Keywords

1 Introduction

There has been a dramatic expand in the world’s population since the last 150–200 years. According to the United Nations, the world’s population is set to reach ~9.7 billion by 2050, which is fourfold the population in 1950 (United Nations 2015). Such drastic increase in population has put a serious pressure on our food resources causing a threat to the food security of many developing countries. Moreover, the environmental crisis like air, water, and soil pollution caused by the growing rate of global industrialization is making things worse for us and our planet (Glick 2015). In agriculture, the overuse of energy-intensive chemical fertilizers to boost crop yield has destroyed our soil ecosystem. Now is the best time to switch from these harmful chemical fertilizers to harmless and sustainable biofertilizers. Biofertilizer is a contraction of the term biological fertilizer, and it is very different from the organic fertilizer. Organic fertilizers contain organic compounds, which directly or indirectly increase soil fertility, whereas biofertilizers contain living organisms that increase the nutrient status of the host plant through their ongoing association with the host plant (Vessey 2003). The biofertilizer is a substance which contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant (Vessey 2003; Meena et al. 2013a; Bahadur et al. 2014; Maurya et al. 2014; Jat et al. 2015; Kumar et al. 2016b).

Apart from chemical fertilizers, the use of chemical pesticides to control the plant pest population is not only destroying our soil and natural environment but also impacting the human health both directly and indirectly. Pesticide is often considered a quick, easy, and inexpensive solution for controlling the pest population, but these benefits are incurred at the cost of our environment (Aktar et al. 2009). Research on the use of much safer, biological control agents against pests is gaining momentum. “Biocontrol” is a commonly used, abbreviated synonym of “biological control.” As defined by Pal and McSpadden Gardener (2006), “Biocontrol refers to the purposeful utilization of introduced or resident living organisms, other than disease resistant host plants, to suppress the activities and populations of one or more plant pathogens.” The organism that suppresses the pest or pathogens are referred to as the “biocontrol agent.” In this chapter, the term biofertilizer and biocontrol agent are used to signify those bacteria that are involved in promoting plant growth and controlling plant pathogen population, respectively (Kumar et al. 2015, 2016a; Ahmad et al. 2016; Meena et al. 2016a, b; Parewa et al. 2014; Prakash and Verma 2016; Jha and Subramanian 2016).

Plant growth-promoting bacteria (PGPB), as their name signifies, are those bacteria that can promote plant growth either directly, by aiding in nutrient acquisition (biofertilization) and moderating the plant hormone levels, or indirectly by acting as biocontrol agents against harmful plant pathogens (Glick 1995). These PGPB include bacteria that are free-living in the rhizosphere, form symbiotic relationships with plants like Rhizobia and Frankia, and can colonize interior tissues of plants (known as bacterial endophytes) (Glick 2012). Among the myriads of PGPB thriving in close association with plants, some spore-forming PGPB, particularly the gram-positive bacilli and streptomycetes, have drawn special attention because of their advantages over nonspore formers in product formulation and stable maintenance (Emmert and Handelsman 1999).

Among these, an agriculturally important microbe vital for present and future sustainable agriculture is “Paenibacillus polymyxa.” P. polymyxa is widely known for its plant growth-promoting (PGP) traits and the ability to thrive in diverse ecological niches. In this chapter, studies are signifying huge potential of P. polymyxa as a biofertilizer and biocontrol agent in sustainable agriculture (Priyadharsini and Muthukumar 2016; Kumar et al. 2017; Meena et al. 2015a, b, f; Raghavendra et al. 2016; Zahedi 2016; Dotaniya et al. 2016; Jaiswal et al. 2016).

2 Brief History of Paenibacillus polymyxa (Formerly Bacillus polymyxa)

History of P. polymyxa dates back to the nineteenth century when Prażmowski (1880) described an organism that closely resembled Clostridium butyricum but was able to grow in the presence of air. Prażmowski reported that this organism is slimy, strongly attacks starch and cellulose, and turns some carbohydrates into carbon dioxide gas (Porter et al. 1937). He designated it as “Clostridium polymyxa.” But, in 1889, Eugéne Macé proposed the species name “ Bacillus polymyxa ” for this bacterium due to its rod-shaped cells (Macé 1889). Bacillus means “a rod” and polymyxa means “much slime.” Macé reported that these bacteria are rod-shaped, produce spores, and can develop on the cooked slices of beets and turnips when exposed to air, and when grown in liquid media, they form a thick, creamy membrane on the surface (Macé 1889). Since then, this bacterium has been isolated frequently from soil and various plant species (Porter et al. 1937; Nakamura 1987) and has been reported to provide a variety of benefits to plants, like fixed nitrogen (N) (Bredemann 1909; Grau and Wilson 1962; Kalininskaya 1968; Seldin et al. 1984); PGP enzymes, viz., β-amylase (Fogarty and Griffin 1975; Hensley et al. 1980) and 2,3-butanediol (Ledingham and Neish 1954); and pathogen protection by secreting antibiotic polymyxin (Porter et al. 1949, Gordon et al. 1973; Skerman et al. 1980). Nakamura (1987) regarded B. polymyxa an agriculturally, industrially, and medically important organism.

