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ABSTRACT
Introduction
Plants are generally associated with diverse or more endophytes (Strobel et al., 1993).
microorganisms. Endophytic organisms are But most likely, there is not a single plant
those that colonize the plant internal tissue species devoid of endophytes. Only a few of
showing no external sign of infection or these plants have ever been completely
negative effect on their host (Schulz & studied relative to their endophytic biology.
Boyle, 2006). Plants constitute vast and Consequently, the opportunity to find new
diverse niches for endophytic organisms. Of and beneficial endophytic microorganisms
the nearly 300,000 plant species that exist on among the diversity of plants in different
the earth, each individual plant is host to one ecosystems is considerable.
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The term endophyte (Gr. endon, within; plants include by helping plants acquire
phyton, plant) was first coined by De Bary nutrients, via nitrogen fixation, phosphate
(De Bary, 1866) and an endophyte is a solubilization or iron chelation, increased
bacterial or fungal microorganism, which drought resistance, thermal protection,
spends the whole or part of its life cycle survival under osmotic stress (Bacon and
colonizing inter- and/or intra-cellularly Hinton, 2006). A particular bacterium may
inside the healthy tissues of the host plant, affect plant growth and development using
typically causing no apparent symptoms of one or more of these mechanisms and may
disease (Wilson, 1995). The presence of use different ones at various times during
endophytes was reported by Vogl in 1898 the life cycle of the plant. In addition to
who revealed a mycelium residing in the these plant-growth-promoting traits,
grass seed Lolium temulentum. endophytic bacteria must also be compatible
with host plants and able to colonize the
Endophytic bacteria have been isolated from tissues of the host plants without being
a large diversity of plants. Organisms like recognized as pathogens (Rosenbleuth and
Bacillus, Enterobacter, Klebsiella, Martinez-Romero, 2006). The survival and
Pseudomonas, Burkholderia, Pantoea, conservation of endophytic communities of
Agrobacterium, Methylobacterium spp. bacteria can also be affected by the type of
constitute the endophytes commonly plant propagation methods used. These
isolated from diverse plants such as rice, bacteria can develop, become distributed
wheat, maize, cotton, clover, potato, throughout the plant as it grows and are then
sugarcane, tomato, cucumber, and wild returned to the soil via crop residues. Any
grasses (Bacon and Hinton, 2006). The plants that are propagated vegetatively have
precise role of endophytes in plants is not an enduring community of bacterial
yet known. However, their capability to colonists that are transferred in successive
thrive within the host tissues away from progeny generations. True seeds can also be
microbial competition and environmental the source of endophytic bacteria in the
degradation has made endophytes potential developing seedling.
candidates for use in agriculture.
Materials and Methods
The role of endophytic microorganisms in
plants can be divided into two categories Collection of samples
based on types of activity: growth promotion
and disease control. Endophytic bacteria are In the present study, Black grapes and
believed to elicit plant growth promotion in Avacado were selected as they are loaded
one of two ways: Directly by producing with antioxidant properties and are less
phytohormones such as auxin or cytokinin probed in terms of endophytic bacteria. The
or by producing the enzyme 1- samples were collected from the regional
aminocyclopropane- 1-carboxylate (ACC) fruit outlet and transported in sterile
deaminase, which lowers plant ethylene polythene bags for further processing.
levels and indirectly by preventing pathogen
infections via antifungal or antibacterial Surface sterilization
agents, by outcompeting pathogens for
nutrients by siderophore production, or by The samples were rinsed with autoclaved
establishing the plant s systemic resistance. distilled water, disinfected with Hydrogen
Other beneficial effects of endophytes to peroxide for 2 minutes. Then were rinsed for
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5 minutes with 70% Ethanol followed by for PGP traits using various assays such as
3% Hypochlorite + Tween 20 (0.1%) and Siderophore production, HCN production,
finally rinsed with autoclaved distilled Ammonia Production and IAA production.
water.
