Research Article Open Access

El-Halmouch et al., J Plant Pathol Microb 2013, 4:10 DOI: 10.4172/2157-7471.1000205

Volume 4 • Issue 10 • 1000205J Plant Pathol MicrobISSN:2157-7471 JPPM, an open access journal

Keywords: Broomrape; Biocontrol; Faba bean; Root exudates; R. leguminosarum; A. chroococcum; Compost tea

IntroductionPhytoparasitic weeds are known as destructive parasites on many

agricultural crops in the Mediterranean region, Eastern Europe and North Africa [1,2]. Orobanche crenata is the most dangerous and the most widespread Orobanche species in the Mediterranean region and Western Asia. It is a major constraint for faba beans, field peas, lentils, vetches and various forage legumes [3]. Unfortunately, several strategies have been employed to control broomrape with little success [4,5]. The potential of natural enemies as biological control agents has received more attention in recent years. The main impact of the biocontrol agent is the reduction of the seed germination [6,7], germ tube-host attachment or seed production, resulting in the prevention of supplementary infestation and seed dissemination, and leading to a reduction of the seed bank in the soil [8]. Germination of Orobanche spp. is stimulated by root exudates from crop hosts, but in the absence of a host, seeds can remain viable for 10 years or more [9], making it difficult for any crop rotation to be efficient. Toxins from soil-borne pathogens that inhibit Orobanche seed germination could prevent attachment of the parasite. The most common soil-borne pathogens isolated from diseased Orobanche plants belong to the genus Fusarium [10].

Information concerning rhizobacteria antagonistic to Orobanche is very few. Zermane et al. [11] identified some Pseudomonas and Ralstonia strains as natural antagonists of Orobanche. Mabrouk et al. [12] showed that symbiosis with some non-pathogenic Rhizobiumleguminosarum strains could induce both better development and

lower susceptibility in pea to O. crenata. Some rhizobacteria referred to as PGPR (plant growth promoting rhizobacteria) or PHPR (plant health-promoting rhizobacteria) have the ability to improve plant growth, and/or root health [13]. Induced resistance in the nodulated pea was characterized by low activity of the root exudates in triggering Orobanche seed germination, and by the induction of necrosis of most of the Orobanche seedlings before and after attachment to host roots [12].

Organic soil amendments, especially composts, can provide a rich source of plant disease suppressive microorganisms and large populations and high diversity of microorganisms with biological control potential [14]. Many microfloral species can be released from the compost, such as Bacillus spp., Enterobacter spp., Flavobacterium balustinum and Pseudomonas spp., and fungi such as Penicillium spp., Gliocladium virens and several Trichoderma spp. that act as biocontrol

*Corresponding author: Yasser El-Halmouch, Biotechnology Department,Faculty of Science, Taif University, Taif 21974, P.O. Box 888, Kingdom of Saudi Arabia, Tel: +966 55 6382034; Fax: +966 27274299; E-mail: halmouch@yahoo.com

Received : October 09, 2013; Accepted October 29, 2013; Published October 31, 2013

Copyright: © 2013 El-Halmouch Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

In the present study, cell-free cultures of four isolates of Rhizobium leguminosarum, an isolate of Azotobacter chroococcum and compost tea were investigated for their biocontrol potential against the root parasitic weed Orobanche crenata. Individual cell-free cultures of Azotobacter chroococcum or Rhizobium sp., dual and mixture of cell-free cultures of Rhizobium spp. or compost tea were applied to infested pots in greenhouse conditions. The treatments showed variable effects on many developmental parameters of both faba bean and broomrape. Significant decrease in the number of broomrape attachments, dry weight of the attached tubercles on faba bean roots and the reduction in percentage of broomrape seed germination were recorded. Compost tea, individual and mixture of R. leguminosarum isolates were more reducing on broomrape germination and growth than A. chroococcum alone did; being the former treatment is the best. The reduction in broomrape incidence by compost tea was due to certain phenotypic mechanisms, which acted alone or in combination. These mechanisms included negative effect of natural stimulant broomrape on seed germination, prevention of radical penetration inside the host roots, parasite yield reduction, and thus increasing the growth and vitality of faba bean. In vitro experiment indicated that seed germination percentage of broomrape was also negatively affected by the combination of root-exudates and compost tea. Radical apexes of the germinated seeds were distorted. These distortions may prevent the radicals to follow up the infestation. In conclusion, the study presents the potential of R. leguminosarum isolates and compost tea in biocontrol of broomrape. More investigations should be carried out with viable bacterial cells on the parasite plant before use in sustainable agricultural systems.

