The amount of biologically available N is dramatically rising in various ecosystems worldwide because of anthropogenic activities (Jiang et al., 2018). Worldwide application of N may have caused significant changes in aboveground plant diversity and production and the composition and diversity of underground microbial community (Zhou et al., 2016; Qin et al., 2022). The efficacy of grassland may be affected by competitions between legume and non-legume plants, due to the changes in availability of macronutrients, particularly phosphorus (P) and N. During plant competition, plant biomass and abundance are inhibited by N enrichment, and P is usually more effective in enhancing the competitiveness of legume plants than N addition (Unger et al., 2021). Furthermore, N-addition may weaken plant-microbe interactions, induce dormancy, and causes indigenous destruction of vulnerable functional grouping of microbes (Kearns et al., 2016; Jiang et al., 2018). In this study, we investigated the interspecific interactions under N-addition and their influence on the colonization of arbuscular mycorrhizal fungi (AMF).

Among the widespread root-associated microbes, AMF are known to be sensitive to N-addition in both pot and field experiments. Interaction between AMF and plants generally plant supply related AMF with carbohydrates, and in return, fungi provide soil P, and possible N to their host plants (Liu et al., 2015; Bahadur et al., 2019b). The mycorrhizal symbiotic association with plant roots may also improve plants ability to protect themselves against several types of biotic and abiotic stresses and root pathogens (Lewandowski et al., 2013; Zhou et al., 2022; Bahadur et al., 2019a). Recently, numerous studies have reported that N-addition will reduce AMF community diversity and abundance (Chen et al., 2017; Williams et al., 2017); nonetheless, positive or even neutral effects of N-addition have also been reported in various studies (Zheng et al., 2014; Unger et al., 2021). For instance, a field experiment conducted in Qinghai-Tibet Plateau reports that AMF spore density is not affected by N-addition, while the extraradical hyphal density dramatically increases (Zheng et al., 2016). Such different opinions and observations propose that AMF performance fluctuates under N-addition and hence necessitates further studies.

Knowledge of plant-fungal processes and mycorrhizal functioning are crucial for the competitive interactions between the host and non-host plant species. For instance, Vicia faba and Brassica napus are the main competitive interactions adopted by farmers in China. These competitive interactors respond differently to AMF, since they have different nutrient acquisition strategies and nutrient demands (Meng et al., 2015; Qiao et al., 2015). N captured by non-host plant B. napus may favor the occurrence of AMF, when B. napus is inter-cropped with the host plant Plantago lanceolate (Hodge, 2003). They are similar in understanding the functioning of local mycorrhizal effects on B. napus under N-addition (Carneiro et al., 2015; Pakpour and Klironomos, 2015). The presence of AMF in and Zea mays growth may both be adversely affected by B. napus (De Souza and Santos, 2018). Previous studies report that competitive interspecific interactions could be altered and regulated by AMF complete reallocating nutrients as reported in numerous host plants (Fellbaum et al., 2014; Bever, 2015), for example the legume and grass, the nutritional competition of legumes is encouraged the presence of AM fungus (Scheublin et al., 2007; Wagg et al., 2011). When grown with Hordeum vulgare, V. faba demonstrates a higher ability to compete nutrients mediated by AMF (Bahadur et al., 2019b). AMF may alter competitive associations between host and non-host plant species by favoring the mycorrhizal plants over the neighboring ones, generally due to the positive mycorrhizal responsiveness.

So far, studies investigating the impacts of AMF on nutrient competition usually focus on the host plants only (Ren et al., 2017; Yang et al., 2018; Bahadur et al., 2019b), it is still not clear how AMF affect the nutrient competition between the host and non-host plant species when they are inter-cropped. Moreover, previous meta-analysis showed that nitrogen addition reduced plant species-diversity, but increase plant productivity (Fu and Zhen-Xi, 2016). Other field warming studies (e.g., Fu et al., 2019; Wang et al., 2021; Zhang et al., 2021) indicated that plant productivity increased with soil nitrogen availability. However, whether and how N deposition regulates AMF affecting the plant nutrient competition is largely unknown. Here, we investigate that under different N-addition levels: (1) how AMF inoculation facilitates the competitive ability and growth performance of host (i.e., V. faba) and non-host (i.e., B. napus) species, and (2) whether the AMF community contributes differently to the plant competitive interactions. We tested the hypothesis that both AMF inoculation and N-addition could promote plant competitive ability. Our results provide essential information to develop strategies for crop management.

