Plant neighbor detection and allelochemical response are driven by root-secreted signaling chemicals

ABSTRACT Plant neighbor detection and response strategies are important mediators of interactions among species. Despite increasing knowledge of neighbor detection and response involving

plant volatiles, less is known about how soil-borne signaling chemicals may act belowground in plant–plant interactions. Here, we experimentally demonstrate neighbor detection and

allelopathic responses between wheat and 100 other plant species via belowground signaling. Wheat can detect both conspecific and heterospecific neighbors and responds by increasing

allelochemical production. Furthermore, we show that (-)-loliolide and jasmonic acid are present in root exudates from a diverse range of species and are able to trigger allelochemical

production in wheat. These findings suggest that root-secreted (-)-loliolide and jasmonic acid are involved in plant neighbor detection and allelochemical response and may be widespread

mediators of belowground plant-plant interactions. SIMILAR CONTENT BEING VIEWED BY OTHERS ABOVEGROUND PLANT-TO-PLANT COMMUNICATION REDUCES ROOT NODULE SYMBIOSIS AND SOIL NUTRIENT

CONCENTRATIONS Article Open access 16 June 2021 HERBIVORY-INDUCED GREEN LEAF VOLATILES INCREASE PLANT PERFORMANCE THROUGH JASMONATE-DEPENDENT PLANT–SOIL FEEDBACKS Article 01 May 2025

INCREASING FLAVONOID CONCENTRATIONS IN ROOT EXUDATES ENHANCE ASSOCIATIONS BETWEEN ARBUSCULAR MYCORRHIZAL FUNGI AND AN INVASIVE PLANT Article Open access 10 February 2021 INTRODUCTION Plants

are capable of detecting and responding to neighboring plants, generating consequences for plant performance and playing important roles in plant coexistence and community assembly1,2,3.

Plant neighbor detection involves both physical and chemical signals, including far-red light reflection, alteration of nutrient availability and plant-released secondary metabolites. These

signals trigger complex plant response strategies such as shade avoidance, root foraging, and chemical defense4,5,6. Much of the research into chemical-mediated neighbor detection and

signaling interactions has dealt with volatiles induced by herbivory or other environmental stressors in initiating defensive responses7,8,9. This focus is primarily driven by the

accessibility of aerial tissues and availability of reliable techniques to detect and identify volatile chemicals. Nevertheless, plant neighbor detection and signaling interactions take

place both aboveground and belowground. Aboveground signaling interactions are well established and mediated by air-borne chemicals including methyl jasmonate, salicylate, benzoate, and

indole, as well as ethylene and several volatile terpenes10,11,12,13. However, the identity of soil-borne chemicals involved in belowground signaling interactions is largely unknown. In

contrast to aboveground signaling chemicals which move freely in air, the transduction of belowground chemicals requires root-soil interactions. A great deal of recent attention has been

paid to belowground signaling interactions among plant species at the root level14,15,16. In particular, root detection and placement patterns may be mediated through root-secreted

chemicals17,18,19,20. These belowground signals are potentially conducted by physical contact of root hairs and tips or conveyed through associated mycorrhizal hyphae21,22. However, it is

extremely difficult to unambiguously isolate chemical-mediated belowground signaling interactions due to the complexity of plant-soil interactions. Specifically, it has been difficult to

identify root-secreted signaling chemicals and determine whether their delivery is dependent on root contact or common mycorrhizal networks. When neighboring plants are detected, focal

plants may respond to neighbors in morphological and biochemical ways23,24. In particular, neighbors influence plant defensive biochemistry in a species-specific fashion, altering the

production of defensive metabolites such as allelochemicals24,25. Allelochemicals can have profound effects on the performance of neighboring plants, i.e., allelopathy, an interference

mechanism in which neighbor plants are chemically suppressed through the release of allelochemicals from focal plants26. However, allelopathy research has primarily focused only on focal

plants and their allelochemicals rather than on the signaling interactions between plants that mediate the interaction27,28,29. Therefore, allelopathic interference and neighbor detection

usually are studied separately, despite their necessary linkage in nature. For two species that coexist, plants first may detect and potentially recognize their neighbors, and then initiate

allelopathic interference to regulate inter-specific or intra-specific interactions. Neighbor detection and allelochemical response are two inseparable processes when one or more plants

occur together and interact30. This pattern may arise through the production and release of signaling chemicals that induce the production of defensive allelochemicals. Accordingly, the

signaling chemicals may ideally be common to most potential competitors. Alternatively, allelochemical production may be constitutive, produced whether or not neighbors are present,

potentially wasting valuable plant resources26. In previous studies we used production of the putative allelochemical DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), which has a

charcterized metabolic profil in soil31,32,33,34, by allelopathic wheat (_Triticum aestivum_) as a model system. We reported that production of DIMBOA was induced by a diverse range of

neighboring plants in a density-dependent manner29,30. Root exudates from a range of species were able to induce DIMBOA production and the plant hormone jasmonate, and to a lesser extent

salicylate, were able to induce DIMBOA production30. Despite these findings, the identity of the soil-borne chemical signals that are responsible for neighbor detection and allelochemical

response in belowground signaling interactions has not been exhaustively characterized and whether such soil-borne chemical signals are common to most plant species remains unknown. Here, we

show that, using new data and data from previous studies we show that wheat can respond to at least 100 other plant species via belowground interactions. We show that (-)-loliolide and

jasmonic acid, present in root exudates are sufficient to induce this response and suggesting that they can mediate this response. RESULTS NEIGHBOR-INDUCED ALLELOCHEMICAL RESPONSES

