Management of root-knot nematode infection by using fly ash and Trichoderma harzianum in Capsicum annum plants by modulating growth, yield, photosynthetic pigments, biochemical substances, and secondary metabolite profiles

A nematicide is a type of chemical pesticide used to kill plant-parasitic nematodes. Nematicides have tended to be broad-spectrum toxicants, possessing high volatility or other properties that promote migration through the soil. In addition, the nematicides used are more expensive and have adverse effects on health and the environment, so it must use more eco-friendly and less expensive alternative methods to control root-knot nematodes (Meloidogyne incognita). Chili (Capsicum annum) suffers from nematode infestation, which reduces its quality and quantity. Therefore, the goal of this research was to assess the effect of different doses of fly ash (FA) mixed soil (5%, 10%, 15 and 20% FA) with two doses of Trichoderma harzianum (1 g and 2 g) for the management of root-knot nematode infection in chili crop. The results showed that significant enhancement in plant growth, yield, chlorophyll, and carotenoid content, protein, carbohydrate, amino acid, tryptophan, indole acetic acid, phenolics, flavonoids, proline, and nitrate reductase content of chili plants was recorded at 10% fly ash with 2 g of T. harzianum (T6). The inoculated plants registered the greatest damage with galling indexes. The lowest galling index was estimated at the T6 treatment. At higher levels of FA + combined with both doses of T. harzianum, nematode could not survive that’s why eighter galls or egg masses were observed. Nematodes may have ceased to function, lost their activity, and hence been unable to resist the stress of fly ash and T. harzianum set. The application of T. harzianum with a lower dose (10%) of fly ash to control the nematode favored plant growth in general. In conclusion, 10% fly ash and 2 g of T. harzianum have the ability to operate as growth promoters and biocontrol agents for M. incognita.


Introduction
The pathogenicity of root-knot nematodes (RKNs) is known to inflict serious economic harm to farmers worldwide and cause serious crop damage (Ahmad et al., 2021a). Meloidogyne incognita, an endoparasite belongs to RKNs are microscopic creatures that dwell in soil and transmit to host plants, infecting and developing feeding mechanisms with styles (Costa et al., 2021) that harms a variety of economically significant crops (Ahmad et al., 2021b). For instance, a study on Lycopersicon esculentum plants found M. incognita caused a reduction in yield and productivity of about 27%, and also caused damage to vegetable crops that is estimated to be 50% worldwide (Kaur et al., 2011). They suck the nutrients from the host roots and cause damage to entire plant thereby reducing crop productivity (Nyaku et al., 2017). As a result, during the infection stage of M. incognita second-stage juveniles, it migrates through the root tip region and invades the plant's vascular system. However, once they've successfully colonized the vascular tissue, nematodes become immobile and spend the rest of their lives in the roots (Elhady et al., 2018).
Wilting, reduction in plant length and fresh and dry weights, enlarged roots, damage to the plasma membrane, and the production of root galls are all symptoms of nematode infection, as well as a decrease in yield (Jang et al., 2014). Moreover, it has been reported that nematode attacks can cause physiological and metabolic disturbances like water balance, mineral and solute transport, cytoplasmic leakage, and photosynthetic efficiency (Seid et al., 2015). Besides, pathogen infections trigger ROS production in plants (Mohamed and Abd-El Hameed, 2014;Sofy et al., 2021a, b). Secondary metabolites produced in plants after infection act as antioxidants and help in the plant's defences under biotic stress conditions Ashry et al., 2018). Resistance to plant pathogens is partly conferred by secondary metabolites, such as flavonoids, proline, and phenols (Aly et al., 2012;Yan et al., 2021). To control nematodes, massive amounts of nematicides are utilized, but the synthetic chemical pesticides cause pollution to the environment, so that, eco-friendly biological control has become a major research priority in recent years (Singh et al., 2020;Yan et al., 2021).
An increasing human population and increased traffic and vehicular traffic have contributed to the accumulation of gaseous and particulate air pollutants over time (El-Beltagi et al., 2020;El-Mahdy et al., 2021).
