Biological control of tomato bacterial spot by seed microbiolization

SHORT COMMUNICATION

Biological control of tomato bacterial spot by seed microbiolization

Carine R. Naue; Dediel J. A. Rocha; Andréa B. Moura

Departmento de Fitossanidade, Universidade Federal de Pelotas, 96010-970, Pelotas, Rio Grande do Sul, RS, Brazil

^{Author for correspondence}

ABSTRACT

Six plant growth promoting rhizobacteria isolates - two Streptomyces (DFs1296 and DFs1315), three Bacillus (DFs1414, DFs1420 and DFs1423) and one Pseudomonas (DFs1421) - were used to microbiolize tomato seeds for the control of bacterial spot caused by Xanthomonas gardneri. Three assays were conducted in a completely randomized design with six replications. In each assay, X. gardneri suspensions (10⁸CFUmL^-1) were spray-inoculated on the leaves. In the first and second assays, three and four leaves, respectively, were assessed for disease severity. In the third assay, three leaves per plant were assessed for the number of lesions per leaf, leaf area and dry weight. Results showed that one of the Bacillus sp. isolate (DFs1420) consistently provided the greatest relative reduction (48%) of tomato bacterial spot severity.

Key words:Solanum lycopersicum, Xanthomonas gardneri, rhizobacteria, PGPR, biocontrol.

Tomato bacterial spot, caused by Xanthomonas species, is of widespread occurrence in Brazil and may cause losses of up to 35% in the yield (Vale et al. , 2004). Losses have been reported in tomato crops cultivated both for fresh market and for industrial processing, especially during warm (>25 ºC) cropping conditions (Lopes & Quezado-Soares, 1997). Eggplants and bell peppers are other economically important hosts for this pathogen (Lopes & Quezado-Soares, 1997). This disease occurs in all aerial tissues of the plant, causing flowers and forming fruits to fall. Symptoms on the leaves are brown circular spots spread on the leaf surface. Circular lesions with slightly raised edges and a corticated aspect are observed on fruits (Kurozawa & Pavan, 2005).

To reduce losses caused by bacteria, integrated practices are recomeneded such as the use pathogen-free seeds and seedlings, roguing, crop rotation, use of less susceptible cultivars and chemical treatment (Lopes & Quezado-Soares, 1997). However, the continuous use of antibiotics, such as streptomycin and oxytetracycline, and copper fungicides has led to the emergence of Xanthomonas spp. populations that are resistant to these products (Quezado-Duval et al. , 2003), in addition to food and environment contamination and human and animal poisoning (Bettiol & Morandi, 2009).

Given the need for alternatives to chemicals that are less harmful to the environment, the development of biological products for the control plant pathogens is being encouraged. Rhizobacteria are among microorganisms used for such a purpose because they can be both antagonists for plant pathogens and promoters of plant growth and yield increase.

Rhizobacteria of the genera Pseudomonas and Bacillus have attractive features for use in biological control programs (Noronha et al., 1995). The potential of these microorganisms as antagonists has been observed in several biological control studies of diseases affecting different crops (Bettiol & Morandi, 2009). In tomatoes, the control of bacterial diseases using rhizobacteria has been tested with success, reaching 100% control in some cases, under controlled conditions and over a short period of time (Moura et al. , 1998; Aysan et al. , 2003; Tan et al., 2006; Moss et al ., 2007). The present study aimed to evaluate the effects of treatment of tomato seeds with rhizobacteria on the control of tomato bacterial spot.

