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: Chenopodium quinoa
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 Kit (Thermo Fisher Scientific, USA) following the
108 manufacturer guidelines. The DNA quality and quantity was measured using a NanoDrop 2000
109 spectrophotometer (Thermo Fisher Scientific, USA). Subsequently, genomic fingerprints of
110 each selected bacteria were determined by PCR amplification of the conserved interspersed
111 repetitive elements (BOX), specifically the boxA region, using the boxA1R primer (Versalovic
112 et al. 1994). PCR reactions were performed as described by Versalovic et al. (1991) using 60
113 ng of purified genomic DNA. PCR amplification condition included an initial denaturation step
114 at 95°C for 5 min, followed by 25 cyclesDraft at 95°C for 45 sec, 53.5°C for 1 min and 65°C for 8
115 min and a final extension step at 65°C for 15 min. The products were evaluated by
116 electrophoresis using 1.5% agarose gel. Genetic diversity analysis based on the BOX-PCR
117 genomic fingerprints profiles was performed using the unweighted pair group method with
118 arithmetic mean (UPGMA) with the program DendroUPGMA (http:
119 genomes.urv.cat/UPGMA/); the similarity calculation was based on the Jaccard coefficient.
120 PCR amplification, sequencing and phylogenetic analysis of the 16S rRNA gene
121 Representative strains for each detected BOX profile were taxonomically identified by
122 amplification of the 16S rRNA gene using the primers fD1 and rD1 (Weisburg et al. 1991).
123 The 1500-bp amplified fragments were purified using the GeneJET PCR kit (Thermo
124 Scientific) following the manufacturer’s guidelines and sent for sequencing to Macrogen Inc.
125 company (Seoul, South Korea). The sequences obtained were processed using the BioEdit
126 program and compared with those in the GenBank database using the BlastN tool (Altschul et
127 al. 1990). The partial 16S rRNA gene sequences of the strains determined in the present study
6 https://mc06.manuscriptcentral.com/cjm-pubs Page 7 of 38 Canadian Journal of Microbiology
128 have been deposited in the National Center for Biotechnology Information database and are
129 available under the accession numbers shown in Table 1. For phylogenetic analysis, the
130 alignments were conducted using MAFFT platform, version 7, (Katoh and Standley 2013) and
131 the phylogenetic trees were constructed using MEGA7 program by the neighbour-joining
132 method, with a calculated evolutionary distance based on the Kimura 2-parameter method.
133 Evaluation of plant growth-promoting traits in non-pathogenic psychrotolerant strains
134 - Indoleacetic acid (IAA) production
135 Production of IAA was assessed using bacteria cultures at exponential phase, grown at 24°C.
136 Each bacterial culture was inoculated at a ratio of 1% v/v in yeast mannitol broth supplemented
137 with L-tryptophan (1g/L) and subsequently incubated at 6°C and 15°C and agitated.
138 Quantification of the phytohormone or its derivatives was performed through Draft 139 spectrophotometry with visible light (A530 mm) in accordance with Gordon and Weber (1951).
140 The concentration of produced IAA (μg/ml) for each strain was calculated using a standard
141 curve in the range of 0–50 µg/ml.
142 - Phosphate salts solubilising activity
143 To detect the capability of the strains to solubilise phosphates, a qualitative analysis was
144 performed. The bacterial inoculum (4 μl) was incubated in NBRIP basal medium modified with
145 dicalcium and tricalcium phosphate (Nautiyal 1999) until the exponential phase was reached.
146 Plates were incubated at 6°C and 15°C for 21 days. The strains that formed a transparent halo
147 around the colony were identified as positive, indicating that they have the solubilising ability.
148
149 - Growth in a nitrogen-free (NF) medium
150 To evaluate whether the strains could grow in an NF medium, bacterial suspensions of 108
151 CFU/ml in PBS buffer were prepared. This buffer was used to reduce the nitrogen traces from
152 the initial culture. Finally, 4 μl of each strain was inoculated in an NF medium (Döbereiner and
7 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 8 of 38
153 Pedrosa 1987) and incubated at 6°C and 15°C for 7 days. The strains that showed growth in
154 this medium were considered positive.
155
156 - Metabolism of 1-aminocyclopropane-1-carboxylic acid (ACC)
157 The consumption ability of ACC as a single source of nitrogen was qualitatively evaluated. For
158 this, a bacterial suspension of 108 CFU/ml in PBS buffer was prepared for each of the evaluated
159 strains. Subsequently, 4 μl of each strain, delivered as a drop, was placed on a Petri dish with
160 the DF medium (Dworkin and Foster 1958). The DF medium was prepared as described by
161 Penrose and Glick (2003) and supplemented with ACC (3 mM). Petri dishes were incubated at
162 6°C and 15°C for 7 days. The strains that showed growth in this medium were considered
163 positive.
164 Draft
165 - Hydrogen cyanide (HCN) production
166 HCN production was evaluated in a qualitative manner in accordance with the methodology
167 described by Nandi et al. (2016). The strains were cultivated in Petri dishes with King’s B
168 medium supplemented with glycine (4.4 g/L); each Petri dish was sealed with a Parafilm for 7
169 days at 6°C and 15°C. The qualitative production of HCN was evaluated using Cyantesmo
170 paper (Machery-Nagel GmbH and Co., Duren, Germany), which turns blue in the presence of
171 HCN.
172
173 - Siderophore production
174 The qualitative determination of siderophores was performed by plating 4 μl of each bacterial
175 inoculum at a concentration of 108 CFU/ml in a CAS culture medium (Louden et al. 2011).
176 The plates were incubated at 6°C and 15°C for 7 days. The appearance of translucent orange
177 halos around the colonies was considered positive for siderophore production.
8 https://mc06.manuscriptcentral.com/cjm-pubs Page 9 of 38 Canadian Journal of Microbiology
178 Evaluation of the effect of plant growth-promoting psychrotolerant strains in quinoa
179 plants
180 Bacterial suspensions of 108 CFU/ml in PBS buffer were prepared for each of the evaluated
181 strains. Furthermore, C. quinoa var. Kancolla was sterilised using alcohol at 70% for 2 minutes
182 and sodium hypochlorite at 3% for 2 minutes and then washed with sterile distilled water.
183 Subsequently, the seeds were inoculated with 30 ml of each bacterial suspension for 20
184 minutes, and a control without inoculum was also prepared. The seeds were placed in
185 germination systems in agar water at 50% and incubated at 22°C. The different sprouts were
186 placed in pots containing a mix of sand, vermiculite and sterile peat (1:1:1). The plants were
187 maintained in a greenhouse at 16°C ± 1°C with light/dark cycles of 12 hours for 90 days. The
188 growth and development with different procedures were evaluated in accordance with different
189 agronomic parameters (aerial and radicularDraft length, aerial and radicular dry/wet weight and
190 number of leaves); eight biological replicas per treatment were made.
191 Plant parameters were measured at the end of the experiment (90 days after sowing). Parametric
192 data were subjected to analysis of variance and Duncan’s multiple range tests with a p-value
193 of <0.05. All analyses were performed using STATGRAPHICS Centurion 18 version 18.1.01
194 statistical package.
