14

CHAPTER 2

LITERATURE SURVEY

Black pepper (Piper nigrum L.) is an important crop plant grown in the Malabar

regions of Karnataka and Kerala in India. It is known for its culinary and medicinal

properties since ancient times. It is a spicy, aromatic plant with carminative and antioxidant

properties, known for its antiperiodic, anti inflammatory and anti cancer effects, (Meghawal

and Goswami, 2012). Though it originated in the Malabar regions of India, it is today grown

in more than 25 countries in the tropics.

Thus the study of rhizospheric microorganisms from the black pepper and the

evaluation of plant growth promoting rhizospheric microorganisms have been taken up and

reviewed.

2.1.Rhizospheric microorganisms

The term rhizosphere signifies the zone of the soil around affected the plant roots.

Rhizospheric soil shows immense microbiological activity than that away from the roots of

plants.

The rhizosphere is a hot spot of microbial interactions as exudates released by plant

roots are a main food source for microorganisms and a driving force of their population

density and activities. The rhizosphere harbors many organisms that have a neutral effect on

the plant, but also attracts organisms that exert deleterious effects, (Jones et al 2004;

Hinsinger et al 2006). The activity of plant roots has an impact on the physicochemical

conditions as well as on the biological activity in the surrounding rhizosphere compartment,

and vice versa. These processes are determining nutrient availability, cycling of nutrients and

solubility of toxic elements for plants and microorganisms, thereby creating the rhizosphere

as a unique micro-ecosystem, which can exhibit completely different properties compared

with the bulk soil, not directly influenced by the activity of roots. (Neumann et al 2009). The

physical architecture and water content play a key role in determining the biogeochemical

ambience of the rhizosphere, via their effect on partial pressures of O2 and CO2, and thereby

on redox potential and pH of the rhizosphere, respectively, (Hinsinger, 2009).

15

The interactions occurring between the plant roots and the microflora in the soil have

been shown to exist in the outer invaded cortical layer on the surface of the roots and the

surrounding soil. As all the nutrients taken up by the plant are essentially the ones in the

rhizosphere, the potential for the microbes to alter these compounds will affect the plant

growth in a great way, (Chanway, 2002). The complexity of the soil system is determined by

the numerous and diverse interactions among its physical, chemical and biological

components as modulated by the environmental conditions, (Buscot, 2005; Barea et al 2005).

Many studies have exhibited the role played by the soil microbes in their interaction

with the plant roots and the soil constituents at the root- soil interface, (Barea, et al 2005).

Plant roots exert strong effects on the rhizosphere through ‘rhizodeposition’ (root exudation,

production of mucilages and release of soughed-off root cells) and by providing suitable

ecological niches for microbial growth (Bais et al 2006; Raajimakers et al 2009), have

evaluated the specific mechanisms involved in the tripartite interactions occurring between

the beneficial microorganisms, pathogens and plants in the rhizospheric soil. The type and

kind of bacteria present in the rhizosphere depend upon the type of bulk soil surrounding and

thrive on the conditions that prevail in the neighbouring soil, (Buee et al 2009). Walker et al

(2003) have conducted experiments on the role of root exudates secreted by the plants and

their effect on the rhizospheric biology. They have inferred from their findings that root-root

and root-microbe communication can either be positive (symbiotic) to the plant, such as the

association of epiphytes, mycorrhizal fungi, and nitrogen-fixing bacteria with roots; or

negative to the plant, including interactions with parasitic plants, pathogenic bacteria, fungi,

and insects.

Bacteria present in the rhizosphere and rhizoplane of Capsicum frutescens at different

stages of plant growth were studied by Oyeyiola et al (2012). The texture of the experimental

soil was loamy sand. The pH of the experimental soil was 6.9 prior to seed sowing but it

ranged from 6.4 to 6.7 during plant growth. The bacteria isolated were Bacillus thuringensis,

Bacillus subtilis, Bacillus alvei, Enterobacter aerogenes, Bacillus badius, Bacillus

macroides, Alcaligenes eutrophus, Bacillus brevis, Proteus vulgaris, Pseudomonas

fluorescens, Azotobacter paspali, Bacillus sphaericus, Pseudomonas aeruginosa,

Acinetobacter calcoaceticus, Aeromonas hydrophila, Flavobacterium ferrugineum,

Micrococcus luteus, Bacillus cereus and Bacillus megaterium.

16

Flavonoids are a diverse class of polyphenolic compounds that are produced as a

result of plant secondary metabolism. They are known to play a multifunctional role in

rhizospheric plant-microbe and plant–plant communication. The presence of microorganisms

undoubtedly influences the quality and quantity of flavonoids present in the rhizosphere, both

through modification of root exudation patterns and microbial catabolism of exudates.

Microbial alteration and attenuation of flavonoid signals may have ecological consequences

for below-ground plant-microbe and plant–plant interaction as reported by Shaw et al (2006).

Noveriza and Qiuimo (2004) have isolated Penicillium, Paecilomyces and Aspergillus

as dominant mycoflora that are known to have antagonistic effect on the soil borne pathogens

in the rhizosphere of black pepper.

A bacterium possessing high ability to solubilize potash was isolated from the

rhizosphere of black pepper by Sangeeth et al (2012). On the basis of biochemical and 16S

rDNA sequence analysis, the bacterium was identified as Paenibacillus glucanolyticus strain

IISRBK2. P. glucanolyticus was found to increase tissue dry mass (ranging from 37.0% to

68.3%) of black pepper in 1g K kg-1wood ash amended soil. In the soil treated with 0.5 -1.5

g K kg-1

, K uptake in live bacterium inoculated black pepper plants increased by 125.0-

184.0% compared to uninoculated control.

The diversity of microbes associated with plant roots is enormous, in the order of tens

of thousands of species. The complex plant-associated microbial community present in the

vicinity of plant roots are also referred to as the second genome of the plant and is crucial for

plant health, (Berendsen et al 2012). A thorough understanding of the principles and

mechanisms that govern the types of microorganisms and their activities in association with

plant roots will provide new opportunities to increase crop production.

The effect of the interaction among rhizospheric microorganisms and plant roots on

plant physiology has been largely investigated by Rosso et al (2010) who studied the PGPR

traits of several microbes in the rhizosphere of sweet pepper and showed the increase in plant

characteristics that included morphometric parameters, flavour profile, amount of vitamins,

carotenoids and sugars in two plant varities of sweet pepper (cv. Corno and cv. Cuneo) grown

in Italy.

17

Panayatov (2006) studied the effect of some effective microorganisms on the

development of pepper seedlings. The experiments were carried out using the pepper

varieties Kurtovska kapia 1619 and Bulgarski rotund. The plants were sown and grew on peat

substrate, which was preliminarily inoculated with the bacteria Bacillus subtilis strain A1,

Pantoeae agglomerans strain B43 and the fungus Trichoderma viridae strain T6 in the

following doses: 50 ml, 100 ml and 150 ml. The second treatment was made ten days after

the pricking. The morphological characteristics of the plants such as weight and volume of

the root system, weight, height and thickness of the stem, weight and number of the leaves

and number of the flower buds were investigated before planting. A well developed root

system was observed when applying 150 ml of Bacillus subtilis А1 and Pantoeaе

agglomerans B43. The plants treated with Bacillus subtilis A1 and Trichoderma viride T6

had the highest stems.

The microbial communities of rhizospheric soil in biofuel crops switchgrass and

jatropha were isolated and studied by Chaudhury et al (2012) who showed that higher

abundance of soil fungi in the rhizosphere increased the biofuel production in these crops.

Silberbush (2013) has reported that recent innovations and the realization that there are gaps

between the expected and actual performance of plant root systems that have emphasized the

need for more realistic solutions. This review analyzes the study of plant roots in view of

developments in soil science, microbiology, botany and plant physiology, and recently the

introduction of molecular biology and computerized imaging.

2.2. Molecular identification of isolates.

Molecular biology techniques are an advantageous approach for obtaining and

characterizing improved PGPB strains (Rodrı´guez and Fraga, 1999; Igual et al 2001).

The analysis of nucleic acids directly extracted from rhizosphere soils provided an

opportunity to study a much broader spectrum of microorganisms residing in the rhizosphere.

Most frequently rRNA gene fragments are amplified from total community DNA and

subsequently analysed by fingerprinting techniques. Cultivation independent fingerprinting

methods clearly showed the influence of the plant species on the structure of the microbial

community, Marschner et al (2004)

18

Phosphate-solubilizing microorganisms are often used as plant growth

promoters. These bacterial strains were characterized by a polyphasic approach using both

phenotypic (API 20 NE) and molecular assays (RAPD, with M13 primer, TP-RAPD, and 16S

rDNA sequencing). TP-RAPD yielded an identical band patterns in the six strains indicating

that they belong to the same bacterial species. The 16S rRNA sequence analysis of a group-

representative strain (P4-22) revealed a sequence similarity value of 99.27% with

Pseudomonas jessenii. These bacterial strains were characterized by a polyphasic approach

using both phenotypic (API 20 NE) and molecular assays (RAPD, with M13 primer, TP-

RAPD, and 16S rDNA sequencing). TP-RAPD yielded an identical band patterns in the six

strains indicating that they belong to the same bacterial species. The 16S rRNA sequence

analysis of a group-representative strain (P4-22) revealed a sequence similarity value of

99.27% with Pseudomonas jessenii, (Valverde et al 2003)

One of the major difficulties that plant biologists and microbiologists face

when studying these interactions is that many groups of microbes that inhabit this

zone are not cultivable in the laboratory. Recent developments in molecular biology methods

are shedding some light on rhizospheric microbial diversity, (Singh et al 2004). Recent

advances in molecular methods and genomics provide an exciting opportunity to redefine the

relationship between plants and the microbes in their rhizosphere. Several techniques have

been developed that allow microbial ecologists directly to correlate a specific metabolic

activity with phylogenetically identifiable units in natural environment. (Gray and Head,

2001). Rapid progress in genomics has led to the availability of full genome sequences of

hundreds of microorganisms, mostly bacteria. Combinations of new molecular

methodology and genomics have been used successfully to link microbial phylogeny with

function in several ecological studies other than those involving plant–microbe interactions,

(Dahllof, I. 2002; DeLong, E.F. 2002; Wagner, M., 2004).

