Biological control of Pectobacterium carotovorum subsp. carotovorum, the causal agent of bacterial soft rot in vegetables, in vitro and in vivo tests

Several chemical bactericides were applied for controlling soft rot bacteria, Pectobacterium carotovorum subsp. carotovorum, which causes the destructive soft rot disease to many economically important vegetables, but because of their toxic hazards on human and environment became limit. The biocontrol was applied to control many plant pathogens. Therefore, this work is aimed to study the antagonistic activity of bacterial agents, i.e. Bacillus subtilis, Bacillus pumilus, Bacillus megaterium and Pseudomonas fluorescens, and fugal agents, i.e. Trichoderma harzianum, Trichoderma viride and Trichoderma virens, to control bacterial soft rot disease under in vitro and in vivo tests. The tested treatments could protect the potato tubers against the development of soft rot. T. viride and T. virens were highly effective in reducing soft rot symptoms on inoculated potato tuber slices, when applied at the same time or 2 h before pathogen inoculation, while B. megaterium and T. harzianum were highly effective when applied at the same time or 2 h after pathogen inoculation. In whole potato tubers technique, B.pumilus highly protected the stored potato tuber under artificially infection conditions, than P. fluorescens, T. harzianum, B. subtilis, T. viride, T. virens and B. megaterium, respectively. Application of fungal agents or specify the bacterial species can play an important role in controlling bacterial soft rot disease in vegetables and increase the stored periods of potato tubers under storage conditions without any toxic effects.


Background
Pectobacterium carotovorum subsp. carotovorum (Syn. Erwinia carotovora subsp. carotovora) causes the destructive soft rot disease to many economically important vegetables such as carrot, cabbage, cucumber, eggplant, garlic, onion, pepper, potato, radish, sweet potato, squash and tomato (Opara and Asuquo 2016), where the disease can be detected in the field, transmit, storage and market. The soft rot bacteria can successfully penetrate the plant, through the wounds or natural openings, causing economic damage to fleshy vegetables by producing many cell wall-degrading enzymes (Péromblon 2002;Bhat et al. 2010). Application of chemical bactericides for controlling soft rot bacteria is not favored because of their non-persistence, side toxic effects, high cost as well as development of resistance in bacterial populations (Jones et al. 1996;Vanneste 2000).
Therefore, biological control may be one of the good crop protection methods for controlling bacterial soft Abd-El-Khair et al. Bull Natl Res Cent (2021) 45:37 rot disease by application of Trichoderma spp., Bacillus spp. or Pseudomonas spp. which widely applied as biological agents against many soil-borne pathogens (Wulff et al. 2003;Alabouvette et al. 2006). Applications of B. cereus, B. subtilis, B. megaterium or B. pumilus showed good activity against P. carotovorum subsp. carotovorum in vitro tests using the disk plate method (Issazadeh et al. 2012). Pseudomonas spp. also controlled the bacterial soft rot disease in Valerian rhizome as well as significantly increased the fresh and dry weights of root (Ghods-Alavi et al. 2012). When neem cake applied in soil with P. putida as seeds treatment, it could reduce E. carotovora infection as well as significantly increased the carrot yield under field conditions (Sowmya et al. 2012). Pseudomonas fluorescens and B. subtilis showed the highest inhibitory effects against E. carotovora subsp. carotovora in vitro and in the pot experiment (Algeblawi and Adam 2013). Bacillus spp. and B. pumilus controlled P. carotovorum subsp. carotovorum in chili in vivo, where all treatments showed a higher reduction in disease severity than the controls (Silva et al. 2014). Trichoderma asperellum also could reduce the pathogenic effect of E. carotovora on the young seedlings of okra (Idowu et al. 2016).
Therefore, this work aimed to test seven biocontrol agents, i.e. four bacterial agents, namely B. subtilis, B. pumilus, B. megaterium and P. fluorescens, and three fungal agents, namely T. harzianum, T.viride and T. virens, to control P. carotovorum subsp. carotovorum in vitro and in vivo tests.

