Early Signals of Physiological Response by Trichoderma asperellum AC.3 induction against Peronosclerospora spp. in Maize

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Introduction
Peronosclerospora is the most species-rich genus of downy mildew (Graminicolous) pathogens, with very high diversity.Many reports show economically important downy mildew species, including P. maydis, P. philippinensis, P. sacchari, and P. sorghi [1].The identification of the three species that cause downy mildew has been described based on cell wall thickness, shape and size of conidia, and has been outlined in an identification key [2].Downy mildew infection decrease maize production, causing significant yield losses widely.All the early symptoms of downy mildew are at the base of the stem or the point of growth, on the basis of the corn plant stem in the morning there is a gutation so that the conidia that sits on the base can reproduce perfectly, tubes germinate and have a great chance of making early penetration on the surface of the leaves [3].Information regarding the prevention and control of downy mildew, especially in Indonesia, still predominantly uses fungicides containing the active ingredient of metalaxyl.However, previous research [4] revealed that a combination of synthetic fungicides and moderately resistant varieties was effective in suppressing the incidence of downy mildew.The use of downy mildew-resistant corn varieties is recommended for controlling downy mildew disease compared to the use of synthetic fungicides because the fungicide applications are less effective and not environmentally friendly [5].The application of biological control agents is an alternative for disease control.Apart from having the potential to suppress disease infections, biological control agents also increase plant production [6].Induced resistance by biological agents is one of the mechanisms for controlling disease and reducing the use of chemicals in the field.
In terms of overcoming environmental stress, especially from pathogen infection, plants develop certain efficient strategies to avoid or tolerate stress for their adaptation to the unfavourable environment.One of the adaptation strategies is through plant physiological responses [7].Each biological agent activates different defense mechanisms in induced resistance pathway, resulting in different symptom suppression from each pathogen inoculation [9].
Pathogen treatment in plants is capable of activating multi-layered Induced Systemic Resistance (ISR) defense responses through rapid increased accumulation of hydrogen peroxide (H2O2) around infection, while hypersensitive reactions (HR) acts as a second layer of defense against pathogens when preinvasive defense responses fail [9].
Reactive oxygen species (ROS) plays a central role in plant pathogen defense.ROS production is one of the earliest cellular responses after successful pathogen infection [10].ROS is used by plants as a weapon against pathogens through direct toxicity and as an important factor in pathogen cell death mechanisms.However, plants also use ROS in signaling as a further defense response.In addition, according to [11] ROS is a versatile compound having toxic or signal effects on a variety of living organisms, including seeds.It is believed that ROS plays an important role in regulating seed flora and dormancy, but action further mechanisms are not fully understood.According to [12], ROS and lignification of mycelium are used as initial parameters for potential activation of plant resistance responses.
The objective of this research is to identify Trichoderma asperellum AC.3 in suppressing downy mildew and inducing early physiological resistance of maize through ROS production in a screenhouse.

Strain and planting materials
T. asperellum AC.3 (GenBank: MK210562) was isolated from maize root from Bone district, South Sulawesi Province.Preparation of suspension and filtrat culture was carried out at the Pest and Disease Laboratory of Ministry of Agriculture.Pulut URI and JH37 seed varieties were used, which were very susceptible and moderately resistant to downy mildew disease.The seeds were obtained from the Ministry of Agriculture.The next morning (four to six o'clock), fresh sporangia was washed with purified water.For source of inoculum, oospora concentration was adjusted to 10 4 oosporas/ml using a hemocytometer.

