- Open Access
Biological control of potato soft rot caused by Erwinia carotovora subsp. carotovora
© The Author(s) 2018
- Received: 10 June 2018
- Accepted: 19 November 2018
- Published: 3 December 2018
Four bioagents, Bacillus subtilis, Pseudomonas fluorescence, P. aeruginosa, and Streptomyces spp., were used in vitro and in vivo against two pathogenic isolates of Erwinia carotovora subsp. carotovora (Ecc1 and Ecc2), the causal agent of potato soft rot. In vitro Streptomyces spp. showed the strongest effect against Ecc1 and Ecc2 and gave the highest values of the inhibition zones, being 37 and 40 mm, respectively followed by P. fluorescence, B. subtilis, and P. aeruginosa, where the inhibition zones reached, respectively, 32, 28, and 24 mm against Ecc1 and 35, 29, and 26 mm against Ecc2. Also, these results confirmed those of the in vivo experiment (in pots) since Streptomyces spp. bioagent exhibited the lowest number of infected tubers followed by P. fluorescence, B. subtilis, and P. aeruginosa, respectively, against the two isolates Ecc1 and Ecc2. Also, disease severity of soft rot caused by each of the two isolates, Ecc1 and Ecc2, was decreased by using bioagents, and the lowest disease severity values were obtained by using Streptomyces spp., P. fluorescence, B. subtilis, and P. aeruginosa, respectively.
- Biological control
- Erwinia carotovora subsp. carotovora
- Potato soft rot
Potato, Solanum tuberosum L., is one of the most important food and crops worldwide, and its production in developing countries increased at the rate of 2.8% annually (CIP 1995). Bacterial soft rot and black leg are probably the most serious diseases in terms of crop losses. Infection by late blight and dry rot pathogens is significant, not only because of the damage they cause to potato tubers, but also because they provide potential avenues of entrance of secondary invasion by Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica (Lui et al. 2005).
Microorganisms that can grow in potato rhizosphere are ideal to be used as biocontrol agents, since the rhizosphere provides the front line defense for roots and tubers against attack by pathogens (Kabeil et al. 2008).
Pseudomonas fluorescence, Bacillus subtilis, and E. herbicola showed activity against E. carotovora subsp. carotovora (Vanneste and Yu et al. 1996). Streptomyces is a well-known genus of the order Actinomycetales family. They usually inhabit soil and commonly enhance soil fertility. These prokaryotes have characteristics which make them useful as biocontrol agents against bacterial plant pathogens (Keiser et al. 2000). Biological control is considered as one of the most important methods to control bacterial soft rot disease in potato tubers (Algeblawi and Adam 2013).
In this study, we aimed to evaluate in vitro and in vivo effectiveness of four bioagents, B. subtilis, P. fluorescence, P. aeruginosa, and Streptomyces spp., against E. carotovora subsp. carotovora (Ecc1 and Ecc2), the causal agent of potato soft rot.
Potato seeds (Solanum tuberosum L., Diamante variety) were obtained from the Horticulture Department, Agricultural Research Center, Giza, Egypt.
Two isolates of E. carotovora subsp. carotovora (Ecc1 and Ecc2) WPP17 were obtained from the Bacterial Disease Research Department, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt.
The antagonistic bacteria and actinomycetes (B. subtilis, P. fluorescence, P. aeruginosa, and Streptomyces spp.) were obtained from Bacteriological Lab, Faculty of Science, Zagazig University, Egypt.
Sensitivity of E. carotovora subsp. carotovora isolates to antagonistic microorganisms (in vitro)
Antagonistic effect of B. subtilis, P. fluorescence, P. aeruginosa, and Streptomyces spp. on two isolates of E. carotovora subsp. carotovora (Ecc1 and Ecc2) was studied. The suspension representing each of the two isolates of E. carotovora subsp. carotovora (1 × 105 CFU/cm3) was spread on the surface of the Czabe-dox agar media in petri dishes by a sterilized L-shaped glass rod spreader followed by placing a 7-mm diameter agar disk cut from the margin of a culture grown in a plate on which the biocontrol strain had been grown for 48 h at 28 °C for bacteria and for 7 days for actinomycetes in the center of each plate. Inhibition zone diameter of two isolates of E. carotovora subsp. carotovora (Ecc1 and Ecc2) was measured after incubation at 28 °C for 48 h, and mean value of three replicates was calculated.
Effect of bacterial bioagents on infected potato tubers with E. carotovora subsp. carotovora (in vivo)
Pots (30 cm diameter) were sterilized by soaking in formalin (5%) for 5 min and left for 1 week to get rid of the poisonous effect of formalin. Pots were filled with autoclaved soil (autoclaved for 3 h for three successive days). Inoculation of both bioagents or pathogenic two isolates of E. carotovora subsp. carotovora (Ecc1 and Ecc2) was prepared by growing each bacterium in a conical flask containing 200 ml autoclaved sucrose-peptone liquid medium incubated at 30 °C for 48 h. Actinomycetes inoculum was prepared by growing Streptomyces sp. in flasks containing autoclaved starch nitrate liquid medium and incubated at 30 °C for 7 days.
