Biological control of kiwifruit and tomato bacterial pathogens




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16th IFOAM Organic World Congress, Modena, Italy, June 16-20, 2008
Archived at http://orgprints.org/view/projects/conference.html


Biological control of kiwifruit and tomato bacterial pathogens

Balestra, G.M.1, Rossetti, A.2 & Quattrucci, A.3

Key words: natural extracts, organic agriculture, Ficus carica, Allium sativum.

Abstract

Biocontrol of bacterial pathogens is effected by using cupric salts associate to appropriate agronomical practices such as seed certification, irrigation and fertilization.

In in vitro and in in vivo tests, aqueous extracts from Allium sativum and Ficus carica fruits reduce the survival and the damages (disease incidence and disease severity) caused by bacterial pathogens of kiwifruit (Pseudomonas syringae pv. syringae, Pseudomonas viridiflava) and of tomato (Pseudomonas syringae pv. tomato) plants. In vitro tests, both vegetal extracts show antimicrobial activity against all bacterial strains utilised at different concentrations (106 – 108 cfu ml-1). In vivo tests Allium sativum and Ficus carica extracts confirm their antimicrobial activity on P. s. pv. tomato reducing DI and DS after two weeks until to 60% and 67% and to 32% and 22%, respectively.

Introduction

Biological control of parasites in organic agriculture crops is based on natural antagonists and substances present in nature. Bacterial diseases are a serious problem in greenhouse and in open field on different plants. Amongst them, Pseudomonas syringae pv. syringae, Pseudomonas viridiflava and Pseudomonas syringae pv. tomato are particularly dangerous on kiwifruit and tomato plants, respectively. At present, to control these bacterial pathogens, especially in organic agriculture, few effective strategies can be adopted. Copper treatments and appropriate agronomical practices, such as seeds certification, irrigation and fertilization, are suggested (Colin et al., 1984; Varvaro et al., 2001).

Due to the recent EU restriction on copper use in organic agriculture (Reg. EU n° 473/2002) and the increased movement of vegetal material among the EU and not EU countries, found effectiveness natural bactericidal/bacteriostatic compounds assume a relevant importance to control these bacterial pathogens especially in organic agriculture. As an alternative to copper compounds, few natural substances have been recently proposed, but further studies need to optimize their effectiveness (De Castro, 2001; Lo Cantore et al., 2004).

As potential natural substances effective against P. s. pv. syringae, P. viridiflava and P. s. pv. tomato, vegetal extracts from A. sativum and F. carica plants were utilised.



A. sativum was chosen for its antimicrobial properties, well known in human healthy, and for its properties to inhibit different enzymes, essential for microbial pathogen infections, by organosulfur compounds (Obagwu and Korsten, 2003).

F. carica was chosen for their richness in phenols and flavonoids effective on different bacteria (Salameh et al., 2004; Zao et al., 2005).

The aims of this study were to verify, in vitro and in vivo, the antimicrobial activity of natural extracts, obtained from Allium sativum and Ficus carica plants, on P. s. pv. syringae, P. viridiflava and P. s. pv. tomato.



Materials and methods

A. sativum and F. carica fruits were sliced into small pieces and blended using twister blender for 10 min at room temperature. The extracts were obtained by centrifuging samples using sorvall RC 5 B (Newton, CT) centrifuge at 8,000 x g for 45 min to remove bigger particles.

In in vitro tests were carried out by the spot tests; aqueous extracts of A. sativum and F. carica fruits were utilised at a concentration of 10 g l-1 and of 60 g l-1 (dry weight), respectively. Spot tests were conducted on NSA medium (nutrient broth 8 g l-1, sucrose 50 g l-1 and agar 18 g l-1). Bacterial strains, characterized by an higher level of virulence and isolated from kiwifruit and tomato plants in Central Italy, were utilised at 106 and 108 colony forming units (cfu)/ml concentration. After distribution of bacterial suspensions (100 µl per Petri dish), natural extracts (4 drops, 30 µl each) were placed on NSA Petri dishes. After incubation at 25  2°C for 48-72 h, eventual inhibition zones, measured in mm without any growth of each bacterial strain, were observed by a stereomicroscope and then measured (Klement et al., 1990). In in vitro spot tests were carried out under laboratory conditions and repeated five times, two replicates each.

In in vivo tests were carried out in greenhouse on tomato plants cv. San Marzano, 1 month old. Greenhouse conditions (temperature, relative humidity) were maintained at day and night temperatures of 25  2°C and 15  2°C, respectively, and relative humidity (RH) between 70-80% during whole experiments. The extracts of A. sativum and F. carica were used at a concentration of 10 g l-1 and of 60 g l-1 (dry weight), respectively. Bacterial strains of P. s. pv. syringae VT2, P. viridiflava VT3 and P. s. pv. tomato VT14 were utilised at 105 cfu/ml concentration.

In greenhouse, tomato plants were sprayed by each natural extract until leaves were homogeneously wet. Considering as preventive treatments by natural substances, 24 h their distribution, bacterial suspension was sprayed on plants with CO2-pressurized hand-held sprayer; 2 h before and 2 h after bacterial inoculation, RH was maintained at 90% by automatic system to favour stomata opening.

