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Article

Assessing the Nutritional Effect of Lupinus montanus on Zea mays HS-2 (Intercropping) and Identification of Nodular Bacteria through the Use of Rhizotrons

by
Juan Espinosa Gonzalez
1,
Vicente Espinosa Hernández
1,*,
Enrique Ojeda Trejo
1,
Julián Delgadillo Martínez
1,
Juan Celestino Molina Moreno
2 and
Francisco Landeros Sánchez
1
1
Departament of Edaphology, Postgraduate College of Agricultural Sciences, Km 36.5 Carretera Mexico-Texcoco, Montecillo C.P. 56230, Edo. De Mexico, Mexico
2
Production of Seeds, Postgraduate College of Agricultural Sciences, Km 36.5 Carretera Mex-Texcoco, Montecillo C.P. 56230, Edo. De Mexico, Mexico
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2023, 14(4), 910-921; https://doi.org/10.3390/ijpb14040067
Submission received: 18 August 2023 / Revised: 29 September 2023 / Accepted: 29 September 2023 / Published: 9 October 2023
(This article belongs to the Section Plant Response to Stresses)
Browse Figures
Versions Notes

Abstract

:
Maize (Zea mays sp.) is one of the most important basic grains in our diets, and it requires high levels of nitrogen and phosphorus for optimum growth. However, phosphorous transitions in forms inaccessible to plants. The Lupinus genus, and more specifically, Lupinus albus, through its root clusters, or proteoid roots, has the ability to solubilize portions of phosphorous when it is found in a limited environment. The objective of the current study was to evaluate the effect of Lupinus montanus under phosphorous stress conditions intercropped with maize, utilizing sandy soils with calcium phosphate bands. Work was conducted in growth chambers using rhizotrons, which allowed the authors to observe the growth and root behavior of both species (Lupinus montanus and maize). The phosphorus analysis in the plant tissue indicated that its concentration in maize was slightly higher in intercropping conditions than in monoculture planting. From this, we concluded that Lupinus montanus is capable of solubilizing portions of phosphorus, making it available for other crops; likewise, we also observed that the proteoid structures did not develop, leaving the study open for other wild species. In conclusion, the use of Lupinus montanus will be as an alternative in favor of more sustainable agricultural methods since it improve soil fertility in phosphorus deficient soils.