Siderophore Production
Processing of Samples and Isolation
Siderophore production was detected by CAS
Two methods were employed in the present assay. This assay was done qualitatively and is
study in order to isolate endophytic bacteria based on competition for iron between a ferric
from the fruits. First method involved complex of chrome azurol S (CAS), an indicator
cutting samples were into 2 halves; and each dye, and a siderophore produced by the
half was impregnated on Nutrient Agar microorganism (Schwyn and Neilands, 1987).
plates and incubated at room temperature for Each endophytic isolate was streaked on the
24h. The second method involved surface of CAS medium and incubated at
macerating the surface sterilized samples room temperature for 1 to 3 days.
using a sterile pestle and mortar. 1 g of the Siderophore production was indicated by orange
macerated sample was serially diluted and halos around the colonies after the incubation.
the dilutions were plated on Nutrient Agar
media and incubated at room temperature HCN Production
for 24h. After overnight incubation, isolated
colonies were selected and used further. Screening of endophytic bacterial isolates for
HCN production was done according to (Lorck,
Morphological Characterization of 1948). The selected isolates were grown in
endophytic bacterial isolates Nutrient Agar supplemented with glycine
(4.4g/l). A Whatman filter paper No.1 soaked in
The selected strains were morphologically 0.5% (w/v) picric acid solution was placed to the
characterized in order to determine the underside of the Petri dish lids. To avoid the
morphology of the bacterial cells upon escape of the gas, the plates were sealed with
observable characteristics such as cell shape, parafilm and incubated at room temperature for
colony color and texture. This was 5-7 days and the production of HCN was
determined by the classical gram staining determined by the change in color of filter paper
method as described by (Cappuccino and from yellow to red-brown.
Sherman, 2002).
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IAA production was determined according Catalase activity of the bacterial isolates was
to (Brick et al., 1991). The endophytic determined by adding H2O2 over the freshly
bacteria were grown in LB broth grown endophytic bacterial cultures in NA
supplemented with L-tryptophan (1µg/ml) plates. The presence of oxygen bubbles gave
for 72 hours. Overnight cultures were the evidence of catalase activity of the
centrifuged at 10,000g for 10 minutes and 1 bacterial isolates (Cappuccino and Sherman,
ml of the supernatant was allowed to react 1992).
with 2 ml of Salkowsky reagent, incubated
for 20 minutes at room temperature Results and Discussion
(appearance of brownish pink color
indicates the presence of IAA) before the Isolation of endophytic bacteria
absorbance was measured at 530nm. The
absorbance of the samples obtained was A total of 6 endophytic bacteria were
plotted against a standard to determine the isolated from avacado and black grapes.
concentration of IAA produced. Among the 6 isolates, 3 isolates namely G1
(N), G2 (N) and G1 (O) were from black
Analysis of Enzyme Activity grapes and other 3 isolates namely SA3,
SA4 and A1 were from avocado.
The endophytic bacterial strains were
screened for various enzyme activities such Morphological Characterization
as Lipase activity, Protease activity and
Catalase activity. The selected endophytic bacterial strains
were morphologically characterized by
Lipase Activity Gram staining (Figure 1). It was observed
that all the isolates from black grapes were
The lipase activity of the selected gram positive short rods, whereas two
endophytic bacterial isolates was determined isolates from avocado were found to be
by supplementing the Nutrient Agar media gram positive cocco bacilli.
with 0.01% CaCl2.H2O, followed by adding
sterilized Tween 80 to the media to give a Screening of endophytic bacteria for PGP
final concentration of 1%. The media was traits
poured into the Petri plates, and presence of
opaque halo zone around the colonies was Among the PGP traits screened, it was
considered as positive (Sierra, 1957). observed that all the six isolates were
positive for Ammonia Production. IAA
Protease Activity production was observed in all the strains
and further quantification was performed.
Proteolytic activity of the cultures was Among the 6 isolates, strain G2 (N), G1 (O),
studied in a medium containing skimmed SA3, SA4 and A1 were positive for
milk. Zone of precipitation of paracasein siderophore production. Likewise HCN
around the colonies in the next 48 hours production was observed only by strain G1
were taken as evidence of Proteolytic (N) and G2 (N). (Table 1)
activity (Vieira, 1999).
