Plant growth promoting rhizobacterial diversity in potato grown soil in the Gwalior region of India.

There seems to be meager studies with regards to rhizo and non-rhizo microbial association with potato plant from the central India. Present study was undertaken to evaluate the microbial diversity of rhizospheric and non-rhizospheric isolates from three varieties of potato viz Kufri sindhuri, Kufri lauvkar and Kufri chipsona-3 procured from the Central Potato Research Station, Maharajpura, Gwalior. A total of 130 bacterial forms were isolated, and amongst these forty isolates were further characterized on their morphological basis, and those showing some of PGPR characteristics were identified to species level using VITEK-2 method. Various bacterial populations were found in potato rhizosphere and dominant presence was those of Bacillus subtilis, Bacillus Megaterium and Lysinibacillus sphaericus. The non-rhizospheric soil was dominant in the forms like Aeromonas salmonicida, Morxella group and Bacillus coagulans. Highest bacterial diversity was found in the rhizosphere soil of different potato cultivars than in the non-rhizospheric soil of potato.

Introduction

Microbial diversity of soils is being increasingly evaluated as indicators of soil fertility. There is an established relationship between microbial diversity and ecosystem sustainability [14]. A layer of soil just surrounding the plant roots is known as rhizosphere and it constitutes the important active area for root activity and metabolism. A large number of microorganisms such as bacteria, fungi, protozoa and algae abound the rhizosphere. Amongst these bacteria are one of the important component one. Certain bacterial species, which showed association with plant rhizosphere, are known to be involved in the enhanced plant growth, yield, and quality [4]. The presence of rhizobacteria in the rhizosphere therefore, can have a neutral, detrimental or beneficial effect on plant growth. Microbial activity may differ in mycorrhizosphere, hyphosphere, rhizosphere and bulk soils [2].

Microbial communities are now known to play key role in controlling biogeochemical cycling of nutrients in soil and as a consequence help plants to grow better [22, 24, 45, 5]. Soil microbial communities are often difficult to characterize, chiefly due to their complex phenotypes accompanied with genotypic diversity. Top soil bacterial populations can attain more than 109 cells per g of soil [42], and most of these may generally be unculturable. Soil microbial diversity is fundamental for sustaining above ground plant diversity and eco condition of a region [1, 27, 40]. Bacterial diversity involve species richness, numerical presence, evenness, and finally the distribution of their bacterial species [34]. This diversity and richness contribute to understanding in immense measure the soil health and sustainability. Thus the present study was to evaluate such microbial diversity around rhizosphere and non-rhizosphere of different potato cultivars.

Materials and methods

Experimental site

Three varieties viz Kufri sindhuri, Kufri chipsona-3 and Kufri lauvkar were assessed for their rhizospheric and non-rhizospheric microbial diversity based on the simple fact that these three varieties can complete their life cycle in three months. These work sites of varieties were generously provided by the Central Potato Research Station, Maharajpura, Gwalior, M.P. (26.22°N 78.18°E). The Institute is spread over an area of nearly 400 acres and is under the professional management of the Indian Council for Agricultural Research, New Delhi.

Isolation of PGPR from rhizosphere and non rhizosphere (bulk) soil of potato varieties

Rhizospheric and non-rhizospheric bulk soil samples were collected from 6 to 25 cm depth from the plant surface using sterilised equipment and packed in sterile plastic bags. These were stored at 4 °C in the lab. Six different fields of three mentioned potato tuber variety with plants were selected for sampling and sampling of the soil was done and plants monitored at an interval of 15 days from sowing till harvesting.

Soil analysis

The soil pH was determined by the mehod of Jackson [21] with a slight modification. 10 gm of soil sample was suspended in 90 ml distilled water. The suspension was shaken vigrously and allowed to settle overnight. systronic pH meter analytic was used to determine pH.

The Electrical conductivity of the soil was determined in the same suspension as used in the pH measurement, with the help of a conductivity meter following modified Jackson [21] method. The EC is expressed in deci Siemens per meter (dSm−1) at 25 °C. systronic analytic and sysmatic, conducting meter was used for the purpose.

Preparation of dilutions, inoculations and observations

In 250 ml capacity of conical flasks, 10 g of soil both from rhizosphere and non- rhizosphere separately were added in the already contained 90 ml of distilled water. The flask was shaken for 10 min on a rotary shaker. One ml of the shaken suspension was added to 9 ml distilled water in a test tube and again shaken for 2 min. This represented 101 dilutions. Similarly, a series up to 107 dilutions was prepared under aseptic conditions. An aliquot of this suspension was spread on the plates of solid Nutrient agar (NA) medium. Plates were incubated for 48hr at 28 °C and the bacterial colonies observed. Isolated single colony was re-streaked on fresh NA medium plate and re-incubated. These Bacterial pure cultures were maintained on the NA medium slants in glass culture tubes. All these isolates were maintained for longer durations at 4 °C in 70% nutrient broth and 30% glycerol in steriliesd vials or eppendrof tube.