1. Introduction

Phosphorus (P), together with nitrogen (N) and potassium (K), form part of the primary plant nutrient group. Phosphorous is a primary component of the systems responsible for enabling, storing and transferring energy and is a basic component of the macromolecular structures of interest, such as nucleic acids and phospholipids, and thus, it can be said that its role is generalized in all physiological processes. In other words, P cannot be substituted for any other nutrient [1]. This means that plants that develop in soil only have access to a small amount of P. In contrast to other elements, the amount of available P in the soil is insufficient for plants, a deficiency that can only be regulated with the application of fertilizers that become less soluble compounds. It is the least mobile element used as a nutrient by plants; therefore, even when in sufficient amounts provided via fertilization, there can occur a P-depleted zone around the root. Also, plants take up P only as orthophosphate ions, and in soil P transitions in inaccessible forms for plants. So, P in particular is reacting with Al and Fe ions that exhibit an increased mobility in acidic pH soils or with Ca in alkaline soils. Since it is likely some plant species are capable of mobilizing P from FePO4, intercropping could be beneficial for the P acquisition of associated non-P-mobilizing plant species. Several studies have already investigated legume/cereal intercropping and suggested that intercropping is advantageous over monocropping due to the high nutrient mobilization capacity of legumes from which the cereals simply benefit [2]. Therefore, P is one of the most limiting macronutrients for plants. On the other hand, among the advantages of introducing intercropping of maize–legumes can improve the yield components [3].
The root provides the plant with physical support, as well as nutrients and water from the soil. Both the development and the growth of the root depend on two factors: environmental stimuli and genetic factors [4]. The roots tend to develop in the direction of water and nutrients. Moreover, there are factors that can affect the growth and the development of the root system (water, temperature, soil compaction, availability of mineral substances, etc.) [5].
The roots principally absorb P through the primary orthophosphate ion in the compound or through the secondary orthophosphate compound [3]. The plants’ response is specific for the deficiency of this nutrient. Some of the characteristics that roots develop for the consumption of P are: (a) a rapid rate of root elongation and a high root biomass [6]; (b) an increase in the ramification of the root and change in their angles, particularly in surface soils and regions rich in nutrients [7]; and (c) root proteoids or root clusters in Proteaceae and in some members of the Fabaceae family such as Lupinus spp. [8,9,10].
The Lupinus species has the ability to improve soil fertility since it has its own source of nitrogen, present in its root clusters or proteoids. It has been shown that Lupinus albus has the ability to mobilize and solubilize P sources that are normally unexploited for other plants [11,12]. The roots also have the ability to absorb metals such as cadmium, lead, zinc, mercury and chrome, thus having phyto-remediating characteristics [13].
The Lupus genus contains more than 600 described species, but only about 300 are recognized [14]: 13 are in the old world—originating in the Mediterranean and Northern Africa—10 are wild, and 3 (Lupinus albus, angustifolius and luteus) are domesticated. Mexican figures indicate that close to 110 species grow as low as sea level (state of Baja California) and as high as 4000 m above sea level (state of Chiapas). Some of the most abundant species are L. montanus, and L. campestris, and L. elegans, and L. hartwegii, and L. mexicanus, and L. polyphyllus, and L. splendens, and L. silvestris and L. stipulatus [15].
It is extremely important to find alternatives to help us reduce the consumption of fertilizers, and improved P uptake is usually achieved with an inoculation/stimulation of mycorrhiza because it is specialized for this, as well as strategies to produce environmentally sustainable foods [16]. The practices of intercropping is an agricultural production strategy that is principally based on the growth of two or more species on a unit of land where a total or partial combination of cycles occurs [17]. In this vein, the maize–leguminous combinations generally exceed the performance of maize monocultures; in other words, maize monocultures require a greater surface area to produce the same yield as one hectare of policulture [3,18,19,20,21,22].
Among research conducted on Lupinus sp-maize intercropping, little to none concentrates on the root system, which is one of the most important organs. One of the biggest problems encountered when studying plant roots is that the majority are destructive, which greatly limits the understanding of their physiology, ecology or architectural structure. Moreover, the use of plastic bags or planters for their study could potentially confound the results of any research, as they confine or limit root growth.
In this research, the main objetives are to investigate the use of Lupinus montanus as an alternative as sustainable agricultural methods and to evaluate the nodulation and fixation of Lupinus montanus, and finally, we used the rhizotrones, or observation chambers, to study the architecture of the roots as well as how the roots behave under different environmental conditions. Particularly in this case, rhizotrons were used to study the behavior of the root in two intercropped species (Lupinus montanus and Maize) and analyze the possibility of nutritional benefits.
The main hypothesis is that probably Lupinus montanus will be able to release phosphorus from calcium phosphate and leave it available to Zea Mays HS-2.

2. Materials and Methods

2.1. Site Description

Research was carried out at the Postgraduate College, Montecillos Campus (Colegio de Postgraduados-Campus Montecillos), in the growth chambers located in the Botany building.

2.2. Plant Material

The first stage began with the collection of Lupinus seeds (Lupinus montanus as identified in the herbarium at the Postgraduate College) the in the State of Mexico at the following coordinates: 19° 10′13.31″ N, 98° 43′ 3.73″ W; 2497 msnm. The maize seeds, which were provided by the Postgraduate College´s Seed Program, are certified, trilinear hybrids, with a physiological maturity close to 150 days.

2.3. Soil

River sand was washed 15 times and sterilized for four hours to eliminate any type of nutrient in the soil. The final product was composed of 20 different samples (Table 1). The samples were analyzed at Posgraduate College, Soils fertility.

2.4. Sources of Phosphorous

Dibasic and tribasic calcium phosphate.

2.5. Glasshouse Experimentation

A randomized complete design (RCBD) with four replicates was used (Table 2).

