First Report of Citrus leprosis virus Nuclear Type in Sweet Orange in Colombia.

Colombia is ranked 18th in the world in citrus production and contributed 0.9% of the total world share. Among four important citrus-producing regions of Colombia, the Orinoco region (3 to 6°N, 68 to 74°W) consists of two citrus-producing states, Meta and Casanare. Citrus leprosis is the most important viral disease of citrus in Colombia (1,3). Three types of Citrus leprosis virus (CiLV) infect citrus, producing leprosis-like lesion symptoms. Two of the three CiLV species, Citrus leprosis virus cytoplasmic type (CiLV-C) and cytoplasmic type 2 (CiLV-C2), produce particles only in the cytoplasm (3). The other species, Citrus leprosis virus nuclear type (CiLV-N), produces particles in both the cytoplasm and nucleus (4). CiLV-C is more prevalent and destructive while CiLV-N has been reported only in Brazil, Panama, and Mexico (4). Interestingly, both CiLV-C and -C2 were reported from the same regions of Meta and Casanare States in Colombia in 2004 and 2012 (1,3). CiLV-C lesions are usually rounded (initially 2 to 3 mm in diameter and extending up to 30 mm), have dark-brown or greenish central chlorotic spots, and are surrounded by yellow halos. CiLV-N lesions have been described as smaller in size and form three well-defined regions including a necrotic center with an intermediate orange color halo and an outer chlorotic halo (2). In 2013, 'Valencia' sweet orange (Citrus sinensis L.) leaves with suspected CiLV-N symptoms were collected from 8 plants in Casanare State and shipped under permit to the USDA-APHIS-PPQ-CPHST, Beltsville, MD. Total RNA from symptomatic and healthy sweet orange leaves were extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). RT-PCR primers specific to CiLV-C, CiLV-C2 (3), and CiLV-N nucleocapsid (N) (CiLV-N-NPF: 5'-ATGGCTAACCCAAGTGAGATCGATTA-3'; CiLV-N-NPR: 5'-AGTTGCCTTGAGATCATCACATTGGT-3') and putative matrix protein (M) genes (CiLV-N-MF: 5'-ATGTCTAAACAGATTAATATGTGCACTGTG-3'; CiLV-N-MR: 5'-CTAACCACTGGGTCCCGC-3') were utilized to identify the CiLV associated with the leprosis-affected leaf samples from Casanare. RT-PCR with CiLV-C primers failed to produce any amplicon, but CiLV-N primers successfully amplified the partial N gene (681 bp) and entire M gene (552 nt) amplicons from multiple leaves of all leprosis samples. In addition, a 795-bp amplicon specific to CiLV-C2 also was amplified from the CiLV-N suspected samples. Similar results were obtained when the vector, flat spider mite (Brevipalpus spp.) total RNA was used as template for RT-PCR. For further confirmation, each amplicon was cloned and sequenced. Sequencing of the N and M gene amplicons of CiLV-N (accession nos. KJ195893 and KJ195894) and coat protein gene of CiLV-C2 showed 97 to 99% nucleotide sequence identity with the CiLV-N M2345 isolate sequence (KF209275) from Mexico (4) and CiLV-C2 L147V1 isolate sequence (JX000024) from Colombia (3), respectively. Phylogenetic analyses of these N and M protein gene sequences confirmed a mixed infection of the same plant with two viruses, one from an unassigned new genus Dichorhavirus (CiLV-N) and another from genus Cilevirus (CiLV-C2). This is the first report of CiLV-N in Colombia, and also the first report of an occurrence of CiLV-N in mixed infection with CiLV-C2. All three known species of CiLV occur in the Orinoco region of Colombia. References: (1) M. G. León et al. Plant Dis. 90: 682, 2006. (2) J. P. R. Marques et al. Anais da Academia Brasileira de Ciências 82:501, 2010. (3) A. Roy et al. Phytopathology 103:488, 2013. (4) A. Roy et al. Genome Announc. 1(4): e00519-13, 2013.

Development of Primers and Probes for Genus and Species Specific Detection of 'Candidatus Liberibacter Species' by Real-Time PCR.

