Short communication

Approach in the identification of rhizospheric rhizobia of rice (Oryza sativa L.) cultivar “INCA LP-7”

0000-0002-5683-6558Arasay Santa Cruz-Suárez^{1  *}  , 0000-0002-5760-816XIonel Hernández-Forte¹ 

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

ABSTRACT

There are few studies about rhizobia rice interaction, in Cuba. The aim of this work was to characterize possible rhizobia from rice plant rhizosphere of cultivar “INCA LP-7”. Sixteen bacterial isolates were characterized from a cultural, morphological and biochemical point of view. All bacterial isolates produced mucous in mannitol yeast medium; however, they had different size and color. Gram stain revealed that most of these isolates were Gram-negative and non-sporulated coccobacilli. Some isolates did not produce for cetolactase enzime, produced acid and grew in nitrogen-free media. It is concluded that in rice plant rhizosphere of cultivar “INCA LP-7”, reside rhizobia populations from Rhizobiaceae family presumably, with potentials in promoting plant growth.

Key words: phenotype; nitrogen fixation; grasses; Rhizobium

INTRODUCTION

In Cuba, an annual average of 72 kg of rice (Oryza sativa L.) is per person consumed, one of the highest in Latin America ¹. Cereal imports cover 66 % of the national demand ². This problem motivated the development of new varieties of rice, more resistant to phytopathogens and with greater yield potential. The rice cultivar “INCA LP-7”, developed at National Institute of Agricultural Sciences (INCA), and it is cultivated in 8 % of total area dedicated to rice cultivation in the country (682 ha) ³.

Rice has the physiological capacity to absorb only 50 kg N ha^{-1 (4}. The rest of the nitrogen fertilizer applied to the crop produces serious contamination problems ⁵. The use of Plant Growth Promoting Bacteria (PGPB) has been used as an alternative to mineral fertilization ⁶. Rhizobia are PGPB that have been studied for their symbiotic association with legume plants ⁷. However, in recent years it has been found that these microorganisms also promote the growth of grasses such as rice ⁸.

Currently, there is no scientific evidence in Cuba about on the presence of rhizobia in the rhizosphere of rice cultivar “INCA LP-7”, which makes it difficult to develop bioproducts based on these microorganisms that allow reducing mineral fertilization and increase yields of this crop. The objective of this research was to characterize possible rhizobia from rhizosphere of rice plant cultivar “INCA LP-7”.

MATERIALS AND METHODS

Sixteen bacterial isolates were used, isolated from the rhizosphere of rice plants cultivar “INCA LP-7” and which presumably belong to rhizobia group ⁹.

Cultural and morphological characterization of rhizospheric bacteria from “INCA LP-7” cultivar rice plants

For the cultural characterization, isolates were cultured by exhaustion on Yeast Mannitol (YM) medium ¹⁰ with Congo red. The cultures were incubated for 48 h at 30 °C. The parameters to take into account were color, mucus and colony size, large 2-4 mm, medium 1-2 mm small 1 mm. The morphological characterization of bacterial isolates was carried out by Gram stain and the cell morphology, response to staining and presence of endospores were determined.

Biochemical characterization of rhizospheric bacteria from cultivar “INCA LP-7” rice plants

Cetolactase test

Bacterial isolates were cultured by exhaustion and in Yeast-Lactose (YL) solid medium ¹¹ and incubated at 30 °C for seven days. Subsequently, 10 mL of Benedict's reagent were added on the bacterial growth and changes of the medium color were observed after 10 min at room temperature. The positive result was considered when the medium changed from blue to yellow, and negative if the medium maintained the blue coloration.

Acid and base production

Bacterial isolates were by cultured exhaustion and in YM solid medium with bromothymol blue indicator (0.5 % in 0.016N NaOH) and the plates were for three days at 30 °C. The change in the color of the medium from green to yellow was interpreted as the acid production, and from green to blue as base production ¹².