2.6. Rhizotron Design

The rhizotron was constructed with wood and sliding glass that permitted the observation of the root system. As shown in Figure 1, it consists of two compartments, each one measuring 28 × 50 cm in width (A), divided by a wooden panel 3.5 cm in thickness (central divider) (B). A 3 mm wooden panel (D) and three wooden strips each 2 × 5 cm (C) were used as support. The lateral walls, which measure 9 × 50 cm with a thickness of 7 mm (D), the base and the central divider all have two tracks: a 3 mm glass panel fits in one track and a 5 mm wooden cover in the other to prevent the passage of light. The 9 × 61 cm base (7 mm in width) has 7 mm perforations placed every 4 cm for drainage (E). The system was then covered with a slab of polystyrene in order to maintain a sterile environment. To ensure that the system had no leaks, it was waterproofed with paraffin. After all of these adaptations, the rhizotron volume measured 1.2 × 10−3 m3.

2.7. Isolation of Atmospheric Nitrogen-Fixing Symbiotic Bacteria

At the field level, Lupinus has the ability to join to bacteria from the genus Bradyrhizobium and Mesorhizobium. As such, the decision was made to isolate the bacteria from its natural environment and inoculate the Lupinus plants in the experiment. To this effect, Lupinus plants were collected from the area of San Pablo Ixayoc, Texcoco, State of Mexico, which has an altitude of 2932 msnm: coordinates 19°26′59.2″ N, 98°46′33.4″. The plants were extracted with the root intact and then transported to the laboratory of Soil Microbiology. Five strains were selected, as they were effective at producing nodules. The inoculum was prepared, and the quality was determined. The bacterial load was 1.8 × 109; and the standard concentration required in the inoculants is 109 viable rhizobia cells per g o mL of support at the date of elaboration [23]; therefore, the inoculant that was prepared in the laboratory satisfied the bacterial load to infect the Lupinus montanus plants.

2.8. Identification of Nodular Bacteria

The extraction of DNA was carried out via the technique described by Doyle and Doyle [24]: The 16S rRNA gene was enhanced with the 8F and 1492R indicators. The sequencing reaction was carried out with the 514F and 1492R indicators.

2.9. Construction of the Phylogenetic Tree

To obtain the consensus sequences, both strains were assembled with the software Bioedit, which were compared with the BLASTNucleotide option from the GenBank database from the National Center for Biotechnology Information (NCBI). The consensus sequences were aligned with the program CLUSTALW [25], including the Mega 5 [26]. The Maxima Parsimonia method was used for the construction of the phylogenetic tree (Figure 2). In order to determine the reliability of the nodules, they were analyzed using the Bootstrap method with 1000 repetitions [27].

2.10. Development of the Experiment

The rhizotrons were filled with river sand to a height of 40 cm, at which point calcium phosphate bands (1 g) were placed. These were then covered with sand to reach a total height of 48 cm. The remaining 2 cm was reserved for carrying out irrigation, as seen in Figure 3a.
Two Lupinus montanus seeds were placed for every maize seed; the spatial arrangement of the system is similar to that found in the field where species are interspersed. Maize was placed in the center of the compartment at 12 cm from the lateral side and Lupinus montanus 7 cm away from the maize (Figure 3a,b), at a depth of 1 cm. Due to the dormancy problems faced by the legume, three seeds were placed at each point (mechanically scarified), and after germination, one was chosen. At 10 days, 1mL of symbiotic bacteria was inoculated. Because Lupinus montanus has a longer growing cycle, it was planted first; the maize seeds followed and were planted after 30 days. Two sources of calcium phosphate were used: dibasic and tribasic. Three fertilizations were carried out with Steiner modified solutions: (a) (g L−1) 0.4929 Mg SO4• 7 H2O, 0.172 Ca SO4• 2 H2O, 0.609 K2 SO4 and (b) (g L−1) 0.944 Ca (NO3)2 • 4 H2O, 0.404 KNO3, 0.261 K2 SO4, 0.492 Mg S04. In the inoculated treatments, a nitrogen-free solution was applied, while the non-inoculated treatments received a nitrogen solution. During the course of the experiment, three fertilizations were carried out. The substrate used is very porous with a high ability to filtrate, and as such, a trickle irrigation system (modified IV system for medical use) was implemented. To clearly view the roots, the rhizotrons were placed at a 30 degree angle (Figure 4), but after the first month, it was noted that this was not sufficient, and the rhizotron was subsequently placed at a 45 degree angle. The research employed a completely random block design with four repetitions with two sources of P, two nutritional solutions and two cultivation systems. Growing chambers were kept at 26 °C with light for 12 h and at 16 °C in darkness. The light emitted by the growth chamber was calculated at 500 µ E s−1 m−2.
In order to manage the growth of the Lupinus roots, acetates that were joined to the crystals of the rhizotron were used. Permanent markers of different colors were used for each measurement. In the case of the maize, the growth was traced over the glass, which allowed us to keep exact track of both species.
The experiment lasted two months, and the maize was only cultivated during the last month. When it was time to harvest, the plants were extracted, and the aerial biomass and root biomass were weighed separately. In the inoculated treatments, the total number of nodules in the Lupinus montanus plants was calculated. The material was washed and dried in a stove at a constant temperature of 70 °C and was then weighed again. In order to determine the nutritional effect on maize plant tissue, the concentration of P was analyzed through the photocolorimetry method via reduction with molibvanadato [28] and N with the semimicro-Kjeldahl technique [29]. The SAS 9.0 program was used for the statistical analysis, and an ANOVA and Tukey Test were performed.

