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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.37 no.4 São Paulo Oct./Dec. 2006

http://dx.doi.org/10.1590/S1517-83822006000400008 

ENVIRONMENTAL AND SOIL MICROBIOLOGY
SHORT COMMUNICATION

 

Antagonism of Serratia marcescens towards Phytophthora parasitica and its effects in promoting the growth of citrus

 

Antagonismo de Serratia marcescens contra Phytophthora parasitica e seu efeito na promoção do crescimentos de citros

 

 

Brigida Pimentel Villar de Queiroz; Itamar Soares de Melo*

Laboratory of Environmental Microbiology - Embrapa Environment

 

 


ABSTRACT

Phytophthora parasitica causes serious widespread, and difficult-to-control root rots in warmer regions. This oomycete is one of the most important pathogen of citrus. This paper reports the biological control of the pathogen by a strain of Serratia marcescens R-35, isolated from citrus rhizosphere. In greenhouse trials, the bacterium suppressed more than 50% of the disease and promoted the plant growth.

Key words: biological control, rhizobacteria, plant growth promotion


RESUMO

Phytophthora parasitica é um oomiceto que causa sérios problemas fitossanitários em diferentes espécies de plantas em regiões tropicais e o controle tem sido difícil. Este patógeno é um dos mais importante à citricultura. Este trabalho relata o controle biológico do patógeno por uma linhagem de Serratia marcescens R-35, isolada da rizosfera de citros. Em condições de casa-de-vegetação, a bactéria reduziu em mais de 50% a incidência da doença, ao mesmo tempo que promoveu o crescimento de plantas.

Palavras-chave: controle biológico, rizobactérias, promoção de crescimento


 

 

Phytophthora parasitica infects many citrus species, causing a widespread disease known as gummosis. This is a prevalent disease in São Paulo State, Brazil. Infected trees generally lack vigor and may die prematurely. The disease has became increasingly important in citrus commercial orchards and nurseries and there is a need for alternate disease management. The use of microorganisms as a means of biological control for this disease is of special interest. Chemical control provided by highly effective Oomycetes-specific fungicides, such as metalaxyl and fosetyl Al, has been successful (2), but is not always desirable because of the high cost of application, potential hazards to the environment, and the development of fungicide-resistant strains. Some rhizobacteria have been reported to be good antagonists of Phytophthora spp. (8,9). Most studies have emphasized bacterial agents such as Pseudomonas spp., Agrobacterium sp., Bacillus subtilis and Serratia marcescens.

The use of plant-growth-promoting rhizobacteria as inoculants depends on their ability to colonize the root system and to compete with indigenous microflora (6). Efficient colonization of roots may present further establishment of pathogens by both physical and nutritional competition (10). Colonization is initiated by the process of bacterial attachment to roots, where such competition phenomena are already operating. We report here the isolation of a Serratia marcescens strain isolated from citrus rhizosphere in São Paulo State and its antagonistic activity in vitro and in vivo against Phytophthora parasitica.

Serratia marcescens strain R3.5 was isolated from washed root surface of healthy plants of commercial crops of citrus. It was obtained following plating of serial dilutions of sample material onto king's B medium (5). Pure culture of S. marcescens was characterized by analysis of 16S rDNA gene.

Antagonism of S. marcescens towards P. parasitica was tested in a dual-plate assay on PDA (potato-dextrose-agar) and KBM, (5) following the following procedure. One disk (6 mm in diameter) of P. parasitica was inoculated equidistantly (60 mm) on PDA and KBM from S. marcescens. It was used four replicates per treatment and the antagonistic activity was measured after 7 days. The level of inhibition was defined as the subtraction of the fungal growth radius of a control culture from the distance of the growth in the direction of S. marcescens. The fungicide metalaxyl was used as positive control. Microscopic observation were done without any citochemical method regions of the interactions between the antagonist and the pathogen were cut off and put in on microscope slides and viewed directly in optic microscope using immersion lens.

S. marcescens was tested for plant-growth-promoting activity and control of P. parasitica towards citrus (C. limonia) in greenhouse trials. As substrate it was used a commercial mixture Plantmax. The mixture was sterilized by autoclaving for two cycles at 127ºC. Thirty-day-old-citrus sterile seedlings were transferred to pots (20 cm diameter, with four plants per pot and five pots per treatment in randomized complete blocks), and each plant was then inoculated by dipping with approximately 108 UFC.mL-1 viable cells of S. marcescens. Half of the pots were previously inoculated with P. parasitica. Phytophthora-colonized wheat seeds (2g) were added to each pot. The incidence of the disease was based in a index on a scale of 0-4, where: 0 healthy plants and 4 dead plant. The experiments carried out in greenhouse conditions were done twice, with similar results.