The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.)Yasser El-Halmouch1,4*, Ahlam Mehesen2 and Abd El-Raheem Ramadan El-Shanshoury3,4

1Botany Department, Faculty of Science, Damanhour University, Damanhour 22511, Egypt 2Soils, Water and Environment Research Institute, Agriculture Research Center, Giza 12619, Egypt3Botany Department, Faculty of Science, Tanta University, Tanta 31527, Egypt4Biotechnology Department, Faculty of Science, Taif University, Taif 21974, Kingdom of Saudi Arabia

Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205

Journal ofPlant Pathology & MicrobiologyJo

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ISSN: 2157-7471

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Volume 4 • Issue 10 • 1000205J Plant Pathol MicrobISSN:2157-7471 JPPM, an open access journal

agents [15]. These beneficial microorganisms can provide nutrients that stimulate the proliferation of antagonistic bacteria and fungi in the rhizosphere [16,17]. Due to the scarce of information about the control of Orobanche by free living and symbiotic nitrogen fixing bacteria, this study aimed to evaluate the potential of individual and mixture of R. leguminosarum isolates, A. chroococcum and compost tea to control faba bean infection by O. crenata.

Materials and MethodsSource of bacterial isolates and herbicide solutions

All Rhizobium leguminosarum isolates (302, 312, 317 and 313), Azotobacter chroococcum and compost tea were obtained from the Micrbiology Department, National Research Centre, Sakha, Egypt.

each containing 150 mL of selective liquid culture medium; Yeast Manitol extract for R. leguminosarum isolates, which consists of the following components in g/L: Yeast extract 1, Mannitol 10, Dipotassium phosphate 0.5, Magnesium sulphate 0.2, Sodium chloride 0.1, Calcium carbonate 1. Basic nitrogen Burk’s medium was also used for Azotobacter that consists of the following components in g/L: Magnesium sulphate 0.2, Dipotassium phosphate 0.8, Monopotassium phosphate 0.2, Calcium sulphate 0.13, Ferric chloride 0.001, Sodium molybdate 0.001

growth appeared on one slant, previously grown at 25°C for 24-48 hrs. Inoculated flasks were incubated at 25 ± 2°C in a rotary shaker at 160 rpm for 24 hrs. Six ml were transferred to 150 ml liquid culture and incubated at 25 ± 2°C for 3 days at 160 rpm on a rotary shaker. Bacterial growth was separated by centrifugation at 4000×g for 20 min, and then the crude cell-free fermentation culture was used as a soil drench throughout the growing season in the green house.

Pot experiment

Pot experiments were performed in 2010 and 2011 in greenhouse of National Research Centre, Sakha, Egypt. O. crenata seeds (4 mg kg-1 of soil; about 1300 seeds) were mixed with a 1:1:1 peat–sand–clay mixture in a 3 L pot. Five infested pots were prepared for each microbial treatment, watered and protected from the light for 1 week at 25°C to obtain preconditioned broomrape seeds; then three seeds of V. faba Misr1 and V. faba Giza 843 cultivars were sown separately into each pot. Each pot recieved 250 ml of crude microbial cell-free culture. Two weeks after faba bean emergence, seedlings were thinned to one per pot. Broomrape-free pots were included as control. The two cultivars were grown under greenhouse conditions at 25°C with 300 μmol m-2s-1 PAR and a 16 h photoperiod. Twelve weeks after sowing, faba bean roots and broomrape attachments were uprooted and observed under a binocular microscope. Broomrape attachments on host roots were counted and the dry weight/plant of the entire parasite was recorded. In addition, some other parameters like dry weight of broomrape, number and weight of of bacterial nodules, total nitrogen and chlorophyll content of faba bean plants (V. faba Misr1 and V. faba Giza 843) were recorded.

