1. bookTom 73 (2022): Zeszyt 1 (March 2022)
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Priming effect of native rhizosphere bacteria on little millet (Panicum sumatrense)

Data publikacji: 28 Sep 2022
Tom & Zeszyt: Tom 73 (2022) - Zeszyt 1 (March 2022)
Zakres stron: 55 - 66
Otrzymano: 15 Sep 2021
Przyjęty: 04 Jan 2022
Informacje o czasopiśmie
License
Format
Czasopismo
eISSN
2719-5430
Pierwsze wydanie
30 Mar 2016
Częstotliwość wydawania
4 razy w roku
Języki
Angielski
Introduction

Little millet (Panicum sumatrense) belongs to the angiospermic family Poaceae. It is a small-seeded cereal used as a staple food by a large number of poor and unprivileged people in rural and hilly areas in India (Verma et al., 2013). The cereal contains valuable nutrients such as carotenoids, phenolic compounds, gamma-aminobutyric acid (GABA), and tocopherols (Guha et al., 2015) and a high amount of minerals such as iron and phosphorus, and vitamins (Rao et al., 2017). The crop shows resistance to adverse agroclimatic conditions (Rao et al., 2017), requires little care and almost no input, and thus, poor and unprivileged farmers find it easier to cultivate it. Its nutrient richness and sturdiness make it a suitable crop to be introduced in dry and open land (horizontal expansion), thus contributing to nutritional and food security, especially because of current climatic change-related threats to food security. The village Tamia (Madhya Pradesh [MP], India) had16,000 ha area under millet (little millet) cultivation in 2015–2016 with an average yield of 203 kg per acre, which is lower than the state average yield of 255 kg per acre (MP Agriculture Statistics, 2019) due to traditional practices. Therefore, native plant growth-promoting rhizobacteria (PGPR) could be of interest in maintaining little millet yield in this rural area.

PGPR can transform unavailable substrates to available growth elements involving biological mechanisms (Backer et al., 2018) and promote growth, shorten the vegetative phase, and promote early onset of flowering (Poupin et al., 2013). PGPR have been found to exhibit some or many of the activities such as non-symbiotic fixation of nitrogen, solubilization of complex phosphate and potassium compounds, production of siderophore, phytohormones, and compounds enabling stress tolerance such as enzymes (1-aminocyclopropane-1-carboxylic acid[ACC]-deaminase and chitinase), exopolysaccharides, rhizobitoxine, and so on, and inhibitory action on pathogenic microbes (Backer et al., 2018; Habibi et al., 2019). The use of PGPR as a biofertilizer to enhance plant growth and productivity is considered a better alternative to costly and unsustainable synthetic fertilizers. They may also be used to reduce the dose of chemical fertilizers substantially if used in combination with chemical fertilizers (Backer et al., 2018).A few bacteria such as Azotobacter sp., Azospirillium sp., Arthrobacter sp., Bacillus sp., Burkholderia sp., Clostridium sp., Enterobacter sp., Pseudomonas sp., and Serratias sp. (Gupta et al., 2015), reported as potential PGPR, have been found to colonize plant roots and exhibit various beneficial effects leading to a positive impact on plant growth and yield. PGPR have been isolated from the rhizosphere of many plants (Dawwam et al., 2013; Castanheira et al., 2016; Habibi et al., 2019), but have not yet been isolated from the rhizosphere of little millet.

Seed priming is a pre-sowing treatment including treatment with PGPR that results in a physiological state facilitating seed germination (Luutts et al., 2016). Among PGPR, various species of Burkholderia constitute very efficient and widespread rhizosphere colonizers (Schroth and Hancock, 1982). Species of this bacterium have been reported from the rhizosphere/root of various plants (Sessitsch et al., 2005; Castanheira et al., 2016). The taxon has been found to have tremendous PGPR activity (Poupin et al., 2013). Species of Burkholderia have not been isolated so far from the root/rhizosphere of little millet.

According to the concept of habitat-adapted symbiosis (Rodriguez et al., 2008), complex microbiomes, and not individual inocula, have been found to offer full beneficial effects to plants, especially under certain stress conditions (Qin et al., 2016). The isolation of native PGPR adapted to local environmental conditions and familiar to the local microbial community is thus a logical step toward designing biofertilizer for a specific crop of a particular area. The major goal was to isolate native PGPR in a little millet-growing area of Tamia and study their priming effect onthe growth and yield of little millet.

Materials and methods
Standard PGPR strain

Culture of Azotobacter chroococcum (NCIM 5576 equivalent to ATCC 9043, DSM 2286), used as standard diazotroph (control), was procured from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune, India. The culture was maintained at 4°C in Burk's N2-free medium containing the following (in grams per liter): sucrose 20.0, dipotassium phosphate 0.64, potassium dihydrogen phosphate 0.16, magnesium sulfate 0.20, sodium chloride 0.20, calcium sulfate 0.05, agar 15.0 supplemented with sodium molybdate (0.05%) 5.0 ml, and ferrous sulfate (0.3%) 5.0 ml; pH adjusted to 7.3 (Wilson and Knight, 1952). Earlier, the bacterial strain has been used as PGPR (Gholami et al., 2012).

Soil and climate characteristics

The soil of the experimental site is shallow sandy loam. The essential physicochemical characteristics of the soil of the site are given in Table 1. The climate of the area is tropical with an average annual rainfall of 1139.3 mm during the southwest monsoon (June–September), and the normal annual mean minimum and maximum temperatures are 18.2°C and 30.6°C, respectively (District Ground Water Bulletin, 2019).