Previously we reported a density dependent increase in DIMBOA concentration in wheat roots when co-cultivated with multiple weed species results29,30. We investigated density-dependence of

the DIMBOA response further by co-cultivating wheat with eight weed species (four of which were examined previously30). When wheat was paired with itself and eight common weeds (_Eleusine

indica, Digitaria sanguinalis, Abutilon theophrasti_, _Bidens frondosa, Lolium perenne, Avena fatua, Alopecurus japonicus_, and _Aegilops tauschii_) that often come into contact with wheat,

allelochemical DIMBOA concentration varied with the density of the neighbors. Significantly increased concentrations of the allelochemical were observed at the 5:4–5:9 (wheat: neighbor)

proportions (Fig. 1; Supplementary Table 1). It appeared that the presence of either conspecific or heterospecifc neighbors could induce the allelochemical response in a density-dependent

manner. To provide a broader context for the neighbor-induced allelochemical response, we combined data from our previous publication where DIMBOA concentration was examined in wheat

co-cultivated with 36 different wheat species29 with newly generated data for additional species. This produced a dataset of wheat biomass and wheat DIMBOA concentration in root and shoot

following co-cultivation with a total of 100 different plant species at ratios of 5:8 and/or 5:5 (Supplementary Table 2). We found markedly increased induction of DIMBOA in heterospecific

combinations, relative to DIMBOA production when competing with conspecifics, particularly in wheat roots (Fig. 2). Significant increases in wheat DIMBOA production occurred in 38/100

species at a 5:5 proportion while 70/100 species at a 5:8 mixture (Supplementary Table 2). This effect was much larger in wheat roots than in shoots, with an effect size in roots nearly

three-fold larger than that of shoots. In sharp contrast to the neighbor-induced allelochemical response, the presence of heterospecific neighbors led to very little changes in wheat biomass

of shoots and roots relative to controls (Fig. 2). Though statistically significant, increases in shoot biomass relative to those in conspecific controls were minimal. The timing of species

interactions also had a significant impact on DIMBOA concentrations. Compared with the simultaneous planting of wheat and neighbors at a 5:5 proportion, the early presence of neighbors led

to a greater increase of DIMBOA concentration, while the later colonization of neighbors did not induce an allelochemical response, being nearly identical to intraspecific controls

(Supplementary Fig. 1). Though significant intraspecific induction occurred in wheat, the responsiveness was by far the least sensitive, showing that neighbor-induced allelochemical

responses are more related to the density and early exposure to heterospecific neighbors, than the identity of the competing neighbor species. CHEMICAL-MEDIATED BELOWGROUND SIGNALING

INTERACTIONS Previously we found that DIMBOA concentration of wheat roots was increased even when roots were segregated using 30 μm mesh that prevents physical contact, but not potential

chemical or bacterial signals30. The mechanisms underlying neighbor detection that generated the allelochemical response were investigated further by belowground segregation experiments

using not only 30 μm mesh, but also 0.45 μm mesh and plastic film (Supplementary Fig. 2). The presence of all four heterospecific neighbors (_E. indica, D. sanguinalis, A. theophrasti_ and

_B. frondosa_) increased DIMBOA concentration in all treatments other than those completely separated with plastic film (Fig. 3). The plastic film completely blocked belowground physical,

chemical and biological interactions so that wheat and neighbor interactions were limited to aboveground. The lack of variation in the concentration of DIMBOA with complete exclusion

indicated that there were not aerial signaling interactions. However, DIMBOA concentration was increased in the presence of all four neighbors even with segregation by fine nylon mesh,

indicating belowground signaling interactions between wheat and neighbors. In particular, the 0.45 μm mesh not only prevented penetration of roots but also blocked mycorrhizal linkages.

Under this treatment, only belowground bacterial and chemical interactions occurred in the experimental pots. These results were verified in experiments with four other neighbors (_L.

perenne, A. fatua, A. japonicas_ and _A. tauschii_). Although these four neighbors did not significantly induce DIMBOA production at a 5:5 mixture, significant induction occurred at a 5:8

mixture, even under belowground segregation with the 0.45 μm mesh (Supplementary Fig. 3; Supplementary Table 3). These results suggest that neighbor-induced allelochemical response was

mediated by root-secreted chemical signals or soil bacteria rather than by root contact and soil mycorrhizal hyphae. ROOT-SECRETED SIGNALING CHEMICALS To confirm that chemicals and not

bacterial interactions were responsible for the observed allelochemical induction, we previously used root exudate extracted from four common weeds and showed that this could induce DIMBOA

production30. We also previously showed that jasmonic acid (JA), and salicylic acid (SA) when supplied at higher concentration, were sufficient to induce the production of DIMBOA30. Here, we

extended this analysis by re-analyzing those samples and adding four additional species. The root exudates from neighbor species were applied to the soil at varying concentrations. When

wheat was exposed to the root exudates after 6 h, the concentration of allelochemical DIMBOA varied with neighbor species identity in a dose-dependent manner (Supplementary Fig. 4).