Preparation of suspension and filtrate culture of T. asperellum AC.3
The procedure for preparing the suspension of T. asperellum fungi was conducted by recultivating the T. aspersellum AC.3 isolate on Potato Dextrose Agar (PDA) medium and incubated for 7 days at 28 o C in an incubator, dispensed into sterile water and the conidia was ajusted to 10 6 cfu/mL.The preparation of the filtrate culture followed the procedure [13] with a slight modification.Briefly, two pieces of mycelia (diameter 0.5 cm) from the T. ascellum AC.3 culture obtained from 12 days of re-culturing on the PDA was put into an Erlenmeyer containing 100 mL Potato Dextrose Broth (PDB) medium.
The suspension was incubated at 28 ± 2 °C for 21 days with occasionally shaking.Trichoderma filtrate was obtained by centrifugation to separate the supernatan from the pellet and then re-filtered the supernatan with Whatman filter paper 42 followed by additional filtration using a 0.45μm filter syringe.The filtered supernatant was incubated in a water bath at 60 °C for 30 minutes.Then the T. asperellum AC.3 filtrat culture was ready to be applied.

Treatments using suspension and filtrate culture of T. asperellum AC.3
The suspension and filtrate culture of T. asperellum was applied through seed treatment, which is by soaking maize seeds in the suspension as well as in the filtrate culture for 30 minutes.PulutURI and JH37 seeds soaked in sterile water for 30 min, respectively, were used as negative controls (without downy mildew inoculation) and positive controls (within downy mildew inoculation).The treated seeds were planted in trays filled with sterile soil, sand, and organic fertilizer in a ratio of 2:1:1.The planting medium is sterilized using the drum sterilization method, namely roasting the soil over a flame (the soil is placed on a drum) at a temperature of 70 o C for 2 hours per day for 3 consecutive days.Media sterilization is carried out so that the soil is free from contamination by harmful microorganisms, including soil-borne diseases.

Inoculum application
The ten-days-old seedlings were vaccinated with a circular inoculation method with Peronosclerospora spp.oospora suspension at a concentration of 10 4 oospores/ml prepared as described above.All test plants were inoculated with Peronosclerospora spp.oospora.In the whorls seedlings, droplets of oospores (Peronosclerospora spp.) were dropped into the leaf test formed by the growing plant and allowed to flow to the base.The spore suspension (inoculum) should be sprayed with the help of hand compression sprayer only and the process should be completed by 6.00 A.M. The plants infected with this pathogen were kept in a screenhouse (90-95% RH, 20-25 °C), and the disease progression was observed [14].

Evaluation of the disease incidence
Observe maximum growth potential by observing the number of plants growing at 7 days after planting (dap), while disease incidence of downy mildew was recorded at 14, 21 days after inoculation (dai), and final observation was conducted at 28 dai.
The maximum growth potential and disease incidence was calculated according to the following formula:

ROS detection
ROS detection was analyzed using diaminobenzidine (DAB) staining.The procedure for making the DAB staining solution was conducted by dissolving 200 mg DAB in 180 mL sterile H2O for a final concentration of 1 mg•mL -1 DAB in a 200 ml Erlenmeyer; 0.2 M HCl was added to dissolve DAB and to reduce the pH to 3.0 using a small magnetic stirrer (the tube was covered with aluminium foil because DAB was sensitive to the light).As many as 100 µL Tween 20 (0.05% v/v) and 10 mL of 200 mM Na2HPO4 were added to the stirred DAB solution.This produced a 10 mM Na2HPO4 DAB staining solution and increased the pH value.Next, coloring the leaves with the DAB solution.
Leaves were collected at the scheduled time manually from the plants and placed in sixwell microtiter plates.Five milliliters of DAB dye solution was added to the leaves in the well holes (adjusting the volume to ensure the leaves were completely submerged), then incubated for 5 hours.After incubation, the DAB dye solution was replaced with a bleach solution (ethanol:acetic acid:glycerol = 3:1:1 v/v/v).The six-well plates were carefully placed into a boiling water bath (at ~90-95 °C) for 15 min.This process bleached the chlorophyll leaving brown precipitation as a form of the DAB reaction with hydrogen peroxide.After 15 ± 5 minutes of boiling, the bleach solution was replaced with new bleach solution and left for 30 minutes.Samples could be stored at 4 °C for 4 days.On the observed leaves, dark brown deposits indicated the accumulation of ROS.The ROS were observed microscopically and captured using a camera (Optilab®).

