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Article

Lotus tenuis in Association with Arbuscular Mycorrhizal Fungi Is More Tolerant to Partial Submergence than to High-Intensity Defoliation

by
Ileana García
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Consejo Nacional de Investigaciones Científicas y Técnicas, Av. Ángel Gallardo 470, Buenos Aires C1405DJR, Argentina
Int. J. Plant Biol. 2025, 16(2), 47; https://doi.org/10.3390/ijpb16020047
Submission received: 18 February 2025 / Revised: 17 March 2025 / Accepted: 25 April 2025 / Published: 29 April 2025
(This article belongs to the Section Plant Response to Stresses)
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Abstract

:
This study aimed to investigate the effect of the association of Lotus tenuis with arbuscular mycorrhizal fungi (AMF) on its development under high defoliation intensity or partial submergence in a P-deficient soil of the Salado River Basin in a pot experiment. L. tenuis mycorrhizal plants showed higher tolerance to partial submergence (91%) than to high defoliation intensity (57%). Shoot biomass was the highest in mycorrhizal non-stressed and submerged plants (11.71 g and 12.06 g, respectively), and decreased by 38% in defoliated plants. Both stress conditions caused a negative effect on root growth of plants with or without AMF. High-intensity defoliation can be considered the most stressful scenario for mycorrhizal L. tenuis plants and AMF play a more marked role in P nutrition. Under submergence, AMF caused a net effect on L. tenuis growth, improving carbon and P resource distribution to sustain shoot growth and elongation. Root AMF colonization and nodulation decreased under submergence. High arbuscular colonization percentages were reached under both stress conditions, indicating that the symbiosis may be functional. L. tenuis roots can act as a reservoir of the fungal community under severe stress conditions, allowing the preservation of the AMF inoculum.

1. Introduction

Overgrazing and environmental conditions are leading to a decline in legume frequency in temperate grasslands around the world [1], affecting forage production. These stressful situations are also common for grasslands of the Salado River Basin (Argentina), which is a very productive area for beef cattle. The forage production of these grasslands is also affected by low phosphorus (P) availability [2,3] and fluctuations in soil water content. In this area, flooding is a common phenomenon during the rainy season (autumn, winter and early spring). In particular, partial submergence, defined as flooding of the entire root system and part of the aboveground organs, is the most common water excess condition occurring in the Salado River Basin [4].
One of the plant strategies to grow under stress conditions is the association with arbuscular mycorrhizal fungi (AMF) [5]. These soil fungi form a symbiotic relationship with 85% of vascular plant species [6]. Several studies have reported that AMF play a crucial role in plant nutrition and growth, mainly in stress conditions, and contribute to improving plant tolerance [7,8]. In this sense, AMF provide the plant with water, soil mineral nutrients (mainly P and nitrogen) and pathogen protection. In exchange, the plant transfers photosynthetic compounds to the fungus [6,7]. Within the soil, the mycelial networks of AMF improve soil aggregation and in turn the porosity and erodibility and facilitate the cycling of nutrients such as nitrogen, carbon, and P [5,6,8]. Field studies have shown that grassland plants can be colonized by AMF under a wide range of soil conditions and types of grassland management [9,10]. Grasslands are ecosystems seriously threatened by agriculture and livestock, and have been among the most degraded ecosystems worldwide over the last 50 years [11,12]. Furthermore, due to global climate change, the frequency and intensity of flooding are expected to increase in the next few decades around the world, also affecting forage production of grasslands [13]. In this sense, AMF are of high value for the functioning and sustainability of grasslands [14,15].
Scenarios such as high intensity of shoot biomass removed by grazing or defoliation and partial submergence are severe stress conditions that affect mainly root growth, in the first case by the excessive loss of photosynthetic tissue and the plant carbon imbalance, and in the second case by the anoxic condition of the soil environment [16,17,18]. Therefore, these stress conditions may also affect the AMF benefits to host plants, altering the plant’s competitive ability to improve plant nutrition and plant growth as a whole [17]. In this sense, AMF may improve the grazing/defoliation tolerance of their host plants depending on the intensity and frequency of grazing or defoliation events and the level of nutrients in the soil [19]. Regarding AMF root colonization, previous studies have shown a wide range of results, from neutral to positive or negative impact of grazing/defoliation on root colonization [19,20,21]. On the other hand, plant tolerance responses to flooding stress depend on the duration and magnitude of the stress period and involve anatomical and morphological changes, such as formation of aerenchyma and development of adventitious roots, which allow acclimation to this stress condition [16,18,22]. With respect to the AMF benefits to host plants under flooding, symbiosis may have an ameliorating effect on plant performance through an improvement of the plant nutrient status by increasing nitrogen and P acquisition in flooded plants [8,23,24]. A recent study has also shown that, in rice, AMF may facilitate the acclimation of plants to different intensities of submergence by mediating gene expression for shoot elongation and sugar concentration in roots [25]. However, flooding has been shown to either decrease or partly inhibit AMF root colonization depending on the extent of the association when flooding occurs [23,26,27].
Legumes are a very important component of temperate grasslands because they increase the forage quality and preserve soil conditions [28,29]. In addition, these species establish symbiotic relationships with AMF and are able to fix atmospheric nitrogen through rhizobia symbiosis [29,30]. In the Salado River Basin, one of the most important naturalized legumes is Lotus tenuis Waldst & Kit. This legume has high plasticity and high-quality forage production [31]. Previous studies performed under controlled conditions have shown that L. tenuis tolerates moderate defoliation intensity (75% of shoot biomass removed) and water excess (waterlogging and partial submergence) and described the morphological and nutritional traits associated [4,32,33,34,35,36]. According to Mendoza et al. [37], L. tenuis plants can be under flooding up to six months. Lotus tenuis is highly dependent on AMF to grow under P-deficient soils and shows a high percentage of AMF root colonization in a wide range of soil conditions and types of environment management [10,38]. Severe stress conditions such as high defoliation intensity (which simulates the loss of shoot biomass removed by overgrazing) and partial submergence represent common stress situations in the Salado River Basin. However, the role of the native AMF community in the tolerance of L. tenuis (especially regarding growth and P nutrition) to stressful conditions is unknown. In these environmental situations, AMF root colonization can result in a factor that acts to the detriment of plant development caused by the extra consumption of carbon compounds [39].
Based on all the above, the aim of this study was to investigate the effect of the association of L. tenuis with native AMF on its development under two stress situations, high defoliation intensity and partial submergence, in a P-deficient soil of the Salado River Basin. Specifically, this study proposed that plant growth and P uptake increase in mycorrhizal and stressed plants compared to non-mycorrhizal plants. This study predicted that the tolerance of mycorrhizal plants to high-intensity defoliation will be lower than that to partial submergence because the AMF community can act as an additional nutritional budget.

