Outline of Chapter 2 - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/79479/11/11_chapter4.pdf · Thaumatin-like proteins (TLP) belong to PR-5 family showing antifungal activity

Outline of Chapter 2

Chapter 2

Elicitation and characterization of ISR in peanut by PGPR against A. niger 65

Introduction

Resistance is the power of a plant to eliminate or defeat, fully or partially, the effect of

damaging factor or pathogen (Agrios 1988; Van loon 1997). Besides the plants’ basal

host defense, further defense mechanisms are inducible (Bonas and Lahaye 2002).

There are mainly two forms of induced resistance, viz. systemic acquired resistance

(SAR) and induced systemic resistance (ISR) wherein plant defense mechanisms are

activated by prior infection or treatment (Jain et al. 2014; Choudhary et al. 2007).

Such biological control is an important way to reduce diseases due to biotic stress in

agro-ecosystem and to overcome the loss of crop yield (Sharma and Johri 2003).

ISR induced by PGPR is a most familiar phenomenon that induces resistance in host

plants to broad spectrum of pathogens (Jain et al. 2014). ISR induced by PGPR was

initially observed in carnation, common bean and in cucumber with reduced

susceptibility to Fusarium wilt, halo blight, and Colletotrichum orbiculare,

respectively. PGPR like Pseudomonas fluorescens, P. putida, Bacillus pumilus, and

Serratia marcescens provide defense to plants against foliar diseases (Choudhary et

al. 2007) by inducing production of phytoalexins and phenolic compounds,

accumulation of pathogenesis related proteins and deposition of structural barriers.

The defense gene products include phenylalanine ammonia-lyase (PAL), peroxidase

(PO), polyphenol oxidase (PPO), lipoxygenase (LOX) and Pathogenesis related

proteins (PR proteins) that include β-1,3-glucanases (PR-2 family) and chitinases

(PR-3 family). Both of these degrade the fungal cell wall and cause lysis of fungal cell

(Ramamoorthy et al 2002). Thaumatin-like proteins (TLP) belong to PR-5 family

showing antifungal activity and enhancing resistance to pathogen infection (Datta et

al., 1999).

As a kind of reactive oxygen species (ROS), hydrogen peroxide (H2O2) plays an

important role in plants under severe environmental conditions, which include various

biotic and abiotic stresses (Dat et al. 2000). H2O2 participates in many resistance

mechanisms, including reinforcement of the plant cell wall, phytoalexin production

and enhancement of resistance to various stresses (Dempsey and klessig 1995). To

some extent, excess H2O2 accumulation can lead to oxidative stress in plants, which

then triggers cell death. The evolution of all aerobic organisms is dependent upon the

development of efficient H2O2-scavenging mechanisms (Arora et al. 2002), enzymes,

Chapter 2


including superoxide dismutase (SOD), catalase (CAT), peroxidase (PO) and many

others.

In this chapter the induction of ISR in two varieties A. hypogaea L. has been studied,

by using isolates RA1 and RR18, to evaluate their usefulness in field or pot conditions

and detailed identification of RA1 and RR18.

Chapter 2


Materilas and methods

Seed imbibiton and sample collection for ISR study

Seed bacterialization was done using two bactrial cultures RA1 and RR18 as

described in the first chapter. Samples were collected at 0, 3, 6 and 9 days form RA1

treated seedlings and 0, 2, 4, 6, 8, and 12 days from RR18 treated seedlings for ISR

study. Germinated seedlings were washed under running tap water, homogenized with

liquid nitrogen and the powder obtained was stored at −20 °C for further use.

ISR related enzymes activity

Extraction of enzymes was carried out following the method of Tonellia et al. (2011).

In this method 1 g of seedlings powder was extracted with phosphate buffer solution

[50 mM Potassium phosphate and 1 mM EDTA, pH 7.8+ 0.5 mM phenyl methane

sulphonyl fluoride (PMSF) + 1 % PVP (polyvinylpyrrolidone)] and then filtered

through a 0.20 mm cheese cloth into a centrifuge tube. The tissue extract was

centrifuged at 12,000g for 20 min at 4 °C. Supernetant was collected and used as

enzyme sources and stored at 4 °C.

A colorimetric assay and UV reading for enzymatic activity were performed with a

Elico SL 171 mini spectrophotometer and Specord 200 plus UV spectrophotometer

respectively. The protein concentration was determined by the method of Lowry et al.

(1951).

Estimation of PAL activity

PAL activity was determined by the mesurament of rate of conversion of L-

phenylalanine to trans-cinnamic acid at 290 nm as described by Dickerson et al.

(1984). Sample containing 0.4mL of enzyme extract was incubated with 0.5 mL of

0.1 M borate buffer, pH 8.8 and 0.5 mL of 12 mM L-phenylalanine in the same buffer

for 30 min at 30 °C. The amount of trans-cinnamic acid synthesized was calculated

using its extinction coefficient of 9630 m−1(Dickerson et al., 1984). Enzyme activity

was expressed as µmol trans-cinnamic acid /min /mg of protein.

Estimation of PO activity

The reaction mixture consisted of 1.5 mL of 0.05 M pyrogallol, 0.5 mL of enzyme

extract and 0.5mL of 1% H2O2. The reaction mixture was incubated at room

Chapter 2


temperature (28 ± 2 °C). The changes in absorbance at 420 nm were recorded at every

30 s intervals for 3 min. The enzyme activity was expressed as changes in the

absorbance/ min /mg of protein (Hammerschmidt et al., 1982).

Estimation of LOX activity

LOX activity was assayed using reaction mixture containing 5 µL of a 25 mM

substrate solution, the enzymatic extract 20 µl and 2.975 mL of phosphate buffer pH

6.0 (10mM) buffer solution to make up the final volume to 3 mL. All experiments

were performed in triplicate. A control assay, containing all the components except

the enzyme preparation, was run in tandem with these trials. LOX activity was

measured at 234 nm (Surry 1964) after one minute of incubation. The specific activity

was defined as μmol of conjugated diene HPOD produced per mg of protein per min,

using a molar extinction coefficient of 25,000 M−1 cm−1 (Patel et al. 2015).

For the determination of isoform profile of LOX native-PAGE (Laemmli 1970) was

performed. For the separation of isoform 5% stacking gel and 8% resolving gel were

used. 50 μg of crude enzyme extract was loaded onto polyacrylamide gels. After

electrophoresis gels were incubated for 1 hour in 0.1 M phosphate buffer pH 7.0,

containing substrate (linoleic acid). Then gel was washed once with distilled water

and then 15% glacial acetic acid containing 5% potassium iodide was added. LOX

band appeared as white band in dark blue gel (Patel et al. 2015).

Estimation of PPO activity

The activity of PPO was detrermined following the method of Archana et al. (2011).

The reaction mixture contained 50 μl of crude extract, 1.6 ml 100mM sodium

phosphate buffer pH 6.5 and 200 μl of 0.01 M catechol. The absorbance values were

taken at 495 nm for 30 sec interval till 3 min. A value of 1300 M-1 cm-1 was employed

for the molar coefficient of o-Quinone (Gauillard et al., 1993). PPO activity was

expressed as μmol of product formed /min/mg protein.

For the determination of isoform profile of PPO native-PAGE (Laemmli 1970) was

performed. For the separation of isoform 5% stacking gel and 8% resolving gel were

used. 50 μg of crude enzyme extract was loaded onto polyacrylamide gels. After

electrophoresis activity of enzyme in gel was detcted by equilibrated the gel for 30

Chapter 2


min in 0.1 % p-phenylalanine diamine followed by addition of 10 mM catechol in 20

mM acetate buffer, pH 4.2 containing 0.03 % H2O2.

Estimation of β-1, 3- glucanase activity

Total β-1, 3-glucanase activity was calorimetrically assayed by the laminarin

dinitrosalicylate method (Pan et al., 1991) with some modification. The reaction

mixture was prepared by mixing 31.25 μL of 4% laminarin and 31.25 μL of enzyme

extract and incubating at 40 °C for 30 min. the reaction was stopped by adding 187.5

μL of dinitrosalicylic acid reagent (prepared by adding 300 ml of 4.5% NaOH to 880

ml containing 8.8g of dinitrosalicylic acid and 22.5g K. Na tartarate) with subsequent

heating for 5 min in a boiling water bath. The resulting colored solution was diluted

with 2.25ml of distilled water and vortexed. Products released were estimated for

reducing groups at 500 nm using glucose as standard. The enzyme activity was

expressed as μg of glucose/ min /mg of protein

Estimation of chitinase activity

Preparation of colloidal chitin

Five grams of crab shell chitin (Sigma, USA) was slowly added into 100 ml of cold

0.25 N HCl with vigorous stirring and kept overnight at 4 °C. The mixture was

filtered through glass wool into 200 ml of ice cold ethanol at 4 °C with rapid stirring.

