Can fungi associated with seeds be an important source for the biological control of invasive plants? Urochloa (=Brachiaria) as a case study

doi:10.21203/rs.3.rs-7861156/v1

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Can fungi associated with seeds be an important source for the biological control of invasive plants? Urochloa (=Brachiaria) as a case study

https://doi.org/10.21203/rs.3.rs-7861156/v1

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The introduction and naturalization of exotic species in Brazil, favored by their high adaptability and the absence of natural enemies, are among the main drivers of biodiversity loss. A notable example is the genus Urochloa, originally introduced for pasture formation but now recognized as one of the major groups of invasive plants capable of suppressing native species regeneration and altering ecological dynamics. In view of the need for sustainable alternatives to manage these species, this study investigated fungi associated with U. decumbens seeds to select isolates with potential for biological control. Seeds collected from three distinct areas were subjected to the Blotter test, resulting in the isolation and identification of 199 fungi, with Bipolaris being the most frequent genus. Three strains of B. gossypina (F11, F13, and F15) were evaluated for pathogenicity on seeds and seedlings of U. decumbens, U. brizantha, and U. ruziziensis, as well as on five native Cerrado species (Cecropia pachystachya, Luehea divaricata, Eugenia dysenterica, Handroanthus ochraceus, and Guazuma ulmifolia). The isolates significantly reduced germination and caused necrosis and death in the invasive grasses, without affecting the native species tested. These findings indicate that fungi associated with the seeds of invasive plants may represent a promising and ecologically safe alternative for the management of Urochloa spp., particularly in areas where herbicide use is restricted.

Bipolaris gossypina

Plant invasion

Mycoherbicide potential

Native flora

Human activities are the primary drivers of biodiversity loss on a global scale. According to the 2020 World Wide Fund for Nature (WWF) report, the most significant environmental threats include species overexploitation, pollution, climate change, invasive species, and changes in land and sea use. Among these, landscape modification, particularly the conversion of natural ecosystems into agricultural areas, stands out as the leading cause of biodiversity loss. This phenomenon is especially evident in Brazil, where the land use systems historically relied on continuous expansion. Land was cultivated until its resources were depleted and subsequently abandoned, leaving behind degraded areas (Boddey et al. 2003). Coupled with this land use system, the deliberate introduction and subsequent spread of exotic species by humans have made areas under intense human influence highly susceptible to biological invasions (Zenni et al. 2024).

A prominent example is the introduction of species from the genus Urochloa P. Beauv. (= Brachiaria). Currently, about 32.5% of Brazilian territory has been converted to agriculture, with pastures accounting for the majority of this transformation, covering over 164 million hectares (MapBiomas 2023). Most of these pastures are dominated by species from the Urochloa, which were introduced due to their high productivity and adaptability to Brazil's environmental conditions (Landau et al. 2020). While these grasses have delivered economic benefits, their rapid adaptation has turned them into invasive plants in various Brazilian ecosystems (Zenni and Ziller 2011; Landau et al. 2020). For example, in Conservation Units in the South, Southeast, and Midwest regions of the country, brachiarias are among the main invasive species displacing native plants (Ziller and Dechoum 2013).

The invasive capacity of Urochloa spp. is attributed to multiple factors, including high resource competition, seed dormancy, allelopathic compound production, and other adaptive traits (Ustarroz et al. 2015; Fares et al. 2020). Consequently, these grasses significantly hinder the development of native species such as Caryocar brasiliense (pequizeiro), Myracrodruon urundeuva (aroeira), and Anadenanthera colubrina (angico-branco) (Feitoza et al. 2020; Ruas et al. 2024). Another critical issue is the increased intensity of fires within the Cerrado biome. Although natural fires are a frequent and integral part of this ecosystem, the presence of these invasive grasses intensifies fire severity (Gorgone-Barbosa et al. 2015). This imbalance favors the establishment of species, like U. decumbens and U. brizantha, which exhibit increased germination rates and greater seedling establishment in fire-affected areas (Gorgone-Barbosa et al. 2016).

