2 ISOLATION, PHENOTYPIC CHARACTERISATION AND SCREENING OF WHEAT INHABITING BACTERIA FOR THEIR PLANT GROWTH PROMOTING EFFECT

2.1 Abstract

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The aim of this study was the isolation of bacteria, which promotes the growth of different plant cultures and controls plant diseases caused by Fusarium species. A large number of bacteria were isolated from root, rhizosphere and phyllosphere of wheat grown in Syrdarya. Determination of phenotypic traits was used to evaluate the presence of the bacterial isolates with similar phenotypic characteristics. Three hundred sixteen strains were discarded due to their similar phenotypic characteristics. In addition, 18 strains isolated in previous studies were screened for their PGPB effect. Based on the plant-inoculation experiments performed on plates, 74 and 154 strains were discarded as potential pathogens and shoot and/or root growth inhibitors, respectively. Of the remaining strains, 111 appeared to have a positive plant growth effect on wheat. These PGPBs were also tested for their ability to inhibit pathogen Fusarium growth selecting 24 isolates. Conventional identification methods identified a number of new plant growth promoting strains as Bacillus sp., Pseudomonas. sp., Azotobacter strains and Micrococcus. Laboratory experiments conducted on wheat under gnotobiotic conditions demonstrated increases in root elongation (up to 50%), root dry weight (up to 31%), shoot elongation (up to 47%) and shoot dry weight (up to 48%) of inoculated wheat seedlings. Based on growth-promoting activity, four isolates were selected and designated as plant growth promoting bacteria. Sand-based seed inoculation with selected PGPB isolates exhibited stimulatory effects on the growth of vegetables, namely cauliflower, cucumber, paprika and tomato with varied response with different plant and PGPB strains, resulting in two universal plant growth promoting bacterial strains, Bacillus licheniformis BL43 and Xanthomonas sp. Xs148.

Key words

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PGPB screening - cauiliflower – cucumber – paprika – tomato – wheat

2.2 Introduction

The microbe-plant interaction in the root, the rhizosphere or phyllosphere can be beneficial, neutral, variable, or deleterious for plant growth. Rhizobacteria that exert beneficial effects on plant development are termed plant growth-promoting rhizobacteria (PGPR) (Kloepper and Schroth 1978, Kloepper et al. 1986). These bacteria significantly influence plant growth by increasing nutrient uptake, suppressing pathogens and may be used in agriculture to minimize the utilization of chemical pesticides and fertilizers (Hartmann and Bashan 2009, Díaz-Zorita and Fernández Canigia 2009). Bacteria species including Pseudomonas, Azospirillum, Azotobacter, Bacillus, Klebsiella, Enterobacter, Xanthomonas and Serratia have been shown to promote plant growth. During the last couple of decades, the use of PGPB for sustainable agriculture has increased. Significant increases in growth and yield of agronomically important crops in response to inoculation with PGPB has been reported (Ruppel 1987, Díaz-Zorita and Fernández Canigia 2009). Biological N2 fixation provides a major source of nitrogen for plants as a part of environmentally friendly agricultural practices. Apart from fixing N2, PGPB can affect plant growth directly improving nutrient uptake, by the synthesis of phytohormones and vitamins, inhibiting plant ethylene synthesis, enhancing stress resistance, solubilising inorganic phosphate, and mineralising organic phosphate (Dobbelaere et al. 2003, Lucy et al. 2004). Plant growth benefits due to the addition of PGPB include increases in germination rate, root growth, yield, leaf area, chlorophyll content, nitrogen content, protein content, tolerance to drought, shoot and root weight, and delayed leaf senescence (Dobbelaere et al. 2003). Siderophore-producing bacteria promote plant growth by an iron uptake resulting in the limited iron in the rhizosphere, especially in neutral and alkaline soils, and thereby reduce its availability for the growth of pathogen (Winkelmann 2002).

Studies have also shown that the growth-promoting ability of some bacteria may be highly specific to certain plant species, cultivars and genotypes (García de Salomone and Döbereiner 1996, Dobbelaere and Okon 2003, Sala et al. 2007, Behl et al. 2007).

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This study focuses on the screening of effective PGPB strains on the basis of their potential for plant growth promoting activity and to study the early phases of bacterial inoculation effect using in vitro methods.

2.3 Materials and methods

2.3.1 Bacteria isolation from root, rhizosphere and phyllosphere of wheat

2.3.1.1 Soil and Plant

Wheat (Triticum aestivum cv. Ziklon) was grown on soil sampled from Syrdarya province, Uzbekistan. Syrdarya soil chemical properties also determined as described in Egamberdieva et al. (2002): soil samples (0-30 cm depth) were taken with a soil probe (3.5 cm diameter). Soil samples were pooled, and sieved (<2 mm mesh) directly after collection. Air-dried samples were analyzed for contents of total C, N, P, and K. Soil chemical analysis was as follows (per 100 g d.w.): 100 mg C; 0.6 mg N; 3.0 mg P; 12 mg K; pH was 7.8. Total C was identified by elementary analysis while total N was determined by Kjeldahl method. The molybdenum blue method was used to determine total P. Potassium was determined using the flame photometric method (Riehm 1985). Soil pH value was measured in H2O (water: soil solution ratio 1:2.0) with a potentiometric glass electrode (measuring range of 0-14 pH with resolution and accuracy of 0.1 pH). The soil water content after planting was approximately 12% water holding capacity and was kept nearly constant throughout the experiment. All soil was sieved (mesh width 3 mm; mesh length 6 mm) prior to use. The soil was placed in 350 ml pots to a bulk density of 1.0 g/cm3.

