Characterization of nodulation capacity with native rhizobia in germplasm of underutilized forage species of Macroptilium (Benth.) Urb.

doi:10.21203/rs.3.rs-3627641/v1

In order to generate reliable characterization and selection schemes in germplasm of Macroptilium for biological nitrogen fixation with native rhizobia, we carried out a trial to evaluate the germplasm variability for initial growth, crude protein in shoots and nodulation capacity with native soil of subtropical environment. The trial was carried out under two conditions, substrate with (N+) and without nitrogen added (N-). The growth variables and nitrogen content were analyzed through a nested ANOVA with the factors species (S), accessions nested within species (ANS), nitrogen level (NL) and the corresponding interactions. Because the plants did not nodulate in the N + treatment, a simple one-way ANOVA was performed in the N- treatment to analyze nodulation capacity. Relationships between variables and accessions were examined using principal component analysis. All accessions evaluated formed effective nodules in N- treatment. Accessions showed different initial growth and nitrogen content at different nitrogen levels. There was variability in the symbiotic response and initial growth, and most of the variance was explained by differences between ANS. Nodule number and nodule weight were related to Macroptilium plant dry matter and crude protein in shoots. Our hypotheses were confirmed in this work. In evaluated germplasm we found variability for nodulation capacity. In the other hand, nodulation capacity was related with biological nitrogen fixation (BNF), since the accessions with higher NC showed higher initial growth and higher protein content in shoot.

promiscuous symbiosis

legume forage

biological nitrogen fixation

germplasm

The Great American Chaco is a vast region extending over an area of more than 1,000,000 km², from tropical to subtropical latitudes, that includes southeastern Bolivia, western Paraguay and central and northern Argentina (Hueck 1972). The Great Chaco is one of the three regions worldwide with the highest percentage of change in land use (deforestation and expansion of annual crops and grass pastures). Much of this deforested area shows soil degradation due to monoculture, drastically reducing the surface stocks of carbon, nitrogen and phosphorus (Baumann et al. 2016; Mosciaro et al. 2022; Durcan 2023).

Cattle production is one of the most important economic activities, with a low stocking rate and the use of natural vegetation as the main source of fodder. South America includes 19.6% of the world’s beef stock (FAO 2020). The member countries of the Great Chaco possess 82% of this stock. Among other issues, livestock in this region require the adoption of leguminous species to increase the quality of their diet, increase the availability of edaphic nitrogen and generate other environmental benefits (Shelton et al. 2005; Nichols et al. 2007; Batello et al. 2008; Schultze-Kraft et al. 2018). Legumes should be included in breeding programs under a new paradigm that makes profitable production compatible with ecosystem services, especially in the face of changes in land use related to climate change (Stuber and Hancock 2008; Brummer et al. 2011; Helgadóttir et al. 2016). Of particular interest is the replacement of chemical fertilizers through biological nitrogen fixation (BNF), which is essential for successful pasture establishment and production (Cooper 1977; Armstrong et al. 1999; Byun et al. 2004; Thrall et al. 2005).

In the Great Chaco, 98 genera, 363 species and 404 specific and intraspecific taxa of legumes have been reported (Morales et al. 2019). Many of them are native or endemic to this region. Historically, species with forage value were surveyed by various authors, such as Rosengurtt (1946), Hawkins and Donald (1963), Covas (1978), Glatzle (1999) and Pensiero et al. (2021). Despite this, successful introduction to cultivation is scarce, and most successful cases have been carried out in other parts of the world, such as Australia, with germplasm collected in Latin America and particularly in the Great Chaco (Hacker et al. 1996; Shelton et al. 2005; Muir et al. 2017). Although there are multiple causes for this, a key problem is the lack of information on the phenotypic and genetic variation of germplasm in seed banks (Shelton et al. 2005; Muir et al. 2014; Rebetzke et al. 2018).