The resultant chitin suspension was centrifuged at 10,000 rpm for 20 min and the

chitin pellets were washed repeatedly with distilled water until pH becomes neutral

this was used as substate for chitinase assay.

Chitinase activity was determined by dinitrosalicylic acid (DNS) method (Miller,

1959). This method works on the concentration of N-acetyl glucosamine (NAG),

which is released as a result of enzymatic action. The 2 ml reaction mixture contained

0.5 ml of 0.5% colloidal chitin in phosphate buffer (pH 5.5), 0.5 ml crude enzyme

extract and 1 ml distilled water. The mixture, after thorough vortexing, was incubated

in a water bath shaker at 50 °C for 1 h. The reaction was stopped by the addition of 3

gm/l DNS reagent followed by heating at 100 °C for 10 min. The colored solution was

centrifuged at 10,000 rpm for 5 min and the absorption was measured at 530 nm along

with substrate and enzyme blanks. N-acetyl glucosamine was used as standerd.

Chapter 2


Estimation od SOD and ctalase activity

Total SOD activity was assayed by monitoring the inhibition of photochemical

reduction of nitro blue tetrazolium (NBT) according to the method of Giannopolitis

and Ries (1977). The 3 ml reaction mixture contained 50 mM potassium phosphate

buffer (pH 7.8), 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 0.1 mM EDTA

and 100 µl enzyme extract. The reaction mixtures were illuminated for 30 min at light

intensity of 5,000 lux. One unit of SOD activity was defined as the amount of enzyme

required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm

(Gao et al. 2008).

SOD gel activity was carried out according to Gao et al. (2008) After completion of

electrophoresis, gels were incubated in 50 mmol phosphate buffer (pH 7.5) containing

28 µmol riboflavin, 28 mmol N,N,N,N-tetramethyl ethylenediamine (TEMED) for 30

min under dark conditions, followed by washing in distilled water for 1 min and

incubation in the same buffer containing 2.45 mmol nitroblue tetrazolium (NBT) for

20 to 30 min exposed to light at room temperature. Isoenzymes appeared as colorless

bands on a purple background.

Catalase activity was assesd using method of Shah et al. (2011) with some

modification. Assay mixture in a total volume of 3 mL contained 2000 µl of 100 mM

KH2PO4 buffer (pH 7.0), 800 µl of 200 mM H2O2 and 200 µl enzyme. Decrease in

H2O2 was monitored at 240 nm (extinction coefficient of 0.036 mM−1 cm−1 ). Enzyme

specific activity is expressed as µmol of H2O2 oxidised/min/mg protein.

Jasmonic acid estimation

Jasmonic acid concentration was measured by the method of Dhandhukia and

Thakkar (2008) using high performance thin layer chromatography (HPTLC). In these

methods 10 µl of the ethyl acetate extract of germinating seedlings was loaded on

Aluminium-backed silica gel 60 F254 TLC foils (20 × 10cm) of 25-mm thickness

(Merck, Darmstadt, Germany) along with standard Jasmonic acid (10 µg/1 µl) using

Linomate 5 applicator (CAMAG, Muttenz,Germany). Mobile phase was isopropanol-

ammonia-water (10:1:1). After development, the plate was dried and peak area of

Jasmonic acid in the sample and standard were quantified by linear scanning at 295

nm.

Chapter 2


Extraction and analysis of salicylic acid (SA)

The SA was extracted from powdered tissues ground in 5 ml of pre-chilled methanol

using pre-chilled mortar and pestle. Collected slurry was kept overnight to let the

complete extraction of SA. Next day it was filtered through 0.2 mm membrane and

concentrated by keeping the vials open at room temperature for 30 min. Quntification

was done using high performance liquid chromatography (HPLC) as described by

Noordin and Chung (2007). SA was separated using Zorbax RP C18 column and

mobile phase of a mixture of methanol (60%) and water (40%). The flow rate used

was 0.8 ml/min and the detection was done at 254 nm. Salicylic acid (1 mg/ml,

Sigma) was used as a standard. The detection was carried out using UV detector at

254 nm. The average retention time of standard SA was found to be 1.2 min, the

corresponding retention time and peak area of the samples were noted and SA

concentration in the samples were calculated.

Estimation of total Phenol content

Seedlings sample (1 g) were homogenized in 10 mL of 80% methanol and agitated for

15 min at 70 °C (Ramamoorthy et al. 2002). 1 mL of the methanolic extract was

added to 5 mL of distilled water and 250 µL of Folin-Ciocalteau reagent (1 N) and the

solution was kept at 25 °C. The absorbance of the developed blue color was measured

using a spectrophotometer at 725 nM. Catechol was used as the standard. The amount

of phenolics was expressed as µg catechol mg/protein.

Total Chlorophyll Estimation

Total chlorophyll in germinating seedlings was estimated by the method described by

Tonelli (2011), in brif, 0.1 g of fresh seedlings are homoginized in mortar and the fine

pulp was added wuth 80% acetone. The resulting extract was collected by filtering

through Whatman filter paper. The final volume was adjusted to 10 ml, and the

optical density of the chlorophyll extract was read with a spectrophotometer at 650

and 665 nm. The amount of total chlorophyll present in the extract was calculated on

the basis of mg of chlorophyll per gram of fresh tissue, according to the following

equation.

Total chlorophyll = 6.45 (Abs 665) + 17:72 (Abs 650)

Chapter 2


Pot study

For experiment, seeds bacterization was carried out through imbibitions, as mentioned

earlier, along with it control seed, challenge inoculated with A .niger alone and

bacterized with A. niger together, 6 seeds/pot were sown. The plant growth

characteristics recorded on day 30 included shoot length, root length, Germination %,

fresh weight, dry weight were measured on harvesting. For further pot study, more

seeds were inoculated with four different treatments as mentioned earlier and

incubated for 45 days and germination assay was carried out after harvesting them.

The experiments were arranged in a completely randomized design with 3

replications/treatment.

Field study

The experiments were conducted at the Valasan village, near Vallabh Vidyanagar

(22° 30' 60'' N 72° 54' 0'' E ~35 meters above sea level), Gujarat, India. For

challenged inoculation and for infection, soil was inoculated with A. niger spores

suspension (1×104 spores/ml) before one day of sowing. Three replicates of 80 seeds

of each of control, bacterized (with RA1), fungus infected and bacteria + fungus

infected seeds were sowed in the field. Germination percentage and disease incidence

were recorded at every week up to 30 days the plant growth characteristics recorded

on day 40 included plant height and dry weight. For checking survivability of

bacteria, germinating seedlings or plants were uprooted every 5 day interval and the

attached soil was collected. Serial dilution of collected soil was prepared and spread

in to King’s B agar. The plates were incubated at 30 °C for 48 h and colony forming

unit was counted.

Identification of RA1 and RR18

Genomic DNA was isolated and 16s rRNA sequence was done of both the culture in

chapter number 1.

Further identification was done using biochemical characterization as per Bergey’s

manual using standard biochemical test protocol. The FAME analysis was done using

Gas Chromatography at BioAxis DNA Research Centre Private Limited, Hyderabad,

Andhra Pradesh, India. Analysis was performed with the help of MIDI Sherlock

software and aerobic library (RTBSA 6.0) as a reference.

Chapter 2


Statistical analysis

All data were analyzed by analysis of variance (ANOVA) using GraphPad Prism 6.0

software. Means of data of effect of various treatments were compared using

uncorrected Fisher’s LSD test (ns or a – not significant, p<0.05=* or b, p<0.005=**

or c, p<0.001=*** or d and p<0.0001=**** or e).

Chapter 2


Results

The chosen bacterial strains (RA1, RR18), found to be gram –ve short rods, (Fig 1b

and d) were examined for their morphological colony characteristics (table 1).

