chapter vi development of bioformulations and field...

Chapter VI Development of Bioformulations

and Field Studies

------------------------ --Chapter VI: Development of bioformulations and field studies

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6.1 Introduction

Even though agricultural production has increased in the past decades, but

pressure is still on to cope up with the increasing population across the world.

Producers became more and more dependent on agrochemicals, as a reliable method

of crop protection with economic stability of their operations. If the credits of

pesticides include enhanced economic potential in terms of increased food production,

than increasing use of chemical inputs causes several negative effects, i.e.,

development of pathogen resistance to the applied chemicals, health implications to

man and his environment, its residual effect on useful soil microorganisms etc. There

is now overwhelming evidence that some of these chemicals do pose potential risk to

humans and other life forms and unwanted side effects to the environment

(Jeyaratnam, 1985; Igbedioh, 1991; Forget, 1993). No segment of the population is

completely protected against exposure to pesticides and the potentially serious health

effects, though a disproportionate burden is shouldered by the people of developing

countries and by high risk groups in each country. The world-wide deaths and chronic

illnesses due to pesticide poisoning number about 1 million per year (Environews

Forum, 1999). The endosulfan tragedy at Kasaragod, Kerala is a fine example of the

impact of pesticides on humans and the environment.

The Indian pesticide industry with 85,000 Mt of production in year 2006-07 is

ranked second in Asia (behind China) and twelfth globally. In value terms, the size of

the Indian pesticides industry was estimated at Rs. 74 billion during 2007, including

exports of Rs 29 billion. The Indian pesticide industry is dominated by insecticides

globally where herbicides and fungicides are the key segments (http://www.

researchandmarkets.com).

Therefore, alternatives to chemical fertilizers and pesticides are much needed.

Hence, the use of beneficial micro-organisms as biofertilizers and biocontrol agents

has become more important in recent years in order to improve plant growth and

manage plant diseases but also to avoid environmental pollution (Fravel et al., 2005).

Plant growth promoting rhizobacteria (PGPR) are known to promote plant growth and

suppress plant diseases by various mechanisms dealt earlier (Chapter IV).


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Source: (IFA, 2009)

Figure 6.1 Top 20 fertilizer consuming countries across world

Most of the reports regarding plant growth promotion and disease protection is

only restricted to laboratory and greenhouse conditions. Under field conditions the

introduced rhizobacteria has to withstand the varying environmental conditions which

may leads to the failure of root colonization or expressing the desired traits which

leads to the failure of field experiments. Hence performance of the selected PGPR

should be analyzed repeatedly and thoroughly under field conditions of different

agroclimatic regions before it is released to farmers for use.

The major factor that goes into success of biocontrol programme is the

effectiveness with which the biocontrol agents are delivered to the environment. This

requires preparation of formulations and is very important for effective

implementation of biocontrol agents against various crop diseases (Prasad and

Rangeshwaran, 2000). Biopesticides and biofertilizer formulations usually prepared as

carrier-based inoculants containing effective microorganisms. Incorporation of

microorganisms in carrier material enables easy-handling, long-term storage and high

effectiveness of biopesticides and biofertilizers. Among various types o f biopesticides

and biofertilizers, bacterial inoculant is one major group which includes rhizobia,

nitrogen-fixing rhizobacteria, plant growth-promoting rhizobacteria, phosphate-

solubilizing bacteria, and so on.

Talc is a natural mineral referred as steatite or soapstone composed of various

minerals in combination with chloride and carbonate. Chemically it is referred as


244

magnesium silicate [Mg3Si4O10(OH)2] and available as powder form from industries

suited for wide range of applications. It has very low moisture equilibrium, relative

hydrophobicity, chemical inertness, reduced moisture absorption and prevents the

formation of hydrate bridges that enable longer storage periods. Owing to the inert

nature of talc and easy availability as raw material from soapstone industries it is used

as a carrier for formulation development (Nakkeeran et al., 2005). In the present study

we selected talc as a carrier material to develop bioformulations because of its above

said properties. According to Jeyarajan and Nakkeeran (2000), characterstics of an

ideal formulation is,

i. Should have increased shelf life.

ii. Should not be phytotoxic to the crop plants.

iii. Should dissolve well in water and should release the bacteria.

iv. Should tolerate adverse environmental conditions.

v. Should be cost effective and should give reliable control of plant

disease.

vi. Should be compatible with other agrochemicals.

vii. Carrier must be cheap and readily available for formulation

development.

The success of a biocontrol agent depends largely on the ability of the

introduced agent to establish itself in the new environment and maintain a threshold

population on the planting material or rhizosphere. The commercial use of biocontrol

agents requires inoculum that retains high cell viability and can easily be transported

and applied to the seed. In most studies requiring bacterial inocula, either liquid

suspensions (Broadbent et al., 1977; Burr et al., 1978; Kloepper et al., 1980) or

bacteria mixed with peat (Roughley and Vincent, 1967; Nair and Fahy, 1976;

Davidson and Reuszer, 1978) have been applied. Kloepper and Schroth (1981)

demonstrated the potentiality of talc to be used as a carrier for formulating

rhizobacteria. Further, different carrier materials such as Talc, Peat, Kaolinite,

Lignite, Vermiculite and Stickers were used by Vidyasekaran and Muthamilan (1995)

to develop a suitable formulation of fluorescent Pseudomonads. Seed-treatment

followed by soil application of talc-based powder formulation effectively checked

chickpea wilt and pigeonpea wilt under field conditions and has increased the yield


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(Vidhyasekaran and Muthamilan, 1995; Vidhyasekaran et al., 1997b). Talcum powder

formulation of P. fluorescens was used to manage rice blast (Krishnamurthy and

Gnanamanickam (1998), rice sheath blight disease (Nandakumar et al. (2000) and

turmeric rhizome rot (Nakkeeran et al., 2004).