A plant growth promoting bacterial isolate (D5/23T) from the phyllosphere of winter

wheat, able to fix atmospheric nitrogen and to produce auxins and cytokinins was

investigated in a polyphasic taxonomy approach by Kampfer et al 2004). Phylogenetic

analyses using the 16S rRNA gene sequence of the strain clearly indicated that the strain

belonged to the family Enterobacteriaceae, most closely related to Enterobacter cloacae with

99.0% and Enterobacter dissolvens with 98.5% sequence similarity.

19

The predominant microorganisms which showed greater phosphate solubilization,

good siderophore production, production of IAA, and catalase with substantial biocontrol

activity were Bacillus subtilis and Aspergillus, and were identified up to molecular level

using 16s rDNA sequencing method (Espinosa-Victoria et al 2009). The method of using

16srDNA sequence analysis for soil microbes has been supported by other workers. The

phylogenetic tree was built using System Software Aligner and the distance matrix was

generated using the Jukes-Counter corrected distance model. The editing of the crude

sequence was done using the manual mode and it was aligned with the reference sequence

obtained from National Center for Biotechnology Information (NCBI) database (Rodriguez et

al 2006). Molecular identification of PSB (Pseudomonas flourescences) using 16srDNA

analysis was carried out by Fankem et al 2006.

Beginning with yeast Saccharomyces cerevisiae, Melo et al (2006), developed a 30-

min DNA isolation protocol for filamentous fungi by combining cell wall digestion with cell

disruption by glass beads. High-quality DNA was isolated with good yield from the hyphae

of Crinipellis perniciosa.

Bacteria in the rhizosphere of rice cultivated in Andosol lowland and upland fields

were analyzed in this study using PCR-DGGE and FISH, in combination with modified

pretreatments. The 16S rDNA band pattern of bacteria in the rhizosphere obtained using

PCR-DGGE indicated different species composition of bacterial community in the two

ecosystems and greater diversity of bacteria in the rhizosphere in upland field. Sequencing of

major 16S rDNA bands identified Bacterium A35 and Clostridium bifermentans as dominant

bacteria in the rhizosphere of rice in lowland fields, (Doi et al 2007).

Genetic diversity and molecular taxonomic identification of PSB was carried out by

Castagno et al (2008).

Microbial community analysis contains a thorough treatise of nucleotide- and PCR

based technologies to study composition and diversity of indigenous bacteria in the natural

rhizosphere, (Sorenson et al 2009). Many secrets of microbial life in the rhizosphere were

recently uncovered due to the enormous progress in molecular and microscopic tools.

Physiological and molecular data on the factors that drive selection processes in the

rhizosphere are presented here by Berg and Smalla (2009). Naz et al (2009) aimed to

20

isolate and characterize plant growth promoting rhizobacteria (Rhizobia) from rhizosphere

(EC: 2300 μS/cm; pH: 8.6) of four halophytes: Sonchus arvensis L., (sow thistle), Solanum

surratense Burm. F., (yellow berried night shade), Lactuca dissecta D. Don., (wild lettuce)

and Chrysopogon aucheri (Boiss.) Stap (golden beared grass) collected from Khewra Salt

Range and the survival efficiency of isolates was measured in culture (colony forming unit / g

soil). The genetic diversity among the isolates assessed by RAPD-DNA finger printing and

PCR was done for the presence of 16S-rRNA gene.

Studies on molecular geno - systematics on soil microorganisms have been done by

Ramezanpour et al 2011) with respect to Pseudomonas as PGPR organism.

Metagenomics is a rapidly developing field that helps in analyzing the complex

genomes of microbial niches through culture independent molecular approaches. In this

study culture-independent molecular techniques, 16S rRNA clone library generation along

with RFLP, sequencing and phylogenetic analysis, were applied to investigate the bacterial

diversity associated with the rice rhizosphere bacterial communities from a paddy field

ecosystem in Kerala, (Arjun and Harikrishnan, 2011).

Rhizobacteria of Bacillus species were isolated from the rhizosphere of soybean plant

of Cirebon, Indonesia, and further examined for plant growth promoting activities by

Wahyudi et al 2011). DNA sequence analysis of 16S rRNA genes of those 12 isolates

revealed that, all of them similar with Bacillus sp. cluster and was separately divided into

four groups.

This study has pointed out 12 isolates of Bacillus sp. that may be applicable as inoculants

according to each supporting characters as growth promoter rhizobacteria.

Large numbers of PGPR were identified by testing the ability of each isolate to

promote the growth of cucumber seedlings. After redundant rhizobacteria were removed via

amplified rDNA restriction analysis, 90 strains were finally selected as PGPR by Kim et al

(2011). On the basis of 16S ribosomal RNA sequences, 68 Gram-positive (76%) and 22

Gram-negative (24%) isolates were assigned to 21 genera and 47 species. Of these genera,

Bacillus (32 species) made up the largest complement.

21

2.3. Plant Growth Promoting Rhizospheric microorganisms.

In the recent years there has been an extensive interest in the use of rhizospheric

microorganisms for inoculation into the soil of agricultural crops which are able to colonize

plant roots and stimulate plant growth as well as increase crop yield. The mechanisms

adapted by these rhizospheric organisms have been studied for the first time by Mahafee and

Kloepper (1994). Rhizobacteria are those bacteria that aggressively colonize plant roots.

Plant growth promoting rhizobacteria (PGPR) are a very small portion of rhizobacteria (2-

5%) that promote plant growth, (Antoun and Kloepper, 2001).

Plant-growth-promoting rhizobacteria (PGPR) are associated with plant roots and

augment plant productivity and immunity, (Yang et al 2009). They help in the reduced use

of chemical fertilizers thus minimizing concentrations of fertilizers in the run-off water and

decreasing the usage of fertilizers by the farmers.

Plant growth promoting microorganisms (PGPM) and biological control agents

(BCA) are shown to possess secondary beneficial effects that would increase their usefulness

as bio-inoculants, regardless of the need for their primary function, this was reported by Alvis

et al 2008) who showed that Rhizobium and Glomus sp. Can promote plant growth and

increased plant productivity and strains of Trichoderma and Pseudomonas were able to

control diseases and potential increased use of these microorganisms afforded by their

multifaceted beneficial effects may further help in reducing problems associated with the use

of synthetic chemicals in agriculture.

The effect of the interaction among rhizospheric microorganisms and plant roots on

plant physiology has been largely investigated by Rosso et al (2010) and it is well known that

they can improve plant nutrition, water efficiency, bioprotection against pathogens, and crop

productivity. Plant-growth-promoting rhizobacteria (PGPR) are associated with plant roots

and augment plant productivity and immunity, this was shown by Yang et al 2009) who

reported that several group studies showed that PGPR also elicit so-called‘induced systemic

tolerance’ to salt and drought.

PGPR use one or more of direct or indirect mechanisms of action to improve plant

growth and health. These mechanisms can probably be active simultaneously or sequentially

22

at different stages of plant growth. P-solubilization, biological nitrogen fixation,

improvement of other plant nutrients uptake and phytohormone production like indole-3-

acetic acid are some examples of mechanisms that directly influence plant growth. Some

PGPR have the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which

hydrolyses ACC, the immediate precursor of ethylene in plants. Plant growth-promoting

bacteria may facilitate plant growth either indirectly or directly. The ability of plant growth-

promoting bacteria to act as biocontrol agents against phytopathogens and thus indirectly

stimulate plant growth may result from any one of a variety of mechanisms including

antibiotic production, depletion of iron from the rhizosphere, induced systemic resistance,

production of fungal cell wall lysing enzymes, and competition for binding sites on the root,

Induction of the systemic resistance against many pathogens, insect and nematodes

(Ramamoorthy et al 2001; Zehnder et al 2001) was studied as indirect mechanism of action

of PGPR. All these traits have been shown how complex and difficult it is to associate the

promotion of plant growth with phosphate solubilization, and they explain in part the reason

of obtaining better responses from plant inoculated with a mixture of PGPR, (Antoun, 2001).

N2-fixing rhizobacteria from pepper rhizosphere were isolated and characterised for

phosphate solubilisation, phytohormones auxin production and plant growth promotion by

Zakry et al 2010). A total of 45 bacterial isolates with prolific growth were successfully

isolated and screened for N2 -fixing activity. Out of 45 rhizobacterial isolates, 14 were

characterised as diazotrophic rhizobacteria, fixing N2 in the range of 0.07 to 0.24 ppm of

culture filtrates. Two out of 14 isolates, namely UPMLH3 and UPMLH13, were able to

solubilise phosphate with solubilisation efficiency at 286% and 563%, respectively. Six

isolates were able to produced indole-3-acetic acid, ranging from 36.71 to 63.05 μg mL-1

.