3. Results and Discussion

The results from the variance analysis showed that there were significant statistical differences (p < 0.05) in the % P in plant tissue, which was greater in the intercropping than in the monoculture. Treatment 2 (T2), 4 (T4) and 8 (T8) refer to the intercropping of maize and Lupinus montanus, as they had the highest P values, indicating that Lupinus montanus releases the nutrients that it finds trapped in the calcium phosphate and makes it accessible for maize (Figure 5). Probably, the highest P values indicate that Lupinus montanus releases the nutrients that it finds trapped in the calcium phosphate and makes it accessible for maize. So, Lupinus montanus released P from calcium. The best concentrations of P in maize were found in T2 (0.032). In this treatment, a N-laced nutrient solution was added, which coincides with a study carried out by [30], which mentioned that good nutrition of N and low levels of P generate a root system that can be fundamental in the solubilization of P. The treatment T4 is relevant because this was where Lupinus montanus was inoculated with the bacteria; it was the second best treatment (0.030). As already mentioned above, Burkholderia is a bacteria capable of creating a symbiosis with certain leguminous plants, and in this study, we observed that the bacteria was capable of nodulating Lupinus montanus. It has been reported that B. cepacia has the ability to adequately solubilize calcium phosphate, iron and aluminum [31]. Burkholderia bacteria have been identified in Lupinus albus, and their presence increased senescent roots, more so in young roots [32].
According to Jones [33], the percentages obtained in maize tissue are less than 0.15%, a criterion that is already considered deficient. None of the treatments approach this value. It is important to remember that the plants were under P and N stress conditions, as well as being in a sandy soil (Table 1, chemical characteristics can be observed). Tribasic calcium phosphate was the best assimilated compound by the maize plants (T2, T4). This is important because the compound has a high level of insolubility, indicating that the legume has the ability to mobilize P that is trapped in the calcium, making it available for maize; this is different to what we find in monoculture circumstances.
Some studies concluded that intercropping wheat and maize with Lupinus albus and Lupinus angustifolius L. improves the concentrations of P in the plant’s biomass [2,9,34,35,36].
In a study with Lupinus albus, [9] mentions that the probable mechanism by which phosphorous moves in the soil/root interface is because of the excretion of citrate ions from the roots of this species. While the authors of [8] were studying the excretion of citric acid and the precipitation of calcium in the Lupinus albus rhizosphere in a limestone soil, they observed that proteoid roots, which are capable of lowering pH, developed in the presence of a P deficiency. Through X-ray spectroscopy, they observed abundant white precipitates of calcium citrate. Citrate in general and acid citrate were highly effective in dissolving tricalcium phosphate, as well as iron and aluminum phosphate. When the authors of [17] studied the influence of carboxylates on the movement of P, Al and Fe, they found citrate and malate in the Lupinus albus rhizosphere. They also found that the movement of P, Al and Fe was attributed to linked exchanges of P by citrate and the solubilization of Al and Fe as carboxylated compounds. In [37], the authors found that L. albus plants in nutrient solutions deficient in phosphorous increased the activity of acid phosphatase.
The majority of intercropping studies use soils of natural origin, but we cannot lose sight of the fact that soils in these conditions are composed of a large amount of bacteria and that many of these bacteria have the ability to solubilize and mobilize P. Bacteria that have this capacity are Pseudomonas, Rhizobium, Burkholderia, Achromobacter, Agrobacterium, Aereobacter, Flavobacterium, Yarowia, Streptosporangium and Erwinia [38]. The importance of the current study lies in the fact that the variable of interest was isolated in such a way that there is no possibility to attribute the solubilization of P to bacteria.
The use of rhizotrons allowed us to observe the behavior of the root system of the Lupinus montanus plant in an environment with P stress. Figure 6 shows the interaction of the Lupinus montanus roots with maize; Figure 5b shows the Lupinus montanus roots that correspond to T2; Figure 5c shows the behavior of the roots subjected to treatment T4. After analyzing the root architecture, we can observe that there are no proteoid structures as observed in L. albus. The Lupinus montanus root system is found in all intercropping treatments. In relation to this, there is clear evidence that Lupinus angustifolius [39] and Lupinus mutabilis [40] do not produce specialized “clusters”, but they do release large quantities of carboxylates. The root structure of this species is very similar to that of a bean.