Although bacterial species belonging to the genus Bacillus have been extensively studied, but it was always acknowledged that the taxonomy of this genus is unsatisfactory. Ash et al. (1991) referring to Bergey’s Manual of Systematic Bacteriology, Vol. 2 (Claus and Berkeley 1986), pointed out that genus Bacillus is phenotypically and phylogenetically heterogeneous with species showing an extremely wide range of nutritional requirements, growth conditions, metabolic diversity, and DNA base composition. Ash et al. (1991) reported this phylogenetic heterogeneity by comparing and analyzing the small-subunit rRNA sequences of all species (51 species) known at that time. They divided these 51 species into five phylogenetically distinct groups and placed B. polymyxa into group 3. Findings reported in this chapter formed the basis for the proposal to reclassify group 3 bacilli comprising of B. polymyxa and ten other close relatives into a new genus Paenibacillus (meaning: almost a Bacillus) under the same family Bacillaceae (Ash et al. 1993). This proposal to form a new genus was officially approved and announced by the International Committee on Systematic Bacteriology (1994) through their official journal. Eventually, genus Paenibacillus was reclassified into a separate family Paenibacilliaceae and was designated as the family’s type genus (Priest 2009; Rawat et al. 2016; Yasin et al. 2016; Meena et al. 2016c; Saha et al. 2016a; Yadav and Sidhu 2016; Meena et al. 2016d; Das and Pradhan 2016; Dominguez-Nunez et al. 2016).

3 P. polymyxa Strains Isolated from Agricultural Crops

The P. polymyxa strains inhabit diverse ecological niches including but not limited to rhizosphere and internal tissues of agricultural crops (von der Weid et al. 2000; Gu et al. 2010) and forest trees (Shishido et al. 1995; Bal et al. 2012), fermented food (Piuri et al. 1998; He et al. 2007), and marine environment (Ravi et al. 2007; Ma et al. 2010). Its ability to survive in a range of environmental conditions can be related to its endospore-forming potential. Agriculturally important P. polymyxa strains that have been isolated from various agricultural sites over the years are listed in Table 6.1. N-fixing P. polymyxa strain B5 was isolated from the rhizosphere of spring wheat (Triticum aestivum L.) growing on a research field (Lindberg and Granhall 1984). Since DNA identification techniques were not available at that time, authors identified the isolates using standard biochemical tests and comparing the results with reference strain ATCC 842. Strain B5 reduced significant amounts of acetylene and exhibited nitrogenase activity in vitro, thus indicating that it is a potent N-fixing bacterial strain (Saha et al. 2016b; Verma et al. 2014, 2015b; Meena et al. 2014a, 2015e; Teotia et al. 2016; Bahadur et al. 2016b).

Table 6.1 List of important Paenibacillus polymyxa strains that have been isolated from agricultural sites
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Another strain of P. polymyxa (CF43) was isolated from the rhizosphere of spring wheat (cv. Castan) growing in a field near Nemours, France (Gouzou et al. 1993). It was determined that population size of strain CF43 present in the wheat rhizosphere ranged from 1 × 105 to 5 × 105 cfu/g dry weight of rhizosphere soil. It was also reported that strain CF43 enhances soil aggregate stability and overall porosity. In another study, bacterial strains were isolated from barley (Hordeum vulgare L.) rhizosphere and screened for their ability to produce selected enzymes and antagonize plant pathogenic fungi (Nielsen and Sørensen 1997). Two strains of P. polymyxa (CM5-5 and CM5-6) were successful in these screening tests, and according to the authors, these strains could be promising biocontrol agents (Sharma et al. 2016; Verma et al. 2015a; Meena et al. 2013b; Shrivastava et al. 2016; Meena et al. 2016e).

