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Article

Effects of Pre-Emergence Application of Organic Acids on Seedling Establishment of Weeds and Crops in Controlled Environments

by
Mattia Alpi
,
Anne Whittaker
,
Elettra Frassineti
,
Enrico Toschi
,
Giovanni Dinelli
and
Ilaria Marotti
*
Department of Agricultural and Food Sciences, University of Bologna, Viale Fanin 46, 40127 Bologna, Italy
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(8), 1820; https://doi.org/10.3390/agronomy15081820
Submission received: 17 June 2025 / Revised: 18 July 2025 / Accepted: 25 July 2025 / Published: 28 July 2025
(This article belongs to the Special Issue Application of Natural Products for Weed Control in Agricultural Systems)
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Abstract

Within the framework of organic acid alternatives to chemical herbicides, pre-emergence weed control research is scarce. Citric acid (CA) and lactic acid (LA), considered significantly less effective than pelargonic acid (PA) and acetic acid (AA) from post-emergence (foliar spraying) studies, have largely been disregarded. This in vitro study was aimed at comparing the effects of 5–20% AA, AA + essential oils, PA, CA, and LA on radicle emergence inhibition (direct spraying of seeds) and shoot emergence inhibition (application to peat) on both weeds (perennial ryegrass, green foxtail, common vetch and chicory) and crops (soft wheat, alfalfa and millet). All tested compounds demonstrated concentration-dependent and species-specific effects on shoot emergence inhibition, with CA and LA (IC50 range: 3.4–19.3%) showing a comparable efficacy to PA and AA (IC50 range: 3.1–35.9%). The results also showed that CA and, to a lesser extent, LA were less inhibitory to soft wheat (CA IC50 = 62.5%; LA IC50 = 35.9%) and alfalfa (CA IC50 = 57.8%; LA IC50 = 44.1%) shoot emergence. CA and LA show potential promise for pre-emergence weed control in field testing, either on a stale seedbed in pre-crop sowing or concurrently with soft wheat and alfalfa sowing. Investigating organic compound herbicidal effects on crops of interest warrants attention.

