Initial growth response of twelve Saccharum Complex genotypes to inoculation using Nitrospirillum viridazoti

ABSTRACT

Sugar and alcohol production in Brazil comes from planting hybrids from the crossing of species belonging to the Saccharum Complex. However, little is known about the growth response of these genera to inoculation using a diazotrophic bacteria Nitrospirillum viridazoti strain BR11145, currently recommended in Brazil as an inoculant for sugarcane growth promotion. This study aimed to evaluate the initial growth of 12 sugarcane genotypes inoculated or not with the strain BR11145. After 109 days, plant growth, biomass and macronutrient accumulation were evaluated. Fresh and dry mass at the end of the trial showed a positive response to inoculation for the genotypes US72-1319, CPDAU 849678, and NG77-122 of S. spontaneum and for Fiji 10 of Miscanthus. A negative response was observed for Q45416 of Saccharum sp. The growth of S. spontaneum US72-1319 is significantly improved by the inoculation with BR11145, which leads to a high accumulation of nutrients, especially N, standing out among the genotypes tested.

Keywords
growth promotion; nitrogen; diazotrophic bacterium

INTRODUCTION

Sugarcane was introduced in Brazil in the year 1,500 by the Portuguese colonizers, and since that time, the crop has spread throughout the territory. It is a perennial Asian crop belonging to the Poaceae family and Saccharum genus. In addition to the Saccharum genus, the Erianthus, Miscanthus, Narenga, and Sclerostachya genera are cultivated like sugarcane and used in genetic improvement. These genera can occasionally hybridize with Saccharum and are referred to as the “Saccharum complex” by breeders (Grivet et al., 2006). Modern sugarcane cultivars are high polyploids, aneuploids (2n = ~ 12x = ~120) derived from interspecific hybridizations between the domesticated sweet species of Saccharum officinarum and the wild species Saccharum spontaneum (Pompidor et al., 2021). The Saccharum genus contains six species, of which four are domesticated: S. officinarum, S. barberi, S. sinensis, and S. edule, and two are wild species: S. spontaneum and S. robustum (Amaral et al., 2015). S. officinarum presents high sucrose content and low fiber, making it the most suitable species for sugar and ethanol production, while S. robustum and S. spontaneum exhibit greater robustness, and relatively low sucrose and high fiber contents, making them suitable for biomass production and as donors of robustness alleles in genetic improvement programs (Alvim, 2015). Additionally, S. spontaneum has slender and erect stalks, high tillering production, and is more resistant to pests and diseases, while S. robustum has longer thick stalks (Pompidor et al., 2021). Genotypes belonging to the Saccharum complex are energy crops in potential as they exhibit less sensitivity to drought stress, require minimal nitrogen for growth, and can be cultivated for biofuel or for producing electricity by burning the stalks, being suitable to be cropped into marginal areas and contaminated soils or even soils of low fertility or slopy areas (Silva, 2017).

Sugarcane is used mostly for sugar, ethanol, animal feed, and biomass production. But nowadays, biotechnological products can also be important for the sugarcane crop industry in Brazil. Based on these multiple uses, Brazil is the largest sugarcane producer, followed by India and China, with a Brazilian harvest forecast of 652.9 million metric tons for 2023/2024, 6.9 % higher than the last harvest (Conab, 2023). To achieve this high productivity, fertilizers are also used at high quantities, especially N and K, which are the most required for sugarcane growth (Otto et al., 2010). Currently, the cost of fertilizer is one of the main constraints for farmers, being nitrogen fertilizer one of the most expensive, usually applied at rates ranging from 60 to 100 kg ha^-1 yr^-1 of N (Robinson et al., 2011; Otto et al., 2016). Typically, the recommendation of N fertilizer for sugarcane considers the expected cane yield, the growth cycle (plant or ratoon), and the environmental factors, which include soil type and climate. Therefore, N fertilization in sugarcane needs to be linked to the agronomic response to be viable. This may also apply to other nutrients.