Growth in nitrogen-free culture media

Bacterial suspensions were prepared. To this, several colonies were re-suspended in 1 mL of sterile NaCl solution (0.9 % (m/v)). One hundred microliters of the suspensions were inoculated into flasks containing 10 mL of the semi-solid nitrogen-free media JMV and Rennie ^13,14. The inoculation was carried out by introducing the tip of a micro pipette inside the culture media. Flasks were inoculated with a suspension of Bradyrhizobium elkanii ICA 8001 strain which is a reference strain in the Biological Nitrogen Fixation (BNF) were used as positive control ¹⁵.

RESULTS AND DISCUSSION

Rhizospheric bacterial populations from plant rice cultivar “INCA LP-7”, similar from the cultural point of view, differ in their morphological characteristics

All isolates produced mucous colonies in YM solid medium. However, they did differ in size and coloration (Table 1). Similar results were found in previous studies with bacterial isolates from Chilean beans (Lablab purpureus (L.) Sweet) and pea (Pisum sativum) ^16,17.

Table 1 Cultural, morphological and biochemical characterization of bacteria isolates from rhizorphere rice plants cultivar “INCA LP-7” 

Isolates	Cultural characteristics	Morphological characteristics	Acid/base production	Growth in semi-solid nitrogen-free media
				JMV	Rennie
4S	Medium, pale pink with dark pink center, mucous	Coccobacilli, G-, not sporulated	acid	+	+
4U	Large, whitish, mucous	Coccobacilli, G-, not sporulated	acid	+	+
1DD1	Medium, whitish, mucous	Coccobacilli, G-, not sporulated	acid	+	+
1DD2	Medium, whitish, mucous	Coccobacilli, G-, not sporulated	acid	+	+
1AA	Large, whitish, mucous	Coccobacilli, G-, not sporulated	acid	+	+
1LL	Large, whitish, mucous	Coccobacilli, G-, not sporulated	ND	ND	ND
3W	Small, pale pink, mucous	Coccobacilli, G-, not sporulated	acid	+	+
5FF1	Medium, whitish, mucous	Coccobacilli, G-, not sporulated	base	+	+
5FF2	Large, whitish, mucous	Coccobacilli, G-, not sporulated	ND	ND	ND
5O	Medium, translucent, mucous	Coccobacilli, G-, not sporulated	acid	+	+
5P1	Small, whitish, mucous	Coccobacilli, G-, not sporulated	acid	+	+
GG1	Large, translucent, mucous	Coccobacilli, G-, not sporulated	acid	+	+
GG2	Medium, pink, mucous	Coccobacilli, G-, not sporulated	acid	+	+
II1	Small, translucent, mucous	Coccobacilli, G-, not sporulated	acid	+	+
II3	Large, translucent, mucous	Coccobacilli, G-, not sporulated	acid	+	+
II2	Large, translucent, mucous	Coccobacilli, G-, not sporulated	acid	+	+

G-, Gram negative; G +, Gram positive; ND, Not determined

The 87.5 % of isolates were coccobacilli Gram negative and not sporulated. Isolates 1LL and 5FF2, which make up the remaining 12.5 %, presented cultural and even some morphological characteristics similar to the rest. However, both isolates were Gram positive.

The rhizobia are generally non-sporulated and Gram-negative coccobacilli ¹⁸. Thus isolates 1LL and 5FF2 isolates were eliminated from the subsequent determinations.

Possible rhizobia with attributes in promoting plant growth reside in rhizosphere in rice plant cultivar “INCA LP-7”

The response to cetolactase test was the third indicator used to determine if the bacterial isolates belong to rhizobia group. This determination was for a long time a reliable phenotypic character, which allowed to differentiate the Rhizobiaceae family from Agrobacterium genus. Rhizobia and the genus Agrobacterium have similar habitats and cultural and morphological characteristics ¹⁹. Agrobacterium forms mucous and semitranslucent colonies, after 2-3 days in YM solid medium and the Gram stain reveals in the form of Gram negative and non-sporulated cells. This genus produces galls or tumors on the roots and stem of plants, very similar to the nodules that form rhizobia in legumes ²⁰.

