Seed Germination Traits

In natural conditions, the majority of seeds are recovered from cattle within the first 24 to 48 h, with recovery continuing up to 72 h and beyond (Gardener et al. Reference Gardener, McIvor and Jansen1993; Xu et al. Reference Xu, Li, Li, Hu, Zhang, Iqbal and Du2022). We observed significant effects of rumen fluid digestion on weed seed germination among the weed species in this study, as seed germination rate progressively declined with increasing duration of incubation (Figure 3). Ipomoea lacunosa seeds were highly affected by rumen fluid incubation, starting with a high germination percentage of 85% at 0 h that fell dramatically to 0% by 24 h of incubation and beyond. This pattern is consistent with previous research, which suggests that prolonged exposure to rumen fluid can adversely affect needle grass (Stipa aliena Keng) seed germination, potentially due to microbial activity and changes in pH levels that inhibit germination (Chen et al. Reference Chen, Huang, Zhang, Wu and Liu2012). Blackshaw and Rode (Reference Blackshaw and Rode1991) reported that downy brome (Bromus tectorum L.) and barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] seeds had 0% germination after 24 h of rumen fluid incubation. Convolvulus arvensis and S. pumila showed a severe and continuous decline in germination rates as the incubation period increased. Germination percentages dropped from 27% at 0 h to 4% at 48 h for C. arvensis, and from 58% at 0 h to 5% at 48 h for S. pumila, with no germination observed beyond this period. Similar findings were reported by Blackshaw and Rode (Reference Blackshaw and Rode1991) for green foxtail [Setaria viridis (L.) P. Beauv.], with seed germination decreasing from 88% to 14% after 24 h of rumen fluid incubation. In all three species (C. arvensis, I. lacunosa, and S. pumila), there was no germination beyond 48 h of incubation. Reduced seed germination can be attributed to the degradation of the embryo due to digestive enzymes in animal gastrointestinal systems. Additionally, microbial attachment in the rumen can further inhibit germination (Obara et al. Reference Obara, Ogura, Shishido and Sugawara2009). The high microorganism content and acidity of rumen fluids can lead to a loss of seed germinability, particularly in seeds with higher seed coat permeability and thin coats (Horiguchi et al. Reference Horiguchi, Takahashi and Kayaba2008).

Figure 3.

Effects of different duration of rumen fluid incubation on seed germination of five weed species. Bars with different letters indicate significant difference among the individual species across durations at P < 0.05. Errors bars indicate standard error of the mean (SEM).

Amaranthus palmeri and S. halepense exhibited a relatively stable germination pattern, although there was a decline in germination over the incubation periods (Figure 3). Notably, these two species were still germinating even after 96 h of incubation, with A. palmeri and S. halepense showing germination rates of 3% and 1%, respectively. Additionally, after 8 h of rumen fluid incubation, A. palmeri seed germination increased to 19% from 13% in control, likely due to the acidic environment affecting the seed coat’s integrity and permeability, similar to the effects of acid scarification, as observed in common lambsquarters (Chenopodium album L.) (Aper et al. Reference Aper, De Cauwer, De Roo, Lourenço, Fievez, Bulcke and Reheul2014; Buhler and Hoffman Reference Buhler and Hoffman1999).

