3.1 Germination energy and rate
The germination energy of A. inebrians seeds was significantly (P < 0.05) affected by Bacillus strains (F = 6.268, P < 0.000), Epichloë endophyte (F = 6.416, P = 0.014) and their interaction (F = 2.372, P = 0.016) (Table 2). The germination energy of EI A. inebrians was significantly (P < 0.05) higher than that of EF plants after inoculation with B154 strain (Fig. 1a). The germination energy of B154(EI), B181(EI), B382(EI), B419(EF), and B453(EF) A. inebrians seeds was significantly (P < 0.05) higher than that of seeds of CK plants (Fig. 1a).
Table 2 Two-way analysis of variance (ANOVA) for the effects of presence or absence of the Epichloë endophyte (E) and Bacillus (B) treatments on seeds germination of A. inebrians
Inoculation with Bacillus strains (F = 3.429, P < 0.010) had significant (P < 0.05) effects on germination rate of A. inebrians seeds, but Epichloë endophyte infection (F = 1.833, P = 0.812) and the interaction between Bacillus strain and Epichloë endophyte (F = 0.652, P = 0.0788) had no significant difference (Table 2). Compared with the CK, inoculation of B419 significantly (P < 0.05) increased the germination rate of A. inebrians seeds, up to 99% (Fig. 1b).
3.2 Length of seedling and radicle
The seedling length was significantly (P < 0.05) affected by Bacillus strains inoculation (F = 4.326, P < 0.000), Epichloë endophyte infection (F = 29.631, P < 0.000), and their interaction (F = 3.681, P < 0.000) (Table 2). The seedling length of EI A. inebrians was significantly (P < 0.05) greater than that of EF plants after inoculation with B10, B15, and B419 (Fig. 2a). The seedling length of B4(EI), B9(EI), B10(EI), B15(EI), B181(EF), and B419(EI) A. inebrians was significantly (P < 0.05) greater than that of CK plants (Fig. 2a).
There were significant (P < 0.05) differences on the radicle length with Bacillus strains inoculation (F = 7.674, P < 0.000), Epichloë endophyte infection (F = 15.495, P < 0.000) and their interaction (F = 2.372, P = 0.030) (Table 2). The radicle length of EI A. inebrians was significantly (P < 0.05) greater than that of EF seedlings after inoculation with B10 and B106 (Fig. 2b). The radicle length of B10(EI), B18(EI), B18(EF), and B419(EI) A. inebrians was significantly (P < 0.05) greater than that of CK seedlings (Fig. 2b).
3.3 Plant height, root length, tiller number and chlorophyll content
Inoculation of two-month-old plants with Bacillus strains (F = 16.228, P < 0.000) and the interaction between Bacillus strains and Epichloë endophyte (F = 6.629, P < 0.000) had significant (P < 0.05) effects on plant height (Table 3). Compared with the CK, inoculated with B4, B9, B18, B154, B181 and B419 all significantly (P < 0.05) increased the plant height, while the treatments of B10, B15, and B106 strains showed significant (P < 0.05) inhibitory effects (Fig. 3a).
Table 3 Two-way analysis of variance (ANOVA) for the effects of presence or absence of the Epichloë endophyte (E) and Bacillus (B) treatments on plant growth of A. inebrians
Inoculation with Bacillus strains (F = 12.900, P < 0.000), Epichloë endophyte infection (F = 9.754, P = 0.003), and their interactions (F = 6.503, P < 0.000), all had significant (P < 0.05) effects on root length (Table 3). The root length of EI A. inebrians was significantly (P < 0.05) greater than that of EF plants under inoculation with B4, B96, B181, B382, B419 and B453 (Fig. 3b). Except for B4(EF), B96(EF), B106(EI), B106(EF), B181(EF), and B453(EF), the other treatments all significantly (P < 0.05) increased the root length of A. inebrians (Fig. 3b).
Inoculation with Bacillus strains (F = 16.067, P = 0.005) and the interaction between Bacillus strains and Epichloë endophyte (F = 2.806, P < 0.000) had significant (P < 0.05) effects on tiller numbers (Table 3). Compared with the CK, except for the B10, B106, and B453, other strains all significantly (P < 0.05) promoted the tiller numbers of A. inebrians (Fig. 3c). The tiller numbers of A. inebrians plants inoculated with strain B419 were significantly (P < 0.05) higher than that of other treatments (Fig. 3c).
