Trichoderma virens mitigates the root-knot disease progression in the chickpea plant

ABSTRACT This study was planned to investigate the efficacy of various concentrations of Trichoderma virens against Meloidogyne incognita in vitro. The five concentrations viz., S, S/2, S/ 10, S/25, S/50 were prepared and planned for in vitro study to test the potential of T. virens against hatching and mortality of second-staged juveniles of M. incognita. It was observed a reduction in second-staged juveniles hatching within all tested aqueous concentrations of T. virens. The second-stage juvenile mortality was also recorded in the above-given concentrations of T. virens. The maximum decrease in second-stage juveniles hatching was found in standard aqueous fungal concentration (S). Moreover, in the same T. virens concentration (S), mortality of juveniles was also recorded as highest, and was followed by S/2, S/10, S/25 and S/50. Additionally, the application of T. virens as an individual, simultaneous, and sequential order with M. incognita was also investigated in pot-grown chickpea plants and found that its use was significantly effective in suppressing root-galling disease and improved the plants' growth and physiological attributes. According to the correlation coefficient analysis, the root-knot index correlated significantly with the per cent reduction of the plants' growth and physiological attributes.


Introduction
Chickpea, also known as chana or gram (Cicer arietinum L.), is a legume crop of the family Fabaceae and an important pulse crop in India. The rank of chickpea is listed as the world's third most productive pulse crop after peas and beans, with about 75% of its production alone coming from India (Khan et al. 2014). Chickpea is susceptible to many endo and ectoparasitic nematodes, including Meloidogyne species (Ali and Askary 2001) and Heterodera species (Sharma et al. 1999). In India, Meloidogyne species, preferably M. incognita and M. javanica, have been noted to cause an estimated loss in chickpea productivity up to 19-40% and 24-61%, respectively (Ali et al. 2010). The root-knot nematode (RKN), M. incognita, is a sedentary endo-parasite and one of the key damaging pests in the agriculture farming system, attacking crops, including pulse, cereals and vegetables. They invade the plant roots at the elongation zone and move to the vascular cylinder region, where they initiate the formation of the gall, which results in the deformation of the vascular tissues (Fuller et al. 2008). It was estimated that nematodes are responsible for the economic loss of 100 billion dollars annually (Coyne et al. 2018).
Due to their extensive host variety, short generation period, and high reproduction potential, their management is difficult (Trudqill and Blok 2001) and is recognised as a severe threat to agricultural production worldwide (Jayasinghe et al. 2003;Eyal et al. 2006). Concerning economic losses produced by RKNs, several strategies have been undertaken for nematode control in agriculture, including applying chemical nematicides. Chemical nematicides are the most reliable management option against plant-parasitic nematodes (PPNs). In recent years, chemicals like 1,3-dichloropropene (1,3-D) and metam sodium have been utilised as alternatives to methyl bromide (Desaeger et al. 2017). However, compounds like metam sodium can have adverse ecological and human health effects. Pruett et al. (2001) suggest that metam sodium can induce both allergic dermatitis and/or asthma in humans. It is, therefore, recently being phased out because of the high negative impact on human and animal health. Consequently, the search for sustainable alternatives to chemical nematicides has become of paramount interest for research. One of the most promising RKNs management strategies is biological control through the action of living organisms defined as biocontrol agents (BCAs). BCAs can directly act as antagonists through antibiosis and competition mechanisms for the nutrients or space or indirectly as inducers of resistance by activating the plant immune system (Molinari and Leonetti 2019;Poveda et al. 2020).

Materials
Chickpea, Cicer arietinum L. cv. Avrodhi was tested as a host cultivar. Seeds were surface sterilised with Sodium hypochlorite (NaOCl) (0.1% v/v) for 15 min, then were rinsed three times with sterile distilled water (DW) for five minutes. The pure culture of the fungus strain, Trichoderma virens (ITCC-7351), was obtained from the Indian Agricultural Research Institute, New Delhi, India. The fungus strain is sub-cultured on Potato Dextrose Agar (PDA) medium. The mass multiplication of T.virens was done by using Richards Medium.