Soft rot pathogen
Fifteen bacterial soft rot strains isolated from some vegetables showing naturally typical bacterial soft rot symptoms were collected from some marketing and storage locations in Egypt (Table 1). All bacterial strains were identified as P.carotovorum subsp. carotovorum according to pathological, cultural, morphological and biochemical characters using standard bacteriological methods in pervious study by Mikhail et al. (2019).

Preparation of soft rot bacteria inoculum
The bacterial inoculums of soft rot bacterial strains were prepared by growing of each bacterial strain on nutrient glucose (2%) agar medium in slant tubes. All inoculated slants were incubated at 28 °C ± 2 for 48 h. The bacterial suspension for each strain was done by scraping the bacterial growth in 5 ml of sterile 0.2 M phosphate buffer (pH 7.2). The bacterial inoculums were adjusted to a standard inoculums density [ca. 10 7-9 colony forming unit (CFU)/ml] by measuring the turbidity using a Prim light spectrophotometer at 610 nm and then kept at cool conditions until used within 12 h (Moh et al. 2012).

Biocontrol agents
Four bacterial agents namely B. subtilis, B. pumilus, B. megaterium and P. fluorescens, and three fungal agents namely T. harzianum, T. viride and T. virens, were tested in this work. All biocontrol agents were obtained from Plant Pathology Department, National Research Centre.

Preparation of the biocontrol agents
Preparation of cultural filtrates of each bacterial biocontrol agent was carried out using sterilized nutrient glucose (2%) broth medium (3 g beef extract, 5 g peptone, 20 g glucose in one liter distilled water, pH 7.2) in 250 ml flasks. Each flask was separately inoculated with 1 ml of 48-h-old-culture of each bacterial agent. Three flasks were used as replicates for each bacterial antagonist. The inoculated flasks were incubated at 28 ± 2 °C for 48 h under static conditions. Each bacterial culture was centrifugated at 2038 × g for 15 min; then, the supernatant was filtered through filter paper (Whatman No.1) and finally sterilized by filtration through sterile 0.45 μm membrane filter (cellulose nitrate, Whatman). The bacterial filtrates were then kept at − 20 °C until used (Abd-El-Khair and Haggag 2007).
Preparation of cultural filtrates of each Trichoderma spp. was made using sterilized potato glucose 2% broth medium (200 ml potato extract and 20 g glucose in one liter distilled water) in 250 ml flasks. Each flask was separately inoculated with 1 cm-diameter disc of one-weekold culture of each Trichoderma spp. The inoculated flasks were incubated at 28 ± 2 °C for one week under static conditions. Then, the Trichoderma spp. mycelial mats were separated by filtration with filter paper (Whatman No.1) and finally the fungal cultural filtrates were sterilized by filtration through sterile 0.45 μm membrane filter. The fungal cultural filtrate of each Trichoderma spp. was separately kept at − 20 °C until used (Abd-El-Khair and Haggag 2007).

Screening for antagonistic activity in vitro tests
The inhibitory activity of cultural filtrates of biocontrol agents against all P.carotovorum subsp. carotovorum strains were tested by using filter paper disc plate method (Thornberry 1950). All tests were laid in complete randomized design with four replicates. Twenty milliliters of nutrient glucose (2%) agar medium were poured in each sterile Petri dish (9 cm-diameter) and allowed to solidify. Each Petri dish was separately inoculated with 0.1 ml of each soft rot bacterial strain suspension (10 7-9 CFU/ml) onto the surface of the plate in the center by pipette. The bacterial inoculums were spread over the surface of the plate using a sterile L-shaped spatula and let for 5 min. The filter paper discs (5 mm-diameter) were immersed individually for 1 min in each cultural filtrate of biocontrol agent which prepared before. For control, the filter paper discs were soaked in sterilized distilled water. Four filter paper discs were used as replicates for each treatment as well as the controls. The inoculated Petri dishes were incubated at 28 ± 2 °C for 48 h. The antibacterial activity was recorded by measuring the diameter of the zones of inhibition around the filter paper disc (Mills et al. 2006;Rashid et al. 2013).

