Introduction

Plant endophytes refer to fungi or bacteria that live in plants (Bacon and White 2016), which are usually distributed in the interstitial space or inside the cells of the plant. Colonized in different tissues of the plant, plant endophytes can often act on the entire plant body (Hurek et al. 1994; Barrow 2003; Verma et al. 2004). Most endophytes and host plants have a mutually beneficial and symbiotic relationship. Although endophytes rely on the nutrients of the plant host for survival, most endophytes will not cause harm to the host plant, but can promote the growth of the host plant (Ali et al. 2017; Jaber and Enkerli 2017). Plant endophytes can secrete and produce substances such as plant growth hormone, cytokinin and gibberellin. Furthermore, endophytes can fix nitrogen and dissolving phosphorus, helping plants resist environmental stress (Montañez et al. 2012; Truyens et al. 2015). Different endophytes produce different effects on plants. Certain endophytic fungi will produce toxic compounds like alkaloids, terpenes, polyketones, phenols, which act to protect plants from insects and pathogenic bacteria. For example, the neurotoxic indole-diterpenoid alkaloids produced by fungi can poison herbivorous insects that ingest them (Siegel et al. 1990). Some endophytic bacteria can produce 1-aminocyclopropane-1- carboxylate (ACC) deaminase, which can reduce the content of ACC and 2-oxobutanoat in plants, inhibiting the synthesis of ethylene and delaying plant senescence (Hardoim et al. 2015).

A variety of endophytic florae are distributed in rice (Walitang et al. 2017). Zhang et al. (2019) used the simple sequence repeat method to study genetic diversity in five rice varieties with different genotypes and used a 16S rRNA gene bank to analyze genetic differences of endophytic flora of rice seeds. They found that endophytic abundance distributions were significantly different. Walitang et al. (2018) crossbred different rice seeds under different growth environments and carried out multiple generations of repeated inbreeding to study the differences in endophytic communities. The endophytic community composition was highly stable and did not significantly change over time. They then used end-restriction fragment length polymorphism analysis to evaluate the distribution of endogenous bacterial communities, and found significant differences in the diversity of endogenous bacteria between the primary generation and the progeny in terms of richness, uniformity, and diversity index.

When plants undergo oxidative stress, they produce a large number of oxidative intermediates, ultimately causing oxidative damage to plants (Yuan et al. 2019). Plants can resist stress conditions by using enzymatic and non-enzymatic antioxidant defense mechanisms. Endophytes enhance the defensive capability of host plants by contributing exogenous antioxidant enzymes and non-enzymatic antioxidants. These antioxidants help avoid excessive accumulation of reactive oxygen species (ROS) in plants, reducing oxidative damage that occurs during biotic and abiotic stress (Khan et al. 2013). Indeed, Qin et al. (2019) isolated 12 endophytic fungi from the roots and leaves of Myricaria laxiflora, and found that Aspergillus fumigatus strain SG-17 exhibited a strong antioxidant capacity both in vivo and in vitro. The main antioxidant in A. fumigatus is (Z)-N-(4-hydroxystyryl) formamide (NFA), an analogue of coumarin. Plant growth under four different conditions were observed: drought, humid, drought+SG-17 crude extract and drought + proline. Qin et al. (2019) found that plants in drought+SG-17 crude extract group could grow normally. The reason is that Aspergillus fumigatus can maintain the integrity of plant cell membranes and regulate the content of NADPH oxidase, antioxidant enzymes and Heat Shock Protein (HSP). Where, NADPH oxidase can directly reduce the production of ROS, antioxidant enzymes and HSP can promote the degradation of ROS. Ullah et al. (2019) isolated two endophytic strains from Solanum nigrum, identified them as Serum bacillus IU01 and enterobacteria IU02 by 16S DNA sequencing, and then placed Brassica juncea plants inoculated with Serum bacillus strains IU01 or IU02 in environments with different Cd concentrations. The results showed that the activities of antioxidant enzymes such as catalase (CAT), polyphenol oxidase (PPO) and peroxidase (POD) in plants exposed to different concentrations of Cd stress were significantly increased when endophytes were not inoculated. However, the antioxidant enzyme activities in plants exposed to different concentrations of Cd stress were significantly reduced and plant tolerance to Cd and antioxidant capacity were significantly improved after inoculation of IU01 and IU02, suggesting that endophytes can reduce the damage of Cd stress on plants.

