Evaluation of Pesticides for Control of Konjac Soft-Rotting Bacteria
1 Introduction
Konjac glucomannan(KGM),derived from the tuber of Amorphophallus konjac C.koch,has traditionally been consumed as rubbery jelly,noodles,and other food products in Asia for centuries.It is also used as food additives in Western countries.Clinical studies have indicated KGM supplement normalizes the mouth-to-cecum transit time and relieves constipation for children and adults[1].In order to meet the increasing demand for konjac,more area is employed to plant A.konjac C.koch.With the increase of plant area,some diseases have appeared,and soft rot is the severest one.Huang et al.[2] showed that konjac soft rot was caused by Erwinia carotovora subsp.carotovora.E.carotovora subsp.carotovora is a plant pathogen responsible for producing soft rots in many vegetable and floricultural crops,and responsible for significant economic losses to the konjac industry each year for the causal agent of tuber soft rot[3].Tuber contamination can occur in field and in storage.Infection may occur at any stage of post-harvest handling including washing,grading and packing.Symptoms may develop slowly during cold storage;as temperature increases and conditions become favorable for disease development,thepathogen proliferates,resulting in rapid tissue breakdown.Contaminated tubers that are used as seed,may cause disease problems in field[4].
Bacterial soft rot caused by E.carotovora subsp.carotovora occurs worldwide and is one of the most destructive diseases of plant's tubers(e.g.,potato,konjac).Many researchers have demonstrated the potential of control of soft rot of potato etc.Several salt compounds were tested in vitro as inhibitors of E.carotovora subsp.Carotovora[4].Acidified Oxine was demonstrated to be potentially effective in preventing potato spoilage(caused by E.carotovora)without any significant risks of chemical residual or change of skin color[5].Harris[6] found a chemical compound of 5-nitro-8-hydroxyquinoline was the most effective in control of potato soft rot.Biological control is an alternative[7].Cladera-Olivera et al.[8] showed that the soft rot bacterium E.carotovora from potato was inhibited by a novel bacteriocin-like substance produced by Bacillus licheniformis P40.Biological control offers an environmentally friendly alternative to the use of pesticides for controlling plant diseases.Unfortunately,growers continue to use chemical control over biological agents,and lack of knowledge often contributes to the downfall of a biocontrol agent.Knowledge of the biological environment in which the agent will be used and of how to produce a stable formulation are both critical to successful biocontrol[9]. And also,biological products may not consistently provide a high level of disease control[10].Therefore agricultural disease is controlled mainly with pesticides.
Numerous pesticides have been tested for efficacy against soft rot E.carotovora on potatoes[11,12],but little was done on the soft rot of konjac tubers.Current control strategies aim to reduce soft rot of konjac tubers in field and storage by employing pesticides.In China,a wide range of pesticides is registered for control of bacterial soft rot of crops.However,there is no special pesticide for konjac soft rot,and most pesticides didn't control konjac soft rot effectively.Therefore,growers were driven to use pesticides blindly.Unreasonable use of pesticides has not only given rise to serious environmental problems,but also caused great waste of resources.The aim of this research was to evaluate seven pesticides and their effect on inhibition of soft-rotting bacterium from Konjac.
2 Materials and Methods
2.1 Tested strains and pesticides
Strains of E.carotovora subsp.carotovora were provided by Hubei Key Laboratory of Natural Product Research and Development,China Three Gorges University,Hubei Province,China.
Pesticides were bought from a pesticide market in Yichang city,Hubei Province,China.The active constituent of each pesticide was Streptomycin,Carbendazim,Kasugamycin,Thiophanate+methyl,Oxadixyl+Mancozeb,Mancozeb or Phosethyl-Al+Mancozeb.