Standard plate count method (CFU)

Standard plate count method was used to enumerate the bacterial cultures. 100 µl inoculum from each sample and dilution was spread across the plate and the colonies that were formed after incubation were counted. The number of bacterial colonies in each were referred to as colony forming units (CFU). Colonies exhibiting good variable growth were selected for further streaking on fresh plates. Further purification and multiplication of isolates was done by streaking on fresh plates. The CFU was determined by the relation [43].

Bacterial identification

Bacterial identifications were done using VITEK-2 method at Supratech Micropath Laboratory, Ahmadabad. VITEK 2 is a fully automated system that performs bacterial identification and antibiotic susceptibility testing. The reagent cards have 64 wells that can each contain an individual test substrate. Each card has a pre-inserted transfer tube used for inoculation. Cards have bar codes that contain information on product type, lot number, expiration date, and a unique identifier that can be linked to the sample either before or after loading the card onto the system.

Four reagent cards are available for the identification of different organism classes as follows:

GN - Gram-negative fermenting and non-fermenting bacilli

GP - Gram-positive cocci and non-spore-forming bacilli

YST - yeasts and yeast-like organisms

BCL - Gram-positive spore-forming bacilli

Results

The physiochemical soil (pH and EC) analysis of different varieties of potato soil in the CPRI, Gwalior showed that the soil pH for different cultivars of potato KS, KC-3 and KL ranged 7.6, 6.8 and 6.3 respectively whereas EC (Electrical conductivity) ranged respectively as 0.35, 0.44 and 0.36 dS/m (Table 1).

Table 1

Soil pH and EC of different potato cultivar.

S/N	Potato varieties	Soil pH	Soil EC (dS/m)
1.	Kufri Sindhuri,	7.6	0.31
2.	Kufri Chipsona-3	6.8	0.44
3.	Kufri Lauvkar	6.3	0.36

Serial dilution and microbial count

In serial dilution from 101 to 106 three sets of the plates that were incubated at 37 °C showed well developed colonies and therefore used for the purpose of colony count. Colonies counts were descended from 101 to 106. These microbial colonies on agar planting at 103 to 105 dilutions were found appropriate for enumeration of colonies. Amongst them 103 dilution showed maximum number of countable colonies. 105 dilutions always showed lower colony count. This was true for soil from all potato cultivars.

Comparatively, rhizospheric soil had more bacterial colony count than the non-rhizospheric one (Figs 1& 2Table 2) this was a trend presented by all the three potato cultivars. Subsequently bacterial density as CFU/g too was higher in the rhizospheric than non-rhizospheric soil of the three potato cultivars.

Fig. 1

Number of countable colonies in the rhizospheric soil of three potato varieties.

Fig. 2

Number of countable colonies in the non-rhizospheric (bulk) soil of three potato varieties.

Table 2

Bacterial density CFU/g of soil in rhizo and non-rhizospheric (bulk) soil in three different varieties of Potato.

S/N	Varieties	CFU/g bacteria in potato varieties
		Rhizo	Non rhizo (bulk)
1	KS	3.9 × 107	2.7 × 107
2	KC-3	5.2 × 107	4.2 × 107
3	KL	6.4 × 107	5.6 × 107

Bacteria morphological characteristic

An approximately 400 colonies were observed. On the basis of their colony structure and morphology 130 bacterial isolates testing on the Pikovskaya agar medium [38] modified with 1% methyl red containing tricalcium phosphate [36] showing phosphorus solubilisation character were used for further studies which they were qualify as PGPR. A total of 40 morphologically distinct isolates were observed. Among these 25 isolates were from rhizosphere and 15 from the non-rhizosphere (bulk soil). Rhizospheric bacteria, presented 90% gram +ve and 10% gram -ve whereas the non-rhizosphere were presently 40% gram +ve and 60% gram -ve. Most of the colonies were off white colonies having both smooth and rough texture. The rest of the other colonies were presented with yellow, pink, brown and peach colorisation, and some either mucoid or non-mucoid showed yellow, green and brown pigmentation (Tables 3 and 4). Gram positive forms were dominantly rod shaped bacillus organized as single, diplobacilli, short cum long chains, and/or as cocci. Gram negative form too were either rod shaped bacillus, cocci, diplococci, and/or as clusters in arrangement (Fig 3a,b,c,d,e).