Induction of DIMBOA was very limited in conspecific exudates when compared to all heterospecific exudates. The root exudates of _A. theophrasti_ significantly increased the DIMBOA

concentration at a low density of 50 plants 500 mL−1, followed by _D. sanguinalis, E. indica_, and _B. frondosa. A. fatua_ and _A. tauschii_ at a medium concentration of 100 plants 500 mL−1.

All root exudates from either wheat itself or other species tested significantly induced the production of DIMBOA at the highest concentrations of 200 plants 500 mL−1 (Supplementary Fig. 

4). Similar effects of the root exudates and the presence of neighbors in a density-dependent manner suggest widespread occurrence of signaling components in the root exudates across

different plant species. To identify which individual root exudate components could elicit DIMBOA production,a bioassay-guided fractionation approach was used (see Methods). Four potential

chemical signals jasmonic acid (JA), salicylic acid (SA), (-)-loliolide and luteolin were identified (Supplementary Table 4). These presence of these potential signals was tested in all 100

heterospecifc neighbors. JA, SA and (-)-loliolide were found in wheat as well as all 100 heterospecifc neighbors, whereas luteolin was less common (Supplementary Table 5). These chemicals

were also detected in the root exudates and rhizosphere soils of the subset of species tested (Supplementary Table 6). There was a significant positive correlation across species between

neighbor-induced DIMBOA and their root-secreted (-)-loliolide concentrations (_r_ = 0.76, _P_ < 0.001), but such a significant correlation was not found for JA, SA or luteolin

(Supplementary Fig. 5). Further, soil incubation showed that JA, SA, (-)-loliolide and luteolin all elicited the production of DIMBOA in a dose-dependent manner. Significant effects were

observed for (-)-loliolide at a low concentration of 5 nmol g−1 dry soil and for JA at a medium concentration of 50 nmol g−1 dry soil. However, SA and luteolin required a higher

concentration of over 100 nmol g−1 dry soil for elicitation (Fig. 4). In particular, the mixture of (-)-loliolide and JA at a low concentration greatly induced allelochemical response but

the joint action was not observed in the mixture of (-)-loliolide and SA or luteolin (Supplementary Fig. 6). Furthermore, there were significant differences in mobility factor among the

chemicals, with (-)-loliolide having the highest mobility in soil (Supplementary Fig. 7). Compared with SA and luteolin, (-)-loliolide and JA were more effective signaling chemicals to

trigger the allelochemical response. Therefore, we suggest that wheat likely detects the presence of neighbors through root-secreted (-)-loliolide and JA in the soil, which can then initiate

the release and production of DIMBOA, resulting in an induction of allelopathy (Fig. 5). DISCUSSION Plants can alter their growth and the production of secondary metabolites in response to

the biotic and abiotic factors to which they are exposed in the environment23,24. Biochemical plasticity frequently occurs when one or more plant species occur together and interact. In

particular, neighboring plants can profoundly affect plant biochemical composition through competition, allelopathy or both28,35,36. However, biochemical responses are strongly related to

neighbor identity mediated through the presence of neighbor detection cues. Most studies have shown that neighbor identity influences plant biochemical responses in a species-specific

fashion14,25. Consistent with our previous work29,30, we report here that allelopathic wheat produces DIMBOA in a density dependent response to neighboring plants and that root exudates are

sufficient to induce this response. Allelopathic wheat appears to be able to detect the presence of plant neighbors when they occur early in development, and respond by increasing

allelochemicals regardless of their neighbor identity. Wheat produces and releases benzoxazolinone allelochemicals, including DIMBOA and its glycosides, at early growth stages. Because of

this, the hydrolysis and metabolic profiling of allelochemical DIMBOA from plants to soils is well-established31,32,33,34. In the current study, the increase in DIMBOA production was only

two-fold or so. From a typical dose/response relationship, a two-fold increase in a toxin that is already present at a significant level may not be expected to have a substantial effect.

However, a more dramatic effect might occur with joint action of other benzoxazolinone compounds of wheat. Although the neighbor-induced allelochemical responses were density-dependent,

these responses occurred primarily at intermediate neighbor densities. Plants growing with a high density of neighbors likely experienced an increase in competition-induced stress.