2.2 In vitro screening trials
Pesticides were prepared according to their instructions using sterile pure water to obtain the maximum concentrations.The in vitro screening trial of antimicrobial activity of seven pesticides was carried out by disk diffusion test[13] using 100μl of suspension containing 108 CFU/ml of bacterium spread on nutrient agar.Small filter disks were generated using a standard 6mm hole puncher andsterilized by autoclaving,and then dried at 80℃.Paper disks were dipped into each solution for 1min in order to assure that paper disks had been saturated with a pesticide solution,whereas those of the controls were dipped into sterile pure water.The inoculated plates with E.carotovora subsp.carotovora were incubated at 27℃ for 24h.The diameter of the clear zone around the disk was measured and expressed in millimeters as its antibacterial activity.The net zone of inhibition was displayed by subtracting the disk diameter(6mm)from the total zone of inhibition around the disk.Five disk per plate and three plates were used,and each test was run in triplicate[14].
2.3 Determination of minimum inhibitory concentration(MIC)and minimum bactericidal concentration (MBC)
The MIC and MBC were determined by a modification of the broth microdilution method as previously described by Fazeli et al.[15].And there were five dose points(by two-fold serial dilutions from maximum concentrations according to the instructions)tested in this study.Pesticide was prepared in sterile Nutrient broth to reach a series of two-fold dilutions.Bacterial cultures were diluted in Nutrient broth from the stock of 108 CFU/ml before they were added to the pesticide preparations.Final concentration of bacteria in individual tubes was 1~5×105 CFU/ml.Control tubes contained no pesticide.After 24h incubation at 28℃ the test tubes were examined for possible growth and MIC of each pesticide were determined as the lowest concentration that ended with no growth.Tubes containing pesticide concentrations above the MIC were streaked onto nutrient agar plates to achieve MBC of pesticide against the tested strain.Each experiment was performed in triplicate.
2.4 Statistical analysis
The data were statistically analysed by using the one-way ANOVA(SPSS 12.0 for Windows),and were expressed as mean±standard deviation(SD).The differences between treatments were also compared.A P value of less than 0.05 was considered statistically significant.
3 Results
3.1 Screening of pesticide for antibacterial activity
According to the results(Table 1),both Oxadixyl-Mancozeb and Mancozeb showed antibacterial activities at the maximum concentrations within the applied concentration range according to the instructions.
The growth of E.carotovora subsp.carotovora was inhibited by Oxadixyl+Mancozeb at concentration of 1μg/ml,and the inhibition zone reached(5.33±0.71)mm.At the concentration of 1μg/ml,Mancozeb inhibited the growth of tested bacteria,and the inhibition zone diameter was(4.22±0.83)mm.Streptomycin,Carbendazim,Kasugamycin,Thiophanate+methyl and Phosethyl-Al+Mancozeb didn't expressed antibacterial activity against E.carotovora subsp.carotovora.
3.2 MIC and MBC of Oxadixyl-Mancozeb and Mancozeb
MIC and MBC of Oxadixyl+Mancozeb and Mancozeb were determined.Oxadixyl+Mancozeb and Mancozeb were found to have the same MICs and the same MBCs.The MICs of both pesticides were 0.25μg/ml,and MBCs were 1μg/ml(Table 2).
Table 1 Antibacterial activities of seven pesticides agaist E.carotovora subsp.carotovora
According to the results(Table 2),both Oxadixyl-Mancozeb and Mancozeb showed strong bactericidal activity with the MBC at 1μg/ml.E.carotovora subsp.carotovora was sensitive to both Oxadixyl+Mancozeb and Mancozeb.
