REVIEW OF LITERATURE

CHAPTER II

REVIEW OF LITERATURE

Plant–microbe interactions that promote plant development and

plant health have been the subject of considerable interest. Plants

constitute vast and diverse niches for endophytic organisms. Among the

microorganisms, endophytic bacteria occupy internal tissues of plants

without causing damage to their hosts. An understanding of the

mechanisms enabling these microorganisms to interact with plants will

be essential to fully achieve the biotechnological potential of efficient

plant–bacterial partnerships for a range of applications. Hence a review

of the work done on endophytic organisms is presented here under.

2.1. History and definition of endophytes

The term endophyte (Gr. endon, within; phyton, plant) was first

coined by De Bary (De Bary, 1866) and an endophyte is a bacterial or

fungal microorganism, which spends the whole or part of its life cycle

colonizing inter- and/or intra-cellularly inside the healthy tissues of the

host plant, typically causing no apparent symptoms of disease (Wilson,

1995). The presence of endophytes was reported by Vogl in 1898 who

revealed a mycelium residing in the grass seed Lolium temulentum. In

1904 in Germany Freeman identified an endophytic fungus, in Persian

darnel (annual grass).

Bacterial endophytes have been known for >100 years. The

presence of bacteria resident within the tissues of healthy plants was

first reported as early as 1926 (Hallman et al., 1997). In 1926, Perotti

recognized endophytic growth as a particular stage in the life of bacteria,

described as an advanced stage of infection and as having a close

relationship with mutualistic symbiosis. Perotti (1926) was the first to

describe the occurrence of non pathogenic flora in root tissues and

Henning and Villforth (1940) reported the presence of bacteria in the

leaves, stems and roots of apparently healthy plants. Since then,

endophytes have been defined as microorganisms that could be isolated

from surface-sterilized plant organs. Since 1940S there have been

numerous reports on endophytic bacteria in various plant tissues

(Hallmann, et al., 1997). In the 1980s endophytic bacteria having

nitrogen fixing ability were found in graminaceous plants (Reinhold-

Hurek and Hurek, 1998b).

These endophytic relationships may have begun to evolve from the

time that higher plants first appeared on Earth hundreds of millions

years ago. Evidence of plant associated microbes has been discovered in

the fossilized tissues of stems and leaves (Taylor and Taylor, 2000). As a

result of these long-held associations, it is possible that some of these

endophytic microbes may have devised genetic systems allowing for the

transfer of information between themselves and the higher plant and vice

versa (Stierle, et al., 1993).

The term endophyte refers to interior colonization of plants by

bacterial or fungal microorganisms. Endophytes have been defined in

several ways and the definitions have been modified as the research in

this field advanced. Tervet and Hollis (1948) defined endophytes as

microorganisms that are able to live inside plants without causing

disease symptoms. ‘Endophytic bacteria’ are the population of bacteria

that reside within the living organism without doing substantive harm or

gaining benefit other than securing residency (Kado, 1992). Endophytic

bacteria or fungi colonize the host tissue internally, sometimes in high

numbers, without damaging the host or eliciting symptoms of plant

disease according to a widely used definition by Quispel (1992).

Microorganisms living within plant tissues for all or part of their life cycle

without causing any visible symptoms of their presence are defined as

endophytes (Wilson, 1993). Bacon and White (2000) defined endophytes

as ‘microbes that colonize living, internal tissues of plants without

causing any immediate, overt negative effects’. According to Schulz &

Boyle (2006) endophytic bacteria can be defined as those bacteria that

colonize the internal tissue of the plants showing no external sign of

infection or negative effect on their host.

Among the definitions given to endophytic bacteria the following by

Hallmann et al., (1997) seems to be the most adequate. Hallmann et al.

(1997) defined endophytic bacteria as all bacteria that can be detected

inside surface sterilized plant tissues or extracted from inside plants and

having no visibly harmful effect on the host plants. This definition

includes internal colonists with apparently neutral behaviour as well as

symbionts. It would also include bacteria which migrate back and forth

between the surface and inside of the plant during their endophytic

phase. The relationship between the endophyte and its host plant may

range from latent phytopathogenesis to mutualistic symbiosis (Sturz et

al., 1997).

In accordance with their life strategies, bacterial endophytes can

be classified as ‘obligate’ or ‘facultative’. Obligate endophytes are strictly

dependent on the host plant for their growth and survival and

transmission to other plants occurs vertically or via vectors. Facultative

endophytes have a stage in their life cycle in which they exist outside

host plants. In the extreme view, bacterial phytopathogens might be

included as (facultative or obligate) endophytes, because they often occur

in avirulent forms in plants. Avirulent forms of plant pathogens should

thus be regarded as endophytes, whereas virulent forms of these

organisms should not be included (Hardoim et al., 2008).

The life cycle of facultative endophytes can be characterized as

biphasic, alternating between plants and the environment (mainly soil).

The vast majority of the microorganisms that can thrive inside plants

probably have a propensity to this biphasic lifestyle. In fact, the observed

microbial diversities inside plants could be explained by the ability of

diverse endophytes to enter into and persist in plants (Rosenbleuth and

Martinez-Romero, 2006).

2.2. Biodiversity of endophytes

Soil microbial communities play an integral and often unique role

in ecosystem functions and are among the most complex, diverse, and

important assemblages in the biosphere (Zhou, et al., 2003). It seems

that the bacteria best adapted for living inside plants are naturally

selected. Endophytes are recruited out of a large pool of soil or

rhizospheric species and clones. Endophytic bacteria can actively or

latently colonise plants locally or systemically and both intercellularly

and intracellularly. Various reports indicate that these bacteria exist in a

variety of tissue types within numerous plant species, suggesting an

ubiquitous existence in most, if not all, higher plants. Endophytic

bacteria in a single plant host are not restricted to a single species but

comprise several genera and species (Ryan et al., 2008).

Endophytic bacteria have been isolated from a large diversity of

plants was reviewed by Sturz et al. (2000). Mundt and Hinkle (1976)

reported as many as 46 different bacterial species from 27 plant species.

In planta and ex planta populations of Pseudomonas species could

be differentiated by biochemical characteristics (van Peer et al., 1990).

McInroy and Kloepper (1991) showed that the endophytic bacterial

populations in corn stems ranged from 1x103 initially to 1 xl0 10cfu/ml

after 10 week.

Mavingui et al. (1992) found that there are different populations of

Bacillus polymyxa in soil, rhizosphere, and rhizoplane and that wheat

roots select specific populations as endophytes.

Sturz et al. (1997) characterized 15 bacterial species from red

clover nodules and estimated endophyte population densities to be in the

range of 104 viable bacteria per gram of fresh nodule.

Germida et al. (1998) found that the endophytic population was

less diverse than the root-surface population and the endophytes

appeared to originate from the latter.

Bacterial endophytes are found in a variety of plants, such as

sugar beet (Dent et al., 2004), prairie plants, agronomic crops (Zinniel et

al., 2002), potato varieties (Sessitsch et al., 2002), wheat (Germida and

Siciliano, 2001), and rice (Sun et al., 2008).

Suman et al. (2001) isolated endophytic bacteria from several

cultivars of Indian sugarcane on LGI medium. In a review by Lodewyckx

et al. (2002), 81 different bacterial species were reported to form

endophytic associations with plants.

Zinniel et al. (2002) isolated 853 endophytic strains from aerial

tissues of four agronomic crop species and 27 prairie plant species. A

majority of the microorganisms isolated (689 strains) were from corn and

sorghum; 45 strains were recovered from soybean and wheat, and 119

strains were obtained from 27 different host species of grasses, forbs,

legumes, and wildflowers. As a whole, fewer isolates were recovered from

perennial plants than from the agronomic crops.

Surette et al. (2003) have reported the isolation of up to 360

endophytic microorganism strains from Daucus carota, which were

classified into 28 genera, with Pseudomonas, Staphylococcus, and

Agrobacterium being predominant.

Bacteria belonging to the genera Bacillus and Pseudomonas are

easy to culture, and cultivation dependent studies have identified them

as frequently occurring endophytes (Seghers et al., 2004).

Rhizobium etli is found as a natural endophyte of maize plants in

traditional agricultural fields in which maize and bean are grown in

association (Gutiérrez-Zamora and Martínez-Romero, 2001). Rosenblueth

and Martínez-Romero (2004) found, both by multilocus enzyme

electrophoresis and by plasmid patterns, that Rhizobium etli strains that

were isolated from inside maize stems were selected subsets of the total

pool of Rhizobium etli found in rhizosphere, roots, or Phaseolus vulgaris

nodules.

Omarjee et al. (2004) isolated endophytic bacteria from the stalks

of sugarcane, Saccharum officinarum, growing in Papua New Guinea.

Endophytic bacterial communities from extracted juice averaged 6x 104

cfu/ml, which is 25 times greater than the number of endophytic

bacteria found in commercial varieties in South Africa. In this case, the

isolated Burkholderia spp. are opportunistic endophytes that are capable

of utilising organic compounds within sugarcane for growth and survival.

They can withstand the physiological conditions encountered within the

plants tissues, suppress pathogen infection of the host and avoid

induction of host defense responses that could eliminate them.

In a large study conducted on potato-associated bacterial

communities, species richness and diversity was lower for fungal

antagonistic bacteria inside roots than in the rhizosphere of potato (Berg

et al., 2005a). N2-fixing endophytic bacterium, Gluconacetobacter

diazotrophicus, was described by Cavalcante and Do¨bereiner as

associated with sugarcane (1988). This bacterium has also been found in

natural association with other host plants. Colonization of sorghum and

wheat after seed inoculation with Gluconacetobacter diazotrophicus

strains PAL 5 and UAP 5541/pRGS561 (containing the marker gene

gusA) was studied by colony counting and microscopic observation of

plant tissues. Inoculum levels as low as 102 cfu per seed were enough for

root colonization and further spreading in aerial tissues (Luna et al.,

2010).

Cho et al. (2007) reported 13 bacterial genera among 63 isolates

from the interior of ginseng roots cultivated in three different areas and

demonstrated marked regional differences in microbial community: the

respective dominant species were high G+C Gram-positive bacteria, low

G+C Gram-positive bacteria and Proteobacteria by 16S rDNA gene

analysis.