Automobiles are the largest source of air pollutants, such as oxides of nitrogen and sulphur. In addition, combustion coal residues (CCRs) such as fly ash, boiler slag, and bottom ash are produced by thermal power plants worldwide due to the use of coal for the generation of electricity (Heidrich et al., 2013). By 2030, the coal consumption used in thermal power plants will rise from 29.9% in 2011 to 46% (Yao et al., 2015). In India, the annual production of coal reaches 160 million tons (Yao et al., 2015). Electric and steam-generating plants produce fly ash, a fine residue from the combustion of pulverized coal. After dispersing the atmosphere, a great deal of fly ash is deposited on the soil and vegetation's surface. Many macro-and micronutrients in fly ash improve soil physical and chemical properties, improving agricultural crop growth and yield (Dahiya and Budania, 2018;Ahmed et al., 2021a). In many crops, the use of fly ash has resulted in an increase of up to 25% in leaves number, plant height, biomass, and yield, thereby serving as a fertilizer and source of plant nutrients for various crops (Ahmed et al., 2021a). Application with sufficient amount of fly ash to soil could increase the availability of B, K, and other nutrients in soil (Ahmed et al., 2021a), and ameliorate the physical and chemical properties of soil, such as improving soil structure, soil water content, and water holding capacity (Ahmed et al., 2021a). Niu et al. (2021) found that composed of fly ash (FA) and polyacrylamide (PAM)and the interaction of FA and PAM all had significant impacts on the percentage of seedling emergence of Artemisia ordosica and total fresh weight.
Trichoderma and other soil-borne fungi are well-known for their capacity to control a wide range of plant diseases (Abd El-Rahman and . Because of the effects of Trichoderma spp. colonization on plant physiology and metabolism, a wide range of defences and growth-promoting metabolites like growth regulators, organic acids, and siderophores are synthesized by the plant (Zhang et al., 2015). Furthermore, by 3 creating enzymes and secondary metabolites (Bhattacharjee and Dey, 2014), Trichoderma species can operate as biological control agents, particularly against Meloidogyne nematodes (Sokhandani et al., 2016) through preventing egg hatching and immobilizing nematode second-stage juveniles (J2) (Feyisa et al., 2015). When it comes to managing root phytopathogen populations, however, various aspects must be considered, including the fungus's origin, compatibility with host plants, and the edaphic characteristics of its site (Al-Hazmi and Javeed, 2016).
Pepper is a Solanaceae plant that is among the major crops in the Mediterranean region, and the lack of water and increased salt stress are major constraints to productivity (Penella et al., 2013(Penella et al., , 2014. Ascorbic acid, carotenoids, and phenolic compounds are among the bioactive antioxidants and minerals found in them (Akladious and Mohamed, 2018). Root-knot nematodes can cause significant damage to this crop if they attack it (Moosavi, 2015). The initial population density of Nematoda at planting affects plant yield reduction (Moosavi, 2015). Plant cultivars and/or nematode strains influence the extent of a nematode's damage to a plant (Kihika et al., 2017). The current research was carried out to determine the potential of a combination of Trichoderma and fly ash to control M. incognita infections on chili plants. The potential for Trichoderma and fly ash to act as biocontrol agents for RKNs was assessed by studying the effect of combination between Trichoderma and fly ash on growth, yield, photosynthetic pigments, nitrate reductase, secondary metabolites, osmolytes and biocontrol against nematodes.

Materials and Methods
Experimental site and plant material The experimental material consists of one cultivars of C. annum (variety: NS 1101) provided by Agricultural University Ludhiana, Punjab, and is highly recommended for the agro-climatic zone of central India. The experimental material was planted in augmented RBD with 5 replications for each treatment at inter and intra row spacing was 45 and 15 cm, respectively. The pots established in the green house of the Botany Department of Aligarh Muslim University, Aligarh, India.

Culturing of nematodes
By collecting infected samples from several locations, the nematode cultures were kept alive in a greenhouse. Meloidogyne incognita was isolated, identified, and kept for research purposes. The egg masses were separated from their roots and placed in distilled water to preserve them. The second-stage juvenile nematodes were collected in double-distilled water after being incubated at 27 °C to stimulate hatching. At the time of inoculation, the nematode's second-stage juveniles were 24 hours old. These were counted and used in experiments using a light microscope ( Figure 1A).
Preparation of Trichoderma culture and inoculum T. harzianum GQ426038 was prepared, maintained in a potato dextrose broth (PDB) medium, and was used for mass culture. A Thoma hemocytometer was used to measure conidia concentrations, and the final concentration was adjusted to 4.0 ×108 conidia mL -1 sterile distilled water for soil inoculation.