At the present, most studies on black rice have targeted bioactive substances such as anthocyanins and flavones (Pal et al. 2019). There are few studies about the effects of endophyte antioxidant activity on black rice (Verma et al. 2017; Greetatorn et al. 2019). Furthermore, the endophytic communities have yet to be characterized for black rice with high nutritional value. In previous studies, 8 endophytes (including 5 fungi and 3 bacteria) have been isolated from black rice seeds. In this paper, we aimed to study the effects of these 8 endophytes on the antioxidant capacity of black rice.

Materials and Methods

Fig. 1: T-AOC of black rice "1C902" treated with 8 entophytes

Different lower- or upper-case letters in each treatment indicate significant differences (P < 0.05 or P < 0.01), the same below

Fig. 2: T-AOC of black rice "1C903" treated with 8 entophytes

Experimental materials

Five types of endophytic fungi in black rice (POB15-1, POB15-11, POB15-15, POB14-7, POB14-10), 3 types of endophytic bacteria in black rice (NOB14-2, NOB15-2, NOB15-9), isolated from black rice seeds and kept separately in the laboratory, were used in this study. Two black rice varieties "1C902" and "1C903", planted in a rice test field in Zhaoan County, Fujian Province, China, were used in this study. Beef extract peptone (NA) medium (pH 7.0) was comprised of 3 g L^-1 of beef extract, 10 g L^-1 of peptone, 5 g L^-1 of sodium chloride, and 15 g L^-1 of agar.

Preparation of endophytes

After activation with the Potato Dextrose Agar (PDA) medium, the 5 types of endophytic fungi were cultured in a 28°C incubator for 7 d, and the fungus lawn was washed with sterile water to dilute the spore fluid concentration to 3×10⁶ CUF mL^-1. After activation with the NA medium, the 3 types of endophytic bacteria were cultured in a 37°C incubator for 1 d, and the bacterial lawn was washed with sterile water to dilute the bacterial solution concentration to 3×10⁶ CUF mL^-1.

Endophyte spraying experiment

Black rice varieties "1C902" and "1C903" were planted via single seed transplant. Endophytes were sprayed at the heading stage of rice, mainly on rice ears and leaves. Each microbial inoculum was sprayed on three plants of each rice variety, and the same type of rice without endophyte spraying was used as a control. After harvesting, the rice seeds were dried in a 32°C drying oven, and then processed into brown rice, crushed and sifted with an 80-mesh sieve for further use.

Determination of total antioxidant capacity and hydroxyl radical-scavenging capacity

Exactly 0.1 g of black rice flour was accurately weighed, 10 mL of 80% ethanol was added, and then the solution was diluted to 10 mL after digestion in a water bath at 80°C for 3 h. The solution was shaken well and then left to stand. The supernatant was stored for later use. Total Antioxidant Capability (T-AOC) and Hydroxyl Radical-Scavenging Capacity (SFRC) of black rice were measured using test kits (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., China), according to instructions. Each measurement was repeated 3 times.

Statistical analysis

The recorded data in this study were statistically analyzed using DPS data processing system.

Results

Effects of spraying endophytes on black rice T-AOC

From Fig. 1 and Table 1, it can be seen that spraying all 8 kinds of endophyte greatly increased the T-AOC of black rice variety "1C902". Increase rate was as follows: 1C902-POB15-15>1C902-POB14-10>1C902-POB15-1>1C902-NOB15-2>1C902-POB15-11>1C902-NOB14-2>1C902-POB14-7>1C902-NOB15-9. The strains POB15-15, POB14-10, POB15-1 and NOB15-2 were found to increase the T-AOC of black rice variety "1C902" by 6.3, 5.6, 4.9 and 4.0 times, respectively, increasing by 832.838, 731.584, 648.786 and 529.187 IU g^-1, respectively. These four endophytes had an excellent effect on improving the T-AOC of the black rice variety "1C902", each of which increased the T-AOC by more than 4 times. Furthermore, after spraying the three strains of NOB15-2, POB15-11 and NOB14-2, although there were no significant differences on T-AOC of black rice variety "1C902" (Fig. 1), there were very significant differences between T-AOC in the other five strains.