Bacteria from Bacillus genus have been reported as maize kernel

endophytes (Rijavec, 2007) and have been isolated not only from maize

(Chelius, 2001, McInroy and Kloepper, 1995) but also from many

different plants such as peas, potatoes, conifers, bananas, and bean

(Elvira-Recuenco and van Vuurde, 2000, Garbeva et al., 2001, Izumi et

al., 2008, Lian et al., 2008, Lo’pez et al., 2010).

Rice endophytic bacteria belong to the following groups;

Pseudomonas sp. (You and Zhou, 1989), Azoarcus sp. (Hurek et al.,

1994), Burkholderia sp. (Engelhard et al., 2000), Herbaspirillium

seropedicae (Olivares et al.,1996), Rhizobium leguminosarum (Yanni et al.,

1997), Serratia sp. (Sandhiya et al., 2005), Klebsiella sp. (Rosenblueth et

al., 2004) and Azorhizobium caulinodans (Engelhard et al., 2000).

Enterobacter has been identified as endophytes of several plants

such as Citrus sinensis, soybean and crop plants (Araújo et al., 2002;

Zinniel et al., 2002; Kuklinsky-Sobral et al., 2004). Other members of

Enterobacteriaceae identified in sugarcane have also been previously

described as endophytes. Endophytic Pantoea was found in sugarcane

(Loiret et al., 2004) and in soybean (Kuklinsky-Sobral et al., 2004).

Furthermore, a completely different bacterial community, dominated by

Pseudomonas, was identified in the leaves of sugarcane (Magnani et al.,

2010). Staphylococcus was found to be associated with sweet pepper

(Rasche et al., 2006). Curtobacterium, the only representative of the

Actinobacteria, was identified in orange, grape, and Pinus (Bell et al.,

1995; Araújo et al., 2002; Idris et al., 2004).

Enterobacter has been found previously as an endophyte of maize

(Rijavec et al., 2007, McInroy and Kloepper, 1995) and other plants such

as rice, cotton, papaya and poplar (Thomas et al., 2007, Taghavi et al.,

2009, Elbeltagy et al., 2001) and its ability to antagonize maize fungal

pathogens within Fusarium genus has also been described (Hinton and

Bacon, 1995). Stenotrophomonas has not been reported before in

association with maize, but it was found as the most abundant genus

present in rice (Sun et al., 2008). Endophytic Stenotrophomonas were

also obtained from potato, coffee and poplar (Garbeva et al, 2001,

Taghavi et al., 2009, Vega et al., 2005).

The presence and taxonomy of endophytic bacteria of the entire

aerial parts of Crocus (Crocus albiflorus) was investigated by Reiter and

Sessitsch (2006). Their results suggest that Crocus supports a diverse

bacterial microbial communities resembling the microbial communities

that have been described for other plants, but also containing species

that have not been described in association with plants before. This

study confirms that the culturable endophytes are a subset of total

endophyte biodiversity.

Thirty five endophytic bacteria were isolated from surface sterilized

stems, root, and nodules of wild and cultivated soybean varieties by

Hung et al. (2007). The genetic variation was more among endophytes

isolated from Glycine max tissues than from G. soja.

The genotypic diversity of indigenous bacterial endophytes within

stem of tropical maize (Zea mays L.) was determined in field and

greenhouse experiments by Rai et al. (2007). Endophytes were found in

most of the growing season at populations ranging from 1.36 – 6.12 x 105

colony-forming units per gram fresh weight (c.f.u./gm fw) of stem.

Bacillus pumilus, B. subtilis, Pseudomonas aeruginosa and P. fluorescens

were the relatively more predominant group of bacterial species residing

in maize stem.

Jha and Kumar (2007) isolated and characterized ten endophytic

diazotrophic bacteria from surface-sterilized roots and culm of a semi-

aquatic grass (Typha australis) which grows luxuriantly with no addition

of any nitrogen source. GR 3, plant growth promoter isolate was

identified as Klebsiella oxytoca.

Seventy-seven endophytic bacterial isolates were isolated from

roots, stems and leaves of black nightshade plants (S. nigrum) grown in

two different native habitats in Jena, Germany by Long et al. (2008).

Aravind et al. (2009) isolated 80 isolates of endophytic bacteria

from different varieties of black pepper (Piper nigrum L.) grown at

different locations in India. Another 30, isolates were obtained from

tissue cultured black pepper plants. These isolates were tentatively

grouped into Bacillus sp. (32 strains), pseudomonads (26 strains),

Arthrobacter sp. (20 strains), Micrococcus sp. (10 strains), Curtobacterium

sp. (one strain), Serratia (one strain) and twenty unidentified strains

based on morphology and biochemical tests.

Stajkovic (2009) isolated 15 endophytic non-rhizobial endophytes

from the surface sterilized root nodules of alfalfa (Medicago sativa L.)

found in Serbian fields and evaluated their influence on alfalfa alone and

its symbiosis with S. meliloti. The strains were identified as Bacillus

megaterium, Brevibacillus chosinensis and Microbacterium

trichothecenolyticum.

One hundred and fifteen putative cultivable endophytic colonizing

bacterial isolates were isolated from leaf samples of seventy two different

plant species collected from northern part of Peninsular Malaysia by

Bhore and Sathisha (2010). Most of the surface decontaminated plant

leaves samples gave 1 or 2 different putative cultivable endophytes and

some had even 3 or 4 types of putative endophytes.

Thirty two isolates of endophytic bacteria were obtained by

Magnani et al. (2010) from Brazilian sugarcane. Most of the bacteria

isolated from the sugarcane stem and leaf tissues belonged to Entero-

bacteriaceae and Pseudomonaceae, respectively, demonstrating niche

specificity. The Enterobacter genus was most frequently found in the

stems of sugarcane according to Magnani et al. (2010).

A total of 264 colonies of endophytic bacteria were isolated from

the interior of young radish leaves and roots by Seo et al. (2010).

Endophytic bacteria from the phylum Proteobacteria were predominant

in the leaf (61.3%) and root (52.1%) samples.

The diversity of bacterial endophytes associated with ginseng

plants of varying age levels in Korea was investigated by Thamizh Vendan

et al. (2010). Although a mixed composition of endophyte communities

was recovered from ginseng based on the results of 16S rDNA analysis,

bacteria of the genus Bacillus and Staphylococcus dominated in 1-year-

old and 4-year-old plants, respectively. Phylogenetic analysis revealed

four clusters: Firmicutes, Actinobacteria, α-Proteobacteria, and γ -

Proteobacteria, with Firmicutes being predominant.

Pereira et al. (2011) investigated bacterial diversity associated with

the roots of maize through the use of culture-dependent and culture-

independent methods and showed that γ-Proteobacteria within

Enterobacter, Erwinia, Klebsiella, Pseudomonas, and Stenotrophomonas

genera were predominant groups. The culturable component of the

bacterial community revealed that the predominant group was

Firmicutes, mainly of Bacillus genus, while Achromobacter, Lysinibacillus,

and Paenibacillus genera were rarely found in association with the roots.

Only two genera within γ-Proteobacteria, Enterobacter and Pseudomonas,

were found in the culture collection. (Pereira et al., 2011).

Yang et al. (2011) isolated a total of 72 endophytic bacteria from

tomato, of which 45 strains isolated from stems and 27 strains from

leaves. The isolation efficiency of bacteria from stems was higher than

from leaves. Based on morphological, physiological and biochemical

properties, 16S rDNA gene sequences and Biolog system analysis, the

isolate W4 was identified and named as Brevibacillus brevis W4.

Patel et al. (2012) isolated and characterized bacterial endophytes

from root and stem of Lycopersicon esculentum plant which was collected

from different regions of Gujarat. Total 18 isolates of endophytic bacteria

were selected in which, Only HR7 endophyte of tomato was identified as

Pseudomonas aeruginosa by 16srDNA analysis.

2.3. Endophytic colonization of plants

2.3.1. Plant colonization by endophytic bacteria

Endophytic bacteria have been considered to originate from the

outside environment and enter the plant through stomata, lenticels,

wounds, areas of emergence of lateral roots and germinating radicles

(Huang, 1986). Many seeds carry a diversity of endophytes (Coombs and

Franco, 2003b; Hallmann et al., 1997). By being seedborne, endophytes

assure their presence in new plants. Methylotrophs are seedborne in

soybean (Holland and Polaco, 1994). Plants that propagate vegetatively

(such as potatoes or sugarcane) can transmit their endophytes to the

next generation and would not require the infection process described

below.

The endophytes often originate from the soil, initially infecting the

host plant by colonizing, for instance, the cracks formed in lateral root

junctions and then quickly spreading to the intercellular spaces in the

root (Chi et al., 2005). Although other portals of entry into the plant

exist, (e.g. wounds caused by microbial or nematode phytopathogens, or

the stomata found in leaf tissue (McCulley, 2001), root cracks are

recognized as the main ‘hot spots’ for bacterial colonization. Hence, to be

ecologically successful, endophytes that infect plants from soil must be

competent root colonizers. Although, it is generally assumed that many

bacterial endophyte communities are the product of a colonizing process

initiated in the root zone (Sturz, et al., 2000), they may also originate

from other source than the rhizosphere, such as the phyllosphere, the

anthosphere, or the spermosphere (Hallman, et al., 1997).

In order to colonize the plant, the bacteria must find their way

through cracks formed at the emergence of lateral roots or at the zone of

elongation and differentiation of the root. Lytic enzymes produced by

these bacteria might also contribute to more efficient penetration and

colonization. Dong et al. (2003) showed that cells of Klebsiella sp/ strain

Kp342 aggregate at lateral-root junctions of wheat and alfalfa. Similarly,

Gluconacetobacter diazotrophicus and Herbaspirillum seropedicae also

colonize lateral-root junctions in high numbers (James and Olivares,

1998). Possible infection and colonization sites have been illustrated by

Reinhold-Hurek and Hurek (1998b). Some rhizospheric bacteria can

colonize the internal roots and stems, showing that these bacteria are a

source for endophytes (Germaine et al., 2004), but also phyllosphere

bacteria may be a source of endophytes (Hallmann et al., 1997).