Figure 1 Morphological characteristics of isolated bacteria (a) Colony characteristics

of RA1 (b) gram’s nature of RA1 (c) Colony characteristics of RR18 and (d) gram’s

nature of RR18

Bacterial

Culture

Size Shape Margin Elevation Surface Consistency Optical

Characteristics

Colour

RA1 Small Round Entire Flat Smooth Viscous Opaque white

RR18 Small Round Entire Slightly

raised

Smooth Viscous Opaque Greenish

yellow

Table 1 Morphological colony characteristics of RA1 and RR18 bacteria

Chapter 2


RA1 and RR18 were found to inhibit growth of A. niger in a in vitro test against A.

niger. RA1 showed 42% and RR18 showed 33.33% of inhibition respectively against

A. niger (Figure 2).

Figure 2 Antifungal activity against A. niger (a) control plate (b) zone of inhibition

shown by RA1 and (c) zone of inhibition shown by RR18.

Germination assay

Effect of RA1

The effect of RA1 bacterial isolate in suppressing collar rot disease was studied by

seed germination assay (Figure 3a and b 4 and 5). Due to RA1 treatment along with

inoculation of A. niger, disease incidence was reduced up to 50% as compared to only

A. niger infected seeds in both the varieties of groundnut. In RA1 treated seedlings,

vigour index was found to be very high compared to control germinating seeds of

groundnut in both varieties. In A. niger infected seedlings, vigour index was found to

be lesser than that of control germinating seeds but in seeds treated with RA1 and

challenged with inoculation of A. niger, vigour index was higher than that of A. niger

treated and control germinating seeds. Germination percentage and seed fresh weights

were also found to be higher in RA1 treated seedlings. In A. niger infected seedlings,

germination % and vigour index were found to be very less in both groundnut

varieties (Table 2).

Chapter 2


Figure 3 Seed germination assay. Control, RA1 treated, RA1 + A. niger and A. niger

inoculated seedlings. (a) GG20 and (b) GG2 varieties.

a

b

Chapter 2


Figure 4 Seed germination assay (a) control, (b) RA1 treated seedlings, (c) A. niger

infected seedlings and (d) RA1 with A. niger inoculated seedlings of GG20 variety.

Figure 5 Seed germination assay (a) control, (b) RA1 treated seedlings, (c) A. niger

infected seedlings and (d) RA1 with A. niger inoculated seedlings of GG2 variety.

Chapter 2


Germination

(%)

Disease

incidence (%)

Vigor index Seed fresh

weight (g)

Control GG20 65.40 ± 1.201 9.33 ± 0.333 1333.33 ±

82.29

1.03 ± 0.031

RA1 GG20 82.33±1.168**** 0.00 ± 0.000 1611.33 ±

54.59**

1.47 ± 0.089**

A. niger GG20 40.62 ±

0.411****

59.95 ±

0.043****

916.00 ±

50.69***

1.07 ± 0.0038 ns

RA1+ A. niger

GG20

76.33 ±

1.168****

32.41 ±

0.707****

1496. 33 ±

12.12ns

1.31 ± 0.069*

Control GG2 82.22 ± 1.111 13.33 ± 1.765 1448.66 ±

25.69

1.00 ± 0.005

RA1 GG2 94.00 ±

1.001****

0.00 ± 0.00 1508.00 ±

48.26ns

1.22 ± 0.062*

A. niger GG2 29.99 ±

0.106****

70.13 ±

0.242****

774.00 ±

13.01****

0.86 ± 0.033 ns

RA1+ A. niger

GG2

62.18 ±

0.243****

40.40 ±

0.400****

1350.33 ±

19.68*

1.11 ± 0.062 ns

Table 2 Effect of treatments of RA1 alone or with A. niger on germination

percentage, disease incidence, vigor index and seedling fresh weight of two the

varieties of groundnut, GG20 and GG2. Data represent means ± SE of three replicates

with 50 germinated seedling in each batch. Different number of asterisk indicate

statistically significant difference according to uncorrected Fisher’s LSD test

(p<0.05=*, p<0.005=**, p<0.001=***, p<0.0001=****and ns- not significant).

Chapter 2


Effect of RR18

Compared to control and A. niger infected seedlings, germination percentage, vigour

index and fresh weight of seedlings were found higher in RR 18 and RR18 + A. niger

treated seedlings of both the varieties (Figure 6a, b, 7 and 8). A. niger treated seeds

showed higher degree of pathogenicity, as it significantly reduced the average

germination compared to control seed, while RR18 treated seeds showed higher

resistance to disease upon challenge inoculation of fungal strain A. niger (Table 3).

These results also showed that RR18 culture possesses antifungal activity besides

PGPR activity.

Chapter 2


Figure 6 Seed germination assay. Control, RR18 treated, RR18 + A. niger and A.

niger inoculated inoculated seedlings. (a) GG20 and (b) GG2 varieties.

a

b

Chapter 2


Figure 7 Seed germination assay (a) control, (b) RR18 treated seedlings, (c) A. niger

infected seedlings and (d) RR18 with A. niger inoculated seedlings of GG20 variety.

Figure 8 Seed germination assay (a) control, (b) RR18 treated seedlings, (c) A. niger

infected seedlings and (d) RR18 with A. niger inoculated seedlings of GG2 variety.

Chapter 2


Table 3 Effect of treatments of RR18 alone or with A. niger on germination

percentage, disease incidence, vigor index and seedling fresh weight of two the

varieties of groundnut varieties GG20 and GG2. Data represent means ± SE. of three

replicates with 50 germinated seedlings in each batch. Different letters indicate

statistically significant difference according to uncorrected Fisher’s LSD test (a- not

significant p<0.05=b, p<0.005=c, p<0.001=d, and p<0.0001=e).

Germination

(%)

Disease

incidence

(%)

Vigour index Seedling

fresh weight

(g)

Control GG20 83.89 ± 0.55a 0.00 ± 0.00 1337.00 ± 18.44 a 1.62 ± 0.06 a

RR18 GG20 95.55 ± 2.77b 0.00 ± 0.00 1414.00 ± 07.11c 1.88 ± 0.05c

A. niger GG20 40.08 ± 1.46e 56.63 ±

3.31a

710.70 ± 05.33e 1.08 ± 0.04e

RR18 + A.

niger GG20

89.55 ± 3.77c 13.33 ±

3.33e

1360.00 ± 29.78e 1.72 ± 0.06d

Control GG2 83.43 ± 1.71a 0.00 ± 0.00 1522 ± 10.89a 1.19 ± 0.04a

RR18 GG2 93.91± 1.95b 0.00 ± 0.00 1544 ± 11.67a 1.48 ± 0.05c

A. niger GG2 32.66 ± 2.67d 68.42 ±

1.71a

813.13 ± 06.66e 1.12 ± 0.03a

RR18 + A.

niger GG2

86.33 ± 3.66e 24.55 ±

4.22e

1378 ± 22.78b 1.29 ± 0.06b

Chapter 2


Induction of defense related enzymes due to RA1 treatments

To study induction of systemic resistance in germinating seedlings due to RA1

treatments, activities of several defense related enzymes were assessed. RA1 was able

to induce PAL activity significantly (p ≤ 0.05) in resistant as well as susceptible

varieties of A. hypogaea L. seedlings (Fig. 9). PAL activity was found to increase

from 3rd day of incubation and reached up to maximum level on 9th day. In only A.

niger inoculated seedlings, up to 6 day, no change was found in PAL activity but it

was increaseed on 9th day of incubation in GG20. In GG2 no changes in PAL activity

was observed.

PO activity was also found to be induced significantly in both the varieties of A.

hypogaea L. due to RA1 treatment (Fig. 10). The maximum PO activity was observed

on 6th day of incubation in both the varieties of groundnut. The activity of PO was

maintained higehr in RA1 and RA1 + A. niger inoculated seedlings. In A. niger

treated seedlings PO activity was aslo found to be higher compared to control

seedlings.

As ISR involves JA as a signaling molecule (Conrath et al. 2001), activity of LOX

levels were investigated and found to be induced due to RA1 treatment (Fig. 11).

