Trichoderma asperellum affects Meloidogyne incognita infestation and development in Celosia argentea

Due to the rise in cost and detrimental health and environmental consequences that accompany the use of nematicides, there is a need for a more eco-friendly and less expensive alternative to control root-knot nematode (Meloidogyne incognita). Nematode infestation reduces the quality and quantity of Celosia argentea Linn. A pot experiment was conducted in a greenhouse to determine the biocontrol efficacy of Trichoderma asperellum against M. incognita in C. argentea. The treatments consisted M. incognita infected C. argentea inoculated with 0, 2.2 × 10, 4.4 × 10, or 6.6 × 10 cfu/pot of T. asperellum. All doses of T. asperellum reduced the root-knot nematode population and root gall index. Growth and development of C. argentea were improved, indicating that T. asperellum has the potential to be used as a biocontrol agent in C. argentea production. The biocontrol activity of T. asperellum in C. argantea increased as the week went by until the plants attained full maturity. Hence, the control of M. incognita by T. asperellum depends on the developmental stage of the plant root system.


Introduction
Celosia argentea Linn. (family Amaranthaceae) is an annual, herbaceous, vegetable whose leaves, tender stems, and young flower spikes are eaten cooked (Daramola et al. 2015). The dried, ripe seed is reported to have medicinal properties because of its β-carotene, vitamin E, folic acid, ascorbic acid, phosphorus, calcium, iron, and protein contents (Tang et al. 2016). The leaves contain amaranthine (betacyanin), oxalic acid, and phytic acid (Tang et al. 2016). The production of C. argentea is challenged by the sedentary endoparasite root-knot nematode (Meloidogyne incognita; Daramola et al. 2015;Fabiyi et al. 2016), which causes 60-80% yield loss of C. argentea (Tang et al. 2016) due in part to stunted growth, rotted, and galled roots (Anwar et al. 2009). Current management of M. incognita in C. argentea production is with synthetic nematicides. Human health risk and environmental consequences accompany the use of synthetic nematicides (Fabiyi et al. 2016; Alori and Babalola 2018). Synthetic nematicides are also very expensive, thereby increasing the cost of C. argentea production. Hence, the need for a more eco-friendly and less expensive alternative that will pose no health risk.
Microbial inoculants are an effective, environmentally safe, alternative method of controlling plant diseases affecting vegetables (Radwan et al. 2012).
Microbial inoculants can improve plant growth and yield and can act as biocontrol agents against many crop pathogens (Zakaria et al. 2013; Alori and Babalola 2018). Whether they are efficacious in the production of C. argentea needs ascertaining. The study therefore evaluated the efficacy of Trichoderma asperellum as a biocontrol agent against M. incognita on C. argentea.

Pot experiment
Twelve plastic pots, 30 cm diameter, filled with 10 kg of sterilized soil were arranged in a completely randomized design in a screenhouse of the Landmark University Teaching and Research Farm. Four pots containing sterilized soil were inoculated with 0, 2.2 × 10 7 , 4.4 × 10 7 , or 6.6 × 10 7 cfu/pot of T. asperellum, replicated 3 times to make 12 pots. Two days after inoculation of the fungus, seeds of C. argentea were planted in pots, and the plants were thinned to two per pot after emergence. The plants were inoculated with approximately 1,000 second-stage juveniles (J2) of M. incognita/pot 1 week after emergence, which were added in 3 cm-deep holes around the plant base using the method of Iheukwumere et al. (1995). The experiment was comprised of the following treatments: 1,000 J2 of M. incognita only, 2.2 × 10 7 cfu/pot of T. asperellum + 1,000 J2 of M. incognita, 4.4 × 10 7 cfu/pot of T. asperellum + 1,000 J2 of M. incognita, or 6.6 × 10 7 cfu/ pot of T. asperellum + 1,000 J2 of M. incognita, each replicated three times. Average temperature of the screenhouse during the experimental period was 25°C. Plants were supplied with tap water to field capacity at 6 am and 6 pm daily with a watering can. Evaluations of responses began 3 weeks after planting (WAP). Data were taken weekly on plant height, number of leaves, and stem diameter. Root gall indices and egg counts were recorded after harvest. The scale used for rating roots for galling, or the root gall index, was based on Taylor and Sasser (1978), where 0 = no infestation, 1 = 1-5% of the root galled, 2 = 6-25% of the root galled, 3 = 26-50% of the root galled, 4 = 51-75% of the root galled, and 5 = 76-100% of the root galled.

Analysis of soil chemical characteristics
Soil chemical properties were determined before and after the experiment, such as soil pH with an electronic soil pH meter (Model 215; Denver Instrument, Colorado, USA), soil particle size analysis by the hydrometer method (Gee and Or 2002), organic matter content was determined using the wet oxidation method (Shamshuddin et al. 1994), and exchangeable bases (K, Mg, Na, and Ca) were determined by ammonium acetate method (Chapman 1965). To determine exchangeable acidity, 5 g of air-dried soil (sieved through 2 mm sieve) was weighed into a 250 mL conical flask. Fifty milliliters of 1 N potassium chloride (KCl) solution was added to the soil sample in the conical flask. The flask was shaken on a reciprocating shaker for 1 h, and the content was filtered through Whatman No. 42 filter paper. Twenty-five milliliters of the filtrate was pipetted into a 100 mL conical flask and 50 mL distilled water was added along with 5 drops of phenolphthalein indicator. The resulting solution was titrated with 0.01 N sodium hydroxide (NaOH) to a permanent pink end point. The volume of the base used was recorded to calculate the total exchangeable acidity (H + Al); the effective cation exchange capacity was determined by the summation of the exchangeable bases (Ca, Mg, Na, and K) and exchangeable acidity expressed in cmol/kg; the total soil nitrogen was determined by Macrokjedahl method (Bremner 1965); and the Bray1 method was used to determine the available phosphorus (Murphy and Riley 1962).