Given the strong association between degraded areas and the presence of Urochloa species, their control has become a priority in environmental restoration projects (Thomas et al. 2019). Practices such as mowing and herbicide application are commonly used to manage these invasive plants, focusing on controlling established individuals in the field to prevent seed deposition into the seed bank and promote native species development (ICMBio 2023). However, significant challenges remain in implementing these methods. Conventional approaches, such as hand weeding and mowing, are labor-intensive and costly, often requiring repeated interventions, sometimes with limited or no effectiveness (Nave et al. 2009; Lisboa et al. 2023). Although herbicides are effective and cost-efficient, their use is restricted in protected areas due to legal limitations stemming from environmental concerns, such as water contamination and impacts on soil microbiota (Thomas et al. 2019; IBAMA 2022; Lima et al. 2023; Weidlich et al. 2020).

In this context, research increasingly seeks efficient and sustainable alternatives for managing degraded areas, aiming to reduce costs and minimize dependency on chemical products (Pywell et al. 2010; César et al. 2013; Cianciaruso et al. 2025). Among these alternatives, biological control stands out as a promising approach. Biological weed control has garnered growing attention in global studies (Winston et al. 2014). Therefore, this study proposes a strategy based on the use of phytopathogens to mitigate the dominance of these grasses, contributing to the development of a novel method for restoring native ecosystems. To this end, fungi naturally associated with U. decumbens seeds were evaluated to assess their potential to control three species of Urochloa.

2.1 Urochloa decumbens Seed Sampling

Seeds of U. decumbens were collected between July 2021 and February 2022 in the municipalities of Monte Carmelo and Canápolis, both in Minas Gerais, Brazil. Sampling sites included the campus of the Federal University of Uberlândia (UFU), a legally protected reserve, and an abandoned pasture in an urban area, representing distinct environmental conditions. The three collection areas, chosen based on the prevalence of the species, were:

Area 1: UFU Campus, Monte Carmelo (18°43'28.9"S, 47°31'30.5"W);
Area 2: Legal Reserve, Monte Carmelo (18°42'49.9"S, 47°33'23.6"W);
Area 3: Abandoned pasture, Canápolis (18°43'01.9"S, 49°12'27.5"W).

Seeds were randomly collected from multiple plants, including healthy and damaged, green and dry seeds, ensuring wide environmental and seasonal variation. Material was gathered from the soil near plants and directly from spikelets cut with pruning shears. Samples were stored in slightly moistened plastic bags during transport and transferred to a freezer upon arrival at the laboratory until further analysis.

2.2 Isolation and Identification of Seed Fungi

Seed health was assessed using the Blotter test (MAPA 2009). Fifty seeds were evenly distributed on moistened filter paper in each gerbox. Prior to incubation, seeds were surface-sterilized with 70% ethanol for 30 s, followed by 1% sodium hypochlorite for 5 min, and rinsed in sterile distilled water. For each collection area, eight gerboxes were prepared, four with disinfected seeds and four with non-disinfected seeds.

Gerboxes were incubated at 25°C in a biochemical oxygen demand (BOD) chamber without photoperiod for seven days. From the seventh day onward, fungal growth was monitored daily under a stereomicroscope. Fungi were isolated directly from infected seeds and transferred to potato dextrose agar (PDA) medium supplemented with rifamycin to suppress bacterial growth. Pure cultures were obtained following standard laboratory procedures and preserved using the Castellani method (Dhingra and Sinclair 1995). Preliminary identification was performed with lactoglycerol-mounted slides and the taxonomic keys of Barnett and Hunter (1987).

2.3 Pathogenicity Assays

From the isolates obtained in the Blotter test, three Bipolaris gossypina strains (F11, F13, and F15) were selected to evaluate their pathogenic potential against three Urochloa species: U. brizantha, U. ruziziensis, and U. decumbens. Seeds of the three species were sourced from commercial lots provided by Semembrás.

