Potential Biofertilizers for Alkaline Soil: Bacteria Isolated from the Rhizosphere of Potatoes

: Root-associated microorganisms, which can be recruited specially by plants to cope with environmental stress under extreme conditions, are one of the major mediators of nutrient exchange between plants and the environment. To obtain more crop-beneficial microbes, rhizosphere bacteria of D é sir é e potatoes cultivated in poor and alkaline soil have been studied. The screening of 83 strains with incomplete identical 16S rDNA sequences showed that 47 strains produced indole acetic acid (IAA), with contents ranging from 0.2 to 42 mg/L, and seven strains were phosphorus-solubilizing, among which six strains significantly increased the growth rate of potato plants. Thirty-seven strains produced siderophore and four strains were zinc-solubilizing, among which three strains significantly alleviated the chlorosis of potato plants. In all of the isolates, the species Variovorax soli (ST98) and Cellulomonas biazotea (ST118) were first found to possess an IAA-secreting ability; the species Leifsonia aquatica (ST172) and Leifsonia naganoensis (ST177) and the genus Sutcliffiella (ST11) were first discovered to be capable of phosphorus solubilization; the species Chryseobacterium daecheongense (ST32) was the first reported to be capable of zinc solubilization; and the species V. soli (ST98), C. biazotea (ST118) and L. naganoensis (ST177) were first found to be capable of plant growth promotion. The discovery of multiple functional bacteria enriched the resources of plant growth-promoting bacteria and provided a foundation for biofertilizer production to improve soil conditions and crop production


Introduction
The potato is the fourth most important staple food crop.It is a food security crop and a possibly decisive weapon in the fight against starvation because of its high yield and great nutritive value, and it is rich in starch, protein and microelements [1].China, as a major potato-planting country, has consistently ranked first in terms of potato-planting area and production volume worldwide [2].Unfortunately, quality and production are under great threat because of the soil environment, pests and diseases.The application of large amounts of chemical fertilizers and pesticides will cause environmental degradation and affect the sustainable development of agriculture.
Plants growing in natural ecosystems live in close association with a multitude of microorganisms [3].As the second genome of plants, the rhizosphere microbiome plays an important role in promoting plant growth and disease resistance [4].In particular, plant growth-promoting rhizobacteria (PGPR), which can benefit plants by producing plant growth hormones [5], convert many plant-unavailable essential nutrients, such as nitrogen, phosphorous, zinc and iron, into available forms, synthesize antibiotics or enhance induced systemic resistance (ISR) to help plants against pathogenic bacteria and fungi [6].
In potatoes, the function of rhizosphere microorganisms is mainly concentrated on the resistance of pathogenic bacteria.Only a few studies have shown that arbuscular fungi, such as Rhizophagus irregularis, Funneliformis mosseae and Claroideoglomus etunicatum, and some bacteria, such as Enterobacter cloacae, Bacillus thuringiensis and Pseudomonas pseudoalcaligenes, can improve increase both the yield and nutritional quality of potatoes [7].
Plant rhizosphere microbial composition is affected by hosts and environment [8].Plants shape rhizosphere microbes by modifying the amount and composition of root exudates to acquire belowground resources in nutrient-limited soil, which is consistent with the "cry for help" hypothesis in disease resistance [9].Plants may shape rhizosphere flora by secreting phenolic substances and recruit related flora to improve the iron solubility in the rhizosphere when planted in iron-deficient soil [10,11], and the result was similar when it experienced phosphorus deficiencies [12].In view of the above research, we hypothesized that when potatoes are grown in poor alkaline soil, special beneficial strains will be recruited to facilitate nutrient absorption.Yunnan is a major province for potato cultivation, but some of the major cultivation areas for potatoes have alkaline soils with extremely low nutrient availability.This is particularly true for Dayao, a small city located in the northwestern part of Yunnan Province.To exploit more microbial biofertilizer to assist the green potato industry development in Yunnan, the rhizosphere bacteria of potatoes cultivated in Dayao were studied.