Table 2 MIC and MBC of Oxadixyl-Mancozeb and Mancozeb against tested bacterium
4 Discussion
Many studies showed that Streptomycin,Carbendazim,Kasugamycin,Thiophanate+methyl,Oxadixyl+Mancozeb,Mancozeb and Phosethyl-Al+Mancozeb could control konjac soft rot caused by E.carotovora subsp.Carotovora[16~19].But,the bacterial soft rot is still a major problem encountered in konjac during postharvest storage and in field.The soft rot bacterium Erwinia carotovora was not inhibited by some pesticides used by people.This problem could be attributed to unreasable use of pesticides,so it is necessary to assess effect of each pesticide before application.Seven pesticides were screened against the soft rot pathogens of konjac tubers.Oxadixyl+Mancozeb and Mancozeb were the only pesticides which completely prevented infection at concentration of 1μg/ml according to their instructions.Nevertheless,Streptomycin,Carbendazim,Kasugamycin,Thiophanate+methyl and Phosethyl-Al+Mancozeb didn't exhibit antibacterial activities against soft-rot causing strain at their higher applied concentrations.Therefore when growers faced severe problems with soft rot,they employed a lot of pesticides to protect konjac tuber,but the disease couldn't be controlled effectively.Oxadixyl+Mancozeb and Mancozeb inhibited soft rot bacterium at 0.25μg/ml,but did not give complete protection against infection for longer time.In order to control konjac soft rot,these pesticides could not be used at a concentration lower than 1μg/ml.According to these observations,we can speculate that Oxadixyl+Mancozeb and Mancozeb could be used to control konjac soft rot in field and storage conditions.Even though Mancozeb had the same MIC and MBC as Oxadixyl+Mancozeb,its diameter of the net zone of inhibition was significant differences from that of Oxadixyl+Mancozeb at 1μg/ml(P<0.05).Oxadixyl+Mancozeb expressed strongerbactericidal activity against E.carotovora subsp.carotovora.Mixtures of certain pesticides may act synergistically to augment the inhibition of disease development[4].Study of the antibacterial activity of compounds in vitro may have application in protecting plant from developing disease[20~22].Pesticides that showed no inhibition of soft rot bacterium in the present study included Streptomycin,Carbendazim,Kasugamycin,Thiophanate+methyl and Phosethyl-Al+Mancozeb.Therefore,although all of these pesticides were always employed to prevent and cure soft rot disease,the loss of soft rot is still severe.When used at the manufacturer's recommended concentration,Oxadixyl+Mancozeb and Mancozeb all displayed antibacterial activities against soft-rot causing bacterium.There is no special pesticide for konjac soft rot in China,and the database(a list of marketing companies is available on the database)does not give details of what problems each pesticide controls from the company that produces the pesticide,in vitro study maybe give the growers suggestions about choosing pesticides.Before growers buy or use any pesticide,ask themselves whether it is really necessary to control the disease that they want to get rid of.Therefore protection of konjac with suitable pesticides becomes a necessity.Chemical pesticides play a great role in eliminating agricultual pathogens,but because pesticides are used blindly and not scientifically,environment are polluted seriously.This paper approached ways of selecting pesticides to control konjac soft rot,using scientifically and effectively chemical pesticides,for the purpose of reducing environmental pollution,and protecting environment.
Although the current study indicates that there is potential for the use of several pesticides as chemical agents to control disease caused by E.carotovora subsp.carotovora,further studies are needed to examine possible inhibitory effects in vivo.More research is also needed on the delivery methods for effective use strategies including spray application during post-harvest handling,application timing at growth stage or application as preservatives in storage facilities[4].
In summary,our data demonstrate that pesticides of Oxadixyl+Mancozeb and Mancozeb inhibited the activity of konjac soft-rot causing bacterium.This disease could be controlled with chemicals such as Oxadixyl+Mancozeb and Mancozeb.Moreover,Oxadixyl+Mancozeb expressed stronger antibacterial activity than Mancozeb.Even though pesticides of various kinds(e.g.,Streptomycin,Carbendazim,Kasugamycin,Thiophanate+methyl,Phosethyl-Al+Mancozeb)have been used on a large scale in China to protect crops from damages inflicted by diseases,none of them were inhibitors of the soft rot pathogen at their recommended concentrations.
Acknowlegements
This work was supported by the Scientific Research Foundation of China Three Gorges University(No.0620060113)and Hubei Provincial Department of Education(No.Q200713002).