Nitrogen in Plant Tissue and the Maize Roots in Sandy Soil

There were no significant differences between the treatments. Similar behavior was presented in the % of N in the root. The objective of inoculating and adding a nutrient solution laced with N was to observe if there were any morphological changes in the root system of the Lupinus montanus. While this would be reflected in the movement of P, no such structures were present (Figure 7e–g). The nutrition of nitrogen can be a determining factor in the formation of proteoid roots in some wild species. In [29], the authors carried out research and examined the effect of the nutrition of N in its different forms: ammonium, nitrate and nitrate fixation under P deficiencies. The number of proteoid roots increased considerably when P was administered at 1 μM, when compared to 50 μM. Furthermore, under phosphate deficiency, NH4+ was the best source of nitrogen, resulting in a high number and biomass of proteoid roots. It is mentioned in [41] that when confronted with P deficiencies, malate dehydrogenase is produced. Malate dehydrogenase participates in the citric acid cycle, and when faced with the deficiency, the synthesis of malate can improve the formation of nodules. However, an excess of this can prevent nitrogen fixation. The authors of [42,43,44,45] evaluated the formation of nodules in Lupinus white proteoid roots with low P concentrations. They observed that at day 21 (Figure 7a–d), the number of nodules was greater in the treatment without P, but at 37 days, the nodules increased under the treatment with (+) P. In this research, the use of rizotrons was very useful when counting legume nodules between T2 and T3 (Table 3, Treatments in intercropping), and there are significant differences between treatments with the application of bacteria Burkholderia, which increased the nodulation.
This results further suggest that Lupinus montanus can mobilize P from calcium phosphate through the rhizosphere. Further, we showed that it can increase its P concentration in the intercropping with Lupinus montanus, indicating that maize HS2 P acquisition benefited from the presence of Lupinus montanus. With this finding, a new research field has been opened, for all of the previously reported work was conducted using treatments via inoculation with Bradyrhizobium, whereas in this research, Burkholderia was used. In Figure 7, the three treatments with the highest percentages of P can be observed; two of them were inoculated with the Burkholderia bacteria.

4. Conclusions

In summary, after analyzing the information in the current research, we can conclude the following:
  • There is an opportunity for a new field of investigation in the nodulation and nitrogen fixation of Lupinus montanus since this research could evaluate nodulation with the Burkholderia bacteria.
  • The use of Lupinus sp. is confirmed as an alternative in favor of more sustainable agricultural methods since it improves soil fertility in phosphorous-deficient soils. This could potentially contribute to the wealth of knowledge used to solve Mexico’s problem of food autonomy.
  • As was observed during the experimental phase, the type and use of rhizotrons that were designed for the current study are only recommended for the evaluation of root systems in leguminous plants since crops with root systems like maize are more complicated and unreliable.

Author Contributions

J.E.G. devised the project, the main conceptual ideas and proof outline. V.E.H. and E.O.T., conducted the survey, recorded data and collected the material for write up. J.D.M., J.C.M.M. and F.L.S. provided critical feedback and helped shape the research, analysis and manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available from the authors upon request.