Soil Sample Collection and Physicochemical Parameter Determination
Soil samples were collected from four major production areas of potatoes in Yunnan Province of China, including Dayao (25.85 • N, 101.25 • E), Xundian (25.63 • N, 102.96 • E), Dehong (24.02 • N, 97.85 • E) and the planting base on campus (24.86 • N, 102.84 • E).The pH of the soils was measured using a pH meter.Plant-available iron and zinc were measured using chelator diethylenetriaminepentaacetic acid (DTPA) extraction [13].Briefly, 10 g of air-dried soil was shaken for 2 h with 20 mL of extracting solution (0.005 M DTPA, 0.1 M triethanolamine and 0.01 M CaCl 2 , pH 7.3).The leachate was centrifuged and filtered through a 0.22 µm membrane.The iron and zinc contents in the leachate were measured using an atomic spectrophotometer.Plant-available phosphorus was measured using NaHCO 3 extraction-the molybdenum antimony resistance colorimetric method [14].

Isolation and Purification of Potato Rhizosphere Bacteria
Samples were collected from the growing region of potato plants that grow in Dayao, Yunnan, China (25.85 • N, 101.25 • E).The whole root system was carefully collected using tweezers, and large soil aggregates and debris were removed by gently shaking.Roots were placed in a 15 mL centrifuge tube and transferred to a laboratory being kept on ice.Isolation of bacteria was carried out through the serial dilution method.Ten grams of root tissues were homogenized using a grinding mill and dissolved in 100 mL of saline solution (0.9% NaCl).One milliliter of suspension was added to a test tube containing 9 mL of saline solution to obtain a suspension with a 10 −2 dilution level.Dilution was performed in the same manner until a 10 −6 suspension was obtained.Subsequently, 0.1 mL of the suspension was grown on Tryptone Soy Agar (TSA) medium in a Petri dish at 30 • C. Single colonies were picked and grown to reisolate and make pure cultures [15].

Sequencing and Phylogenetic Analysis
To confirm the taxonomic status of bacterial isolates, 16S ribosomal Deoxyribo Nucleic Acid(16S rDNA) with a length of 1.5 kb was amplified and sequenced with the primers 8F/1492R (8F, AGAGTTTGATCCTGGCTCAG; 1492R, GGTTACCTTGTTACGACTT) [16].The sequences of the selected strains were compared and aligned with those of other strains from GenBank using BLAST (http://www.ncbi.nlm.nih.gov/blast,accessed on 4 May 2023).A phylogenetic tree was constructed using the neighbor joining method with MEGA.

Indole Acetic Acid (IAA) Production
Each bacterial strain was grown in TSB solution under shaking at 180 rpm at 30 • C for 3 days, and supernatants were collected to determine the auxin IAA.The production of IAA by bacteria was tested using Salkowski reagent (1.2% FeCl 3 and 37% H 2 SO 4 ) and measured at 530 nm wavelength using a spectrophotometer.The concentration of IAA was calculated using a standard curve of known concentrations of synthetic IAA [17].

Siderophore Production
Siderophore production by the bacterial isolates was detected using the universal chrome azurol 'S' (CAS) agar plate assay.A single colony of isolated bacteria was cultured in TSB solution overnight at 30 • C, and then 10 µL of bacterial solution was inoculated on CAS plates and incubated at 30 • C for 72 h [18].The diameters of the colony and orange halo were detected, and the solubilization index (SI) was calculated as follows: SI = Halozone Diameter(mm) /Colony Diameter(mm) (1)