It is well known that sugarcane is capable of associating with nitrogen-fixing microorganisms, which can obtain from 40 kg ha^-1 yr^-1 (Urquiaga et al., 2012) to 58 kg ha^-1 yr^-1 of N (Resende et al., 2006) to complement its annual nitrogen demand. This benefit from Biological Nitrogen Fixation (BNF) varies according to the degree of a cultivar’s association with diazotrophic bacteria. Several bacteria species have been described as promoters of nitrogen gains by the crop (Souza et al., 2016), and part of this highly diverse community are diazotrophs (Fischer et al., 2012; Teheran-Sierra et al., 2021).

One of these benefic bacteria is Nitrospirillum viridazoti, a species that can associate with several plants, including sugarcane, and can provide part of the nitrogen required for its development, as well as presenting other mechanisms for promoting plant growth (Reis et al., 2020). N. viridazoti was first isolated from native plants of the Amazon region and several types of grasses in Rio de Janeiro State, formally classified as Azospirillum amazonense by Magalhães et al. (1983), but later reclassified as N. viridazoti (Baldani et al., 2024). This species presents characteristics that distinguish it from the genus Azospirillum, including smaller cell size, better growth at pH between 5.8 and 6.8, sensitivity to alkaline pH, and the use of malate and sucrose as carbon sources (Lin et al., 2014). N. viridazoti can also be capable of secreting indole-3-acetic acid (IAA), producing biofilms and bacterial phytochromes, and other means capable of modifying plant development (Schwab et al., 2018). This species was used as an inoculant in several field trials, associated with four other species of diazotrophs (Oliveira et al., 2006; Schultz et al., 2014, 2017), and growth promotion was also described by Santos et al. (2017). The strain BR11145 (= CBAmC) is currently used as an inoculant recommended for sugarcane application (Reis et al., 2020; Sica et al., 2020). The genome of this strain was characterized by Schwab et al. (2018) and the plant colonization was described by Schwab et al. (2023).

This bacterium can be considered a model for sugarcane response, but the growth promotion was only tested in commercial cultivars. Previous studies evaluated the growth response of different cultivars to inoculation, but no other representative of the Saccharum complex was ever tested. This study aimed to identify which of the 12 genotypes of the bacteria N. viridazoti strain BR11145 is most responsive in promoting plant growth after 109 days of growth using a substrate with 0.02 % nitrogen and no further fertilizer addition.

MATERIALS AND METHODS

Three experiments were conducted in a temperature-controlled greenhouse to evaluate the response of 12 sugarcane genotypes to inoculation with the diazotrophic bacteria N. viridazoti strain BR11145 (=CBAmC). The experiments took place at Embrapa Agrobiologia (Seropédica-RJ Brazil; 22° 44’ S, 43° 42’ W, and altitude of 26 m). Plant material originated from a collection of the National Center for Genetic Resources Preservation/ ARS/ USDA, Fort Collins, CO, USA. After undergoing quarantine, this plant material was micropropagated and then planted in an Active Germplasm Bank of Saccharum Complex at Embrapa Tabuleiros Costeiros, located at Jorge Prado Sobral, Nossa Senhora das Dores city, Sergipe-Brazil (10° 29’ 27’’ S; 37° 11’ 34’’ W). S. spontaneum genotypes originated from plants collected at Embrapa Clima Temperado, Pelotas-RS.

Plant material

Sugarcane genotypes evaluated in the first experiment were: Sacharum officinarum IJ76-470, US72-1319 and SP791011; Sacharum robustum US76-414; Mischantus sp. Fiji 10 (five genotypes). In the second experiment, the tested genotypes were: Sacharum spontaneum CP Dau 849678, NG77-042, NG77-122, and Arundinoid B; Sacharum officinarum Hinahina; Sacharum arundinaceus IJ76-364 and Sacharum sp. Q 45416 (seven genotypes). Origin acronyms: IJ – Purari, Papua New Guinea; US – United States; CP – Canal Point, Florida-USA; NG – Papua New Guinea; RB – Ridesa, SP – Copersucar, Brazil (currently CTC - Centro de Tecnologia Canavieira); Q – Queensland, Australia. The acronyms represent the countries of origin of this germplasm. All plant material was planted at Embrapa Agrobiologia’s local germplasm bank for 12 months before the experiments were initiated. Individual buds (2 cm long) were selected from the stalk and separated by size.

