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Cultivos Tropicales

versión impresa ISSN 0258-5936versión On-line ISSN 1819-4087

cultrop vol.41 no.2 La Habana abr.-jun. 2020  Epub 05-Abr-2020

 

Original article

Isolation and characterization of Stenotrophomonas asociated to maize (Zea Mays L.) rhizosphere

1Instituto Nacional de Ciencias Agrícolas (INCA), carretera San José-Tapaste, km 3½, Gaveta Postal 1, San José de las Lajas, Mayabeque, Cuba. CP 32 700

2Departamento de Ingeniería Biológica. Instituto Universitario de Tecnología, Lyon, Francia

3Universidad Estatal de Milagro (UNEMI), Facultad de Ciencias e Ingeniería. Vía Milagro km 26. Milagro, Ecuador

4Centro de Investigaciones Biotecnológicas del Ecuador. Escuela Superior Politécnica del Litoral, Guayaquil, Ecuador

ABSTRACT

The inoculation of plant growth-promoting rhizobacteria in agricultural interest crops such as maize constitutes an economically and environmentally friendly alternative to chemical fertilization. Stenotrophomonas is a common bacterial genus in many soil types and rhizospheres of different crops, and presents mechanisms for growth promotion such as biological nitrogen fixation, mineral solubilization and phytohormones production. Consequently, the present work aimed to characterize and molecularly identify Stenotrophomonas strains from the maize rhizosphere. For this, an isolation was performed from rhizospheric and rhizoplane soil and colonies with cultural characteristics similar to those described for this genus were selected. The microscopic characteristics and the ability to perform the biological nitrogen fixation solubilize different phosphate and potassium sources and the antagonistic activity against Fusarium oxysporum were also taken into account. Molecular identification was performed by 16S rDNA sequencing. Twenty isolates with cultural, morphological and physiological characteristics coinciding with the genus in question were obtained. From these 15 solubilized at least one of the phosphate sources used, two solubilized the potassium sources and six showed antagonism against the pathogen. Seven isolates were identified as Stenotrophomonas and the rest were included in three genera, also belonging to the Proteobacteria phylum. The integral isolate in terms of positive attributes evaluated was INCA-FRr1 identified as Stenotrophomonas. This could be a promising inoculant for maize and other crops.

Key words: PGPR; FBN; solubilization; antagonism; biofertilization

INTRODUCTION

Corn is an important component in human and animal nutrition. It is cultivated in the most diverse edaphic and ecological conditions given its high plasticity; and its production and consumption worldwide, reach the highest figures compared to other crops 1. However, the availability of certain nutrients, generally caused by mineral fertilization, mainly nitrogen, potassium and phosphorous, is essential to obtain acceptable yields. Although the application of these inputs makes it possible to obtain representative yields, they also constitute a high cost for the production process and negatively influence the agroecosystem 2.

The use of biofertilizers based on Plant Growth Promoting Rhizobacteria (PGPR) is a viable alternative to decrease the load of mineral fertilizer applied every year in agriculture. These biopreparations are harmless to the environment, increase crop yields and decrease production costs 3.

The Stenotrophomonas genus has been as a rhizospheric microbiota of different crops described, including corn 4-7, and it is considered a PGPR due to the different mechanisms of promotion of plant growth it presents. These include Biological Nitrogen Fixation (FBN), solubilization of phosphate salts, production of indoles 6) and synthesis of ACC deaminase 8.

In Cuba, there are no studies related to the Stenotrophomonas-corn interaction, nor with the use of the genus for agricultural exploitation, therefore, the present work intends to isolate, identify and characterize Stenotrophomonas from the corn rhizosphere, as well as to determine the effect of their inoculation under controlled conditions.

MATERIALS AND METHODS

Sampling

The sampling was carried out at the El Mulato farm in San José de las Lajas municipality, Mayabeque. The corn was cultivated in Ferralitic Red Leachate Typical eutric 9) with Phaseolus vulgaris L. as the preceding crop. Five random sampling points were established and two plants were from each of them taken, for 10. The extraction was carried out taking a volume of soil of approximately 20 cm3. The samples were placed in separate polyethylene bags, and before two hours were stored in the laboratory at 4 ºC until use.