Plant-Bacteria Interaction
To investigate the effect of the strains on plant growth under greenhouse conditions, the strains were cultured overnight, washed and resuspended in 0.9% NaCl.Three-weekold potato plants grown in 1/2 Hoagland medium were inoculated with a cell suspension of strains at a final concentration of 5 × 10 7 CFU mL −1 .To further verify whether related strains can promote mineral element absorption in plants, potato plants were grown in 1/2 Fe/P/Zn-deficient Hoagland medium with insoluble FeCl 3 (pH 7.3)/Ca 3 (PO 4 ) 2 /ZnO.The plant height and root length were measured every week to calculate the growth rate.The relative growth rates (RGRs) were calculated as follows: RGR = height on the 7th/14th/21st day(mm) − height on the 1st day(mm) height on the 1st day(mm) (3)

Detection of Chlorophyll and Mineral Elements in Plants
Chlorophyll extraction and quantification were adapted from reference [20].Samples were prepared by a hole puncher from 10 mm 2 of leaf tissue.Chlorophyll was extracted by adding 1 mL Dimethyl sulfoxide (DMSO) and incubating samples at 65 • C with shaking for 45-60 min until plant tissue was transparent and chlorophyll was completely extracted.The absorbance of the tissue-free chlorophyll extract was measured at 652 nm on a spectrophotometer (NanoDrop One, Thermo Scientific, Waltham, MA, USA).The youngest fully expanded leaves were collected for drying and digestion by a microwave digestion system, and the content of mineral elements was measured by an atomic spectrophotometer.

Data Analysis
Statistical analysis of the data was carried out by GraphPad Prism 6 software.When the normality and homogeneity of variance hypotheses were satisfied, a one-way analysis of variance (ANOVA) was used to compare the effect of these bacterial isolates on potato growth performance.A posteriori multiple comparisons were carried out using Tukey's range tests at p ≤ 0.05.All results are the means of three to five independent replicates, as specified above.

Nucleotide Sequence Accession Number
The GenBank accession numbers for the 16S rDNA gene sequence of all the strains are attached in Table S1.

Physicochemical Parameters of Soils
The pH and available iron, zinc and phosphorus of soil samples collected from four areas of potato crops in Yunnan Province were measured.All the soil samples were neutral (6.5 < pH < 7.5) or alkaline (7.5 < pH < 8.5).The soil of Dehong contained high contents of iron and phosphorus, followed by Xundian and the planting base in campus.The soil of Dayao contained the lowest iron and phosphorus contents, possibly because of its higher pH (Figure 1).

Identification and Phylogenetic Analysis of Bacteria Isolated from the Potato Rhizosphere
One hundred and fifty-three strains were isolated and identified from the roots (mixed with six plants) of cultivated potato cultivated in Dayao and named following ST (Solanum tuberosum).Based on the sequencing results of the 16S rDNA gene, the nucleo-

Rhizosphere Bacterial Strains Promote Plant Growth by Producing IAA
IAA is one of the most physiologically active auxins in plants and can regulate the growth of stems, buds and roots.Forty-seven isolates could produce IAA, among which seven strains had concentrations greater than 10 mg/L, nine strains had concentrations between 5 and 10 mg/L and 18 strains had concentrations ranging from 1 to 5 mg/L (Table 1).Twenty strains that were highly IAA-producing strains were inoculated into threeweek-old potato plant roots.The results showed that the growth rates of plants inoculated with strains, V. soli ST98, Agromyces sp.ST139, C. biazotea ST118, Cupriavidus sp.ST9 and Cellulosimicrobium sp.ST85 were significantly increased on the 21st day (Figure 3a,b).We also observed the growth of roots.Interestingly, plants whose above-ground growth was

