20

3.0. REVIEW OF LITERATURE

The following section shows some examples of natural products

obtained from endophytic microbes and their potential in the pharmaceutical

and agrochemical arenas.

3.1. Biodiversity of Endophytes

Verma et al. (2001) reported that a study of the diversity of

endophytic bacteria present in seeds of a deepwater rice variety revealed the

presence of seven types of BOX-PCR fingerprints. In order to evaluate the

plant growth promoting potential, the presence of nitrogenase, indole acetic

acid production and mineral phosphate solubilization were estimated in the

representative BOX-PCR types. The seven representatives of BOX-PCR .The

endophytic colonization this strain was genetically tagged with the reporter

gene, gusA. Histochemical analysis of the seedling grown in hydroponics

showed that, the tagged strain colonized the root surface, root hairs, root cap,

points of lateral root emergence, root cortex and the stelar region. Treatment

of the roots with 2,4-D produced short thickened lateral roots which showed

better colonization by Pseudomonas agglomerans.

21

Bussaban et al. (2001) reported that endophytic fungi were isolated

from apparently healthy organs of the wild ginger Amomum siamense Criab.

Endophytes were relatively common with an isolate prevalence of 70%-83%

at the two sites sampled in the wet and dry seasons. The endophyte

assemblages from the two sites were diverse and comprised 7 Ascomycetes

and 26 mitosporic fungi. Glomerella spp., Xylariaceous fungi, and Phomopsis

spp. were consistently dominant as endophytes on Amomum siamense, but

many rare species and mycelia sterilia were also recorded. Two new

Ascomycetes species. Gaeumannomyces amomi and Leiosphaerella amomi, were

discovered from leaves and rhizomes, respectively, and four species of

Pyricularia, including three new species, were isolated from leaves.

Gamboa et al. (2002) reported that measuring diversity of endophytic

fungi in leaf fragments: does size matter. Since endophytic fungi are poorly

known, especially in the tropics, current estimates of fungal species are

probably conservative. Here we tested strategies for sampling endophytic

fungi in tropical plants. We compared the number of fungi isolated from 400

mm2 leaf pieces that were divided into increasingly small fragments. Leaf

pieces were surface-sterilized, cut into fragments and plated on culture

media. For a given area, cutting leaf pieces into smaller fragments

significantly increased the number of fungal morpho species recovered.

There was a strong linear relationship between size of fragments and

22

number of fungi isolated. By extrapolation, an estimated 16 +/- 3 fungi could

be recovered from a 2 x 2 cm leaf piece, using infinitely small fragments. This

represents a large part of the fungal diversity estimated to exist in leaf

endophytes in a population. We conclude that reducing the size and

increasing the number of leaf fragments will increase the number of fungal

species isolated. This strategy will help to estimate real values of endophytic

fungal diversity.

Krechel et al. (2002) reported that the abundance and diversity of

bacteria isolated from the rhizosphere, phyllosphere, endorhiza, and

endosphere of field grown potato was analyzed. Culturable bacteria were

obtained after plating on R2A medium. The endophytic populations

averaged 103 and 105 CFU.g-1 (fresh wt.) for the endosphere and endorhiza

respectively, which were lower than those for the ectophytic

microenvironments, with 105 and 107 CFU.g-1 (fresh wt.) for the phyllosphere

and rhizosphere, respectively. The occurrence and diversity of potato-

associated bacteria was additionally monitored by a cultivation-independent

approach using terminal restriction fragment length polymorphism analysis

of 16S rDNA. The antagonistic potential of potato-associated bacteria, a total

of 440 bacteria was screened by dual testing for in vitro antagonism towards

the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. The

proportion of isolates with antagonistic activity was highest for the

23

rhizosphere (10%), followed by the endorhiza (9%), phyllosphere (6%), and

endosphere (5%). All 33 fungal antagonists were characterized by testing

their in vitro antagonistic mechanisms, including their glucanolytic,

chitinolytic, pectinolytic, cellulolytic, and proteolytic activity, and by their

BOX-PCR fingerprints. They were screened for their biocontrol activity

against Meloidogyne incognita. Nine isolates belonging to Pseudomonas and

Streptomyces species were found to control both fungal pathogens and

M. incognita and were therefore considered as promising biological control

agents.

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

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

Host range greenhouse studies demonstrated that, 26 of 29 endophytes were

recoverable from at least one host other than corn and sorghum at levels of

up to 5.8 log10 CFU.g-1 (fresh weight). Three prairie and three agronomic

endophytes exhibiting the most promising levels of colonization and an

ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium,

and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and

carbon source utilization analyses.

24

Endophytic fungi were isolated from the stems, petioles, midribs, and

leaves from Doi Suthep-Pui National Park, Thailand. Endophytes were also

isolated from all tissue samples investigated, and taxa included five

ascomycetes, eight anamorphic taxa, and numerous sterile mycelia. Twenty-

six strains were tested for their ability to produce cellulase, mannanase,

proteinase, and xylanase. The ability to produce these enzymes was

distributed amongst the strains tested. Rainforest seedlings supported

a diverse array of endophytes that have a wide range of enzymatic activities

(Lumyong et al., 2002).

Fungal endophytes associated with leaves of woody angiosperms are

especially diverse. Transmitted endophytes of woody angiosperms are

thought to contribute little to host defense. Inoculation of endophyte-free

leaves with endophytes isolated frequently from naturally infected,

asymptomatic hosts significantly decreases both leaf necrosis and leaf

mortality when T. cacao seedlings are challenged with a major pathogen.

Endophyte-mediated protection was greater in mature leaves, which bear

less intrinsic defense against fungal pathogens than do young leaves.

Protection may be mediated by direct interactions of endophytes with foliar

pathogens (Arnold et al., 2003).

25

Verma et al. (2004) reported that colonization ability of the two

endophytic bacteria, isolated from surface sterilized seeds of Jaisurya variety

of deep-water rice viz., Pantoea sp. and Ochrobactrum sp., was compared after

genetically tagging them with a constitutively expressing green fluorescent

protein gene (gfp). Confocal laser scanning microscopy (CLSM) of

hydroponically grown seedlings of Jaisurya rice, inoculated with gfp-tagged

endophytes, revealed that both Pantoea sp. and Ochrobactrum sp. colonized

the intercellular spaces in the root cortex when inoculated separately.

Colonization by gfp-tagged Ochrobactrum sp. was severely inhibited when

co-inoculated with an equal number (105 CFU. ml -1) of wild type Pantoea sp.,

but the converse was not true. Pantoea sp. was a more aggressive endophytic

colonizer of its host than Ochrobactrum sp.

Endophytic and epiphytic bacteria were isolated from two soybean

cultivars (Foscarin and Cristalina). Significant differences were observed in

bacterial population densities in relation to season of isolation, soybean

growth phase and the tissues from which the isolates were obtained. The

isolates were identified by partial 16S rDNA sequence analysis, with most of

the isolates belonging to the Pseudomonaceae, Burkholderiacea and

Enterobacteriaceae groups. We also found that 60% of endophytic and 69%

of epiphytic isolates that produced IAA and solubilized mineral phosphate

were also able to fix nitrogen in vitro. The soybean-associated bacteria

26

showing characteristics related to plant growth promotion were identified as

belonging to the genera Pseudomonas, Ralstonia, Enterobacter, Pantoea and

Acinetobacter (Kuklinsky-Sobral et al., 2004).

Endophytic fungi associated with cacti in Arizona 21 cactus species

were screened for the presence of fungal endophytes. 900 endophyte isolates

belonging to 22 fungal species were isolated. Cylindropuntia fulgida had the

maximum endophyte species diversity, while C. ramosissima harboured the

maximum number of endophyte isolates. Alternaria sp., Aureobasidium

pullulans, and Phoma spp. were isolated from several cactus species. The

diversity of the endophyte assemblages was low and no host specificity

among endophytes was observed. However, the frequencies of colonization

of the few endophyte species recovered were high and comparable to those

reported for tropical plant hosts. Species of Colletotrichum, Phomopsis, and

Phyllosticta, which are commonly isolated as endophytes from plants of more

mesic habitats, were absent from these cacti (Suryanarayanan et al., 2005).