It has been proposed that cellulolytic and pectinolytic enzymes

produced by endophytes are involved in the infection process (Hallmann

et al., 1997), as in Klebsiella strains, pectate lyase has been implicated to

participate during plant colonization (Kovtunovych et al. 1999). The cell

wall–degrading enzymes endogluconase and polygalacturonase seem to

be required for the infection of Vitis vinifera by Burkholderia sp.

(Compant et al., 2005).

Guo et al. (2002) inoculated the roots of hydroponically grown

tomato plants with Salmonellae at around 4.55 log CFU ml–1 and, the

next day, found that hypocotyls, cotyledons, and stems had around 3 log

CFU g–1. The systematic spread of an endophytic Burkholderia strain to

aerial parts of Vitis vinifera seems to be through the transpiration stream

(Compant et al., 2005).

Endophytes can also play an active role in colonization. Azoarcus

sp. type IV pili are involved in the adherence to plant surfaces, an

essential step towards endophytic colonization (Dörr et al.,1998). Two

Klebsiella strains differ significantly in their invasion capacity in different

plant hosts (Medicago sativa, Medicago truncatula, Arabidopsis thaliana,

Triticum aestivum, and Oryza sativa). One of them (Kp342) was a better

colonizer in all hosts and only needed a single cell to colonize the plants

substantially a few days after inoculation (Dong et al., 2003). The plant

hosts also differed in their ability to be colonized endophytically by the

same bacterium, further suggesting an active host role in the

colonization process.

Colonization of wheat by Azorhizobium caulinodans and

Azospirillum brasilense were stimulated by flavonoids (Webster et al.,

1998), as was the colonization by Azorhizobium caulinodans of two

Brassica napus (oilseed rape) varieties (O’Callaghan et al., 2000). Strain

to strain variation in colonizing capabilities have been found among

Rhizobium etli strains (Rosenblueth and Martínez-Romero, 2004). In

general, endophytic isolates were capable of colonizing or recolonizing the

inside plant tissues in higher numbers than isolates from the root

surface (van Peer et al., 1990; Rosenblueth and Martínez-Romero, 2004).

Changes in plant physiology can lead to the development of a distinct

endophytic population (Hallmann et al., 1997).

Colonization does not depend on the nitrogen-fixing ability of the

bacteria, as Nif– mutants of Gluconacetobacter diazotrophicus or

Herbaspirillum seropedicae, were able to colonize as well as Nif+ strains

(Sevilla et al., 2001). In contrast, Azoarcus mutants affected in pili were

incapable of systemic spread into rice shoots (Dörr et al., 1998); this is

also the case with mutants unable to produce a secreted endoglucanase

(Reinhold-Hurek et al., 2006). Non motile mutants of Salmonella enterica

were incapable of colonizing or had only a reduced invasion capacity in

Arabidopsis thaliana (Cooley et al., 2003). The data gathered by Asis et al.

(2004) showed that the number of endophytic bacteria in the stem of

sugarcane cultivars ranged from 104 to 109 cells / ml juice and varied

among sites and cultivars.

2.3.2. Plant location

Endophytic bacteria are found in roots, stems, leaves, seeds, fruits,

tubers, ovules, and also inside legume nodules (Hallmann et al., 1997;

Sturz et al., 1997). In most plants, roots have the higher numbers of

endophytes compared with above-ground tissues (Rosenblueth and

Martínez-Romero, 2004). Endophytic bacteria are able to penetrate and

become systemically disseminated in the host plant, actively colonizing

the apoplast, conducting vessels (Hallmann et al., 1997), and

occasionally the intracellular spaces.

Colonization of plant tissues has now been shown for several

Gram-negative diazotrophs, including Azoarcus sp. in kallar grass and

rice (Reinhold Hurek and Hurek, 1998b) and Acetobacter diazotrophicus

and Herbaspirillum seropedicae (James and Olivares, (1998) in sugar

cane. These microorganisms share the ability to penetrate deeply into

plants, being found in the stele and xylem vessels as well as in the outer

cortex layers. Once inside the plant, they can spread systemically and

reach aerial tissues, probably via xylem vessels (James and Olivares,

(1998). Colonization of stems and leaves appears to be especially

pronounced in sugar cane for A. Diazotrophicus and for the

phytopathogenic Herbaspirillum rubrisubalbicans.

In Kallar grass and rice, it was demonstrated that the bacteria

Azoarcus sp. BH72 were present in vessels of roots in gnotobiotioc

cultures by immunogold-labeling using genus-specific antibodies (Hurek

et al., 1991). Intercellular spaces and xylem vessels are the most

commonly reported locations for endophytic bacteria (Reinhold-Hurek

and Hurek 1998a).

Microscopical studies using immunological approaches and

reporter genes have clearly shown similar colonization patterns for

several nitrogen-fixing grass endophytes, such as Azoarcus sp. BH72, H.

seropedicae, Gluconacetobacter diazotrophicus and certain strains of

Azospirillum sp. (Hurek et al., 1994; James and Olivares, 1998; Olivares

et al., 1996; Schloter and Hartmann, 1998). In plants showing no

symptoms of disease, Azoarcus sp. BH72 colonizes the original host

Kallar grass and also rice seedlings in a similar manner: outer cell layers

(exodermis, sclerenchyma) and the root cortex are colonized inter and

intracellularly within 2–3 weeks, the aerenchyma which forms in

waterlogged plants being the main site for large microcolonies ( Hurek et

al., 1994).

By immunogold labelling and electron microscopic analysis,

Quadt- Hallmann and Kloepper (1996) observed a systemic colonization

of Enterobacter asburiae JM22 in cotton plants specifically in the roots

surface, within epidermal cells, and inside intercellular spaces of the root

cortex close to the conducting elements.

A Burkholderia sp. strain was found in xylem vessels and

substomatal chambers in Vitis vinifera plants (Compant et al., 2005). By

inhabiting similar niches as vascular pathogens, endophytes may be

used as competing bacteria for disease control (Hallmann et al., 1997).

Surface-washing of sprouts was not an effective way to eliminate

Salmonella enterica and Escherichia coli strains from alfalfa sprouts or

seeds, indicating that these bacteria are located in a protected niche

(Cooley et al., 2003). By introducing the green fluorescent protein, their

location was defined; Salmonella enterica colonized seed coats and roots,

while Escherichia coli colonized only roots (Charkowski et al., 2002).

2.3.3. Factors influencing endophytic colonisation

The variations in the endophytic bacterial communities can be

attributed to plant age, plant source, tissue type, time of sampling, and

environment condition (Kobayashi and Palumbo, 2000). ThamizhVendan

et al. (2010) showed that the age of the plant could largely influence the

variation in the endophytic community of ginseng plants. Also, variation

in endophytic diversity might be a function of the different maturation

stages specific to each plant, which might influence the different types

and amounts of root exudates (Ferreira et al., 2008).

Competition experiments with endophytes have shown that some

endophytes are more aggressive colonizers and displace others. This was

observed with Pantoea sp. out-competing Ochrobactrum sp. in rice

(Verma et al., 2004) and with different Rhizobium etli strains in maize

(Rosenblueth and Martínez-Romero, 2004). However, when the host

range of a large diversity of endophytes was analyzed, a seeming lack of

strict specificity was observed (Zinniel et al., 2002). The presence of

different endophytic species in soybean depended on the plant genotype,

the plant age, the tissue sampled, and also on the season of isolation

(Kuklinsky-Sobral et al., 2004). The soil type determined to a large extent

the endophytic population in wheat (Conn and Franco, 2004).

Correlations to growth promotion of tomato plants were observed with

inocula levels that promoted endophytic populations but not rhizospheric

populations (Pillay and Nowak, 1997).

The population density of endophytes is highly variable, depending

mainly on the bacterial species and host genotypes but also in the host

developmental stage, inoculum density, and environmental conditions

(Pillay and Nowak, 1997; Tan et al., 2003). A diminished colonization of

sugarcane by Gluconacetobacter diazotrophicus was observed in plants

under a high nitrogen-fertilization regime as opposed to low N

fertilization. In rice, a rapid change of the nitrogen-fixing population was

observed within 15 days after nitrogen fertilization (Tan et al., 2003).

Organic amendments to plants also influence the endophytic populations

(Hallmann et al., 1997).

2.3.4. Isolation of bacterial endophytes

In general endophytic bacteria occur at lower population densities

than rhizospheric bacteria or bacterial pathogens (Hallmann et al. 1997;

Rosenblueth and Martínez-Romero 2004). The endophytic niche offers

protection from the environment for those bacteria that can colonize and

establish in planta. These bacteria generally colonize the intercellular

spaces, and they have been isolated from all plant compartments

including seeds (Posada & Vega, 2005). Endophytic bacteria have been

isolated from both monocotyledonous and dicotyledonous plants, ranging

from woody tree species, such as oak and pear, to herbaceous crop

plants such as sugar beet and maize. Studies on the diversity of bacterial

endophytes have focused on characterization of isolates obtained from

internal tissues following disinfection of plant surfaces with sodium

hypochlorite or similar agents (Miche and Balandreau, 2001). Lodewyckx

et al. (2002) has highlighted the methods used to isolate and characterize

endophytic bacteria from different plant species. In plant tissue in

general, endophytic bacterial populations have been reported between

102 to 104 viable bacteria per gram (Kobayashi and Palumbo, 2000).

The surface sterilisation protocol followed for endophyte isolation is

as follows. The plants were thoroughly washed to remove all soil from

the root mass. Washing included a sonication step to dislodge any soil

and organic matter from the roots. The roots were excised and subjected

to a three-step surface sterilization procedure: a 60-s wash in 99 %

ethanol, followed by a 6-min wash in 3.125 % NaOCl, a 30 s wash in 99

% ethanol, and a final rinse in sterile reverse osmosis-treated (RO) water.

The surface-sterilized roots were then aseptically sectioned into 1-cm

fragments and distributed onto the isolation media, followed by

incubation at 27°C for up to 4 weeks (Schulz et al., 1993).

2.4. Functional diversity of endophytes

It has not been resolved whether plants benefit more from an

endophyte than from a rhizospheric bacterium or if it is more

advantageous for bacteria to become endophytic compared with

rhizospheric; nevertheless, benefits conferred by endophytes are well

recognized. The intimate association of bacterial endophytes with plants

offers a unique opportunity for their potential application in plant

protection and biological control.