Among the forage legumes of the Great Chaco, the Macroptilium (Benth.) Urb. genus is highlighted. Macroptilium includes annual or perennial herbaceous species with a spring-summer-autumn cycle (Covas 1978; Nichols et al. 2007) and a high forage value and BNF capacity (Crew et al. 2018; Borges et al. 2019; Reiss and Drinkwater 2022). Cover crops can provide soil health benefits, symbiotic nitrogen fixation and a significant increase in the total organic carbon of soil (Kinshua et al. 2019; Mahanta et al. 2020). Despite this, no species of Macroptilium are used as forage or cover crops in the Great Chaco. The species with the highest forage value for this region are Macroptilium bracteatum (Nees & C. Mart.) Maréchal & Baudet, Macroptilium erythroloma (Mart. ex Benth.) Urb. and Macroptilium lathyroides (L.) Urb. (Pensiero et al. 2021). Macroptilium bracteatum is a short-lived perennial species. Two cultivars were released in Australia, “Cadarga” and “Juanita” (Dalzell et al. 1997; Jones and Rees 1997; Nichols et al. 2007). Macroptilium erythroloma is a perennial species that is not currently used as forage, although in our preliminary evaluations, it was highlighted for its production and regrowth capacity. Macroptilium lathyroides is an annual or biennial herbaceous species that is very palatable and shows good reseeding, with a cultivar released in Australia called "Murray" (Milford 1967; Cameron 1985b; Tobisa and Nakano 2019).

In relation to the establishment of symbiotic associations with soil bacteria, Macroptilium atropurpureum (Moq. & Sessé ex DC.) Urb has the ability to associate with a wide range of rhizobial strains, where associations with the genera Bradyrhizobium, Ensifer and Rhizobium have been reported (Bromfield and Barran 2011; Yuhashi et al. 2000). With rhizobia isolated in the trials of this work, among other trials, Toniutti et al. (2015) reported symbiotic rhizobia of M. atropurpureum and M. bracteatum, which were suggested to belong to the genus Bradyrhizobium, and Fornasero et al. (2018) observed characteristics coincident with the genus Bradyrhizobium among M. erythroloma and M. lathyroides rhizobia.

Domesticated legume species were selected without taking into account the complex genetic relationships between them and soil microorganisms. Later, breeding for an increase in BNF was carried out considering the need for inoculation with effective strains of rhizobia (Sinclair and Vades 2002; Herridge 2003; Muller et al. 2021). Although there are few in-depth studies that relate the BNF capacity between wild and domesticated germplasm, in various species, there are reports that wild germplasm is more promiscuous than domesticated germplasm (Liu et al. 2020). Legume genotypes with more effective nodulation ability can be obtained by selecting the best combination between host genotype and nodule bacteria (Rengel 2002). The selection of Macroptilium genotypes for forage production together with effective nodulation capacity with native rhizobia strains would improve pasture establishment and reduce its cost because inoculation with rhizobia would not be needed. In addition, it would improve the use of these genotypes in ecological restoration strategies or as a cover crop (Crews and Peoples 2004). The Germplasm Bank “Ing. Agr. José Mario Alonso” of the Universidad Nacional del Litoral (Argentina) (GBJMA) has a unique collection of leguminous forage germplasm.

There is not a documented selection strategy in Macroptilium to improve BNF with native rhizobia and initial growth. To generate reliable characterization and selection schemes for germplasm of Macroptilium for effective nodulation ability with native rhizobia, we carried out a trial to evaluate the variability of initial growth, nitrogen content in plants and NC in a set of Macroptilium germplasm. Our hypotheses are: a) there is genetic variability in conserved Macroptilium germplasm for nodulation capacity with native rhizobia and b) higher nodulation capacity will be associated to a higher initial growth and better nitrogen status in the plant.

We used the work of Sylvester-Bradley and Kipe-Nolt (1988) as a reference for trial design. We used a soil with nitrogen and phosphorus deficiency that came from an old Rhodes grass (Chloris gayana) pasture of more than 10 years. In this work, we also include M atropurpureum as a control with high nodulation capacity. Macroptilium atropurpureum is a perennial species that is the most commonly used forage in tropical environments (Cameron 1985a; Oram, 1990; Bray and Woodroffe, 1994). This species is not used in subtropical environments due to its sensitivity to low temperatures.