T. harzianum was injected into flasks containing PDB medium using a sterile inoculation needle. The inoculated flasks were incubated for about 15 days at 25 °C. The medium was filtered with filter paper when the fungus had grown sufficiently. On the filter paper, a fungal mycelia mat was obtained, which was collected ( Figure 1B). The mycelia mat was blotted with blotting sheets to remove nutrients and excess water. In a flask, 100 g of fungal mycelia was blended in 1000 mL of double distilled water (DDW) in a flask so that 10 ml of suspension contained 1g of mycelia. For inoculation of fungus, different concentrations of suspension were 4 standardized, which contained 1 g fungus 10 mL -1 of dry weight (DW) and 2 g fungus 20 mL -1 of DW that were inoculated in the pots 8-10 days prior to inoculation of J2s of M. incognita for colonizing the rhizosphere to prevent root-knot nematode infection.  Table 1. The soil was sandy loam containing 66% sand, 24% silt, 8% clay, 2% organic matter, and pH7.7. Before utilization, the soil was autoclaved.

Physico-chemical properties of soil
The soil texture was determined using the soil texture triangle method (Chopra and Kanwar, 1991). Soil Testing Kit Model 161E was used to calculate the electrical conductivity (EC) and cation exchange capacity (CEC) of the soil and fly ash (Jackson 1973). The water-holding capacity of the soil samples was calculated using the methodology given by Black et al. (1965). The Carter and Ball (1993) method was used to calculate porosity (St), which is the percentage of soil volume filled by pore spaces. Tensiometers were used to determine the moisture content (Nunes Instruments). One of the most essential aspects of soil that makes up a soil sample is particle density. It was accomplished using the approach previously stated by Blake (2008).
According to Jackson (1973), the soil pH was evaluated using a pH meter (PCE Instruments). Dickman and Bray (1940) method was used to estimate phosphorus (P) from sample extract using calorimeter after the addition of HClO4. A flame photometer was used to estimate potassium (K) in soil and fly ash samples. The nitrogen content was determined using the Kjeldahl (1883) method. Chopra and Kanvar (1982) method were used to determine magnesium. The carbonates and bicarbonates in samples were determined using Richard (1954) technique. Jackson (1973) method was used to determine the chloride and sulphate content in samples. Finally, an atomic absorption spectrophotometer was used to determine Zn and Mn in soil. Determination of elemental status Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX) was used to determine elements.

Autoclaving of soil and preparation of pots
The soil was sealed in gunny bags and steam sterilized for 20 minutes at 20Ib pressure in an autoclave. To achieve a varied ratio, the autoclaved soil was dried and mixed with fly ash. The test plant was chosen to be the chili. In 10-inch clay pots, the certified seeds (variety: NS 1101) were planted. Seedlings were transplanted to different pots for several studies once the four-leaf stage emerged.

Treatments
The earthen pots of 12 inches height were filled with 3 kg (3000 g) of each type of soil mixture. The treatments were 5% FA (2850 g soil + 150 g fly ash); 10% FA (2700 g soil + 3000 g fly ash); 15% FA (2550 g soil + 450 g fly ash); 20% FA (2400 g soil + 600 g fly ash). Each ration of soil and fly ash were mixed with 1 and 2 g of T. harzianum culture mat. The following treatment sets were taken in this experiment. All plants were harvested after 3 months on 15 February 2020, and during the experiment, plants were examined regularly for symptoms, photographs of growth, and yield. In addition, the following parameters were measured after termination. The length, fresh and dry weights (wt.) were measured. The number of branches and leaves were counted on each plant. Five average-sized leaves from each treatment were traced on tracing paper to estimate leaf area. The areas occupied by these drawings were measured with the help of a planimeter. Also, the number of flowers and fruits per plant were counted. Five average-sized fruits were collected from each treated plant, and their lengths, fresh and dry weights were measured.