Fungal endophytes in five medicinal plant species from Kudremukh

Range, Western Ghats of India was reported. Eighteen species of endophytic

fungi were isolated from bark, stem and leaf segments. The dominant

endophytic fungal species isolated from these plant species were Curvularia

clavata, C. lunata, C. pallescens and Fusarium oxysporum. The highest species

27

richness as well as frequency of colonization of endophytic fungi was found

in the leaf segments, rather than the stem and bark segments, of the host

plant species. The greatest number of endophytic fungal species were found

within Callicarpa tomentosa (11 species), whereas Lobelia nicotinifolia harbored

the lowest number of fungal endophytes (5 species) (Raviraja, 2005).

Salgado-Salazar and Cepero de Garcia (2005) reported that

endophytic fungi associated with rose plants (Rosa hybrida) in Colombia.

Endophytic fungi were isolated from healthy leaves of ten ornamental roses

plants from gardens cultured in malt extract, peptone, yeast extract agar

plates (MPY). We sampled 560 leaves fragments, 56 per sample. Endophytic

fungi comprised 92 isolates (16.4%); of these isolates, 41 were classified as

sterile mycelium (without reproductive structures that allowed their

identification), 31 isolates were identified to genus or to species, and 20

isolates could not be identified at all. Nigrospora oryzae, Aureobasidium spp,

Acremonium spp were the identified endophytic fungi. The fungi

Nodulisporium sp, Gliocladium virens, Cladosporium sp, Alternaria sp, Phoma sp

and Chaetomium globosum were represented by one isolate each.

Vega et al. (2005) reported that eighty-seven culturable endophytic

bacterial isolates in 19 genera were obtained from coffee plants collected in

Colombia (n = 67), Hawaii (n = 17), and Mexico (n = 3). Both Gram positive

28

and Gram negative bacteria were isolated, with a greater percentage (68%)

being Gram negative. Tissues yielding bacterial endophytes included adult

plant leaves, various parts of the berry (e.g., crown, pulp, peduncle and

seed), and leaves, stems, and roots of seedlings. Some of the bacteria also

occurred as epiphytes. The highest number of bacteria among the berry

tissues sampled was isolated from the seed, and includes Bacillus,

Burkholderia, Clavibacter, Curtobacterium, Escherichia, Micrococcus, Pantoea,

Pseudomonas, Serratia, and Stenotrophomonas.

Fungal endophytes were isolated from the inner bark segments of

ethnopharmaceutically important medicinal tree species, namely Terminalia

arjuna, Crataeva magna, Azadirachta indica, Holarrhena antidysenterica,

Terminalia chebula, and Butea monosperma (11 individual trees), growing in

different regions of southern India. Forty-eight fungal species were

recovered from 2200 bark segments. Mitosporic fungi represented a major

group (61%), with ascomycetes (21%) and sterile mycelia (18%) the next major

groups. Species of Fusarium, Pestalotiopsis, Myrothecium, Trichoderma,

Verticillium, and Chaetomium were frequently isolated. Exclusive fungal taxa

were recovered from five of the six plant species considered for the study of

endophytic fungi (Tejesvi et al., 2006).

29

Burch and Sarathchandra (2006) reported that the genera,

abundance, and activities of endophytic bacteria in field-grown white clover

(Trifolium repens) and the fate of introduced antibiotic-tolerant bacteria in

white clover tissues were investigated. Pseudomonas, Pantoea, and

Corynebacterium were the most frequently isolated endophytic bacteria

genera, whereas Xanthomonas, Microbacterium, and Cellulomonas occurred less

frequently. The average bacterial populations in stolons and roots were

approximately 100,000 colony-forming units (CFU). The strains used,

sourced from white clover (endophytic and rhizoplane) and organic

compost, had previously shown growth promotion potential of white clover

seedlings by increasing plant mass and decreasing nematode numbers. The

main route of bacterial entry into seedlings was through stomata and that

bacterium remained in the aerial parts of plants rather than migrating to the

roots.

3.2 Endophytic Microbial Products as Antibiotics

Antibiotic are defined as low-molecular weight organic natural

products made by microorganisms that are active at low concentration

against other microorganisms. Often endophytes are a source of these

antibiotics. Natural products from endophytic microbes have been observed

to inhibit or kill a wide variety of harmful disease-causing agents including

30

but not limited to phytopathogens, as well as bacteria fungi, viruses and

protozoan that affect humans and animals (Demain, 1981).

Cryptosporiopsis quercina is the imperfect stage of Pezicula cinnamomea,

a fungus commonly associated with hardwood species in Europe. It was

isolated as an endophyte from Tripterigeum wilfordii, a medicinal plant native

to Eurasia. On Petri plates, C. quercina demonstrated excellent antifungal

activity against some important human fungal pathogens – Candida albicans

and Trichophyton spp. A unique peptide antimycotic, termed cryptocandin

was isolated and characterized from C. quercina (Strobel et al., 1999). This

compound contains a number of peculiar hydroxylated amino acids and a

novel amino acid 3 hydroxyl hydroxyl methyl praline. The bioactive

compound is related to the known antimycotics the echino candins and the

pneumocandins. As it is generally true not one but several bioactive and

related compounds are produced by a microbe. Thus, other antifungal agents

related to cryptocandin are also produced by C. quercina. Cryptocandin

is also active against a number of plant-pathogenic fungi, including

Sclerotinia sclerotionum and Botrytis cinerea . Crytocandin and its related

compounds are currently being considered for use against a number of fungi

causing diseases of skin and nails (Walsh, 1992).

31

Cryptocin, a unique tetramic acid, is also produced by C. quercina.

These unusual compounds possess potent activity against Pyricularia oryzae

as well as a number of other plant-pathogenic fungi. The compound was

generally ineffective against a general array of human pathogenic fungi.

Nervertheless, with MICs of this compound for P. oryzae being 0.39 µg.ml-1.

This compound is being examined as a natural chemical control agent for

rice blast and is being used as a base model to synthesize other antifungal

compounds (Li et al., 2000).

The ecomycins are produced by Pseudomonas viridiflava. Pseudomonas

viridiflava is a member of a group of plant-associated fluorescent bacteria. It

is generally associated with leaves of many grass species and is located on

and within the tissues (Miller et al., 1998). The ecomycins represent a family

of novel lipopeptides and have molecular weights of 1,153 and 1,181. Besides

common amino acids such as alanine, serine, threonine and glycine, such

unusual amino acids are also involved in the structure of the ecomycins

including homoserine and human-pathogenic fungi as Cryptococcus

neoformans and Candida albicans.

Another group of antifungal compounds is the pseudomycins,

produced by a plant-associated Pseudomonad (Harrison et al., 1991). The

pseudomycins represent a family of lipopetides that are active against a

32

variety of plant and human-pathogenic fungi. Some of the notable target

organisms include Candida albicans, Cryptococcus neoformans and a variety of

plant-pathogenic fungi including Ceratocystis ulmi (the Dutch elm disease

pathogen) and Mycosphaerella fijiensis (the casual agent of Black Sigatoka

disease of banana). The key conserved part of the pseudomycins is a cyclic

nonopeptide. The terminal carboxyl group of D-chlorothreonine closes the

macro cyclic ring on the OH group of the N-terminal serine. Variety is added

to this family of compounds by virtue of N-acetylation by one of a series of

fatty acids, including 3, 4-dihydroxydecanoate or 3-hydroxytetradecanoate

and others (Ballio et al., 1994). The pseudomycins contain several

nontraditional amino acids including D-chlorothreonine D-hydroxy aspartic

acid and both D- and L-diaminobutryic L-hydroxy aspartic acid and both D-

and L-diaminobutryic acid. The molecules are candidates for use in human

medicine especially after structural modification has successfully removed

mammalian toxicity (Zhang et al., 2001). Although the pseudomycins are also

effective against a number of ascomycetous fungi, they are also being

considered for agricultural use.