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isolates, four isolates exhibited siderophore activity for 3 enzymes: Lipase, Catalase and
activity. Several reposts from the past have Protease. Except for A1 all the isolates were
confirmed that siderophore producing found to be Lipase test while catalase was
bacteria significantly help in the uptake of being produced by all the endophytic
various metals such as Fe, Zn and Cu by bacterial isolates. Though, protease assay
plants (Carrillo- Castenada et al., 2003; was positive for strains G1 (N) and G1 (O)
Egamberdiyeva, 2007; Gururani et al., only. Buchenauer (1998) also stated that
2012). HCN production was observed only lytic enzymes secreted by bacteria are
by 2 strains among the 6 strains and suspected to play an important role in
Ammonia production was observed in all the suppression of pathogens. Further molecular
strains which were found to be in parallel characterization and their effects on plant
with those seen by Sudhir Allu et al., 2014. growth under pot and field conditions would
The endophytic bacterial isolates were help us to understand the plant microbe
further studied for their hydrolytic enzyme interaction in detail.
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G2 (N) 30.26
G1 (O) 24.51
SA 3 54.83
SA 4 34.75
A1 49.97
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All plants are hosts to one or more bacilli. According to Jacobs et al., (1985)
endophytic microorganisms. However the the most common taxa of endophytic
endophyte plant interaction is one of the bacteria recovered include Bacillus,
least studied biochemical systems in Enterobacter, Erwinia, Pseudomonas and
nature. Endophytes include the fungi, Flavobacterium. McInroy and Kloepper
bacteria, and actinomycetes that primarily (1995) reported that the endophytic
reside in the tissues beneath the epidermal bacterial diversity spanned over 40 genera,
cell layers and the host tissues are with predominance of Pseudomonads and
transiently symptomless and Bacillus. Hallmann et al., (1997) have
inconspicuous (Stone et al., 2000). There reported that the former Pseudomonas
is ample evidence that many endophytic group (Pseudomonas, Burkholderia) and
bacteria have beneficial effects on plants Enterobacteriacea (Enterobacter,
(Hallmann et al., 1997). Growth Klebsiella) are the common taxa found in
promotion of plants may be achieved by tomato, potato, cotton, soybean, rice and
bacterial production of plant growth maize. These taxa were predominantly
regulators such as auxins, cytokinins and found in the present study.
gibberellins; nitrogen or other nutrients
may be provided by biological nitrogen The selected bacterial isolates were tested
fixation or mobilized as is the case for for PGP traits. All the six strains were
phosphorus; moreover, endophytes may found to produce IAA. IAA, a member
confer plant protection against pathogens belonging to the group of phytohormones,
by induction of plant defense mechanisms, is generally considered to be the most
pathogen-antagonistic substances or important native auxin (Strzelczyk and
through competition for colonization sites Pokojska, 1984). Further quantification of
and nutrients. The present work is carried IAA produced was performed. It was
out to study the diversity of endophytes of observed that maximum production of
fruits such as Avacado and Black Grapes IAA was exhibited by strain SA 3 (54.83
which are known to have high antioxidant µg/ml). IAA production by endophytes
property. isolated from sweet potato which could
influence host growth in fertile soil has
The isolates selected in the present study been reported by Zareen Khan et al., 2005.
were chosen for their dominance as well as In the present study among the six isolates,
uniqueness or differences with others in four isolates exhibited siderophore
colony morphology. Six isolates were activity. Several reposts from the past have
selected from Avacado and Black Grapes confirmed that siderophore producing
and were named according to the host bacteria significantly help in the uptake of
plant. All the bacterial isolates were found various metals such as Fe, Zn and Cu by
to be Gram positive. Earlier workers have plants (Carrillo- Castenada et al., 2003;
reported a predominance of Gram negative Egamberdiyeva, 2007; Gururani et al.,
bacteria in the tissues of various plants 2012). HCN production was observed only
(Sudhir allu et al., 2014). However, Toldi. by 2 strains among the 6 strains and
O et al., (2009) reported an equal presence Ammonia production was observed in all
of Gram negative and Gram positive the strains which were found to be in
bacteria in strawberry. The bacterial parallel with those seen by Sudhir Allu et
isolates in the present study were found to al., 2014. The endophytic bacterial isolates
be either Bacillus short rods or cocco- were further studied for their hydrolytic
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