After spraying 8 kinds of endophytes on black rice variety "1C903", there were very significant differences in the effect on T-AOC (Fig. 2 and Table 1). 3 types of endophytes significantly increased the T-AOC of black rice variety "1C903". Increase rate was as follows: POB15-15> POB14-7> POB14-10. There was a relative increase of 110.2%, 69.6% and 19.7% for respectively, with T-AOC increasing by 946.455, 597.738 and 169.549 IU g^-1, respectively. After spraying NOB15-9, no significant difference was observed in the T-AOC and CK of black rice variety "1C903". Furthermore, the other four endophytes (POB15-1, POB15-11, NOB14-2 and NOB15-2) lowered the T-AOC of black rice variety "1C903". That is, spraying these four endophytes was unfavorable for T-AOC of black rice variety "1C903".

Table 1: Variance analysis of T-AOC and SFRC in black rice "1C902" or "1C903" treated with 8 entophytes

***, significant at P ≤ 0.001; ns: Non significant

Effects of spraying endophytes on black rice SFRC

After spraying 8 different kinds of endophytes, there were very significant differences in the SFRC of black rice variety "1C902" (Fig. 3 and Table 1). Where, 6 strains were found to significantly increase the SFRC of black rice variety "1C902", as follows: NOB15-2>POB15-11>NOB14-2>POB14-10>POB15-15>NOB15-9. The first four strains had increase rates above 50%, the highest of which was 1.3 times, showing increase amounts of 1853.348, 1069.956, 862.725 and 807.013 IU g^-1, for respectively. These four endophytes significantly improved the SFRC of black rice variety "1C902". However, spraying the strains POB14-7 and POB15-1 were found to lower the SFRC of black rice variety "1C902", indicating that the spraying of these two endophytes is unfavorable for this rice variety.

Spraying 8 kinds of endophytes had significantly different effects on SFRC of black rice "1C903" (Fig. 4 and Table 1). Where, 4 strains can significantly increase SFRC of black rice "1C903", whose increase rate follows the order of: POB15-1> NOB15-2> POB15-11> NOB14-2. The first three strains have an increase rate of above 40%, the highest of which is 82.1%, and the increase amounts are 848.189, 566.398 and 444.608 IU g^-1, respectively. These three endophytes were better for improving the SFRC of black rice variety "1C903" than all other varieties. Spraying the remaining 4 strains (NOB15-9, POB14-10, POB15-15 and POB14-7) were found to decrease the SFRC of the black rice variety "1C903", and spraying strain POB14-7 will significantly lower SFRC of black rice "1C903".

Discussion

In this study, 8 endophytes were sprayed to two varieties of black rice at the heading stage, some of these endophytes obviously improved the T-AOC and SFRC of black rice. This indicated that spraying the black rice with endophytes can promote their production of antioxidants and their ability to resist stress.

Fig. 3: SFRC of black rice "1C902" treated with 8 entophytes

Fig. 4: SFRC of black rice "1C903" treated with 8 entophytes

The interaction between endophytes and host plants is primarily used for three purposes: 1) mitigation of the abiotic stress of host plants (such as drought and heavy metals), 2) protection of host plants against the threat of biotic stress (pathogens and herbivores), and 3) promotion og the growth of host plants by adding inorganic substances such as nitrogen, phosphorus and iron (Sessitsch et al. 2012).

Plant endophytes can produce antioxidants such as PPO, POD, CAT and superoxide dismutase (SOD), which can help host plants improve their ability to resist stress (Ullah et al. 2019). In addition to exogenous antioxidant enzymes, endophytes can also produce some non-enzymatic antioxidants, such as polysaccharides (Chen et al. 2011), and lectins that can regulate the activity of antioxidant enzymes (Alen Kina et al. 2018). Chen et al. (2011) isolated a exopolysaccharides homogenate (As1-1) consisting of mannose and a small amount of galactose from the medium of mangrove endophytic fungus Aspergillus Y16. Through in vitro experiments, they found that like vitamin C and other antioxidants, As1-1 could remove DPPH free radicals and superoxide radicals. Alen kina et al. (2018) studied the ability of two lectins (Sp7 and Sp245) produced by Azospirillum brasilence to regulate the activities of POD, CAT and SOD in wheat seedling roots under short-term, low and high temperature environmental stress conditions. Results demonstrated that these two lectins could significantly change the activities of related enzymes within the first few minutes under stress conditions. Both lectins could increase the activities of POD and SOD in wheat roots under stress, thereby enhancing the wheat's adaptability to severe environments.