LOX activity started to increase from 3rd day onwards to throughout the experimental

period in both GG20 and GG2 germinating seedlings and its activity was found to be

maximum on 9th day of germination. PPO activity was also found to be significantly

(p ≤ 0.05) higher in RA1 treatment in both varieties of peanut (Fig. 12). PPO activity

was also found to increase from 3rd day of incubation and reached maximum on 9th

day of germination. In only A. niger inoculated seedlings, PPO was also elevated from

3rd compared to control but it remain less compared to RA1 treated seedlings. In A.

hypogaea L. seedlings challenged with A. niger, activities of PAL, PO and LOX and

PPO levels increased after 6 days.

The early induction of these defense parameters in RA1 treated plants shows evidence

of induced systemic resistance (ISR). Activation of β-1,3-Glucanase (PR 2) and

chitinase (PR 3) were found to be induced rapidly and significantly (p ≤ 0.05) (within

3 days) in RA1 treated plants compared to plants challenged with A. niger (after 6

days). Their activities were higher in GG20 variety of A. hypogaea L. than in GG2

Chapter 2


variety but ISR was evident in both the varieties (Fig. 13 and 14). Both enzymes

reached maximum on 6th day of incubation and than it was reduced.

The levels of CAT activity in seedlings was found to increase rapidly after RA1

treatments. CAT activities in all the treatments were significantly higher than that in

the control on 6th day of incubation (Fig. 16). The levels of CAT activity in the

germinating seedlings treated with RA1 and RA1 with A. niger inocultaed seedlings

were induced significantly (p ≤ 0.05). There was not detectable changes observed in

SOD activity in the germinating seedlings of both varieties (Fig. 15). The levels of

SOD activity was found higher in RA1 treatments and peaked on 6th day in GG20 and

on 9th day in GG2.

Furthermore, the chlorophyll content of seedlings inoculated with RA1 was found to

be higher significantly (P ≤ 0.05) in both the varieties of A. hypogaea L. From 3rd day

of incubation, it remianed higher up to last day of germination (Fig. 17). In A. niger

inoculated seedling chlorophyll content was reduced compared to control. Studies on

induction of defense mechanisms revealed that higher accumulation of phenolics was

observed in RA1 inoculated seedling of both varieties (Fig. 18). Accumulation of

phenolics started at third day of incubation. The maximum accumulation was

observed on the 6th day of inoculation. Seedlings inoculated with the pathogen alone

also showed increased accumulation of phenolics. Moreover, the accumulation of

phenolics was lesser in A. niger treated seedlings compared to seedlings treated with

RA1 and challenged with the pathogen.

Initially up to 3 days, no changes were found in Jasmonic acid concentration in in

germinating seedlings. After 3 days of incubation, jasmonic acid concentration was

found to increase gradually in RA1 and RA1 along with of A. niger treated seedlings

of both the varities (Fig. 19). SA content was also found to higher in 6 day

germinating seedling treated with RA1 and RA1 along with A. niger in both varities.

After 6 day it’s contain was reduced in all treatments.

Chapter 2


Figure 9 PAL activity of germinating seedlings of groundnut GG20 and GG2

varieties. Values were mean of three replicates.

Figure 10 PO activity of germinating seedlings of groundnut GG20 and GG2

varieties. Values were mean of three replicates. Errors represent SE. Bar with

different number of asterisk indicate statistically significant difference according to

uncorrected Fisher’s LSD test (p<0.05=*, p<0.005=**, p<0.001=*** and

p<0.0001=****).

Chapter 2


Figure 11 LOX activity of germinating seedlings of groundnut GG20 and GG2


Figure 12 PPO activity of germinating seedlings of groundnut GG20 and GG2




p<0.0001=****).

Chapter 2


Figure 13 β-1, 3-glucanase activity of germinating seedlings of groundnut GG20 and

GG2 varieties. Values were mean of three replicates.

Figure 14 Chitinase activity of germinating seedlings of groundnut GG20 and GG2




p<0.0001=****).

Chapter 2


Figure 15 Catalase activity of germinating seedlings of groundnut GG20 and GG2


Figure 16 SOD activity of germinating seedlings of groundnut GG20 and GG2




p<0.0001=****).

Chapter 2


Figure 17 Chlorophyll content of germinating seedlings of groundnut GG20 and GG2


Figure 18 Total phenols content of germinating seedlings of groundnut GG20 and

GG2 varieties. Values were mean of three replicates. Errors represent SE. Bar with



p<0.0001=****).

Chapter 2


Figure 19 Jasmonic acid content of germinating seedlings of groundnut GG20 and


Figure 20 salysilic acid content of germinating seedlings of groundnut GG20 and




p<0.0001=****).

Chapter 2


Isoform patterns of induced enzymes LOX, PPO and SOD

Seedlings samples from 3, 6 and 9 days were selected for LOX, PPO and SOD

isozymes detection. Native PAGE revealed 8 isoforms of LOX designated as LOX1-

LOX8. All 8 isofoms were observed only in RA1 and RA1 + A. niger inoculated

seedlings of GG20 variety at 6 day (Fig. 21a). Seven bands of LOXes were observed

in 3rd day seedlings of RA1 + A. niger treatments. LOX2 was detected early in RA1 +

A. niger treated 3rd day old seedlings. In GG2 only 7 bands of LOXes were observed

in RA1 and RA1 + A. niger inoculated seedlings visible on 3rd day (Fig. 21b). After

3rd day LOX isofoms expression slowed down in control and RA1 treated seedlings of

GG2.

Native PAGE of PPO gel showed four isoforms designated as PPO1-PPO4 (Fig. 22a

and b). All 4 PPO isoforms were observed in both the varieties of GG20 and GG2

seedlings. PPO isoforms were more clearly observed in 6 day old germinating

seedlings. Higher levels of induction of PPO isoforms were detected in RA1 and RA1

+ A. niger treated germinating seedlings of both varieties. After 6 days the level of

PPO isoforms were reduced and some faint bands disappeared in extract of 9 day old

germinating seedlings of GG20 and GG2. In other treatments including A. niger

inoculated and in control mild induction of PPO with less intensity was found. In only

A. niger treated seedlings of GG20 and GG2, PPO isoforms were not observed on 3rd

day.

Non denaturing PAGE and SOD staining revealed that 3 different isoforms of SOD

which are referred as SOD1-SOD 3 were observed. One constitutive SOD-band

(SOD1) was clearly found in all seedlings in both varieties (Fig. 23a and b). SOD3

was found only in GG20 and in 6 day germinating seedlings; its intensity was higher

in RA1 and RA1 + A. niger treated seedlings compared to control and A. niger

infected seedlings. SOD2 intensity was found very high in RA1 + A. niger treated

seedlings compared to other three treatments in GG2 at 6 day interval.

Chapter 2


Figure 21 Isoform pattern of LOX enzyme in Native-PAGE analysis (a) GG20

seedlings and (b) GG2 seedlings. Where 1= control 3rd day, 2= RA1 treated 3rd day,

3= A .niger infected 3rd day, 4= RA1 + A. niger treated 3rd day, 5= control 6th day, 6=

RA1 treated 6th day, 7= A. niger infected 6th day, 8= RA1+ A. niger treated 6th day, 9=

control 9th day, 10= RA1 treated 9th day, 11= A. niger infected 9th day and 12= RA1 +

A. niger treated 9th day.

b

a

Chapter 2


Figure 22 Isoform pattern of PPO enzyme in Native-PAGE analysis (a) GG20






a

b

Chapter 2


Figure 23 Isoform pattern of SOD enzyme in Native-PAGE analysis (a) GG20






a

b

Chapter 2


Induction of defense related enzymes due to RR18 treatments

Because RR18 coated seeds could reduce the disease incidence by more than 50 % in

germinated seedlings of both the varieties of groundnut, investigation of induction of

systemic resistance (ISR) due to RR18 was studied by analyzing activities of several

defense related enzymes in germinated seedlings. PAL activity was found to be

induced by A. niger infection in GG20 variety in 8 day old germinated seedlings but

not in GG2 variety, however it was found to be significantly induced (p ≤ 0.05) in

both the varieties by RR18 treatment from 2 days up to end of experiments, and

higher induction was found compared to A. niger infection (Fig. 24). It was found that

higher induction of PAL activity was observed in RR18 + A. niger treated in 6 and 8

day old seedlings of GG20.