195
196 Results
197
198 Evaluation and selection of bacterial strains tolerant to low temperatures
199 The colony size of 51 strains was evaluated at 4°C, 6°C, 15°C and 24°C (Fig. 1). Moreover,
200 73% (37) of the strains could grow at 4°C and 92% could grow at 6°C; most of them showed
201 a large colony level of 2, 3 or 4. At 15°C, all strains were able to grow and most of them showed
202 a large colony level of 3 or 4. According to these results, in total, 37 strains could grow at 4°C–
9 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 10 of 38
203 24°C, with 24°C as the optimal growth temperature; therefore, the studied microorganisms
204 were psychrotolerant or psychrotrophic bacteria.
205 BOX-PCR for molecular genotypic analysis
206 BOX-PCR genotypic analysis of the 37 psychrotolerant isolates generated a distinct variation
207 in the banding pattern, and these were classified in a total of 12 clusters. For the molecular
208 taxonomic identification, one representative isolate was selected from each cluster.
209 Molecular taxonomy identification
210 From the sequencing analysis of the 16S rRNA gene of a representative strain from each
211 distinct BOX profile (Table 1, Fig. 3), the presence of the beta- and gamma-proteobacteria
212 classes was detected; the latter was the dominant and most diverse at the genus level including
213 Enterobacter (43.2% of the total), Pseudomonas (21.6%) and Acinetobacter (21.6%), followed
214 by beta-proteobacteria with the BurkholderiaDraft genus as the single representative (13.5%).
215 The Pseudomonas genus showed a higher diversity at the species level. The representative
216 strains ILQ215 (cluster A), ILQ103 (cluster B), ILQ219 (cluster C), JUQ307 (cluster I) and
217 JUQ310 (cluster H) were found to be related to different species such as P. silesiensis, P.
218 brassicacearum, P. corrugata, P. plecoglossicida and P. oryzihabitans, respectively, with
219 similarity levels of >99%. In contrast, the ILQ201 (cluster E), JUQ409 (cluster D) and ILQ104
220 (cluster K) strains were related to the Enterobacter genus (similarity > 99%); the JUQ304
221 (cluster J) and JUQ303 (cluster L) strains were related to the Acinetobacter genus, with 93.36%
222 and 99.57% similarity, respectively and ILQ211 (cluster F) and ILQ216 (cluster G) were
223 identified as Burkholderia contaminans with a 100% similarity. Identified psychrotolerant
224 bacterial species not reported as human pathogens were selected for the following analysis of
225 plant growth-promoting traits.
10 https://mc06.manuscriptcentral.com/cjm-pubs Page 11 of 38 Canadian Journal of Microbiology
226 Plant growth-promoting traits in psychrotolerant strains
227 The plant growth-promoting traits of the psychrotolerant species P. silesiensis (ILQ215 strain),
228 P. brassicacearum (ILQ103 strain) and P. plecoglossicida (JUQ307 strain) were evaluated
229 using in vitro trials at 15°C and 6°C (Table 2). These species are not reported to cause diseases
230 in humans and could be considered a safe plant inoculum for the following traits.
231 All strains, except ILQ103 (P. brassicacearum), produced 0.7–20.83 μg/ml of IAA at 15°C.
232 Conversely, at 6°C, the ILQ215 strain (P. silesiensis) produced 16.73 μg/ml of IAA. In terms
233 of the phosphate salts solubilising ability at low temperatures, all the Pseudomonas strains
234 could generate halos of solubilisation at 15°C, with diameters of 5.77–7.57 mm in a bicalcium
235 medium and 5.17–6.37 mm in a tricalcium medium. At 6°C, the halos produced had a diameter
236 of 5.87–7.1 mm in a dicalcium medium; however, in a tricalcium medium, ILQ215 and
237 JUQ307 strains showed activity with halosDraft of 4.83 and 5.5 mm, respectively. The Pseudomonas
238 strains grew at 15°C and 6°C in an NF medium. They could also metabolise ACC as a single
239 source of nitrogen at 15°C; it was observed that ILQ103 and ILQ215 could also consume this
240 substrate at 6°C (Table 2). In the same way, all the Pseudomonas strains produced siderophores
241 at 15°C, and ILQ307 (P. plecoglossicida) stood out owing to its ability to produce siderophores
242 at 6°C. Furthermore, ILQ103 strain (P. brassicacearum) could produce HCN at 6°C and 15°C.
243 Effect of plant growth-promoting psychrotolerant strains on the growth of C. quinoa
244 The effect of strains ILQ215 (Pseudomonas silesiensis), JUQ307 (P. plecoglossicida) and
245 ILQ103 (P. brassicacearium) on the growth and development of quinoa plants in a greenhouse
246 was evaluated (Table 3, Fig. 4). ILQ215 and JUQ 307 showed a significantly positive effect on
247 the growth of quinoa plants compared with the control without inoculum. Both strains were
248 found to increase plant aerial (about 50%) and radicular length in 112% and 79%, respectively,
249 aerial wet weight in 172% and 213%, respectively, radicular wet weight in 243% and 318%,
250 respectively, aerial dry weight in 236% and 326%, respectively, and radicular dry weight in
11 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 12 of 38
251 440% and 366%, respectively. Contrary to expectations, inoculation treatment with ILQ103
252 totally inhibited the emergence of all inoculated seeds.
253
254 Discussion
255 In this study, psychrotolerant bacteria strains belonging to the Pseudomonas genus were
256 isolated from the soil of the Peruvian Andean Plateau where quinoa (C. quinoa) is grown. These
257 strains exhibit different plant growth-promoting abilities at low temperatures and have a
258 positive effect on the growth and development of quinoa plants.
259
260 Their psychrotolerant ability was evaluated with a total of 51 isolated bacteria obtained from
261 the rhizosphere of quinoa plants; it was found that a total of 37 strains were capable of growing
262 at 4°C, 6°C, 15°C and 24°C, showingDraft optimal levels of growth at 24°C. These results were
263 similar to those reported by Ortiz-Ojeda et al. (2017), who reported that endemic bacteria from
264 the high Andean regions were capable of growing at 6°C, 12°C and 28°C, with the highest
265 levels of growth at 28°C. Furthermore, Przemieniecki et al. (2014) isolated bacteria that were
266 capable of growing at 8°C, 10°C and 28°C from the rhizosphere of wheat crops, and Anwar et
267 al. (2019) isolated bacteria that were capable of growing between 5°C and 35°C from the
268 rhizosphere of pea plants, classifying them as psychrotolerant or psychrotrophic bacteria. These
269 bacteria are capable of growing at low temperatures and have an optimal growth at
270 approximately 20°C, unlike the psychrophiles whose optimal growth temperature is above
271 15°C or less (Moyer et al. 2017; Morita 1975). Therefore, the 37 bacterial strains studied here
272 were psychrotolerant or psychrotrophic.
273 Conversely, the 12 different BOX genomic fingerprints detected (Fig. 2) in these
274 psychrotolerant bacteria evidenced the presence of a relative diversity among the groups in the
275 study. Amplification of the 16S rRNA gene sequence was performed to study the phylogeny
12 https://mc06.manuscriptcentral.com/cjm-pubs Page 13 of 38 Canadian Journal of Microbiology
276 of the selected bacteria. However, the use of other housekeeping genes could be useful to
277 improve the reliability of phylogenies. Genes like gyrB or rpoD could be suitable phylogenetic
278 markers for Pseudomonas identification (Watanabe et al., 2001; Yamamoto et al., 2000).