Biochemical analysis

Total nitrogen content: 0.3 gm of finely ground oven dried plant samples were digested by 2 ml of concentrated sulfuric acid (H2SO4), then potassium sulphate, 8 g; copper sulphate, 1 g and mercuric oxide, 1 g were added as catalyst. Digestion was done using Micro-Kjeldahl technique with continuous heating until the mixture turned colorless. The final volume was made up to 25 ml with distilled water in a volumetric flask. 5 ml of this volume was analyzed by steam distillation

Chlorophyll content: Extracts of plant material were obtained from green leaves of faba bean by direct immersion of faba bean sample (500 mg) in N,N-dimethylformamide (DMF), as recommended by Moran and Porath [19]. Immersed sample was mixed in a Sorvall Omni-Mix

stored in the dark for one to two days at 4°C, prior to spectroscopic examination. Spectrophotometric measurements were made by means of Varian Techtron model 635 UV-VIS scanning spectrophotometer, calibrated at 703 nm, using the 0.2 nm band width measuring beam and 1 ml cuvette having a path length of 1 cm [20].

Root exudates collection and in vitro germination of O. crenata

Two seeds of each faba bean cultivar were sown and grown in a pot filled with sand. Root exudates were collected using the double pot technique, as described by Parker et al. [21]. An aliquot of 100 ml of root exudates was collected from each pot and sterilized by filtration through 0.22 µm bacterial filters before storage for several weeks at -20°C. Pre-conditioned surface-sterilized broomrape seeds were transferred to small Petri dishes (3.5 cm), containing mixture of 250 μl root exudates plus 250 μl of microbial culture-filtrate or compost tea filtrate, and then incubated at 25°C for one week. Viability and germination tests were performed according to Linke [22]. Germination was determined after observation of radical emergence under a binocular microscope.

Statistical analysis

All results were subjected to one-way ANOVA and the means were compared according to the Student–Newman–Keuls (SNK) multiple range test (P ≤ 0.05).

ResultsIn the two pot experiments, many parameters were used to evaluate

the resistance of faba bean cultivars to O. crenata; two specific for the parasite (number of broomrape attachments and dry weight), and six specific for the host (faba bean dry weight, bacterial nodules number and dry weight, total nitrogen and chlorophyll a and b content). Although none of the tested microbial treatment was absolutely inhibitory to the parasite, remarkable variations were observed in faba bean response to broomrape, according to the treatment.

First pot experiment

Effect of the microbial treatment on parasite: In the first pot experiment done in 2010, significant variations among the treatment were obtained in broomrape number (Figure 1). Resistant cultivar Misr 1 always exhibited lower number of broomrape tubercles than the susceptible cultivar Giza 843. Microbial treatment and compost tea reduced broomrape attachments fixed in both faba bean cultivars.

In Misr 1, a significant reduction in total number of broomrape tubercles was recorded by all microbial treatments and compost tea, except Azotobacter chroococcum. About 86.5% of reduction in broomrape attachments was observed using compost tea as a biocontrol agent (Figure 1). Among the bacterial treatments, the mixture of R. leguminosarum 313 and 317 isolates exhibited the inhibitoriest effect

Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205

in the presence of NaOH (40%) and ammonia were in boric acid (10%), plus three drops of indicator containing; 6 ml methyl red (0.16% in 95% ethyl alcohol); 12 ml bromocresol green (0.04% in water) and 6 ml of 95% alcohol. Titration was made with 0.01 M HCl, until the indicator turns from green to pink. Then, total nitrogen was calculated as mg in the sample [18].