Isolation of Stenotrophomonas from the Corn Rhizosphere

Isolation was made from rhizospheric soil and rhizoplane from corn plants following the methodology proposed by Granada 5. For rhizospheric soil isolation, 1 g samples were weighed and serial dilutions of 10-1 to 10-6 were made in sterile distilled water. The 100 µl of the suspensions (10-4-10-6) were by dissemination in LB medium, taken and sown with three replicates each. The plates were at 28 °C for 48 hours incubated.

For rhizoplane isolation, roots were into 1 cm long portions cut and placed in Erlenmeyers bottles with 10 mL of sterile distilled water. These were kept in agitation for 1h at 150 rpm and the same rhizospheric soil methodology was applied to them.

The cut roots were with LB medium plated and incubated at 28 °C for 48 h. Subsequently, roasts were taken from the bacterial growth that was visualized around and on the roots and isolated in the same medium with the same conditions.

Characterization of Stenotrophomonas isolated from the corn rhizosphere

Cultural and morphological characterization

For the cultural characterization, the coloration, mucus and morphology of the colony were taken into account. Successive striating in LB medium purified those with translucent coloration, light mucus, smooth edges and flat elevation 5. Then Gram staining was performed and gram-negative isolates with bacillary or cocobacillary morphology and non-sporulated were selected, which were subsequently punctured in the nitrogen-free media, Rennie and JMV. The isolates that grew in these media were as nitrofixers classified.

Characterization as PGPR

In the test, the capacity of the isolates to solubilize phosphorus and potassium was evaluated, in addition the antagonistic activity against Fusarium oxysporum was determined.

Phosphate solubilization was determined in NBRIP medium using the bromocresol purple indicator, In addition, three different sources of inorganic phosphorus were tested: tricalcium phosphate (Ca3(PO4)2), aluminum phosphate (AlPO4) and iron phosphate (FePO4). The bacteria were by spots around 0.5 cm in diameter seeded, at the rate of five isolates per plate, and three replicates were for each one established. The plates were at 28 °C for 48 hours incubated. Those isolates that showed a yellow halo around the growth were taken as positive results, indicating the solubilization of the phosphate salts.

Following the same procedure, the solubilization capacity of potassium in the Alexandrov medium was determined using dipotassium hydrogen phosphate (K2HPO4) and potassium oxide (K2O) as potassium sources. The plates were at 28 °C incubated for 48 h and the isolates that had a solubilization halo around the colonies were as positive results taken.

To determine the antagonistic activity against the Fusarium oxysporum EC20-E-GM strain (belonging to the strain of the plant pathology laboratory of the Center for Biotechnological Research of Ecuador), the mycelium was cut with a sterile scalpel. This was in a Falcon tube placed with 20 ml of sterile distilled water and subsequently sterilized steel pellets, 4 mm in diameter were added. The bottle was shaken vigorous and manually for 5 min and the contents were filtered with a 40-µm sieve mounted on a sterile Falcon tube. The 200 µl of the sieved solution were seeded in Potato Dextrose Agar, and the bacterial isolates were spotted on this point at the rate of four isolates per plate. Incubation was at 28 °C for 7 days. The isolates that showed a fungal growth inhibition halo with a radius greater than 0.5 mm were as positive results for the antagonistic activity taken.

Identification by sequencing of the 16S rDNA gene

Genomic DNA extraction

The extraction of the genetic material was carried out by alkaline lysis. A colony was from the axenic culture taken and placed in Eppendorf with 40 μl of NaOH at 0.20 M. It was then at 10 %, power of 700 W for one minute microwaved and immediately cooled on ice for 5 min. The lysed cells were for 10 min at 10,000 gravities spun. The supernatant was taken and the nucleic acid quantification was performed by spectrophotometry using the NanoDropTM 2000 at 260 nm, and the ratio 260/280 and 260/230 were calculated in search of determining the concentration and quality of the DNA, respectively.

Amplification and sequencing of 16S rDNA

For the amplification, 25 µl of the PCR mixture containing 1 µl of the crude extract containing DNA, 1 µl (10 µM) of the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-) were used. 3') (10), which allow obtaining amplicons of around 1500 bp, and GoTaq® Green Master Mix (Promega Corporation, USA). Amplification was performed in an MS mini thermocycler (Major Science, USA) under the following parameters: initial denaturation 95 °C for 10 minutes and additional denaturation for 1 minute at 92 °C, 35 cycles of 72 °C each, for 2 minutes. Amplification products were verified by 1 % agarose gel electrophoresis (1 g agarose in 150 mL of 1X TAE buffer) at 80 V for 45 min. The reaction products were purified using the prior to sequencing, was performed by the Sanger method at the Macrogen® Company (Republic of Korea)

Editing sequences and identification

A quality analysis was performed of the sequences obtained using the FinchTV program (ver. 1.4.0) (Geospiza Inc.) using as a criterion of acceptance a quality value (Q) equal to or greater than 20 per base. They were subsequently analyzed using the BLASTn program 11. The cutoff value used was 1 x 10-5, with a minimum coverage of approximately 80 %. The debugging and alignment of the obtained sequences were performed using the ClustalW multiple sequence alignment tool 12 in the MEGA-X program (ver. 10.0.4) 13.