Statistical analysis
Analysis of variance from screenhouse and laboratory test data was calculated using Microsoft Excel software.Similarly, the calculation of the Least Significance Difference (LSD) was done at a 95% confidence level.

Evaluation of disease incidence
There was a significant difference between the growth performance of maize plants treated with T. asperellum AC.3 suspension on JH37 (SR) variety at 7 dap.However, there was no significant treatment effect shown by the PulutUri variety (Figure 1).Spore suspension of T. asperellum TaspHu1 with high concentrations can promote growth and increase the resistance of tomato seedlings to pathogens [15].Fig. 1 Means percentage of maximum growth potential on JH37 and PulutUri maize varieties inoculated with downy mildew at 7 dap.
The pooled mean and standard error values of 5 replicates from the experiment is presented for each treatment.Different letters indicate significant differences among treatments according to the LSD test (P ≤ 0.05).
The filtrate culture and suspension of T. asperellum AC.3 treatments did not have a toxic effects on maize seeds, so it did not have a negative impact on the plant growth.In fact, the suspension treatment (SR) was able to stimulate plant growth in the JH37 variety, which was observed on growth potential of 96%, which was significantly different from the control treatment (WR) of 79% on the same variety.Seed treatment using Trichoderma filtrate culture was able to increase plant growth parameters in millet [16].
The highest incidence of downy mildew was observed in only Peronosclerospora spp.inoculated (WR and WS) compared to the treatments using filtrate culture and suspension of T. asperellum AC.3 with downy mildew inoculated (Figure 2).According to [17] Trichoderma sp.isolates potentially as biocontrol agents with an emphasis on the development of pathogens Bipolaris maydis and Curvularia sp. in maize.Metabolite compounds produced by T. asperellum AC.3 isolate was able to induce defense mechanism against downy mildew.[18] reported that air signals produced by Trichoderma spp.were considered by plant tissues as a mediator of fungal-plant communication and as an inducer of plant resistance.The pooled mean and standard error values of 5 replicates from the experiment is presented for each treatment.Different letters indicate significant differences among treatments according to the LSD test (P ≤ 0.05) The filtrate culture treatment (FR) on the JH37 variety was observed at 28 days to have a disease incidence of 23%, which was significantly different from the control treatment (WR) of 36%.This might be related to the production of ROS produced by plants due to the filtrate treatment given.The effectiveness of glucohexaose treatment in inducing resistance to downy mildew pathogen infection in cucumber was related to the increase of ROS production [15].