Chemical characteristics of the experimental soil

Tabelle 1. Chemische Eigenschaften des Versuchsbodens

Site pH EC (ds per m) Organic carbon (%) Nitrogen (%) Phosphorus (kg per ha) Potassium (kg per ha)
Tamia 6.9 0.53 0.91 0.154 14.71 190.11

Values (except for EC and pH) indicate average values of soils used to collect PGPR; pH and EC values were similar in all the fields.

PGPR: plant growth-promoting rhizobacteria

Sample collection

The rhizospheric soil samples from little millet, wheat, paddy, maize, chickpea, and common bean (Phaseolus vulgaris) (all crops at the flowering stage) were collected from 0–5 cm depth of corresponding fields in Tamia area (22°24′40″ N, 78°45′28″ E to 22°39′97″N, 78°70′25″E) of district Chhindwada (Figure 1), Madhya Pradesh, and carried in a sterile polyethylene bag. Soil loosely adhered to the roots was removed, and soil that adhered strongly was collected with the help of a sterile plastic brush. The soil was then sieved (~ 2 mm) and stored in sterile plastic bags at 4°C.

Figure 1

Geographical position of the experimental site

Abbildung 1. Geografische Position des Versuchsstandorts

Analysis of experimental soil

The experimental soil was analyzed for the chemical properties including total nitrogen by the Kjeldahl method and organic carbon, phosphorus, potassium, pH, and electrical conductivity by standard methods (Peech, 1965).

Bacterial isolation

Prospective PGPR were isolated from soil samples using Jensen's nitrogen-free medium containing the following (in grams per liter): sucrose 20.0, dipotassium phosphate 1.0, magnesium sulfate 0.50, sodium chloride 0.50, ferrous sulfate 0.10, sodium molybdate 0.005, calcium carbonate 2.0, agar 20.0; pH 7.2 (Jensen, 1951) at an incubation temperature of 30°C. Isolation was carried out applying a serial dilution technique and maintained on the same medium at 4°C for further study. Fast-growing colonies were selected.

Testing plant growth-promoting (PGP) activities
Efficiency of nitrogen fixation

Erlenmeyer flasks (250 ml) containing 50 ml Jensen's broth were used to inoculate 0.1 ml fresh seed culture of the bacterium. Inoculated flasks were incubated on a rotary shaker for 7 days at 30°C ± 2°C. Fixed total nitrogen in the broth was determined by applying the semi-micro-Kjeldahl method according to BF and OF in FCO (1958). Nitrogen fixation efficiency was determined in terms of total nitrogen fixed per gram sucrose consumption according to the method given earlier (Din et al., 2019).

Efficiency of phosphate solubilization

The bacterial isolates were tested for their phosphate solubilization efficiency (PSE) using Pikovskaya's medium supplemented with 2.4 mg/ml bromophenol blue (Pikovskaya, 1998). The inoculated medium was incubated for 48 h at 30°C and was observed for the formation of a yellowish halo around the colony due to the utilization of tricalcium phosphate present in the medium. PSE was calculated applying the formula of Nguyen et al. (1992): PSE=DiameterofhalozoneDiameterofcolony×100 {\rm{PSE}} = {{{\rm{Diameter}}\;{\rm{of}}\;{\rm{halo}}\;{\rm{zone}}} \over {{\rm{Diameter}}\;{\rm{of}}\;{\rm{colony}}}} \times 100

Efficiency of siderophore production

Siderophore-producing ability was estimated by universal Chrome Azurol Sulphonate (CAS) assay (Schwan and Neelands, 1987). Reddish-orange halos around the colonies after 48 h of incubation indicated siderophore production. Quantitative estimation of siderophores (siderophore production efficiency) was carried out by the method described earlier (Payn, 1994), where percent decolorization was calculated using the following formula: Siderophoreunits(%)=ArAsAr×100 {\rm{Siderophore}}\;{\rm{units}}\;\left( \% \right) = {{{\rm{Ar}} - {\rm{As}}} \over {{\rm{Ar}}}} \times 100 where Ar = absorbance of reference at 630 nm (CAS reagent) and As = absorbance of the sample at 630 nm.

Indole acetic acid production

Indole acetic acid (IAA) production was qualitatively assayed by the method given earlier (Gordon and Weber, 1951). Bacterial cultures were grown in a Luria–Bertani broth supplemented with tryptophan (5 mM) for 3–4 days at 30°C on a rotator shaker. Cultures were centrifuged at 2800×g for 20 min. Two milliliters of supernatant were mixed with two drops of orthophosphoric acid and 4 ml of Salkowski reagent. Tubes were incubated at room temperature for 25 min. Development of pink color indicated IAA production. For quantification, the absorbance was recorded at 530 nm and the quantity of IAA was extrapolated from IAA standard curve.

Inoculum preparation

On the basis of higher PGP activities (data not shown), isolates DUM4 (from maize rhizosphere soil) and FKK5 (from little millet rhizosphere soil) were selected for studying their effect on the growth and productivity of little millet. Stock cultures of FKK5 and DUM4 were maintained on Jensen's medium at 4°C. The inoculum was prepared in 500 ml Erlenmeyer flasks using Jensen's medium (pH 7.2) without agar. Other growth conditions were temperature 30°C, shaking speed 300 rpm, and incubation period 24 h.