All isolates selected in this work as possible rhizobia, taking into account the cultural and morphological characteristics, were cetolactase negative test. Scientific evidence has allowed atoxonomic redistribution of some bacteria species, since they constitute exceptions to rule. For example, according to the Bergey Manual, species that were previously identified as Agrobacterium rhizogenes, Agrobacterium rubi, and Agrobacterium vitis are cetolactase negative and Agrobacterium tumefaciens is cetolactase positive ²¹.

Molecular biology methods have allowed a greater clarification of these issues, so that these three species of Agrobacterium are currently within the genus Rhizobium^22,23. Taking into account these and other scientific evidences it is necessary to use, in addition to conventional phenotypic tests, 16S rRNA sequencing to identify bacterial isolates associated with the rice plants cultivar “INCA LP-7”.

Acid/base production was another phenotypic criteria studied in this research. Except isolate 5FF1, the rest produced acid in the culture medium. Some microbial acids act as siderophores, which capture iron from the soil ²⁴. Others allow the phosphorus solubilization and increases its availability of plants ²⁵. Indole-3-acetic acid and salicylic acid are also acids that produce PGPB, which increase plant growth ²⁶ and induce defensive responses against pathogens ²⁷, respectively.

On the other hand, the growth of bacterial isolates in nitrogen-free culture media was used as an approach to their ability to perform the biological nitrogen fixation (BNF). The 100 % of isolates grew on JMV and Rennie media (Table 1). These results are similar to recent studies with those from other authors ²⁶, with bacteria associated with rice plants.

Nitrogen-free semi-solid media are used isolate diazotrophic microorganisms from soil, rhizosphere, or interior of plant ²⁷. Thus, it is necessary to apply techniques such as the amplification of the nifH gene ²⁸⁾ and the Acetylene Reduction Assay (ARA) ²⁹, which allow confirming the bacteria ability to fix nitrogen.

Biological Nitogen Fixation is one of the direct mechanisms used by PGPB to promote the plant growth ³⁰. Rhizobia have been studied primarily for their ability to fix nitrogen, symbiosis with legumes ³¹. The presence of this attribute in rhizobia isolates studied here would allow their use as biofertilizer allowed the reduction of nitrogen fertilizer to the crop.

CONCLUSIONS

In this work phenotypic aspects of Rhizobiaceae family were identified. In bacterial isolated from rhizosphere of rice plant cultivar “INCA LP-7” characterization allow that this isolated belong to Rhizobium, Ensifer or Shinella genera. However, it is necessary to use molecular techniques to confirm it. Molecular methods and phenotypic studies and constitute what is now known as polyphasic taxonomy, a tool that allows a greater comprehensiveness of microorganism taxonomic studies.

BIBLIOGRAFÍA

1.  MINAG. El Arroz en La Economía Y Sociedad Cubana [Internet]. Cuba y la Economía. 2016 [cited 21/12/2020]. Available from: https://cubayeconomia.blogspot.com/2016/10/el-arroz-en-la-economia-y-sociedad.html1.

2.  Rendón T. Cuba por aumentar producción de arroz y evitar importaciones [Internet]. Agencia Cubana de Noticias. [cited 21/12/2020]. Available from: http://www.acn.cu/economia/22803-cuba-por-aumentar-produccion-de-arroz-y-evitar-importaciones2.

3.  MINAG. Plan 2018-2019 de Producción de semilla de arroz en Cuba [Internet]. Ministerio de la Agricultura República de Cuba. 2019 [cited 21/12/2020]. Available from: https://www.minag.gob.cu/node/1673.