Competition can alter plant allocation strategies, resulting in a shift from defense to growth37,38. Allelopathic plant species often increase allelochemical concentrations in response to

low or intermediate levels of competition, but a decrease of allelochemicals at higher levels of competition where resource limitation may prevent such allocation39. Furthermore, the timing

of plant exposure to competition may strongly mitigate allelochemical responsiveness. In the current study, induction of DIMBOA only occurred when wheat was exposed to competitors very early

in development, with no response to later exposure. This suggests that there is a critical temporal window for detection of competition that also has large implications for species

coexistence. In the crop-weed model system explored here, allelochemical induction early in development may aid the performance of plants exposed to competition. However, later germinating

competitors will pose a much lower competitive risk because of their smaller size relative to the wheat, and therefore allocation to allelochemicals would be less beneficial to plant

fitness. A similar strategy would be an advantageous response to competition in non-crop systems as well. Although any stress caused by neighboring plants as a result of resource depletion

or alteration of microbial communities might also induce an allelochemical response40,41, direct neighbor-induced allelochemical responses are critical to plant-plant interactions. Plant

neighbor detection usually is mediated by direct contact or chemical release4,11. In belowground plant-plant signaling interactions, the detection of neighbors may occur through contact of

root tips, soil microbes, especially common mycorrhizal networks21,22, or root-secreted signaling chemicals14,42. However, the mediators of belowground signaling interactions are still

controversial due to inadequate methodology and our poor knowledge of the diversity of soil interactions. This study addressed the mechanism of belowground signaling through a series of

experiments with and without root, mycorrhizal, and soil chemical segregation. This segregation-based approach clearly distinguished aboveground and belowground signaling interactions in

generating allelochemical responses. Allelochemical production was induced by competitors with root segregation with both sizes of nylon mesh but not with complete segregation, where only

aboveground signals would occur. Neighbor-induced allelochemical responses still occurred with the 0.45 μm mesh that would have segregated soil mycorrhizal hyphae but still allowed bacterial

and chemical contact. Furthermore, allelochemical production was experimentally induced by the root exudates of neighboring plants, isolating the effects of chemicals from soil bacteria.

These results indicatethat allelopathic wheat can detect neighboring plants purely through root-secreted signaling chemicals rather than via root contact, common mycorrhizal networks, or

soil bacteria. Plants may detect neighbors through plant volatiles as air-borne signals10,11, while root exudates drive belowground plant–plant signaling interactions16,18. Until recently,

most studies on the signaling chemicals in plant–plant interactions have focused on jasmonic acid (JA) and salicylic acid (SA)30,43,44,45. JA and SA are two ubiquitous signaling chemicals

which elicit the production of defensive plant metabolites against microbes, herbivores, or competitors46,47. However, information on other signaling chemicals involved in belowground

plant–plant signaling interactions has remained scarce. Root-secreted strigolactones were described as signaling chemicals between parasitic plants and their hosts several decades ago48.

Strigolactones also act as chemical signals for fungal symbionts and parasitic weeds in plant roots49. However, strigolactones do not appear to have a universal role in belowground

plant-plant signaling interactions and there may be other, more specific, root-secreted signaling chemicals awaiting identification. JA and SA may play some roles in belowground chemical

communications. However, most studies have tested this with exogenous addition of JA and SA at arbitrary concentrations, rather than through quantification in the root exudates and

rhizosphere soils43,45. A few studies have documented root-secreted JA and SA and their roles in rhizosphere signaling interactions30,44. In this study, besides the commonly studied JA and

SA, (-)-loliolide and luteolin contributed to neighbor-induced allelochemical responses. In particular, (-)-loliolide and JA strongly induced allelochemical production at very low

concentrations, whereas luteolin and SA required greater concentrations. Although these concentrations were greater when compared with the quantities of (-)-loliolide and JA as determined in

the root exudates and rhizosphere soils from interacting weeds tested, we propose that such bioactive phytochemicals provided over a long time period at low concentrations might be able to

induce substantial effects. Additionally, soil extractions would have diluted the chemical signals over large soil volumes while the concentrations of (-)-loliolide and JA detected would be

locally much higher in intact soils. Furthermore, there was a joint action in the mixture of (-)-loliolide and JA at low concentrations. Thus, even if the actual concentration of

(-)-loliolide and JA in soil was still substantially lower than the necessary concentration to elicit wheat allelochemical production, an effect would still be expected. Similar to SA,

phenolic luteolin is a signal in the resistance response of plant to microbes50,51. However, (-)-loliolide has never been reported in participating in any plant–plant or plant–microbe

signaling interactions. More importantly, (-)-loliolide was detected in all of the 101 plant species tested while luteolin only occurred in 79 plant species. Actually, (-)-loliolide is the

most ubiquitous lactone that occurs in many plant families and marine alga52. In the current study, (-)-loliolide was found in every monocot and dicot plant species tested and could be

secreted from roots into rhizosphere soils. (-)-loliolide had a higher soil mobility and could easily be moved from the rhizosphere to bulk soil. Therefore, (-)-loliolide may be a

ubiquitous, soil-borne signaling chemical that can trigger plant defensive responses in belowground plant–plant interactions. Growth-inhibiting allelochemicals and signaling chemicals

involved in plant–plant interactions profoundly affect the performance of plants, altering the consequences of intra-specific and inter-specific interactions in ecosystems19,20,35. It is now

clear that plant-derived signaling chemicals contribute to plant neighbor detection and defensive responses. In general, chemicals produced by the competitor species that are xenobiotic to

the focal plants might represent a better, species-specific signal, just like strigolactones as signaling chemicals of parasitic plants to the host48,49. However, (-)-loliolide and JA found

in this study are ubiquitous signaling chemicals among plants, and are produced by the focal allelopathic wheat as well. In fact, signaling chemicals such as ethylene, JA, SA, and their

