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African Journal of Microbiology Research Vol. 6(2), pp. 326-334, 16 January, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.917 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Characterization and application of microbial antagonists for control of Fusarium head blight of wheat caused by Fusarium graminearum using single and mixture strain of antagonistic bacteria on resistance and susceptible cultivars Mahsa Alimi¹*, Mohammad Javad soleimani2 and Meysam Taghinasab Darzi3 1

Department of Plant Pathology, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2 Department of Plant Pathology, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran. 3 Department of Plant Pathology, Faculty of Agriculture, Gorgan University of Agricultural science and natural resources, Gorgan, Iran. Accepted 9 December, 2011

Fusarium head blight (FHB) caused by Fusarium graminearum leads to significant yield losses of wheat (Triticum aestivum L.) in many provinces of Iran, especially in the northern provinces in the margin of Caspian Sea. One strategy for the control of FHB is to use antagonistic bacteria that are plant growth promoting. In order to assess the potential of phyllospheric microorganisms in biological control of FHB, one hundred and ninety isolates of Pseudomonas, Erwinia and Bacillus spp. were collected from phyllosphere of healthy and infected wheats. Among them, eight isolates were selected and purified with the most antagonistic ability against the growth of pathogenic fungal species (F. graminearum) using the dual culture method. According to the results of biochemical and physiological tests, they were identified as three biovar of Pseudomonas fluorescens, one isolate of them was Erwinia herbicola and two species of Bacillus such as Bacillus subtilis and Bacillus cereus. Production of antifungal substances and volatile metabolites, siderophores and secretion of lytic enzymes such as protease and cellulase were evaluated as the inhibitory mechanisms in vitro. Furthermore, the effects of antagonistic bacteria were studied on severity and incidence of disease caused by F. graminearum in greenhouse conditions. Statistical analysis of data indicated that wheat spikes treatment with antagonistic bacteria not only reduced the severity and incidence of disease compared with the control but also showed positive influence on growth and yield of wheat cultivars. In general, multiple isolates such as B. subtilis 1 and P. fluorescens bv. 1 and 4 together were determined as the most effective strains in reducing incidence of disease. It can be concluded that multiple antagonistic isolates have better effects of control than single isolate. Key words: Biological control, Fusarium head blight, Fusarium graminearum, Pseudomonas fluorescens, Erwinia herbicola and Bacillus subtilis.

INTRODUCTION The pathogenic Fusarium graminearum strains cause air-

*Corresponding author. E-mail: [email protected]. Tel: +98 171 5529313. Fax: 98 171 4420982.

borne diseases of wheat, including pink and mouldy spots on rachis and glumes of spikletes. This disease is one of the most widespread diseases of wheat, especially in areas with warm and wet condition during growing season and it is economically so important. The pathogen is often localized in top, middle, or bottom of head. It may