4.  Hernández I. Rizobios asociados a plantas de arroz Oryza sativa L.) cultivar INCA LP-5. Efecto en el crecimiento del cultivo. [Tesis en opción al título académico de Máster en Ciencias en Microbiología]. Universidad de La Habana, Instituto Nacional de Ciencias Agrícolas; 2015.

5.  Villarreal-Núñez JE, Barahona-Amores LA, Castillo-Ortiz OA. Efecto de zeolita sobre la eficiencia de fertilizantes nitrogenados en el cultivo de arroz. Agronomía Mesoamericana. 2015;26(2):315-21.

6.  INTAGRI. Los Biofertilizantes en la Agricultura [Internet]. 2017 [cited 21/12/2020]. Available from: https://www.intagri.com/articulos/agricultura-organica/biofertilizantes-en-agricultura6.

7.  Elzanaty AM, Hewedy OA, Nagaty HH, Abd Elbary MI. Molecular and Biochemical Characterization of Some Egyptian Genotypes Rhizobium Vicia Faba Isolates. Journal of Bioengineering & Biomedical Sciences. 2015;5(1):1.

8.  Santa Cruz A. Efecto de inoculantes a base de rizobios sobre el crecimiento y el desarrollo de plantas de arroz Oryza sativa L. cultivar INCA LP-5. [Tesis de Diploma]. [Mayabeque]: Universidad Agraria de la Habana, Instituto Nacional de Ciencias Agrícolas; 2018.

9.  Betancourt C. Aislamiento y caracterización de poblaciones rizosféricas y endófitas de semillas de plantas de arroz Oryza sativa L. cultivar INCA LP-7. [Tesis en opción al Título Académico de Licenciada en Ciencias de la Microbiología]. Universidad de La Habana, Instituto Nacional de Ciencias Agrícolas; 2018.

10.  Vincent JM. A manual for the practical study of root-nodule bacteria. International Programme Handbook (No. 15). Oxford, England; 1970.

11.  Thies JE, Singleton PW, Bohlool BB. Modeling symbiotic performance of introduced rhizobia in the field by use of indices of indigenous population size and nitrogen status of the soil. Applied and Environmental Microbiology. 1991;57(1):29-37.

12.  Sosa A, Elías A, García OA, Sarmiento M. Aislamiento y caracterización fenotípica parcial de cepas de rizobios que nodulan leguminosas rastreras. Revista Cubana de Ciencia Agrícola. 2004;38(2):197-201.

13.  Rennie RJ. A single medium for the isolation of acetylene-reducing (dinitrogen-fixing) bacteria from soils. Canadian Journal of Microbiology. 1981;27(1):8-14.

14.  Baldani JI, Pot B, Kirchhof G, Falsen E, Baldani VLD, Olivares FL, et al. Emended description of Herbaspirillum; inccusion of Pseudomonas rubrisubalbicans, a mild plant pathogen, as Herbaspirillum rubrisubalbicans comb. Nov.; and classification of a Group of Clinical Isolates (EF Group 1) as Herbaspirillum species 3. International Journal of Systematic and Evolutionary Microbiology. 1996;46(3):802-10.

15.  Nápoles MC, Martínez J, Costales D, Morales B, Martínez E, Rogel MA. Avances en la reidentificación de la cepa ICA 8001 Bradyrhizobium japonicum como perteneciente a Bradyrhizobium elkanii. Revista Cubana de Ciencias Biológicas. 2011;20(1-2):43-6.

16.  Ángeles Cumpa AA, Tesén Villanueva JF. Infectividad y efectividad de rizobios en Lablab purpureus (L.) Sweet "Frijol chileno". [Tesis para optar el título profesional de licenciado en biología microbiología - parasitología]. [Lambayeque]: Universidad Nacional Pedro Ruiz Gallo; 2019.

17.  Singh AK, Nandan R, Singh PK. Genetic variability and association analysis in rice germplasm under rainfed conditions. Crop Research. 2015;47(1, 2 & 3):7-11.

