CHEMICAL CONTROL OF SOFT ROT OF VEGETABLES CAUSED BY ERWINIA CAROTOVORA
Antibacterial activity of five chemicals namely vinegar, copper compound (Cupravit), potassium chloride, calcium chloride and boric acid was evaluated in vitro against the soft rot causing bacterial pathogen, Erwinia carotovora subsp. carotovora. Among them vinegar, Cupravit and boric acid showed highly antibacterial activity. Complete inhibition (100%) of the growth of test pathogen was observed in vinegar at 1.0 and 2.0%, Cupravit at 0.05 and 0.1% and boric acid at 0.1% concentrations of the formulated product. These three chemicals were tested as pre-storage treatment to control soft rot of tomato, cucumber and okra in storage. Each of the fresh vegetables were treated by dipping in 0.5% solution of vinegar, 0.1% solution of Cupravit and 0.1% solution of boric acid individually for 30 min and inoculated with the soft rot bacterial suspension by single needle puncture method. All the three vegetables under untreated control were completely damaged within 9 days of inoculation. Vinegar was found more effective in controlling soft rot of the three vegetables compared to Cupravit and boric acid. On 3rd day of treatment with chemicals soft rotted area ranged from 1.2 to 2.5%, while in control it was 3.0, 7.5 and 13.4% for tomato, cucumber and okra, respectively. On 6th day of treatment soft rotted area ranged from 3.33 to 7.66%, 3.5 to 7.5% and 6.0 to 11.6% in tomato, cucumber and okra, respectively. Soft rotted area under control was 33.33, 27.5 and 33.0% for tomato, cucumber and okra, respectively. On 9th day of treatment soft rotted area in tomato was 40.0, 71.66 and 80.0% vinegar, Cupravit and boric acid, respectively. Soft rotted area in cucumber treated with vinegar, Cupravit and boric acid was 22.5, 76.0 and 80.0%, respectively. The soft rot area of okra was 70.0, 90.0 and 98.0% with vinegar, Cupravit and boric acid, respectively. Among the three chemicals vinegar showed the best performance to control soft rot of tomato, cucumber and okra caused by E. carotovora subsp. carotovora.
Generally, shelf-life of vegetables is maximum of 7-14 days at 5ºC depending on crop species and quality. The shelf-life is reduced due to endogenous biochemical and physiological changes in the produces as well as the attack of spoilage microorganisms (Garcia Gimeno and Zurera Cosano 1997, Heard 1999, 2002). Vegetables contains high quantity of water resulting in a high water activity (>99%). Tomato, cucumber and okra are common fruit vegetables in Bangladesh. They are grown in the country almost throughout the year. Vegetables carry natural non-pathogenic epiphytic microflora. During plant growth, harvest, transportation, handling and post harvest processing, the produce may be physically damaged. Post harvest bacterial soft rot is the prime cause of such losses. The disease may cause 15-30% loss of harvested crops. Due to lack of proper management practices during harvesting, storage and marketing, the damage may be greatly extended. Wounded areas on plant tissues provide a suitable substrate for microbial growth providing sufficient nutrients available (King et al. 1991, Zagory 1999). Bacterial soft rot causing bacteria have high potentiality for spoilage of vegetables because they produce pectolytic enzymes, which have capacity to degrade pectin, a major constituent of the primary plant cell wall and middle lamella (Brummell 2006). Species of Erwinia belonging to the carotovora group are usually referred to as the soft rot bacteria (Lelliot and Dickey 1984). Many scientists recommended various chemicals to control the soft rot bacteria (Chen and Lin 2000, Khair 2004, Wright et al. 2005). Dipping potato tubers in acetyl-salicylic acid (ASA) increases resistance against E. carotovora subsp. carotovora (Bokshi et al. 2003). Salt treatments can inhibit plant pathogens or suppress toxin production by pathogens (McGuire and Kelman 1986, Olivier et al.1998). Saleh and Huang (1997) reported that benzoic acid and sodium benzoate at 1, 5 and 10 mM inhibited soft rot bacterial growth. Effectiveness of acetic acid, boric acid and bleaching powder against soft rot bacteria of onion Burkholderia cepacia was reported by Rahman et al. (2013). The above research findings suggested that chemical treatment of vegetables may be effective method to control post-harvest bacterial soft rot disease of vegetables. The present investigation was carried out to find out effective chemicals to control soft rot disease of vegetables in storage.