Seeds of A. palmeri are reported to survive digestive tracts of various birds (De Vlaming and Proctor Reference De Vlaming and Proctor1968; Farmer et al. Reference Farmer, Webb, Pierce and Bradley2017) and ruminant animals (Blackshaw and Rode Reference Blackshaw and Rode1991; Yu et al. Reference Yu, Blair, Hardel, Chandler, Thiede, Cortilet, Gunsolus and Becker2021). Amaranthus palmeri is an aggressive invader by nature due to several advantageous adaptive traits, including its seed morphophysiology and diverse dispersal routes. Its high fecundity, tolerance to extreme temperatures and drought, and outcrossing nature due to dioecy leading to high genetic diversity, along with its ability to develop herbicide resistance, make it a formidable weed (Franssen et al. Reference Franssen, Skinner, Al-Khatib, Horak and Kulakow2001; Ward et al. Reference Ward, Webster and Steckel2013). These traits, combined with its prolonged survival in rumen fluid, suggest a high potential for the spread of herbicide-resistant populations. Similarly, S. halepense is a persistent invasive species with high seed dormancy and can thrive more aggressively under stress conditions (Acciaresi and Guiamet Reference Acciaresi and Guiamet2010; Ghersa et el. Reference Ghersa, Arnold and Martinez-Ghersa1992). Seeds of S. halepense need fluctuating diurnal temperatures for seed dormancy release (Taylorson and McWorther Reference Taylorson and McWorther1969). Upon animal grazing, seeds of S. halepense showed enhanced germination when retrieved from fecal materials in 24 to 48 h, primarily due to loosening of external bracts on seeds from scarification processes in the gut passage (Abbas et al. Reference Abbas, Al-Kahtani, Abdelazeem Mousa, Badry, Hassaneen, Ezzat-Ahmed, Mancilla-Leytón and Castillo2020; Grande et al. Reference Grande, Mancilla-Leytón, Vicente and Delgado-Pertíñez2016; Kaur and Soodan Reference Kaur and Soodan2017). Moreover, S. halepense can hybridize with other Sorghum species, creating resilient perennial and rhizomatous hybrids that thrive in harsh environments and establish in new habitats (Klein and Smith Reference Klein and Smith2021).

Days to First Germination, the MTG

The time to first germination remained constant at 3 d for all incubation periods up to 48 h and 12 h, after which no germination was observed in C. arvensis and I. lacunosa, respectively (Table 1). In A. palmeri, the initial germination occurred rapidly within 3 d up to 8 h of incubation. This duration extended to 5 d up to 72 h and further increased to 7 d at 96 h of incubation. In S. halepense, the time to first germination was consistently 5 d for most periods of incubation, except for 48 and 72 h, where it increased to 7 d, and 96 h, where it significantly increased to 13 d. The decrease in speed of germination in A. palmeri and S. halepense may be due to genetic variability in seed coat impermeability, with more impermeable seeds surviving longer in rumen fluids and emerging more slowly in germination tests. This delay likely stems from solute leakage and membrane damage caused by exposure to rumen fluid disrupting osmotic balance and germination processes. Gama et al. (Reference Gama, Brum, Martins, Silva and Dias2021) reported that water deficits can significantly slow down germination rates, which may be a survival strategy in arid conditions. Early-germinated seeds may have a higher risk of dying from factors like desiccation, pathogens, or predators (Traveset et al. Reference Traveset, Rodríguez-Pérez and Pías2008) as observed in I. lacunosa and C. arvensis (Figure 3). Highly permeable seed coats in these species are likely digested by rumen fluids, suggesting that seed coat permeability plays a crucial role in seed survival.

Table 1.

Percent final germination, number of days to first germination, and the mean time to germination (MTG) of the weed seeds after various durations of incubation in rumen fluid (hours).