279 Representative strains corresponding to each BOX fingerprint (Fig. 3) enabled the detection of
280 the presence of bacteria belonging to the beta- and gamma-proteobacteria classes. Within the
281 gamma-proteobacteria class, a wide diversity of species of the genus Pseudomonas such as P.
282 silesiensis, P. brassicacearum, P. corrugata, P. plecoglossicida and P. oryzihabitans were
283 found. Moreover, P. silesiensis had been isolated for the first time from a pesticide industry
284 wastewater treatment plant. This species is capable of growing at 4°C, and its optimal
285 temperature is 15°C–30°C, which is closely related to that of the P. mandelii subgroup
286 (Kaminski et al. 2018), which comprises psychrotrophic species capable of growing at 4°C,
287 with an optimal temperature of 25°C Draft(Hong et al. 2012). In addition, P. brassicacearum can
288 grow at 4°C–37°C (Ivanova et al. 2009), and its ability as a biocontrol agent of phytopathogenic
289 fungi has been reported owing to its ability to produce antifungal compounds (Mandryk-
290 Litvinkovich et al. 2017) and its phytotoxic action, which is involved in the inhibition of
291 processes such us germination (Chung et al. 2008). Conversely, P. corrugata is a ubiquitous
292 bacterium and has been isolated from a wide variety of sources like the Himalayan soils
293 (Pandey et al. 2002). This species was reported and described as the causal agent of pith
294 necrosis in different crops (Catara, 2007). Furthermore, P. plecoglossicida is capable of
295 growing at 10°C (Nishimori et al. 2000). Strains of this species have a beneficial effect in plant
296 development and growth (Dharni et al. 2014; Rahmoune et al. 2017). This species belongs to
297 the P. putida group and is capable of interacting with plant roots, thus generating positive
298 effects such as promotion of plant growth (Cheng et al. 2012). Finally, P. oryzihabitans has
299 been considered a potential nosocomial pathogen, especially in immunocompromised hosts
13 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 14 of 38
300 (Woo et al. 2014) or in patients with surgical site infections, mastitis and wound infections
301 after bites or trauma (Tena and Fernandez 2015).
302 Conversely, the presence of the Enterobacter genus in the soil is common; it is one of the most
303 isolated bacteria from the rhizosphere of different crops such as corn, rice, cotton, peanut,
304 broccoli and sweet potato (McInroy and Kloepper 1995; Zakria et al. 2008; Morales-García et
305 al. 2011). Its presence has also been reported in the Himalayan soil (Kandasamy et al. 2013);
306 however, E. cancerogenus and E. hormoachei have been reported as pathogenic species.
307 Another genus present was Acinetobacter, and A. soli has psychrotolerant ability because it can
308 grow in freezing environments such as the Siberian soil (Suzuki et al. 2002). This genus is
309 known to cause blood infections in immunocompromised newborns and can be spread among
310 patients (Pellegrino et al. 2011). Within the beta-proteobacteria class, the presence of
311 Burkholderia genus was identified withDraft the species B. contaminans, which has been reported
312 to be pathogenic and is catalogued as an infectious respiratory agent affecting patients with
313 cystic fibrosis (Martina et al. 2013).
314 The reported ability of all the described bacterial genera to grow at low temperatures support
315 their presence in the soils of the Peruvian Andean Plateau, where temperatures can range from
316 17°C to less than 0°C (Andrade 2018). Non-pathogenic psychrotolerant species majorly belong
317 to the Pseudomonas genus. It is important to highlight that this was the genus that showed the
318 highest species diversity. Different species of this genus are capable of growing at low
319 temperatures and have presented beneficial properties through their growth-promoting
320 activities in different crops (Yarzábal et al. 2018; Balcazar et al. 2015; Das et al. 2003).
321 The plant growth-promoting traits at low temperatures were evaluated for the psychrotolerant
322 strains identified as species that are not reported to be phytopathogenic or pathogenic for
323 human beings; these belonged to the Pseudomonas genus. ILQ215 (P. silesiensis), JUQ307 (P.
324 plecoglossicida) and ILQ103 (P. brassicacearum) strains exhibited different plant growth-
14 https://mc06.manuscriptcentral.com/cjm-pubs Page 15 of 38 Canadian Journal of Microbiology
325 promoting traits (Table 2). These strains produce IAA at low temperatures (15°C), with ILQ215
326 (P. silesiensis) also being capable of producing this phytohormone at 6°C. The production of
327 this phytohormone is an important attribute in the promotion of radicular development of
328 plants, and strains of this genus have been reported to produce them at low temperatures such
329 as at 15°C (Selvakumar et al. 2009; Yarzábal et al. 2018). Furthermore, these strains were also
330 able to solubilise phosphate salts at 15°C, generating solubilisation halos of diameter > 5 mm
331 both in dicalcium and tricalcium mediums. ILQ215 (P. silesiensis) and JUQ307 (P.
332 plecoglossicida) also showed activity at 6°C (Table 2) with halos of diameter >5 mm and 3
333 mm in dicalcium and tricalcium mediums, respectively. These results are similar to those
334 obtained by Ortiz-Ojeda et al. (2017), who reported that strains of Pseudomonas isolated from
335 the Andean Plateau soil related to Lepidium meyenii showed activity at 6°C and 12°C by
336 producing solubilisation halos in dicalciumDraft and tricalcium mediums, with diameters of >5 and
337 2 mm at 12°C, respectively, and >5 mm in both medium at 6°C. Besides, it was also reported
338 that strains isolated from the Himalayan soil showed this ability at low temperatures (Trivedi
339 and Pandey 2007; Mishra et al. 2011). Recently, Rondón et al. (2019) found that strains isolated
340 from glaciers showed the ability of solubilising phosphate salts at 15°C.
341
342 ILQ215, JUQ307 and ILQ103 strains could grow in an NF medium and consume 1-
343 aminocyclopropane-1-carboxylic acid (ACC) as a single source of nitrogen at low temperatures
344 (Table 2). ACC deaminase metabolises ACC, a precursor of the ethylene stress hormone, at an
345 optimal temperature of approximately 25°C–30°C (Glick 2014). However, Cheng et al. (2007)
346 reported a strain of Pseudomonas that showed activity at 10°C and promoted the growth of
347 canola plants even under saline stress conditions. Moreover, P. brassicacearum is capable of
348 producing ACC deaminase, which acts as a growth promoter in tomatoes (Belimov et al. 2007).
349 Li et al. (2017) reported strains of P. plecoglossicida with ACC deaminase activity. The
15 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 16 of 38
350 presence of enzymatic activity even at low temperatures shows metabolic versatility of the
351 Pseudomonas strains that are being studied.
352 Siderophore production has also been acknowledged within the genus; a strain of P. mandelii
353 (a species phylogenetically related to P. silesiensis) can produce the siderophore pyoverdine,
354 whereas P. plecoglossicida can produce siderophores of a different nature (Meyer et al. 2002)
355 and has been reported to be positive for the production of IAA, solubilisation of phosphates,
356 and antagonistic activity against phytopathogenetic fungi (Jha et al. 2009). In accordance with
357 the above, the presence of metabolic activity and plant growth-promoting properties at low
358 temperatures makes these strains of psychrotrophic Pseudomonas relevant targets to study their
359 effect in Andean Plateau crops such as quinoa.