RESULTS AND DISCUSSION

Isolation of Stenotrophomonas from the corn rhizosphere

The 59 total isolates were obtained, of which 72.9 % came from the rhizoplane and 27.1 % from the rhizospheric soil of corn plants. The nutritional wealth that the rhizoplane represents makes it an attractive area for the establishment of microbial populations. Radical exudates, whose concentrations are highest at the rhizoplane level, constitute important sources of carbon for many edaphic microorganisms 14.

Characterization of isolated Stenotrophomonas from the corn rhizosphere

Cultural and morphological characterization

The 59 isolates obtained presented flat, translucent, mucous colonies with smooth edges, characteristics similar to those described by other author 5. From these, 86.4 % (51 isolates) were gram-negative and, in turn, 94.1 % (48 isolates) coincided with the proposed morphological specifications for the genus Stenotrophomonas: gram-negative bacilli or cocobacilli not sporulated. Many bacterial genera develop in the soil, however, in absolute numbers; there is a predominance of gram-negative bacteria over gram-positive bacteria 15. In the particular case of the rhizosphere, the roots have a direct influence on the composition and density of the soil microbiota. The release of organic plant substances favors the establishment of heterotrophic microbial populations that, for the most part, comprise gram-negative bacilli and actinomycetes 15.

The genus Stenotrophomonas presents the ability to perform the FBN, so the Rennie and JMV media were used to rule out those isolates that do not perform this function; and 41.7 % (20 isolates) grew in the media. Red Ferralitic soils are the most common in the Habana-Matanzas plain, an area that includes the sampled area. In general, the productivity of these soils is high due to the depth and characteristics of their horizons; however, they are poor in organic matter and have low fertility 9, which can translate into poor nitrogen concentrations. These characteristics could favor the proliferation and activity of diazotrophic microorganisms such as Stenotrophomonas. FBN occurs as a function of the concentration of the element in the medium, responding to the principle of cellular economy. The presence of high concentrations of nitrogenous compounds inhibits the synthesis of nitrogenase, the enzyme responsible for the process, by repressing the expression of the nif genes that encode it. In this way, the microorganism preferably uses these elements as a nitrogen source, instead of carrying out a process that involves high-energy consumption, such as FBN. On the other hand, it is argued that any deficiency in organic and inorganic nitrogenous compounds stimulates N2 microbial fixation (16, 17).

The means used in the isolation of nitrofixing bacteria were completely devoid of nitrogen sources and included in its composition, sodium molybdate salt dihydrate (Na2MoO4.2H2O) as a source of molybdenum, a structural component of the enzyme nitrogenase 17. Both aspects favored the synthesis and activity of the enzyme, and therefore allowed microbial growth under these conditions.

Characterization as PGPR

The 20 isolates that showed the ability to perform the FBN, grew in the NBRIP medium. The carbon source present in said medium is glucose, which constitutes the sugar most used as a carbon source in culture media; and there are few microorganisms with the inability to metabolize it 17.

The 75 % (15 isolates) of these isolates developed a solubilization halo against the different phosphorus sources used (Table 1).

Table 1 Qualitative characterization as PGPR. Solubilization of phosphorus, potassium and antagonistic activity against Fusarium oxysporum 

Isolated PO4 -2 source K+ source Antagonism
Ca3(PO4)2 AlPO4 FePO4 K2HPO4 K2O
INCA-FRr1 + + + + + +
INCA-FRr2 + - + - - +
INCA-FRr3 + + + nc nc +
INCA-FRr4 - - + nc nc -
INCA-FRr5 + - + + + -
INCA-FRr6 + + + - - -
INCA-FRr7 + + + nc nc +
INCA-FRr8 + - + nc nc +
INCA-FRr9 + + + nc nc -
INCA-FRr10 - - - - - -
INCA-FRr11 + + + nc nc -
INCA-FRr12 + + + - - -
INCA-FRr13 + - + nc nc -
INCA-FRr16 + + + nc nc -
INCA-FRc1 - - - - - -
INCA-FRc4 - - - - - -
INCA-FRc8 - - - - - -
INCA-FRc16 + + + - - -
INCA-FRc19 - - - - - -
INCA-FRc24 - - + - - +

