Nematicide-enriched seed encrustation enhances early growth without compromising microbial viability in Urochloa brizantha

ABSTRACT

The use of nematicides in cover crops offers promising results in nematode management, but some treatments may interfere with the efficacy of biological nematicides, such as seed coating, which uses high temperatures in the process. This study aimed to evaluate the germination and initial development of Urochloa brizantha (syn. Brachiaria brizantha) seedlings from seeds encrusted with biological and chemical nematicides. Seed encrustation was carried out by Matsuda, with the first layer consisting of nematicide abamectin, thiodicarb, fluopyram, Bacillus amyloliquefaciens + B. subtilis + B. pumilus, Purpureocillium lilacinum + Pochonia chlamydosporia, Trichoderma harzianum + T. asperellum + B. amyloliquefaciens, B. firmus, B. velezensis, standard encrusted seed, and non-encrusted seeds. The seeds were sown in polystyrene trays, and assessments made based on germination percentage, shoot height, and root length, as well as the recovery of biological control organisms from the seeds at 30, 60, 90, and 120 days after treatment. No significant differences were observed between treatments relating to seedling emergence, but seedling development was superior in treatments with biological nematicides. Furthermore, the brachiaria seed coating process did not affect the viability of the biological control organisms applied in the seed treatment which, consequently makes it a promising alternative for nematode management.

Keywords:
biological control; chemical control; nematode management; seed coating

Introduction

Over the years, the most widely cultivated forage grasses in Brazil have included several species of brachiaria, such as Urochloa decumbens Stapf., U. ruziziensis (R.Germ.& Evrard) Crins, U. humidicola (Rendle) Morrone & Zuloaga, and U. brizantha (Hochst. ex A.Rich.) R.D.Webster, primarily grown in the central-western and northern regions of the country (Boddey et al., 2004). In these areas, particularly in the cerrado, large populations of phytonematodes, such as root-lesion nematodes (Pratylenchus spp.), are commonly found. These nematodes multiply in forage grasses and persist in the soil, posing a significant threat to subsequent crops. This is especially concerning when grains are planted next, as Pratylenchus spp. can severely impact plant development, and lead to reduced yield and grain quality (Machado, 2014).

To manage nematodes, strategies such as the use of resistant cultivars, crop rotation with non-host species, and the application of chemical and biological nematicides are recommended (Machado, 2022a). In recent years, Brazil has seen a rapid expansion in the use of biological nematicides (Machado, 2022b), opening up new possibilities for nematode control, including their application in cover crops like brachiaria (Machado, 2022a). Treating both the cover crop and the succeeding soybean crop can enhance nematode management (Soares et al., 2022). Thus, understanding the interaction between biological control agents and different plant species, as well as determining the most effective application methods for each cover crop, is crucial to optimizing nematode management in Brazil (Dias-Arieira et al., 2023).

Treatments on cover crop seeds can affect biocontrol agent effectiveness, as their compatibility with agrochemicals like nematicides, insecticides, fungicides, and herbicides is still debated, with possible negative impacts on beneficial microorganisms (Ribeiro et al., 2025). For brachiaria, encrustation standardizes seed size and weight which improves planting and early development by adding nutrients and fungicides. However, it can also reduce germination and seedling emergence (Brites et al., 2011; Derré et al., 2016).

Despite the widespread use of encrusted brachiaria seeds, little is known about their effects on biological control agents of nematodes. Therefore, this study aimed to evaluate the germination and early development of U. brizantha plants grown from encrusted seeds treated with biological and chemical nematicides. In addition, the effect of the seeds’ encrustation process on the biological control agents was evaluated by monitoring the viability and recovery of fungi and bacteria over time after encrustation.

Materials and Methods

The experiments were conducted in a greenhouse at Embrapa Soja, in Londrina, in the state of Paraná (23°00’10.5" S, 51°10’05.3" W, 630 m altitude), under controlled temperature conditions. The first trial took place from May to June 2023, with average temperatures in the range of 19 °C ± 5 °C, while the second was carried out between Oct and Nov, with average temperatures of 24 °C ± 5 °C. The experimental design followed a randomized block layout, consisting of 200 plots (four blocks of 50 cells) for each treatment.

For the experiments, ten 200-cell Styrofoam seed trays (each cell measuring 2.5 cm in diameter × 3 cm in depth) were filled with a substrate composed of 80 % sand, 5 % silt, and 15 % clay. Seeds (one per cell) were sown at a depth of 2 cm in the center of each cell using tweezers. To maintain soil moisture at field capacity, trays were irrigated twice daily throughout the study.