PO enzyme was found to be activated due to A. niger infection (in both the varieties)

as well as by RR18 treatment. PO activity was induced significantly at a higher level

by RR18 than in infected plants. PO activity increased gradually, it showed peak on

6th day and then it showed reduction gradually in RR18, A. niger and RR18 + A. niger

treatments compared to control (Fig. 25).

LOX activity was induced much earlier and at a higher level in GG20 variety than in

GG2 variety in RR18 treated seedlings. In A. niger infected seedlings delayed (after 4

days) and lesser LOX activity was found (Fig. 26). LOX activity was also induced in

GG2 seedlings treated with RR18 after 4 day and reached at peak on 12 day. However

in GG20 seedlings treated with RR18, it was induced much rapidly and reached peak

on 8th day. Higher activity of LOX was found in RR18 treated seedlings from all other

treatments in both varieties. PPO activity was also induced by A. niger infection as

well as by RR18 treatment, but RR18 could induce much higher activity than A. niger

infection, also PPO activity was higher in GG20 variety than in GG2 variety (Fig. 27).

It reached at peak on 8th day in both GG20 and GG2. Higher activity of PPO was

noted in RR18 + A. niger treated seedlings on 6th day and in RR18 treated seedlings

on 8th day of germination.

β-1,3-Glucanase (PR 2) and chitinase (PR 3) were found to be induced rapidly,

significantly (p ≤ 0.05) in RR18 treated seedlings compared to the ones challenged

with A. niger. Their activities were higher in GG20 variety of A. hypogaea L. than in

Chapter 2


GG2 variety but ISR was evident in both the varieties (Fig. 28 and 29). β-1,3-

Glucanase activity was found to be highest on 8th day but chitinase activity was found

to be highest on 6th day of germinating seedlings of both GG20 and GG2.

Catalase activity was gradually found to be increased in germinating seedlings treated

with the RR18 and with RR18 + A. niger. CAT activities reached at peak in 8 day old

germinating seedlings in both GG20 and GG2 (Fig. 30). The levels of CAT activity in

the germinating seedlings treated with RR18 and RR18 + A. niger inocultaed were

induced significantly (p ≤ 0.05) in both varieties. There was not detectable changes

observed in SOD activity in the germinating seedlings of both varieties (Fig. 31). The

levels of SOD activity was found higher in RR18 and RR18 + A. niger treatemest in

GG20 and GG2.

Furthermore, the chlorophyll content of seedlings inoculated with RR18 was found to

be higher and significant (P ≤ 0.05) in both the varieties of A. hypogaea L.

chlorophyll content was gradually increased up to last day of germination in both

bactrial treatment (Fig. 32). In A. niger inoculated seedlings, chlorophyll content was

reduced compared to control. Due to RR18 treatments higher accumulation of phenol

was observed in both varieties of groundnut (Fig. 33). Accumulation of phenolics

started at 4 day of incubation. The maximum accumulation was observed on the 12th

day of inoculation. Seedlings inoculated with the pathogen alone also recorded

increased accumulation of phenolics in GG2 seedlings only at 12 day, but no

accumulation was observed in GG20 seedlings. Moreover, the accumulation of

phenolics was lesser in A. niger infected seedlings compared to RR18 challenged with

the A. niger.

Initially up to 3 days, no changes were found in Jasmonic acid levels in germinating

seedlings. After 3 days of incubation, jasmonic acid concentration gradually increased

in RR18 and RR18 challanged inoculation of A. niger in both varieties of groundnut

germinating seedlings (Fig. 34). SA content was also found to higher in 6 day

germinating seedling treated with RR18 and RR18 along with A. niger in both

varities. Higher activity found in GG2 varitety compared to GG20.

Chapter 2


Figure 24 PAL activity of germinating seedlings of groundnut GG20 and GG2


Figure 25 PO activity of germinating seedlings of groundnut GG20 and GG2


different latter indicate statistically significant difference according to uncorrected

Fisher’s LSD test (a- not significant p<0.05=b, p<0.005=c, p<0.001=d, and

p<0.0001=e).

Chapter 2


Figure 26 LOX activity of germinating seedlings of groundnut GG20 and GG2


Figure 27 PPO activity of germinating seedlings of groundnut GG20 and GG2




p<0.0001=e).

Chapter 2


Figure 28 β-1,3-glucanase activity of germinating seedlings of groundnut GG20 and


Figure 29 Chitinase activity of germinating seedlings of groundnut GG20 and GG2




p<0.0001=e).

Chapter 2


Figure 30 CAT activity of germinating seedlings of groundnut GG20 and GG2


Figure 31 SOD activity of germinating seedlings of groundnut GG20 and GG2




p<0.0001=e).

Chapter 2


Figure 32 Chlorophyll content of germinating seedlings of groundnut GG20 and GG2


Figure 33 Total phenols content of germinating seedlings of groundnut GG20 and




p<0.0001=e).

Chapter 2


Figure 34 Jasmonic acid content of germinating seedlings of groundnut GG20 and


Figure 35 salysilic acid content of germinating seedlings of groundnut GG20 and




p<0.0001=e).

Chapter 2


Isoform patterns of LOX, PPO and SOD

Seedlings of 3, 6 and 9 days old were selected for LOX isozymes detection and 6 day

old seedling samples were used to detect PPO and SOD isoenzymes. Native PAGE

revealed that 7 isoforms of LOX were observed, designated as LOX1-LOX7. All 7

isofoms were observed only in RR18 and RR18 + A. niger inoculated seedlings of

GG20 variety on 6th day (Fig. 36a). Only four bands of LOX were observed in 3rd day

old seedlings that is LOX1, LOX3, LOX5 and LOX7. In GG2 only 5 bands of LOX

were observed in 6 day old RR18 and RR18 + A. niger inoculated seedlings (Fig.

36b). After 6th day LOX isoforms expression was slowed down in control as well as

in all treatments seedlings of GG2.

Native PAGE of PPO gel showed three isoforms designated as PPO1-PPO3 (Fig. 37a

and b). All 3 PPO isoforms were observed in both the varieties of GG20 and GG2

seedlings. PPO isoforms were more clearly observed in extract of 6 old day

germinating seedlings. Higher levels of induction of PPO isoforms were observed in

RR18 and RR18 + A. niger treated germinating seedlings compared to control or only

A. niger infected seedlings of both varieties.

Non denaturing PAGE and SOD staining revealed that 3 different isoforms of SOD,

SOD1-SOD 3 were observed in GG20 and only 2 bands of SOD were observed in

GG2 (Fig. 38a and b). SOD1 was not found to be induced in A. niger inoculated

seedlings of GG20 and its intensity was higher in RR18 + A. niger treatments

compared to control and A. niger alone. SOD3 induction was also much higher in its

intensity in RR18 and RR18 + A. niger treatments compared to control and A. niger

inoculated seedlings. In GG2 seedlings SOD 1 was clearly induced only in RR18 and

RR18 + A. niger inoculated seedlings. SOD2 was found in all germinating seedlings

but SOD2 intensity was found very high in RA18 with A. niger treated seedlings

compared to other three treatments of GG2.

Chapter 2


Figure 36 Isoform pattern of LOX enzyme in Native-PAGE analysis (a) GG20

seedlings and (b) GG2 seedlings. Where 1= control 3rd day, 2= RR18 treated 3rd day,

3= A .niger infected 3rd day, 4= RR18 + A. niger treated 3rd day, 5= control 6th day, 6=

RR18 treated 6th day, 7= A. niger infected 6th day, 8= RR18+ A. niger treated 6th day,

9= control 9th day, 10= RR18 treated 9th day, 11= A. niger infected 9th day and 12=

RR18 + A. niger treated 9th day.

b

a

Chapter 2


Figure 37 Isoform pattern of PPO enzyme in Native-PAGE analysis (a) GG20

seedlings and (b) GG2 seedlings where 1= control 6th day, 2= RR18 treated 6th day,

3= A .niger infected 6th day, 4= RR18 + A. niger treated 6th day.

b

a

Chapter 2


Figure 38 Iosform pattern of SOD enzyme in Native-PAGE analysis (a) GG20

seedlings and (b) GG2 seedlings where 1= control 6th day, 2= RR18 treated 6th day,

3= A .niger treated 6th day, 4= RR18 + A. niger treated 6th day.