1. Introduction

Two interconnected compounding issues challenging global agricultural productivity are weed-induced losses to crop yields and the intensive use of chemical herbicides [1,2]. Weeds are currently the most important biotic limitation to global agricultural production. The average yield losses of economically significant global crops (including cereals [wheat, maize, barley and rice, millet, sorghum], sugar beet, beans, peanut, and vegetable crops) are estimated at 28–31.5% worldwide [1,2]. Consequently, chemical herbicide application has become an integral part of modern agriculture, with herbicides accounting for the highest percentage (47.5%) of the current global pesticide usage [3]. However, synthetic herbicides pose significant negative impacts on the environment, biodiversity and food safety, with resultant toxicity, carcinogenicity, and respiratory effects, to mention a few, on both animals and humans [2,3,4]. The aforementioned impacts combined with the significant increase in chemical herbicide-resistant weeds has rendered agricultural systems more vulnerable and less sustainable [2,3].
Within the framework of the European Union (EU), the overall use of synthetic pesticides is a contentious issue, involving debates about environmental and human health risks, agricultural practices, and the balance between food security and sustainability [5]. The Integrated Pest Management and the European Biodiversity Strategy are currently aimed at reducing the use of chemical crop protection products in order to safeguard the environment [2,6]. Aside from respecting the directives restricting chemical inputs, enormous challenges facing agricultural producers include meeting the projected food needs of a rapidly expanding human population [6,7]. Additionally, the impact of weeds on major crops is predicted to become exacerbated in the Mediterranean basin under decreased precipitation [1]. Hence, there is a requisite for the implementation of eco-friendly strategies, which include the removal of herbicide contaminants from the environment [4], the implementation of well-recognized sustainable weed-control farming practices [7,8], the introduction of innovative weed-control farming practices [7,9] and the use of non-chemical herbicide alternatives [2,7,10,11,12,13,14].
Focusing on chemical herbicide alternatives, bioherbicides are broadly defined as eco-friendly, biological herbicide control products, predominantly made from living microbial organisms (bacteria, viruses, and fungi) or phytotoxins derived from microbes, insects, or plant extracts [10]. Overall, bioherbicides have a shorter environmental persistence and are biodegradable, with fewer risks to non-target organisms and ecosystems [13]. However, microbial bioherbicides are specifically shown to display a narrow control spectrum, pose a risk of genetic exchange, and are subject to climate-dependent environmental factors that affect the stability and spread of phytopathogens [11]. Instead, organic herbicides are defined as contact, non-selective (knockdown), broad-spectrum herbicides derived from plant- or mineral-based ingredients that act as natural eco-friendly alternatives to chemical herbicides [12]. Of particular interest to the present investigation are organic acid-based compounds, including acetic acid (AA), pelargonic acid (PA), citric acid (CA), and lactic acid (LA). In the EU, only PA has been registered as a plant protection product with recognized herbicidal use in agricultural crops (Reg. (EC) No. 1107/2009) [15]. Although AA (vinegar) is not officially registered in the EU as a herbicide, it is used for non-selective herbicidal activity and can be found in many advertised commercial “weed killer” formulations. CA and LA are not officially recognized or registered as herbicides under EU regulations.
Post-emergence herbicides are administered in the control of germinated weed plants to disrupt cell membranes of vegetative tissues, whereas pre-emergence herbicides are administered to target the early stages of weed growth, preventing the development of the embryonic radicle, the shoot, or both, thereby inhibiting seedling establishment [3,13]. Research on post-emergence foliar applications has indicated that CA is less effective than AA and PA [16,17,18], and, as a consequence, CA has received less consideration due to this lower efficacy. Furthermore, there is a lack of research specifically examining the pre-emergence herbicidal activity of CA and LA on weed seeds. Our interest in examining the potential pre-emergence herbicidal effectiveness is because seed banks are reservoirs of both newly shed weed seeds and persistent older seeds that contribute to the persistence of invasive plant infestations [7]. Given the increasing interest in sustainable herbicide research within the context of integrated weed management strategies, the use of organic compounds could be administered to a stale seedbed for pre-emergence control of seedling establishment before sowing the crop plants. A stale seedbed involves preparing the soil for the germination of weeds, followed by a herbicidal method for weed control just before the sowing of the crop. The organic compounds with herbicidal activity are generally applied to the vegetative tissues for post-emergence control [3,19,20,21,22]. Alternatively, organic compounds could be administered to reduce weed seedling establishment concurrently with crop sowing. The latter strategy would necessitate examining the impact of these organic compounds on crop plant seeds, for which information on many crop species is still lacking.
The present study was, therefore, aimed at investigating the in vitro effects of the administration of different concentrations (5%, 10%, and 20%) of AA, AA + eugenol and geraniol (AA+EG), CA, LA, and PA in inhibiting radicle and shoot emergence of four common weeds (perennial ryegrass, common vetch, green foxtail, and common chicory) and three well-recognized crops (soft wheat, millet, and alfalfa [lucerne]). The objective was to evaluate whether the organic acid compounds possess potential for future field trials, and more specifically, whether they could potentially be used in a stale seedbed technique to, firstly, control weed shoot emergence prior to sowing the crop plants or whether there is a possibility of using them concurrently with crop sowing. With the exception of a single article on wheat treated with AA [21], the potential herbicidal impacts of organic compounds on soft wheat, millet, and alfalfa seedling establishment have not been investigated. Of particular interest are soft wheat, which is one of the most cultivated cereals in the EU [2], and perennial ryegrass, which has emerged as being resistant to multiple synthetic herbicides and as the most problematic global early weed competitor for wheat [23]. Green foxtail and common vetch have also developed resistance to synthetic herbicides [23], requiring the use of organic compounds as alternatives to synthetic herbicides. Inclusion of adjuvants such as essential oils to increase the post-emergence herbicidal efficacy of AA has been shown [24,25]. The need for additional scientific information on adjuvants was recommended [19,24], and as such, EG was included as an adjuvant to AA in the present study.

2. Materials and Methods

2.1. Materials

Seeds from the weed species Lolium perenne L. (perennial ryegrass), Vicia sativa L. (common vetch), Setaria viridis L. (green foxtail), and Cichorium intybus L. (common chicory) were obtained from the germplasm collection available at the Seed Research and Test Laboratory (LaRAS) of the Department of Agricultural and Food Sciences, University of Bologna. Crop seeds of Triticum aestivum L. var. Verna (soft wheat) and Panicum miliaceum L. var. Blond (millet) were purchased from Arcoiris (Modena, Italy). Seeds of Medicago sativa L. var. Minerva (alfalfa [lucerne]) were also obtained from LaRAS. All three crops are cultivated in the region of Emilia-Romagna, which is one of the largest agricultural regions in Italy. The Great Life project (an ongoing EU project), led by the University of Bologna, is involved in the study of old local soft wheat varieties [26]. Emilia-Romagna is also a region where proso millet is being studied as a potential alternative to maize [26]. Moreover, the region is a major producer of alfalfa, primarily for feeding dairy cattle for the production of Parmigiano Reggiano cheese [27]. The Great Life project is conducted on several farms, including one near Cadriano-Granarolo dell’Emilia (Bologna). Analysis of the weed populations in Cadriano showed that perennial ryegrass, common vetch, green foxtail, and common chicory were the most abundant weed species (unpublished results), and for this reason, all of them were selected as the weed species of interest.
AA was purchased as a 20% concentrated vinegar formulation (extracted from wine and fruit) from Flortis (Milan, Italy), whereas PA (nonanoic acid), CA, LA, eugenol, and geraniol were purchased in pure form from Merck (Darmstadt, Germany). The agar was also purchased from Merck. The commercial peat substrate was obtained from VigorPlant (Lodi, Italy).