Seed Viability

Overall, seed viability was significantly affected by rumen fluid incubation across the species (Figures 4 and 5). Although the initial germination percentage varied across the weed species, the initial viability was more than 95%, except for C. arvensis (89%). Similar to germination reduction, there was a gradual decrease in seed viability with increased duration of rumen fluid incubation. Simao Neto and Jones (Reference Simao Neto and Jones1987) observed an inverse relationship between incubation time and seed viability in six pasture species (signal grass [Urochloa decumbens (Stapf) R. Webster; syn.: Brachiaria decumbens Stapf], common carpetgrass [Axonopus fissifolius (Raddi) Kuhlm.; syn. Axonopus affinis Chase], perennial soybean [Neonotonia wightii (Wight & Arn.) Lackey], Kenya clover [Trifolium semipilosum Fresen.], cheesy-toes [Stylosanthes hamata (L.) Taubert] and shrubby stylo [Stylosanthes scabra Vog.]). However, this decline was not gradual for all species in our data. The viability of I. lacunosa seeds decreased sharply from 100% at 0 h to 34% by 24 h, and ultimately to 0% after 48 h. Similarly, the viability of C. arvensis seeds dropped from 96% at 0 h to 54% by 24 h, and finally to 0% after 96 h; and S. pumila began with a viability of 99% and maintained viability above 80% up to 24 h, declining sharply to 5% by 96 h. Haidar et al. (Reference Haidar, Gharib and Sleiman2010) observed a similar trend, with a steep decrease in viability of C. arvensis seeds in the first 24 h of rumen fluid incubation from 80% at 0 h to 30% by 24 h. Similar observations were made in case of kochia [Bassia scoparia (L.) A.J. Scott], for which viability sharply declined from 94% at 0 h to 15% after 24 h of incubation (Blackshaw and Rode Reference Blackshaw and Rode1991). This rapid loss of viability suggests that this species is highly sensitive to the conditions created by rumen fluid. Ipomoea spp. are highly sensitive to flood-induced anaerobic conditions, as high temperatures and humidity in rumen fluid triggered the water gap (a specialized area in the seed coat that facilitates the entry of water into the seed) to open, as seen in the case of rumen fluid (Jayasuriya et al. Reference Jayasuriya, Baskin, Geneve and Baskin2007; Siahmarguee et al. Reference Siahmarguee, Gorgani, Ghaderi–Far and Asgarpour2020) which inhibited their germination.

Figure 4.

Effect of different duration of rumen fluid incubation on seed viability (germinated + dormant but viable seeds) of five weed species. Letters on the data points indicate significant difference (P < 0.05) among the species within each observation timing. Asterisks (*) indicate significant difference (P < 0.05) between two consecutive observation timings for an individual species. Errors bars indicate standard error of the mean (SEM).

Figure 5.

(A) Viable and (B) dead seed of Amaranthus palmeri; (C) viable and (D) dead seed of Setaria pumila, after a tetrazolium test under a Leica EZ4 Stereo Microscope at 35× magnification.

On the other hand, A. palmeri maintained relatively high viability throughout the incubation period, starting at 99% and decreasing to 47% by 96 h. Similarly, S. halepense started at 83% and decreased to 27% by 96 h. The slower decline in viability suggests that A. palmeri and S. halepense may have mechanisms like hard seed coats and high seed tannin content (Adjesiwor et al. Reference Adjesiwor, Liu, Felix and Alder2024; Bagavathiannan and Norsworthy Reference Bagavathiannan and Norsworthy2013; Burt Reference Burt1954; Egley and Chandler Reference Egley and Chandler1978; Schutte et al. Reference Schutte, Klypin and Shukla2016) that allow them to withstand the inhibitory effects of rumen fluid longer than other species. Blackshaw and Rode (Reference Blackshaw and Rode1991) found that seeds of redroot pigweed (Amaranthus retroflexus L.) and C. album retained viability rates of 45% and 52%, respectively, even after 24 h of rumen fluid incubation. This remnant viability suggests that even after being subjected to harsh conditions, such as digestion in the rumen, these seeds retain the post-excretion potential for germination upon onset of favorable environmental conditions. This adaptive strategy is critical for their survival and proliferation in competitive agricultural ecosystems.

Time to 50% Germination Reduction (G₅₀)

All species exhibited a significant reduction in seed germination compared with the control (Figure 6). Amaranthus palmeri and S. halepense demonstrated a gradual decrease in germination over time, with the greatest reduction occurring within the first 24 h. In contrast, C. arvensis, I. lacunosa, and S. pumila showed a more rapid decline in germination, reaching 50% within the first 12 h. The time required to reduce germination by 50% (G₅₀) also differed significantly (P < 0.001) among the species (Figure 6). The G₅₀ values for individual weed species reflect their unique physiological and ecological adaptations to environmental conditions, particularly in response to rumen fluid treatment. The rapid G₅₀ value for I. lacunosa of only 5 h indicates a high sensitivity to the inhibitory effects of rumen fluid. Convolvulus arvensis and S. pumila G₅₀ values (11 and 12 h, respectively) suggest a moderate sensitivity to rumen fluid. The longer G₅₀ for A. palmeri (42 h) and S. halepense (24 h) suggests a high level of resilience to the inhibitory effects of rumen fluid. The differences in G₅₀ values indicate that different weed species exhibit varying degrees of sensitivity and resilience to rumen fluid treatment that ultimately dictate their establishment speed and success under natural conditions. The decline in germination and viability might be attributed to alterations in membrane and protein structures, as well as a rise in embryo mortality (Hendricks and Taylorson Reference Hendricks and Taylorson1976). Haidar et al. (Reference Haidar, Gharib and Sleiman2010) found that seed germination and viability of various weed species decreases with longer incubation in the rumen due to microbial activity. Additionally, volatile fatty acids from fermentation can inhibit germination by altering osmotic potential, as noted by Cordeiro et al. (Reference Cordeiro, Cabrita, Oliveira, Maia, Rodrigues, Fonseca and Valente2022).