Rhizosphere Bacterial Strains Promote Plant Growth by Producing IAA
IAA is one of the most physiologically active auxins in plants and can regulate the growth of stems, buds and roots.Forty-seven isolates could produce IAA, among which seven strains had concentrations greater than 10 mg/L, nine strains had concentrations between 5 and 10 mg/L and 18 strains had concentrations ranging from 1 to 5 mg/L (Table 1).Twenty strains that were highly IAA-producing strains were inoculated into three-week-old potato plant roots.The results showed that the growth rates of plants inoculated with strains, V. soli ST98, Agromyces sp.ST139, C. biazotea ST118, Cupriavidus sp.ST9 and Cellulosimicrobium sp.ST85 were significantly increased on the 21st day (Figure 3a,b).We also observed the growth of roots.Interestingly, plants whose above-ground growth was promoted had a decrease in the amount of root (Figure 3c), which may be related to the availability of nutrition, because root morphology and growth are regulated by environmental nutrition, and in an environment with adequate nutrition, not too many roots are needed for nutrient acquisition [21,22].environmental nutrition, and in an environment with adequate nutrition, not too many roots are needed for nutrient acquisition [21,22].

Rhizosphere Bacterial Strains Promote Plant Zinc Absorption by Zinc Solubilization
Four isolates, Lysobacter sp.ST103, C. daecheongense ST32, Stenotrophomonas sp.ST34 and Cellulosimicrobium sp.ST85, could improve the solubility of insoluble zinc.ST103 showed significant solubilizing capacity, and large clearance rings with diameters greater than two appeared around the colony (Figure 6a, Table 4).Plants inoculated with Lysobacter sp.ST103 and Stenotrophomonas sp.ST34 had higher chlorophyll in leaves when planted in Zn-deficient Hoagland medium with ZnO (Figure 6b,c).6a, Table 4).Plants inoculated with Lysobacter sp.ST103 and Stenotrophomonas sp.ST34 had higher chlorophyll in leaves when planted in Zn-deficient Hoagland medium with ZnO (Figure 6b,c).

Discussion
Plant growth and health are closely related to rhizosphere microorganisms.Plants regulate rhizosphere microbiota through exudate secretion to shape and utilize specific microbial communities to help them respond to external stressors by secreting hormones, siderophores, organic acid antibiotics, lytic enzymes, volatile organic compounds and so on [9].Our object was to isolate and screen more plant-promoting bacteria from potato rhizosphere soils with low iron, phosphorus and zinc.Our research showed that Microbacterium, which is one of the most common PGPR, was determined to be the dominant genus [6].Bacteria in this genus promoted plant growth in a variety of ways, such as auxin, salicylic acid and gibberellin secretion [25], siderophore production [26], phosphorus solubilization [26], volatile substances production [27], nitrogen fixation [28] and zinc solubilization [29].
PGPR can promote plant growth by directly secreting plant hormones or by influencing the hormone synthesis process of plants [5].The production of IAA has been recognized as an important factor in direct plant growth-promoting abilities of rhizosphere bacteria.They stimulate the proliferation of lateral roots that increase nutrient-absorbing surfaces and result in the better assimilation of water and nutrients from the soil [30].In our study, more than 50% of strains produced IAA, among which Chryseobacterium sp.