Bacteria living inside plant tissues form associations ranging from

pathogenic to symbiotic. Multitudes of bacteria reside in leaves, stems,

roots, seeds, and fruits of plants that are seemingly neutral in terms of

plant health (Surette et al., 2003). However, several studies have also

suggested that many endophytic associations are not neutral at all, but

are beneficial to plants (Barka et al., 2002; Bailey et al., 2006).

The growth stimulation by the microorganisms can be a

consequence of nitrogen fixation (Hurek et al., 2002; Iniguez et al., 2004;

Sevilla et al., 2001) or the production of phytohormones, biocontrol of

phytopathogens in the root zone (through production of antifungal or

antibacterial agents, siderophore production, nutrient competition and

induction of systematic acquired host resistance, or immunity) or by

enhancing availability of minerals (Sessitsch et al., 2002; Sturz et al.,

2000). The elucidation of the mechanisms promoting plant growth will

help to favor species and conditions that lead to greater plant benefits.

Volatile substances such as 2-3 butanediol and aceotin produced by

bacteria seem to be a newly discovered mechanism responsible for plant-

growth promotion (Ryu et al., 2003).

2.4.1. Nitrogen fixation by endophytes

In 1986, Brazilian scientists (Cavalcante and Dobereiner, 1988)

discovered in the sugarcane stem N2-fixing endophytic bacteria called

Gluconacetobacter diazotrophicus. Their pioneering work was confirmed

by other scientists in USA, UK, and Germany and led to the identification

of two other N2-fixing endophytes, Herbaspirillum seropedicae and H.

rubrisubalbicans (Boddey et al., 1995). Endophytic diazotrophs seem to

constitute only a small proportion of total endophytic bacteria (Barraquio

et al,. 1997; Martínez et al., 2003). Such microbes include Azospirillum

lipoferum, Klebsiella pnemoniae and Azorhizobium caulinadans (Schloter

et al., 1994). Endophytic diazotrophic bacteria that have been discovered

in other plants include some specific diazotrophs, Glucanoacetobacter

diazotrophicus in sugarcane, sweet potato, and pineapple (Silva-Froufe,

2009) Herbaspirillum sp. in sugarcane and rice, and Azoarcus sp. in rice

and Kallar grass (James, 2000).

Endophytic bacteria are found in legume nodules as well. In red

clover nodules, some species of rhizobia were found, including Rhizobium

(Agrobacterium) rhizogenes, in addition to R. leguminosarum bv. trifolii,

which is the normal clover symbiont (Sturz et al., 1997). Some γ-

Proteobacteria are co-occupants with the specific rhizobia in Hedysarum

plant nodules (Benhizia et al., 2004). In most cases, the endophytic

bacteria are unable to form nodules.

Kallar grass grows in N-poor soils in Pakistan and a diversity of

Azoarcus sp. have been recovered from it (Reinhold-Hurek et al., 1993).

Inside wheat, Klebsiella sp. strain Kp342 fixes N2 (Iniguez et al., 2004),

and it has been reported that it increases maize yield in the field (Riggs et

al., 2001). Similarly, nitrogen fixing endophytes seem to relieve N

deficiencies of sweet potato (Ipomoea batatas) in N-poor soils (Reiter et

al., 2003). Nitrogen-fixing bacteria were identified in sweet potato in N-

poor soils with an analysis that consisted of amplifying nitrogenase (nifH)

genes by polymerase chain reaction (Reiter et al., 2003). The resulting

sequences, presumably derived from endophytes, resembled those from

rhizobia, including Sinorhizobium meliloti, Sinorhizobium sp. strain

NGR234, and Rhizobium etli. Other detected bacteria were Klebsiella sp.

and Paenibacillus odorifer (Reiter et al., 2003). In culture-dependent

studies, it seems that fast growing γ-Proteobacteria out-grow slower-

growing α-Proteobacteria such as rhizobia.

High N2-fixing activities have been estimated in the high or

excessive carbohydrate producing plants, like as sugarcane, pineapples

and sweet potato. Rice endophytic bacteria which fix N2 belong to the

following groups; Pseudomonas sp. (You and Zhou, 1989), Azoarcus sp.

(Hurek et al., 1994), Burkholderia sp. (Engelhard et al., 2000),

Herbaspirillium seropedicae (Olivares et al.,1996), Rhizobium

leguminosarum (Yanni et al., 1997), Serratia sp. (Sandhiya et al., 2005),

Klebsiella sp. (Rosenblueth et al., 2004) and Azorhizobium caulinodans

(Engelhard et al., 2000).

The isolation of diazotrophic endophytes Pantoea agglomerans by

Asis and Adachi (2003) and Klebsiella oxytoca by Adachi et al. (2002)

apparently support the findings of Yoneyama et al. (1998) on the possible

contribution of biological nitrogen fixation in Japanese sweetpotato

cultivars. Yoneyama et al. reported (1998) based on natural 15N

abundance method, the estimated amount of nitrogen derived through

biological nitrogen fixation ranged from 26 to 44% in field grown sweet

potato.

Out of the many N2-fixing endophytes isolated from sugarcane, it

has not been clearly defined which are responsible for fixing N inside the

plant. However, there is controversy on the level of N fixed by endophytes

and the proportion contributed to the plant (Giller and Merckx, 2003).

These estimates vary widely in different reports and range from 30 up to

80 kg N/ha/year (Boddey et al., 1995).

In a study by Asis et al. (2004) in sugarcane cultivars in

Philippines the number of N2-fixing endophytes ranged from 102 to 103

cells/ml of apoplast solution. Among the commercial cultivars, the

population density of N2-fixing endophytes ranged from 103 to 106

cells/ml apoplast solution. They concluded that the stem apoplast

solution could provide energy and carbon sources for the endophytes as

it contains high amounts of organic acids and sugar compounds.

Moreover, 15N dilution and 15N abundance studies showed that BNF

contributed significantly to the N uptake of sugarcane.

Grasses growing in nutrient-poor sand dunes contain members of

genera Pseudomonas, Stenotrophomonas as well as Burkholderia. It

seems that the Burkholderia endophytes could contribute N to the

grasses, because nitrogenase was detected with antibodies in roots

within plant cell walls of stems and rhizomes (Dalton et al., 2004).

During a survey in Tamil Nadu with sugarcane varieties, four isolates

belonging to the genus Burkholderia were studied. Burkholderia

vietnamiensis was found more active in reducing acetylene than the

others (Govindarajan et al., 2006).

Jha and Kumar (2007) isolated and characterized endophytic

diazotrophic bacteria from a semi-aquatic grass (Typha australis) which

grows luxuriantly with no addition of any nitrogen source. Amplification

of nifH by polymerase chain reaction (PCR) and detection of

dinitrogenase reductase by western blot confirmed the diazotrophic

nature of an isolate,GR-3.

2.4.2. Phosphate solubilisation by endophytes

Endophytic bacteria possess the capacity to solubilize phosphates,

and it was suggested by the authors that the endophytic bacteria from

soybean may also participate in phosphate assimilation (Kuklinsky-

Sobral et al., 2004).

Seventy-seven endophytic bacterial isolates were isolated from

roots, stems and leaves of black nightshade plants (S. nigrum) grown in

two different native habitats in Jena, Germany by Long et al. (2008) and

six isolates were able to solubilize inorganic phosphate.

Thamizh Vendan et al. (2010) reported that 9 out of 18 endophytic

isolates from gingseng plants had phosphate solubilizing ability by

detecting extracellular solubilization of precipitated tricalcium phosphate

with glucose as sole source of carbon.

Out of 18 endophytic isolates obtained from tomato by Patel et al.

(2012), 8 showed phosphate solubilisation activity. Results revealed that

majority of the PGPR strains do have phosphate solubilizing activity.

2.4.3. Production of plant growth-regulators by endophytes

Research has been conducted on the plant growth-promoting

abilities of various endophytic bacteria. They increase plant growth

through the improved cycling of nutrients and minerals such as nitrogen,

phosphate and other nutrients. These include phosphate solubilisation

activity (Verma et al., 2001; Wakelin et al., 2004), indole acetic acid

production (Lee et al., 2004) and the production of a siderophore (Costa

and Loper, 1994). Endophytic organisms can also supply essential

vitamins to plants (Pirttila et al., 2004). Moreover, a number of other

beneficial effects on plant growth have been attributed to endophytes and

include osmotic adjustment, stomatal regulation, modification of root

morphology, enhanced uptake of minerals and alteration of nitrogen

accumulation and metabolism (Compant et al., 2005). The recent areas

where these plant growth-promoting bacterial endophytes are being used

are in the developing areas of forest regeneration and phytoremediation

of contaminated soils.

Adhikari et al. (2001) reported potentiality of endophytic bacterial

strains for controlling the seedling disease of rice and promoting the

plant growth.

Harish (2005) found that application of endophytic bacterial

strains significantly increased the growth parameters viz., pseudostem

height, girth, number of leaves and physiological parameters viz.,

chlorophyll stability index, stomatal resistance and transpiration in

banana plants both under greenhouse and field conditions.

Jha and Kumar (2007) also reported the presence of IAA in the

culture filtrate of endophytes from Typha australis in which seven of 10

endophytic isolates were positive for IAA production.

Harish et al., (2008) isolated forty endophytic bacteria from the

corm and roots of banana plants and assessed for their efficacy on plant

growth promotion. Endophytic bacterial isolates EPB5, EPB22 and

EPB31 were found to increase the vigour index of rice seedlings

significantly in both roll towel and pot culture methods. The maximum

vigour index (5002) was recorded in rice seedlings treated with EPB22

suspension, followed by EPB5 (4680) whereas less vigour index (791) was

recorded from untreated control. The rhizobacterial strains Pf1 and

CHA0 also recorded a higher vigour index of 4528 and 4298, respectively.

Twelve endophytic isolates from gingseng plants produced

significant amounts of IAA in nutrient broth supplemented with

tryptophane as precursor (Thamizh Vendan et al., 2010).

Endophytic bacteria were isolated from surface sterilized stems,

root, and nodules of wild and cultivated soybean varieties by Hung et al.