360 The effect of inoculation of ILQ215, JUQ307 and ILQ103 strains on the growth of quinoa
361 plants was evaluated. ILQ103 strain (DraftP. brassicacearum) was the only one that inhibited the
362 emergence of quinoa sprouts in their totality. This species possessed biocontroller activity
363 because of its nematicidal effect, with HCN as the main active agent (Nandi et al. 2016).
364 However, a phytotoxic effect was reported from a strain of this species by inhibiting the
365 germination and development of radish sprouts (Chung et al. 2008). Therefore, the HCN
366 production of ILQ103 strain could have been an inhibitory factor in the emergence of the
367 quinoa. Conversely, inoculation of ILQ215 (P. silensiensis) and JUQ307 (P. plecoglossicida)
368 strains showed a significant increase in the growth and biomass (about 170% and 210%,
369 respectively, compared with the control without inoculation) of quinoa plantlets 90 days after
370 sowing. Such an effect is comparable with that reported by Mishra et al. (2008), who reported
371 that a psychrotolerant strain originating from the north-east of the Himalayas could
372 significantly promote the increase in the biomass of 30-day-old wheat plants.
373 Different genus strains could improve the nutrient intake of 60-day-old wheat plants when
374 compared with the control without inoculation (Mishra et al. 2011). Yarzábal et al. (2018)
16 https://mc06.manuscriptcentral.com/cjm-pubs Page 17 of 38 Canadian Journal of Microbiology
375 reported the plant growth-promoting effect of Pseudomonas strains in the germination and
376 growth of wheat at 15°C. Recently, the ability of different species of Pseudomonas isolated
377 from glaciers in Venezuela to stimulate lengthening of the roots and stem of wheat plants has
378 also been reported (Rondón et al. 2019). In accordance with the above, the abilities of ILQ215
379 and JUQ307 to stimulate and improve the development of quinoa plants show their relevance
380 as potential biofertilizers for this Andean Plateau crop. The psychrotolerant ability of these
381 strains suggests that they can be successfully used in the Peruvian Andean Plateau, which is
382 characterised by severe seasonal meteorological phenomena such as frost that leads to an abrupt
383 decrease in the ambient temperature. The temperature of the soil during frost is between 2°C
384 and 10°C, which could affect introduced inoculants based on mesophyll species (Trivedi et al.
385 2012). In this context, use of psychrotrophic bacteria as inoculants is suitable. Thus, the
386 psychrotolerant ability showed by theDraft isolated Pseudomonas suggests that they could be
387 successfully used as inoculants in the Peruvian Andean Plateau, where frost is a common
388 meteorological phenomenon that affects plant growth.
389 In conclusion, this study demonstrated the presence of psychrotolerant bacteria in Peruvian
390 Andean Plateau soil associated with C. quinoa. Among all isolates, two non-pathogenic
391 species, P. silensiensis and P. plecoglossicida, exhibited important plant growth-promoting
392 characteristics in vitro at low temperatures and were also capable of positively influencing the
393 growth of quinoa seedlings under greenhouse conditions. This indicates the potential of both
394 strains to be used as inoculants to improve the development of quinoa cultivated in the Andean
395 regions, where low temperature is a limiting factor for agricultural production.
396
397 Acknowledgements
398 This work was supported by Consejo Nacional de Ciencia y Tecnología (CONCyTEC) and
399 Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECyT) [Contract No. 105-
17 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 18 of 38
400 2015, and Contract No. 009-2017]. The authors would like to thank Ernesto Ormeño Orrillo
401 (UNALM) for his suggestions and critical editing of the manuscript, Jesús Arcos (INIA-Puno,
402 Peru) for coordinating the sampling and for providing quinoa seeds, Alexandra Florián for
403 conducting the sampling and Lee-Anne Maningas for improving the use of English in the
404 manuscript.
405
406 Declarations of interest
407 None.
408
409
410
411 Draft
18 https://mc06.manuscriptcentral.com/cjm-pubs Page 19 of 38 Canadian Journal of Microbiology
412 References
413
414 Abugoch, L.E., Romero, N., Tapia, C.A., Silva, J., and Rivera, M. 2008. Study of some
415 physicochemical and functional properties of quinoa (Chenopodium Quinoa Willd)
416 protein isolates. J. Agric. Food Chem. 56(12), 4745–4750. doi:10.1021/jf703689u
417 Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic local
418 alignment search tool. J. Mol. Biol. 215(3), 403–410. doi:10.1016/S0022-2836(05)80360-
419 2
420 Andrade, M. 2018. Clima y eventos extremos del Altiplano Central perú-boliviano /
421 Climate and extreme events from the Central Altiplano of Peru and Bolivia 1981-2010. J.
422 Chem. Inf. Model (Geogr) Geogr. Bernensia. doi:10.4480/GB2018.N01
423 Anwar, M.S., Paliwal, A., Firdous,Draft N., Verma, A., Kumar, A., and Pande, V. 2019. Co-
424 culture development and bioformulation efficacy of psychrotrophic PGPRs to promote
425 growth and development of Pea (Pisum sativum) plant. J. Gen. Appl. Microbiol. 65(2),
426 88–95. doi:10.2323/jgam.2018.05.007
427 Balcazar, W., Rondón, J., Rengifo, M., Ball, M.M., Melfo, A., Gómez, W., et al. 2015.
428 Bioprospecting glacial ice for plant growth promoting bacteria. Microbiol. Res. 177, 1–7.
429 doi:10.1016/j.micres.2015.05.001
430 Belimov, A., Dodd, I., Safronova, V., Hontzeas, N., and Davies, W. 2007. Pseudomonas
431 brassicacearum strain Am3 containing 1-aminocyclopropane-1-carboxylate deaminase
432 can show both pathogenic and growth-promoting properties in its interaction with tomato.
433 J. Exp. Bot. 58(6), 1485–1495. doi:10.1093/jxb/erm010
434 Calvo, P., and Zúñiga, D. 2010. Caracterización fisiológica de cepas de bacillus spp.
435 aisladas de la rizósfera de papa (Solanum tuberosum). Ecología Aplicada. 9(1–2), 31-39.
436 doi: 10.21704/rea.v9i1-2.393
19 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 20 of 38
437 Catara, V. 2007. Pseudomonas corrugata: plant pathogen and/or biological resource?
438 Mol. Plant. Pathol. 8(3), 233–244. doi:10.1111/j.1364-3703.2007.00391.x
439 Cheng, Z., Park, E., Glick, B.R. 2007. 1-Aminocyclopropape-1- carboxylate deaminase
440 from Pseudomonas putida UW4 facilitate the growth of canola in the presence of sat.
