rRNA operons and genome size of 'Candidatus Liberibacter americanus', a bacterium associated with citrus huanglongbing in Brazil.

Huanglongbing is one of the most severe diseases of citrus worldwide and is associated with 'Candidatus (Ca.) Liberibacter africanus' in Africa, 'Ca. Liberibacter asiaticus' in Asia and the Americas (Brazil, USA and Cuba) and 'Ca. Liberibacter americanus' (Lam) in Brazil. In the absence of axenic cultures, genetic information on liberibacters is scarce. The sequences of the entire 23S rRNA and 5S rRNA genes from Lam have now been obtained, using a consensus primer designed on known tRNAMet sequences of rhizobia. The size of the Lam genome was determined by PFGE, using Lam-infected periwinkle plants for bacterial enrichment, and was found to be close to 1.31 Mbp. In order to determine the number of ribosomal operons on the Lam genome, probes designed to detect the 16S rRNA gene and the 3' end of the 23S rRNA gene were developed and used for Southern hybridization with I-CeuI-treated genomic DNA. Our results suggest that there are three ribosomal operons in a circular genome. Lam is the first liberibacter species for which such data are available.

Liberibacters Associated with Citrus Huanglongbing in Brazil: 'Candidatus Liberibacter asiaticus' Is Heat Tolerant, 'Ca. L. americanus' Is Heat Sensitive.

In São Paulo State, Brazil, 'Candidatus Liberibacter americanus' and 'Candidatus Liberibacter asiaticus' are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with 'Ca. L. asiaticus' or 'Ca. L. americanus' was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any 'Ca. L. americanus'-infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, 'Ca. L. asiaticus'-infected trees had high titers at 32 and 35°C, but not at 38°C, while 'Ca. L. americanus'-infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of 'Ca. L. asiaticus' is not yet affected at 35°C, while a temperature of 32°C is detrimental to 'Ca. L. americanus'. Thus, 'Ca. L. americanus' is less heat tolerant than 'Ca. L. asiaticus'. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

A phytoplasma closely related to the pigeon pea witches'-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil.

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of São Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.

The tufB-secE-nusG-rplKAJL-rpoB gene cluster of the liberibacters: sequence comparisons, phylogeny and speciation.

The rplKAJL-rpoBC operon or beta operon is a classic bacterial gene cluster, which codes for proteins K, A, J and L of the large ribosomal subunit, as well as proteins B (beta subunit) and C (beta' subunit) of RNA polymerase. In the early 1990s, the operon was obtained as a 2.6 kbp DNA fragment (In-2.6) by random cloning of DNA from periwinkle plants infected with the Poona (India) strain of the huanglongbing agent, later named 'Candidatus (Ca.) Liberibacter asiaticus'. DNA from periwinkle plants infected with the Nelspruit strain (South Africa) of 'Ca. L. africanus' was amplified with a primer pair designed from In-2.6 and yielded, after cloning and sequencing, a 1.7 kbp DNA fragment (AS-1.7) of the beta operon of 'Ca. L. africanus'. The beta operon of the American liberibacter, as well as the three upstream genes (tufB, secE, nusG), have now also been obtained by the technique of chromosome walking and extend over 4673 bp, comprising the following genes: tufB, secE, nusG, rplK, rplA, rplJ, rplL and rpoB. The sequence of the beta operon was also determined for a Brazilian strain of 'Ca. L. asiaticus', from nusG to rpoB (3025 bp), and was found to share 99 % identity with the corresponding beta operon sequences of an Indian and a Japanese strain. Finally, the beta operon sequence of 'Ca. L. africanus' was extended from 1673 bp (rplA to rpoB) to 3013 bp (nusG to rpoB), making it possible to compare the beta operon sequences of the African, Asian and American liberibacters over a length of approximately 3000 bp, from nusG to rpoB. While 'Ca. L. africanus' and 'Ca. L. asiaticus' shared 81.2 % sequence identity, the percentage for 'Ca. L. americanus' and 'Ca. L. africanus' was only 72.2 %, and identity for 'Ca. L. americanus' and 'Ca. L. asiaticus' was only 71.4 %. The approximately 3000 bp nusG-rpoB sequence was also used to construct a phylogenetic tree, and this tree was found to be identical to the known 16S rRNA gene sequence-based tree. These results confirm earlier findings that 'Ca. L. americanus' is a distinct liberibacter, more distantly related to 'Ca. L. africanus' and 'Ca. L. asiaticus' than 'Ca. L. africanus' is to 'Ca. L. asiaticus'. The dates of speciation have also been estimated.

First Report of a Huanglongbing-Like Disease of Citrus in Sao Paulo State, Brazil and Association of a New Liberibacter Species, "Candidatus Liberibacter americanus", with the Disease.