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different 'Candidatus Liberibacter species', 'Ca. Liberibacter asiaticus', 'Ca. Liberibacter americanus', and 'Ca. Liberibacter africanus', which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of 'Ca. Liberibacter' at the genus level, providing increased convenience. Recent molecular analyses of 'Ca. Liberibacter species' and other bacteria suggest that the rpoB gene (encoding the ß-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus 'Ca. Liberibacter species', enabling detection of 'Ca. Liberibacter' at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected 'Ca. Liberibacter solanacearum', a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

Development and optimization of a real-time detection assay for Xanthomonas fragariae in strawberry crown tissue with receiver operating characteristic curve analysis.

Angular leaf spot of strawberry is caused by the bacterium Xanthomonas fragariae. The disease is transmitted primarily through systemically infected nursery stock. This creates problems for nurseries wishing to export plants to Europe because of quarantine restrictions. Currently, field inspections for symptoms are used to certify plants free of X. fragariae, but visual inspection is not useful for detecting plants infected systemically. To detect systemic infections, polymerase chain reaction (PCR) is the desired tool because of its sensitivity, specificity, and ease of use. In this study, we developed three sets of real-time PCR primers and probes and determined optimal reaction conditions for use of these primers for the detection of the bacterium X. fragariae in strawberry crown tissue. Real time detection proved to be both more sensitive and specific than standard PCR. Moreover, the detection of X. fragariae in crown tissue extract was possible with real-time PCR but not with standard PCR which is a significant improvement over standard PCR. The information on sensitivity and specificity of the primer sets was used to evaluate the performance of these primers with receiver operating characteristic (ROC) curve analysis under different tolerances. The results of this analysis can be used to provide guidance on threshold selection to manage disease below unacceptable levels. The results of this research may be useful to regulators and inspectors who must certify that plants meet European and Mediterranean Plant Protection Organization standards.

First Report of Xylella fastidiosa in Avocado in Costa Rica.

Since the late 1990s, chlorotic mottling, marginal scorch, deformation of leaves, defoliation, shortening of internodes, and branch dieback have been observed in avocado trees (Persea americana Mill.) in Costa Rica. The symptoms are not uniformly distributed in the tree, so some branches are symptomatic while others are not. These symptoms are similar to several leaf scorch diseases caused by the bacterium Xylella fastidiosa Wells (2,4). This bacterium has been detected in coffee and citrus plants in Costa Rica. Of 227 avocado trees tested by double-antibody sandwich (DAS)-ELISA with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) from 2000-2004, 188 were positive. Results of ELISA tests of individual trees varied with the season and branches tested. Fifteen greenhouse-grown, ELISA-negative avocado seedlings were grafted with budwood from an ELISA-positive tree. Eight of these developed scorch symptoms and one also showed chlorotic mottling and deformation, showing that the disease is graft transmitted. All of these features are characteristic of diseases caused by X. fastidiosa (2,4). Transmission electron microscopy of leaf petioles from three field trees positive by ELISA, revealed rod-shaped bacilli approximately 1.6 to 2.0 µm long and 0.3 µm in diameter with a rippled cell wall inside xylem vessels and embedded in a matrix; morphology and measurements that are consistent with those reported for X. fastidiosa (2). DNA extraction and PCR attempts have been limited by mucilaginous sap from avocado. Positive PCR results (approximately 472-bp band) were obtained from two of the grafted seedlings and seven field trees from two distinct geographical locations (Alajuela and San José provinces) with DNA extractions from the plant sap using DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) following a modified protocol (1) and nested PCR (3). Four of the PCR products, including one from the grafted seedlings, were cloned and sequenced in duplicate. GenBank sequences EU021997 to EU022000 present 99 to 100% sequence identity to a Pierce's disease strain from California (Temecula1) and 94 to 95% to a citrus variegated chlorosis strain from Brazil (Found-5). Several attempts have been made to isolate the bacterium in 'periwinkle wilt' and buffered cysteine-yeast extract media with negative results, probably because of the rapid production of mucilaginous sap when the avocado tissues were sampled. To our knowledge, this is the first report of X. fastidiosa in avocado trees. References: (1) M. J. Green et al. Plant Dis. 83:482, 1999. (2) S. S. Hearon et al. Can. J. Bot. 58:1986, 1980. (3) M. R. Pooler and J. S. Hartung. Curr. Microbiol. 31:377, 1995. (4) A. H. Purcell et al. Phytopathology 89:53, 1999.

