Seed biopriming with Parachlorella, Bacillus subtilis, and Trichoderma harzianum alleviates the effects of salinity in soybean

doi:10.21203/rs.3.rs-4954003/v1

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Seed biopriming with Parachlorella, Bacillus subtilis, and Trichoderma harzianum alleviates the effects of salinity in soybean

https://doi.org/10.21203/rs.3.rs-4954003/v1

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Background

Seed conditioning with bioinputs (biopriming) offers a promising and sustainable alternative to mitigate the adverse effects of salt stress on soybeans. This study aims to evaluate the potential of isolated or combined biopriming using microalgae and different microorganisms in alleviating salinity-induced stress in soybeans.

Methods

Seeds were subjected to five biopriming treatments: microalgae Parachlorella sp., Bacillus subtilis, Trichoderma harzianum, Parachlorella sp. + B. subtilis, and Parachlorella sp. + T. harzianum, along with a control group without biopriming. Subsequently, the seeds were exposed to two conditions: i) control (0.0 MPa), and ii) salinity induced by NaCl (-0.8 MPa). Germination, photochemical indicators, and seedling performance were assessed.

Results

Salinity impaired root protrusion and seed physiology, resulting in a high percentage of abnormal seedlings, thus creating a stressful condition. However, biopriming alleviated the negative effects of salinity, particularly with T. harzianum, Parachlorella sp. + B. subtilis, and Parachlorella sp. + T. harzianum, which led to high germination rates and normal seedlings. All biopriming treatments, especially the combined ones, reduced the suppression of non-photochemical quenching, thereby enhancing the maximum yield of photosystem II. Seedlings under salt stress without biopriming exhibited short lengths and low fresh and dry mass, whereas those bioprimed with Parachlorella sp. + B. subtilis and Parachlorella sp. + T. harzianum showed significantly higher values.

Conclusion

Seed biopriming, especially with Parachlorella sp. combined with B. subtilis or T. harzianum, effectively alleviates the stressful effects of salinity on soybeans.

bioinputs

microalgae

osmotic potential

photochemical yields

salt stress

synergism

The current scenario of global climate change is a highly debated topic among physiologists and other professionals in the agricultural sector. Among various agricultural commodities, the soybean production chain (Glycine max L.) stands out due to its diverse uses and significant importance in the world market. However, stress factors significantly affect soybean production.

Salt stress is an adverse condition that has increased in recent years due to the excess of ions such as Na⁺ and Cl^– in the soil. These ions have toxic effects, impairing seed germination, seedling performance, and vigor [1, 2]. They make water absorption difficult and intensify osmotic stress, leading to water deficits [3, 4]. In addition to reducing osmotic potential, salt stress hinders respiratory and reserve metabolism for the embryonic axis [5].

Consequently, adopting practices that alleviate the effects of salinity stress has become a research focus. Seed conditioning with biological inputs, known as biopriming, is a promising strategy for mitigating the negative effects of osmotic and oxidative stress. Biopriming improves seed germination and the morphophysiological performance of seedlings [6], aligning with sustainable development goals (SDGs).

Among bioinput alternatives, microalgae, such as the genus Parachlorella sp., have gained prominence for their role in maintaining physiological homeostasis [7, 8]. Microalgae enhance amino acid levels and activate protection systems [9, 10]. Additionally, they contribute to the synthesis of phytohormones and other non-antioxidant compounds [11].

Other bioinputs with potential to mitigate salt stress include multifunctional microorganisms, particularly Bacillus subtilis and Trichoderma harzianum [12, 13]. These microorganisms stimulate the synthesis of phytohormones, enzyme activity, and antioxidant compounds [14–16]. They also enhance nutrient availability and stimulate germination and seedling physiology [17]. However, there remain gaps in understanding the protective mechanisms of these microorganisms, whether isolated or associated with microalgae, in the biopriming of soybean seeds.

During seedling formation, unifoliate leaves are emitted, still largely dependent on cotyledon reserves. We believe this biopriming affects kinetic fluorescence homeostasis, enhances seedling performance, and induces tolerance to abiotic stress. The hypothesis here is that (i) exposure of seeds to salinity affects photochemical yields in these leaves even at the seedling stage, dissipating energy and reducing photochemical yields, and (ii) although soybean is sensitive to salt stress, inoculation with the microalgae Parachlorella, alone or combined with B. subtilis and T. harzianum, mitigates the negative effects of this condition.