Figure 6.

Percent germination response curve for five weed species over the incubation period at 3 wk after treatment with rumen fluid. The horizontal red dashed line in the plot represents 50% of the relative germination percentage. Data normalized to 100% according to control.

The R² values for the fitted regression models indicate a strong relationship between incubation period and germination for all species, suggesting that rumen fluid treatment effectively inhibits seed germination in these weed species (Figure 6). The nonlinear model (Equation 1) provided a good fit to the germination data for all five weed species, as indicated by the high R² values (0.79 to 0.97). However, the model’s predictive accuracy varied among species, as reflected in the root mean-square error (RMSE) values (Figure 6). RMSE values were higher for C. arvensis (12.33), S. halepense (17.00), and A. palmeri (14.05), suggesting that the model’s predictions for these species were less precise. In contrast, I. lacunosa and S. pumila had the lowest RMSE values (5.77 and 5.76, respectively), indicating a higher degree of model accuracy for these two species.

Seed Coat (Membrane) Integrity

Membrane integrity of seed coat was significantly affected by different periods of rumen fluid incubation among the weed species (Figures 7 and 8). The seed coats of I. lacunosa and C. arvensis were highly damaged after 48 h of incubation and were completely disintegrated at 96 h. The conductivity values for C. arvensis seeds increased from 31.09 µS cm⁻¹ at 0 h to a peak of 70.71 µS cm⁻¹ at 72 h, before slightly decreasing to 69.93 µS cm⁻¹ at 96 h. This gradual increase in conductivity suggests a decline in membrane integrity over time, indicating leakage of solutes from the seed, which ultimately diminishes seedling vigor. Ipomoea lacunosa displayed the highest conductivity values, starting at 111.50 µS cm⁻¹ and peaking at 408.73 µS cm⁻¹ at 48 h, before declining to 133.95 µS cm⁻¹ at 96 h. Declining value suggests that seeds were fully exhausted of ions and completely dead following 72 h of incubation. Sorghum halepense seeds had the least damage, even after 96 h of rumen fluid incubation. Amaranthus palmeri seeds were significantly damaged after 96 h of incubation, but plenty of seeds survived the rumen fluid. Amaranthus palmeri showed relatively low conductivity values, ranging from 4.62 µS cm⁻¹ at 0 h to 11.19 µS cm⁻¹ at 96 h. The low values indicate good membrane integrity and higher seed vigor, suggesting that this species is more resilient to stress conditions.

Figure 7.

Membrane integrity test to examine damage to seed coat due to incubation in rumen fluid. Letters on the data points indicate significant difference (P < 0.05) among the species within each observation timing. There was no significant difference between two consecutive observation timings for an individual species. Errors bars indicate standard error of the mean (SEM).

Figure 8.

Seed morphology after incubation in rumen fluid at different time intervals under a Leica EZ4 Stereo Microscope at 35× magnification. Red arrows show the damage and cracks on seed coats with increasing duration of rumen fluid incubation.