The purpose of the present work was to develop a suitable bioformulation of

selected rhizobacteria and their evaluation under greenhouse and field conditions for

their plant growth promoting and fungal disease suppressing ability (Fusarium wilt

and Early blight) which can be used as alternative to chemicals in sustainable tomato

production.


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6.2. Materials and Methods

6.2.1 Plant materials

For greenhouse experiments, seeds of tomato cultivar PKM-1 obtained from

local seed agencies, Mysore. Seeds were surface-sterilized with 1% sodium

hypochlorite for 30 s and then rinsed in sterile distilled water, blot dried and used for

the experiments. Where as for field experiments, cultivar Suruchi F1 hybrid was used

after surface sterilization as explained above.

6.2.2 Microorganisms and cultural conditions

Three rhizobacterial isolates (Bacillus subtilis PSIRB2, Pseudomonas

aeruginosa 2apa, and Serratia marcescens Pan-9/c) were originally isolated from

rhizospheric soil samples of tomato collected from different tomato growing regions

of Karnataka. These isolates were characterized for their varied PGPR traits (chapter

IV) and maintained in Microbial culture collection of the Department of Studies in

Biotechnology, University of Mysore, Mysore. All selected isolates were maintained

on NA slants at 4°C for routine work. For long term storage rhizobacterial isolates

were maintained in 40% glycerol at -80°C.

Detailed explanation of these rhizobacterial isolates has been given in chapter

IV (Part – B) (Bacillus subtilis PSRIB2), Part – C (Pseudomonas aeruginosa 2apa)

and Part - D (Serratia marcescens Pan-9/c). Growth conditions, conidial suspension

of Fusarium oxysporum f. sp. lycopersici and Alternaria solani was done as explained

in chapter III.

6.2.3 Inoculum preparation

Rhizobacterial inoculum was prepared as explained in early chapters and the

bacterial concentration was adjusted spectrophotometrically to 1x108 cfu/ml for

routine seed treatment and to prepare bioformulations concentration was adjusted to

1x1016 cfu/ml.

6.2.4 Preparation of bioformulation

Talcum powder formulation was developed by following modifying the

method of Vidhyasekaran and Muthamilan (1995) using a mixture of 10 g of

carboxymethyl cellulose (CMC) and 1 Kg of talc. The pH was adjusted to 7.0 by

adding calcium carbonate (CaCO3) and placed in a metal tray followed by thorough


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mixing. The mixture was autoclaved for 45 min at 121.5°C (15 lb/inch2) on each of

two consecutive days. Rhizobacterial inoculum was grown in LB broth and

concentration was adjusted as explained above. Five hundred ml of bacterial

suspension containing 1x1016 cfu/ml, was added to 1 Kg of the talc material and

mixed well under sterile conditions to get an inoculum density of 1x108 cfu/g talc

powder. Formulation was packed in polythene bags, sealed and stored at room

temperature with a moisture content of 35%.

6.2.5 Determining the shelf life of the developed formulation

To determine the efficacy of selected PGPR strains to survive in carrier

material under room temperature, one gram of the formulation was drawn at regular

time intervals viz., 0, 15, 30, 45, 60, 75, 90, 105 and 120 days after storage. Each

formulation was serially diluted and suitable dilutions were spread plated onto defined

medium amended with suitable antibiotics to determine the concentration of bacterial

population. The bacterial colony in each plate was counted and tabulated as cfu/g

formulation.

6.2.6 Seed bacterization

Seed bacterization was done with regular methods as explained in previous

chapters. To treat the tomato seeds with bioformulations. The tomato seeds were first

wetted with water and mixed thoroughly with talc-based formulation (10 g/kg seed) of

each bacterial isolates. Seeds were then spread on a blotter sheet, shade-dried and

used for sowing purpose. Seeds treated with talcum powder amended with CMC serve

as control.

6.2.7. Greenhouse experiments

6.2.7.1 Efficacy of talcum formulation on plant growth and fruit yield of tomato

Plant growth promotion studies under laboratory and greenhouse conditions

were conducted using standard procedures as explained in the chapter III.

Further to analyze the plant growth at later stage and fruit yield, treated and

control seedlings were grown in 96 well crate containing coir pith as potting media,

for 25 days. Two days before transplantation each seedlings were drenched with 25

ml suspension of formulation (1 g talcum powder formulation dissolved in 10 ml of

tap water). Twenty five day-old seedlings were transplanted into earthern pots


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containing sterilized potting mixture, soil: FYM: coir pith at the ratio 2:1:1 (v/v/v).

Seedlings were maintained under greenhouse conditions and watered at regular

intervals.

Plant height, fresh weight and chlorophyll content for each treatment were

determined using 75 day-old-seedlings (Bansal et al., 1999). Briefly, 100 mg of fresh

leaves were crushed in 20 ml of 80% acetone and the extract centrifuged for 10 min at

1000 rpm. Absorbance of the supernatant was recorded at 645 and 663 nm in a

spectrophotometer. Total chlorophyll content was expressed as mg/g of each sample.

Total chlorophyll (mg/g tissue) = [20.2 (A645) – 8.02 (A663) x VW]/1000

where A=absorbance at the given wavelength, W = weight of fresh leaf

sample, V = final volume chlorophyll solution.

The index leaflets were collected from different treatments, dried and

subjected for nutrition uptake studies. In order to analyze yield, fruits were picked just

before ripening separately from each treatment, the numbers of fruits were counted

and weighed. The average numbers of fruits per plant and average weight of fruit per

treatment was calculated. For each treatment there were twelve plants per replicate

and six replicates were maintained. The experiment was performed twice.

