Dr.Sandhya Vardharajula Assistant Professor- Microbiology

Agri Biotech Foundation, PJTSAU Campus, Rajendranagar, Hyderabad, Telangana State,

India Email:- sandhyarao28@gmail.com

Website:- www.abfindia.org

Endophytic bacteria as plant growth promoters under biotic and abiotic conditions

4th Asian PGPR Conference May 3rd-6th,2015 Hanoi, Vietnam

Major challenge for 21st century sufficient food - 10 billion by 2050. The challenge - boost plant productivity -environmentally sustainable manner.

Management & maintenance of natural resources, environment -satisfy human needs

Stresses threats to agriculture, resulting in deterioration of environment Plants dependent on environment, hence constrained to biotic and abiotic stresses. Reducing yields by more than 50%.

High light - photosynthetic reaction-accumulation of ROS

High temperature denatures proteins and lipids Drought interferes metabolism, ROS

Soil salinity-ion cytotoxicity and uptake of nutrients

Air pollution with oxidizing species (OZ & H2SO4)-oxidative damage

Mechanical damage by biotic and abiotic

Cold stress interferes enzyme activity, damage tissues, water deficit.

Microbial communities that interact with plants - health, productivity, biodiversity.

Plant growth and health through mechanisms as PGPR.

Growing interest as biofertilizers /biocontrol

Aid N & P

Protect from diseases,pests

Abiotic stresses

Fossil records > 400 million years

Endophytes within Plants

Endophytes

Plant-Microbe Interaction under abiotic stress

Climate change and use of marginal land for crops needs- innovative management systems adapted to stressful environments.

Beneficial plant–microbe interactions that promote plant growth is subject of study.

Organism Crop Type of stress Mechanism References P. polymyxa and Rhizobiumtropici

Common bean Drought

Change in hormone balance and stomatal conductance Figueiredo et al. 2008

Pseudomonas sp. Pea Drought Decreased ethylene production Arshad et al 2008 Pseudomonas mendocina and Lettuce Drought Improved antioxidant status Kohler et al 2008 Glomus intraradices Pseudomonas sp. AMK-P6 Sorghum Heat Induction of heat shock proteins Ali et al 2009

Pseudomonas putida P45 Sunflower Drought Improved soil aggregation due to EPS production

Sandhya et al. 2009a;2009b

Bacillus megaterium and Glomus sp. Trifolium Drought IAA and proline production Marulanda et al. 2010 Pseudomonas fluorescence Synthesis of ACC-deaminase Ali et al. 2014 Bacillus spp. Drought Soil Aggregation Sandhya et al. 2014

Pseudomonas spp.

Drought, High temperature, Salinity EPS Production Sandhya et al. 2015

Table 1 Microorganisms conferring abiotic stress tolerance in crop plants

Rhizobacteria from rhizosphere/rhizoplane of plants promoting plant growth- direct and indirect mechanisms.

Fungal endophytes Type of stress Host plant/cultivar Reference

Neotyphodium sp. Drought Festuca pratensis, Perennial Ryegrass Morse, et al. 2002

Trichoderma hamatum Drought Cacao Ravel, et al.1997 Fusarium culmorum Drought Tomato,Rice

Colletotrichum magna Drought Pepper,Tomato Singh et. al. 2011, NAAS 2014

Phialophora sp. Drought F. pratensis Malinowski, et al. 1997 Curvularia protuberate Heat Dichanthelium lanuginosum Redman, et al. 2002

C. protuberate Drought Oryza sativa, Triticum aestivum Watermelon Rodriguez, et al. 2008

Table 2 Fungal endophytes that conferred abiotic stress tolerance

Singh et al.2011

Associative bacteria Plants Pathogenic Fungi/Bacteria

P. fluorescens EP Sugarcane C falcatum

P. putida Oak Ceratocystis fagacearum

P. fluorescens Tomato F. oxysporum f. sp. radicis-lycopersici

P. fluorescens pea Pythium ultimum and F. oxysporum f. sp.

Bacillus pumilus SE Pea F. oxysporum f. sp. Pisi

Bradyrhizobium Sp. Arabidopsis transcriptome analysis based study

Paenibacillus alvei K A. thaliana Verticillium dahlia Actinobacteria A. thaliana Quantitative PCR analysis based study