Nematode inoculum and maintenance
The RKN, M. incognita, was maintained on eggplant grown in a glasshouse. For J2s collection, egg masses detached from the infected roots (Hussey and Barker 1973) and kept in sterile water for hatching within a Biological Oxygen Demand (BOD) Incubator (28 ± 2°C) for four days to allow the hatching process. After four days, hatched J2s were collected. These freshly hatched J2s were considered nematode inoculum for this study.

Meloidogyne species identification using Scanning Electron Microscopy (SEM)
Species identification was performed using SEM analysis and characterised based on perineal pattern features (Sasser and Carter 1982). A mature female of M. incognita was separated from the infected eggplant root. The proposed method for preparing perineal patterns was followed (Abrantes and Santos 1989). The perineal pattern was coated with 14 nm gold, and images were captured using SEM (JSM 6510 LV Jeol-Japan). The morphology of the perineal pattern was studied to characterise the RKN species (Figure 1). The angularly oval structure with a high dorsal arch in a typical pyriform was seen, and striations were in distinct waves that bent towards lateral lines without interruption. These striations were straight with an oval appearance in the ventral regions. The above-obtained features of the perineal pattern confirm the RKN species, M. incognita.

Cultural filtrate preparation of T. virens
For the mass production of T. virens, Richards's medium was utilised (Riker and Riker 1936). The fungal mycelia mat on filter paper was washed in sterile water, and extra water and nutrients were removed with the help of blotting paper. Ten grams of mycelia mat (fungal inoculum) were mixed in 100 mL of DW followed by blending in a waring blender (10,000 RPM) for 30 s. The inoculum collected was labelled as Standard suspension (S), and consecutive concentrations such as S/2, S/10, S/25, S/50 were prepared using DW (Mukhtar et al. 2013). 10 mL of 'S' concentration of fungal inoculum were used to inoculate chickpea plants.

T. virens for J2s hatching test
The inhibitory effect of T. virens on J2s hatching of M. incognita was tested using different concentrations (S, S/2, S/10, S/25, S/50) through the egg mass dipping method. Four egg masses were placed into Petri dishes containing 10 mL of each prepared concentration of T. virens. Petri dishes were covered with parafilm to prevent evaporation and then placed in a BOD incubator (28°C). Each treatment was repeated five times, excluding control. The experiment was conducted twice under the same conditions, and the mean of the two was calculated. The hatching value was calculated by counting the number of J2s hatched per replication after four days of incubation and calculated per cent inhibition using the mentioned formula (Khan et al. 2019).
Where, C 0 = Number of J2s hatched from the egg masses in DW (control), T α = Number of J2s hatched from the egg masses in each concentration of T. virens.

T. virens for J2s mortality test
Similar five concentrations of T. virens were used to test J2s mortality of M. incognita. For the mortality test, 1 mL of DW containing 100 J2s was poured into Petri dishes by adding 9 mL of different concentrations of T. virens. Petri dishes with only water were labelled as control. Each treatment had five replications. Petri dishes were sealed with the help of a lid, wrapped in parafilm, and incubated at 28°C in BOD incubator. The dead and alive J2s were counted separately after 8, 16, and 24 h of incubation using a stereoscopic microscope. The per cent mortality of J2s was noted accordingly with the mean percentage of dead nematodes. Those J2s that looked like flexible or winding shapes were declared alive (El-Rokiek and El-Nagdi 2011), and if J2s did not move and the outline of their body appeared as straight, they were considered dead. The experiment was conducted twice under the same conditions, and the mean of the two was calculated. The per cent mortality was calculated using the following formula (Sun et al. 2006).

