First Report of a 16SrI-B Group Phytoplasma Associated with a Yellows-Type Disease Affecting Tomato Plants in the Baja California Peninsula of Mexico.

Since 2000, a phytoplasma-like disease (locally known as "permanent yellowing") was observed on tomatoes (Lycopersicon esculentum Mill.) grown in the Valle de San Quintín in northern Baja California Peninsula. Affected plants showed general chlorosis, severe stunting, upwardly rolled leaves, bronzing of mature leaves, purple discoloration of veins, "little leaf", abnormal floral structures, and excessive branching of axillary shoots. Total DNA extracted from symptomatic and asymptomatic plants was used in nested (n)-PCR assays driven by phytoplasma-universal primer pair P1/P7 (3), followed by primer pair R16F2n/R16R2 (1) targeting the 16S ribosomal RNA gene of the putative phytoplasma. PCR conditions (direct and nested) were conducted as previously described (l,3). Restriction fragment length polymorphism (RFLP) patterns of nPCR-amplified products (≈ 1.25-kbp 16S rDNA fragments) digested with enzymes AluI, MseI, HhaI, and HpaII showed that 85% (17 of 20) of PCR-positive tomato samples had restriction patterns typical of phytoplasmas belonging to the aster yellows group, subgroup B (16SrI-B) "Candidatus Phytoplasma asteris" (2). Only 10% (2 of 20) of the samples were associated with a phytoplasma related to the 16SrXIII-A Mexican periwinkle virescence group (formerly group 16SrI, subgroup I). None of the symptomless plants tested positive. Subsequently, these results were confirmed by nPCR using 16SrI specific primer pair P1/Aint (4) and specific primers rp(I-B)F1/rp(I-B)R1 that amplify the ribosomal protein (rp) gene operon of aster yellows phytoplasma subgroup B (16SrI-B[rp-B]) (1). The presence of the phytoplasmas in symptomatic plants was confirmed by scanning electron microscopy. Characteristic yellow symptoms could be experimentally reproduced by graft inoculation of tomato seedlings (cv. Maya) with tissue of field-infected plants. Symptoms similar to those of field-grown diseased plants were observed consistently in most of the plants, and when graft transmitted from tomato to periwinkle (Catharantus roseus (L.) G. Don), symptoms of virescencent, small flowers were observed. In contrast, no symptoms were observed on plants grafted with tissues from healthy plants. In Baja California, it appears that at least two distinct phytoplasmas are involved in the disease complex. To our knowledge, this is the first molecular evidence of the presence of a phytoplasma associated with yellows-type diseases in the major tomato cultivation areas of the peninsula. References: (1) I.-M. Lee et al. Phytopathology 93:1368, 2003. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) B. Schneider et al. Page 369 in: Molecular and Diagnostic Procedures in Mycoplasmology. Academic Press, San Diego, CA, 1995. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Bacterial Canker Caused by Clavibacter michiganensis subsp. michiganensis on Tomato in the Baja California Peninsula of Mexico.

During the 2005 tomato-growing (Lycopersicon esculentum Mill.) season, an apparent bacterial disease with cankers on the stems and bird's eye lesions on the fruit appeared in commercial fields and greenhouses in the San Quintin and San Simon areas (a 60-mile long coastal plain) near the central region of the Baja California Peninsula of Mexico. The disease was found in midseason, especially when plants were flowering, and the mature and ripe stage. Incidence ranged from 4 to 46%, which represented an important loss in field and greenhouse production. Symptomatic plants showed reddish brown cavities in the stem, discoloration, and water soaking of vascular tissue. Diseased tissues were washed with phosphate buffer and placed on semiselective Clavibacter medium (SCM) (1), and a gram-positive, nonmotile, nonspore-forming, aerobic, curved rod bacterium was consistently isolated and morphologically characterized. Twenty-eight isolates were identified as Clavibacter michiganensis subsp. michiganensis by polymerase chain reaction (PCR) technology with primers CMM5/CMM6 to amplify a fragment of approximately 6.2 kb (2). The isolates were also identified by REP-PCR (repetitive extragenic palindromic PCR) genomic fingerprinting techniques (3) with REP and BOX primer sets (4). Pathogenicity tests consisting of three replicates of 4-week-old tomato seedlings (cv. Tequila) were performed by spraying (twice, 2 days apart) inocula at 108 CFU/ml. Control seedlings were sprayed with sterile water. Inoculated plants previously covered in polyethylene bags were incubated in a growth chamber at 25°C for 48 h. Within 3 weeks, symptoms of reddish-brown cavities, water-soaked lesions, and asymmetrical wilting appeared on inoculated plants and were similar to those symptoms observed in the field. No symptoms were observed on control plants. Confirmation of the causal agent was done by culturing the bacteria on SCM and PCR analysis. Occurrence of the disease in San Quintin Valley is relevant because the disease is one of the five most serious tomato diseases in the peninsula. Moreover, the potential spread of the pathogen by tomato seedlings represents a permanent risk to other pathogen-free areas in the peninsula. Although bacterial canker has been observed in Baja California (Punta Colonet, Vicente Guerrero, San Quintin, and San Simon), to our knowledge, this is the first confirmation of C. michiganensis subsp. michiganensis in Baja, Mexico. References: (1). C. Alarcon et al. Phytopathology 88:306, 1998. (2) J. Dreier et al. Phytopathology 85:462, 1995. (3) F. J. Louws et al. Phytopathology 88:862, 1998. (4) J. Versalovic et al. Methods Mol. Cell. Biol. 5:25, 1994.