The rhizosphere gives support to many active microbial populations capable of

exerting beneficial, neutral or detrimental effects on plant growth (Whipps, 2001, Wahyudi,

2011). Rhizobacteria of Bacillus species were isolated from the rhizosphere of soybean plant

of Cirebon, Indonesia, and further examined for plant growth promoting activities. A total of

118 isolates identified as Bacillus sp., 90 isolates (76.3%) among them positively produced

phytohormone indole acetic acid (IAA). 12 isolates of Bacillus sp. were found to be

applicable as inoculants which could support as growth promoter rhizobacteria.

23

Parvatha Reddy (2013) has reported that the addition of compost and compost teas

promote existing PGPR and may introduce additional helpful bacteria to the field. The

absence of pesticides and the more complex organic rotations likely promote existing

populations of these beneficial bacteria.

Edi Husen (2003) isolated soil bacteria including two strains of Azotobacter

vinelandii Mac 259 and Bacillus cereus UW 85 and showed that they had the PGPR traits.

The microbes were tested for IAA production, phosphate solubilization, dinitrogen fixation

and siderophore production in vitro.

Plants not only provide nutrients for microorganisms, but some plant species also

contain unique antimicrobial metabolites in their exudates. Many of them are used as

medicinal plants, for example chamomile, thyme and eucalyptus. The existing huge diversity

of plant species was estimated to range from 310 000 to 422 000 species (Pitman &

Jorgensen, 2002). Screening of soil bacteria for their plant growth promoting effects was

done by Husen, (2003); Berg and Smalla, (2009).

The mechanisms by which PGPRs promote plant growth are not fully understood, but

are thought to include - the ability to produce phytohormones, (Egamberdiyeva, 2007;

Shaharoona et al 2006; Gholami et al 2009) . PGPR strains of Bacillus subtilis strain GBO3

and Bacillus amyloliquefaciens strain IN937a were used in the field trials as transplant

amends conducted by Kokalis-Burelle et al 2006) in Florida in the rhizosphere of Capsicum

annuum. They found that the use of PGPR applied to the potting media established stable

populations in the rhizosphere that persisted throughout the growing season, increased the

plant growth and reduced disease incidence in a detached leaf assay, indicating that systemic

resistance was induced by the PGPR treatments.

Avis et al (2008) have reported that plant growth promoting microorganisms (PGPM)

and biological control agents (BCA) are shown to possess secondary beneficial effects that

would increase their usefulness as bio-inoculants, regardless of the need for their primary

function. They have inferred that the increased use of these microbes in agriculture will yield

multi- faceted beneficial effects that may further help in reducing problems associated with

the use of synthetic chemicals.

24

Plant-microbe interactions may be beneficial or harmful, depending on the

characteristics of the microbes involved and the ways in which they interact with plants.

Among such microbes, plant growth-promoting rhizobacteria (PGPR) are distributed on plant

roots or in the surrounding soil and have beneficial effects on plants. PGPR may promote

plant growth, thus providing high crop yields, and they also function as biocontrol agents

against plant diseases caused by phytopathogenic microorganisms, (Lugtenberg and

Kamilova. 2009; Kim et al 2011).

Microbial populations are key components of the soil- plant system where they are

immersed in a framework of interaction affecting plant development. Plant Growth

promoting bacteria (PGPB) can benefit plant growth by different mechanisms, (Bashan and

de Bashan, 2005).

The exact mechanism by which PGPR plant growth are not fully understood but are

thought to include the ability to produce or change the concentration of plant growth

regulators like indole acetic acid, gibberellic acid, cytokinins and ethylene and antagonisms

against phytopathogenic soil microbe by production of siderophores (Arshad and

Frankenberger, 2005; Sher and Baker, 2006).

The beneficial activities of PGPR bacteria have been established in many crops

including black pepper by several workers, (Sarma et al 2003; Lucy et al 2004; Vestberg et al

2004; Paul et al 2005). Study of PGPR in the rhizosphere soils of black pepper with respect

to Pseudomonas species has been extensively done by Paul et al 2005) who have shown the

antagonistic effect of Pseudomonas species on P. capsicii.

The effect of a Pseudomonas fluorescens strain (Aur 6) isolated from Lupinus

hispanicus on pepper seedlings (Capsicum annum cv. Roxy) was studied by Garcia et al

2003) who showed that the PGPB effects could be related to auxin and siderophore

production, as strain Aur 6 produced substances of both classes in pure culture.

The growth of red pepper plug seedlings was promoted by Bacillus cereus MJ-1, B.

macroides CJ-29, and B. pumilus CJ-69 isolated from the rhizosphere by Joo et al (2005),

who reported the growth promoting effects of gibberllins, GA3, GA4 and GA7 in the

organisms.

25

Tizzard (2006) showed that inoculation with Bacillus licheniformis resulted in

enhanced growth of oak seedling, reflected by significant increases in shoot surface area,

shoot length and shoot dry weight.

Datta et al (2011) have shown that the plant growth promoting rhizobacteria (PGPR)

can enhance the growth and productivity by exerting beneficial effects through direct

and indirect mechanisms. They have isolated 15 bacterial species from chilli rhizosphere and

their morphological, biochemical, plant growth promoting, and biocontrol characteristics

were elucidated. The results clearly showed the rhizocompetence and plant growth

enhancing efficacy of these strains.

Mehta, et al (2010) have isolated Bacillus circulans from the rhizosphere of apple and

showed the plant growth promoting activities of the strain. Mishra, et al (2010) have isolated

of plant growth promoting rhizobacteria (PGPR) which were isolated from the rhizosphere

soil of Pyrethrum (Chrysanthemum cineraefolium) designated as MA-2 and MA-4, and

identified as Bacillus subtilis and Pseudomonas fluorescens on the basis of cultural as well as

biochemical testing. They gave excellent result on the productivity of Pelargonium

graveolens, increased herb yield over control by 9 and 27.6% respectively.

Hayat, et al (2010) have reported the presence of free-living soil bacteria beneficial to

plant growth, usually referred to as plant growth promoting rhizobacteria (PGPR), are

capable of promoting plant growth by colonizing the plant root. Free-living nitrogen-fixing

bacteria or associative nitrogen fixers, for example bacteria belonging to the species

Azospirillum, Enterobacter, Klebsiella and Pseudomonas, have been shown to attach to the

root and efficiently colonize root surfaces.

The diversification of PGP rhizospheric microorganism was studied by Joshi and

Bhatt (2011) in the rhizospheres of wheat in Uttarkashi district in northern Himalayan region.

On the basis of biochemical characterization 44 % were Bacillus sp. and 24% belong to

Pseudomonas sp. Microorganisms that colonize the rhizosphere can be classified according

to their effects on plants and the way they interact with roots, some being pathogens whereas

other trigger beneficial effects. So, the bacteria inhabiting the rhizosphere and beneficial to

plants are termed PGPR, (Saharan and Nehra, 2011). Growth promoting activities of Bacillus

subtilis and B. amyloliquefaciens have been studied by Idris et al 2004) who showed through

26

the bioassays using diluted cultures of the organisms that the enhancement in growth

occurred as a result of combined effects of more than one PGPR trait.

PGPR have been subjected to numerous investigations focused on biotechnological

applications in agriculture, horticulture, forestry and environmental protection (Zahir et al

2004). It was reported that PGPR strains are broadly distributed among many taxa including

Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes and Proteobacteria (Tilak et al

2005), such that determination of the background population size and activity of PGPR in

resident microbial communities is difficult to assess based on analysis of microbial

community structure or abundance of a particular taxonomic group.

Beatti (2006) reported that most rhizobacteria are commensals in which bacteria

establish an innocuous interaction the effect of which is not seen visibly on the plant growth.

In addition to parasitic and disease causing organisms, such bacteria include those that

produce phytotoxic substances, such as hydrogen cyanide or ethylene that inhibit root

growth. Counter to these deleterious bacteria are PGPR, which exert a positive effect on plant

growth by direct mechanisms such as solubilization of nutrients, nitrogen fixation, production

of growth regulators, etc., or by indirect mechanisms such as stimulation of mycorrhizae

development, competitive exclusion of pathogens, or removal of phytotoxic substances that

are produced by deleterious bacteria and plant roots under stress condition mechanisms

(Beattie, 2006; Bashan and de-Bashan, 2010).

Classification terms have been established to describe the activities and mechanisms

by which the functioning of the PGPR organisms is achieved. In general, direct mechanisms

are those affecting the balance of plant's growth regulators, enhancing plant's nutritional

status and stimulating systemic disease resistance mechanisms (Zahir et al 2004; Glick et al

2007). Indirect mechanisms are related to biocontrol, including antibiotic production,

chelation of available Fe in the rhizosphere, synthesis of extracellular enzymes that hydrolyze

the fungal cellular wall and competition for niches within the rhizosphere (Zahir et al 2004;

Glick et al 2007). Antagonistic isolates from the bamboo rhizosphere soil was isolated and

characterized for biocontrol and plant growth promotional property by Niveditha et al (2008).

Martinez- Viveros et al (2010) reported that Microorganisms having mechanisms that

facilitate nutrient uptake or increase nutrient availability or stimulate plant growth are

27

commonly referred to as biofertilizers. Biofertilizers are considered as an alternative or

complement to chemical fertilization to increase the production of crops in low input

agricultural systems. There are some PGPR that can fix nitrogen, solubilize mineral nutrients

and mineralize organic compounds. The well-studied PGPR considered biofertilizers

correspond to nitrogen fixation and utilization of insoluble forms of phosphorus.