In experiments 1 and 2, the treatments were: the control without inoculation and inoculated plants using the strain BR11145, with six replicates distributed in a completely randomized design. Each pot received two buds, and after 45 days, one was maintained until the final harvest. Three buds of each genotype were also used to measure the nutrient content of the explant used for propagation.

Experiments were conducted using plastic pots filled with 3 kg of soil: sandy substrate 2:1 (v/v). The soil was collected at Embrapa Agrobiologia field station, using the subsoil (below horizon A) of soil classified as Ultissol (Argissolo Vermelho Amarelo), with medium texture (sandy loam) (Santos et al., 2018). Chemical analyses of the substrates were: experiment 1 - pH(H₂O), 6.34; C (%) 0.23; in cmol_c dm^-3 - Al³⁺, 0.00; Ca²⁺, 1.73; Mg²⁺, 0.53; H+Al - 1.25; in mg L^-1, K - 21.10, P - 8.02; and N (%) 0.02. Experiment 2: pH(H₂O), 6.09; C (%) 0.53; in cmol_c dm^-3 - Al³⁺, 0.00; Ca²⁺, 1.47; Mg²⁺, 0.58; H+Al, 0.99; in mg L^-1, K – 16.71, P – 8.47; and N (%) 0.02; analyss using the procedures of Embrapa (1997).

Bacterial preparation and inoculation procedure

The bacterium used in the study was obtained from the CRB Johanna Döbereiner Culture Collection (Seropédica-RJ, Brazil = BR strain): Nitrospirillum viridazoti strain BR11145 (= CBAmC) (Baldani et al., 2024). The strain was cultivated using a DYGS-rich medium (Baldani et al., 2014), of which 10 µL were transferred to tubes containing 5 mL of LGI medium using sucrose as a carbon source, and with a final pH 6.0 with 100 µg L^-1 of yeast extract (Baldani et al., 2014). Cultures grew for 24 h at 170 rpm in a rotary shaker at 30 °C. This culture was transferred to Erlenmeyer of 125 mL containing 50 mL of BP medium (Scheidt et al., 2020). For the inoculation procedure, the bacterial suspension was mixed with a polymerized formula as described by Fernandes et al. (2021) and used to maintain cell numbers. For that, 50 mL of polymeric inoculant was transferred to polyethylene plastic vials (high-density polystyrene PEAD, 0.05 × 0.05 × 0.10 m with screw cap; total volume of 190 mL and useful volume of 150 mL). The inoculant was kept at 20-22 °C in a controlled chamber for at least one week before use in the experiments. Before the plant assay was installed, bacterial numbers were measured by plating counting using LGI solid medium as described by Baldani et al. (2014). Bacterial counting of the inoculant reached: exp. 1 - 4.5 x 10⁸ UFC mL^-1; exp. 2 - 1.1 × 10⁹ UFC mL^-1.

Inoculation of individual buds of each plant genotype was done as described by Santos et al. (2017) by immersing the buds in the inoculant solution diluted in water in a proportion of 1:100 (v/v) for 30 min. Control plants were immersed in water.

Plant measurement

Number of leaves, height (measured from the base until the end of the highest leaf), and stem neck diameter (base of the stem – 1 cm above the soil) were measured using a ruler and digital pachymeter at the final harvest at 109 days after planting (Exp. 1 and 2).