Wheat seeds were obtained from the University of Agriculture of Uzbekistan, Tashkent.

2.3.1.2 Collecting samples

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Five plants were harvested. To collect samples, 21 days after sowing plant in soils (Syrdarya, Uzbekistan), and the plants were removed from the soil. Subsequently, the plant was shaken carefully and soil still tightly adhering to the roots was defined as rhizosphere soil. Roots were washed in running tap water to remove adhering soil, cut into 1 cm pieces and surface sterilised in 0.7% NaOCl solution for 30 minutes. To isolate phylosphere bacteria, 1 g of youngest part of the leave and stem of seedlings were cut. All samples were kept separated in Erlenmeyer flasks.

2.3.1.3 Bacteria isolation

To isolate the root or phylosphere microorganisms samples were placed in Erlenmeyer flasks containing 95 ml of 0.1% sterile sodium pyrophosphate solution and 10 g grit and shaken on a rotary shaker at 200 rpm for 20 min. Tenfold-serial dilutions of the suspensions were made with 0.1% sodium pyrophosphate and plated in triplicate on glycerin peptone agar for total bacterial counts. To count the total number of bacteria, 100 µl of resulting suspensions were sprayed over the surface of glycerin peptone agar (GPA). The plates contained cyclohaximide in a concentration of 100 mg l-1 to inhibit fungal growth. The total number of bacteria was established after 7 days of incubation. The bacterial isolates grown in “master plate” were transferred to fresh Petri dishes containing the same medium. This process was repeated 3 times to purify diazotrophic bacteria isolates and they were then stored in tubes containing GPA medium for further examination. Determination of diazotrophic bacteria was performed using nitrogen free medium Ashby agar. Ten serial dilutions of bacterial isolates were sprayed on the Ashby agar. Survived isolates were then stored in tubes containing Ashby agar medium and regarded as diazotrophic bacterial isolates.

2.3.1.4 Morphological characterization and identification of bacteria

The identification of strains relied on standard biochemical and physiological tests according to the classification of Bergey (Holt and Krieg 1984). Cultures were grown on nutrient broth and nutrient agar for morphological characterization, i.e. gram staining, study colonial forms, motility. Gram reaction of each culture was determined by the rapid KOH test (Ryu 1938). Colony morphologies were examined after 24 h, 48 h, and 72 h growth on glycerin peptone agar at 28oC. Cell morphologies were examined with phase contrast microscopy or after staining with methylene blue. The activity of catalase was tested by suspending a loopful of cells in a 10% (vol/vol) H2O2 solution. Formation of a fluorescent pigment was observed on King B medium. The oxidation and fermentation of glucose was performed according to the method of Hugh and Leifson (1953).

2.3.2 Screening of bacterial isolates for their effect on wheat growth

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Wheat (Triticum aestivum cv. Ziklon) seeds were obtained from of Tashkent Agriculture University, Uzbekistan. In addition to the isolated strains, we used diazotrophic isolate BL43 (identified as Bacillus licheniformis in our study). These strains were obtained from Microorganism collection Institute of Microbiology, Uzbekistan Academy of Sciences.

2.3.2.1 Bacteria suspension preparation

Pure cultures of the diazotrophic bacterial isolates were grown in Ashby broths on a rotary shaker (150 rpm) at 28°C for 72h. The pH of medium was adjusted to 7.0 before autoclaving. The bacterial suspensions were centrifuged at 7.000 rpm for 10 min. Growth medium was discarded and the bacterial pellet was resuspended in 0.05M NaCl buffer. Cell densities of bacterial suspensions used for seed inoculation were counted by dilution plating and CFU counts. The bacterial cell densities in the inoculant material were 107-108.

2.3.2.2 Screening in Petri dishes

Petri dish and pot experiments were conducted on wheat to screen the diazotrophic isolates for their effect on wheat root and shoot growth, respectively. The first screening was performed in Petri dishes. Wheat seeds were surface sterilized by momentarily exposing to 95% ethanol and immersing in 0.2% HgCl2 solution for 3 min. The seeds were then subjected to six washings with sterile distilled water. Thoroughly washed seeds of wheat were sown on sterilized filter paper sheets placed in Petri plates. Six seeds were sown in each Petri dish with four replicates. Two ml of bacterial suspension were applied on seeds present in each dish with the sterilized pipette. The control group seedlings were immersed in sterile 0.05M NaCl. Sterilized distilled water (10 ml) was added to each Petri dish to wet the filter paper sheets and the seeds were covered with another sterilized filter paper sheet. The dishes were incubated in a growth room at 24°C. After 2 weeks, the wheat seedlings were examined for shoot, root growth (length, fresh weight). The isolates which have shown significant plant growth promoting effect were selected for the further screening step.

2.3.2.3 Screening in pot experiments

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Based on the performance of rhizobacteria in the Plate experiments, nine effective plant growth promoting isolates (WR101, WR2, WR9, WR22, BL43, WPh45, WPh138, WR109, Xs148) were selected and used in pot trials. For pot experiment, a loamy soil sample with pH 7.7 was collected, air-dried, sieved (2-mm) before filling the pots. Inoculation suspension preparation and seed inoculation were performed as described for Petri experiments. Four inoculated and non-inoculated seeds of wheat were sown in pots (400 g soil per pot). Plants were supplied with half-strength Hoagland solution (Hoagland and Arnon 1950) receiving nutrient inputs of potassium/nitrogen/phosphorus in a ratio of 0.4:1:0.6. Two seedlings were maintained in each pot after germination. The pots were arranged in complete randomized design with six repeats. Plants were grown under greenhouse conditions with a temperature of 26°C to 28°C during the day and 16°C to 18°C at night. Four weeks after germination, plants were sampled to assess effects of inoculation. Six replications were harvested, roots and shoots were separated and soil particles were carefully removed from the roots under a gentle stream of tap water and were used to measure plant fresh mass, total root length and plant dry mass.