As mentioned elsewhere, Pestalotiopsis microspora is a common

rainforest endophyte (Strobel et al., 2002). It turns out that enormous

biochemical diversity does exist in this endophytic fungus, and as such there

seem to be many secondary metabolites produced by a myriad of strains of

33

this widely dispersed fungus. One such secondary metabolite is ambuic acid,

an antifungal agent which has been recently described from several isolates

of P. microspora found as representative isolates in many of the world’s

rainforests (Li et al., 2001). In fact, this compound and another endophyte

product, terrain, have been used as models to develop new solid-state

nuclear magnetic resonance (NMR) tensor methods to assist in the

characterization of molecular stereochemistry of organic molecules

(Harper et al., 2001).

A strain of P. microspora was also isolated from the endangered tree

Torreya taxifolia and produces several compounds that have antifungal

activity, including pestaloside, an aromatic β glucoside and two pyrones:

pestalopyrone and hydroxypestalopyrone (Lee et al., 1995). These products

also possess phytotoxic properties. Other newly isolated secondary products

obtain from P. microspora includes two new caryophyllene sesquiterpenes-

Pestalotiopins A and B (Pulici et al., 1996). Other novel sesquiterpenes

produced by this fungus are 2-∞-hydroxydimeninol and a highly

functionalized humunlane. Variation in the amount and kinds of products

found in this fungus depends on both the cultural conditions of the

organisms as well as the original plant source from which it was isolated.

34

Newly described species of Pestalotiopsis, namely, Pestalotiopsis jesteri,

from the Sepik River area of Papua New Guinea produces jesterone and

hydroxyl-jesterone, which exhibit anti fungal activity against a variety of

plant-pathogenic fungi (Li and Strobel, 2001). Jesterone, subsequently has

been prepared by organic synthesis with complete retention of biological

activity (Hu et al., 2001).

Phomopsichalasin, a metabolic from an endophytic Phomopsis sp.,

represents the first cytochalasin-type compound with a three-ring system

replacing the cytochalasin macrolide ring. This metabolite mainly exhibits

antibacterial activity in disk diffusion assays (at a concentration of 4 µg/disk)

against Bacillus subtilis (12-mm zone of inhibition). Salmonella enterica serovar

gallinarum (11-mm zone of inhibition) and Staphylococcus aureus (8-mm zone

of inhibition). It also displays a moderate activity against the yeast Candida

tropicalis (8-mm zone of inhibition) (Horn et al., 1995).

An endophytic Fusarium sp. from the plant Selaginella pallescens,

collected in the Guanacaste Conservation Area of Costa Rica, was screened

for anti fungal agent was isolated from the culture broth of the fungus and

showed potent activity against C. albicans in agar diffusion assays performed

in fungal lawns (Brady and Clardy, 2000).

35

Colletotric acid a metabolic of Colletotriclum gloeosporioides and

endophytic fungus in Artemisia mongolica displays antimicrobial activity

against bacteria as well as against the fungus Helminthsporium sativum

(Zou et al., 2000). Another Collectotrichum sp. isolated from Artemisia annua,

produces bioactive metabolites that showed varied antimicrobial activity as

well. A. annua is a traditional Chinese herb that is well recognized for its

synthesis of artemisinin (an antimalarial drug) and its ability to Chum sp.

found in A. annua produced not only metabolites with activity against

human-pathogenic fungi and bacteria but also metabolites that were

fungistatic to plant-pathogenic fungi (Lu et al., 2000).

In addition of plant such as A.annua producing antimalarial

compounds, some endophytes have shown powerful activity against

protozoan diseases as well. Wide-spectrum antibiotics are produced by

Streptomyces sp. strain NRRL 30562 an endophytein K. nigricans. These

antibiotics called munumbicins, posses widely differing biological activities

depending on the target organism. In general, the munumbicins demonstrate

activity against gram-positive bacteria such as Bacillus anthraces and

multidrug-resistant M. tuberculosis as well as a number of other drug-

resistant bacteria. However, the most impressive biological activity of

any of the munumbicins in that of munumbicin D against the malarial

parasite Plasmodium falciparum, for which the 50% inhibitory concentration is

36

4.5 0.07ml (Castillo, 2002). The munumbicins are highly functionalized

peptide, each containing threonine, aspartic acid (or asparaginic) and

glutamic acid (or glutamine). Since the peptides are yellowish orange in

color, they also contain one or more chromophoric groups. Their masses

range from 1.269 to 1.326 Da. The isolation of an endophytic Streptomyces sp.

strain NRRl 30562, represents an important clue in providing one of the first

examples of plants serving as reservoirs of actinomycetes, which are the

world’s primary source of antibiotics. However, in the past, virtually all of

them used for modern antibiotic production had been isolated from soils.

Now, more than 30 of these are on hand as endophytes and many possess

antibiotic activity. In fact, endophytic actinomycetes are now being tested

and seriously considered for use in controlling plant disease (Kunoh,

2002).

Another endophytic streptomycete (NRRL 30566) from a fern-leaved

Grevillea tree (Grevillea pteridifolia) growing in the Northern Territory of

Australia produces novel antibiotics called kakadumycins. Each of these

antibiotics contains by virtue of their amino acid compositions alanine,

serine, and an unknown amino acid. Kakadumycin A has wide spectrum

antibiotic activity similar to that of munumbicin D especially against gram-

positive bacteria and it generally displays better bioactivity than

echinomycin. For instance, the MIC of kakdumycin A for B. anthracis strains

37

is 0.2 to 0.3 µg per ml in contrast to that of echinomycin which is 1.0 to 1.2 µg

per ml. both echinomycin and kakdumycin A have impressive activity

against P. falciparum with 50% lethal does in the range of 7 to 10 ng.ml-1.

Kakdumycin A and echinomycin are related by virtue of their very similar

chemistries (amino acid content and quinoxaline rings) but differ slightly

with respect to their elemental compositions, aspects of their spectral

qualities and biological activities. This is yet another example of an

endophytic acntinomycete having promising antibiotic properties

(Castillo et al., 2003).

Rodrigues et al. (2000) reported that, culture broth extracts of

Guignardia sp., Phomopsis sp. and Pestalotiopsis guepinii fungal endophytes

from Spondias mombin (Anacardiaceae) were tested against fourteen

organisms, including actinomycetes, Gram-negative and Gram-positive

bacteria, yeast, and filamentous fungi. All fungal extracts inhibited

actinomycete growth. Guignardia sp. was also active against Escherichia coli,

Staphylococcus aureus, Saccharomyces cerevisiae, Geotrichum sp. and Penicillium

canadensis. Culture extracts of P. guepinii were active against S. cerevisae,

while strains of Phomopsis sp. showed a pronounced antifungal effect against

Cladosporium elatum, Mycotypha sp. and S. cerevisae.

38

3.3 Antiviral Compounds

Another fascinating use of antibiotic products form endophytic fungi

is the inhibition of viruses. Two novel human cytomegalovirus protease

inhibitors, cytonic acids A and B have been isolated from the solid-state

fermentation of the endophytic fungus Cytonaema sp. their structures as

p-tridepside isomers were elucidated by mass spectrometry and NMR

methods. It is apparent that, the potential for the discovery of compounds

from endophytes having antiviral activity is in its infancy. The fact, however,

that some compounds have been found is promising. The main limitation in

compound discovery is probably related to the absence of appropriate

antiviral screening systems in most compound discovery programs

(Guo et al., 2000).