Inoculation and treatments

Seeds of little millet (variety JK4) were procured from Regional Agriculture Research Station, Dindori, affiliated to Jawaharlal Nehru Kristi Vidhyalay, Jabalpur, MP. Broth having Colony Forming Units (CFU) 109 per ml serving as inoculum was counted before the inculcation BF and OF in FCO (1958). Black polyethylene bags (2.5 kg capacity, 20 cm height, and 15 cm wide) were filled with 2.0 kg of semi-sterilized (autoclaved at 121°C) soil from a little millet field. Provision of drainage was made in the bags. Four types of treatment including no inoculation (negative control), inoculation with FKK5, DUM4 (tests), and A. chroococcum 5576 (positive control) were assessed for the growth of little millet under semi-sterile poly-house conditions. The seeds were inoculated with the bacterial suspension before sowing. The experiment was carried out in a randomized complete block design with three replications (Sharma et al., 2012).

Growth conditions and parameters

Plants were grown under greenhouse conditions from July to September (temperature range 25°C–34°C and relative humidity [RH] 38%–63%) for 90 days. Planted bags were watered regularly with sterile water to the field capacity to maintain soil moisture level. Pre- and post-harvesting growth parameters of plants, such as plant height, leaf number, stem diameter, panicle number, panicle length, root length, weight of total biomass, and grains weight per plant, were measured by the conventional method.

Inoculation effectiveness

Inoculation effectiveness (IE) of plant yield by the application of native and non-native inocula was calculated against the control by using the following formula: IE=SYiSYnSYn×100 {\rm{IE}} = {{{\rm{SYi}} - {\rm{SYn}}} \over {{\rm{SYn}}}} \times 100 where SYi = seed yield (inoculated plant) and Syn = seed yield (non-inoculated plant).

Identification of the isolates
Morpho-biochemical characterization

The isolates DUM4 and FKK5 were characterized morphologically and physiologically applying standard procedures (Bergey et al., 1994). Cyst-forming ability was assayed by growing the isolates in media containing Burk's nitrogen-free salts supplemented with n-butanol (0.2% v/v) (Sadoff, 1975).

Molecular characterization

Preliminary molecular identification of the isolates FKK5 and DUM4 was carried out based on 16S rDNA sequence characteristics. Polymerase chain reaction (PCR) amplification of 16S rDNA and sequencing were outsourced at the National Center for Microbial Resource (Pune, India) following the protocol of Yoon et al. (2016). The sequences were used to retrieve closely related sequences of type strains from National Center for Biotechnology Information (NCBI) database applying BLASTn. Altogether, 20 sequences showing > 99% identity with query sequence (FKK5 and DUM4) were retrieved. Phylogenetic analysis was carried out applying the neighbor-joining (NJ) method (Saitou and Nei, 1987), and evolutionary distances were determined applying the maximum composite likelihood method (Tamura et al., 2004) in MEGA6 (Tamura et al., 2013). The analysis was carried out in conjunction with the bootstrap method with 1000 replicates.

Statistical analysis

The experiments were carried out in triplicates, and the results were expressed as mean values with standard error. Analysis of variance (ANOVA) was carried out in case of the effect of inocula on various growth and yield parameters of little millet, applying one-way ANOVA in GraphPad Prism 9 software; p ≤ 0.05 was considered significant. Post-hoc least significant differences (LSD) test was carried out at 5% (p ≤ 0.05) probability level (Williams and Abdi, 2010).

Results
Rhizospheric bacterial isolation

The rhizospheric soil from the rhizosphere of selected crops was used to isolate bacteria on Jensen's medium (Figure 2a). Fast-growing bacterial colonies showing distinct morpho-types were selected. As many as 48 bacterial isolates were obtained from the rhizosphere of the six crops from different fields on nitrogen-free medium.

Figure 2

Rhizobacterial isolates on Jensen's nitrogen-free medium (a) and PGPR activities such as IAA production (b), phosphate solubilization on Pikovskaya's medium (c), and siderophore production (d) exhibited by selected rhizobacterial strains, DUM4 and FKK5; IAA: indole acetic acid; PGPR: plant growth-promoting rhizobacteria

Abbildung 2. Rhizobakterielle Isolate auf Jensens stickstofffreiem Medium (a) und PGP-Aktivitäten wie IAA-Produktion (b), Phosphat-Solubilisierung auf Pikovskaya-Medium(c) und Siderophor-Produktion (d) bei ausgewählten Rhizobakterien-Stämmen DUM4 und FKK5

Growth-promoting activities

All the 48 isolates were screened for their selected PGP activities (data not shown). Of them, two isolates (FKK5 and DUM4) exhibiting all the selected PGP activities, namely, N2 fixation, phosphate solubilization, and siderophore and auxin production at the highest levels, were selected for further study (Figure 2b–d). Potential isolates FKK5 and DUM4 showed nitrogen-fixing efficiency of 14.1 and 11.2 mg per g sucrose consumption, respectively. The isolates FKK5 and DUM4 exhibited phosphate-solubilizing efficiency (%) of 221 and 89, respectively. The siderophore production (%)by FKK5 and DUM4 was 41.9 and 35.6, respectively. Both the isolates were also auxin producers; FKK and DUM4 yielded 21 and 18 μg per ml of IAA, respectively, in the presence of tryptophan.