6.2.7.2 Efficacy of talcum formulation on Fusarium wilt and early blight

incidence in tomato under greenhouse conditions

Seedlings from control and treated seeds were raised as explained above, after

transplantation 30 day-old-seedlings were challenge inoculated with conidial

suspension (1x105 conidia/ml) of F. oxysporum. Wilt incidence was recorded up to 60

days after challenge inoculation. Conidial suspension (5x104 conidia/ml) was sprayed

onto leaf of 30 day-old seedlings until runoff for Early blight disease. Appearance of

typical leaf spot symptoms was recorded up to 30 days after challenge inoculation.

For each experiment four pots per replication were maintained with eight replications

and the experiment was repeated twice.

6.2.7.3 Population dynamics of introduced PGPR’s on tomato roots

Seed treatment with formulations and seedlings were raised as explained

earlier. After transplantation of 25 day-old-seedlings from crate to pot. For root

colonization studies, seedlings were harvested 30 days after transplantation. Plants

were uprooted carefully without damaging to the root system, shaken thoroughly to


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remove loosely adhering soil, pooled, and then separated into different groups: upper

tap root, middle tap root and lower tap root, similarly, upper lateral roots, middle

lateral roots and lower lateral roots. Each group was divided into five replicates. One

gram of root sample was homogenized in 10 ml of saline and serially diluted up to

10-8 dilutions and spread plated on the selective medium. To analyze the population of

isolate 2apa, King’s B medium amended with 100 μg/ml rifampcin; for isolate

PSRIB2, modified Pikovskaya’s medium was used; for strain Pan-9/c, Nutrient agar

medium amended with 80 μg/ml rifampcin was used as mentioned in chapter IV.

Bacterial colonies with their defined characters on selective medium was counted and

expressed as cfu/g root tissue. For each set of experiment, three replicates of 30 pots

each was and arranged in randomized design. For each analysis, three plants were

maintained randomly and pooled together. The whole experiment was repeated twice.

6.2.8 Field trials

To analyze the efficacy of formulations of Pseudomonas aeruginosa 2apa and

Bacillus subtilis PSRIB2, field trials were conducted in farmer’s fields. Serratia

marcescens Pan-9/c was not considered for the field studies because, based on reports

stating that S. marcescens is an opportunistic human pathogen in nature.

6.2.8.1 Location and field histories

Eight field trials were conducted during June – October 2008 and 2009 at two

locations in Pandavapura, Mandya District and Lakshmipura, Mysore district,

Karnataka. The same season was selected during both the years because, in mansoon

season the disease spreads and is expressed better than other season. The properties of

the soils of the different location are given in the table 6.1. Selected fields in the both

the locations were continuously used to grow solanaceous crops such as, chilli, brinjal

and tomato, and paddy and sugarcane also grown as crop rotation. These plots were

monitored for two continuous years for the Fusarium wilt and Early blight incidence

and selected for the present study (Table 6.1 and 6.2).

6.2.8.2 Treatments and raising of seedlings

Seeds treated with formulations were sown in crates and grown for 30 days

under greenhouse conditions. Two days before transplantation seedlings were

drenched with suspension of formulation with tap water (25 ml/seedling). While


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transplanting, whole seedling along with coir pith was removed from crate and sown

into soil. Watering was done by channel irrigation for every 4 days. Application of

organic fertilizers (farm yard manure) and chemical pesticides were done as shown in

table 6.6. For every trial a farmer’s control was maintained where all agricultural

practices such as, raising of seedlings from chemical treated seeds, application of

chemical fertilizers and chemical fungicides, watering etc. was done according to

farmer’s practices.

6.2.8.3 Plot design and methods of disease assessment and yield analysis

All trials were randomized complete block designs with 40 subplots/acre

which were permanent till the end of the experiment. Each sub plot was of 3 x 3 m

which approximately contains 30 plants/subplot. The number of healthy plants and

diseased plants were recorded in a subplot up to 60 days after transplantation. Disease

incidence was calculated by observing the typical symptoms such as, yellowing of

lower leaves, drooping, development of adventitious roots on lower stem part,

complete wilting and discoloration of vascular tissue for Fusarium wilt and for Early

blight, leaf spots, concentric rings (circles) of necrotic lesions on leaf, stem and fruits

were counted. Yield analysis was also done at each picks and mean yield was

expressed as Kg/acre.

6.2.8.4 Population dynamics of introduced PGPR’s on tomato roots

Experiments were conducted under field conditions as explained above (under

greenhouse conditions) with minor modifications. Here seedlings were harvested at

30 days after transplantation. For each analysis three seedlings were harvested

randomly in the field excluding the subplot marked to analyze disease incidence.

Remaining analysis, such as grouping and determination of cfu/g root tissue were

done as explained earlier.

6.2.9 Soil, potting mixture and plant analysis

Total N was determined by the Kjeldahl method (Bremner and Mulvaney,

1982). Soil pH was measured in water (1:1 v/v), and available nutrients were

extracted and measured according to the Mechlich 3 method (Mehlich, 1984).

Organic matter was estimated by a modified Walkley and Black method (Mc Keague,

1978). Plant tissue was digested in a mixture of 15 ml of Perchloric acid (HClO4) and


251

5 ml of Nitric acid (HNO3), and phosphorus was determined spectrophotometrically

by the vando-molybdate method (Tandon et al., 1958). Other elements were

determined by atomic absorption spectrophotometry (Gaines and Mitchell, 1979). All

the above said analysis was conducted in Soil Analysis Lab, V.C. Farm, Gandhi

Krishi Vignayan Kendra (GKVK), University of Agriculutral Sciences, Mandya,

Karnataka.

6.2.10 Experimental designs and statistical analyses

Field plots or pots in greenhouse/growth chamber were designed/arranged at

randomized block design. Data from plant growth promotion yield and disease

incidence obtained from greenhouse and field conditions were analyzed for significant

differences by analysis of variance (ANOVA) with mean separation using the least

significant difference (LSD) (P≤0.05) by Fisher’s protected LSD test, employing the

statistical tool SPSS version 16.0.