Epichloë endophyte infection (F = 18.909, P < 0.000) and inoculation with Bacillus strains (F = 65.686, P < 0.000) significantly (P < 0.05) affected chlorophyll content (Table 3). Compared with the CK, the Bacillus strains, except for strain B96, significantly (P < 0.05) increased chlorophyll content of A. inebrians (Fig. 3d). After inoculation with B18 and B106, the chlorophyll content of EI A. inebrians was significant (P < 0.05) higher than of EF plants (Fig. 3d). The chlorophyll content of A. inebrians inoculated with B18 strain was significantly (P < 0.05) higher than that of other treatments (Fig. 3d).
3.4 Fresh and dry weight
The shoot fresh weight of A. inebrians was significantly (P < 0.05) affected by the treatment of Bacillus strains (F = 26.885, P = 0.000), Epichloë endophyte infection (F = 8.829, P = 0.004) and their interaction (F = 2.139, P = 0.030) (Table 3). After inoculation with strains B18 and B419, the shoot fresh weight of EI A. inebrians was significantly (P < 0.05) higher than of EF plants (Fig. 4a). Except for B4(EF), B9(EF), B10(EF), B106(EI), B106(EF), B453(EI), and B453(EF), the other treatments all significantly (P < 0.05) increased the shoot fresh weight of A. inebrians (Fig. 4a).
Bacillus strains inoculation (F = 54.498, P < 0.000) and the interaction between Bacillus strains and Epichloë endophyte (F = 6.419, P < 0.000) had significant (P < 0.05) effects on shoot dry weight (Table 3). Compared with the CK, except for the strain B106, other strains all significantly (P < 0.05) enhanced the shoot dry weight of EI and EF A. inebrians (Fig. 4b). The shoot dry weight of EI A. inebrians was significantly (P < 0.05) higher than of EF plants under strain B419 treatment, while the shoot dry weight of EI A. inebrians was significantly (P < 0.05) lower than of EF plants under B106 B181, and B453 treatments (Fig. 4b).
There were significant (P < 0.05) differences on root fresh weight with Bacillus inoculation (F = 15.221, P < 0.000), Epichloë endophyte infection (F = 23.814, P < 0.000) and their interaction (F = 4.202, P < 0.000) (Table 3). The root fresh weight of EI A. inebrians was significantly (P < 0.05) higher than of EF plants under B15, B18, B96, and B419 treatments (Fig. 4c). The root fresh weight of B15(EI), B18(EI), B18(EF), B96(EI), B154(EI), B419(EI), and B419(EF) was significantly (P < 0.05) higher than of CK plants, and the root fresh weight of B18(EI) was significantly (P < 0.05) higher than B18(EF), B154(EI), and B419(EF) plants (Fig. 4c).
Inoculation with Bacillus strains (F = 14.812, P < 0.000), endophyte infection (F = 6.419, P < 0.000), and their interaction (F = 3.245, P = 0.002), all had significant (P < 0.05) effects on root dry weight (Table 3). The root dry weight of EI A. inebrians was significantly (P < 0.05) higher than of EF plants under B15 and B96 treatments (Fig. 4d). The root dry weight of B15(EI), B18(EI), B18(EF), B96(EI), B154(EI), B181(EF), B419(EI), and B419(EF) was significantly (P < 0.05) higher than of CK plants, and the root dry weight of B18(EI) was significantly (P < 0.05) higher than B154(EI) and B181(EF) plants (Fig. 4d).
3.5 Membership function value and rank of A. inebrians growth parameters
As shown in Table 4, the average membership function value is the average value of 4 germination experiment indicators and 8 pot experiment parameters. The larger the average membership function value is, the better is the comprehensive evaluation of the strain. According to the rank of the average membership function value, three strains were found to have excellent growth promoting ability, specifically B419 > B18 > B181. These three strains showed excellent growth promoting ability both in the seedling stage and the mature stage of the plant, and so could be used in the research of microbial fertilizer. There were three strains with a poorer comprehensive evaluation, namely B106 < B453 < B10. Because the membership function value of their pot experiment parameters was low, the comprehensive evaluation of these three strains was poor.
Table 4 The membership function value and rank of Bacillus strains to the growth parameters of A. inebrians
Note: GE, GR, RL1, SL, PH, RL2, TN, SFW, SDW, RFW, RDW and Y respectly represent the germination energy, germination rate, radicle length, seeding length, plant height, root length, tiller number, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight and chlorophyll content.