(+) Production of solubilization halo or inhibition halo in case of antagonism; (-) bacterial growth without halo formation of solubilization or inhibition in case of antagonism; (nc) there was no bacterial growth

Aluminum phosphate was the least consumed source, with nine isolates; while tricalcium phosphate and iron phosphate were more successful with 13 and 15 isolates respectively. The iron content in Red Ferralitic soils is high 9, making it more likely that phosphate will establish bonds with ferric ions compared to other ions, and in turn, the microorganisms present in these soils are more familiar with these compounds.

The formation of the solubilization halos occurs because of the cation exchange carried out by organic acids secreted by the bacteria. These compounds convert insoluble phosphate to soluble forms of the salt 18.

On the other hand, the presence or not of the halo around the colony is not a sufficient criterion to classify the microorganisms as phosphorus solubilizers 19. The solubilization of phosphorous salts could occur by the binding of a chelating agent to the cations, displacing the phosphate groups. These reactions, although they allow the phosphate to be solubilized, do not produce halo in the culture medium. Therefore, although the possibility that some of the isolates studied here may not have the ability to solubilize any of the phosphate salts used, it cannot be ruled out that there may be isolates with solubilizing potentialities and, due to the methodology used, it has not been possible to determine 20.

Regarding the solubilization of potassium, 60 % (12 isolates) of the isolates grew in Aleksandrov's medium and of these, 16.6 % (2 isolates) developed a halo of solubilization. This medium has as its carbon source and the main component sucrose. Disaccharide that not constitute the most attractive carbon source for most microorganisms.

Some authors associate the solubilization of potassium with the production of acids of microbial origin 21. This could have been the mechanism used to form the solubilization halo. However, as in the phosphate solubilization, there are studies that indicate that this occurs without the release of acids into the medium, and therefore without the decrease in pH 22.

The antagonistic activity against Fusarium oxysporum was positive in 30 % (6 isolates) of the isolates. A large number of pathogens that cause significant economic damage to its production attacks corn in tropical environments. These include species of the genus Fusarium23 that cause ear rot, which, in addition to reducing yield, cause deterioration and poor quality of grains, and due to the ability to produce mycotoxins, they are also to diseases in their diners related 24.

Identification by sequencing of the 16S rDNA gene

Four bacterial genera belonging to the Phylum Proteobacteria were identified, a group especially abundant in the rhizosphere during the different phenological stages of maize 25,26. These included Enterobacter, Pseudomonas, Rhizobium and Stenotrophomonas with a percentage of identity greater than or equal to 97 in 95 % of cases (Table 2).

Table 2 Comparison of the sequences obtained, with the NCBI database using the BLASTn tool 

Isolated Putative species Identity
INCA-FRr16 Enterobacter bugandensis 99 %
INCA-FRr5 Pseudomonas brenneri 98 %
INCA-FRr3 Pseudomonas gessardii 99 %
INCA-FRr2 Pseudomonas graminis 99 %
INCA-FRr4 Pseudomonas hibiscicola 97 %
INCA-FRr8 Pseudomonas putida 100 %
INCA-FRr9 Pseudomonas sp. 100 %
INCA-FRr11 Pseudomonas sp. 99 %
INCA-FRc4 Rhizobium aegyptiacum 100 %
INCA-FRc1 Rhizobium mesosinicum 99 %
INCA-FRc8 Rhizobium mesosinicum 99 %
INCA-FRr10 Rhizobium sp. 88 %
INCA-FRc19 Rhizobium sp. 99 %
INCA-FRr12 Stenotrophomonas maltophilia 100 %
INCA-FRr13 Stenotrophomonas maltophilia 100 %
INCA-FRc16 Stenotrophomonas maltophilia 98 %
INCA-FRr7 Stenotrophomonas pavanii 98 %
INCA-FRc24 Stenotrophomonas pavanii 99 %
INCA-FRr1 Stenotrophomonas rhizophila 99 %
INCA-FRr6 Stenotrophomonas rhizophila 100 %

The most representative genera were Stenotrophomonas and Pseudomonas for 35 % of the total in each case (7 isolates). Similar results were obtained in the Daucus carota L. rhizosphere 6. To a lesser extent, the genera Rhizobium and Enterobacter were identified with 25 % (5 isolates) and 5 % (1 isolate) of appearance respectively.