Determination of biochemical substances of chili plant
The biochemical substances of the vegetative part of chili plant were determined in terms of their chlorophyll content, 0.5 g sample of fresh chili leaf was homogenized with 80% acetone. Each sample was homogenized and pestle in a 10 mL solution of acetone (80%), and then, the sample mixture was subjected to centrifugation at 12.000 rpm for 20 min. After centrifugation, the supernatants were collected for the estimation of chlorophyll and carotenoid contents by measuring the absorbance at 470, 649, and 665 nm on a UV-Vis spectrophotometer (UV-3200). Then, chlorophyll a, b (Bruinsma, 1963) and carotenoids were calculated by the formulae given by Lichtenthaler and Wellburn (1983). Slyke et al. (1941), used to determine total free amino acids content in chili leaves that were pulverized in a mortar and pestle in 5 ml of 80% ethanol (v/v). The mixture was heated for 10 min and centrifuged at 2000 rpm for 10 min, and the supernatant was collected and using ninhydrin reagent to measure amin acids.
Protein content was determined using Folin-Ciocâlteu reagent according to Lowery et al. (1951) technique, the optical density read at 750 nm and bovine serum as a standard.
The quantity of carbohydrate in the chili leaf was calculated by the anthrone reagent method (Dubols et al., 1956). Two hundred milligrams of dried chili leave were homogenized in 96% v/v ethanol. For 10 min, the samples had been centrifuged at 3500 ×g. Three millilitres (150 mg anthrone + 10 mL (v) sulfuric acid) from a freshly prepared anthrone reagent was reacted with 0.5 mL of the supernatant for 10 min by heating in a bath of water. The cooling process was completed, and the mixture was absorbed using a spectrophotometer at 652 nm. Indole acetic acid was determined according to Beffa et al. (1990) method.
The method of Sadasivam and Manickam (1996) was used for the extraction as well as for the quantitative of amino acid tryptophan. 500 mg of chili sample was homogenized with 10 ml of 80% ethanol and centrifuged at 10,000 g for 20 minutes. The supernatant was preserved, and the extraction was repeated twice (10 ml+10 ml) with the residue. The pooled supernatants were collected and evaporate it to dryness on a boiling water bath and dissolve the residue in 5 ml of 0.2M citrate buffer (pH 5.0). Pipette 2 ml of the above sample preparation in a test tube. For a reagent blank, take 2.0 ml of 0.2M citrate buffer (pH 5.0) in place of the sample preparation. In another set of tubes, take graded concentrations of tryptophan (0-100 µg) and make the total volume to 2 ml with 0.2M citrate buffer (pH 5.0). Add 1ml of KCN-acetone Ninhydrin reagent and mix thoroughly. Keep the test tubes in boiling water bath for 20 min, cool under running tap water and make the volume to 10 ml with distilled water and the intensity of the purple colour developed was read using spectrophotometer (Spectronic-20, Japan) at 570 nm, with reagent blank.
Two grammes of chili leaves were extracted with 20 mL methanol (80%) and filtered through filter paper No. 1 by shaking at 150 rpm for 12 h. The methanol extract filtrate will be used to determine total phenols. 50 μL of the methanol extract was mixed with 100 μL Folin-Ciocalteu reagent, 850 μL of methanol and allowed to stand for 5 min at ambient temperature. A 500 μL of 20% sodium carbonate was added and allowed to react for 30 min. Absorbance was measured at 750 nm according to Ruanma et al. (2010). In addition, 0.5 mL solution of each plant extracts in methanol were separately mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water, and left at room temperature for 30 minutes. The absorbance of the reaction mixture was measured at 415 nm with a 7 double beam Perkin Elmer UV/Visible spectrophotometer (USA) to determine total flavonoids according to Van acker et al. (1996). Sadasivam and Manickam (1971) described the ninhydrin technique for estimating proline content. 3% sulphosalicylic acid was used to homogenize 0.3 g of fresh leaves. In a test tube held in a water bath at 100 °C for 1 hour, the filtrate was acted in response to 1 mL each of glacial acetic acid and acid ninhydrin. The reaction was stopped by putting the test tube in the freezer. The optical density of the mixture was measured at 520 nm using a spectrophotometer. The activity of nitrate reductase (NRA) was assessed using Jaworski, (1971) technique. 1.0 g of fresh leaves were milled into a powder in liquid N2 and stored at a temperature of 80 °C. The powder was thawed in a 250 mM Tris-HCl buffer for 10 minutes at 4 °C before homogenization. The homogenate was centrifuged for 30 minutes at 10,000 ×g at 4 °C. NR activity was analysed by adopting the method of Nakagawa et al. (1984).