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6.3. Results

6.3.1 Preparation of bioformulation and shelf life determination

Bioformulation of all three selected rhizobacterial isolates were prepared using

talc powder as carrier material (Fig. 6.2). Initial rhizobacterial population was 1x108

cfu/g formulation as determined by serial dilution method on defined medium.

Shelf life of all the selected PGPR was found to be decreasing along with the

storage period. The initial high population of rhizobacteria in the talc formulation was

not necessarily sustained during storage. Population of PSIRB2 decreased gradually

throughout the experimental period. After the storage of 120 days at room

temperature, population of PSIRB2 decreased to 0.25x107 cfu/g formulation. Whereas

in case of isolate 2apa and Pan-9/c the stability of population was maintained up to 60

days of storage, after that a drastic decline in the bacterial population was observed

and at the end of the experimental period the cfu was 0.9 x 105 and 1.7 x 104 /g

formulation for isolate Pan-9/c and 2apa respectively (Fig. 6.3).

6.3.2 Efficacy of bioformulations in increasing the plant growth and yield of

tomato under greenhouse conditions

Seed treatment with bioformulations of isolate 2apa and PSIRB2 significantly

(P≤0.05) increased the root, shoot length and vigor index of tomato seedlings when

compared to isolate Pan-9/c and untreated control (Table 6.3).

Similarly, under greenhouse conditions when 30-day-old seedlings were

analyzed, a significant increase in root length, shoot length, fresh weight and dry

weight was recorded with all the rhozobacterial bioformulations in comparison with

untreated control (Table 6.3). In 75 day-old-seedlings, increased plant height, fresh

weight was noticed with bioformulations treatments. Yield analysis revealed that,

PSIRB2 was most significant (P≤0.05) in the form of both fresh culture and

bioformulations followed by strain 2apa. In the case of Pan-9/c, mean fruit weight and

number fruits per plant was not significantly different from control (Table 6.4). But

under both laboratory and greenhouse conditions, fresh cultures were found more

effective in increasing the early and later plant growth, and also yield, than

bioformulations and control (Table 6.3 and 6.4).

The accumulation of nutrients in tomato seedlings was also studied (Table

6.5). Isolate PSRIB2 and 2apa in the form of both fresh culture and bioformulations


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significantly (P≤0.05) stimulated the accumulation of P, N and K in shoot dry matter

of tomato plants. But the accumulation of these nutrients in case of Pan-9/c treatment

was almost equal to that of control seedlings. In case of Ca and Mg a similar pattern

of accumulation was observed. Both fresh cultures and bioformulations performed

similarly in case of nutrient accumulation.

Colonization of the introduced rhizobacteria into rhizosphere was analyzed by

sampling the rhizosphere soil samples at 30 days after transplantation. Even though

the initial bacterial application was same in all the treatments, but at 30 days after

transplantation a varied level was observed between the three isolates studied.

Different selected root zones of tomato rhizosphere for the analysis of bacterial

dynamics was represented in the fig. 6.4. From the results it was clear that introduced

rhizobacterial strain preferentially colonize the lower tap root and lateral roots rather

than upper and middle tap roots. Bacterial population was usually found higher on

lateral roots than on tap roots in case of all rhizobacteria-root colonization studies

(Fig. 6.5 and 6.6).

The bioformulations of isolate 2apa significantly (P≤0.05) controlled fusarium

wilt and early blight disease (12% and 17%) over control (76%, Fusarium wilt and

80%, Early blight). Isolate Pan-9/c significantly (P≤0.05) reduced the Fusarium wilt

incidence up to 21%, but failed to reduce the Early blight. Formulation of isolate

PSIRB2 completely failed to offer protection against both the fungal disease studied

under greenhouse conditions. In all cases, performance of both fresh cultures and

bioformulations were not significantly different (P≤0.05) (Table 6.4).

------ - Chapter VI: Development of bioformulations and field studies

254

Figure 6.2 Bioformulations, a. Fresh culture, b. Talcum powder

formulation, c. Preparation of Talcum powder formulation under aseptic

conditions

0.000001

0.00001

0.0001

0.001

0.01

0 15

30

45

60

75

90

105

120

Days after storage

Lo

g c

fu/g

fo

rmu

lati

on

Serratia marcescens Pan-9/c

Pseudomonas aeruginosa 2apa

Bacillus subtilis PSIRB2

Figure 6.3 Survival of selected PGPR’s in talcum powder formulation at room

temperature


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Table 6.1. Some physical and chemical characters of soil from two locations and potting medium used in the present study

Location Soil type pH

%

Organic

matter

Available elements WHC

(%) N

(mg/g)

P

(μg/g)

K

(μg/g)

Mg

(μg/g)

Ca

(mg/g)

Potting mixture (Greenhouse)

Soil: FYM: Coir pith

(2:1:1) 7.0 6.04 2.48 132 329 487 7.1 69.7

Plot-I, Pandavapura (Mandya) Sandy Loam 7.2 3.63 1.84 78 212 368 6.7 61.4

Plot-II, Pandavapura (Mandya) Sandy Loam 7.15 3.48 1.85 75 220 345 6.7 62.5

Plot-I, Lakshmipura (Mysore) Sandy Loam 7.1 2.85 1.75 62 188 332 6.9 59.9

Plot-II, Lakshmipura (Mysore) Sandy Loam 7.1 2.80 1.82 65 187 340 6.9 60.0

FYM: Farm yard manure, N: Nitrogen, P: Phosphorus, K: Potassium, Mg: Magnesium, Ca: Calcium, WHC: Water holding capacity.