2.2. Preparation of Treatment Concentrations

Solutions of AA, PA, CA, and LA were prepared individually by adding distilled water to obtain final concentrations of 5%, 10%, and 20% (v/v or w/v), as used previously [19,21]. The 0% concentration treatment contained only distilled water and represented the untreated control (CTRL). The essential oils were added as adjuvants at a concentration of 1.5% (v/v) eugenol + 1.5% (v/v) geraniol to each of the different AA treatment concentrations (5%, 10%, 20%).

2.3. Growth Chamber Parameters and Experimental Design

To investigate the pre-emergent herbicidal effects of the organic compounds on seedling establishment, two different experimental trials were adopted. The first experimental trial was conducted in Petri dishes to assess either the presence or absence of radicle emergence on agar after directly spraying the seeds with the organic compounds. In the second trial, the organic compounds were sprayed on moistened peat in pots and not directly on the seeds. The presence or absence of shoot emergence (seedling establishment) was then assessed. Both experimental trials were conducted in a growth chamber during the period from March to September 2023.

2.3.1. Growth Chamber Parameters

Experiments were performed in a growth chamber at the Department of Agricultural and Food Sciences (DISTAL—University of Bologna). Above the bench surface, there were three fluorescent tube lights (FH21830, 21 W, warm white 830, 85 cm, 116 W; OSRAM SpA, Milan, Italy), installed at a height of 60 cm from the positioning of the Petri dishes and pots. From a spectrometric analysis (Miniature Fibre Optic, USB2000+UV–VIS, Ocean Optics, Milan, Italy), light quality was characterized by two major spectral profile peaks in green and red and a minor peak in blue. Mean irradiance or photon flux density (PPFD) was 71.65 µmol m−2 s−1 (portable luxmeter, Delta OHM, Padova, Italy). The lighting was set to a 16/8 h (day/night) photoperiod. The internal temperature was maintained at 23 °C, and the relative humidity was 70–75%.

2.3.2. Experimental Design for the Assessment of Radicle Emergence in Petri Dishes

For the assessment of the presence or absence of a radicle, tests were performed in 90 mm diameter glass Petri dishes with 15 mL of 1.5% (w/v) agar. The agar in the Petri dishes was sterilized for 30 min inside a VWR® PCR Workstation (containing 2 internal UV tubes [254 nm, 25 W each] and 1 UV tube in a UV Air Recirculator [254 nm, 8 W]) offering decontamination action by UV inactivation of airborne and surface-bound contaminants (Avantor, Milan, Italy). Seeds of all species tested were rinsed in distilled water containing 5% sodium hypochlorite for 3–5 min, followed by rinsing in distilled water, prior to being placed on the agar. Each Petri dish contained 20 seeds. Treatments were carefully applied to each Petri dish at an application rate of 1000 L/ha using a portable 2 L manual pressure sprayer (LeroyMerlin.it, ref. No. 84438959, Bologna/Milano, Italy). The untreated CTRLs were sprayed with water only. After 7 days, seeds with radicles were counted following treatment/water exposure.
The experimental trial was composed of 7 plant species (3 crops and 4 weeds), comprising 5 different organic compound (AA, AA+EG, CA, LA, and PA) treatments at 4 different concentrations (0%, 5%, 10%, 20%) with 3 replicates (for each species, treatment and concentration), representing a total of 420 Petri dishes. As such, the experiments were performed separately for each treatment at a time. The positioning of the Petri dishes for the species, concentrations, and replicates (84 samples) was according to a completely randomized design.

2.3.3. Experimental Design for the Assessment of Shoot Emergence in Pots

For the assessment of the percentage of shoot emergence, seeds were sown in pots (6 cm diameter) with a commercial peat substrate that was wet with distilled water. All the seeds were sown at a depth of 1–2 cm in the peat. Prior to sowing, the seeds were incubated for 5 min in distilled water containing 5% sodium hypochlorite for 3–5 min, followed by rinsing in distilled water. Each pot contained 20 seeds. The treatments were sprayed on day 1 at an application rate of 1000 L/ha using a portable 2 L manual pressure sprayer. The untreated CTRLs were sprayed with water only. Water was sprayed on all the pots to wet the peat after 5 days to ensure germination. After 10 days, the numbers of apical tips that had emerged from the peat after exposure to the treatments/water were recorded [28].
As with the Petri dish trial, the peat trial was performed on the 7 plant species, comprising 5 different herbicide treatments at 4 different concentrations with 3 replicates. The experiments were performed separately for each treatment at a time. Similarly to the Petri dish trials, the positioning of the pots for species, concentrations, and replicates (84 samples) was performed according to a completely randomized design.

2.4. Statistical Analyses

Statistical analyses were conducted using the Statistica 6.0 software (2001, StatSoft, Tulsa, OK, USA). Radicle/shoot emergence inhibition was calculated as a percentage, with the control group (CTRLs) set as the baseline at 0% inhibition. A two-way analysis of variance (ANOVA) was performed between plant species (all seven species) and organic compound percentages (5%, 10%, 20%) for the Petri dish and soil experiments. Tukey’s honest significant difference was used to determine the differences between means at p < 0.05.
The concentration required for 50% radicle and shoot emergence inhibition (IC50) was estimated for each organic acid treatment applied to each species. Using the individual percentage radicle and shoot emergence values for each different treatment (5%, 10%, and 20% compared to the untreated baseline 0%), the absolute IC50 values (expressed in %) were estimated using the regression equation of the concentration–response curve as reported previously [29].