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cause head to be bleached; over time, the premature bleaching of the spikelets may progress the entire head (Strange and Smith, 1971). If the environment is warm and moist, aggregation of pink spores and sporodochia may appear on the glumes. Later in the season, bluishblack spherical bodies are appeared but are sexual structures of the fungus known as perithecia. The fungus causes grains to shrink and wrinkle inside the head. Often infected kernels have a rough, wilted appearance, ranging in color from pink and soft gray to light brown. Losses are estimated $ 1 billion annually in lost yields, especially in North Dakota, Midwestern and Eastern states of the USA, Central and Eastern Canada, Brazil and Canada (Sutton, 1982; Gilbert and Tekauz, 2000). Fusarium head blight (FHB) is particularly concerned because of the ability of the Fusarium species to produce mycotoxins in the grain that are harmful for human and animal consumers. The predominant mycotoxins within cereals are the trichothecenes, mainly deoxynivalenol, nivalenol and their acetylated derivatives, T-2, HT-2, diacetoxysciropenol and neosolaniol. In order to reduce the application of chemicals for diseases control, especially air-borne fungal pathogens, some efforts have been conducted in biological control of F. graminearum by antagonistic microorganisms (Parry et al., 1999; Khan et al., 2004). Phyllosphere bacteria should be isolated and tested as biocontrol agents against air-borne pathogenic fungi on spikes. After testing antagonistic ability of isolates, they should be treated in greenhouse and field condition for valuation of their adaption to natural condition (Schisler, 2004). A successful antagonist agent efficiently suppresses the pathogen growth and reduces disease incidence. Biocontrol agents act by different forms of antagonism as competition for nutrients and space, antibiosis, hyperparasitism and resistance induction. Fluorescent Pseudomonas species have the ability of colonization of organs and production of secondary metabolites such as antibiotics, volatile compounds, HCN, enzymes, phytohormones and siderophore (Gupta et al., 2001). The secreted siderophores bind to the Fe3+ that is available in the rhizosphere, thereby effectively prevent growth of pathogens in that region. It sequesters iron for plant and promotes plant growth. They also can protect plants against wide range of important agronomic fungal diseases such as blotch root-rot of tobacco, root-rot of pea and wheat, damping-off of sugar beet (Ramesh et al., 2002). Antagonistic fungi and bacteria have been studied in order to control FHB, fungi such as Trichoderma spp., Chaetomium spp. and Gliocladium roseum (Knudsen et al., 1995; Xue et al., 2008) and bacteria such as Pseudomonas putida, Paenibacillus macerans and Sporobolomyces roseus (Bleakley et al., 2000; Schisler et al., 2006). Some other plant growth promoting bacteria (PGPB) synthesize antifungal antibiotics, e.g. P. fluorescens produces 2,4-diacetyl phloroglucinol, which inhibits

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growth of phytopathogenic fungi (Knudsen et al., 1995). Certain PGPB degrade fusaric acid and mycotoxins produced by Fusarium spp., causal agent of wilt, and thus prevent the pathogenesis. In addition, some PGPB can produce enzymes and lyse fungal cells (Schisler et al., 2002). The objective of the present research is to determine the effects of antagonistic bacteria by using single and multiple of determined antagonistic isolates, e.g. fluorescent Pseudomonads, Erwinia herbicola and some species of Bacillus, on reducing the severity and incidence of disease. The production of mycotoxins in spikes caused by F. graminearum in vitro and in vivo and their characterization in terms of antagonistic mechanism used to control pathogen and conditions for growth similar to those present in the field. MATERIALS AND METHODS Isolation of F. graminearum species Spikes and rachises of wheat were collected from several infected fields of Golestan Province that has been located in the margin of Caspian Sea; these samples had symptoms of pink spots on spikes, small and shrinked seeds, and white heads. The samples were submerged in 0.5% sodium hypochlorite for 3 to 5 min. After this treatment, they were extensively washed by sterile distilled water, placed on Petri dishes containing potato dextrose agar (PDA) and incubated at 24°C for one week. F. graminearum was the most prevalent fungi on medium. Their recognition was based on some characters such as long falcate macroconidia (Cappellini and Peterson, 1965).

Preparation of fungal inoculum and pathogenicity test 200-g sterile wheat stubble and barley were inoculated by four pieces of 5 mm mycelia disk from five days old culture of fungal species in Erlenmeyer. They were incubated at room temperature for 96 h on rotative shaker (150 rpm/min). The colonies of fungi were developed on wheat and barley. For pathogenicity test, fresh inoculum was isolated and sprayed on the spikes in 1% (w/w).

Isolation, selection and identification of bacteria Antagonistic bacteria were isolated from infected and healthy wheat spikes. Spike segments were washed under tap water and then 0.5 g of them was added to 50 ml of 1% peptone and shaked for 30 min. 0.1 ml of each bacterial suspension was spotted on Nutrient Agar (NA) medium, incubated at 24°C. Isolates were cultured on King's B (KB) for identification of Fluorescent Pseudomonads (Nelson et al., 1983). Antagonistic bacteria were identified by biochemical, physiological and biological tests according to the methodology of Schaad (Schaad et al., 2001). The purified isolates were pre-evaluated against the isolates of F. graminearum by using dual culture in Petri dishes containing PDA. The percentage of fungal growth inhibition were determined and calculated by using the fallowing formula (Sivan et al., 1987). Eight isolates were selected and re-purified which had the most inhibition percentage. % Inhibition = (1- [fungal growth / control growth]) × 100

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Afr. J. Microbiol. Res.