formylates are ubiquitous plant–plant signals rather than xenobiotic chemicals that may indicate identity10,11. Plant neighbor detection and allelochemical response in generating coexistence

heavily depended on neighbor density rather than neighbor identity, reminiscent of plant neighbor detection mediated in a dose-dependent fashion. The (-)-loliolide or JA secreted from

either conspecific or heterospecifc roots could elicit the production of allelochemicals and appears to be a signal for belowground competition, potentially common to all species. We

speculate that variation in level of soil signaling chemicals produced across species has the potential to lead to variable responses in natural systems, though these chemical signals could

still be general indicators of competition. We assume that there is an additional, species-specific signaling chemical that inhibits the induction of allelochemicals in conspecific

interactions. This could be a much less common soil chemical, one more representative of the species identity and also much less likely to be detected by our methods which focused on

chemicals that generated an allelochemical response. Fractionation-guided bioassays that focus on the inhibition of (-)-loliolide responses would be necessary to identify this soil signal.

The importance of belowground signaling interactions to plant neighbor detection and response strategies, as well as the mechanisms underlying such communications, has been a major focus of

the science of plant interaction in recent years14,16,42. From a model system of allelopathic wheat and 100 interacting plant species, this study suggests that plant neighbor detection and

allelochemical response mediated by root-secreted signaling chemicals are a general phenomenon. In particular, this study identified (-)-loliolide as a soil–borne chemical signal that could

act to signify the presence of competitors and enhance allelochemical production. In conclusion, the discovery of (-)-loliolide as a general soil-borne signaling chemical common to all the

plants tested here, improves our understanding of plant neighbor detection and response strategies. Studies on the behavior of (-)-loliolide in the soil and its molecular biology, could lead

to new insights into plant sensing and communication. Although a generic response to (-)-loliolide was detected here, there are likely to be additional signaling mechanisms that could allow

species-specific responses. In addition, the ability of the signaling chemical to induce allelopathic responses in plants needs to be explored in other plant systems to test if they respond

similarly to allelopathic wheat. METHODS PLANT MATERIALS AND SOILS Wheat produces and releases benzoxazinoids against various pests, most notably in affecting the growth of plant

competitors. Among benzoxazinoids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) is a putative and dominant allelochemical33,53. Accordingly, a DIMBOA-rich winter wheat cultivar

(Jing411) was used in this study and its seeds obtained from the Chinese wheat germplasm collection. Combining data from our previous study29 with new data, wheat plants were challenged with

100 plant species (Supplementary Table 2) which were selected on the basis of their occurrence and distribution in the local wheat industry or being ecologically relevant weeds and crops in

agricultural ecosystems. Seeds were collected from local fields or obtained from germplasm collections in China. Soil for use in experiments was collected from the surface (0–10 cm) of a

wheat field at the Shangzhuang Experimental Station of China Agricultural University (Beijing, China). Soil samples were air-dried, then sieved (2 mm mesh) to remove plant tissues. The soil

is a Hapli-Udic Cambisol (FAO classification) with a pH of 6.52, an organic matter content of 1.65%, and a nutrient content of available N of 71.41 mg kg−1, available _P_ of 70.12 mg kg−1,

and available _K_ of 94.51 mg kg−1. WHEAT–NEIGHBOR INTERACTIONS Three experiments for wheat–neighbor interactions were carried out in plastic pots (11 cm diameter × 12 cm height) that

contained a central cylinder (7.5 cm diameter, 12 cm height) where a barrier could be inserted (Supplementary Fig. 2). The experiments were conducted in a completely randomized design with

three replicates for each treatment or control. Seeds of wheat and other plants were sterilized with 75% alcohol for 3 min, followed with 3% sodium hypochlorite for 12 min. Sterilized seeds

were rinsed with distilled water and then transferred to Petri dishes (9 cm diameter) with moistened filter paper and were pre-germinated at 28 °C in the dark. The first experiment

investigated the allelochemical response of wheat to eight commonly interacting weeds (_E. indica, D. sanguinalis, A. theophrasti_, _B. frondosa, L. perenne, A. fatua, A. japonicus_, and _A.

tauschii_) at different densities. Pre-germinated seeds of wheat and neighbor pairs were each sown into the plastic pot containing 800 g of soil with no barrier. Five wheat seeds were

spaced uniformly in the central cylinder of each pot, while neighbor seeds were sown surrounding the wheat. Proportions of wheat to eight common neighbor species ranged from 5:1 to 5:10 per

pot (wheat:neighbor), while trials between wheat and the remaining 92 species (Supplementary Table 2) were conducted at 5:8 and/or 5:5 mixture proportions. The plant:competitor ratios were

found to generate sufficient responses (Fig. 1). Pots with wheat monocultures in the same planting patterns and mixture proportions served as the controls for all analyses. A second

experiment was run to determine the influence of time of association on allelochemical response of wheat to eight commonly interacting weeds at the 5:5 mixture. A series of experimental pots

containing 800 g of soil with no barriers and were planted with five wheat seeds sown into the central cylinder and five neighbor seeds sown in the surrounding area. Plantings occurred