1

1

2

2 3

b

a

Chapter 2


Effect of RA1 and RR18 on plant growth

Field and Pot assays were conducted to check effect of bacteria (RA1 and RR18) and

fungal pathogen on groundnut plants of GG2 and GG20 varieties.

In field assay also, plants treated with RA1 bacterial culture showed the highest

germination percentage compared to controls in both the varieties of A. hypogaea L.

(Fig. 39). Plants pre-treated with RA1 bacteria showed significantly (p ≤ 0.05) lesser

disease incidence upon challenge with A. niger compared to only A. niger treated

plants (Table 4). Root length and shoot length of RA1 treated plant was found to be

very high in both GG20 and GG2 varieties. Leaf number was also more in RA1

treated plants. Weight of plant was found to be more in RA1 treated plants. In only A.

niger inoculated plants, root length, shoot length and weight of plant was less

compared to control plants (Table 4). Yield of peanut was also found to be very high

in RA1 treated plants (Fig. 39).

About 1×108 CFU/mL of RA1 was inoculated in the rhizosphere of A. hypogaea L.

plants and after 15 days 6×102 CFU/mL RA1 were found - RA1 culture survived up

to 15 days in the field.

In pot study, plants were treated with RR18 and observations were made at 45 days

interval time. RR18 treated seeds showed significantly higher germination than

control seeds, however A. niger treated seeds showed only 53.77% and 44.44% of

germination in GG20 and GG2 respectively (Tables 5). Disease incidence was found

very high in A. niger inoculated seeds and it was reduced to 50% in RR18 + A. niger

inoculated plants. Number of leaves, fresh weight and dry weight of the plants were

found higher in RR18 treated GG20 and GG2 plants (Fig. 40). From pot study and

field study it was concluded that RA1 and RR18 bacterial strains possess very good

PGPR characteristics.

Similar inoculum (1×108 CFU/mL) of RR18 was inoculated in root of plant in the pot

study and it was found that it is survived (4 ×103 CFU/mL) up to 18 days.

Chapter 2


Figure 39 (a) Effect of RA1 on growth of GG20 plants: Control, RA1 treated, RA1 + fungal

treated and A. niger treated plant (b) Effect of RA1 on growth of GG2 plants: Control, RA1

treated, RA1 + fungal treated and A. niger treated plants.

b

a

Table 4 Effect of treatments of RA1 alone or with A. niger on germination percentage, disease incidence, Root length, Shoot length and biomass dry weight of two varieties of groundnut plants GG20 and GG2. Data represent means ± SE of three replicate with 6 plants each. Different number of asterisk indicate statistically significant difference according to uncorrected Fisher's LSD test (p < 0.05=*, p < 0.005=**, p < 0.001=*** , p < 0.0001=****and ns- not significant)

Germination (%)

Disease incidence (%) Root length (cm)

Shoot length (cm)

No. of leaves Biomass dry weight (g)

Control GG20 83.30 ± 9.653 0.00 ± 0.00 16.26 ± 2.272 6.83 ± 0.441 530 ± 23.56 1.40 ± 0.327

RA1 GG20 99.30 ± 0.066 ns 0.00 ± 0.00 23.23 ± 4.699 ns 10.46 ± 1.370* 865 ± 74.34*** 2.53 ± 0.524 ns

A. niger GG20 55.53 ± 5.539* 50.66 ± 0.067**** 13.10 ± 1.007 ns 7.10 ± 0.413 ns 400 ± 45.87ns 1.68 ± 0.318 ns

RA1+ A. niger GG20

83.30 ± 9.653 ns 33.86 ± 0.567**** 19.53 ± 0.826 ns 9.30 ± 0.589 ns 689 ± 32.33* 1.83 ± 0.339 ns

Control GG2 72. 16 ± 5.573 0.00 ± 0.00 9.66 ± 1.833 6.66 ± 0.472 430 ± 54.65 1.10 ± 0.144

RA1 GG2 83.30 ± 9.653 ns 0.00 ± 0.00 16.96 ± 0.634** 10.53 ± 0.618** 520 ± 38.12ns 1.58 ± 0.206*

A. niger GG2 44.43 ± 5.573* 64.00 ± 1.501**** 9.56 ± 0.470 ns 8.33 ± 0.869 ns 352 ± 10.97** 0.96 ± 0.130 ns

RA1+ A. niger GG2

77.73 ± 11.14* 45.53 ± 2.736**** 15.10 ± 2.044* 11.13 ± 0.713** 478 ± 42.13ns 1.52 ± 0.023 ns

Chapter 2


Figure 40 (a) Effect of RR18 on growth of GG20 plants and (b) Effect of RR18 on growth of

GG2 plants.

a

b

Germination (%)

Disease incidence (%)

Shoot length (cm) Root length (cm) No. of leaves Fresh weight (g)

Control GG20 82.67 ± 2.66a 0.00 ± 0.00 17.69 ± 1.15a 09.27 ± 0.13a 19.00 ± 1.00a 2.27 ± 0.13a

RR18 GG20 97.33 ± 3.22e 0.00 ± 0.00 20.95 ± 1.02c 10.53 ± 0.26d 22.33 ± 1.33b 2.57 ± 0.23c

A. niger GG20 53.72 ± 1.85d 50.33 ± 2.12a 15.61 ± 0.19a 08.46 ± 0.23b 15.67 ± 0.66b 1.21 ± 0.06e

RR18 + A. niger GG20

86.22 ± 2.11c 20.66 ± 1.01e 18.13 ± 0.93b 11.22 ± 0.11e 23.00 ± 2.00b 2.49 ± 0.14a

Control GG2 72.22 ± 1.11a 10.00 ± 0.00 16.67 ± 0.66a 09.13 ± 0.06a 19.67 ± 0.33a 1.42 ± 0.11a

RR18 GG2 85.55 ± 2.77d 00.00 ± 0.00 17.97 ± 0.51 b 14.46 ± 0.23e 25.33 ± 0.60c 1.85 ± 0.07b

A. niger GG2 44.44 ± 2.22c 55.55 ± 1.21a 14.55 ± 0.27a 10.62 ± 0.31d 13.67 ± 0.33c 1.04 ± 0.02b

RR18 + A. niger GG2

74.44 ± 3.21b 25.55 ± 0.98c 17.33 ± 0.33a 13.37 ± 0.18e 24.67 ± 1.33c 1.50 ± 0.32b

Table 5 Results of various treatments on germination percentage, disease incidence, shoot length, root length No. of leaves and plant fresh and dry weight on both varieties on 45 day old plants. Data represent means ± SE of three replicate with 6 plants each. . Different alphabets indicate statistically significant difference according to uncorrected Fisher's LSD test (a- not significant p < 0.05=b, p < 0.005=c, p < 0.001=d, and p < 0.0001=e)

Chapter 2


Detailed identification of RA1 and RR18

From the genomic DNA of RA1 and RR18, 16S rRNA gene was amplified and its

sequence obtained was subjected to BLASTn analysis on NCBI as well as on

EzTaxon. BLASTn results showed that RA1 (accession no KC818478) to be 99%

homologous with P. plecoglossicida, P. monteilii, P. putida and P. mosselii and

99.57% homologous with P. guariconensis (Fig. 41) and RR18 (accession no

KM387316) showed 99% similarity with Burkholderia cenocepacia, B. cepacia, B.

metallica, B. seminalis, B. ambifaria, and B. anthina in NCBI data base and 100%

similarity with B. cepacia EzTaxon (Fig. 42).

However, biochemical tests for identification as per Bergey’s Manual showed RA1 to

be different compared to P. plecoglossicida, P. monteilii, P. putida and P. mosselii

in terms of nitrate reduction, urease production and mannitol utilization (Table 7).

Also fatty acid metabolite profile of RA1 was found to be different with all other

Pseudomonas species (Table 8), which showed that RA1 is a novel strain, to which

we called strain ‘mungfali’ as it was isolated from the field of mungfali (means

groundnut). This new strain was deposited in Microbial type culture collection and

gene bank (MTCC), Institute of microbial technology, Chandigarh, India and its

MTCC accession number was given as 12267.

Chapter 2


Figure 41 16S rRNA gene sequences of closely related sp. were taken from NCBI

data base. Neighbor joining tree constructed using MEGA 5.2 Distances and

clustering in the neighbor joining tree contained Bootstrap values based on 1000

replications listed as per percentages at the branching points.