3.4 Volatile Antibiotics from Endophytes

Muscodor albus is a newly described endophytic fungus obtained

from small limbs of Cinnamomum zeylanicum (cinnamom tree)

(Worapong et al., 2002). This xylariaceaous (non-spore-producing) fungus

effectively inhibits and kills certain other fungi and bacteria by producing a

mixture of volatile compounds. The majority of these compounds have been

identified by gas chromatography-mass spectrometry, synthesized or

acquired, and then ultimately made into an artificial mixture. This mixture

39

mimicked the antibiotic effects of the volatile compounds produced by the

fungus. It was also used to gain positive identification of the ingredients of

the fungal volatile compounds (Strobel et al., 2001). Each of the five classes of

volatile compounds produced by the fungus had some inhibitory effect

against the test fungi and bacteria but none was lethal. However, collectively

they acted synergistically to cause death in a broad range of plant and

human pathogenic fungi and bacteria. The most effective class of inhibitory

compounds was the esters of which isoamyl acetate was the most

biologically active. The ecological implications and potential practical

benefits of the mycofumigation effects of M. albus are very promising given

the fact that soil fumigation utilizing methyl bromide will soon be illegal in

the United States. The potential use of mycofumigation to treat soil, seeds

and plants may soon be a reality. In fact, this organism is already on the

market for the decontamination of human wastes.

Using M. albus as a screening tool, it has now been possible to isolate

other endophytic fungi that produce volatile antibiotics. The newly described

Muscodor rosens was twice obtained from tree species growing in the

Northern Territory of Australia. This fungus is just as effective in causing

inhibition and death of test microbes in the laboratory as M. albus (Worapong

et al., 2001). In addition, for the first time, another muscodor species, a

Gliocladium sp. was discovered to be a volatile antibiotic producer.

40

The volatile components of this organism are totally different from those of

either M. albus or M. roseus. Infact, the most abundant volatile inhibitors is

annulene, formerly used as a rocket and fuel and discovered for the first time

as a natural product in an endophytic fungus. The bioactivity of the volatile

compounds of Gliocladium sp. is not as good or comprehensive as those of the

Muscoldor spp (Stinson et al., 2003).

Ezra et al., (2004) reported that Muscodor albus, an endophytic fungus

originally isolated from Cinnamomum zeylanicum, produces a mixture of

volatile organic compounds (VOCs) in culture and its spectrum of

antimicrobial activity is broad. Using the original isolate of M. albus as a

selection tool to find other culturally and biochemically unique wild-type

isolates including Grevillea pterifolia (fern-leafed grevillea), Kennedia

nigriscans (snake vine) and Terminalia prostrata (nanka bakarra) .Interestingly,

none of the new isolates had a culture morphology that was identical to the

original isolate, nevertheless each possessed hyphal characteristics that

resembled that isolate. Furthermore, their ITS-5.8S rDNA sequences were

96-99% identical to that of M. albus and the isolates were considered M. albus

on the basis of the DNA sequence data. However, the VOCs produced by

these new isolates greatly differed in quality from the original strain by

virtue of the production of naphthalene, naphthalene, 1,1'-oxybis-, and one

or more other compounds. In bioassays with a range of test micro-organisms,

41

including fungi and bacteria, each isolate possessed biological activity but

the range of activity was great. Artificial mixtures of some of the VOCs

mimicked the effects of the VOCs of the fungus.

3.5 Endophytic Fungal Products as Anticancer Agents

Strobel (2003) reported that an increase in the number of people in the

world having health problems caused by various cancers, drug-resistant

bacteria, parasitic protozoans, and fungi is a cause for alarm. An intensive

search for newer and more effective agents to deal with these disease

problems is now under way and endophytes are a novel source of potentially

useful medicinal compounds.

Paclitaxel and some of its derivatives represent the first major group

of anticancer agents that is produced by endophytes. Paclitaxel, a highly

functionalized diterpenoid is found in each of the world’s yew species

(Suffness, 1995). The mode of action of paclitaxel is to preclude tubulin

molecules for depolymerizing during the processes of cell divisions. This

compound is the world’s first billion-dollar anticancer drug. It is used to

treat a number of other human tissue proliferating diseases as well. The

presence of paciltaxel in yew species prompted the study of their

endophytes. By the early 1990s, however, no endophytic fungi had been

isolated from any of the world’s representative yew species. After several

42

T. andreanae, was discovered in T. brevifolia (Strobel et al., 1993). The most

critical line of evidence for the presence of paclitaxel in the culture fluids of

this fungus was the electro spray mass spectrum of the putative paclitaxel

isolated form T. andreanae. In electro spray mass spectroscopy, paclitaxel

usually gives two peaks, one at a mass of 854, which is (M + H), and the

other at 876 which is (M +Na) and fungal paclitaxel had a mass spectrum

identical to that of authentic paclitaxel. Then C labeling studies irrefutably

showed the presence of fungus derived paclitaxel in the culture medium

(Stierle et al., 1993). This early work set the stage for a more comprehensive

examination of the ability of other Taxus species and other plants to yield

endophytes producing pacitaxel.

Some of the most commonly found endophytes of the world’s yews

are Pestalotiopsis spp. One of the most commonly isolated endophytic species

is P. microspora. An examination of the endophytes of Taxus wallichiana

yielded P. microspora (Strobel, 2002) and a preliminary monoclonal antibody

test indicated that it might produce paclitaxel. After preparative thin-layer

chromatography, a compound was isolated and shown by spectroscopic

techniques to be paclitaxel. Labeled shown by spectroscopic techniques to be

paclitaxel. Labeled paciltaxel was produced by this organism from several

precursors that had been administered to it (Strobel et al., 1996).

Furthermore, several other P. microspora isolates were obtained from bald

43

cypress in South Carolina and also shown to produce paciltaxel

(Li et al., 1996). This was the first indication that endophytes residing in

plants other than Taxus spp. were producing paclitaxol. Therefore, a specific

search was conducted for paclitaxel producing endophytes on continents not

known for any indigenous Taxus spp. This included an examination of the

prospects that paclitaxel producing endophytes exist in South America and

Australia. From the extremely rare, and previously thought to be extinct

Wollemi pine (Wollemia nobilis). Pestalotiopsis guepini was isolated, which was

shown to produce paclitaxel (Strobel et al., 1997). Also, quite surprisingly, a

rubiaccous plant Maguireothamnus speciosus, yielded a novel fungs,

Seimatoantlerium tepuiense, that produces paclitaxel. This endemic plant

grows on the tops of the tepuis in the Venzuelan-Guyana region in

southwestern Venezuela. Furthermore, fungal paclitaxel production has also

been noted in a Periconia sp. and in Seimatoantlerium nepalense, another novel

endophytic fungal species (Bashyal et al., 1999). Simply it appears that, the

distribution of those fungi making paclitaxel is worldwide and not confined

to endophytes of yews. The ecological and physiological explanation for the

wide distribution of fungi that make paclitaxel seems to be related to the fact

that paclitaxel is a fungicide and the organisms with the most sensitivity to it

are plant pathogens such as Phythium spp. and Phytophthora spp. These

pythiaceous organisms are some of the world’s most important plant

44

pathogens and are strong competitors with endophytic fungi for niches

within plants. In fact, their sensitivity to paclitaxel is based on their

interaction with tubulin in a manner identical to that in rapidly dividing

human cancer cells (Schiff et al., 1980). Thus, bonafide endophytes may be

producing paclitaxel to protect their respective host plant from degradation

and disease caused by these pathogens.