The diversity of P. polymyxa strains colonizing the rhizosphere and the surrounding soil is huge. Mavingui et al. (1992) studied the diversity among 130 strains of P. polymyxa isolated from rhizosphere soil, non-rhizosphere soil, and rhizoplane of wheat. Phenotypic and genotypic characterization tests revealed that there is higher diversity within P. polymyxa strains isolated from non-rhizosphere and rhizosphere soil as compared to the strains isolated from the rhizoplane (Meena et al. 2017). In another study conducted with Zea mays plants growing in Cerrado soil, which is found commonly in tropical belts of the world, ~70 different isolates of P. polymyxa were harvested from the rhizosphere of corn plants (von der Weid et al. 2000; da Mota et al. 2002). Isolation of two strains designated as B5 and B6 from soil around the peanut (Arachis hypogaea L.) roots was performed, and by using genomic identification techniques, strains were identified as P. polymyxa (Haggag and Timmusk 2008).

The strains were able to suppress the activity of pathogenic fungus Aspergillus niger (causes crown rot disease of peanut) both in vitro and in vivo (greenhouse and field experiments). P. polymyxa strains have also been isolated from watermelon (Citrullus lanatus) and muskmelon (Cucumis melo L.). Fusaricidin-type compound-producing strain SQR-21, exhibiting antagonistic activity against pathogenic fungus, Fusarium oxysporum f. sp. niveum, was isolated from watermelon plants (Raza et al. 2009). On the other hand, a strain of P. polymyxa (G-14) was isolated from soil samples collected from muskmelon fields in Changji, Xinjiang, China (Shi et al. 2012). It was reported that strain G-14 can produce antibiotic compounds that can antagonize the activity of pathogenic bacteria, Pseudomonas syringae pv., Lachrymans, and Acidovorax avenae subsp. citrulli, that cause bacterial spot disease in muskmelon. Apart from living in the rhizosphere of a plant, P. polymyxa strain has also been reported to inhabit agricultural soils. P. polymyxa strain JSa-9 was isolated from soil collected from the farmland of Nanjing (Jiangsu province, China) and was reported to show antagonistic activity against local plant pathogens (Deng et al. 2011; Velazquez et al. 2016; Meena et al. 2014b, 2015c; Sindhu et al. 2016; Singh et al. 2016; Masood and Bano 2016).

Similarly, three strains of P. polymyxa (SC09-21, SR04-02, SR04-16) were isolated from soil samples collected from 30 different locations within fields (where Chinese cabbage, garlic, and orrice were cultivated) in Samcheok, Gangwon Province, Korea (Xu and Kim 2014). Isolated strains showed a range of PGP traits both in vitro and in vivo. In another study, two diazotrophic (N-fixing) strains EG2 and EG14 of P. polymyxa were isolated from agriculturally used land in Poland (Górska et al. 2015). P. polymyxa strain was also isolated from phyllosphere of wheat cultivated in a field located at Tongzhou near Beijing City, China (Gu et al. 2010). A study reviewed in this section clearly proves the potential of P. polymyxa strains to inhabit a range of agricultural crops and soil at a variety of locations all around the world (Meena et al. 2013c, 2015d; Singh et al. 2015; Bahadur et al. 2016a).

4 Complete Genome Sequencing of P. polymyxa Strains

Complete genome sequencing determines the complete DNA sequence of an organism’s genome at a single time. This technique is crucial to identify the genes that are responsible for different traits of a PGPB. For instance, N-fixing trait of a PGPB is related to the presence of nif genes. Genome sequencing helps to link the field/lab observed characteristics of a PGPB with the genes responsible for exhibiting those PGP traits. Although complete genomes of a large number of PGPB have been sequenced, very few studies have reported the complete genome sequence of P. polymyxa strains. To date, complete genomes of only six strains of P. polymyxa are available in the NCBI database (https://www.ncbi.nlm.nih.gov), and amazingly all six strains were isolated from agricultural sites and were reported to possess PGP abilities (Table 6.2). Korean researchers were the first to sequence and report the complete genome of a P. polymyxa strain (E681) (Kim et al. 2010). P. polymyxa E681 is an endospore-forming bacterium that was isolated from the rhizosphere of winter barley in South Korea (Ryu and Park 1997). Based on sequence investigations, Kim et al. (2010) reported that strain E681 possesses genes responsible for synthesizing antibiotic compound polymyxin, antifungal compound fusaricidin, and phytohormone auxin. Subsequently, the complete genome of another P. polymyxa strain (SC2) originally isolated from pepper (Capsicum annuum L.) (Zhu et al. 2008) was sequenced and reported (Ma et al. 2011). Genome sequencing revealed that this strain possesses genes that are involved in antibiotic biosynthesis like fusaricidin-synthetic gene, polymyxin-synthetic gene cluster, and antibiotic-synthetic gene cluster; thus, it can be concluded that this bacterial strain will exhibit a broad-spectrum antimicrobial activity (Ma et al. 2011). Till now, the one and only endophytic strain of P. polymyxa (M-1) whose complete genome has been sequenced (Niu et al. 2011) was isolated from internal tissues of wheat roots (Yao et al. 2008).