The in vitro antagonism assays showed that the strain R3.5 of S. marcescens is an antagonist to P. parasitica. S. marcescens showed the highest activity against P. parasitica in KB medium when compared to PDA medium reducing the mycelial growth in 40% in KB and 37% in PDA. The inhibition of fungal growth in KB (a Fe (III) - poor medium) is likely to be mediated by production of siderophores besides antibiotics (4). The fungicide metalaxyl, used to control plant pathogens from the Peronosporales order, completely inhibited the mycelial growth of P. parasitica. The bacterium exhibited an antagonistic activity through antibiosis. Micelium of P. parasitica appeared red due the red pigments produced by S. marcescens (Figure 1) and, when in contact to Phytophthora, Serratia lysed the oosporos of the pathogen. The oospore germination of P. parasitica was affected by the culture filtrate of S. marcescens (data not shown).

 

 

In test for PGPR activity, S. marcescens resulted in increased growth (in terms of dry weight of root and shoot) (Table 1). The bacterium reduced the seedling infection in 50%. It was not investigated the mechanism by which S. marcescens biologically control the gummosis in citrus. Biocontrol of a pathogen generally has been attributed to one individual compound or mechanism, though the roles of other possible factors have been discussed. Phytophthora species have been biologically controlled by different rhizobacteria, including Brevibacterium linens, Bacillus thuringiensis and Bacillus subtilis active against P. vignae (3); Pseudomonas vignae active against P. citrophthora and P. parasitica (1); and Enterobacter aerogenes against P. cactorum (7).

 

 

Evidence to date has shown that S. marcescens is capable of inducing plant growth and suppresses P. parasitica, and also to establish in the rhizosphere. Thus, all these criteria make it a potential biocontrol agent of gummosis disease.

 

REFERENCES

1. Amorin, E.P.R.; Melo, I.S. Ação antagônica de rizobactérias contra Phytophthora parasitica e P. citrophthora e seu efeito no desenvolvimento de plântulas de citrus. Rev. Bras. de Fruticult., 24: 565-568, 2002.         [ Links ]

2. Davis, R.M. Control of Phytophthora root and foot rot citrus with systemic fungicides metalaxyl and phosethyl aluminium. Plant Dis., 66: 218-220, 1982.         [ Links ]

3. Fernando, W.G.D.; Linderman, R.G. Inhibition of Phytophthora vignae and stem and root rot of cowpea by soil bacteria. Biol. Agric. and Horticult., 12: 1-14, 1995.         [ Links ]

4. Hebbar, P.; Berge, O.; Heulin, T.; Singh, B.P. Bacterial antagonists of sunflower (Heliantus annus) fungal pathogens. Plant and Soil., 133: 131-140, 1991.         [ Links ]

5. King, E.O.; Ward, M.K.; Raney, D.E. Two simple media for the demonstration of pyocyanin and fluorescin. Journal of Laboratory and Clinical Medicine, St. Louis, 44(2): 301-307, 1954.         [ Links ]

6. Kloepper, J.W.; Beanchamp, C.J. A review of issues related to measuring colonization of plant roots by bacteria. Can J. Microbiol., 38: 1219-1232, 1992.         [ Links ]

7. Levesque, C.A.; Holley. J.D.; Utkhede, R.S. Individual and combined effects of Enterobacter aerogenes and metalaxyl on apple tree growth, and Phytophthora crown and root rot symptom development. Soil biology and biochemistry, 25: 975-979, 1993.         [ Links ]

8. Steddom, K.; Menge, J.A.; Growdey, D.; Borneman, J. Effect of repetitive applications of the biocontrol bacterium Pseudomonas putida 06090-rif/nal on citrus soil microbial communities. Phytopathology, 92(8): 857-862, 2002.         [ Links ]

9. Turney, J.K. Biocontrol of Phytophthora citrophthora root rot of citrus. Phytopathology (abstract) 83: 1348, 1993.         [ Links ]

10. Weller, D.M. Biological control of soil-borne plant pathogens in the rhizosphere with bacteria. Ann. Rev. Phytopathol., 26: 379-407, 1988.         [ Links ]

 

 

Submitted: March 14, 2006; Returned to authors for corrections: April 27, 2006; Approved: October 13, 2006

 

 

* Corresponding Author. Mailing address: Laboratory of Environmental Microbiology - Embrapa Environment - Rodovia SP 340 km 127.5 cep 13820-000. Jaguariuna, SP - Brasil. E-mail: itamar@cnpma.embrapa.br

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