Analysis of Defense-Related Gene Expression in Citrus Hybrids Infected by Xylella fastidiosa.

Resistance to Xylella fastidiosa was evaluated in 264 hybrids of crosses between Murcott tangor (Citrus reticulata × Citrus sinensis) and Pera sweet orange (C. sinensis) under field conditions. Uninfected hybrids were grafted with buds collected from Pera sweet orange plants infected with X. fastidiosa, forming a plant with two scions (i.e., hybrid branches and Pera sweet orange branches). From these plants, we chose 10 genotypes with three biological replicates. We evaluated gene expression, bacterial multiplication, and citrus variegated chlorosis (CVC) symptom development in both scions. X. fastidiosa was not detected in most hybrid scions and none showed disease symptoms. In contrast, all Pera sweet orange scions were infected with X. fastidiosa and expressed symptoms of CVC. We quantified the expression of 12 defense-related genes by qPCR comparing resistant to susceptible scions. We suggest that some of these genes are involved in resistance of the hybrids to X. fastidiosa, since their expression was significantly higher in the resistant hybrid scions than in tolerant hybrids and scions originated from CVC symptomatic Pera sweet orange buds. However, we note that these data should be interpreted carefully, as the plant genotypes tested are related but necessarily distinct (hybrids of C. reticulata and C. sinensis, in relation to a C. sinensis control). A principal component analysis revealed a relationship between the expression of these genes and hybrid scions, and between scions that originated from infected buds and the presence of the bacteria and plant symptoms. Multiyear field trials are necessary to develop plant resistance to X. fastidiosa. While the experimental design used here had limitations, it allowed us to identify a set of genes potentially involved in Citrus sp. resistance to this pathogen. Future work on the role of these genes in plant defenses to X. fastidiosa infection is necessary to confirm their importance in the displayed resistance phenotype.

Evaluation of the genetic variability of orchid fleck virus by single-strand conformational polymorphism analysis and nucleotide sequencing of a fragment from the nucleocapsid gene.

The variability of a fragment of the nucleocapsid gene of orchid fleck virus (OFV) was investigated by single-strand conformational polymorphism (SSCP) analysis and nucleotide sequencing. Forty-eight samples of 18 genera of orchids were collected from Brazil, Costa Rica and Australia. The SSCP analysis yielded six different band patterns, and phylogenetic analysis based on the nucleotide fragment sequence obtained in this work and six available in GenBank showed two different groups, one with isolates 023Germany and So-Japan, and other with the rest of the isolates. None of the analyses showed geographic correlation among the Brazilian strains. The data obtained in this study showed a low genetic variation in this region of the genome; the d(N)/d(S) ratio of 0.251-0.405 demonstrated a negative selective pressure that maintains the stability of the analyzed fragments.

Analysis of 16S rDNA Sequences from Citrus Huanglongbing Bacteria Reveal a Different "Ca. Liberibacter" Strain Associated with Citrus Disease in São Paulo.

Citrus huanglongbing (HLB, ex greening) is one of the most serious and destructive citrus diseases in the world. It is caused by a phloem-limited and nonculturable bacterium, "Candidatus Liberibacter". The disease occurs in some Asian and African countries and recently has been reported in the state of São Paulo, Brazil. Analysis of the 16S ribosomal (r)DNA of the HLB bacteria from orchards in São Paulo revealed the presence of two distinct strains of "Ca. Liberibacter". One of them, named LSg1 (Liberibacter sequence group 1), was 100% identical to strains from Japan (GenBank accessions AB038369 and AB008366), the Asian forms of the bacteria. The other, LSg2, is genetically distant from the Asian (96.1 to 96.3% identity) and African (95.8 to 96.1% identity) strains. Comparison of the 16S rDNA sequences from the LSg2 and the Asian strain revealed the presence of INDELs and point mutations. Specific primers designed for this Brazilian Liberibacter strain revealed that it is more widely distributed throughout the São Paulo orchards compared with the LSg1 strain. The HLB symptoms caused by both strains are almost identical and, interestingly, both strains were found in the same sample, revealing mixed infection in a citrus plant.