Amaranthus palmeri’s hard seed coat may have contributed to its survival in rumen fluid, unlike I. lacunosa’s larger seed size and quickly imbibed seed coat due to the opening of the water gap in rumen fluid. Amaranthus spp. seeds have a high lignin content (Calabrò et al. Reference Calabrò, Oteri, Vastolo, Cutrignelli, Todaro, Chiofalo and Gresta2022) and peptides with antimicrobial activity (Lipkin et al. Reference Lipkin, Anisimova, Nikonorova, Babakov, Krause, Bienert, Grishin and Egorov2005), which primarily provides structural integrity and impermeability to seed coats. Additionally, lignin may protect seeds from microbial pathogens and various biotic and abiotic stresses (Tobimatsu et al. Reference Tobimatsu, Chen, Nakashima, Escamilla-Treviño, Jackson, Dixon and Ralph2013). On the other hand, S. halepense relies on its substantial hemicellulose and cellulose content for protective functions. The hard, impermeable seed coat blocks rumen fluid from entering the seed and acts as a barrier against microbial enzymes, ensuring seed survival (Michael et al. Reference Michael, Steadman, Plummer and Vercoe2006). The hard and impermeable seed coat of small-flowered mallow (Malva parviflora L.) helped it to survive rumen digestion (Michael et al. Reference Michael, Steadman, Plummer and Vercoe2006). On the other hand, Olson and Wallander (Reference Olson and Wallander2002) found that in some species, the seed coat softens over time in rumen fluid and becomes susceptible to microbial digestion.

Seedling Vigor

Seedling length and weight, which control the seedling vigor indices, decreased significantly over incubation time in most of the weed species but were not much affected in the case of A. palmeri (Figure 9). Seedling vigor, which indicates the strength of a seed or seedling to thrive and establish a healthy plant, was significantly affected by rumen fluid, and there was a gradual decrease in seedling vigor with increased duration of incubation for all the species (Figures 10 and 11). The initial vigor index of I. lacunosa was high, but it decreased to 0 after 24 h of exposure to rumen fluid. Similarly, S. pumila began with the highest seedling vigor index among all the species, which decreased to 0 by 72 h. This rapid decline suggests that, despite their higher initial vigor, both species are highly susceptible to the conditions imposed by rumen fluid. Although both species exhibited similar trends, S. pumila maintained its vigor for a longer duration. Similarly, the other three species showed lower initial values but sustained their vigor for a longer period. Although A. palmeri and S. halepense exhibited low initial vigor, they were able to maintain their vigor for the longest duration among all the species. Notably, A. palmeri and S. halepense retained substantial vigor even after 96 h of rumen fluid incubation, demonstrating their high resistance and adaptability to stress conditions. While A. palmeri and S. halepense are resilient, prolonged exposure to rumen fluid significantly impairs their growth potential. Early seedling vigor, a key seed-quality attribute, ensures rapid, uniform germination and strong seedling establishment under various environmental conditions, with high-vigor seeds providing better resistance to stresses compared with low-vigor seeds (Bourgeois et al. Reference Bourgeois, Munoz, Fried, Mahaut, Armengot, Denelle, Storkey, Gaba and Violle2019; Foolad et al. Reference Foolad, Subbiah and Zhang2007). Rapid seedling growth, positioning S. halepense as the leading species in terms of seedling size among dominant warm-season grasses, contributes significantly to its invasive success (Reichmann et al. Reference Reichmann, Schwinning, Polley and Fay2016).

Figure 9.

Effect of different durations of rumen fluid incubation on (A) seedling length and (B) seedling weight of five weed species. (A) Letters on the data points indicate significant difference (P < 0.05) among the species within each observation timing. There was no significant difference between two consecutive observation timings for an individual species. (B) Letters on the data points indicate significant difference (P < 0.05) between two consecutive observation timings for an individual species. There was no significant difference (P < 0.05) among the species within each observation timing. Errors bars indicate standard error of the mean (SEM).

Figure 10.

Effect of different duration of rumen fluid incubation on seedling vigor index I of five weed species. Bars with different letters indicate significant difference (P < 0.05) between two consecutive observation timings for an individual species. Errors bars indicate standard error of the mean (SEM).

Figure 11.

Effect of different duration of rumen fluid incubation on seedling vigor index II of five weed species. Bars with different letters indicate significant difference (P < 0.05) between two consecutive observation timings for an individual species. Errors bars indicate standard error of the mean (SEM).