2.3.3 Antagonistic activity of bacteria isolated from wheat root, rhizosphere against pathogenic Fusarium isolates

2.3.3.1 Bacterial and fungal isolates.

Bacterial isolates originated from wheat root and rhizosphere, altogether 111 isolates, which have shown the stimulatory effect on wheat growth in plate experiments, were included in screening for their antagonistic activity.

In this study, Fusarium culmorum, Fusarium solani, and  F. avenaceum were used as pathogenic Fusarium isolates. Monoconidial cultures of these isolates were stored in sterile soil tubes at 4°C. Active cultures were obtained from small aliquots of a soil culture plated on potato-dextrose agar (PDA). Fungal cultures were incubated at 25°C.

2.3.3.2 Selection of bacteria for ability to inhibit in vitro growth of Fusarium culmorum.

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A total of 111 bacterial isolates were assayed in dual cultures on PDA for their ability to inhibit in vitro growth of F. culmorum. All bacteria-fungi combinations were examined on 15 ml of PDA in 9 cm Petri dish with 3 replications. A bacterial isolate per plate were spotted 1 cm from an edge of the plate, and was first incubated in the dark at 28°C. After 48 h, a 6-mm plug from the leading edge of a 5-day-old culture of F. culmorum on PDA was placed in 1 cm from the opposite edge of the plate. For control, PDA agar was inoculated with pathogen alone. Plates were incubated at 28°C. After 5 days, the length of hyphal growth toward the bacteria (Tinoc) and that on a control plate (Tcontrol) were measured. Inhibition of fungal growth was recorded as the relative growth ratio R = Tinoc/Tcontrol (Hamdam et al. 1991). There were 3 replicated plates in a completely randomized design for each bacterium–fungal isolate combination.

2.3.3.3 Specificity of bacterial antagonistic activity against Fusarium isolates.

Bacterial isolates selected from dual-culture assay with F.culmorum, were used to determine the degree of their antagonistic specificity against Fusarium solani and  F. avenaceum. Fungal growth inhibition was assayed in a dual culture experiment on PDA as described previously.

2.3.4 The influence of beneficial bacteria isolated from wheat rhizosphere on growth promotion of some vegetable plants

2.3.4.1 Plants and bacterial strains

The experiments were carried out using quartz sand. Cauliflower (Brassica oleracea L. cv. Fremont), cucumber (Cucumis sativus L. cv. Corona F1), paprika (Capsicum annuum L. cv. Rosita F1), and tomato (Lycopersicon esculentum [Mill] L. cv. Counter F1) were used as test plants for the inoculation experiments. Seeds of these plants were obtained from the Institute of Vegetable and Ornamental crops, Grossbeeren, Germany. 

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Bacterial strains Bacillus licheniformis BL43, Xanthomonas sp. Xs148 and Bacillus sp. WR2, Bacillus sp. WR9, and Bacillus sp. WR22 were tested for their effect on plant growth of vegetable plants listed above.

2.3.4.2 Pot experiment setup

Based on the performance of bacterial isolates in the pot experiments with wheat, five effective PGPB isolates (WR2, WR9, WR22, BL43 and Xs148) were selected and used to test the effect of PGPB isolated from wheat on vegetable plants. Surface-sterilized seeds were sown on trays for 10 days. Uniformly germinated seeds were selected for transplantation to the pots containing sand to eliminate the variation in growth contributed by different endogenous germination rate/potential of the seeds. For pot experiments, plastic containers were filled with 800 g sand and half-strength Hoagland solution (Hoagland and Arnon 1950) was applied to provide nutrition to the plants.

Plant seedlings were divided into six treatment groups. Seedling roots were inoculated with respective bacteria inoculation suspension for 2 minutes. Control seedlings received 1 ml 0.05M NaCl solution. The inoculation treatments were set-up in a randomized design with six replicates. The seedlings were re-inoculated by applying 1ml bacterial suspension the plants rhizosphere soil 2 days after transplantation. Two seedlings were planted per pot and after germination; plants were thinned to one per pot. The pots were incubated in the growth chambers. The pots were placed on plates and, thus, nutrient loss through leaching was prevented. A climate chamber conditions were set as given Tab. 2, and a relative humidity of 70% day/80% night. Light intensity provided by lamps (Agro Son T 400, Phillips, Hamburg, Germany) was between 450 and 600 μmol·m-2·s-1 at different positions in the chamber. Pots were re-arranged in regular intervals. Pots were always arranged in a completely randomised design.

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Tab. 2: Physical conditions of the pot experiment in growth chambers.

Light intensity in 24 hour

Temperature C°

2 h

8h

2h

12h

Day

Night

Cauliflower

200

400

200

-

20

18

Cucumber

400

600

400

-

25

20

Paprika

400

600

400

-

25

20

Tomato

300

500

300

-

23

18

2.3.4.3 Preparation of inoculation material

The bacteria were grown in glycerol-peptone-medium. Tubes were secured on a rotary shaker (120 rpm; 28°C) and agitated for 48 h. Tubes were centrifuged for 4 min at 7000 rpm/min.