Table 3 Biocontrol activity of associative bacteria

Jha et al. 2013

Carvalho et al. 2013, Naveed et al. 2013

Endophytes Host plant/cultivar

Mechanism Crop Reference

Rahnella sp. Rhodotorula graminis, Rhizobium tropici Sphingomonas

Poplar & Willow IAA,N fixation Corn, tomato, pepper, sunflower,

Khan et al.2012

Associative bacteria/endophytes

Rice & Sugarcane

N-fixation, Hormones

Sugarcane, grapevine, tomato, pea, A. thaliana

Jha et al. 2013

Bacillus,Lysinibacillus, Pseudomonas, Achromobacter Brevibacterium

Prosopis strombulifera (halophyte)

Siderophore, IAA, Gibberellic acid

Plant growth traits

Sgroy et al. 2009

Folier bacterial Endophyte

Pinus radiata anti-fungal properties

Munters 2014

B.Subitilis, B. pumilus, B.licheniformes , B.cereus, B.amiloliquefascens

Maize leaf Taxonomic studies

Figueiredo et al. 2009

Burkholderia phytofirmans PsJN & Enterobactersp. FD17

Maize roots, Vegetable crops

Improving physiology, growth

Maize under drought

Naveed et al.2014,

Invade plant tissues

Endophytes Epidermis/ apoplastic intercellular spaces

Site of injury in epidermis Root tips Site of lateral root

emergence

Table 4 Endophytes as plant growth promoters

b) Uninoculated - stressed & Sphingomonas yanoikuyae, willow bacterial endophyte are healthier and greener

a. Uninoculated -stunted , chlorotic & Rhodotorula graminis, poplar yeast endophytes are healthy with expanded leaves

b Pepper Plants a Tomato Plants

C) Colletotrichum sp.endophyte influence on Sclerotinia;1. Control, 2.Enhanced disease, 3.Reduced disease d),i) BCA + key endophyte. ii) BCA - key endophyte. Colletotrichum – induced resistance. Immunizing the plant. Endophytes influence the activity of biocontrol agents

1 3 2

c

Microbe interactions-Microscopic

d

Glasshouse trials – assess disease development on commercial grasses

Khan et al.2012, Nature/Microbiology

i ii

Nitrogen stress Nitrogen stress

Understanding endophytic interactions in plants to abiotic stress tolerance (drought, salinity and heavy metal toxicity)….. focus on

Molecular Plant-Endophyte interactions?

Signaling, Adhesion, Competence, Overcoming plant defense, Interactions under abiotic stress

Elucidation

mechanisms

Understand which genes- turned on or off during interaction under stress-in host plants inoculated with PGP endophytes.

Knowledge contributes new ideas -improving the traits using PGP-Endophytes -

efficient plant growth under abiotic stresses- improving stress tolerance of

agriculturally important plants

Perception

MAMPs

Signal Transfer Receptor oligomerization,

Phosphorylation, Ca2+ influx

Intracellular Receptors

Other sensors ROS, Trehalose,

Hexokinase

Signal Transduction

Gene Expression

Effector Genes HSPs, LEA,

dehydrins, ROS, Osmolytes, Hormones

Gene

coding for TFs

Cell surface Receptors

(PRRs)

Plant cell surface-PRRs recognize cell/organism-associated molecular patterns (MAMPS)

Protective mechanisms - response to stresses -regulated by alterations in the expression levels of stress-responsive genes.

Association involves mutual recognition which requires molecular dialogue between plant and organism

Responses Function Adjustment of chlorophyll antenna size Photo destruction Thermal dissipation of light energy

Reduction in photosynthetic electron transport, Uncoupling of photophosphorylation and electron transport.

Xanthophyll cycle, Water–water cycle Protection against ROS generated in chloroplasts Stomatal closure Reduced hydraulic conductance Delay in stomatal closure

Prevention of water loss through transpiration, Maintenance of photosynthetic activity under stress

Altered source–sink relations Carbon partitioning

Induction of root growth, inhibition of shoot growth, Osmolyte synthesis

Alternative oxidase pathway Uncoupling proteins, NADPH dehydrogenases

Uncoupling of oxidative phosphorylation and electron transport

Prohibitins Maintenance of protein structure in inner mitochondrial membranes

Antioxidant enzymes and substrates Scavenging ROS Synthesis of osmotically active solutes Osmotic adjustment

ABA biosynthesis Stomatal closure, regulation of aquaporin activity, inhibition of ethylene accumulation

Table 5 Responses contributing to drought tolerance in plants

Sujata Bhargava & Kshitija Sawant 2013

Endophytes influence the physiology and expression of stress responsive

genes in plant (maize) under abiotic stress (drought)

Whether endophytic bacteria Stimulate plant growth. Shows antagonistic activity. Elicit stress responsive genes, Enhance stress tolerance in plants (Maize).