Thus, this study aims to evaluate the potential of isolated or combined biopriming of microalgae and different microorganisms in alleviating the stressful effects of salinity on soybeans.

Experiment site

The experiment was conducted at the Seed Technology Laboratory, Faculty of Agricultural Sciences, Federal University of Grande Dourados (UFGD), Dourados, MS. The cultivar 64i63 RSF IPRO (2023–2024 harvest) from the S1 generation was used, with a thousand-seed weight of 154 g on a 6.25 sieve.

Seed biopriming and salinity induction

Seeds were subjected to five biopriming treatments: microalgae Parachlorella sp., Bacillus subtilis, Trichoderma harzianum, Parachlorella sp. + B. subtilis, and Parachlorella sp. + T. harzianum, in addition to a control without biopriming (CK). Subsequently, the seeds were exposed to two conditions: i) control (0.0 MPa, distilled water) and ii) salinity, with an osmotic potential of -0.8 MPa induced by sodium chloride (NaCl). The experimental design was completely randomized, with treatments arranged in a 6 x 2 factorial scheme and four replications of 50 seeds.

Characterization of bioinputs and inoculation

The microalgae Parachlorella sp. (Fig. 1a and 1c) used in this study was produced in a 4,000-L Raceway photobioreactor (Fig. 1b) in a culture medium adapted from Ribeiro et al. [18], housed in a closed greenhouse with natural light, a 12/12 photoperiod (light/dark), and constant aeration. The Parachlorella sp. contained 5 × 10⁶ cells mL^–1, had a pH of 7.5, and included 0.62% nitrogen and 2.02% organic carbon, along with amino acids and phytohormones [19]. Trichoderma harzianum isolate IB19/17 (2 x 10⁹ viable conidia g^–1) and Bacillus subtilis UVF S1 strain (1 x 10⁸ CFU mL^–1) were used.

The products were diluted in distilled water (5.0 g L^–1 for T. harzianum and 45 L ha^–1 for B. subtilis) as recommended by the manufacturers. A dose of 10 mL of solution per kilogram of seed was applied, based in work of Santos et al. [19]. For combined biopriming, 5 mL of microalgae and 5 mL of either T. harzianum or B. subtilis were added. The seeds were placed in plastic packaging, inoculated with the corresponding treatment using a micropipette, homogenized, and left to rest for 45 minutes before sowing.

Assessments

Germination

Conducted with four replications of 50 seeds, arranged on Germitest® paper rolls moistened with water equivalent to 2.5 times the mass of the dry paper. The rolls were placed in a BOD chamber at 25°C with a 12-hour photoperiod for eight days after sowing, assessing normal and abnormal seedlings according to the Rules for Analysis of Seeds [20].

Root protrusion and first count

Conducted as part of the germination test. Two days after sowing, the number of seeds with radicles ≥ 1.5 cm was counted. The first count of seedling percentage was recorded five days after sowing in accordance with the Rules for Seed Analysis [20].

Phenotypic responses

Photographic records of shoot and root appearance were taken five and eight days after sowing.

At 8 days after sowing, kinetic fluorescence and vigor analyzes based on seedling performance were carried in ten seedlings.

Photochemical indicators

Kinetic imaging fluorescence was assessed after subjecting seedlings to dark conditions for 30 minutes to ensure open reaction centers. Unifoliate leaves were then placed in a Closed FluorCam FC 800-C (Fig. 2), utilizing LED panels with actinic light intensity above 2,500 µmol m^–2 s^–1 and a charge-coupled device (CCD). Maximum yield values of photosystem II after dark adaptation (QY_Max) and suppression of non-photochemical quenching (NPQ_Lss) were recorded.

Seedling length

Measured from the base to the apical bud, and root length was measured from the insertion to the root cap of the primary root using a millimeter ruler.

Fresh and dry biomass

Seedlings were separated into shoots and roots. Fresh mass was weighed on a precision scale (0.0001 g). The materials were then dried in an oven at 60 ºC for 72 hours and dry biomass was determined.

Data analysis

Root protrusion, first count, and germination data were subjected to the Shapiro-Wilk normality test. All data were analyzed using analysis of variance (ANOVA). When significant by the F-test (p ≤ 0.05), means were compared using the Scott-Knott test for seed biopriming with bioinputs and the Bonferroni t-test for salinity conditions, both at p ≤ 0.05 ± standard error.

