IN VITRO COINOCULATION BETWEEN ACTINOBACTERIA AND DIAZOTROPHIC NODULATING BACTERIA FROM THE SEMIARID

Purpose: To evaluate the potential of actinobacterial strains from the Brazilian semiarid to establish facilitation relationships with native rhizobia from the same region. Theoretical framework: The study is based on the ecological and biotechnological importance of soil actinobacteria, producers of biosurfactants and enzymes, and of nitrogen-fixing rhizobia in legumes. Method: 50 strains of actinobacteria were isolated from soils from Ceará with different levels of anthropization and 19 strains of rhizobia using cowpea. The morphological, cultural and micromorphological characterization of the strains was performed, as well as the evaluation of their enzymatic profiles. In vitro facilitation tests were conducted between cellulolytic actinobacteria and non-cellulolytic rhizobia. Results: The soil areas presented a similar composition of actinobacteria, but strains from the anthropized area showed higher enzymatic activity. Two Streptomyces strains promoted the growth of non-cellulolytic rhizobia in vitro , indicating potential application as bioinoculants in microbial consortia. Conclusions: The study contributes to the knowledge of the interaction between beneficial microbial groups from the semiarid region and their possible biotechnological use in agriculture. Originality/value: Works on coinoculation between actinobacteria and rhizobia from semiarid soils are scarce.


INTRODUCTION
The beneficial microorganisms present in agricultural systems play an important role, helping to increase production and reducing the use of chemical fertilizers, pesticides and herbicides.These benefits are made possible through phytohormone production (Velrajan and Bhagawati, 2018), siderophore production (Kumar et al., 2018), phosphorus solubilization (Saeid et al., 2018) and nitrogen fixation (Pinheiro et al., 2019).
Microorganisms can be affected by the use and characteristics of soil, plants present and environmental factors such as temperature, humidity and nutrient availability, modifying the composition, interaction and enzymatic production of the microbial community (Xu et al., 2014).In research carried out by Houfani et al., (2019) it was observed that forest and garden soils present greater microbial diversity, and higher endocellulose activity, as well as higher lignocellulose decomposition potential, when compared to agricultural and desert soils.Amongst these microorganisms, actinobacteria and rhizobia stood out.
Actinobacteria, in addition to diversity and ecological importance, have a unique potential due to secondary metabolism and the ability to produce a multitude of bioactive molecules with extensive medical, industrial and agricultural applications (Lewin et al., 2016), while rhizobia are recognized for their ability to produce multiple enzymes, which allow the use of different substrates (Menéndez et al., 2016).
Diazotrophic bacteria are present in the form of bacilli, aerobic, mobile, gram-negative and can be found both in free form and associated with legumes.The benefits that rhizobia can bring to agriculture are widely reported, such as nitrogen supply by symbiotic fixation of N2, which is the most important source of N in agroecosystems because it is renewable and environmentally sustainable (Zheng et al., 2019).Some mechanisms assist in this symbiosis, and the importance of the cell wall degradation enzyme cellulase in the primary infection process and its importance in the secondary symbiotic infection and in the strict regulation of its production to establish an effective symbiosis is reported (Robledo et al., 2015).
Actinobacteria are Gram-positive, filamentous bacteria, found abundantly in the soil, can interact with plants through nitrogen fixation, hormone production, and protection against infection (Singh and Gaur, 2016); in addition, the degradability of actinobacteria is important for the carbon cycle and humus formation in the environment, which increases soil nutrition for plant growth.They also actively participate in the degradation of natural lignocellulose, as a result of their ability to synthesize extracellular polymer hydrolases, such as cellulases, hemicellulases, xylanases, chitinases, pectinases, amylases, peptidases, proteases, and keratinases (Saini et al., 2016).
Cellulase is the most abundant organic polymer on earth, present in plant cell walls, and is mainly found in combination with hemicellulose and xylans.The degradation of cellulose is an essential ecosystem service and has a key value for a greater understanding of the breakdown of cellulose in the carbon cycle.Efficient cellulose degradation requires a wide range of cellulolytic enzymes (de Vries et al., 2015).
The joint inoculation of plant growth-promoting actinobacteria, rhizobia, and rhizobacteria results in increased uptake of nitrogen, phosphorus, and potassium, improving soybean yield and soil quality (Amule et al., 2018), while the symbiosis between Bradyrhizobium and Streptomyces griseoflavus promotes significant increases in plant growth, nodulation, nitrogen fixation, NPK uptake, and seed production in mung beans and soybeans (Htwe et al.,201 9).
This research has the hypothesis that actinobacteria coming from the semi-arid region have the capacity to act in facilitating, in vitro, nodulliferous diazotrophic bacteria coming from the same biome.The objective was to compare the ability of actinobacteria, isolated from semiarid soils under different anthropization conditions, to establish positive interrelations with nodulliferous diazotrophic bacteria.The results obtained help to clarify the positive interactions that occur in the soil community, as well as highlighting the potential of the joint use of strains of actinobacteria and rhizobia as bioinoculants futures.