MATERIALS AND METHODS
In vitro evaluation of chemicals against soft rot bacteria
A preliminary experiment was conducted to evaluate five chemicals namely vinegar, boric acid, calcium chloride, potassium chloride and Cupravit (a copper compound) for their bactericidal activity against a standard isolate P 138 of soft rot bacteria, Erwinia carotovora subsp. carotovora under in vitro conditions. Boric acid, calcium chloride, potassium chloride and Cupravit were tested at 0.02%, 0.05% and 0.10% (w/v), and vinegar was tested at 0.5%, 1.0% and 2.0% concentrations. A standard bacterial growth medium, yeast extract peptone dextrose agar (YPDA) was used as basic medium. After cooking, the YPDA medium was amended with each chemical at the selected concentrations and mixed thoroughly. The amended medium was autoclaved for 20 min at 121ºC under 1.1 kg/cm² pressure. The sterilized medium was poured into sterilized Petri dishes at 20 ml/plate and allowed to solidify under sterile condition. Five plates (replications) were used for each treatment.
After solidification, the amended YPDA in Petri plates were spot inoculated with E. carotovora subsp. carotovora P 138 suspension (ca. 108 cfu/ml) and incubated at 30ºC in an incubator. To prepare the inocula of E. carotovora subsp. carotovora P 138 causing soft rot in vegetables, bacterial cells were collected from 24 hr old fresh culture on YPDA and suspended in sterilized distilled water. Additional plates without any chemical were maintained, which served as control. Growth of the test bacterium was observed up to 14 days of inoculation and antibacterial activity of the chemical was determined.
Effect of the chemicals on bacterial colony growth was recorded in terms of percent inhibition of radial colony diameter based on colony growth on control plates. The percent inhibition was computed using a standard formula (Sundar et al. 1995) as shown below:
Efficacy of the chemicals to control soft rot of vegetables in storage
Based on the results of the in vitro test, vinegar, Cupravit and boric acid were selected to evaluate their efficacy to control soft rot of tomato, cucumber and okra in storage. Each of the fresh vegetables were dipped in 0.5% suspension of vinegar, 0.1% suspension of Cupravit and 0.1% suspension of boric acid individually for 30 min prior to inoculation with soft rot bacterial (E. carotovora subsp. carotovora) suspension. Three fruits of each vegetable were tested for each treatment. After dipping in chemical suspensions, the vegetables were air dried at room temperature. Chemical treated vegetables were inoculated with the bacterial inocula by single needle puncture method. Fresh cultures of soft rot bacteria grown on YPDA at 28ºC for 24 hr were used as inocula. Inoculated vegetables were kept in plastic basket and stored at room temperature for 9 days. Untreated control was maintained for each vegetable, which was inoculated with the pathogen but not treated with any chemical. Incidence of soft rot was recorded on 3rd, 6th and 9th day of inoculation. The severity was measured in terms of percentage of surface area diseased.
RESULTS AND DISCUSSION
In vitro evaluation of five chemicals against soft rot bacteria
Among five chemicals tested in-vitro, calcium chloride and potassium chloride did not show any antibacterial activity against E. carotovora subsp. carotovora (data not included in table). Out of three effective chemicals, vinegar was found most effective against. Complete inhibition (100%) of colony growth was observed at two higher concentrations of vinegar (1.0 and 2.0%), while 90% growth inhibition was recorded at 0.5% concentration. Complete inhibition (100%) of colony growth was also found with two higher concentrations (0.05 and 0.1%) of Cupravit. At 0.02% of Cupravit, inhibition was 70%. In case of boric acid, complete inhibition (100%) of the bacterial colony was recorded only from the highest concentration of 0.1%. Growth inhibition with boric acid at 0.02 and 0.05% concentration was 50 and 80%, respectively (Table 1). Laboratory evaluation of these three chemicals has given encouraging results, indicating their potential for control of bacterial soft rot disease of vegetables. Based on the results of the in vitro test these three chemicals were selected to evaluate their effectiveness to control soft rot disease of vegetables in storage.