Galls, egg masses, and gall index
The roots were carefully cleaned with water and immersed in phlox in B (0.15 g L -1 tap water) solution for 15 min to stain the egg masses. Then, the number of galls and egg masses of each root were counted. For the gall index, the scale of Taylor and Sasser (1978) was followed.

Statistical analysis
The values were entered into a one-way analysis of variance (ANOVA) using R (x64 2.14.1). Sigma Plot was used to create the graphs (11.0). Duncan's multiple range test (DMRT) at 0.05% was used to detect variations between treatments and compare them to mean values. The average mean of five replicates of the data was used to calculate each value. The experiment was carried out in a completely random manner (CRD).

Results
Colour and shape of fly ash particle Figure 2 A-C shows images of fly ash particles taken with SEM. Some of the particles appeared solid, while others appeared hollow spheres made up of several smaller particles. The colour of the fly ash that we studied in this research can be dark grey or grey, and even black.

Element's status of fly ash
Fly ash is classified according to its elemental composition by (SEM-EDX) in Figure 2D. Elements as SiO2 and Al2O3 are found in the fly ash, along with trace amounts of oxides of Al, Fe, and traces of Mg, P. According to the American Society for Testing and Materials, the fly ash used in this study is classified as class F.
Interactive effect of different concentrations of fly ash and T. harzianum on growth and yield Figure 3, and Figure 4A  and (23.08%), (19.27%) and (20.43%), (16.55%) and (19.69%) respectively as compared to both control that is without nematode and fly ash (only soil) as well as nematode inoculated plant (nematode and soil). Above 10% level of fly ash and both levels of T. harzianum combination (T7, T8, T9, and T10), the chl a, b, total chl, and carotenoid contents were also greater than both control but less than the T6 treatment ( Figure 5). Each value is a mean (±SD) of five replicates. Different letters on the same bars show significant differences according to Duncan's multiple range test at p < 0.05 using R-software Interactive effect of different concentrations of fly ash and T. harzianum on amino acids, protein, and carbohydrates The data in Figure 6A shows that inoculation with 2000J2 of M. incognita caused a significant decrease in amino acids (13.4%), protein (14.3%), and carbohydrates (25.6content in chili plants as compared to uninfected plants. In comparison to both control (T1) and inoculated plants, amino acid (21.23%, 13.45%), protein (16.23%, 18.19%), and carbohydrate content (17.34%, 20.39%) were found to be significantly higher in plants grown in T6 combination. At higher levels of fly ash, T. harzianum, and M. incognita combinations (T7, T8, T9, and T10), the amino acid, protein, and carbohydrate content were also more significant than the control but less than the contents of the T6 combination.
Interactive  The phenolic and flavonoids content of chili plants grown in soil inoculated with 2000J2 of M. incognita was significantly inhibited as compared to control plants (Figure 7). Furthermore, when compared to T1 and T2, the concentration of phenolic and flavonoids was considerably (P ≤ 0.05) higher in plants grown in the T10 combination by about (16.89%, 18.67%) and (21.90%, 32.22%) respectively. On the other hand, treatment with T4, and T5 had a non-significant effect on total phenolic and flavonoid contents in chili plants compared to control plants, and T3 and T4 show a non-significant effect on flavonoid content compared with inoculated plants. In addition, T6 caused a significant increase in phenolic content (10.7, 24%) and flavonoids content (18.8, 33.3%) as compared to T1 and T2 respectively.  Figure 8 A-B. The proline and NRA content were significantly higher in plants grown in T10 by about (20.60%, 33.16) and (14.07%, 16.16) as compared to both controls, T1 and T2. On the other hand, T5 shows a non-significant effect on chili plants proline and NRA content compared to control plants.