Effect of individual, sequential, and simultaneous application of T. virens and M. incognita on chickpea
The experiment was laid out in a glasshouse. The clay pots were filled with 1 kg sterilised soil mixed with farmyard manure in a ratio of 3: 1 (sandy loam: farmyard manure). The pots were autoclaved at twentypound pressure at 121°C for twenty minutes. Five to seven sterilised seeds of chickpea cv. 'Avarodhi' were sown in pots. The water was sprayed through the sprinkler when necessary in pots for germination. When the seedlings grew into two sets of leaves, the plants were thinned in each pot. Healthy and stable seedlings were selected per pot, and the remaining ones were removed, including in control. The experiment was conducted with five treatment replications in a completely randomised design (CRD). 2500 hatched J2s of M. incognita and 'S' concentration of T. virens (10 mL) were inoculated around the roots of chickpea plants. However, 10 mL of DW were used in control plants instead of T. virens inoculum. The experiment was terminated at approximately 60 days. All the tested plants were washed in running tap water to separate soil adhered, and then assessments were performed. The experiment was conducted twice under the same conditions and the mean of the two was calculated. The plant growth and physiological and pathological parameters of chickpea were considered and presented in tables and figures.

Experimental design
The following experimental set-up was designed.
(1) Tv: Inoculated with T.virens alone ( The plant growth, yield, and physiological attributes were analysed at termination. The growth attributes, including plant length, plant fresh weight, the number of pods and nodules per plant and physiological attributes, including nitrate reductase activity (μmh −1 g −1 ), chlorophyll content (mg/g), and carotenoid content (mg/g) were determined following the methods described by Jaworski (1971), Mackinney (1941) and MacLachlan and Zalik (1963), respectively.

Statistical analysis
The data analysis was performed by applying R software (2.14.1). The Duncan's Multiple Range Test (DMRT) were calculated at p = 0.05 to show the significant differences between the treatments. However, the principal component analysis (PCA) showed the variability among studied attributes by using Origin software [version 2019b (9.65)]. The coefficient of correlation was determined by using Microsoft excel.

T. virens for J2s hatching and mortality test
T. virens significantly inhibited the J2s hatching and found that all prepared concentrations (S, S/2, S/10, S/25, and S/ 50) of the fungal strain potentially inhibit J2s hatching of M. incognita. The per cent inhibition was maximum in standard concentration (S), followed by S/10, S/25, and S/50, respectively (Figure 2). It was also found that inhibition in J2s hatching was directly proportional to the strength of fungal concentration during the hatching test.
In the mortality test, J2s were exposed to different concentrations of T. virens viz., S, S/2, S/10, S/25, and S/50. The J2s mortality was analysed in each treatment after 8, 16, and 24 h of incubation. The per cent mortality of J2s was 83.63% in standard concentration (S) at 24 h of incubation (Table 1). The S/2, S/10, S/25, and S/50 concentrations also showed 68.72%, 58.18%, 47.18%, and 38.72% J2s mortality at 24 h of incubation, respectively. However, the per cent mortality of J2s decreased as the incubation time declined (Table 1). All the concentrations significantly killed the J2s compared to the control.

Effect of individual, sequential, and simultaneous application of T. virens and M. incognita on chickpea
We found a significant improvement in the growth of chickpea plants when the different sequences of treatments of T. virens and M. incognita were applied. The results revealed that different sequences of treatments modified the growth attributes of chickpeas. The application of T. virens alone showed the most significant  (p < 0.05) improvement in plant growth attributes. It was followed by T. virens when treated 15 days before M. incognita inoculation (Tv prior to Mi), inoculated with T. virens and M. incognita simultaneously (Mi + Tv), and M. incognita inoculated 15 days before T. virens treatment (Mi prior to Tv). The highest reduction was noticed in growth attributes when M. incognita was inoculated alone. The suppression in growth attributes was found in the order of (J2s only) > (Mi prior to Tv) > (Mi + Tv) > (Tv prior to Mi) > (Tv only) (Figure 3). According to the correlation coefficient analysis, the RKI is significantly correlated with plant growth attributes; including plant length, plant fresh weight, number of pods and nodules, chlorophyll and carotenoid content, and nitrate reductase activity (Figure 4). The scattered points in the graphs represent whether or not the two variables have a relationship. RKI has a strong linear relationship with plant length (R 2 = 0.91), plant fresh weight (R 2 = 0.98), number of pods (R 2 = 0.99), number of nodules (R 2 = 0.95), chlorophyll content (R 2 = 0.94), carotenoid content (R 2 = 0.99) and nitrate reductase activity (R 2 = 0.99). As the relation is positive, the surge in RKI increased the per cent reduction of chickpea attributes was observed ( Figure 4). The applied different sequence of treatments significantly (p < 0.05) enhanced the physiological parameters of chickpea plants. The application of T. virens alone significantly increased the chlorophyll and carotenoid content and NR (nitrate reductase) activity. It was followed by T. virens treatment given 15 days before M. incognita inoculation (Tv prior to Mi), inoculation with T. virens and M. incognita simultaneously (Mi 15 + Tv), and M. incognita inoculated 15 days before T.virens treatment (Mi prior to Tv). However, the inoculation of M. incognita alone caused the highest reduction in physiological attributes. The suppression of physiological parameters was found in the order of (J2s only) > (Mi prior to Tv) > (Mi + Tv) > (Tv prior to Mi) > (Tv only) ( Figure 5).
In the case of the pathogenic effect of M. incognita, applying different sequences of treatments