The seed encrustation process was conducted industrially by Matsuda Group. Before encrustation, the seeds were standardized in terms of size, weight, and color, then subjected to sulfuric acid scarification. This chemical cleaning process involves treating the seeds with sulfuric acid 98 %, followed by thorough washing with water. During this stage, a brief temperature increase occurs due to the acid-water reaction, but lasts only a few seconds. At the end of scarification, seed purity reaches 97 %.

The encrustation process follows a structured sequence: first, a fungicide is applied, followed by an adhesive and powder-based coating. When required, a third layer containing an insecticide is added, and finally, the seeds receive a polymer coating. However, in this study, the standard treatment did not include fungicides or insecticides; instead, the seeds were coated with adhesive and powder products only. In treatments involving nematicides, the first layer contained the respective nematicide, with no insecticides included.

During encrustation, temperatures exceed 55 °C to facilitate drying of the coating. However, because the seeds are encased in multiple layers, they experience minimal temperature-related impact. After this stage, seed purity reaches 99.5 %. The experiment used Urochloa brizantha cv. Marandu (Matsuda), a cultivar recommended for medium- to high-fertility soils. The treatments consisted of the following: 1) non-encrusted seeds; 2) encrusted seeds; 3) encrusted seeds with abamectin (Avicta®, Syngenta) at 100 mL 100 kg⁻¹ seeds; 4) encrusted seeds with thiodicarb (Cropstar®, Bayer) at 700 mL 100 kg⁻¹ seeds; 5) encrusted seeds with fluopyram (Ilevo®, BASF) at 200 mL 100 kg⁻¹ seeds; 6) encrusted seeds with Bacillus amyloliquefaciens + B. subtilis + B. pumilus (BFN 2022-06 RET 1650/2022, Ballagro) at 100 g 100 kg⁻¹ seeds; 7) encrusted seeds with Purpureocillium lilacinum + Pochonia chlamydosporia (Nemat Stellus®, Ballagro) at 200 mL 100 kg⁻¹ seeds; 8) encrusted seeds with Trichoderma harzianum + T. asperellum + B. amyloliquefaciens (Pardella®, Ballagro) at 50 g 100 kg⁻¹ seeds; 9) encrusted seeds with B. firmus (Votivo Prime®, BASF) at 300 mL 100 kg⁻¹ seeds; 10) encrusted seeds with B. velezensis (Arvatico®, Syngenta) at 800 mL 100 kg⁻¹ seeds.

Seedling emergence was recorded 21 days after sowing, and the germination percentage was calculated based on the number of emerged plants. At the same time, plant height and root length were measured.

In addition to these agronomic evaluations, the study assessed the viability of the biological control microorganisms applied to the seeds. These microorganisms were recovered from seeds treated at 30, 60, 90, and 120 days after treatment to determine the impact of encrustation on their survival. The Bacillus species were recovered using the plating method, while Trichoderma harzianum, P. lilacinum, and P. chlamydosporia were analyzed via quantitative polymerase chain reaction (qPCR). The qPCR analysis was performed by Ballagro Agrotecnologia, which provided specific molecular markers for each strain contained in the biological nematicides evaluated.

For the plating of Bacillus-treated seeds, 50 seeds per treatment were placed in 0.85 % saline solution and subjected to orbital agitation at 2.33 Hz at 28 °C for 20 min, resulting in a 10⁹ dilution. Serial dilutions were then performed (1: 9 ratio), transferring 1 mL of the 10⁹ suspension into 9 mL of 0.85 % saline solution to obtain a 10⁻¹ dilution, repeating the process until reaching 10⁻². Subsequently, 100 µL of each dilution and a control sample (100 µL of 0.85 % saline solution) were plated onto nutrient agar (NA) culture medium. Plates were incubated in the dark at 28 °C for 16-24 h in a biochemical oxygen demand (B.O.D.) chamber. Colony counts were then performed, and the number of colony-forming units (CFU) was calculated as follows: CFU = number of colonies × dilution factor.

The phytotechnical data were analyzed for normality (Shapiro-Wilk test) and homogeneity of variances (Bartlett's test), followed by analysis of variance (ANOVA). The means were grouped using the Scott-Knott test at the 5 % significance level. Statistical analyses were carried out using R software (v. 4.4.2), with the agricolae (Mendiburu, 2023) packages.

Results

In Experiment 1, no significant differences were observed between treatments in connection with seedling emergence (Table 1). The emergence rate for non-encrusted seeds was 41 %, while the treatments ranged from 37 % to 60 %. In Experiment 2, seedling emergence for non-encrusted seeds was 67 %, with values varying between 55 % and 69 % across treatments. The seeds treated with B. velezensis exhibited emergence rates closest to those of non-encrusted seeds (Table 2).