3. Results

3.1. Effect of the Organic Compounds on the Radicle Emergence Inhibition of the Three Crop and Four Weed Species in Petri Dishes

There was a minimal percentage inhibition of radicle emergence (ca. 5–15%) on soft wheat following the administration of both CA and LA (5–20%) (Figure 1C,D). In contrast, CA and LA inhibited radicle emergence by 80–100% at all concentrations in millet (Figure 1H,I) and between 65–100% for alfalfa (Figure 1M,N). At 10–20% PA, more than 90% of soft wheat radicle emergence was inhibited (Figure 1E). PA (5%) resulted in a minimal inhibition of alfalfa radicle emergence, but inhibition exceeded 90% at 10–20% PA (Figure 1O).
Examining the weed species, CA and LA at 5–20% were comparably effective at significantly inhibiting radicle emergence of perennial ryegrass, common chicory, green foxtail, and common vetch (Figure 2C,D,M,N,R,S). The efficacy of PA was comparable to that of CA and LA at inhibiting the radicle emergence of perennial ryegrass, green foxtail, and common vetch (Figure 2E,O,T). AA+EG at 5% was ineffective at inhibiting radicle emergence of ryegrass, common chicory, and vetch (Figure 2B,G,Q). AA with added essential oils was less effective than AA alone at 5% in inhibiting ryegrass (Figure 2A) and at 5–20% for common vetch (Figure 2Q), respectively. At 5–20%, both AA and AA+EG were equally effective at inhibiting radicle emergence in green foxtail (Figure 2K,L).
Of relevance, soft wheat was the only seed type that was resistant to the direct spraying of CA and LA between 5–20% (IC50 = 72.0%) (Table 1). Overall, AA, AA+EG, CA, LA, and PA were effective in significantly inhibiting the radicle emergence of the weeds, as well as those of millet and alfalfa (Table 1).

3.2. Effect of the Organic Compounds on the Shoot Emergence Inhibition of the Three Crop and Four Weed Species in Peat Pot Trials

CA showed minimal shoot emergence inhibition (5–20%) on soft wheat and alfalfa (Figure 3C,M), but was more phytotoxic to millet (Figure 3H). LA also showed a minimal inhibitory effect on the alfalfa shoot emergence (Figure 3M) and a relatively reduced inhibition of both soft wheat and millet (Figure 3D,I). In contrast to the non-inhibitory effect of PA on soft wheat shoot emergence (Figure 3E), this compound was phytotoxic to millet and alfalfa (Figure 3J,O). AA and AA+EG were phytotoxic to all the 3 crops (Figure 3A,B,F,G,K,L).
Collectively, the organic acid compounds at 5–20% showed both non-dose-dependent and dose-dependent inhibition type responses on the shoot emergence of the 4 weed species (Figure 4). Effects were also species-specific, with CA, LA, and PA showing a significantly higher inhibition of the shoot emergence of perennial ryegrass compared to AA and AA+EG (Figure 4A–E). In their turn, AA, AA+EG, and CA were phytotoxic to common chicory seed establishment, with PA significantly less phytotoxic (Figure 4F–J).
The IC50 was similarly calculated using all the data (0–20%) of each compound to estimate the percentage required to inhibit the shoot emergence of 50% of the seedlings. PA was shown to be suitable for use in soft wheat cultivation, with significant inhibitory effects on the weeds (Table 2). For the first time, CA was shown to have a minimal inhibitory effect on both soft wheat and alfalfa shoot emergence, with significant control on weed seedling establishment (Table 2). LA showed minimal inhibitory effects on alfalfa shoot emergence, with significant control of weed seedling establishment. LA demonstrated increased inhibition of soft wheat shoot emergence compared to CA. Nonetheless, the percentage of LA required to remove 50% of the weeds ranged from 3.4% to 8.8%, which was significantly lower than that required to inhibit soft wheat (Table 2). The present results show potential for CA and LA in pre-emergence weed control concurrently with soft wheat and alfalfa sowing (Table 2).