Six rhizobacteria isolates were involved, namely: two Streptomyces spp. (DFs 1296 and DFs1315) (Moura, 1996); three Bacillus spp. (DFs1414, DFs1420 and DFs 1423) and one Pseudomonas sp. (DFs1421) (Deuner, 2004). These bacteria were identified through sequencing of rDNA 16S gene portion at the Centro de Genômica e Melhoramento of the Universidade Federal de Pelotas (Pelotas, RS, Brazil). Sequencing was performed using the Megabace 1000 (Gene ID). The nucleotide sequence was aligned and compared with those deposited in the NCBI database using the ClustalW program. The sequences were deposited in GenBank and were given the access numbers KC815306 and KC815307 for the streptomycetes and KC815303, KC815304, KC815305 and KC815308 for the remaining rhizobacteria.The pathogenic isolate used was provided by the Laboratório de Bacteriologia Universidade Federal de Viçosa (UFV). The species was determined through rDNA 16S sequencing in the Laboratório de Genômica (Bioagro, UFV, Viçosa, MG, Brazil). Sequencing was performed as previously described. The sequenced fragment allowed identification of the Xanthomonas isolate as X. gardneri (data not shown).

Three assays were conducted in this study. For this purpose, tomato seeds of the SuperMarmande cultivar (Gaúcho/Maçã) were kept immersed for 4 h with stirring at 4ºC in a cell suspension (10⁸ CFU.mL^-1) of each rhizobacteria in a saline solution (0.85% NaCl) with 24h growth in a Kado & Heskett medium (1970) (A₅₄₀= 0.50). Seeds immersed in saline solution served as a control. After the period of exposure of seeds to rhizobacteria, five seeds were immediately deposited in plastic cups (700 mL) containing Plantmax^® substrate. After germination, only one seedling was kept per cup, forming an experimental unit. The plants were sprayed with a cell suspension of the pathogen (10⁸ CFUmL^-1) 30 days after sowing, and they were kept in a moist chamber for 24 h before and 48 h after inoculation.

For the first two assays, evaluations were performed 12 days after inoculation (onset of symptoms). In the first and second assays, three and four leaves were assessed for severity, respectively, with the aid of a diagrammatic scale (Melo et al. ,1997). For the third assay, the number of lesions per leaf was determined on three leaves per plant at 13, 15, 17 and 19 days after inoculation. These values were used to calculate the area under the number of lesions progress curve (AULPC) using specific software (Maffia, 1995). In the third assay, the leaf area of the three leaves was estimated using LI-3100 area meter (LI-COR). Subsequently, the leaves were kept in a drying oven at 60ºC for three days, and then weighed to determine their mass.

The assays were arranged in a completely randomized design with six replications (portions of a plant). Data from the AULPC obtained in the third assay were transformed into

and subjected to analysis of variance. The means were compared using Tukey's test at 5% using Winstat statistical software (Machado & Conceição, 2007). For the other variables (dry weight and foliar area) and for data obtained in the first and second assays, original data (non-transformed) were used.

The overall mean (three assays) percent control of tomato bacterial spot for DFs1420 was 48% and yielded a consistent result in contrast with the other isolates evaluated (Table 1). In the first assay, DFs1296 and DFs1420 controlled bacterial spot in 60% which was the best performance among the six rhizobacteria tested. In the second assay, only the isolate DFs1420 was efficient in reducing the disease (39%). In the third assay, the DFs1420 isolate also provided the greatest disease reduction (50%) with the lowest AULPC value, promoting the growth of the plants and leading to greater dry weight (Table 1).

The percentages of disease control obtained in the present study were higher than those found by Byrne et al. (2005) and Kavitha & Umesha (2007) for the same pathosystem. These authors reported 36.4% disease control using Pseudomonas syringae and 20 to 30% using P. fluorescens, respectively . Conversely, inconsistent result, such as that observed for DFs1296 (only effective in the first assay), is not uncommon and can be attributed to several factors (Ludwig & Moura, 2007).

Several studies have shown both biocontrol and growth promotion effect induced by the same bacterial isolate, such as for DFs1420. Growth-promoting rhizobacteria are also known to effectively control fungal tomato diseases, such as Fusarium wilts and seedling damping-off caused by Rhizoctonia (Domenech et al., 2006). Bacterial diseases that affect tomato, such as Ralstonia solanacearum, were controlled by growth promoters such as actinomycetes (El-Abyad et al., 1993; Moura & Romeiro, 2000) and Pseudomonas (Peixoto et al., 1995). Recently, Kavitha & Umesha (2007) showed that growth-promoting Pseudomonas also controlled bacterial diseases of the shoot, such as bacterial spot caused by Xanthomonas vesicatoria.