2. Materials and Methods

2.1. Experimental Set Up

The experiment was conducted using a factorial arrangement with a completely randomized design. The main factors were native AMF (two levels: with and without AMF) and stress condition (three levels: non-stress, high intensity defoliation and partial submergence). Four replicates were assigned to each combination of AMF treatment and stress condition. Soil was collected from the top 15 cm layer of a grassland of the Salado River Basin (35°37′ S, 58°50′ W, Province of Buenos Aires, Argentina). The soil was classified as Typic Natraquoll and its chemical characteristics were pH 6.11 (1:2.5 water), electrical conductivity 0.71 dS·m−1, available P 3.96 mg·kg−1 (Bray I), exchangeable K+ 1.26 meq·100 g−1, exchangeable sodium percentage 4.51%, and field water capacity 40% (w/w) [10]. The soil sample was sieved through a 2 mm mesh screen and sterilized by solarization [40]. After sterilization, the available P (Bray I) was 11.65 mg·kg−1.
Closed-bottom pots (1.6 L) were filled with 1150 g of air-dried soil. The pots were divided into two groups, with and without AMF source. The soil sampled was used as the source of native AMF. The AMF source contained 97 spores per 100 g of dry soil. The taxonomic identification of AMF spores has been previously described in García et al. [10] (IN1 site). The pots were assembled following the method provided by Chippano et al. [38]. The AMF source was added as a thin layer of 50 g, 4 cm below the soil surface. Non-mycorrhizal pots received the AMF source previously sterilized by microwave for 10 min at 900 W and 25 mL of AMF filtrate to equalize the microbial community. The AMF filtrate was prepared by filtering a dilution (1:3) of the soil used as AMF source through a 35 μm sieve to remove AMF spores and root fragments but allowing other soil microorganisms, including soil pathogens. Mycorrhizal pots received the AMF source and 25 mL of the AMF filtrate previously sterilized in a boiling water bath for 15 min to equilibrate the nutrients added to all pots.
Seeds of L. tenuis cv. Esmeralda were superficially sterilized and pre-germinated in sterile conditions. Five seedlings per pot were planted and the soil surface was covered with 1 cm of sterilized sand to minimize water evaporation.