4. Discussion

The mode of action of the most recognized organic post-emergent products, namely PA and AA, has been attributed to the disruption of plant cell membranes and consequent tissue desiccation and inhibition of metabolic processes following direct contact with the shoots of established weeds [2,12,16,19]. At the same time, information pertaining to the effects of pre-emergent organic acid compounds is scarce. There is also a need to understand the impacts of pre-emergent compounds on the seedling establishment of crop plants that germinate simultaneously with specific weeds. Therefore, the present work was a preliminary in vitro study aimed at identifying candidate organic acid compounds, effective at inhibiting the seedling establishment of the four weed species, and at determining whether these compounds can be considered for future field studies, to be administered either before crop sowing or simultaneously with crop sowing. The efficacy of the compounds tested for inhibiting radicle and shoot emergence will be considered for the weed species, followed by the crop species. The potential use of these compounds in sustainable agriculture as part of an integrated herbicide approach will also be considered.
The organic compounds used in the present study demonstrated both dose-dependent and non-dose-dependent inhibitory effects on weed radicle and shoot inhibition in the 5–20% concentration range. Moreover, weed species-specific effects were observed. PA, CA, and LA were specifically effective at inhibiting perennial ryegrass shoot emergence (IC50 range: 3.2–5.1%), whereas AA and AA+EG (IC50 range: 35.9–38.1%) were significantly less effective. PA was previously shown to significantly reduce rigid ryegrass biomass as a post-emergent herbicide [20,30]. In their turn, AA and LA (IC50 range: 3.7–4.6%) were more effective at inhibiting common chicory shoot emergence compared to CA and PA (IC50 range: 13.1–19.3%). Differing impacts of AA, PA, and CA alone and/or in combinations with essential oils on weed control were, similarly, noted previously for post-emergence treatments both in vitro [16,19,25,31] and in the field [19,24,30,31,32,33]. The present results on AA+EG corroborated previous results showing no synergistic benefits of adding essential oils to AA in the pre-emergence control of different weeds [19].
The present study showed for the first time that CA and LA (5–20%) inhibit weed radicle and shoot emergence to an extent equivalent to that of PA and CA. These results contrast with those shown in post-emergence studies. CA (10%) alone, when used in post-emergence, had an efficacy of 0–20% in controlling ryegrass and green foxtail, which was significantly lower than that observed for AA [16]. Post-emergence efficacy in weed control was previously ranked in descending order for PA, AA, and CA based on the necrotization (disruption of cellular membranes) of vegetative tissues [17]. Similar results were obtained from comparing CA to PA [18]. More recently, CA (14%) was used in combination with AA (23%) to increase post-emergence efficacy against weeds [34]. The most direct use of LA as a potential herbicide (Organo-sol®; 2% CA and 1.8% LA) was reported on several legume weeds [35]. LA (1%) used alone was reported to show minimal post-emergence weed control [36]. No comparative reports exist for the concentrations of LA used in the present study, as the use of LA for herbicidal effects has not been well-studied [36]. The comparable pre-emergence weed control efficacy of AA, PA, CA, and LA with regard to seeds in this preliminary study suggests different mechanisms of control from those previously shown on vegetative tissues. Notably, higher application rates were also used to investigate pre-emergence weed control. Previous work indicated that the application of higher dose ranges of AA (1600 L/ha) just prior to sowing soft wheat improved the herbicidal efficacy against weeds with no effect on soft wheat [18].
Regarding the effects of the natural compounds of interest, the present results indicate PA to be a potentially safe herbicide for use against weeds during soft wheat sowing, with no effect on soft wheat shoot emergence (IC50 = 89.7%). The results also showed minimal effects of the pre-emergence application of CA on soft wheat seeds on both radicle (IC50 = 71.8%) and shoot emergence (IC50 = 62.5%). Although LA (IC50 = 35.9%) had a significantly higher inhibitory impact on shoot emergence than PA and CA, this value was significantly higher than the 3.4–8.8% IC50 range of LA required to control weed seeds. To the best of our knowledge, neither CA nor LA has been investigated as potential compounds with herbicidal activity in soft wheat cultivation. Notably, both CA and LA produced significantly fewer inhibitory effects on alfalfa shoot emergence (IC50 = 57.8% and 44.1%, respectively) compared to PA, AA, and AA+EG, which were significantly more phytotoxic to alfalfa seedling establishment. All the organic compounds tested were considered phytotoxic to millet seeds. To the best of our knowledge, there were no previous reports on the herbicidal impacts of these organic compounds on alfalfa and millet crops.
The present results indicated potential for field testing. In vitro, AA, CA, LA, and PA showed approximately 90% radicle emergence inhibition and 60–90% shoot emergence inhibition of the weed species at 1000 L/ha. Field administration of AA (20%) for the control of weeds at an application rate of 935 L/ha was shown to be effective in controlling weeds prior to crop sowing [19], as was 10% AA three days before sowing soft wheat (at an application rate of 1600 L/ha), with no negative effects on soft wheat [21]. Post-emergence herbicide control of PA applied to a stale seedbed in field studies was also reported [20,22]. It is feasible that pre-emergent weed control with AA, PA, LA, and CA on a stale seedbed may represent a potentially cost-effective alternative to post-emergent weed control and warrants field investigations. Notably, CA did not significantly inhibit seedling establishment of either soft wheat or alfalfa, showing potential for the suppression of weed establishment at the time of crop sowing. LA can also potentially be applied for weed control at the time of sowing of alfalfa. Since PA is more costly [20] and also inhibitory to alfalfa, CA and LA warrant testing in the field to investigate their potential as more cost-effective pre-emergence alternatives to PA. Although LA was shown to be more phytotoxic to soft wheat shoot development than PA and CA, if potentially used at concentrations that ensure weed control without negative impacts on soft wheat shoot development, it may similarly show potential in field testing. The preliminary results from this study indicated that none of the weed control compounds warrant consideration during millet sowing. Given that weed control compounds impacted the seedling establishment of the three crops tested differently, more preliminary studies are a requisite to investigate impacts on crop species of interest prior to implementing field studies.
The mechanisms of action of the tested organic compounds on radicle and shoot emergence were not considered. This aspect represents a limitation of the present study, requiring future attention. The following aspects warrant further attention. Of interest, increased inhibition of soft wheat shoot emergence (40%) with 20% LA was not evident for CA. Whilst direct contact of the seeds (by spraying) with a wide range of LA and CA contents may have ranged from a stimulatory to non-inhibitory mode of action on soft wheat radicle emergence (albeit toxic to weeds), the sensitivity of shoots to LA may have been attributable to cell membrane disruption by LA, which is more lipid-soluble than citric acid based on the dissociation constants (pKa of 3.86 and 2.92 for LA and CA, respectively). By extension, this mechanism may similarly explain the more severe inhibitory effect of AA on both soft wheat root and shoot emergence. These aspects remain to be evaluated. An explanation as to why CA and LA inhibited the radicle emergence of grass weeds but not soft wheat also remains to be investigated. Seed coat characteristics (permeability) can affect how herbicides impact plants, with some stimulating germination and others inhibiting germination [37].