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.

Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants.

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003. Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.

Adult Citrus Leafminers (Phyllocnistis citrella) Are Not Efficient Vectors for Xanthomonas axonopodis pv. citri.

Interest in the interaction between the citrus leafminer (Phyllocnistis citrella) and citrus bacterial canker, caused by Xanthomonas axonopodis pv. citri, has increased as a greater incidence and severity of canker-diseased plants was observed in groves infested with the citrus leafminer. To determine whether adults of the citrus leafminer could act as vectors of citrus canker, we investigated two potential mechanisms for direct spread by leafminer adults using experimental microcosms. First, adult leafminers were raised on canker-infected foliage and were allowed to mate and lay eggs on healthy plants. These plants then were observed for development of citrus canker symptoms. In a second set of experiments, adults raised on healthy plants were given free access to canker-diseased plants during the period in which they mated and laid eggs on healthy plants. In all, 3,119 mines were produced by developing larvae on a total of 2,384 leaves examined for citrus canker symptoms. No symptoms of citrus bacterial canker disease were observed on any of the healthy test plants in 37 independent experimental trials conducted to test these two potential mechanisms of spread of citrus canker, and the pathogen was not recovered from insects exposed to symptomatic Rangpur lime plants. The upper limit on the rate of transmission was estimated to be less than 0.2% per oviposition event based on the binomial probability distribution. However, when adult P. citrella insects were artificially contaminated with high levels of X. axonopodis pv. citri, transmission to Rangpur lime plants with the induction of citrus canker was observed. This suggests that the ability of P. citrella to transmit X. axonopodis pv. citri is limited by the rate at which it can acquire inoculum from infected plants. The results support the conclusion that adult citrus leafminers are not efficient vectors for citrus canker bacteria, and the disease is unlikely to be spread this way.

First Report of Oleander Leaf Scorch Caused by Xylella fastidiosa in Texas.

Bacterial leaf scorch caused by Xylella fastidiosa has been reported on oleander in California (3) and Florida (4). In June 2002, leaf scorch symptoms including chlorotic mottling of leaves, necrosis on leaf tips or whole leaves, defoliation, and shortened internodes were observed in oleander plants at various locations in Texas, including Galveston, Harlingen, Austin, San Antonio, and El Campo. The symptomatic varieties Calypso, Commandant Barthelemy, Lane Taylor Sealy, Little Red, Mrs. George Roeding, Mrs. Runge, Scarlet Beauty, and Petite Salmon, as well as symptomless varieties, Turner's Shari D and Sugarland from Moody Gardens in Galveston, Texas were sampled. All samples were tested for the presence of X. fastidiosa using enzyme-linked immunosorbent assay (ELISA) and bacterial isolation according to the methods of Huang and Sherald (2). All symptomatic varieties reacted positively in ELISA, and colonies characteristic of X. fastidiosa were isolated from all eight symptomatic varieties 10 to 15 days after incubation at 28°C. The colonies were confirmed to be X. fastidiosa using polymerase chain reaction (PCR) (2). ELISA tests and bacterial isolations from the symptomless Shari D and Sugarland varieties were negative. Membrane entrapment immunofluorescence (MEIF) (1) using the antibody CREC 26 to X. fastidiosa was also done on three of the symptomatic varieties and one asymptomatic variety obtained from Moody Gardens, and fluorescing bacteria were found only in the three symptomatic varieties. Symptomatic samples of Petite Salmon, one pink variety and one red variety obtained from a residential area west of Galveston, and a red oleander in Harlingen, TX, also tested positive with MEIF. Other ELISA-positive samples were obtained from symptomatic oleanders from Austin, San Antonio, and El Campo, TX. The X. fastidiosa bacteria isolated from the variety Lane Taylor Sealy were used to inoculate three red oleander plants by making an 8-cm-long vertical cut into the stem and then in 15 locations injecting approximately 15 µl of a X. fastidiosa suspension (108-9 cells per ml). The cut area was wrapped with Parafilm after inoculation, and the plants were kept at 29°C in a greenhouse. Three healthy red oleanders were inoculated with periwinkle wilt liquid medium for controls. Approximately 3 months after inoculation, chlorotic mottling along the edges of leaves was observed in the oleanders inoculated with X. fastidiosa, and the bacterium was reisolated from symptomatic leaves as described above. No symptoms were observed on the control plants, and bacterial isolation from the control plants was also negative. To our knowledge, this is the first report to show the causal role of X. fastidiosa in oleander leaf scorch and the presence of the disease in different locations in Texas, extending the geographic range of this important bacterial disease. References: (1) R. H. Brlansky et al. Plant Dis. 74:863, 1990. (2) Q. Huang and J. L. Sherald. Curr. Microbiol. 48:73, 2004. (3) A. H. Purcell et al. Phytopathology 89:53, 1999. (4) R. L. Wichman and D. L. Hopkins. Plant Dis. 84:198, 2000.