As described by Meng et al. (2015) and Qiao et al. (2015), farmers in China mainly used V. faba and B. napus as competitive interactors. However, little is known about the influence of AMF communities and N-addition on this competitive interaction. Most previous studies (both field and glasshouse studies) have also reported that N enrichment, mainly N-addition at high levels, reduces AMF functions and shifts the mycorrhizal symbiosis toward parasitism (Liu et al., 2015; Jiang et al., 2018).

The current results demonstrate that AMF in mixed-culture are directly essential for the survival of host plant. Mycorrhizal inoculation enhanced the competitive ability of V. faba, with a greater aggressivity index and competitiveness than B. napus. In contrast, N-addition had less impact on interspecific interactions than AMF symbionts. Previous findings by Veiga et al. (2013) and Zhang et al. (2012) demonstrated the benefits of AMF inoculation on mycorrhizal plants ability for interspecific competition with regard to biomass and nutrients. Another study has revealed that the AMF community might induce variable plant growth responses (Munkvold et al., 2004). Considering our study, in both harvests, V. faba is the stronger competitor than B. napus in terms of plant growth responses, which reveals that AMF inoculation contributes to reduce the growth rate in non-host plants (Jordan et al., 2000; Facelli et al., 2010). Mycorrhizal inoculation improved the competitive ability of V. faba, which is consistent with previous study (Daisog et al., 2011). In contrast to these findings, different competing plants also indicated a decreasing trend in biomass in the presence of AMF than the NAMF treatments (Van Der Heijden et al., 2006). In the absence of competition or companionship, AMF generally reduces the growth performance of non-host plants (Muthukumar et al., 1997; Daisog et al., 2011), while it enhances the growth rate of host plant species (Ayres et al., 2006); however, exceptions are always there. For instance, previous findings suggested that both N-addition and inoculum from non-mycorrhizal plants reduced the AMF functions and growth of the mycorrhizal plant (De Souza and Santos, 2018). Mycorrhizal inoculation of non-host plant roots might develop into parasitic associations by altering plant defense mechanisms (Allen et al., 1989; Giovannetti and Lioi, 1990). Therefore, the AMF colonization was presumed in non-host plants, as detected in numerous species of Brassicaceae (Hirrel et al., 1978; Veiga et al., 2013), should ideally be categorized based on AMF to value the importance of these plant-AMF interactions. However, the underlying mechanism of such a phenomenon is still unknown.

In our study, in both harvests, maximum AMF colonization was reported in V. faba in the presence of high concentration of N. The potential competitive association of host plants is not just directly impacted by AMF colonization (Daisog et al., 2011; Lin et al., 2015) but also indirectly by altering the AMF community structure linked with the roots (Zhang et al., 2014; Qiao et al., 2015). Besides the impact of AMF on competitive association of host plant, the addition of N would improve the ability of plant photosynthesis, increasing the amount of C contribution into the fungal network, therefore enhancing colonization, and boosting C-P trade advantages (Evans, 1989; Unger et al., 2016). Consistent with these findings, other studies reported a significant competitive shift toward improved grass performance with increasing N levels (Hacker et al., 2015; Grygierzec et al., 2020). Contrary to these findings, the infectivity ability of AMF has been extensively decreased in different natural ecosystems by high levels of nitrogen (Smith and Read, 2008; Diepen et al., 2010; Hodge and Storer, 2014; Chen et al., 2017). The reduction of AMF colonization in monocultures under N-addition supporting the Johnson (2010) report that stated the C consumptions by mycorrhizal fungus. Apart from this, in the current study, N-addition unexpectedly had no effect on biomass of either plant species, signifying that N-addition does not offer any growth advantages.

