crop and also as an export commodity. It is the second most important vegetable crop in Bangladesh with respect to acreage and production [1]. However, production and yield rate of eggplant in Bangladesh is abysmal compared to other countries like India, Japan, China, etc., due to infection by different diseases. Eggplant cultivation is significantly impaired by wilt complex caused by Fusarium oxysporum, Ralstonia solanacearum and Meloidogyne spp [2] [3]. Nematodes facilitate fungi and bacteria to cause infection by creating wounds. The fungus and bacteria infect the vascular system of the plant and disrupt water and nutrient transportation resulting in wilt incidence. Nemic wilt shows knot/gall formation in the root system and the plants become weak due to the hindrance of water and nutrient uptake. The interaction of fungus, bacteria and nematode is called wilt complex and could be seen even in the same plant.
Farmers often face substantial crop loss due to the wilt complex resulting in severe economic losses. In case of bacterial wilt, 100% of the affected plants may collapse in the kitchen gardens of Bangladesh [4] while the severity is 10% -90% in field conditions [5] [6]. For fungal wilt, the fruit yield is reduced by 20% -30% [7], and it may turn into epidemic by complete crop failure in the winter season [8]. Nemic wilt (Meloidogyne spp.) solely may cause 27.2% yield loss of eggplant [9], and it may rise to 80% in solanaceous crops [10].
The standard control measures against wilt complex include the use of resistant varieties, crop rotation, agronomic practices, biological control and integrated management [11]. However, cultural practices and crop rotation may provide limited control of Ralstonia solanacearum [12] [13]. Discriminate uses of chemical pesticides create a harmful effect on the environment and ecology that leads the eggplants with residual effects [14]. Due to this, the export of eggplants from Bangladesh is restricted, which is alarming for the host country [15].
Integration of plant health management strategies (soil amendments, biocontrol agent and judicious use of fungicide, bactericide and nematicides) is now considered an effective approach emphasizing the environment, economics and social acceptance compared to conventional management. Trichoderma spp. is a well-documented biocontrol agent against a lot of soil and seed-borne pathogens for its antagonism, mycoparasitism and competition [16] [17] [18]. T. harzia-N. Akter et al. num and T. viride reported complete inhibition of mycelial growth of Fusarium oxysporum [19]. Bactericides and biocontrol agents are reported effective against Ralstonia solanacearum [20]. Organic residues supply plant nutrients and increase natural suppressiveness of the soil against soil-borne pathogens and improve physio-chemical and biological characteristics [21]. Suppression of Ralstonia solanacearum is seen in poultry manure amended soil [22]. Carbofuran is the most effective nematicide in controlling Meloidogyne sp. in warm weather conditions. Moreover, integration of poultry manure, bioagent and carbofuran considerably reduce gall index and give higher fruit yield over control [23]. The present research work has been designed to formulate an integrated approach using those eco-friendly options for the management of wilt complex of eggplant.

Isolation and Identification of Pathogens
Fusarium oxysporum and Ralstonia solanacearum 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).

In Vitro Assay of Fungicide and Bactericide
The fungicide Autostin 50WP (Carbendazim) (Supplementary Table 1) was assayed against Fusarium oxysporum 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 Fusarium oxysporum 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 Fusarium oxysporum 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 Ralstonia solanacearum 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.

Pathogenicity Test for the Causal Pathogens
The root-dip assay modified from Ramaiah and Garampalli [19] was followed for pathogenicity test of Fusarium oxysporum. Seedlings with wounded roots were submerged for 10 min in the conidial suspension (1 × 10 6 ml −1 ) obtained from 7 days old culture of Fusarium oxysporum on. In the case of Ralstonia solanacearum, 5 ml spore suspension (10 5 CFU ml −1 ) was injected into   In each case, uninoculated plants served as a positive control.

Application of Treatments
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 Trichoderma harzianum. was prepared by scraping the 10 -12 days old culture, maintaining the concentration 1 × 10 8 conidia ml −1 solution. The roots of seedlings were soaked with Trichoderma suspension. The specific pit soil was treated with black gram based Trichoderma formulation (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).  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].

Pathogenicity Test
The pathogenicity of Fusarium oxysporum, Ralstonia solanacearum and Meloidogyne spp. was satisfied the Koch's postulates (Figure 8). In Fusarium oxysporum, 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 previous Meloidogyne spp.