Adhesive properties of a LamB-like outer-membrane protein and its contribution to Aeromonas veronii adhesion.

AIMS: To identify and characterize nonfimbrial proteins from Aeromonas veronii involved in the attachment to epithelial cells in vitro. METHODS AND RESULTS: Two Aer. veronii mucin- and lactoferrin-binding proteins with molecular masses of 37 and 48 kDa were identified by Western blot analysis. According to its N-terminal amino acid sequence, the 48-kDa protein was identified as Omp48, an outer-membrane protein similar to LamB of Escherichia coli. LamB is a well-known porin involved in maltose transport across the outer membrane in E. coli. In a microtitre plate assay, Omp48 bound to the immobilized extracellular matrix proteins collagen and fibronectin, and the mucin- and lactoferrin-binding activity was confirmed. Adhesion of Omp48 to mucin, lactoferrin and collagen was diminished by preincubation with homologous glycoproteins or other carbohydrates, suggesting a putative Omp48 lectin-like binding domain. Anti-Omp48 antiserum significantly inhibited the Aer. veronii adhesion to confluent HeLa cell monolayers and pretreatment of cells with purified Omp48 elicited competitive inhibition of adhesion. Similarly, cross-inhibition of Aer. hydrophila and Aer. caviae adhesion was achieved with the same treatments, indicating the existence of a conserved surface protein among these species. CONCLUSIONS: Taken together, these data indicate that Omp48 is involved in Aer. veronii adhesion to epithelial cells and might be an alternative adhesion factor of this micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesive potential of Aeromonas spp. is correlated with pathogenicity; however, the adhesion mechanism is complex and not well understood. This study provides evidence of a putative adhesion factor that might be contributing to pathogenicity of Aer. veronii and could be used for vaccine development.

Molecular cloning, sequencing and characterization of omp48, the gene encoding for an antigenic outer membrane protein from Aeromonas veronii.

AIMS: To clone, sequence and characterize the gene encoding the Omp48, a major outer membrane protein from Aeromonas veronii. METHODS AND RESULTS: A genomic library of Aer. veronii was constructed and screened to detect omp48 gene sequences, but no positive clones were identified, even under low stringency conditions. The cloned gene probably was toxic to the host Escherichia coli strain, so the cloning of omp48 was achieved by inverse PCR. The nucleotide sequence of omp48 consisted of an open reading frame of 1278 base pairs. The predicted primary protein is composed of 426 amino acids, with a 25-amino-acid signal peptide and common Ala-X-Ala cleavage site. The mature protein is composed of 401 amino acids with a molecular mass of 44,256 Da. CONCLUSIONS: The omp48 gene from Aer. veronii was cloned, sequenced and characterized in detail. BLAST analysis of Omp48 protein showed sequence similarity (over 50%) to the LamB porin family from other pathogenic Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial diseases are a major economic problem for the fish farming industry. Outer membrane proteins are potentially important vaccine components. The characterization of omp48 gene will allow further investigation of the potential of Omp48 as recombinant or DNA vaccine component to prevent Aer. veronii and related species infections in reared fish.