Impact of biological products on soybean seed treatment: evaluation of early performanceABSTRACT:
In agriculture, biological products are an important sustainable alternative for reducing environmental and human health impacts. However, using biologicals in seed treatment may influence early seed performance. This study aimed to evaluate soybean seeds treated with commercial biological products registered for this purpose, applied individually or in combination. Water content, germination, first count, and seedling length were measured using traditional methods, and seedling length, vigor and uniformity via Vigor-S software. Evaluations were carried out immediately after treatment and after 40 days of storage of the treated seeds. The tests conducted indicated that treatments with the Bacillus amyloliquefaciens (BV03) and Trichoderma asperellum (BV10) strains may negatively affect the early growth and development parameters of seedlings under laboratory conditions. This may be associated with testing conditions, such as the absence of substrates and organic compounds, high humidity, and lack of pathogens; with product-related factors, such as metabolite production by each strain, dose, and formulation ingredients; or with seed-related factors, including initial physiological quality and storage.
Index terms:
automated analysis; beneficial microorganisms; Glycine max L.; seed deterioration; vigor
RESUMO:
O uso de práticas sustentáveis na agricultura, com menor impacto ambiental ou na saúde humana, tem no uso de produtos biológicos uma alternativa importante. Porém, o uso de produtos biológicos no tratamento de sementes pode influenciar no desempenho inicial das sementes. Este trabalho objetivou avaliar sementes de soja tratadas com produtos biológicos comerciais. As sementes foram tratadas com produtos comerciais biológicos registrados para o tratamento de sementes, de forma isolada ou em mistura. Foram realizadas avaliações de teor de água, germinação, primeira contagem, comprimento de plântulas na metodologia tradicional e comprimento de plântulas, vigor e uniformidade por meio do software Vigor-S. As avaliações foram realizadas logo após o tratamento e após 40 dias de armazenamento das sementes tratadas. Os testes avaliados mostram que tratamentos com as cepas Bacillus amyloliquefaciens (BV03) e Trichoderma asperellum (BV10) podem afetar negativamente os parâmetros de crescimento e desenvolvimento inicial de plântulas nos testes de laboratório. Isso pode estar associado às condições destes testes como: ausência de substratos e outros compostos orgânicos, elevada umidade, ausência de patógenos; condições relacionadas aos produtos comerciais: produção de metabólitos em cada cepa, dose, outros ingredientes da formulação; e condições relacionadas às sementes, como a qualidade fisiológica inicial e o armazenamento.
Termos para indexação:
análise automatizada; microrganismos benéficos; Glycine max L.; deterioração de sementes; vigor
INTRODUCTION
Soybean (Glycine max (L.) Merril) is one of Brazil’s most economically important oilseed crops, with grain production in the 2023/2024 growing season reaching 146.52 tonnes, cultivated across 45.235 million hectares (CONAB, 2024). Crop productivity depends on various factors, including the establishment of plant stands in the field, which is directly related to seed quality (Decarli et al., 2021). Even under stress, crops obtained from high-quality seeds exhibit superior agronomic performance and result in higher yields (Krzyzanowski et al., 2018).
In the field, different biotic and abiotic factors can compromise seed performance during germination and early seedling development (Marcos-Filho, 2015; França-Neto et al., 2016). Pathogenic microorganisms, insect pests and nematodes are significant potential threats to reducing plant stands in the field. Seed treatment with fungicides, insecticides and nematicides is an alternative to mitigate these losses (Hitaj et al., 2020), since the primary objective is to enhance or preserve seed quality (Cunha et al., 2015).
However, the routine use of seeds treated with chemical products raises concerns regarding socioeconomic factors, human health, and environmental issues (Fadel et al., 2021), such as declining bee populations (Main et al., 2020) and disruptions in the reproductive processes of wild birds (Fernández-Vizcaíno et al., 2022). Biological products are an important sustainable alternative for reducing environmental and human health impacts. These products may contain active ingredients such as microorganisms, their metabolic by-products, and/or plant and algae extracts (Lamichhane et al., 2022).
Some microorganisms can enhance plant development and defend against diseases and abiotic stress. Considered beneficial, they include primarily plant growth-promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF) (Paravar et al., 2023). Recent studies on biological products indicate yield increases, particularly when used at the recommended dose (Costa et al., 2019). Effects are even more pronounced in low-vigor seed lots (Feliceti et al., 2023).
Whether applied individually or in combination, using biological products to treat seeds can influence their initial performance, justifying investigations to clarify and measure these effects in commercial products. Research has indicated potential negative impacts on the physiological quality of treated seeds, with reports of biologicals minimal influence on high-vigor seed lots (Feliceti et al., 2023); reduced germination in treated and stored seeds, particularly in those with lower vigor (Lourenço et al., 2022); and effects dependent on the cultivar and seed physiological quality (Lima et al., 2021).