Although the endophytes could improve the antioxidant capacity of black rice, there is no static law concerning the effects of endophytic bacteria and endophytic fungi on the antioxidant properties of rice. Indeed, the effects of endophytes on antioxidant properties of different varieties of rice appear to vary. This is similar results found by Afridi et al. (2019), who planted two varieties of wheat (Khirman and Pasban 90) under salt stress and inoculated endophytes the Kocuria rhizophila (14ASP) and Cronobacter sakazakii (OF115). They found that Pasban 90 had significantly higher antioxidant activity (POD, SOD and CAT) than Khirman, indicating that the effects of endophytes on the antioxidant properties of different wheat varieties differ.

Spraying different endophytes to black rice had different variation in T-AOC and SFRC in this study. After spraying with the 8 endophytes, the T-AOC of black rice variety "1C902" was greatly improved, the highest increase of which was 6.3 times. However, the black rice variety "1C903" had less of an increase in T-AOC than variety "1C902", as the highest increase was only 1.1 times. Furthermore, 4 kinds of endophytes reduced the T-AOC of black rice variety "1C903" after spraying. The reason for this may be that black rice variety "1C903" has much higher T-AOC (859.063 IU g^-1) than "1C902" (130.589 IU g^-1). Therefore, the improvement effect of T-AOC after spraying endophyte is obviously inferior in the former, and T-AOC may even be reduced. SFRC of black rice also exhibits a similar response pattern. Except for when strain POB15-1 was applied, black rice "1C902" generally had higher increase in SFRC than "1C903" after endophyte spraying. This demonstrates two things. First, different endophytes have significantly different effects on the antioxidant capacity within the same rice variety. Second, the same endophyte can have very different effects on the antioxidant capacity in different varieties of rice.

Conclusion

Spraying endophytes to black rice at the heading stage can improve the antioxidant capacity of black rice. But there are significant differences in the effects of different endophytes on the antioxidant capacity of black rice and there are significant differences in the effects of endophytes on the antioxidant capacity of different rice varieties. In this study, eight endophytes were found to significantly increase the T-AOC of the black rice variety "1C902" (up to 6.3 times higher than in untreated rice). Furthermore, 6 strains could significantly increase the SFRC of the black rice variety "1C902" (up to 1.3 times higher than in untreated rice). Three strains could also significantly increase the T-AOC of the black rice variety "1C903" (up to 1.1 times higher than in untreated rice). Finally, 4 strains could significantly increase SFRC of the black rice variety "1C903" (up to 82.1% higher than in untreated rice). So, in order to improve the antioxidant capacity of different variety of black rice, different endophytes should be chosen to infect it.

Acknowledgments

This study was supported financially by the Special Fund for Science and Technology Innovation of Fujian Agriculture and Forestry University (CXZX2019) and the National Key Research and Development Program (2017YFD0100100) in China.

Author Contributions

JZ and ZH designed the study; YX and YZ performed the experiments, conducted the analysis, and wrote the paper; QW assisted in analyzing the data and interpreted the results; XC assisted in performing the experiments. All authors read and approved the final manuscript.

References

Afridi MS, Amna, Sumaira, T Mahmood, A Salam, T Mukhtar, S Mehmood, J Ali, Z Khatoon, M Bibi, MT Javed, T Sultan, HJ Chaudhary (2019). Induction of tolerance to salinity in wheat genotypes by plant growth promoting endophytes: Involvement of ACC deaminase and antioxidant enzymes. Plant Physiol Biochem 139:569–577

Alen Kina SA, NI Romanov, VE Nikitina (2018). Regulation by Azospirillum lectins of the activity of antioxidant enzymes in wheat seedling roots under short-term stresses. Braz J Bot 41:579–587

Ali S, TC Charles, BR Glick (2017). Endophytic Phytohormones and their role in plant growth promotion. In: Functional Importance of the Plant Microbiome, pp:89–105. Doty SL (Ed.). Springer, Cham, Switzerland

Bacon CW, JF White (2016). Functions, mechanisms and regulation of endophytic and epiphytic microbial communities of plants. Symbiosis 68:87–98

Barrow JR (2003). Atypical morphology of dark septate fungal root endophytes of Bouteloua in arid southwestern USA rangelands. Mycorrhiza 13:239–247

Chen Y, W Mao, H Tao, W Zhu, X Qi, Y Chen, H Li, C Zhao, Y Yang, Y Hou, C Wang, N Li (2011). Structural characterization and antioxidant properties of an exopolysaccharide produced by the mangrove endophytic fungus Aspergillus spp. Y16. Bioresour Technol 102:8179–8184