2.3.1 Pre-germination Assay

For the pre-germination assay, 10 g of seeds from each Urochloa species were surface-sterilized (30 s in 70% ethanol, followed by 5 min in 1% sodium hypochlorite, and rinsed three times in autoclaved distilled water) and dried for 24 h at 55°C. Seeds were then arranged in a single layer in Petri dishes in direct contact with actively growing mycelium of each fungal strain. Control seeds underwent the same sterilization and drying procedures but were placed in Petri dishes without mycelium. After 48 h of exposure, 20 seeds from each treatment were sown in 50 mL plastic cups containing a sterilized 2:1:1 (v/v/v) mixture of red latosol, sand, and substrate. Germination was recorded daily for 10 days as the percentage of seeds sown, and seedling development was visually assessed. The experiment followed a completely randomized design (CRD) with six replicates per treatment.

2.3.2 Post-germination Assay

For the post-germination assay, seeds were treated with Bravonil® (chlorothalonil) for 10 min at the manufacturer’s recommended concentration, rinsed in distilled water, dried for 12 h at 55°C, and sown in 250 mL plastic cups containing the same sterilized soil mixture. Each cup received 20 seeds and was maintained under greenhouse conditions (Federal University of Uberlândia – Monte Carmelo campus) until emergence. Fifteen days after emergence, seedlings were thinned to five per cup. Inoculation was performed by placing 5 cm-diameter PDA discs containing actively growing mycelium onto the adaxial surface of leaves. In the control treatment, PDA discs without mycelium were used. Plants were kept in a moist chamber for 48 h at 25º C to promote infection. Disease symptoms were evaluated daily for seven days. Incidence was quantified per cup, and severity was assessed using a scale adapted from the Sociedade Brasileira da Ciência das Plantas Daninhas (SBCPD, 1995) (Table 1). The experiment followed a CRD with six replicates per treatment.

Table 1
Note scale for assessing weed control efficacy
Concept	%	Description
A	86–100	Excellent or total control of the target species
B	66–85	Good control, acceptable for area infestation
C	41–65	Moderate control, insufficient for area infestation
D	0.1–40	Poor or negligible control
E	0	No control
* adapted from SBCPD (1995)

2.3.3 Tests on Native Species

To assess disease incidence in native Cerrado species, five species were selected: Cecropia pachystachya Trécul (embaúba), Luehea divaricata Mart. (açoita-cavalo-miúdo), Eugenia dysenterica (Mart.) DC. (cagaita), Handroanthus ochraceus (Cham.) Mattos (ipê-amarelo), and Guazuma ulmifolia Lam. (mutambo). For each species, four plants were used, each assigned to one treatment: control (PDA discs without mycelium), isolate F11, isolate F13, or isolate F15. On each plant, 5 cm-diameter discs corresponding to the assigned treatment were placed on all leaf surfaces. Evaluations were conducted at 2 and 5 days after inoculation (DAI) to record leaf lesion formation and compare with the control.

2.4 Statistical analysis

Statistical analyses for all tests were performed using the Kruskal–Wallis test followed by Dunn’s post hoc test, with a significance level of p < 0.05. Analyses were conducted in R (version 4.5.1) via RStudio (version 2025.05.1 + 513).

A total of 199 fungal isolates were obtained from the three study areas (Table 2). Among these, 10 fungal genera were identified, while 25 isolates could not be identified and were categorized under a group labelled as 'N.I.' (Not Identified). Among the identified isolates, Bipolaris was the most frequent genus across the three analysed areas, accounting for 18.09% of the total isolates. This was followed by the genera Alternaria and Fusarium, which also showed high occurrences associated with the seeds, representing 12.06% and 11.56% of the isolates, respectively. The other genera had occurrences ranging from 8% to 2%. The distinction between green and dry seeds, as well as between damaged and healthy seeds, did not significantly influence the diversity of isolated fungi. It was observed that the application of surface disinfestation slightly reduced the occurrence of fungi associated with the seeds.