Antagonistic mechanisms of antagonistic bacteria

(Maurhofer et al., 1995). Each experiment considering single and multiple bacteria was run four replicates and repeated four times.

Production of volatile metabolites 100 μl of each bioantagonistic bacterial suspension (5 × 109 cfu/ml) were scattered on Petri dish containing King'S B (KB) medium. A 5 mm- disk of five days old pure culture of F. graminearum was placed in the center of another Petri dish containing PDA. Both plates were placed and sealed face to face preventing any physical contact between the pathogen and the bacterial suspension. Plates were incubated at 22 to 25°C for one week. Growth of pathogen was measured and compared to control that was developed in the absence of antagonists. Single isolate and multiple bacterial isolates were run four replicates and experiments were repeated three times. Results were expressed as means of percentage inhibition ± Standard Deviation (SD) of the growth of F. graminearum in the presence and absence of any bacterial isolates.

Cellulose production The medium was provided including K2 HPo4 (1 g), NaNo3 (0.5 g), MgSo4 (0.5 g), KCl (0.5 g) and FeSo4 (0.01 g) in 1L distilled water. A piece of filter paper in dimension of 1 × 9 cm2 was put in tubes containing 9 ml of medium. After sterilization, 1 ml of each bacterial suspension was added to tubes and incubated at 25°C. Until 3 weeks, the color changing of filter papers was traced daily. In control, 1 ml of MgSo4 solution (0.1 M) was added instead of bacterial isolate.

Greenhouse experiments Preparation of bacterial inocula

Production of diffusible antifungal substances According to Montealegro (Montealegre et al., 2003), 100 μL antagonist bacterial suspension (5 × 109 cfu/ml) were inoculated in the center of PDA plates; then they were covered with a cellophane membrane. Grown bacterial isolate and membrane were completely removed after incubation for 72 h at 22°C. A 5 mm- disk of a pure culture of F. graminearum was inoculated in the middle of plates and was incubated at 22°C for one week and growth of pathogen was measured. For controls, bacterial suspension was replaced by sterile distilled water and inoculated with F. graminearum. Results were expressed as means of percentage inhibition ±SD of growth of F. graminearum in the presence and absence of antagonistic bacterial isolate. Effect of Fe3+ on antagonism levels This effect was tested according to Weller and Cook (1983), by placing a 0.5 mm- disk of fungi in the face of each bacterial isolate on King B medium and King B plus 1000 μm FeCl3 incubating one week at 22°C. After this period, the fungal growth was compared for both media. In addition, inoculating bacterial strains on Chrome Azurol S (CAS) medium and changing the color of medium from blue to orange were considered as siderophore production (Schwyn and Neilands, 1987).

Hydrogen cyanide production At first, 100-μL suspension from pure culture of each bacteria were scattered on NA medium. Some pieces of filter paper were suspended in HCN indicator solution (5 mg cupper ethyl acetoacethate and 5 mg 4, 4-methylene-bis-(N,N-dimethyl aniline) in 2 cm3 chloroform). Changing the color of the filter papers placed on Petri dish’s caps after 3 to 18 h indicated the HCN production (Casteric and Casteric, 1983). Each experiment considering a single and multiple bacterial isolate was run four replicates and repeated four times.

Protease production Petri dishes contained Skim Milk Agar (SMA) culture medium including milk powder (15 g), yeast extract (5 g), blood agar (4 g) and agar-agar (13.5 g) were inoculated with bacterial isolates and incubated at 27°C for 24 h. Production of a colorless hallow around each bacterial colony indicated protease activity of that strain

Cells of antagonistic bacteria were grown on King's Medium B Broth (KMB) for greenhouse experiments. They were shaked in 150 rpm at 25°C. Cells were harvested by centrifugation (3000 rpm/min, 10°C, 15 min.). Samples were washed twice and re-suspended in MgSo4.7H2 o solution (0.1 M). The bacterial suspension was adjusted about 107 to 109cfu ml-1 by using haemocytometre for each experiment. The bacterial cells suspensions were used for seed coating and soil drenching.

