Thyme and suico essential oils: promising natural tools for potato common scab control.

Potato common scab is a worldwide disease mainly caused by Streptomyces scabiei. It seriously affects potato crops by decreasing tuber quality. Essential oils (EO) are natural products with recognised antimicrobial properties. In this research, the antibacterial activities of thyme, oregano, suico and mint EO against S. scabiei were analysed. Infected tubers and soil samples were used for bacterial isolation; the obtained isolates were genetically identified. The chemical composition of the EO was determined by GC-MS. The broth microdilution method was used to analyse antibacterial properties of EO. Thirty-one bacterial isolates were obtained. The isolate chosen for antibacterial assays was morpho-physiologically and genetically identified as S. scabiei. Thyme EO was mainly composed of thymol and o-cymene; suico EO of dihydrotagetone, trans-tagetone and verbenone; oregano EO of trans-sabinene hydrate, thymol and É£-terpinene; and mint EO of menthone and menthol. All the EO tested were effective against S. scabiei, but thyme and suico EO were the most successful, with a minimum inhibitory concentration of 0.068 g·l-1 and 0.147 g·l-1 , respectively, and a minimum bactericidal concentration of 0.137 g·l-1 and 0.147 g·l-1 , respectively. Scanning electron microscopy showed similar damage caused by both thyme and suico EO to the bacterial envelope. Total phenolic content of EO was not related to their antibacterial activity. Thyme and suico EO are effective antibacterial agents against S. scabiei, impeding bacterial viability and disturbing the bacterial cell envelope. These EO are promising tools for control of potato common scab.

Genotypic and Phenotypic Characterization of Streptomyces Species Causing Potato Common Scab in Uruguay.

Isolation and characterization of common scab (CS) pathogen Streptomyces spp. from Uruguayan potato tubers and soil samples were done in response to significant economic losses due to CS on potato in autumn 2010. Seventy of the 331 isolates were classified as pathogenic owing to their ability to induce necrosis on tuber disks and stunting of radish seedling. Streptomyces spp. causing CS on potato in Uruguay were found to represent a range of different species by virtue of their diverse morphological and physiological traits as well as rep-PCR, rpoB phylogenetic analysis, and multi-locus sequences analysis. We identified isolates primarily as Streptomyces scabiei, S. acidiscabies, and S. europaeiscabiei. However, some of the pathogenic isolates still remain to be identified at the species level. This highlights the need for improved methods for discrimination among pathogenic Streptomyces species. The presence of Streptomyces pathogenicity island (PAI) genes was analyzed, including genes encoding for thaxtomin synthetase (txtA, txtB), tomatinase (tomA), and a necrosis protein (nec1). Among the isolates that were pathogenic, 50% contained the four pathogenicity genes, 33% had an atypical composition of PAI marker genes, and 17% did not contain any genes. The absence of the genes reported to be involved in thaxtomin biosynthesis (txtA, txtB) was confirmed by whole-genome sequencing of two representative strains of this group. This finding suggests the participation of other virulence factors in plant pathogenicity.

First Report Regarding Potato Scab Caused by Streptomyces acidiscabies in Uruguay.

Potato scab disease is caused by gram-positive filamentous bacteria in the genus Streptomyces. A great variety of species cause this disease, but Streptomyces scabies is the most ancient of these pathogens and can be found in a worldwide distribution, whereas S. turgidiscabies and S. acidiscabies are newly emerged pathogens (2). During the autumn of 2010, potato (Solanum tuberosum) crops had large economic losses by common scab, corresponding to 29% of the total potato-cultivation area (according to our survey), which was unusual in Uruguay. Specifically, the disease was very aggressive and the tubers showed particularly deep scab lesions. We isolated the Streptomyces species present in these particular scab lesions of tubers collected in July 2010 from one of the three potato cultivation areas (San José). A total of 19 Streptomyces spp. strains were isolated and identified using classical and molecular techniques. Morphological characteristics of colonies and microscopic structure of the mycelium were observed (1). Molecular characterization by conventional PCR was carried out using primers directed to specific regions of the 16S rRNA gene for the genus Streptomyces, Aci1: (5'-TCACTCCTGCCTGCATGGGCG-3') and Aci2: (5'-CGACAGCTCCCTCCCACAAG-3'). Also, regions of two pathogenicity genes, namely txtAB and nec1, were amplified and confirmed by sequencing (2). Additionally, melanin production and pathogenicity of the isolates was determined by inoculation of potato discs (1). Six of the 19 strains succeeded in PCR amplification with primers specific to Streptomyces acidiscabies, which has white, aerial hypha and flexuous spore chains. These strains did not produce melanin on tyrosine agar media. The amplified fragments for 16S rRNA and pathogenicity genes from one representative strain 61 were sequenced. BLASTn analysis of the 16S rRNA gene sequence obtained of the strain 61 (Accession No. JN206667) showed the highest similarity (100%) with S. acidiscabies type strain 84-01-182 (GenBank Accession No. FJ007427.1). Pathogenicity of the isolate was tested on tuber slices. The isolate was grown on YME for 5 to 7 days at 28°C and agar plugs from the sporulating colonies were inverted onto excised tuber tissue. Disks were incubated at 28°C in the dark and the presence of necrosis was evaluated after 5 days (1). All tuber slice assays were repeated three times. The noninoculated control tuber slices did not show any necrosis, while those inoculated with the strain did. To our knowledge, this is the first report of S. acidiscabies causing potato scab disease in Uruguay. References: (1) D. H. Park et al. Plant Dis. 87:1290, 2003. (2) L. A. Wanner. Phytopathology 96:1361, 2006.