simultaneously, neighbors planted after the wheat emerged, or wheat planted after the neighbors had emerged. Again, pots with wheat monocultures for each group served as the controls. The

third experiment evaluated allelochemical response of wheat to eight commonly interacting weeds with varying levels of belowground segregation (Supplementary Fig. 2). A series of pots

containing 800 g of soil had central cylinders that were: open (full contact), covered with 30 μm mesh (prevented penetration of roots but allowed chemical and microbial interactions),

covered with 0.45 μm mesh (prevented penetration of both roots and common mycorrhizal hyphae but allowed chemical and bacterial interactions) or covered with plastic film (complete

separation). As previously described, wheat and neighbor pairs at 5:8 and/or 5:5 proportions were sown simultaneously in the pots with or without belowground segregation. Monocultures of

wheat-wheat (5:5 or 5: 8) in a pot for each group or treatments served as the controls. All pots from the experiments described above were placed in a greenhouse with 20–30 °C night and

daytime temperatures and 65–90% relative humidity, watered daily and their positions randomized once a week. Any emerging plants, other than wheat or sown neighbor species were hand removed

as soon as they were detected. Seedlings were harvested after 4 weeks and separated carefully into wheat and neighbor plants. In addition, rhizosphere soils from the third experiment were

collected by shaking soil off from roots from treatments separated by nylon mesh according to the procedure of Guo et al.54. Plant and soil samples were taken for the quantification of

allelochemical DIMBOA and signaling chemicals as described in the quantitative analysis section below. POTENTIAL SIGNALING COMPONENTS FROM ROOT EXUDATES To obtain substantial amounts of

potential signaling components, a larger scale collection of the root exudates was carried out with a modified continuous root exudate-trapping system55 in a greenhouse (Supplementary Fig. 

8). Three-thousand seedlings of each of eight commonly interacting weeds at the 3-leaf or 5-leaf stage were transplanted into culture containers (2 m × 1 m × 0.3 m) with 1/2 Hoagland’s

solution. Chemical trapping was done within a column (5 cm × 50 cm) with 1000 g Amberlite XAD-4 resin (Sigma-Aldrich Co., St. Louis, MO, USA) packed into it. This was connected to the

reservoir with a pump and the culture solution was circulated for 2 h though it once a day. After 10 days, the column was detached. The resin was continuously washed with distilled water for

24 h to purge inorganic ions and carbohydrates. The resin was then eluted with methanol. After filtration, the exudates were evaporated to dryness in vacuo by using a rotary evaporator at

ambient temperature. The dried root exudates (100 g) were successively partitioned three times with petroleum ether (PE), followed by methylene chloride (CH2Cl2). The CH2Cl2 extract was

concentrated, and subjected to silica gel column chromatography (5 cm × 80 cm) by eluting stepwise with a series of mixtures of PE, CH2Cl2 and MeOH (10:0:0, 8:2:0, 6:4:0, 4:6:0, 2:8:0,

0:10:0, 0:9:1, 0:8:2, 0:7:3, 0:6:4, 0:5:5, and 0:0:10, v/v/v). The fractions were screened using a bioassay-driven fractionation approach56 as described in the soil incubation section below,

resulting in four fractions that induced the production of allelochemical DIMBOA in wheat. The first fraction was further purified by silica gel column chromatography (2.5 cm × 40 cm) with

a mixture of CH2Cl2 and MeOH (5:5, v/v), and yielded luteolin. The second fraction was further purified by Sephadex LH-20 (20–150 μm, 1 cm × 25 cm) with MeOH, resulting in liquid jasmonic

acid (JA). The third fraction was further purified by ODS (YMC 120 A 50 μm, 1 cm × 25 cm) with H2O containing increasing amounts of MeOH to obtain a green solid that was recrystallized with

CH2Cl2 to yield (-)-loliolide. The fourth fraction was further purified by silica gel column chromatography (2.5 cm × 40 cm) with a mixture of CH2Cl2 and MeOH (3:7, v/v), resulting a crude

solid that was recrystallized with _n_-hexane-MeOH (4:6; v/v) mixture and gave salicylic acid (SA). All four chemicals, JA, SA, (-)-loliolide and luteolin, were identified by spectroscopic

analysis (Supplementary Table 4) and chromatographic co-elution with authentic standards (Sigma-Aldrich Co., St. Louis, MO, USA). SOIL INCUBATION Induction activities of root exudates and

their signaling components on the production of DIMBOA were verified using a pot-culture study. The root exudates of wheat and eight commonly interacting weeds were collected in a hydroponic

experiment. Fifty, one or two hundred seedlings at the 3-leaf stage for each species were respectively inserted into holes in a Styrofoam float and transplanted into a hydroponic container

with 1/2 Hoagland’s solution (500 ml). The container was placed in a sterile environmental chamber at 28 ± 1 °C with a 12 h photoperiod. The hydroponic solution in the container was kept at

a constant level by adding distilled water daily. After 7 days the hydroponic solution was filtered with sterile filter papers, and the filtrate was collected to yield the root exudates. Ten

wheat seeds were sown into a series of 5 cm × 5 cm plastic pots with 100 g of soil. At the 2-leaf stage these were thinned to fivee plants per pot. The root exudates described above, one of

