QUANTITATIVE CHANGES OF ENZYME ACTIVITY IN WHEAT INDUCED BY STREPTOMYCES SP. STRAIN HU2014

Biocontrol microorganism have a diverse range of activities and they have been focused on potential biocontrol agents in agriculture. They can induce plant defensed response and enhance plant disease resistance. Streptomyces sp. produce active metabolites that can inhibit the growth of phytopathogens. Induced resistance is usually indicated by the activity of Peroxidase (POD), Polyphenoloxidase (PPO)

The enzymatic activities of PPO, PAL, -1,3-glucanase and chitinase were significantly enhanced in the rice treated with antifungalmycin N2 plus Rhizoctonia solani Kühn (Zhang et al., 2020). Streptomyces rochei A-1 treatment significantly increased the activities of POD, superoxide dismutase (SOD), catalase (CAT) and PAL, effectively induced the resistance of apple fruit to ring rot (Zhang et al., 2016).
The V76-12 isolate was the most effective treatment tested in reducing leaf spot disease of oil palm seedlings, due to its ability to enhance PAL, POD and PPO activities in the oil palm leaves (Sunpapao et al., 2018). SA from the strain of Streptomyces diastatochromogenes KX852460 induced the high activity of glutathione reductase (GST), catalase (CAD), PAL and PPO in tobacco against R. solani AG-3 (Ahsan et al., 2019).
Six endophytes stimulated systemic resistance which was evaluated by seed treatments in pathogen inoculated chickpea (Singh et al., 2017). Streptomyces rubrogriseus HDZ-9-47 enhanced the activity of PPO, POD, PAL and SOD in tomato roots (Jin et al., 2016 Streptomyces HU2014, kindly provided by Dr. Hu Linfeng of Henan Institute of Science and Technology (HIST), was precultured on potato dextrose agar (PDA) medium at 4 ℃ before experiment. The mycelia discs were transferred to sterile GPY broth in 250 ml flasks, incubated at 30 ℃ with shaking at 150 r. min -1 for 28 days. The fermentation broth was centrifuged (8000 r min -1 , 4 ℃) for 15 minutes to separate the supernatants. The supernatants were filtrated through 0.45 m candle filters, and then the extracellular filtrate (EF) was stored at 4 ℃. The mycelia were incubated for 7 days, filtrated through nylon filter, washed with sterile water, dried with filter paper and stored at 4 ℃.
Wheat cultivar of "BN4199" afforded by Breeding Center of HIST was used in this study. The seeds were disinfected with H2O2 (30 %) for 2 min and washed thoroughly with sterile distilled water. The seeds were germinated for 12 hours in plastic tray (30 cm  20 cm  3 cm) covered with wet gauze. Subsequently, the germinated seeds were transferred in the pot (12 cm  12 cm  9 cm), 15 seeds per pot, placed into growth chamber with the conditions of light (12 h/day) and temperature (27 ℃  2 ℃).
2.2. Assay of defense enzymes. The EF was considered as original broth concentration, and five concentrations were set up by adding sterile water to the EF: 10-fold dilution, 10 2 -fold dilution, 10 3 -fold dilution, 10 4 -fold dilution and 10 5 -fold dilution. An aliquot was applied by soil drench of 50 ml with 3 replications. The gradient concentration of the M was 0.1 mg/ ml, 0.01 mg/ml, 0.001 mg/ml, 0.0001 mg/ ml and 0.00001 mg/ml. Sterile water was untreated control. Each experiment had randomized design. Sterile water was untreated control. In ten days after soil drench treatment, leave tissues were collected and weighted, 0.1 g per aliquot for one enzyme activity assay. Then tissues were immediately submerged in liquid nitrogen. Material was ground in mortar with a pestle under liquid nitrogen, transferred into centrifugal tube. The enzyme activity was determined by POD colorimetry ( Results. Induced resistance is usually indicated by the activity of POD, PPO and PAL or other defense enzymes. To estimate the quantitative changes in the plant defense-related enzyme activities of POD, PPO, and PAL in wheat leaves, our pot experiment was conducted under different concentrations of the M and EF with soil drench treatment. With the treatment of the EF (Table 1, Fig. 1), the POD and PAL activities at the concentration of 10 3 -fold dilution of the EF increased significantly to some extent compared to control, by 173.86 % (P  0.05) and 71.92 % (P  0.05), respectively. It was shown that EF of S. sp. strain HU2014 can significantly induced the activity of these enzymes in wheat at low concentrations.  Although the activity of PPO at the concentrations of 10 2fold dilution and 10 4 -fold dilution increased compared to untreated control, by 16.99 % (P  0.05) and 16.48 % (P  0.05) respectively, the difference was non-significant. With the treatment of the M (Table 2, Fig. 1), the activities of POD and PPO enhanced with the increasing of dilution ratio in the range of different concentration, but the difference was not significant.  The activity of PAL (with the exception of 0.01 mg/ml) increased compared to untreated control by 10.16 %  21.23 % (P  0.05), but the difference was non-significant as well.
Discussion. In Moreover, in this experiment the increase of induced enzyme activity was higher. Therefore, it was determined that the S. sp. strain HU2014 could significantly improve the resistance of wheat seedlings. The level of enzyme activity with the M was lower than that of the EF. The main reason would be that the mycelia need a certain time to colonize in the rhizosphere and gradually metabolize the active components.
Conclusion. It was found that POD and PAL enzymes had high level of activity at a low dilution of the EF of S. sp. strain HU2014 within certain concentration range. Activities of POD and PPO were enhanced with the increasing of dilution ratio in the range of different concentration of M, but the difference was not significant. Characteristics of S. sp. strain HU2014 growth in soil need further research. The changes of defense enzymes are related to the induction of plant disease resistance.
In our experiment, S. sp. strain HU2014 could induce the enzyme activity without plant pathogenic fungi, so the induction of the strain would follow Jasmonate signaling pathway. Otherwise, the enzyme activities measured at the physiological level of plants were easily affected by some factors, which need to be further verified at the molecular level. Further studies should be focused on wheat disease resistance with RT-PCR assay, metabolic pathways and transcriptomics research.