Control (untreated tubers planted in sterilized soil),
Tubers coated by each of the antagonistic microorganisms alone (bacteria or actinomycetes) and planted in soil,
Tubers planted in soil inoculated by E. carotovora subsp. carotovora (Ecc1),
Tubers planted in soil inoculated by E. carotovora subsp. carotovora (Ecc2),
Tubers coated by each of the antagonistic bacteria or actinomycetes and planted in soil inoculated by E. carotovora subsp. carotovora (Ecc1)
Tubers coated by each of the antagonistic bacteria or actinomycetes and planted in soil inoculated by E. carotovora subsp. carotovora (Ecc2).
0 = superficial flack (no rot)
1 = 1–24% of the surface decayed
2 = 25–49% of the surface decayed
3 = 50–74% of the surface decayed
4 = 75% or more of the surface decayed
The decay index (DI) for each treatment was obtained as follows:
Experimental design and statistical analysis
All treatments in this study were arranged in a complete randomized design. The obtained data were subjected to analysis of variance using the general linear module procedure of Anonymous (1985), where appropriate treatment means were separated using Duncan’s multiple range test (Duncan 1955) and all percentages were transferred to the analysis before statistical analysis.
In vitro antagonistic effect of bacterial bioagents on two isolates of E. carotovora subsp. carotovora
In vitro antagonistic activity of Bacillus subtilis, Pseudomonas fluorescence, Pseudomonas aeruginosa, and Streptomyces spp. with two isolates of Erwinia carotovora subsp. carotovora (Ecc1 and Ecc2)
Inhibition zone (mm)
Erwinia carotovora (Ecc1)
Second strain (Ecc2)
28 ± 0.47e
29 ± 0.57e
32 ± 0.35d
35 ± 0.45c
24 ± 0.47g
26 ± 0.52f
37 ± 0.35b
40 ± 0.42a
In vivo antagonistic effect of bacterial bioagents against the two isolates of E. carotovora subsp. carotovora
In vivo biological control of potato soft rot caused by two isolates of Erwinia carotovora subsp. carotovora by using Bacillus subtilis, Pseudomonas fluorescence, Pseudomonas aeruginosa, and Streptomyces spp.
No. of healthy tubers
No. of infected tubers
Infection % to control
Tubers only (control)
Tubers + Ecc1
Tubers + Ecc1 + B. subtilis
Tubers + Ecc1 + P. fluorescence.
Tubers + Ecc1 + P. aeruginosa
Tubers + Ecc1 + Streptomyces spp.
Tubers + Ecc2
Tubers +Ecc2 + B. subtilis
Tubers + Ecc1 + P. fluorescence
Plant + Ecc1 + P. aeruginosa
Plant + Ecc1 + Streptomyces spp.
The obtained results show that soft rot disease caused by the two isolates of E. carotovora subsp. carotovora (Ecc1 and Ecc2) can be biologically controlled by Streptomyces spp., B. subtilis, P. fluorescence, and P. aeruginosa, respectively. These results are in agreement with those recorded by Ryan et al. (2001) who reported that B. subtilis GBO3 and B. amyloliquefaciens IN937a were able to promote plant growth indirectly through induced systematic resistance (ISR); this happens through secretion of volatiles which in turn activate an ISR pathway in Arabidopsis seedlings challenged with the soft rot pathogen E. carotovora subsp. carotovora. Lemessa and Zeller (2007) showed that using antagonistic isolates like B. subtilis and P. macerans has potential in potato bio protection or as a part of an integrated disease management package for bacterial diseases. Seaf Elyazel (2008) revealed that Streptomyces spp. (gram-positive filamentous bacteria) can produce and secrete a biologically active compound including antibiotics, ionophores, hydrolytic enzymes (protease, nuclease, lipase, and a variety of enzymes hydrolyzing polysaccharides), and enzyme inhibitors. These characteristics make Streptomyces spp. attractive candidates for biological control agents against soil-borne plant pathogens. Salem and Askora (2012) confirmed that the brown rot disease in Egyptian potato tubers caused by Ralstonia solanacearum can biologically be controlled by using the bioagents P. fluorescence, B. subtilis, P. aeruginosa, and Streptomyces spp., and the latter gave the effective results in controlling the brown rot in potato tubers. Algeblawi and Adam (2013) reported that the bioagents, i.e., P. fluorescence, B. subtilis, and B. thuringiensis, reduced soft rot disease in potato tubers caused by E. carotovora subsp. carotovora in pot experiment. The best results were obtained when isolates of P. fluorescence and B. subtilis were applied against E. carotovora subsp. carotovora compared to control treatment.
The results of the current study indicated that the usage of four bacteria as bioagents was effective in decreasing the severity of E. carotovora subsp. carotovora (Ecc1 and Ecc2) infection in potato tubers. It is worthy to note that Streptomyces spp. showed more pronounced effects against E. carotovora subsp. carotovora (Ecc1 and Ecc2) in either in vivo or in vitro studies than the other three bacterial bioagents.
The authors are grateful to the Horticulture Department, Agricultural Research Center, Giza, Egypt; Bacterial Disease Research Department, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt; and Bacteriological Lab, Faculty of Science, Zagazig University, Egypt, for the facilities provided for conducting the study.
We are working on our own fund.
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The dataset supporting the conclusions of this article are included within the article.
First author EA is responsible for the implementation and conception or design of the experimental work and for designing and supervising the study. Second author YM is responsible for revising the paper scientifically, checking analysis, and interpreting data. Both authors read and approved the final manuscript.
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