Background

Eggplant (Solanum melongena L.) is a tropical and subtropical vegetable from the solanaceous (Rajan et al. 2022). It is widely cultivated, but key producers are in South East Asia including Pakistan, India, Japan, and China (Alam and Salimullah 2021). S. melongena faces enmity from both biotic and abiotic stresses including environmental hazards, pests including pathogens like (fungi, viruses, bacteria, root-knot nematode, and insects, i.e., jassids, mites, thrips, fruit and shoot borer, mealybug, aphids, and whitefly (Elkelany et al. 2020).

Root-knot nematodes (RKN) are considered among the top five phytopathogens. RKN’s genera are the tenth most prevalent genera of phyto-parasitic nematodes globally (El Aimani et al. 2022). Meloidogyne species are obligate sedentary endoparasitic RKNs that attack the underground plant tissues including the root system. They are polyphagous pests and are supposed to be cosmopolitan in nature. They also interact with several other phytopathogens that make them even more lethal and thus are responsible for hindering the high yield of S. melongena (Giri et al. 2022).

The J2 stage is highly active and feeds within the cortex. RKN infection initiates with a root incursion by the J2 that is hatched from the eggs in the soil. The J2 punctures the epidermis of the cell wall with the help of stylet. It constantly moves around in the root intercellular spaces in search of establishing a suitable feeding site. Also, it remains in the vascular tube and the feeding site is established in giant cells due to the defense system activation from the host plant (Niu et al. 2022).

The RKN management plans are staggeringly more intricate than other phytopathogens because habitually parasitic nematodes conceal underground inhabitation in soil and root tissues, and thus escape the grower eye visibility. The chemical control is not feasible because of its low penetration into the RKN ovum, fast leaching down into the water table, and degradation issues. The high price of commercial nematicides makes it economically unfeasible to be utilized by the growers for the management of RKN. Over the past few decades, biological control gained interest to tackle the pest problem. The concept of biological control is considered a reliable substitute for toxic pesticides (Saad et al. 2022). Scientists have explored several antagonistic microorganisms that could be utilized as bio-control agents against the RKNs. Nematophagous fungi had a core action against the RKNs as they directly influence them by minimizing their population. Furthermore, these fungi may have a key role in the host plant growth promotion and could induce systemic resistance in plants (Yu et al. 2022). The use of biocontrol is highly effective because it could be simultaneously used with other control practices in the Integrated Pest Management (IPM). Keeping in view the significance of biocontrol agents for the management of RKNs, present in vitro and planta experiments were conducted to investigate the antagonistic properties of fungi, viz. Trichoderma harzianum, Aspergillus niger, Penicillium chrysogenum, Pochonia chlamydosporium against M. incognita in the laboratory and greenhouse on eggplant.

Methods

Six treatments including four fungi (Trichoderma harzianum, Aspergillus niger, Pochonia chlamydosproium, and Penicillum chrysogenum @ 2.5 g/1000 ml of water) were carried out. Positive check included the nematicide, Carbofuran (@ 1 g per plant). A negative check received simple distilled water (SDW) with nematode eggs and juveniles. Figure 1 shows microscopic photographs of the applied fungal treatments.

Fig. 1
figure 1

Microscopic photographs of A Trichoderma harzianum B Aspergillus niger C Penicillium chrysogenum D Pochonia chlamydosporium

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Host plant, fungi, and root-knot nematode (RKN)

Eggplant cv. Local-Peshawar was utilized as the host plant against the RKN. Seeds of eggplant were surface sterilized in Sodium hypochlorite (NaOCl) 1 percent solution for about 20 min and gently washed with SDW. The fungal suspensions of the selected biocontrol agents were provided by the Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture Peshawar Pakistan. A fungal culture was maintained on the freshly prepared Potato Dextrose Agar (PDA) medium under sterile conditions. The suspensions of fungi were identified based on conidium, color, colony texture, and shape (Darwesh et al. 2019). The Richards Liquid Medium (Fuchs et al. 2002) was used for the mass production of fungal bio-control agents. M. incognita was used as a pathogen of the eggplant host.

Pure culture maintenance of Meloidogyne incognita

Pure culture of M. incognita was maintained on eggplant cultivar (Local-Peshawar) through single egg mass inoculation in the green house (Fatima et al. 2022). The swollen female body patterns (perennial patterns) were used for the identification (Fig. 2) (Sasser et al. 1983). Infected galls of eggplant collected from Malakand Division were isolated from the roots dipping the roots in 0.05 percent NaOCl for 5 min (Barker and Hussey 1976). Then, eggs were collected and washed with SDW on 25-μm sieves. The egg masses were allowed to hatch in an incubator for the collection of J2s about 5 days later for their use in in vitro and in-planta studies.