Microbial isolates were cultured in Erlenmeyer flasks (250 mL),

and glucose 20 as carbon source. Flasks were inoculated with the

for 3 min and then centrifuged at 27×10 Xg for 10 min. The extract 3

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Figure 1: Effect of microbial cultures and compost tea on total number of O. crenata tubercles fixed on Vicia faba cultivar roots in experiment done in 2010.

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Figure 2: Influence of microbial treatment and compost tea on dry weight of O. crenata fixed on faba bean plant in 2010. Different letters on the column for each parameter (V. faba Misr 1 and V. faba Giza 843) are differ significantly at p ≤ 0.05.

Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205

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Volume 4 • Issue 10 • 1000205J Plant Pathol MicrobISSN:2157-7471 JPPM, an open access journal

of broomrape attacked Misr 1. However, more than 77% reduction in broomrape tubercles was recorded. Broomrape tubercles fixed on Giza 843 varied significantly and ranged between 5.6-33 per faba bean plant. Compost tea produced about 87% reduction in number of broomrape tubercles and considered the most effective biocontrol agent.

Dry weight of broomrape treated with the microbial strains and compost tea was significantly lower than those of non-treated control (Figure 2). Most of the mixed microbial treatments were more effective than individual treatments. The treatments with R. leguminosarum 312+302 and compost tea were inhibitorier to broomrape growth on Misr 1 roots than individual treatment. Compost tea was the best inhibitory, that caused 88% and 94% dry weight reduction in broomrape fixed on Misr 1 and Giza 843, respectively (Figure 2).

Effect of the microbial treatment on hosts: All faba bean growth and metabolic parameters varied significantly by microbial and compost tea treatments. In V. faba Misr 1, utilization of compost tea as a biocontrol agent significantly increased the shoot dry weight, total nitrogen and chlorophyll b content by 97%, 179% and 94%, respectively, compared to the infested control (Table 1). The mixture of R. leguminosarum isolates increased nodule number and their dry weight by 5.3 and 3.3 times, respectively, over the infested control. Chlorophyll a was increased two times by dual culture filtrates of isolates 312 and 317, compared to the infested control.

significantly in all microbial and compost tea treatments, in comparison with the infested control. An increase in shoot dry weight, total nitrogen and chlorophyll b content was recorded by 3, 2 and 1.6 times, over the infested control, respectively, in case of compost tea treatment. The mixture of all R. leguminosarum isolates enhanced both nodule number and dry weight by eight and two folds, respectively.

Second pot experiment

Effect of the microbial treatment on parasite: In the second experiment done in 2011, expression of resistance in faba bean by number of broomrape tubercle showed a considerable variation among microbial and compost tea treatments for the two cultivars (Misr1 and Giza 843), that ranged between 2.6 to 18.2 for Misr 1 and from 15.4 to 48.8 for Giza 843.

In Misr 1, dual inhibitory effect of isolates 312+317 and 312+313, and compost tea significantly varied compared to other treatments on broomrape tubercles number (Figure 3). The highest percentage of reduction was obtained by the treatment with R. leguminosarum 312+313 and compost tea (80% and 61% respectively). No reduction was recorded by the treatment with R. leguminosarum 313+317, R. leguminosarum 302+317 and Azotobacter. In contrast, these treatments induced the broomrape attachment. However, broomrape tubercles fixed on Giza 843 roots varied significantly according to the treatment (Figure 3). The dual treatment with 312 and 302 isolates induced broomrape attachment, compared with the control. R. leguminosarum 312, R. leguminosarum 317, dual isolates of 302 and 317 and compost

about 51%, 52% and 60% reduction, respectively. Cultivar resistance to Orobanche based on broomrape dry weight varied significantly among the treatments. About 93% and 96% reduction in broomrape dry weight were obtained by compost tea in Misr 1 and Giza 843, respectively (Figure 4).

R.