Endophytic bacteria from surface sterilized and macerated root

tissues (Qadri et al. 2013)

Screening for PGP Properties (Sandhya et al.

2009)

Biocontrol activity against major plant pathogens

(Meera and Balabaskar, 2012)

Genomic Characterization

Growth Promotion on Maize (Sandhya et al. 2010)

Fig. 1 Isolation of endophytic bacteria from root tissues of various cropping systems

Crop Village District Plant part PFA TSA Total NF F NF F

Maize Reddipalli Anantapur Root Tissue 2 1 2 - 4 Okra Reddipalli Anantapur Root Tissue 2 1 1 1 5

Roselle Reddipalli Anantapur Root Tissue - 2 2 1 5 Red gram Reddipalli Anantapur Root Tissue 1 - 1 - 2

Maize Bodam Srikakulam Root Tissue - - 2 - 2 Brinjal Kothavalasa Srikakulam Root Tissue - 1 2 - 3 Okra Kothavalasa Srikakulam Root Tissue 1 - 1 - 2

NF, Non Fluorescence, F, Fluorescence

Table 5 Endophytes from root tissues of various cropping systems

PFA, Pseudomonas Fluorescence Agar;TSA,Trypticase Soya Agar

S.No. Crop Variety Plant Part

PFA

TSA

Total

NF F NF F 1 Maize NK6240 seed 1 - 1 - 2 2 Maize 30V92 seed 1 - 1 - 2 3 Maize DHM 117 seed 2 - 2 - 4 4 Maize 900M seed 1 - 2 - 3 5 Maize BML-7 seed 1 - 1 - 2 6 Maize HYBRID seed 1 - 1 - 2

Twenty three isolates selected from seven different crop root tissues. Screening- colony characters, pigmentation and fluorescence. Among 23, 7 showed fluorescence and 16 were non-fluorescent both on PFA and TSA. Endophytic bacteria were also isolated from different maize seed varieties, 15 non-fluorescent isolates were selected from six different maize seed varieties .

Table 6 Endophytic bacteria from of maize seeds varieties

PFA, Pseudomonas Fluorescence Agar; TSA,Trypticase Soya Agar NF, Non Fluorescence, F, Fluorescence

A B C D E F G H I J K L M

A - FBK1 B – FGR3 C – FTR1 D - NFTR1 E – NFMZR2 F - FBK2 G – NFRGR1 H – FMZR7 I – FMZR2 J – FMZR1 K- MZ30V92 L – FBK3 M – NFMZR3

Molecular characterization of Endophytic Bacteria

16s rDNA gene band (1500bp), PCR amplified product

DNA bands for the selected 13 isolates

Sequencing under process…..

S.NO. Isolate Ammonia Phosphorous Solubilization

(Index) Siderophore Hydrogen

Cyanide IAA

μg mg−1 protein

1 PFOR 1 ++ - + 0.995±0.06

2 PFOR 2 + - + 1.143±0.090 3 PFOR 3 ++ - + 0.996±0.060 4 PFGR 1 + - + 1.201±0.13 5 PFGR 2 ++ - + 1.419±0.29 6 PFGR 3 + 225 - + 1.529±0.34 7 PFBK 1 + 226 - 2.529±0.25 8 PFBK 2 + 227 - 3.529±0.14 9 PFBK 3 + 228 - 4.529±0.37

10 PFMZR 1 + 229 - 5.529±0.41 11 PFMZR 2 + 230 - 6.529±0.22 12 PFMZR 3 + - + 1.161±0.32 13 PFMZR 7 + 185 + 2.003±0.23 14 PFTR 1 +++ 222 + + 1.3185±0.25 15 PNFOK ++ - - 1.188±0.31 16 PNFOR 2 + - - 1.043±0.36 17 PNFTR +++ 285 + + 1.298±0.33 18 PNFRGR 1 + 120 + 2.365±0.41 19 PNFMZR 1 - - - 1.207±0.28 20 PNFMZR2 ++ 170 + 1.424±0.24

Table 3 Plant growth promoting properties of endophytic bacteria from root tissues

Ammonia Production HCN Production

Phosphate solubilization

Siderophores production

Twenty isolates tested for PGP traits. 13 solubilized phosphate with solubilizing index ranging between 111 to 285 11 isolates produced siderophores 3 showed HCN production IAA production: variability was observed among the isolates, Isolates FTR 1 and NFTR showed all the PGP traits. Isolates FGR 7 and FGR 8 produced higher amount of IAA 6.52 μg mg−1 protein

and P-solubilization (285).