441 Can. J. Microbiol. 53(7), 912–918. doi:10.1139/W07-050
442 Cheng, Z., Woody, O.Z., McConkey, B.J., and Glick, B. R. 2012. Combined effects of
443 the plant growth-promoting bacterium Pseudomonas putida UW4 and salinity stress on
444 the Brassica napus proteome. Appl. Soil. Ecol. 61, 255–263.
445 doi:10.1016/j.apsoil.2011.10.006
446 Chung, B.S., Aslam, Z., Kim, S.W., Kim, G.G., Kang, H.S., Ahn, J.W., et al. 2008. A
447 bacterial endophyte, Pseudomonas brassicacearum YC5480, isolated from the root of
448 Artemisia sp. producing antifungalDraft and phytotoxic compounds. Plant Pathol. J. 24(4),
449 461–468. doi:10.5423/PPJ.2008.24.4.461
450 Das, K., Katiyar, V., and Goel, R. 2003. ‘P’ solubilization potential of plant growth
451 promoting Pseudomonas mutants at low temperature. Microbiol. Res. 158(4), 359–362.
452 doi:10.1078/0944-5013-00217
453 Dharni, S., Srivastava, A.K., Samad, A., and Patra, D. D. 2014. Impact of plant growth
454 promoting Pseudomonas monteilii PsF84 and Pseudomonas plecoglossicida PsF610 on
455 metal uptake and production of secondary metabolite (monoterpenes) by rose-scented
456 geranium (Pelargonium graveolens cv. bourbon) grown on tannery slud. Chemosphere.
457 117(1), 433–439. doi:10.1016/j.chemosphere.2014.08.001
458 Döbereiner, J. and Pedrosa F. O. 1987. Nitrogen-Fixing Bacteria in Nonleguminous Crop
459 Plants. p. 155. In: Brock/Springer Series in Contemporary Biosciences. Science Tech
460 Publishers, Madison, WI.
461 Dworkin, M., and Foster, J.W. 1958. Experiments with some microorganisms which
20 https://mc06.manuscriptcentral.com/cjm-pubs Page 21 of 38 Canadian Journal of Microbiology
462 utilize ethane and hydrogen. J. Bacteriol. 75(5), 592–603. doi: 10.1128/JB.75.5.592-
463 603.1958
464 Glick, B. R. 2014. Bacteria with ACC deaminase can promote plant growth and help to
465 feed the world. Microbiol. Res. 169(1), 30–39. doi:10.1016/j.micres.2013.09.009
466 Gómez, L., Aguilar, E. 2016. Guía de cultivo de la quinua. (U. N. A. La Molina, Ed.) (2th
467 ed.). Lima: FAO y Universidad Nacional Agraria La Molina. Available from
468 http://www.fao.org/3/a-i5374s.pdf [accessed 17 January 2020].
469 Gordon, S., and Weber, R. 1951. Colorimetric estimation of indoleacetic acid. Plant
470 Physiol. 26(1), 192–195. doi:10.1104/pp.26.1.192
471 Hong, S., Lee, C., and Jang, S.H. 2012. Purification and properties of an extracellular
472 esterase from a cold-adapted Pseudomonas mandelii. Biotechnol. Lett. 34(6), 1051–1055.
473 doi:10.1007/s10529-012-0866-y. Draft
474 Ivanova, E.P., Christen, R., Bizet, C., Clermont, D., Motreff, L., Bouchier, C., et al. 2009.
475 Pseudomonas brassicacearum subsp. neoaurantiaca subsp. nov., orange-pigmented
476 bacteria isolated from soil and the rhizosphere of agricultural plants.
477 Int. J. Syst. Evol. Microbiol. 59(10), 2476–2481. doi:10.1099/ijs.0.009654-0.
478 Jha, B.K., Gandhi Pragash, M., Cletus, J., Raman, G., and Sakthivel, N. 2009.
479 Simultaneous phosphate solubilization potential and antifungal activity of new
480 fluorescent pseudomonad strains, Pseudomonas aeruginosa, P. plecoglossicida and P.
481 mosselii. World J. Microbiol. Biotechnol. 25(4), 573–581. doi:10.1007/s11274-008-9925-
482 x.
483 Josine, T.L., Ji, J., Wang, G., and Guan, C. F. 2011. Advances in genetic engineering for
484 plants abiotic stress control. Afr. J. Biotechnol. 10(28), 5402–5413. Available from
485 https://www.ajol.info/index.php/ajb/article/view/94312/83693 [accessed 17 January
486 2020].
21 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 22 of 38
487 Kaminski, M.A., Furmanczyk, E.M., Sobczak, A., Dziembowski, A., and Lipinski, L.
488 2018. Pseudomonas silesiensis sp. nov. strain A3 T isolated from a biological pesticide
489 sewage treatment plant and analysis of the complete genome sequence. Syst. Appl.
490 Microbiol. 41(1), 13–22. doi:10.1016/j.syapm.2017.09.002.
491 Kandasamy, P., Chaturvedi, N., Sisodia, B.S., Shasany, A.K., Gahoi, S., Marla, S.S., et al.
492 2013. Expression of CspE by a psychrotrophic bacterium Enterobacter ludwigii PAS1,
493 isolated from Indian Himalayan soil and in silico protein modelling, prediction of
494 conserved residues and active sites. Curr. Microbiol. 66(5), 507–514.
495 doi:10.1007/s00284-013-0304-y.
496 Katoh, K., and Standley, D. M. 2013. MAFFT Multiple Sequence Alignment Software
497 Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 30(4), 772–780.
498 doi:10.1093/molbev/mst010. Draft
499 Kumar Meena, R., Kumar Singh, R., Pal Singh, N., Kumari Meena, S., and Singh Meena,
500 V. 2015. Isolation of low temperature surviving plant growth–promoting rhizobacteria
501 (PGPR) from pea (Pisum sativum L.) and documentation of their plant growth promoting
502 traits. Biocatal. Agric. Biotechnol. 4(4), 806–811. doi:10.1016/j.bcab.2015.08.006.
503 Li, H.B., Singh, R.K., Singh, P., Song, Q.-Q., Xing, Y.-X., Yang, L.-T., et al. 2017.
504 Genetic diversity of nitrogen-fixing and plant growth promoting Pseudomonas species
505 isolated from sugarcane rhizosphere. Front. Microbiol. 8, 1–20.
506 doi:10.3389/fmicb.2017.01268.
507 Louden, B. C., Haarmann, D., and Lynne, A. M. 2011. Use of blue agar CAS assay for
508 siderophore detection. J. Microbiol. Biol. Educ. 12(1), 51–53.
509 doi:10.1128/jmbe.v12i1.249
510 Mandryk-Litvinkovich, M.N., Muratova, A.A., Nosonova, T.L., Evdokimova, O.V.,
511 Valentovich, L.N., Titok, M.A., et al. 2017. Molecular genetic analysis of determinants
22 https://mc06.manuscriptcentral.com/cjm-pubs Page 23 of 38 Canadian Journal of Microbiology
512 defining synthesis of 2,4-diacetylphloroglucinol by Pseudomonas brassicacearum BIM
513 B-446 bacteria. Appl. Biochem. Microbiol. 53(1), 31–39.
514 doi:10.1134/S0003683817010124.
