Inoculation of Herbaspirillum Seropedicae Increases Biomass in Maize Roots in the Early Stages of Plant Development

Herbaspirillum seropedicae is a plant growth-promoting bacteria isolated from diverse plant species. In this work, the main objective was to investigate the eciency of H. seropedicae strain SmR1 in colonizing and increasing maize growth in the early stages of development under greenhouse conditions. Inoculation with H. seropedicae resulted in 10.51 and 19.43% in mean of increase of root biomass concerning non-inoculated controls, mainly in the initial stages of plant development, at 21 days after emergence (DAE). Quantication of H. seropedicae in roots and leaves was performed by quantitative PCR.. H. seropedicae was detected only in maize inoculated roots by qPCR, and a slight decrease in DNA copy number g −1 of fresh root weight was observed from 7 to 21 DAE, suggesting that there was initial effective colonization on maize plants. H. seropedicae strain SmR1 eciently increased maize root biomass exhibiting its potencial to be used as inoculant in agricultures systems.

Herbaspirillum seropedicae is an endophytic PGPB, aerobic, Gram-negative, diazotrophic, β-Proteobacteria (Baldani et al., 1986) that has been isolated from many grass species, such as maize, sorghum, rice, sugar cane, wheat, and other forage plants (Monteiro et al., 2012;Olivares et al., 1996). The most studied strain of H. seropedicae is strain SmR1, a spontaneous mutant of strain Z78 (ATCC 35893) isolated from sorghum by Baldani et al. (1986) Alves et al. (2015) under two growing conditions (greenhouse and eld), 21 strains of Herbaspirillum in two maize varieties were evaluated. The strain that presented the best results for both conditions was H. seropedicae ZAE94, demonstrating that it can increase maize production up to 34% and provide 37% of the nitrogen (N) demand by plants by BNF. Assessing the effect of Azospirillum brasilense and Herbaspirillum seropedicae inoculation on maize nitrogen metabolism in the presence of two nitrogen levels, Breda et al. (2016) observed that H. seropedicae contributed to the change on the N metabolism and promoted maize plants development, as well as higher accumulation of phosphorus (P) and potassium (K) in the shoots when compared with A. brasilense.
An essential requirement for PGPB to stimulate plant growth is the inoculant survival used as biofertilizer in the plant surface in a su cient cell number to establish satisfactory colonization (Berninger et  The bacterial inoculant survival can be affected by biotic and abiotic factors before, during, and after plant application (Berninger et al., 2018). Furthermore, under eld conditions, inoculants may behave differently from that observed in greenhouses or axenic conditions (Sammauria et al., 2020). Therefore, quantifying H. seropedicae DNA in maize plants when grown in non-sterile soil and with the inoculated seed would make it possible to evaluate bacteria in plant tissues and relate the bene ts of plant growth and environment interaction. Therefore, the objective of the work was to evaluate the establishment and e ciency of diazotrophic bacteria H. seropedicae strain SmR1 in the early stages of the development of a maize cultivar.

Bacterial growth conditions
Herbaspirillum seropedicae strain SmR1 (strain Z78 ATCC 35893 Sm R ) was grown in NFbHPN medium supplemented with 5 g L −1 malic acid (Klassen et al., 1997) at 30 ºC under aeration and 120 rpm shaking until OD 600 0.8, corresponding to ~10 8 CFU mL −1 . A correlation was obtained between the optical density (OD) measured at 600 nm using Hitachi U2910 Spectrophotometer (Tokyo, Japan) and the number of colony forming units obtained by plate counting in NFbHPN agar medium after ten-fold serial dilution (0.9% saline solution). Plate counting was performed after incubation for 48h at 30°C.

Maize inoculation and growth conditions
Seeds of maize variety DKB 390 were surface sterilized in biological safety cabinet by 3 washing in autoclaved distilled water, followed by submersion in 70% ethanol for 5 min, and 20 min shaken in 1% sodium hypochlorite plus 0.01% Tween 20 solution. Then seeds were rinsed 5 times with sterile distilled water and treated with 10% sucrose solution (300 mL 50 kg −1 of seeds) for inoculant adherence.

Assessment of plant growth parameters
Maize seedlings from each treatment were randomly collected on the 7th, 14th, and 21st day after emergence (DAE). Plant growth parameters of root and shoot length were determined with the aid of a graduated ruler. Dry weight was determined by drying of root and shoot at 65 ºC for 72h until constant weights were achieved. Nitrogen content was determined by sulfuric digestion of shoot tissue according to Tedesco et al. (1995). The remained sampled tissues were frozen in liquid nitrogen and stored in -80 ºC freezer until DNA extraction.

Statistical analysis
Statistical analysis was performed using Sisvar 5.3 software (DEX-UFLA). Data was rst evaluated for normality and homogeneity using Lilliefors and Bartlet tests followed by the variance analysis and Tukey test (P ≤ 0.05), used to determine statistically signi cant differences between plant growth parameters and quanti cation in maize by qPCR.