Interactive effect of different concentrations of fly ash and T. harzianum on root-knot disease The level of 10% FA + in combination with 2g T. harzianum was significantly inhibited the hatching ability of M. incognita. At this level, only some galls appeared, but none of the egg mass and eggs was observed. At higher levels of FA + both doses of T. harzianum, nematode could not survive. That's why neither galls nor egg mass was observed. As a result, nematodes may have ceased to function and so been unable to tolerate the stress of fly ash. Thus, soil application of fly ash at 10% level + 2.0 g T. harzianum is useful for chili crops, and it will also manage the root-knot disease on the host plant (Table 2). Ahmad G et al. (2022). Not Bot Horti Agrobo 50 (1)

Discussion
In the present investigation, chili plant growth and production increased significantly at the lower levels of fly ash (T6 = 10% FA + 2 g T. harzianum + 2000J2 of M. incognita). There have been similar beneficial effects on various crops such as pumpkins and carrots (Ahmad et al., 2017;Haris et al., 2019). Due to its high K, Mg, and S content, fly ash is a valuable source of essential plant nutrients and is used as fertilizer to boost crop growth (Ahmed et al., 2021a). Fly ash alone had higher physical properties like water-holding capacity, moisture content, CEC, EC, and porosity than soil (Table 1). Inorganic components in fly ash and increased soluble macro-and micronutrients and metals released with the ash to the soil may be responsible for the increase in EC (Ahmed et al., 2021a). Because it is rich in salts and trace elements, fly ash affects soil properties, increasing its water-holding capacity (Ahmed et al., 2021a). Fly ash boosts the soil's ability to retain water because it alters its texture and structure. Soil can retain water depending on the surface area, pore space, and pore space continuity (Panda and Biswal, 2018). As well as phosphorus and potassium, the amounts of Mg, Mn, and Zn in the soil were also steadily boosted when fly ash was added to the soil. Because fly ash contains enough of these elements (Ahmed et al., 2021b). Besides enriching nutrients, it also improves soil pH and makes it more conducive to plant growth (Haris et al., 2019).
Trichoderma species can generate secondary metabolites such as indole acetic acid, a hormone that induces growth in meristematic tissues and improves the efficiency of cysteine, which alters root architecture and improves root growth (Samolski et al., 2012). Plant metabolism depends on the production of organic acids, such as gluconic acid and fumaric acid, which decrease the pH of soil solution and enable the dissolution and solubilization of phosphates and micronutrients and minerals cations such as Fe, Mn, and Mg (Hermosa et al., 2013). Trichoderma strain effectively suppressed root-knot nematodes (RKNs) in tomatoes (Yan et al., 2021). Fly ash contains elements such as Ca, Fe, Mg, and K, which are essential for plant growth, and other minerals such as B, Se, and Mo, which are toxic to plants. Yu et al. (2019) showed that plant biomass boosted about 11.6-29% when fly ash was applied at low concentrations (< 25% of soil mass) but decreased by about 44.8% at high concentrations (50-100%) due to heavy metal toxicity (Mohamed, 2011;El-Beltagi et al., 2020;Moustafa-Farag et al., 2020). Soil fungi Trichoderma spp. form symbioses with many plants and are commonly found in the natural environment (Druzhinina et al., 2011). Plant development, yield, nutrient cycling, and energy conversion in soil are all dependent on the relationship between the plant rhizosphere and soil microbes (De et al., 2015). Plant root exudates promote the colonization of rhizosphere microorganisms, while soil microorganisms improve plant growth and increase soil nutrient levels by utilizing plant photosynthates to the fullest extent possible (De et al., 2015). Trichoderma applied led to a dramatic boost in the soil's N and P  15 content due to the ability of Trichoderma to degrade soil macromolecular nutrients into a form that plants can use, thereby stimulates soil nutrient cycling and energy flow (Halifu et al., 2019). Adding T. harzianum to fly ash was the main cause of the nitrogen overabundance in the soil because fly ash itself does not contain nitrogen (Ahmad et al., 2021a). For plants to absorb light energy, they need chlorophyll (Katz et al., 1978). Neals (1956) found that plants need minerals to produce chlorophyll molecules. Nematodes infected chili plants reduced the photosynthetic pigments content. The findings of our study are validated by Khanna et al. (2019) who reported that the total chlorophyll content in Lycopersicon esculentum was reduced after infection with M. incognita.
The reduction of photosynthetic pigments may be due to a decrease in enzymatic activities involved in the violaxanthin cycle, which disrupted the photosynthetic apparatus's stabilization, as well as the development of galls in the plant roots, which hindered the water balance of the entire plant due to gall formation in the roots, which effected on the chlorophyll synthesis (Khanna et al., 2019). Treatment with 10% FA + 2 g T. harzianum + 2000J2 of M. incognita caused a significant increase in photosynthetic pigments as compared to control and inoculated plants. Fly ash is a good source of macro and microelements like sulfate, magnesium, and zinc, and its application to the soil increases the absorption of these metals by plants and thereby increases the photosynthetic pigments Ahmed et al., 2021a).