2.2. Growing Conditions

Mycorrhizal and non-mycorrhizal plants were grown in a greenhouse and maintained near field capacity for 55 days (initial time). After that initial period, four pots of mycorrhizal and non-mycorrhizal plants were harvested and the remaining pots were subjected to high defoliation intensity, partial submergence or near field capacity (non-stress, control condition), replicated four times. The high defoliation intensity treatment consisted of removing 78% of the above-ground biomass with respect to the pots harvested at the initial time. The partial submergence consisted of maintaining a water level of 5 cm above the soil surface. The non-stress condition consisted of non-defoliated plants growing at near field capacity. The duration and levels of stress imposed were based on previous studies [32,33]. The water status of the pots was examined daily. Mycorrhizal and non-mycorrhizal plants of the three treatments (non-stressed, high intensity defoliated and partial submerged plants) were grown for an additional period of 25 days and then harvested (final time). The experimental pots were placed in a greenhouse with a mean relative humidity of 65 ± 9%, day mean temperature of 30 ± 3 °C, night mean temperature of 20 ± 3 °C, a photoperiod of 10–12 h and a photon flux density of 900–1300 μmol.m−2s−1. Pots were randomized and daily rotated to minimize potential gradient effects.

2.3. Plant Yield and Analytical Determinations in Tissue

After each harvest (initial and final time), the plant biomass of each treatment was separated into shoots and roots, oven-dried at 70 °C for 48 h and weighed for subsequent determination of shoot and root dry weight (DW) and tissue P concentration. The biomass of the clipped shoots was weighed, and later included in the shoot fraction of the corresponding plants at the end of the additional period of 25 days to determine shoot yield. A portion of fresh shoot was used to measure the concentration of total chlorophyll [41]. A portion of fresh root was used to measure total root length and AM root colonization. Root length was determined by the line intercept method [42], specific root length (SRL) was calculated as the root length per unit of root DW, and the root mass fraction (RMF) was calculated as the root DW divided by the total plant DW.
For both mycorrhizal and non-mycorrhizal plants, stress-tolerance efficiency (STE) in each stress condition was calculated using the following equation [34]:
STE (%) = (dry biomass of stressed plant/dry biomass of non-stressed plant) × 100
For shoot and root tissue of mycorrhizal and non-mycorrhizal plants, a susceptibility index was computed using the following equation [32]:
SI = 1 − (stressed plant/non-stressed plant)
SI is positive when a stress treatment decreases the value of shoot or root DW with regard to its non-stressed value, and is negative when the opposite holds true.
Dry shoots and roots were digested separately in a nitric–perchloric acid mixture (3:2) to determine P using the molybdovanadophosphoric acid method [43]. The P allocation to roots was expressed as the percentage of quotient between root P content (mg) and plant P content (mg). The specific P uptake (SPU) was calculated as the plant P content (mg) per unit of root length (m) for mycorrhizal and non-mycorrhizal plants grown under each stress conditions [38].

2.4. Arbuscular Mycorrhizal Fungi Colonization and Root Nodulation

AMF root colonization was measured in fresh roots cleared in 10% KOH for 12 min at 90 °C and stained in 0.05% lactic–glycerol–Trypan Blue [44]. Thirty-five root segments per plant sample were examined under a microscope at ×200 magnification. The percentage of total root length colonized by AMF, the fraction of colonized roots containing arbuscules and vesicles were determined by the method of McGonigle et al. [45]. Since arbuscules are structures where nutrient exchange occurs, this estimate represents the functionality of the mycorrhizal symbiosis [6].
The total DW and total P content of mycorrhizal and non-mycorrhizal plants from each stress treatment were used to estimate mycorrhizal growth response (MGR) and mycorrhizal P response (MPR), respectively, according to the equations presented by Cavagnaro et al. [46]
MGR = 100 × ((total DW M − mean total DW NM)/mean total DW NM)
MPR = 100 × ((P content M − mean P content NM)/mean P content NM)
where M is the total DW (Equation (3)) or total P content (Equation (4)) per pot of mycorrhizal plants and NM is the mean of the total DW (Equation (3)) or total P content (Equation (4)) per pot of the corresponding non-mycorrhizal plants.
Rhizobia nodules were counted in whole fresh root systems under a binocular stereomicroscope (×7.5).

2.5. Statistical Analyses

Plant variables were analyzed through a two-way ANOVA with AMF treatment and stress condition as the first and second factors, respectively. AMF root colonization, MGR and MPR were analyzed through a one-way ANOVA. Means were separated by the Tukey test. The normality and homogeneity of variances were previously verified. Root nodulation data were transformed to logarithms for comparison among treatments. Relationships among shoot and root DW, RMF, SRL, P in shoot and root tissue and SPU of mycorrhizal and non-mycorrhizal plants under the two stress conditions were evaluated by principal component analysis (PCA). Statistical analyses were performed with the INFOSTAT version 2019 [47].