The initial growth and nodulation ability (NA) were analyzed in a set of Macroptilium accessions of GBJMA (Table 1) under two culture conditions. Seeds were sown in 475 cc pots with soil from an old Rhodes grass (Chloris gayana) pasture from Chaco Province, Argentina (S -27.1925; W -61.4222). Sample soil was collected from the superficial horizon (first 30 cm). This soil showed deficiencies in nitrogen (N) and phosphorus (P). The soil nutrient contents were 0.11% total N and 8 ppm extractable P. The trial was carried out under two nitrogen supply conditions. The soil was sieved through 2 mm mesh. A mixture of 100 g of soil and 25 g of perlite was used in each pot, which gave a volume/substrate ratio of 1:3. The first conditions corresponded to the substrate without nitrogen added (N-), and the second was composed of a substrate with nitrogen fertilization (N+). For the N + treatment, the fertilizer used was potassium nitrate, whose application was carried out with irrigation in a staggered manner. The accumulated dose was 77 ppm N per pot. This high dose of nitrogen met the needs for this macronutrient.

Table 1

Germplasm (accessions) of the Macroptilium genus analyzed in the trial.
Species	ID	Origin	Trial ID
M. atropurpureum	AusTRCF 321058	Australia (cv. Aztec)	MA1
M. atropurpureum	AusTRCF 82306	Cuba	MA2
M. atropurpureum	AusTRCF 82307	Cuba	MA3
M. atropurpureum	AusTRCF 91957	Cuba	MA4
M. atropurpureum	AusTRCF 91973	Cuba	MA5
M. bracteatum	ARG0006	Argentina	MB6
M. bracteatum	AusTRCF 68892	Australia (cv. Juanita)	MB9
M. bracteatum	AusTRCF 93100	Brazil	MB35
M. bracteatum	AusTRCF 11833	Paraguay	MB11
M. bracteatum	AusTRCF 39099	Paraguay	MB41
M. erythroloma	AusTRCF 319162	Paraguay	ME16
M. erythroloma	AusTRCF 319482	Argentina	ME42
M. erythroloma	ARG0043	Argentina	ME43
M. erythroloma	ARG0044	Argentina	ME44
M. erythroloma	AusTRCF 49752	Brazil	ME46
M. lathyroides	ARG0054	Argentina	ML54
M. lathyroides	AusTRCF 320971	Australia (cv Murray)	ML23
M. lathyroides	AusTRCF 49799	Brazil	ML56
M. lathyroides	AusTRCF 39095	Paraguay	ML25
M. lathyroides	AusTRCF 319166	Paraguay	ML62

AusTRCF: FAO code, germplasm of Australian Tropical Crops and Forage Collection.

ARG: FAO code, accessions of the “Ing. Agr. José Mario Alonso” Germplasm Bank, Universidad Nacional del Litoral (Argentina).

The trial was carried out in a growth chamber under controlled temperature conditions (25°C+-2) and a 16-hour photoperiod, with a light intensity of 400 mmol/m/s PAR radiation provided by LED lamps. Five disinfected (with 20% sodium hypochlorite solution, 20 min) and mechanically scarified seeds were sown in each pot, leaving 2 plants per pot after emergence. The pots were arranged in a completely randomized design in the growth chamber. Ten pots per accession and treatment were analyzed. All pots were irrigated regularly with distilled water (every two days) and with Jensen solution without nitrogen (twice a week).

Forty-five days after emergence, the plants were removed, and the roots were washed under running water. After manual extraction, all root nodules were counted, and the abundance, size and distribution of nodules on roots were recorded according to a reference table (CIAT 1988). Additionally, the presence of leghemoglobin in the nodules was assessed by examining 20% of the nodules of each plant for visual signs of leghemoglobin concentration (Santana et al. 1998; Golluscio et al. 2006). The nodules, roots and shoots were then placed in paper bags and placed in a forced circulation chamber (65–70°C) until a constant weight was reached to determine the dry matter weight. In addition, the concentration of total N in plant shoots was determined according to the Kjeldahl method (Helrich 1990), and the percentage of crude protein (CP) was estimated from the concentration of total N (%) by a factor of 6.25.