Discussion
Plant growth and health are closely related to rhizosphere microorganisms.Plants regulate rhizosphere microbiota through exudate secretion to shape and utilize specific microbial communities to help them respond to external stressors by secreting hormones, siderophores, organic acid antibiotics, lytic enzymes, volatile organic compounds and so on [9].Our object was to isolate and screen more plant-promoting bacteria from potato rhizosphere soils with low iron, phosphorus and zinc.Our research showed that Microbacterium, which is one of the most common PGPR, was determined to be the dominant genus [6].Bacteria in this genus promoted plant growth in a variety of ways, such as auxin, salicylic acid and gibberellin secretion [25], siderophore production [26], phosphorus solubilization [26], volatile substances production [27], nitrogen fixation [28] and zinc solubilization [29].
PGPR can promote plant growth by directly secreting plant hormones or by influencing the hormone synthesis process of plants [5].The production of IAA has been recognized as an important factor in direct plant growth-promoting abilities of rhizosphere bacteria.They stimulate the proliferation of lateral roots that increase nutrient-absorbing surfaces and result in the better assimilation of water and nutrients from the soil [30].In our study, more than 50% of strains produced IAA, among which Chryseobacterium sp.ST32 reached the highest with a yield of 43.16 mg/L under natural culture conditions.The genus of Chryseobacterium has been widely reported for IAA production and a growth-promoting effect on crops, and most of them were tryptophan-dependent [5,31].In addition, the genera of Microbacterium (ST25, ST49, ST144, ST43) [25], Arthrobacter (ST7, S21) [32], Variovorax (ST98) [33], Agromyces (ST139) [34], Cellulomonas (ST118) [35], Cupriavidus (ST9) [36], Cellulosimicrobium (ST85) [37] and Ensifer (ST46) [38] were also reported to possess the ability to secrete IAA, while the species V. soli (ST98) and C. biazotea (ST118) were first found to be capable of that in the present study.
Siderophores are common microbial secondary metabolites that can chelate metal ions such as iron and zinc from the environment and transport them into cells through specific receptors [39], and the bacterial siderophore-Fe complex can be utilized by plants with higher efficiency than EDTA-Fe [40].Studies have shown that, in addition to serving as a medium to chelate iron, siderophores can also act as signaling molecules [41].Siderophores produced by some bacteria can induce other strains to produce homologous iron carriers, thereby enhancing the use of iron in the soil.In addition, some strains can secrete siderophores to deplete limited iron in the environment, thereby inhibiting the growth of pathogenic bacteria from the same ecological niche [42] or inducing other genera of bacteria to produce many antibacterial substances to resist invasion by plant pathogens [43].According to our research, half of the isolates from potato roots planted in Dayao were siderophore producers, which may be involved in the low iron property of the soil.Most of the strains identified as high-siderophore-producing strains belong to Microbacterium (ST51, ST35, ST40, ST49, ST43), and the species M. foliorum (ST51) [44], M. phyllosphaerae (ST40) [45] and M. hydrocarbonoxydans (ST49) [46] have been reported to produce a high amount of siderophores and promote plant growth.Siderophores produced by P. fluorescens (ST18) have been extensively studied and demonstrated to play roles in growth promotion, biocontrol and pheromones [47].The kinds and biosynthetic gene clusters of siderophores produced by A. tumefaciens (ST81) have also been identified [48].In addition to these common species, the genera Cellulosimicrobium (ST85) [29], Pseudoxanthomonas (ST106) [49] and Bordetella (ST65) [50] have also been reported to produce siderophores.
According to relevant reports, appropriate amounts of IAA can promote plant growth, and P is an essential element for plant growth, so the relative growth rate was selected as the target for the function verification of IAA-producing and phosphorus-solubilizing strains.Most strains isolated from potato roots could produce a certain concentration of IAA, but only strains with IAA concentrations ranging from 5 to 15 mg/L could promote the root growth of potato plants.This indicated that the promotive effect of IAA on plants was limited to a certain concentration range, which was consistent with reports [58].Among the screened strains, the species V. soli (ST98), C. biazotea (ST118) and L. naganoensis (ST177) were first found in the present study to promote plant growth.Both iron and zinc are directly related to chlorophyll synthesis, and the chlorosis of leaves is an obvious symptom of iron and zinc deficiency [59].Therefore, chlorophyll content was selected as the functional target of iron-and zinc-solubilizing strains.Only two isolates with a high siderophores yield and one isolate with a zinc-solubilizing function could improve the chlorosis of plants significantly, which may be related to the colonization ability of the strains.
According to our research, none of these strains can dissolve iron, phosphorus and zinc simultaneously, indicating that a single strain is unlikely to possess multiple functions.Only multiple strains that coexist and collaborate can help plants achieve a balanced nutrient absorption and resist pathogen invasion in complex soil environments.Therefore, research on and the preparation of synthetic microbial communities (SynComs) that contain carefully chosen microbial species to produce the desired microbiome function has become the trend.In recent years, more and more studies have focused on the use of SynComs to improve plant nutrient efficiency and health [60,61].In contrast to selecting microbes based on single microbial in vitro activities or taxonomy, SynComs development considers multiple attributes, including microbes associated with desirable plant phenotypes and microbial traits, such as the production of metabolites, robust biofilm formation and the ability to chemically trigger plant defense mechanisms that will make microbes fit to persist in different environments and to colonize plants.