Characterization of Bacillus subtilis as a PGPR organism was done by Mehta et al

2010) in tomato rhizosphere. Plant growth promoting bacteria can directly facilitate plant

growth. They may fix atmospheric nitrogen and supply it to plants; synthesize siderophores

which can sequester iron from the soil and provide it to plant cells which can take up the

bacterial siderophore–iron complex and synthesize phytohormones such as auxins, cytokinins

and gibberellins, which can act to enhance various stages of plant growth; solubilize minerals

such as phosphorus, making them more readily available for plant growth., (Amora-Lazcano

et al 2010).

Rhizobacteria of Bacillus species were isolated from the rhizosphere of soybean plant

of Cirebon, Indonesia, and further examined for plant growth promoting activities, (Wahyudi

et al 2011).

Similar to PGPR (plant growth promoting rhizobacteria), some rhizosphere fungi able

to promote plant growth upon root colonization are functionally designated as 'plant-growth-

promoting-fungi’ (PGPF), (Pandya and Saraf, 2010). Recent studies indicate that PGPR are

able to boost plant tolerance to abiotic stresses such as salt and drought, (Yang et al 2009).

This suite of benefits has led to the increasing application of PGPR in arable agriculture. The

bacteria can be used to replace chemical fertilizers and pesticides that are agents of pollution,

(Adesemoye and Kloepper, 2009; Kim et al 2011).

Isolation of fungal species for the plant growth promoting abilities in the soil and

their capacity to produce phytohormones was shown by Nenwani et al (2010). Many

experiments have demonstrated the growth stimulation of plant crops in the greenhouse,

resulting in increased yield parameters and in the control on soil-borne pathogenic organisms.

However, the replication of successful results of PGPR applications under field conditions

has been limited by the lack of knowledge about their ecology, survival and activity in the

plant rhizosphere, (Martines- Viveros et al 2010).

28

Kumar et al (2012) have identified the role of Pseudomonas, Azospirillum,

Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus,

Rhizobium and Serratia in enhancing the plant growth and acting as PGPR in the rhizosphere

of French bean. Study of the diversity of Bacillus spp. isolated from tomato rhizosphere and

their evaluation as plant growth promoter was conducted by Kumar et al (2012). Bacillus

showed multiple plant growth promotion (PGP) attributes such as production of Indole acetic

acid (IAA), siderophore, ammonia, HCN and phosphate solubilization and was studied for its

diversity pattern using carbon utilizing profiling and RFLP.

The composition and quantity of essential oil from a particular species of thyme plant

could be markedly affected by environmental and agronomical factors. Application of bio

fertilizers can improve environmental conditions and human health. Azotobacter is one of the

plant growth promoting rhizobacteria (PGPR). Sharafzadeh et al (2012) have investigated the

effect of Azotobacter inoculation as PGPR in the rhizosphere of thyme and have shown the

increase in the essential oil content of the plant as a result of the inoculation.

Bhattacharya and Jha, (2012) have studied the effect of PGPR on the growth of many

plants including pepper, and have shown that can enhance plant growth by a wide variety of

mechanisms like phosphate solubilization, siderophore production, biological nitrogen

fixation, rhizosphere engineering, production of 1-Aminocyclopropane-1- carboxylate

deaminase (ACC), quorum sensing (QS) signal interference and inhibition of biofilm

formation, phytohormones production, exhibiting antifungal activity, production of

volatile organic compounds (VOCs), induction of systemic resistance, promoting beneficial

plant-microbe symbioses, interference with pathogen toxin production etc.

Ramyasmruthi et al (2012) have tested the efficacy of 18 bacterial isolates on the

Rhizosphere of Solanaceae family namely brinjal, capsicum, chilli and screened for the

production of chitinase enzyme. The main isolate R was identified as Pseudomonas

fluorescens and showed Concurrent production of siderophore, IAA, HCN, phosphate

solubilisation, NH3 and catalase coupled with anti-fungal activity suggests the plant growth

promotion and broad spectrum biocontrol potential of this isolate.

Roopa et al (2012) have evaluated the efficacy played by the PGPR and rhizobial

inoculants in enhancing the growth productivity by increase in the nodulation in chick peas.

29

They have reported that the increase in the nodulation in the roots of chick pea plant was

shown in response to co inoculation of the plant with rhizobial and PGPR organisms.

Plant growth promoting abilities of phosphate solubilizing bacteria in the rhizosphere

of Parthenium hysterophorus was studied by Dugar et al (2013). They have analysed the

ability the effect of these microorganisms in providing a new dimension to the existence of

weeds in agriculture and their role in enhancing the plant growth.

Prathiba and Siddalingeshwara (2013) have studied the plant-growth promoting

rhizobacteria (PGPR) such as Pseudomonas fluorescens and Bacillus subtilis in Sorghum

bicolor and were used to study the effect on seed germination and nutritional qualities such as

total protein, carbohydrate and peroxidase activity and showed that PGPR strains,

Pseudomonas fluorescens and Bacillus subtilis were effective in improving seed quality such

as seed germination, vigour index and nutritional quality such as protein content and

carbohydrate content.

Singh et al (2013) have evaluated PGPR function in three different ways synthesizing

particular compounds for the plants facilitating the uptake of certain nutrients from the soil

and lessening or preventing the plants from diseases. Rhizobium has been noted as PGPR in

almost all review articles. Some of Plant growth promoting characteristics such as Indole

acetic acid (IAA) capacity, ability to produce ammonia (NH3) as sole nitrogen source and

production of hydrogen cyanide were evaluated in three Rhizobacteria isolated from

rhizosphere of pigeon pea. HCN production was observed in 33% isolates, among test

isolates. In this study, all the isolates were able to produce Indole acetic acid (IAA) and

Production of ammonia was commonly detected in the isolates of Rhizobium (66%).

2.3.1. Phosphate solubilization

As cultivation of many plants was emphasized it was seen that the organic fertilizers

containing phosphates were only partially converted into soluble phosphates and the rest was

being converted into insoluble forms and was being deposited in the soils, (Turan et al 2007).

It was made clear that the presence of higher numbers of PSM in the rhizospheric soil was

more important than in the non rhizospheric soil, (Chailharn et al 2008). Taalab and Badr

(2007) have experimented with the efficacy of application of rock phosphates in the

30

biological-organic sector of agriculture and the need for inexpensive phosphate in developing

countries. They worked on the application of rock phosphates in sorghum plants and have

shown that Inoculation of phosphate dissolving bacteria increased sorghum dry matter yields

and total N and P 4 uptake when applied along with NH +-N combined with rock phosphate

and gave almost yield similar to N plus super phosphate.

Chen et al (2006) investigated the ability of a few soil microorganisms to convert

insoluble forms of phosphorus to an accessible form which is an important trait in plant

growth-promoting bacteria for increasing plant yields. They isolated, screened and

characterized several strains of PSBs and showed that the use of phosphate solubilizing

bacteria as inoculants increases the P uptake by plants.

Studies of phosphate solubilization of tricalcium phosphate have been done with

various microbes indicating the solubilization of phosphate source used. Phosphate

solubilizing microbes are detected in vitro studies and solubilization of insoluble phosphates,

which in turn is mediated via the production of organic acid in the surrounding medium have

been shown by Yasmin and Bano, (2011).

Plant can absorb phosphate only in soluble form. The transformation of insoluble

phosphate into soluble form is carried out by a number of microbes present in the soil. A

large fraction of soil microbes can dissolve insoluble inorganic phosphates present in the soil

and make them available to the plants, (Sharma et al 2011).

The fact that microorganisms isolated from the soil are able to dissolve different kinds

of rock phosphates in the soil as well as in the liquid culture medium was investigated by

Ivanova et al (2006). Phosphate solubilizing bacteria from the rhizospheric zone of pepper

was studied by Ramachandran et al (2003) who have reported the presence of Pseudomonas

and Azospirillum as efficient PSB showing more than 20% solubilization of phosphorus.

Biofertilizers have been used as sources to improve plant nutrients in sustainable

agriculture. Experiments were conducted by Han et al (2007) to evaluate the potential of

phosphate solubilizing bacteria (PSB) Bacillus megaterium var. phosphaticum and potassium

solubilizing bacteria (KSB) Bacillus mucilaginosus inoculated in nutrient limited soil planted

with pepper and cucumber and showed that Combined together, rock materials and both

31

bacterial strains consistently increased further mineral availability, uptake and plant growth

of pepper and cucumber, suggesting its potential use as fertilizer.

Nopparat et al (2007) studied phosphorus replenishment, particularly in smallholder

agriculture lands and inferred that select phosphate solubilizing soil fungi from Kanchanaburi

area showed good levels of phosphate solubilization with excellent phosphatase activity by

soil fungi like Aspergillus sp.

A study was undertaken to investigate the occurrence of phosphate solubilizing

bacteria (PSB) and nitrogen-fixing bacteria (NFB) from soil samples of Wamena Biological

Garden (WbiG) by Suliahsih and Sri Widavati, (2005) who isolated Bacillus sp., B.

pantothenticus, B. megatherium, Flavobacterium sp., F. breve, Klebsiella sp., K. aerogenes,

Chromobacterium lividum, Enterobacter alvei, E. agglomerans, Pseudomonas sp., Proteus

sp. and as NFB i.e. Azotobacter sp., A. chroococcum, A. paspalii, Rhizobium sp., and

Azospirillum sp.