Table 3

Rhizospheric bacterial (PR) colony characteristics.

S/N	Isolates	Gram stain	Color	Shape	Elevation	Surface	Opaquenes
1	PR1	+ve	Peach	Small, Round	Raised	Smooth	Opaque
2	PR2	+ve	Off white	Oval	Raised	Smooth, Shiny	Opaque
3	PR3	+ve	Pink	Small, round	Raised	Smooth	Opaque
4	PR4	+ve	Off white	Irregular	Middle flat edge swollen	Rough	Opaque
5	PR5	+ve	Off white	Oval	Raised	Smooth, Mucoid	Opaque
6	PR6	+ve	Off white	Irregular,	Flat	Smooth, shiny	Opaque
7	PR7	-ve	Light brown with pigmentation	Oval	Raised	Rough	Opaque
8	PR8	+ve	Off white	Irregular	Flat	Rough	Opaque
9	PR9	+ve	Off white	Irregular	Flat	Smooth	Transparent
10	PR10	+ve	Yellow	Round	Raised	Smooth, Mucoid	Opaque
11	PR11	+ve	Peach	Small, oval	Raised	Smooth	Opaque
12	PR12	+ve	Off white	Irregular	Flat	Smooth Mucoid,	Opaque
13	PR13	+ve	Off white with brown pigmentation	Round	Flat	Rough	Opaque
14	PR14	+ve	Off white	Round	Flat	Rough	Opaque
15	PR15	+ve	Off white	Irregular	Flat	Smooth, shiny	Opaque
16	PR16	+ve	Off white	Circular	Middle flat edge swollen	Rough	Opaque
17	PR17	+ve	Off white	Circular	Flat	Smooth	Opaque
18	PR18	-ve	Peach	Round	Raised	Smooth, shiny	Opaque
19	PR19	+ve	Yellow	Oval	Flat	Smooth	Opaque
20	PR20	-ve	Off white	Irregular with brown pigmentation	Flat	Rough	Opaque
21	PR21	+ve	Off white	Irregular	Flat	Smooth	Opaque
22	PR22	+ve	Off white	Circular	Flat	Smooth, shiny	Opaque
23	PR23	+ve	Off white	Circular with brown pigmentation	Flat	Smooth	Opaque
24	PR24	+ve	Purple	Round with brown pigmentation	Raised	Rough	Opaque
25	PR25	+ve	Brown with pigmentation	Oval	Raised	Smooth	Opaque

Table 4

Non-rhizospheric bacterial (PB) colony characteristics.

S/N	Isolates	Gram stain	Color	Shape	Elevation	Surface	Opaquenes
1	PB1	+ve	Off white	Irregular	Flat	Rough	Opaque
2	PB2	-ve	Yellow	Round,	Raised	Smooth, shiny	Opaque
3	PB3	+ve	Off white	Circular	Flat	Smooth	Opaque
4	PB4	+ve	Off white	Circular	Flat	Rough	Opaque
5	PB5	-ve	Light peach	Round	Raised	Smooth	Opaque
6	PB6	-ve	Light yellow,	Round	Raised	Smooth	Transparent
7	PB7	-ve	peach	Circular	Flat	Smooth	Opaque
8	PB8	+ve	Light yellow	Round	Slightly raised	Smooth	Opaque
9	PB9	+ve	Off white	Circular	Flat	Smooth	Opaque
10	PB10	+ve	Off white	Irregular	Flat	Smooth	Opaque
11	PB11	-ve	Off white	Circular	Slightly raised	Smooth	Opaque
12	PB12	-ve	Off white	Irregular	Flat	Smooth, Mucoid	Opaque
13	PB13	-ve	Off white	circular	Flat	Smooth	Opaque
14	PB14	-ve	Off white with brown pigmentation	Round	raised	Rough	Opaque
15	PB15	-ve	Light yellow	Circular	slightly raised	Smooth	Opaque

Fig. 3

Various morphological forms a,b,c: Gram (-) rod, cocci, diplococci respectively d,e: Gram (+)rod, cocci, forms from rhizo and non-rhizo soil of potato cultivars.

All the 40 isolates were further subjected to charactersation and identification at Supratech Micropath Laboratory, Ahmadabad. Based on the numerical probability and calculation of confidence of similarities the form identification probability are present in the Tables 5 and 6. Therefore, the probability range from 99% to 86% and confidence are referred in their descending order, as excellent, very good, good, acceptable, and low. Based on this therefore, the generic distribution tentatively was shown to be as these of Bacillus with 20 form, Neisseria, Pseudomonas, Sphingobacterium and actinomycetes with one form each in the rhizosphere and Bacillus with 6, Aeromonas with 3, Morxella with 2 and Pseudomonas, Sphingomonas, Sphingobacterium with single form each in non rhizospheric soil.