As a consequence of a higher amount of chlorophyll, more carbohydrates and other chemicals are synthesized, which improves fruit output (Paradikovic et al., 2011). Likewise, Abd El-Rahman and  found that using T. harzianum to control faba bean chocolate spot disease caused by Botrytis fabae caused enhancement in both chlorophyll production and photosynthetic activity by boosting water absorption, which eventually resulted in an enhanced of the photosynthetic pigments. Trichoderma and fly ash may up-regulate enzymes related to photosynthetic pigments in plants during nematode attack, resulting in increased photosynthetic activity. In addition, Trichoderma increases soil organic matter and essential minerals, such as nitrogen, potassium, phosphorus, and magnesium, which stimulates the development of photosynthetic pigments in plants (Sofy et al., 2021b).
The present study showed that levels of osmoprotectants such as amino acids (13.4%), protein (14.3%), carbohydrates (25.6%), and proline (23.5%) were significantly decreased in chili plants under nematode treatment. On the other hand, treatment with fly ash, T. harzianum, and 2000J2 of M. incognita caused a significant increase in amino acids, protein, carbohydrates, and proline compared to plants applied with the nematode. Those findings are similar to those in treating nematode-infected tomato plants with T. harzianum and Serratia marcescens induced systemic resistance in the plants. In addition, the polyphenol oxidase and -1,3glucanase activity triggered the production of sugar and amino acids, which increased the expression of defence genes in the plants (Abd-Elgawad and Kabeil, 2012). The current study also observed a rise in the secondary metabolite's contents, which can be related to an increase in the protein synthesis of enzymes that participate in secondary metabolism. As a result, Trichoderma-induced creation of defence metabolites and antioxidant compounds may provide physical and chemical protection against RKNs (Ji et al., 2019).
Furthermore, de novo synthesis decreased utilization and decreased degradation hydrolysis of proteins could also contribute to proline accumulation. In addition to acting as an outstanding osmolyte, proline also acts as a metal chelator and an antioxidant molecule under stressful conditions, including stabilization of subcellular structures, membranes, and proteins (Sofy et al., 2021a). As reported by several researchers, plants exposed to biotic and abiotic conditions may develop stress tolerance by maintaining osmotic stability or cell viability and protecting cell functions by neutralizing ROS, which would reduce the risk of the oxidative burst in plants (Sofy et al., 2021 b).
Organic nitrogen compounds, amino acids have traditionally been regarded as precursors and components of proteins (Azher et al., 2011;El-Beltagi et al., 2019). As a growth regulator and a cell differentiation modulator, it may impact general metabolism and morphogenesis (Basu et al., 1989;Mohamed et al., 2016). The amino acids also play an important role in developing multiple shoots (Vasanth et al., 2006).
In every aspect of plant growth and development, proteins play a key role. These processes include chemical reactions, membrane transport, intracellular structure, and energy-generating reactions requiring electron transport. Low protein levels in plants result from structural carbohydrates that make up the majority of plant structure (Vierstra, 1993). In-plant growth, development, and stress response, carbohydrates play a critical role. A wide range of factors influences species, organs, growth conditions. However, carbohydrates are indicative of the relationship between photosynthesis and growth. For many crops, biomass and yield are also heavily dependent on photosynthetic carbon fixation and carbohydrate metabolism (Azher et al., 2011).
Chili plants tryptophan and indole acetic acid (IAA) contents significantly increased after treatment with T. harzianum with fly ash. A precursor to IAA, tryptophan is one of the most studied amino acids. As a result of higher levels of tryptophan, the synthesis of IAA (auxin) increases, resulting in increased plant growth (Wurtman and Anton-Tay, 1969;Ghonaim et al., 2021). As a precursor to auxin and cytokinin, tryptophan plays an essential role in the plant metabolic process (Law, 1987). Therefore, higher levels of tryptophan in plants may aid in phytohormone synthesis, resulting in increased plant growth. Trichoderma strains generated considerable amounts of IAA and boosted the fresh weight of Arabidopsis seedling shoots (Nieto-Jacobo et al.,

2017).