Table 6.2. Previous history of disease occurrence and crop rotation of selected fields

Location Crops commonly grown Fusarium wilt

(% incidence)

Early blight

(% incidence)

Yield

(Kg/acre)

Plot-I, Pandavapura (Mandya) Tomato, Chilli, Brinjal, Beans 11 34 16,200

Plot-II, Pandavapura (Mandya) Tomato, Sugarcane, Paddy 1 38 16.,240

Plot-I, Lakshmipura (Mysore) Toamto, Chilli, Brinjal, Paddy 9 28 16,550

Plot-II, Lakshmipura (Mysore) Tomato, Beans Sugarcane, Paddy 0 31 17,120


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Table 6.3. Effect of seed treatment with formulation on early plant growth of tomato under laboratory and greenhouse

conditions

Isolates Treatment

Under laboratory conditions Under greenhouse conditions

MRL

(cm) MSL

(cm) Germination

(%) VI

MRL

(cm) MSL

(cm) FW

(g/s) DW (g/s)

Control 6.6±0.11b 5.8±0.17b 76±2.15a 942±12.1b 13.6±0.34c 11.3±0.17b 0.425±0.04b 0.050±0.005b

Pan-9/c FC 7.8±0.40a 7.1±0.34a 75±2.33a 1117±38.6a 15.6±0.34ab 13.5±0.23a 0.540±0.07ab 0.067±0.004ab

FO 7.8±0.17a 8.1±0.23a 77±1.15a 1224±22.8a 15.6±0.17ab 13.7±0.28a 0.580±0.04ab 0.065±0.002ab

2apa FC 8.0±0.28a 7.1±0.17a 79±2.30a 1193±45.2a 14.0±0.34bc 13.0±0.28a 0.632±0.01a 0.073±0.001a

FO 8.0±0.23a 8.0±0.28a 78±3.46a 1248±23.5a 15.5±0.17ab 13.5±0.17a 0.601±0.04ab 0.065±0.001ab

PSIRB2 FC 8.2±0.11a 7.7±0.05a 78±1.73a 1240±39.4a 15.9±0.51a 13.6±0.11a 0.574±0.01ab 0.063±0.002ab

FO 8.1±0.05a 7.9±0.23a 78±3.46a 1248±13.8a 13.9±0.57bc 13.2±0.05a 0.479±0.08ab 0.053±0.007b

MRL: Mean root length, MSL: Mean shoot length, VI: Vigor index, FW: Fresh weight, DW: Dry weight, FC: fresh culture; FO: formulation. Values are the mean with in the column sharing the same letters are not significantly different according to Tukey’s HSD at P≤0.05.


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Table 6.4. Effect of seed treatment with formulations of different selected PGPR on plant growth, yield and Fusarium wilt and

early blight incidence under greenhouse conditions

Isolates Treatment

Plant

height

(cm)

Fresh

weight

(g/plant)

Total

chlorophyll

(mg/g tissue)

Number of fruits/

Plant

Mean fruit

weight

(g)

Fusarium wilt

incidence

(%)

Early blight

incidence

(%)

Control 119.5±6.70b 199.5±5.48b 16.5±0.11a 27±0.57b 36±1.57c 76±2.30a 80±1.73a

Pan-9/c FC 129.0±3.45a 229.0±5.19a 16.5±0.28a 32±1.15a 38±1.15ab 19±1.15b 71±0.57a

FO 127.0±4.00a 223.7±2.13a 16.7±0.057a 30±3.46ab 37±3.46bc 21±2.02b 74±4.04a

2apa FC 129.0±3.46a 230.6±3.23a 16.5±0.17a 31±2.30a 39±2.30ab 14±0.00b 19±1.15b

FO 129.0±5.71a 230.0±5.36a 16.5±0.08a 32±1.15a 40±1.73a 12±1.15b 17±1.73b

PSIRB2 FC 131.0±2.60a 240.3±4.53a 16.7±0.40a 34±2.30a 41±3.46a 74±3.46a 81±3.46a

FO 131.5±2.96a 239.3±3.06a 16.7±0.17a 34±1.73a 40±2.45a 72±4.04a 80±3.46a

P: Phosphorus, Rhizobacteria was applied as seed treatment and an additional soil drench treatment was give just before transplantation. To analyze plant height, fresh weight, total chlorophyll and total P content 75 day-old-seedlings were used. FC: fresh cultures, FO: formulation, Number of fruits Plant

-1 and mean fruit weight was analyzed separately for each treatment.


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Table 6.5. Effect of seed treatment with formulations of different selected PGPR on

nutrient accumulation in tomato plants under greenhouse conditions

Isolates Treatment Dry matter (Mg/g)

N P K Ca Mg

Control 12.4±0.23c 1.9±0.23b 20.1±1.15b 20.4±1.50a 4.2±0.11b

Pan-9/c FC 14.7±0.40bc 2.0±0.11b 19.4±0.23b 21.0±0.57a 4.3±0.05b

FO 14.5±0.28bc 1.9±0.08b 20.2±0.69b 21.1±1.50a 4.2±0.20b

2apa FC 21.1±2.07a 2.0±0.17b 23.8±0.98ab 24.5±2.59a 4.8±0.18ab

FO 18.0±1.50ab 2.0±0.11b 24.0±0.98ab 22.8±2.25a 4.6±0.14ab

PSIRB2 FC 21.7±0.98a 2.4±0.17a 29.0±1.15a 27.8±1.61a 5.2±0.11a

FO 20.0±1.15a 2.3±0.17a 29.0±2.03a 26.9±1.97a 5.1±0.13a

P: Phosphorus, Rhizobacteria was applied as seed treatment and an additional soil drench treatment was give just before transplantation. To analyze plant height, fresh weight, total chlorophyll and total P content 75 day-old-seedlings were used. FC: fresh cultures, FO: formulation, Number of fruits Plant

-1 and mean fruit weight was analyzed separately for each treatment.

Figure 6.4 Different root parts to analyze the dynamics of rhizobacterial population.