Huanglongbing (HLB) (ex-greening) is one of the most serious diseases of citrus. The causal agent is a noncultured, sieve tube-restricted α-proteobacterium, "Candidatus Liberibacter africanus" in Africa and "Candidatus Liberibacter asiaticus" in Asia (2). The disease has never been reported from the American continent. However, Diaphorina citri, the Asian psyllid vector of HLB, is found in South, Central, and North America (Florida and Texas). Early in 2004, leaf and fruit symptoms resembling those of HLB were observed in several sweet orange orchards near the city of Araraquara, Sao Paulo State. Leaf mottling on small and large leaves was the major symptom. Shoots with affected leaves were yellowish. Fruits were small and lopsided, contained many aborted seeds, and appeared more severely affected than were plants infected with classic HLB. Forty-three symptomatic samples and twenty-five samples of symptomless sweet orange leaves from five farms were analyzed for the presence of the HLB-liberibacters using polymerase chain reaction (PCR) with two sets of HLB-specific primers for amplification of 16S rDNA (2,3) and ribosomal protein genes (1). None of the 43 symptomatic leaf samples gave a positive PCR amplification, while HLB-affected leaves from the Bordeaux HLB collection produced the characteristic amplicons with both sets of primers. The 43 symptomatic and the 25 symptomless leaf samples were then analyzed using PCR with universal primers for amplification of bacterial 16S rDNA (4). All symptomatic leaf samples, but none of the symptomless leaf samples, yielded the same 16S rDNA amplification product, indicating the presence of a bacterium in the symptomatic leaves. This was confirmed using the observation of a sieve tube restricted bacterium by electron microscopy. The 16S rDNA product was cloned, sequenced, and compared with those of "Ca. L. africanus" and "Ca. L. asiaticus". While the 16S rDNAs of these two liberibacter species have 97.5% sequence identity, the 16S rDNA sequence of the new bacterium shared only 93.7% identity with that of "Ca. L. asiaticus" and 93.9% with that of "Ca. L. africanus". The 16S rDNA sequence of the new bacterium had a secondary loop structure characteristic of the α subdivision of the proteobacteria and possessed all the oligonucleotide signatures characteristic of the liberibacters. For these reasons, the new bacterium is a liberibacter and is sufficiently different phylogenetically from known liberibacters to warrant a new species, "Candidatus Liberibacter americanus". Specific PCR primers for amplification of the 16S rDNA of the new species have been developed. They were able to detect "Ca. L. americanus" in 214 symptomatic leaf samples from 47 citrus farms in 35 municipalities, while the "old" species, "Ca. L. asiaticus", has been found only four times within the 47 farms. References: (1) A. Hocquellet et al. Mol. Cell. Probes, 13:373, 1999. (2) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Sudden Death of Citrus in Brazil: A Graft-Transmissible Bud Union Disease.

Citrus Sudden Death (CSD), a new, graft-transmissible disease of sweet orange and mandarin trees grafted on Rangpur lime rootstock, was first seen in 1999 in Brazil, where it is present in the southern Triângulo Mineiro and northwestern São Paulo State. The disease is a serious threat to the citrus industry, as 85% of 200 million sweet orange trees in the State of São Paulo are grafted on Rangpur lime. After showing general decline symptoms, affected trees suddenly collapse and die, in a manner similar to trees grafted on sour orange rootstock when affected by tristeza decline caused by infection with Citrus tristeza virus (CTV). In tristeza-affected trees, the sour orange bark near the bud union undergoes profound anatomical changes. Light and electron microscopic studies showed very similar changes in the Rangpur lime bark below the bud union of CSD-affected trees: size reduction of phloem cells, collapse and necrosis of sieve tubes, overproduction and degradation of phloem, accumulation of nonfunctioning phloem (NFP), and invasion of the cortex by old NFP. In both diseases, the sweet orange bark near the bud union was also affected by necrosis of sieve tubes, and the phloem parenchyma contained characteristic "chromatic" cells. In CSD-affected trees, these cells were seen not only in the sweet orange phloem, but also in the Rangpur lime phloem. Recent observations indicated that CSD affected not only citrus trees grafted on Rangpur lime but also those on Volkamer lemon, with anatomical symptoms similar to those seen in Rangpur lime bark. Trees on alternative rootstocks, such as Cleopatra mandarin and Swingle citrumelo, showed no symptoms of CSD. CSD-affected trees did recover when they were inarched with seedlings of these rootstocks, but not when inarched with Rangpur lime seedlings. These results indicate that CSD is a bud union disease. In addition, the bark of inarched Rangpur lime and Volkamer lemon seedlings showed, near the approach-graft union, the same anatomical alterations as the bud union bark from the Rangpur lime rootstock in CSD-affected trees. The dsRNA patterns from CSD-affected trees and unaffected trees were similar and indicative of CTV. CSD-affected trees did not react by immunoprinting-ELISA using monoclonal antibodies against 11 viruses. No evidence supported the involvement of viroids in CSD. The potential involvement of CTV and other viruses in CSD is discussed.

Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.

Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.

Catharanthus roseus, an Experimental Host Plant for the Citrus Strain of Xylella fastidiosa.

We verified by pathogenicity tests that the herbaceous plant Catharanthus roseus (Madagascar periwinkle) can be used as an experimental host for the strain of Xylella fastidiosa that causes citrus variegated chlorosis (CVC). Plants were mechanically inoculated with CVC strain 9a5c, the genome of which was recently sequenced. Plants were inoculated with the virulent 8th passage (9a5c-8) and the 51st passage (9a5c-51). Leaf deformation and stunting were seen 2 months after inoculation on 18 of 21 plants with 9a5c-8 and 8 of 21 plants with 9a5c-51. The plants were infected with X. fastidiosa as shown by polymerase chain reaction. The bacterium could be reisolated from all plants tested, showing that CVC-X. fastidiosa multiplied and moved systemically in C. roseus plants causing dysfunction in plant growth. The disease symptoms evolved within 4 months post-inoculation to a severe leaf chlorosis in all inoculated plants. The localization of X. fastidiosa in the xylem was verified by immunofluorescence. Genes coding for proteins with homologies to plant sterol-C-methyltransferase, a transketolase-like protein, subunit III of photosystem I, and a desiccation protectant protein were found to be differentially expressed in symptomatic C. roseus plants as a response to infection with X. fastidiosa in comparison to healthy plants. A tentative correlation between the pattern of expression of these C. roseus genes with the mechanism of pathogenicity of X. fastidiosa is discussed.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.