Evaluating the disease intensity on wheat For this purpose, the effects of fungal species were evaluated on health wheat growth. The prepared fungal spores’ suspension was sprayed on spikes in 1% (w/w) in each pot (on phyllospheric organs). Disease intensity was scored on infected plants for 3 to 4 weeks after spraying the suspension (2 × 105 cfu ml-1) in the stage of flowering. Disease intensity was scored using Horsfall-Barrett scale (Stack and McMullen, 1998). As described in Table 1, four replications were maintained for each treatment. The pots were arranged in factorial design. The trials were repeated at least twice and showed similar results.

Efficacy of the suspension spraying of antagonistic bacterial isolates In greenhouse conditions, wheat seeds (cvs. Shanghaghay and Falat) were sterilized in 0.5% sodium hypochlorite for 5 min, and then air dried under laminar hood. Suspensions of antagonistic bacteria were sprayed two times by total concentration of 107109cfu/ml on phyllospheric bodies in boot stage. Fungal suspensions were sprayed for 72 days by total concentration of 2 × 105 cfu/ml (F. graminearum) on the spikes in pot. It was performed five times during a week at stage of flowering. Pots were maintained in greenhouse at 25°C and 90% relative humidity condition. Control treatments were inoculated with sterile distilled water. The symptoms were recorded for 3 to 4 weeks after spraying fungi. The effects of treatments were assessed on wheat growth factors such as the weight of 100 seeds for each plant and means of height of wheat, severity and incidence of disease on two wheat cultivars (Shanghaghay and Falat).

Statistical analysis Obtained data were subjected to analysis of variance. The means were checked by using Duncan’s Multiple Range Test and ANOVA.

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Table 1. Assessment of disease severity (Horsfall-Barrett scale).

Disease severity levels Clean Slight Moderate Hard Severe Strongly severe

% area with discoloration 0-7 14 - 21 33 - 50 50 - 66 79 - 90 100%

100 grains

Mean weights of

6 5

The weight of 100 seed in

4

shanghay cultivars The weight of 100 seed in falat cultivars

3 2 1

til is .c er eu s C on tr ol B

ub

P. f P. .4+ f . P. 4. P.f. flu + E 5 or . h es . P. ce flu ns or es 1 E. c e n .h s P. e r b 4 ic flu o .o re la sc en s B .s 5

0

Figure 1. The infeluence of bacterial teatments on the weight of 100 seeds in shanghay and falat cultivars (Resistance and suceptible cultivars).

RESULTS

as Bacillus subtilis and Bacillus cereus.

Isolation and identification of the fungal species The prevalent agents of Fusarium head blight of wheat was identified as F. graminearum (simple lateral phialides, branched conidiophores and some isolates of this fungi have floccose aerial mycelium and rose to coral pigmentation) in wheat fields of the margin of Caspian Sea, Iran. Pathogenicity test by inoculation of spikes by F. graminearum showed significant reduction on weight of 100 seeds and means of height of both wheat cultivars (Shanghaghay and Falat). In the case of effects on severity and incidence of disease, the results showed significant reduction on both of them (Figure 1). Selection and identification of antagonistic bacteria A total of eight antagonistic bacteria with the most inhibition percentage against F. graminearum was identified as: three biovars of P. fluorescens (1, 4 and 5), one isolate of E. herbicola and some species of Bacillus such

Antagonistic bacteria

mechanisms

of

the

antagonistic

Six single isolates and two multiple isolates were positive in production of volatile and diffusible antifungal metabolites against the fungal species in vitro. Volatile compounds were provided by multiple isolates, showing the most effect, followed by B. cereus. Volatile compounds provided by P. fluorescens bv. 5 and E. herbicola indicated the least inhibitory effect on growth rate of F. graminearum (Table 2). Antifungal substances produced by multiple isolates of F. graminearum were the most effective, followed by P. fluorescens bv. 1, P. fluorescens bv. 4 and 5 (Table 2). Totally, the inhibitory effects of antifungal substances on the mycelia growth were more than those of volatile compounds. In addition, three biovars of P. fluorescens altogether with B. subtilis were able to produce siderophores. Only P. fluorescens bv. 5 produced hydrogen cyanide (HCN). Biovars of P. fluorescens secreted both protease and

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Table 2. Inhibition percentage of antagonistic bacterial strains on the mycelia growth rate of the fungal species.