Plant-growth parameters
Maize DKB 390 variety inoculated with H. seropedicae strain SmR1 under greenhouse conditions resulted in increased root biomass in the early stages of plant development, compared to the non-inoculated control for both experiments conducted in different periods.
For the rst greenhouse experiment, shoot dry mass and root length were not in uenced by treatments (inoculated or non-inoculated High N and Low N controls), just by the growth period ( Figure 1A and 1B).
In this assay, shoot length has increased by the inoculation with H. seropedicae strain SmR1 4.93% in mean compared to the non-inoculated High N control ( Figure 1C), regardless of the growth period. Root dry mass was also signi cantly in uenced by treatments and the inoculation increased root biomass by 19.43% in mean, at 21 DAE, compared with High N and Low N non-inoculated controls ( Figure 1D). Inoculation with H. seropedicae strain SmR1 in uenced the nitrogen content of the maize shoot only at 14 DAE (Figure 2), increasing in mean 20% of this content compared with High N and Low N noninoculated controls. Besides it, a signi cant decrease in N was observed during the growth period (7 to 21 DAE).
For the second greenhouse experiment, shoot length and shoot dry mass were not in uenced by treatments ( Figure 3A and 3B). Regardless of the growth period, the inoculation with H. seropedicae strain SmR1 has increased root length ( Figure 3C) and root dry mass ( Figure 3D) in mean by 8% and 10.51%, respectively, in comparison to the non-inoculated Low N control. Inoculation treatment has shown no signi cant difference of non-inoculated High N control. qPCR reaction parameters for H. seropedicae strain SmR1 quanti cation Reaction parameters (e ciency, slope and correlation coe cient) of the qPCR assay using HERBAS1 were determined based on two independent standard curves obtained from DNA isolated from H. seropedicae strain SmR1 pure culture ( Figure 4). The reaction parameters were calculated by plotting the Cq values against the log10 of the genome copy number. H. seropedicae strain SmR1 standard curves. E ciency varied from 89 to 92% and sloped ranged from -3.61 to -3.53 to standard curve performed for quanti cation of control samples ( Figure 4A) and inoculated samples or soil ( Figure 4B), respectively.
Limit of detection (LOD) of a qPCR is de ned as the lowest amount of DNA that can be reliably detected  (Table S1). Quanti cation results of inoculated roots showed that bacterial DNA copy number per gram of fresh root weight ranged from 3.48 ± 1.48 x 10 5 (7 DAE) to 3.83 ± 0.9 x 10 4 (21 DAE) ( Table 1).  shown the potential of the Herbaspirillum-maize association, however, a differential response between genotypes has been reported (Vacheron et al., 2013), so that further studies on the factors that in uence this interaction are needed. Molecular methods have been widely used to detect and monitor the survival of microbial populations due to its high sensitivity and speci city. In the present study, we evaluated the effects of plant growth-promoting bacteria H. seropedicae strain SmR1 in association with hybrid maize variety DKB 390 and monitored the bacterial survival during association using molecular tools such as quantitative PCR. The inoculation of hybrid maize variety DKB 390 with H. seropedicae strain SmR1 increased plant shoot length concerning the control treatments in the rst greenhouse experiment performed ( Figure 1C), however, this increase was not observed in the second experiment ( Figure 3A). Similarly, signi cant differences in root length were observed only in the second experiment in response to inoculation ( Figures  1A and 3C), with additions compared to the non-inoculated Low N control. Inoculation with H. seropedicae strain SmR1 did not demonstrate signi cant differences in the shoot dry mass evaluation on the experiments performed ( Figures 1B and 3B). In order to establish a reliable assay of qPCR for H. seropedicae DNA quanti cation in maize root and leaves, reaction parameters for SYBR Green assay were de ned using standard curves obtained from diluted DNA isolated from pure bacterial culture. According to Zhang and Fang (2006), a reliable standard curve should present slope values between -3.9 and -3.0, corresponding to qPCR e ciency between 80 and 115%, and R² values higher than 0.95. In this work, standard curves presented suitable reaction parameters with e ciency values ranging from 89 to 92% (Figure 4). Similar results were already observed in qPCR assay using HERBAS1 for H. seropedicae quanti cation: 85 -99% of e ciency and R² = 0.99 (Pereira et al., 2014).
Regarding the colonization of maize tissues ( Table 1) On the other hand, we must also consider that the inoculation in this work was performed in maize seeds, and quanti cation results were based on the period after the plants emergence. This inoculation performed directly into the seeds may have hampered the colonization of maize roots concerning inoculation performed into pre-germinated seeds as observed in Pereira et al (2014) and do Amaral et al (2014).
In our study, even though bacterial population decreased, at 21 DAI the inoculated plants showed signi cantly higher root biomass increments compared to the non-inoculated plants. In conclusion, H.
seropedicae strain SmR1 has shown that inoculation of maize plants cultivar DKB 390 occurred quickly and effectively, exhibiting its potential to be used as an inoculant. However, due to the variation of results