Biological activity of chitosan inducing resistance efficiency of rice (Oryza sativa L.) after treatment with fungal based chitosan

Reduced pathogen resistance and management of the left-over rice stubble are among the most important challenges faced in rice cultivation. A novel and eco-friendly strategy to synthesise ‘Fungal Chitosan’ (FC) from Aspergillus niger using rice straw could serve as a sustainable treatment approach to improve both disease resistance and yields, while also effectively managing the rice stubble waste. The FC treatment promoted germination as well as growth parameters in rice varieties, TN1 (high yielding-susceptible) and PTB33 (low yielding-resistant) better than a commercial chitosan (PC). Treatments of exogenously applied FC to plants produced direct toxicity to Xoo, and reduced the BLB disease index by 39.9% in TN1. The capability of FC to trigger a cascade of defense pathways was evident from the measurable changes in the kinetics of defense enzymes, peroxidase (POD) and polyphenol oxidase (PPO). FC treatment increased levels of POD in TN1 by 59.4%, which was 35.3% greater than that of untreated PTB33. Therefore, the study demonstrated the effectiveness of FC treatments for use in agriculture as a potential biostimulant as well as protective agent against bacterial leaf blight, BLB, of rice (Oryza sativa) that could be produced from stubble waste and improve rice stubble management strategies.


Results
Chitosan characterization. The extracted fungal chitosan, FC, was analysed in FT-IR and compared with the standard chitosan. The same patterns of peaks were observed in both product chitosan and extracted fungal chitosan. The peaks obtained were between the wavelength at 3200-3400 cm −1 presence of H-bonded NH 2 and OH stretching and 2850-3100 cm −1 attributes to CH stretching vibrations which corresponds presence of aliphatic group (Figs. 1 and 2).
Both chitosans (FC and PC) examined under the scanning electron microscope exhibited morphologically similarities (Fig. 2).

Bio-stimulant effect of chitosan on rice seeds. A significant variability in germination parameters
was observed in treated seeds from both varieties. Percentage of emergence of rice varieties treated with fungal chitosan (FC) or the product chitosan (PC) across 12 day period, post treatment was found to be higher than that of control rice varieties A-TN1, B-PTB33 (Fig. 3A,B). However, seed treated with fungal chitosan emerged 17 h before that of seeds treated with PC. Both the treatments reduced the mean germination time, promoting the earlier emergence of TN1 that was reduced to 4.54 and 3.8 DAP by PC and FC respectively (F 4,20 = 8.87; P ≤ 0.0001). At 50 ppm treatment concentration, PC reduced MGT to 4.86 DAP while FC reduced MGT to 4.26 DAP over untreated control seeds (F 4,20 = 8.79; P ≤ 0.0001) (Fig. 3C).
Subsequently there was an increase in germination percentage, GP, over untreated control. The GP of PC and FC treated TN1 seeds at 25 and 50 ppm treatment concentrations were 87.4 and 95.4% and 90.4 and 96.8% respectively (F 4,20 = 37.71; P ≤ 0.0001). A likewise increase in germination percentage of PTB33 was also observed at the same treatment concentrations of PC, 86.2% and FC, 94.2% (F 4,20 = 44.82; P ≤ 0.0001) (Fig. 3D).
Bio-stimulant effect of chitosan on germination energy, plant growth and biomass. Increased biomass reported as germination energy, GE, was found to be enhanced by both the treatments over untreated control (26%) (Fig. 3E). PC treatment on TN1 seeds displayed a GE of 59.4% and the GE of FC treated seeds was 61.76 cm at 50 ppm treatment concentration (F 4,20 = 102.36; P ≤ 0.0001). A similar effect was also observed in PTB33, resulting in GE of 52.8 and 57.46% respectively treated with 50 ppm PC and FC (F 4,20 = 54.86; P ≤ 0.0001).