Table 1
Number of emerged seedlings and germination percentage, height and root length (± standard deviation) of seedlings at 21 days after sowing in Experiment 1.

Treatments	Number of emerged seedlings	Germination	Height of seedlings1	Root length1
		%
1) Non-encrusted seeds	82	41 a	5.1 ± 1.4 d	4.0 ± 0.7 b
2) Encrusted seeds	74	37 a	3.2 ± 0.7 e	4.8 ± 2.3 b
3) Abamectin	78	39 a	2.6 ± 0.8 e	3.1 ± 1.6 b
4) Thiodicarb	91	46 a	3.3 ± 1.1 e	4.4 ± 1.7 b
5) Fluopyram	96	48 a	3.7 ± 0.7 e	5.6 ± 1.2 b
6) Bacillus amyloliquefaciens + B. subtilis + B. pumilus	74	37 a	8.2 ± 0.8 b	6.5 ± 0.8 a
7) Purpureocillium lilacinum + Pochonia chlamydosporia	87	44 a	9.0 ± 0.6 b	6.9 ± 2.4 a
8) Trichoderma harzianum + T. asperellum + B. amyloliquefaciens	119	60 a	8.8 ± 0.6 b	7.8 ± 2.4 a
9) Bacillus firmus	84	42 a	6.6 ± 0.9 c	6.7 ± 1.6 a
10) Bacillus velezensis	92	46 a	10.3 ± 0.8 a	6.2 ± 1.3 a

• ¹
  Values are means of six replicates.
• Means followed by the same letter in columns did not differ significantly according to Scott-Knott test at a significance level of 5 %.

Table 2
Number of emerged seedlings and germination percentage, height and root length (± standard deviation) of seedlings at 21 days after sowing in Experiment 2.

Treatments	Number of emerged seedlings	Germination	Height of seedlings1	Root length1
		%
1) Non-encrusted seeds	135	67 b	8.5 ± 0.4 b	5.0 ± 0.4 c
2) Encrusted seeds	110	55 e	10.8 ± 1.4 b	5.1 ± 0.4 c
3) Abamectin	127	63 d	9.2 ± 0.8 b	4.3 ± 0.9 c
4) Thiodicarb	130	65 c	9.6 ± 2.7 b	4.0 ± 0.6 c
5) Fluopyram	129	64 c	10.3 ± 0.9 b	4.3 ± 0.4 c
6) Bacillus amyloliquefaciens + B. subtilis + B. pumilus	124	62 d	12.8 ± 0.7 a	6.3 ± 0.6 b
7) Purpureocillium lilacinum + Pochonia chlamydosporia	112	56 e	12.9 ± 1.9 a	5.9 ± 0.4 b
8) Trichoderma harzianum + T. asperellum + B. amyloliquefaciens	127	63 d	14.1 ± 1.6 a	7.3 ± 1.9 a
9) Bacillus firmus	127	63 d	13.4 ± 1.1 a	6.0 ± 1.4 b
10) Bacillus velezensis	138	69 a	12.5 ± 1.6 a	5.8 ± 1.4 b

• ¹
  Values are means of six replicates.
• Means followed by the same letter in columns did not differ significantly according to Scott-Knott test at a significance level of 5 %.

As regards plant development, in Experiment 1, encrustation with biological nematicides led to an increase in seedling height from 1.5 cm (23 %) to 5.2 cm (102 %) and in root growth from 2.2 cm (35 %) to 3.8 cm (95 %) compared to non-encrusted seeds (Table 1). Among treatments, B. velezensis produced the tallest seedlings, whereas non-encrusted seeds had the lowest height values. The biological nematicides B. amyloliquefaciens + B. subtilis + B. pumilus, P. lilacinum + P. chlamydosporia, T. harzianum + T. asperellum + B. amyloliquefaciens, and B. firmus exhibited higher intermediate values, whereas chemical nematicides were grouped with lower intermediate values (Figure 1).

Figure 1
Urochloa brizantha seedlings 21 days after sowing, treated with chemical and biological nematicides, compared with non-encrusted and encrusted seeds.