Table 6.2 List of Paenibacillus polymyxa strains whose complete genomes have been sequenced to date
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In another report, the complete genome of a PGPB (P. polymyxa CR1), isolated from corn rhizosphere, was sequenced (Eastman et al. 2014a). Genome sequencing revealed that this strain possesses genes responsible for N fixation (nif genes), indole-3-acetic acid (IAA) synthesize, biomass degradation, and antimicrobial production, thus confirming their previous infield observations. Li et al. (2014) and Rybakova et al. (2015) have also reported complete genomes of P. polymyxa strains SQR-21 and Sb3-1, respectively. These strains also possess a variety of PGP traits. Eastman et al. (2014b) was the first to conduct a comparative genomic analysis of P. polymyxa strains. Their work highlighted that plant growth promotion by P. polymyxa is mediated largely through phytohormone production, increased nutrient availability, and biocontrol mechanisms. Similar study comparing the genomes of nine P. polymyxa strains and five other Paenibacillus spp. isolated from diverse ecological niches and geographic regions was conducted recently (Xie et al. 2016). Authors concluded that genes relevant to PGP traits, i.e., phosphate solubilization, N fixation, IAA production, and antibiotic synthesis, are well conserved or have evolved with diversity in P. polymyxa and its closely related species.

5 Plant Growth Promotion by Paenibacillus polymyxa Strains

Generally, P. polymyxa strains promote plant growth either directly by helping in nutrient acquisition (like biological N fixation), producing plant growth regulators (like auxin, cytokinins, gibberellins), controlling plant ethylene levels, and enhancing root permeability and soil porosity or indirectly through biocontrol of major plant pathogens. Primary PGP characteristics of P. polymyxa strains have been presented in Fig. 6.1. Various reports about the direct and indirect benefits provided by prominent P. polymyxa strains to agricultural crops have been listed in Table 6.3.

Fig. 6.1
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Primary plant growth-promoting (PGP) characteristics of Paenibacillus polymyxa strains

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Table 6.3 List of prominent Paenibacillus polymyxa strains reported to provide a variety of benefits to agricultural crops
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5.1 Biofertilization : Direct Plant Growth Promotion

P. polymyxa strains possess many PGP characteristics through which direct plant growth promotion is achieved (Fig. 6.1). This includes biological N fixation by either living in the rhizosphere or internal tissues of the plant; positively affecting the physical structure of rhizosphere soil; enhancing nutrient uptake from the soil, thus increasing the plant length and biomass along with the overall crop yield; and secreting plant growth hormones. Lindberg et al. (1985) observed the N fixation activity of two P. polymyxa strains B1 and B2 isolated from field-grown wheat (Lindberg and Granhall 1984) by using acetylene reduction assay (ARA). Results of ARA indicated that strains B1 and B2 can produce 10.6 and 3.3 nmol C2H4plant−1 h−1, respectively, thus establishing their N-fixing ability. Besides fixing N, these strains were involved in increasing the seedling biomass and shoot length. To observe the endophytic colonization by these strains in wheat roots, authors used transmission electron microscopy technique and found that these strains can colonize intercellular and intracellular spaces of root epidermal cells. In later studies, production of cytokinin by strain B2 was also assessed using the chromatography techniques (Timmusk et al. 1999). Strains B1 was tagged with GFP to analyze the endophytic colonization ability and pathway through which it enters the plant tissues (Timmusk et al. 2005). Fluorescence microscopy and electron scanning microscopy revealed that this strain colonizes predominantly the root tip, where it forms biofilms and then invade the plant roots. This was the first study in which invasion and colonization of plant roots by a P. polymyxa strain were reported in detail. Some studies have implied the physical and microbial approach to analyze the effects of P. polymyxa inoculation on soil aggregation. In one such study, P. polymyxa strain CF43, isolated from wheat rhizosphere, increased the mass of soil adhering to the wheat roots by 57% when grown in a glasshouse (Gouzou et al. 1993). When aggregate size distribution was compared, it was observed that inoculated rhizosphere soil has a more porous structure as compared to the uninoculated control. Increased aggregation of rhizosphere soil by CF43 was also confirmed by Bezzate et al. (2000) using molecular techniques. Bezzate et al. (2000) hypothesized that levan, which is a fructosyl polymer produced by strain CF43, is responsible for enhanced soil aggregation. A mutant strain, SB03, was constructed by silencing the sacB gene (responsible for encoding levan) of strain CF43. Inoculation with CF43 significantly increased the wheat root dry mass and root-adhering soil dry mass as compared to inoculation with SB03 strain, thus signifying the importance of levan produced by the strain CF43 in increasing the soil aggregation. In a recent study, N-fixing ability of two P. polymyxa strains (EG2 and EG14) isolated from agricultural soils was assessed (Górska et al. 2015). The genome of these strains was found to carry nif genes which encode individual components of the nitrogenase complex. In vitro tests of nitrogenase activity by using ARA revealed that EG2 and EG14 can produce 2.9 and 0.4 nM C2H4ml−1 h−1.

