Role of endophytes in Agriculture.

ENDOPHYTES

 Plants are living in diverse environments (aquatic, terrestrial) and interacting with

a diverse group of microbes i.e. Bacteria, fungi, nematodes etc.

 Plant microbes interaction may be parasitic or symbiotic

 Amongst these Endophytes are those microorganism that can reside within plant

tissues without any external sign of infection or other harmful effects on the host

plants.

 Endophytes are present in all known plant species and can inhabit different

compartments of a plant such as leaf, stem, root, seeds and flower

 Bacterial endophytes are highly diverse and polyphyletic in nature

 The most commonly-occurring bacterial genera include Pseudomonas, Bacillus,

Burkholderia, Stenotrophomonas, Micrococcus, Pantoea, and Microbacterium.

 Fungal endophytes constitute important component of plant endophytic community.

Most of the reported endophytic fungi belong to the phyla, Ascomycota, Basidiomycota

and Mucoromycota .

 Diversity and abundance of endophytes have been reported in many plants species with

respect to low and high temperature, drought, pH and salinity.

 Endophytes transmission may occur vertically through the seeds and pollen grains or

horizontally via the rhizospheric soil or airborne spores

 Their entry into the host plant can occur through the cracks in the lateral root junction,

wounds caused by phytopathogens, root hairs, stomata and lenticel (spaces between

epidermal cells)
 Applications of endophytes in agriculture
Bacterial and fungal Endophytes have the potential to bring rapid and revolutionary changes

in the field of ecofriendly agriculture practices, that offer several benefits to the host plant. i.e

 Growth promotion

 Biocontrol

 Disease suppression

 Stress tolerance.
Endophytes help plants directly, either by aiding in the acquisition of nutrients such as

 Phosphate solubilization

 Siderophore Production

 Nitrogen fixation

 Production of phytohormones such as IAA, gibberellic acid, and

salysalic

 Metabolites production i.e. EPS, Flavonoids and phenolics

 Isolation of Endophytes
 Plant parts will cut into small sizes and washed several times with running tap water to
remove surface contaminant and dust.

 Plant parts will be Surface sterilized with sodium hypochlorite's and ethanol in
Laminar Flow Hood and rinse several time with autoclave distal water .

 The sterilized parts will be placed on surface of NA/PDA plate and incubated for 3-7
days at 28 °C .

 To purified the bacterial isolates, morphologically different bacterial colony were

picked from plates by sterilized loop and streaked on nutrient agar plate, and placed in
incubator for 24 hrs. at 28C.
 Screening of Endophytes for Phytohormones production
IAA Production
 selected bacterial colonies were inoculated on Nutrient broth and were incubated in shaking
incubator for three days at 180 ppm and 28c.after three days of incubation, the bacterial
culture were centrifuged for 15 minutes at 6000 rpm and cell free supernatant were transfer
to clean falcon tube.
 IAA production was estimated my mixing 2ml Salkowski’s reagent with 1 ml of cell free
supernatant and incubated in the dark for 25 minutes at 30 °C. development of pink-red
color in the reaction mixture indicated the presence of IAA. The optical density was
recorded at 535 nm using a spectrophotometer.
 GA3 production

 Five days old bacterial culture, grown on Nutrient broth at 30°C were centrifuged for 15

minutes at 10000 rpm to separate cell free supernatant.

 pH of the supernatant was kept at 2.5 by using 3.75 N HCl.

 Ethyl acetate was used to extract gibberellic acid

 The aquas phase was transfer to separated funnel.

 Absorbance was recorded at 254 nm.

 Salicylic Acid production

 36 hrs. bacterial culture in broth will be centrifuged at 6000 rpm for 10 minutes.

 pH of the supernatant was maintained at 2.0 by using 1N HCl.

 Salicylic acid was extracted by adding 1ml ethyl acetate into 2 ml supernatant.

 One volume of 2.5 mM FeCl3 was added to the ethyl acetate phase.

 Consequently, the purple Fe–SA complex developed in the aqueous phase indicated the

presence of salicylic acid.

 the absorbance of this complex was measured at 520 nm.

 Siderophores production
 100ul of bacterial inoculum containing 108 cfu/ml, from freshly grown bacterial
culture will be added to 50 ml nutrient broth and incubated for 48 h at 30 c and 180
rpm.
 Cell free supernatant will obtained by centrifuging the bacterial culture at 10000
rpm for 12 minutes
 the supernatant will transfer to iron free flask. One ml of each bacterial culture
supernatant will be loaded into separate wells of 24 wells microplate followed by
adding 1 ml autoclave CAS-HDTMA reagent and incubated for 30 minutes.
 After incubation, microplate reader will used to record the optical density of each
sample at 630 nm and % siderophore unit (psu) production will be estimated.
 Phosphate Solubilization Assay
 0.1 ml freshly grown bacteria will be inoculated in 20 ml PVK broth containing 5 mg/L tricalcium

phosphate and incubated at 30 °C and 150 rpm for 3 days in shaking incubator.

 three-day old cultured will be centrifuged at 12000 rpm for 15 minutes to obtained the

supernatant.

 0.5ml Barton’s reagent will be mixed with 0.2ml supernatant and the volume of the reaction

mixture will be made up to 10 ml with double distilled water.

 After 10 minutes of incubation the intensity of yellow color will be read with spectrophotometer at

430 nm.
 Molecular Identification of endophytic bacteria

The selected bacterial isolates were identified up to species level by using 16s rDNA gene

sequencing.

 DNA Extraction: genomic DNA will be extracted from bacterial strain by modified

chillax method.

 Gel Electrophoresis: Genomic DNA extraction was confirmed by running the DNA in

1% agarose gel.
PCR Base amplification of 16s ribosomal DNA :

 27F forward (5”-AGAGTTTGATCMTGGCTCAG- 3”) and 1492R reverse (5”-

GGTTACCTTGTTACGACTT-3”) universal primers were used to amplified 16s ribosomal

gene.

 A total of 50 µl reaction mixture for each bacterial isolate composed of 25 µl 2X green master

max, 4 ul reverse primer, 4 ul forward primer, 4 ul of template DNA and 13 ul PCR grade

water was used for PCR amplification of

 Initial denaturing of template DNA for 5 minutes at 94ºC, denaturation of DNA for 1 minute at

94ºC, annealing of primer at 53 ºC for 1 minute, and extension for 1 minute at 72 ºC.
 Sequence Alignment and Construction of Phylogenetic Tree

 Finally, 20 µl of the PCR product for each bacterial isolate was placed in an ice-box pack
and sent for sequencing at Inqaba Biotechnical Industries (Pty) Ltd, Pretoria of South
Africa.

 The Basic Local Alignment Search Tools (BLAST) program of the nucleotide sequences
on the National Center for Biotechnology Information (NCBI) was employed to determine
bacterial isolate sequence similarities and identities.

 The sequenced data were further analyzed by subjecting to multiple sequence alignment
by ClustalW using a Bio-Edit program. MEGA-X online program was used to construct
the phylogenetic tree.
 References
Burragoni, S. G., & Jeon, J. (2021). Applications of endophytic microbes in agriculture,
biotechnology, medicine, and beyond. Microbiological Research, 245, 126691.