The following variables were recorded: a) number of nodules per plant (NNP); b) nodule dry weight (NDW); c) shoot dry weight (SDW); d) root dry weight (RDW); e) total dry weight (TDW) and f) crude protein (%) in the shoot (CP).

The variables SDW, RDW, TDW and CP were analyzed through a nested ANOVA with the factors species (S), accessions nested within species (ANS), nitrogen levels (NL) and the corresponding interactions (SxNL and ANSxNL). Because the plants did not nodulate in the N + treatment, a simple one-way ANOVA was used to assess the effect of S and ANS on NNP and NDW in the N- treatment. Variance components of principal factors were included in the analysis. The relationships between variables and accessions were examined using multivariate analysis. Means of variables for the twenty accessions in both treatments were used in a principal component analysis (PCA). The statistical analyses were carried out with the statistical package INFOSTAT (Di Rienzo et al. 2020).

The effectiveness of nodulation in the accessions was also calculated based on a classification proposed by the International Center for Tropical Agriculture (Sylvester-Bradley and Kipe-Nolt 1988). Specifically, the accessions that presented little difference in the protein content of the plants between the treatments with and without nitrogen were classified as effective (i.e., higher capacity to establish an effective symbiosis with native strains of rhizobium), those that presented an intermediate difference were classified as semieffective, and those that presented a great difference between the two treatments were classified as ineffective. The effective groups were considered unable to benefit from inoculation with rhizobia. For the purposes of this work, we separated efficient accessions from the rest. We performed a two-sample t test (p > 0,1) to determine whether means differed depending on nitrogen level. No difference between the N + and N- treatments indicated effective accessions.

The NN per plant was 8.1 and 9.4 for M. lathyroides and M. atropurpureum, respectively, showing significant differences from M. bracteatum and M. erytroloma, which presented 13.0 nodules per plant, while the NN in the main root/plant was 4.0 for all species. Nodular structures presented an oblong shape and spherical meristem and were of the desmoid type with lenticels, showing typical characteristics of determinate-growth nodules. The size was medium (2–5 mm), and the internal coloration of nodules was pink to red in more than 85% of the plants (Fig. 1).

There were statistically significant effects (p < 0.0001) of the S, ANS, NL and ANSxNL interaction variables. This implies that the accessions showed different initial growth at different nitrogen levels. To analyze growth variables and NC, a simple one-way ANOVA was used to assess the effects of S and ANS on variables in the N- treatment. In this analysis, there were statistically significant effects (p < 0.0001) of S and ANS. The variance components showed that most of the variance was explained by ANS (Table 2). For the variables NNP, CP and TDW, the sum of variance components for S and ANS was greater than that of the error.

Table 2

Variance components (%) in the N- treatment according to simple one-way ANOVA. Number of nodules per plant (NNP), weight of dry nodules (DWN), shoot dry weight (SDW), root dry weight (RDW), total dry weight (TDW) and crude protein (%) in the shoot (CP). Species (S), accessions within species (ANS) and error (E).
Factors	NNP	NDW	SDW	RDW	TDW	CP
S	3.5	4.7	6.0	5.1	6.7	4.1
ANS	58.2	32.1	40.3	42.6	51.8	57.2
E	38.3	63.2	53.7	52.3	41.5	38.7

Principal component analysis showed that 81.7% of the total variability could be explained by two main components (PCs). Table 3 shows eigenvectors and eigenvalues for the two main components. For PC1 (55.1% of the total variation), total, shoot and root dry weights in the N- treatment and nodule weight and protein content in the N-treatment were the variables with the greatest weight. The variation in PC2 (26.6% of the total variation) was mainly a result of differences in total, shoot and root dry weights in the N + treatment. In the biplot diagram (Fig. 2), PC1 discriminates accessions of different species based on growth variables (TDW, SDW and RDW) and protein content (PC) in the N- treatment and nodule dry weight (NDW). For example, accessions ME46 and ML62 were distinguishable from ML54 and ME16 by their greater TDW, SDW, RDW, and PC values in the N- treatment and NNP. DWN was correlated with TDW N- (r = 0.98; P < 0.0001), SDW N- (r = 0.98; P < 0.0001), RDW N- (r = 0.81; P < 0.0007) and PC N- (r = 0.82; P < 0.0001). NNP was correlated with TDW N- (r = 0.71; P < 0.0005), SDW N- (r = 0.7; P < 0.0005) and PC N- (r = 0.68; P < 0.0009). Dispersion graphics for these variables are shown in Fig. 3 and Fig. 4.