Presence of Xylella fastidiosa in Sweet Orange Fruit and Seeds and Its Transmission to Seedlings.

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.

Citrus and Coffee Strains of Xylella fastidiosa Induce Pierce's Disease in Grapevine.

Xylella fastidiosa causes citrus variegated chlorosis (CVC) disease in Brazil and Pierce's disease of grapevines in the United States. Both of these diseases cause significant production problems in the respective industries. The recent establishment of the glassy-winged sharpshooter in California has radically increased the threat posed by Pierce's disease to California viticulture. Populations of this insect reach very high levels in citrus groves in California and move from the orchards into the vineyards, where they acquire inoculum and spread Pierce's disease in the vineyards. Here we show that strains of X. fastidiosa isolated from diseased citrus and coffee in Brazil can incite symptoms of Pierce's disease after mechanical inoculation into seven commercial Vitis vinifera varieties grown in Brazil and California. Thus, any future introduction of the CVC strains of X. fastidiosa into the United States would pose a threat to both the sweet orange and grapevine industries. Previous work has clearly shown that the strains of X. fastidiosa isolated from Pierce's disease- and CVC-affected plants are the most distantly related of all strains in the diverse taxon X. fastidiosa. The ability of citrus strains of X. fastidiosa to incite disease in grapevine is therefore surprising and creates an experimental system with which to dissect mechanisms used by X. fastidiosa in plant colonization and disease development using the full genome sequence data that has recently become available for both the citrus and grapevine strains of this pathogen.

Transmission of the Citrus Variegated Chlorosis Bacterium Xylella fastidiosa with the Sharpshooter Oncometopia nigricans.

Citrus variegated chlorosis (CVC) is an economically important, destructive disease in Brazil and is caused by the bacterium Xylella fastidiosa Wells. The bacterium has been found to be transmitted in Brazil by sharpshooter leafhoppers (Cicadellidae). Sharpshooters are present in most citrus growing areas of the United States. The sharpshooter leafhopper, Oncometopia nigricans Walker, frequently is found feeding on citrus in Florida. This sharpshooter transmits the X. fastidiosa strains that cause Pierce's disease of grape and ragweed stunt. Research was initiated to determine if O. nigricans was capable of vectoring the X. fastidiosa that causes CVC. In 59 different transmission tests, using 1 to 57 insects per test, transmission of the bacterium was observed 12 times (20.3%). Symptom development in the greenhouse was not a reliable indicator of transmission. Transmission was verified by specific polymerase chain reaction-based assays. Individual insects were able to transmit the bacterium. This information on sharpshooter transmission of CVC is needed to assess the threat posed by the CVC disease to the citrus industries in the United States.

Construction of a shuttle vector and transformation of Xylella fastidiosa with plasmid DNA.

We have isolated, cloned, and sequenced a 5823-bp cryptic plasmid from a strain of Xylella fastidiosa. This plasmid encodes five open reading frames (ORF) greater than 400 nucleotides each. ORF 2 encodes a protein with 37% amino acid identity to the replication initiator protein of plasmid pECB2 from Pseudomonas alcaligenes. This RepA protein from X. fastidiosa contains both a leucine zipper and helix turn helix motif characteristic of proteins involved in DNA replication. The sequence 5' of ORF 2 has all of the features characteristic of plasmid origins of replication as well as regulatory elements required for transcription of ORF 2. Open reading frame 2, along with the upstream origin of replication, was cloned as an EcoRI fragment into pUC19 to create a shuttle vector. This construct was introduced into Xylella fastidiosa by electroporation, with selection for carbenicillin resistance. Transformation was verified by both PCR and Southern hybridization experiments. Frequency of transformation was low, but increased ten-fold when the plasmid was grown in X. fastidiosa rather than Escherichia coli prior to transformation. This work represents the first step towards the development of a system for genetic analysis of this important plant pathogen of citrus, grapevines, and other horticultural crops.