Study of PSB in a buffered medium was conducted by Joseph and Jisha, (2009) who

experimented with the efficiency of phosphate solubilization with tricalcium phosphate in

buffered and non buffered media and showed that the buffering capacity of the medium

reduced the effectiveness of PSBs in releasing P from tricalcium phosphates.

The efficacy of Aspergillus tubingensis and Aspergillus niger to solubilize rock

phosphate (RP) and to improve the growth of maize (Zea mays) in rock phosphate amended

soils was studied (Richa et al 2007; Pandya and Saraf, 2010).

It was stated by Mehrvarz and Chaichi (2008) that the biofertilizers are considered

among the most effective plant assistants to supply phosphorus at a favorable level in

phosphorus poor and unfertilized soil and have shown that use of PSB and PSF increase the

efficiency of the soil in providing available phosphorus to the plants.

The effect of phosphate solubilization microorganisms (PSM) and plant growth

promoting rhizobacteria (PGPR) on yield and yield components of corn Zea mays (L. cv.

SC604) an experiment was conducted at research farm of Sari Agricultural Sciences and

Natural Resources University, Iran by Yazdani et al (2007) . The results showed that all

32

fertilizer treatments application of PSM and PGPR together could reduce P application by

50% without any significant reduction of grain yield.

Field study was conducted by Balakrishnan et al (2007) to evaluate the effect of three

halophytic composts in combination with farmyard manure and phosphate solubilising

bacteria (Bacillus megaterium) on soil microflora and enzyme activities. The results showed

that among the application of Suaeda compost in combination with farmyard manure and

phosphate solubilising bacteria (T9) significantly increased the soil microflora such as

bacteria, fungi and actinomycetes and soil enzyme activities such as dehydrogenases, alkaline

phosphatase, cellulase and urease in soil cultivated with Arachis hypogaea.

Phosphatase activity was assayed according to the method by (du Plessis et al 2002).

The study phosphatase activity by the PSB in vitro was conducted by Ponmurugan and Gopi

(b, 2006). The role of phosphatase enzyme in the mineralization of phosphorus in the soil

was shown by Rahmansyah et al (2009).

The activity of acid phosphatases under controlled conditions were studied by Baghel

et al (2009) in Cantharellus tropicalis grown in axenic cultures and showed that the cultures

grew well in optimum pH of 4 and at an optimum temperature of 40◦C.

Turan et al (2007) have experimented with Bacillus (FS-3) strain obtained originally

from pepper rhizosphere for its capacity to solubilize phosphorus in laboratory conditions.

Three efficient inorganic-phosphate solubilizing bacteria (PSB) were isolated from a

phosphate rock deposit of a Moroccan mine by Mardad et al (2013). The phosphate

solubilization index of these isolates, determined in National Botanical Research Institute's

phosphate (NBRIP) medium supplemented with tribasic calcium phosphate, ranging from 2.8

to 4.4. The medium pH dropped from 7.0 to 3.5 units after growth under continuous agitation

for seven days. PSB6, the most efficient PSB, closely related to Enterobacter hormaechei

subsp. steigerwaltii strain NM23-1, permitted the recovery of the maximum soluble

orthophosphate concentration in the medium (505 mg/L) after a growth period of 60 to 72 h.

The capacity to solubilize inorganic phosphate by these PSB can be attributed to the secretion

of organic acids, to determine their presence in the cultures supernatant, reverse-phase high

33

performance liquid chromatography was performed. The presence of 9 identified and three

unidentified organic acids was consequently demonstrated.

Plant growth promoting traits which include indoleacetic acid (IAA), ammonia,

siderophore and hydrogen cyanide (HCN) production were assessed in two phosphate

solubilizing bacterial (PSB) isolates (Pantoea agglomerans and Burkholderia anthina) and

their effect on growth and phosphorous uptake of tomato plants was investigated with a pot

experiment conducted under green house conditions by Walpola and Hoon (2013). The pots

were arranged in a completely randomized block design with three replications per treatment.

Under green house conditions, both strains remarkably enhanced plant height, root length,

shoot and root dry weight, phosphorous uptake and available phosphorous content of soil

compared to the control. The increases were more pronounced in co-inoculation of PSB

strains with TCP. Based on the results, it could be concluded that the strains possess great

potential to be developed as biofertilizers to enhance soil fertility and plant growth.

2.3.2. Production of siderophores

Many microorganisms utilize an efficient system of utilizing iron present in form of

ferric salts in the environment. It is composed of low molecular compounds with high affinity

towards iron and is called siderophores (Mahmoud and Abd-Alla, 2001). Siderophores bring

about the inhibition of pathogens in the soil by sequestering iron from the pathogens, thus

limiting their growth (de Villegas, 2002). Studies of microorganism siderophore producers

have received much attention because of the clinical applications and potential utilization of

these chelators in agriculture (Machuca and Milagres, 2003). Rhizosphere microorganisms

produce a variety of biologically active substances among which growth-promoting

compounds represent a keen interest (Asghar et al 2002; Rodríguez, 2006; Ramezanpour et al

2011).

Siderophores are also known to function as virulence factors and are commonly

produced by pathogenic fungi especially the hydroxymate type, (Hossain et al 2007; Nenwani

et al 2010).

Dastager et al (2011) have isolated Bacillus tequilensis NRRL B-41771T (99.5%).

Strain NII-0943 was able to produce good amount of indole acetic acid (IAA) and positive

34

for siderophore production from the rhizosphere of pepper grown in Western Ghats. It has

also shown good phosphate solubilizing capacity and has been shown to have a potential to

be deployed as a plant growth promoting inoculant to attain the desired results of

bacterization.

Lacava et al 2008 have studied the production of siderophores by endophytic bacteria

Methylobacterium spp., which occupy the same ecological niche as Xylella fastidiosa subsp.

pauca (Xfp) in citrus plants and have shown from their experiments that the production of

siderophores by the bacterium have increased the in vitro growth of Xfp.

Chaiharn et al (2009) have isolated several bacterial organisms from the rhizosphere

of bacteria that were able to produce siderophores and inhibit the mycelia growth I certain

pathogenic fungi like Alternaria sp., Fusarium oxysporum, Pyricularia oryzae and

Sclerotium sp., the causal agent of leaf spot, root rot, blast and stem rot in rice.

Mehta et al (2010) observed that siderophore production was one of the important

traits along with the production of IAA, phosphate solubilization that was exhibited by

Bacillus circulans in the rhizosphere of apple.

2.3.3. Production of IAA

Indole acetic acid (IAA) is a common natural auxin and is a product of L -tryptophan

metabolism in microorganisms. Approximately 80% of rhizosphere bacteria can secrete

IAA (Bhavdish et al. 2003; Khamna et al 2011). Effect of IAA is seen on the apical

dominance, phototropism, gravitropism, prevention of leaf and fruit abscission, and in

induction of adventitious roots, (Sachdev et al 2009).

Hasan (2002) has shown through his experiments that species of Aspergillus flavus, A.

niger, Fusarium oxysporum, Penicillium corylophilum, P. cyclopium, P. funiculosum and

Rhizopus stolonifer have the ability to produce gibberellins and indole acetic acid in presence

of calcium and sodium ions

Ahmad et al (2004) have isolated 21 different bacterial isolates from the rhizosphere

of different plants and characterized them for their production of IAA and pant growth

promoting activities. IAA has since been implicated in virtually all aspects of plant growth

35

and development Woodward & Bartel, (2005) and Teale et al (2006). It was later found that

not only plants but also microorganisms including bacteria and fungi are able to synthesize

IAA (Costacurta & Vanderleyden, (1995). In plant cells, auxins [specifically indole-acetic

acid (IAA)] are largely formed by de novo synthesis from tryptophan via oxidative

deamination or decarboxylation reactions. In microorganisms the three known pathways for

IAA production are also initiated by the tryptophan precursor and many bacteria possess

more than one pathway for IAA production. Interestingly, pathogenic strains tend to produce

IAA via a different route than nonpathogenic or symbiont strains (Patten and Glick 2002;

Tizzard, 2006).

Ahmad et al 2005 have reported the role of IAA producing Pseudomonas and

Azotobacter species from Aligarh which showed the capacity to stimulate the growth in

Sesbania aculeate and Vigna radiata in their experiments.

Microorganisms can produce a range of cytokinins similar to those produced by

plants - including kinetin, zeatin, and isopentyladenine (Tsavkelova et al 2006).

Ordhookhani (2011) has shown through his investigations with Ocimum basilicum

that PGPR treatments with Pseudomonas putida strain 41, Azotobacter chroococcum strain 5,

and Azosprillum lipoferum strain OF have significantly increased antioxidant activity of

essential oil and Fe, Mn and Cu contents of sweet basil plant.

Interactions between IAA-producing bacteria and plants lead to diverse outcomes on

the plant side, varying from pathogenesis to phytostimulation. Reviewing the role of bacterial

IAA in different microorganism–plant interactions highlights the fact that bacteria use this

phytohormone to interact with plants as part of their colonization strategy, including

phytostimulation and circumvention of basal plant defense mechanisms, (Spaean et al 2007).

Inoculation with IAA producing PGPR has been used to stimulate seed germination,

to accelerate root growth and modify the architecture of the root system, and to increase the

root biomass. In recent studies, Tsavkelova et al, (2007) have extended beyond individual

strains as inoculants and reported an increase in the germination of orchid seeds (Dendrobium

moschatum) inoculated with Sphingomonas sp. and IAA producing Mycobacterium sp. In

addition to stimulating root growth, IAA producing bacteria can also be used to stimulate

36

tuber growth. Swain et al. (2007) reported a positive effect of Bacillus subtilis IAA

producing strains on the edible tubercle Dioscorea rotundata L in one of their studies.