3. Results

3.1. Plant Yield

The shoot and root DW of L. tenuis was affected by the AMF treatment, stress condition and interaction of both factors (Figure 1a,b; Table S1). Lotus tenuis showed higher tolerance to partial submergence than to high defoliation intensity when plants were mycorrhizal (Figure 2a). The stress tolerance efficiency of mycorrhizal plants decreased by 10% compared to non-mycorrhizal plants under high defoliation intensity (Figure 2a). The stress tolerance efficiency of mycorrhizal plants and non-mycorrhizal plants under partial submergence was 91% and 80%, respectively. Shoot DW was higher in mycorrhizal plants regardless of the growing condition (Figure 1a). Biomass was the highest in non-stressed and submerged mycorrhizal plants (11.71 g and 12.06 g, respectively), and decreased by 38% in mycorrhizal defoliated plants. Under the high defoliation intensity treatment, the susceptibility index showed a negative effect on shoot growth of 29% and 38% in non-mycorrhizal and mycorrhizal plants, respectively, whereas in the partial submergence treatment, this index showed a positive effect on shoot growth in mycorrhizal plants (3%) and a negative effect in non-mycorrhizal plants (10%) (Figure 2b). Root DW was higher in non-stressed mycorrhizal plants than in the two stress conditions (Figure 1b; Table S1). The susceptibility index showed a negative effect of both stress conditions on the root growth of mycorrhizal and non-mycorrhizal plants (Figure 2c).
The root mass fraction was affected by the AMF inoculation, stress treatment and combination of both factors (Figure 1c; Table S1). The root mass fraction decreased in non-mycorrhizal and mycorrhizal plants under both stress conditions compared to non-stressed plants. The mycorrhizal plants reached the minimum value of specific root length independent of the stress condition, and this value was the maximum for non-mycorrhizal plants under non-stress or high defoliation intensity conditions (Figure 1d; Table S1).

3.2. P in Tissue

Shoot and root P concentration was affected by the AMF treatment, stress condition and interaction of both factors (Figure 3a,b; Table S1). The highest P concentration was reached by mycorrhizal plants under high-intensity defoliation, whereas this value decreased in mycorrhizal plants under partial submergence or non-stress condition. The root P concentration was always higher in mycorrhizal plants regardless of the stress conditions (Figure 3b). The allocation of P to roots was reported as the minimum value in mycorrhizal and non-mycorrhizal defoliated plants and non-mycorrhizal submerged plants (Figure 3c; Table S1). The specific P uptake was affected by the AMF treatment, stress condition and interaction of both factors (Figure 3d; Table S1). The specific P uptake in mycorrhizal plants was higher than that in non-mycorrhizal plants under non-stress conditions and high defoliation intensity and showed no differences between mycorrhizal and non-mycorrhizal plants under partial submergence.

3.3. Photosynthetic Pigments

Chlorophyll concentration was affected by the AMF treatment, stress condition and interaction of both factors (Figure 4; Table S1). This parameter reached the maximum in mycorrhizal defoliated plants.

3.4. Mycorrhizal Growth and P Response

The mycorrhizal growth response was highest under partial submergence (63.01%) and decreased to 43.05% and 32.55% under non-stress and defoliation conditions, respectively (Figure 5a). The mycorrhizal P response was highest under defoliation (112.9%) and decreased to 53.52% and 36.94% under non-stress and submergence conditions, respectively (Figure 5b).

3.5. Mycorrhizal Root Colonization and Nodulation

No AMF colonization was observed in non-mycorrhizal plants of all stress conditions. AMF colonization was maximum in non-stressed and defoliated plants (68.66% and 73.31%, respectively) and decreased to 43.62% in submerged plants (Figure S1a). Arbuscular colonization was maximum in defoliated plants and minimum in submerged plants (Figure S1b). Vesicle colonization reached 34.91% in non-stressed plants and showed similar values in stressed plants (14.18% and 9.86% in defoliated and submerged plants, respectively) (Figure S1b). Both mycorrhizal and non-mycorrhizal plants were nodulated and the number of nodules was reported as the maximum in mycorrhizal defoliated plants (Figure S1c).

3.6. Relationships Between Mycorrhizal and Non-Mycorrhizal Plants Under Severe Stress Conditions

The PCA explained 84.4% of the accumulated variance (Figure 6). The first PCA component (PC1) explained 51.9% of the variance, whereas the second PCA component (PC2) explained 32.5%. The analysis showed that mycorrhizal non-stressed plants were associated with an increase in the shoot and root biomass production arrows. Non-mycorrhizal non-stressed and defoliated plants were associated with the RMF and SRL arrows. On the other hand, mycorrhizal submerged plants were associated with an increase in P in root tissue and the SPU arrows. Non-mycorrhizal plants were associated with an increase in the shoot P arrow. Mycorrhizal defoliated plants were related to P in the shoot and root tissue and SPU arrows (Figure 6). The biplot shows mycorrhizal and non-mycorrhizal treatments separately.