Place of Study
The strains of actinobacteria were isolated from the soil originating from the Do Not Leave Me, Farm, located in the municipality of Quixadá, Ceará -BR.The geographical location is 4º49'34" S and 38º 58'9" W, with 210 m altitude.The climate of the municipality of Quixadá is identified as Tropical Hot Semi-arid (BSh, according to Köppen-Geiger Climate Classification), with average rainfall of 717.5 mm, centralized in the period from February to April, and average temperature of 26.6°C.The farm soil is classified as Argissolo, equivalent to Lixisol (WRB -FAO, 2014).The soil sampling was carried out in two areas of the farm, an anthropolized area lying fallow and a conserved area, belonging to the Private Permanent Heritage Reserve (RPPN) recognized by the Brazilian Institute for the Environment and Renewable Natural Resources (IBAMA).

Micro-Organisms
The strains of actinobacteria were isolated from the soil by the spread plate technique, using the medium casein dextrose agar (CDA) (Clark, 1965).The rhizobium strains were isolated using caupi beans [Vigna unguiculata (L.) Walp.] as a bait plant and after this period the nodules obtained were macerated in the middle yeast mannitol agar (YMA) (Vincent, 1970).The isolates obtained were authenticated by planting and inoculating the caupi bean.The 50 strains of actinobacteria, 22 coming from the anthropized area and 28 from the conserved area, and 19 strains of rhizobia are deposited in the Culture Collection of the Environmental Microbiology Laboratory of the Federal University of Ceará (UFC).

Soil Chemical Analysis
Chemical analysis of soil was performed according to Teixeira et al. 2017.The soil attributes assessed were pH, which was measured in H2O (1:2,5) by potentiometry; potential acidity (H+Al), which was extracted with buffered calcium acetate at pH 7,0 and determined by titrometry; Ca 2+ and Mg2+,which were extracted with KCl 1M solution and determined by atomic absorption spectrometry; exchangeable aluminum (Al 3+),which was extracted with KCl 1M solution and determined by titrometry.Phosphorus (P), sodium (Na+) and potassium (K+) were extracted with Mehlich 1 solution, with P determined by colorimetry and K+ and Na+ by flame photometry.

Cultural Characterization
Chromogenic characterization was performed by observing aerial and reverse mycelia after the growth of strains in petri plates, according to Wink (2012), with the use of the color chart RAL, and the classification of colonies according to Augustine et al., (2013), in velvety, concentric, radial, umbonate and convex.Micromorphological characterization was carried out by means of microcultivation according to Kern & Blevins (2003), with modifications.The culture was inoculated on one side of a cube of medium CDA contained in a slide and covered with a lamina, along with cotton moistened and then incubated for 14 days at 28ºC, after which the lamina was relocated to a new slide, colored with cotton blue and observed under a Zeiss light microscope at a magnification of 1000.

Enzymatic Profile
The production of amylase (Alariya et al., 2013), xylanase (Kumar et al., 2012) and cellulase (Couri and Farias 1995) was evaluated, where the degradation of the compounds: starch, xylans and cellulose contained in the medium was observed.Actinobacterial strains were incubated at temperatures of 28°, 39°, 41°, 43° and 45 °C to determine the effect of temperature on enzyme activity.
The enzymatic index (IE) was calculated using the equation: IE = Diameter in mm of the hydrolysis halo/ Diameter in mm of the colony halo.

Facilitation in Vitro
The strains of actinobacteria and rhizobia were separately inoculated in a culture medium containing carboxymethylcellulose as the sole source of carbon and energy (Couri and Farias 1995), the presence of hydrolysis zones around the colonies was recorded as a positive response.Each experiment was carried out independently.The strains of actinobacteria that presented a statistically distinct enzymatic index along with the strains of non-cellulolytic rhizobes were selected for the facilitation test.
Actinobacterial strains were separately inoculated into carboxymethylcellulose agar in spots and incubated at 28 °C for 10 days.One milliliter of culture of each rhizobium strain previously cultivated in YM medium for seven days was transferred to microtubes, centrifuged at 9659.52 G for 10 minutes and the precipitate resuspended twice.One drop of each purified rhizobium culture was distributed around each actinobacterium colony at a distance of 2 cm.It was considered a positive result when the growth of rhizobium colonies occurred.
The Compatibility Index was calculated with the number of matched pairs detected divided by all possible pairs: CI = number of matched pairs / all peer interactions.