Diazotrophic (N-fixing) strains have immense potential as biofertilizers since N is believed to be the most important mineral nutrient required for plant growth and maintenance (Robertson and Vitousek 2009). Bal et al. (2012) isolated an endophytic diazotroph, P. polymyxa P2b-2R , from stem tissues of lodgepole pine (Pinus contorta). Strain P2b-2R was able to grow on N-free medium [combined carbon medium (CCM; Rennie 1981)] and consistently reduced significant amounts of acetylene in ARA (Bal et al. 2012). By using a more accurate method of determining the amount of N fixed (15N foliar dilution assay), Bal and Chanway (2012a), Anand et al. (2013), and Yang et al. (2016) reported P2b-2R’s remarkable ability to derive up to 79% of N from the atmospheric pool when inoculated into lodgepole pine. In a subsequent report, it was observed that strain possesses nif genes required to encode the nitrogenase enzyme (Anand and Chanway 2013c). GFP-tagged P2b-2R strain was constructed to evaluate the endophytic colonization sites in lodgepole pine, and it was reported to colonize both intercellular and intracellular spaces of lodgepole pine interior tissues (Anand and Chanway 2013a).

P2b-2R was able to colonize internal tissues of stem and root of another gymnosperm tree species, western red cedar (Thuja plicata), and significantly enhance seedling length and biomass along with fixing considerable amounts of N from the atmosphere (Bal and Chanway 2012b; Anand and Chanway 2013b; Tang et al. 2017; Yang et al. 2017). Puri et al. (2015) hypothesized that strain P2b-2R can provide similar benefits to agricultural crops through rhizospheric and endophytic colonization. Puri et al. (2015) used corn as the model crop to test this hypothesis. P2b-2R colonized rhizosphere and internal root tissues of corn seedlings with a population size of 105 cfu/g dry root or fresh tissue in just 10 days. P2b-2R also fixed up to ~20% of N from the atmosphere and increased seedling length by ~35% and biomass by 30% in 30-day long trials (Puri et al. 2015). P2b-2R successfully colonized an important oilseed crop species, canola (Puri et al. 2016a), and vegetable crop species, tomato (Padda et al. 2016a). Similar benefits were provided by P2b-2R to these crop species indicating that P2b-2R can symbiotically associate and provide benefits to a broad range of hosts (Table 6.4). Padda (2015) reported an astonishing discovery with the GFP-tagged P2b-2R (P2b-2Rgfp) constructed by Anand and Chanway (2013a). Padda (2015) compared the GFP-tagged strain with the wild-type strain of P2b-2R in terms of their ability to fix N and enhance seedling length and biomass.

Table 6.4 Plant growth promotion (seedling length, seedling biomass, and foliar N concentration enhancement) and nitrogen fixation (% nitrogen derived from the atmosphere) by Paenibacillus polymyxa strain P2b-2R when inoculated into important agricultural crops
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P2b-2Rgfp inoculation significantly enhanced corn seedling growth (length and biomass) as compared to the wild-type P2b-2R inoculation. This was the first report in literature where GFP-tagging of a bacterial strain related to the Bacillus (and Paenibacillus) genus enhanced its growth-promoting abilities. The ability of P2b-2Rgfp to perform better than the wild-type strain was also confirmed in canola and tomato (Padda et al. 2016a). Benefits of inoculating this P. polymyxa strain and its GFP-tagged counterpart in a long-term trial were also evaluated, and the results were even better than the previous studies (which were of shorter duration) (Puri et al. 2016b; Padda et al. 2016b). In Fig. 6.2, a clear difference can be seen in canola plant growth when seeds were, either inoculated with P2b-2Rgfp or P2b-2R or not inoculated (controls) (reproduced from Padda et al. 2016b). The increased PGP efficiency of P2b-2R after GFP-tagging is still a mystery, although in an unpublished study, it has been determined that GFP-tagging of P2b-2R leads to overexpression of nifH, nifD, and nifK genes, which play a major role in N fixation activity of a bacterial strain.

