Canadian Journal of Microbiology Bioprospection of native psychrotolerant plant growth- promoting rhizobacteria from the Peruvian Andean Plateau soils associated with Chenopodium quinoa Journal: Canadian Journal of Microbiology Manuscript ID cjm-2020-0036.R2 Manuscript Type: Article Date Submitted by the 01-Jun-2020 Author: Complete List of Authors: Chumpitaz-Segovia, Carolina; Universidad Nacional Agraria La Molina Alvarado, Débora; Universidad Nacional Mayor de San Marcos Ogata-Gutiérrez, Katty; Universidad Nacional Agraria La Molina Zúñiga-Dávila,Draft Doris; Universidad Nacional Agraria, Biology Psychrotolerant, low temperatures, PGPR, Peruvian Andean Plateau, Keyword: <i>Chenopodium quinoa</i> Is the invited manuscript for consideration in a Special Rhizosphere 5 Issue? : https://mc06.manuscriptcentral.com/cjm-pubs Page 1 of 38 Canadian Journal of Microbiology 1 Bioprospection of native psychrotolerant plant growth-promoting rhizobacteria from 2 the Peruvian Andean Plateau soils associated with Chenopodium quinoa 3 4 Carolina Chumpitaz-Segovia1, 2, Débora Alvarado2, Katty Ogata-Gutiérrez1, Doris Zúñiga- 5 Dávila1* 6 1Laboratorio de Ecología Microbiana y Biotecnología, Departamento de Biología, Facultad de 7 Ciencias, Universidad Nacional Agraria La Molina, Av. La Molina S/N, 15024 La Molina, 8 Lima, Peru, Tel: (+51) 16147800 ext. 274 9 2 Lab. Molecular Microbiology & Biotecnology. Facultad de Ciencias Biológicas, Universidad 10 Nacional Mayor de San Marcos, Calle Germán Amézaga N° 375 - Edificio Jorge Basadre, 11 Ciudad Universitaria, 15081, Lima, Peru, Tel: (+51) 16197000 ext 1530. 12 ∗Corresponding author: Doris Zúñiga-Dávila,Draft Laboratorio de Ecología Microbiana y 13 Biotecnología, Departamento de Biología, Facultad de Ciencias, Universidad Nacional Agraria 14 La Molina, Av. La Molina S/N, 15024 La Molina, Lima, Peru, Tel: (+51) 975286657, (+51) 15 16147800 ext. 274; E-mail address: [email protected] 16 17 18 1 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 2 of 38 19 Abstract 20 The Peruvian Andean Plateau, one of the main production areas of native varieties of 21 Chenopodium quinoa, is exposed to abrupt decreases in environmental temperature, affecting 22 crop production. Plant growth-promoting rhizobacteria that tolerate low temperatures could be 23 used as organic biofertilizers in this region. We aimed to conduct bioprospecting of native 24 psychrotolerant bacteria from the quinoa rhizosphere of this region that show plant growth- 25 promoting traits. Fifty-one strains belonging to the quinoa rhizosphere were characterised, and 26 73% of the total could grow at low temperatures (4°C, 6°C and 15°C), from which genetic 27 diversity based on DNA amplification of interspersed repetitive elements (BOX) showed 12 28 different profiles. According to the 16S rRNA sequence, bacterial species belonging to the 29 classes beta- and gamma-proteobacteriaDraft were identified. Only three (6%) isolates identified as 30 non-pathogenic bacteria exhibited PGP activities like IAA production, phosphate 31 solubilisation, growth in a nitrogen-free medium and ACC deaminase production at 6°C and 32 15°C. ILQ215 (Pseudomonas silesiensis) and JUQ307 (P. plecoglossicida) showed 33 significantly positive plant growth effects in aerial length (about 50%), radicular length (112% 34 and 79%, respectively) and aerial and radicular weight (above 170% and 210%, respectively) 35 of quinoa plants compared with the control without bacteria. These results indicate the potential 36 of both psychrotolerant strains to be used as potential organic biofertilizers for quinoa in this 37 region. 38 39 Keywords: psychrotolerant, low temperatures, plant growth-promoting rhizobacteria, 40 Chenopodium quinoa, Peruvian Andean Plateau 41 42 43 2 https://mc06.manuscriptcentral.com/cjm-pubs Page 3 of 38 Canadian Journal of Microbiology 44 Introduction 45 The Peruvian Andean Plateau region in South America is characterised by a particular 46 environmental phenomenon, which restricts the development of different native crops, limiting 47 their growth and development. This region is the setting of an intermittent meteorological 48 phenomenon known as frost, characterised by an abrupt reduction in the ambient temperature 49 to critical levels at approximately 0°C (Snyder and Melo-Abreu 2010). Although frost is 50 seasonal, its frequency has changed over the years owing to climate change. Chenopodium 51 quinoa (Quinoa) is an Andean grain, whose main production zone in Peru lies in the high 52 Andean Plateau region. It has a high nutritional value owing to its elevated protein and essential 53 amino acids content (Abugoch et al. 2008). Frost can produce damage in sensitive phenological 54 states of the plant, affecting its growth and production (Gómez and Aguilar 2016). 