Thus, the aim of this study was to evaluate the effects of different microorganisms, in both commercial seed treatment products and those with known potential, and clarify their influence on seed physiological quality.
MATERIAL AND METHODS
The experiment was carried out in the Seed Laboratory of the Instituto Federal de Goiás (Urutaí Campus), Using seeds of the Nidera Sementes 8397 IPRO soybean cultivar. A preliminary germination test revealed a germination rate of 94%. The seeds were then placed in paper bags and stored in a climate-controlled environment with an average temperature of 19.8 °C and 40% relative humidity.
Next, eight registered commercial biological products for soybean seed treatment and three combinations of two of these products were applied to the seeds, in addition to a Trichoderma spp. isolate obtained from the institution’s plant pathology laboratory. These 12 subsamples and the untreated control correspond to the 13 treatments in the experiment (Table 1).
Each subsample consisted of 500 g of seeds. The treatments were applied using the highest label-recommended doses of each product and the maximum spray volume indicated for soybean (Table 1), adjusting the product dosages proportionally to the 500 g seed samples. The products were measured using a precision scale (for solids) or an automatic pipette (for liquids). A sufficient volume of water was added to beakers to make up the difference between the spray volume and the label-recommended product dose. The seeds were placed in transparent plastic bags and, after adding the treatment solution, the contents were mixed until the sample was homogeneous.
The Trichoderma spp. isolate was quantified in a Neubauer chamber at a concentration of 3.6.107 spores mL-1 and applied by moistening the germination substrate (paper). In stage 1 all the treatments were tested immediately after seed treatment and in stage 2, after forty days of storage.
Stage 1: Immediate physiological quality evaluation of seeds treated with biological products.
Moisture content: determined using the oven-drying method at 105 ± 3 °C for 24 h (Brasil, 2009), with two subsamples of 10 seeds per treatment. The results were expressed in percentage on a wet basis.
Germination test: 200 seeds per subsample were used, divided into four replications of 50 seeds each. The seeds were sown on paper rolls moistened with water at 2.5 times the dry weight of the paper and placed in a germinator at 25 °C. The final count of normal seedlings was performed on the eighth day after test initiation (Brasil, 2009) and the results were expressed as the percentage of normal seedlings.
First count test: The first count was performed on the fifth day of the previously described germination test.
Seedling length test - traditional method: four replications of 20 seeds each were used. The seeds were arranged in two rows on the paper roll and placed in a germination chamber at 25 °C for 4 days. Next, the root (RL) and shoot length (SL) of each normal seedling were measured in centimeters with a graduated ruler and summed to obtain total seedling length (TSL).
Seedling length, growth, vigor and uniformity were obtained by automated analysis using the Vigor-S system: four replications of 20 seeds per lot were used, distributed on moistened paper and maintained under the same conditions described for the germination test. Three days after sowing, the seedlings were transferred to blue ethylene-vinyl acetate (EVA) foam sheets (30 x 22 cm), corresponding to the useful area of the scanner. A flatbed scanner (Epson Perfection V19) adapted for seedling image analysis was used to capture the images, with a resolution of 300 dpi. Vigor-S® software was used for automated analysis of seed vigor, providing data on seedling length, uniformity, growth, and vigor index.
Stage 2: seed physiological quality evaluation 40 days after treatment with biological products
The thirteen paper bags containing the seeds were stored side by side on a shelf in a climate-controlled environment for 40 days, at an average temperature of 19.8 °C and 40% relative humidity. The maximum and minimum temperatures were 22.8 °C and 18.0 °C, respectively, with maximum relative humidity of 46% and a minimum of 36%. Next, the seeds were tested using the previously described methodology for moisture content, germination, first germination count, traditional seedling length, and seedling length, growth, vigor, and uniformity via the Vigor-S system.
Experimental design and data analysis
All the tests were conducted using a completely randomized design in a 13 x 2 factorial arrangement (seed treatments x storage).
For the traditional and Vigor-S-based seedling length tests, linear analysis of variance (ANOVA) models were fitted according to the factorial scheme for the factors “Treatment” and “Storage” and the variables “Shoot Length”, “Root Length”, and “Total Seedling Length”. To meet the assumptions of the model, optimal power transformations were applied using the Box-Cox method. Means were then back-transformed and presented on the original data scale. The Scott-Knott test was applied for mean separation at 5% significance.
For moisture content, germination, and first count results and comparison between stored and non-stored seeds, residual normality and homogeneity of variances were tested for each parameter analyzed, based on the same factorial design. When assumptions were met, ANOVA was performed, and when significant differences were identified, means were compared using the Scott-Knott at 5% significance.