The flow-through was discarded and the cells were washed with 0.05M NaCl solution three times. Bacterial cells were resuspended with 0.05M NaCl. Seedlings of these plants were inoculated with 1 ml of the bacterial suspension that resulted in an inoculum’s density of 108 – 109 CFU ml-1.

2.3.5 Harvest and plant analysis

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Eight weeks after transplanting, the roots were separated from shoot, washed from the substrate with running cold water using a set of sieves (smallest sieve size 1 mm). The fresh weight (FW), length of plant shoot and root were recorded. Both shoot and root are dried at 80°C for two days, and dry weight (DW) was recorded separately.

2.3.6 Statistical analysis

 Comparison of mean values of six replicates plant growth measurements, respectively, was performed using Student’s t - test at a P-level of ≤ 5 %. All statistical analyses were performed using STATISTICA 6.0 (StatSoft, Tulsa, OK, U.S., 2001). 

2.4 Results and Discussion

2.4.1 Bacteria isolation from root, rhizosphere and phyllosphere of wheat

2.4.1.1 Isolation and phenotypic characterization of diazotrophic bacteria

Basic phenotypic tests based on the colony formation at the surface of medium, cell shape, colony type, pigmentation on different media discarded 316 apparently sibling isolates (the isolates apparently belonging to same species) out of 780 isolates counted on Master plate. Isolates showing plant growth promoting effect were further identified based on utilization of specific carbon substrates. Natural colonisation of the studied isolates in wheat root, phylosphere and rhizosphere is given in Tab. 3. Studies based on the cultivation techniques provides useful information regarding microbial diversity in environmental samples, such as plant root, rhizosphere and phyllosphere samples, however, this studies suffers from bias, resulting from the media and cultivation condition applied, and from the inability to grow and isolate approx. 99% of the natural microbial community. These limitations have been overcome to some extent by using bacteria rRNA gene analysis for microbial population and colonization analysis. In our further studies, we have studied the colonization ability of certain bacteria in plant root and shoot using real-time PCR approaches (chapters 4, 6).

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We identified the strains at genus level only. Majority were found to be facultative anaerobic, catalase positive spore-forming rods and identified as Bacillus sp. The remaining isolates were identified as Pseudomanas, Burkholderia, Klebsiella and others. Diazotrophic Bacillus sp. and Pseudomonad sp. are commonly isolated from wheat roots (Nelson et al. 1976, Ruppel 1987). Trials with plant growth promoting Bacillus species showed yield increases in rice (Sudha et al. 1999), wheat (de Freitas 2000), canola (de Freitas et al. 1997), maize (Pal 1998), sugar beet (Cakmakcı et al. 1999), sugarcane (Sundara et al. 2002), and conifer species (Bent et al. 2002).

Tab. 3: Natural colonisation of the studied isolates in wheat root, phylossphere and rhizosphere as counted on the master plate with a visual inspection.

Isolate

CFU 10 6

Origin

Used as

Bacillus sp. WR2

4

root

PGPB

Bacillus sp. WR9

15

rhizosphere

BCA/PGPB

Pseudomonas sp. WR12

1

rhizospere

BCA

Pseudomonas sp. WR14

4

rhizosphere

BCA

Bacillus sp. WR17

8

rhizospere

BCA

Bacillus sp. WR22

3

rhizosphere

PGPB

Pseudomonas sp. WPh45

1

phylossphere

PGPB

Azotobacter sp. WRh101

1

rhizosphere

PGPB

Pseudomonas sp. WR109

1

root

PGPB

Pseudomonas sp. WPh138

1

phylossphere

PGPB

Xanthomonas sp. WR148 (Xs148)

2

root

PGPB

Bacillus strains increased root and shoot dry weight, as well as total nutrient uptake, including N, by plants (Canbolat et al. 2006) by different plant growth promoting mechanisms, such as nitrogen fixation (Coelho et al. 2003), P solubilisation (de Freitas et al. 1997), antibiotic production (Rosado and Seldin 1993), cytokinin production (Timmusk et al. 1999), and increased root and shoot growth (Sudha et al. 1999). Some reports have shown the effect of plant growth promoting substances, such as phytohormones, produced by PGPB (Gutierrez Manero et al. 2001). Inoculation with Bacillus megaterium reduced the required P fertilisation of sugarcane by 25% (Sundara et al. 2002). Pseudomonas inoculants significantly increased root dry weight in spring wheat (Walley and Germida 1997) and promoted the growth of spinach (Urashima and Hori 2003).

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A group of N2-fixing bacteria isolated from the surface sterilized roots of wheat were identified as Xanthomonas based on a 114-bp 16S rRNA and 240-bp 16S-23S ISR sequences (chapter 3). Although Xanthomonas is generally regarded as a potential plant pathogen (Van den Mooter and Swings 1990, Van Sluys et al. 2002, Succstorf and Berg 2003), species of Xanthomonas have shown positive effect on growth effect on sunflower (Helianthus annus L.) growth (Fages and Arsac 1991). Moreover, xanthan produced by Xanthomonas sp. was reported to improve aggregate formation (Chaney and Swift 1986).

2.4.2 Screening of bacterial isolates for their effect on wheat growth

A series of plate and pot experiments were conducted to assess the potential of various wheat bacterial isolates for improving growth and yield of wheat (Triticum aestivum L.).