Earlier investigators have also made observations on paclitaxel

production by a Tubercularia sp. the discovery of paclitaxel production by a

Tubercularia sp. isolated from southern Chinese yew in the Fujian province of

southeastern China. At least three endophytes of T. wallichiana produce

paclitaxel, including Sporomia minima and a Trichothecium sp. (Shrestha et al.,

2001). By the use of high-performance liquid chromatography and

electrospray mass spectroscopy, paclitaxel has been discovered in Corylus

avellana cv. Gasaway . Several fungal endophytes of filbert produce paclitaxel

production by all endophytes of filbert produce paclitaxel in culture. It is

important to note, however, that paclitaxel production by all endophytes in

culture is in the range of submicrograms to micrograms per liter. Also

commonly endophytic fungi will attenuate paclitaxel production in

attenuated cultures if certain activator compounds are added to the medium

(Li et al., 1998). Efforts are being made to determine the feasibility of making

microbial paclitaxel a commercial possibility.

45

Torreyanic acid, a selectively cytotoxic quinine dimmer (anticancer

agent) was isolated from a P. microspora strain. This strain was originally

obtained as an endophyte associated with the endangered tree T. taxifolia

(Floria torreya) as mentioned above (Lee et al., 1996). Torreyanic acid was

tested in several cancer cell lines, and it demonstrated 5 to 10 times more

potency in those lines that are sensitive to protein kinase C against and

causes cell death by apoptosis. Recently, a complete synthesis of torreyanic

acid has been successfully completed using the application of a biomimetic

oxidation-dimerization cascade (Li et al., 2003).

The alkaloids are also commonly found in endophytic fungi. Such

fungal genera as Xylaria, Phoma, Hypoxylon and Chalara are representative

producers of a relatively large group substances known as the cytochalasins,

of which over 20 are now known. Many of these compounds possess

antitumor and antibiotic activities, but because of their cellular toxicity they

have not been developed into pharmaceuticals. Three novel cytochalasins

have recently been reported from a Rhinocladiella sp. as an endophyte on

Tripterygium wilfordii. These compounds have antitumour activity and have

been identified as cyhtochalasins. Thus, it is not uncommon to find one or

more cytochalasins. Thus, it is nor uncommon to find one or more

cytochalasins in endophytic fungi and workers in the field need to be alerted

to the fact that redundancy in discovery does occur with certain groups of

46

organisms on which previous studies have already established the chemical

identity of major biologically active compounds. For instance, as with the

cytochalasins, they are commonly associated with the xylariacaeous fungi

(Wagenaar et al., 2000).

Huang et al., (2001) reported that antitumor and antifungal activities

in endophytic fungi isolated from pharmaceutical plants Taxus mairei,

Cephalataxus fortunei and Torreya grandis. Antitumor activity was studied by

the MTT assay and antifungal activity was determined by observing fungal

growth inhibition. 13.4% of endophytic fungi fermentation broths displayed

cytotoxic activity on HL-60 cells at and below a dilution of 1:50, and 6.4% on

KB cells. 52.3% of endophytic fungi fermentation broths displayed growth

inhibition on at least one pathogenic fungi, such as Neurospora sp.,

Trichoderma sp. and Fusarium sp. Among all endophytic fungi isolated, the

genus Paecilomyces sp. has the highest positive rate of antitumor and

antifungal activity. These results indicate that endophytic fungi could be a

promising source for antitumor and antifungal bioactive agents.

Liu et al. (2007) reported that bioactivity of endophytic actinomycetes

from medicinal plants and secondary metabolites from strain D62. Based on

genetic recombination of the endophytes novel natural products are

screened. Antimicrobial and antitumor activities of 165 actinomycetes

47

isolated from medicinal plants collected from Xishuangbanna were tested by

agar diffusion method and WST-8 assay respectively. The results showed

that over 42% of the isolates exhibited antagonism against pathogenic

strains, and 54.5% displayed excellent inhibition against mouse melanoma

cell line B16 or/and human alveolar epithelial cell line A549. D62 was

separated from isolated Streptomyces spp. by resin adsorption, silica-gel

column and sephadex chromatography, etc. Based on all these results, it is

convinced that endophytic actinomycetes are a promising resource for

bioactive natural product discovery.

3.6 Products from Endophytes as Antioxidants

Two compounds pestacin and isopestacin have been obtained from

culture fluids of P. microspora, an endophyte isolated from a combretaceaous

plant. Terminalia morobensis growing in the Sepik River drainage of Papua

New Guinea. Both pestacin and isopestacin display antimicrobial as well as

antioxidant activity. Isopectacin was suspected of antioxidant activity based

on its structural similarity to the flavonoids. Electron spin reasonance

spectroscopy measurements confirmed this antioxidant activity; the

compound is able to scavenge superoxide and hydroxyl free radicals in

solution (Strobel, 2002). Pestacin was later described from the same culture

fluid, occurring naturally as a racemic mixture and also possessing potent

48

antioxidant activity. Proposed antioxidant activity of pestacin arose

primarily via elevage of a usually reactive C—H bond and to a lesser extent,

though O—H abstraction. The antioxidant activity of pestacin is at least 1

order of magnitude greater than that of trolox a vitamin E derivative

(Harper et al., 2003).

3.7 Products of Endophytes with Insecticidal Activities

Bioinsecticides are only a small part of the insecticide field but their

marker is increasing (Demain, 2000). Several endophytes are known to have

anti-insect properties. Nodulisporic acids, novel indole diterpenes that

exhibit potent insecticidal properties against the larvae of the blowfly, work

by activating insect glutamate-gated chloride channels. The first nodulisporic

compounds were isolated from an endophyte, a Nodulisporium sp., from the

plant Bonia daphnoides. This discovery has since resulted in an intensive

search for more Nodulisporium spp. or other producers or more-potent

nodulisporic acid analogues. Insect toxins have also been isolated from an

unidentified endophytic fungus from wintergreen (Gaultheria procumbens).

The two new compounds, 5-hydroxy-2 (1-hydroxy, 5-methyl-4-hexenyl)

benzofuran both show toxicity to spruce budworm and the latter is also toxic

to the larvae of spruce budworm. Another endophytic fungus Muscodor

vitigenus, isolated from aliana (Paulina paullinioides) yields naphthalene as its

49

major product. Naphthalene the active ingredient in common mothballs is a

widely exploited insect repellant. M. vitigenus shows promising preliminary

results as an insect deterrent and has exhibited potent insect repellency

against the wheat stem sawfly (Ceplus cincus) (Daisy et al., 2002). As the

world becomes worry of ecological damage done by synthetic insecticides

endophytic research continues for the discovery of powerful, selective and

safe alternatives.

3.8 Antidiabetic Agents from Rainforest Fungi

A nonpeptidal fungal metabolite was isolated from an

endophytic fungus (Pseudomassaria sp). Collected from an African rainforest

near Kinshasa in the Democratic Republic of the Congo. This compound acts

as insulin mimetic and, unlike insulin, is not destroyed in the digestive tract

and may be given orally. Oral administration of L-783, 281 to two mouse

models of diabetes resulted in significant lowering of blood glucose levels.

These results may lead to new therapies for diabetes (Zhang, 1999).

3.9 Immunosuppressive compounds from Endophytes

Immunosuppressive drugs are used today to prevent allograft

rejection in transplant patients and in the future they could be used to treat

autoimmune disease such as rheumatoid arthritis and insulin-dependent

diabetes. The endopytic fungus Fusarium subglutinana, isolated from

50

T. wilfordii, produces the immunosuppressive but noncytotoxic diterpene

pyrone subglutinol A and B. Subglutinol A and B are equipotent in the

mixed lymphocyte reaction assay and thymocyte proliferation assay with a

50% inhibitory concentration of 0.1µM. In the same assay systems the famed

immunosuppressant drug cyclosporine is roughly as potent in the mixed

lymphocyte reaction assay and 10-4 more potent in the thymocyte

proliferation assay. Still, the lack of toxicity associated with subglutinols

A and B suggests that they should be explored in greater detail

(Lee et al., 1995).

