Biological control of damping-off and plant growth promotion in soybean using Trichoderma virens

Rhizoctonia solani is soil borne pathogen that causes damping-off in legumes including soybean. To reduce disease infection of R. solani in soybean, seven isolates of Trichoderma virens were used as seed treatments. Soil was artificially infected using R. solani grown in organic media. Agronomic parameters and total phenolics were recorded at vegetative phase. Treatments with T. virens T.v6, T.v4, T.v7, and T.v3 showed lower disease incidence (22% to 34%) than that of the control (46%). T. virens T.v6 induced higher shoot and root length compared to plants grown in sterile soil. Phenolic in T. virens T.v7 treated plants showed the highest content (2.69 mg GAE/g) and the increase of this content was 13.7% compared to the control. Higher number of normal seedling growth and lower disease incidence than the control were observed in these treated plants. Another treatment with T. virens T.v6 showed lower amount of phenolic content (2.53 mg GAE/g) and lower increase of this content (7.2%) than those of T. virens T.v7 treated plants. However, the T. virens T.v6 treated plants performed higher normal seedling growth, lower disease incidence than the control. T. virens T.v6 and T.v7 were promising for plant growth promotion and biological control agents.


Introduction
Soil borne diseases may infect many crops which cause host growth problems.Several soilborne pathogens can form resistant structures such as sclerotia which function as inoculum for adaptation to the unfavourable environment.This inoculum is difficult to be controlled using chemicals.Rhizoctonia solani Kuhn infects several crops including soybean, alfalfa, peanut, and mung bean [1][2].
R. solani may limit legume production because this pathogen causes blight, damping off and rotting disease [1,3].Infection on soybean may occur at any growth stage and severe damage can be found at seedling growth.The symptoms appear as seed rot and root rot.The symptoms are distributed uneven in the field.Shrinking, reddish brown lesion which turns to dry when decayed can be observed on the roots and hypocotyl of soybean seedlings.The reddish brown lesion extends into the stem base on the older soybean plants and the disease occurrence is supported by warm and wet climate [4].
Since there are no commercial soybean cultivars resistance to R. solani available, applications of fungicides such as sedaxane, fluxapyroxad and penthiopyrad are carried out as one of the controls to reduce the pathogen infection.These active ingredients belong to succinate dehydrogenase inhibitor which are used in broad spectrum fungicides [5].Repeated application of fungicides triggers resistance in pathogen population.Therefore, an alternative control that is more environmentally friendly approach should be considered.Applications of beneficial fungi including genera of Trichoderma to reduce soil pathogen infection have been conducted in several cultivated plants.
Trichoderma virens has been reported effective to control plant pathogens specifically pathogenic fungi.Action modes of this species include mycoparasitism, hydrolytic enzyme production, volatile and non-volatile compound secretion [2,6].A previous study showed that applications of T. virens T.v1 to T.v7 isolated from different rhizosphere of the plant species were effective to suppress soilborne pathogen growth in mung bean [2,3].Hyperparasitism and hydrolytic enzyme production particularly cellulase and chitinase of these beneficial fungi were observed during interaction with R. solani [2].
The interaction between Trichoderma and plants affects biochemical changes in the plants.A study conducted by Yusnawan et al. (2019) [7] showed that phenolic contents in soybean plants increased during the Trichoderma-soybean interaction, whereas peroxide activity was not affected.Selection of effective T. virens isolates is necessary to obtain superior biocontrol agents against the targeted pathogen considering different isolates having different suppression ability.Therefore, this present study aims to select effective T. virens isolate as effective biocontrol agents to suppress disease incidence caused by R. solani and as plant growth promoters in soybean crops.

Treatment of seeds with conidia
Soybean seeds were sterilized using two chemicals: ethanol and sodium hypochlorite, and rinsed with sterile distilled water as conducted by Yusnawan et al. (2019) [7].Conidia of T. virens (T.v1 to T.v7) obtained from potato dextrose agar culture was suspended in sterile water (10 6 CFU per mL).Soybean seeds were dipped into the conidia suspension for 30 minutes.