(2007). Except nine, all from G. max, IAA production was observed in

the rest 56 endophytes. Fifteen produced IAA of more than 25 μg ml−1 in

the presence of the precursor tryptophan.

Seventy-seven endophytic bacterial isolates were isolated from

roots, stems and leaves of black nightshade plants (S. nigrum) grown in

two different native habitats in Jena, Germany by Long et al. (2008).

They were all characterized for their ability to produce ACC deaminase;

synthesize the phytohormone IAA; solubilize phosphate; and colonize

seedlings, since these traits are associated with plant growth promotion.

One isolate was able to produce IAA without supplementation of Trp and

28 were able to produce IAA with supplementation of Trp.

Three plant-growth-promoting isolates of endophytic bacteria from

sugar beet roots produced indole-3-acetic acid (IAA) in vitro in a

chemically defined medium and significantly increased plant height,

fresh and dry weights and number of leaves per plant, as well as levels of

phytormones compared with control plants (Yingwu Shi et al., 2009).

Vetrivelkalai et al. (2010) reported that on seed bacterization with

nineteen endophytic bacterial isolates, four isolates viz., EB3, EB16,

EB18 and EB19 significantly enhanced the germination percentage,

shoot and root length and vigour index of bhendi seedlings by roll towel

technique and pot culture studies.

All the 18 isolates of endophytic bacteria isolated from roots and

stems of tomato (Lycopersicon esculentum) plant which was collected

from different regions of Gujarat exhibited a significant amount of IAA

production (Patel et al., 2012).

2.5. Interactions of endophytes with pathogens

Endophytic bacteria are able to lessen or prevent the deleterious

effects of certain pathogenic organisms. Diseases of fungal, bacterial,

viral origin and in some instances even damage caused by insects and

nematodes can be reduced following prior inoculation with endophytes

(Sturz et al., 2000; Berg and Hallmann, 2006). The widely recognized

mechanisms of biocontrol mediated by PGPB are competition for an

ecological niche or a substrate, production of inhibitory allelochemicals,

and induction of systemic resistance (ISR) in host plants to a broad

spectrum of pathogens (Bloemberg and Lugtenberg, 2001) and/or abiotic

stresses.

Endophytic bacterial biocontrol agents can be divided into two

groups: (i) strains that extensively colonize the internal plant tissues and

suppress invading pathogens by niche occupation, antibiosis, or both,

and (ii) strains that primarily colonize the root cortex where they

stimulate general plant defense/resistance mechanisms. More extensive

and continuous colonization of plants might be required for endophytes

of the first type because coincidence with pathogen propagules would be

necessary for antagonism.

According to Backman et al. (1997), the effectiveness of endophytes

as biological control agents (BCAs) is dependent on many factors. These

factors include: host specificity, the population dynamics and pattern of

host colonization, the ability to move within host tissues, and the ability

to induce systemic resistance.

It is believed that certain endophytic bacteria trigger a

phenomenon known as induced systemic resistance (ISR), which is

phenotypically similar to systemic-acquired resistance (SAR). SAR

develops when plants successfully activate their defence mechanism in

response to primary infection by a pathogen, notably when the latter

induces a hypersensitive reaction through which it becomes limited in a

local necrotic lesion of brown desiccated tissue (van Loon et al., 1998).

ISR is effective against different types of pathogens but differs from SAR

in that the inducing bacterium does not cause visible symptoms on the

host plant (van Loon et al., 1998).

2.5.1. Effect of endophytes on pathogens

The first record of an endophyte affecting a plant disease was that

by Shimanuki (1987) who showed that timothy (Phleum pratense) plants

infected with the choke fungus, Epichloe typhina, were resistant to the

fungus Cladosporium phlei. In some cases, they can also accelerate

seedling emergence and promote plant establishment under adverse

conditions and enhance plant growth and development (Pillay and

Nowak, 1997). Furthermore, several antagonistic endophytic bacterial

species have been isolated from the xylem of lemon roots (Citrus

jambhiri), including Achromobacter sp., Acinetobacter baumannii, A.

lwoffii, Alcaligenes- Moraxella sp., Alcaligenes sp., Arthrobacter sp.,

Bacillus sp., Burkholderia cepacia, Citrobacter freundii, Corynebacterium

sp., Curtobacterium flaccumfaciens, Enterobacter cloacae, E. aerogenes,

Methylobacterium extorquens, Pantoea agglomerans, Pseudomonas

aeruginosa, and Pseudomonas sp. against root pathogens (Araújo et al.,

2001, and Lima et al., 1994).

Jetiyanon (1994) established that cabbage colonized by endophytes

in the greenhouse had season-long reduced black rot in the field due to

induction of defense mechanisms. Non-treated cabbage plants reached

the economic threshold (symptoms of systemic disease) approximately 33

days after inoculation with Xanthomonas campestris pv. campestris while

the disease progressed slower in plants treated with log 9.0 CFU/ml of

either a low virulence isolate of Xanthomonas campestris pv. campestris

or the non-compatible pathogen Xanthomonas campestris pv.

malvacearum. Cabbage colonized by endophytes did not reach the

economic threshold for the disease until approximately 50 days after

inoculation, which coincided with harvest maturity.

Several bacterial endophytes have been reported to support growth

and improve the health of plants (Hallmann et al., 1997) and therefore

may be important sources of biocontrol agents. Erwinia caratovora, for

example, is inhibited by numerous endophytic bacteria, including several

strains of Pseudomonas sp., Curtobacterium luteum, and Pantoea

agglomerans (Sturz et al., 1999). In oak, endophytic bacteria biologically

active against the oak wilt pathogen Ceratocystis fagacearum have been

isolated (Brooks et al., 1994). Furthermore, Wilhelm et al. (1997)

demonstrated that Bacillus subtilis strains isolated from the xylem sap of

healthy chestnut trees exhibit antifungal effects against Cryphonectria

parasitica causing chestnut blight.

Interestingly, the ability to inhibit pathogen growth by endophytic

bacteria, isolated from potato tubers, decreases as the bacteria colonize

the host plant’s interior, suggesting that bacterial adaptation to this

habitat occurs within their host and may be tissue type and tissue site

specific (Struz et al.,1999). For example, Pseudomonas sp. strain PsJN,

an onion endophyte, inhibited Botrytis cinerea Pers. and promoted vine

growth in colonized grapevines, demonstrating that divergent hosts could

be colonized (Barka et al., 2002). Colonization of multiple hosts has been

observed with other species of endophytes and plants. For example:

Pseudomonas putida 89B-27 and Serratia marcescens 90-166 reduced

Cucumber Mosaic Virus in tomatoes and cucumbers (Raupach et al.,

1996) as well as anthracnose and Fusarium wilt in cucumber (Liu et al.,

1995).

Sturz et al. (1999) found that 61 of 192 endophytic bacterial

isolates from potato stem tissues were effective biocontrol agents against

Clavibacter michiganensis subsp. sepedonicus. Researchers concluded

that Bacillus mycoides isolate BacJ (Bargabus et al., 2002) and Bacillus

pumilis isolate 203-7 (Bargabus et al., 2004) suppressed Cercospora leaf

spot in sugar beets. An increased diversity of bacterial endophytes was

found in Erwinia carotovora–infected potatoes in comparison with non-

infected control plants (Reiter et al., 2003).

The frequent isolation of Curtobacterium flaccumfaciens as

endophytes from asymptomatic citrus plants infected with the pathogen

Xylella fastidiosa suggested that the endophytic bacteria may help citrus

plants to better resist the pathogenic infection (Araujo et al., 2002).

Endophytes from potato plants showed antagonistic activity against fungi

(Berg et al., 2005a; Sessitsch et al., 2004) and also inhibited bacterial

pathogens belonging to the genera Erwinia and Xanthomonas (Sessitsch

et al. 2004). Some of the endophytic isolates produced antibiotics and

siderophores in vitro (Sessitsch et al., 2004).

Inhibition of the oak wilt pathogen Ceratocystis fagacearum was

obtained with 183 endophytic bacteria of 889 isolates tested (Brooks et

al., 1994). Of 2,648 bacterial isolates analyzed from the rhizosphere,

phyllosphere, endosphere, and endorhiza, only one, a root endophyte

corresponding to Serratia plymuthica, was a highly effective fungal

antagonist against Xanthomonas sp. in Brassica seeds (Berg et al.,

2005b). Endophytic actinobacteria are effective antagonists of the

pathogenic fungus Gaeumannomyces graminis in wheat (Coombs et al.,

2004), and several endophytes showed antagonism against Rhizoctonia

solani (Parmeela and Johri, 2004).

The first actinobacterial endophyte isolated, belonging to the genus

Frankia, is a nitrogen-fixing actinobacterium that forms actinorhizae

with eight families of angiosperms (Provorov et al., 2002). A number of

the biologically active endophytes and root-colonizing microorganisms

that have been isolated or detected belong to the actinobacterial phylum,

specifically the genus Streptomyces (Coombs, and Franco. 2003a,

Sessitsch et al., 2004).

A number of endophytic actinobacteria were isolated by culture

dependent methods, with the major genera being Streptomyces,

Microbispora, Micromonospora, and Nocardioides (Coombs and. Franco,

2003a). Many of these isolates were capable of suppressing fungal

pathogens of wheat in vitro and in planta, including Rhizoctonia solani,

Pythium spp., and Gaeumannomyces graminis var tritici, indicating their

potential use as biocontrol agents (Coombs et al., 2004).

Aravind et al. (2009) isolated, characterized and evaluated

endophytic bacteria against Phytophthora capsici in black pepper. Three

isolates, IISRBP 35, IISRBP 25 and IISRBP 17 were found effective for

Phytophthora suppression in multilevel screening assays which recorded

over 70% disease suppression in greenhouse trials. These endophytic

bacteria were identified as effective antagonistic endophytes for biological

control of Phytophthora foot rot in black pepper.

The endophytes Herbaspirillum seropedicae and Clavibacter xylii

have been genetically modified to produce and excrete the δ-endotoxin of

Bacillus thuringensis to control insect pests (Downing et al., 2000).

