Pseudomonas syringae pv. tabaci in papaya seedlings

Pseudomonas syringae pv. tabaci em plântulas de mamoeiro

ARTICLES

Pseudomonas syringae pv. tabaci in papaya seedlings

Pseudomonas syringae pv. tabaci em plântulas de mamoeiro

Luís Otávio S. Beriam^*; Irene M.G. Almeida; Suzete A.L. Destéfano; Eunice Grabert; Denise M. Balani; Mariana Ferreira; Júlio Rodrigues Neto

Instituto Biológico, Caixa Postal 70, 13001-970, Campinas, SP, Brazil

ABSTRACT

The natural occurrence of Pseudomonas syringae pv. tabaci causing leaf spot symptoms in papaya seedlings is reported. The pathogen was identified through biochemical, physiological, serological, and molecular assays and artificial inoculations in papaya plants. It was also shown that the strains were pathogenic to bean and tobacco plants. The restriction patterns obtained with Afa I, Alu I, Dde I, Hae III, Hpa II, Hinf I, Sau 3A I and Taq I of the PCR-RFLP of 16S-23S DNAr were identical to the P. s. pv. tabaci patterns. Primers corresponding to hrpL gene of P. syringae were also tested and the results grouped the papaya strains with P s. pv. tabaci. Bacterial strains were deposited at Coleção de Culturas IBSBF, Instituto Biológico, Campinas, Brazil, under access numbers 1687 and 1822.

Additional keywords: papaya bacterial diseases, 16S-23S rDNA.

RESUMO

É relatada a ocorrência natural de Pseudomonas syringae pv. tabaci causando sintomas de lesões foliares em plântulas de mamoeiro. O patógeno foi identificado por meio de testes bioquímicos, fisiológicos, serológicos e moleculares, além de ensaios de patogenicidade em plantas de mamoeiro, feijoeiro e fumo. Os padrões de restrição obtidos com as enzimas Afa I, Alu I, Dde I, Hae III, Hpa II, Hinf I, Sal 3A I e Taq I, utilizando-se a técnica de PCR-RLFP da região espaçadora 16S-23S do DNA ribossômico, foram idênticos àqueles apresentados para P. s. pv. tabaci. Primers correspondentes ao gene hrpL de P. syringae foram também testados e os resultados obtidos permitiram agrupar as linhagens isoladas de mamão com P. s. pv. tabaci. Linhagens bacterianas estão depositadas na coleção de culturas IBSBF, Instituto Biológico, Campinas, sob n. 1687 e 1822.

Palavras-chave adicionais: bacteriose do mamoeiro, 16S-23S DNAr.

Brazil is the most important producer of fresh papaya (Carica papaya L.) in the world, with a planted area of approximately 30,000 ha and is responsible for an annual production of 1,6 millions of ton of fresh papaya. The main region of exportation is located at the State of Espírito Santo which represents 87.6% of the total exported fruits (24).

Papaya production may be affected by many factors such as the occurrence of phytopathogenic agents, like bacterial diseases, which could causes serious losses. Several bacterial species could infect papaya plants. In 1956, Robbs (19) reported a bacterial disease in Brazil causing symptoms of water soaked and angular spots on papaya leaves, naming the pathogen as Pseudomonas caricapapayae. Nelson & Alvarez (13) described in 1976 a disease causing symptoms of "purple stain" in papaya fruits in Hawaii, which causal agent was identified as Erwinia herbicola. In 1979, Erwinia cypripedii causing black rot on seedlings, trees and fruits of papaya was observed in Taiwan (10). Two other bacterial diseases caused by Erwinia spp. and called "erwinia mushy canker" and "erwinia decline" occurring respectively in trees and seedlings of papaya were reported by Trujillo & Schroth (23) in Hawaii. Other diseases induced by Erwinia sp. were also described by Webb (25) in Saint Croix, U.S. Virgin Islands, causing canker on papaya trees and by Frossard et al. (5) who reported a papaya disease caused by Erwinia belonging to the "amylovora" group.

An Erwinia strain associated with Papaya Ringspot Virus inducing symptoms of bud rot and causing severe damage on papaya plants was described by Robbs et al. (20) in the South of Brazil and further investigations identified it as E. carotovora subsp. atroseptica¹.

Recently, the causal agents of the diseases described by Trujillo & Schroth (23), Web (25) and Frossard et al. (5) were identified as a new bacterial species named Erwinia papayae (6).

Enterobacter cloacae was another bacterial species described in papaya, inducing symptoms of internal yellowing of fruits in Hawaii (14). Strains of E. cloacae were also isolated from papaya fruits in Brazil¹.