5. Conclusions

Overall, CA and LA, as compounds with herbicidal activity, were not shown to be less effective than PA and AA in inhibiting radicle and shoot emergence of weeds in vitro. Pre-emergent weed control with AA, PA, LA, and CA on a stale seedbed may represent a potentially cost-effective alternative to post-emergent weed control and warrants further investigations in the field. Moreover, CA and, to a lesser extent, LA had a significantly lesser impact on soft wheat and alfalfa seedling establishment. Hence, CA and LA show potential promise as effective pre-emergent weed control compounds during soft wheat and alfalfa sowing. The next step would be to conduct field trials during soft wheat and alfalfa cultivation to validate the efficacy of CA and LA for the range of naturally growing weeds under non-controlled environmental conditions. More research is warranted to understand the mechanisms of action of these organic compounds and their differential effects on weeds and crops.

Author Contributions

Conceptualization, I.M. and G.D.; methodology, I.M., G.D. and M.A.; validation, I.M., G.D. and M.A.; formal analysis, M.A., E.F. and E.T.; investigation, M.A., E.F. and E.T.; resources, I.M. and G.D.; data curation, I.M., G.D., M.A. and A.W.; writing—original draft preparation, I.M., M.A. and A.W.; writing—review and editing, I.M., M.A. and A.W.; visualization, I.M., G.D., M.A., E.F., E.T. and A.W.; supervision, I.M. and G.D.; project administration, I.M. and G.D.; funding acquisition, I.M. and G.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original data presented in the study are openly available in Zenodo at DOI 10.5281/zenodo.15681895.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AAAcetic acid
AA+EGAcetic acid + eugenol and geraniol
CACitric acid
EUEuropean Union
IC50Inhibitory concentration at 50%
LALactic acid
PAPelargonic acid