Statistical Analysis
All tests were performed in quadruplicate, with two trials, totaling eight repetitions.Qualitative data on the characterization of actinobacteria and rhizobia were submitted to the Chi-square test.The normality of the data was evaluated using the Kolmogorov-Smirnov test and the homogeneity of variance using the Levene test, made to verify the assumptions of the applied statistical tests.The enzyme indices were submitted to a Studart T-test for unpaired data to compare the mean values of each area and a variance analysis to compare the means between enzymes and between strains, using Tukey's test as a post hoc.
Data on the effects of temperature on enzyme activity were evaluated using Pearson's Correlation Coefficient.A 95% confidence interval was established.

Soil Properties
The soil in the two areas presented a low pH, classifying them as strongly acidic soils, and a low cation exchange capacity (T), the anthropized area fits into low fertility and susceptible to desertification (Table 1).

Characterization
The 50 selected strains were characterized culturally by the color of the aerial and reverse mycelium, where, in both areas, there was a hegemony of the colors white and gray in the aerial mycelium, while in the reverse mycelium there is a primacy of the color yellow in the anthropized area and gray in the conserved area.In the two areas there was a domain of radial texture and of the genus Streptomyces (Table 2).There was no significant difference between the strains of the two areas analyzed in the characteristics morphology (p = 0.307), color of the aerial mycelium (p = 0.396), color of the reverse mycelium (p = 0.066), texture (p = 0.907) and genus (p = 0.851).--------------------------cmol c kg -1 ------------------------------------%---------  In this work, 50 strains of actinobacteria were isolated, of which 22 come from the anthropized area and 28 from the conserved area.The diversity of genera found did not show any difference between the areas (Figure 1; p = 0.851), with a predominance of the genus Streptomyces in both, and the genus Actinomadura, identified only in the conserved area.8 The 19 strains of rhizobia plus the seven standard strains were analyzed through phenotypic characteristics, where 78% neutralized or alkalized the culture medium and 67% showed butyric or gummy consistency, 85% of the strains were tolerant to high temperatures and high concentrations of salts, but in relation to pH despite tolerating well the alkalinity showed susceptibility to acidity of the medium (Table 3).By comparing the 16S rRNA sequences of bacteria with those available in the GenBank® database, 18 strains were identified as Bradyrhizobium while one was identified as Rhizobium tropici [38].The strains did not show any statistical difference between them in the characteristics analyzed (p= 0.395).The rhizobia strains showed intrinsic resistance to several antibiotics and 14 strains (eight strains isolated and six standard strains) were capable of producing the amylase enzyme.However, no strain was able to produce xylanase or cellulase (Table 4).The strains that did not show the capacity to produce cellulase were selected for the facilitation tests with the actinobacteria.

Enzymatic Profile
Among the strains of actinobacteria analyzed, 47 showed amylolytic activity (94%), 37 showed xylanolytic activity (74%) and 32 showed cellulolytic activity (64%).There was no statistical difference in cellulolytic activity (p = 0.360) between the two areas, while amylolytic activity (p = 0.000) and xylanolytic activity (p = 0.005) were higher between the strains of the anthropized area (Figure 2).The less expressed enzymes were amylolytic and cellulolytic, while the xylanolytic showed the highest values of Enzymatic Indices (EI), peaking at 5.8 and standing out from the other activities statistically (p = 0.000).

Rhizobium Isolates Antibiotics
Cellulase Amylase Xylanase In Vitro Coinoculation Between Actinobacteria and Diazotrophic Nodulating Bacteria from the Semiarid 10 The production of the enzyme cellulase and amylase showed a low positive Pearson correlation coefficient, r = 0.133 and r = 0.172 respectively (p = 0.000), according to the temperature increase, while the enzyme xylanase showed a very strong negative correlation coefficient (r = -0.703,p = 0.000) (Figure 3), only three strains were able to present xylanolithic activity at the temperature of 45ºC (QX 15 -Nocardia, QX 1 9 -Streptomyces, Qx 29 -Nocardia) all coming from the conserved area.