Similarly, in Experiment 2, biological treatments promoted seedling growth, increasing height by 4 cm (51 %) to 5.6 cm (65 %) and root growth by 0.8 cm (26 %) to 2.3 cm (46 %) compared to non-encrusted seeds (Table 2). The treatment containing T. harzianum + T. asperellum + B. amyloliquefaciens resulted in the tallest seedlings, while non-encrusted seeds had the shortest. Other biological nematicides (B. amyloliquefaciens + B. subtilis + B. pumilus, P. lilacinum + P. chlamydosporia, B. firmus, and B. velezensis) showed higher intermediate values, whereas chemical nematicides were grouped from those with lower intermediate values.

In Experiment 1, root growth was significantly greater in treatments with biological nematicides compared to chemical nematicides, non-encrusted seeds, and the standard encrustation treatment (Table 1). This trend continued in Experiment 2, where T. harzianum + T. asperellum + B. amyloliquefaciens resulted in the longest roots. Other biological nematicides (B. amyloliquefaciens + B. subtilis + B. pumilus, P. lilacinum + P. chlamydosporia, B. firmus, and B. velezensis) also exhibited higher intermediate values. In contrast, chemical nematicides, non-encrusted seeds, and the standard encrustation treatment were grouped from those with the shortest root lengths (Table 2).

Although chemical nematicides did not enhance seedling height nor root system development, they also had no negative effect on plant growth. Their results were statistically similar to those of non-encrusted seeds and the standard encrusted treatment.

Recovery of fungal species from treated seeds via qPCR demonstrated that Trichoderma spp. propagules remained viable at a concentration of 10³ CFU g⁻¹ seed from 30 to 120 days after treatment, indicating that encrustation did not affect their survival or development in brachiaria seeds (Table 3). The concentration of P. lilacinum remained stable at 10⁴ CFU g⁻¹ seed throughout the evaluation period. However, P. chlamydosporia showed a decline, decreasing from 10⁵ CFU g⁻¹ seed at 30 and 60 days to 10³ CFU g⁻¹ seed at 90 and 120 days (Table 3).

Table 3
Number of colony-forming units of fungi and bacteria obtained from Urochloa brizantha seeds, after 30, 60, 90, and 120 days with the biological nematicides via seed encrustation in Experiment 1.

Method	Organism	30 DAT	60 DAT	90 DAT	120 DAT
qPCR	Trichoderma harzianum + T. asperellum	1.4 × 104	2.0 × 104	5.0 × 103	4.3 × 103
qPCR	Pochonia chlamydosporia	2.6 × 105	2.6 × 105	4.0 × 103	5.3 × 103
qPCR	Purpureocillium lilacinum	8.3 × 104	8.9 × 104	4.4 × 104	1.4 × 104
Plating	Bacillus amyloliquefaciens	2.3 × 106	3.0 × 106	2.8 × 106	5.1 × 106
Plating	Bacillus subtilis + B. amyloliquefaciens + B. pumilus	3.4 × 107	3.2 × 107	3.7 × 107	4.7 × 107
Plating	Bacillus firmus	2.7 × 104	1.7 × 104	1.9 × 104	3.0 × 104
Plating	Bacillus velezensis	9.4 × 103	1.1 × 104	1.1 × 104	1.1 × 104

• Values are means of three replicates. DAT = days after treatment; qPCR = quantitative polymerase chain reaction.

With regard to bacterial viability, plating revealed that B. amyloliquefaciens (present in the formulation with Trichoderma species) maintained a concentration of 10⁶ CFU g⁻¹ seed from 30 to 120 days after treatment. Similarly, B. subtilis + B. amyloliquefaciens + B. pumilus remained stable at 10⁷ CFU g⁻¹ seed throughout the evaluation period, and B. firmus maintained a concentration of 10⁴ CFU g⁻¹ seed. In contrast, B. velezensis exhibited an increase, rising from 10³ CFU g⁻¹ seed at 30 days to 10⁴ CFU g⁻¹ seed at 60, 90, and 120 days after treatment (Table 3).

Discussion

The application of biological nematicides on cover crops, such as brachiaria, has been increasing in Brazilian agriculture over recent years. In addition to the nematode control, this practice can enhance plant development and bring other benefits to the crop system. Previous study has demonstrated that applying Purpureocillium lilacinum and Trichoderma harzianum to crop residues of U. ruziziensis reduced by 45 % to 98 % the total population of Meloidogyne javanica (Soares et al., 2022). This beneficial interaction between grasses and biological control agents may be linked to the high carbon-to-nitrogen (C:N) ratio in brachiaria straw, which slows residue decomposition and increases organic matter and humic substance content (Souza et al., 2019). This environment supported the development and persistence of saprophytic microorganisms.