259

bb

a

bcbc

ab

c

b

a

0

1

2

3

4

5

6

7

8

9

10

Upper roots Middle roots Lower roots

Lo

g C

FU

/g r

oo

t ti

ssu

e



Bacillus subtilis PSIRB2

Figure 6.5 Colonization of tomato tap root by different selected PGPR’s 30 days after

transplanting under greenhouse conditions. Different letters on the bars indicated significant difference among different locations of roots at P≤0.05. Vertical bars indicate standard error of means of three experiments.

cb

a

c c

a

c

ab

a

0

2

4

6

8

10

12


Lo

g C

FU

/g r

oo

t ti

ssu

e



Bacillus subtilis PSRB2

Figure 6.6 Colonization of tomato lateral root by different selected PGPR’s strains 30

days after transplanting under greenhouse conditions. Different letters on the bars indicated significant difference among different locations of roots at P≤0.05. Vertical bars indicate standard error of means of three experiments.


260

Table 6.6. Experimental design for field studies two consecutive years 2008 and 2009

Location Treatment Bioformulations@ Organic

fertilizers#

Chermical pesticides$

Fungicide Bactericide Insecticide

Plot-I,

Pandavapura (Mandya)

Farmers practice - + + + + Control - + - - +

2apa + + - - + PSIRB2 + + + + +

Plot-II,

Pandavapura (Mandya)

Farmers practice - + + + +

Control - + - - + 2apa + + - - +

PSIRB2 + + + + +

Plot-I, Lakshmipura

(Mysore)

Farmers practice - + + + +

Control - + - - + 2apa + + - - +

PSIRB2 + + + + +

Plot-II, Lakshmipura

(Mysore)

Farmers practice - + + + + Control - + - - +

2apa + + - - + PSIRB2 + + + + +

*All other agricultural practices followed were common for all treatments, in all locations in both the years. @

Bioformulation treatment: seed treatment followed by soil drench to crate 2 days before transplantation. # Organic fertilizers: Farmyard manure was applied once during the time of field preparation.

$Chemical pesticides: Application of chemical pesticides were only by foliar spray, root treatment or soil drench was avoided.

Harvesting: Ripe fruits were picked at regular intervals, total yield was calculated as Kg/acre.


261

6.3.3 Efficacy of bioformulations in suppressing the fungal disease and increasing the

yield of tomato under field conditions

Field experimental design was as shown in the Table 6.6 (Fig. 6.9). Field studies were

conducted in two locations (Lakshmipura, Mysore and Pandavapura, Mandya), and

experimental plots were selected based on their previous history of cropping pattern and

disease incidence (Table 6.2). For the field experiments only two isolates (PSIRB2 and 2apa)

were selected.

In 2008, Fusarium wilt and Early blight incidence was significantly reduced by

bioformulations treatment (2apa). In plot I (Mandya) regular farmars practice showed an

incidence of 7% Fusarium wilt incidence which is reduced to 3% with isolate 2apa

bioformulations. Similarly in case of Early blight the disease incidence was significantly

reduced from 30 (Farmers practice) to 22 (2apa bioformulations treatment). In plot I

(Mysore) a similar kind of reduction of Fusarium wilt and Early blight disease was recorded

with 2apa bioformulations. A similar kind of observation was made in the year 2009. Where

2apa bioformulations significantly reduced the Fusarium wilt and Early blight incidence in

both the location studied. In both the years increased plant growth promotion and reduced

disease incidence resulted in increased yield was recorded over farmers practice in all four

plots studied, in both two years of field experiment (Table 6.7). Isolate PSIRB2 as

characterized earlier failed to protect the tomato plants from root and foliar fungal disease

such as Fusarium wilt and Early blight. This may affects it nutrient uptake ability finally on

yield. Hence even though the isolate PSIRB2, under greenhouse conditions showed a

significant increase in fruit yield. But under greenhouse conditions yield was lesser than the

strain 2apa bioformulations treatment.

In both the seasons in all the plots studied, control plots, which contains seedlings

raised from untreated seeds and that which did not receive any chemical fertilizer or chemical

fungicide application showed a significantly reduction in fruit yield. Whereas as fruit yield

from farmers practice was significantly (P≤0.05) lesser that the bioformulations treatments

(Table 6.7).


262

6.3.4 Population dynamics of PGPR’s at rhizosphere under field conditions

When the rhizosphere of tomato plants 30 days after transplantation was analyzed for

the colonization of introduced rhizobacterial isolates, we observed the colonization of

introduced isolates with varied level. When colonization pattern was observed both isolates

prefers the young growing regions of tap root as well as lateral roots. Comparatively a less

population of the rhizobacterial isolate was observed in the upper portion of the tap root, and

lateral roots had the higher population of the rhizobacterial in comparison with tap root (Fig.

6.11 and 6.12). In Location Pandavapura (Mandya), PSIRB2 found at its higher level of

9.95x1010 cfu/g root was recorded in lower later roots followed by 9.7x1010 cfu/g root by

isolate 2apa at lower lateral roots. Where as least bacterial population was recorded at the

upper taproot, 3.2x1010 and 4.6x1010 cfu/g root of PSIRB2 and 2apa respectively. Similarly

in location Lakshmipura (Mysore), a highest of PSIB2 population was found at its higher

level of 9.56x1010 cfu/g root on lower later roots followed by 9.3x1010 cfu/g root on middle

lateral roots. Where as least bacterial population was recorded at the upper taproot, 3.74x1010

and 4.3x1010 cfu/g root of PSIRB2 and 2apa respectively.