In September 2001 seedlings of papaya cv. Golden showing symptoms of brown colored leaf spots, with, sometimes surrounded by a diffuse chlorotic halo, that may progress to large necrotic areas, were observed in commercial nurseries located at Linhares county, State of Espírito Santo, Brazil. Bacterial strains that belong to Pseudomonas syringae group (LOPAT Ia) were isolated from these necrotic lesions in a preliminary study (2).

The objective of the present study was to identify these papaya strains at pathovar level through biochemical, serological, pathological, and molecular tests.

MATERIAL AND METHODS

Pathogen isolation

Small pieces of diseased leaf tissues were excised from the lesions and macerated in sterile distilled water. The resultant suspension was streaked on plates containing Nutrient Agar (NA) (11) or King's B (9) media and then incubated at 28⁰C for 48h. Individual colonies were cultured and used in hypersensitivity tests on tobacco leaves.

Bacterial strains

Besides the papaya isolates, other bacterial strains were included in this study for comparative purposes (Table 1). The strains were recovered from freeze-dried cultures and grown on NA at 28⁰C for 48h.

Pathogenicity assays

Papaya seedlings cv. Golden, tobacco (Nicotiana tabacum L.), bean (Phaseolus vulgaris L.) and poinsettia (Euphorbia pulcherima Willd.) plants were inoculated by infiltration with bacterial cell suspensions of papaya strain IBSBF 1687 containing ca. 10⁸cfu/mL from 48-72h-old NA cultures, under moisture chamber conditions. Besides leaves, fruits, stems and flowers of papaya were also inoculated. Negative controls were inoculated with sterile distilled water. All the inoculated and controls were maintained in a greenhouse (25 - 30ºC) and examined daily for disease development. Papaya plants were also inoculated with P. caricapapayae strain IBSBF 361 for comparative purposes.

Biochemical and physiological assays

Biochemical tests for the identification at pathovar level were carried out according to Young & Triggs (27) and Schaad et al. (21).

Serological assays

Bacterial suspensions (ca. 10⁹ cfu/mL) obtained from 48 h-old NA cultures as well as membrane protein complex (MPC) (22) were used as antigens. Microscopy slides for double diffusion tests were prepared with 3 mL of 1% purified agar in phosphate buffered saline 0.1M, pH 7 with 200 ppm sodium azide. Papaya bacterial strains were tested with antisera against P. syringae strains [P.s pv. syringae (AS-375), P. s. pv. tabaci (AS-761), P. savastanoi pv. phaseolicola (AS-736) and P. s. pv. lachrymans (AS-961)] obtained from the Antisera Collection of the Laboratório de Bacteriologia Vegetal (LBV), Instituto Biológico, Campinas, SP, Brazil. All the antigen fractions were also tested against normal serum.

DNA extraction and amplification

Genomic DNA from papaya strain (IBSBF-1687), P. syringae pv. garcae (IBSBF 248^P), P. s. pv. lachrymans (IBSBF 1258^P), P. s. pv mori (IBSBF 1419^P), P. s. pv. pisi (IBSBF 1418^P), P. s. pv. syringae (IBSBF 451^P), P. s. pv. tabaci (IBSBF 758) and P. caricapapayae (IBSBF-361) were extracted (16) and the concentrations were estimated by comparison of the intensity of fluorescence emitted by known concentrations of the bacteriophage lambda DNA in an ethidium bromide-stained 0.6% agarose gel. Amplification of the 16S-23S spacer region was carried out using the primers pHr (12) and p23Suni322-anti (8). All PCR reactions were performed in a total volume of 25 mL using 100 ng of genomic DNA, 1.0 U Taq polymerase (Amersham Biosciences), 1 X Taq buffer, 200 mM dNTPs mixture, and 0.4 mM each primer. The PCR protocol consisted of a denaturating cycle of 95ºC for 2 min, followed by 25 cycles at 94ºC for 1 min, 60ºC for 30 s and 72ºC for 3 min, and a final extension of 72ºC for 5 min, in a thermocycler (GeneAmp PCR system 9700; Perkin-Elmer Corporation, Norwalk, Conn).

The primer set pshrp1F/2R, corresponding to hrpL gene of P. syringae pathovars morsprunorum, pisi and syringae (4) was also tested. PCR was performed under the same conditions of the spacer regions and the amplifications were carried out by using an initial denaturation step of 95ºC for 2 min, followed by 25 cycles at 94ºC for 1 min, 55ºC for 30 s and 72ºC for 1 min, and a final extension period of 72ºC for 3 min. The amplification fragments were observed by electrophoresis in 1% agarose gels in 1X TAE buffer (0.04M tris-acetate, 0.001M EDTA). The gels were stained with 0.1mg/mL of ethidium bromide and photographed under UV light using the Alpha Innotech 2200 Digital System.