Continued work with endophytic bacteria also holds potential for

developing biocontrol agents that may be self-perpetuating by colonizing

hosts and being transferred to progeny much as is the case with

associative nitrogen-fixing PGPB on sugarcane (Boddey, 2003) or the

nonsymbiotic endophyte bacterium Burkholderia phytofirmans PsJN

(Sharma and Nowak,1998).

2.5.2. Defense mechanisms of Intrinsic Systemic Resistance (ISR) mediated by endophytes

Endophytes triggered ISR fortifies plant cell wall strength and

alters host physiology and metabolic responses, leading to an enhanced

synthesis of plant defense chemicals upon challenge by pathogens

and/or abiotic stress factors (Nowak and Shulaev, 2003). After

inoculation of tomato with endophytic P. fluorescens WCS417r, a

thickening of the outer tangential and outermost part of the radial side of

the first layer of cortical cell walls occurred when epidermal or

hypodermal cells were colonized (Duijff et al., 1997).

The type of bacterized plant response induced after challenge with

a pathogen resulted in the formation of structural barriers, such as

thickened cell wall papillae due to the deposition of callose and the

accumulation of phenolic compounds at the site of pathogen attack

(Benhamou et al., 1998). Biochemical or physiological changes in plants

include induced accumulation of pathogenesis-related proteins (PR

proteins) such as PR-1, PR-2, chitinases, and some peroxidises (M’Piga et

al., 1997).

Kloepper et al. (2004) reported that combination of B. subtilis

strain GB03, B. amyloliquefaciens strain IN937a and B. subtilis strain

IN937b together with chitosan resulted in significant growth promotion

that was correlated with induced resistance in tomato, bell pepper,

cucumber and tobacco.

The ability of four Bacillus sp. isolated from vegetable crops to

colonize Theobroma cacao seedlings and reduce the severity of black pod

rot (Phytophthora capsici was evaluated by Melnick et al. (2008). Of the

Bacillus sp. tested, application of B. cereus isolates BT8 (from tomato) or

BP24 (from potato) together with the polysilicon surfactant Silwet L-77

(0.24% vol/vol) resulted in long-term (>68 days) stable colonization of

cacao leaves. These newly developed, non-colonized leaves from colonized

plants exhibited disease suppression, which supports a probable disease

suppression mechanism of induced systemic resistance for the BT8

isolate (Melnick et al., 2008).

Harish et al., (2008) reported that the tissue culture banana plants

treated with mixtures of rhizobacterial and endophytic bacterial

formulations viz., EPB5 + EPB22 + Pf1 + CHA0 was significantly effective

in reducing Banana Bunchy top virus under field conditions recording

33.33% infection with 60% reduction over control. The expression of

defense-related enzymes and pathogenesis related proteins were more in

the plants treated with rhizosphere and endophytic bacterial

formulations than the control plants.

2.5.3. Synthesis of allelochemicals by endophytes

Offensive endophyte colonization and defensive retention of

rhizosphere niches are enabled by production of bacterial

allelochemicals, including iron-chelating siderophores, antibiotics,

biocidal volatiles, lytic enzymes, and detoxification enzymes (Glick,

1995).

2.5.3.1. Competition for iron and the role of siderophores.

Iron is an essential growth element for all living organisms. The

scarcity of bioavailable iron in soil habitats and on plant surfaces

foments a furious competition. Under iron-limiting conditions

endophytes produce low-molecular-weight compounds called

siderophores to competitively acquire ferric ion (Whipps, 2001). Although

various bacterial siderophores differ in their abilities to sequester iron, in

general, they deprive pathogenic fungi of this essential element since the

fungal siderophores have lower affinity (Loper and Henkels., 1999). In in

vitro assays by Jafra et al. (2009), 35 out of 565 hyacinth-associated

endophytic bacterial isolates produced antimicrobial substances against

Dickeya zeae, and 35 produced siderophores. Iron sequestering

siderophore production was detected in seven isolates out of the 18

endophytic isolates from Genseng plants by Tamizh Vendan et al. (2010).

2.5.3.2. Hydrolytic enzyme production

Biocontrol activity of microorgansims involving synthesis of

allelochemicals has been studied extensively for endophytic bacteria

(Lodewyckx et al., 2002), since they can synthesize metabolites with

antagonistic activity toward plant pathogens (Chen et al., 1995). A variety

of endophytes also exhibit hyperparasitic activity, attacking pathogens by

excreting cell wall hydrolases (Chernin and Chet, 2002). The ability to

produce extracellular chitinases is considered crucial for the endophyte,

Serratia marcescens to act as antagonist against Sclerotium rolfsii

(Ordentlich et al., 1988), and for Paenibacillus sp. strain 300 and

Streptomyces sp. strain 385 to suppress Fusarium oxysporum f. sp.

cucumerinum. It has been also demonstrated that extracellular chitinase

and laminarinase synthesized by the endophytic Pseudomonas stutzeri

digest and lyse mycelia of F. solani (Lim et al., 1991). Aino et al. (1997)

have also reported that the endophytic P. fluorescens strain FPT 9601

can synthesize DAPG and deposit DAPG crystals around and in the roots

of tomato, thus demonstrating that endophyte can produce antibiotics in

planta. Chitinase produced by S. plymuthica C48 inhibited spore

germination and germ-tube elongation in Botrytis cinerea (Frankowski,

2001).

Castillo et al. (2002) demonstrated that munumbicins, antibiotics

produced by the endophytic bacterium Streptomyces sp. strain NRRL

30562 isolated from Kennedia nigriscans, can inhibit in vitro growth of

phytopathogenic fungi, P. ultimum, and F. oxysporum. Subsequently, it

has been reported that certain endophytic bacteria isolated from field-

grown potato plants can reduce the in vitro growth of Streptomyces

scabies and Xanthomonas campestris through production of siderophore

and antibiotic compounds (Sessitsch et al., 2004).

Sixty endophytic bacterial isolates from sugarcane were used for in

vitro inhibition tests against Clavibacter michiganensis subsp. insidiosus

(a relative of the causative agent of ratoon stunting disease), Fusarium

napiforme, F. proliferatum and Ustilago scitaminea (smut). None of the

isolates showed inhibition of Fusarium. Thirteen belonged to the genus

Burkholderia, as shown by 16S sequencing; among these, four inhibited

the growth of Ustilago scitaminea and seven inhibited the growth of

Clavibacter michiganensis. Six of the Burkholderia isolates from

sugarcane showed strong chitinase activity. (Omarjee et al., 2004).

Hung et al. (2007) isolated endophytic bacteria from soybean

cultivars and all the 65 endophytes were screened for their ability to

produce cellulase and pectinase enzymes. Seventeen isolates from G.

max and three from G. soja gave a clear zone of hydrolysis on pectin agar

plate. When grown on cellulase medium, 28 of the endophytes from G.

max and 16 of G. soja were able to grow, utilizing the C-source with the

production of cellulase enzyme. In this study, 33% of the isolates

secreted pectinases, and 70% produced cellulases.

Rhizosphere and endophytic bacterial isolates from the roots and

corms of banana were tested for their biocontrol efficiency against

Banana bunchy top virus (BBTV) by Harish et al. (2009). Bio-

formulations of mixtures of the rhizobacterial isolate Pseudomonas

fluorescens (Pf1) and endophytic Bacillus sp. (EPB22) were effective in

reducing the incidence of BBTV under green-house (80%) and field

conditions (52%).

Seo et al. (2010) reported that the endophytic bacteria from young

radish plants belonging to Enterobacter sp. YRL01 and B. subtilis YRL02

had the highest amount of inhibitory action against human pathogenic

bacteria, while B. subtilis YRR10 had an inhibitory action against plant

pathogenic fungi.

Yang et al. (2011) isolated 72 endophytic bacteria from healthy

tomato stems and leaves from field-grown plants, and the strain W4

identified and named as Brevibacillus brevis, gave strongly inhibitory

effect on Botrytis cinerea Pers, with the inhibition rate 78% in dual

culture assay and 100% using fermentation filtrate diluted 20 times.

About 16 endophytic bacteria were isolated from roots of Pongamia

glabra by Jalgaonwala (2011) which were subjected to in vitro screening

for antagonism. The strong antifungal activity of B.megaterium was found

against pathogenic fungi A.niger and T.konningi and potent activity

against other pathogenic fungi such as Aspergillus avamori, Penicillium

chrysosporium, Penicillium fumicalsuri and Fusarium oxysporium.

Thirty endophytic bacteria were isolated from various plant species

growing near Saint-Petersburg, Russia by Malfanova et al. (2011).

Metabolites possibly responsible for these plant-beneficial properties

were identified as the hormone gibberellin and (lipo) peptide antibiotics

respectively. The antibiotic properties of strain HC8 are similar to those

of the commercially available plant-beneficial strain Bacillus

amyloliquefaciens FZB42 (Malfanova et al., 2011). In a study conducted

by Patel et al. (2012) 17 out of 18 isolates of tomato (Lycopersicon

esculentum) from Gujarat revealed the production of pectinase and 5

showed chitinase activity.

2.6. Molecular characterization of nif genes of endophytic diazotrophs

The isolation and study of endophytic nitrogen fixing bacteria from

several grasses (Baldani et al., 1997) represent an exciting phase in the

field of biological nitrogen fixation. Endophytic diazotrophs usually live

within the root apoplast, and interact more closely with the host with

less competition for carbon sources and a more protected environment

for N2 fixation (Reinhold Hurek and Hurek, 1998b). Endophytic

diazotrophs have been proposed to be responsible for the supply of

biologically fixed N to their host plant (Boddey et al., 1995).

According to Dobereiner et al. (1995), endophytic diazotrophs, by

inhabiting the interior of the plants, can avoid the competition with

rhizospheric bacteria and derive nutrients directly from the host plants.

In return, as the plant interior may provide an environment conducive to

N2 fixation by being low in O2 and relatively high in carbon, the bacteria

can fix N2 more efficiently to the host (James and Olivares, 1998).

Endophytic diazotrophs, such as Acetobacter, Azoarcus and

Herbaspirillum, mainly isolated from graminaceous plants, have occurred

within plant tissues and have been shown to fix significant amounts of

nitrogen (James and Olivares, 1998; Reinhold-Hurek and Hurek, 1998b).