Screening for antagonistic activity in vivo tests a. Potato slices method
The inhibitory activity of cultural filtrates of applied biocontrol agents, against high pathogenic P.carotovorum subsp. carotovorum strains, i.e. Pcc 3 , Pcc 4 and Pcc 5 , were tested by using potato tuber slices method. Healthy potato tubers cv. Diamond was surface-sterilized by sodium hypochlorite solution at concentration of 5% for 3 min. The potato tubers were washed in serial of sterile distilled water and left for drying. The potato tubers were cut into slices (2 cm-thick) by sterile knife under sterile conditions. One potato slice was put in each Petri dish containing sterilized filter paper and impregnated with 3 ml of sterile distilled water. Then, each potato slice was separately inoculated with 0.1 ml of each soft rot bacteria suspension onto the center. Each cultural filtrate of biocontrol agent was applied at the same time of pathogen inoculation and 2 h before or after pathogen inoculation. Potato slices were separately treated with pathogenic bacteria suspension and distilled water for controls. Three potato slices were used as replicates for each treatment. Inoculated potato slices were incubated at 30 °C ± 2 for 72 h. The soft rot symptoms were evaluated according to the scale described by Bartz (1999), as the following:no rotting; + restricted rot < 1 cm; + + small active rot 1-2 cm and + + + highly active rot > 2 cm.

b. Potato tubers method
The inhibitory activity of cultural filtrates of applied biocontrol agents against the highest pathogenic P. carotovorum subsp. carotovorum (Pcc 3 strain) were tested by using potato tubers method. Healthy potato tubers cv. Diamond were surface sterilized by sodium hypochlorite solution as mentioned before. Then, each potato tuber was wounded in three places using sterile knife (cross hale). Firstly, potato tubers were separately treated with each cultural filtrate by spraying with atomizer and then allowed 2 h for drying. Then, the treated tubers were inoculated with bacterial pathogen inoculums by spraying with atomizer and then air-dried. For controls, potato tubers were either treated with bacterial pathogen inoculums or distilled water. Ten inoculated potato tubers were weighed and stored in plastic net at room temperature. Three batches were employed as replicates for each treatment as well as the controls. Data of soft rot incidence were weekly recorded for 3 months. Weight of soft rot infected tubers were recorded and expressed in percentage using the following modified formula described by Abd-El-Khair and Haggag (2007).

Statistical analysis
Data were subjected to analysis of variance using Computer Statistical Package (CO-STATE) version 3.03, Barkley Co., USA, and means were compared using the Least Significant Difference (LSD) test at P = 0.05 (Snedecor and Cochran 1980). The significance of the treatment effects, concentration, exposure time and their interactions were also analyzed.