Increased plant growth (height) as an indicator of biomass, shown as germination energy, GE, post treatment with both (PC and FC) was observed at the greatest treatment concentration (50 ppm The FC treatment on PTB33 stimulated growth parameters at a comparatively higher rate than that of TN1 enhancing the biomass of rice plants. The treatments augmented the plant biomass together in both varieties, increasing the FW from 11.08 to 20.41 and 28.88 g (F 4,20 = 33.83; P ≤ 0.0001) with a corresponding DW of 6.628 and 11.266 g (F 4,20 = 67.9; P ≤ 0.0001) in TN1 treated with 50 ppm of PC and FC respectively (Fig. 6A,B). FC at 50 ppm also induced FW and DW increase from 9.76 to 25.4 g (F 4,20 = 28.84; P ≤ 0.0001) and 1.245 to 8.54 g (F 4,20 = 37.63; P ≤ 0.0001) respectively which was 31.73 and 48.94% higher than that prompted by PC in PTB33 (P ≤ 0.005) (Fig. 6).
Effect of chitosan spray on BLB disease. The effect of chitosan spray on BLB disease was assessed in terms of mean lesion length and disease incidence percentage. There was a significant reduction in lesion length prompted by chitosan sprays on infected plants (Fig. 8A,B). The lesion length was reduced from 3.9 to 2.8 and 2.4 mm F 4,20 = 26.59; P ≤ 0.0001) by PC and FC sprays (50 ppm) in TN1 (Fig. 8A). Control untreated PTB33 plants developed lesions of size 3 mm that were significantly reduce in the the chitosan treatment sprays, PC and  Effect of chitosan spray assay on induction of defense enzymes. The effect of chitosan sprays on POD and PPO titers was analysed for across 7 timepoints, starting on day 0 when treated and for 6 days (144 h after spray) (HAS). The enzyme levels differed variably with respect to treatment type, concentration and induction. POD levels treated with 25 ppm PC followed a similar kinetics with that of 50 ppm PC spray in both plant varieties (Fig. 9A,B). POD levels induced by 50 ppm PC spray on TN1 increased 24 h after spray (HAS) from 9.108 to 12.03 (F 4,20 = 25.13; P ≤ 0.0001) and continued to increase to 18.401 U/mg FW till 72 h (F 4,20 = 77; P ≤ 0.0001) after which it started to decrease to 15.08 U/mg FW at 144 HAS (F 4,20 = 230.74; P ≤ 0.0001) which was still 44.36% higher than control (Fig. 9B). A likely increase in POD levels after 25 and 50 ppm FC sprays displayed an increase in POD levels for 120 HAS after which the levels remained constant till 144 HAS in both plant varieties (Fig. 9A,B). POD levels brought by 50 ppm FC spray on TN1 increased 24 HAS from 9.108 to 13.687 (F 4,20 = 25.13; P ≤ 0.0001) and continued to increase to 22.44 U/mg Fresh weight (FW) till 120 HAS (F 4,20 = 181.02; P ≤ 0.0001) and staying constant till 144 HAS which was still 32.754% higher than 50 ppm PC spray (Fig. 9A).

Discussion
Sustainable agricultural practices are of great importance to establish and maintain food security in any country. Use of treatments that improve crop health, reduce pests and pathogens, but that are also environmentally and economically feasible can greatly aid efforts to build sustainable cropping systems. Chitosan demonstrating their capacity to provide an eco-friendly agronomic strategy to improve crop yield and resistance to pathogens and pests 20,[28][29][30] . New methods of chitosan production are providing a renewable and sustainable source of this valuable compounds 15,16,23,24 . www.nature.com/scientificreports/ Among them, ligno-cellulose residues are profusely available as an economical viable, natural chitosan resource 31 . Copiously available ligno-cellulose from management of rice stubble using the hydrolytic activity of the fungi, A. niger produces an excellent source for the post treatment production of chitosan 32 . This study   www.nature.com/scientificreports/ further shows the benefits from A. niger produced chitosan as a seed priming agent to induce improvements in germination capabilities of low yielding, disease resistant, rice variety PTB33. Chitosan produced from A. niger treated rice stubble, also demonstrates their capacity to induce BLB disease resistance in high yielding, susceptible TN1 rice variety.