Treats P. fluorescens bv 4+ P. fluorescens bv5 P. fluorescens bv 4+E. herbicola P. fluorescens bv 1 P. fluorescens bv 4 P. fluorescens bv 5 B. subtilis B. cereus E. herbicola Control

Volatile Comp. 81.7a ±0.064 76.4 b ±0.0163 62.0e ±0.163 50.5 g ±0.374 50.0f ±0.412 66.7d ±0.053 72.0c ±2.081 41.7h ±0.043 00.00i

PDA Antifungal Sub. 81.8a±0.112 80.0b ±0.141 81.8a ±0.043 72.7c ±0.099 67.7d ±0.172 72.7c ±0.086 72.7c ±0.095 65.4e ±0.016 0.00f

NA Volatile Comp. 75.0a±5.099 58.3d ±0.057 66.7c ±0.053 58.3d ±0.075 36.7g ±0.028 58.3d ±0.045 68.3b ±0.014 43.3f ±0.021 0.00 h

Antifungal Sub. 83.3a ±0.112 72.7b ±0.081 71.2c ±0.074 70.9c ±0.043 56.4 f ±0.149 54.5g ±0.266 69.0d ±0.074 68.8e ±0.029 0.00h

Table 3. Influence of the interactions of antagonistic bacterial and fungal species on the weight of 100 seeds cv.shanghay and Falat.


Fusarium graminearum Shanghay Falat 3.7cd ±0.125 5.1a ±0.136 3.0e ±0.169 4.4b ±1.290 2.6f ±0.545 3.7cd ±0.139 3.0e ±0.178 3.7cd ±0.137 3.3e ±0.102 3.0e ±0.171 4.0c ±0.208 3.0e ±0.165 2.6f ±0.026 3.0e ±0.198 2.3f ±0.169 3.6d ±0.195 3.6d ±0.192 2.4f ±0.094

cellulose enzymes, whereas B. subtilis and B. cereus isolates produced only protease.

means of height crop of the both wheat cultivars, (Shanghaghay and Falat) (Table 3).

Influence of the interaction of antagonistic bacteria and fungal species on wheat growth factors

Influence of the antagonistic bacteria on Fusarium head blight disease

Results of the co-inoculation of wheat with antagonistic bacterial and fungal species indicated that spraying the suspension of antagonistic bacteria caused the reduction of severity and incidence of disease as well as caused considerable positive effects on wheat’s growth factors (Table 3). The factors such as the means of height, dry weight and severity and incidence of disease were affected significantly by interaction between fungi and bacteria (Tables 3 to 5). Spraying with multiple isolates and after P. fluorescence bv. 1 and 5 and B. subtilis showed positive effects on the yield components of wheat cultivars such as the weight of 100 grains germination and dried weight of aerial parts of the plant. Inoculation of spikes to F. graminearum not only caused significant reduction effect on severity and incidence of disease, but also had a significant effect on seed germination and

Data presented in Tables 4 and 5 regarding of the coinoculation of wheat with the antagonistic bacterial and fungal species indicated that spraying with the antagonistic bacteria suspension have reduced the severity and incidence of disease on both cvs Shanghaghay and Falat. In greenhouse experiments, interaction between the fungal species and the antagonistic bacteria affected significantly on severity and incidence of disease (Figure 2). DISCUSSION Application of fluorescent Pseudomonas and other antagonistic bacteria have drawn attention worldwide because of the production of the secondary metabolites

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Table 4. Efficacy of antagonistic bacteria on disease severity caused by fungal species on cvs. Shanghay and Falat.