Discussion
In vitro experiment, the tested concentrations viz., S, S/2, S/10, S/25 and S/50 of T. virens were effectively inhibited J2s hatching and caused J2s mortality of M. incognita. Standard concentration 'S' was found to be highly effective in reducing J2s hatching and showed the highest toxicity towards J2s of M. incognita, followed by S/2, S/10, S/25, S/50 ( Figure 2; Table 1). However, contrary findings were also reported by Moo-Koh et al. (2022), they reported in their study that 50% concentration of T. virens showed only 22% mortality of J2s of M. incognita. Singh and Mathur (2010) found that T. viride and T. harzianum caused mild inhibition in J2s hatching and showed least J2s mortality compared to other applied fungal BCAs. The nematicidal effect of fungal inoculum was increased when exposure time extended. Meyer et al. (2004) and Elbadri et al. (2008) reported in their study that the impact of fungal inoculum varied from concentration to concentration, thus confirming these findings. In our study, the concentration and incubation period were important factors. Sharon et al. (2001) reported that the nematicidal activity of T. viride is due to chitinase and protease enzymes that infect nematode larvae and eggs. Abo-Elyousr et al. (2010) noted that Trichoderma spp.
produced chitinase enzyme in the culture that can inhibit the egg hatching of nematodes.
In the pot experiment, inoculation of T. virens either individually, simultaneously, or sequentially with M. incognita on chickpea was performed and found that all the treatments showed significant improvement in growth and physiological attributes and the reduction in pathological parameters. The highest reduction in RKI and nematode populations was found in those plants treated with T. virens given 15 days prior to M. incognita. Because, T. virens were got sufficient time to colonise the root system and making it less susceptible to nematode, lower penetration to J2s of M. incognita or released compounds which have an antagonistic effect on M. incognita ( Figure 6). However, contrary findings were also reported by Meyer et al. (2001). According to their study, the role of T. virens in reducing the numbers of eggs and J2s on the root of bell pepper is comparatively less than Burkholderia   in roots. Soil application of T. virens in chickpea plants minimised the population of J2s in soil due to the colonising action of T. virens near the roots (Figure 3). However, contrary findings were also reported by Zhang et al. (1996). They reported that T. virens did not suppress the reproduction of M. incognita on cotton. Analysis of correlation coefficient exhibited that RKI positively correlated with plant length, plant fresh weight, chlorophyll and carotenoid content, NR activity and number of pods and nodules (Figure 4). Scattered points which are existing in graphs show whether two variables have a relationship or not. Maximum scattered points with minimum correlation were found between the RKI and plant length (R 2 = 0.91) with positive correlation and maximum correlation with highly condensed points observed between RKI and number of pods, carotenoid content, chlorophyll content and NR activity (R 2 = 0.99) (Figure 4). Our finding confirmed with Rich et al. (1984), reported that significant positive correlations were observed between nematode numbers and plant yield of tobacco. The colonisation of T. virens may create adverse conditions for the J2s to penetrate the plant roots. In addition, it was possible that toxic secretions produced by T. virens may create a suppressive effect on nematodes and make the plants less susceptible to the attack of nematodes. After colonisation, toxic secretions released by the applied BCAs induced a suppressive effect on M. incognita and improved the atmospheric N2 accessibility to the plants (Bashan and Holguin 1997). Furthermore, M. incognita inoculation 15 days prior to T. virens showed the least improvements in growth attributes of chickpea, as firstly applied J2s had sufficient time for multiplication and caused infection in the root system of plants (Figure 3). Contrary findings were also reported by Fan et al. (2020). They reported that T. citrinoviride treated plants increased shoot length, root length, root fresh weight, and root dry weight by 15.61, 23.32, 35.08, and 