RESULTS AND DISCUSSION
In moisture content determination after seed treatment, higher values were obtained in treatments 6, 8, 10 and 11. After storage, none of the treatments exhibited statistically significant differences (Table 2). This is due to the hygroscopic equilibrium reached after storage, whereby seeds absorb or release water vapor from or to the air depending on the temperature and relative humidity of the storage environment (Silva, 2019).
The first germination count is a physiological test to determine vigor. Because seed vigor declines before germination rates drop, vigor tests are more sensitive in detecting differences in physiological quality between seed lots, complementing the information obtained from germination tests (Marcos-Filho, 2020).
Before storage, the percentage of normal seedlings was significantly higher in treatments 4, 6, 8, 10 and 11 and lower in treatment 9 (Table 2). Nascimento and Anjos (2023) studied the effect of biostimulants on the physiological quality of maize seeds and found that the influence of these substances depends on the composition and number of elements present in the commercial solution.
Treatments 2 and 9 exhibited worse performance after storage. This may be related to the findings of Montalvão et al. (2020), who observed that Trichoderma can rapidly develop on the seed coat and cause rotting in tomato seeds under high humidity conditions and the absence of organic substrate.
A comparative analysis of treatment 2 before and after storage showed a significantly lower percentage of normal seedlings following storage (Table 2). Strahl et al. (2021) evaluated low-vigor canola seed lots and found that T. asperellum did not increase seed germination when compared to the control, whereas other species improved germination. This suggests that T. asperellum may not be beneficial for low-vigor seeds, which reflects the results obtained in our study.
Prior to storage, only treatment 9 exhibited the smallest number of normal seedlings, whereas the lowest value post-storage was recorded in treatment 2, followed by treatments 1 and 9 with intermediate values (Table 2).
In line with the results obtained in treatments 1 and 9, Pereira et al. (2019) reported reduced germination in wheat seeds treated with B. amyloliquefaciens.
As observed in treatments 1 and 2 in the present study, Strahl et al. (2021) found that a T. asperellum-based product did not increase germination or first count in low-vigor canola seeds, indicating that this fungal species may not be beneficial when compared to results obtained with T. harzianum-based formulations.
As occurred in the first count test, a comparison of treatment 2 pre- and post-storage showed significantly lower germination in the latter evaluation, while treatments 5 and 9 exhibited lower values before storage (Table 2).
For traditional seedling length assessed before storage, the longest shoots were observed in treatment 8 and the shortest in treatments 2 and 12 when compared to the control. Following storage, 1, 2, 3, 9, 11 and 12 exhibited the shortest shoots (Table 3). Plants with well-developed shoots achieve a better photosynthetic rate, resulting in more photoassimilates being translocated to growing or storage organs in later developmental stages (Taiz and Zeiger, 2004).
The lowest root lengths recorded before storage were in treatments 2, 9 and 12, and post-storage in 2, 9 and 11 (Table 3). Treatments with Bacillus spp. were less frequently associated with the shortest root lengths. B. amyloliquefaciens is characterized as an auxin-producing rhizobacterium capable of stimulating root growth by producing plant hormones (Asari et al., 2017).
The lowest total seedling lengths were recorded in treatments 2, 9 and 12 before storage and 1, 2, 9, and 11 after storage (Table 3).
A general observation of all the growth indicators demonstrated worse performance across all indicators in treatment 11, no treatments with greater seedling length than the control after storage, and that treatments 2, 9 and 12 were frequently among those with the lowest values. It should be noted that the presence of Trichoderma sp. is a common factor among these treatments.
Many Trichoderma spp. species are widely known for their growth-promoting effects in different crops and Trichoderma asperellum has been characterized as capable of increasing root length in relation to untreated plants (Ghosh and Panja, 2021). However, similar findings to those obtained here are also reported in the literature. Strahl et al. (2021) found that T. asperellum was less efficient in promoting shoot growth in low-vigor canola seeds.
Pedroso et al. (2018) attributed the limited effect of bioproducts on carrot seeds to reduced antagonistic efficiency due to the saprophytic activity of Trichoderma spp. present in the commercial products which, when combined with phytopathogens, may compromise emergence capacity. Similarly, Ethur et al. (2008) studied T. harzianum isolates in tomato seedlings and noted that seeds should be treated shortly before sowing because the fungus can act as a seed-decaying agent in highly humid environments.
The effectiveness of Trichoderma spp-based bioproducts is influenced by local biotic (living organisms) and abiotic factors (soil type, moisture content, pH and temperature) (Lucon, 2009). Thus, the environmental conditions that the organisms were exposed to may not have resembled their natural habitat (Guzmán-Guzmán et al. 2019). Additionally, factors such as application methods, product formulation, water stress, and the duration of seed-fungus contact influence emergence and crop growth (Cadore et al., 2018).