Greetatorn T, S Hashimoto, S Sarapat, P Tittabutr, N Boonkerd, T Uchiumi, N Teaumroong (2019). Empowering rice seedling growth by endophytic Bradyrhizobium sp. SUTN9-2. Lett Appl Microbiol 68:258–266

Hardoim PR, LSV Overbeek, G Berg, AM Pirttila, S Compant, A Campisano, M Doring, A Sessitsch (2015). The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes. Microbiol Mol Biol Rev 79:293–320

Hurek T, B Reinhold-Hurek, MV Montagu, E Kellenberger (1994). Root colonization and systemic spreading of Azoarcus spp. strain BH72 in grasses. J Bacteriol 176:1913–1923

Jaber LR, J Enkerli (2017). Fungal entomopathogens as endophytes: can they promote plant growth? Biocontr Sci Technol 27:28–41

Khan AL, M Waqas, M Hamayun, A Al-Harrasi, A Al-Rawahi, IJ Lee (2013). Co-synergism of endophyte Penicillium resedanum LK6 with salicylic acid helped Capsicum annuum in biomass recovery and osmotic stress mitigation. BMC Microbiol 13:51-63

Montañez A, AR Blanco, C Barlocco, M Beracochea, M Sicardi (2012). Characterization of cultivable putative endophytic plant growth promoting bacteria associated with maize cultivars (Zea mays L.) and their inoculation effects in vitro. Appl Soil Ecol 58:21–28

Pal S, TB Bagchi, K Dhali, A Kar, P Sanghamitra, S Sarkar, M Samaddar, J Majumder (2019). Evaluation of sensory, physicochemical properties and Consumer preference of black rice and their products. J Food Sci Technol 56:1484–1494

Qin W, C Liu, W Jiang, Y Xue, G Wang, S Liu (2019). A coumarin analogue NFA from endophytic Aspergillus fumigatus improves drought resistance in rice as an antioxidant. BMC Microbiol 19; Article 50

Sessitsch A, P Hardoim, J Döring, F Friedrich, T Hurek, M Rahalkar, AB Reinhold-Hurek (2012). Functional Characteristics of an Endophyte Community Colonizing Rice Roots as Revealed by Metagenomic Analysis. Mol Plant Microb 25:28–36

Siegel MR, GC Latch, LP Bush, FF Fannin, DD Rowan, BA Tapper, CW Bacon, MC Johnson (1990). Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response. J Chem Ecol 16:3301–3315

Truyens S, N Weyens, A Cuypers, J Vangronsveld (2015). Bacterial seed endophytes: genera, vertical transmission and interaction with plants. Environ Microbiol Rep 7:40–50

Ullah I, BO Al-Johny, KMS AL-Ghamdi, HAA Al-Zahrani, Y Anwar, A Firoz, N AL-Kenani, MAA Almatry (2019). Endophytic bacteria isolated from Solanum nigrum L., alleviate cadmium (Cd) stress response by their antioxidant potentials, including SOD synthesis by sodA gene. Ecotoxicol Environ Saf 174:197–207

Verma SC, A Singh, SP Chowdhury, AK Tripathi (2004). Endophytic colonization ability of two deep-water rice endophytes, Pantoea spp. and Ochrobactrum spp. using green fluorescent protein reporter. Biotechnol Lett 26:425–429

Verma SK, K Kingsley, I Irizarry, M Bergen, RN Kharwar, JF White (2017). Seed-vectored endophytic bacteria modulate development of rice seedlings. J Appl Microbiol 122:1680–1691

Walitang DI, CG Kim, S Jeon, Y Kang, T Sa (2018). Conservation and transmission of seed bacterial endophytes across generations following crossbreeding and repeated inbreeding of rice at different geographic locations. Microbiologyopen 8; Article e00662

Walitang DI, K Kim, M Madhaiyan, YK Kim, Y Kang, T Sa (2017). Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of rice. BMC Microbiol 17; Article e00662

Yuan X, H Wang, J Cai, Y Bi, D Li, F Song (2019). Rice NAC transcription factor ONAC066 functions as a positive regulator of drought and oxidative stress response. BMC Plant Biol 19; Article 278

Zhang J, C Zhang, J Yang, R Zhang, J Gao, X Zhao, J Zhao, D Zhao, X Zhang (2019). Insights into Endophytic Bacterial Community Structures of Seeds Among Various Oryza sativa L. Rice Genotypes. J Plant Growth Regul 38:93–102