515 Martina, P., Bettiol, M., Vescina, C., Montanaro, P., Mannino, M.C., Prieto, C.I., et al.
516 2013. Genetic diversity of burkholderia contaminans isolates from cystic fibrosis patients
517 in Argentina. J. Clin. Microbiol. 51(1), 339–344. doi:10.1128/JCM.02500-12.
518 Meyer, J.-M., Geoffroy, V.A., Baida, N., Gardan, L., Izard, D., Lemanceau, P., et al.
519 2002. Siderophore typing, a powerful tool for the identification of fluorescent and
520 nonfluorescent Pseudomonads. Appl. Environ. Microbiol. 68(6), 2745–2753.
521 doi:10.1128/AEM.68.6.2745-2753.2002.
522 Mishra, P., Bisht, S., Ruwari, P., Selvakumar, G., Joshi, G., Bisht, J., et al. 2011.
523 Alleviation of cold stress in inoculatedDraft wheat (Triticum aestivum L.) seedlings with
524 psychrotolerant Pseudomonads from NW Himalayas. Arch. Microbiol. 193(7), 497–513.
525 doi:10.1007/s00203-011-0693-x.
526 Mishra, P.K., Mishra, S., Selvakumar, G., Bisht, S.C., Bisht, J.K., Kundu, S., et al. 2008.
527 Characterisation of a psychrotolerant plant growth promoting Pseudomonas sp. strain
528 PGERs17 (MTCC 9000) isolated from North Western Indian Himalayas. Ann. Microbiol.
529 58(4), 561–568. doi:10.1007/BF03175558
530 McInroy, J. A. and Kloepper, J. W. 1995. Population dynamics of endophytic bacteria in
531 field-grown sweet cron and cotton. Can. J. Microbiol. 41(10), 895–901. doi:10.1139/m95-
532 123
533 Morales-García, Y., Juárez-Hernández, D., Aragón-Hernández, C., Mascarua-Esparza,
534 M., Bustillos-Cristales, M., Fuentes-Ramírez, L., et al. 2011. Growth response of maize
535 plantlets inoculated with. Rev. Argent. Microbiol. 43, 287–293. Retrieved from
536 https://www.redalyc.org/articulo.oa?id=213021188009.
23 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 24 of 38
537 Morita, R.Y. 1975. Psychrophilic bacteria. Bacteriol. Rev. 39,144–167.
538 Morita, R. Y., and Moyer, C. L. (2001). Psychrophiles, Origin of. In Encyclopedia of
539 Biodiversity. Elsevier.4:917–924. doi: 10.1016/B0-12-226865-2/00362-X
540 Moyer, C. L., Eric Collins, R., and Morita, R. Y. (2017). Psychrophiles and
541 Psychrotrophs. In Reference Module in Life Sciences. Elsevier. pp. 1-6. Available from
542 https://doi.org/10.1016/B978-0-12-809633-8.02282-2 [accessed 09 March 2020]
543 Nandi, M., Berry, C., Brassinga, A. K.C., Belmonte, M.F., Fernando, W. G.D., Loewen,
544 P.C., et al. 2016. Pseudomonas brassicacearum strain DF41 kills Caenorhabditis elegans
545 through biofilm-dependent and biofilm-independent mechanisms. Appl. Environ.
546 Microbiol. 82(23), 6889–6898. doi:10.1128/AEM.02199-16.
547 Nautiyal, C. 1999. An efficient microbiological growth medium for screening phosphate
548 solubilizing microorganisms. FEMSDraft Microbiol. Lett. 170(1), 265–270.
549 doi:10.1016/S0378-1097(98)00555-2.
550 Nishimori, E., Kita-Tsukamoto, K., and Wakabayashi, H. 2000. Pseudomonas
551 plecoglossicida sp. nov., the causative agent of bacterial haemorrhagic ascites of ayu,
552 Plecoglossus altivelis. Int. J. Syst. Evol. Microbiol. 50(1), 83–89. doi:10.1099/00207713-
553 50-1-83.
554 Odoh, C. 2017. Plant growth promoting rhizobacteria (PGPR): A bioprotectant
555 bioinoculant for sustainable agrobiology. A Review.
556 Int. J. Adv. Res. Biol. Sci. (IJARBS). 4(5), 123–142. doi:10.22192/ijarbs.2017.04.05.014.
557 Ogata-Gutiérrez, K., Alvarado, D., Chumpitaz-Segovia, C., and Zúñiga-Dávila, D. 2016.
558 Characterization of plant growth promoting rhizobacteria isolated from the rhizosphere of
559 Peruvian Highlands native crops. Int. J. Plant Soil Sci. 11(1), 1–8.
560 doi:10.9734/IJPSS/2016/24573.
561 Ortiz-Ojeda, P., Ogata-Gutiérrez, K., and Zúñiga-Dávila, D. 2017. Evaluation of plant
24 https://mc06.manuscriptcentral.com/cjm-pubs Page 25 of 38 Canadian Journal of Microbiology
562 growth promoting activity and heavy metal tolerance of psychrotrophic bacteria
563 associated with maca (Lepidium meyenii Walp.) rhizosphere. AIMS Microbiol. 3(2),
564 279–292. doi:10.3934/microbiol.2017.2.279.
565 Pandey, A., Palni, LMS., Mulkalwar, P., Nadeem. 2002. Effect of temperature on
566 solubilization of tricalcium phosphate by Pseudomonas corrugata. J. Sci. Ind. Res. 61,
567 457–460. Available from http://nopr.niscair.res.in/handle/123456789/26363 [accessed 17
568 Junuary 2020].
569 Pellegrino, F.L.P.C., Vieira, V.V., Baio, P.V.P., dos Santos, R.M.R., dos Santos, A.L.A.,
570 de Barros Santos, N.G., M., et al. 2011. Acinetobacter soli as a cause of bloodstream
571 infection in a neonatal intensive care unit: Table 1. J. Clin. Microbiol. 49(6), 2283–2285.
572 doi:10.1128/JCM.00326-11.
573 Penrose, D.M., and Glick, B. R. 2003.Draft Methods for isolating and characterizing ACC
574 deaminase-containing plant growth-promoting rhizobacteria. Physiol. Plant. 118(1), 10–
575 15. doi:10.1034/j.1399-3054.2003.00086.x.
576 Przemieniecki, S.W., Kurowski, T.P., Korzekwa, K., and Karwowska, A. 2014. The
577 effect of psychrotrophic bacteria isolated from the root zone of winter wheat on selected
578 biotic and abiotic factors. J. Plant Prot. Res. 54(4), 407–413. doi:10.2478/jppr-2014-0061.
579 Rahmoune, B., Morsli, A., Khelifi-Slaoui, M., Khelifi, L., Strueh, A., Erban, A., et al.
580 2017. Isolation and characterization of three new PGPR and their effects on the growth of
581 Arabidopsis and Datura plants. J. Plant Prot. Res. 12(1), 1–6.
582 doi:10.1080/17429145.2016.1269215.