Evaluation of Chemicals against Isolated Pathogens
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 Fusarium oxysporum and Ralstonia solanacearum, respectively. Autostin 50WP (0.1%) and Krosin 10SP (0.05%) were found to be effective and gave 81% and 87% reduction of growth of Fusarium oxysporum and Ralstonia solanacearum, respectively (Table 1 & Table 2). Autostin 50 WP was found to be promising in the reduction of the growth of Fusarium oxysporum. Amini et al., [34] observed that Carbendazim (Autostin 50 WP) was effective in controlling Fusarium oxysporum in in vitro and field condition. Murakoshi and Takahashi [35] and Svetlana Milijasevic et al. [36] stated that antibiotics like Streptocyclin found to be very effective in controlling Ralstonia solanacearum, 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.

Effect of Different Treatments on Wilt Complex and Yield of Eggplant
The incidence of fungal wilt with the application of different treatments singly or in combination varied sharply ranging from 0 to 13.33% against Fusarium oxysporum. Plants in control treatments showed the highest disease incidence (13.33%) while no wilt incidence was noticed in case of treatments T 1 , T 4 , T 5 , T 6 , T 7 , T 8 , T 9 , T 12 , T 13 , T 14 and T 15 (Table 3). In case of incidence of bacterial wilt, treatment T 2 , T 3 , T 4 , T 7 , T 8 , T 12 , T 13 , T 14 and T 15 exhibited complete reduction of Ralstonia solanacearum compared to control (33.33%). Nemic wilt incidence was also greater in control plots (13.33%) than in treatment T 1 , T 6 , T 9 and T 12 (6.67%) whereas no nemic wilt incidence was observed in case of treatment T 2 , T 4 , T 7 , T 8 , T 13 , T 14 and T 15 . 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 T 15 where Krosin, Furadan, Trichoderma formulation and Poultry manure were applied combinedly. The minimum yield was obtained in control treatment T 16 (8.02 t·ha −1 ) ( Table 3).

Effect of Different Treatments on Plant Growth Parameters against Wilt Complex of Eggplant
The was observed in case of treatment T 15 followed by the treatments T 13 , T 14 and T 1 (Table 4).

Effect of Treatments on Growth Parameters and Yield Over Control
Treatment T 15 (Krosin 10SP + Furadan 5G + Trichoderma formulation + Poultry manure) was found to influence the number of branches, leaves, fruits, fruit  Figure 10).

Benefit Cost Ratio (BCR)
The benefit cost ratio (BCR) of treatment T 15 (Krosin + Furadan + Trichoderma formulation + Poultry manure) was the highest (5.68) followed by T 14 (5.56) and T 4 (5.48) ( Table 5).  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 Fusarium oxysporum, 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 Ralstonia solanacearum 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 Trichoderma harazianum was effective in controlling Meloidogyne incognita and Ralstonia solanacearum causing wilt complex of eggplant under field condition [39]. The integration of Streptocylin @0.5 g/L + COC 50% WP (1 g/L) in-American Journal of Plant Sciences tercropped 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 Ralstonia solanacearum [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 Ralstonia solanacearum. Thus the integrated use of Krosin, Furadan, Trichoderma formulation, and Poultry manure showed the maximum performances in yield and yield contributing characters. Though treatment T 13 comprised of Trichoderma formulation and Poultry manure, it gave a lower yield than T 14 . It might be due to the presence of component Autostin 50 WP which has antifungal properties on Trichoderma formulation.

Conclusions
In the current study, the morphological and cultural characteristics of F. oxysporum, R. solanacearum and Meloidogyne sp. have been described to provide key information for identification. The pathogenicity test confirmed the virulent nature of these pathogens which caused wilt of eggplant plants. Further, in vitro assay suggests that carbendazim can control F. oxysporum while streptomycin sulphate with tetracycline hydrochloride act against R. solanacearum effectively even at low concentrations.
From the field experiment, it can be concluded that integration of Streptomycin Sulphate with Tetracycline Hydrochloride (Krosin 10SP), Carbofuran (Furadan 3G), Poultry manure and Trichoderma harzianum formulation may be an alternative approach to control wilt complex of eggplant with higher economic return and lower impact on the environment. The feasibility of using this treatment in controlling wilt complex of eggplant needs to be tested both in other solanaceous vegetables irrespective of Agro-Ecological Zone (AEZ) of the country.