The complete genome sequence of 'Candidatus Liberibacter americanus', associated with Citrus huanglongbing.

Liberibacter spp. form a Rhizobiaceae clade of phloem-limited pathogens of limited host range. Two obligately parasitic species have been sequenced: 'Candidatus Liberibacter asiaticus', which causes citrus huanglongbing (HLB) worldwide, and 'Ca. L. solanacearum', which causes potato "zebra chip" disease. A third (proposed) species, Liberibacter crescens, was isolated from mountain papaya, grown in axenic culture, and sequenced. In an effort to identify common host determinants, the complete genomic DNA sequence of a second HLB species, 'Ca. L. americanus' strain 'São Paulo' was determined. The circular genome of 1,195,201 bp had an average 31.12% GC content and 983 predicted protein encoding genes, 800 (81.4%) of which had a predicted function. There were 658 genes common to all sequenced Liberibacter spp. and only 8 genes common to 'Ca. L. americanus' and 'Ca. L. asiaticus' but not found in 'Ca. L. solanacearum'. Surprisingly, most of the lipopolysaccharide biosynthetic genes were missing from the 'Ca. L. americanus' genome, as well as OmpA and a key regulator of flagellin, all indicating a 'Ca. L. americanus' strategy of avoiding production of major pathogen-associated molecular patterns present in 'Ca. L. asiaticus' and 'Ca. L. solanacearum'. As with 'Ca. L. asiaticus', one of two 'Ca. L. americanus' prophages replicated as an excision plasmid and carried potential lysogenic conversion genes that appeared fragmentary or degenerated in 'Ca. L. solanacearum'.

Citrus Sudden Death Is Transmitted by Graft-Inoculation and Natural Transmission Is Prevented by Individual Insect-Proof Cages.

Citrus sudden death (CSD) transmission was studied by graft-inoculation and under natural conditions. Young sweet orange trees on Rangpur rootstock were used as indicator plants. They were examined regularly for one or two characteristic markers of CSD: (i) presence of a yellow-stained layer of thickened bark on the Rangpur rootstock, and (ii) infection with the CSD-associated marafivirus. Based on these two markers, transmission of CSD was obtained, not only when budwood for graft-inoculation was taken from symptomatic, sweet orange trees on Rangpur, but also when the budwood sources were asymptomatic sweet orange trees on Cleopatra mandarin, indicating that the latter trees are symptomless carriers of the CSD agent. For natural transmission, 80 young indicator plants were planted within a citrus plot severely affected by CSD. Individual insect-proof cages were built around 40 indicator plants, and the other 40 indicator plants remained uncaged. Only two of the 40 caged indicator plants were affected by CSD, whereas 17 uncaged indicator plants showed CSD symptoms and were infected with the marafivirus. An additional 12 uncaged indicator plants became severely affected with citrus variegated chlorosis and were removed. These results strongly suggest that under natural conditions, CSD is transmitted by an aerial vector, such as an insect, and that the cages protected the trees against infection by the vector.

Distribution and quantification of Candidatus Liberibacter americanus, agent of huanglongbing disease of citrus in São Paulo State, Brasil, in leaves of an affected sweet orange tree as determined by PCR.

Huanglongbing (HLB), an insect-transmitted disease of citrus, known for many years in Asia and Africa, has appeared in the state of São Paulo State (SSP), Brazil, in 2004, and the state of Florida, USA, in 2005. HLB endangers the very existence of citrus, as trees infected with the bacterial pathogen, irrevocably decline. In the absence of curative procedures, control of HLB is difficult and only based on prevention. Even though not available in culture, the HLB bacterium could be shown to be Gram-negative and to represent a new candidate genus, Candidatus Liberibacter, in the alpha subdivision of the Proteobacteria. Three Candidatus (Ca.) L. species occur: Ca. L. africanus in Africa, Ca. L. asiaticus in Asia, SSP, and Florida, and Ca. L. americanus in SSP. The liberibacters occur exclusively in the phloem sieve tubes. On affected trees, HLB symptoms are often seen on certain branches only, suggesting an uneven distribution of the Liberibacter. Occurrence of Ca. L. americanus, the major HLB agent in SSP, has been examined in 822 leaf samples from an affected sweet orange tree by two conventional PCR techniques and a newly developed real time (RTi) PCR, also used for quantification of the Liberibacter in the leaves. Even though RTi-PCR was able to detect as few as 10 liberibacters per gram of leaf tissue (l/g), no liberibacters could be detected in any of the many leaf samples from a symptomless branch, while in blotchy mottle leaves from symptomatic branches of the same tree, the Liberibacter titer reached values as high as 10(7)l/g. These results demonstrate the uneven distribution of the Liberibacter in HLB-affected trees.

'Candidatus phytoplasma phoenicium' sp. nov., a novel phytoplasma associated with an emerging lethal disease of almond trees in Lebanon and Iran.