Our study showed that passage of weed seeds through the digestive tract of cattle reduces the viability of weed seeds. Consequently, cattle grazing on weeds can help in decreasing the number of seeds entering the weed seedbank, further aided by the mechanical damage caused by their teeth (Stanton et al. Reference Stanton, Piltz, Pratley, Kaiser, Hudson and Dill2002). However, some seeds remain viable after digestion, depending on the weed species (Asaduzzaman et al. Reference Asaduzzaman, Piltz, Koetz, Hopwood, Shephard and Wu2022; Wang et al. Reference Wang, Lu, Waly, Ma, Zhang and Wang2017). Weed seeds are generally less affected by ingestion by cattle compared with sheep (Ovis spp.) or birds (Farmer et al. Reference Farmer, Webb, Pierce and Bradley2017; Gardener et al. Reference Gardener, McIvor and Jansen1993; Haidar et al. Reference Haidar, Gharib and Sleiman2010; Michael et al. Reference Michael, Steadman, Plummer and Vercoe2006). Thus, viable weed seeds in cattle excreta can potentially lead to weed infestations in fields (Wilson et al. Reference Wilson, Brusa, Christensen, Strack, Alto, Allen, Cortus, Modderman and Becker2022).

In our study, A. palmeri and S. halepense seeds showed a high tolerance to rumen fluid compared with I. lacunosa, C. arvensis, and S. pumila. Specifically, I. lacunosa seeds were dead after 48 h of incubation, with no germination observed after 24 h. Seeds of C. arvensis and S. pumila were almost dead after 96 h of incubation, with no germination occurring after 72 h. Amaranthus palmeri is particularly concerning due to its prolific seed production, with a single female plant capable of producing up to 600,000 seeds (Keeley et al. Reference Keeley, Carter and Thullen1987). Additionally, looking at the herbicide-resistance scenario in these two weeds (Heap Reference Heap2024), there is a risk of these seeds being spread by cattle and a potential risk of herbicide resistance spread. Results indicated that, unlike soft seeds, some hard-coated seeds remained viable in the digestive system of cattle for more than 4 d. Use of manure compost piles by farmers in the U.S. Southeast might contribute unknowingly to the spread of weeds and, consequently, herbicide resistance. Consequently, farmers should avoid grazing cattle in areas infested with weeds like A. palmeri and S. halepense, especially after the formation of viable seeds. Cattle that have grazed in such infested fields should not be moved to new, uninfested areas, and their fresh manure should not be used on arable lands. This is crucial, because cattle can inadvertently transport viable weed seeds through their manure, which can lead to the establishment of these weeds in previously noninfested fields.

Effective grazing management is crucial for herders aiming to mitigate weed invasion while optimizing livestock productivity. Cattle can consume various weeds like C. album, Canada thistle [Cirsium arvense (L.) Scop.], Setaria spp., and S. halepense. Marten and Anderson (Reference Marten and Andersen1975) observed that A. retroflexus, a species similar to A. palmeri, has forage nutritive value similar to high-quality alfalfa (Medicago sativa L.) when harvested at the vegetative stage. Strategic grazing timing can significantly affect weed phenology, particularly preventing flowering stages of certain weed species. Grazing before the flowering of summer annuals such as common ragweed can reduce local weed occurrence effectively; however, this has been shown to be context dependent and influenced by various factors throughout the growing season. Rotational grazing practices tailored to specific weed phenologies further enhance this effect by allowing herders to manage the grazing of different areas over time and avoid periods that favor weed reproduction.

Furthermore, the treatment of manure before field application can be an effective strategy to mitigate the risk of weed seed dispersal. Composting manure at high temperatures (≥60 C) can kill most of the viable weed seeds, thereby reducing the potential for these seeds to germinate and establish in agricultural fields (Larney and Blackshaw Reference Larney and Blackshaw2003). High temperature in combination with organic acids reduces seed viability during composting (Liu et al. Reference Liu, Wang, Awasthi, Chen, Awasthi, Duan and Zhang2020). This practice is particularly relevant for farmers who rely on manure as a fertilizer, as it allows them to utilize this resource without inadvertently introducing weed seeds into their cropping systems.