For cultivar Giza 843, compost tea increased shoot dry weight and nitrogen content by 279% and 143%, respectively, compared with the infested control. High increases of nodule number, nodule dry weight, nitrogen and chlorophyll a contents were observed due to treatment

Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205

tea were the most inhibitory for broomrape attachment. They gave

Effect of the microbial treatment on hosts: All faba bean growth parameters (shoot dry weight, nodule number, nodule dry weight, total nitrogen and chlorophyll a contents) for the two cultivars (Misr 1 and Giza 843) varied significantly by the treatments, except chlorophyll b content in V. faba Giza 843. In V. faba Misr 1, a highly significant reduction in all parameters was obtained in infested non-treated control, compared with non-infested non-treated control. In contrast, significant increases in shoot dry weight, nitrogen content, chlorophyll a and chlorophyll b contents, were recorded by 81.2%, 165%, 94.1% and 86.4%. respectively, using compost tea (Table 2). Moreover, nodule number and dry weight were significantly higher due to the treatment with the mixture of all R. leguminosarum isolates than the infested control (57.57 and 460 gm/plant, respectively). They increased by 4.49 and 3.41 times over than those of infested non-treated control. More than 100% of chlorophyll b content was induced in Misr 1 using leguminosarum 312+317.

Treatments Shoot dry weight(gm/plant)

Nodule number/plant

Nodule dry weight(mg/plant)

Total nitrogen content (mg/plant)

Chlorophyll a content (mg/cm2)

Chlorophyll a content (mg/cm2)

V. faba Misr1

V. faba Giza 843

V. faba Misr1

V. faba Giza 843

V. faba Misr1

V. faba Giza 843

V. faba Misr1

V. faba Giza 843

V. faba Misr1

V. faba Giza 843

V. faba Misr1

V. faba Giza 843

Non-infested control 6.51e 8.28e 18.03g 17.51e 165d 138g 51.58f 56.88h 0.31e 0.27d 0.27b 0.17 d

Infested control 5.89e 4.36f 9.82h 5.31f 118e 238ef 36.98g 47.74j 0.22f 0.18g 0.17e 0.15d

R. leguminosarum 312 6.74e 10.57d 27.52e 26.33 d 255c 273e 54.52e 76.72e 0.44b 0.21f 0.29ab 0.16d

R. leguminosarum 317 9.12d 9.45d 31.06de 25.51d 245c 225f 71.45d 72.23g 0.38bc 0.24e 0.23c 0.16d

R. leguminosarum 302 7.20e 9.67d 32.82cd 26.02d 333b 248ef 84.89b 91.15b 0.41b 0.23ef 0.29ab 0.16d

R. leguminosarum 313 8.37d 10.79d 29.53de 25.05d 338b 235ef 70.19d 65.07i 0.31e 0.22ef 0.22d 0.17d

R. leguminosarum 312+317 10.15b 11.62c 35.05c 32.01bc 333b 418b 88.92b 71.69g 0.46a 0.29c 0.29ab 0.20bc

R. leguminosarum 313+317 9.14d 12.33b 41.51b 30.04c 353b 375c 86.18b 71.13g 0.35cde 0.27d 0.27b 0.18cd

R. leguminosarum 312+302 8.65d 12.68b 42.04b 34.04b 323b 360c 86.19b 74.08f 0.33de 0.29c 0.21cd 0.21b

R. leguminosarum 312+313 8.77d 12.40b 41.51b 33.83b 338b 320d 87.39b 77.99e 0.35cde 0.30b 0.21cd 0.25a

R. leguminosarum 302+317 8.84d 12.43b 43.06b 31.54bc 320b 320d 75.75c 80.69d 0.34cde 0.26d 0.20d 0.23ab

Mixture 10.53b 13.41b 52.09a 43.57a 390a 468a 91.55b 83.56c 0.41b 0.35a 0.26b 0.24a

Azotobacter chroococcum 9.66c 12.65b 23.11f 19.51e 225c 248f 70.05d 70.72g 0.36bcd 0.28cd 0.22cd 0.24a

Compost tea 11.58a 14.44a 28.51e 25.04d 328b 315d 103.13a 96.59a 0.41b 0.24e 0.33a 0.24a

Different letters on the column for each parameter (V. faba Misr 1 & V. faba Giza 843) are differ significantly at p≤ 0.05. Table 1: Influence of different microbial treatments and compost tea on shoot dry weight, nodule number, nodule dry weight, total nitrogen content and chlorophyll a and chlorophyll b contents in faba bean in experiment done in 2010.