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Figure 1. Radicle emergence inhibition of the crop seeds placed on agar in Petri dishes. Soft wheat (A–E), millet (F–J), and alfalfa (K–O) were evaluated 7 days following treatments of acetic acid (A,F,K), acetic acid with eugenol + geraniol (B,G,L), citric acid (C,H,M), lactic acid (D,I,N), and pelargonic acid (E,J,O) at concentrations of 5%, 10%, and 20%, respectively (eugenol + geraniol at 1.5%). Radicle emergence inhibition was calculated as the mean percentage and standard deviation. The control groups (CTRLs) were set to a baseline value of 0% radicle inhibition for zero treatment, which was taken into consideration when calculating the values for the treatments. The average radicle emergence inhibition of untreated soft wheat, millet, and alfalfa was 5%, 0%, and 5%, respectively. The different letters (a–f) represent significant differences between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), PA (pelargonic acid), and the CTRLs (controls).
Figure 1. Radicle emergence inhibition of the crop seeds placed on agar in Petri dishes. Soft wheat (A–E), millet (F–J), and alfalfa (K–O) were evaluated 7 days following treatments of acetic acid (A,F,K), acetic acid with eugenol + geraniol (B,G,L), citric acid (C,H,M), lactic acid (D,I,N), and pelargonic acid (E,J,O) at concentrations of 5%, 10%, and 20%, respectively (eugenol + geraniol at 1.5%). Radicle emergence inhibition was calculated as the mean percentage and standard deviation. The control groups (CTRLs) were set to a baseline value of 0% radicle inhibition for zero treatment, which was taken into consideration when calculating the values for the treatments. The average radicle emergence inhibition of untreated soft wheat, millet, and alfalfa was 5%, 0%, and 5%, respectively. The different letters (a–f) represent significant differences between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), PA (pelargonic acid), and the CTRLs (controls).
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Figure 2. Radicle emergence inhibition of the weed seeds placed on agar in Petri dishes. Perennial ryegrass (A–E), common chicory (F–J), green foxtail (K–O), and common vetch (P–T) were evaluated 7 days following treatments of acetic acid (A,F,K,P), acetic acid with eugenol + geraniol (B,G,L,Q), citric acid (C,H,M,R), lactic acid (D,I,N,S), and pelargonic acid (E,J,O,T) at concentrations of 5%, 10%, and 20%, respectively (eugenol + geraniol at 1.5%). Radicle emergence inhibition was calculated as the mean percentage and standard deviation. The control groups (CTRLs) were set to a baseline value of 0% radicle inhibition for zero treatment, which was taken into consideration when calculating the values for the treatments. The average radicle emergence inhibition of untreated perennial ryegrass, common chicory, green foxtail, and common vetch was 2%, 20%, 5%, and 5%, respectively. The different letters (a–f) represent significant differences between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), and PA (pelargonic acid).
Figure 2. Radicle emergence inhibition of the weed seeds placed on agar in Petri dishes. Perennial ryegrass (A–E), common chicory (F–J), green foxtail (K–O), and common vetch (P–T) were evaluated 7 days following treatments of acetic acid (A,F,K,P), acetic acid with eugenol + geraniol (B,G,L,Q), citric acid (C,H,M,R), lactic acid (D,I,N,S), and pelargonic acid (E,J,O,T) at concentrations of 5%, 10%, and 20%, respectively (eugenol + geraniol at 1.5%). Radicle emergence inhibition was calculated as the mean percentage and standard deviation. The control groups (CTRLs) were set to a baseline value of 0% radicle inhibition for zero treatment, which was taken into consideration when calculating the values for the treatments. The average radicle emergence inhibition of untreated perennial ryegrass, common chicory, green foxtail, and common vetch was 2%, 20%, 5%, and 5%, respectively. The different letters (a–f) represent significant differences between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), and PA (pelargonic acid).
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Figure 3. Shoot emergence inhibition of the crop seeds sown in peat in pots. Soft wheat (A–E), millet (F–J), and alfalfa (K–O) were evaluated 10 days following treatments of acetic acid (A,F,K), acetic acid with eugenol + geraniol (B,G,L), citric acid (C,H,M), lactic acid (D,I,N), and pelargonic acid (E,J,O) at concentrations of 5%, 10%, and 20%, respectively (eugenol + geraniol at 1.5%). Shoot emergence inhibition was calculated as the mean percentage and standard deviation. The control groups (CTRLs) were set to a baseline value of 0% shoot inhibition for zero treatment, which was taken into consideration when calculating the values for the treatments. The average shoot emergence inhibition of untreated soft wheat, millet, and alfalfa was 10%, 5%, and 13%, respectively. The different letters (a–l) represent significant differences between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), and PA (pelargonic acid).
Figure 3. Shoot emergence inhibition of the crop seeds sown in peat in pots. Soft wheat (A–E), millet (F–J), and alfalfa (K–O) were evaluated 10 days following treatments of acetic acid (A,F,K), acetic acid with eugenol + geraniol (B,G,L), citric acid (C,H,M), lactic acid (D,I,N), and pelargonic acid (E,J,O) at concentrations of 5%, 10%, and 20%, respectively (eugenol + geraniol at 1.5%). Shoot emergence inhibition was calculated as the mean percentage and standard deviation. The control groups (CTRLs) were set to a baseline value of 0% shoot inhibition for zero treatment, which was taken into consideration when calculating the values for the treatments. The average shoot emergence inhibition of untreated soft wheat, millet, and alfalfa was 10%, 5%, and 13%, respectively. The different letters (a–l) represent significant differences between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), and PA (pelargonic acid).