Table 2
Number of fungal isolates obtained from *Urochloa decumbens* seeds collected in three areas
	Area 1		Area 2		Area 3
Genus	N.D.	W.D.	N.D.	W.D.	N.D.	W.D.	Total
Alternaria	12	1	5	5	1	0	24
Bipolaris	10	5	6	5	5	5	36
Epicoccum	5	1	6	0	1	0	13
Trichoderma	4	3	2	1	0	0	10
Fusarium	7	3	6	0	4	3	23
Cladosporium	5	3	5	0	1	0	14
Chaetomium	0	5	2	2	3	2	14
Aspergillus	0	0	3	1	0	0	4
Nigrospora	0	4	0	3	7	2	16
Penicillium	1	1	0	1	3	3	9
N.I.	0	0	5	9	13	9	36
Total	44	26	40	27	38	24	199
* N.D. = non-disinfected; W.D. = with disinfected

In the pre-germination assay, after 48 h of seed exposure to the three fungal isolates, a significant reduction in germination was observed for all three Urochloa species when compared with the control group (Fig. 1, Fig. 2). However, no statistically significant differences were detected among the isolates for any of the Urochloa species. Visual inspection of the seeds also revealed that colonization by isolate F15 after 48 h was less intense than that observed for isolates F11 and F13, although their effects on germination were comparable. In some cases, even when germination occurred, seedlings failed to establish and subsequently died.

Figure 1 Response of Urochloa brizantha, U. ruziziensis, and U. decumbens after inoculation with three Bipolaris gossypina strains (F11, F13, and F15) compared to the non-inoculated control, based on the pre-germination (A) and post-germination (B) assays. Different letters indicate significant differences among treatments (p < 0.05) according to Dunn’s post hoc test; identical letters indicate no significant difference

Figure 2 Germination response of Urochloa brizantha, U. ruziziensis, and U. decumbens (from left to right) after inoculation with Bipolaris gossypina strains F11, F13, and F15, compared with the non-inoculated control

In the post-germination assay, the first symptoms appeared two days after inoculation and stabilized on the fifth day. Across all Urochloa species inoculated with the phytopathogens, the disease symptoms initially appeared as chlorosis on the leaf area in contact with the mycelial disc and at the leaf apex, progressing toward the leaf base. This could lead to necrosis and, in severe cases, plant death (Fig. 3). In treatments with fungal isolates, the management ratings ranged from B to A, representing good suppression, acceptable for area infestation, to excellent, with total suppression observed. In contrast, no symptoms were observed in the control group, which was rated as E, indicating no management. Statistical analysis confirmed that management ratings for fungal isolates differed significantly from the control (p < 0.05), but no significant differences were found among isolates (Fig. 1).

Figure 3 Severity of symptoms in seedlings of Urochloa brizantha, U. ruziziensis, and U. decumbens (from left to right) after inoculation with Bipolaris gossypina strains F11, F13, and F15, showing tissue necrosis and plant death compared with the non-inoculated control

In the evaluation of native Cerrado species, no lesions were observed on the leaf blade of any species in contact with the mycelial discs of the fungal isolates (Fig. 4). The foliar aspect remained comparable to that of the control plants in both evaluations, two and five days after inoculation.

Figure 4 Specificity test of Bipolaris gossypina isolates on five native species. From left to right: control, strain F11, strain F13, and strain F15. (A1–A4) Cecropia pachystachya; (B1–B4) Luehea divaricata; (C1–C4) Eugenia dysenterica; (D1–D4) Handroanthus ochraceus; (E1–E4) Guazuma ulmifolia. No statistical differences (p < 0.05) were observed between the control and inoculated treatments according to Dunn’s post hoc test, due to the absence of symptoms

Urochloa species play a central role in the replacement of native flora across several Brazilian biomes. Consequently, the restoration of degraded areas and the preservation of threatened ecosystems directly depend on the effective control of these invasive grasses. In the field, current management strategies emphasize the removal of established plants, the prevention of new seed deposition into the soil, and the promotion of native vegetation recovery (ICMBio 2023). However, it is not enough to simply remove the plants in the field, it is essential that removal occurs before flowering to prevent the continuous enrichment of the seed bank. This dynamic is clearly illustrated in areas invaded by U. brizantha, where high seed density is strongly linked to its efficient reproductive strategy, marked by continuous and abundant seed production throughout the year (Dairel and Fidelis 2020). While these practices are indispensable, complementary approaches are required to accelerate the depletion of invasive seeds in the soil seed bank. Since the seed bank is regulated by both input (seed deposition) and output (germination, mortality, or loss of viability), its reduction can be enhanced through the action of microorganisms (Lai et al. 2021).