The following parameters were evaluated: fresh and dry mass of shoots and roots, and buds used for plant propagation (initial and final). A complete analysis of the plant tissue and soil was performed to calculate the amount of nutrients accumulated during the period. All plant tissue was dried in a forced-air oven at 65 °C to a constant weight, after which the samples were ground using a Wiley mill, and the total N, P, K, Ca, and Mg concentrations were analyzed (experiments 1 and 2). Chemical analysis of the plant material was made as described in Nogueira et al. (2005). Nutrient concentrations (kg kg^-1 dry mass) were multiplied by the corresponding dry mass of the plant, and the respective amount of each nutrient was expressed in terms of mg pot^-1. Millet (Panicum miliaceum) was seeded in a pot containing 300 g of the same soil used in the experiment to estimate the amount of soil available N to the plants. Ten seeds were added per pot, and plants were harvested after all leaves began green pale to yellow, indicating strong N deficiency. Nitrogen accumulated by millet plants was considered the soil available N to cane plants.

Data analysis

Inoculated versus non-inoculated (control) treatments for each genotype were compared by using the F-test (p<0.05) from ANOVA. In all cases, normality (Lilliefors test) and homogeneity of errors (Cochran’s test 1941) were tested. The analyses were carried out using SISVAR and R-Studio software.

RESULTS

Growth, biomass, and nutrient accumulation of each genotype were compared to inoculated plants with the uninoculated control. Comparisons between genotypes were not performed as each showed a different growth curve. The biometric approach showed differences between genotypes (Table 1). Leaf number varied from 3 to 6 per plant after 109 days of growth. Plant height varies from 7.75 cm for IJ76-470 (S. officinarum) to 79.63 cm for US72-1319 (S. spontaneum). Inoculation improved the plant height of US72-1319 and NG77-122, and the stem diameter, of the first one and Hinahina.

Table 1
Biometric evaluation of 12 sugarcane genotypes belonging to the Saccharum complex and inoculated with Nitrospirillum viridazoti BR11145. Plants were evaluated 109 days after planting

Saccharum species	Genotypes	Inoculation	Leaf number	Height	Stem diameter
			Unit	cm	mm
Miscanthus sp.	Figi 10	Control	5.50	8.25	7.13
		Inoculated	5.25	8.50	6.25
S. arundinaceus	IJ76-364	Control	5.50	8.13	3.88
		Inoculated	5.75	9.25	5.00
S. officinarum	IJ76-470	Control	4.75	10.13	7.63
		Inoculated	5.00	7.75	6.63
	SP701011	Control	5.00	8.00	6.60
		Inoculated	5.50	8.38	6.88
	Hinahina	Control	5.50	13.75	4.88
		Inoculated	6.00	10.50	6.38 *
S. robustum	US76-414	Control	4.50	10.38	6.50
		Inoculated	5.00	9.88	6.75
S. spontaneum	US72-1319	Control	5.75	40.75	5.25
		Inoculated	5.00	79.63 *	7.13 *
	Arundinoid B	Control	4.50	12.88	7.75
		Inoculated	5.00	13.88	7.75
	CP Dau 899678	Control	4.50	13.38	6.75
		Inoculated	4.25	15.48	6.75
	NG77-042	Control	5.00	11.67	4.83
		Inoculated	4.75	14.75	5.25
	NG77-122	Control	4.75	8.00	5.75
		Inoculated	5.00	12.65 *	5.00
Saccharum sp.	Q 45416	Control	4.75 *	9.80	4.88
		Inoculated	3.00	10.50	4.83
	Mean	Control	5.00	12.93	5.99
		Inoculated	4.93	17.51	6.21

• Mean value of six replicates.
• ^*
  F test at p<0.05.

Of the twelve genotypes evaluated, one responded expressively to inoculation by increasing all biomass parameters evaluated and foliar area by 94.9 % (not shown), the US72-1319 with an increment in shoot and root dry mass by 253 and 200 %, respectively (Figure 1b). Inoculation response was lower in the other 11 genotypes, only three presented positive increments in root dry mass, and it was significant only for NG77-122 (Figure 1a). Shoot dry mass (SDM) was reduced upon inoculation treatment in two genotypes, NG77-122 and Q 45416 (Figure 1a). Biomass accumulation between the three S. officinarum and five S. spontaneum showed a lack of growth response in the S. officinarum genus, compared to the S. spontaneum, especially in the shoot fresh and dry mass (Figure 2).

