Biochemical and Molecular Characterization, and Bioprospecting of Drought Tolerant Actinomycetes from Maize Rhizosphere Soil

Drought is a major limitation to maize cultivation around the globe. Seven actinomycetes strains were isolated from maize rhizosphere soils in Mahikeng, North-West Province, South Africa. The isolates were biochemically characterized and identified with 16S rRNA gene sequence analysis. Isolates were also screened in vitro for abiotic stress tolerance to different concentrations of NaCl, pH, and polyethylene glycol (PEG 8000), as well as for biosynthesis of drought tolerance genes namely Glutathione peroxidase (GPX), Glycine-rich RNA binding protein (GRP), Desiccation protectant protein (DSP), Guanosine triphosphate binding protein (GTP) and plant growth-promoting genes:1-aminocyclopropane-1-carboxylate deaminase (accd) and siderophore biosynthesis (Sid). About 71.43% of isolates were of the genus Streptomyces (99-100% similarity), while 14.29% belong to the genus Arthrobacter (R15) and 14.29% to the genus Microbacterium (S11) respectively (99% similarity). Five isolates had their optimum growth at 35°C. Arthrobacter arilaitensis (R15) grew and tolerated 5%, 10%, and 20% PEG at 120 h. Root length increased by 110.53% in PEG treated maize seeds (−0.30 MPa) inoculated with Streptomyces pseudovenezuelae (S20) compared to the un-inoculated control. Likewise, germination percentage and vigor index increased by 37.53% and 194.81% respectively in PEG treated seeds inoculated with S20 than the un-inoculated PEG treated seeds. ACC deaminase gene was amplified in all the isolates, while the gene for siderophore biosynthesis was amplified in 85.71% of the isolates. Genes for the synthesis of GPX, GRP, DSP and GTP were amplified in Arthrobacter arilaitensis (R15) and Streptomyces pseudovenezuelae (S20) which lacked GTP. The amplification of drought-tolerant and plant growth-promoting primers indicates the possible presence of these genes in the isolates. These isolates have the potential for use as bio-inoculants, not only to improve drought tolerance in maize but also to be utilized as biofertilizers and biocontrol agents to facilitate growth promotion.


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As the world's climatic conditions change because of massive increases in the world's 44 population and global industrialization, more agricultural land is being lost to drought. Loss of 45 arable land due to drought is a problem that is becoming common in many regions of the world The seven selected actinomycetes isolates were tested for their ability to utilize various carbon 139 compounds as energy sources using ISP-medium 9 as recommended by Shirling and Gottlieb

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Acid production by the isolates from the above carbon sources was also studied. The positive 144 utilization of a carbon source was considered when the growth of an isolate on a tested carbon 145 source was significantly better than the growth on the basal medium without a carbon source

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[33], while growth similar or less than growth on basal medium without a carbon source was 147 considered a negative utilization [28].

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Catalase production test

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Sequences obtained were deposited in GenBank.

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The evolutionary history was deduced by using the Maximum Likelihood approach based 201 on the Tamura-Nei model [40]. The tree with the highest log likelihood (-6740.13) was obtained.

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The percentage of trees in which the related taxa grouped together appears next to the branches.  DNA samples were prepared by mixing 7 µl of DNA template, and 3 µl of 6x DNA loading dye (Fermentas), 5 µl PCR products and 2 µl DNA ladders (1 kb or 100 bp) were also taken. Samples The effect of pH on bacterial growth was determined by growing 10 µl of each bacterial culture 275 in test tubes containing 10 ml of sterilized ISP-1 medium supplemented with 5% PEG 8000. The 276 pH of the media was adjusted to 3, 5, 7, 9, and 11 using 1 N HCl and 1 N NaOH before    To study the drought tolerance capacities of bacterial isolates used in this study, a 5 ml (3.8 x 10 6 289 CFU ml -1 ) aliquot of freshly prepared cultures were inoculated into 150 ml cotton plugged flasks

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R11+PEG, plate 9: S11+PEG, and plate 10: M+PEG. Seeds were left to grow for 12 days after 318 which 5 best seedlings were selected from each plate for measurement of growth parameters.
The experiment was performed in triplicate. The percentage germination and vigor index of 320 seedlings were estimated as previously described by Chukwuneme, Babalola [26] as follows:

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Where n is the number of germinated seeds after 10 days, and N is the total number of seeds  which showed moderate growth on the medium. After 10 days' incubation period, colonies were 336 observed to be white, brownish-white, and gray ( Table 1). The biochemical properties disclosed 337 that all tested bacterial isolates had a positive reaction for catalase activity, nitrate reduction, 338 starch hydrolysis, and the utilization of glucose as a carbon source. Moreover, only two tested 339 bacterial isolates (S4 and S7) hydrolyzed casein. From our results, four isolates (S12, S4, S11, 340 and S20) utilized D-galactose, four (R15, S7, S4, and R11) utilized the D-xylose, five (R15, S12, obtained also showed that all the isolates used fructose except isolate S7, while this isolate was

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isolates were submitted to GenBank (NCBI) with the following accession numbers: MG547867

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(S11) and MG640369 (S12) ( Table 3). Phylogenetic analyses using the Maximum Likelihood technique were computed with the 16S the bacterial isolates with the closest reference bacteria species. The 16S rRNA sequences of the closely related taxa as retrieved from GenBank data library, and Pseudomonas gessardii strain OBE3 (MN685265) was used as the out-group (Fig 1) lengths of the primer sequences were moderate, assisting in precise binding to the target genes.

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The excellent specificity of the primers in the Gene Infinity Platform were confirmed by in silico 402 PCR. Also, primer specificity performed in the NCBI Primer-BLAST gave the target drought 403 tolerance genes. The PCR amplification of the GPX gene yielded the expected band size of 215 404 bp for isolates R15, S20, S7, and R11 while isolates S11, S12 and S4 yielded no amplification.

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( Supplementary Fig S1A). In the case of GRP ( Supplementary Fig S1B), isolates R11, S20, 406 S12, and R15 yielded the expected band size of 220 bp while isolates S4, S7, and S11 did not 407 amplify. Isolates S20 and R15 amplified the DSP gene at the expected product size of 920 bp 408 while no amplification observed for the other isolates at this size and the GTP gene was only 409 amplified in isolate R15 at the expected band size of 668 bp ( Supplementary Fig. S1C).

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The ACC deaminase (accd) gene was amplified in all seven isolates ( Supplementary Fig S1A) 412 with the reference primers, as shown in Supplementary Table S1. The amplification of 413 siderophore (Sid) gene ( Supplementary Fig S1B) for the seven isolates was performed by PCR this gene.  Table 5. Bacterial growths were determined by measuring the optical density (OD)

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of each bacterium at 600 nm. All tested bacterial isolates grew at various temperatures, although 424 there were variations in the growth pattern of each isolate. Isolate S4 had optimum growth at 425 35°C (0.65 ± 0.11) and its least growth at 25°C (0.29 ± 0.13) which was significantly lower than 426 the growth recorded for other isolates at 25°C with optimum growth recorded for isolate S11

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(0.68 ± 0.12). At 30°C, optimum bacterial growth was recorded for isolates S20 (0.56 ± 0.32), 428 S12 (0.55 ± 0.16), and R15 (0.53 ± 0.19) which were not significantly different from one another 429 but significantly higher than the growth obtained for other isolates with the least growth recorded 430 in isolate S4 (0.34 ± 0.19). At a temperature of 35°C, isolate R15 had its optimum growth rate 431 (0.91 ± 0.04) which was significantly (p < 0.001) higher than the growth recorded for other 432 isolates, followed closely by isolate S12 (0.89 ± 0.15) with S20 having the least growth (0.27 ± 433 0.12). At this temperature (35°C), isolates R11 and S4 had their optimum growth rates (0.64 ± 434 0.12) and (0.65 ± 0.11), respectively. Most isolates had their optimum growth at 35°C except 435 isolates S11 and S20 (Table 5). At the highest temperature of 40°C, a significant decrease in 436 growth was observed for most isolates (R15, S11, S20, and S7). Nonetheless, isolate R11 had the Effect of pH on bacterial growth 443 The level of microbial activity in the soil is usually affected by the pH of the soil. In the present 444 study, isolated actinomycetes strains were grown at different pH ranges of 3, 5, 7, 9, and 11

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There were no significant differences in the growth rate of other isolates at pH 9 ( Effect of sodium chloride (NaCl) on bacterial growth 460 Bacterial isolates S4, S7, R11, S11, S12, R15, and S20 were assessed for their ability to  Table 7). Significant differences were observed in the growth of bacterial 463 isolates at the different concentrations of NaCl except at 0.8% NaCl. At 0.2% NaCl, isolates S4

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(1.13 ± 0.13) was significantly highest at 0.4% NaCl than the other isolates, which had low