33.33%, respectively, compared to those of with untreated plants. Inoculation of T. virens either individually, simultaneously, or sequentially with M. incognita showed significant improvement in physiological attributes compared to J2s only ( Figure 5). However, contrary findings were also reported by Singh et al. (2017). They reported that T. harzianum showed the highest chlorophyll content compared to carbofuran treated plants. Multiple action mechanisms of Trichoderma spp. were recorded to contribute to the biological control, including competition for space and nutrients, antibiosis, myco-parasitism, and induction of systemic resistance in plants (Lombardi et al. 2018). The mechanisms of Trichoderma in promoting plant growth include the production of auxin-like compounds, increased availability of nutrients, affecting the root system, and inducing systemic resistance to plants (Li et al. 2015;Marra et al. 2019). The reduction in the roots galls may be due to the failure of most J2s of M. incognita to enter the host plant roots. The BCAs applied in the roots of host plants provide a physical barrier for the penetration of J2s of M. incognita and enhance root growth and nutrient uptake (Wickramaarachchi and Ranaweera 2008). Trichoderma spp. has increased systemic resistance to plant diseases via root colonisation which activates the plant defence mechanisms (Forghani and Hajihassani 2020). The obtained results revealed that T. virens have antagonistic activity for M. incognita. In pots-grown chickpea, T. virens reduced the root-galling infestation by killing the infective J2s of M. incognita. Thus, the potential of T. virens could be considered for better crop growth by reducing the disease infestation. However, the use of T. virens must be extended to field experiments to gather the maximum data for considering the antagonistic potential against pests and diseases. The obtained results data were based on in vitro and pot experiments within this study. Therefore, findings from this study have shown the potential for using T. virens as an ecological safe option to manage the RKNs, M. incognita in agricultural practices. It would also minimise the use of chemical nematicides in the farming system. Environmental Science in the College of Natural and Health Sciences at Zayed University, United Arab Emirates. Dr. Manar's research interests include air quality, environmental sustainability, plant protection, pesticide residues, environmental pollution, and waste management.
Hera Nadeem, is a research scholar in the Department of Botany, Aligarh Muslim University, Aligarh. Presently, she is working on managing root-knot nematode disease in vegetables and pulses through the microbial-based compound.
Lukman Ahamad did his Ph.D. in Botany with a specialization in Plant Pathology from the Aligarh Muslim University, Aligarh (UP), India. He received his M.Phil. and M.Sc. in Botany from the same University. He also qualified National Eligibility Test in Plant Pathology conducted by ICAR-ASRB in 2014-15 and Currently working with quarantine plant pathogens at the Regional Plant Quarantine Station, Kolkata, India.
Mohamed Hashem, is a professor of microbiology at King Khalid University, Saudi Arabia, and Assiut University, Egypt. He was awarded as a distinguished professor at KKU in 2015. His research interest includes mycology, plant pathology, biological control, microbial biotechnology, bioenergy, and bio-nanotechnology. He supervised fifteen Ph.D. and master students to completion of their studies. He published more than 130 scientific papers in international journals and implemented 20 projects in collaboration with international scientists.
Saad Alamri, is a professor of microbiology at King Khalid University, Saudi Arabia. He occupied many administrative positions, and his last position was as vice-president of KKU. His research interest includes bacteriology, environmental toxicology, microbial biotechnology, waste management, and bionanotechnology. He supervised ten M.Sc. thesis in the field of interest. He published more than 100 scientific papers in international journals and implemented many funded local and international projects.
Rishil Gupta, is a Ph.D. student of Plant Pathology/Nematology who researches on nematicidal properties of secondary plant metabolites.