Fig. 2
figure 2

A Scanning electron microscope (SEM) photographs of the perennial pattern present on the swollen female of M. incognita

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In-vitro bioassay

The in vitro investigation was evaluated against the RKN egg masses and J2s in Petri plates. The Completely Randomized (CR) design was adopted and total of five replicates of each treatment was taken. The egg hatch inhibition and J2s mortality were recorded at the incubation periods of 24, 48, and 72 h by using the following formulae (Sikandar et al. 2020).

$$\begin{aligned} & {\text{Percentage}}\;{\text{of}}\;{\text{egg}}\;{\text{hatch}}\;{\text{inhibition}} = \left( {{\text{No}}\;{\text{of}}\;{\text{eggs}}\;{\text{hatched}}} \right){/}\left( {{\text{initial}}\;{\text{eggs}}} \right) \times {1}00. \\ & {\text{Percentage}}\;{\text{of}}\;{\text{J2}}\;{\text{mortality}} = \left( {{\text{No}}\;{\text{of}}\;{\text{dead}}\,{\text{J2s/total}}\;{\text{J2s}}} \right) \times {1}00\% . \\ \end{aligned}$$

Serial dilutions of each bio-control agent (1:1, 1:10, 1:100 V/V) were prepared in SDW. A total of 2 ml of SDW containing 6 egg masses and 200 freshly hatched J2s decanted into Petri plates having 8 ml of each dilution of the tested bio-control agents. SDW was used as negative control, while positive control received the nematicide and carbofuran. The Petri plates were covered with lids and enclosed in parafilm. Petri plates were kept at 28 °C (Khan et al. 2022a).

Greenhouse experiment

Seedling raising and transplantation

The eggplant seeds were sown in sterilized soil beds (sand: clay; 1:2). The two-week-old eggplant seedlings were transplanted to 50-cm diameter earthen pots (Dennis 2022).

Application of fungal biocontrol agents in a greenhouse experiment

The in-planta studies were laid-out in the glasshouse of Plant Pathology Section, Agricultural Research Institute (ARI) Turnab Farm Peshawar. The experiment was set up in a Completely Randomized (CR) design, having eight replications. The glasshouse condition was maintained at 25 °C, and RH was about 95–100%.

Approximately, 2200 J2s were inoculated to the earthen pots containing eggplant seedlings. The fungal suspension (1000 ml; containing 2.5 g mycelium + spores) was applied with the help of a sterile micro-pipette as a soil drench method (Naz et al. 2013). The eggplant plants inoculated with J2s were raised for two months and then uprooted for recording the data regarding RKN and plant growth parameters before experiment termination. The plants were uprooted and gently washed with SDW for removing debris of the soil particles. The Taylor and Sasser (1978) galling index (GI) was followed which was given below. The plants having no galls were indexed as 0, while the plant having over 100 galls was indexed as 5 (Oyetunde et al. 2022). The data on the eggplant growth parameters i.e., root and shoot lengths/ weights, and no. of flowers, and RKN parameters i.e. egg masses and females were recorded.

Statistical analysis

Data regarding the egg hatch inhibition and J2 mortality were corrected with Abbot Formula (Chen et al. 2023); then, percent inhibition and mortality were calculated with the help of mentioned formulae. A statistical software STATISTIX (8.0 EL USA) was used for the analysis of the data. The least significant difference (LSD) test at p = 0.05 was used to find the F-ratio statistical variation among the fungal biocontrol agents..

Results

In vitro bioassays

In vitro bioassay for egg hatch inhibition and J2s mortality showed that the effectiveness of all the treatments increased with the passage of time as well as concentration. All the treatments were found statistically significant at p = 0.05% over a check with eggs and juveniles.

The means comparison of different fungal biocontrol agents at 24 h revealed that T. harzianum inhibited 37.6% of RKN eggs, followed by the standard carbofuran (32.4%) and then by P. chlamydosporium, which inhibited 27.1% of eggs. A. niger (20.6%) was the least number of eggs inhibited (Table 1).

Table 1 Percentage of egg hatching inhibition by various fungal biocontrol agents at varying concentrations at 24 h
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The means comparison of different fungal biocontrol agents at 48 h revealed that T. harzianum inhibited 57.8% of RKN eggs, followed by the standard carbofuran (50.4%) and then by P. chlamydosporium, which inhibited 41.6% of eggs. The least number of eggs was inhibited by A. niger (30.8%), followed by P. chrysogenum (23.2%) (Table 2).

Table 2 Percentages of egg hatching inhibition by various fungal biocontrol agents at varying concentrations at 48 h
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The means comparison of the results of different fungal biocontrol agents at 72 h indicated that T. harzianum inhibited 64% RKN eggs, followed by the standard carbofuran (58.8%) and then by P. chlamydosporium that inhibited 53.0% eggs. The followed treatments were A. niger and P. chrysogenum which inhibit 42.6 and 35.6% RKN eggs, respectively, at a p = 0.05% significance level (Table 3).