55 In general, low temperatures are a stressDraft factor that result in a negative impact on the plants, 56 affecting their physiology and biochemistry (Josine et al. 2011), as well as the biological 57 activity of the microbial communities present in the soil (Robertson and Grandy 2006). 58 Bacterial species capable of tolerating low temperatures are called psychrotolerants or 59 psychrotrophs and are characterised by exhibiting growth at 5°C or lower. Their optimal and 60 maximal growth temperature can range above 20°C (Morita and Moyer 2001). This adaptive 61 characteristic has been of great interest from a biotechnological perspective for microbial 62 inoculants. Many psychrotolerant species have been reported in the literature as biofertilizers 63 because of their plant growth-promoting abilities (Mishra et al. 2011; Anwar et al. 2019). In 64 this context, plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms that 65 establish different interaction mechanisms with the plant roots, thereby increasing their 66 development and growth (Odoh 2017). These mechanisms include the production of different 67 growth phytohormones, solubilisation of inorganic phosphates, nitrogen fixation and 68 production of plant-protective metabolites. Some PGPRs also have the ability to tolerate biotic 3 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 4 of 38 69 or abiotic stress factors, such as low temperatures (Kumar et al. 2015; Ogata-Gutiérrez et al. 70 2016; Ortiz-Ojeda et al. 2017). For these reasons, the isolation and study of PGPR with 71 psychrotolerant traits is of interest to find a potential bioinoculant to increase the production of 72 native crops from the Peruvian Andean Plateau. 73 In this study, we aimed to conduct bioprospecting of psychrotolerant rhizospheric bacteria with 74 potential plant growth-promoting activity from a group of strains obtained from the rhizosphere 75 of quinoa plants native to the Peruvian Andean Plateau. This study holds importance because 76 it enables the identification of bacteria that can be used as biofertilizers in native crops in the 77 Peruvian Andean Plateau whose production can be affected by abiotic stress owing to low 78 temperatures. Draft 4 https://mc06.manuscriptcentral.com/cjm-pubs Page 5 of 38 Canadian Journal of Microbiology 79 Materials and methods 80 Bacterial strain selection 81 In this study, 51 bacterial strains isolated in 2016 from the rhizosphere of quinoa plants (C. 82 quinoa) grown in the Peruvian Andean Plateau were selected. Strains were obtained from 83 quinoa rhizosphere samples that were collected from two fields: one placed in Ilave 84 (16°04’08.9’’S 69°39’24.4’’W and 16°04’27.6’’S 69°38’59.9’’W) and the other in Juli 85 (16°09’00.1’’S 69°33’38.8’’W and 16°10’18.0’’S 69°32’28.3’’W), both located in Puno, Peru. 86 Three composite rhizosphere samples were obtained from the roots of five randomly selected 87 quinoa plants. The rhizosphere soil was separated from the bulk soil by cleaning the roots until 88 only remaining soil particles stayed near the roots. The rhizosphere soil was removed using a 89 sterile brushpaint and poured into a sterileDraft 0.85% NaCl solution. Isolation was made in nutritive 90 agar using the serial dilution technique. Plates were exposed to −5°C for 3 h and then incubated 91 at 6°C for 20 days. All strains were preserved in the strain Collection of the Laboratory of 92 Microbial Ecology and Biotechnology in Universidad Nacional Agraria La Molina (Lima, 93 Peru). Pure bacterial cultures were revived on nutrient agar (NA) to be used in subsequent 94 trials. 95 Colony size at different temperatures 96 To determine the psychrotolerant nature of the strains, bacterial cultures were prepared in 97 nutrient broth to obtain a concentration of 108 CFU/ml. Subsequently, 5 μl of each culture was 98 delivered in drops on NA plates and were incubated at 4°C, 6°C, 15°C and 24°C (optimal 99 temperature) for 14 days until bacterial colonies became visible. The colony size of each strain 100 was evaluated according to Calvo and Zúñiga (2010) and grouped using four levels. Large 101 colony levels were determined grouping the ratios (%) obtained from the bacterial diameter at 102 the tested temperatures compared with that at the optimal temperature (24°C). Levels were 5 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 6 of 38 103 classified as follows: 0: no colonies showed growth; 1: 0%–25%; 2: 26%–50%; 3: 51%–75% 104 and 4: 76%–100%. 105 BOX-PCR for molecular genotypic analysis 106 The total genomic DNA of the psychrotolerant bacterial strains was obtained using the 107 GeneJET Genomic DNA Purification