In Brazil, more than 70 species of phytopathogenic fungi have been documented in association with Urochloa (Mendes et al. 2019). Additionally, analyses of seed lots from different regions of the country revealed a high incidence of associated fungi, reflecting the poor phytosanitary quality of these seeds (dos Santos et al. 2024). In line with this, the present study identified considerable fungal diversity in U. decumbens seeds, with 199 isolates obtained. These findings indicate that microorganisms naturally occur in the studied areas and actively contribute to seed degradation. This highlights the relevance of seeds as a model for exploring phytopathogen-based weed biocontrol strategies. In addition, the blotter test proved to be an efficient method for detecting and isolating fungi from seeds. It allowed clearer identification of the diversity already associated with them and facilitated the isolation process when compared to pathogens obtained from leaf lesions.

Unlike the results of dos Santos et al. (2024), who reported high frequencies of Phoma and Curvularia in Urochloa seed lots, these genera were not detected in the present study. By contrast, the genus Bipolaris showed high occurrence across the three evaluated areas, corroborating previous records. Fungi of these genera are known for their rapid growth and prolific sporulation, characteristics that allow them to colonize seeds, reduce viability, decrease germination rates, and often cause seed mortality (Mallmann et al. 2013; Jeromini et al. 2020). Moreover, environmental conditions common in Brazil, such as average temperatures between 20°C and 30°C and fluctuating humidity levels, are highly favorable for the development of these pathogens (da Silva et al. 2019). In this study, the pre-germination assay further confirmed this trend, in which seeds of three Urochloa species colonized by B. gossypina strains exhibited a significant reduction in germination. An important observation was the rapid colonization of seeds within 48 hours, particularly by strains F11 and F13. Such speed underscores the potential applicability of these fungi in field control, since seeds can act as primary sources of inoculum, ensuring pathogen persistence in the environment. A remarkable parallel is found with B. sorokiniana, for which seeds serve as the main inoculum source (Marchi et al. 2010; Kobayasti and Pires 2011). Additionally, after germination, some seedlings failed to develop properly and eventually died. Which can be attributed to the high transmission rates of these isolates from seeds to seedlings, leading to symptoms such as leaf spots, leaf desiccation, and seedling mortality (Corrêa et al. 2017).

The symptoms observed in seedling assays resemble those described for B. cynodontis in U. brizantha in 2005, where necrosis began at the leaf apex and progressed to the base, culminating in complete plant desiccation (Macedo and Barreto 2007). The severity of symptoms caused by the three selected strains of B. gossypina, ranging from 80% to 100%, reinforces their ability to control the three Urochloa species evaluated. These results indicate that seed-associated isolates can achieve a high degree of suppression over invasive plants. It is worth emphasizing that symptoms appeared as early as two days after inoculation, with marked progression until the fifth day, when they were fully developed. This short timeframe is striking when compared to the control period of certain herbicides tested under similar conditions on Urochloa seedlings at 15 days of age. Herbicides such as sethoxydim, haloxyfop-methyl, and fluazifop-p-butyl showed high control efficacy against U. decumbens, with visible lesions appearing around the fourth day and complete plant death by day 14 (Marques et al. 2011).

