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: Plant Growth-Promoting Endophytic Bacteria and Their Potential Benefits in Asian Countries

NITTAYA PITIWITTAYAKUL1 and SOMBOON TANASUPAWAT2

'Department of Agricultural Technology and Environment,

Faculty of Sciences and Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima Campus,

Nakhon Ratchasima 30000, Thailand

2Department of Biochemistry and Microbiology,

Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand

INTRODUCTION

Endophytic bacteria are those bacteria that inhabit inside plant tissues and generally do not cause apparent disease or deleterious effect to their host (Wilson, 1995; Ryan et al., 2008). They are able to colonize the spaces that exist between adjacent cells, and they have been found in all components of plant as well as seeds (Posada and Vega, 2005). Generally, endophytic plant growth-promoting bacteria (PGPB) can stimulate plant growth by direct and indirect mechanisms. Direct beneficial mechanisms are either assisting in resource acquisition (nitrogen fixation and enhancing mineral uptake) or production of phytohonnones. Indirect mechanisms involve the ability of PGPB to suppress the growth of various plant pathogens and produce biocontrol agents (Glick, 2012; Ahemad and Kibret, 2014). Nowadays, sustainable agriculture is an alternative way of maintaining high productivity, together with maintaining ecosystems and biodiversity. PGPB are used as the potential eco-friendly and sustainable aspect for plant growth promotion. PGPB may replace or reduce the use of chemical fertilizers and capable of improving the growth and productivity of many plant species, increase agronomic efficiency, and ecological significance. Asian countries have rich plant biodiversity, and there are a veiy large number of plant species. Endophytic PGPB are ubiquitous as they can be isolated in all plant species, residing in a latent state or actively colonizing plant tissues. Moreover, each individual plant can be inhabited with one or more endophytes. The relative between only a few of these plants and their endophytic biology has ever been completely studied. As a result, the opportunity to discover novel and beneficial endophytic microorganisms among the diversity of plants in different ecosystems is considerable. In this chapter, we described the isolation, identification, diversity, plant growth-promoting activities, and enzyme, including other products of endophytic PGPB in Asian countries.

ISOLATION AND IDENTIFICATION

The isolation of endophyte bacterial species directly involves the surface sterilization by treating the plant material with a strong oxidant or general disinfectant for a period, followed by a sterile rinse. The most generally used as a surface sterilant is household bleach (NaOCl) diluted in water to a concentration of 2-10% or similar agents (Miche and Balandreau, 2001; Zhang et al., 2006). Furthermore, ethanol (EtOH) (70-95%) can be used as a wetting agent combining with surface sterilization for improving the efficacy of surface sterilization. Lodewyckx et al. (2002) reviewed the procedures for isolation and characterization of endophyte from diverse plant species and concluded that no protocol of surface disinfection results in the complete killing of surface bacteria on 100% of samples without penetrating interior tissues and thereby killing internal colonists. Therefore, comparisons between different studies should be carefully evaluated, taking into account the different surface sterilization methods and conditions used. Moreover, total populations of bacteria accompanying within host plants may depend on the growth media used for isolation. There have been relatively few studies that have analyzed the influence of the culture medium type on the diversity of bacterial isolates. According to Okubo et al. (2009), nodulation-dependent communities of culturable bacterial endophytes from stems of field-grown soybeans were analyzed. In this study, three isolation media, R2A, NA, and PDA, were used to examine the impact of medium type on the diversity of bacterial isolates. The results revealed that the R2A medium exhibited the highest diversity indexes, whereas the PDA medium showed the lowest. Some methods and media for endophytic bacteria isolation that have been reported are shown in Table 3.1. However, the most information on endophytic bacterial diversity that was obtained by using culture-dependent approaches has been so far due to the unknown condition for growth requirements of many bacteria and the presence of cells that are in a viable but noil- cultivable state (Shen et al., 2010).