In vivo tests a. Potato tuber slices method
Results showed that the cultural filtrates of B. subtilis, B. pumilus, B. megaterium, P. fluorescens, T. harzianum, T. viride and T. virens could reduce the incidence of soft rot disease on potato slices when inoculated with pathogenic strains, viz. Pcc 3 , Pcc 4 and Pcc 5 , at the same time of inoculation and 2 h before or after inoculation (Table 4). Results showed that B. subtilis when applied at 2 h before the bacterial pathogen inoculation (strains Pcc 4 and Pcc 5) produced highly active rot symptoms on  -El-Khair et al. Bull Natl Res Cent (2021) 45:37 inoculated potato slices, while Pcc 3 strain produced small active rot symptom. Bacillus subtilis when applied at the same time or 2 h after the pathogen inoculation strains Pcc 3 , Pcc 4 and Pcc 5 could produce highly active rot symptoms. Results revealed that when B. pumilus applied at 2 h before the pathogen inoculation, no soft rot symptoms were occurred on inoculated potato slices with Pcc 3 and Pcc 4 strains, while highly active rot symptoms were produced by strain Pcc 5 . Bacillus pumilus when applied at the same time of soft rot pathogen inoculation, strains Pcc 3 and Pcc 5 could produce highly active rot symptoms, while no soft rot symptoms were recorded with Pcc 4 strain. Strains Pcc 4 and Pcc 5 produced highly active rot symptoms, while no soft rot symptoms were recorded with strain Pcc 3 , when B. pumilus applied after 2 h the pathogen inoculation. Results showed that no rotting symptoms were recorded by Pcc 3 and Pcc 4 on inoculated potato slices when B. megaterium applied 2 h before or at the same time of pathogen inoculation, while strain Pcc 5 produced highly active rot symptoms. Bacillus megaterium when applied after 2 h of pathogen inoculation; strains Pcc 3 and Pcc 5 could produce highly active rot symptoms, while no soft rotting was recorded with Pcc 4 . Pseudomonas fluorescens, when applied 2 h before pathogen inoculation, could prevent the soft rot symptoms with all applied soft rot strains as well as when it applied at the same time of pathogen inoculation, except Pcc 5 strain which could produce highly active rot symptoms. Pseudomonas fluorescens when applied after 2 h of pathogen inoculation; no soft rotting, symptoms were recorded with strains Pcc 3 , Pcc 4 and Pcc 5 , respectively (Table 4).
Results revealed that T. harzianum when applied 2 h before pathogen inoculation, no soft rotting was recorded with Pcc 4 and Pcc 5 strains on inoculated potato slices, while Pcc 3 could produce highly active rot symptoms. Trichoderma harzianum also when applied at the same time or 2 h after pathogen inoculation, Pcc 3 and Pcc 4 could not produce any soft rot symptoms, while Pcc 5 could produce highly active rot symptoms. Trichoderma viride when applied 2 h before pathogen inoculation strains Pcc 3 and Pcc 5 could produce highly active rot symptoms, while no rotting was recorded with strain Pcc 4 . Results showed that no soften symptoms were recorded using T. viride when applied at the same time or 2 h after the pathogen inoculation with tested soft rot bacteria, except Pcc 3 and Pcc 5 which could produce highly active rot symptom, respectively. Trichoderma virens when applied 2 h before the pathogen inoculation with strains Pcc 3 or Pcc 4 could produce highly active rot symptoms, while no soft rotting was recorded with strain Pcc 5 . T. virens when applied at the same time or 2 h after of the pathogen inoculation where no soft rotting was recorded, except Pcc 3 and Pcc 5 which could produce highly and small active rot symptom at above periods, respectively (Table 4).

b. Whole potato tubers method
Results revealed that the cultural filtrates of B. subtilis, B. pumilus, B. megaterium, P. fluorescens, T. harzianum, T. viride and T. virens could differently protect the whole potato tubers against soft rot disease incidence, under artificially infection in storage, as shown in Table 5. The cultural filtrates of bacterial agents and fungal agents could reduce the soft rot disease incidence in the ranges of 4.5-12.5% and 3.2-21.9%, compared to the ranges of 5.5-83.0% in the untreated control during three months of storage, respectively. Results revealed that B. pumilus could protect potato tubers against the soft rot disease during storage period. On the other hand, B. subtilis could protect the potato tubers until the 9th week, and then, the soft rot disease incidence reached to 6.0% at the 10th week, followed by 12.2% at the two last weeks of storage. Application of B. megaterium could protect potato tubers to the 8th week, and the disease incidence ranged from 5.1 to 12.5% at the four last weeks of storage. Pseudomonas fluorescens could protect the potato tubers until the 10 th week, and then, the disease incidence was 4.5% at the two last weeks of storage (Table 4). Application of T. harzianum could protect the potato tubers until the 10th week of storage, and then, the disease incidence ranged from 8.0 to 21.9% at the two last weeks of storage, respectively. Both T. viride and T. virens could protect the potato tubers until the 7 th week of storage and then the disease incidence was 3.2% at the four weeks of storage (Table 4).

Discussion
The bacterial soft rot disease, caused by P. carotovorum subsp. carotovorum, causes severe losses in many vegetables in fields, storages and transit, where the losses ranged from 15 to 30% in harvested vegetables and reached 60%