The interaction between conditions and seed biopriming significantly affected all evaluated characteristics (p ≤ 0.05) (Table 1). Salinity induced by NaCl negatively impacted the phenotypic responses of soybean seedlings; under this condition, seedlings exhibited low vigor after five days (Fig. 3). However, biopriming with Parachlorella sp. + B. subtilis and Parachlorella sp. + T. harzianum resulted in seedlings with better lateral root and root development. In the control condition, at five days, seeds bioprimed with T. harzianum and Parachlorella sp. + B. subtilis showed greater root length and volume.

Table 1

Results of the analysis of variance of the effects of salinity and seed biopriming and the interaction on the characteristics evaluated in soybean seeds and seedlings
Characteristic	p-valor
Characteristic	Salinity (S)	Biopriming (B)	S × B	C.V. (%)
RP	0.0408	< 0.0001	0.0012	4.62
FC	0.0001	0.1705	0.0137	5.60
NS	0.6711	0.0001	0.0407	8.76
AS	0.9021	0.0001	0.0509	49.44
NPQ_Lss	0.0009	0.0001	< 0.0001	28.16
QY_Max	0.0001	< 0.0001	< 0.0001	8.74
SL	< 0.0001	< 0.0001	0.0071	9.36
RL	0.0385	0.0003	0.0372	13.34
TL	0.0003	< 0.0001	0.0105	9.87
SFM	0.2534	0.0013	0.0042	5.76
RFM	< 0.0001	< 0.0001	< 0.0001	14.92
SDM	0.2534	0.0013	0.0042	5.76
RDM	< 0.0001	< 0.0001	< 0.0001	14.92

RP: root protrusion, FC: first count, NS: normal seedlings, AS: abnormal seedlings, QY_Max: maximum yield of the photosystem II after dark adaptation, NPQ_Lss: non-photochemical quenching suppression, SL: shoot length, RL: root length, TL: total length, SFM: shoot fresh mass, RFM: root fresh mass, TFM: total fresh mass, SDM: shoot dry mass, RDM: root dry mass, TDM: total dry mass.

The negative effects of salinity persisted at eight days, leading to low phenotypic responses. Biopriming with T. harzianum, Parachlorella sp. + B. subtilis, and Parachlorella sp. + T. harzianum resulted in seedlings with enhanced shoot vigor, including the opening of cotyledons, emission of the first twin leaves, and more expressive formation of main and lateral roots (Fig. 4).

Regarding root protrusion under salinity, the highest values were observed for T. harzianum, Parachlorella sp. + B. subtilis, and Parachlorella sp. + T. harzianum, with no statistical difference compared to the control for the latter two bioprimings (Fig. 5a). Although no statistical difference was found for the first count between the control condition and salinity in CK seeds, biopriming in the control condition contributed positively. Under NaCl, biopriming with T. harzianum, Parachlorella sp. + B. subtilis, and Parachlorella sp. + T. harzianum favored the first count, differing from other bioprimings under the same condition (Fig. 5b).

The percentage of abnormal seedlings increased markedly under salt conditions in CK seeds, differing from those with isolated or combined biopriming (Fig. 5c). Regarding normal seedlings, there was no statistical variation from the control condition, but under salinity, only CK seeds resulted in a low value (Fig. 5d).

The maximum yield values of photosystem II after dark adaptation (QY_Max) did not vary statistically in the control condition, indicating a stable condition. However, under salinity, QY_Max values were markedly reduced in CK seeds, while those inoculated with isolated bioinputs, especially the association of Parachlorella sp. with B. subtilis or T. harzianum were higher (Fig. 6a).

Regarding the suppression of non-photochemical quenching (NPQ_Lss), there was an increase in seedlings bioprimed with B. subtilis and Parachlorella sp. + B. subtilis, while T. harzianum showed low values under non-stressful conditions (Fig. 6b). Under salt stress, CK seedlings exhibited higher NPQ_Lss compared to bioprimed seedlings under the same condition and the control without biopriming. Seedlings from biopriming with B. subtilis, either isolated or associated with microalgae, presented lower values under salinity compared to the control condition.