Conclusions
In this study, 153 bacterial strains were isolated and identified from potato roots cultured in alkaline soil.Among the 83 bacterial strains with incompletely identical 16S rDNA sequences, 47 isolates produced different concentrations of IAA, 37 isolates produced siderophores and seven and four isolates produced phosphate and zinc solubilizers, respectively.Five isolates significantly increased the growth rate of potato plants and four isolates significantly alleviated the chlorosis of leaves.Among all strains, the species V. soli (ST98) and C. biazotea (ST118) were first found to possess the ability to secrete IAA; the species L. aquatica (ST172) and L. naganoensis (ST177) and the genus Sutcliffiella (ST11) were first discovered to be phosphorus-solubilizing; the species C. daecheongense (ST32) was first reported to be zinc-solubilizing; and the species V. soli (ST98), C. biazotea (ST118) and L. naganoensis (ST177) were first found to be plant growth-promoting.Although the isolates represented only part of large plant rhizosphere bacteria population, the new functions discovered in some strains has enriched the resources of plant growth-promoting bacteria.The validation of growth-promoting functions under hydroponic conditions is the initial step for biofertilizer production for field applications.Future research on colonization mechanisms and optimization conditions, and competition with other soil microbial communities for exploiting multifunctional microbial fertilizers (i.e., SynComs) are needed.

Figure 1 .
Figure 1.The pH and available mineral element contents of soil from different areas of Yunnan Province: (a) pH of soil from different areas of Yunnan Province; (b) available mineral element contents of soil from different areas of Yunnan Province; and (c) potato plants cultivated in Dayao (left) and Dehong soil (right).

Figure 1 .
Figure 1.The pH and available mineral element contents of soil from different areas of Yunnan Province: (a) pH of soil from different areas of Yunnan Province; (b) available mineral element contents of soil from different areas of Yunnan Province; and (c) potato plants cultivated in Dayao (left) and Dehong soil (right).

Figure 2 .
Figure 2. Phylogenetic tree of strains isolated from potato rhizosphere.

Figure 3 .
Figure 3. Growth promotion by IAA-producing strains: (a) partial plant growth state on the 21st day; (b) relative height growth rate on the 21st day; and (c) root dry weight on the 21st day.* p ≤ 0.05 (Student's t test).

Figure 6 .
Figure 6.Solubilization of zinc: (a) clearance zone presented on medium with insoluble ZnO; (b) chlorophyll contents of plans; and (c) plant growth state cultured in Zn-deficient Hoagland medium with ZnO.* p ≤ 0.05 (Student's t test).

Figure 6 .
Figure 6.Solubilization of zinc: (a) clearance zone presented on medium with insoluble ZnO; (b) chlorophyll contents of plans; and (c) plant growth state cultured in Zn-deficient Hoagland medium with ZnO.* p ≤ 0.05 (Student's t test).

Table 1 .
IAA production by strains.

Table 2 .
Solubilization index of siderophores produced by strains.

Table 3 .
Solubilization index of strains on phosphorus.Lysobacter sp.ST103, C. daecheongense ST32, Stenotrophomonas sp.ST34 and Cellulosimicrobium sp.ST85, could improve the solubility of insoluble zinc.ST103 showed significant solubilizing capacity, and large clearance rings with diameters greater than two appeared around the colony (Figure

Table 4 .
Solubilization index of strains on zinc.

Table 4 .
Solubilization index of strains on zinc.