The association between Urochloa species and fungi of the genus Bipolaris, in Brazil, has been recorded since the last century (Mendes and Urben 2023). However, regarding the native flora, there is a gap in information about the occurrence of diseases caused by species of this fungal genus. In this study, the inoculation of three strains of B. gossypina on five native species did not result in any foliar lesions, demonstrating the safety of using these isolates in areas designated for natural protection, since the aim is to control the invasive target species without compromising the development of native plants. Another relevant aspect refers to the first report of B. gossypina, recorded in Kenya in 1985, when the species was described as the causal agent of leaf spot on cotton (Gossypium sp.) (Sivanesan 1985). Among the native species evaluated in the present study, two belong to the family Malvaceae (L. divaricata and G. ulmifolia), which reinforces the inability of the tested strains to cause disease in native plants related to cotton. In addition, there are records of B. gossypina on barley, which raised the hypothesis of a host transition from native species to the crop (Rodrigues et al. 2024). However, it is plausible that this transition actually occurred from weeds, such as U. decumbens, rather than from native species. Although the host range of B. gossypina is not yet fully clarified, the species is currently considered restricted to a single host (Manamgoda et al. 2014; Muniz et al. 2025).

Despite the promising results, biological control of invasive plant seed banks remains rarely documented in the literature and has so far been explored only in agricultural contexts (Kremer 2000; Lundgren 2009). Moreover, research specifically addressing the biological control of Urochloa species is still scarce, particularly regarding the use of microorganisms such as fungi. To date, no consolidated or widely disseminated programs exist (whether classical, natural, or mycoherbicide-based) using Bipolaris species for invasive plant management, nor are there initiatives worldwide specifically targeting Urochloa (Winston et al. 2014). Nevertheless, studies on Bipolaris have long demonstrated their potential for controlling significant invasive plants (Winder and Dyke 1990; Nechet et al. 2006; Zhang et al. 2022). Furthermore, the use of phytopathogenic fungi as biological control agents has shown both high field establishment and strong effectiveness in suppressing invasive plants (Schwarzländer et al. 2018). Overall, the results highlight the potential of the three B. gossypina strains for the biocontrol of the evaluated Urochloa species. Nevertheless, further studies addressing host specificity, environmental persistence, and field-scale efficacy are required to support their safe and effective application.

The evaluation of U. decumbens seeds provided insights into the biological control of species within this genus. The effectiveness of the three B. gossypina strains selected was evidenced by the significant reduction in seed germination, as well as by the rapid development of disease symptoms in established plants, occurring within two days of inoculation, which ultimately resulted in plant death. Beyond target suppression, the results also highlight the specificity of the isolates in controlling the invasive grass without inducing symptoms in the native species assessed in this study. In this context, the seed bank emerges as a strategic component for the development of more efficient and sustainable management practices aimed at reducing the occurrence of these invasive grasses, especially in areas where chemical herbicide use is limited or prohibited. Overall, this research emphasizes the unexplored potential of targeting weed seed banks for biological control and advances scientific understanding of sustainable strategies for suppressing invasive plant populations.

Acknowledgements

The authors express their gratitude to the Federal University of Uberlândia, Monte Carmelo campus, and the Federal University of Viçosa, Rio Paranaíba campus, for providing the facilities and infrastructure necessary for conducting this research. Appreciation is also extended to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) code no. APQ-02656-18, and to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship and financial support provided to this project.

Funding

This work is the result of the research project and master's dissertation with FAPEMIG code no. APQ-02656-18, supported by the Minas Gerais State Research Support Foundation (FAPEMIG)

Author Contributions

Material collection and data acquisition were performed by Vinícius A. de Oliveira, who also prepared the first draft of the manuscript. André L. Firmino was responsible for data curation, as well as the review and editing of the final version. Both authors read and approved the final manuscript.

Competing Interests

The authors have not disclosed any conflict of interests.

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Can fungi associated with seeds be an important source for the biological control of invasive plants? Urochloa (=Brachiaria) as a case study

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Abstract

Figures

1 INTRODUCTION

2 MATERIALS AND METHODS

2.1 Urochloa decumbens Seed Sampling

2.2 Isolation and Identification of Seed Fungi

2.3 Pathogenicity Assays

2.3.1 Pre-germination Assay

2.3.2 Post-germination Assay

2.3.3 Tests on Native Species

2.4 Statistical analysis

3 RESULTS

4 DISCUSSION

5 CONCLUSIONS

Declarations

References

Status:

Version 1