Table 3 Percentages of egg hatching inhibition by various fungal biocontrol agents at varying concentrations at 72 h
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The means comparison of percentage of J2s mortality results of different fungal biocontrol agents at 24 h of incubation indicated that T. harzianum caused maximum mortality (56.2%), followed by the standard carbofuran (52.3%) and then by P. chlamydosporium that caused (47.8%) J2s mortality. The, following treatments were A. niger and P. chrysogenum causing 36.7 and 23.2% RKN J2s mortality, respectively, at a p = 0.05% significance level (Table 4).

Table 4 Percentages of J2 mortality by various fungal biocontrol agents at varying concentrations at 24 h
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The means comparison of the percentage of J2s mortality results of different fungal biocontrol agents at 48 h of incubation indicated that T. harzianum caused the maximum mortality (65.4%), followed by the standard carbofuran (61.8%) and then by P. chlamydosporium that caused 55.6% J2s mortality. The followed treatments were A. niger and P. chrysogenum causing 44.4 and 32.5% RKN J2s mortality, respectively, at a p = 0.05% significance level (Table 5).

Table 5 Percentages of J2 mortality by various fungal biocontrol agents at varying concentrations at 48 h
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The means comparison of the percentage of J2s mortality results of different fungal biocontrol agents at 72 h of incubation indicated that T. harzianum caused the maximum mortality (72.4%), followed by the standard carbofuran (68.1%) and then by P. chlamydosporium that caused 61.4% J2s mortality. The followed treatments were A. niger and P. chrysogenum causing 47.3 and 40.0% RKN J2s mortality, respectively, at a p = 0.05% significance level (Table 6).

Table 6 Percentages of J2 mortality by various fungal biocontrol agents at varying concentrations at 72 h
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Greenhouse experiment

The in-planta results about number of galls revealed that control (negative) had the most number of galls (206.8), followed by P. chrysogenum having 75.1 galls per root system. T. harzianum-treated plants had the least number of galls 22.2 (Figs. 3, 4). All the treatments significantly lowered the RKN parameters, i.e., egg masses and swollen females. T. harzianum had the least number of egg masses (35.7), followed by the carbofuran (standard) having 40.0 egg masses. The followed treatment was P. chlamydosporium (57.1). Maximum no. of egg masses was found in the check (control negative) (224.1) per root system (Fig. 3). Likewise, the control negative had a maximum no of adult females (182.7), while the least swollen females were found in T. harzianum and carbofuran (standard) having 21.1 and 22.75 swollen females per root system, respectively (Fig. 3).

Fig. 3
figure 3

Effects on the Meloidogyne incognita parameters in the eggplant root system inoculated with various fungal Biocontrol agents at the in-planta conditions

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Fig. 4
figure 4

Galled roots of A Control negative and B Best treatment (Trichoderma harzianum)

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Data regarding the eggplant growth parameters revealed that different fungal biocontrol agents significantly enhanced the eggplant growth condition over the check (negative) at p = 0.05% (Fig. 5). The highest shoot weight (fresh) was achieved in T. harzianum-treated plants (29.8 g), followed by carbofuran (standard) (22.7 g) and P. chlamydosporium having 18.7 g fresh shoot weight at p = 0.001% significance (Fig. 4). The highest shoot weight (dry) was recorded in T. harzianum treated plants (19.1 g), followed by carbofuran (standard) (15.3 g) and P. chlamydosporium having 11.8 g fresh shoot weight at p = 0.001% significance (Fig. 5). Maximum shoot length was recorded in case of T. harzianum-treated plants (41.3 cm), followed by P. chlamydosporium (34.8 cm). All the treatments had significantly high shoot length over check (control negative) (11.8 g fresh shoot weight at p = 0.001% significance) (Fig. 5). Likewise, all the treatment had significantly raised the root length than the control (negative) (Fig. 5).

Fig. 5
figure 5

Effects on the eggplant growth parameters after the addition of various fungal biocontrol agents inoculated with Meloidogyne incognita at the in-planta conditions

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Discussion

Root-knot nematode (RKN) is a small worm that is responsible for causing substantial damage to a variety of plant species including vegetables, cash crops, and ornamental plants. Management of RKNs is extremely disputing because of its concealed below-ground activities. Farmers opt for chemical control, but due to its percolation into the water table, its application is useless mostly. Thus, biological control research was carried out to explore the most reliable and long-lasting control method of RKN in eggplant.