Priming effect on little millet

Pre-harvest data indicated that plants inoculated with rhizobacteria (FKK5, DUM4, and A. chroococcum 5576) had an increased number of leaves, reproductive tillers, stem diameter, and length of panicle, compared to control (noninoculated) plants (Figure 3). The highest length of panicle (13.23 ± 0.52 cm) and the highest number of reproductive tillers (4.66 ± 0.33) were observed in plants inoculated with FKK5, while the highest plant and root lengths were shown by DUM4-primed plants (Table 2). The post-harvest data also showed enhanced root length, root dry weight, total biomass, and seed weight per plant as a result of inoculation. The highest root length (17.00 ± 1.82 cm) and root dry weight (0.279 ± 0.01g) were observed in plants inoculated with DUM4 (Table 3), but the highest seed yield was shown by FKK5-primed plant. The native inocula in all the cases, however, exhibited significantly higher enhancement effect over non-native ones (Table 3).

Figure 3

Effect of different bioinocula (a = control, b = FKK5, c = DUM4, and d = Azotobacter chroococcum) priming on the vegetative growth of little millet. The best among three of every treatment is shown

Abbildung 3. Wirkung verschiedener Bioinokula (a = Kontrolle, b = FKK5, c = DUM4 und d = A. chroococcum) Priming auf das vegetative Wachstum kleiner Hirse. Die Beste unter den Dreien jeder Behandlung wird gezeigt.

Effect of native and non-native bioinocula priming on important growth characteristics (mean ± SE, n = 3) of little millet after 85 days of sowing before harvest in pot trials

Tabelle 2. Wirkung von Priming nativer und nicht nativer Bioinokula auf wichtige Wachstumsmerkmale (Mittelwert ± SE, n = 3) von Hirse nach 85 Tagen Aussaat vor der Ernte in Topfversuchen

Treatment Length Plant height (cm) Stem diameter (mm) No. of leaves No. of tillers (per plant) Panicle length (cm)
Non-inoculated 62.5±2.47b 1.96 ± 0.13a 09.66 ± 1.20a 3.66± 0.33a 11.33 ± 0.52a
Azotobacter chroococcum 57.6 ± 2.04a 2.66 ± 0.14b 14.66 ± 1.45b 4.33 ± 0.33b 11.66 ± 0.29b
FKK5 69.0 ± 2.17c 2.93 ± 0.17d 15.40 ± 1.76c 4.66 ± 0.33d 13.23 ± 0.52d
DUM4 72.0 ± 2.62d 2.76 ± 0.19c 17.00 ± 1.15d 4.53 ± 0.33c 13.10 ± 0.64c
LSD (p ≤ 0.05) 6.16 0.44 4.04 1.38 1.54

Values within the same column followed by different superscript letters are significantly different at p ≤ 0.05 level. LSD: least significant differences; SE: standard error

Effect of native and non-native bioinocula priming on important growth characteristics (mean ± SE, n = 3) of little millet at the harvest

Tabelle 3. Einfluss des Primings von nativen und nicht nativem Bioinokulen auf wichtige Wachstumsmerkmale (Mittelwert ± SE, n = 3) von Hirse bei der Ernte

Treatment Root length (cm) Root dry weight (g) Total plant biomass (g) Seed weight per plant (g)
Non-inoculated 10.30 ± 1.27a 0.163 ± 0.02a 1.01 ± 0.07a 0.360 ± 0.01a
Azotobacter chroococcum 12.90 ± 0.47b 0.197 ± 0.01b 1.19 ± 0.01b 0.433 ± 0.03b
FKK5 13.60 ± 0.55c 0.206 ± 0.01c 1.24 ± 0.06d 0.460 ± 0.02d
DUM4 17.00 ± 1.82d 0.279 ± 0.01d 1.22 ± 0.03c 0.449 ± 0.01c
LSD (p ≤ 0.05) 3.21 0.05 0.07 0.08

Values within the same column followed by different superscript letters are significantly different at p ≤ 0.05 level. LSD: least significant differences; SE: standard error

The IE data showed the priming effect of various inocula on grain yield; inoculation of little millet withFKK5 and DUM4 resulted in enhancement of yields by 28.14% and 24.72%, respectively, which were better than those obtained with the non-native PGPR A. chroococcum (20.43%) (Table 4). All the data presented here were significant at the level of p ≤ 0.05.

Priming effect of native and non-native bioinocula on grain and biomass yield enhancement (IE) (mean ± SE, n = 3)

Tabelle 4. Priming-Effekt von nativen und nicht nativem Bioinokula auf Getreide- und Biomasseertragssteigerung (IE) (Mittelwert ± SE, n = 3)

Inoculum Grain (seed) yield enhancement (%) Total biomass yield enhancement (%)
FKK5 28.14 ± 8.93c 23.08 ± 2.23c
DUM4 24.72 ± 3.62b 21.87 ± 5.58b
Azotobacter chroococcum 20.43 ± 9.78a 19.09 ± 9.34a

Values are the means ± SE. Values within the same column followed by different superscript letters are significantly different at p ≤ 0.05 level. IE: inoculation effectiveness; SE: standard error

Identification of PGPRs

Both the isolates shared many common features; both were rod shaped, gram negative, motile, and catalase and oxidase positive. However, while FKK5 could utilize mannitol, DUM4 could not. The selected morpho-physiological features are given in Table 5. The BLASTn search showed more than 99% sequence similarities with the species of Burkholderia. Phylogenetic dendrogram involving sequences from type strains of the 20 most closely related species (sharing 99.34%–97.5% sequence similarity) of Burkholderia, applying the NJ method, showed DUM4 and FKK5 (Figure 4) clustering together, but forming separate lineage with 99% bootstrap support. Although sharing a common ancestor, the two isolates DUM4 and FKK5 seem to have diverged quite earlier in the evolution. The sequences have been submitted to GenBank (www.ncbi.nlm.nih.gov) with the accession numbers MT 176443 (FKK5) and MT176479 (DUM4).