Molecular approaches based on the amplification of the 16S rDNA have been performed for bacterial community analysis in host plants to overcome the limitations of classic isolation procedures that are a culture- based method for isolation of bacteria (culture-dependent approaches). Furthermore, molecular approaches based on 16S rDNA such as amplified rDNA restriction analysis (ARDRA), denaturing gradient gel electrophoresis (DGGE), and restriction fragment length polymorphism (RFLP) can be applied to study population composition including the population dynamics of endophytic bacteria. Additionally, the specific amplification of housekeeping genes such as atpD and glnll, or genes encoding important traits of endophytic bacteria, for example the nif genes required for nitrogen fixation; the nod gene required for synthesis and secretion Nod factors relating with the root nodule formation of leguminous plants, can be applied to determine the ability of the endophytic population to participate in important processes within host plant. The diversities of endophytic bacteria in Caragana microphylla grown in the desert, rice (Oryza saliva L.) roots, peanut roots, wild alpine-subnival plant species and narrow leaf cattail (Typha angustifolia L.) have been estimated by using 16S rRNA gene sequence analysis together with ARDRA (Sun et al., 2008; Li et al., 2011; Wang et al., 2013; Dai et al., 2014). RFLP based on 16S-23S internal transcribed spacer (ITS) region was used for characterized the bacterial strains that isolated from nodules of eighteen Vida species in China (Lei et al., 2008). Chen et al. (2015) characterized the endophytic bacteria isolated from Astragalus species as traditional Chinese medicine source by the PCR-RFLP of 16S rRNA gene and symbiotic genes as well as the phylogenetic analysis while Kang et al. (2016) characterized the communities of bacteria associated with surface-sterilized pepper plants by using PCR-DGGE (culture-independent) together with culture- dependent (plating). Moreover, nested polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) with novel specific 16S

Endophytic

Species

Host Plant Species

Isolation Method

Identification Method

References

1Rhizobium lemnae

lRhizobium

paknamense

Duckweed (.Lemna aequinoctialis)

Surface-sterilized plants with 10% sodium hypochlorite (NaOCl) and a few drops of Tween 20, were rinsed with sterilized distilled water (DW) five times, and then ground in sterilized DW. The suspension was placed on 1/10 strength tryptic soy agar (TSA) and incubated at 30°C for 7 days.

  • • 16S l'RNA gene sequence
  • • Housekeeping recA and atpD gene
  • node and nifH gene

Kittiwongwattaua etal., 2013; Kittiwongwattaua etal., 2014

1Rhizobium smilacinae

Traditional Chinese medicinal plant (Snulacma japonica)

Tlie surface-sterilized leaf tissue was cut into small fragments and macerated using a sterile pestle and mortal' in sterile DW. The diluted macerated samples in sterile DW were spread onto R2A agar supplemented with 50 pg ml'1 of imazalil and incubated at 25°C for 2 weeks.

  • • 16S l'RNA gene sequence
  • • Housekeeping dnaK. glnll, and recA gene

Zhang et al., 2014a

1Bradyrhizobium sp.

Rice (Oiyza saliva)

Rice roots and paddy soils were collected and immediately transferred to the laboratory in polyethylene boxes at 4°C. The roots and stems were chemically sterilized in 3% NaOCl (5 min) and soaking in 70% EtOH (5 min) and were thoroughly rinsed with sterilized DW (at least 5 times), cut into 4- to 5-cm-loug sections and placed on plate count agar (PCA). The 200 pi of water from the final rinse was spread PCA, as a control to check superficial contamination for each plant.

  • • 16S l'RNA gene sequence
  • • Housekeeping recA and atpD gene
  • gin В gene
  • • BOX-AIR nod A, nodB, node, pufM, bchL

Piromyou et al., 2015a

1Novosph ingobium sediminicola and 1Ochrobactiwn intern edium

Sugarcane

Tlie samples were surface sterilized by sequential washing in 50% EtOH for 1 min, 2% NaOCl for 3 miu. and 50% ethanol for 30 sec and then rinsed twice with sterile DW. Each sample was ground in

• 16S l'RNA gene sequence

Ddbereiuer et al., 1972; Muangthong etal., 2015

Endophytic

Species

Host Plant Species

Isolation Method

Identification Method

References

a sterile mortar, suspended in an N-free broth (0.1 g K,HPC>4, 0.4 g KH,P04, 0.2 g MgS04, 0.1 g NaCl, 0.02 g CaCl,, 0.01 g FeClr 0.002 g NaMoO,. and 10 g glucose in 1000 ml distilled water) at 30°C for 24 h. Ten-fold serial dilutions of the suspensions (10_1 to 10“J) were spread on Nitrogen Free Agar (NFA) in triplicate and incubated for 72 h.

  • 1Rhizobium,
  • *Entewbacter, Stenotrophomonas

Pteris vittata

The sterilized roots (0.2 g) were ground in an autoclaved mortar pestle containing 10 ml sterile DW. Serial dilutions of samples were spread on sucrose- minimal salts low-phosphate (SLP) medium (sucrose 1%, (NH4),S04 0.1%, K,HP04 0.05%, MgS04 0.05%, NaCl 0.01%, yeast extract 0.05%, CaCOj 0.05%, pH 7.2) along with 10 mg L"1 as sodium arsenate and incubated at 28°C for 72 h.