Facilitation in Vitro
No strain of rhizobium was able to produce cellulase (Table 4), while 32 strains of actinobacteria showed a cellulose degradation halo.The enzymatic index (EI) was calculated by the ratio between the diameter of the halo and the diameter of the colony, and the result was submitted to an analysis of variance (p = 0.000), where two strains stood out in relation to the others, QX 59 and QX 67.
The strains QX 59 and QX 67 belong to the genus Streptomyces and come from the anthropized area and were selected to be cultivated together with the 19 strains of rhizobia that did not show any production of cellulase.The results obtained revealed 17 positive relations.
The rhizobia L20 (B.yuanamingense) and L27 (B.iriomontense) that did not show facilitation activity with the QX 59 strain (Streptomyces) were capable of showing this activity with the QX 67 strain(Streptomyces), while the rhizobia L9 (Rhizobium tropici), L14 (Bradyrhizobium sp.) and L15 (B.japonicum) presented facilitation activity QX 59 strain only (Figure 4).This lack of facilitation can be attributed to the low intrinsic resistance to antibiotics shown by these rhizobia strains, since in vitro tests of the 15 antibiotics tested showed sensitivity to ten.
In relation to soil fertility, the conserved area has a higher concentration of nutrients for plants and organisms, expressed by a greater sum of bases (SB) and the capacity of cation exchange (T), its limitation is in acidity (greater H + Al).Although the low pH may decrease the availability of nutrients, causing the anthropogenic area to appear to be more fertile than the natural one, this effect is due to the correction of the pH and preparation of the soil.
The formation of all types of aerial hyphae of actinobacteria depends on the characteristics of the species, nutritional conditions or environmental factor, so there is this difference in color expression between the areas, but chromogenesis is not sufficient for the classification of the species, it is necessary to add parameters such as morphology of the spore chain, spore shape and colony, which are basic for the taxonomy of the genera (Thampi & Bhai, 2017).The results found coincide with those expected for arid environments, as found in the desert in the plateau of Tibet (Ding et al., 2013), presented white, gray, yellow and brown colors, with predominance of the genera Streptomyces, Micromonospora and Streptosporangium, while in soils of the Brazilian semi-arid there is presence of the genera Streptomyces and Nocardia (Silva et al., 2019).
Locally isolated strains have the capacity to withstand the environmental stresses present, which could otherwise limit their productivity.Rhizobes isolated from Egypt by Yanni et al., (2016) were used for biofertilizer production and provided significant increases in seed yield and in the agronomic efficiency of beans (Phaseolus vulgaris) under field conditions.Ali et al., (2019) isolated strains of soy nodules in Bangladesh and found that the local strains were effective and resistant to conditions of physical stress, pH, salinity and temperature.
The selection of antibiotic-resistant rhizobia strains results in greater competitive capacity.Anand et al., (2012) showed that inoculation of soybeans with antibiotic-and phageresistant Bradyrhizobium has a high nitrogen fixation capacity, providing increased soybean production in Indian soil.
The antagonism between rhizobes and actinobacteria has been studied for some time (Jha et al., 2020), so a preliminary study was needed to assess the intrinsic resistance of strains to antibiotics.Among the enzymes analyzed, the QX 67 strain (Streptomyces) originating from the anthropized area showed high enzyme indices in all three activities.The strains where the highest levels of enzymatic index were observed, for each activity, in their majority come from the anthropogenic area, this occurs because the release of enzymes in the soils is related to the metabolic demands and available nutrients, more than the diversity of organisms.
The capacity of microbial communities to maintain functional diversity through disturbance, stress or succession may be more important for ecosystem productivity than taxonomic diversity, this reflects in cellulolytic, amylolytic and xylanolytic activities that are strongly influenced by nutrient deprivation (Chroni et al., 2009) In addition, the physical-chemical characteristics of the soil may influence the production of xylanase by fungi, bacteria and actinobacteria (Ramanjaneyulu et al., 2017), where the highest concentration of these organisms occurred in a soil with pH 5.1, consistent with the results found, where the conserved area has a more acidic pH and presented a lower concentration of strains of actinobacteria producing the xylanase enzyme.
The origin of actinobacteria influences their ability to produce functional enzymes under high stress conditions, strains originating in warmer environments, such as arid and semi-arid, have a capacity to produce enzymes even when submitted to high temperatures (Nithya et al., 2017).Chroni et al. (2009) observed that there is an increase in the halo indicative of cellulose degradation with the increase in temperature, as well as Minotto et al. (2014) which observed the same for cellulase and amylase enzymes.According to Nithya et al., (2017) enzyme production will depend mainly on strain, composition of the environment, cultivation methods, growth, nutritional requirements, pH, temperature and incubation time.
The optimal temperature for enzyme production of actinobacteria can vary depending on the origin, e.g.Chaudhary and Prabhu (2016) described 55ºC as optimal temperature for amylase and cellulase production.While working with actinobacteria from marine environments, they reported that the optimum temperature for amylase production is between 25 and 30ºC, with a decrease in production when it passed this range (Krishnakumar et al., 2015).
Despite reports in the literature about actinobacteria that show xylanolytic activity at temperatures up to 80ºC (Rahmani et al., 2018), the strains studied showed strong sensitivity to temperature changes, only five strains showed activity at a temperature of 39ºC.Sanjivkumar et al., (2017), where strains of actinobacteria produced xylanase up to 40º C, decreasing after that temperature.
Many extracellular metabolites are produced by strains of actinobacteria, especially Streptomyces, which can suppress phytopathogens and act as regulators in plant growth (Saif et al., 2014) These characteristics, the ability to produce various enzymes and better performance at high temperatures, make actinobacteria ideal candidates to be used as microbial inoculants for agricultural use and in the recovery of degraded areas.
The compatibility index of the two strains of actinobacteria, QX 59 and QX 67, was 0.89 and 0.84 respectively, showing that there was no difference in the capacity to act as facilitators for the rhizobium strains, but it is a high index, confirming the hypothesis that these microorganisms have the capacity to act in facilitating in vitro nodulous diazotrophic bacteria.
The use of microbial consortia is an emerging field that allows microorganisms to perform complex functions that are impossible for a single organism and provides solutions to environmental stress factors.The co-inoculation of two different bacterial species has stronger effects on plant growth than the inoculation of a species, suggesting that the synergistic functions of multiple strains are more effective in plant-bacterial interactions (Santiago et al., 2017).
The use of actinobacterial co-inoculation with rhizobia is still incipient, but has been documented with promising results.Soe and Yamakawa, (2013), showed that co-inoculation of Bradyrhizobium yuanmingense and Streptomyces griseoflavus caused increased nodulation, nitrogen fixation and seed yield in different varieties of soybean, while LePlexet al., (2016) reported thatSinorhizobium meliloti co-inoculated with Streptomyces, inoculated in alfalfa, increased the plant's dry weight by up to 30%.
The study of antagonistic interactions between actinobacteria and rhizobia has been widely reported (Lima et al., 2017), but there are few reports of a synergistic relationship between these groups.In addition, species of actinobacteria and rhizobia from the same environment interact among each other and over time promote coexistence, with both antagonistic and mutualistic interactions (Silva et al., 2019).

