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Isolation, Characterization, and Identification of Biological Control Agent for Potato Soft Rot in Bangladesh

M. M. Rahman
Department of Plant Pathology, Bangabadhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh

M. E. Ali
Nanotechnology and Catalysis Research Center, Universiti Malaya, 50603 Kuala Lumpur, Malaysia

A. A. Khan
Department of Plant Pathology, Bangabadhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh

A. M. Akanda
Department of Plant Pathology, Bangabadhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh

Md. Kamal Uddin
Institute of Tropical Agriculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Malaysia

U. Hashim
Institute of Nano Electronic Engineering, Universiti Malaysia Perlis, 01000 Kangar, Malaysia

S. B. Abd Hamid
Nanotechnology and Catalysis Research Center, Universiti Malaya, 50603 Kuala Lumpur, Malaysia

Abstract/ Executive Summary:

A total of 91 isolates of probable antagonistic bacteria of potato soft rot bacterium Erwinia carotovora subsp. carotovora (Ecc) were extracted from rhizospheres and endophytes of various crop plants, different soil varieties, and atmospheres in the potato farming areas of Bangladesh. Antibacterial activity of the isolated probable antagonistic bacteria was tested in vitro against the previously identified most common and most virulent soft rot causing bacterial strain Ecc P-138. Only two isolates E-45 and E-65 significantly inhibited the in vitro growth of Ecc P-138. Physiological, biochemical, and carbon source utilization tests identified isolate E-65 as a member of the genus Bacillus and the isolate E-45 as Lactobacillus sp. The stronger antagonistic activity against Ecc P-138 was found in E-65 in vitro screening and storage potatoes. E-65 reduced the soft rot infection to 22-week storage potatoes of different varieties by 32.5–62.5% in model experiment, demonstrating its strong potential to be used as an effective biological control agent for the major pectolytic bacteria Ecc. The highest (62.5%) antagonistic effect of E-65 was observed in the Granola and the lowest (32.7%) of that was found in the Cardinal varieties of the Bangladeshi potatoes. The findings suggest that isolate E-65 could be exploited as a biocontrol agent for potato tubers.

Keywords: Identification of Biological Control, Potato, Soft Rot, Bangladesh

Location: Department of Plant Pathology, Bangabadhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Potato, Diseases

Objectives:

Considering the above facts, we extensively searched for an effective biological control agent for the most common potato soft rot strain Ecc P-138 of Bangladesh. The antagonistic activity against soft rot of potato strain Ecc P138 was tested in in vitro and storage experiments, and the strain identification was performed through physiological and biochemical identification schemes.

Methodology:

2.1. Isolation of Bacteria Antagonistic to Soft Rot Ecc P-138. As a potential source of antagonistic bacteria of potato soft rot Ecc, rhizospheres and endophytes of different crop plants as well soils and atmosphere of major potato farming areas in Bangladesh were selected. For the isolation of rhizospheric bacteria, soil samples were collected from rhizosphere of potato, onion, papaya, rice, tomato, garlic, zinger, and turmeric. Dilution plate techniques were followed, and yeast peptone dextrose agar (YPDA) was used as a basic medium. Ten gram (10 g) of soil was taken from each rhizosphere soil samples in a beaker and was mixed thoroughly in 100 mL distilled water on a rotary shaker (250 rpm). Then, the suspension was allowed to settle down to soil for 10 min. After sedimentation, the soil suspension was taken from the upper part and diluted to 103–104 x in distilled water. The diluted soil suspension was streaked on petri dishes containing YPDA and incubated at room temperature for 24–48 h. At the end of the incubation, different types of bacterial colonies that appeared on the medium were selected and restreaked for pure culture. The pure culture of selected bacterial isolates was preserved in test tubes containing sterilized water for antibacterial evaluation. To isolate endophytic bacteria, fresh and diseased plant specimens of root, stem, and leaves of various plants including potatoes and onions were collected from different locations of Bangladesh. Desired bacteria were isolated following streak-plate technique using the medium YPDA. After 24– 48 h of incubation, bacterial colonies were observed on the medium. Isolated bacterial colonies were restreaked on YPDA media for obtaining pure culture. Colonies selected from isolated plates were transferred into test tubes containing 5 mL sterilized distilled water. The tubes with the bacterial suspension were preserved at room temperature. Some bacterial isolates were isolated from soil mixed with compost following the procedures as described in case of rhizospheric bacteria. Several bacteria were isolated from atmosphere following air trapping. Three petri dishes containing YPDA were placed in the field and also in the laboratory of Bangabandhu Sheikh Mujibor Rahman University (BSMRAU) and were kept for 5, 10, and 15 min. The petri dishes were covered with the lids and incubated at room temperature for 24–48 h. The bacterial colonies developed on the medium were restreaked for pure culture and preserved in test tubes containing sterilized distilled water. 2.2. Antagonistic Activity Test. Antagonistic activity of the bacterial isolates was tested in vitro using plate chloroform method. Briefly, one loop full of 1-2-day-old probable antagonistic bacterial colony grown in YPDA medium was transferred to the center of a petri dish containing 20 mL of YPDA. The plates were incubated at 30⁰C for 2 to 3 days. When the bacteria colonies were formed as several millimeters in diameter, the plate was turned upside down. A sheet of filter paper was placed in the petri dish lid with 0.5 mL of chloroform. The dish was kept at room temperature for 2 h. After complete evaporation of chloroform vapor, 5 mL suspension of indicator bacteria (ca. 108 cfu/mL) was overlaid on each plate. Here, the soft rot bacterial strain, E. carotovora subsp. carotovora (Ecc) P-138, was used as an indicator bacterium. The plate thus prepared was incubated at 30⁰C for 2 days. When an inhibition zone appeared, its diameter was measured to evaluate the antibacterial activity of the probable antagonistic bacteria. The bacterial isolates which showed antagonistic effect against indicator bacteria were selected for the further studies. 2.3. Biological Control of Soft Rot Disease under Storage Conditions. To evaluate the effectiveness of the selected antagonistic bacteria in reducing soft rot infection in storage potatoes, 700 g of fresh tubers of each three potato varieties, Cardinal, Diamant, and Granola, were dipped in suspensions of antagonistic bacterium E-65 (ca. of 107–108 cfu/mL), for 30 min and air-dried separately. The treated potato tubers were inoculated with soft rot bacteria E. carotovora subsp. carotovora P-138 by spraying with inoculum suspensions (107–108 cfu/mL) with an atomizer. Inoculated potato tubers bulbs were air-dried and stored separately in net bags at room temperature. Data on soft rot incidence was recorded after 2, 6, 10, 14, 18, and 22 weeks of inoculation. Number and weight of soft rot infected tubers were recorded.

Source of Information: The Scientific World Journal; Volume 2012, Article ID 723293, 6 pages

Article Link (Online Journal): doi:10.1100/2012/723293

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Two antagonistic bacterial isolates E-65 and E-45 of the common soft rot bacterial pathogen were isolated from the endophytes of potato and papaya of Bangladesh. The isolate E-65 was a strong antagonist of soft rot bacteria E. carotovora subsp. carotovora P-138. This was confirmed through the in vitro and storage experiments. Biochemical and physiological tests identified E-65 as a Bacillus sp. and E-45 as a Lactobacillus sp. The ability of these isolates to suppress the growth of various phytopathogenic bacteria makes them potential biocontrol agents.

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