A number of studies reported that sequential applications of Pochonia chlamydosporia in U. ruziziensis resulted in superior nematode control (Dias-Arieira et al., 2023). However, some reports indicated that rapid decomposition of certain cover crop residues can release high concentrations of chemical compounds into the soil, as the monocrotaline produced by Crotalaria spp., negatively affects fungal hyphae and bacterial propagules (Vilchis-Martínez et al., 2013; Miamoto et al., 2021; Soares et al., 2022).

In our study, biological nematicides, particularly bacterial strains, improved seedling root system development. However, chemical nematicides did not show significant statistical differences compared to non-encrusted seeds or the standard encrustation treatment. Overall, germination percentage, plant height, and root length were higher in Experiment 2 than in Experiment 1. This outcome is likely due to the higher average temperature in Experiment 2 (24 °C), which is more favorable to Brachiaria seedling development, compared to 19 °C in Experiment 1. The optimal temperature for U. brizantha seed germination is 25 °C (Chiodini and Cruz-Silva, 2013). Despite the less favorable conditions in Experiment 1, biological nematicides still promoted initial seedling growth.

The encrustation process of brachiaria seeds did not cause mortality in the evaluated Bacillus species and did not appear to have compromised the viability of the fungal biological control agents included in the seed treatment. Bacillus species are known for their ability to form endospores, which provides a significant advantage in extending the shelf life of bionematicides formulated with these bacteria. This characteristic also allows their application under adverse soil and climate conditions (Machado, 2022b). Since endospore-based formulations are more tolerant to environmental stress, they are likely less affected by the encrustation process and the mixing of products on the seeds, which aligns with the successful recovery of Bacillus spp. from encrusted brachiaria seeds in this study.

However, the efficacy of Bacillus spp. in nematode control can be influenced by the specific combination of products applied to seeds. Studies conducted by our group (non-published data) demonstrated that certain polymers, when combined with clothianidin, carbendazim/thiram, and chlorantraniliprole in corn seed treatment, reduced the effectiveness of B. firmus against Meloidogyne incognita. On the other hand, B. firmus remained stable and effective in controlling M. incognita for up to 100 days after seed treatment when mixed with a polymer that did not inhibit bacterial viability.

The primary Bacillus species used for nematode biological control is classified as plant growth-promoting rhizobacteria (PGPR) (Machado, 2022b). These bacteria exhibit an endophytic lifestyle or associate closely with plant tissues, and stimulate the production of growth regulators, particularly phytohormones. Additionally, they contribute to nitrogen fixation regulation, indole acetic acid (IAA) synthesis, phosphorus solubilization, and reduced evapotranspiration (Santos et al., 2018). Certain Bacillus species also influence root system architecture and growth, and thereby enhance root development (Grover et al., 2021). These attributes help explain the improved root growth observed in brachiaria seedlings in this study.

Similarly, Trichoderma spp. promote plant growth and vigor, enhance stress tolerance, and improve nutrient absorption and rhizosphere bioremediation. They provide plants with beneficial secondary metabolites, enzymes, and pathogenesis-related proteins (Kumar, 2013). Purpureocillium lilacinum and P. chlamydosporia also exhibit an endophytic lifestyle, contributing to increased plant growth and development (Zavala-Gonzalez et al., 2015; Ahmed and Monjil, 2019). Like Bacillus spp., these fungal agents played a role in the enhanced development of brachiaria seedlings observed in this study.

Among the fungal agents, P. chlamydosporia was the most affected by encrustation, with a decline in concentration over time. In the absence of nematodes in the soil, P. chlamydosporia can survive saprophytically by utilizing organic matter as a food source (Lopez-Lima et al., 2014). However, when applied directly to seeds without an immediate food source, the survival conditions for P. chlamydosporia become less favorable. This challenge is particularly significant because commercial bionematicide formulations in Brazil contain fungal propagules such as hyphae and conidia (Machado, 2022b) rather than survival structures such as chlamydospores, which are more resistant to environmental stress (Krug et al., 2004).

This study did not evaluate the interaction of biological nematicides with chemical fungicides or insecticides. Therefore, further research is necessary to assess whether these interactions could compromise the viability and effectiveness of biological treatments.

• Declaration of use of AI technologies
  No AI technologies were used.

Data availability statement

Data will be available under request.

Acknowledgments

Fifth author would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the grants awarded.

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Edited by

• Edited by:
  Fabrício de Ávila Rodrigues https://orcid.org/0000-0002-3091-0000

Publication Dates

• Publication in this collection
  14 Aug 2026
• Date of issue
  2026

History

• Received
  18 June 2025
• Accepted
  25 Sept 2025