Introduction

Plants require nitrogen in great quantities because this element is an essential component of proteins, nucleic acids, and other cellular constituents (^{Shridhar, 2012}). The elevated nitrogen quantity in the Earth atmosphere (close to 80% in N₂ gaseous form) cannot be used directly by the biological systems in the production of the necessary chemical compounds for their growth and reproduction (^{Shin et al., 2016}). The reduction of N₂, commonly known as “nitrogen fixation” is performed chemically or biologically (^{Shin et al., 2016}). Biological nitrogen fixation (BNF) is one of the most important processes that provides the greatest external N source for the different ecosystems and agroecosystems and constitutes an important option for recovery and maintenance of soil fertility (^{Figueiredo et al., 2013}). Some of the bacteria that live in soil are called free-living because they do not depend on plant root exudates for their survival (^{Sharma et al., 2014}). The microorganisms that perform BNF are a limited group of free-living symbiotic bacteria called diazotroph, which have the capacity of reducing and transforming atmospheric nitrogen (N₂) to ammonium (NH₄ ⁺), a form of readily assimilated nitrogen for plants (^{Bano and Iqbal, 2016}). Among the free-living nitrogen-fixing bacteria (FLNFB), some are obligate or facultative anaerobic, such as Azospirillum sp., Clostridium pasteurianum, Klebsiella spp., Desulfovibrio sp.; other obligate aerobic, such as Azotobacter spp., Beijerinckia sp., and photosynthetic, such as purple sulfur, non-sulfur bacteria, and green sulfur bacteria (^{Allan and Graham, 2002}). Some FLNFB may promote plant growth by means of synthesis and the release of phytohormones, such as auxins, gibberellins, cytokinins and abscisic acid (^{Hernández-Rodríguez et al., 2014}). Some Azotobacter strains, for example, have the capacity of producing amino acids that promote plant growth; they are also capable of producing siderophores, which may provide iron to plants (^{Jnawali et al., 2015}). The results of previous studies on incorporating FLNFB to soil have shown high yields in a great diversity of crops, such as rice, maize, bean, and tomato, minimizing the use of chemical fertilizers, especially nitrogenous (^{Hernández, 1998}; ^{Bonilla et al., 2000}; ^{Meunchang et al., 2005}). Some reports have shown that inoculation with Azospirillum lipoferum in rice plants under greenhouse conditions increased yield above 6.7 g plant^-1 (^{Mirza et al., 2000}). Cotton plant height and dry biomass increased with Azospirillum brasiliense inoculation under greenhouse conditions (^{Bashan, 1998}).

In Mexico, agave plants have had and still have a great economic and cultural importance for numerous indigenous and mixed-race populations that have used them for 7000 years as source of food, medicine, fuel, shelter, ornament, fiber, manure, housing construction, agricultural tool production, and mainly in the production of different types of alcoholic beverages, such as mezcal and tequila (^{García-Mendoza, 2007}). Recent studies have indicated that agave plants also have a high potential for fixing carbon (^{García-Moya et al., 2010}; ^{Núñez et al., 2010}). In the State of Oaxaca, around nine agave species are used, of which Agave angustifolia Haw. is the one with the greatest demand and the only one cultivated significantly in semi-arid soils, where approximately 8422.7 ha are cultivated with this agave (^{OEIDRUS-SAGARPA, 2011}). The other eight agave species were collected from wild or semi-cultivated populations, mainly in living fences with little or null cultural management. Such is the case of Agave potatorum Zucc., colloquially known as maguey papalomé or maguey tobalá, a species whose mezcal is highly demanded (^{Sánchez-López, 2005}) and distinguished for its top quality because of the proportion of the volatile aromatic compounds it contains (^{Vera et al., 2009}). In 2018, 5.9 million L of mezcal were produced at national level. Oaxaca contributed with 92.3% of this production, of which 3.3% was made with A. potatorum (^{CRM, 2019}). This agave develops in low fertility arid and semi-arid soils, estimating 42 ha of such species in the regions of Valles Centrales and Sierra Sur of Oaxaca, which represents 0.45% of the total plants of the genus Agave that exist in the state (^{OEIDRUS-SAGARPA, 2011}). Because it is a wild species, not much information is available with respect to its agronomic management; thus it is necessary to carry out research in different aspects, including its nutrition within an agro-ecologic context. The analysis of sugars in the stems or agave “piñas” (stem without leaves) is important because the alcohol obtained in the fermentation depends on the quantity of reducing sugars. In the tequila and mezcal distilleries, two type of analyses are made, such as measuring °Brix (°Bx) degrees and determining reducing sugars by using Fehling reagent produced in the laboratory. °Brix represents an arbitrary scale to measure sugar densities in solutions and is equivalent to the percentage in weight of the soluble solids in one sample, which are mainly sugars. For example, the stems without leaves (piña) of A. tequilana should have at least 24% of total sugars to be considered of good quality (^{Bautista-Justo et al., 2001}). In this study, the effect of FLNFB on plant growth and total solid soluble content of the stem (SSC) in plants of A. potatorum under semi-controlled conditions was evaluated.