The variability in the efficacy of control of bacterial spot for the isolates used in this work (except for DFs1420) was previously registeded by Ludwig & Moura (2007) and it was associated with the stage of plant development, geographical areas (Byrne et al. , 2005) or cultivars (Kavitha & Umesha, 2007). The stability of the control by the microorganism is usually associated with the performance of multiple mechanisms of action (Guetsky et al. , 2001) and with an aggressive colonization of the tissues of the host plant (Kloepper & Beauchamp, 1992). In the present study, the mechanisms of action of the DFs1420 isolate were not studied here but some of its capabilities are already known. The DFs1420 rhizobacteria, previously known as B403R, colonizes roots of different tomato cultivars and has an inhibitory capacity due to production of in vitro antibiotics against Alternaria solani (Ell & Mart), R. solanacearum, Rhizoctonia solani (Kuhn), Sclerotinia sclerotiorum (Lib.) De Bary and Stemphylium solani (Weber) (Deuner et al., 2004). Considering the promising results obtained for DFs1420 in this work, its efficiency in controlling bacterial spot should next be evaluated in the field.

ACKNOWLEDGEMENTS

To the graduate program in Plant Sanitation of Universidade Federal de Pelotas and CAPES for the scholarship granted to the first author, to Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul-FAPERGS for a junior research scholarship granted to the second author, and to the Conselho Nacional de Desenvolvimento Científico e Tecnológico -CNPq for a research fellowship granted to the senior author.

Submitted: 6 May 2013

Revisions requested: 13 May 2013

Accepted: 9 May 2014

Portion of the Master's thesis of the first author presented at the Universidade Federal de Pelotas. Pelotas, Rio Grande do Sul, RS, Brazil. 2009.