Figure 4

Dendrogram showing the evolutionary relationship of DUM4 and FKK5 with type strains of closely related taxa in the NCBI database, applying neighbor-joining method. The branch length indicates evolutionary distance. Maximum composite likelihood method was used to calculate the evolutionary distance, with units used being the number of base substitutions per site. Sequence accession number is given before the name of taxon. The numbers at the nodes represent bootstrap percent values from 1000 replicates. All positions containing gaps and missing data were eliminated. The analyses were carried out in MEGA6. Dendrogram was condensed with cut-off value 50% for the tree

Abbildung 4. Dendrogramm, das die evolutionäre Beziehung von DUM4 und FKK5 mit Typstämmen eng verwandter Taxa in der NCBI-Datenbank unter Anwendung der Neighbor-Joining-Methode zeigt. Die Astlänge zeigt die evolutionäre Distanz an. Die Maximum-Composite-Likelihood-Methode wurde verwendet, um die evolutionäre Distanz zu berechnen, und die verwendeten Einheiten sind die Anzahl der Basensubstitutionen pro Stelle. Die Sequenzzugangsnummer wird vor dem Namen des Taxons angegeben. Die Zahlen an den Knoten stellen Bootstrap-Prozentwerte von 1000 Replikaten dar. Alle Stellen mit Lücken und fehlenden Daten wurden eliminiert. Die Analysen wurden in MEGA6 durchgeführt. Das Dendrogramm wurde mit einem Cut-Off-Wert von 50% für den Baum verdichtet.

Selective morpho-physiological characteristics of the potential isolates

Tabelle 5. Selektive morpho-physiologische Eigenschaften der potentiellen Isolate

Character Azotobacter chroococcum 5576 FKK5 DUM4
Gram reaction
Cyst formation +
Pigmentation Brown Brown Brown
Motility + + +
IAA production + + +
NH3 production + + +
Starch formation + + +
Urease activity + + +
Catalase activity + + +
Oxidase activity + + +
Growth on sucrose + + +
Growth on maltose + + +
Growth on mannitol + +
Growth on citrate + + +
KOH + + +

+ indicates positive reaction, −indicates negative reaction. IAA: indole acetic acid

Discussion

The rural area selected for the study has low fertile shallow sandy soil, which, due to continuous cropping of the same crop year after year, is losing productivity. Monocropping has earlier been found to reduce microbial richness and activity (Misra et al., 2019; Tayyab et al., 2021). Although the agricultural practice followed here is not characterized by heavy use of chemicals, the geoclimatic conditions and agriculture together may be responsible for this decline in productivity. To explore the possibility of restoring the fertility of the soil by changing the relative proportion of local soil microbiome by the addition of selected native PGPR, the study was undertaken. Extensive screening finally led to the identification of two very efficient PGP bacterial isolates, namely, FKK5 and DUM4, from the rhizosphere of little millet and maize, respectively. Potential PGPR have earlier been isolated and described from the rhizosphere of various plants (Dawwan et al., 2013; Castanheira et al., 2016; Backer et al., 2018) including finger millet (Sekar et al., 2018) and foxtail millet (Niu et al., 2018). However, the little millet rhizosphere has been explored for the first time to isolate PGPR.

Of various PGP activities, N2 fixation was considered to be of foremost importance. Nitrogen in a fixed form is a critical nutrient for the plant, as it is required in large quantities during various phases of growth. Ensuring a continuous supply of nitrogen in the soil in the presence of a microbial denitrification system itself is a challenge. PGPR with N2 fixation ability can easily handle this challenge. Both the isolates FKK5 and DUM4 exhibited N2 fixation ability, although at a variable rate or with variable efficiency. Both, however, performed better than known PGPR A. chroococcum (Gholami et al., 2012). Bacteria with different N2 fixation rates/efficiency have been reported earlier (Gupta et al., 2015; Habibi et al., 2019). The difference is generally regarded as a species-/strain-dependent function. The better N2 fixation efficiency of native isolates FKK5 and DUM4 than non-native-known PGPR is attractive and similar to earlier findings (Misra et al., 2017). The compatibility of native isolates with the microbiome is the most possible reason for the better performance of native isolates.

Solubilization of complex phosphates in the soil is the second most important PGP activity after N2 fixation, which may reduce or abolish the need for applying synthetic phosphate fertilizer (Castanheira et al., 2016; Backer et al., 2018). The isolates FKK5 and DUM4 with their phosphate-solubilizing activity (Figure 2c) make them important PGPR. PGPR with even higher PSE have been reported (Dawwan et al., 2013); however, FKK5 and DUM4 with their additional PGP activities make them more attractive. The soil in the tropics and subtropics is generally low in soluble phosphate (Ae et al., 2010), requiring supplementation of phosphate to support optimum crop production. PGPR with phosphate-solubilizing ability is thus important for the soil in these regions. The siderophore produced by microbes is another PGP activity that may serve as Fe nutrition for plants, overcoming soil heavy metal stress and suppressing plant pathogens (Castanheira et al., 2016; Backer et al., 2018). A range of siderophores with very high iron affinity is produced by soil bacteria that reduce the availability of iron in the soil, thus suppressing many types of soil pathogens. The siderophore production by the isolates FKK5 and DUM4 (Figure 2d) is thus considered an important PGP activity. Besides these, auxin, which is an important plant growth regulator affecting many plant growth activities such as photo and geotropism, root and shoot growth, organogenesis, and vascularization (Sekar et al., 2018), is also a very important PGP stimulant. IAA is the most common and important auxin found in plants (Backer et al., 2018). Both the isolates produce IAA in the presence of tryptophan (Figure 2b); the latter is used as a precursor for auxin production by many PGPR.