Fig. 6.2
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Paenibacillus polymyxa strain P2b-2Rgfp inoculated seedling (left), P. polymyxa strain P2b-2R inoculated seedling (center), and uninoculated control seedling (right) of canola (Brassica napus L.) harvested 3 months after sowing and inoculation. Obvious differences in length, biomass, number of floral buds and pods, and plant health can be seen (Reproduced from Padda et al. 2016b)

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Plant hormones , like cytokinins, gibberellins, IAA, ethylene, etc., play a vital role in plant growth and development and in the response of plants to their environment (Glick 2012). PGPB can produce or modulate plant hormone levels, thereby affecting the plant’s hormonal balance and its response to abiotic and biotic stress. P. polymyxa strains isolated from different proximity to wheat roots were evaluated for production of indolic and phenolic compounds like IAA, indole-3-ethanol, indole-3-lactic acid, indole-3-carboxylic, and benzoic acid (Lebuhn et al. 1997). Authors concluded that the presence of P. polymyxa strains at different proximities to roots indicates distinct potentials to produce indolic and phenolic compounds. Phi et al. (2008) reported that a number of genes are involved in the regulation of IAA biosynthesis by P. polymyxa, and a change in IAA regulation directly affects the growth of inoculated plants. In another study, in vitro production of IAA, siderophore, and other phytohormones and phosphate solubilization by P. polymyxa strains SC09-21, SR04-02, and SR04-16 isolated from agricultural soil was reported (Xu and Kim 2014). Greenhouse pot trials revealed that inoculation with these P. polymyxa strains can enhance tomato shoot and root length, shoot and root fresh weight, shoot and root dry weight, and chlorophyll content. In vitro production of phytohormones like ammonia, cellulase, indole-3-acetic acid, protease, and siderophores and phosphate solubilization by P. polymyxa strain SC09-21 was confirmed in a subsequent study (Xu and Kim 2016). Xu and Kim (2016) also inoculated pepper with strain SC09-21 in a 2-week-long greenhouse trial and found that inoculated pepper plants were longer and had more fresh weight, biomass, and chlorophyll content, thus establishing that P. polymyxa strain SC09-21 could be an effective biofertilizer which can associate with a variety of agricultural crops. Although regarded as a micronutrient, iron is a major limiting factor in plant growth and development. Microbe-induced iron assimilation in a plant by P. polymyxa strain BFKC01 was recently reported (Zhou et al. 2016). Based on their findings, authors proposed a model: “productions of IAA by strain BFKC01 activates auxin-mediated signaling pathways and promotes lateral root formation in Arabidopsis plants, thus plants efficiently absorb iron from the rhizosphere. Strain BFKC01 also regulates plant iron uptake by integrating the mechanisms of both enhancement of iron deficiency responses and increased secretion of iron-mobilizing phenolic compounds.”

5.2 Biocontrol: Indirect Plant Growth Promotion

Biocontrol of plant pathogens is an effective and environmentally safe alternative to chemical pesticides. P. polymyxa can produce two types of peptide antibiotics, one type is only active against bacteria and the other is active against fungi, gram-positive bacteria, and actinomycetes (Beatty and Jensen 2002). This antagonistic potential is the base for effective applications of P. polymyxa strains against a wide set of fungal and bacterial plant pathogens. The possible mechanisms that enable P. polymyxa strains to control a variety of plant pathogens have been reviewed extensively (Raza et al. 2008). Timmusk and Wagner (1999) used a known PGPB, P. polymyxa strain B2, along with other P. polymyxa strains (B3 and B4) isolated by Lindberg and Granhall (1984) to assess their abiotic and biotic stress response when tested in Arabidopsis thaliana. Challenges by either the pathogenic bacteria, Erwinia carotovora (biotic stress), or induction of drought (abiotic stress) revealed that P. polymyxa inoculated plants were more resistant than control plants.

Authors also suggested that genes and/or gene classes associated with plant defenses against abiotic and biotic stress may be co-regulated. In another study, strains of P. polymyxa (B5 and B6), isolated from peanut rhizosphere, were reported to show in vitro antagonism against pathogenic fungus, Aspergillus niger (Haggag 2007; Haggag and Timmusk 2008). A. niger causes crown rot disease of peanut, which is the most important disease in Egypt and several other temperate countries (Haggag and AboSedera 2000). Strains B5 and B6 densely colonized the roots of peanut as visualized by scanning electron microscopy and suppressed the activity of A. niger in peanut, thereby decreasing the crown rot disease development. It was also reported that these strains increase the activity of plant defense enzymes including β-1,3-glucanase and chitinase, which might be the reason behind the suppression of pathogen activity (Haggag 2007). The results of two field trials indicated that these strains significantly reduce the incidence rate of crown rot disease in peanut; thus, they could be used as an effective biocontrol agent against A. niger at farm level (Haggag and Timmusk 2008).