Many important plant-microbial interactions center on the production of auxins, IAA

being the main plant auxin. The ability to synthesize IAA has been detected in many

rhizobacteria as well as in pathogenic, symbiotic and free living bacterial species (Tsavkelova

et al 2006; Martínez-Viveros et al 2010). They have reported that microbes use Indole 3-

acetic acid (IAA) to interact with plants including phytostimulation and circumvention of

basal plant defence mechanism.

The effects of incubation time, temperature, pH, and agitation on indole-3- acetic acid

and gibberellic acid production in Aspergillus niger were studied by Bilkay et al (2010) who

exhibited the requirement of different temperature and pH requirements for the production of

optimum levels of IAA in vitro.

Khamna et al (2010) have isolated species of Streptomyces from the rhizosphere of

Thai medicinal plants which produced sufficient levels of IAA and enhanced the growth of

plants.

Phytohormones such as IAA may indirectly improve P acquisition by plants by

increasing root growth (Marschner et al 2011).

PGPR bacilli strains which have multiple mechanisms by which they promote plant

growth have attracted considerable interest by microbiologists; biofertilizers containing

Bacillus (Acuna et al 2011). Their experiments revealed the production of IAA and phytase

activity by the rhizospheric bacilli Bacillus and Paenibacillus to be characterized by low pH

and high total P.

Idris et al (2007) have shown that biosynthesis of IAA in the PGPR Bacillus species

affects its ability to promote plant growth in a positive way.

2.3.4. Production of Catalase

Lately it was shown that several rhizospheric bacteria could stimulate the plant

metabolic process and implicitly the plant growth (Stefan et al 2005). Enzymes produced by

37

soil microorganisms catalyzed biochemical processes involved in nutrient cycling in soil and

may provide an index of total microbial activity.

Soil catalase was used to characterize soil microbial activities. The mechanisms of

PGPR-mediated enhancement of plant growth and yields of many crops are not yet fully

understood. However, the possible explanations include: ability to synthesize hormones like

indole acetic acid (IAA) (Patten and Glick, 2002).

Kumar et al (2012) have shown that the activity of the lytic enzymes, hydrogen

cyanide, catalase and siderophore or through competition for nutrients and space can improve

significantly plant health and promote growth, as evidenced by increases in seedling

emergence, vigor, and yield.

Anandraj (2003) has reported the use of biocontrol agents in the prevention

and management of soil borne diseases in black pepper, cardamom and ginger and the role

played by PGPRs in suppressing the diseases of foot rot on a war footing has been

established.

Soil enzymes can affect decomposition of organic matter in soil and soil fertility

improvement and plant growth. Soil enzymes are specific catalyst for different organic matter

in soil. Because soil enzyme is involved in the biochemical processes of decomposition of

organic matter, and soil has the metabolic capacity (Cao et al 2003; Wen-Quan Niu, 2012).

The occurrence of high catalase and other enzymatic activity in the rhizospheric and

endophytic microbes has been shown.

2.3.5. Biological control of pathogens

Control of plant diseases has remained a challenge to mankind. Conventional

strategies of disease control were replaced with the use of chemical fungicide. However,

these fungicides affected soil fertility and the ecosystem. With root rot and spot, the efficacy

of fungicides and plant genetic resistance is determined by the interaction of environmental

and cultural conditions (Dean et al 2005). To overcome this problem, biocontrol agents have

been and are being, investigated. Research has repeatedly demonstrated that phylogenetically

38

diverse microorganisms can act as natural antagonists of various plant pathogens (Cook,

2000). The interactions between microorganisms and plant hosts can be complex. Interactions

that lead to biocontrol can include antibiosis, competition, induction of host resistance, and

predation (Gardener and Fravel, 2002).

Bio-control agents are easy to deliver, improve plant growth, and activate resistance

mechanism in the host, and increase biomass production and yield. These antagonists act

through antibiosis, secretion of volatile toxic metabolites, mycolytic enzymes, and parasitism

and through competition for space and nutrients (Nakkeeran et al 2005.)

Biological control is the viable strategy for sustainable disease management. Efficient

strains of P. fluorescens reduced the foliar infection caused by P. capsicii significantly. It has

been observed that the level of piperine, the pungent principle in black pepper, is increased to

significant levels upon root bacterization of the black pepper vines. In addition to it, piperine

(Sigma) inhibited the mycelial growth of P. capsicii, in vitro, demonstrating the direct

fungicidal activity of this alkaloid. Paul and Sarma (2004) have reported the role of

rhizobacteria in the induction of piperine in black pepper.

Ann (2012) isolated and selected indigenous soil Bacillus bacteria capable of

developing multiple mechanisms of action related to the biocontrol of phytopathogenic fungi

affecting pepper vines (Piper nigrum). The screening procedure consisted of antagonism tests

against a panel of phytopathogenic fungi, in vitro detection of the antifungal products and

root colonization assay. Four isolates, identified and designated as Bacillus amyloliquefaciens

(WW6), Bacillus atrophaeus (MPB), Bacillus subtilis (CBF) and Bacillus vallismortis

(WW14) were selected for further studies. All bacterial isolates obtained where effective for

the in vitro control of the growth of phytopathogenic fungi, where the control mechanisms

used by the bacteria involved the secretion of protease and cellulase enzyme that are

responsible for fungal cell wall hydrolysis. The bacteria also produced volatile as well as

diffusible substances.

Shashidhara et al (2008) have reported the occurrence of foot rot disease caused by

Phytophthora capsicii as one of the major diseases affecting the black pepper crop in

Karnataka, India. It has been claimed that the disease was put under control with the use of

39

biocontrol agents like Pseudomonas flourescences and Trichoderma harzianum by Jahgirdar

et al (2000).

Anandraj (2000) reported that in the rhizosphere of black pepper several antagonistic

microorganisms occur which help in the biocontrol of pathogenic fungi. Studies on foot rot

disease of black pepper, caused by Phytophthora capsicii L., are mainly focused on the

occurrence of rhizospheric microorganisms that can effectively suppress the disease,

(Noweriza and Quimio, 2004).

Paul and Sharma (2006) have evaluated the role of Pseudomonas flourescences in the

biocontrol of foliar infections caused by P. capsicii through mycelia growth inhibition and

shown that the bacterium increases the piperine content of the plant as well. Efficient strains

of P. fluorescens reduced the foliar infection caused by P. capsicii significantly. It has been

observed that the level of piperine, the pungent principle in black pepper, is increased to

significant levels upon root bacterization of the black pepper vines. In addition to it, piperine

inhibited the mycelial growth of P. capsicii, in vitro, demonstrating the direct fungicidal

activity of this alkaloid.

Fluorescent pseudomonads that produce biosurfactants with zoosporicidal activities

were isolated from the black pepper rhizosphere in Vietnam, and their genotypic diversity

and potential to control Phytophthora capsicii root rot was determined by Tran et al (2008).

Rhizobacteria that can provide biocontrol of disease or insect pests (biopesticides) are

considered an alternative to chemical pesticides (Zahir et al 2004). Phytophthora blight of

peppers is an important disease worldwide. The disease can affect plants at any growth stage,

and the damping-off syndrome can kill seedlings within 5 days of infection. The pathogen

can also cause crown, leaf and fruit blight, wilting of the whole plant and dark purplish

discoloration of the stem. Akgul and Mirik (2008) have seen into the possibility of using

phosphate-solubilizing bacteria which were inoculated with the pathogens into the

rhizosphere of pepper plants and their biological control properties were studied.

Chae et al (2006) have evaluated the compost sustaining capacity of chitinase

producing bacteria from the rhizosphere of pepper (Capsicum annuum L.) and their role in

the control of disease caused by P. capsicii.

40

Hoon et al (2007) have demonstrated the ability of bacterial entophytes that

have the capacity to promote the growth of pepper seedlings and protect pepper plants against

a bacterial pathogen. Two bacterial isolates obtained from the tissues of the pepper plant

Pseudomonas rhodesiae and Pantoea ananatis, respectively, were drenched on the pepper

seedlings promoted significant growth of peppers, enhancing their 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.

Bacterial spot disease caused by Xanthomonas axonopodis pv. vesicatoria (X.

axonopodis pv. vesicatoria) is a devastating pepper (Capsicum annuum) disease in Turkey.

Biological control of Xanthomonas axanopodis pv. vesicatoria was studied by Mirik et al.,

(2008) who have studied the phenomenon of biological control using three different strains of

Bacillus as biocontrol agents to check the disease.

Ragab et al (2009) have reported the inhibitory effect of the antagonistic bioagents,

chemical plant resistance inducers and some essential oils against the linear growth of two

isolates of F. oxysporum the wilt pathogen of pepper (Capsicum annum L.) was evaluated in

vitro. The antagonistic microorganisms, Trichoderma harzianum, T. viride, T. aureiviride,

Bacillus subtilis and Pseudomonas fluorescens were tested. Also, the tested chemical

inducers were Sodium benzoate, Potassium bicarbonate, Potassium sorbate and Chitosan.