263

Figure 6.7. Tomato plants severely infected with Fusarium wilt disease caused by

Fusarium oxysporum (Plot I, Pandavapura, Mandya)

Figure 6.8 Tomato plants severely infected with Early blight disease caused by

Alternaria solani (Plot II, Lakshmipura, Mysore)


264

Figure 6.9 a and c. Tomato seedlings Cv. Suruchi F1 hybrid raised from the seeds treated with bioformulations, b. and d.

transplantation of tomato seedlings at Pandavapura (Mandya) at Lakshmipura (Mysore)


265

Table 6.7 Efficacy of bioformulation against Fusarium wilt and Early blight incidence and fruit yield of tomato under field

conditions

Location Treatments

Year 2008 Year 2009

Fusarium wilt

(% incidence)

Early blight

(% incidence)

Yield

(Kg/acre)

Fusarium wilt

(% incidence)

Early blight

(% incidence)

Yield

(Kg/acre)

Plot-I,

Pandavapura

(Mandya)

Farmer practice 7± 0.57a 30±2.30a 16,900±2651b 11±0.57a 32±1.15ab 17,100±2690a

Bioformulation 2apa 3± 0.55b 22±1.15a 17,850±2950a 03±1.15bc 23±1.73bcd 18,000±2990a

Bioformulation PSIRB2 9±0.57a 30±2.88a 17,570±2851a 10±1.44a 29±2.09abcd 18,000±3000a

Plot-II,

Pandavapura

(Mandya)

Farmer practice 2±0.00b 30±1.15a 16,490±2509b 03±0.86bc 31±0.57abc 16,.640±2540a

Bioformulation 2apa 1±0.57b 24±1.17a 17,400±1254a 01±0.57bc 21±0.57d 17,690±2900a

Bioformulation PSIRB2 3±0.00b 28±1.17a 17,000±1985a 04±0.57b 30±2.88abcd 17,540±2710a

Plot-I,

Lakshmipura

(Mysore)

Farmer practice 8±1.73a 35±0.577a 17,500±3240a 09±0.63a 38±1.15a 17,375±2800a

Bioformulation 2apa 3±0.57b 23±2.30a 17,720±2389a 02±0.00bc 24±2.03bcd 17,920±3050a

Bioformulation PSIRB2 8±1.73a 33±0.577a 17,950±2559a 09±1.15a 30±1.73abcd 18,000±2015a

Plot-II,

Lakshmipura

(Mysore)

Farmer practice 0±0.00b 27±1.15a 16,800±3521b 00±0.00c 32±1.73ab 17,100±3280a

Bioformulation 2apa 0±0.00b 16±2.88a 17,400±2661a 00±0.00c 22±2.30cd 18,010±2178a

Bioformulation PSIRB2 0±0.00b 34±1.73a 17,910±2801a 00±0.0c 29±2.30abcd 18,270±2587a


266

Figure 6.10 Field experiments

a. Healthy tomato plants raised from bioformulations (isolate 2apa) treatment at Mandya, b. A women worker picking the

tomato fruits from experimental field, c. Farmer grading the fruits and analyzing the yield, d. Plants showing early blight

symptoms, e. tomato plant severely infected with early blight disease, f. Tomato plant showing the typical wilt symptoms and

g. Local farmer showing discoloration of vascular region of wilted plant.

-------------------------- Chapter VI: Development of bioformulations and field studies

267

e

d

c

b

cd

bc

a

a

bc

b

aa

0

2

4

6

8

10

12

2apa PSIRB2 2apa PSIRB2

Tap roots Lateral roots

Lo

g C

FU

/g r

oo

t ti

ssu

e


Figure 6.11 Colonization of tomato tap and lateral roots by

different selected PGPR’s 30 days after transplanting under field

conditions in the location Pandavapura (Mandya). Data

represented here are the average of two seasons. Different letters

on the bars indicated significant difference among different

locations of roots at P≤0.05. Vertical bars indicate standard error

of means of two experiments.

d

c

b

a

cd

c

a a

bb

a a

0

2

4

6

8

10

12

2apa PSIRB2 2apa PSIRB2

Tap roots Lateral roots

Lo

g C

FU

/g r

oo

t ti

ssu

e


Figure 6.12 Colonization of tomato tap and lateral roots by

different selected PGPR’s 30 days after transplanting under field

conditions in the location Lakshmipura (Mysore). Data

represented here are the average of two seasons. Different letters

on the bars indicated significant difference among different

locations of roots at P≤0.05. Vertical bars indicate standard error

of means of two experiments.


268

6.4 Discussion

Several attempts have been made previously to prepare formulations of

rhizobacteria (Hagedorn et al., 1993) and also talc based formulation of specific strain

of fluorescent pseudomonas (Hofte et al., 1991). Kloepper and Schroth (1981)

demonstrated the potentiality of talc to be used as a carrier for formulating

rhizobacteria. The fluorescent Pseudomonads did not decline in talc mixture with 20%

xanthum gum after storage for two months at 4°C. According to the report by Mathre

et al. (1999), Bacillus spp. may be the only genus of bacteria that meets the shelf life

standard required by a commercial microbial product. In addition to long term

viability, strain Bacillus spp. have become commercially successful due to their

ability to effectively colonize plant roots, produce antifungal compounds, and secrete

volatile substances that can directly stimulate plant growth (Mc Spadden and Fravel,

2002). Being Gram negative bacteria, isolate 2apa and Pan-9/c were unable to survive

in talc powder for longer period. But according to studies of Vidyasekaran and

Muthamilan (1995), P. fluorescens isolate Pf1 survived up to 240 days in storage. The

initial population of Pf1 in talc-based formulation was 37.5 x 107cfu/g and declined to

1.3 x 107cfu/g after 8 months of storage.

In the present study, we attempted to develop effective bioformulations by

using well characterized rhizobacterial isolates which were previously known to

increase the plant growth and suppress the plant disease to various extents (from the

studies of chapter III, VI and V). All the three isolates were used to develop

bioformulations using talc powder as carrier material. When bioformulations were

analyzed for their shelf life period, all isolates were found stable up to 60 days after

storage and after a drastic decline in the population of isolate 2apa and Pan-9/c was

observed and being a gram +ve bacteria maintained higher population of up to the end

of storage period (120 days).

