Priming effect of native rhizosphere bacteria on little millet (Panicum sumatrense)

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.

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.

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.