Materials and Methods

The experiment used three FLNFB strains, previously isolated from the rhizosphere of A. angustifolia cultivated in the District of Tlacolula, Oaxaca, Mexico in the communities of Magdalena Teitipac (16° 54´ N and 96° 33´ W), San Baltazar Guelavila (19° 80´ N and 96° 29´ W) and San Juan del Río (16° 53´ N and 96° 09´ W). Based on the Analytical Profile Index (API20 NE, bioMérieux, Inc., Durham, NC, USA) system, the identity of Burkholderia cepacia was confirmed with the phenotypical characteristics of gram negative bacteria, large white-colored colonies, entire borders, circular mucoid shape with yellow pigments in the center; Flavobacterium sp., gram negative bacteria, yellow-colored colonies with entire borders, circular shape with greenish excreted pigment; and Paenibacillus amylolyticus, gram positive bacteria, translucid colonies with entire borders and circular shape. These FLNFB were selected because of their high efficiency to fixate atmospheric N in vitro by quantifying the NH₄ ⁺ ion. The diazotrophic capacity obtained was 1.10 µg N-NH₄ ⁺ mL^-1 for B. cepacia; 1.54 µg N-NH₄ ⁺ mL^‑1 for Flavobacterium sp.; and 1.68 µg N-NH₄ ⁺ mL^-1 for Paenibacillus amylolyticus.

Results and Discussion

The ANOVA and multiple comparison of means test (Tukey, P ≤ 0.05) revealed that with respect to control, B. cepacia increased RV 322.2%, ULN 42.6%, and SSC 72.9%; P. amylolyticus increased SDB 317.1% (Figure 1). B. cepacia, Flavobacterium sp., and P. amylolyticus increased PSD approximately 50.3% (Figures 1 and 5), ROD 48.6%, LA 127.2%, and PH 51.8% (Figures 2 and 3). Flavobacterium sp. increased TB 164.8% (Figure 2).

Different small letters on each bar indicate the inoculant effect (Tukey’s test, P ≤ 0.05).

Figure 1: Mean value ± standard error in some growth variables and total solid soluble content of the stem or “piña” (stem without leaves) of the Agave potatorum Zucc. plants inoculated with free-living nitrogen-fixing bacteria in semi-controlled environments after 48 weeks of assessment.  

Different small letters on each bar indicate the inoculant effect (Tukey’s, test P ≤ 0.05).

Figure 2: Mean value ± standard error in some plant growth variables of Agave potatorum Zucc. inoculated with free-living nitrogen-fixing bacteria in semi-controlled environments after 48 weeks of assessment. 

Divisions are in scale = 1 mm.

Figure 3: Effect of free-living nitrogen-fixing bacteria on plant height of Agave potatorum Zucc. in semi-controlled environments after 48 weeks of assessment. 

From the assessed growth variables, RD was the only one that did not show statistically significant changes among the inoculated plants and the control group. The increase in agave plant growth variables could be attributed to different mechanisms performed by the FLNFB previously reported as plant growth promoters (^{Requena et al., 1997}); those found are (1) increase in elongation and cellular multiplication because of a better nutrient absorption as N; (2) phytohormone production, among those, auxins, cytokinins, gibberellins, and abscisic acid; (3) siderophore production, which may provide iron to plants; (4) enzyme synthesis, such as 1-aminocyclopropane-1-carboxylate deaminase, which decreases ethylene levels in plants (^{Lucy et al., 2004}; ^{Podile and Kishore, 2006}; ^{Appanna, 2007}).