4. Discussion

The results of this study show that the association of L. tenuis with native AMF improved its tolerance efficiency to partial submergence compared to its non-mycorrhizal counterpart in a P-deficient soil. However, the tolerance efficiency to high defoliation intensity was lower in mycorrhizal plants than in non-mycorrhizal plants. In this stress situation, the AMF community can act as an additional nutritional budget, showing a strong negative effect on root growth of mycorrhizal plants.
Despite the discrepancy in the stress tolerance efficiency of L. tenuis plants, both severe stress conditions have in common that they most affected the root system. This decrease in root biomass is coincident with previous studies for both stress situations [4,33,35].

4.1. Mycorrhizal Growth and P Response

High-intensity defoliation caused a similar negative effect on the shoot growth of mycorrhizal and non-mycorrhizal plants; however, this stress condition had a substantial negative impact on the root system of mycorrhizal plants compared to that on non-mycorrhizal plants. Mycorrhizal and non-mycorrhizal plants were not able to recover the clipped biomass compared to their non-stressed counterparts, as was expected according to a previous study, in which the aerial biomass of L. tenuis was clipped 0.5 cm above the soil surface, removing 96% of shoot biomass [33]. It is important to note that the regrowth of L. tenuis is mainly due to the sprouting of basal auxiliary buds of the old stems [48]. In the present study, the high proportion of removed shoot biomass led to a carbon and nutritional imbalance. Therefore, shoot regrowth would be dependent on the use of the remaining nutrient reserves and the ability of the plant to absorb nutrients efficiently from the soil, rather than on bud sprouting. Carbon resources of mycorrhizal and defoliated L. tenuis plants are invested to support not only shoot regrowth but also to maintain AMF symbiosis, showing a net decrease in root tissue and root mass fraction compared to non-mycorrhizal and non-stressed plants. Then, carbon and P in mycorrhizal plants are preferentially transferred to shoots (RMF, 15%; P allocation to roots, 17%) as a physiological adjustment to support shoot regrowth and recover the leaf area lost. According to the PCA diagram, the highest specific P uptake of mycorrhizal defoliated plants was associated with an increase in P in tissue, which was also associated with the highest mycorrhizal P response (112.9%). The ability of AMF to explore a greater volume of soil than the root system and absorb P beyond the limits of the P depletion zone [6] allowed for a reduction in the impact of soil P deficiency and improvement in plant P nutrition compared to non-mycorrhizal defoliated plants.
Regarding partial submergence, mycorrhizal L. tenuis plants showed better performance than non-mycorrhizal plants. In particular, mycorrhizal submerged plants reached an amount of shoot biomass similar to that of mycorrhizal non-stressed plants. However, this stress situation decreased root biomass compared to non-stressed plants, but this decrease was more marked in non-mycorrhizal plants, contrary to the case of defoliated plants. Both stress situations can trigger the consumption of reserves and this consumption is related to the severity of the stress suffered by plants [49]. Previous studies have shown that L. tenuis escapes from submergence using root carbon reserves (in particular from the crown) and mobilizes these reserves to shoot elongation [50]. In the present study, partial submerged plants (mycorrhizal and non-mycorrhizal) showed the greatest mobilization of carbon reserves towards the shoot tissue (RMF, 13%) in all stress conditions, and in shoots, more allocation to stems than to leaves. The latter resulted in increased internode length and root crown diameter as a strategy to escape from submergence. These results are consistent with previous observations in L. tenuis plants growing in different media [4,35]. It is important to note that, in the case of mycorrhizal submerged plants, the carbon resources are allocated not only to shoot growth and elongation, but also to the maintenance of the AMF symbiosis. In this sense, mycorrhizal and non-mycorrhizal plants transfer the same percentage of carbon compounds to shoots, and then mycorrhizal plants may limit the carbon flux to AMF root colonization in favor of sustaining shoot growth and elongation. It is also important to note that, in a previous study, the shoot growth of L. tenuis submerged plants was lower than that of non-stressed plants but the plants were not inoculated with AMF [4]. In this sense, these previous results are coincident with the results of non-mycorrhizal plants of the present study and highlight the importance of studying the tolerance of a species in conjunction with symbiotic microorganisms, mainly those related to nutrition and stress tolerance, to obtain holistic results that could be applied in future field assays.
On the other hand, non-mycorrhizal submerged plants showed a higher specific root length than mycorrhizal submerged plants, as a strategy to increase P uptake (which was coincident with non-mycorrhizal plants under non-stress and defoliation situations). In this sense, the shoot P concentration decreased and root P concentration increased in mycorrhizal plants compared to non-mycorrhizal plants under partial submergence. Mycorrhizal plants allocated 31% of P to roots, while non-mycorrhizal plants allocated 20% of P with similar values of specific P uptake. According to the PCA diagram, the increase in P in root tissue was mainly related to an increase in specific P uptake for mycorrhizal submerged plants. It is important to note that flooding may increase the soil P availability [32,34,37,51]. This great P availability in the soil may justify the increase in P uptake by submerged plants compared to non-stressed plants, and together with a differential distribution of P to shoot tissue to sustain shoot growth. In addition, in submerged plants, the mycorrhizal growth response was the highest of all stress conditions, and the mycorrhizal P response was similar to that of non-stressed plants. In the partial submergence situation, the AMF community caused a net effect on L. tenuis growth, improving carbon and P resource distribution to sustain shoot growth and elongation. In this regard, Xu et al. [25] reported that AMF facilitate the acclimation of rice to submergence, mediating the genetic expression to enhance rice shoot elongation and the sugar concentration in roots as a result of reduced competition for carbon between rice and AMF. In the case of the legume tree Pterocarpus officinalis, AMF colonization contributes to flooding tolerance by improving plant growth and P acquisition in leaves [24]. Previous studies have also shown that L. tenuis tolerance to partial submergence is also associated with morphological traits such as the formation of aerenchyma in shoot and root tissue to maintain the oxygen transport from shoot to roots and production of adventitious roots [4,35]. These previous reports are in line with the present observation of aerenchymatic tissue in mycorrhizal and non-mycorrhizal submerged plants.