In the control condition, there was no statistical difference between bioprimings for seedling length (Fig. 7). Generally, CK seedlings showed shorter lengths under salt conditions compared to the control. The highest root length values under salinity were observed in seedlings from biopriming with Parachlorella sp. + B. subtilis (Fig. 7a), followed by other bioprimings in the same condition, which were higher compared to CK.

Moreover, shoot and total length followed a similar response pattern, with seedlings from biopriming with Parachlorella sp. + B. subtilis and Parachlorella sp. + T. harzianum showing the highest values, followed by T. harzianum and isolated microalgae, not differing from the control condition in the same biopriming (Fig. 7b). In total length, only seedlings from CK seeds had low values (Fig. 7c).

For root and shoot fresh mass of seedlings in the control condition, the highest values were for biopriming with Parachlorella sp. isolated, Parachlorella sp. + B. subtilis, and Parachlorella sp. + T. harzianum (Fig. 8). Under salinity, all fresh masses decreased, but bioprimed seedlings maintained high values, especially when the microalga was associated with B. subtilis or T. harzianum, differing from CK seeds (Figs. 8a and 8c).

Root dry mass under salt conditions was higher in seedlings from seeds bioprimed with Parachlorella sp. + T. harzianum, even larger than those not stressed, differing from CK seeds (Fig. 8b). Although stressed seedlings had lower values compared to the control condition, seedlings conditioned with isolated microalgae and B. subtilis, as well as microalgae associated with B. subtilis and T. harzianum, showed values that did not differ from the control condition.

In the control, biopriming with B. subtilis and T. harzianum isolates contributed to increased shoot dry mass (Fig. 8d), while under salinity, biopriming with Parachlorella sp. + B. subtilis and Parachlorella sp. + T. harzianum resulted in higher values, differing statistically from other bioprimings under the same condition. However, they did not differ from the control. Notably, the biomass accumulation values of seedlings from biopriming with B. subtilis isolates and T. harzianum were smaller than those associated with Parachlorella sp., but all were larger than CK.

Soybean is sensitive to salinity, which reduces germination, photochemical indicators, and seedling performance. However, biopriming alleviates the adverse effects of this stress, maintaining values close to or statistically similar to non-stressed plants (control), confirming the initial hypothesis. Furthermore, the microalga Parachlorella sp. showed synergy with B. subtilis and T. harzianum, positively contributing to soybean seeds and seedlings.

NaCl-induced salt stress drastically decreases osmotic potential and cell turgor [21, 22], impairing plant vigor, particularly the physiological characteristics of seeds and seedlings. This impact on phenotypic responses of shoots and roots was observed in this study and corroborates the findings of Soares et al. [23], where soybean seedlings were adversely affected by salt stress.

Exposure of seeds to salinity hinders imbibition, damaging germination potential due to reduced osmotic potential [24]. It also obstructs the release of reserves for the embryonic axis [5], altering the vigor pattern of normal seedlings. These effects are caused by excess Na⁺ and Cl^–, leading to ionic, osmotic, and oxidative stress [25]. These stresses trigger the overproduction of reactive oxygen species (ROS) that affect respiratory metabolism, seed physiology, and genetic integrity [26, 27].

Under salt stress, cell membrane integrity is compromised, as evidenced by an increased rate of electrolyte leakage due to ROS overproduction [28]. This condition alters kinetic fluorescence dynamics, indicated by increased NPQ_Lss. However, bioinputs favored the homeostasis of the seedlings' photochemical apparatus, suggesting improved electron utilization, minimizing dissipation, and increasing QY_Max.

The increase in germination indicators with biopriming demonstrates that bioinputs create better physiological conditions for seeds to germinate and invest in biomass accumulation even under adverse conditions (NaCl). The bioinputs assessed, as per to Hashem et al. [14] and Alghuthaymi et al. [15], modulate amino acid synthesis, favoring osmoregulation and enhancing antioxidant enzyme activity, leading to detoxification, cellular repair, and a reduction in abnormal seedlings, as observed in this study.

The effects of bioinputs in this study are varied and involve different mechanisms of action. The microalga Parachlorella sp. contains phytohormones such as abscisic and gibberellic acids, which, although not contributing in isolation to germination indicators, efficiently influenced seed physiology, promoting cell elongation and root growth [11, 29, 30].