In line with this discussion, the Trichoderma spp. isolates used in commercial formulations vary considerably, meaning that these effects may not occur with all products. T. harzianum is the most widely marketed species globally, found in 38.8% of single-species formulations, 50% of products containing a mixture of Trichoderma spp. species, and 60% of those containing a mixture of Trichoderma spp. and other fungi, bacteria and mycorrhizae (Bettiol et al., 2019).
Finally, the root, shoot and total seedling lengths measured before storage were higher than the post-storage values obtained, confirming findings reported in the literature that storage alone negatively influences seedling length.
Shoot length determined by Vigor-S software before storage indicated that treatments 4 and 8 obtained the highest values and 12 the lowest, whereas the longest shoots post-storage occurred in treatments 3, 4, 6, 8, 10 and 13 (Table 4).
The shortest roots prior to storage were recorded in treatment 12 and following storage in 1, 2 and 9 (Table 4). With respect to total seedling length, the lowest pre-storage values were recorded in treatment 12 and post-storage in 1, 2, 7, 9 and 11.
Overall, pre-storage length values were higher than those obtained after storage for all the length assessments (Figures 1 and 2), except for root and total seedling length in treatment 9, where no significant difference was observed. Except for treatment 12, the lowest significant length values were recorded after seed storage. No treatment showed significantly greater length than the control, except shoot length before storage, which exhibited no statistically significant difference.
Seedlings from seeds treated with biological products before storage, submitted to automated analysis via Vigor-S software.
Seedlings from seeds treated with biological products and analyzed after storage using Vigor-S software.
Most of the results obtained via Vigor-S software were also recorded with the traditional length method.
Along with growth, the uniformity of seedling development is used as the basis for calculating the vigor index. Uniformity is evaluated based on the difference in length of each seedling in the sample relative to a predefined maximum seedling length (Rodrigues et al., 2020).
For this vigor indicator provided by the software, pre-storage values were lower in treatment 12 (546.47) than in the remaining treatments, with intermediate values in 2 and 9 (688.75 and 694.75, respectively), whereas after storage treatment 2 obtained the lowest value (595.57) and 7 and 9 intermediate values (700.60 and 668.60, respectively) (Table 5). No treatment obtained a higher vigor index than that observed in treatment 13 (control).
The computerized analysis system reports seedling uniformity and vigor index on a scale from 0 to 1000, with higher values indicating greater vigor. When interpreting the results, samples with low uniformity and vigor combined with shorter average seedling lengths are considered to have lower physiological vigor (Gomes-Junior, 2020).
When pre- and post-storage values were compared, most treatments showed higher vigor before storage, except for 9, 10 ,12 and 13, which exhibited greater post-storage vigor (Table 5).
In uniformity testing before storage via Vigor-S software, treatments 2, 9 and 12 obtained lower values in relation to other treatments, with similar values across all treatments after storage. No treatment showed greater uniformity than that of the control. The uniformity index reflects the consistency of seedling development and is generally associated with high-vigor seed lots in germination and vigor tests. This information is important because the establishment of an adequate initial stand depends on the rapid uniform emergence of normal seedlings (Rodrigues et al., 2020). Ribeiro et al. (2024) concluded that the Vigor-S analyses made after the accelerated aging test allow ranking of the seed lots in a similar way to the seedling emergence test.
When comparing treatments before and after storage, 2, 3, 4, 9, 10, 11 and 12 showed increased uniformity after storage (Table 5). However, the uniformity index alone does not necessarily indicate high vigor, since uniformly underdeveloped seedlings can also produce high uniformity values, 2020). Therefore, the increase in uniformity after storage in these treatments may be due to a uniform reduction in physiological quality across the seed lot, as reflected in the pre- and post-storage vigor index values.
Computerized image analysis of seedlings offers advantages over traditional vigor tests, since the process is manual, thus ensuring greater precision and speed via computer vision and specific algorithms. Additionally, the data are collected by software, providing a standardized analysis with less human interference and fewer interpretation errors (Gomes-Junior, 2020).
CONCLUSIONS
The conducted tests indicated that treatments with the Bacillus amyloliquefaciens (BV03) and Trichoderma asperellum (BV10) strains may negatively affect the early growth and development parameters of seedlings under laboratory conditions. This may be due to testing conditions such as the absence of substrates and organic compounds, high humidity, and lack of pathogens; product-related factors, such as metabolite production by each strain, dose, and formulation ingredients; or seed-related factors, including initial physiological quality and storage.
ACKNOWLEDGMENTS
To the Federal Institute of Goiás (IF Goiano) and the Research Support Foundation of Goiás State (FAPEG)
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Publication Dates
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Publication in this collection
04 July 2025 -
Date of issue
2025
History
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Received
26 Sept 2024 -
Accepted
23 May 2025