The in vitro results revealed that the concentrations of 1:1, 1:10, and 1:100 of the fungal inoculum successfully inhibited RKN eggs. It was also found statistically effective against the J2s mortality. The 1:1 concentration was highly effective against egg hatch inhibition and J2s mortality, followed by 1:10 and 1:100. Uddin et al (2019) reported that nematicidal efficacy increases with the increasing concentration. The order of treatment’s efficacy was T. harzianum ˃ Carbofuran (Control positive) ˃ P. chlamydosporium ˃ A. niger ˃ P. chrysogenum ˃ Control (negative) against the RKN eggs and J2s. The current best performance of T. harzianum against the RKN eggs and J2s was also reported by Khan et al (2022a, b) who reported that T. harzianum provides significant management against the RKN in in-vitro conditions. The T. harzianum conidia gather around the egg masses of the RKN, and thus, there is a possibility of breaking the defense mechanism of geletenious-matrix and leading to the ill effects on the eggs of RKN. P. chlamydosporium effective results indicated that it produced appressoria that could have parasitized the RKN eggs in in vitro bioassay. Similar findings are also reported by Bouchagier (2018), who stated that P. chlamydosporium alone was effective against the egg inhibition of the RKN. Literature suggested that A. niger produces oxalic acid and nitric acid that parasitizes the RKN eggs and J2s. However, its less effective influence of it could be due to the less exposure time (72 h of incubation) (Jang et al. 2016).

The in-planta studies were carried out to check the influence of nematicidal activities of the fungal biocontrol agents against the M. incognita and their effects on eggplant growth parameters. The findings revealed that T. harzianum, followed by P. chlamydosproium, remained persistent by efficiently lowering the RKN growth parameters, i.e., less no. of eggs, swollen females and less GI. These findings are in line with the previous results of Balardin et al. (2021) who reported that Trichoderma efficiently controlled at the glasshouse conditions the growth parameters of RKN and inclining the growth parameters including the yield of tomato and eggplant. The standard (Carforburan) used in the present experiment results revealed that only T. harzianum was better than the standard, while slightly better results were exhibited by the standard against the P. chlamydosproium by improving the plant growth parameters and declining the RKN productivity and stability. These results are per the results El-Nagdi et al (2019), who reported that the performance of T. viride and T. harazianum was better than Carbofuran and other applied treatments. Trichoderma produces an elevated amount of protease and chitinolytic enzymes which could have been parasitized eggs of nematodes (Khan et al. 2022b). The reduction in nematode eggs could have ultimately resulted in a reduction in the final population of the nematodes in the soil. Trichoderma also possesses a special organ called appressoria through which it can press its prey and thus, creating puncture holes and that’s how hyphae enter. The mortality of egg mass could be due to the parasitization that occurred by Trichoderma on egg masses. It is also evident that Trichoderma also produces several lytic enzymes like proteases, lipases, and glucanases for the cell wall breaking (Tyskiewicz et al. 2022). P. chlamydosproium is thought to be an egg parasite. P. chlamydosporia produces branched mycelial structures (Finetti-Sialer and Manzanilla-López. 2022). According to Dallemole-Giaretta et al. (2012), its efficiency is depending on two factors, temperature and the larval stage of the embryo within the egg. Most importantly in a biological control system is the establishment of biocontrol agents in the rhizosphere, for further interaction with the plant or nematode. The nutritional values and organic fertility of the soil can shackle this establishment, and ultimately, root colonization will not occur. Egg shells of RKNs are penetrated by penetration special organ appressoria and thus disintegrate chitin of the eggs (Siddiqui et al. 2004). The chemical activity of enzymes is considered very important in causing infection. Literature suggested that infection could not be possible without the combination and presence of proteases and chitinases. This combination is considered necessary to initiate infection. Aspergillus species commonly occur in soils in warmer climates, in compost, decaying plant material, and stored grains, and many of them are known to produce a variety of secondary metabolites. Some Aspergillus species have also been reported for their biocontrol potential against root-knot nematodes (He et al. 2020).

The cultural filtrate produced in a bioreactor lowered the viability of J2s of M. incognita in a concentration-dependent manner. Oxalic acid is known to be the key metabolite of A. niger and is responsible for J2s mortality (Sikandar et al. 2020). A. niger is imputed to its parasitic nature and production of nematicidal serine proteases, which ultimately destroy eggshell and check egg hatching. The juvenile cuticle is mainly composed of proteins that could be degraded by the proteolytic activities of fungi (Hussain et al. 2017). Some fungi have spiny structures or other trapping devices that could facilitate the mechanical penetration of eggs as well as J2s. Eggs have surface binding components in a gelatinous matrix, which might facilitate fungal spore attachment, germination, and penetration (Gamalero and Glick 2020).

Conclusions

The fungal biocontrol agents colonizing the eggplant roots and the myco-metabolites may suppress the growth and production of pathogens including M. incognita and also upraise the plant growth. The present findings expressed that fungal biocontrol agents have hazardous repercussions against the egg mass, J2 and adult females of the M. incognita. It was concluded T. harzianum and P. chlamydosporium were very effective against RKN and could be utilized as a substitute for toxic commercially available nematicides.