Additionally, Chandra et al. [31] highlight that the polysaccharides in microalgae act as functional groups connecting with essential microelements in seedling nutrition, suggesting a biofertilizing action. The microalga extract also contains amino acids that aid in osmoregulation [10], making it an important tolerance strategy.

Inoculation with B. subtilis regulates plant growth, particularly by promoting root growth [32]. This bacterial group enhances auxin synthesis and secretes enzymes that protect metabolism and induce root expansion [33–35].

T. harzianum aids in seed germination [36, 37]. Studies indicate that activating jasmonic acid, auxin, and other pathways induces tolerance to abiotic stress [38, 39]. T. harzianum also stimulates the production of osmolytes and increases antioxidant enzyme activity, protecting against oxidative stress by eliminating ROS and recycling ascorbate and glutathione, previously oxidized by stressors [40–42].

Considering the beneficial effects of the association of microalgae with other microorganisms, we pose the following question: how do microalgae Parachlorella sp., B. subtilis, and T. harzianum act synergistically? According to Solomon et al. [43], this relationship is beneficial as microalgae produce O₂ and extracellular products such as carbohydrates and proteins. In return, the other microorganisms provide microalgae with CO₂, vitamins, nutrients, and other essential constituents, maintaining a symbiotic relationship. These synthesized constituents benefit seedlings, promoting growth and inducing tolerance.

The best results with bioinputs, especially under salt conditions, underscore their mitigating role through oxidative protection mechanisms, along with biofertilizing and biostimulating effects. Gul et al. [44] reported increased biomass of Triticum aestivum L. under salt stress, attributing this response to signaling and hormonal synthesis, which contribute to cell differentiation and the accumulation of photoassimilates.

In a practical context, biopriming enhances seed physiology, germination, and seedling emergence, leading to faster establishment and better stress tolerance mechanisms. The bioinputs assessed here are widely available, accessible, and manageable for rural producers.

This study demonstrates that the bioinputs assessed, particularly the combination of microalgae with other microorganisms, are beneficial and often superior to isolated applications. They provide protection against adversities like salinity and promote tolerance, sustainable development goals (SDGs), and bioeconomy. These positive responses are due to biomolecules synthesized by these bioinputs, resulting in better seedling performance.

Further studies are recommended to determine the activity of antioxidant enzymes and better understand the modes of action of these bioinputs in inducing tolerance to salinity in the studied cultivar.

Soybean cv. 64i63 RSF IPRO exhibits responsiveness and sensitivity to NaCl-induced salt stress, resulting in reduced seedling vigor. Biopriming mitigates the adverse effects of salinity, particularly when the microalga Parachlorella sp. is associated with T. harzianum or B. subtilis. This combination effectively regulates photochemical yields and contributes to improved indicators of seed germination and seedling performance.

Bacillus subtilis

CCD

charge-coupled device

without biopriming

microalgae Parachlorella sp

NaCl

sodium chloride

NPQ_Lss

suppression non-photochemical quenching

QY_Max

maximum yield of photosystem II after dark adaptation

ROS

reactive oxygen species

Trichoderma harzianum

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Funding

Not applicable

Acknowledgments

The authors thank CAPES and CNPq, for granting the scholarships, and the FUNDECT, for financial support. We thank the members of the Plant Ecophysiology Study Group (GEEP) who contributed to the experiment.

Competing Interests

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Author Contributions

Conceptualization and methodology, C.C.S and D.M.M.S.; data collection, D.M.M.S., F.E.W., L.O.M.M., J.P.A.O., and O.A.S.; writing–original draft preparation C.C.S., D.M.M.S., F.E.W., L.O.M.M., J.P.A.O., O.A.S., D.M.R., and S.P.Q.S.; validation, analysis, and visualization, C.C.S and D.M.MS.; writing-review and editing, C.C.S., D.M.M.S., F.E.W., L.O.M.M., J.P.A.O., and O.A.S.; revision, C.C.S. All authors have read and agreed to the published version of the manuscript.

Availability of data and materials:

All data generated or analyzed during this study are included in this published article.

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Seed biopriming with Parachlorella, Bacillus subtilis, and Trichoderma harzianum alleviates the effects of salinity in soybean

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Material and Methods

Experiment site

Seed biopriming and salinity induction

Characterization of bioinputs and inoculation

Assessments

Data analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1