Preparation of R. solani and disease incidence
Rhizoctonia solani which infected legumes was cultured on slant PDA for 10 days and this fungus was propagated on rice husk for 21 days.Inoculation with the propagule of R. solani on sterile soil was conducted five days prior to seed sowing.Modification of seed germination was conducted as performed by Yusnawan et al. (2019) and Mastouri et al. (2010) [7][8].Seeds treated with T. virens conidia were sowed on the infected soil.Seeds sown on infected soil without treatment with conidia of T. virens (C0) and on sterile soil (C2) were used as controls.Each treatment was conducted in plastic trays with 50 seeds per each tray.The treatments were arranged in a completely randomized design with three replications.Sterile water was used to keep soil moisture.The number of germinate, infected seeds and abnormal germination were recorded at 7 and 14 days after planting (dap).Disease incidence (% DI) was calculated as number of infected plants divided by numbers of total plants.For biochemical change of soybean plant experiments, seeds without conidia treatment sown on infected soil were used as a control (C).

Shoot and root development
The development of shoot and root of soybean plants were monitored and measured at 14 dap [8].To measure the length of roots, the soybean plants were harvested and the roots were washed carefully preventing root damage.

Peroxidase activity
Peroxidase activity was determined according to Kuvalekar et al. (2011) and Köksal (2011) [9][10] with slight modifications.Briefly, source of enzyme was obtained from the first trifoliate of soybean leaves by grinding the leaves in 0.1 M phosphate buffer saline (PBS).Peroxidase activity was measured by reacting the enzyme with 0.1 M PBS containing 20 mM guaiocol and hydrogen peroxide.The change of absorbance values which represented enzyme activity was recorded every 30 seconds using a spectrophotometer at 436 nm.

Estimation of phenolic content
Total phenolic quantification was carried out using a spectrophotometer [7].Briefly, supernatant of ground trifoliate leaves in 80% methanol were mixed with Folin Ciocalteu's reagent and reacted with sodium carbonate.After being incubated, absorbance value was recorded at 765 nm.

Seedling growth as affected by T. virens treatment
The seed treatment application reduced the number of abnormal and infected seedlings up to 14 dap (Figure 1).Normal seedlings from T. virens T.v3, T.v4, T.v6, and T.v7 applications showed higher quantity (66-78%) than other treatments, although those numbers were less than seeds grown in sterile soil (94%).It is not surprising that number of infected seedlings was significant in infected soil (46 %) at 14 dap.Treatments with isolates of T.v6, T.v4, T.v7, and Tv.3 showed similar low disease incidence (22, 32, 34, and 34 %, respectively) (Figure 2).A significant increase of disease incidence occurred in T. virens T.v1 treatment from 7 to 14 dap.T. virens have been reported effective to reduce the growth of soil borne pathogens in several crops such as reduction of mung bean (Vigna radiata) damping-off caused by R. solani [2], suppression of collar rot (Sclerotium rolfsii) which infected chickpea (Cicer arietinum) and lentil (Lens culinaris) [11] and inhibition of Fusarium wilt in tomato [12].Previous studies showed the inhibitory mechanisms of T. virens against plant pathogenic fungi through mycoparasitic, hydrolytic enzymatic activities, volatile and non-volatile metabolite production [2,6].