The microbiology Department at Sandoz Ltd. developed a computer –

aided evaluation program to screen and evaluate fungi for bioactivity. The

program can recognize and eliminate from study common fungi producing

known compounds and thereby direct attention to the evaluation of rare

samples which are more likely to produce metabolites with novel bioactivity.

This approach resulted in the discovery of the fungus Tolypocladium inflatum

from which cyclosporine, a hugely beneficial immunosuppressant, was

isolated (Borel and Kis, 1991). This example perfectly depicts the current aim

of many investigators to seek out rate endophytes from interesting and

uncommon hosts and environments.

51

3.10 Molecular Biological studies

Of some compelling interest is an explanation as to how the genes

for paclitaxel production may have been acquired by P. microspora

(Miller et al., 1998). Although the complete answer to this question is no at

hand. Some other relevant genetic studies have been done with this

organism. P. microspora Ne 32 is one of the most easily genetically

transformable fungi that have been studies to date. In vivo addition of

telomeric repeats to foreign DNA generates extra chromosomal DNAs in this

fungus (Long et al., 1998). Repeats of the teloemeric sequence5’-TTAGGG-3’

were appended to nontelomeric transforming DNA termini. The new DNAs

Carrying foreign genes and the telomeric repeats replicated independently of

the chromosome and expressed the information carried by the foreign genes.

The addition of telomoeric repeats to foreign DNA is unusual among fungi.

This finding may have important implications in the biology of P. microspora

since it explains at least one mechanism by which new DNA can be captured

by this organism and eventually expressed and replicated. Such a

mechanism may being to explain how the enormous biochemical variation

may have arisen in this fungus (Li et al., 1996). Also, this initial work

represents a framework to aid in the understanding how this fungus may

adapt itself to the environment of its plant hosts and suggests that the uptake

of plants DNA into is own genome may occur.

52

Iniguez et al. (2005) reported that bacterial endophytes reside within

the interior of plants without causing disease or forming symbiotic

structures. Some endophytes, such as Klebsiella pneumoniae 342 (Kp342),

enhance plant growth and nutrition. Several lines of evidence are presented

here to support the hypothesis that plant defense response pathways

regulate colonization by endophytic bacteria. Addition of ethylene, a signal

molecule for induced systemic resistance in plants, decreased endophytic

colonization in Medicago spp. This ethylene-mediated inhibition of

endophytic colonization was reversed by addition of the ethylene action

inhibitor, 1-methylcyclopropene. Mutants lacking flagella or a component of

the type III secretion system of Salmonella pathogenicity island 1 (TTSS-SPI1)

colonize the interior of Medicago spp., Flagella act primarily through SA-

independent responses (compromising SA accumulation still affected

colonization in the absence of flagella). Removal of a TTSS-SPI1 effector

resulted in hypercolonization regardless of whether the genotype was

affected in either SA-dependent or SA-independent responses.

Berg et al. (2005) reported that differences between endophytic and

ectophytic bacterial communities with stress on antagonistic bacteria, were

studied by comparing the composition of communities isolated from the

rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato

plants using a multiphasic approach. Terminal restriction fragment length

53

polymorphism analysis of 16S rDNA of the bacterial communities revealed

discrete microenvironment-specific patterns. To measure the antagonistic

potential of potato-associated bacteria, a total of 2648 bacteria were screened

by dual testing of antagonism to the soilborne pathogens Verticillium dahliae

and Rhizoctonia solani. The rhizosphere and endorhiza were the main

reservoirs for antagonistic bacteria and showed the highest similarity in their

colonisation by antagonists. The most prominent species of all

microenvironments was Pseudomonas putida, and rep-PCR with BOX primers

showed that these isolates showed microenvironment-specific DNA

fingerprints. Evaluation of the bacterial isolates for biocontrol potential based

on fungal antagonism and physiological characteristics resulted in the

selection of five promising isolates from each microenvironment. The most

effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.

3.11. Transformation in endophytes

Endophyte taxa that are most frequently reported tend to belong to

fungal groups composed of morphologically similar endophytes and

parasites. Thus, it is plausible that endophytes are known (i.e., described)

parasites in a latent phase within the host. If this null hypothesis were true,

endophytes would represent neither additional fungal diversity distinct from

parasite diversity nor a symbiont community likely to be novel ecologically.

54

To be synonymous with parasites of the host, endophytes should at least be

most closely related to those same parasites. Ganley et al. (2004) reported

that, seven distinct parasites of Pinus monticola do not occur as endophytes.

The majority of endophytes of P. monticola (90% of 2,019 cultures) belonged

to one fungal family, the Rhytismataceae. However, not a single

Rhytismataceous endophyte was found to be most closely related by

sequence homology to the three known Rhytismataceous parasites of

P. monticola. Similarly, neither endophytic Mycosphaerella nor endophytic

Rhizosphaera isolates were most closely related to known parasites of

P. monticola. Morphologically, the endophytes of P. monticola can be

confounded with the parasites of the same host. However, they are actually

most closely related to, but distinct from, parasites of other species of Pinus.

If endophytes are generally unknown species, then estimates of 1 million

endophytes (i.e., approximately 1 in 14 of all species of life) seem reasonable

(Ganley et al., 2004).

Promputtha et al., (2007) reported that fungal endophytes and

saprotrophs generally play an important ecological role within plant tissues

and dead plant material. Several reports based solely on morphological

observations have postulated that there is an intimate link between

endophytes and saprotrophs. Valuable insight as to whether some

endophytic fungi manifest themselves as saprotrophs upon host decay.

55

Molecular data suggest there are fungal taxa that possibly exist as

endophytes and saprotrophs. Isolates of Colletotrichum, Fusarium, Guignardia,

and Phomopsis, which are common plant endophytes, have high sequence

similarity and are phylogenetically related to their saprotrophic

counterparts. This provides evidence to suggest that some endophytic

species change their ecological strategies and adopt a saprotrophic lifestyle.

3.12. Bioremediation by endophytes

Use of plants and bacterial to clean up environmental pollutants has

gained momentum in past years. A limitation to phytoremediation of

solvents has been toxicity of the compounds to plants, and the uncertainty as

to the fate of many of the compounds. Newman and Reynolds (2005)

investigated that, engineered endophytes have been shown to increase plant

tolerance to toluene, and to decrease the transpiration of toluene to the

atmosphere. This type of work has the potential to increase the use of

phytoremediation by decreasing toxicity and increasing degradation of

toxins.

Lemons et al. (2005) reported that plant-microbial mutualism affects

the rate of leaf litter decomposition using the widespread interaction

between tall fescue grass (Lolium arundinaceum) and the fungal endophyte

Neotyphodium coenophialum. In grasses, fungal endophytes live symbiotically

56

in the aboveground tissues, where the fungi gain protection and nutrients

from their host and often protect host plants from biotic and abiotic stress. In

a field experiment, decomposition rate depended on a complex interaction

between the litter source (collected from endophyte-infected or endophyte-

free plots), the decomposition microenvironment (endophyte-infected or

endophyte-free plots), and the presence of mesoinvertebrates (manipulated

by the mesh size of litter bags). Over all treatments, decomposition was

slower for endophyte-infected fescue litter than for endophyte-free litter.

When mesoinvertebrates were excluded using fine mesh and litter was

placed in a microenvironment with the endophyte, the difference between

endophyte-infected and endophyte-free litter was strongest. In the presence

of mesoinvertebrates, endophyte-infected litter decomposed faster in

microenvironments with the endophyte than in microenvironments lacking

the endophyte, suggesting that plots differ in the detritivore assemblage.