4.2. Photosynthetic Pigments

In general terms, grazing or defoliation and submergence decrease the photosynthetic activity caused by the loss of green biomass and by a limited availability of oxygen, respectively [17,25]. However, the association with AMF stimulates the synthesis of photosynthetic pigments [52,53]. In the present study, defoliated mycorrhizal plants showed an increase in chlorophyll concentration with respect to non-mycorrhizal plants. The increase in chlorophyll content in plants associated with AMF could be related to a greater uptake of nutrients essential for chlorophyll biosynthesis [54,55]. On the other hand, chlorophyll concentration in submerged plants showed no differences compared with their non-mycorrhizal counterparts, but these values were lower than those in non-stressed plants. In this sense, Mishra et al. [56] found that the decrease in chlorophyll content in flooded rice influenced the photochemical efficiency of photosystem II. The flooding stress contributed to a higher degree of degradation in the reaction centers of the photosystems [57], decreasing the photosynthetic activity. However, in the present study, a non-significant trend of an increase in chlorophyll concentration was observed in mycorrhizal non-stressed plants.

4.3. Mycorrhizal Root Colonization and Nodulation

In general, the amount of carbon and nutrients that move from plants to fungi can be measured, and varies with the environmental context [8], plant and fungal species involved [58]. AMF are estimated to receive c. 13% of the carbon assimilated by the plant [59]. Similarly, AMF can deliver almost all the P and c. 20% of the nitrogen needed by plants [60,61]. Previous studies have reported that severe defoliation causes a decrease in AMF root colonization and AMF benefits in grassland species [19,62,63]. This negative effect of defoliation on AMF symbiosis has been ascribed to a reduced photosynthetic capacity of plants, which, in turn, limits the carbon supply to roots and AMF, reducing fungal colonization and mycorrhizal benefits [19,20,21]. Contrary to the predicted in the present study, the AMF colonization did not decrease with respect to non-stressed plants, and this colonization was mainly given by arbuscules and the mycorrhizal P response was the highest. The high proportion of roots colonized by arbuscules (64%) suggests that L. tenuis and AMF may establish a functional symbiosis even when the plants have suffered an intensive loss of shoot biomass. In addition, the vesicular colonization significantly decreased from 34.91% in non-stressed plants to 14.18% in defoliated plants. Vesicle formation is associated with storage structures; it has been reported that the mobilized carbohydrates and lipids are stored in vesicles to form structures like spores, which consume a considerable amount of carbon compounds [64]. In the present study, the high proportion of roots colonized by arbuscules, the decrease in vesicle formation, the increase in chlorophyll concentration, the highest mycorrhizal P response and the lack of differences in root growth of defoliated mycorrhizal and non-mycorrhizal plants would indicate that the strategy of L. tenuis and AMF consists of investing carbon compounds to the growing shoot and the fungal symbiont to preferentially retain arbuscular colonization and guarantee P nutrition to the detriment of root growth.
On the other hand, previous studies have shown a general trend towards a reduction in AMF colonization with increasing water availability due to the development of anaerobic conditions [37,65,66]. Furthermore, a previous study has proposed that the decrease in AMF colonization in L. tenuis roots under waterlogging may be associated with the increase in soil P availability or P in tissue [32]. In line with previous studies, in the present study, the AMF colonization in mycorrhizal submerged L. tenuis plants decreased. According to the PCA diagram, submerged plants were related to an increase in P in roots and specific P uptake. In addition, L. tenuis root colonization was mainly formed by arbuscules (30.92%) and lower vesicular colonization (9.86%), while the root P concentration increased markedly. An increase in root P concentration may negatively affect the AMF root colonization level in L. tenuis, probably due to a reduction in the permeability of cell membranes leading to a decreased availability of carbon to the fungi [67]. In the present study, the high proportion of roots colonized by arbuscules suggests that L. tenuis and AMF may establish a functional symbiosis independently of the level of P in tissue or in the soil and the anoxic condition. The regulation of root colonization may be based on the plant carbon reserve control to avoid the delivery of carbon to AMF and then improve plant tolerance [39]. The strategy of L. tenuis and AMF would consist of investing carbon and P to sustain shoot growth and elongation, limit root colonization and preferentially retain arbuscular colonization to guarantee mycorrhizal benefits.
Finally, previous studies have shown that nodulation in L. tenuis roots is not affected by defoliation intensity with an opposite effect under water excess [32,33,37]. In the present study, mycorrhizal defoliated plants reached the highest number of nodules, but this value decreased in mycorrhizal submerged plants. Although the number of nodules is not directly associated with their functionality, according to previous reports, in the case of defoliated plants, a high nodulation could contribute to nitrogen metabolism and then plant growth [33,34]. Previous studies have also reported that most of the nodules in L. tenuis roots under water excess were inefficient to fix atmospheric nitrogen [32,37]. In general terms, nitrogen fixation in legume roots is usually adversely affected by flooding depending on the intensity and duration of the stress condition [23,68]. This may be attributed to the lower energy status of flooded roots and the energy costs of nitrogen uptake and assimilation. In the case of L. tenuis, more information is needed to elucidate the effect of submergence on root nodulation of mycorrhizal plants and the ability to fix nitrogen.