Figure 1
Increment (%) of root and shoot dry mass (RDM, SDM) and foliar area (FA) [(I – NI) / NI × 100] of 11 genotypes belonging to the Saccharum Complex (a) and root and shoot fresh mass (RFM, SFM) dry mass (RDM, SFM) and total mass accumulation (TFM, TDM) of S. spontaneum US72-1318 (b). Inoculation used N. viridazoti BR11145. * Differ at p<0.05 using F-test (n = 6).

Figure 2
Biomass accumulation between S. officinarum (n = 3) and S. spontaneum (n = 5) initial growth inoculated with N. viridazoti BR11145 (INOC).

Nutrient content was influenced by the inoculation (Figure 3). Nitrogen content was increased in 21.6 % of plants of IJ76-364 due to inoculation compared to its control. The increment of K content of Fiji 10 increased by 10.83 and 49.15 % in NG77-122. Phophorus content of IJ76-470 was 50 % higher than the control, and a similar increase (42.1 %) was observed for the Mg content of Hinahina (Figure 3).

Figure 3
Increment (%) of four macronutrient content evaluated in the shoots [(I – NI) / NI × 100] of 12 genotypes belonging to the Saccharum Complex. Inoculation used N. viridazoti BR11145. * Differ at p<0.05 using F-test (n = 6).

The macronutrient accumulation in the shoots of the 12 genotypes accompanied the gains of shoot biomass. Notably, the S. spontaneum US72-1319 genotype presented the most expressive response to inoculation with the BR11145 strain, standing out in all evaluations (Figure 4). We can consider a genotype of great interest in studying positive interaction between the plant and this bacterium. The opposite occurred with CPDau 849678, in which inoculation reduced the N accumulated in the shoots by 27.7 %, and it was observed for NG77-042 with a reduction of 11 %. Hinahina accumulated more K in the control plants. Even though the total amount of P in the aerial tissue was about 10 times less than potassium, inoculation improved its accumulation in three genotypes, one of S. officinarum IJ76-470 (29 %), one of S. robustum US76-414 (18 %), and one of S. spontaneum US72-1319 (107 %), but reduced in other two S. spontaneum, CPDau 849678 and NG77-122. Calcium accumulation was also higher in the control plants of two S. spontaneum, Arundinoid B and CP DAU 849678. Irrespective of the low soil fertility, the order of macronutrient accumulation in the 12 genotypes followed what is expected for sugarcane, with K > N > Ca > Mg > P (data not shown). The experiment was conducted without fertilization, and this very low N content in the soil seemed not to influence the accumulation of the five nutrients.

Figure 4
Increment (%) of five macronutrient accumulations evaluated in the shoots [(I – NI) / NI × 100] of 12 genotypes belonging to the Saccharum Complex. Inoculation used N. viridazoti BR11145. * Differ at p<0.05 using F-test (n = 6).

One important part of the plant is the bud used for germination. As all these genotypes accumulated very low amounts of sugar but possess a high fiber content, it is expected that the biomass of the bud influences the initial growth of the plants. As three buds were also collected to measure the dry mass and nutrient content used to propagate the plants, we can use this data to quantify the contribution of each genotype in nutrients that will be partially mobilized for the plant to grow.