There was variability in the symbiotic response (measured as the difference in protein content between the N + and N- treatments) (Table 3). T tests showed that five accessions were effective at nodulation with native rhizobial strains (MB6, MB9, ML23, ME42 and ME46).

Macroptilium sp. plants presented spherical nodules with a defined growth habit. According to Bergersen (1982) and Sprent (2008), desmoid nodules are spherical with lenticels on the outside and infected tissue of uniform age, similar to those obtained in this study. Likewise, nodulated roots of Argentine plants of the tribe Phaseoleae, family Fabaceae, present desmoid nodules (Bianco 2020). In this work, all the accessions evaluated formed nodules with pink to red internal coloration, which suggests the presence of rhizobial strains effective for Macroptilium sp. plants in the soil. Fornasero et al. (2018) reported similar native rhizobia with the ability to nodulate on M. erythroloma plants in soils with different edaphic characteristics and different chemical properties.

Table 3. Eigenvectors and eigenvalues of the first 2 principal components of 10 quantitative variables in accessions of Macroptilium species. Shaded cells show coefficients with the greatest weighting in each eigenvector. Number of nodules per plant (NNP), weight of dry nodules (DWN), shoot dry weight (SDW), root dry weight (RDW), total dry weight (TDW) and crude protein (%) in the shoot (CP). N- and N+ treatments.
Variables	Eigenvector
Variables	PC1	PC2
RDW N+	0.22	0.5
SDW N+	0.26	0.47
TDW N+	0.22	0.51
RDW N-	0.36	0.02
SDW N-	0.4	-0.15
TDW N-	0.41	-0.13
NNP N-	0.29	-0.35
DWN N-	0.41	-0.13
PC N+	-0.08	0.21
PC N-	0.34	-0.2
Eigenvalue	5,5	2,6

Table 4. Effectiveness of nodulation in accessions of Macroptilium species. CP (mean crude protein of shoots in %). N+/N- treatments. Accessions with no significant differences between CP means are considered effective. Between parenthesis standard error (SE).

Accession	CP% (SE) N+	CP% (SE) N-
MB6	25.16 (1.1)	23.59 (2.6) ^ns
ME42	22.88 (1.2)	21.24 (2.7)^ns
ME46	25.88 (2.1)	22.76 (2.7)^ns
MB9	25.34 (1.7)	20.89 (2.9)^ns
ML23	25.62 (3.1)	20.69 (1.9)^ns
MA2	27.48 (0.8)	21.88 (1.2)^*
MA5	27.27 (0.9)	21.12 (1.3)^*
MB41	27.1 (1.3)	20.64 (1.6)^*
MA4	31.1 (1.3)	23.47 (1.5)^*
MB35	25.48 (0.7)	17.81 (1.1)^*
MA3	27.4 (2.9)	19.57 (0.6)^*
ML56	24.29 (1.2)	16.09 (0.9)^*
ME43	28.89 (0.9)	19.8 (1.0)^*
MA1	29.92 (1.0)	19.89 (0.4)^*
ME44	27.77 (1.0)	17.62 (0.8)^*
MB11	28.7 (1.6)	18.39 (0.7)^*
ML25	25.2 (1.5)	14.53 (0.8)^*
ML62	26.77 (0.9)	11.17 (0.6)^*
ME16	33.57 (1.4)	16.5 (1.0)^*
ML54	30.9 (1.7)	12.52 (0.7)^*
NS (not significant, p value > 0.1 in the T test). * (p value <0.1 in the T test). -

Evaluations of nodule formation on plant roots (number, aspect and size) can indirectly indicate the nitrogen fixation potential of subtropical legumes and contribute to the evaluation and interpretation of rhizobium–legume symbiotic associations (Peoples and Herridge 1990). Thus, nodule number and nodule weight, two characters commonly used in this type of study (Peoples and Herridge 1990; Herridge and Rose 2000), were related to Macroptilium plant dry matter and N content. The dry weight of nodules showed stronger correlations with biomass and protein content than with nodule number. As NDW is an easier trait to evaluate, it could be used when analyzing nodulation ability in large plant populations in breeding programs (Gwata et al. 2004).