Genetic Diversity and Aggressiveness of Ralstonia solanacearum Strains Causing Bacterial Wilt of Potato in Uruguay.

Bacterial wilt, caused by Ralstonia solanacearum, is a major disease affecting potato (Solanum tuberosum) production worldwide. Although local reports suggest that the disease is widespread in Uruguay, characterization of prevalent R. solanacearum strains in that country has not been done. In all, 28 strains of R. solanacearum isolated from major potato-growing areas in Uruguay were evaluated, including 26 strains isolated from potato tubers and 2 from soil samples. All strains belonged to phylotype IIB, sequevar 1 (race 3, biovar 2). Genetic diversity of strains was assessed by repetitive-sequence polymerase chain reaction, which showed that the Uruguayan strains constituted a homogeneous group. In contrast, inoculation of the strains on tomato and potato plants showed, for the first time, different levels of aggressiveness among R. solanacearum strains belonging to phylotype IIB, sequevar 1. Aggressiveness assays were also performed on accessions of S. commersonii, a wild species native to Uruguay that is a source of resistance for potato breeding. No significant interactions were found between bacterial strains and potato and S. commersonii genotypes, and differences in aggressiveness among R. solanacearum strains were consistent with previously identified groups based on tomato and potato inoculations. Moreover, variation in responses to R. solanacearum was observed among the S. commersonii accessions tested.

First Report of Iris yellow spot virus on Onion in Uruguay.

From October to December 2005, onion (Allium cepa) plants in seed-production fields in south Uruguay (Canelones) had symptoms suggestive of those caused by Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae). Symptoms included diamond-shaped lesions on seed stalks (scapes), each 1 to 5 cm long with a necrotic border, green center, and sometimes a second necrotic area in the center of the diamond (2,3). Necrotic lesions with more irregular shape were also associated with diseased plants. In 2006, scape samples with these symptoms were collected from four onion seed crops and assayed for IYSV using an IYSV-specific antiserum (Agdia Inc., Elkhart, IN) in a double-antibody sandwich-ELISA. IYSV was detected in all four onion seed crops monitored in 2006. IYSV incidence, expressed as the number of plants with symptoms, ranged from <1% (1 of 120 plants evaluated) to 20% (24 of 120 plants). Two fields were monitored in 2007, in which IYSV incidence increased from 2 and 3% in October to 7% (198 of 2,768 plants) and 40% (253 of 638 plants) in December, respectively. The highest incidence was observed in the same farm in 2006 and 2007. Scapes were sampled from the field with the highest incidence of symptoms in 2007 and tested for IYSV with IYSV-specific primers (3). Total RNA was extracted from 100 mg of symptomatic tissue, with green tissue adjacent to typical lesions, following a Trizol-based protocol (1). A reverse transcriptase-PCR assay with nucleocapsid gene-specific primers was used (3). A PCR product of approximately 26 bp was obtained, coincident with the expected length for IYSV. The PCR product was cloned and sequenced. The tospovirus N sequence of the isolate in Uruguay (Accession No. GU550518) had maximum identity (97%) with an Australian IYSV isolate (Accession No. AY345227), and >87% identity only with IYSV N protein sequences in GenBank. Because of the presence of IYSV in Brazil, Chile, and Peru, this first documentation, to our knowledge, of IYSV in onion crops in Uruguay suggests that the threat of IYSV to onion is increasing in South America. References: (1) P. Chomczynski and K. Mackey. Biotechniques 19:942, 1995. (2) D. H. Gent et al. Plant Dis. 90:1468, 2006. (3) H. R. Pappu et al. Plant Dis. 92:588, 2008.