583 Robertson, G. and Grandy, A. 2006. Soil system management in temperate regions. In
584 Biological Approaches to Sustainable Soil Systems. Edited by N. Uphoff, A. Ball, E.
585 Fernandes, H. Herren, O. Husson, M. Laing, C. Palm, J. Pretty, P. Sanchez, N.
586 Snanginga, and J. Thies. CRC Press. pp. 27–39. doi:10.1201/9781420017113
25 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 26 of 38
587 Rondón, J.J., Ball, M.M., Castro, L.T., and Yarzábal, L. A. 2019. Eurypsychrophilic
588 Pseudomonas spp. isolated from Venezuelan tropical glaciers as promoters of wheat
589 growth and biocontrol agents of plant pathogens at low temperatures. Environ.
590 Sustainability. 2(3), 265–275. doi:10.1007/s42398-019-00072-2.
591 Selvakumar, G., Joshi, P., Nazim, S., Mishra, P.K., Bisht, J.K., and Gupta, H.S. 2009.
592 Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 (MTCC
593 8984), a psychrotolerant bacterium isolated from a high altitude Himalayan rhizosphere.
594 Biologia. 64(2), 239–245. doi:10.2478/s11756-009-0041-7.
595 Snyder, R., and Melo-Abreu, J. 2010. Protección contra las heladas: fisiología y
596 temperaturas críticas. Protección contra las heladas: fundamentos, práctica y economía
597 (Vol. 1). Rome: FAO. Available from http:// http://www.fao.org/3/y7223s/y7223s00.htm
598 [accessed 17 January 2020]. Draft
599 Suzuki, T., Nakayama, T., Kurihara, T., Nishino, T., and Esaki, N. 2002. Primary
600 structure and catalytic properties of a cold-active esterase from a psychrotroph,
601 Acinetobacter sp. strain no. 6. isolated from Siberian soil. Biosci. Biotechnol. Biochem.
602 66(8), 1682–1690. doi:10.1271/bbb.66.1682.
603 Tena, D., and Fernández, C. 2015. Pseudomonas oryzihabitans: an unusual cause of skin
604 and soft tissue infection. Infect. Dis. 47(11), 820–824.
605 doi:10.3109/23744235.2015.1034170
606 Trivedi, P., and Pandey, A. 2007. Application of immobilized cells of Pseudomonas
607 putida strain MTCC 6842 in alginate to solubilize phosphate in culture medium and soil.
608 J. Plant Nutr. Soil Sci. 170(5), 629–631. doi:10.1002/jpln.200625193.
609 Trivedi, P., Pandey, A., and Palni, L. M. S. 2012. Bacteria in Agrobiology: Plant
610 Probiotics. In Bacteria in agrobiology: Plant probiotics (Vol. 9783642275). Edited by D.
611 K. Maheshwari. Springer Berlin Heidelberg, Berlin, Heidelberg. doi:10.1007/978-3-642-
26 https://mc06.manuscriptcentral.com/cjm-pubs Page 27 of 38 Canadian Journal of Microbiology
612 27515-9.
613 Versalovic, J., Koeuth, T., and Lupski, R. 1991. Distribution of repetitive DNA sequences
614 in eubacteria and application to finerpriting of bacterial enomes. Nucleic Acids Res.
615 19(24), 6823–6831. doi:10.1093/nar/19.24.6823.
616 Versalovic J., Schneider M., de Bruijn F.J., and Lupski J. 1994. Genomic fingerprinting of
617 bacteria using repetitive sequence-based Polymerase Chain Reaction. Methods Mol. Cell
618 Biol. 5, 25–40.
619 Watanabe, K., Nelson, J., Harayama, S. & Kasai, H. 2001. ICB database: the gyrB database
620 for identification and classification of bacteria. Nucleic. Acids. Res. 29(1), 344–345. doi:
621 10.1093/nar/29.1.344
622 Weisburg, W., Barns, S., Pelletier, D., and Lane, D. 1991. 16S ribosomal DNA
623 amplification for phylogenetic study.Draft J. Bacteriol. 173(2), 697–703.
624 doi:10.1128/jb.173.2.697-703.1991.
625 Woo, K.-S., Choi, J.-L., Kim, B.-R., Kim, J.-E., Kim, K.-H., Kim, J.-M., & Han, J.-Y.
626 2014. Outbreak of Pseudomonas Oryzihabitans Pseudobacteremia Related to
627 Contaminated Equipment in an Emergency Room of a Tertiary Hospital in Korea. Infect.
628 Chemother. 46(1), 42. doi:10.3947/ic.2014.46.1.42
629 Yamamoto, S., Kasai, H., Arnold, D. L., Jackson, R. W., Vivian, A. & Harayama, S.
630 2000. Phylogeny of the genus Pseudomonas: intrageneric structure reconstructed from the
631 nucleotide sequences of gyrB and rpoD genes. Microbiology. 146(10), 2385–2394. doi:
632 10.1099/00221287-146-10-2385
633 Yarzábal, L.A., Monserrate, L., Buela, L., and Chica, E. 2018. Antarctic Pseudomonas
634 spp. promote wheat germination and growth at low temperatures. Polar Biol. 41(11),
635 2343–2354. doi:10.1007/s00300-018-2374-6.
636 Zakria, M., Ohsako, A., Saeki, Y., Yamamoto, A., and Akao, S. 2008. Colonization and
27 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 28 of 38
637 growth promotion characteristics of Enterobacter sp. and Herbaspirillum sp. on Brassica
638 oleracea. Soil Sci. Plant Nutr. 54(4), 507–516. doi:10.1111/j.1747-0765.2008.00265.x.
Draft
28 https://mc06.manuscriptcentral.com/cjm-pubs Page 29 of 38 Canadian Journal of Microbiology
640 Tables
641
642 Table 1. Identification of psychrotolerant strains representative of each BOX cluster (A-L) based on the 16S rRNA gene sequence
643
BOX Psychrotolerant Genbank Closest related strain in terms of the 16S rRNA gene Similarity
cluster† strains isolated accession (%)
No.