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Figure 4. Shoot emergence inhibition of the weed seeds sown in peat in pots. Perennial ryegrass (A–E), common chicory (F–J), green foxtail (K–O), and common vetch (P–T) were evaluated 7 days following treatments of acetic acid (A,F,K,P), acetic acid with eugenol + geraniol (B,G,L,Q), citric acid (C,H,M,R), lactic acid (D,I,N,S), and pelargonic acid (E,J,O,T) at concentrations of 5%, 10%, and 20%, respectively (eugenol + geraniol at 1.5%). Shoot emergence inhibition was calculated as the mean percentage and standard deviation. The control groups (CTRLs) were set to a baseline value of 0% shoot inhibition for zero treatment, which was taken into consideration when calculating the values for the treatments. The average shoot emergence inhibition of untreated perennial ryegrass, common chicory, green foxtail, and common vetch was 5%, 25%, 10%, and 10%, respectively. The different letters (a–l) represent significant differences between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), and PA (pelargonic acid).
Figure 4. Shoot emergence inhibition of the weed seeds sown in peat in pots. Perennial ryegrass (A–E), common chicory (F–J), green foxtail (K–O), and common vetch (P–T) were evaluated 7 days following treatments of acetic acid (A,F,K,P), acetic acid with eugenol + geraniol (B,G,L,Q), citric acid (C,H,M,R), lactic acid (D,I,N,S), and pelargonic acid (E,J,O,T) at concentrations of 5%, 10%, and 20%, respectively (eugenol + geraniol at 1.5%). Shoot emergence inhibition was calculated as the mean percentage and standard deviation. The control groups (CTRLs) were set to a baseline value of 0% shoot inhibition for zero treatment, which was taken into consideration when calculating the values for the treatments. The average shoot emergence inhibition of untreated perennial ryegrass, common chicory, green foxtail, and common vetch was 5%, 25%, 10%, and 10%, respectively. The different letters (a–l) represent significant differences between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), and PA (pelargonic acid).
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Table 1. IC50 estimated from the concentrations (0%, 5%, 10%, 20%) of each organic compound (required to inhibit radicle emergence by 50% in the four weed and three crop species).
Table 1. IC50 estimated from the concentrations (0%, 5%, 10%, 20%) of each organic compound (required to inhibit radicle emergence by 50% in the four weed and three crop species).
Plant SpeciesIC50 AA (%)IC50 AA+EG (%)IC50 CA (%)IC50 LA (%)IC50 PA (%)
Common vetch 8.08 bc8.4 c3.6 c3.8 c8.3 bc
Perennial ryegrass6.6 c12.2 b3.6 c3.6 c3.8 c
Green foxtail5.1 c4.1 c3.6 c3.34 c3.2 c
Common vetch8.00 bc22.5 b3.9 c5.0 bc3.9 c
Alfalfa4.5 c4.8 c3.4 c4.2 c10.3 bc
Millet6.2 c8.3 c3.3 c3.1 c7.2 bc
Soft wheat11.5 bc13.2 a71.8 a71.8 a4.1 c
The different letters (a–c) denote significantly different values between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), and PA (pelargonic acid).
Table 2. IC50 estimated from the concentrations (0%, 5%, 10%, 20%) of each organic compound (required to inhibit shoot emergence by 50% in the four weed and 3 crop species).
Table 2. IC50 estimated from the concentrations (0%, 5%, 10%, 20%) of each organic compound (required to inhibit shoot emergence by 50% in the four weed and 3 crop species).
Plant SpeciesIC50 AA (%)IC50 AA+EG (%)IC50 CA (%)IC50 LA (%)IC50 PA (%)
Common vetch 3.7 f14.4 d–f19.3 d–f4.6 f13.1 d–f
Perennial ryegrass35.9 c–e38.1 cd5.1 f4.7 f3.8 f
Green foxtail5.1 f4.1 f3.4 f3.4 f3.2 f
Common vetch8.3 f7.6 f14.3 d–f8.8 f14.3 d–f
Alfalfa16.1 d–f14.9 d–f57.8 b44.1 c19.1 d–f
Millet6.4 f15.5 d–f12.8 d–f6.9 f11.5 ef
Soft wheat10.1 ef15.8 d–f62.5 b35.9 c–e89.9 a
The different letters (a–f) denote significantly different values between the species and treatments (p < 0.05, Tukey’s least significant difference test). Abbreviations are AA (acetic acid), AA+EG (acetic acid + eugenol + geraniol), CA (citric acid), LA (lactic acid), and PA (pelargonic acid).
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Alpi, M.; Whittaker, A.; Frassineti, E.; Toschi, E.; Dinelli, G.; Marotti, I. Effects of Pre-Emergence Application of Organic Acids on Seedling Establishment of Weeds and Crops in Controlled Environments. Agronomy 2025, 15, 1820. https://doi.org/10.3390/agronomy15081820

AMA Style

Alpi M, Whittaker A, Frassineti E, Toschi E, Dinelli G, Marotti I. Effects of Pre-Emergence Application of Organic Acids on Seedling Establishment of Weeds and Crops in Controlled Environments. Agronomy. 2025; 15(8):1820. https://doi.org/10.3390/agronomy15081820

Chicago/Turabian Style

Alpi, Mattia, Anne Whittaker, Elettra Frassineti, Enrico Toschi, Giovanni Dinelli, and Ilaria Marotti. 2025. "Effects of Pre-Emergence Application of Organic Acids on Seedling Establishment of Weeds and Crops in Controlled Environments" Agronomy 15, no. 8: 1820. https://doi.org/10.3390/agronomy15081820

APA Style

Alpi, M., Whittaker, A., Frassineti, E., Toschi, E., Dinelli, G., & Marotti, I. (2025). Effects of Pre-Emergence Application of Organic Acids on Seedling Establishment of Weeds and Crops in Controlled Environments. Agronomy, 15(8), 1820. https://doi.org/10.3390/agronomy15081820

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