Specific genes from the potato brown rot strains of Ralstonia solanacearum and their potential use for strain detection.

Ralstonia solanacearum is the agent of bacterial wilt infecting >200 different plant species covering >50 botanical families. The genus R. solanacearum can be classified into four phylotypes and each phylotype can be further subdivided into sequevars. The potato brown rot strains of R. solanacearum from phylotype IIB, sequevar 1 (IIB1), historically known as race 3, biovar 2 strains, are responsible for important economic losses to the potato industry and threaten ornamental crop production worldwide. Sensitive and specific detection methods are required to control this pathogen. This article provides a list of 70 genes and 15 intergenes specific to the potato brown rot strains of R. solanacearum from phylotype IIB1. This list was identified by comparative genomic hybridization on microarray and subsequent polymerase chain reaction validation with 14 IIB1 strains against 45 non-IIB1 strains that covered the known genetic diversity in R. solanacearum. The microarray used consisted of the previously described microarray representative of the phylotype I strain GMI1000, to which were added 660 70-mer oligonucleotides representative of new genomic islands detected in the phylotype IIB1 strain IPO1609. The brown rot strain-specific genes thus identified were organized in nine clusters covering 2 to 29 genes within the IPO1609 genome and 6 genes isolated along the genome. Of these specific genes, 29 were parts of mobile genetic elements. Considering the known instability of the R. solanacearum genome, we believe that multiple probes are required to consistently detect all IIB1 strains and we recommend the use of probes which are not part of genetic mobile elements.

Characterization of Penicillium Isolates Associated with Blue Mold on Apple in Uruguay.

Blue mold caused by Penicillium spp. is the most important postharvest disease of apple in Uruguay. Fourteen isolates of Penicillium were recovered from rotten apple and pear fruit with blue mold symptoms, and from water from flotation tanks in commercial apple juice facilities. Phenotypic identification to species level was performed, and the isolates were tested for sensitivity to commonly used postharvest fungicides. Genetic characterization of the isolates was performed with restriction fragment length polymorphism of the region including the internal transcribed spacer (ITS) ITS1 and ITS2 and the 5.8SrRNA gene (ITS1-5.8SrRNA gene-ITS2) ribosomal DNA region and with random amplified polymorphic DNA (RAPD) primers. Both techniques were able to differentiate these isolates at the species level. RAPD analysis proved to be an objective, rapid, and reliable tool to identify Penicillium spp. involved in blue mold of apple. In all, 11 isolates were identified as Penicillium expansum and 3 as P. solitum. This is the first report of P. solitum as an apple pathogen in Uruguay.

Overproduction of the rbo gene product from Desulfovibrio species suppresses all deleterious effects of lack of superoxide dismutase in Escherichia coli.

In an attempt to isolate the superoxide dismutase (SOD) gene from the anaerobic sulfate-reducing bacterium Desulfoarculus baarsii, a DNA fragment was isolated which functionally complemented an Escherichia coli mutant (sodA sodB) deficient in cytoplasmic SODs. This region carries two open reading frames with sequences which are very similar to that of the rbo-rub operon from Desulfovibrio vulgaris. Independent expression of the rbo and rub genes from ptac showed that expression of rbo was responsible for the observed phenotype. rbo overexpression suppressed all deleterious effects of SOD deficiency in E. coli, including inactivation by superoxide of enzymes containing 4Fe-4S clusters and DNA damage produced via the superoxide-enhanced Fenton reaction. Thus, rbo restored to the sodA sodB mutant the ability to grow on minimal medium without the addition of branched amino acids, and growth on gluconate and succinate carbon sources was no longer impaired. The spontaneous mutation rate, which is elevated in SOD-deficient mutants, returned to the wild-type level in the presence of Rbo, which also restored aerobic viability of sodA sodB recA mutants. Rbo from Desulfovibrio vulgaris, but not Desulfovibrio gigas desulforedoxin, which corresponds to the NH2-terminal domain of Rbo, complemented sod mutants. The physiological role of Rbo in sulfate-reducing bacteria is unknown. In E. coli, Rbo may permit the bacterium to avoid superoxide stress by maintaining functional (reduced) superoxide sensitive 4Fe-4S clusters. It would thereby restore enzyme activities and prevent the release of iron that occurs after cluster degradation and presumably leads to DNA damage.