Meanwhile, the tested essential oils were Cinnamon, Clove, Thyme, Lemon grass, Lemon,

Mint, Pepper mint and Mustard. The obtained results indicate that the antagonistic bioagents,

T. viride, B. subtilis, P. fluorescens showed superior inhibitory effect against the growth of

pathogenic fungi compared with T. harzianum and T. aureiviride. The fungal mycelial

growth reduced gradually by increasing of tested concentrations to reach complete reduction

(100%) at the concentrations of 4% for Potassium bicarbonate and Sodium benzoate and at

6% for Potassium sorbate.

Bacillus amyloliquefaciens strain KPS46, a plant growth promoting rhizobacterium

isolated from soyabean was investigated for the secretion of compounds that might be

involved in plant growth promotion by Buensanteai et al 2008. The analysis revealed a

number of proteins which may be involved in plant growth promotion by acting as plant

growth regulators, stimulating metabolism or functioning in defense against stress factors.

41

Utilization of microbial antagonists against plant pathogens in agricultural crops has

been proposed as an alternate to chemical pesticides. Fluorescent pseudomonads and Bacillus

species play an active role in suppression of pathogenic microorganisms. These bacterial

antagonists enforce suppression of plant pathogens by the secretion of extracellular

metabolites that are inhibitory at low concentration, Fernando et al 2005.

Pandya and Saraf (2010) have reported a huge array of microbes like bacteria and

fungi reside inside the plant tissues and interact with them. Of these, a range can be isolated

from apparently healthy tissues, many plants that have never been documented to be

associated with disease; others may cause disease when environmental conditions change.

Madhanraj et al (2010) isolated the genus Aspergillus, Penicillium and Trichoderma,

most frequently from the rhizosphere of Banana plants and were shown to produce metabolic

products that had antagonistic effect against Fusarium solanii.

Biocontrol treatments containing Azospirillum/ Azotobacter (Nitroxin),

Azospirillum/Bacillus subtilis/ Pseudomonas fluorescens (Super Nitro Plus), Glomus

intraradices (Mycorrhizal inoculant), Pseudomonas fluorescens, Glomus intraradices /

Pseudomonas fluorescens, Azospirillum/ Azotobacter/ Glomus intradica / Pseudomonas

fluorescens and a control was performed on the Hyssop (Hyssopus officinalis) by Tabrizi et al

(2008) who showed that the plant showed an increase in growth parameters significantly.

Cumin rhizobacteria were isolated from four locations of north-west India and

diversity of antifungal isolates in terms of inhibition of growth of different fungus isolates in

laboratory medium, PGPR activity and their phylogeny was studied by Nisha Kumari and

Deshawal (2012). They showed that Pseudomonas and Bacillus found in the rhizosphere of

cumin had potential as biocontrol agents and could inhibit fungal significantly in different

fungal isolates.

Adhikari et al (2013) isolated twenty one antagonistic bacterial isolates representing

biovars of Pseudomonas fluorescens (biovars I, II, III, and V) from the rhizosphere of okra,

chilli, ground nut, brinjal, cabbage and tomato from different agro-ecological regions of West

Bengal and subjected them to evaluate their antifungal activity under in vitro condition

against Rhizoctonia solani, the most important soil-borne plant pathogen two isolates, PF-8

42

and PF-7 effectively inhibited the mycelial growth of Rhizoctonia solani (72.05 and 68.25%,

respectively) in dual culture method. Rhizobacterial isolate PF-8 was found to be effective as

seed and soil treatment for management of root rot disease of okra. The antagonistic nature of

fluorescent pseudomonads against fungal pathogens can be assigned to design a potential

candidate for development of agriculture sector to be used in biological control of soil borne

plant pathogens.

2.4. Antimicrobial spectrum

The antiseptic qualities of aromatic and medicinal plants and their extracts have been

long recognized and attempt to characterize these properties in the laboratory date back to

early 1900s.

The plant based drugs form ingenuous pharmacological products used in India since

centuries. These active principles and volatile oils have provided vast number of chemical

formulations and have aided the discovery of newer drugs in the pharmaceutical industry,

(Lokhande et al 2007). Dr. K.M. Nadkarni in his book mentions the medicinal properties of

black pepper as a hot, acrid, pungent, carminative which can be used as an anti- periodic,

rubifacient, stimulant, used as a cubeb, on skin and mucous membranes. Pepper is elaborately

described for its medicinal uses in Ayurveda. It is described as a drug used to cure colds,

coughs, dyspnea, diseases of the throat, fever, colic, used as a digestive and for various

ailments, (Prabhakaran Nair, 2011).

The traditional uses include analgesic, antipyretic, CNS depressant, anti-

inflammatory, antioxidant, anticonvulsant, anti-bacterial, anti-tumor and hepato-protective

activities, (Madhavi et al 2009). Pharmacological basis for use of black pepper in Indian

medicines have been analysed by Mehmood and Gilani, (2010). Numerous classes of

phytochemicals including the isoflavones, anthocyanins and flavonoids are found associated

with the spices.

The role of terpenes in the formulation of the medicinal, culinary and fragrant uses of

aromatic and medicinal plants and their importance has been studied by various investigators.

Numerous phytochemicals (such as alkaloids, tannins, flavonoids and terpenes) present in

active extracts, tannins and flavonoids are thought to be responsible for antidiarrhoeal activity

by increasing colonic water and electrolyte reabsorption, (Palombo, 2006) Others act by

inhibiting intestinal motility. As some of the active ingredients are potentially toxic, there is a

43

need to evaluate the safety of plant preparations. Dorman and Deans (2008) gave the

importance of the presence of branched five carbon isoprene units in these terpenes. The

presence of oxygen in the frame work of terpenes increases their anti microbial properties.

The study conducted by Trivedi et al (2011) reveals relevant pharmacognostic,

phytochemical, physicochemical, chromatographic and antimicrobial data of two piper

species namely Piper nigrum and Piper longum. Black pepper oil contains β and α-pinenes,

δ-Iimonene and β-caryophyllene as major components. Caryophyllene is the substance with

sweet floral odours, whereas oils with high pinene content give turpentine like off-odours

(Mann, 2011). The major compounds in the fresh pepper are trans – linalool oxide and α-

terpineol. Pepper has long been recognized as a carminative, (a substance that helps prevent

the formation of intestinal gas), a property likely due to its beneficial effect of stimulating

gastric acid secretion by piperine, an alkaloid found in pepper.

Shan et al (2005) studied 26 different spice extracts and found that the major

constituents were high levels of phenolics and demonstrated high antioxidant capacity. Wide

variation in TEAC values (0.55−168.7 mmol/100 g) and total phenolic content (0.04−14.38 g

of gallic acid equivalent/100 g) was observed in these extracts. Major types of phenolic

constituents identified in the spice extracts were phenolic acids, phenolic diterpenes,

flavonoids, and volatile oils (e.g., aromatic compounds).

2.4.1. Antibacterial activity

Antibacterial potential of aqueous decoction of black pepper (Piper nigrum L.), was

studied by Masood et al (2006) against 176 bacterial isolates belonging to 12 different genera

of bacterial population isolated from oral cavity of 200 individuals. Overall aqueous

decoction of black pepper was the most bacterial-toxic exhibited 75% antibacterial activity.

The use of natural products for oral diseases and their perspectives were studied by Dr.

Amrutesh, (2011).

The susceptibility of pathogens to antibiotics greatly affects its ability to successfully

treat patients empirically. Plant derived products have been used for medicinal purposes

many centuries. At present it has been estimated that about 80% of the world population rely

on botanical preparations as medicine to meet the needs as they are considered safe and

provided to be effective against certain ailments, (Joe et al 2009).

44

The problem of drug resistance amongst microbes and the outlook for the use of

antimicrobial drugs in the future is still uncertain. Thus it is essential to combat this problem

and control the use of antibiotics and to continue studies to develop new drugs, (Nascimento

et al 2000).

Chaudry and Tariq (2006) have investigated the aquaeous extracts of pepper for their

anti-bacterial potential. Their investigation focused on antibacterial potential of aqueous

decoction of black pepper (Piper nigrum L.), bay leaf (Laurus nobilis L.), aniseed

(Pimpinella anisum L.), and coriander (Coriandum sativum L.) against 176 bacterial isolates

belonging to 12 different genera of bacterial population isolated from oral cavity of 200

individuals. It was shown that black pepper had the most bacterial toxicity.

Pingale and Ravindra (2013) have evaluated the anti- tubercular properties of Piper

nigrum in their studies and have shown the optimal sustained release formulation of

isoniazid microspheres and the effect of Piper nigrum (Black Pepper) on its in-vitro release.

Microspheres containing isoniazid were prepared using double emulsification and complex

co-acervation methods. The critical formulation variables were concentration of polymer,

drug - polymer ratio, cross-linking agent concentration and cross-linking time. The time to

release 85% of the contents of the microspheres (t85) was used as the measure for the release

time of the drug. The microspheres were optimized on the basis of their particle size,

percentage yield, entrapment efficiency, bio-adhesion study and in vitro drug release. The in

vitro release of the optimized batches of isoniazid microspheres was enhanced to the extent

of 100-107%, by co-administration of 10 or 15 mg of bioenhancer.

Joe et al (2009) have evaluated the antimicrobial effect of the extracts of the three

widely used spices in South India such as garlic (Allium sativum), ginger (Zingiber officinale)

and pepper (Piper nigrum). Dr. Amrutesh (2011) has reported the anti microbial properties of

black pepper with respect to dental infections as depicted in Ayurvedic scriptures and

formulations.