Indeed, the presence of the endophyte in plots shifted the composition of

Collembola, with more Hypogastruridae in the presence of the endophyte

and more Isotomidae in endophyte-free plots. In a separate outdoor pot

experiment, the litter source or the soil microbial/microinvertebrate

community on decomposition doesnot have strong effects, which may reflect

differences between pot and field conditions or other differences in

methodology.

57

Moore et al. (2006) reported that the diversity of endophytic bacteria

found in association with poplar was investigated as part of a larger study to

assess the possibility and practicality of using endophytic bacteria to enhance

in situ phytoremediation. Endophytic bacteria were isolated from the root,

stem and leaf of two cultivars of poplar tree growing on a site contaminated

with BTEX compounds. They were further characterised genotypically by

comparative sequence analysis of partial 16S rRNA genes and BOX-PCR

genomic DNA fingerprinting, and phenotypically by their tolerance to a

range of target pollutants, heavy metals and antibiotics. One hundred and

21 stable, morphologically distinct isolates were obtained, belonging to

21 genera. The endophytic bacteria exhibited marked spatial

compartmentalisation within the plant, suggesting there are likely to be

species-specific and non-specific associations between bacteria and plants.

This study demonstrates that within the diverse bacterial communities found

in poplar several endophytic strains are present that have the potential to

enhance phytoremediation strategies.

Rosenblueth and Martínez-Romero (2006) reported that endophytes

promote plant growth and yield, suppress pathogens, may help to remove

contaminants, solubilize phosphate, or contribute assimilable nitrogen to

plants. Some endophytes are seedborne, but others have mechanisms to

colonize the plants that are being studied. Bacterial mutants unable to

58

produce secreted proteins are impaired in the colonization process. Plant

genes expressed in the presence of endophytes provide clues as to the effects

of endophytes in plants. Molecular analysis showed that plant defense

responses limit bacterial populations inside plants. Some human pathogens,

such as Salmonella spp., have been found as endophytes, and these bacteria

are not removed by disinfection procedures that eliminate superficially

occurring bacteria. Delivery of endophytes to the environment or

agricultural fields should be carefully evaluated to avoid introducing

pathogens.

Germaine et al. (2006) reported that bacterial endophyte-

enhanced phytoremediation of the organochlorine herbicide 2, 4-

dichlorophenoxyacetic acid. Persistence of the herbiside residues residues

migrate in the soil and contaminated soil and groundwater. This compound

particularly toxic to the broad-leaved plants, such as the poplar (Populus)

and willow (Salix), which are often used in phytoremediation projects. They

describe the inoculation of a model plant, the pea (Pisum sativum), with a

genetically tagged bacterial endophyte that naturally possesses the ability to

degrade 2, 4-dichlorophenoxyacetic acid. Inoculated plants showed a higher

capacity for 2, 4-dichlorophenoxyacetic acid removal from soil and showed

no 2, 4-dichlorophenoxyacetic acid accumulation in their aerial tissues. This

demonstrates the usefulness of bacterial endophytes to enhance the

59

phytoremediation of herbicide-contaminated substrates and reduce levels of

toxic herbicide residues in crop plants.

Ryan et al. (2008) reported that endophytic bacteria have been found

in virtually every plant studied, where they colonize the internal tissues of

their host plant and can form a range of different relationships including

symbiotic, mutualistic, commensalistic and trophobiotic. Most endophytes

appear to originate from the rhizosphere or phyllosphere; however, some

may be transmitted through the seed. Endophytic bacteria can promote plant

growth and yield and can act as biocontrol agents. Endophytes can also be

beneficial to their host by producing a range of natural products that could

be harnessed for potential use in medicine, agriculture or industry. In

addition, it has been shown that they have the potential to remove soil

contaminants by enhancing phytoremediation and may play a role in soil

fertility through phosphate solubilization and nitrogen fixation. There is

increasing interest in developing the potential biotechnological applications

of endophytes for improving phytoremediation and the sustainable

production of nonfood crops for biomass and biofuel production.

Sziderics et al. (2007) reported that endophytes are nonpathogenic

plant-associated bacteria that can play an important role in plant vitality and

may confer resistance to abiotic or biotic stress. The effects of 5 endophytic

bacterial strains isolated from pepper plants showing 1-aminocyclopropane-

60

1-carboxylate deaminase activity were studied in sweet pepper under in vitro

conditions. Plant growth, osmotic potential, free proline content, and gene

expression were monitored in leaves and roots under control and mild

osmotic stress conditions. All indole acetate producers promoted growth in

Capsicum annuum L. 'Ziegenhorn Bello', from which they were isolated.

Differential gene expression patterns of CaACCO, CaLTPI, CaSAR82A, and

putative P5CR and P5CS genes during moderate stress were observed,

depending on the bacterium applied. Inoculation with 2 bacterial strains,

EZB4 and EZB8 (Arthrobacter sp. and Bacillus sp., respectively), resulted in a

significantly reduced upregulation or even downregulation of the stress-

inducible genes CaACCO and CaLTPI, as compared with the gene

expression in noninoculated plants. This indicates that both strains reduced

abiotic stress in pepper under the conditions tested.

Liu et al., (2007) reported that bacterial endophyte-enhanced

phytoremediation of the organochlorine herbicide 2, 4-

dichlorophenoxyacetic acid. Leave samples of Paeonia lactiflora and Trifalium

repens were collected for the study their endophytic actinmycetes. The

samples were plated on agar media of TWYE, HV, YECD, NA and WA,

followed by incubation at 28 degrees C for 2 - 4 weeks. 15 actinomycetes

strains were isolated from the plates. All the strains were assigned to

12 different genotypes, based on comparison of cultural features and finger

61

printing analysis. A combination of morphological and 16S rRNA gene

sequence data showed that except strains C4 and C5, which belonged to

Pseudonocardia, 13 of the isolates were streptomycetes. In the tests of

antimicrobial activity against 7 bacteria, 3 fungi and 1 yeast, 11 isolates were

positive in one or more tests, and 55% of the positive ones could inhibit the

growth of Rhizoctonia solani, a significant pathogen of plants.

Ryan (2008) reported that bacterial endophytes; recent development

and applications. Endophytic bacteria form a range of different relationships

including symbiotic, mutualistic, commensalistic and trophobiotic. Most

endophytes appear to originate from the rhizosphere or phyllosphere;

however, some may be transmitted through the seed. Endophytic bacteria

can promote plant growth and yield and can act as biocontrol agents.

Endophytes can also be beneficial to their host by producing a range of

natural products that could be harnessed for potential use in medicine,

agriculture or industry. In addition, it has been shown that they have the

potential to remove soil contaminants by enhancing phytoremediation and

may play a role in soil fertility through phosphate solubilization and

nitrogen fixation. There is increasing interest in developing the potential

biotechnological applications of endophytes for improving

phytoremediation and the sustainable production of nonfood crops for

biomass and biofuel production.

62

3.13. Industrial enzymes from endophytes

Endophytes comprise mainly microorganisms that colonize inner

plant tissues, often living with the host in a symbiotic manner. Several

ecological roles have been assigned to endophytic fungi and bacteria.

Endophytes are viewed as a new source of genes, proteins and biochemical

compounds that may be used to improve industrial processes.The gene EglA

was cloned from a citrus endophytic Bacillus strain. The EglA encodes a beta-

1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions.

Characteristic is an important feature for further applications of this enzyme

in biotechnological processes in which temperatures of 50-60 degrees C are

required over long incubation periods (Lima et al., 2005).