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It was also observed that time influenced the tolerance capacities of these isolates as better 482 growth on PEG medium was observed with an increase in time (Fig 3A-G). The result obtained 483 for each concentration was compared with that of the control (without PEG). Isolate S12 had 484 maximum tolerance to PEG treatment at 5% PEG (1.53 ± 0.18) after 120 h of growth, with the 485 lowest growth recorded at 20% PEG after 24 h (Fig 3A). However, for isolate R11, maximum length increased by 114.75% more than the un-inoculated watered maize seeds (M+H20) and by 511 40.37%, 13.26%, and 14.58% more than the root lengths of watered maize seeds inoculated with 512 isolates S11, S12 and R11, respectively. Likewise, isolates S12 and S20 treated with PEG 8000 (-513 0.30 MPa) also produced longer root lengths than the PEG treated maize seeds inoculated with 514 other isolates and the un-inoculated PEG treated maize seeds (Fig 4A). Root length was 515 increased by 110.53% in PEG treated maize seeds inoculated with isolate S20 (S20+PEG) than 516 the un-inoculated PEG treated maize seeds (M+PEG). PEG treated maize seeds inoculated with 517 isolate S12 (S12+PEG) also had long root length which was just 11.11% shorter than the root 518 length of seeds inoculated with isolate S20, but 86.53% and 65.89% longer than the roots of PEG 519 treated seeds inoculated with isolates S11 and R11 respectively. Nevertheless, no significant 520 difference was recorded in the root length of maize seeds inoculated with isolate S11 (1.93±0.19 521 cm) and that of the un-inoculated maize seeds (1.90 ± 0.15 cm). Highly significant (p < 0.001)

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differences were also observed in the shoot lengths of all inoculated maize seeds treated with 523 PEG compared to the un-inoculated seeds (Fig 4A). Shoot length was significantly highest in 524 watered maize seeds, and PEG treated maize seeds inoculated with isolate S20 than the other 525 treatments. Shoot length increased by 44.82% and 115.61% than the un-inoculated watered 526 maize seeds, and PEG treated maize seeds, respectively. Generally, the root and shoot lengths 527 were shorter in the un-inoculated watered, and PEG treated maize seeds.

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Regarding germination percentage and vigor index, highly significant (p < 0.001), differences 530 were obtained among the treatments (Fig 4B). The germination percentage of the PEG treated 531 maize seeds inoculated with isolate S20 was significantly highest than other PEG treated seeds higher than the un-inoculated PEG treated maize seeds by 37.53%. The same trend was observed 534 in the watered maize seeds inoculated with isolate S20, which had significantly higher 535 germination percentage than the watered seeds inoculated with isolates S11, S12, and R11.
Germination percentage in the S20 inoculated watered seeds was 23.86% higher than the un-537 inoculated watered seeds (M+H20). Vigor index in the watered treatments was significantly 538 highest in maize seeds inoculated with isolate S20 (S20+H20). This was 108.71% higher than the 539 control seeds (M+H20). Maize seeds inoculated with the same isolate (S20) at water stress (-0.30 than the un-inoculated maize seeds treated with sterile water and PEG medium ( Fig 4B).

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The phylogenetic analysis of the drought-tolerant actinomycetes isolates used in the present 559 study revealed that these isolates expressed a very high similarity value (99-100%), which was 560 above the 70% borderline degree of relatedness or identity as suggested by Wayne,Brenner (48).

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Furthermore, the similarities expressed by these isolates with the reference taxa belonging to  which may be due to their ability to withstand high temperature, hence may be regarded as high-604 temperature-tolerant strains.

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The pH of an environment influences bacterial survival and growth. For most soil bacteria, the 606 specific pH range is usually between 4 and 9, with the optimum being 6.5 to 7.5

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In this study, the bacterial isolates were assessed for tolerance to salinity stress by growing in 618 different concentrations of NaCl. The isolates were able to grow at all concentrations of NaCl.

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This confirms their ability to withstand various salinity stress conditions and suggests their 620 applicability for salt tolerance improvement in plants. It is also an indication of halotolerant

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This resulted in greater plant biomass and physiological parameters, especially the chlorophyll 656 content which is relevant in yield increase in plants.

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The study on maize seedlings exposed to water stress revealed that significant growths were 658 observed in the root and shoot lengths, germination percentage, and vigor index of the seedlings 659 obtained from seeds inoculated with the bacterial isolates S20, S12, R11, and S11 compared to    932 S12, and Lane 7: S11.  with either water or PEG 8000. M = maize seeds, H20 = water, S20, S12, R11, S11 = maize seeds inoculated with 985 bacterial isolates, and PEG = PEG treated seeds either inoculated or un-inoculated. All values are means of triplicate