5. Conclusions

High-intensity defoliation can be considered the most stressful scenario for mycorrhizal L. tenuis plants. In this study, high defoliation intensity had a positive effect on L. tenuis mycorrhizal growth and P response and a strong negative effect on root growth. In this sense, the AMF community plays a more marked role in P nutrition. On the other hand, AMF improved mainly shoot growth and elongation and supported root growth under partial submergence. Based on the results, the hypothesis proposed was accepted. It is important to note the capacity of L. tenuis roots to maintain the AMF inoculum through a high percentage of root colonization mainly formed by arbuscules, independently of the severity of the stress condition. The results of the present study indicate that, even under intense carbon stress caused by high defoliation intensity or submergence conditions, the benefits of AMF association exceeded the costs in a P-deficient soil and improved L. tenuis growth and P nutrition.
In order to maintain the ability of L. tenuis to conserve the AMF inoculum in its roots over time and reduce the negative effect on the root system, it is proposed to avoid agronomic practices such as excessive removal of shoot biomass due to poorly controlled grazing (overgrazing). Finally, in the face of increasingly frequent flooding and submergence scenarios caused by global climatic change around the world, L. tenuis is an excellent option to maintain the benefits of the native AMF community of grassland soils.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijpb16020047/s1: Figure S1: Mycorrhizal colonization (a), arbuscular colonization (b) and number of nodules (c) in Lotus tenuis grown under non-stress, high-intensity defoliation and partial submergence. Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in (c) graph, * p < 0.05, ** p < 0.01, ns p > 0.05.; Table S1: Results of two-way ANOVA (F and p values) for AMF inoculation (AMF), stress treatment (ST) and the interaction between AMF and ST on different variables.

Funding

This research was funded by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina), through PIP 0670, and Agencia Nacional de Promoción Científica y Tecnológica (Argentina), through PICT 2020-01901.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The author declares no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AMFArbuscular mycorrhizal fungi
MWith arbuscular mycorrhizal fungi
NMWithout arbuscular mycorrhizal fungi
STStress treatment
DWDry weight
RMFRoot mass fraction
SRLSpecific root length
SPUSpecific P uptake
MGRMycorrhizal growth response
MPRMycorrhizal P response