Cho et al. (2007) reported that interference of quorum sensing and

virulence of the rice pathogen Burkholderia glumae by an engineered

endophytic bacterium. They isolate Burkholderia spp., Among 44 putative

endophytic isolates isolated from surface-sterilized rice roots. KJ006 was

selected for further study because it given that the major virulence factor of

Burkholderia glumae is controlled in a population depedent manner (quorum

sensing) N-acyl-homoserine lactonase (aiiA) gene from Bacillus thuringiensis

was introduced into Burkholderia sp., It control the seedling rot and grain rot

of rice which caused by Burkholderia glumae. The result of engineered strain

KJ006 (pKPE-aiiA) inhibited production of quorum-sensing signals by

Burkholderia glumae in vitro and reduced the disease incidence of rice

63

seedling rot caused by Burkholderia glumae in situ. These results indicate the

engneered bacterial endophyte with aiiA gene can be used as a novel

biological control agent against pathogenic Burkholderia glumae.

Cho et al. (2007) reported that endophytic bacterial community in

ginseng and their antifungal activity against pathogens. The diversity of

bacterial endophytes associated with ginseng roots cultivation. Because they

do not exert adverse effects. Sixty-three colonies were isolated from the

interior of ginseng roots. Phylogenetic analysis based on 16S rRNA gene

sequences showed that the isolates belonged to three major phylogenetic

groups at the three different ginseng growing areas. The high G+C

Gram-positive bacteria (HGCGPB), low G+C Gram-positive bacteria

(LGCGPB), and the Proteobacteria at Jinan (61.9%). Most cellulase-,

xylanase-, and pectinase-producing colonies among the isolates belong to the

LGCGPB group, except for Pectobacterium carotovora which belonged to the

Proteobacteria. The 13 isolates belonging to LGCGPB and Proteobacteria

were assessed for their antifungal activity against phytopathogenic fungi

such as Rhizoctonia solani. Among them, Paenibacillus polymyxa GS01,

Bacillus sp. GS07, and Pseudomonas poae JA01 show potential activity as

biocontrol agents against phytopathogenic fungi.LGCGPB showed

cellulolytic enzyme activity against phytopathogeic microorganisms which is

not found in HGCGPB and proteobacteria.

64

Cho et al. (2007) reported that plant roots are associated with diverse

communities of endophytic bacteria which do not exert adverse effects. The

diversity of bacterial endophytes associated with ginseng roots cultivated in

three different areas in Korea was investigated. Sixty-three colonies were

isolated from the interior of ginseng roots. Phylogenetic analysis based on

16S rRNA gene sequences showed that the isolates belonged to three major

phylogenetic groups. Cellulase-, xylanase-, and pectinase-producing colonies

among the isolates belong to the LGCGPB group.

3.14. Endophytes in crop improvement

Bandara et al. (2006) reported that Interactions among endophytic

bacteria and fungi: effects and potentials. The study experimentally showed

that endophytes isolated from rice (Oryza sativa) used as the test plant

produced two types of interactions; biofilms (bacteria attached to mycelia)

and mixed cultures with no such attachments. Production of acidity and

indoleacetic acid like substances (IAAS) of biofilms was higher than that of

fungi alone, bacteria alone or the mixed cultures. There was a negative

relationship between IAAS and pH of the biofilms, indicating that IAAS was

the main contributor to the acidity. Microbial acid production is important

for suppressing plant pathogens. Thus the biofilm formation in endophytic

environment seems to be very important for healthy and improved plant

65

growth. The mixed cultures of effective microbes may not give the highest

microbial effect, which may only be achieved by biofilm formation.

Kang et al., (2007) reported that two bacterial endophytes eliciting

both plant growth promotion and plant defense on pepper (Capsicum

annuum L.). They isolated 150 Bacteria from healthy stems of peppers. In that

23 putative endophytic isolates were subjected to phenotypic

characterization and partial 16s rDNA sequence analysis , the isolates were

identified as species of Ochrobacterium, Pantoea, Pseudomonas, Sphingomonas,

Janthinobacterium, Ralstonia, Arthrobacter, Clavibacter, Sporosarcina, Acidovorax,

and Brevundimonas. Among them, two isolates, PS4 and PS27, were selected

because they showed consistent colonizing capacity in pepper stems at the

levels of 106-107 CFU.g-1 tissue, and were found to be most closely related to

Pseudomonas rhodesiae and Pantoea ananatis, respectively, by additional

analyses of their entire 16S rDNA sequences. These two strains were

enhancing the pepper root fresh weight by 73.9% and 41.5% respectively.

The two strains also elicited induced systemic resistance of plants against

Xanthomonas axonopodis pv. vesicatoria.

Thomas et al. (2007) reported that fourteen distinct bacterial clones

were isolated from surface-sterilized shoot tips (approximately 1 cm)

of papaya (Carica papaya L. 'Surya') planted on Murashige and

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Skoog (MS)-based papaya culture medium (23/50 nos.) during the 2-4 week

period following in vitro culturing. These isolates were ascribed to six

Gram-negative genera, namely Pantoea (P. ananatis), Enterobacter (E. cloacae),

Brevundimonas (B. aurantiaca), Sphingomonas, Methylobacterium

(M. rhodesianum), and Agrobacterium (A. tumefaciens) or two Gram-positive

genera, Microbacterium (M. esteraromaticum) and Bacillus (B. benzoevorans)

based on 16S rDNA sequence analysis. Pantoea ananatis was the most

frequently isolated organism (70% of the cultures) followed by

B. benzoevorans (13%), while others were isolated from single stocks.

Bacteria-harboring in vitro cultures often showed a single organism. Pantoea,

Enterobacter, and Agrobacterium spp. grew actively on MS-based

normal papaya medium, while Microbacterium, Brevundimonas, Bacillus,

Sphingomonas, and Methylobacterium spp. failed to grow in the absence of host

tissue. Supplying MS medium with tissue extract enhanced the growth of all

the organisms in a dose-dependent manner, indicating reliance of the

endophyte on its host. Inoculation of papaya seeds with the endophytes (20 h

at OD550=0.5) led to delayed germination or slow seedling growth initially.

However, the inhibition was overcome by 3 months and the seedlings

inoculated with Pantoea, Microbacterium or Sphingomonas spp. displayed

significantly better root and shoot growths.

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Jha and Kumar (2007) reported that to isolate and characterize

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

which grows luxuriantly with no addition of any nitrogen source. Ten

endophytic diazotrophic bacteria from surface-sterilized roots and culm of

T. australis were isolated and screened for plant growth-promoting activities

employing standard methods. It 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. Roots and culm of T. australis harbour several

endophytic diazotrophic bacteria. One root isolate, identified as K. oxytoca

GR-3, seems to be an efficient plant growth-promoting bacterium. Plant

growth-promoting properties of GR-3 suggest that this promising isolate

merits further investigations for potential application in agriculture.

Shin et al., (2007) reported that Endophytic bacteria associated with

the roots of coastal sand dune plants. Ninety-one endophytic bacterial

isolates were collected and assigned to 17 different genera of 6 major

bacterial phyla based on partial 16S rDNA sequence analyses.

Gammaproteobacteria represented the majority of the isolates (65.9%), and

members of Pseudomonas constituted 49.5% of the total isolates. Testing for

antagonism towards plant pathogenic fungi, 25 strains were antagonistic

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towards Rhizoctonia solani, 57 strains were antagonistic towards Pythium

ultimum, 53 strains were antagonistic towards Fusarium oxysporum, and 41

strains were antagonistic towards Botrytis cinerea. Seven strains were shown

to produce indole acetic acid (IAA), 33 to produce siderophores, 23 to

produce protease, 37 to produce pectinase, and 38 to produce chitinase. The

broadest spectra of activities were observed among the Pseudomonas strains,

indicating outstanding plant growth-promoting potential. The isolates from

C. kobomugi and M. sibirica also exhibited good plant growth-promoting

potential.

Govindarajan et al. (2008) reported that survey of endophytic

diazotrophic bacteria associated with different rice varieties in Tamilnadu,

some "endophytes" were obtained. Thirteen bacterial isolates from surface-

sterilized roots and shoots were obtained in pure culture, which produced

indole acetic acid (IAA) and reduced acetylene to ethylene.