FINAL CONSIDERATIONS
The level of anthropization did not influence the phenotypic, taxonomic and enzymatic profile of actinobacteria, showing that strains are able to survive the anthropic effects that occurred in the environment.The human action for preparing and cultivating the soil did not reduce the viability and capacity of these strains, indicating potential for their use in agricultural areas.
Highlighted were the strains QX 59 and QX 67, identified as Streptomyces and coming from the anthropized area, which showed the highest values of enzymatic index, and were selected for the facilitation test.

Figure 1 :
Figure 1: Percentage of genders by area of study Source: Prepared by the authors (2021)

Figure 3 :
Figure 3: Enzyme index (EI) of cellulase, amylase and xylanase influenced by temperature, environment, 39º, 41º, 43º and 45ºC, of strains of actinobacteria isolated from the semi-arid northeast of Brazil.Source: Prepared by the authors (2021)

Figure 4 :
Figure 4: Facilitation between nineteen strains of rhizobia and two strains of actinobacterias, isolated microorganisms from the northeastern semi-arid region, Brazil.Source: Prepared by the authors (2021)

Table 1 :
Chemical attributes of soils, from two areas, originating from Fazenda Não Me Leave Me, Quixadá, CE -northeastern semi-arid region, Brazil.

Table 2 :
Morphological characteristics and identification of genus of actinobacteria.

Table 3 :
Phenotypic and physiological characteristics of rhizobia strains † The symbol (+) indicates a positive result while (-) indicates a negative result.

Table 4 :
Intrinsic resistance to antibiotics and enzymatic profile of rhizobia strains.
Source: Prepared by the authors(2021)