The RV (Figure 4), ULN, and LA in agave plants increased with B. cepacia inoculation. The results that agree with those established by ^{Argüello-Navarro and Moreno-Rozo (2014)}. These authors inoculated cacao plants with FLNFB, such as Burkholderia sp., and after 120 days of nursery assessment, they found increments in leaf number, LA, TB, and root length in plants inoculated compared with the control group. Similarly, ^{Roesch et al. (2005)} evaluated the inoculation effect of diazotrophic bacteria from the genus Azospirillum in wheat and observed an increase in size and number of radicle hair compared with the control group. Numerous studies have shown that diazotrophic bacteria may promote plant growth, mainly by changing the root system morphology, improving development and productivity of several crops of economic interest (^{Bashan et al., 2004}; ^{Somers et al., 2004}).

Divisions in scale = 1 mm.

Figure 4: Effect of free-living nitrogen-fixing bacteria (FLNFB) on root volume of Agave potatorum Zucc. in semi-controlled environments after 48 weeks of assessment. 

Figure 5: Effect of free-living nitrogen-fixing bacteria on stem or “piña” (stem without leaves) diameter of Agave potatorum Zucc. in semi-controlled environments after 48 weeks of assessment. Division in scale = 1 mm. 

Flavobacterium sp. increased TB of agave plants; other studies have also reported that inoculation with FLNFB, such as Herbaspirillum seropedicae in rice plants, increased TB in 71.5% with respect to the control group. Under the conditions of pot cultivation, the bacteria of the genus Paenibacillus sp. RFNB4 increased the PH and TB of canola (Brassica campestris) plants significantly in 42.3 and 29.5%, respectively (^{Islam et al., 2009}). ^{Kifle and Laing (2016)} found that inoculation with diazotrophic bacteria of the genus Bacillus, Pseudomonas, and Enterobacter in maize plants under greenhouse conditions, increased TB when compared with those non-inoculated. ^{Govindarajan et al. (2006)} assessed inoculation with Burkholderia vietnamiensis MG43 in the varieties Co 86032 and Co 86027 of micro-propagated sugar cane and found increments of 20 and 19% in the production of dry matter of Co 86032 and Co 86027, respectively.

Some authors have reported that when plants established these associations with endophytic fungi or FLNFB, their sugar content and development increased (^{Obledo et al., 2003}; ^{Ruiz et al., 2011}). ^{De la Torre-Ruiz et al. (2016)} inoculated Agave americana plants with Rhizobium daejeonense, Acinetobacter calcoaceticus and Pseudomonas mosselii and found positive effects on TB, PSD, leaves number, root length and sugar content in stems without leaves (piñas) when compared with those non-inoculated agave plants. These results agree with those reported in this study where B. cepacia inoculation increased SSC (°Bx) in agave stems (piñas). The °Brix are equivalent to the percentage in solid soluble weight of one sample, which are mainly sugars (^{Bautista-Justo et al., 2001}). The sugar content in agave plants may also vary with the plant physiological stage (^{Arrizon et al., 2010}). The young A. tequilana plants, for example, possess higher levels of monosaccharides (e.g. glucose, fructose) than adult plants, which accumulate fructans starting from 8-10 years of age (^{Cedeño, 1995}).

Conclusions

- Inoculation with free-living nitrogen-fixing bacteria in Agave potatorum plants had a positive effect on the evaluated plant growth variables of 90%. Burkholderia cepacia increased the root volume 322.2%; number of unfolded leaves 42.6%; and solid soluble content of the stem 72.9%. Paenibacillus amylolyticus increased stem dry biomass 317.1%. B. cepacia, Flavobacterium sp. and P. amylolyticus increased plant stem diameter approximately 50.3%; plant rosette diameter 48.6%; leaf area 127.2%; plant height 51.8%. Flavobacterium sp. increased total biomass 164.8%.

- The results in this study suggest that these bacteria are capable of promoting A. potatorum growth. Therefore, they can be an eco-friendly and economic technology for the production of this agave species in the State of Oaxaca, Mexico and other places with similar management conditions.