Table 5

Bacterial isolates from rhizospheric soil with comparative confidence levels using Vitek −2 system for tentative identification.

Isolates	Identify Bacteria	Varieties	Probability	Confidence
PR1	Bacillus subtilis	KS	91%	Good
PR2	Bacillus subtilis	KL	93%	Very good
PR3	Micrococcus Luteus	KC	99%	Excellent
PR4	Lysinibacillus sphaericus	KC	85%	Low
PR5	Bacillus subtilis	KS	89%	Good
PR6	Bacillus vallismortis	KC	86%	Acceptable
PR7	Neisseria animaloris	KL	94%	Low
PR8	Bacillus subtilis	KC	86%	Acceptable
PR9	Bacillus subtilis	KL	86%	Low
PR10	Bacillus subtilis	KC	88%	Acceptable
PR11	Lysinibacillus sphaericus	KL	91%	Low
PR12	Lysinibacillus sphaericus	KL	91%	Good
PR13	Brevibacillus bervis	KS	86%	Acceptable
PR14	Bacillus Megaterium	KC	95%	Very good
PR15	Bacillus Megaterium	KC	89%	Good
PR16	Bacillus Megaterium	KL	88%	Acceptable
PR17	Bacillus Megaterium	KC	90%	Good
PR18	Pseudomonas stutzeri	KL	86%	Low
PR19	Bacillus Megaterium	KL	97%	Excellent
PR20	Sphingobacterium thalpophilum	KC	96%	Excellent
PR21	Alicyclobacillus acidoterrestris	KL	97%	Excellent
PR22	Bacillus Megaterium	KL	92%	Good
PR23	Lysinibacillus sphaericus	KL	91%	Low
PR24	Brevibacillus bervis	KS	91%	Good
PR25	Aerobic actinomycetes spp.	KS	——	Very low

Table 6

Bacterial isolates from non-rhizospheric soil with comparative confidence levels using Vitek −2 system for tentative identification.

Isolates	Identify Bacteria	Varieties	Probability	Confidence
PB1	Bacillus lentus	KC	87%	Acceptable
PB2	Aeromonas salmonicida	KL	98%	Excellent
PB3	Bacillus pumlus	KC	96%	Excellent
PB4	Bacillus vallismortis	KL	90%	Good
PB5	Morxella group	KS	99%	Low
PB6	Aeromonas salmonicida	KC	97%	Low
PB7	Morxella group	KS	99%	Excellent
PB8	Bacillus coagulans	KC	85%	Acceptable
PB9	Bacillus coagulans	KS	85%	Low
PB10	Bacillus smithii	KL	98%	Excellent
PB11	Pseudomonas stutzeri	KC	99%	Excellent
PB12	Acinetobacter lwoffii	KS	86%	Low
PB13	Sphingomonas paucimobilis	KL	90%	Low
PB14	Sphingobacterium thalpophilum	KL	96%	Excellent
PB15	Aeromonas salmonicida	KL	98%	Excellent

Note:- In table PR represent rhizospheric form and PB represent the non-rhizospheric. KS, KC-3, KL represent the three potato cultivars of Kufri sindhuri, Kufri chipsona-3 and Kufri lauvkar respectively.

Bacterial diversity distribution

The highest bacterial population was found in rhizosphere soil of Kufri luvakar and lowest in Kufri sindhuri whereas in non rhizosphere soil highest bacterial population was found in Kufri luvakar and lowest in Kufri chipsona-3. The count wise highest count numbers shown were those of Bacillus subtilis and Bacillus Megaterium followed by Lysinibacillus sphaericus in the rhizospheric soil and in non-rhizospheric soil the highest bacterium count was presented by Aeromonas salmonicida followed by Bacillus coagulans and Morxella sp. (Table 7).

Table 7

Formwise distribution of various bacteria in the rhizospheric and non-rhizospheric soil of the three potato cultivars.