An Evaluation of the Genetic Diversity of Xylella fastidiosa Isolated from Diseased Citrus and Coffee in São Paulo, Brazil.

ABSTRACT Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Café, respectively, were indistinguishable based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of São Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.

Coffee Leaf Scorch Caused by a Strain of Xylella fastidiosa from Citrus.

Citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS) are two economically important diseases in Brazil caused by the bacterium Xylella fastidiosa. Strains of the bacterium isolated from the two plant hosts are very closely related, and the two diseases share sharpshooter insect vectors. In order to determine if citrus strains of X. fastidiosa could infect coffee and induce CLS disease, plant inoculations were performed. Plants of coffee, Coffea arabica 'Mundo Novo', grafted on Coffea canephora var. robusta 'Apuatão 2258' were mechanically inoculated with triply cloned strains of X. fastidiosa isolated from diseased coffee and citrus. Three months postinoculation, 5 of the 10 plants inoculated with CLS-X. fastidiosa and 1 of the 10 plants inoculated with CVC-X. fastidiosa gave positive enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Eight months postinoculation, another six plants inoculated with CVC-X. fastidiosa gave positive PCR results. The two X. fastidiosa strains were isolated from the inoculated plants and showed the same characteristics as the original clones by microscopy, ELISA, and PCR. None of the plants inoculated with sterile periwinkle wilt (PW) medium as controls gave positive reactions in diagnostic tests, and none developed disease symptoms. Six months postinoculation, seven plants inoculated with CLS-X. fastidiosa and eight inoculated with CVC-X. fastidiosa began to develop characteristic CLS symptoms, including apical and marginal leaf scorch, defoliation, and reductions of internode length, leaf size, and plant height, terminal clusters of small chlorotic and deformed leaves, and lateral shoot dieback. We have demonstrated that X. fastidiosa from citrus plants is pathogenic for coffee plants. This has important consequences for the management of CLS disease and has implications for the origin of citrus variegated chlorosis disease.

16S rDNA sequence analysis of Xylella fastidiosa strains.

The 16S rDNA encoding the small subunit ribosomal RNA were amplified by PCR, cloned, and sequenced from 16 strains of Xylella fastidiosa originating from nine different hosts. In pair-wise comparisons, X. fastidiosa strains showed a maximum variation of 1.0% or 14 nucleotide positions. When all 16 sequences were considered as a set, 54 variable positions were found. Analysis of the sequence data indicated that the X. fastdiosa strains formed three rDNA groups. Group one includes Pierce's disease and mulberry leaf scorch strains; Group two, periwinkle wilt, plum leaf scald, phony peach, oak leaf scorch, and elm leaf scorch strains; and Group three, citrus variegated chlorosis and coffee leaf scorch strains. All X. fastidiosa strains exhibited significantly higher levels of sequence heterogeneity (63 to 83 nucleotide positions) when compared to species from Xanthomonas and Stenotrophomonas. Our data demonstrate that 16S rDNA sequence data could provide valuable information for future classification of X. fastidiosa at the sub-species level.

Strains of Xylella fastidiosa rapidly distinguished by arbitrarily primed-PCR.

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0. 872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors.

Distribution of Xylella fastidiosa in Citrus Rootstocks and Transmission of Citrus Variegated Chlorosis Between Sweet Orange Plants Through Natural Root Grafts.