After bacterial contamination, tomato plants were monitored daily for 15 days and disease incidence (DI) (n° of diseased leaflets/plant) and disease severity (DS) (n° of necroses/cm2 leaflets) were analysed according to Steel et al. (1997).

In in vivo tests were repeated five times; for each combination (bacterial pathogen/natural extract) 60 tomato plants were used: 15 plants treated with A. sativum extract, 15 with F. carica extract, 15 with copper oxychloride (28%) as positive control and 15 untreated as negative control. All data obtained were statistically analysed using GraphPad Prism 4 software for analysis of variance (ANOVA), and the significance of the treatments were determined using Tukey’s HSD test (P 0.05).



Results

In in vitro spots tests, both natural extracts inhibit the growth of the different bacterial strains utilised.



A. sativum fruit extract resulted effectiveness on all strains utilised, with highest effects against P. s. pv. syiringae VT2 at both bacterial concentrations (106 and 108 cfu ml-1) (Tab. 1).

F. carica extract showed better effects than A. sativum extract on P. s. pv. tomato VT14, and light effects on P. s. pv. syiringae VT2 and P. viridiflava VT3 at both concentration (106 and 108 cfu ml-1) (Tab. 1).

In in vivo tests the use of both natural extracts confirmed their biocontrol effect on P. s. pv. tomato. Using A. sativum extract, considering untreated control values recorded, DI was reduced until to 60% and DS by 67,7%; by using F. carica extract, DI was reduced until to 32% and DS by 22% after 15 days (Fig.1).



Discussion and conclusions

The natural extracts tested seem to be useful for a biocontrol of P. s. pv. syringae, P. viridiflava and P. s. pv. tomato bacterial pathogens.



A. sativum and F. carica extracts successfully reduced disease incidence and disease severity caused by P. s. pv. tomato, and none negative effect was recorded on tomato plants.

The use of these natural substances appear to be particularly interesting to protect tomato plants in greenhouse. The antimicrobial activity of these natural substances showed to be effectiveness at least for 10 days, giving interesting opportunities to substitute or to be associated to copper compounds treatments normally used in organic agriculture.

Further studies are in progress to evaluate field-doses of these natural substances and to characterized their active principles.

Acknowledgments

This research was supported by the Italian Ministry of the Agricultural, Food and Forest Policies (MIPAAF), (N° 893/2006).



References

Colin J., Gerard M. and Laabari H. (1984): Influence du type d’irrigation sur la moucheture bacterienne chez la tomate au Maroc. Parasitica 40:3-12

De Castro S. L. (2001): Propolis: biological and pharmacological activities. Therapeutic uses of this bee-product. Annual Review on Biological Sciences 3:49-83

Klement Z., Rudolph K. and Sands D. C. (1990): Methods in phytobacteriology. Ed. Akademiai Kiado, Budapest. 657p.

Lo Cantore P., Iacobellis N. S., De Marco A., Papasso F. and Senatore F. (2004): Antibacterial activity of Coriandrum sativum L. and Foeniculum vulgare Miller Var. vulgare (Miller) essential oils. Journal of Agriculture and Food Chemistry 52:862-7866

Obagwu J. and Korsten L. (2003): Control of citrus green and blue moulds with garlic extracts. European Journal of Plant Pathology 109:221-225

Salameh M. M., Ibrahim S. A., Seo C. W. (2004): Antimicrobial activity of figs and fig extract on the growth of Escherichia coli O157:H17 and Salmonella typhimurium. (Paper presented at the 104th General Meeting of American Society of Microbiology, New Orleans, USA)

Steel R. G. D., Torrie J. H. and Dickey D. A. (1997): Principles and procedures of statistics: A biometrical approach. 3rd ed. McGraw-Hill Book Co Eds; New York, 666 p.

Varvaro L., Antonelli M., Balestra G. M., Fabi A. and Scermino D. (2001): Control of phytopathogenic bacteria in organic agriculture: cases of study. Journal of Plant Pathology 83: 44

Zao A., Wu S. and Du G. (2005): Experiment study of antibacterial constituents of Ficus carica leaves. Ziran Kexueben 3:37-40



Tab. 1: In vitro antibacterial activity in spot agar tests by A. sativum and by F. carica extracts on Pss VT2, Pv VT3 and Pst VT14 on NSA medium after 72 h of incubation at 25 ± 2 °C

1Values expressed are mean ± S.D. of five experiments. Means within row followed by different letters are significantly different at P ≤ 0.05 according to Tukey’s HSD test.



Figure 1: Disease incidence (DI) on tomato plants contaminated by Pseudomonas syringae pv. tomato VT14 by using A. sativum (1%) and by F. carica (6%) vegetal extracts.

1 Dipartimento di Protezione delle Piante, Università degli Studi della Tuscia, Via S. Camillo de Lellis 01100 Viterbo, Italy,

E-Mail: balestra@unitus.it



2 As Above

3 As Above



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