The effectiveness of strains B5 and B6 along with strain B2 was tested against common oomycete plant pathogens, Phytophthora palmivora and Pythium aphanidermatum (Timmusk et al. 2009). These oomycete pathogens cause one of the most devastating groups of diseases. Almost all plants are susceptible to root rot disease caused by these pathogens, and the disease is difficult to control once the rot has begun. Strains B2, B5, and B6 showed clear antagonism against oomycete pathogens in the in vitro experiment (using agar plates and liquid medium). Using Arabidopsis thaliana as the model plant system antagonism against oomycete pathogens was also studied in a soil medium, and it was found that the survival rate of P. polymyxa inoculated plants was significantly higher than the control plants. P. polymyxa strain E681 isolated from barley in South Korea showed in vitro antagonism against Rhizoctonia solani, P. ultimum, and F. oxysporum f. sp. Cucumerinum (Ryu et al. 2005a). When E681 strain was inoculated into cucumber (Cucumis sativus cv. Shinpung), incidence of damping-off disease caused by the abovementioned pathogens was significantly reduced (Ryu et al. 2005a).

In another study, Ryu et al. (2006) reported that strain E681 shows in vitro antagonism against nine different pathogens, viz., P. debaryanum, R. solani, F. oxysporum, Botrytis cinerea, B. allii, Cladosporium fulvum, P. ultimum, P. capsici, and Aspergillus sp. Ryu et al. (2006) also established that E681 is an effective biocontrol against damping-off disease caused by pathogens in a month-long field trial conducted with sesame (Sesamum indicum L.). In a subsequent study, strain E681 was screened for fusaricidin compounds (Lee et al. 2013). Pre- and posttreatment of a 3-week-old red pepper (Capsicum annuum L.) with fusaricidin compound through soil drench or foliar spray application greatly reduced the disease severity caused by P. capsici. Similar effects were reported in tobacco (Nicotiana tabacum) and Arabidopsis thaliana (Lee et al. 2013).

Brown stem rot disease of soybean (Glycine max L.) caused by the soilborne fungus, Phialophora gregata, is one of the most disastrous soybean diseases in the USA and Japan (Bachman and Nickell 2000). Biocontrol effectiveness of P. polymyxa strain BRF-1 isolated from the rhizosphere of diseased soybean seedlings was tested (Zhou et al. 2008). The severity of brown rot disease in BRF-1 inoculated and control soybean plants was significantly decreased after 62 days of fungal inoculation, thus establishing its biocontrol efficacy against the P. gregata fungus. In vitro antifungal activity by BRF-1 against a range of fungal pathogens was also reported by Chen et al. (2010). Authors also isolated and identified the antifungal peptide of this strain, which is active against a range of pathogens. Raza et al. (2009) isolated P. polymyxa strain SQR-21 from the rhizosphere of healthy watermelon plants growing in a heavily wilt-diseased field and evaluated its biocontrol potential against F. Oxysporum (the causative agent of Fusarium wilt disease of watermelon) in a greenhouse experiment.

Strain SQR-21 combined with organic fertilizer significantly decreased the disease incidence (by 70%) and increased the plant biomass by ~113%. Subsequently, Ling et al. (2011) attempted to understand the plant-microbe communications that take place when watermelon plants are inoculated with either SQR-21 strain or pathogenic fungi F. oxysporum f. sp. niveum. When treated with SQR-21 watermelon plants produced root exudates that significantly reduced the conidial germination of F. oxysporum. Another strain of P. polymyxa (WR-2) showed a similar antagonistic effect against F. oxysporum f. sp. niveum (Raza et al. 2015b). Strain WR-2 inhibited the growth fungal pathogen by ~40% in three different media (agar, sterilized soil, and natural soil), and this inhibitory effect was increased to about 60% when organic fertilizer was added. Raza et al. (2015b) also reported that strain WR-2 produces seven different volatile organic compounds, viz., benzothiazole, benzaldehyde, undecanal, dodecanal, hexadecanal, 2-tridecanone, and phenol that inhibit the growth of F. oxysporum. In another study, P. polymyxa strain CF05 showed in vitro antagonism against F. oxysporum f. sp. lycopersici (the causative agent of Fusarium wilt of tomato).

Greenhouse experiment confirmed this finding, where CF05 suppressed the Fusarium wilt disease by ~78% in tomato. It was also reported that strain CF05 induces systemic resistance in the tomato plant, thereby stimulating the release of plant defense enzymes and protecting the plant from the pathogen.