Chapter 2


Figure 42 16S rRNA gene sequences of closely related sp. were taken from NCBI

data base. Neighbor joining tree constructed using MEGA 5.2 Distances and

clustering in the neighbor joining tree contained Bootstrap values based on 1000

replications listed as per percentages at the branching points

Chapter 2


Figure 43 GC spectra of RA1.

Chapter 2


Table 6 Fatty acid composition of RA1 culture

Chapter 2


Biochemical test RA1 P.

plecoglossicida

P.

monteilii

P.

putida

P.

mosselii

Growth at 4 0C - - - + -

Growth at 41 0C - - - - -

Growth at 5% NaCl + + - - +

Nitrate reduction - + - - -

D-glucose

fermentation

- - - - ND

Urease production - - + - ND

Mannitol - + - + +

Table 7 Biochemical test of RA1 and closely related Pseudomonas strains.

Chapter 2


Fatty acid RA1 P. guariconensis P. plecoglossicida P. monteilii P. putida P. mosselii

C10:0 3-OH 4.17 ----- 8 3.6 3.3 4.4

C12:0 3-OH 3.54 ----- 4.4 4.0 3.9 4.2

C12:1 3-OH 0.21 ----- 1.6 0.2 0.1 0.2

C12:0 2-OH 4.00 ----- 4.3 2.9 3.4 2.4

C12:0 3.04 ----- 2.0 3.2 3.0 3.1

C14:0 0.45 ----- 0.2 0.2 0.2 0.2

C16:0 24.38 25.7 23.3 18.9 25.1 17.4

C18:0 0.72 ----- 0.5 1.1 0.7 1.3

C18:1 ω7c 23.56 20.4 19.9 31.6 22.4 42.3

C17:0 cyclo 1.13 11.5 5.2 0.7 1.0 0.7

C17:1 ω8c 0.10 ----- ----- ----- 0.1

C19:0 cyclo ω8c 0.18 ----- ----- ------ ----- ------

Summed

feature 3*

32.69 10.8 29.2 32.4 36.4 23.2

Table 8 Weight percent of cellular fatty acid composition of RA1 strain and other

related strains analyzed by Gas Chromatography and MIDI Sherlock software.

Chapter 2


Discussion

The potential of PGPRs, P. guariconensis (RA1) and B. cepacia (RR18) to protect the

plants by activating the ISR was studied in the present work. The use of PGPR has

been reported for the control of various fungal pathogens (Gutterson 1990; Wei et al.

1991; Nandakumar et al. 2001; Jain and Choudhary 2014). However no promising

PGP rhizobacteria were found to be reported for A. hypogaea L. plants in our

literature study.

Results obtained from seed germination assay revealed that P. guariconensis (RA1)

and B. cepacia (RR18) treated groundnut seeds germinated more vigorously

compared to untreated control seedlings with a reduction in the disease incidence by

50 % (Table 2 and 3). Increase in germination in ground nut plants challenged with A.

niger has been reported to be exhibited by Methylobacterium (Madhaiyan et al. 2006)

and B. subtilis AF1 (Sailaja et al. 1998). However successful field studies have not

been reported. In the present study, it was found that P. guariconensis (RA1) treated

plants in field showed higher percentage of germination and better growth

characteristics like root and shoot length and biomass dry weight. B. cepacia (RR18)

treated seeds also showed higher germination and better growth of plants in the pot

studies. Both RA1 and RR18 were found to produce phytohormones (IAA, GA3,

ABA) and solubilize N, P, K and Zn. Disease incidence was also reduced up to 66%

in field due to pre-treatment of P. guariconensis (RA1) and up to 40% in pot study

due to pretreatments of B. cepacia (RR18), that supports its indirect plant growth

promoting activities of found in the lab studies like siderophore, HCN and fungal cell

wall degrading enzymes production abilities (Chapter 1, table 2). Yuttavanichakul et

al. (2012) also reported 50% reduction in disease incidence and severity of crown-

and root-rot of peanut by co-inoculation of B. subtilis, B. megaterium and

Psuedomonas species in pot studies.

RA1 and RR18 were found to survive for fifteen and eighteen days respectively in

field. By this time, infected as well as control seeds already had germinated.

However, the number were reduced to 600 CFUs from 1x 108 for RA1 and 400 CFUs

from 1x 108 for RR18. Since A. niger infection affects seed germination, seed

bacterization was considered the best method for inoculation of RA1 and RR18 to

control collar rot disease of A. hypogaea L.

Chapter 2


Plants are endowed with defense mechanisms, which can be activated in response to

attack by pathogens and insect. The defense genes which are quiescent in healthy

plants are required to be induced. Activation of these genes by various factors (PGPR,

plant elicitor or plant compound), induce systemic resistance against disease.

Induction of defense in groundnut by RA1 and RR18 are largely associated with the

production or increase in defense related enzyme activities like PAL, PO, LOX, PPO,

β-1, 3-glucanase and chitinase and reactive oxygen species scavenging enzymes SOD

and catalase. All these enzymes are related to activation of defense of plants against

pathogen and decrease the level of reactive oxygen species.

PAL plays an important role in the biosynthesis of various defense chemicals by

phenylpropanoid metabolism. PAL catalyzes the elimination of ammonia from

phenylalanine to produce trans-cinnamic acid. Trans-cinamic acid serves as substrate

for the formation of phenolics like salicylic acid, phytoalexins and antibiotics. Both

phenolics and phytoalexin compounds are important in plant defense. Tonelli et al.

(2011) reported induction of PAL activity in peanut by PGPR Pseudomonas sp.

BREN6 and Bacillus sp. CHEP5. Madhaiyan et al. (2006) also reported induction of

PAL activity in Methylobacterium treated groundnut plants. In our experiments also,

PAL activity was found to be induced in both varieties of groundnut upon treatment

with RA1 and RR18 (Fig. 9 and 24).

PO activities are linked to lignification and the generation of hydrogen peroxide at a

later stage of infection, which inhibit pathogen directly. Generation of free radical

with antimicrobial effects is another mechanism which restricts the development of a

phytopathogen. Increased activity of cell wall bound PO has been elicited in different

plants such as cucumber (Chen et al. 2000) and tobacco (Ahl Goy et al. 1992) due to

pathogen infection. PO activity was observed to be induced in groundnut seedlings

due to A. niger inoculation. However, inoculation of RA1 and RR18 with A. niger

showed higher and rapid induction of PO activity in both the varieties compared to

only A. niger infected seedlings (Fig. 10 and 25). Similar results were observed in

groundnut plants treated with Methylobacterium and Methylobacterium with

challenging inoculation of A. niger and S. rolfisi up to 72 h and afterward it was

decreased in groundnut (Madhaiyan et al. 2006).

Chapter 2


LOX activity has been implicated in the generation of reactive oxygen species,

mediation of lipid peroxidation and plays an important role in the synthesis of JA. JA

is a signaling molecule in ISR. The product of LOX such as 13-HPODE and 13-

HPOTE also show significant antifungal activity against A. niger. The early release of

antifungal substances or molecules that induce resistance in bacterized seedling could

control A. niger in groundnut (Sailaja et al. 1998). Our study showed that RA1 and

RR18 alone and RA1 and RR18 with A. niger seeds of both groundnut varieties

showed increase in LOX activity at early stages of seed germination (Fig. 11 and 26),

which shows induction of defense by PGPR. Induced LOX isoforms were also found

in bacterial treatments (Fig. 21 and 36). Mariutto et al. (2011) observed increase of

transcription level of two isoforms of LOX due to Pseudomonas putida BTP1 in

tomato. Increase in LOX activity due to fungal infections is reported in many plants.

ISR could be marked by activation of JA and ethylene and then activation of gene

related to ISR. Genes induced during ISR are responsible for production of antifungal

compounds and PR proteins (Conrath et al. 2001). JA acts as a signaling compound in

induction of defense against pathogen. In the present study, higher induction of JA

was observed in RA1, RR18 and RA1 and RR18 with A. niger treated seeds of both

groundnut varieties (Fig. 19 and 34). Induction of JA in RA1 and RR18 treatments

indicate the induction of ISR and resistance in RA1 and RR18 treated germinating

seedlings of groundnut. Treatment of tobacco roots with P. fluorescens CHA0

activates accumulation of SA-inducible PR proteins in the leaves (Maurhofer et al.,

1994). Similar result was also observed in our study.