A (2) ILQ215 * MN826142 Pseudomonas silesiensisDraft A3T (NR156815.1) 99.79
B (1) ILQ103 * MN826144 Pseudomonas brassicacearum subsp. Neaurantiaca CIP 109457T 99.72
(NR116299.1)
C (2) ILQ219 MN826143 Pseudomonas corrugata (NR117826.1) 99.3
D (3) JUQ409 MN826154 Enterobacter ludwigii EN-119T (NR042349.1) 100
E (3) ILQ201 MN826153 Enterobacter cancerogenus LMG 2693T (NR044977.1) 99.09
F (4) ILQ211 MN826150 Burkholderia contaminans J2956T (NR104978.1) 99.93
G (1) ILQ216 MN826151 Burkholderia contaminans J2956T (NR104978.1) 99.93
H (2) JUQ310 MN826146 Pseudomonas oryzihabitans NBRC 102199T (NR114041.1) 99.93
29 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 30 of 38
I (1) JUQ307 * MN826145 Pseudomonas plecoglossicida NBRC 103162T (NR114226.1) 99.79
J (6) JUQ304 MN826148 Acinetobacter soli B1 T (NR044454.1) 93.36
K (10) ILQ104 MN826152 Enterobacter hormaechei subsp. xiangfangensis 10.17T (NR126208.1) 99.42
L (2) JUQ303 MN826149 Acinetobacter johnsonii ATCC 17909T (NR117624.1) 99.57
644 *Strains identified without reports of pathogenic activity
645 † (#): Number of strains belonging to the BOX cluster
646
647 Draft
648
649
650
651
30 https://mc06.manuscriptcentral.com/cjm-pubs Page 31 of 38 Canadian Journal of Microbiology
652 Table 2. Plant growth-promoting characteristics of in vitro non-pathogenic psychrotolerant strains from the Pseudomonas genus isolated from the
653 C. quinoa rhizosphere
654
Strain Taxon * IAA Phosphate Growth ACC HCN Siderophore
production solubilisation (mm) in Nfb metabolis productio s production
(µg/ml) CaHPO4 Ca3(PO4) m n
2
6°C 15°C 6°C Draft15° 6°C 15° 6° 15° 6° 15°C 6°C 15°C 6°C 15°C
C C C C C
ILQ215 Pseudomonas silesiensis 16.7 20.8 5.8 7.57 4.8 5.47 ++ +++ ++ ++ − − − +
3 ± 3 ± 7 3
0.51 0.42
ILQ103 Pseudomonas 0 0 7.1 5.77 0 5.17 ++ +++ ++ ++ + ++ − +
brassicacearum 0
31 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 32 of 38
JUQ30 Pseudomonas 0 0.73 6.4 6.53 5.5 6.37 + ++ − ++ − − + ++
7 plecoglossicida ± 3 0
0.32
655 ± DS: Standard deviation obtained from three biological replicas
656 +: positive result, −: negative result
657 Rotation intensity and/or growth: none (−), slight (+), high (++), and very high (+++). 658 Draft
32 https://mc06.manuscriptcentral.com/cjm-pubs Page 33 of 38 Canadian Journal of Microbiology
659 Table 3. Effect of inoculation of psychrotolerant strains of the Pseudomonas genus on the growth of C. quinoa plants in a greenhouse
660
Treatment No. of Length (cm) Wet weight (g) Dry weight (g)
leaves
Aerial Radicular Aerial Radicular Aerial Radicular
Control (without inoculant) 9 ± 1 a 7.75 ± 1.3 10.8 ± 2.3 a 0.75 ± 0.153 0.061 ± 0.05 ± 0.006 ± 0.001
a a 0.023 a 0.012 a a
ILQ215 (P. silesiensis) 12 ± 2 b 11.91 Draft ± 22.8 ± 5.2 c 2.043 ± 0.209 ± 0.169 ± 0.033 ± 0.017
1.7 b 0.512b 0.096 b 0.057 b b
JUQ307 (P. plecoglossicida) 13 ± 2 b 11.50 ± 19.3 ± 3.9 bc 2.346 ± 0.254 ± 0.214 ± 0.028 ± 0.009
2 b 0.560 b 0.073 b 0.053b b
ILQ103 (P. brassicacearum) 0 0 0 0 0 0 0
661
662 ± DS: Standard deviation from a total of six biological replicas
663 Different letters indicate statistically significant differences between the treatments in accordance with the Duncan multiple range test,
664 with a confidence level of 95.0%.
33 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 34 of 38
665 Figure captions
666
667 Figure 1. Percentage of all strains isolated from the rhizosphere of C. quinoa plants from
668 Peruvian Andean Plateau at different temperatures (4°C, 6°C, 15°C and 24°C) grouped by large
669 colony levels (0–4): 0, no colonies appeared; 1, 0%–25%*; 2, 26%–50%*; 3, 51%–75%* and
670 4, above 75%–100%*
671 (*) Percentage of colony size at each of the tested temperatures with respect to the colony size
672 at control temperature (24°C)
673
674 Figure 2. Dendrogram UPGMA constructed from BOX-PCR fingerprints of psychrotolerant
675 strains isolated from the rhizosphere of C. quinoa from the Peruvian Andean Plateau. Cluster
676 groups are A–L. Jaccard cogenetic correlationDraft coefficient (CP) = 0.991.
677
678 Figure 3. Phylogenetic tree based on 16S rRNA gene sequence from psychrotolerant strains
679 isolated from the rhizosphere of C. quinoa from the Peruvian Andean Plateau. Psychrotolerant
680 strains were selected from a different cluster of the BOX-PCR detected. A phylogenetic tree
681 was constructed by the neighbour-joining method using Kimura-2-parameters model.
682 Bootstrap values of >70% are shown. The bar represents 0.020 substitutions per site.
683
684 Figure 4. Effect of the psychrotolerant ILQ215 (Pseudomonas silesiensis) and JUQ307
685 (Pseudomonas plecoglossicida) strains in the development of 90-day-old quinoa seedlings in a
686 greenhouse
34 https://mc06.manuscriptcentral.com/cjm-pubs Page 35 of 38 Canadian Journal of Microbiology
Figure 1.
100 4°C 6°C 15°C 24°C
80
60
40
Strains (%) Draft
20
0 0 1 2 3 4 Large colonie levels
https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 36 of 38 Figure 2.
Draft
https://mc06.manuscriptcentral.com/cjm-pubs Page 37 of 38 Canadian Journal of Microbiology
Figure 3
ILQ216 (MN826151) 86 ILQ211 (MN826150) 75 Βeta )B. contaminans J2956T (NR104978.1) Burkholderia 100 Proteobacteria B. arboris R-24201T (NR 042634.1) B. cepacia NBRC 14074T (NR113645.1) ILQ104 (MN826152) E. hormaechei subsp. xiangfangensis 10.17T (NR126208.1) 100 ILQ201 (MN826153) 81E. cancerogenus LMG 2693T (NR044977.1) Enterobacter JUQ409 (MN826154) 100 E. ludwigii EN-119T (NR042349.1) ILQ215 (MN826142) 99 P. silesiensis A3T (NR156815.1) 68 90 P. mandelii NBRC 103147T (NR114216.1) P.lini DLE411JT (NR029042.2) 72 ILQ219 (MN826143) 100 P. migulae NBRC 103157T (NR114223.1) P. corrugata (NR 117826.1) Ganma ILQ103 (MN826144) Draft Proteobacteria 92 98 P. brassicacearum subsp. Neaurantiaca CIP 109457T (NR116299.1) P. fluorescens CCM 2115T (NR 115715.1) Pseudomonas 99 JUQ307 (MN826145) P. plecoglossicida NBRC 103162T (NR114226.1) 99 T 100 P. putida NBRC 14164 (NR 113651.1) JUQ310 (MN826146) 100 P. oryzihabitans NBRC 102199T (NR114041.1) P. psychrotolerans C36T (NR 042191.1) 98 P. aeruginosa DSM_50071T (NR 117678.1)
100 JUQ304 (MN826148) A. soli B1 T (NR044454.1) 100 JUQ303 (MN826149) Acinetobacter 100 A. johnsonii ATCC 17909T (NR117624.1)
0.020
https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 38 of 38
Draft
Effect of the psychrotolerant ILQ215 (Pseudomonas silesiensis) and JUQ307 (Pseudomonas plecoglossicida) strains in the development of 90-day-old quinoa seedlings in a greenhouse
1757x2022mm (72 x 72 DPI)
https://mc06.manuscriptcentral.com/cjm-pubs