IAA Production

Macrophomina phaseolina

Rhizoctonia solani

Sclerotium rolfsii

Fusarium oxysporum

Botrytis cinerea

Inhibitory percentage

PFGR 3 98 82.7 84.7 78.7 80.3

PFBK 1 77.5 81.2 75.1 89.6 92.1

PFBK 2 82 78.7 88.2 620 78.9

PFBK 3 76.3 86.5 86.5 79.5 70.1

PFMZR 1 77.9 84.0 85.4 64.4 96.3

PFMZR 2 77.0 93.2 87.1 97 81.6

PFMZR 7 82.1 90.6 62.1 69.2 88.5

PFTR 1 91.1 84.8 78.9 81.8 91.9

PNFTR 92.4 81.6 84.3 86.4 86.9

PNFRGR 1 80.9 80.2 76.6 77.4 92.5

PNFMZR2 89.8 78.9 95.1 88.2 82.7

PNFMZR 3 80.2 91.1 85.0 97.5 72.6

PMZ 30V92 87.6 92.2 96.3 71.2 95.1

Table Antagonistic activity of endophytic bacteria

Endophytic bacteria showing antagonistic activity

Fig. Pot studies, Plant growth promotion of Maize. Control and endophytes inoculated maize seedlings.

Based on plant growth promoting traits, the best PGP produces were selected for plant growth promotion of maize under green house conditions.

Inoculation of plant tissue endophytes significantly increased total root, shoot length and dry biomass in maize seedlings compared to control (Table 3).

S.No Isolate Root Length (cm)

Shoot length (cm)

Dry Biomass (g)

1 PFGR 3 43±0.09 41.2±0.30 0.39±0.03 2 PFBK 1 36.2±0.11 38.8±0.26 0.37±0.01 3 PFBK 2 41.8±0.10 42.1±0.13 0.40±0.01 4 PFBK 3 43.2±0.13 47.9±0.14 0.49±0.04 5 PFMZR 1 42.1±0.07 39.8±0.07 0.42±0.03 6 PFMZR 2 37.6±0.13 41.2±0.11 0.39±0.02 7 PFMZR 7 32.3±0.09 36.7±0.06 0.25±0.03 8 PFTR 1 38.9±0.01 39.8±0.02 0.35±0.05 9 PNFTR 34.4±0.08 39.6±0.07 0.39±0.03

10 PNFRGR 1 41.4±0.05 42.3±0.04 0.44±0.02 11 PNFMZR2 34.3±0.39 35.7±0.09 0.31±0.01 12 PNFMZR 3 30.5±0.22 34.1±0.13 0.33±0.01 13 PMZ 30V92 45.9±0.18 43.5±0.02 0.47±0.02

Table 3 Plant growth promotion of maize inoculated with endophytic bacteria

Isolated:Brinjal/eggplant

Isolated: Maize

Plant growth promotion using endophytes under drought stress

Endophytes as elicitors for expression of drought stress responsive genes

Transcriptomic approach

To understand endophytes mediated drought

tolerance in maize/plants

Plant genomics helps in understanding how plants

tailor the responses to endophytes

PGP Endophytes-elicited induce systemic tolerance (IST) to drought stress is focused

Understanding endophytes associated with crops at gene level assumes importance in agriculture.

Taking into account the potential of such organisms in PGP, protection against biotic and abiotic stresses….

Lead to their large scale applications in environmentally unfavourable conditions,

minimize risk to farmers.

Inoculation of endophytic bacteria improved plant growth promotion of maize seedlings as compared to uninoculated seedlings.

By this way a most effective endophytic bacteria can be identified for managing

crop yield and crop diseases under abiotic stress

Thankful to Department of Biotechnology, Govt.of India for providing the financial assistance under DBT-Bio-CARE Women scheme.

Acknowledgement

Dr. Sk.Z.Ali, Assistant Professor, Microbiology, Agri Biotech Foundation

G.V.S.Kishore, Junior Research Fellow, Agri Biotech Foundation

S.S.K.Prasad Junior Research Fellow, Agri Biotech Foundation

Prof G.Pakki Reddy, Executive Director, Agri Biotech Foundation,

Hyderabad, Telangana State, India

With an overwhelming sense of gratitude I extend my thanks to Asian PGPR Committee.

Encourage The Use Microbes To Improve Environment Rather Than Killing It With Chemicals

Microbes we need them

Thank you