Almonds (Prunus amygdalus) represent an important crop in most Mediterranean countries. A new and devastating disease of almond trees in Lebanon was recently reported, characterized by the development of severe witches'-brooms on which no flowers or fruits developed, and leading to tree death within a few years. A phytoplasma was detected in diseased trees by PCR amplification of rRNA operon sequences, and RFLP patterns of amplified DNA indicated that the phytoplasma belonged to the pigeon pea witches'-broom (PPWB) group. In the present work, the presence of a phytoplasma in symptomatic plants was confirmed by electron microscopy; this phytoplasma was graft-transmissible to almond, plum and peach seedlings. The phytoplasma was characterized by sequence analysis of rRNA genes and was shown to be different from the phytoplasmas previously described in the PPWB group. A 16S rDNA phylogenetic tree identified the almond tree phytoplasma as a member of a distinct subclade of the class Mollicutes. Oligonucleotides have been defined for specific detection of the new phytoplasma. The almond phytoplasma from Lebanon was shown to be identical to a phytoplasma that induces a disease called 'almond brooming' in Iran, but different from another PPWB-group phytoplasma that infects herbaceous annual plants in Lebanon. Based on its unique properties, the name 'Candidatus Phytoplasma phoenicium' is proposed for the phytoplasma associated with almond witches'-broom in Lebanon and Iran.

Spiroplasma citri, a plant pathogenic molligute: relationships with its two hosts, the plant and the leafhopper vector.

Spiroplasma citri, the type species of the genus Spiroplasma (Spiroplasmataceae, Mollicutes), is restricted to the phloem sieve tubes and transmitted by phloem sap-feeding insects, as is characteristic of the phytopathogenic mollicutes. The spiroplasmas are the only mollicutes showing motility and helical morphology, apparently mediated by a contractile fibrillar cytoskeleton bound to the inner surface of the spiroplasmal membrane. MreB genes, which are involved in cell-shape determination, have been identified in S. citri. Identified genes of other functional groups are those involved in the transmission of S. citri by the leafhoppers and genes coding for lipoproteins, including spiralin, bound to the outer surface of the spiroplasma membrane. S. citri mutants that are unable to use fructose induce only mild and delayed symptoms. Fructose utilization by the sieve tube-restricted wild-type spiroplasmas is postulated to deprive the companion cells of fructose, thereby impairing sucrose loading into the sieve tubes.

Spatial and temporal analyses of citrus sudden death as a tool to generate hypotheses concerning its etiology.

ABSTRACT Citrus sudden death (CSD), a new disease of unknown etiology that affects sweet orange grafted on Rangpur lime, was visually monitored for 14 months in 41 groves in Brazil. Ordinary runs analysis of CSD-symptomatic trees indicated a departure from randomness of symptomatic trees status among immediately adjacent trees mainly within rows. The binomial index of dispersion (D) and the intraclass correlation (k) for various quadrat sizes suggested aggregation of CSD-symptomatic trees for almost all plots within the quadrat sizes tested. Estimated parameters of the binary form of Taylor's power law provided an overall measure of aggregation of CSD-symptomatic trees for all quadrat sizes tested. Aggregation in each plot was dependent on disease incidence. Spatial autocorrelation analysis of proximity patterns suggested that aggregation often existed among quadrats of various sizes up to three lag distances; however, significant lag positions discontinuous from main proximity patterns were rare, indicating a lack of spatial association among discrete foci. Some asymmetry was also detected for some spatial autocorrelation proximity patterns, indicating that within-row versus across-row distributions are not necessarily equivalent. These results were interpreted to mean that the cause of the disease was most likely biotic and its dissemination was common within a local area of influence that extended to approximately six trees in all directions, including adjacent trees. Where asymmetry was indicated, this area of influence was somewhat elliptical. Longer-distance patterns were not detected within the confines of the plot sizes tested. Annual rates of CSD progress based on the Gompertz model ranged from 0.37 to 2.02. Numerous similarities were found between the spatial patterns of CSD and Citrus tristeza virus (CTV) described in the literature, both in the presence of the aphid vector, Toxoptera citricida. CSD differs from CTV in that symptoms occur in sweet orange grafted on Rangpur lime. Based on the symptoms of CSD and on its spatial and temporal patterns, our hypothesis is that CSD may be caused by a similar but undescribed pathogen such as a virus and probably vectored by insects such as aphids by similar spatial processes to those affecting CTV.

Effect of polyclonal, monoclonal, and recombinant (single-chain variable fragment) antibodies on in vitro morphology, growth, and metabolism of the phytopathogenic mollicute Spiroplasma citri.

Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed.

Fructose operon mutants of Spiroplasma citri.

Fructose-negative mutants of Spiroplasma citri wild-type strain GII-3 were selected by two methods. The first method is based on the selection of spontaneous xylitol-resistant mutants, xylitol being a toxic fructose analogue. Five such mutants were obtained, but only one, xyl3, was unable to use fructose and had no phosphoenolpuryvate:fructose phosphotransferase system (fructose-PTS) activity. Amplification and sequencing of the fructose permease gene of mutant xyl3 revealed the presence of an adenylic insertion leading to a truncated permease. The second method is based on inactivation of fruA and/or fruK by homologous recombination involving one crossing-over between the chromosomal genes and inactivated genes carried by replicative plasmids. Fructose-negative mutants were obtained at a frequency of about 10%. Fructose-PTS activity and 1-phosphofructokinase activity were not detected in four representative mutants that were characterized (H31, H45, E38 and E53). In strain H31, Southern blot analysis and PCR showed that the result of homologous recombination was, as expected, the presence in the chromosome of two mutated fruA-fruK copies with the plasmid sequence in between. Only the mutated copy, under control of the fructose operon promoter, was transcribed. This work describes for the first time the use of two methods to obtain fructose-auxotrophic mutants of S. citri. The method involving homologous recombination is a general procedure for gene disruption in S. citri.