Using the N data collected from the plant analysis, and the soil available N estimated from millet, it was possible to calculate an N balance for both experiments as well as an index called “Inoculant effect” (Table 2). Initial N varies from 7.24 (Hinahina) to 32.82 mg pot^-1 (US72-1319), depending on the plant used for the vegetative propagation of the genotypes. Nitrogen mobilized for new plant tissue (MNPG - shoots, and roots) also differs between the genotypes, with a very low contribution of 1.98 in inoculated Q 45416 to 25.9 mg pot^-1 in IJ76-470 without inoculation. Nitrogen was mobilized differently depending on the biomass accumulation of the genotype, and inoculation did not play a role in this N use, except for US72-1319 (Table 2). As soil available N was the same for all plants to access, and after 109 days the pot was completely explored by the roots, and the rest of the nitrogen was obtained from the original bud N mobilization except for four genotypes, with the respective order of magnitude: Hinahina > US72-1319 > CP DAU 849678 and Q 45416. In the other eight genotypes, this short period and using a substrate with a low content of several nutrients, including nitrogen, the plant growth was slow or even limited.

Table 2
Nitrogen balance of 12 genotypes of the Saccharum Complex inoculated with N. viridazoti BR11145 and evaluated 109 days after planting

Saccharum	Gentotypes	Inoculation	Available	INB	FNB	MNPG	NR	NAT	NR+AT	Ratio MNPG/ NR+AT	INOC effect
Miscanthus sp.	Figi 10	Control	457.56	14.65	4.52*	10.13	3.59	13.46	17.05
		Inoc	457.56	14.65	3.15	11.50	5.08	12.24	17.32	1.68	-0.18
S. arundinaceus	IJ76-364	Control	457.56	8.94	5.69	3.25	3.64	10.88	14.52	1.51
		Inoc	457.56	8.94	4.04	4.90	2.24	12.28	14.52	4.47	-1.50
S. officinarum	IJ76-470	Control	457.56	31.14	5.17	25.97	3.87	12.64	16.51	2.96
		Inoc	457.56	31.14	6.25	24.89	3.92	11.36	15.28	0.64	-0.02
	SP701011	Control	457.56	32.27	7.16	25.11	5.46	11.54	17.00	0.61
		Inoc	457.56	32.27	8.02	24.25	5.81	10.60	16.41	0.68	0.00
	Hinahina	Control	457.56	7.24	3.49	3.75	5.59	13.31	18.90	0.68
		Inoc	457.56	7.24	5.21	2.03	4.55	16.68	21.23	5.04	5.42
S. robustum	US76-414	Control	457.56	32.82	8.57	24.25	5.73	13.66	19.39	10.46
		Inoc	457.56	32.82	9.51	23.31	4.57	15.82	20.39	0.80	0.08
S. spontaneum	US72-1319	Control	457.56	26.76	7.04	19.72	6.44	14.14	20.58	0.87
		Inoc	457.56	26.76	12.68*	14.08	12.33*	44.16*	56.49	1.04	2.97
	Arundinoid B	Control	457.56	20.84	16.56	4.28	10.44	21.13	31.57	4.01
		Inoc	457.56	20.84	17.36	3.48	11.23	16.89	28.12	7.38	0.70
	CP Dau 899678	Control	457.56	14.01	7.44	6.57	6.73	21.53*	28.26	8.08
		Inoc	457.56	14.01	9.98	4.03	7.20	15.57	22.77	4.30	1.35
	NG77-042	Control	457.56	8.94	4.36	4.58	5.07	17.78*	22.85	5.65
		Inoc	457.56	8.94	5.19	3.75	3.83	15.83	19.66	4.99	0.25
	NG77-122	Control	457.56	4.7	5.19	ND	3.96	11.99	15.95	5.24
		Inoc	457.56	4.7	2.60	2.10	4.39	10.94	15.33	ND	ND
Saccharum sp.	Q 45416	Control	457.56	9.48	6.63	2.85	3.95	15.14	19.09	7.30
		Inoc	457.56	9.48	7.50	1.98	4.49	11.48	15.87	6.70	1.32
	Mean	Control		17.65	6.82	11.86	5.37	14.77	20.14	8.02
		Inoc		17.65	7.62	10.03	5.80	16.15	21.95	3.43

• INB: Initial N in the bud; FNB: Final N in the bud; MNPG: Mobilized N to plant growth; NR: N in the root; NAT: N in the aerial tissue; NR + NAT: N in the root plus N in the aerial tissue. #: initial N in the bud was lower than the final; ND: N-balance was not reliable.
• ^*
  F teste at p<0.05 (n = 6).