Fusarium graminearum Shanghay Falat 1.9e ± 2.986 6.5c ± 7.365 1.9e ± 2.986 6.5c ± 7.365 4.2d ± 2.986 7.7b ± 8.655 4.2d ± 2.986 7.7b ± 8.655 4.2d ± 2.986 9.2a ± 4.787 4.2d ± 2.986 7.7b ± 8.655 4.2d ± 2.986 7.7b ± 8.655 4.2d ± 2.986 9.2a ± 4.787 4.2d ± 2.986 10.0a ± 0

Table 5. Efficacy of antagonistic bacteria on disease incidence caused by fungal species on cvs. Shanghay and Falat.


Fusarium graminearum Shanghay Falat 22.3 h ± 3.862 64.9 bcde ±1.519 42.6 g ± 1.971 66.7 bcd ±0.977 50.2 fg ± 12.903 82.3 a ±1.050 55.5 def ±8.009 69.63 b ±1.261 55.2 def ± 0.753 87.9 a ±1.351 56.9 cdef ± 5.389 82.3 a ±1.050 46.63 fg ± 10.445 68.5 bc ±0.367 54.3 efg ± 5.822 85.4 a ±9.837 52.2 fg ± 8.436 92.2 a ±3.172

100 90 80 70 60 50 40 30 20 10 0

Disease severity in shanghay cultivar Disease severity in falat cultivar

P. flu

or Co es t c e r ol P. E n flu . h s e 5 or r e s bi c c e ol ns a P. flu B or . s 1 e s ub c e til is n P. s f. 4+ 4 B. E. h ce . P. re u f. 4 s +P . f. 5

Means of disease severity

Means followed by a common letters in a column are not significantly different according to Duncan’s Multiple Range Test (p=0.01). Distribution of data were normalized with √X+1/2 ± means SE or standard error among repeats of a treatment.

Figure 2. The influence of bacteria on disease severity in shanghay and falat cultivars (Resistance and suceptible cultivars)

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Bacterial effect on disease incidence in shanghay cultivar Bacterial effect on disease incidence in falat cultivar

P. f

P

P. f

.4 +E . 4 . h. + p. f.5

100 90 80 70 60 50 40 30 20 10 0

.f C lu ot or es rol ce ns E. 5 he rb ic o B . s la P u bt .f P lu ili .f o re s lu sc or B . c esc ens e er 1 eu ns 4 s

Means of ID

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Figure 3. The influence of bacteria on disease incidence severity in shanghay and falat cultivars (Resistance and suceptible cultivars).

such as siderophore, antibiotics, volatile compounds, hydrogen cyanide (HCN), enzymes and induction of systemic resistanc (Kloepper et al., 1991; Maurhofer et al., 1995; Van Loon et al., 1998). Pseudomonas spp., Erwinia spp. and Bacillus spp. are well known antagonistic bacteria. The potential ability of biocontrol of P. fluorescens, E. herbicola and Bacillus species caused they serve as biocontrol agent of Fusarium head blight disease (Van et al., 1998; Xue et al., 2009). The results obtained from current experiments also indicated similar antagonistic potential. Comparing in vitro antagonism and in vivo disease suppression, it appears that in vitro test has some predictive value for disease suppression by P. fluorescens, E. herbicola and Bacillus spp. strains. This case especially accounts for multiple Pseudomonas and Bacillus strains. Different disease-suppressive mechanisms are involved in enhancing the disease suppression, for example competition and consequently disease suppression (Kragelund and Nybroe, 1996; Foroutan et al., 2005). It demonstrated that a positive relationship exists between population size of the biocontrol strain on phyllosphere and disease suppression (Johnson, 1994; Smith et al., 1997; Petti et al., 2010). In general, competition for nutrients supplied by roots and seeds and occupation of sites favored for colonization are probably responsible for a small or moderate degree of disease suppression by most PGPB and are primary importance in some strains. It is likely that the production of volatile compounds could be responsible for enhancing the disease suppressions (Table 2). However, it has been reported that in the case of antagonistic mechanism of B. cereus, result has been demonstrated that there is some evidence to suggest that its potential for providing both protease and cellulase