Utilizing the natural cellulosic substrate of rice straw as the source to increase the biomass of A. niger and their subsequent chitosan concentration, fungal chitosan was successfully extracted and shown to be comparable in efficacy with commercially available sea-shell chitosan product. Extracting the chitosan from the fungal mycelia using SSF provided the maximal production of hydrolytic enzymes such that rice straw utilization to produce chitosan is economically feasible 33 . The chitosan quality was further supported by analyses using FT-IR spectrum which indicates the strong similarity in the produced and purchased chitosan compositions. Similar analyses using spectral uniformity between commercial and fungal chitosan extracted from Auricularia sp., previously reported similar results 34 .
Seed treatment with the chitosan, FC, produced using A. niger, conclusively caused improvements in seed germination GP, and energy GE, traits for both varieties, TN1 and PTB33, when using concentration of FC (50 ppm). When using FC priming of PTB33 seeds early germination was observed, reducing the MGT from  44.89% greater lengths, with a corresponding 45% increase in root-shoot lengths, and a 61.57% increase in plant biomass (FW) compared to untreated disease resistant seeds. The FC treatment prompted the growth of PTB33 to be 31.28% greater than untreated TN1. The plant biomass increases and early establishment provoked by activation of various biochemical processes has found to enrich the grain nutrient status 38 40,41 . The accomplishment of chitosan as a successful seed dress is attributed to their higher molecular weight conferring physical protection 42 . Furthermore, the capability of chitosan to induce the activities of lipase, gibberellic acid and indole acetic acid are endorsed for their active priming properties 40 .
The ability of chitosan to inhibit Xoo under in vitro conditions produced a positive outcome. The higher treatment concentrations of FC and PC produced significant differences in bactericidal activities, with FC performing better than PC. Kulikov et al. reported that differences among bioactivities of chitosan rely upon their source, degree of polymerization or type 43 . However, the antibacterial activity of PC and FC did not differ significantly at lower concentrations (25 and 50 ppm). Chitosan was previously documented with antibacterial activities against Escherichia coli, Staphylococcus aureus along with Bacillus sp 44 .
The effect of exogenous application of chitosan to confer resistance against BLB was analysed. The disease index of the chitosan sprayed plants was considerably reduced. The disease control mechanism is an indication of the activation of innate plant defense systems as confirmed by the secretion of pathogenesis related enzymes (POD and PPO). The POD level in TN1 increased by 59.4% when treated with FC, which was 35.3% higher than that of resistant PTB33 at the end of 144 HAS. Analogous increase in PPO levels was also observed in TN1 plants treated with FC. At 50 ppm FC concentration, the PPO level increased 67.3% compared to control and was only 16.78% less than levels in the resistant PTB33 at 144 HAS. The treatments also altered the enzyme kinetics, displaying a continuous rise till 120 HAS and remaining stable after that in both plant varieties. Meanwhile the enzyme levels in leaves treated with PC started to decrease after 120 HAS.
A corresponding chitosan induced disease resistance was reported in treated wheat seeds against Fusarium blight reducing the severity of the disease 45 47 . The effect of chitosan to modulate plant defense systems in response to various pathogens has been reported and characterised by the accumulation of phytoalexins, pathogenesis related proteins, along with proteinase inhibitors 44 . Apart from being directly toxic to pathogens, chitosan was also found to enhance host resistance in date palm against the wilt pathogen by increasing the synthesis of POD and PPO 47 . Based upon the results, the ability of chitosan treatments were shown to improve the germination capabilities along with disease resistance of rice plants. In addition to managing the peril of rice stubble, the fungal based chitosan production system clearly improved resistance as a bio-stimulant, and elicitor of the plant defense pathway, producing a better response than the commercial crustacean-based chitosan treatment. Therefore, the application of fungal chitosan in agricultural systems could diminish the undesirable influence of disease-causing pathogens on the produce and quality of rice and other crops, along with providing economic relief to growers using rice stubble for fungal chitosan production as a sustainable agricultural system that is more effective and profitable. The fungus was re-cultured in PDA, maintained in the laboratory (4 °C). Production inoculum was prepared by inoculating fungal spores in Potato Dextrose Broth (PDB-30 ml, pH 5) and incubated (28 °C; 72 h). The spores were harvested and adjusted to 2 × 10 7 Spores ml −1 using 0.1% tween 80 in sterile distilled water by haemocytometer counting 48 . (Figure 10). Xoo culture from previous experiments was used in this study 49 . The bacteria were cultured (Nutrient broth; 48 h), centrifuged (8000×g-15 min) and bacterial count was adjusted to 3 × 10 5 CFU/ml (sterile distilled water; haemocytometer counting).