All the tested growth parameters of V. faba Giza 843 varied

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Figure 3: Effect microbial cultures and compost tea on total number of O. crenata tubercles fixed on Vicia faba cultivar roots in experiment done in 2011. Different letters on the column for each parameter (V. faba Misr 1 and V. faba Giza 843) are differ significantly at p ≤ 0.05.

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Figure 4: Influence of microbial treatment and compost tea on dry weight of O. crenata fixed on faba bean plant in 2011. Different letters on the column for each parameter (V. faba Misr 1 and V. faba Giza 843) are differing significantly at p ≤ 0.05.

Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205

Page 6 of 9

Volume 4 • Issue 10 • 1000205J Plant Pathol MicrobISSN:2157-7471 JPPM, an open access journal

with the mixture of R. leguminosarum isolates, compared with the infested control. The highest chlorophyll a content (0.46 mg/cm2) was obtained using R. leguminosarum 312+317. On the other hand, chlorophyll b content varied insignificantly among all treatments and the controls. Germination of O. crenata seeds was strongly stimulated by faba bean root exudates. About 81% and 76.5% were obtained by root exudates of Misr 1 and Giza 843, respectively (Figure 5).

In vitro assay of microbial isolates on the broomrape germination: To test the inhibitory effects of different treatments on broomrape germination, broomrape seeds were mixed separately with root exudates (1:1) and their effects were analyzed. Broomrape seed germination percentage reached more than 75% by the two tested root exudates. All microbial culture filtrates significantly decreased broomrape germination, except those of A. chroococcum, compared to

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Figure 5: Influence of microbial culture filterate and compost tea on broomrape germination in presence of Faba bean root exudates. Different letters on the column for each parameter (V. faba Misr 1 & V. faba Giza 843) are differ significantly at p ≤ 0.05.

Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205

Treatments Shoot dry weight(gm/plant)

Nodule number/plant

Nodule dry weight(mg/plant)

Total nitrogen content (mg/plant)

Chlorophyll a content (mg/cm2)

Chlorophyll b content (mg/cm2)

V. faba Misr1

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V. faba Giza 843

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Non-infested control 9.13d 8.14g 19.33e 17.83d 213d 150g 47.81h 51.61g 0.26b 0.28b 0.31 c 0.17 a

Infested control 7.27e 4.04h 12.82f 8.25e 135e 243ef 38.41i 40.82h 0.17e 0.19c 0.22 d 0.16 a