DISCUSSION

This study aimed to measure the growth promotion of different genotypes belonging to the Saccharum Complex in response to a single application of the bacterium N. viridazoti BR 11145. However, comparing the growth of different genotypes that were not bred for any purpose proved to be challenging within the limited time frame. Even though these materials are robust and effective in utilizing the natural soil fertility of a sandy substrate, plants accumulated between 1.67 to 3.93 g pot^-1 during the assay, except for the US72-1319 genotype that reached 10.15 g pot^-1 in the inoculated plots (Figure 1b). There was also variation in the root dry mass, with an average accumulation of 1.35 g pot^-1 in the control and an additional 22 % in the inoculated treatment, starting with 0.70 g pot^-1 and reaching 4.26 g pot^-1 in US72-1319, followed by 3.25 g pot^-1 of inoculated Arundinoid B genotype.

One reason for this limited influence observed could be attributed to the short duration of the assays. Additionally, using a sandy substrate with a nitrogen content of only 0.02 %, and the absence of fertilizer addition might have further impeded biomass accumulation. Notably, these wild genotypes do not accumulate significant amounts of sugars in their stems, which also affects bacterial survival and subsequently influences bud germination and plant response. To fully understand the factors affecting genotype response in ancestral Saccharum species, it is necessary to address the observed limitations and make improvements in several conditions.

Despite these limitations, the US72-1319 genotype demonstrated a significant 2.97-fold increase in shoot biomass accumulation compared to the control, without altering the N content. It implies that the inoculation of BR11145 did not act by the biological nitrogen fixation process, even the auxin response was not the reason for such a specific growth response of 2.97-fold improvement (Figure 1b; Table 2). Another mechanism must be addressed to prove the expected response of this genotype. This finding presents an opportunity to further explore this genotype using other strains and evaluate the plant performance throughout an entire crop life cycle. As a wild species, little or very little information about its growth is available. Urquiaga et al. (1992) employed the balancing approach to assessing the BNF effectiveness in various materials, comprising various commercial hybrids and an S. spontaneum called Krakatau and one S. barberi namely Chunee. This investigation revealed that Krakatau displayed the highest BNF contribution in non-inoculated plants and the greatest total nitrogen accumulation, surpassing all tested hybrids and S. barberi. These two studies did not utilize inoculation, and all the N derived from the air was attributed to the natural population of diazotrophs associated with this genotype, reaching 70 % of the total N accumulated by the plants. However, when these materials are inoculated with a single strain of diazotrophic bacteria as done in this study, it can modify this intrinsic contribution and ultimately lead to significant biomass gains, even under conditions characterized by extremely limited nutrient availability in the soil (Figure 1 and Table 1).

In general, commercial sugarcane commercial hybrids respond positively to the application of diazotrophic bacteria. Chaves et al. (2015) evaluated the initial development of two sugarcane hybrids inoculated with diazotrophic bacteria and concluded that N. viridazoti BR11145 increased the germination speed index and shoot dry mass in both cultivars. Additionally, they found that the differences in genotypes influenced the sugarcane response to inoculation.

The growth promotion of plants inoculated with different strains of diazotrophic bacteria can be attributed to various factors. A comparison of bacterial endophytes present in the roots of four Saccharum ancestral including S. robustum and S. spontaneum showed that these two species harbor different microbiomes than S. officinarum, S. barberi or two commercial hybrids collected in China (Dong et al., 2018). These findings supported that sugarcane germplasm influences the bacterial community, and somehow the bacterial response to an exogenous inoculated species.