Acknowledgments

The authors thank Carlos Trejo, Josue Kohashi and Miguel for their assistance at Botany Department Postgraduate College. The authors are grateful for the cooperation of Jenny Zehemer for the comments on the last version of this manuscript. This research was supported by National Council of Science and Technology (CONACYT).

Conflicts of Interest

The authors declare no conflict of interest.

Significance Statements

Through the use of calcium phosphate, this study highlighted an enhanced phosphorus turnover in the intercropping maize and lupins. The capability of some plant species to mobilize phosphorus (P) from poorly available soil P fractions can improve P availability for P-inefficient plant species in intercropping. These findings might help researchers and farmers to propose and apply agriculture, because the P-mobilization-based facilitation by lupins to enhance P acquisition of cooccurring plant species is determined by both the available P concentration and P sorption capacity of soil, and the root intermingling capacity among the two plant partners enables rhizosphere overlapping.

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Figure 1. The apparatus called “rhizotron” which was used to evaluate the intercropping Lupinus montanus and Zea Mays.
Figure 1. The apparatus called “rhizotron” which was used to evaluate the intercropping Lupinus montanus and Zea Mays.
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Figure 2. Phylogenetic tree constructed using the Maxima Parsimonia method. The obtained sequences were compared with the baseline sequences from the Genbank database.
Figure 2. Phylogenetic tree constructed using the Maxima Parsimonia method. The obtained sequences were compared with the baseline sequences from the Genbank database.
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Figure 3. Rhizotrons filled with calcium phosphate bands and river sand to evaluate the intercropping Lupinus montanus and Zea mays. Two lupins seeds and one maize seed was placed at depth of 1 cm. (a,b) showed just the spatial arrangement of the system of intercropping (two Lupinus montanus and one Maize seed).
Figure 3. Rhizotrons filled with calcium phosphate bands and river sand to evaluate the intercropping Lupinus montanus and Zea mays. Two lupins seeds and one maize seed was placed at depth of 1 cm. (a,b) showed just the spatial arrangement of the system of intercropping (two Lupinus montanus and one Maize seed).
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Figure 4. This picture show the rhizotrons placed at a 30 and 45 degree vertical angle in the growth root system during the two month period.
Figure 4. This picture show the rhizotrons placed at a 30 and 45 degree vertical angle in the growth root system during the two month period.
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Figure 5. Effect of concentrations of phosphorus in maize plant tissue. (T1) FA SA C1; (T2) FA SA C2; (T3) FA SB C1; (T4) FA SB C2; (T5) FB SA C1; (T6) FB SA C2; (T7) FB SB C1; (T8) FB SB C2. FA: tribasic calcium phosphate; FB: dibasic calcium phosphate. SA: Steiner nutrient solution (+) nitrogen; SB: Steiner nutrient solution (−) nitrogen. C1: monoculture; C2: intercropping. * Figures followed by the same letters are statistically equal (Tukey, α = 0.05). a and b are the differences between concentrations of phosphorus.
Figure 5. Effect of concentrations of phosphorus in maize plant tissue. (T1) FA SA C1; (T2) FA SA C2; (T3) FA SB C1; (T4) FA SB C2; (T5) FB SA C1; (T6) FB SA C2; (T7) FB SB C1; (T8) FB SB C2. FA: tribasic calcium phosphate; FB: dibasic calcium phosphate. SA: Steiner nutrient solution (+) nitrogen; SB: Steiner nutrient solution (−) nitrogen. C1: monoculture; C2: intercropping. * Figures followed by the same letters are statistically equal (Tukey, α = 0.05). a and b are the differences between concentrations of phosphorus.
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Figure 6. Interaction of Lupinus montanus root and the maize. In this figure (a–c) the observations in the rizotron showed the differences between treatments focus to behavior of the root system.
Figure 6. Interaction of Lupinus montanus root and the maize. In this figure (a–c) the observations in the rizotron showed the differences between treatments focus to behavior of the root system.
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Figure 7. (a–d) The formation of nodules in the roots of Lupinus albus without (−P) and with P (+P) at day 21; (e,f) formation of nodules in Lupinus montanus roots with Burkholderia at day 60 under P stress conditions; (g) root system of Lupinus montanus with N, at day 60. The last reports of symbiotic Agrobacterium radiobacter strains (LMR670 and LMR 676) showed that when used in inoculation, they are effective biological fertilizers for common bean cultivation [46].
Figure 7. (a–d) The formation of nodules in the roots of Lupinus albus without (−P) and with P (+P) at day 21; (e,f) formation of nodules in Lupinus montanus roots with Burkholderia at day 60 under P stress conditions; (g) root system of Lupinus montanus with N, at day 60. The last reports of symbiotic Agrobacterium radiobacter strains (LMR670 and LMR 676) showed that when used in inoculation, they are effective biological fertilizers for common bean cultivation [46].
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Table 1. Analysis of Sand.
Table 1. Analysis of Sand.
Parameter Technique
pH (ratio 1:2)7.6Potentiometer in the saturation extract
Phosphorus (mg kg−1) 2.6Olsen et al., 1965
Total Nitrogen (%)0.01Micro-Kjeldahl
Table 2. Treatments of experiment.
Table 2. Treatments of experiment.
Phosphorus SourceDose (gr)Crop SystemSteiner Solution
Tribasic Calcium phosphate1MA
Tribasic Calcium Phosphate1M/LB
Tribasic Calcium Phosphate1MA
Tribasic Calcium Phosphate1M/LB
Dibasic Calcium Phosphate1MA
Dibasic Calcium Phosphate1M/LB
Dibasic Calcium Phosphate1MA
Dibasic Calcium Phosphate1M/LB
M = monoculture; Maize HS-2; M/L = intercropping (Maize HS-2/Lupinus montanus); A = nitrogen, B = no nitrogen.
Table 3. Nodulation in Lupinus montanus plants.
Table 3. Nodulation in Lupinus montanus plants.
(+)InoculumLupinus montanus
Number of Nodules		(−)InoculumLupinus montanus
Number of Nodules Total
Plant 1Plant 2 Plant 1Plant 2
R1152136141226
R2151833121537
R327194615823
R422153716925
X = 38 a X = 25.5 b
Mean = 38 a; mean = 25.5 b; R = replicate; same letters are statistically equal (Tukey, alpha = 0.05).
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Espinosa Gonzalez, J.; Espinosa Hernández, V.; Ojeda Trejo, E.; Delgadillo Martínez, J.; Molina Moreno, J.C.; Sánchez, F.L. Assessing the Nutritional Effect of Lupinus montanus on Zea mays HS-2 (Intercropping) and Identification of Nodular Bacteria through the Use of Rhizotrons. Int. J. Plant Biol. 2023, 14, 910-921. https://doi.org/10.3390/ijpb14040067