enzymes seems to be involved in regards (El-Tarabily et al., 1996; Khan and Doohan, 2009). In addition, three biovars of P. fluorescens altogether with B. subtilis were able to produce siderophores. Methalophores or siderophores are low-molecular-weight molecules that are secreted by microorganisms to take up iron from the environment, and their mode of action in suppression of disease are thought to be solely based on competition for iron with the pathogen (Andrews, 1992; Srivastava and Shalini, 2009). Production of metabolites such as antifungal substances, siderophores and hydrogen cyanide is the primary mechanisms of biocontrol. Therefore, the disease suppression by these strains, which been reported here, might be due to involvement of this mechanism. Production of secondary metabolites such as phenazine-1-carboxilic acid (PCA), 2,4-diacetyl phloroglucinol (phl), pyoleutorin (plt), pyrrolnitrin, oomycin and Hydrogen Cyanide (HCN) are also characteristic features of biocontrol agents (Knudsen et al., 1995; Jousset et al., 2011). Probably, accumulation of HCN produced by P. fluorescens bv. 5 on the phyllosphere region due to inoculation of spikes with high population of this strain is resulted in negative influence of this strain on growth and yield components of wheat. Spraying phyllospheric bodies with E. herbicola, and P. fluorescens bv. 1 showed positive influence on the most yield components of wheat cultivars (Table 3). Also comparing disease severity and incidence in wheat cultivars confirmed differences between sensitivity of cultivars to pathogenic agents such as the mentioned fungi (Figure 3). Since in these experiments, cv. Shanghay was more resistant, they exhibit milder symptoms spots and shrinked grains, comparing with cv. Falat (Tables 4 and 5). Numerous biotic and abiotic factors are likely to

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contribute to this inconsistent performance of biocontrol microorganisms (Khalid et al., 2004; Shaukat et al., 2006). In conclusion, these results indicate that, specific antagonistic bacterial agents can influence disease suppression. They could be considered as part of a disease control strategy like integrated pest management, which offers a successful approach for deployment of both agro-chemicals and biocontrol agents. However, there is not uniformity in results of using biocontrol agents especially in natural situation and fields. It should be emphasized the necessity of the continual study in this regard as well as their ecological interactions. In general, multiple isolates such as B. subtilis1 and P. fluorescens bv. 1 and 4 were determined as the most effective strains in reducing incidence of disease caused by the fungi. Statistical analysis showed that the correlation was significant (r = 0.64) among the weight of 100 seeds and infection of wheat to F. graminearum. At last, the treatments of bacteria did not show any remarkable difference in prevention of disease on resistant cultivars as compared with control, but for susceptible cultivars, treatment of bacteria showed significant difference in the level of 0.01. The multiple bacteria had better effect than single bacteria, whereas, for multiple isolates, homogenous had better effect than isolates with different genera. It should be pointed that in both cases, multiple isolates showed better effect than single bacteria and all of the treatments on susceptible cultivars had significant difference as compared to control. Therefore, we suggest using mixture isolates for biological control in future. ACKNOWLEDGEMENT Authors gratefully acknowledged Research Council of Bu-Ali Sina University, Hamadan, Iran, for financial support. REFERENCES Bleakley BH, Draper MA, Ruden KR (2000). Control of Fusarium head blight whit biological antagonists. In the proceedings of the 2000 National Fusarium head blight Froum, pp. 75-76. Cappellini RA, Peterson JL (1965). Macroconidia formation in submerged cultures by non-sporulating strain of Gibberella zea. Mycologia, 57:963-966. Casteric KF, Casteric PA (1983). Method for rapid detection of cyanogonic bacteria. Appl. Environ. Microbiol., 54: 701-702. El-Tarabily KA, Sykes ML, Kurtbuke ID, Hardy G, Dekker RFH (1996). Synergistic effects of a cellulose-producing Micromonospora carbonacea and an antibiotic-producing Stereptomyces violasence on the suppression of Phytophthora cinnamomi root-rot of Banksia grandis. Can. J. Bot., 74: 618-624. Foroutan AAR, Rahimian H, Alizadeh azizi E (2005). Effect of antagonistic bacteria on Fusarium Head Blight of wheat. Iranian J. Plant Pathol., 41(3): 455-473 Gilbert J, Tekauz A (2000). Recent development in research on Fusarium head blight of wheat in Canada. Can. J. Plant Pathol., pp. 22:1-8.

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