the fractions or mixtures and potential signaling components (i.e., jasmonic acid, salicylic acid, (-)-loliolide or luteolin) that were isolated and identified as describe in the section

above, were each added to the treated pots at different concentrations. Control pots received distilled water only. All treatments and controls were replicated three times and were placed in

an environmental chamber with a temperature of 25 °C and 65–90% relative humidity. The pots were randomly sampled at 6 h after soil-incubation. This sampling time was based on a significant

increase in wheat DIMBOA observed in an incubation experiment involving the addition of weed root exudates29. Wheat seedlings were collected for the quantification of DIMBOA as described in

the quantitative analysis section below. SOIL THIN LAYER CHROMATOGRAPHY (TLC) Soil TLC was performed by a combination of two methods57,58. Soils as described above were ground and sieved to

125 μm. The soil was suspended in a dioxane/water (1:1, v/v) solvent to make a slurry which was then spread as a 0.7 mm thick layer on a 10 cm × 20 cm glass plate. The plates were air-dried

at 20–25 °C and stored in a desiccating chamber until used for chromatographic tests. Four potential signaling chemicals, JA, SA, (-)-loliolide or luteolin were each sampled with a

microsyringe at 2.5 cm from the bottom edge of the plates. Distilled water in sampling served as the control. After the spots had been deposited, the plates were allowed to develop in a

closed glass chamber using distilled water as solvent. A sheet of filter paper dipping into the developing water fed water continuously to the substrate at the base of the plate, thus

leading to a relatively uniform flow. During development with water, the whole device was held in a horizontal position. Water migration occurred at a distance 17.5 cm from the baseline. The

migration lasted between 1 h and 5 h depending on the samples. The plates were dried at 20–25 °C, and the soil layer of drying the developed TLC plates with three replicates for each

chemical was cut into segments of 1.5 cm each. To avoid microbial degradation and transformation, chemical residue in each segment was quantified immediately by ultra performance liquid

chromatography (UPLC) described below. Mobility factor (Rf) value of each chemical was calculated according to the formulae _R_f = _∑R_i × _M_i /_R_w × _∑M_i, where, _R_w was remove distance

of water from start point, i was number of segments, Ri was distance of segment _i_ from start point, _M_i was chemical content in segment _i_59. QUANTITATIVE ANALYSIS OF ALLELOCHEMICAL AND

SIGNALING CHEMICALS Quantification of the allelochemical DIMBOA was performed by liquid extraction/solid-phase extraction, followed by high-performance liquid chromatography (HPLC). The

wheat tissues (roots or shoots) were freeze-dried and ground with liquid nitrogen. Then 500 mg of the resulting powder was homogenized with 50 ml of 25% aqueous MeOH and extracted by

ultrasonic oscillator for 30 min at a temperature of 25 °C. The extract was filtered, and the filtrate was evaporated to dryness individually under nitrogen gas. Dry residues were dissolved

in 2.5 ml of 0.05% acetic acid (HOAC) in a MeOH-H2O mixture (60:40 v/v) and then loaded onto reverse-phase C18 Sep-Pak cartridges (Waters Co., Milford, MA, USA) equilibrated with water,

which eluted with acidified MeOH (1% HOAC) and then MeOH. The MeOH fraction was concentrated with nitrogen gas to a final volume of 100 μl. The concentrated samples were subsequently

subjected to an HPLC-1260 instrument (Agilent, Palo Alto, CA, USA) equipped with a C18 reverse-phase column (Hypersil 4.6 mm × 150 mm, 5 μm) and a diode array UV detector at 280 nm. Elution

was performed with a mixture of 0.5% acetic acid and MeOH (70:30, v/v) at a constant flow rate of 1.0 ml min−1 at 40 °C. The peak of DIMBOA was identified by its retention time (9.8 min) and

coelution with an authentic standard (Regenstauf, Germany). DIMBOA was quantified by regression analysis of the peak areas against standard concentrations (limit of quantification, 10 μg 

g−1; recovery rates at concentrations of 200–1500 μg g−1, 66.5–82.4%). Four potential signaling chemicals JA, SA, (-)-loliolide and luteolin were quantified by ultra-performance liquid

chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The plant tissues, root exudates or rhizosphere soils were each freeze-dried and ground with liquid nitrogen. An amount of

250 mg of the resulting powder was extracted with 10 ml of MeCN (acetonitrile)-H2O-HOAC mixture (90:9:1, v/v/v), vortexed for 5 min at 25 °C and then NaCl was added and immediately vortexed

vigorously for 1 min. After the solution was centrifuged at 2800×_g_ for 10 min, the supernatant was filtered with a 0.22 μm nylon syringe filter (Sterlitech, Kent, WA, USA). Analyses of

four chemicals were carried out on a triple–quadrupole mass spectrometer (TQD, Waters Co., Milford, MA, USA) equipped with an electrospray ionization (ESI) source operating in positive mode

for (-)-loliolide and luteolin and negative mode for JA and SA. Instrument control and data acquisition were performed using MassLynx software (version 4.1). Chromatographic separation was

performed using an Acquity UPLC-BEH C18 column (50 mm × 2.1 mm, 1.7 μm) at 40 °C. The injection volume was 5 μl. The elution gradient was carried out with a binary solvent system consisting

of 0.2% HOAC in H2O (solvent A) and MeCN (solvent B) at a constant flow rate of 0.3 ml min−1. Simultaneous separations were completed using a gradient elution of 0.0 min/90% A, 2 min/10% A,