There are several reports on the association of nitrogen-fixing bacteria

such as Azospirillum sp. with wheat (Baldani et al., 1983), Herbaspirillum

seropedicae with maize (Baldani et al., 1986), Gluconacetobacter

diazotrophicus with sugarcane (Cavalcante and Do¨ bereiner, 1988) and

G. johannae and G. azotocaptans with coffee plants (Fuentes-Ramirez et

al., 2001). Also Herbaspirillum seropedicae, Burkholderia vietnamiensis,

Rhizobium leguminosarum bv. trifolii, Azoarcus, Serratia marcescens and

innumerable species of Pseudomonas have been found in association

with rice plants. Endophytic diazotrophs have been proposed to be

responsible for the supply of biologically fixed N to their host plant

(Boddey et al., 1995).

Nitrogenase, the enzyme highly essential for reducing nitrogen to

ammonia, is composed of Fe (dinitrogenase) and Mo-Fe protein

(dinitrogenase reductase), which is encoded by nif gene. A substantial

molecular diversity of N2 fixing bacteria has been detected in field grown

rice based on retrieval of nif H or nif D gene fragments from root DNA (da

Rocha et al., 1986). Since the nif H gene only occurs in nitrogen fixing

microorganisms, it has been used to monitor the presence of these

diazotrophs in pure cultures (Frank et al., 1998), in soil (Widmer et al.,

1999) and plants. For studying the evolution of nitrogen-fixing

population in the environment, analysis of nifH, the gene encoding

nitrogenase reductase, has been used with various PCR primers that

amplify this gene from both microorganisms and environmental samples

(Widmer et al., 1999). nifH encoding dinitrogenase reductase is

evolutionarily conserved and has often been used to detect nitrogen-

fixing microorganisms in natural microbial communities (Satoko et al.,

2002). Numerous researchers have employed various PCR primers

specific for segments of nifH sequences from diazotrophic pure cultures

and from various environmental samples, including marine plankton,

termite hindguts, microbial mats and aggregates, terrestrial soils, and

the rhizoplanes of rice (Oryza sativa) and of shoal grass (Halodule

wrightii). These studies have yielded a diverse array of nifH sequences

representing many, mostly unknown, lineages of diazotrophic Bacteria

and Archaea (Charles et al., 2000).

Nitrogenase has been found in phylogenetically diverse groups of

prokaryotes and has been highly conserved through evolution. The

implementation of molecular techniques such as PCR amplification has

greatly facilitated the study of diazotrophs in bacterial communities.

Various PCR primers specific for segments of nifH, the structural gene

encoding dinitrogenase reductase, to amplify partial nifH sequences from

various environmental samples and from diazotrophic pure cultures have

been employed in several studies (Betancourt, 2008). Rapid and

unambiguous identification of diazotrophs has greatly benefited from

recent advances in DNA fingerprinting based on the Polymerase Chain

Reaction (PCR), Random Amplified Polymorphic DNA PCR (RAPD-PCR)

and the interspersed repetitive sequences PCR (rep-PCR) (Berg et al.,

1994). The nifH sequence database is rapidly expanding and is currently

composed of over 1500 sequences, most of which have been obtained

from environmental samples (Zehr, 2003). Several studies have also

addressed the importance and contribution of biological nitrogen fixation

in ecologically unique habitats by focussing on the diversity of nifH

sequences (Zehr, et al., 2003).

A substantial molecular diversity of N fixing bacteria has been

detected in field grown rice based on retrieval of nif H or nif D gene

fragments from root DNA (Ueda et al., 1995). Palus et al. (1996) used

PCR amplification for the identification of a diazotrophic bacterial

endophyte, Klebsiella, isolated from the stems of Zea mays L. by

amplifying portions of nif H and 16 S rRNA genes from this organism.

The nif H gene, which codes for dinitrogenase reductase, from this

organism is closely related to nif H from K. pneumoniae. Stoltzfus et al.

(1997) developed highly conserved DNA primers for PCR mediated

detection of nif D genes and used to screen the collection for the presence

of nif genes. Perret and Broughton (1998) employed Targeted PCR

Fingerprinting (TPF) using primers specific for the nif H and rec a genes

to discriminate between Rhizobium species NGR 234 and R. fredii USDA

257, the closely related bacteria in which the symbiotic loci are 98%

homologous.

Kumari Sugitha (2003) identified nif H genes of heterotrophic and

endophytic diazotrophs isolated from rice by TPF using nif primers. The

nif H primers produced informative and reproducible genetic markers in

standard Rhizobium culture (BMBS 1) and eight (HDPY 1, HDC 8, HDM

7, HDT 1, EDM 2, EDA 1, EDC 5 and EDA 2) of the thirteen diazotrophic

isolates tested, confirming the presence of nif H in these isolates.

Muthukumaraswamy et al., (2005) showed that nitrogen-fixing

acetic acid bacteria are found in natural association with rice plants. The

acetic acid bacteria isolates recovered from rice and capable of fixing

nitrogen were assigned to the species G. diazotrophicus and A.

peroxydans. The presence of nifH genes in A. peroxydans was confirmed

by PCR amplification with nifH specific primers.

In a study by Mirza et al., (2006) the amplification of partial nifH

(360 bp) from the endophytic bacterial strain K1 identified as

Pseudomonas sp. in rice confirmed (in addition to acetylene reduction

activity) the presence of a nitrogen fixation ability in this strain. The nifH

sequence showed highest similarity to those of Azotobacter chroococcum.

PolF and PolR primers amplify a 360 bp region between sequence

positions 115 and 476 (referring to Azotobacter nifH coding sequence

M20568).

Wei et al. (2007) searched for novel endophytic nitrogen-fixing

bacteria and demonstrated the diversity of endophytic diazotrophs living

in bamboo. Forty isolates of endophytic diazotrophs were obtained from

the root, leaves and stem samples of Bambusa blumeana. The expected

nifH gene fragments (about 360 bp) were amplified by PCR in all isolates

and in the positive control strain Azospirillum lipoferum DSM 1691T, but

not in the blank control.

2.7. Applications of endophytes in agriculture

2.7.1. Endophytes in nursery technology

2.7.1.1. Rooting and establishment of Horticultural crops

High quality of planting material is a critical input for success of

any horticulture venture. To ensure the quality of the planting materials,

an effective production and protection system is of paramount

importance. Propagating material used in nurseries includes rooted

cuttings and nodal cuttings. Naturally occurring plant associated

endophytic bacterial species can be delivered stem or rooted cuttings of

horticultural plants. Such a delivery mechanism for endophytic bacteria

during early stage of its development would ensure better rooting of the

planting material. Several methods of delivery of endophytic bacteria are

reported which includes seed treatment (seed biopriming), bacterisation

of plant propagation material, soil application and even foliar application.

For vegetatively propagated plant species, endophytic bacteria can be

directly delivered into the succulent plant system prior to the planting in

the soil. In these plants shoots are amenable for bacterisation by

endophytic bacteria.

Endophytic bacteria from poplar trees, representative of the

dominantly observed genera Enterobacter, Serratia, Stenotrophomonas,

and Pseudomonas, were tested for their capacities to improve rooting of

their poplar host by Taghavi et al. (2009). After endophytic inoculation

and subsequent growth in soil, we noticed that the root systems of

inoculated poplar cuttings were often denser with many fine roots

compared to those of the noninoculated control plants. Root formation

was very slow for noninoculated plants. In contrast, for cuttings that

were allowed to root in the presence of the selected endophytes, root

formation was initiated within 1 week, and shoot formation was more

pronounced compared to that of the noninoculated plants. After 10

weeks, root formation for the noninoculated controls was still poor;

however, for plants inoculated with endophyte, roots and shoots were

well developed.

An experiment was conducted to study the influence of planting

time and IBA (Indole Butyric Acid) on rooting and vegetative growth on

cuttings of pomegranate ‘Ganesh’ by Singh et al. (2009). The respective

cuttings were treated with varying concentrations of IBA i.e., 0, 50, 100

and 200 ppm for slow dip and 1000, 1500 and 2000 ppm for quick dip

techniques. IBA 100 and 2000 ppm were found to be most efficacious in

encouraging rooting and invigorating shoot growth. The cuttings treated

with IBA 100 ppm (slow dip) and 2000 ppm (quick dip) exerted positive

effect with regard to sprouting percentage, rooting percen¬tage, number

of roots per cutting, longest root, root weight, plant height and shoot

girth.

In a study by Kurd et al. (2010) semi-hardwood cuttings of olive

(Oleo europea) variety Coratina were treated with 3000, 4000 and 5000

ppm IBA solution for 3-4 seconds. Highest rooting percentage (60%) was

obtained in the cuttings treated with 3000 ppm. The maximum average

number of roots per cutting (4.443) and average root length (5.687 cm)

were recorded with 4000 ppm IBA treatment.

A study was conducted to investigate the influence of IBA at 0,

1000, 1500 and 2000 ppm concentration on rooting potential of

hardwood cuttings of four varieties of Bougainvillea by Singh et al.

(2010). The result obtained indicated that both IBA concentration and

variety had significant effect on sprouting, rooting, callusing and

establishment of cuttings. Louise Wathen cuttings treated with 1000

ppm IBA were found superior with 85.39 % sprouting 75.46 % rooting,

80.78 % callusing, no of primary roots (19.73), secondary roots (28.8)

and tertiary roots (49.19) per cutting, length of longest root (39.9 cm) and

diameter of thickest root (6.61 mm). Establishment (100 %) was also

found best in this treatment.

Effect of IBA on seed germination, sprouting and rooting in

cuttings for mass propagation of Jatropha curcus L strain DARL-2 was

studied by Kumari et al. (2010). In case of clonal propagation 200 ppm

IBA treatment was found to be the best for sprouting and rooting in

cuttings taken from basal, middle and top portion of one year old branch.

Basal portion of the branch responded best (76.7 % sprouting and 72 %

rooting) to IBA treatment (50 ppm).

Propagation of an endangered species, Celastrus paniculata by

hardwood cuttings using different concentrations of IBA treatment has

been reported by Sharma et al.(2010).