2.4.2.1 First screening of isolates for their effect on the plant growth.

First screening results of all bacterial isolates picked from Petri dishes with Ashby agar showed that 24 % (111 out of 464) bacterial isolates significantly increased plant growth with variable degree of stimulation (30% higher growth patterns: shoot and loot length, fresh mass) compared to non-inoculated plants and 25% (116) of tested bacterial strains showed inhibitory effect on plant growth. Before testing for plant growth promoting activities of 111 isolates using saline Syrdarya soils, they were screened for their ability to inhibit plant-pathogen Fusarium species in dual-culture assay.

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Tab. 4: The effect of bacterial strains on plant growth and development

Effects

Proportion of

bacterial isolates

Bacterial strains, used for study

100% (464)

Stimulators

24 (111)

Inhibitors

Shoot

12 (56)

Root

13 (98)

Neutral

34 (158)

2.4.2.2 Antagonistic activity of bacteria isolated from wheat root, rhizosphere against pathogenic Fusarium isolates.

Antagonistic root-associated bacteria are an important functional group of beneficial bacteria responsible for the control of soilborne pathogens (Weller 1988, Sørensen 1997). The goal of this work was to test if wheat-growth-simulating bacteria have also the ability to protect the plant from plant pathogens, namely Fusarium species. Our reasoning is that plant growth promotion by bacteria can be partly due to their ability to protect the plant of pathogenic organisms in plant rhizosphere. In order to test the bacterial isolates of wheat for their ability to inhibit the growth of soilborne pathogens of wheat, 111 root-associated bacteria were evaluated using a combination of two screening steps.

2.4.2.3 Selection of bacteria for ability to inhibit in vitro growth of Fusarium culmorum.

As a result of the first screening, isolates were found to produce detectable inhibition zones against Fusarium culmorum on agar. 22% (24 of the 111) bacterial isolates from the wheat root and rhizosphere inhibited the in vitro hyphal growth of F. culmorum with R values lower than 0.7, while the majority had a neutral effect. The production of clear inhibition zones in dual culture screens is due to the production of antibiotics, toxic metabolites or siderophores as mechanisms for biological control (Swadling and Jeffries 1996). The presence and size of the zone of inhibition have been used as evidence of the production of antibiotics by the bacteria (Rothrock and Gottlieb 1981, Jackson et al. 1991, Crawford et al. 1993). Another possibility is that the bacterial isolates depleted the nutrient in the agar surrounding them and thereby inhibited the growth of F. culmorum. However, the PDA medium used for dual cultures is rich in nutrients and thus competition for them might be excluded. These observations from bioassays in dual cultures suggest that production of antibiotics and/or other antifungal substances by these bacteria may be involved in the inhibition of mycelial growth of fungal isolates. In most cases, bacteria effective as biocontrol agents of fungal plant diseases belong to the genera Bacillus, Pseudomonas and Streptomyces (Edwards et al. 1994). The 24 antagonistic bacterial isolates of the above belonged to genus Bacillus (11 isolates), Microccoccus (3 isolates), Pseudomonas (5 isolates) and others (5 isolates). These bacterial isolates were selected as antagonists for subsequent assays. In our study, the bacterial isolates from the wheat root and rhizosphere with the greater inhibitory capacity against F. culmorum are Bacillus sp., while isolates of Pseudomonas sp. showed a lesser inhibitory capacity against F. culmorum in dual cultures. Isolates showing a lesser ability to inhibit F. culmorum were not selected for further assays.

2.4.2.4 Specificity of bacterial antagonistic activity against isolates of Fusarium. 

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The antagonism of selected isolates towards other fungal pathogens was assessed because under field conditions synergistic interactions of pathogens occurred (Scholte and Jacob 1989). Biological control agents which can control more than one pathogen are extremely interesting. In our study, the majority of the bacteria tested in the second step was also active against other fungal pathogens, only a small number of selective F. culmorum antagonists was found (Tab. 5).

Tab. 5: Relative inhibition of growth of Fusarium species by selected strains.

Isolate

F. culmorum

F. avenaceum  

F. solani

Pseudomanas sp. WR12

0.77

0.56

0.65

Pseudomanas sp. WR14

0.73

0.77

0.65

Bacillus sp. WR9

0.83

0.88

0.55

Bacillus sp. WR17

0.92

0.93

0.83

Note: Inhibition of fungal growth is expressed as the ratio of the radius of mycelial growth in the direction of the bacteria relative to the radius of growth on a control plate on which no bacteria were spotted. The values shown are means of 3 plates.

Pseudomonas sp. (WR12, RW14) and Bacillus sp. (WR9, WR17), which inhibited in vitro growth of F. culmorum, also inhibited growth of F. avenaceum, F. solani and nonpathogenic Fusarium. Bacterial isolates differed in the extent of growth inhibition of the fungal isolates, with Bacillus sp. WR17 showing the strongest activity. In our other studies, isolates Pseudomonas sp. WR12 and RW14 showed the strong antagonistic activity against tomato Fusarium dry rot in vivo (Juraeva et al. 2004) and tomato Fusarium wilt caused by F.o. lycopersici (unpublished data). Antagonistic bacteria may effect against a beneficial rhizosphere fungi. Fravel (1988) discussed the possibility of deleterious effects of antibiotic and antibiotic-like compounds, produced by biocontrol agents, on beneficial microorganisms. The inhibitory effect of antagonistic bacteria that inhibit pathogenic F. culmorum on suppression of beneficial/nonpathogenic Fusarium should be investigated.