First Report of the Causal Agent of Huanglongbing ("Candidatus Liberibacter asiaticus") in Brazil.

Huanglongbing (ex-greening) disease is one of the most serious diseases of citrus. It is caused by the phloem-limited, gram-negative bacterium "Candidatus Liberibacter spp.". This bacterium is not well characterized mainly because it is still uncultured. There are two known strains, Asian ("Candidatus Liberibacter asiaticus") and African ("Candidatus Liberibacter africanus") that cause severe damage to citrus plants including twig dieback, decline, and death. Symptoms first appear as leaf mottling and chlorosis occurring in one shoot or sector of trees. Later, leaf symptoms resemble nutritional deficiencies (Zn, Ca, and N) that vary depending on the strains, with more severe symptoms caused by "Ca. L. asiaticus". The Asian strains are transmitted by the Asian citrus psyllid (AsCP), Diaphorina citri, which is present in Brazil. The bacterium has been detected in citrus plants in many geographic locations including China, Japan, Thailand, India, the Philippines, the Arabian Peninsula, and Africa. In 2004, plants showing Huanglongbing symptoms were observed in the Araraquara County, a central region of the State of Sao Paulo, the largest citrus-producing area in Brazil. To verify the presence of "Ca. L. spp." in these plants, leaf samples of sweet orange cvs. Hamlin and Valencia were used for DNA extraction and polymerase chain reaction amplification using the specific OI1 and Oi2c primers (1). Amplification of the 16S rDNA was positive for 2 (cvs. Hamlin and Valencia) of 10 analyzed plants. The amplified fragments were cloned and sequenced. The amplicons obtained from both plants showed the same sequence, which differed from "Ca. L. africanus", utilized as the positive control in the amplification experiment (27 divergent bases in 1,160). The sequences were used for BLAST searches, and the results showed identities ranging from 94.71 to 100% with "Ca. L. spp." sequences available at the National Center for Biotechnology Information database (on-line publication). The highest scores were obtained with "Ca. L. asiaticus sequences. These analyses confirmed the presence of such agent in the State of Sao Paulo. To our knowledge, this is the first report of "Ca. L. asiaticus" in Brazil as well as elsewhere in the Americas. The significance of this report relates to the potential damage that this pathogen could cause to the citrus industry in the largest citrus-producing country in the world. It remains unclear how and when the pathogen entered Brazil. Reference: (1) S. Jagoueix et al. Mol. Cell Probes 10:43, 1996.

Primers based on the rpf gene region provide improved detection of Xanthomonas axonopodis pv. citri in naturally and artificially infected citrus plants.

AIMS: To have a PCR-based detection method for Xanthomonas axonopodis pv. citri (Xac) using primers designed in a specific region of its genome. METHODS AND RESULTS: A Xac-specific region was identified inside the rpf gene cluster of strain IAPAR 306 in an analysis of its complete genomic sequence. Two primers were designed, Xac01 and Xac02, which, when used in a standard PCR assay, direct the amplification of a 581 bp fragment from DNA of strains belonging to Xac from different regions around the world including unusual American and Asian strains. This product was not observed when DNA from strains of the closely related X. a. aurantifolli and X. a. citrumelo were used as templates. Extracts prepared from 28 xanthomonads of other species, and epiphytic bacteria isolated from citrus also failed to produce products with these primers. Amplification was obtained from cells grown in vitro, from extracts of both fresh and dried citrus canker lesions and from washes of inoculated but asymptomatic leaf surfaces. In sensitivity tests, this PCR technique detected as few as 100 cells. CONCLUSIONS: Primers Xac01 and Xac02 provide specific and sensitive detection of Xac in all citrus tissues where the pathogen is found. SIGNIFICANCE AND IMPACT OF THE STUDY: This PCR-based diagnostic test is suitable for monitoring asymptomatic plants in areas where the bacteria is endemic, in plant quarantine and regulatory situations, and also for obtaining an accurate diagnosis in a very short time. These are important characteristics for any assay to be used for the management of citrus canker disease.

Differentiation of strains of Xylella fastidiosa by a variable number of tandem repeat analysis.

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although mono- and dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Nei's measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a H value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.