AMA Style

Espinosa Gonzalez J, Espinosa Hernández V, Ojeda Trejo E, Delgadillo Martínez J, Molina Moreno JC, Sánchez FL. Assessing the Nutritional Effect of Lupinus montanus on Zea mays HS-2 (Intercropping) and Identification of Nodular Bacteria through the Use of Rhizotrons. International Journal of Plant Biology. 2023; 14(4):910-921. https://doi.org/10.3390/ijpb14040067

Chicago/Turabian Style

Espinosa Gonzalez, Juan, Vicente Espinosa Hernández, Enrique Ojeda Trejo, Julián Delgadillo Martínez, Juan Celestino Molina Moreno, and Francisco Landeros Sánchez. 2023. "Assessing the Nutritional Effect of Lupinus montanus on Zea mays HS-2 (Intercropping) and Identification of Nodular Bacteria through the Use of Rhizotrons" International Journal of Plant Biology 14, no. 4: 910-921. https://doi.org/10.3390/ijpb14040067

APA Style

Espinosa Gonzalez, J., Espinosa Hernández, V., Ojeda Trejo, E., Delgadillo Martínez, J., Molina Moreno, J. C., & Sánchez, F. L. (2023). Assessing the Nutritional Effect of Lupinus montanus on Zea mays HS-2 (Intercropping) and Identification of Nodular Bacteria through the Use of Rhizotrons. International Journal of Plant Biology, 14(4), 910-921. https://doi.org/10.3390/ijpb14040067

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