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Figure 1. Shoot and root dry weight (DW) (a,b), root mass fraction (c) and specific root length (d) of Lotus tenuis plants grown with (M) or without (NM) arbuscular mycorrhizal fungi (AMF) under three stress conditions (non-stress, high-intensity defoliation and partial submergence). Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in each graph, * p < 0.05, *** p < 0.001.
Figure 1. Shoot and root dry weight (DW) (a,b), root mass fraction (c) and specific root length (d) of Lotus tenuis plants grown with (M) or without (NM) arbuscular mycorrhizal fungi (AMF) under three stress conditions (non-stress, high-intensity defoliation and partial submergence). Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in each graph, * p < 0.05, *** p < 0.001.
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Figure 2. Stress tolerance efficiency (a), shoot and root susceptibility index (b,c) of Lotus tenuis plants grown with (M) or without (NM) arbuscular mycorrhizal fungi (AMF) under three stress conditions (non-stress, high-intensity defoliation and partial submergence). Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in each graph, ** p < 0.01, *** p < 0.001, ns p > 0.05.
Figure 2. Stress tolerance efficiency (a), shoot and root susceptibility index (b,c) of Lotus tenuis plants grown with (M) or without (NM) arbuscular mycorrhizal fungi (AMF) under three stress conditions (non-stress, high-intensity defoliation and partial submergence). Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in each graph, ** p < 0.01, *** p < 0.001, ns p > 0.05.
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Figure 3. Shoot and root P concentration (a,b), allocation of P to roots (c) and specific P uptake (d) of Lotus tenuis grown with (M) or without (NM) arbuscular mycorrhizal fungi (AMF) under three stress conditions (non-stress, high-intensity defoliation and partial submergence). Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in each graph, * p < 0.05, *** p < 0.001.
Figure 3. Shoot and root P concentration (a,b), allocation of P to roots (c) and specific P uptake (d) of Lotus tenuis grown with (M) or without (NM) arbuscular mycorrhizal fungi (AMF) under three stress conditions (non-stress, high-intensity defoliation and partial submergence). Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in each graph, * p < 0.05, *** p < 0.001.
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Figure 4. Chlorophyll concentration of Lotus tenuis plants grown with (M) or without (NM) arbuscular mycorrhizal fungi (AMF) under three stress conditions (non-stress, high-intensity defoliation and partial submergence). Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in each graph. *** p < 0.001.
Figure 4. Chlorophyll concentration of Lotus tenuis plants grown with (M) or without (NM) arbuscular mycorrhizal fungi (AMF) under three stress conditions (non-stress, high-intensity defoliation and partial submergence). Values are means ± SE. Different letters indicate significant differences among treatments according to the Tukey test. Statistically significant treatment effects are shown in each graph. *** p < 0.001.
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Figure 5. Mycorrhizal growth response (MGR) (a) and mycorrhizal P response (MPR) (b) of Lotus tenuis plants grown under non-stress, high-intensity defoliation and partial submergence. Values are means ± SE. Different letters indicate significant differences among treatments (p < 0.05) according to the Tukey test.
Figure 5. Mycorrhizal growth response (MGR) (a) and mycorrhizal P response (MPR) (b) of Lotus tenuis plants grown under non-stress, high-intensity defoliation and partial submergence. Values are means ± SE. Different letters indicate significant differences among treatments (p < 0.05) according to the Tukey test.
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Figure 6. Principal component analysis diagram for shoot DW, root DW, root mass fraction (RMF), specific root length (SRL), P in shoot, P in root and specific P uptake (SPU) of Lotus tenuis plants with (M) and without (NM) arbuscular mycorrhizal fungi (blue circles) grown under non-stress, high-intensity defoliation and partial submergence. Variables are represented by red circles and lines.
Figure 6. Principal component analysis diagram for shoot DW, root DW, root mass fraction (RMF), specific root length (SRL), P in shoot, P in root and specific P uptake (SPU) of Lotus tenuis plants with (M) and without (NM) arbuscular mycorrhizal fungi (blue circles) grown under non-stress, high-intensity defoliation and partial submergence. Variables are represented by red circles and lines.
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MDPI and ACS Style

García, I. Lotus tenuis in Association with Arbuscular Mycorrhizal Fungi Is More Tolerant to Partial Submergence than to High-Intensity Defoliation. Int. J. Plant Biol. 2025, 16, 47. https://doi.org/10.3390/ijpb16020047

AMA Style

García I. Lotus tenuis in Association with Arbuscular Mycorrhizal Fungi Is More Tolerant to Partial Submergence than to High-Intensity Defoliation. International Journal of Plant Biology. 2025; 16(2):47. https://doi.org/10.3390/ijpb16020047

Chicago/Turabian Style

García, Ileana. 2025. "Lotus tenuis in Association with Arbuscular Mycorrhizal Fungi Is More Tolerant to Partial Submergence than to High-Intensity Defoliation" International Journal of Plant Biology 16, no. 2: 47. https://doi.org/10.3390/ijpb16020047

APA Style

García, I. (2025). Lotus tenuis in Association with Arbuscular Mycorrhizal Fungi Is More Tolerant to Partial Submergence than to High-Intensity Defoliation. International Journal of Plant Biology, 16(2), 47. https://doi.org/10.3390/ijpb16020047

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