TPP-2013-0068

Section Editor: Wagner Bettiol

• Aysan Y, Karata A, Cinar O (2003) Biological control of bacterial stem rot caused by Erwinia chrysanthemi on tomato. Crop Protection 22:807-811.
• Bettiol W, Morandi MAB (2009) Biocontrole de doenças de plantas no Brasil: uso e perspectivas. Jaguariúna, SP, Brazil. Embrapa Meio Ambiente.
• Byrne JM, Dianese AC, Ji PJ, Campbell HL, Cuppels DA, Louws FJ, Miller SA, Wilson M (2005) Biological control of bacterial spot of tomato under field conditions at several locations in North America. Biological Control 32:408-418.
• Deuner CC (2004) Isolamento e seleção de rizobactérias promotoras de crescimento de tomateiro (Lycopersicon esculentum Mill). M.Sc. Dissertation, Universidade Federal de Pelotas. Pelotas, RS, Brazil.
• Deuner CC, Moura AB, Romeiro RS (2004) Biocaracterização parcial de rizobactérias pré-selecionadas para promoção de crescimento de tomate: II-antibiose e colonização de raízes Fitopatologia Brasileira 29:86.
• Domenech J, Reddy MS, Kloepper JW, Ramos B, Gutierrez-Manero J (2006) Combined application of the biological product LS213 with Bacillus, Pseudomonas or Chryseobacterium for growth promotion and biological control of soil-borne diseases in pepper and tomato. BioControl 51:245-258.
• El-Abyad M, El-Sayed MA, El-Shanshoury AR, El-Sabbagh SM (1993) Towards the biological control of fungal and bacterial diseases of tomato using antagonistic Streptomyces spp. Plant and Soil 149:185-195.
• Guetsky R, Shtienberg D, Elad Y, Dinoor A (2001) Improving biological control by combining biocontrol agents with several mechanisms of disease suppression. Phytopathology 92:976-985.
• Kado CI, Heskett MS (1970) Selective media for isolation of Agrobacterium, Corynebacterium, Erwinia, Pseudomonas and Xanthomonas Phytopathology 60:969-976.
• Kavitha R, Umesha S (2007) Prevalence of bacterial spot in tomato fields of Karnataka and effect of biological seed treatment on disease incidence. Crop Protection 26:991-997.
• Kloepper JW, Beauchamp CJ (1992) A review of issues related to measuring colonization of plant roots by bacteria. Canadian Journal of Microbiology 38:1219-1232.
• Kurozawa C, Pavan MA (2005) Doenças do tomateiro. In: Kimati H, Amorim L, Rezende JAM, Bergamim-Filho A, Camargo LEA (Eds). Manual de Fitopatologia - Doenças das Plantas Cultivadas. 4th Ed. São Paulo, SP, Brazil. Agronômica Ceres. p. 607-626.
• Lopes CA, Quezado-Soares AM (1997) Doenças bacterianas das hortaliças: diagnose e controle. Brasília, DF, Brazil. Embrapa CNPH.
• Ludwig J, Moura AB (2007) Controle biológico da queima das bainhas em arroz pela microbiolização de sementes com bactérias antagonistas. Fitopatologia Brasileira 32:381-386.
• Machado AA, Conceição AR (2007) WinStat - Sistema de análise estatística para windows.
• Maffia LA (1995) Programa para cálculo de área abaixo da curva do progresso da doença (AACPD) Gw-basic 3.20. Viçosa, MG, Brazil, Dep. de Fitopatologia, Universidade Federal de Viçosa.
• Melo SCM, Takatsu A, Lopes CA (1997) Escala diagramática para avaliação da mancha bacteriana do tomateiro. Fitopatologia Brasileira 22:447-448.
• Moss WP, Byrne JM, Campbell HL, Ji P, Bonas U, Jones JB, Wilson M (2007) Biological control of bacterial spot of tomato using hrp mutants of Xanthomonas campestris pv . vesicatoria Biological Control 41:199-206.
• Moura AB, Romeiro RS (2000) Actinomicetos pré-selecionados para controle de Ralstonia solanacearum como promotores de crescimento de tomateiro. Revista Ceres 47:613-626.
• Moura AB, Romeiro RS, Neves MCP (1998) Bioensaio para avaliação massal de actinomicetos antagonistas a Ralstonia solanacearum, em tomateiro. Pesquisa Agropecuária Brasileira 33:2065-2072.
• Moura AB (1996) Actinomicetos como agentes potenciais de controle biológico da murcha bacteriana (Pseudomonas solanacearum) e como promotores de crescimento de tomateiro. D.Sc. Thesis, Universidade Federal de Viçosa. Viçosa, MG, Brazil.
• Noronha MA, Michereff SJ, Mariano RLR (1995) Efeito do tratamento de sementes de caupi com Bacillus subtilis no controle de Rhizoctonia solani Fitopatologia Brasileira 20:174-178.
• Peixoto AR, Mariano RLR, Michereff SJ, Oliveira SMA (1995) Ação antagônica de Pseudomonas aeruginosa a Pseudomonas solanacearum e efeito no desenvolvimento de plântulas de tomate. Summa Phytopathologica 21:219-224.
• Quezado-Duval AM, GazzotoFilho A, Leite Júnior RP, Camargo LEA (2003) Sensibilidade a cobre, estreptomicina e oxitetraciclina em Xanthomonas spp. associadas à manchabacteriana do tomate para processamento industrial. Horticultura Brasileira 21:670-675.
• Tan HM, Cao LX, He ZF, Su GJ, Lin B, Zhou SN (2006) Isolation of endophytic actinomycetes from different cultivars of tomato and their activities against Ralstonia solanacearum in vitro World Journal Microbiology & Biotechnology 22:1275-1280.
• Vale FXR, Zambolim L, Zambolim EM, Alvarenga MAR (2004) Manejo integrado das doenças do tomateiro: epidemiologia e controle. In: Alvarenga MA (Ed.) Tomate: Produção de Campo, em Casa-de-Vegetação e em Hidroponia. Lavras, MG, Brazil. Editora UFLA. p. 217-318.

Publication Dates

• Publication in this collection
  31 Oct 2014
• Date of issue
  Oct 2014

History

• Received
  06 May 2013
• Accepted
  09 May 2014
• Reviewed
  13 May 2013