Treatment with fly ash, T. harzianum, and 2000J2 of M. incognita boost phenolic and flavonoids content in chili plants. As fly ash contains various toxic elements, it becomes detrimental for root-knot nematodes to survive thus results in their death. The substantial decrease in the egg masses and galling shows that the fly ash has caused a direct inhibitory response on the survival and multiplication of M. incognita (Ahmad and Khan, 2016). Similarly, Ahmad and Khan (2016) observed that the soil amendment with different levels of fly ash inhibits the penetration of the M. incognita juveniles in the roots of pumpkin. Rootknot disease in terms of root gall index, no. of egg masses and eggs/egg mass was highest in untreated inoculated control. In fly ash + nematode combinations, galls were formed but less than inoculated control which gradually decreased to 10%, 20% and 30% levels of fly ash. However, galls were completely absent at 50% level of fly ash (Haris et al., 2019). The phenolic compounds have a positive effect on nematode infection (Patel et al., 2017) through impacts on gall production, egg hatching, and second-stage nematode proliferation (Patel et al., 2017).
Flavonoids have also been shown to diminish the nematode's negative effects by slowing worm mobility, reducing nematode activity, limiting nematode migration, and eventually killing nematodes. Using T. harzianum in soil improved growth and yields and significantly reduced M. incognita galling, egg production, and soil population when applied to the soil before planting. Trichoderma can inhibit egg hatching and create host resistance in plants (Khan, 2016). Trichoderma produced chemical metabolites that can also inhibit nematode feeding, penetration and increased crop yield (Khan et al., 2020). In addition, Yan et al. (2021) found that tomato plants treated with T. harzianum have significantly reduced root-knot disease and boosted plant growth.
Egg formation was reduced by 60% in C. annuum after treatment with T. virens (Meyer et al., 2001), and egg production was repressed by 86% and 84% in S. lycopersicum treated with T. brevicompactum and T. longibrachiatum, respectively (Zhang et al., 2015). Trichoderma biocontrol mechanisms against nematodes may be based on their ability to produce lysis enzymes that affect the cuticle and the viability of nematode eggs (Zhang et al., 2015). The importance that the nematode did not reach the mature stage and, as a result, no crop loss is critical from an agricultural standpoint. Recently, Haris et al. (2019) also observed that all the levels of fly ash (10-50%) significantly reduced M. incognita on carrots and hatching, mortality, and penetration on pumpkin (Ahmad and Khan 2016). It was found that the maximum inhibition occurred at 50% fly ash content.
Juvenile M. incognita larvae were significantly suppressed in the pumpkin roots in all fly ash and soil mixtures. Trichoderma increase the level of extracellular enzymes like chitinase and protease, which allow the penetration of the fungus into the eggs by directly affecting very abundant structural components of the eggshell, thus reducing the number of eggs capable of hatching and therefore, the number of infective J2. Specifically, T. longibrachiatum has a strong inhibitory effect on the hatching of cysts produced by Heterodera avenae, since the spores completely cover the surface of these structures, causing their destruction, which is probably due to the production of enzymes (e.g., chitinases) that caused physiological alterations to the cysts (Zhang et al., 2014). T. longibrachiatum also showed an effect on females and on the development of both eggs and J2s of H. avenae (Zhang et al., 2017), hence T. longibrachiatum can be used as a BCA for the management of H. avenae in selected crop species. Within the cyst-forming nematodes, the specie Globodera pallida has also a high agronomic impact. Figure 9. represents the possible mechanisms of the effects of Trichoderma and fly ash on chili plants. increased plant photosynthetic pigments, phenolic compounds, and osmolyte levels ( Figure 9). After being exposed to nematode infection and then treated with Trichoderma or fly ash, plants accumulate metabolites, showing that this is an effective strategy to combat the nematode infection. Therefore, 2 g of Trichoderma and 10% of fly ash play an important role in nematode-stressed chili plant stress reduction. Using Trichoderma and fly ash is a cost-effective and promising way to improve the growth and productivity of plants infected with nematode pests. But more research is needed to understand the mechanisms behind Trichoderma and fly ash nematicidal activity. All authors read and approved the final manuscript.
Ethical approval (for researches involving animals or humans) Not applicable.