Plant Growth-Promoting Microbiome Network


Department of Molecular Biology and Genetics,

Gebze Technical University, Kocaeli-41400, Turkey


Bacteria and plants have a huge relevance stemming from symbiotic relationships that continue for a long time and have an influence on their evolutionary process. To date, endophytes have been pervasively found to exist almost in eveiy plant tissue (Guerin et al., 1898; Zhang, 2006; Staniek et al., 2008). Bacterial endophytes not only appear in plant micro- biome just as rhizospheric and epiphytic microbiota but also internal tissues (endophytic) (Hardoim et al., 2015). Regardless of the number of bacteria in a special soil sample, bacteria may affect plants in three ways; beneficial, harmful, or neutral (when viewed from the plant standpoint).

The bacteria that can promote plant growth, i.e., plant growth-promoting rhizobacteria (PGPR), include those that free-living and form certain symbiotic relationships with plants (e.g., Rhizobia spp. and Frankia spp.) while bacterial endophytes can colonize some or a part of the internal tissues of the plant. Despite the differences between these bacteria, they all use the same mechanisms. Plant growth-promoting bacteria (PGPB) can generally promote plant growth by either facilitating direct source or modulating plant gr owth regulator levels, or indir ectly by acting as biocontrol bacteria via reducing the inhibitory effects of various pathogenic agents on plant growth and development (Glick et al., 1995).

Since the beginning of 21st century, the probability to investigate possible advantages and applications of PGPB's or endophytic microbes in the fields of medicine, pharmacy, and agriculture has become practicable due to intensive research and exploration of plant-microbe interaction systems as they produce a diverse range of biologically active secondary metabolites (Strobel et al., 2004; Gunatilaka, 2006; Zhang et al., 2006; Staniek et al., 2008) that supply host plant tolerance against various biotic and abiotic (heat, salt, disease, and drought) stresses (Stone et al., 2000; Redman et al., 2002; Rodriguez and Redman, 2008). However, up to now, commercially ‘sustained production’ of these pharmaceutically valuable metabolites could not be achieved (Kusari et al.,

2014). Thus, the interaction between endophyte and host organism needs more attention. With these aims, endophytes starting from its definition, impact, and localization in its host plant together with its potential, will be reviewed in this chapter.


Endophytes can be defined as “a group of microorganism that infects the internal tissues of the plant regardless of any important symptom of infection and/or any visible sign of disease and lives in mutualistic relationships with plants at least in a part of their life cycle” (Bacon and White, 2000). The term of “endophyte” was introduced to the literature initially by de Baiy (1866), and since then this term is used to describe a large group of organisms including bacteria, fungi, and insects (Feller, 1995; Mailer et al., 1999; Kobayashi and Palumbo, 2000; Stone et al., 2000).

Plant-associated bacteria include endophytic, rhizospheric, and phyl- lospheric bacteria. Since these endophytic bacteria can proliferate inside the plant tissue, they interact closely with their hosts; they compete less for nutrients and are protected more than the negative changes in the environment when compared to the bacteria in the rhizosphere or in the phyllosphere (Reinhold-Hurek and Hurek, 1998; Beattie, 2007). Phyl- losphere can be called the external regions of plant parts such as stalks, flowers, fruits, and leaves. Rhizospheric bacteria live directly vicinity of the roots and, therefore, root exudates are thought to be a major influence on the diversity of microorganisms in the rhizosphere (Kloepper et al., 1991).

Endophytic colonization of bacteria could be observed in whole plant tissues or organs (Turner et al., 2013), stalling from the root to meristematic cells (Pirttila et al., 2000), including pollen (Madmony et al., 2005). Plants containing a large number of microbial species are complex microecosy- tems (Mclnroy and Kloepper, 1995), some of which may predominantly be found (Van Peer et al., 1990). Differently, in some cases, only one endophyte could be found in its host plant (Seker et al., 2017), which may be evaluated as species-specific. Sometimes endophytic species cannot be isolated because they are present in plant tissues at veiy low concentrations or unculturable.

Different methods have been used by many investigators to isolate bacterial endophytes (Hallmann et al., 1997). Endophytes are isolated from the initial surface sterilization after culturing from a ground tissue extract or by direct culturing of plant tissues or by using appropriate media for bacteria (Rai et al., 2007). Traditionally, to define endophytic bacteria, the morphological characteristics of them have been examined, and biochemical tests have been carried out. Reinhold-Hurek co-workers (1998) reported the criteria for identifying “true” endophytic bacteria, and accordingly, it should be isolated from surface-sterilized tissues together with its microscopic evidence that shows “tagged” bacteria in the inner part of the plant. When the second criterion cannot be fulfilled, it is proposed to use the term ‘putative endophyte.’ It is also possible to identity true endophytes by their capability to re-infect seedlings that have been surface sterilized.

Ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequence analysis is generally utilized for the determination of microorganisms. rDNA ITS offers considerable evidence for revealing phylogenetic associations as genera or species (Youngbae et al., 1997). Along with recent advances in biotechnology, more molecular studies have been carried out with endophytes, by using DNA markers, DNA cloning, and expression studies. The denaturing gradient gel electrophoresis (DGGE) profiles of the 16S rRNA gene sequencing were utilized to settle on various endophytic bacteria that could not be cultured compared to the band profiles obtained from culturable endophytes from the citrus plant (Araujo et al., 2002). Bacterial automatic ribosomal intergenic spacer analysis (B-ARISA) and pyrosequencing were also used to study bacterial endophytic populations of potato (Manter et al., 2010).

Metagenomic studies could also be utilized to find microorganisms that could not be easily detected from diverse environments. For instance, a 1-aminocyclopropane-l-carboxylate deaminase gene (aCtrlS) operon was determined from an uncultured endophytic microorganism colonizing potato by this approach. In addition, metagenomic analysis can be complementary to PCR-based methods and supply information on whole operon profiles (Nikolic et al., 2011). The complete genome of endophytic plant growth-promoting gainma-proteobactenum Enterobacter sp. 638 isolated from the stem of poplar, was sequenced (Taghavi et al., 2010). According to sequencing result, it has 4,518,712 bp chromosome and a 157749 bp plasmid (pENT638-l) that contain genes expressed for adaptation (i.e., colonization/establishment inside the plant, root adhesion, biotic stress protection, and enhanced plant growth). The techniques such as genome sequencing, comparative genomics, microarray, next-generation gene sequencing, metagenomics, and metatranscriptomics are used to find out the host-endophyte relationship (Kaul et al., 2016).

Thanks to molecular biology techniques, more than 200 bacterial genera from 16 phyla including Actinobacteria, Aquificae, Acidobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Deinococcus-Thermus, Cholo- robi, Fusobacteria, Verrucomicrobiae, Fmnicutes, Nitrospira, Proteobac- teria, Gemmatimonadetes, Planctomycetes, and Spirochaetes (Berg and Halhnann, 2006; Sessitsch et al., 2012) have been indicated as endophytes since their reliable initial isolation (Samish et al., 1959; Mundt and Hinkle,

1976). Among these phyla, Actinobacteria, Fmnicutes, and Proteobacteria contain the members of Enterobacter, Herbaspirillum, Azoarcus, Bacillus, Burkholderia, Pseudomonas, Streptomyces, Gluconobacter, Stenotroph- omonas, and Serratia (Suzuki et al., 2005; Krause et al., 2006; Bertalan et al., 2009; Ryan et al., 2009; Taghavi et al., 2009, 2010; Deng et al., 2011; Pedrosa et al., 2011; Weilhaiter et al., 2011), were notified as predominant. As species of these genera omnipresent in the soil/rhizosphere, they could be nominated as the main source of endophytic colonizers (Halhnann and Berg, 2006). Phyllosphere, the anthosphere, and seeds could also represent the other possible endophyte sources (Compant et al., 2010). Moreover, endophytic bacteria can also be used as a vector for the transfer of interested genes into plants. In genetic studies, plasmids of endophytic bacteria can be used as transmission vectors instead of the whole organism (Berde et al., 2010).

Endophytes can accelerate the plant growth by (i) supplying the necessary nutrients, e.g., nitrogen fixation, phosphate solubility or iron chelation, (ii) preventing pathogenic infections through antifungal or antibacterial agents, (iii) outcompeting pathogens for nutrients by sidero- phore production, or (iv) establishing the plant’s systemic resistance (SR) through four interrelated mechanisms: phytostimulation, biofertilization, bioremediation, and biocontrol (Bloemberg and Lugtenberg, 2001) (Figure 2.1).

Schematic diagram of the common mechanisms in plant-bacterial endophyte interactions

FIGURE 2.1 Schematic diagram of the common mechanisms in plant-bacterial endophyte interactions.


Endophytes are mostly intercellular in terms of location in host cells and tissues. It has been found that endophytic bacteria can colonize different plant parts, including roots, tubers, stems, leaves, ovules, and seeds, and also immature flower buds (Mishagi and Doimdelinger, 1990; Benhizia et al., 2004). However, most plants have a higher number of endophytes in their roots than other tissue parts (Rosenblueth and Martinez-Romero,

