Effects of Microbial Fertilizer on the Growth, Physiology, and Chlorophyll Fluorescence Response of Spinach Seedlings

We investigated the effects of different types of bacillus on the growth, physiological characteristics, and chlorophyll uorescence of spinach seedlings in the soil environment of a Qinxi demonstration garden in Taibai County. Five different fertilization treatments were conducted in the growing room: no fertilizer (CK), addition of Bacillus subtilis (Bs, T1), addition of Bs and Bacillus mucilaginosus (Bm) (T2), addition of Bs and Bacillus amyloliquefaciens (Ba) (T3), and addition of Bs and Ba (T4). There were signicant differences in the plant height (PH), leaf length (LL), and leaf weight (LW) of the spinach seedlings (F=54.37, 13.30, and 46.03, respectively; P<0.01). The growth and physiological characteristics of the spinach seedlings attained a maximum under the Bs and Bm treatments. There were differences in the activities of the PSII reaction centers between the ve treatments. Under the Bs and Bm treatments, the ABS/RC, TR 0 /RC, and ET 0 /RC increased signicantly, while the DI 0 /RC decreased. The OJIP curve increased under different types of fertilization, and the growth trends under the Bs and Bm treatments were the largest. The leaf light response curve (LC) increased signicantly under the Bs and Bm treatments. The plant growth characteristics (LL, LW, PH) were positively correlated with the J-I-P test chlorophyll uorescence parameters (PI ABS , φP 0 , φE 0 , ψ 0 , TR 0 /RC, and ET 0 /RC), and negatively correlated with φD 0 and DI 0 /RC. The leaf physiological characteristics (SP, SC, Chla, Chlb, Chla+b, Chla/b, and WP) were positively correlated with the J-I-P test chlorophyll uorescence parameters (PI ABS , φP 0 , φE 0 , ψ 0 , ABS/RC, TR 0 /RC, and ET 0 /RC), and negatively correlated with φD 0 and DI 0 /RC. The leaf MDA was signicantly positively correlated with φD 0 and DI 0 /RC, and positively correlated with other J-I-P test chlorophyll uorescence parameters. The Bs and Bm treatments promoted the growth of the spinach seedlings and improved the adaptability of the crops to soil by enhancing the effective phosphorus utilization rate.


Introduction
In recent years, with the rapid development of the vegetable industry in China, fertilization has become the main strategy for improving yields. With the cultivation of vegetables gradually increasing over the years, the excessive application of chemical fertilizers has led to progressively serious soil problems. Therefore, microbial fertilizers have gradually replaced chemical fertilizers as a good method to improve soil quality, while increasing vegetable yields. Microbial fertilizers are also referred to as bacterial fertilizer, biological fertilizer, and microbial inoculant (Shen et al., 2011), which contain bene cial microorganisms that provide fertilization for crops through their activities. The metabolic activities of microorganisms can improve soil fertility and crop quality, while increasing soil microorganism populations, reduce plant diseases, and enhance plant root activities. Cao et al. (2011) and Manjurul et al. (2012) showed that biological fertilizers rich in Trichoderma could increase mustard and tomato yields, while reducing the need for the excessive use of N, P, and K in crop cultivation.
Bacillus is a type of growth-promoting bacteria that can produce phytase at the rhizosphere of plants. It has strong resistance to ultraviolet light, high salt, high acid, high heat, and radiation, with the capacity to inhibit bacteria, prevent disease, and increase production (Cavalcanti et al., 2018;Guo et al., 2009). Bacillus subtilis can improve the stress resistance of plants (Pliego and Ramos., 2010) and the availability of nitrogen and phosphorus in soil (Idriss and Borriss., 2002). Bacillus mucilaginosus converts the unusable phosphorus in soil to available phosphorus for plants. Simultaneously, Bm can secrete substances that promote plant growth and development, such as growth hormones, gibberellin, and more (Liu et al., 2016;Zhao et al., 2009). Bacillus amyloliquefaciens has a certain antagonistic effect on bacterial diseases. Arrebola et al. (2009) showed that Bacillus amylolyticus PPCB004 could inhibit the mycelial extensions of Penicillium fungi.
Photosynthesis is a process through which plants convert captured light energy into biochemical energy. Chlorophyll uorescence can re ect the processes of light reactions in plants (Goltsev et al., 2009). The chlorophyll uorescence technique can accurately analyze the distribution of light energy without harming the leaves of plants, and is considered a rapid and nondestructive probe for measuring leaf photosynthesis. Through the analyses of chlorophyll uorescence parameters, a further elucidation of the light energy absorption, utilization, transfer, and dissipation of plant chloroplast PSI and PSII processes can be obtained (Li et al., 2005).
The chlorophyll uorescence technique has also been widely investigated for the detection of The purpose of this study was to explore the effects of different types of Bacillus on the growth, physiology, and chlorophyll uorescence characteristics of spinach seedlings, and subsequently, to make reasonable plans for improving the quality and yields of spinach.

Experimental materials
These experiments employed the soil of a greenhouse at the Qinxi demonstration garden in Taibai County, Baoji County, Shaanxi Province as the test soil. The spinach (Spinacia oleracea L.) variety was big-leaf spinach, which belongs to the spinach genus Chenopodiaceae (annual herbaceous plant), and the seeds were provided by the demonstration garden. The Bacillus subtilis, Bacillus mucilaginosus, and Bacillus amyloliquefaciens selected the original strains produced by Lvlong Biotecnology Co., Ltd., which contained > 200 million live bacteria per gram of fertilizer.The fertilizer components are all the Bacillus and its metabolites, and the rate of miscellaneous bacteria < 3%.