The isolates have been tested for their growth-enhancement activity in the case of little millet under semi-controlled conditions. Both the isolates showed significant (p ≤ 0.05), but differential enhancement in pre-harvest and (Table 2) post-harvest (Table 3) growth parameters. Among the pre-harvest growth parameters tested, an increase in the number of leaves, reproductive tillers, stem diameter, and length of panicle in both the isolates compared to control (non-inoculated) plant (Figure 3) was seen; the highest increase in the length of panicle and the number of reproductive tillers was found with FKK5 inoculation, while the highest plant and root length was found with DUM4 priming (Table 2). Similarly, among the post-harvest growth parameters tested, higher increase in root length, root dry weight, total biomass, and seed weight per plant was seen in inoculated plants compared to non-inoculated control plant; the highest increase in root length and root dry weight was observed in plants inoculated with DUM4 (Table 3), but the highest seed yield was shown by FKK5-primed plant. It is important to note that a positive effect on various growth characteristics was shown more by native inocula than non-native ones (Table 3). Similarly, the native PGPR (FKK5 and DUM4)-primed plants showed more grain yield and biomass yield compared to those inoculated with non-native A. chroococcum 5576 (positive control) (Table 4).

A closer look at the data indicates that DUM4 promotes vegetative growth, such as shoot and root length and root biomass, more, while FKK5 stimulates reproductive growth, such as tillering and seed yield, more. The differential performance of the two isolates may be attributed to their variable PGP activities (e.g., N2 fixation and phosphate solubilization rate and/or auxin production characteristics) as estimated under in vitro conditions. Tailoring a plant for better tillering that usually results in more seed yield has been one of the important objectives of crop breeders. In this regard, FKK5 is an important bioresource, whose activity analysis may reveal more insight into the physiology of tillering in this crop. PGPR-stimulated enhancement of growth in the case of rice (Govindarajan et al., 2008), sweet potato (Radziah and Zulfikar, 2003), and potato (Dawwan et al., 2013) has earlier been reported. Grain yield is at the center of all the efforts of breeders as well as farmers. The native PGPR (FKK5 and DUM4)-primed plants showed 3%–5% more grain yield and 4%–7% more biomass yield compared to those inoculated with non-native A. chroococcum 5576 (positive control). FKK5-primed little millet plant seems to have acquired idealized appearance or ideotype (more tiller, less height, and less overall vegetative growth) that favors higher grain yield. A similar positive effect on growth and yield has been shown by PGPR-inoculated cucumber (Islam et al., 2016) and oil palms (Ai'shah et al., 2009).

Morphological, partial physiological data (Table 4) and molecular analysis (Figure 3) indicated that the isolates belong to Burkholderia sp. Although almost 100 species of Burkholderia are known (Eberl and Vandamme, 2016), only 20 type strains of closely related species showing 97.5%–99.34% sequence similarity could be retrieved from the NCBI database. They all belong to Burkholderia cepacia complex (Bcc strains), indicating the isolates’ affinity with the members of this complex. But at the same time, their clustering into separate subclade renders them “closely related separate taxa.” In this connection, hardly, there is a consensus over the “threshold value” for percent sequence dissimilarity to define species (Rajendharan and Gunasekaran, 2011); authors, in many instances, suggested about a 0.5%–1% difference (99%–99.5% similarities) to designate a separate species (Rajendharan and Gunasekaran, 2011) and a 1%–5% difference (99%–95%) to define a genus. Alternatively, 5–15 bp difference in 16S rDNA sequence or < 1.0% rDNA sequence difference in combination with a clear phenotypic uniqueness may be used to define new species (Roth et al., 2003). These suggestions and NJ dendrogram (Figure 4) provide strong support to designate the isolates as separate species.

The species of Burkholderia with multiple PGP traits have earlier been described (Poupin et al., 2013), exhibiting significant growth enhancement in corn and soybean. Burk holderia sp. has also been reported to exist as endophytes in various plants (Sessitsch et al., 2005) and potentially help in nodulation because of the presence of nodulation genes (Backer et al., 2016). One of its species, Burkholderia phytofirmans, has been reported to protect plants from pathogens and augment capacity to resist various stresses such as drought, salinity, and low temperature (Poupin et al., 2013). Both the current isolates (FKK5 and DUM4), however, exhibit close affinity with the members of B. cepacia complex (Bcc strains), which are opportunistic pathogens. Although these isolates have not been tested for their pathogenic behavior, their close affinity with the pathogenic group is a matter of concern. In this regard, the point of relief is that many of the strains in this group show attenuated virulence and good biocontrol potential (Eberl and Vandamme, 2016).

The viability of isolates FKK5 and DUM4 was also tested using lantana (Lantana camera) charcoal as a carrier and A. chroococcum as the control PGPR. Viable count was determined after 6 months as earlier (Mankar et al., 2021 in press) (data not shown). Both DUM4 and FKK5 showed parallel performance, further indicating the potential of the two strains for commercial application as PGPR.