The talcum powder developed was evaluated for their plant growth promoting

and disease protecting ability under greenhouse conditions. Among the isolates

selected PSIRB2 and 2apa were found to promoting the plant growth well under

laboratory and greenhouse conditions over control. The isolate PSIRB2 was know to

produce IAA and also solubilize the phosphate by producing organic acid and enzyme

phytase (Hariprasad et al., 2009) and isolate 2apa also characterized as a producer of

IAA and siderophore (Chapter IV). These unique characters make these isolates to


269

promote early growth of tomato plants. Indole acetic acid and phosphate solubilizing

rhizobacteria were frequently being reported as plant growth rhizobacteria (Fuentes-

Ramirez et al., 1993; Leinhos and Nacek, 1994; Patten and Glick 2002). Further when

formulations of these isolates were evaluated for their disease protecting ability,

isolate PSIRB2 completely failed to protect tomato seedlings from Fusarium wilt and

Early blight disease. Where as isolate Pan-9/c offered significant protection against

fusarium wilt and but not against early blight disease. Isolate 2apa significantly

reduced the incidence of both root and foliar fungal pathogen of tomato studied. As

previously studied, isolate PSIRB2 did not have any mechanism to inhibit fungal

growth and was ISR negative (Hariprasad et al., 2009, Chpter IV). Isolate Pan-9/c

was characterized as chitinase positive rhizobacteria which produce a significant

quantity of chitinase which is sufficient to inhibit the fungal growth at rhizosphere

(Chapter IV). Where as isolate 2apa is known to produce an antibiotics with wide

range of antimicrobial properties – Phenazine, along with the strain can induce

systemic resistance against foliar pathogen. Hence the disease protection revealed that

these mechanisms of the selected rhizobacterial strain are playing an important role in

determining their disease suppressing ability under greenhouse conditions.

The bottom line for biocontrol is whether it works under production conditions

or not. Biocontrol agents are being tested more often in the production system for

which they are intended, rather than relying solely on experiments done in vitro, on

detached leaves, on plantlets, or in the greenhouse on non-greenhouse crops.

They are often being tested in multiple locations and seasons and tested in

locations naturally infested with pathogens. In a field naturally infested with

Verticillium dahliae and Phytophthora cactorum, the chitinolytic bacterium Serratia

plymuthica suppressed disease caused by both pathogens and increased strawberry

yield by 60% compared with the nontreated control (Kurze et al., 2001). Trichoderma

harzianum and the mycorrhizal fungus Glomus intradices were tested in a field

naturally infested with the tomato root and crown rot pathogen Fusarium oxysporum

f. sp. radicis-lycopersici (Datnoff et al., 1995). Similarly in our studies too we

selected farmers field which were naturally infested with fusarium wilt and early

blight at two locations (Figs. 6.7 and 6.8; Tables. 6.1 and 6.2). Based on the results

obtained under greenhouse conditions we designed our experiment (Table 6.6) which

was based on the characters of the selected rhizobacterial strain. For Pseudomonas


270

aeruginosa 2apa treatment and additional application of chemical fertilizer was done

as this isolate was found negative for P solubilization and N fixation. Similarly, for

Bacillus subtilis PSRIB2 treatment an additional application chemical pesticides was

done, as this isolate was characterized as not offering any protection to plant disease.

Hence here we tried to improve the performance of these isolates under field

conditions by amending the chemical fertilizer or pesticide application.

For field studies, Serratia marcescens Pan-9/c was not considered because,

according the previous literature survey these isolates were some times acts as

opportunistic human pathogen (Sehdev, 1999; Bennett, 2000). Results from field

studies revealed that the isolate 2apa significantly increased the fruit yield and also

suppressed the root and foliar disease (Fusarium wilt and Early blight) of tomato.

Isolate PSRIB2 also showed an increased yield over farmers practice. Hence, from the

results we can conclude that these PGPR strains can used as talc formulation under

field conditions as seed treatment along with the chemical fertilizers and pesticides. In

our study we noted that, throughout the experimental period the chemical pesticides

were not in direct contact with the PGPR studied.

Similar studies were previously conducted by several researchers with talc

formulation of PGPR such as, Talc based formulation of Pseudomonas fluorescens

was found to be effective as seed treatment and foliar applications in the control of

rust and leaf spot of groundnut (Meena et al., 2002). Radjacommare et al. (2002)

tested the efficacy of Pseudomonas fluorescens (Pf1) and Pseudomonas fluorescens

(FP7) talc-based formulation with and without chitin amendment in rice plants against

sheath blight infection. The application of Pf1+chitin and FP7+chitin recorded low

PDI of 22 and 23, respectively, compared to untreated control (75 PDI) in field

conditions.

Our findings are in accordance with that of Jayaraj et al. (2007) who attempted

an integrated approach of damping-off management employing dual inoculation of

PfT-8 in seed and soil coupled with soil application of organic amendments including

poultry manure or FYM under field conditions. They reported a significantly reduced

damping-off incidence up to 90% and further significantly increased healthy plant

stand, plant biomass and plant rhizosphere population of Pseudomonas fluorescens in

tomato.


271

Although reports are available for combined application of biocontrol agents

(consortia) could improve the ability of formulation against plant disease. But, in the

present study we tried to co-cultivate these two isolates (2apa and PSIRB2),

Pseudomonas aeruginosa isolate 2apa suppressed the growth of isolate PSRIB2 by

producing phenazine (Data not shown). Hence, the talc formulations of b iocontrol

agents 2apa either alone or in combination with organic fertilizers can be

recommended to the farmers as one of the crop protection strategies for the

management of fungal disease in tomato and its practice may also be extended to

other solanaceous crops. Further we are aiming our work to use organic manures

instead of chemical fertilizers along with PGPRs.

chapter vi development of bioformulations and field...

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