Table 1. Antibacterial activity of vinegar, Cupravit and boric acid against soft rot bacterium E. carotovora subsp. carotovora P 138 in vitro
|Chemical||Concentration (%)||% inhibition|
Efficacy of selected chemicals to control soft rot disease of three vegetables
Pre-storage treatment of tomato, cucumber and okra with the suspensions of vinegar, Cupravit and boric acid at different concentrations reduced the severity of soft rot disease in storage. Mean percentage of surface area rotted of the three vegetables was the lowest in case of vinegar treatment, which was followed by Cupravit and boric acid. In tomato, on 3rd day of treatment surface diseased area ranged from 1.33 to 2.0%, while it was 3.0% under control. On 6th day of treatment, soft rot area increased to 33.33% under control, while it was 3.33, 7.66 and 6.66%, respectively in vinegar, Cupravit and boric acid treatment. On 9th day of treatment, tomato was destroyed completely under control, while SDA was 40.0, 71.66 and 80.0% under vinegar, Cupravit and boric acid, respectively (Fig. 1).
In cucumber, on 3rd day of treatment SDA in different treatments ranged from 1.5 to 2.5% but in control it was 7.5%. On 6th day, SDA ranged from 3.5 to 7.5%, while it was 27.5% under control. On 9th day SDA of cucumber increased drastically with 100% under control, while it was 22.5, 76.0 and 80.0% under vinegar, Cupravit and boric acid treatment, respectively (Fig. 2).
In okra, on 3rd day of treatment SDA ranged from 1.2 to 1.8% under different treatments but it was 13.4% under control. On 6th day of treatment SDA ranged from 6.0 to 11.6% under three treatments while it was 33.0% under control. On 9th day of treatment the SDA was 70.0, 90.0 and 98.0% under vinegar, Cupravit and boric acid, respectively while it was 100% under control (Fig. 3).
The results suggested that vinegar, Cupravit and boric acid can control soft rot disease of tomato, cucumber and okra to some extent in storage. Other researchers also reported similar results. Mills et al. (2006) tested several salt compounds as inhibitors of E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora causing bacterial soft rot of potato. In-vitro studies with sodium metabisulphate, propyl paraben, alum, potassium sorbate, calcium propionate and copper sulphate pentahydrate showed that these chemicals were completely inhibitory at lowest concentration (0.002M). Salts including calcium propionate and calcium chloride, potassium sulfate and ammonium phosphate reduce tissue maceration of potato tubers caused by E. carotovora (McGuire and Kelman 1986, Hajhamed et al. 2007). Farrar et al. (2009) reported antimicrobial agents such as peroxy-acetic acid and hydrogen peroxide, applied as a final rinse in the packing process, are effective in reducing the potato tuber surface populations of soft-rot organisms, resulting in less post-harvest loss due to Erwinia rot. Rahman et al. (2013) found bleaching powder, boric acid and acetic acid effective to control the soft rot disease of potato and onion in storage. So, the results of the present study partially agreed to the findings of Farrar et al. (2009) and Rahman et al. (2013). Among the three effective chemicals, vinegar is generally used as preservative for different types of jam, jelly and pickles. Use of vinegar as chemical treatment of vegetables may not create health hazards. Therefore, vinegar may be used to control soft rot disease of vegetables caused by E. carotovora. The residual effect of Cupravit and boric acid in different vegetables is unknown. Therefore, use of Cupravit and boric acid in treating vegetables needs further study to know the residual effect of these chemicals.
The authors express their gratefulness to the Research Management Committee of Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur for providing the fund to conduct the research work.
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