ROS detection
ROS production is an early sign during plant defence response triggered by plant pathogen infection.In this study, the influence of T. asperellum AC.3 in ROS accumulation was observed in infected maize leaves by Peronosclerospora spp.before (0h) and after inoculation (6 dan 12h).This study showed that ROS was detected in all treatments of JH37 variety with the presence of brown color in the tested leaf tissue, while the control treatment did not show ROS (Figure 3).ROS production represented by the darkest color concentration was observed in the filtrate culture treatment (FR) followed by the treatment with T. asperellum AC.3 suspension (SR).According to [16].plant response induction by Trichoderma is time-and concentration-dependent process, during the first hours of interaction, a SAR-like reaction can occur as a result of trichodermal colonization, when fungi are applied to plant roots in high concentrations (10 7 cfu/ml).production was detected at 6 hours and for up to 12 hours on plants infected by downy mildew.These results showed that T. asperellum AC.3 triggered more production of ROS in the presence of downy mildew, therefore, maize crops could improve plant defence against Peronosclerospora spp.However, different results were observed by [20], in which T. asperellum inhibited the general production of ROS against pathogen infection in tomato plants.Although ROS is usually correlated with a successful resistance response to disease, some pathogens can induce ROS production for their own benefit.Thus, ROS is produced as part of a complex signal tissue that responds to pathogen infection and mediates various responses, sometimes with opposite effects or in response to different pathogens.ROS plays an important role in plant-pathogen interactions; ROS is used by plants as a weapon against pathogens through direct toxicity.For diseases where ROS supports pathogenicity, potential artificial antioxidant induction can be used to weaken the disease [21].
Based on the area size (Table 2), ROS production triggered by the filtrate culture treatment (FR) on JH37 variety had the largest area of 243 mm 2 followed by the suspension treatment (SR), then the treatment of filtrate (FS) and suspension (SS) of PulutUri variety.All of which had been inoculated with downy mildew.Meanwhile, ROS in plants that were only inoculated with Peronosclerospora spp.(WR and WS) had the smallest area because oxygen reactive was only produced in response to pathogen infections.Treatment of T. asperellum AC.3 had a larger area of ROS.This could be induced by an elisitor that activated plant resistance signals.A study conducted by [19] showed that T. pseudokoningii produced locally and systematically Trichoconin metabolite compounds, induced ROS production and triggered the accumulation of phenolic compounds at the application site in tobacco plants.culture treatment (FR) on JH37 variety had the largest ROS area with the lowest disease incidence compared to the other treatments.Therefore, ROS could act as an initial signal induced by microorganisms.
It is difficult to differentiate ROS in their cytotoxic or signal-induced role because although ROS causes cell death, this is a necessary process to provide resistance to stress.According to [22]. the stress-induced ROS activation response should occur rapidly with the onset of stress and should decrease when the stress disappeared.From a signalling point of view and taking into account the hypothesis of ROS is used as initial signals to detect lifesupporting oxygen levels, it is not surprising that ROS is required for the maintenance of key biological processes.ROS is very important for cell proliferation in microorganisms, but if there are excess or deficit, ROS has a bad impact on plants [23].[24] added that the development of various basic biological processes related to cell differentiation, proliferation, and cell death is associated with ROS-induced redox signaling, which is previously thought to be due to oxidative damage caused by ROS.ROS-induced redox signaling plays an important function in various physiological responses.
The most famous phytotoxic anti-infectious effect that relies on ROS is Hypersensitive Reaction (HR).HR is the rapid death of the plant cells attacked which results in death of a parasite or the cessation of its development.The T. asperellum AC.3 fungal isolate which was infiltrated on the leaves of 8-week-old tobacco plants showed hypersensitivity reaction after 96 hours.(Fig. 5).The HR occurred to restrict the growth of pathogens and was very effective against biotropic patogens, because with host cell death, the supply of nutrients was reduced.HR is often ineffective against necrotrophic pathogen because this pathogen usually kills host cells to feed on them [21].

Conclusion
Filtrate culture of T. asperellum AC.3 treatment (FR) on the JH37 maize variety significantly reduced disease incidence and induced rapid accumulation of ROS around infected area as a plant defence response to downy mildew.

Fig. 2
Fig. 2 Means percentage of disease incidence in maize treated with Peronosclerospora spp.and T. asperellum AC.3.The pooled mean and standard error values of 5 replicates from the experiment is presented for each treatment.Different letters indicate significant differences among treatments according to the LSD test (P ≤ 0.05)

Figure 4
Figure 4 shows significant accumulation of ROS in PulutUri variety that was treated with T. asperellum AC.3 filtrate (FS) and suspension (SS) with downy mildew inoculated.ROS

Fig. 4
Fig. 4 Differences in accumulation of ROS in PulutUri maize variety before (0hour) and after infection, as shown with brown colour (6 and 12hours).

Table 1 .
The treatments were arranged in a completely randomized block design.Each treatment consisted of 3 replications.2traysper replication with 32 seeds/tray.The treatment composition is shown in Table1.Composition of the treatment used in the test

Table 2 .
Large area of ROS produced by plants.Based on the area of ROS produced by plants, there was a negative correlation between the area of ROS and the incidence of downy mildew in maize.It was observed that the filtrate