Rogers (2012) investigated the effect of inoculating hard wood

cuttings of Poplar with the endophyte, Enterobacter sp. 638. After 17

weeks, plants inoculated with Enterobacter sp. 638 had 55 % greater

total biomass than un-inoculated control plants. There was markedly

increased root growth in poplar inoculated with endophytes, and

marginally significant increase in root to shoot ratio.

Several rhizo and endophytic bacterial communities have been

identified in black pepper that can be exploited for production of

plantlets of black pepper in a nursery (Aravind et al., 2009). Pre-plant

bacterisation was reported as a strategy for delivery of beneficial

endophytic bacteria and production of disease-free plantlets of black

pepper (Piper nigrum L.) by Aravind et al. (2012). In this study, the

sprouting behaviour of black pepper stem cuttings was superior when

treated with the endophytes P. aeruginosa, B. megaterium and C. luteum

to the untreated stem cuttings. Pre-plant root bacterisation of black

pepper plantlets had positive effect on growth performances such as

height of plantlets, number of leaves, root biomass and total biomass.

Root bacterisation with P. putida significantly increased all the growth

promoting parameters.

2.7.1.2. Effect of endophytes on the growth and establishment of tissue culture plants

Although endophytic bacteria are ubiquitously inhabiting most

plant species and have been isolated from a variety of plants (Lodewyckx

et al., 2002), the shoot meristems regions have been considered virtually

free-of-microbial cells (Pirttila¨ et al., 2000). Nevertheless, the presence of

bacteria in micropropagated plants is commonly mentioned as microbial

contamination, which must be prevented and eliminated (George et al.,

2008). Inoculants seem to be successful in micropropagated plants, as

there are few or no other microorganisms with which to compete. There

could be enormous benefits to be gained through the inoculation of

microorganisms into soil-less mixes in which plants are transplanted at

an early stage in their growth. In such cases, when the plantlets were

inoculated, they were more vigorous and had increased drought

resistance, an increased resistance to pathogens, less transplanting

shock, and lower mortality (Barka et al., 2000; Martínez et al., 2003).

Recently, the presence of such bacteria was reported in peach

palm plants (Almeida et al., 2009). Moreover, studies that confirm and

characterize the presence of beneficial endophytic microorganisms in

‘‘axenic’’ plant cultures are even rarer (Almeida et al., 2009; Dias et al.,

2009; Pirttila¨ et al., 2000). Endophytic bacteria sometimes remain covert

or latent with no obvious growth on tissue culture medium (Ewald et al.,

2000) but they can also reduce growth rate, retard rooting, and even

cause plant death (Leifert and Waites, 1992). On the other hand, several

bacterial endophytes have been reported to support growth and increase

the vitality of plants (Glick et al., 1998). The association of beneficial

endophytic bacteria and micropropagated plants may be more frequent

than it is reported, and can lead to positive effects on micropropagation

and cell culture studies (Dias et al., 2009; Pirttila¨ et al., 2000). However,

although the potentiality of these bacteria is extremely high to improve

the micropropagation and acclimatization of micropropagated plants,

only a few attributions are made for endophytic bacteria in such plants.

Dias et al. (2009) has shown that these bacteria can promote the growth

of strawberry plants during the acclimatization process in greenhouse.

Recently, high densities of Paenibacillus spp. in tissue cultures

from different woody plants that had been micropropagated for at least

five years was reported (Ulrich et al. 2008a and b). Endophytic bacteria

belonging to this Paenibacillus group were found in different in vitro

cultures of various poplar, larch, and spruce clones (Ulrich et al. 2008a

and b). Poplar microcuttings inoculated with this Paenibacillus strain

showed significantly more roots and higher root length compared with

control plants (Ulrich et al. 2008a).

Biopriming refers to the use of microbial inoculants, primarily

bacterial and mycorrhizal as propagule priming agents both as in vitro

co-cultures and on transplanting (Nowak and Pruski, 2002). Upon

exposure to stress, the pre-sensitized plant adapt better and faster than

non-primed plants enhancing resistance to plant pathogens in several

economically important horticultural crops (Sharma and Nowak, 1998;

Barka et al., 2002). Similarly, biohardening of tissue culture banana

plantlets with Pseudomonas spp. significantly improved plant growth and

reduced disease severity under field conditions (Smith et al., 2003).

Metabolic profiling via gas chromatography coupled to mass

spectrometry was used to investigate the influence of endophytic bacteria

on shoots of in vitro-grown poplar plants free from culturable endophytic

bacteria by Scherling et al. (2009). The results demonstrate that the

occurrence of an endophytic Paenibacillus strain strongly affects the

composition of the plant metabolites of in vitro-grown poplars.

Biohardening of tissue culture banana plantlets with Pseudomonas

spp. significantly improved plant growth and reduced disease severity

under field conditions (Smith et al., 2003; Jaizme-Vega et al., 2004). In a

study by Harish et al. (2008) micropropagated banana plantlets were

tested for the presence of Banana Bunchy Top Virus by ELISA, DIBA and

PCR and the uninfected plants were biohardened with two rhizobacterial

(Pseudomonas fluorescens, Pf1, CHA0) and endophytic bacterial (EPB5,

EPB22) strains. Plants treated with mixtures of rhizobacterial and

endophytic bacterial formulations viz., EPB5 + EPB22 + Pf1 + CHA0 was

significantly effective in reducing BBTV under field conditions recording

33.33% infection with 60% reduction over control.

2.7.2. Endophytes in field application

Some research has been directed to find endophytes that could

significantly increase the yields in different crops after their inoculation.

To reveal the effects of endophytes, inoculation experiments have been

performed, but it has been a problem to eliminate resident or indigenous

endophytes from plants in order to have bacteria-free plants or seeds.

Functional redundancy of resident endophytes and added inocula may

limit the effects observed from inoculation. Very complex microbial

community-plant interaction, poor rhizosphere competence with

endogenous microorganisms, and bacterial fluctuations with

environmental conditions may also limit the applicability of endophyte

inoculation in the field (Sturz et al., 2000). Furthermore, in the field, the

large abundance and diversity of soil bacteria may be a rich source of

endophytes and, for this, inoculation effects may not be observed. Since

surface disinfection does not remove endophytes, procedures such as

warming and drying seeds have been assayed to diminish bacterial

populations inside (Holland and Polacco, 1994). Tissue culture has also

been used to eliminate or reduce endophytes (Holland and Polacco, 1994;

Leifert et al. 1994).

Three of the 14 endophytes improved soybean nodulation and

plant weight when co-inoculated with B. japonicum (Bai et al. 2002).

Hung et al. (2007) studied the effect of endophytes on soybean

growth and development, I-15 and I-110 had positive effects on root

weights. Maximum shoot dry weight was given by I-73 (213 mg per plant)

of G. max followed by I-109 (203 mg per plant) of G. soja. Both of these

isolates enhanced the total plant biomass by more than 80% over that of

uninoculated control. The percent increase ranged from 9 to 83 with

inoculation.

Jha and Kumar (2007) isolated and characterized endophytic

diazotrophic bacteria from a semi-aquatic grass (Typha australis). The

diazotrophic isolate, GR-3 was tagged with gusA fused to a constitutive

promoter and the resulting transconjugant was inoculated onto

endophyte-free rice variety Malviya dhan-36 seedlings to express cross-

infection ability which resulted in a significant increase in root/shoot

length and chlorophyll a content.

In a report by Long et al. (2008) a Solanum nigrum seedling vigor

assay was carried out to screen the endophytic bacterial isolates for their

PGP ability, using the isolates’ effects on seed germination, root and

hypocotyl growth; 37 of 77 isolates increased seedling vigor. Of these 37

isolates, 22 significantly enhanced seed germination–up to 100%–

compared with untreated controls.

Harish et al. (2009) studied the effect of bio-formulations of

mixtures of the rhizobacterial isolate Pseudomonas fluorescens (Pf1) and

endophytic Bacillus sp. (EPB22) on banana and recorded significant

increase in the yield (53.33%) in the bacterized plants when compared to

the control plants. The mixtures of bacteria also exhibited higher yields

(11.5 kg bunch-1), when compared to chemical (8.8 kg bunch-1) treated

and untreated control (7.5 kg bunch-1). There was a significant increase

(P < 0.05) in the plant morphological and physiological characters like

pseudostem height, pseudostem girth, leaf area, phyllocron, chlorophyll

content, relative water content and rate of transpiration in the

endophytic bacteria treated plants. Populations of endophytic bacteria

also remained high and stable throughout the growing period. Thus,

application of mixtures of rhizosphere and endophytic bacteria increases

yield and has a potential role in inducing resistance against Banana

bunchy top virus.

A study was performed by Stajković et al. (2009) to assess the

effects of non-rhizobial endophytes from the surface sterilized root

nodules of alfalfa (Medicago sativa L.) on alfalfa growth. Co-inoculation of

all non-rhizobial strains with S. meliloti positively influenced nodule

number of alfalfa, but was without significant effect on growth

parameters with respect to inoculation with S. meliloti alone. However,

single inoculation with non-rhizobial strains caused significant increase

in shoot and root parameters compared to uninoculated plants,

indicating that non-rhizobial strains possess some plant growth

promoting potential.

One of the bacterial endophyte, Bacillus subtilis HC8, isolated from

the giant hogweed Heracleum sosnowskyi Manden, significantly

promoted plant growth and protected tomato against tomato foot and

root rot (Malfanova et al., 2011).

2.8. Perspectives

The natural condition of plants seems to be in a close interaction

with endophytes. Endophytic fungi and bacteria evolved biochemical

pathways, resulting in the production of each of the five classes plant

growth hormones (auxins, abscisins, ethylene, gibberellins,and kinetins).

In the endophyte - host interactions the minimum contribution of the

plant to the endophyte is one of providing nutrition. However, the plant

may provide compounds critical for the completion of the life cycle of the

endophyte (Metz et al., 2000).

Endophytes seem promising to increase crop yields, remove

contaminants, inhibit pathogens, and produce fixed nitrogen or novel

substances. The challenge and goal is to be able to manage microbial

communities to favour plant colonization by beneficial bacteria. This

would be amenable when a better knowledge on endophyte ecology and

their molecular interactions is attained. The contributions of this

research field may have economic and environmental impacts

(Rosenbleuth and Martinez Romero, 2006).