R. leguminosarum 312 8.23d 9.38f 27.31d 27.52c 258cd 255ef 61.30g 73.23f 0.29ab 0.23b 0.44 ab 0.17 a

R. leguminosarum 317 8.45d 11.12d 32.33c 27.33c 268c 225f 82.33de 76.84e 0.23c 0.26b 0.38 ab 0.18 a

R. leguminosarum 302 7.21e 10.12d 36.11c 27.03c 250cd 235f 85.61f 72.52f 0.35ab 0.23b 0.43 ab 0.19 a

R. leguminosarum 313 7.43e 11.23d 32.51c 25.52c 253cd 275ef 70.80f 76.42e 0.22cd 0.24b 0.13 c 0.18 a

R. leguminosarum 312+317 10.33c 11.93c 26.51e 35.53b 340b 393b 88.91c 86.11b 0.29ab 0.32b 0.46 a 0.21 a

R. leguminosarum 313+317 9.35d 10.73d 42.52b 34.04b 390b 338cd 86.81d 81.11d 0.27b 0.33b 0.35 b 0.21 a

R. leguminosarum 312+302 8.33d 10.81d 43.81b 36.06b 363b 382bc 84.72de 82.51c 0.21cd 0.31b 0.33 c 0.22 a

R. leguminosarum 312+313 8.89d 9.61ef 46.33b 34.30b 360b 320d 85.42de 83.23c 0.21cd 0.29b 0.35 b 0.24 a

R. leguminosarum 302+317 9.03d 11.52d 42.11b 33.16b 343b 335cd 77.81e 89.63b 0.23d 0.27b 0.34 b 0.24 a

Mixture 10.5b 13.21b 57.57a 47.55a 460a 443a 93.52b 96.93a 0.26b 0.35a 0.41 ab 0.24 a

Azotobacter chroococcum 9.66d 10.33d 22.82e 19.35d 248cd 245ef 71.23f 75.13e 0.22cd 0.28b 0.36 b 0.24 a

Compost tea 13.17a 15.23a 34.33c 23.53c 280c 298de 101.71a 99.53a 0.33a 0.27b 0.41 ab 0.24 a

Different letters on the column for each parameter (V. faba Misr 1 and V. faba Giza 843) are differ significantly at p ≤ 0.05. Table 2: Influence of different microbial treatments and compost tea on shoot dry weight, nodule number, nodule dry weight, total nitrogen content, chlorophyll a and chlorophyll b contents in faba bean in experiment done in 2011.

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the treatment with root exudates of Misr 1 alone (as a control). The lowest percentage of germination was obtained by compost tea.

In the presence of Giza 843 exudates, relatively lower percentage of germination of the parasite was observed for all microbial culture filtrates, except those of Azotobacter sp. Culture filtrate of Azotobacter sp. was slightly effective in triggering broomrape germination. Significantly, the lowest percentages of broomrape germination (60.5%) were observed for compost tea.

On the other hand, microscopic examination of germinated broomrape seeds treated by the compost monitored certain malformations in the form of germ tubes (Figure 6).

DiscussionNon-pathogenic rhizobacteria can induce a systemic resistance

in plants against many pathogens like fungi, bacteria and viruses. The present study confirm that using rhizobacteria filtrates induced resistance of faba bean against root-parasitic weed, as shown by the significant decrease in the number of broomrape attachments, dry weight of the attached tubercles on faba bean roots and the reduction in percentage of broomrape seed germination. R. leguminosarum play a very important role in agriculture by inducing the number and activity of nitrogen-fixing nodules on the roots of legumes. This symbiosis can reduce the requirements for added nitrogenous fertilizer during the growth of leguminous crops [23,24]. This may give a power for faba bean to de tolerate the infectivity of broomrape. The defense elicited by the tested isolates of R. leguminosarum and compost tea is strongly efficient. Our results are in agreement with Mabrouk et al. [12], who showed that symbiosis with some non-pathogenic R. leguminosarum strains could induce in pea both better development and lower susceptibility to O. crenata. Pseudomonas spp. is typical rhizosphere bacteria with high root-colonizing abilities [25]. These species have been identified as the major group of rhizobacteria with potential as effective candidates for biological control of weeds [26]. In this connection, Zermane et al. [27] showed high biocontrol activity against both O. crenata and O. foetida species and positively influenced faba bean growth by five isolates of P. fluorescens. The nitrogen-fixing bacterium Azospirillum brasilense

has been reported to inhibit germination and radical growth of O. aegyptiaca [28] and Striga hermonthica [29].

The statistical difference in the total number of O. crenata tubercles recorded in 2010 (Figure 1) and 2011 (Figure 3) may be due to some of individual genetic variations in the host, which lead to variation in many metabolic processes. This assumption was confirmed by the studies of Appels and Dvorak [30] and Saghai-Maroof et al. [31], who found clear variations in the ribosomal classes among individuals in wheat and barley, respectively.