It is common to find reports of pathogenicity associated with the genera Stenotrophomonas, Pseudomonas and Enterobacter, in humans, animals and even plants 27-30. However, on many occasions, the presence of virulence factors and the pathogenicity of microorganisms are restricted to species, and even to specific strains within a genus 17. The pathogenic character that some of these genera may present does not detract from the biostimulant potentialities that they develop in plants; although they are not exempt from the eco-toxicological tests established for the agricultural exploitation of microorganisms.

The genus Pseudomonas have been widely reported as PGPR of various plants including grasses 5,6,15,31. Populations of Pseudomonas frequently stand out above other diazotrophic genera due to their short latency period, rapid growth rate, and metabolic versatility 32. On the other hand, Enterobacter has been described as a rhizospheric microorganism and, above all, as an endophyte of different plant species; standing out for its growth promoting characteristics 33,34. In the case of the Rhizobium genus, isolations have been made as endophytes of corn 35 and to a lesser extent of the rhizosphere 36; mainly in soils where the crop rotation system is applied with some legume species. The use of Phaseolus vulgaris L. as a rotation culture in this study, may have favored the establishment of rhizobia populations, since this legume presents greater susceptibility to colonization and symbiosis with species of the Rhizobium genus with respect to other genera rhizobia 37.

Stenotrophomonas has been isolated from various environments, from different soil types to animal intestinal tracts 38, which highlights the ubiquitous character of this bacterium. The Stenotrophomonas pavanii species is in bioremediation processes at industrial 39 and agricultural 40 levels used, while Stenotrophomonas maltophilia has a contrasting behavior in the different habitats it occupies. This species is common in hospital settings and some strains are as powerful nosocomial agents described; however, it is described as part of the normal edaphic microbiota and is associated with many crops of agricultural interest without causing damage to them 41. Likewise, it occurs with Stenotrophomonas rhizophila, which shows great potential to promote the growth of plants such as soybeans 42, cotton, tomato and pepper, among others; and it is considered a planktonic microbiota of marine environments 43.

In this study, the most comprehensive isolate in terms of the plant growth promotion mechanisms evaluated was INCA-FRr1, identified as Stenotrophomonas rhizophila. The ability to solubilize different sources of phosphorus and potassium, to perform FBN and antagonistic activity against phytopathogens, in addition to other positive characteristics that were not in this study demonstrated but are known 6; they represent clear advantages over other rhizospheric bacterial genera 43. On the contrary, they use a strain of Stenotrophomonas rhizophila that does not present the ability to perform the FBN and still obtain important results in terms of development of variables such as height, stem and root length, dry area and leaf biomass in basil plants. This could suggest the presence of other mechanisms for promoting plant growth. In co-inoculation tests of Stenotrophomonas rhizophila with Arbuscular Mycorrhizal Fungi (AMF), compared to simple inoculations, an increase in plant growth and chlorophyll content is observed 44; consequently, the bacterium is also classified as a mycorrhizal enhancer (term known as Mycorrhiza Helper Bacteria, MHB) 45,46.

The success of bioproduct use lies, among other aspects, in obtaining strains compatible with the crop in question 47 and efficient for achieving the desired yields. In this way, the use of native PGPRs as inoculants promotes ecological-sustainable management of agroecosystems as well as, could improve corn production. The ability of native strains to interact positively with the resident soil microbiota and their adaptability to local climatic and agro-ecological conditions often enhances their performance compared to non-native strains.

CONCLUSIONS

  • Diazotrophic bacterial populations coexist in the genus Stenotrophomonas, Pseudomonas, Rhizobium and Enterobacter. In an integral way, the INCA-FRr1 isolate corresponding to the genus Stenotrophomonas, stands out for its characteristics that promote plant growth and could constitute a promising inoculant for crops of agricultural interest and for the cultivation of corn itself.

  • The study of the rhizosphere and the knowledge of its microbial composition is key to the conception of efficient and compatible biological products within a sustainable agriculture

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Received: October 14, 2019; Accepted: April 05, 2020

*Author for correspondence: riny@inca.edu.cu

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