18.  Zhang X-X, Gao J-S, Cao Y-H, Sheirdil RA, Wang X-C, Zhang L. Rhizobium oryzicola sp. nov., potential plant-growth-promoting endophytic bacteria isolated from rice roots. International journal of systematic and evolutionary microbiology. 2015;65(9):2931-6.

19.  Lloret L, Martínez-Romero E. Evolución y filogenia de rhizobia. Revista Latinoamericana de Microbiología. 2005;47(1-2):43-60.

20.  Murugesan S, Manoharan C, Vijayakumar R, Panneerselvam A. Isolation and characterization of Agrobacterium rhizogenes from the root nodules of some leguminous plants. Intl J Microbiol Res. 2010;1:92-6.

21.  Whitman WB. Bergey's manual of systematics of Archaea and Bacteria. New York, United States of America: Wiley; 2015. 503 p.

22.  Young JM, Kuykendall LD, Martinez-Romero E, Kerr A, Sawada H. A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. International Journal of Systematic and Evolutionary Microbiology. 2001;51(1):89-103.

23.  Peix A, Ramírez-Bahena MH, Velázquez E, Bedmar EJ. Bacterial associations with legumes. Critical Reviews in Plant Sciences. 2015;34(1-3):17-42.

24.  Niehus R, Picot A, Oliveira NM, Mitri S, Foster KR. The evolution of siderophore production as a competitive trait. Evolution. 2017;71(6):1443-55.

25.  Kudoyarova GR, Vysotskaya LB, Arkhipova TN, Kuzmina LY, Galimsyanova NF, Sidorova LV, et al. Effect of auxin producing and phosphate solubilizing bacteria on mobility of soil phosphorus, growth rate, and P acquisition by wheat plants. Acta physiologiae plantarum. 2017;39(11):253.

26.  Singh AP, Dixit G, Mishra S, Dwivedi S, Tiwari M, Mallick S, et al. Salicylic acid modulates arsenic toxicity by reducing its root to shoot translocation in rice Oryza sativa L. Frontiers in Plant Science. 2015;6:1-28. doi:10.3389/fpls.2015.00340

27.  Largia MJV, Pothiraj G, Shilpha J, Ramesh M. Methyl jasmonate and salicylic acid synergism enhances bacoside A content in shoot cultures of Bacopa monnieri (L.). Plant Cell, Tissue and Organ Culture (PCTOC). 2015;122(1):9-20.

28.  Quim N, Cerra A, Chamorro L, Pérez A. Resistencia a cadmio (Cd) de bacterias endófitas y bacterias rizosféricas aisladas a partir de Oriza sativa en Colombia. Revista Colombiana de Ciencia Animal-RECIA. 2017;9(2):281-93.

29.  de la Fe Pérez Y, de la Osa AD, Franco GMR, Baldani VL, Rodríguez AH. Diversidad de bacterias diazotróficas asociativas potencialmente eficientes en cultivos de importancia económica/Diversity of associative diazotrophic bacteria and potential use in economic important crops. Revista Cubana de Ciencias Biológicas. 2015;4(1):17-26.

30.  López SMY. Análisis de las alteraciones estructurales y/o regulatorias en los genes de nodulación y fijación de nitrógeno, en aislados de Bradyrhizobium japonicum que difieren en su capacidad de fijar nitrógeno. [Tesis Doctoral]. Universidad Nacional de La Plata, Instituto de Fisiología Vegetal; 2015.

31.  Bernabeu P. Caracterización de la colonización y promoción del crecimiento vegetal por Burkholderia tropica en gramíneas [Tesis para optar al grado Doctor de la Facultad de Ciencias Exactas]. Universidad Nacional de La Plata, Centro de Investigación y Desarrollo en Fermentaciones Industriales (CINDEFI-CONICET-CCT La Plata); 2017.

Received: October 14, 2019; Accepted: December 09, 2020

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