Growth conditions and experiment design
The experiments were carried out in growth chambers at the College of Geography and Environment of Baoji University of Arts and Sciences under a 12 h day/night photoperiod at temperatures of 25/15 °C, respectively, a photosynthetically active radiation (PAR) of 300 µmol/(m 2 ·s), and humidity of 60%. The spinach seeds were soaked in deionized water for 12 h and placed in a 4 °C refrigerator for 24 h to promote germination. The germinated seeds were placed on a seedling tray, covered with nutritious soil, and germinated in the dark in a 25 °C incubator. Following seven days of treatment, the seedlings were transferred to pot (Ø25 cm x 20 cm high) lled with greenhouse soil 18 cm deep, and 10 seedlings were planted in each pot. The soil nutrients were comprised of 33.35 g/kg organic matter, 2.79 g/kg total nitrogen, 1.51 g/kg total phosphorus, and 10.75 mg/kg available phosphorus, and a pH of 5.2. For this experiment, 90 kg/hm 2 of phosphate fertilizer (potassium dihydrogen phosphate) was used as base fertilizer with microbial fertilizer 0.5 kg/hm 2 . There were four treatments and three repeats ( Table 1).

Measurement of physiological characteristics
The leaf soluble protein (SP), soluble carbohydrate (SC), and water potential (WP) were closely related to plant metabolism. Chlorophyll (Chl) is an important substance in plant photosynthesis, and malondialdehyde (MDA) can re ect the degree of membrane lipid peroxidation. Within seven to 10 days of fertilization, the SP was determined by the Coomassie brilliant blue (CBB) method at 595 nm using a DR6000 spectrophotometer (Bradford, 1976), whereas the SC was determined using an anthrone method at 620 nm (Jermyn, 1975 Relative variable uorescence at the I-step To compare the OJIP curve with the normalized PF transient curve between OJ and OK, the following formula was used: The leaf light-response curve (LC) measurement, based on pulse modulated uorometry (PAM) was designed using seven photosynthetic photon ux densities (PPFD) (10,20 50 100 300 500, and 1000 µmol/(m 2 ·s)) to acquire chlorophyll uorescence parameter changing curves (Ft, QY) relating the rate of photosynthesis.

Statistical analysis of data
All collected data were subjected to one-way ANOVA analysis using SPSS (SPSS software version 22.0, Chicago, Illinois, USA). Differences between means were compared by Tukey's HSD test at P < 0.05. The correlations between parameters were determined using Pearson's simple correlation test function in SPSS.

Effects of microbial fertilizer on growth and physiological characteristics of spinach seedlings
There were signi cant differences in the plant height (PH), leaf length (LL), leaf weight (LW), and other growth parameters of the spinach seedlings under the ve different treatments (F = 54.37, 13.30, and 46.03, respectively; P < 0.01), where the growth characteristics attained a maximum under the T2 treatment (Table 3). Except for Chla + b and Chla/b, there were signi cant differences in the physiological characteristics of the leaves. Among them, the T2 treatment had the greatest impact on the physiological leaf responses. Compared with the F value, the difference in soluble sugar (SC) was the highest (F = 118.35; P < 0.01).  However, there was no signi cant difference in the ABS/RC and ET 0 /RC between the treatments. The quantum yield (φP 0 ), e ciency (φE 0 ), and ψ 0 values were highest under the T2 treatment, and with the exception of ψ 0 , there were signi cant differences (P < 0.01). On the contrary, the value of φD 0 was lowest under the T2 treatment.

Transient analysis of prompt uorescence OJIP of spinach seedlings by adding microbial fertilizer
By measuring the transient curve of OJIP as shown in Fig. 1, the OJIP curves of the ve treatments were similar to those reported by Strasser et al. (2004). As can be seen from Fig. 1(A), compared with CK, the uorescence trend of the other treatments gradually increased, among which the uorescence value of the T2 treatment was the largest, which signi ed that it had a greater effect on the photosynthetic chemical rate of the leaves. As shown in Fig. 1(B), the difference of relative variable uorescence (Vt) at point J (2 ms) was the largest. This is better illustrated in Fig. 1(C (Table 5). Table 5 Correlations between leaf physiological characteristics and chlorophyll uorescence parameters. In this research, microbial fertilizer was found to signi cantly 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). The φP 0 (Fv/Fm) represents the maximum quantum yield of PS , and the ψ 0 value re ects the electron transfer e ciency, from QA − to QB, whereas φE 0 re ects the quantum yield of electron transport. The increase of φP 0 , ψ 0 , and φE 0 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 uorescence 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 re ect 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 ( 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 uorescence 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 ow rate to the electron receptors at the end of PSI.
Combined with PI ABS , the higher PI ABS values under the Bs and Bm treatment indicated increases in the density of active PSII centers, the e cacy of photoreactions, and e ciency of biochemical dark redox reactions, as well as the production and utilization of NADPH (Habib et al., 2016).

Conclusions
The main results of our study are as follows: Bacillus promoted the growth of spinach seedlings and improved the adaptability of crops to soil by improving the effective phosphorus utilization rate. Simultaneously, the chlorophyll content, soluble protein and other physiological characteristics, chlorophyll uorescence, and PSII photochemical activity were improved to a certain extent.
The results revealed that under the Bs and Bm treatment, the growth conditions for the spinach seedlings were best, and the physiological index was higher. According to this study, it is suggested that inorganic fertilizer and Bacillus may be used together for soil fertilization in greenhouses. It can not only promote the utilization of mineral elements in the soil by plants to improve crop quality, but also prevent soil acidi cation caused by the excessive accumulation of mineral elements. Future research should endeavor to control the fertilization concentrations of microorganisms, so as to provide an improved theoretical basis for agricultural fertilization.
Declarations Figure 1 Shape change of uorescence transient curve measured through then addition of microbial fertilizer in the ve different treatments. Each curve represents the average of three independent measurements.