2.4.3 Screening of bacterial isolates for their plant growth promoting effect

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The data summarised in Tab. 6 demonstrate that seed inoculation with nine selected diazotrophic PGPB isolates significantly effected the growth of wheat under greenhouse conditions (Tab. 6). All the treatments, except for Pseudomonas WR109 and Pseudomonas WPh138, enhanced shoot dry weight as compared to the control. Shoot weight enhancement was greatest in response to Xanthomonas sp. Xs148 (46% more than the control) and Bacillus sp. WR9 (44% more than the control) whereas maximal root weight resulted from Xanthomonas sp. Xs148 (31% more than the control) followed by Bacillus licheniformis BL43 (28% more than the control) (Tab. 2). Of the bacterial inoculations, Xanthomonas sp. Xs148 inoculation produced the highest total weight (39% more than control) followed by Bacillus sp. WR9, Bacillus licheniformis BL43, Bacillus sp. WR2, Bacillus sp. WR22, all increasing root dry weight significantly compared to non inoculated plants.

A number of studies (Abdel-Wahab and El-Sharouny 1979, Abdel-Waheb 1980, Rennie and Larson 1979, Rennie et al. 1983) found that wheat specifically harbored nitrogen fixing Bacillus in the rhizosphere. They found that nitrogen fixation by this bacterium and Azospirillum could account for 14% - 63% of the plant N, as tested by non-isotopic methods in vitro and in the field, and by isotopic methods in vitro. Some of these yield increases, however, may not be due to nitrogen fixation, but to bacterial production of plant growth substances (Gutierrez-Manero et al. 2001, Dobbelaere et al. 2003). The present experiment revealed that inoculation with diazotrophic bacteria Xanthomonas sp. Xs148, Bacillus sp. WR9, Bacillus licheniformis BL43, Bacillus sp. WR2, Bacillus sp. WR22 was an effective treatment for improving the parameters measured, especially with reference to the increase in shoot and root dry weight in nonsterilised soil. The plant growth promoting ability of these bacteria is further tested with vegetable plants.

In the present study, we investigated the diazotropic bacterial isolates that are positively tested from the wheat experiments for their effect on different vegetable plants growth. All parameters for all test plants inoculated with certain bacteria used in this study were increased relative to control. The significant effect of inoculation on plant growth differed depending on plant type. The most effective inoculation effect on all test plants were observed with Xanthomomas sp. Xs148 (Fig. 1).

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Tab. 6:  The effect of inoculation of wheat with diazotrophic PGPB on the length and weight of shoots and roots in nonsterile soil 4 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.

Bacterial strains

Shoot length

(cm plant-1)

Root length

(cm plant-1)

Shoot d. wt

(g plant-1)

Root d. wt

(g plant-1)

Control

17.60 ± 0.73

14.07± 0.92

0.031± 0.0020

0.0170± 0.0014

Bacillus sp. WR2

24.46± 0.87*

20.44± 1.64*

0.039± 0.0014*

0.0214± 0.0013*

Bacillus sp. WR9

23.93± 0.96*

21.00± 0.48*

0.045± 0.0018*

0.0204± 0.0011*

Pseudomonas WPh45

23.40± 1.12*

19.18± 2.37*

0.039± 0.0019*

0.0190± 0.0019

Azotobacter sp. WR101

23.93± 1.07*

14.98± 2.39

0.040± 0.0018*

0.0196± 0.0016

Bacillus sp. WR22

25.87± 0.27*

19.88± 2.03*

0.040± 0.0007*

0.0194± 0.0009*

Pseudomonas WPh138

22.70± 0.98*

19.74± 1.04*

0.033± 0.0022

0.0170± 0.0012

Xanthomonas sp. Xs148

25.69± 1.87*

17.50± 0.51*

0.045± 0.0017*

0.0223± 0.0014*

Pseudomonas WR109

20.24± 1.92

14.84± 3.55

0.033± 0.0023

0.0207± 0.0024

Bacillus licheniformis BL43

24.77± 1.33*

20.33± 1.12*

0.039± 0.0019*

0.0215± 0.0011*

Fig 1: The effect of diazotrophic bacteria Xanthomonas sp. Xs148 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.

2.4.4 The influence of beneficial bacteria isolated from wheat rhizosphere on growth promotion of some vegetable plants

In all test plants, all growth parameters tested shown to be significantly increased (Tab. 7-10). This indicates that the bacterium does not distinguish between plants. Only exception is that the number of leaves in cauliflower, cucumber and paprika was not significantly different than control plants. Also, root length of tomato was not significantly different than control plants.

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The second most effective strain was Bacillus licheniformis BL43. Bacterial inoculation resulted in significantly increased growth of plant for all tested plants (Fig. 2). In cauliflower, cucumber and paprika, shoot length, shoot fresh weight and number of leaves, respectively, did not show significant response to the inoculation (Tab. 7 -10).

Fig. 2: The effect of diazotrophic bacteria Bacillus licheniformis BL43 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.

Inoculation with Bacillus sp. WR2 showed the best effect on cauliflower growth (Fig. 3). Except number of leaves, all measured growth parameters reached the significant level (Tab. 8). Paprika plants also showed the significantly positive response to the inoculation with Bacillus sp. WR2 (Fig. 3). For cucumber plants, no significant effect on plant root was observed (Tab. 8). In tomato plants, plant shoot weight increased relatively to control, the rest of the traits tested were significant (Tab. 10).

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Fig. 3: The effect of diazotrophic bacteria Bacillus sp. WR2 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.