3.0 min/10% A, 4 min/90% A, and 5.0 min/90% A. Separation and stabilization were achieved in 5.0 min. The typical conditions were capillary voltage, 3.0 kV; source temperature, 120 °C; and

desolvation temperature, 350 °C. The cone and desolvation gas were set at flow rates of 50 and 600 l h−1, respectively. Multi-reaction monitoring (MRM) mode was operated for each chemical.

All parameters for the MRM transitions, cone voltage and collision energy were optimized to obtain the highest sensitivity and resolution. For JA: retention time, 1.60 min; parent ion,

_m_/_z_ 209; product ion, _m_/_z_ 58.9; cone voltage, 35 V. For SA: retention time, 1.40 min; parent ion, _m_/_z_ 136.8; product ions, m/z 92.9; cone voltage, 26 V. For (-)-loliolide:

retention time, 1.25 min; parent ion, _m_/_z_ 197; product ion, _m_/_z_ 178.7; cone voltage, 23 V. For luteolin: retention time, 1.35 min; parent ion, _m_/_z_ 287; product ion, _m_/_z_ 153;

cone voltage, 20 V. Quantification of four chemicals was each conducted by the addition method with authentic standards. Limit of detection (LOD) for the chemicals is the concentration that

produces _S_/_N_ (signal-to-noise ratio) = 3, estimated from the chromatogram corresponding to the lowest concentration used in the calibration, whereas limit of quantification (LOQ) is

defined based on S/N = 10. The LOD were estimated at 0.006 nmol g−1 for loliolide, 0.004 nmol g−1 for luteolin, 0.004 nmol g−1 for JA and 0.006 nmol g−1 for SA. The LOQ were estimated at

0.02 nmol g−1 for loliolide, 0.01 nmol g−1 for luteolin, 0.01 nmol g−1 for JA and 0.02 nmol g−1 for SA. The recoveries of the four chemicals ranged in 86.2–102.6% (RSD, 4.56–10.88) at the

spike level of 0.01–150 nmol g−1. DATA ANALYSIS For the survey of 100 species, data were presented as means ± standard error (SE) from each of independent experiments with three replicates.

The data were analyzed using Student’s _t_-test or analysis of variance (ANOVA). Tukey post-hoc tests were used for multiple comparisons when ANOVA terms were significant using SPSS 16.0 for

Windows (SPSS Inc. Chicago, Illinois, USA). Regression lines were fitted as linear or quadratic model results using Sigmaplot 10.0 (Systat Software Inc. San Jose, California, USA). A simple

Pearson’s correlation was used to test whether the allelochemical DIMBOA induction across samples correlated with signaling components of the root exudates also using SPSS. While we did not

control for multiple comparisons in the response survey of 100 species, we calculated binomial probabilities for obtaining the number of significant individual tests. To provide a single

overview of this survey, we calculated Ln-transformed response ratios relative to the wheat:wheat treatment in each species for both the DIMBOA concentration and biomass of roots and shoots.

From these 100 ratios, we present means ±95% confidence intervals as a measure of effect size. DATA AVAILABILITY All relevant data supporting the findings of this paper are available from

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388, 206–213 (2007). Article  ADS  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (31171865; 31672040)

and the Special Fund for Agro-scientific Research in the Public Interest, China (201403030). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * College of Resources and Environmental Sciences,

China Agricultural University, 100193, Beijing, China Chui-Hua Kong, Song-Zhu Zhang, Yong-Hua Li, Zhi-Chao Xia & Xue-Fang Yang * Department of Biological Sciences, Eastern Illinois

University, Charleston, IL, 61920, USA Scott J. Meiners * Institute of Applied Ecology, Chinese Academy of Sciences, 110016, Shenyang, China Peng Wang Authors * Chui-Hua Kong View author

publications You can also search for this author inPubMed Google Scholar * Song-Zhu Zhang View author publications You can also search for this author inPubMed Google Scholar * Yong-Hua Li

View author publications You can also search for this author inPubMed Google Scholar * Zhi-Chao Xia View author publications You can also search for this author inPubMed Google Scholar *

Xue-Fang Yang View author publications You can also search for this author inPubMed Google Scholar * Scott J. Meiners View author publications You can also search for this author inPubMed 

Google Scholar * Peng Wang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS C.H.K. conceived and designed the experiments. S.Z.Z., Y.H.L.,

and Z.C.X. performed the experiments. C.H.K., X.F.Y., S.J.M., and P.W. analyzed the data. C.H.K. wrote the manuscript and S.J.M. edited the manuscript. CORRESPONDING AUTHOR Correspondence to

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detection and allelochemical response are driven by root-secreted signaling chemicals. _Nat Commun_ 9, 3867 (2018). https://doi.org/10.1038/s41467-018-06429-1 Download citation * Received:

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