Usually, four criteria are used to recognize a potential PGPR, namely, the ability to survive in the rhizosphere, protect crops from pathogens, promote the growth of crops, and exhibit compatibility with the native microbiome (Deketelaere et al., 2017). Among the four, native PGPR have a natural capacity to exhibit compatibility with the native soil microbiome. The isolates DUM4 and FKK5 were, in addition, able to survive in the rhizosphere and cause positive growth enhancement in the case of little millet. Based on fulfillment of three of the four criteria, we suggest that the isolates FKK5 and DUM4 are potential PGPR for little millet.

Conclusion

In summary, we isolated 48 native rhizobacteria from the rhizosphere of different crops. Two (DUM4 and FKK5) from little millet and maize rhizosphere, respectively, were studied for their PGP activities, and their priming impact on the growth of little millet was compared with that of non-native PGPR. The locally adapted isolates identified as Burkholderia sp. have better impacts on plant growth and yield, compared to non-native strain and control in the pot culture. Though the two isolates belong to the same species, they have some variation in PGP activities and also in the priming impact on little millet growth, with DUM4 promoting more vegetative growth and FKK5 stimulating more reproductive growth. This is the first report of isolation of Burkholderia sp. from the rhizosphere of little millet and its potential effect on the enhancement of growth and yield of little millet. The results may have a far-reaching impact on food security, especially because of the current climate change-related threat to food security and the furtherance of the country's priority to apply science and technology for inclusive growth.

Figure 1

Geographical position of the experimental siteAbbildung 1. Geografische Position des Versuchsstandorts
Geographical position of the experimental siteAbbildung 1. Geografische Position des Versuchsstandorts

Figure 2

Rhizobacterial isolates on Jensen's nitrogen-free medium (a) and PGPR activities such as IAA production (b), phosphate solubilization on Pikovskaya's medium (c), and siderophore production (d) exhibited by selected rhizobacterial strains, DUM4 and FKK5; IAA: indole acetic acid; PGPR: plant growth-promoting rhizobacteriaAbbildung 2. Rhizobakterielle Isolate auf Jensens stickstofffreiem Medium (a) und PGP-Aktivitäten wie IAA-Produktion (b), Phosphat-Solubilisierung auf Pikovskaya-Medium(c) und Siderophor-Produktion (d) bei ausgewählten Rhizobakterien-Stämmen DUM4 und FKK5
Rhizobacterial isolates on Jensen's nitrogen-free medium (a) and PGPR activities such as IAA production (b), phosphate solubilization on Pikovskaya's medium (c), and siderophore production (d) exhibited by selected rhizobacterial strains, DUM4 and FKK5; IAA: indole acetic acid; PGPR: plant growth-promoting rhizobacteriaAbbildung 2. Rhizobakterielle Isolate auf Jensens stickstofffreiem Medium (a) und PGP-Aktivitäten wie IAA-Produktion (b), Phosphat-Solubilisierung auf Pikovskaya-Medium(c) und Siderophor-Produktion (d) bei ausgewählten Rhizobakterien-Stämmen DUM4 und FKK5

Figure 3

Effect of different bioinocula (a = control, b = FKK5, c = DUM4, and d = Azotobacter chroococcum) priming on the vegetative growth of little millet. The best among three of every treatment is shownAbbildung 3. Wirkung verschiedener Bioinokula (a = Kontrolle, b = FKK5, c = DUM4 und d = A. chroococcum) Priming auf das vegetative Wachstum kleiner Hirse. Die Beste unter den Dreien jeder Behandlung wird gezeigt.
Effect of different bioinocula (a = control, b = FKK5, c = DUM4, and d = Azotobacter chroococcum) priming on the vegetative growth of little millet. The best among three of every treatment is shownAbbildung 3. Wirkung verschiedener Bioinokula (a = Kontrolle, b = FKK5, c = DUM4 und d = A. chroococcum) Priming auf das vegetative Wachstum kleiner Hirse. Die Beste unter den Dreien jeder Behandlung wird gezeigt.