The inoculation with Bacillus sp. WR9 was the most effective on paprika and tomato increasing all growth parameters significantly, except tomato shoot fresh weight (Fig. 4). In cauliflower plants, only increases in root growth of cauliflower were statistically significant (Tab. 7). On the contrary, cucumber plant root growth stayed unaffected by inoculation (Tab. 8).

The influence of the inoculation with Bacillus sp. WR22 was the most effective for paprika plants resulting in significantly increased growth pattern (Fig. 5). The least effected plant was cucumber showing only significant difference from control plants in shoot length and number of leaves (Tab. 8). In cauliflower, plant fresh and dry weight was shown to be significantly increased (Tab. 7). The response of tomato plants to inoculation was shown in root growth (length, fresh and dry weight) (Tab. 10).

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Tab. 7: Cauliflower shoot, root length and dry matter 8 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.

Parameters

Control

Bacillus sp. WR2

Bacillus sp. WR9

Bacillus sp. WR22

B. licheniformis BL43

Xanthomonas sp. Xs148

Shoot length

(cm plant-1)

19.1 ± 0.43

21.2* ± 0.30

21.2 ± 0.75

21.1 ± 0.64

20.9 ± 0.55

21.7* ± 0.71

Root length

(cm plant-1)

16.6 ± 0.92

20.3* ± 0.70

19.7 ± 0.62

20.0 ± 0.85

22.0* ± 1.00

21.2* ± 0.84

Number of leaves ( >3 cm)

5.80 ± 0.20

6.00 ± 0.00

6.20 ± 0.20

6.00 ± 0.00

6.00 ± 0.00

6.00 ± 0.00

Shoot f. wt

(g plant-1)

14.6 ± 0.92

16.5* ± 0.26

16.0 ± 0.26

18.7* ± 0.67

18.4* ± 0.66

19.3* ± 0.52

Root f. wt

(g plant-1)

2.53 ± 0.07

3.20* ± 0.15

3.45* ± 0.27

3.16* ± 0.17

3.09* ± 0.07

4.00* ± 0.17

Shoot d. wt

(g plant-1)

1.70 ± 0.08

1.95* ± 0.02

1.87 ± 0.04

2.03* ± 0.09

2.02* ± 0.07

2.05* ± 0.08

Root d. wt

(g plant-1)

0.27 ± 0.01

0.36* ± 0.01

0.39* ± 0.01

0.33* ± 0.01

0.38* ± 0.01

0.37* ± 0.02

Tab. 8: Cucumber shoot, root length and dry matter 8 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.

Parameters

Control

Bacillus sp. WR2

Bacillus sp. WR9

Bacillus sp. WR22

B. licheniformis BL43

Xanthomonas sp. Xs148

Shoot length

(cm plant-1)

31.4 ± 2.44

43.0* ± 2.60

42.7* ± 2.95

40.4* ± 3.69

42.1* ± 1.90

42.7* ± 3.33

Root length

(cm plant-1)

20.5 ± 1.51

21.5 ± 1.78

23.4* ± 1.88

22.5 ± 1.28

26.7* ± 1.30

30.3* ± 1.65

Number of leaves ( >3 cm)

9.8 ± 0.58

11.4* ± 0.24

11.6* ± 0.24

11.2* ± 0.37

12.4* ± 0.24

11.8 ± 0.49

Shoot f. wt

(g plant-1)

39.9 ± 2.11

45.8* ± 2.55

46.5* ± 2.00

42.8 ± 3.18

45.3 ± 1.42

47.6* ± 2.15

Root f. wt

(g plant-1)

6.94 ± 0.87

9.17 ± 0.68

8.35 ± 0.81

9.28 ± 0.59

9.84* ± 0.76

10.4* ± 0.72

Shoot d. wt

(g plant-1)

4.05 ± 0.31

4.57 ± 0.23

4.70 ± 0.11

4.35 ± 0.29

4.80* ± 0.16

5.02* ± 0.13

Root d. wt

(g plant-1)

0.65 ± 0.08

0.79 ± 0.10

0.71 ± 0.03

0.75 ± 0.04

0.91* ± 0.05

0.92* ± 0.04

Tab. 9: Paprika shoot, root length and dry matter 8 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.

Parameters

Control

Bacillus sp.

WR2

Bacillus sp.

WR9

Bacillus sp.

WR22

B. licheniformis BL43

Xanthomonas sp. Xs148

Shoot length

(cm plant-1)

12.6 ± 0.29

14.1* ± 0.47

14.3* ± 0.01

14.9* ± 0.78

14.7* ± 0.32

14.4* ± 0.25

Root length

(cm plant-1)

14.4 ± 0.40

16.7* ± 0.14

18.0* ± 1.34

15.8* ± 0.66

17.4* ± 0.50

17.4* ± 0.74

Number of leaves ( >3 cm)

10.8 ± 0.20

11.4 ± 0.24

11.4 ± 0.24

12.4 ± 0.92

11.4 ± 0.24

11.2 ± 0.20

Shoot f. wt

(g plant-1)

9.95 ± 0.35

11.9 ± 0.70

12.5* ± 0.74

11.9* ± 0.22

12.3* ± 0.25

11.8* ± 0.19

Root f. wt

(g plant-1)

2.69 ± 0.14

3.29* ± 0.12

3.23* ± 0.09

3.45* ± 0.18

3.60* ± 0.07

3.24* ± 0.07

Shoot d. wt

(g plant-1)

1.35 ± 0.04

1.66* ± 0.09

1.62* ± 0.06

1.63* ± 0.03

1.67* ± 0.03

1.51* ± 0.02

Root d. wt

(g plant-1)

0.29 ± 0.01

0.42* ± 0.01

0.41* ± 0.01

0.42* ± 0.01

0.47* ± 0.01

0.41* ± 0.01

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Tab. 10: Tomato shoot, root length and dry matter 8 weeks after planting. Plants were either non-inoculated with bacteria, or were inoculated with one of the bacteria given in the list. Effects of the bacterial treatment were tested with one-way ANOVA. Asterisks (*) denote significant differences between means of non-inoculated plants as determined by the Student-Newman-Keuls test (P<0.05). Values are means of 6 observations ± SE.