We found variability in the effectiveness of legume–rhizobium symbiosis in the germplasm evaluated. Five of twenty accessions were classified as effective. The variability found corresponds mainly to intraspecific differences rather than differences between species. The effective accessions belong to three evaluated species (M. bracteatum, M. erytrholoma and M. lathyroides) with different origins. Two of them are cultivars developed in Australia. It is necessary to analyze whether the genotypes of cultivars were not unconsciously selected due to their promiscuity with native rhizobia. Effective accessions showed a higher nodulation capacity than M. atropurpureum accessions. This species has been used in several studies as a "trap" plant for studies of uncharacterized rhizobia (Rodrigues-Rodrigues et al. 2021; Hernández-Oaxaca et al. 2022; Freitas Costa et al. 2023). In the future, M. bracteatum, M. erytrholoma and M. lathyroides should be used as trap plants for the study of rhizobial diversity in different environments of the Gran Chaco. Similarly, Pulver et al. (1985) carried out a successful breeding program in soybean with the objective of developing promiscuous cultivars that have an effective nodulation ability with native strains from various regions of Africa (Mpepereki et al. 2000). Although it was later shown that these cultivars are favored under inoculation with effective rhizobial strains, the BNF gap is much smaller compared to that of cultivars that are not selected for promiscuity (Vanlauwe et al. 2019).

Our hypotheses were confirmed in this work. In evaluated germplasm we found variability for nodulation capacity. In the other hand nodulation capacity was related with BNF, since the accessions with higher NC showed higher initial growth and higher protein content in shoot. The germplasm set evaluated represented 40% of the “Macroptilium” germplasm conserved in our germplasm bank. Later, we used this screening strategy as part of the breeding program of our group, and some accessions with higher nodulation capacity with native rhizobia were part of the genetic background of the first two cultivars of “Macroptilium” released in Argentina, cv Don Augusto of M. erytroloma and cv Mancebo of M. lathyroides (https://gestion.inase.gob.ar/consultaGestion/gestiones). These cultivars have shown great nodulation capacity without inoculation in different field trials (Oscar Peman and Asoc. S. A, personal communication). According to the “Genesys” platform (https://www.genesys-pgr.org/), there are at least 300 other accessions of M. bracteatum, M. erythroloma and M. lathyroides conserved in different germplasm banks. This indicates that most of the conserved germplasm has not been characterized in relation to its effectiveness of symbiosis with native rhizobia.

For native forage species, this breeding approach is innovative and should continue to be studied, combining advances in the understanding of the molecular basis of BNF with the paradigm shift in agriculture to reduce the use of technologies based on the consumption of fossil fuels. This screening strategy could be used in the future to a) characterize agronomically different aspects of the nodulation ability in Macroptilium genus, b) analyze variability in symbiosis effectiveness of different germplasm with native rhizobia in different soil types and c) understand the genetic basis of effective symbiosis capacity with native rhizobium strains.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

This study was funded by Fondo para la Investigación Científica y Tecnológica (FONCyT), (grant number PICT 2022).

Author Contribution

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by all authors. The first draft of the manuscript was written by Juan Marcelo Zabala and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript

Acknowledgment

The authors would like to acknowledge Australian Pastures Genebank (APG), http://www.pir.sa.gov.au/research/australian_pastures_genebank. Australia to provide accessions analyzed in this work.

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No competing interests reported.

Characterization of nodulation capacity with native rhizobia in germplasm of underutilized forage species of Macroptilium (Benth.) Urb.

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Version 1

Abstract

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Introduction

Materials and Methods

Results

Discussion

Conclusion

Declarations