4.1 Physiological characteristics of growth and leaves
Spinach is a type of Chenopodiaceae plant that is extensively planted in facility agriculture, which has strong environmental adaptability (Chen et al., 2016; Long et al., 2013). In terms of plant phenology and biological characteristics, plant growth, leaf chlorophyll content, and other physiological characteristics are critical influencing factors (Bennie et al., 2016). The results of Yan et al. (2017) revealed that the application of microbial fertilizer could effectively improve the plant height of pakchoi. In this study, the addition of microbial fertilizer effectively improved plant height, leaf length, and leaf weight, among which the growth status of plants with the addition of Bs and Bm was more dominant. This was similar to the results of Samia et al. (2014).
In this research, microbial fertilizer was found to significantly enhanced the physiological characteristics of spinach seedling leaves (SP, SC, Chla, Chlb, Chla + b, Chla/b, and WP). Wang et al. (2018) found that the application of microbial fertilizer could effectively increase the chlorophyll content of winter wheat at the overwintering, jointing, and booting stages. The application of phosphate fertilizer can promote the synthesis of chlorophyll in leaves (Li et al., 2002).
For this study, the chlorophyll content of leaves treated with Bs and Bm was at a higher level. It may be possible that the Bs and Bm convert ineffective phosphorus into available phosphorus in the soil, which improves the availability of phosphorus and enhances the ability of plants to absorb it, thus promoting the synthesis of ATP and NADPH in leaves. When plants are under stress, the MDA content is typically an important indicator of membrane lipid peroxidation, which reflects the harmful effects of stress on plant cells and tissues (Han et al., 2013). In this study, the concentration of MDA in leaves treated with microbial fertilizer decreased.
4.2 Leaf chlorophyll fluorescence
In this research, the values of F0 and Fm were enhanced, which indicated that the Bs and Bm promoted an increase in the size of leaf PSII antenna and a decrease in the non-radiative dissipation of chlorophyll in PSII antenna, thus increasing the capacity of leaves to capture light energy (Oukarroum et al., 2016). We observed higher Fv and Fv/F0 (nFv) values under the Bs and Bm treatments, which signified an increase in the efficiency of supplying electrons to PSII RCS and the photosynthetic quantum conversion rate of PSII RCS. This translated to less energy being used for non-photochemical dissipation in the dark adaptation process (Spoustova et al., 2013).
In this experiment, the ABS/RC values of the leaves increased, indicating that the Bs and Bm promoted the increased size of active RCS, which led to a higher number of active PSII reaction centers and enhanced dark accumulation (Pooja et al., 2010). Under the action of the Bs and Bm, the TR0/RC value increased, which reflected the higher electron capture rate of RC, where more QA was converted to QA−. This resulted in an increase in the electron transfer energy (ET0/RC), thus reducing the energy dissipated by non-photochemical activities (DI0/RC) (Yiotis and Manetas, 2010; Tang et al., 2017).
The φP0 (Fv/Fm) represents the maximum quantum yield of PS Ⅱ, and the ψ0 value reflects the electron transfer efficiency, from QA− to QB, whereas φE0 reflects the quantum yield of electron transport. The increase of φP0, ψ0, and φE0 indicated that the Bs and Bm promoted the redox reaction following QA, which resulted in an increase of the electron transfer rate between QA− and QB (Lebkuecher et al., 1999).
The change of the chlorophyll fluorescence curve is closely related to the physiological morphology of plants (Malaspina et al., 2015). Changes in the O-J segment are related to an increased number of inactive reaction centers, or the energy transfer from the LHCII to PSII reaction center (Tomar and Jajoo, 2013). The K and L bands reflect the connection between the S state of the PSII oxygen evolution complex (OEC) and PSII unit, as well as the energy connection between the PSII units (DaBrowski et al., 2016; Paunov et al., 2018).
An increase in the J-I segment can reflect a decrease in the relative number of active PQ molecules that is reduced by each active RC of PSII ((Paunov et al., 2018). Further, changes in the I-P segment are closely related to the pool of electron receptors (ferredoxin and NADPH) at the end of the PSI, signifying the kinetic flow rate to the electron receptors at the end of the PSI (Tsimilli Michael and Strasser, 2013). In this experiment, the OJIP transient curves of the spinach seedlings were affected by microbial fertilizer (Fig. 1).
Under the treatment of Bs and Bm, the relative fluorescence intensity of chlorophyll in the O-J segment exhibited a larger value, indicating that the population of active PSII centers decreased, whereas the QA− accumulated massively. Figures 1E G show that there were obvious K bands and positive L bands under the Bs and Bm treatment, which indicated that the PSII had an inhibitory effect on the OEC. This resulted in a weakening of the connectivity between PSII and OEC and a decreased energy connection between the PSII units.
This may have been due to the variable light and ventilation that was present at different locations in the incubator, which may have led to PSI receptor side damage and chlorophyll protein denaturation in some plant leaves (Bertamini and Nedunchezhian, 2003). In addition, the fluorescence of J-I and I-P segments remained large under the Bs and Bm treatment, which indicated that the relative number of PQ decreased, while there was an increase in the pool of electron receptors (ferredoxin and NADPH) at the end of PSI, which led to a higher kinetic flow rate to the electron receptors at the end of PSI.
Combined with PIABS, the higher PIABS values under the Bs and Bm treatment indicated increases in the density of active PSII centers, the efficacy of photoreactions, and efficiency of biochemical dark redox reactions, as well as the production and utilization of NADPH (Habib et al., 2016).