PCR-RLFP of the 16S-23S spacer region and hrpL gene

PCR products (5 mL) were digested individually with each of the following restriction endonucleases Afa I, Alu I, Dde I, Hae III, Hpa II, Hinf I, Sau 3A I and Taq I under conditions specified by the manufacturer (Amersham Biosciences) and the restriction fragments separated by electrophoresis in 3% agarose gels using 1X TAE buffer. The gels were stained with ethidium bromide and visualized under UV. The molecular weights of the fragments were determined by comparison with a 100 bp DNA ladder (Amershan Biosciences).

RESULTS AND DISCUSSION

Cultural, morphological, physiological and biochemical tests previously carried out by Beriam et al.(2) showed that the papaya strains belong to Pseudomonas syringae species. In this study, these strains were investigated in order to classify them at the pathovar level.

P. syringae causes diseases in a large number of plants and according to Young et al. (26) this species includes more than 50 pathovars, circumscribed on the basis of distinct host range. In addition, Young & Triggs (27) showed that physiological and biochemical determinative tests could be used to differentiate Pseudomonas syringae at the pathovar level.

In this study, the papaya strains were pathogenic to tobacco, bean, and poinsettia. Bean and tobacco are natural hosts of P.s. pv. syringae and P. s. pv. tabaci, but only P. s. pv. tabaci was also described causing disease in poinsettia (18), suggesting that the papaya strains could be allocated as P. syringae pv. tabaci. The determinative tests described in the literature (27) for identification at the pathovar level were very useful in this study (Table 2), corroborating the results of pathogenicity assays.

Another evidence that reinforced the identification of the papaya strains as P. s. pv. tabaci was the presence of precipitin bands only between the papaya isolates and P. s. pv. tabaci antisera in the results of the serological assays (Figure 1).

Besides biochemical, serological and pathological results, the molecular tests also confirmed the papaya strains as P. s. pv. tabaci. The amplification of the 16S-23S spacer region of different pathovars of P. syringae (P.s. pv. garcae, P. s. pv. lachrymans, P.s. pv. mori, P. s. pv. pisi, P. s.pv. syringae and P. s. pv. tabaci) resulted in a single product for all strains. The size of the product was approximately 1.1 kilobase (kb). Only fragments ranging from 90 to 1100 base pairs (bp) obtained from restriction endonucleases experiments were considered for analysis. Several restriction enzymes were tested, but only Dde I yielded distinct profiles for each pathovar tested, which allowed to group the papaya strains with P. s. pv. tabaci (Figure 2 and Table 3).

The amplifications with the pshrp 1F/2R primers set were carried out with P.s. pv. syringae (IBSBF 451^T), P.s. pv.tabaci (IBSBF 758 and 974) and the papaya strain (IBSBF 1687) and yielded a fragment about 450 bp. No amplification was observed with P. caricapapayae strains. In the Alu I, Hae III, Hpa II, Hinf I, and Taq I digestions, P.s. pv. syringae could clearly be differentiated from P. s. pv. tabaci while the papaya strains showed identical profiles to P.s. pv. tabaci (Figure 3 and Table 4).

Specific primers have been widely used as a rapid method for identification of phytopathogenic bacteria like Erwinia amylovora (1), P. savastanoi pv. phaseolicola (17), Xanthomonas albilineans (15), X. axonopodis pv. citri (7), and others. In this study, the pshrp 1F/2R primer set exhibited specificity, discriminating P.s. pv. syringae and P.s. pv. tabaci from P. caricapapayae. Although the amplification has occurred for both P.s. pv. syringae and P.s. pv. tabaci, the restriction profiles clearly differentiated these pathovars, confirming the identification of the papaya strains as P.s. pv. tabaci.

According to Bradbury (3), strains of P. s. pv. tabaci can be transmitted by seeds. Denardin² isolated P.s. pv. tabaci from papaya seed lots. Herein, the seed infection probably could be the source of primary inoculum since the papaya seedlings showed cotyledonary leaf lesions, suggesting bacterial seed transmission, which represents an important vehicle of dissemination of the disease over considerable distances.

Data de chegada: 01/09/04. Aceito para publicação: 23/06/05.

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1

Robbs,.C.F. Data not published.

2

N.Denardin (Universidade de Passo Fundo/RS). Data not published.

Publication Dates

• Publication in this collection
  23 Oct 2006
• Date of issue
  Mar 2006

History

• Accepted
  23 June 2005
• Received
  01 Sept 2004