Apart from above findings, B. napus showed negative responsiveness to AMF in mixed-culture, while stronger MGD was estimated for V. faba in monoculture at 2^nd harvest. In support of this, numerous prior works reported that AMF preferred legumes when competing against grass species (Klabi et al., 2014; Van Der Heijden et al., 2016; Bahadur et al., 2019b). AMF increase the availability of N and P to plants and improve the photosynthesis, ultimately leading to increased biomass (Yang et al., 2018; Begum et al., 2019; Vasan et al., 2021). Plant species with greater more mycorrhizal responsiveness generally have better nutrient uptake ability than less responsive species, which usually shows slow growth in the competition (Zabinski et al., 2002; Bahadur et al., 2019b). Medicago sativa showed high biomass production and broad canopy, which may deplete the biomass of nearby species, the below-ground drivers might be further exacerbated by aboveground competition (Klabi et al., 2014; Zhou et al., 2022). The involvement of AMF in plant nutrients uptake has been debated in different harvest periods with different N concentration, and the effect was varied from positive to negative (Schroeder-Moreno et al., 2012; Jiang et al., 2018). At high level of N concentration, the plant tissue N concentration was significantly enhanced by AMF inoculation, whereas it remained unaffected by a low level of N concentration in the greenhouse (Jackson et al., 2002; Yang et al., 2018). Here our study, AMF inoculation likely induced the flexible plant growth response, extending from beneficial to detrimental interaction. The MGDs ranged from highly positive to neutral, showing different contributions of AMF inoculation to plant growth. Our results recommended that the low P uptake by B. napus in N-addition might facilitate V. faba. Supporting our findings, previous studies showed that the growth of the host plant depends on the competitive pattern, AMF species, and nutrient availability (Hodge, 2003; Daisog et al., 2011). Therefore, the AMF community significantly contributes to plant growth performance and tissue N and P contents (both host and non-host plant) compared to a single AMF species.

Unlike previous studies (Höpfner et al., 2015; Bahadur et al., 2019b; Zhou et al., 2022), in this study the effect of competition on the AMF and N-addition were weak. In the current study, the inoculum contained the community of AMF, not the single-species of AMF. However, earlier research has confirmed that the mixed AMF inoculum treatment is more beneficial to the host than a single-species inoculum (Boyer et al., 2015; Armada et al., 2016). Due to the differences in growth strategies and nutrient acquisition by the competing plant species, it is hard to unravel the mechanisms by mycorrhizal fungi, which might alter plant performance in interspecific competition (Smith and Read, 2008; Unger et al., 2021). Recently, the specific mechanisms related to AMF colonization and its effects on the host and non-host plants to compete with N-addition have not been well approached. Explanations from this study point out that N-addition might alter the mycorrhizal colonization of the companion and host plants. Numerous studies have stated that the suppression of AMF colonization might impact the growth of host plants, leading to alteration in plant interactions (Qiao et al., 2016). In line with these findings, many studies have showed that higher competitive ability is associated with more excellent mycorrhizal growth responses of interspecific competitive interaction (Jansa et al., 2008; Johnson, 2010; Unger et al., 2021), consequently the simultaneous responses by N-addition and AMF and their interactions can affect plant nutrient uptake and acquisition.

This study reports the AMF community inoculation and N-addition patterns in quantified the competitive interactions in V. faba and B. napus. It was observed that AMF inoculation positively influenced the interspecific competitive ability of its host plant V. faba. Also, from the results it is evident that AMF might substantially impact the competitive interactions, growth performance, and tissue nutrient uptake between the host and non-host plant species. Furthermore, competitive interaction status depends on the presence of N-addition levels. However, V. faba increased shoot and root biomass when in mixed-culture, whereas AMF inoculation increased the N:P ratio in B. napus in mixed-culture under both N-addition treatments than NAMF plants at the 1^st harvest. Notably, little effects of N-addition on colonization growth were detected regardless of both harvests. In general, our results showed that N-addition reduced mycorrhizal growth dependency of B. napus, potentially weakening non-host competitiveness in N-addition treatments. Collectively, these findings provide information on how AMF functions in the competitive interaction in the absence of mycotrophic host plants with N-enrichments. To further increase understanding of plant competitive interactions under environmental change conditions, more research is required to determine how N deposition affects the function of AMF.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AB, SJ, and JP conceived and designed the research. HF, YL, and QZ supervised the study. AB, SJ, MU, and JP performed greenhouse experiments. AB performed the microscopic study. AB, WS, and FN analyzed the data. WZ and MZ assisted with data valuation. AB, MU, and FN wrote the manuscript. TC and HF edited the manuscript writing. All authors contributed to the article and approved the submitted version.