2004). They can become intracellular and enter the cytoplasm of the host cell or placed on the periplasmic surface (Thomas and Sekliar, 2014; White et al., 2014a). Passive and active several colonization routes have been identified according to the strain (Hallmann, 2001). The progression of these pathways allows the bacteria to migrate from the rhizoplane to the cortical cell layer (Mercado-Bianco et al., 2014). Xylem vascular system is the principal transport way for systemic colonization of inner plant parts for endophytes that can pass the endodennis (James et al., 2002). However, others could also locate in intercellular areas. It has been demonstrated that bacteria are able to localize xylem vessels, and dimensions of the perforations plates between the xylem elements are enough to provide bacterial passage (James et al., 2002) even the bacterial progression was so slow in the xylem. In the xylem tissues and substomal chambers of grape, a Burkholderia sp. strain was identified (Compant et al., 2005). Moreover, detection of bacteria inside reproductive organs (i.e., flowers), fruits, and seeds of grapevines (Compant et al., 2010) and pumpkin (Fttmkranz et al., 2012), together with the pollen of pine (Madmony et al., 2005) was also possible. Colonization by bacterial endophytes has been declared for different plants, including Angiospermae, Bryophytes, Gymnospermae, and Pteridophytes (Compant et al., 2010).

When endophytic bacterial are inside the plant, according to the response of the plant, competent endophytes may induce the necessary stimulation to start the endophytic life cycle and spreading to other tissues of the root cortex and beyond. Endoglucanases and endopolygalacturonidases play a role in this process. Competent endophytes often quickly multiply inside the plant as soon as they reach high cell numbers (Barraquio et al., 1997). Endophytic population sizes correlate positively with the developmental stage of the plant and progressively increase and reach maximal stages of the seedling stage (Van Overbeek et al., 2008). However, it is remained unknown whether endophytes need to reach a specific organ or tissue for optimal performance of their functions.

As previously mentioned, the isolation of bacteria from surface- sterilized plant tissues does not prove that it is a “true endophyte,” so it is necessary to demonstrate that the bacteria are in plants by means of various labeling techniques such as immunological detection of bacteria, fluorescence tags, confocal laser scanning microscopy and immuno-gold labeling in combination with transmission electron microscopy, specific oligonucleotide probes (Chelius and Triplett, 2000; Hartmann et al., 2000) or bacterial marking with green fluorescent protein (GFP) (Venna et al., 2004). It has been observed that bacterial cells initially colonize the rhizosphere via microscopic visualization that permits the analysis of strains labeled by (i) gfp- or gusA (Charkowski et al., 2002), (ii) immu- nomarkers, or (iii) fluorescence in situ hybridization (FISH) (Gamalero et al., 2003; Loy et al., 2007). Transformation of microorganisms with plasmids bearing GFP or insertion of GFP into their genomes ensures a helpful experimental tool for the detennination of the behavior of specific microbes when interacting with host tissues and cells (Chalfie et al., 1994; Valdivia et al., 1996). GFP is nontoxic; therefore, it does not interfere with cell function, and it provides a unique and visual phenotype for studying the behaviors of microorganisms inside the plant tissues. For example, after the introduction of GFP-tagged Sinorhizobium meliloti into rice, it was reported that its growth under sterile conditions and the endophyte population densities within plants were very high (Chi et al., 2005). After inoculation with poplar trees, Germaine et al. (2004) found that various GFP-labeled endophytes were determined in the inner tissues of the poplar tree. Pantoea agglomerans 33.1, tagged with the GFP gene, was observed to have colonized Eucalyptus roots, primarily in intercellular spaces, stems, and xylem vessel (Ferreira et al., 2008).

The presence of bacterial endosymbionts was demonstrated using electron microscopy in the cells of in vitro peach palm shoots (De Almeida et al., 2009). In another study, the presence of non-culturable endosymbiont bacteria located in plant tissues were revealed by molecular biologic analysis such as DGGE and 16S rDNA sequencing (Abreu-Terazi et al., 2010). That showed a high similarity of Moraxella sp., Brevibacillus sp., and cyanobacterium. Following surface sterilization, Abreu-Terazi et al. (2010) isolated DNA from axenic cultures of pineapple (five-year-old) and identified bacteria belonging to families of Actinobacteria, Alphap- roteobacteria, and Betaproteobacteria by similar techniques. In the same study, it was discovered that different bacterial communities could be found in various plant organs. Moreover, microbiological analysis, visualization techniques, and sequencing methods revealed out the evidence of vast microbiome including known, unknown bacteria and fungi within regenerated Atriplex sp. leaves and roots (Lucero et al., 2011). In meri- stematic explants of Aglaonema cultivars, bacteria that belong to 13 different genera were identified by Fang and Hsu (2012), and 30% of these isolates were Pseudomonas aeruginosa. In the contaminant plant cultures detected from many laboratories in Poland, 108 isolates were observed from many genera, most commonly Bacillus, Metliylobacte- rium, and Pseudomonas (Kaluzna et al., 2013). All of the isolates in this study, except Staphylococcus, were located inside plant tissues without showing any detrimental effect.

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