Shoot and root growth
The application of T. virens T.v6 induced longer shoot and root length compared to plants grown in sterile soil (Figure 3).The treatment of T. virens T.v1 triggered root length only, however, was unable to promote shoot length.Surprisingly, survival plants grown in infected soil produced shoot and root length similar to T. virens T.v6 treated plants.Although those both parts of plants from T. virens T.v5 were not as long as T. virens T.v6 treatment, these plants produced high fresh biomass as T.v6 plant treatment (Figure 4).The genera of Trichoderma are well known as plant growth promoter beside having antagonistic properties against plant pathogens.According to Subramaniam et al. (2022) [13], Trichoderma involvement in stimulating growth relates to the activation of single or more processes.Growth promoter of Trichoderma relies on the reduction of plant pathogen infection, the increase of photosynthetic rate, the increase of nutrient uptake, delaying in senescence, enhancement of drought tolerance and production of phytohormones.These processes may occur in host plants either directly or indirectly.In a previous study, the applications of T. virens T.v1 to T.v7 triggered soybean plant growth in the absence of R. solani infection [7], besides having antagonistic activity against R. solani which infected mung bean [2].In unfavourable environment such as in saline environment, T. virens was able to increase chlorophyll content during vegetative growth phase and higher indole-3-acetic acid [14].

Change of peroxide activity
The change of peroxide activity was influenced by T. virens isolates, for instance T.v1 and T.v5 treatments showed lower peroxide activity compared to that of control, whereas activity of peroxidase in T.v3 treatment was higher.Plant interaction with microbes influences peroxide activity.Both beneficial microbes and plant pathogenic microorganisms altered peroxide activity, however, the alteration of this enzyme in plant root systems was more prominent in plants interact with plant pathogenic microbes [15].The change of peroxide activity due to interactions between plants and microbes depends on the plant age and time of elicitation as observed in [16] study.In their experiment, reduction in peroxide activity occurred during interaction between corn roots and T. virens at the fifth days of interaction, suggesting the fungal colonization did not trigger elevation of immune responses at this plant stage.

Change of phenolic content
Total phenolics in T.v7 isolate treated plants performed the highest content (2.69 mg GAE/g) and the increase of this content was 13.7 % compared to the control (Figure 6).Higher normal seedling growth and lower disease incidence than the control were observed in these treated plants.Another treatment with T. virens T.v6 showed lower amount of phenolic content (2.53 mg GAE/g) and lower increase of this content (7.2 %) than those in T. virens T.v7 treated plants.However, the T. virens T.v6 treated plants performed higher normal seedling growth, lower disease incidence, higher shoot and root lengths than the control.The increase of about 14% phenolic content was reported on phenolic biostimulant-treated lettuce plants with T. virens (TG41) and consortium of TG41 with a vegetal biopolymer-based biostimulant [17].Another study showed that the increase of phenolic content in tomato treated plants with T. asperelloides either as protective or curative treatments was two to four-folds higher than the control.Moreover, in infected tomato plants by a damping-off pathogen, phenolic increasement was lower than the Trichoderma treated plants, however, still higher than the control [18].Plant response to biotic stress particularly pathogen infection involves rapid aggregation of phenolic compounds at the infection spots which delays pathogen development since phenolic compounds have antimicrobial, antioxidant activities and photoreceptor [19].Accumulation of phenolic compounds in crops interacted with Trichoderma may attribute to electron and hydrogen movements which prevent the interacted tissue from destruction due to oxidative process caused by infection [20], which may explain alteration of phenolics in soybean plants in this current study.

Conclusions
Application of T. virens as seed treatment reduced damping-off infection caused by R. solani and promoted plant growth in soybean.Interactions between crops and several T. virens caused the fluctuation of phenolic contents in soybean seedlings.The ability of T. virens T.v6 and T.v7 to reduce R. solani damping-off infection and to induce soybean seedling growth was promising for biological control agents as well as plant growth promoting fungi.

Figure 1 .
Figure 1.Number of normal seedlings (N), abnormal seedlings (A) and ungerminated or infected seedlings (I) on soybean as affected by T. virens (1-7 = T.v1-T.v7) on infected soil by R. solani at 7 days after planting (dap) and 14 dap.C0 = control plants on infected soil, C2 = control plants on sterile soil.Bars show standard deviation.

Figure 5 .
Figure 5. Activity of peroxide in soybean as affected by T. virens (1-7 = T.v1-T.v7).Straight and dotted lines refer to absorbance values of the control at 60 and 90 seconds.C = control plants in infected soil by R. solani.a-c = absorbance values every 30 second interval.