Data analysis
All data from the experiment were subjected to analysis of variance (ANOVA) using IBM SPSS statistical package (2012). Means were separated using Duncan's multiple range test (P ≤ 0.05).

Results
3.1 Effects of T. asperellum on the population of nematode on gall index in C. argentea infected with M. incognita Table 1 shows the population of M. incognita in infected C. argentea inoculated with T. asperellum. M. incognita was found to be the highest in plants treated with 1,000 second-stage juveniles (J2) of M. incognita only and lowest in plants that received 1,000 second-stage juveniles (J2) of M. incognita + 6.6 × 10 7 cfu/pot of T. asperellum. The C. argentea that received 1,000 second-stage juveniles (J2) of M. incognita only had the highest mean for gall index at 3.3 and this mean was significantly different (P ≤ 0.05) from those plants treated with T. asperellum suspension except for those that received 1,000 second-stage juveniles (J2) of M. incognita + 2.2 × 10 7 of T. asperellum suspension.
3.2 Effect of T. asperellum on plant height of C. argentea infected with M. incognita Table 2 reports the impact of T. asperellum on the height of C. argentea infected with M. incognita. It was observed that at 5 and 6 WAP, M. incognita-infected C. argentea inoculated with T. asperellum at the rate of 6.6 × 10 7 cfu/mL had significantly higher height than the untreated control. However, at 7 and 8 WAP, the application of T. asperellum at the rate of 2.2 × 10 7 , 4.4 × 10 7 , and 6.6 × 10 7 cfu/mL resulted in significantly higher height of C. argentea when compared to the control that was not treated with T. asperellum.

Effect of T. asperellum on number of leaves of C. argentea infected with M. incognita
The effect of T. asperellum on the number of leaves of C. argentea infected with M. incognita is presented in Table 3. At weeks 5 and 6, the application of T. asperellum at 6.6 × 10 7 cfu/mL significantly increases the number of leaves compared with the uninoculated C. argentea in the presence of M. incognita. However, at weeks 7 and 8, the application of T. asperellum at 2.2 × 10 7 , 4.4 × 10 7 , and 6.6 × 10 7 increased the number of leaves of C. argentea infected with M. incognita. Table 4 reports the impact of the interactive effects of T. asperellum and WAP on the growth parameters of C. argentea infected with M. incognita. The treatment (inoculants, WAP) effects on measured plant growth parameters were significant (P ≤ 0.05). It was observed that inoculation of M. incognita-infected C. argentea with T. asperellum significantly increased the plant height and the number of leaves. Likewise, the interactive effect (inoculant × weeks) on measured growth parameters was significant. This implies that the interaction between T. asperellum with the C. argantea seedling root increases as the week goes by.

Effects of T. asperellum on chemical composition of the soil used
The result in Table 5 shows that the application of microbial inoculants caused a significant change in the mineral composition of the soil after harvesting. The pH of the soil where plants were treated with M. incognita only increased by 13%, while the soil where plants were treated with M. incognita + 2.2 × 10 7 cfu/pot of T. asperellum, M. incognita + 4.4 × 10 7 cfu/pot of T. asperellum, and M. incognita + 6.6 × 10 7 cfu/pot of T. asperellum caused up  to 7% reduction in pH. There was an increase in all the mineral contents of the soil of plants treated with M. incognita + 2.2 × 10 7 cfu/pot of T. asperellum, M. incognita + 4.4 × 10 7 cfu/pot of T. asperellum, and M. incognita + 6.6 × 10 7 cfu/pot of T. asperellum when compared with the composition at the start of the experiment ( Table 5).

Interactive effects of T. asperellum and WAP on growth parameters of C. argentea infected with M. incognita
Increased plant height and number of leaves could be attributed to the reduction in nematode population and galling index ( Table 1). The improved growth recorded in this research could also be attributed to increased nutrient status of the soil because of the application of T. asperellum as seen in Table 5. Alori and Babalola (2018) reported that microbial inoculants effectively control plant pathogens by either direct or indirect mechanism. They improved plant growth by the production of plant growth hormones. However, T. asperellum spp. increased plant growth due to increased nutrient uptake (Harman 2006). The interaction between T. asperellum and WAP depends on the developmental stage of the plant root system, which improves with the age of the plant.

Effects of T. asperellum on the chemical composition of the soil used
A reduction in pH recorded in pots treated with T. asperellum probably accounts for the reduction in nematode population and root gall index. Also, increase in all the mineral contents of the soil when compared with the composition at the start of the experiment as recorded in Table 5 agrees with the findings of many researchers; Marathe et al. (2011) reported that microbial inoculant application resulted in improved soil fertility status and plant nutrient uptake. A significant increase in organic carbon content of the soil was noted by reason of inoculation of groundnut with microbial inoculants such as T. viride, T. harzianum, and Bacillus megaterium (Prasad et al. 2017). This could be due to the ability of these organisms to accelerate decomposition of organic matter and the mineralization of both micro-and macronutrients in the soil (Xu and Li 2017; Table 5).

Conclusion
Trichoderma asperellum improved the growth and development of C. argentea infected with M. incognita, indicating that T. asperellum has the potential to be used as a biocontrol agent in C. argentea production. The biocontrol activity of T. asperellum in C. argantea increased as the week went by until the plants attained full maturity. Hence, the control of M. incognita by T. asperellum depends on the developmental stage of the plant root system.