Parameters

Control

Bacillus sp.

WR2

Bacillus sp.

WR9

Bacillus sp.

WR22

B. licheniformis BL43

Xanthomonas sp. Xs148

Shoot length

(cm plant-1)

18.7 ± 0.60

21.0* ± 0.82

21.2* ± 0.37

21.4* ± 0.92

22.3* ± 0.69

21.4* ± 0.50

Root length

(cm plant-1)

16.8 ± 0.72

28.2* ± 2.93

27.3* ± 0.73

28.3* ± 1.09

25.0* ± 1.89

19.6 ± 1.36

Number of leaves ( >3 cm)

21.6 ± 1.86

29.4* ± 1.43

28.6* ± 0.40

33.2* ± 1.68

33.2* ± 0.73

31.0* ± 2.58

Shoot f. wt

(g plant-1)

13.3 ± 1.65

14.3 ± 0.89

16.6 ± 0.46

17.5 ± 1.07

18.9* ± 0.72

17.7* ± 0.13

Root f. wt

(g plant-1)

3.94 ± 0.53

5.62* ± 0.35

5.56* ± 0.16

5.51* ± 0.15

5.78* ± 0.26

5.50* ± 0.23

Shoot d. wt

(g plant-1)

1.53 ± 0.19

1.89 ± 0.10

2.07* ± 0.05

1.91 ± 0.07

2.04* ± 0.03

2.14* ± 0.05

Root d. wt

(g plant-1)

0.31 ± 0.03

0.52* ± 0.04

0.57* ± 0.02

0.48* ± 0.02

0.54* ± 0.03

0.52* ± 0.04

Fig. 4: The effect of diazotrophic bacteria Bacillus sp. WR9 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.

Fig. 5: The effect of diazotrophic bacteria Bacillus sp. WR22 on shoot root length, fresh and dry weight of vegetables 8 weeks after planting. SL – shoot length, RL – root length, NL – number of leaves, SFW – shoot fresh weight, RFW – root fresh weight, SDW – shoot dry weight, RDW – root dry weight.

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The different responses of plants to PGPB include simulation of root branching and root hair development, stimulation of total nutrient uptake (especially that of nitrogen), and an increase in biomass accumulation.

It has been suggested that certain PGPB are plant-species-specific (Chanway and Holl 1993). And therefore, one of the effective strategies for initial selection and screening of rhizobacteria is the consideration of host plant specificity. Inoculation with the strains used in this study has been carried out because of its capacity to increase the growth of wheat. Bacterial inoculation affected the early plant growth of tomato, cucumber, paprika and cauliflower grown in quartz sand. Paprika seedlings were more responsive to treatment with all used bacteria than were other test plants (Tab. 8). The establishment of inoculated PGPBs in the root system, showing a closer interaction between the bacteria and paprika roots, is a precondition for beneficial plant growth-promoting effects (Lucas-Garcia et al. 2003, Lugtenberg et al. 2001, Wiehe and Höflich, 1995). Characteristic quantifiers and qualifiers of root exudation play a fundamental role in the colonisation, as do the root structure/architecture and dynamics (e.g. flat rooting versus deep rooting) (Brimecombe et al. 2001, Neumann and Römheld 2001, Uren 2001). In this respect, the root system of paprika presents more surface contact than the root system of other plants tested, example tomato, which develops a main root with fewer branches.

Since, plants were grown in sand and supplied with half-strength Hoagland solution, it emphasizes that the bacteria tested can influence plant growth even in the presence of a nutrient solution. These findings suggest that plants may be grown with lower amounts of applied fertilizers and implies (1) a reduction in the cost associated with growing plants and (2) a reduction in the pollution associated with agricultural practices.

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The importance of bacterial strain Bacillus licheniformis BL43 and Xanthomonas sp. Xs148 is particularly significant regarding its beneficial effects on the plant growth and suppression the growth of plant pathogens. Therefore, bacterial isolates Bacillus licheniformis BL43 and Xanthomonas sp. Xs148 are selected for further studies.

2.5 Conclusion

A set of wheat bacterial isolates was characterized based on their effect on plant growth promotion and pathogen growth inhibition. The isolates that tested positive for wheat plant growth were further studied for their effect on some vegetable plants. Thus the characterization and screening of PGPR has helped in the selection of Bacillus licheniformis BL43 and Xanthomonas sp. Xs148 as potent strains in stimulating growth promotion in both cereal and vegetables.

Based on the results described above it can be concluded that the bacterial strains isolated in this study mainly belong to two genus, namely Bacillus and Pseudomonas, and that they stimulate the growth of wheat, and that PGPBs of wheat tested were not strictly plant-specific stimulating the different vegetable species.

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Chapter 3. Evaluation of 16S rRNA and 16S-23S ISR sequence-based analyses as part of a polyphasic approach to identify plant-inhabiting bacteria


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