Several previous studies have suggested that plant productivity increases are because of these bacteria acting on the solubilization of phosphorus (Crespo et al., 2011; Bernabeu et al., 2016), zinc, and other nutrients (Glick et al., 2012) besides others; but one of the most easily identifiable factors, especially for species of the genus Azospirillum and related bacteria like Nitrospirillum, is the production of auxin (Cassán et al., 2014; Chaves et al., 2015). In this study, the P accumulation was one of the four nutrient accumulations in the shoots with a higher influence of the inoculation using BR11145, which improved its accumulation of the shoot of US76-470 (29.3 %), and US76-414 (18.6 %), besides US72-1319 (107.9 %) (Figure 4).

In a previous study conducted by Santos et al. (2017), the growth of pre-germinated sugarcane roots inoculated with diazotrophic bacteria was evaluated. Researchers observed that the inoculation with five selected strains of diazotrophic bacteria resulted in improved initial growth and seedling production. Similarly, Gírio et al. (2015) found that pre-sprouted sugarcane seedlings of RB867515 inoculated with the same five strains of diazotrophic bacteria, including BR11145, exhibited a positive physiological effect on the initial growth of the plants. Although the inoculant used did not increase the dry mass of the roots, it did promote greater root length. Three of the 12 genotypes tested also improved the root dry mass, especially the NG77-122 (50 %), followed by CPDau 849678 (21.6 %) and Fiji 10 (18.3) (Figure 1a). None of the S. officinarum genotypes tested improved the root and shoot fresh and dry mass, even foliar area (Table 1), one normal growth promotion effect observed in the new commercial hybrids tested by others (Chaves et al., 2015; Gírio et al., 2015; Santos et al., 2017). This lack of growth response was observed also in the shoot accumulation, although the S. officinarum accumulated 76 % more fresh shoot mass (FSM) than the S. spontaneum genotypes in the control plants and 43% more when the plants were inoculated with BR11145 (Figure 2).

Among the species within the Saccharum complex, S. spontaneum is notable for its widespread presence in the wild (Tai and Miller, 2002). This species is likely to possess the greatest potential for genetic variation, making it well-suited to adapting to challenging environments for biomass production (Aitken and Mcneil, 2010). This genetic variability grants the plant significant adaptability, which may explain the superior growth response observed during the initial 109-day phase, particularly in genotypes like US72-1316, CPDau 849678, and NG77-122. Among the species evaluated in this study, S. spontaneum holds the most promise for introducing genetic variation in closely related sugarcane species and is commonly used in varietal crosses. Therefore, incorporating wild cane into sugarcane breeding programs has the potential to enhance drought tolerance (Munawarti et al., 2014). One reason for the rusticity could be attributed to a higher capacity to associate with beneficial bacteria. These findings provide some insights into the plant’s growth response.

CONCLUSION

Growth and nutrient accumulation by the 12 genotypes belonging to the Saccharum complex is poorly stimulated by applying Nitrospirillum viridazoti strain BR11145. Genotypes belonging to the S. spontaneum species, especially genotype US72-1319 improved biomass, nutrient accumulation, and leaf area when inoculated with BR11145.

ACKNOWLEDGMENTS

The authors express their gratitude to the Coordination of Improvement of Higher Education Personnel – CAPES (Grant n. 001) of APL da S. Finep [grant number 01.13.0295.00] and fellowships of LFC da S, BJRA and VMR. To FAPERJ – fellowship CNE of BJRA and VMR.

• How to cite: Silva APL, Silva LFC, Alves BJR, Reis VM. Initial growth response of twelve Saccharum Complex genotypes to inoculation using Nitrospirillum viridazoti. Rev Bras Cienc Solo. 2025;49:e0240056. https://doi.org/10.36783/18069657rbcs20240056

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Edited by

• Editors:
  José Miguel Reichert https://orcid.org/0000-0001-9943-2898 and Marco Aurélio Carbone Carneiro https://orcid.org/0000-0003-4349-3071

Publication Dates

• Publication in this collection
  21 Feb 2025
• Date of issue
  2025

History

• Received
  20 Mar 2024
• Accepted
  19 Aug 2024