Straw pre-treatment and solid-state fermentation (SSF). The process of SSF was carried out for chitosan extraction from pre-treated straw by modifying the methods of Rane and Hoover and Crestini et al. 50,51 . Rice stubble was collected from local field (Alwarkurichi, Tamil Nadu, India). Appropriate permission was obtained from the agriculture land owners before collecting the stubble. The rice straw was rinsed with tap water, shaken of excess water and then pulverized (size 1-2 cm). A synthetic medium (0.2% yeast extract, 1.0% peptone and 2.0% glucose) was developed to humidify the straw 60% water content 52 . The substrates were autoclaved at 121 °C for 20 min, inoculated with spore suspension in sterile plastic bags and closed with cotton plugs to avoid contamination by preventing air flow, then maintained at 30 °C for 15 days.   www.nature.com/scientificreports/ were extracted using 2% acetic acid at 95 °C for 12 h. The slurry was centrifuged at 11,600×g for 15 min and the acid insoluble fraction was discarded. The supernatant was collected, adjusting to a pH of 10 using 2 N NaOH producing precipitated alkali insoluble chitosan. The precipitated chitosan was collected and air dried at 60 °C to a calculated weight and deacetylated as in Zhang et al. 53 .  Bio-stimulant effect of chitosan. For the germination assays, 100 seeds of each variety per treatment-FC, PC and control were soaked in 25 ml of FC, PC and sterile distilled water (24 h). Filter paper method was used to analyse the germination parameters inclusive of emergence, germination percentage (GP), mean germination time (MGT) and germination energy (GE) 54 . The experiments were replicated five times to obtain the raw data before processing the statistical analysis.

Preparation of chitosan. Fungal chitosan extracted from
In germination assay the emerging hypocotyls were recorded every day and the mean germination time (MGT) was premeditated 55,56 by calculating the time taken for 1, 10, 25, 50, 75 and 100% of the seeds to germinate (expressed as days).
where n = number of germinated seeds at time T (25 °C). T = hours from the beginning of the germination test. Σn = final germination.
The germination percentage (GP) was calculated using the following formula Seed Germination Energy (GE) was calculated according to the formula Effect of seed treatment under green-house. Rice seeds, TN1 and PTB33 were sown (5 seeds/treatment; 0.5 L pots). The potting soil and experimental conditions were followed by the method of Kalaivani et al. 49 After 20 days of sowing (DOS), growth parameters (Total plant height, root and shoot length in cm) and biomass (fresh and dry weight-oven drying-40 °C for 2 days) of plants was determined.
Antibacterial activity of chitosan against Xoo. Bacterial suspension (10 µl; 3 × 10 5 CFU/ml of Xoo) was used in disc diffusion method to determine the antibacterial activity of FC, PC (25, 50 and 100 ppm) and sterile distilled water (control) 57 . Inhibition zones were measured in diameter (mm) post incubation (28 ± 2 °C; 48 h). Tetracycline (1 mg/ml) is used as positive control and control (0.1% acetic acid) were used in the assay. Three replication of samples were loaded in respective prelabelled wells to record the zone of incubation. The plates were kept upright position and incubated at 37 °C for 24 h.
Induced resistance by spray treatment of chitosan against BLB. Xoo inoculation. Xoo was inoculated on rice plants grown under greenhouse conditions mentioned for seed treatment assay (27-33 °C; 12 h L: D, 90% RH), 28 DAS by scissor-dip method 58 . Symptoms of BLB were observed 7 days post inoculation (DPI).
Exogenous application of chitosan. FC and PC (25 and 50 ppm), 15 ml, were sprayed uniformly on the inoculated rice plants rice plants at the maximum seedling stage in green house condition on 15th day after planting. Plants sprayed with sterile distilled water served as untreated control.
Disease assessment. Lesion length was measured on 15 DPI and data for one treatment was obtained from 40 inoculated leaves. Subdual of BLB was evaluated in terms of reduction in the mean BLB lesion length 59 .
Effect of spray treatments on the induction of defense related enzymes. Leaves