6 Field Studies and Commercial Availability of Paenibacillus polymyxa Strains

The PGPB inoculated crops signify a minor segment of current global agricultural practice, but lately the interest in the infield usage of PGPB that promotes plant growth and yield has increased. A number of these bacteria are now being used commercially as aides to promote sustainable agriculture. Numerous studies underlining the plant growth-promoting effects of various P. polymyxa strains under field conditions have been reported. Ryu et al. (2006) conducted experiments under field conditions to evaluate the antagonistic effect of P. polymyxa strain E681 in two types of soilborne diseases: preemergence and postemergence damping-off in sesame seeds. Seed pelleting technique was used in this study to improve the ability of strain E681 as a biocontrol agent. Experiments were conducted at GSNU Research Farm, Daegok, Jinju, where sesame had been cultivated for 2 successive years with serious yield loss. Seed pelleting with strain E681 enhanced emergence rate by ~92%, whereas emergence rate of untreated sesame seeds was less than 30%. Combined treatment of pelleting and strain E681 resulted in a greater percentage of healthy stand (92%) than pelleting alone (40%) or non-pelleted seeds treated with E681 (24%) when evaluated 2 months after sowing. These results suggest that pelleting combined with P. polymyxa strain E681 can be used to control damping-off disease caused by complex organisms in the field. The effect of P. polymyxa strains B5 and B6 on pod yield and control of crown rot disease in peanut caused by A. niger was investigated in two successive field experiments (Haggag 2007; Haggag and Timmusk 2008).

Peanut plants treated with P. polymyxa strains displayed decreased incidence of crown rot disease triggered by A. niger. Plant growth and yield of seeds treated with strain B5 were found to be significantly higher in comparison to seeds treated with strain B6 and untreated plants. Biocontrol activity of P. polymyxa strain G-14 against bacterial spot diseases of muskmelon caused by two pathogens, Pseudomonas syringae pv. Lachrymans and Acidovorax avenae subsp. citrulli, was examined under field conditions (Shi et al. 2012). G-14 strain significantly inhibited the development of pathogens and suppressed the incidence of bacterial spot diseases. Inoculation with an N-fixingP. polymyxa strain increased sugar beet (Beta vulgaris cv. Loretta) root yields by 12% and barley seed yields by 15% when evaluated by in-field studies conducted at two different sites in Turkey (Çakmakçi et al. 1999). These results were also confirmed by a subsequent field study (Çakmakçi et al. 2006).

A yearlong field investigation (for three seasons—autumn, spring, and summer) was conducted to determine the effects of P. polymyxa strain EBL-06 on the growth of tea (Camellia sinensis) plantations (4 years old) in semitropical uplands, Hunan, China (Xu et al. 2014). Inoculation with EBL-06 increased tea plant yield (~17%) and tea quality by enhancing the level of green tea extracts by about 6% and tea polyphenols by ~10%. Thus, it was concluded that this strain could be a successful biofertilizer for tea plants that might be used for organic tea production in the future to enhance tea yield and quality. Field studies play an important role in determining the effects of a particular bacterial strain in actual conditions and open the doors for their use as commercial biological inoculants . Due to numerous field studies that have reported rigorous testing by scientists, many P. polymyxa strains are now available commercially in several countries.

7 Concluding Remark and Future Prospective

Since the first isolation and characterization more than a century ago, significant advances have been made in understanding how P. polymyxa affects plant growth. P. polymyxa is now known to fix nitrogen, secrete phytohormones, and produce antibiotic and antifungal compounds. The most common antifungal compound produced by P. polymyxa is fusaricidin which has been reported to suppress many strains of Fusaricidin oxysporum in a variety of plants species both in vitro and in vivo. It is believed that microorganisms with two or more PGP traits which are able to colonize and provide benefits to a wide range of crops may be effectively used for commercial and large-scale agriculture. It is an effective biocontrol agent against a wide range of plant pathogens like Aspergillus sp., B. allii, B. cinerea, C. fulvum, F. oxysporum, P. capsici, P. debaryanum, P. ultimum, and R. solani. Complete genome sequencing indicated that P. polymyxa E681 could produce antibiotic polymyxin and antifungal fusaricidin compounds, revealing the genetic evidence behind its ability to antagonize plant pathogens. Another example is P. polymyxa P2b-2R, which was reported to fix significant amounts of N directly from the atmosphere in crop species like corn, canola, and tomato. Possibly through N fixation and some other linked mechanisms, strain P2b-2R also promoted plant growth (length and biomass) and crop yield. These PGP properties together with its endospore-forming potential enable it to thrive in a wide range of environmental conditions, making it an important and promising biofertilizer and biocontrol agent for current and future sustainable agriculture. Through continuing research, agricultural scientists are making important inroads to understand the biology and ecology of P. polymyxa strains that could ultimately result in more commercially viable and environmentally friendly bio-inoculants for use in agriculture.