Bacterial form rhizospheric soil	Potato cultivar			Total
	Kufri sindhuri	Kufri luvakar	Kufri chipsona-3
Bacillus subtilis	2	2	2	6
Brevibacillus bervis	2	0	0	2
Aerobicactinomycetes spp.	1	0	0	1
Micrococcus Luteus	0	0	1	1
Lysinibacillus sphaericus	0	3	1	4
Bacillus vallismortis	0	0	1	1
Bacillus Megaterium	0	3	3	6
Sphingobacterium thalpophilum	0	0	1	1
Neisseria animaloris	0	1	0	1
Pseudomonas stutzeri	0	1	0	1
Alicyclobacillus acidoterrestris	0	1	0	1
Total	5	11	9	25
Bacterial form non rhizospheric soil	Potato cultivar			Total
	Kufri sindhuri	Kufri luvakar	Kufri chipsona-3
Morxella group	2	0	0	2
Bacillus coagulans	1	0	1	2
Pseudomonas stutzeri	1	0	0	1
Bacillus lentus	0	0	1	1
Bacillus pumlus	0	0	1	1
Aeromonas salmonicida	0	2	1	3
Acinetobacter lwoffii	0	0	1	1
Bacillus vallismortis	0	1	0	1
Bacillus smithii	0	1	0	1
Sphingomonas paucimobilis	0	1	0	1
Sphingobacterium thalpophilum	0	1	0	1
Total	4	6	5	15

Discussion

[32] have said that the relation between biodiversity, which can simply be defined as the numerical presence of species in a certain system, relates to the functional dynamics of the soil and therefore, is part of prime concern to be conserved so as to maintain its role in a functional biosphere. In the present study therefore, higher no of countable colonies of bacterial forms in neutral pH and lower in number in low pH needs, considered attention in potato cultivar soils. pH, as is known, determines the availability of nutrients, thus can have a strong effect on physiological processes, such as root exudations containing signal molecules which consequently, affect the microbial communities in the plant rhizosphere.

Soil pH, aeration, and physicochemical characteristics are jointly responsible for creating specific soil environment, thereby, the rhizosphere microbial communities [11, 17, 29] [13]. have reported reduced bacterial diversity with increase in soil pH. Role of soil salinity induced bacterial diversity, is suggested as a cause of environmental stress [8]. Bacterial identification by VITEK-2 method as employed in this study has been used by other workers too for bacterial identification [12, 25, 26, 31, 35].

The diverse microbial community was found in three different potato cultivars KS, KC-3 and KL at different time intervals [20]. and [18] have reported that the, rhizospheric bacterial community of varieties 'Monalisa' and 'Asterix' were phylogeneticlly more similar at the early first and second samplings. Thus suggesting that their root signals may be selecting similar bacterial groups. Multisplicity of groups shown by the other cultivars, influence rhizosphere associated microbial communities during early development and then diversifying subsequently as shown by the present three cultivars.

[6] and [9] have produced detailed reviews regarding biotic and abiotic factors such as soil type, seasons, plant developmental stage, proximity to root, root architecture, plant species, and cultivars that can affect the structure of microbial communities in the rhizosphere. Various other studies have established that the influence on rhizospheric microbial communities is a synergic effect of both the plant species and the plant genotypes [3, 6, 41, 44, 46, 47]. This observation therefore, can also aptly explain the PGPR community structure variation in the present cultivars too.

There are distinct differences in bacterial form between bulk (non rhizosphere) and rhizosphere soil [7, 10, 30] and the probable reason seems that in rhizosphere, roots exudate chemicals which are helping the bacteria to flourish abundantly. In the present study Bacillus sp. was shown to be abundant both in rhizospheric soil than in the bulk soil. This being followed by Aeromonas, Morxella sp. and Pseudomonas [28]. have reported Bacillus as a dominant form genus in the tuber rhizosphere of sweet potato. Various species of Bacillus constitute major populations in the rhizospheres of chrysanthemum [15], of barley [33], and that of grass [16].

In this study too high number of colonies was found in rhizosphere compared to non-rhizosphere (bulk) soil and also different species of Bacillus, Pseudomonas and other forms.

70 bacteria species were reported isolated from the rhizosphere of potato cv [23]. [39]. too have reported 25 morphologically distinct bacterial isolates belonging both to Gram+ve and Gram−ve groups from the mana potato field. They further reported that in potato these beneficial microbes mainly belonged to the genera of Bacillus, Pseudomonas, and Penicillium, along with actinomycetes and yeast. This study too recorded that on morphological basis viz shape of colonies, color and elevation and then surface distinctions, the bacterial isolates belonging to Gram+ve and Gram−ve bacteria. Bacteria belonging to the Bacillus and Pseudomonas groups are known to be growth promoters [19, 37].

Conclusion

The study shows the presence of bacterial diversity in the rhizospheric and non- rhizospheric soil of different potato cv. KS, KC-3 and KL. The extent of the microbial diversity in soil is consistent with the soil health and quality. Soil microorganisms, such as bacteria, play important roles in soil fertility and promoting plant health, and can be employed and tested to be PGPR consortium potato plant.

Declaration of Competing Interest

All authors declares that they have no conflict of interest