The inhibitory effect of the antagonistic bioagents, chemical plant resistance inducers

and some essential oils against the linear growth of two isolates of F. oxysporum the wilt

pathogen of pepper (Capsicum annum L.) was evaluated in vitro. The antagonistic

microorganisms, Trichoderma harzianum, T. viride, T. aureiviride, Bacillus subtilis and

45

Pseudomonas fluorescens were tested by Ragab et al (2012). They also the tested the effect

of chemical inducers like Sodium benzoate, Potassium bicarbonate, Potassium sorbate and

Chitosan and essential oils of cinnamon, clove, thyme, lemon grass, lemon, mint, pepper

mint and mustard on the pathogens. Results also showed that thyme, lemon grass,

peppermint, clove and mint oils had higher inhibitor effect on fungal mycelial growth than

lemon, cinnamon and mustard oils. Fungal mycelial growth decreased significantly as the

concentrations of essential oils were increased, to reach the fungal growth’s minimum at the

highest concentration used. Complete reduction (100%) in mycelial growth of two fungal

isolates was recorded at concentration of 6% of all tested essential oils.

The antibacterial activity of the n-hexane, acetone/dichloromethane, ethanol and

aqueous extracts of twelve common medicinal plants from the Philippines obtained through

pounding and solvent extraction was evaluated by Penecillo and Magno (2011) using disc

Agar diffusion. The microorganisms tested were: Staphylococcus aureus, Bacillus subtilis,

Escherichia coli, and Pseudomonas aeruginosa. The common medicinal plants which

showed highly positive activity were Psidium guajava (guava), Eucalyptus globulus,

Mangifera indica (Indian mango), Nasturtium officinale (Watercress), Pterygospermum

oleiferum (Moringa), Carmona retusa (Wild tea), Citrus aurantifolia (Lemon), Citrus

sinensis (Orange), Allium sativum (garlic), and Allium cepa (onion). Preliminary

phytochemical screening revealed the presence of flavonoids, tannins, alkaloids, glucosides,

saponins and steroids/terpenes. The results suggest that the different plant extracts contain

bioactive constituent(s) particularly tannins, flavonoids, terpenoids and other glycosides with

very strong antibacterial activity and validates the ethno-medical use in the treatment of

bacterial skin diseases and other forms of bacterial infections.

2.4.2. Antifungal activity

The pepper extracts and volatile oils of other plants were studied for their anti

microbial properties by Dorman and Deans, (2000) and Indu et al 2006). The anti-fungal

activities were identified with several species of Aspergillus and bacterial isolates from the

rhizosphere of pepper, (Ghai et al 2007, Sadeghi-Nejad, 2010). The antibacterial activities of

piper longum have been studied by Lokhande et al (2007).

46

Pundir and Jain (2010) have evaluated the antimicrobial activity of extracts of pepper

and curcumin on B.subtilis and other bacterial and fungal strains of Rhizopus and Mucor.

Joe et al (2009) have reported the anti microbial properties of black pepper ethanol

extract against E. coli and Staphylococcus aureus.

The antibacterial susceptibility testing of piper extracts using broth MIC assay has

been done by Pessini et al (2003). The MIC methods have been further supported by

Cavalieri et al (2005). Antifungal properties of extracts from eight Brazilian plants

traditionally used in popular Brazilian medicine were tested against five clinically relevant

Candida species, Cryptococcus neoformans, and Sporothrix schenckii by Johann et al (2007).

Results demonstrate that almost all extracts exhibited antifungal activity, at least against one

of the microorganisms tested. The ethanolic extract from the leaves of Schinus

terebinthifolius exhibited potential antifungal activity against C. glabrata and S. schenckii.

Preliminary phytochemical analysis of extract from S. terebinthifolius showed the presence of

biologically active compounds, namely saponins, flavonoids, triterpenes, steroids and

tannins.

2.4.3. Piperine structure and HPLC

Piperine in recent studies is known to increase thermo genesis in GI tract and make

available a broad range of nutrients and water soluble vitamins, fat soluble β- carotene as

well as seleno- amino acids. Dried fruits of Piper nigrum (black pepper) are commonly used

in gastrointestinal disorders. The study was conducted by Mehmood and Gilani (2010) to

rationalize the medicinal use of pepper and its principal alkaloid, piperine, in constipation and

diarrhea using in vitro and in vivo assays. Piperine exhibited a partially atropine-sensitive

laxative effect at lower doses, whereas at higher doses it caused antisecretory and

antidiarrheal activities that were partially inhibited in mice pretreated with naloxone

(1.5 mg/kg), similar to loperamide. This study illustrates the presence of spasmodic

(cholinergic) and antispasmodic (opioid agonist and Ca2+ antagonist) effects, thus providing

the possible explanation for the medicinal use of pepper and piperine in gastrointestinal

motility disorders.

Piperine is a solid that is essentially insoluble in water. It is weak base which is

tasteless but leaves a burning sensation. Piperine belongs to vanilloid family of compounds,

47

which also includes capsaicin, the pungent formula in the chilli peppers. Its molecular

formula is C17H19NO3 and has a molecular weight of 285.34 daltons. It is the trans - trans

stereoisomer of 1- piperoylpiperidine, (Badmaev et al 2000).

Pepper extract was obtained with 95% ethanol. The extract was purified with acetone

and KOH, as given by Lin et al 2001) who have obtained a patent for the preparation of

piperine. The percentage of piperine in the extract was determined using high pressure liquid

chromatography (HPLC) analysis, Reshmi et al 2010). The fluorescence and HPLC

standardization of Piper nigrum fruits was conducted by Jain et al 2007). The pepper extracts

were isolated and formulated into alginate beads by Madhavi et al 2009).

2.5. Anti-oxidant activity for pepper extracts

Antioxidants terminate these chain reactions by removing free radical intermediates,

and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants

are often reducing agents such as thiols, ascorbic acid or polyphenols. Plants have developed

an array of defense strategies (antioxidant system) to cope up with oxidative stress. The

antioxidative system includes both enzymatic and non-enzymatic systems. The function of

this antioxidant system is to scavenge the toxic radicals produced during oxidative stress and

thus help the plants to survive through such conditions (Mandal et al 2009). There are

number of herbal drugs and formulation available to withstand stress and strain of life

without altering physiological functions of the body.

Molecules containing unpaired electrons are known as free radicals that cause tissue

collapse by means of DNA, protein, and lipid damage (Guha et al 2010). Free radicals, such

as superoxide anion, hydroxyl radicals, and hydrogen peroxide, are known as reactive oxygen

species (Rajkumar et al 2010). They reported that antioxidants play an important role in

inhibiting and scavenging free radicals, thus providing protection to humans against

infections and degenerative diseases.

Natural plant compounds show a wide range of activities like anti-cancer, anti-

inflammatory, and antiageing. Ageing is due to complicated biochemical processes. This

review was given by Sharazadeh (2013) on some natural compounds serve as anti-ageing

materials. Some materials such as phenolics, carotenoids, terpenoids or alkaloids may have

an important role as antioxidant compounds and as free radical scavengers.

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Keservani et al (2010) have reported the need for those within the industry to become

more vigilant in their use of terminology with increasing consumer awareness as it applies to

terms such as nutraceutical, functional, medical and novel foods. Dietary supplements have

also been developed to manage a variety of diseases. Recognition of variation in functional

food and nutraceutical composition provide opportunity for the industry to give consumers a

variety of new products that can be developed for niche or specialized markets. Pepper has

been classified as a nutraceutical plant material by these workers.

Diverse medicinal plants have been screened and assessed for their ability to agonize

free radical- induced oxidative stress, (Zhang, 2004).

Spices and herbs are recognized as sources of natural antioxidants and thus play an

important role in the chemoprevention of diseases and aging, Khalaf et al (2008). The

antioxidant and radical scavenging activities of black pepper (Piper nigrum Linn.) seeds have

been well reported, as reported by Qulcin, (2005). Natural antioxidants that are present in

herbs and spices are responsible for inhibiting or preventing the deleterious consequences of

oxidative stress. Spices and herbs contain free radical scavengers like polyphenols,

flavonoids and phenolic compounds, (Khalaf et al 2008).

Antioxidant activity of pepper oil and extracts were determined by three different

methods- 2, 2-Diphenly 1-picryl hydrazyl (DPPH) mentioned according to Bandoniene,

(2002) and superoxide dismutase scavenging method by Llop et al 2000), and scavenging

by nitric oxide, (Nenadis et al 2004).

The antioxidant and stability of hexane solutions during storage of black and white

pepper essential oils along with essential oils of other spice plants was studied by Misharina

et al 2009).

Effect of heat treatment and storage on antioxidant activity of black pepper, allspice

and oregano was studied by Harvathova et al (2007) by determination of antiradical activity,

reducing power, thiobarbituric acid number and content of total phenolic substances.

Thermal treatment at 130°C for 5 minutes caused significant decrease of all

antioxidant activity parameters with the exception of increased content of phenolic

substances in black pepper.

49

Anti oxidant and radical scavenging activity of pepper was studied by Gulcin, (2005).

The anti oxidant activity of PET extract of pepper was given by Singh et al (2008).

The anti oxidant and anti inflammatory properties of black pepper, P. nigrum, Linn.

have been reported by Singh and Duggal, (2009). The anti-oxidant activity of Trikatu, an

Ayurvedic preparation containing Piper nigrum fruits has been studied and reported to have

good potential by Jain and Mishra, (2011). The antioxidant properties were determined by

DPPH and super oxide radical scavenging methods. P.nigrum Linn. was found to have good

antioxidant properties in synergism with P.longum Linn. and Zinziber officinalis Rosc.