• 16S lRNAgene sequence

Tiwari et al., 2016

-Enterobacter

cloacae

Other:

Paenibacillus

xylanexedens,

Bacillus

Date palm

(.Phoenix dactylifera L.)

Seedling roots growing in each soil sample were separated from the plants, pooled, surface sterilized. and the endophytic bacteria were eluted from the tissues using Ringer’s solution as described. Tire eluted Ringer’s solutions were diluted, and 10 pi aliquots were spread onto King’s В agar (Kg) (20 g proteose peptone 3, 10 ml glycerol, 1.5 g K,HP04, 1.5 g MgS04 7H,0 and 15 g agar- in 1 1 water). Luria agar (LA) and tryptic soy agar- (TSA) rich media.

• 16S rRNAgene sequence analysis

Rashid et al., 2012; Yaish et al., 2015

Endophytic

Species

Host Plant Species

Isolation Method

Identification Method

References

2Klebsiella four species Others:

Microbacterium, two species of Paenibacillus, three Bacillus species

Korean rice cultivars

Plant tissue samples were surface sterilized with 70% EtOH (1 min) and shaken in 1.2% (w/v) NaCIO solution (15 min). Samples that washed three times with sterile DW with shaking (15 min each) were gr ound with sterilized mortal' and pestle and inoculated on nitrogen-free semi-solid agar media. After incubation at 30°C for 2 days, the inoculants were transferred to fresh nitrogen-free media and then incubated at 30°C for 2 days.

  • nifH genes
  • • 16S l'RNAgeue sequence analysis

Ji et al., 2014

Kigrobacterium tumefaciens 1Sphingopyxis chilensis -Enhydrobacter aerosaccus

Pepper plants

(Capsicum annuum L. cv. Nokkwang)

The root, stem, and leaf par ts were aseptically cut into pieces. Surface disinfection was done by stepwise washing in 70% EtOH (1 min), 1% NaOCl (1 min), and 70% EtOH (30 sec), followed by three rinses with sterile DW for 1 min each. The other disinfected pieces (0.5 g) were macerated by grinding in sterile mortars. Ground plant material was transferred into a sterile culture tube with 3 mL 120 mM sodium phosphate buffer (19.9 g Na,HP04 H,0, 1.27 g NaH,P04-2H,0, H,0 11, pH 8.0) and agitated at 120 rpm for 2 h. The suspension was serially diluted 10-fold to 10~5 and spread onto 0.3% TSAwith 100 pg mL'1 cycloheximide and incubated at 2S°C for 7 days.

  • • 16S l'RNAgene sequence analysis
  • • DGGE of PCR-amplified 16S l'DNAs

Kang et al., 2016

-Pseudomonas sp., -Enterobacter sp. Others:

Bacillus sp.

Aloe vera

The leaves, stems, and roots were detached with a sterile knife, washed with sterile DW, and left to drain (10-15 min). Pieces of tissue (2-3 cm) were surface sterilized (Azevedo et al., 2000) with

• 16S l'RNAgeue sequence analysis

Azevedo et al., 2000; Akinsanya et al., 2015

Endophytic

Species

Host Plant Species

Isolation Method

Identification Method

References

modifications, immersed in 90% EtOH (5min),

3% NaOCl solution (2 min), and into 75% EtOH (3 min). The samples were raised three tunes in sterile DW and drained in a laminar flow hood. They were cut longitudinally with a sterile scalpel and laid, with the exposed inner surface facing downwards, on NA plates and incubated at 30°C for 36-48 h to obtain the cultures.

  • 1Novosphingobium sp.
  • 1Asticcacaulis sp.
  • 1Rhodobacter sp.
  • 1Rhizobiian sp. ^Burkholdena vietnamiensis lB. tropica ‘B. kururiensis lB. ambifavia lHeibaspinl!tmi sp.

Nipa Palm (Nypa fmticans)

Root samples were first washed with 70% EtOH (30 sec) and then sur face-sterilized with diluted commercial bleach containing 0.5% NaOCl (5 min), followed by rinsing three times with sterilized water for 10 min each tune, hi view of the relatively high concentration of sucrose in the sap of the Nipa palm, surface-sterilized root cut vertically in 10 mm long was inoculated into a gellan gum soft gel medium that contained 0.2% w/v sucrose as sole carbon source.

• 16S rRNA gene sequence analysis

Tang et al.. 2010

lAlphaproteobacteria;2 Go mm aproteobactena; lBetapmteobacteria.

rDNA-targeted primers were successfully used to characterize the endophytic diversity in Dendrobium officinale from three different sources in China. Consequently, the good alternative for investigation of communities and roles of endophytes was the nested PCR-DGGE method based on the novel primers (Yu et al., 2013).

 
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