Figure 4

Dendrogram showing the evolutionary relationship of DUM4 and FKK5 with type strains of closely related taxa in the NCBI database, applying neighbor-joining method. The branch length indicates evolutionary distance. Maximum composite likelihood method was used to calculate the evolutionary distance, with units used being the number of base substitutions per site. Sequence accession number is given before the name of taxon. The numbers at the nodes represent bootstrap percent values from 1000 replicates. All positions containing gaps and missing data were eliminated. The analyses were carried out in MEGA6. Dendrogram was condensed with cut-off value 50% for the treeAbbildung 4. Dendrogramm, das die evolutionäre Beziehung von DUM4 und FKK5 mit Typstämmen eng verwandter Taxa in der NCBI-Datenbank unter Anwendung der Neighbor-Joining-Methode zeigt. Die Astlänge zeigt die evolutionäre Distanz an. Die Maximum-Composite-Likelihood-Methode wurde verwendet, um die evolutionäre Distanz zu berechnen, und die verwendeten Einheiten sind die Anzahl der Basensubstitutionen pro Stelle. Die Sequenzzugangsnummer wird vor dem Namen des Taxons angegeben. Die Zahlen an den Knoten stellen Bootstrap-Prozentwerte von 1000 Replikaten dar. Alle Stellen mit Lücken und fehlenden Daten wurden eliminiert. Die Analysen wurden in MEGA6 durchgeführt. Das Dendrogramm wurde mit einem Cut-Off-Wert von 50% für den Baum verdichtet.
Dendrogram showing the evolutionary relationship of DUM4 and FKK5 with type strains of closely related taxa in the NCBI database, applying neighbor-joining method. The branch length indicates evolutionary distance. Maximum composite likelihood method was used to calculate the evolutionary distance, with units used being the number of base substitutions per site. Sequence accession number is given before the name of taxon. The numbers at the nodes represent bootstrap percent values from 1000 replicates. All positions containing gaps and missing data were eliminated. The analyses were carried out in MEGA6. Dendrogram was condensed with cut-off value 50% for the treeAbbildung 4. Dendrogramm, das die evolutionäre Beziehung von DUM4 und FKK5 mit Typstämmen eng verwandter Taxa in der NCBI-Datenbank unter Anwendung der Neighbor-Joining-Methode zeigt. Die Astlänge zeigt die evolutionäre Distanz an. Die Maximum-Composite-Likelihood-Methode wurde verwendet, um die evolutionäre Distanz zu berechnen, und die verwendeten Einheiten sind die Anzahl der Basensubstitutionen pro Stelle. Die Sequenzzugangsnummer wird vor dem Namen des Taxons angegeben. Die Zahlen an den Knoten stellen Bootstrap-Prozentwerte von 1000 Replikaten dar. Alle Stellen mit Lücken und fehlenden Daten wurden eliminiert. Die Analysen wurden in MEGA6 durchgeführt. Das Dendrogramm wurde mit einem Cut-Off-Wert von 50% für den Baum verdichtet.

Effect of native and non-native bioinocula priming on important growth characteristics (mean ± SE, n = 3) of little millet at the harvestTabelle 3. Einfluss des Primings von nativen und nicht nativem Bioinokulen auf wichtige Wachstumsmerkmale (Mittelwert ± SE, n = 3) von Hirse bei der Ernte

Treatment Root length (cm) Root dry weight (g) Total plant biomass (g) Seed weight per plant (g)
Non-inoculated 10.30 ± 1.27a 0.163 ± 0.02a 1.01 ± 0.07a 0.360 ± 0.01a
Azotobacter chroococcum 12.90 ± 0.47b 0.197 ± 0.01b 1.19 ± 0.01b 0.433 ± 0.03b
FKK5 13.60 ± 0.55c 0.206 ± 0.01c 1.24 ± 0.06d 0.460 ± 0.02d
DUM4 17.00 ± 1.82d 0.279 ± 0.01d 1.22 ± 0.03c 0.449 ± 0.01c
LSD (p ≤ 0.05) 3.21 0.05 0.07 0.08

Effect of native and non-native bioinocula priming on important growth characteristics (mean ± SE, n = 3) of little millet after 85 days of sowing before harvest in pot trialsTabelle 2. Wirkung von Priming nativer und nicht nativer Bioinokula auf wichtige Wachstumsmerkmale (Mittelwert ± SE, n = 3) von Hirse nach 85 Tagen Aussaat vor der Ernte in Topfversuchen

Treatment Length Plant height (cm) Stem diameter (mm) No. of leaves No. of tillers (per plant) Panicle length (cm)
Non-inoculated 62.5±2.47b 1.96 ± 0.13a 09.66 ± 1.20a 3.66± 0.33a 11.33 ± 0.52a
Azotobacter chroococcum 57.6 ± 2.04a 2.66 ± 0.14b 14.66 ± 1.45b 4.33 ± 0.33b 11.66 ± 0.29b
FKK5 69.0 ± 2.17c 2.93 ± 0.17d 15.40 ± 1.76c 4.66 ± 0.33d 13.23 ± 0.52d
DUM4 72.0 ± 2.62d 2.76 ± 0.19c 17.00 ± 1.15d 4.53 ± 0.33c 13.10 ± 0.64c
LSD (p ≤ 0.05) 6.16 0.44 4.04 1.38 1.54

Priming effect of native and non-native bioinocula on grain and biomass yield enhancement (IE) (mean ± SE, n = 3)Tabelle 4. Priming-Effekt von nativen und nicht nativem Bioinokula auf Getreide- und Biomasseertragssteigerung (IE) (Mittelwert ± SE, n = 3)

Inoculum Grain (seed) yield enhancement (%) Total biomass yield enhancement (%)
FKK5 28.14 ± 8.93c 23.08 ± 2.23c
DUM4 24.72 ± 3.62b 21.87 ± 5.58b
Azotobacter chroococcum 20.43 ± 9.78a 19.09 ± 9.34a

Selective morpho-physiological characteristics of the potential isolatesTabelle 5. Selektive morpho-physiologische Eigenschaften der potentiellen Isolate

Character Azotobacter chroococcum 5576 FKK5 DUM4
Gram reaction
Cyst formation +
Pigmentation Brown Brown Brown
Motility + + +
IAA production + + +
NH3 production + + +
Starch formation + + +
Urease activity + + +
Catalase activity + + +
Oxidase activity + + +
Growth on sucrose + + +
Growth on maltose + + +
Growth on mannitol + +
Growth on citrate + + +
KOH + + +

Chemical characteristics of the experimental soilTabelle 1. Chemische Eigenschaften des Versuchsbodens

Site pH EC (ds per m) Organic carbon (%) Nitrogen (%) Phosphorus (kg per ha) Potassium (kg per ha)
Tamia 6.9 0.53 0.91 0.154 14.71 190.11

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