PPO has been implicated in plant defense, specifically against necrotrophic pathogen

infection (Constabel, 2008). PPO oxidizes phenolics to highly toxic quinones and is

speculated to be involved in the terminal oxidation in the diseased plant tissue which

was attributed for its role in disease resistance. Similar to other defense enzymes, PPO

activity was induced by bacterial isolates RA1 and RR18 in the germinating seedlings

(Fig. 12 and 27). We also recorded higher induction of PPO isoforms in PGPR

treatments (Fig. 22 and 37). Chen et al. (2000) reported that many rhizobacteria and

P. aphanidermatum induced the PPO activity in cucumber root tissues. Thipyapong

and Steffens (1997) reported increased activity of PPO in young leaves of tomato

when matured leaflets were injured. Constable et al. (2000) reported the expression

PPO due to action of methyl jasmonate in poplar plant upon wounding. Bacterial

Chapter 2


isolates RA1 and RR18 might have elicited the induction of PPO gene through

increased JA response. Similar result was reported by Jain and Choudhary (2014) who

reported that increased activity of PPO enzymes was found due to Carnobacterium

sp. SJ-5 upon challenge inoculation of Fusarium oxysporum in soybean plants.

β-1,3-glucanase (PR-2) and chitinase (PR-3) have potential to hydrolyze β-1,3-

glucanase and chitin respectively, which are a major components of fungal cell wall,

leading to direct inhibition of several fungi. Maurhofer et al. (1994) reported that the

induction of systemic resistance by P. fluorescens was correlated with the

accumulation of β-1,3-glucanase and chitinase. In the present study, activities of β-

1,3-glucanase and chitinase were found to be significantly increased in ground nut

seeds treated with RA1, RR18 alone and RA1 and RR18 with A. niger in both

varieties, which showed peak on 6th day (Fig. 13, 14, 28 and 29). Significant increase

in β-1,3-glucanase activity was also reported in peanut seeds treated with PGPR

strains and PGPR strains with challenged inoculation of S. rolfsii by Tonelli et al.

(2011). Madhaiyan et al. (2006) reported that induction of β-1,3-glucanase activity in

groundnut treated with Methylobacterium inoculated with A. niger and S. rolfsiiup

upto 72h and then a decrease at 96h. Ramamoorthy et al. (2002) reported induction of

β-1,3-glucanase and chitinase activity in tomato plants treated with PGPR stains P.

fluorescens Pf1 and P. fluorescens Pf1 challenged with pathogen F. oxysporum f. sp.

Lycopersici, which showed peak on 5th day.

SOD and CAT are most important antioxidant enzymes with the ability to repair

oxidative damage caused by ROS. SOD is a necessary component and work as the

first line of plant defense against oxidative damage under various stress because it

dismutase two O2− to hydrogen peroxide (Cakmak and Hors 1991). CAT, which is

involved in the degradation of hydrogen peroxide into water and oxygen, is the most

effective antioxidant enzymes in preventing oxidative damage (Willekens et al. 1995;

Mittler 2002). In the present study both enzyme activities were found to be higher in

PGPR treatments. Wang et al. (2012) reported induction of SOD activity in cucumber

plant by consortium of three plant growth-promoting rhizobacterium strains for

induction of draught tolerance. Increased levels of SOD, PO, CAT enzymes were

observed in root and leaf of cucumber treated with Bacillus megaterium strain L8

inoculated with P. aphanidermatum (Liang 2011).

Chapter 2


Chlorophyll a content is an important parameter directly related to the amount of

photosynthesis in the plants and hence in turn to the final production. The higher N

also contributed to the formation of chlorophyll, which consequently, increased the

photosynthetic activity. In our result we found that due to RA1 and RR18 treatments

chlorophyll content was increased in germinating seedlings of both varieties because

inoculated bacteria improved the mineral nutrition, and in general, health of the plants

thereby leading to an increase in the chlorophyll content. Bal et al. (2012) found

increased concentration (50%) of chlorophyll due to PGPR treatments. Arif et al.

(2012) reported an increase of 43% in chlorophyll content in response to Zn

fertilization.

Phenolic compounds improve the mechanical strength of host cell wall and also

inhibit the invading pathogenic organisms as they are mycotoxic in nature. Induction

of phenolics occurs due to induction of shikimic acid pathway. Increase in phenolic

content in plants has been correlated with increased resistance to pathogens

(Velazhahan and Vidhyasekaran, 1994). In the present study accumulation of

phenolics was observed in seedling treated with RA1 and RR18. Singh et al. (2003)

found that foliar application of Pseudomonas fluorescens strain Pf4 and P. aeruginosa

strain Pag in chickpea, increase phenolics (gallic, chlorogenic and cinnamic acids)

within 24 h after application. Foliar application of a plant growth-promoting

rhizobacterium, P. fluorescens strain Pf1 significantly increased phenolic content was

observed in groundnut (Meena et al. 2000).

The RA1 and RR18 proved to be capable of promoting growth in groundnut plants

and also reduce disease incidence. The PGPR treated groundnut plants were found to

be in enhanced resistance state. RA1 and RR18 are gram negative, non spore forming

short rod bacteria.

RA1 produces fluorescent pigment in King's B agar medium. 16S rRNA gene analysis

revealed that this microorganism was related closely to members of Pseudomonas sp.

Further phylogenetic analysis indicated that levels of similarity with all the other

Pseudomonas sp. were around 99% (Fig. 41). The isolated strain RA1 forms a

common cluster with Pseudomonas stutzeri. RA1 like P. stutzeri was found to be

originated from the root of the neighbor joining tree, unlike other Pseudomonas sp.

Chapter 2


The 1,408 bp sequence of 16S rRNA gene of the isolated RA1 bacterium has been

submitted to Gen Bank at NCBI and its accession no. is KC818478. To confirm its

evolutionary relationship with other Pseudomonas sp., all the biochemical test as per

the Bergey's manual were performed and it was found that RA1 was different from

other Pseudomonas sp. in some aspects: RA1 is not able to utilize mannitol unlike

other Pseudomonas sp.; RA1 could not to grow at 4°C unlike Pseudomonas putida.

Also RA1 showed differences in Nitrate reduction and Urease production (Table 3).

Furthermore the FAME analysis also showed that RA1 was different from other

closely related Pseudomonas sp. It was found that there is difference in its fatty acid

composition and in summed feature 3, C14:0, and C16:0. It seemed from phylogenetic

analysis and capability of RA1 to resist A. niger that RA1 could have evolved earlier,

from which other Pseudomonas sp. would have originated. RA1 was found to be

99.57 % similar to P. guariconensis. As there were differences in the FA profile of

this organism as well as its important role in protecting groundnut plants from A.niger

infection, we named RA1 as strain ‘mungfali’ of P. guariconensis. This strain name

was given considering the fact that it was isolated from the field of groundnut (means

mungfali in Gujarati language). No other sp. of Pseudomonas has been reported to

resist A. niger growth.

RR18 also was able to produce dark yellow pigment in king’s B medium. It showed

100% similarity with B. cepacia (Fig. 42) and its sequence was submitted to the NCBI

data base (KM387316). Karthikeyan et al. (2006) used Pseudomonas fluorescens for

coconut plant growth promotion. A number of reports describe PGP and ISR activity

of Pseudomonas sp. (Cattelan et al. 1999; Shaharoona et al. 2006; Mayak et al. 1999;

Babalola et al. 2003) PGPR strains, particularly Pseudomonas fluorescens and

Bacillus subtilis are finest recorded as the most promising member of indirect

stimulation (Damayanti et al. 2007). Poupin et al. (2013) used Burkholderia

phytofirmans PsJN for Arabidopsis thaliana growth promotion. Burkholderia cepacia

has been observed to possess biocontrol characteristics against Fusarium spp., while,

it could stimulate growth of maize under iron-poor conditions via siderophore

production (Bevivino et al. 1998). Rice plants inoculated with Burkholderia spp

promote the growth of plant (Baldani et al. 2000).

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Outline of Chapter 2 - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/79479/11/11_chapter4.pdf · Thaumatin-like proteins (TLP) belong to PR-5 family showing antifungal activity