Orobanche requires the presence of host root-exuded germination stimulants in order to germinate and orientate its radical to the host root [32,33]. In this study, minimization the impact of stimulant producing plants (faba been) by microbial culture filtrate or compost tea solution could be a suitable option for reducing O. crenata infection. In this context, our investigation showed a reduction in broomrape seed germination by R. leguminosarum isolates. These results are in agreement with Mabrouk et al. [34], who stated that the germination of O. crenata seeds in vitro decreased significantly after inoculation with P.SOM and P.1236 Rhizobium strains. Similarly, bacteria isolates obtained from the soil reduced Striga seeds germination significantly [29,35]. However, there are other reports of effective inhibition of broomrape germination by microorganisms without active destruction of the seed. For example, purified toxins produced by Fusarium species significantly reduced O. ramosa germination [36,37]. Combination of P. fluorescens and P. putida recovered from suppressive soils in sorghum fields in Nigeria inhibited the seed germination of Striga hermonthica [38]. Composts can play a promising role in the biological control of plant diseases. Generally, they provide the slow release of adequate quantities of bio-available energy sustaining the introduced biocontrol agents. Composts affect the release of nutrients to plants directly through their nutrients content or indirectly by their effect on the cation-exchange capacity [39]. Composts may offer further advantages over pesticides and herbicides, in addition to their nutrient contents. They introduce many biological control agents (BCAs) to the soil and establish themselves in the ecosystem.

There are many reports that demonstrate the ability of compost tea to suppress a wide range of both air- and soil-borne plant pathogens [40,41]. Direct application of compost tea on tomato plants significantly reduced disease symptoms caused by three tomato pathogens: Alternaria alternata, Botrytis cinerea and Pyrenochaeta lycopersici [42]. Bharathi et al. [43] reported that PGPR-microorganisms involving in compost tea, can induce the systemic resistance in the plants against different pathogens. In the present study, in addition to the reduction in broomrape seed germination by compost tea, abnormal germ tubes were formed in the presence of natural stimulants. In accordance with our finding, Miche´ et al. [29] reported that abnormal germ tubes of Striga were formed by some bacterial isolates obtained from sorghum soil fields in the presence of the synthetic germination stimulant (GR24). Our work suggested that the reduction in broomrape incidence by compost tea may be due to certain phenotypic mechanisms which acted alone or in combination, including negative effect of natural stimulant broomrape seed germination, radical deformation which hinders the ability to penetrate faba bean root. All these deleterious effects on O. crenata increased growth and vitality of faba bean. These are in agreement with other workers who stated that the compost tea enhance crop fertility by introducing microorganisms that might aid in soil nutrient retention and extraction, and by adding soluble nutrients, further adding to their potential value as a part of an integrated crop management plan [40,44-46]. In conclusion, the present study has

(d) (c

(a) (b)

)

Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205

Figure 6: Effect of faba bean root exudates on germination of broomrape seeds in vitro: (a) Pure faba bean root exudates (b, c and d) mixture of faba bean root exudates and compost tea (v/v). Note: different radical malformation is formed by adding compost tea to faba bean root exudates after 7 days of pre-conditioning period (par 0.5 mm).

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Acknowledgements

The authors thank Dr. W. Al Rodeny for providing faba bean seeds and T. El-Sakhawy for greenhouse culture management.

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Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205

shown the potential use of the mixture of R. leguminosarum isolates and compost tea to suppress O. crenata in area cultivated with V. fabaFurther detailed investigations are needed to better understand the mechanisms of microbial induction to the resistance in faba bean plants to broomrape.

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Citation: El-Halmouch Y, Mehesen A, El-Shanshoury AR (2013) The Potential of Cell-free Cultures of Rhizobium leguminosarum, Azotobacter chroococcum and Compost Tea as Biocontrol Agents for Faba Bean Broomrape (Orobanche crenata Forsk.). J Plant Pathol Microb 4: 205. doi:10.4172/2157-7471.1000205