To study translocation of Xylella fastidiosa to citrus rootstocks, budsticks from citrus variegated chlorosis (CVC)-affected cv. Pera sweet orange (Citrus sinenesis (L.) Osb.) were top grafted on 15 citrus rootstocks. Disease symptoms were conspicuous 3 months later on all 15 rootstocks tested. The presence of X. fastidiosa was confirmed by light microscopy, double-antibody sandwich enzyme-linked immunosorbent assays, and polymerase chain reaction in rootlets and main roots of CVC-symptomatic Pera sweet orange in 11 of the 15 rootstocks tested. These results suggest that bacterial translocation from the aerial plant parts to the root system occurs but is not essential for X. fastidiosa to induce symptoms in the aerial parts. Bacterial translocation to the roots was not correlated with CVC leaf-symptom severity in the Pera scion. To determine if CVC disease could be transmitted by natural root grafts, two matched seedlings of each of four sweet orange cultivars (Pera, Natal, Valencia, and Caipira) were transplanted into single pots. One seedling rootstock of each pair was inoculated by top grafting with a CVC-contaminated budstick while the other seedling rootstock was cut but not graft inoculated. Transmission of X. fastidiosa from an inoculated plant to a noninoculated plant sharing the same pot was observed in all four sweet orange cultivars tested. Transmission was confirmed by observation of natural roots grafts between the two plants, presence of X. fastidiosa in the root grafts, and disease development in the uninoculated plants. This is the first report of transmission of CVC disease through natural root grafts.

Coffee Leaf Scorch Bacterium: Axenic Culture, Pathogenicity, and Comparison with Xylella fastidiosa of Citrus.

Symptoms of coffee leaf scorch (CLS) appear on young flushes of field plants as large marginal and apical scorched areas on recently mature leaves. Affected leaves drop, shoot growth is stunted, and apical leaves are small and chlorotic. Symptoms may progress to shoot dieback. Only scorched leaves which could not be related to other known agents consistently contained bacteria and bacterial agglomerates when observed with light microscopy. Only plants with these symptoms were positive in enzyme-linked immunosorbent assay (ELISA) tests using antiserum to Xylella fastidiosa Wells et al. The bacterium Xylella fastidiosa Wells et al. was isolated in November 1995 from coffee (Coffea arabica) leaves with scorch symptoms on supplemented periwinkle wilt medium. Colonies were circular, dome-shaped, white, and 0.5 to 1.5 mm in diameter. Two of 10 young coffee seedlings stem-inoculated with a suspension of the isolated X. fastidiosa in January 1996 showed leaf scorch symptoms 3 to 5 months later, contained bacteria in xylem extracts, and reacted positively in ELISA using antiserum to the citrus variegated chlorosis (CVC) strain of X. fastidiosa. ELISA-positive bacteria were reisolated from this plant. None of the symptomless plants, including controls, revealed bacteria on microscopic examinations, ELISA, or isolation attempts. Antisera developed against cultured bacteria from both CLS and CVC plants reacted positively against plant extracts of both diseases in dot immunobinding assays (DIBA). The level of detection was about 5 × 105 bacteria ml-1 for both homologous and heterologous reactions. The polymerase chain reaction amplification products produced by CLS and CVC strains of X. fastidiosa were indistinguishable. Geographical distribution of these strains is not the same. CLS is widespread and usually occurs if coffee is adjacent to CVC-affected citrus. However, CVC does not always occur when citrus is grown adjacent to CLS-affected coffee. The bacteria are closely related, if not identical.

Sequence analysis of a 1296-nucleotide plasmid from Xylella fastidiosa.

A cryptic plasmid from Xylella fastidiosa strain ATCC 35868 was cloned, sequenced, and the sequence entered into GenBank (U71220). The plasmid is 1296 nucleotides in length with 55% GC content and three open reading frames. A plasmid with sequence homology was found in only one other strain of X. fastidiosa, ATCC 35878. Searches of the GenBank reveal nucleotide sequence homology with plasmid pNKH43 from Stenotrophomonas maltophilia, and amino acid sequence homology with phage Pf3 from Pseudomonas aeruginosa, plasmid pAP12875 from Acetobacter pasteurianus, and plasmid pVT736-1 from Actinobacillus actinomycetemcomitans.

Detection of Xylella fastidiosa in potential insect vectors by immunomagnetic separation and nested polymerase chain reaction.

A sensitive and specific assay for detecting Xylella fastidiosa in potential insect vectors was developed. This assay involves immunomagnetic separation of the bacteria from the insect, followed by a two-step, nested polymerase chain reaction (PCR) amplification using previously developed oligonucleotide primers specific to X. fastidiosa. A total of 347 leafhoppers representing 16 species were captured and sampled from American elm (Ulmus americana L.) trees growing in a nursery where bacterial leaf scorch caused by X. fastidiosa occurs. Two of these leafhopper species, Graphocephala coccinea and G. versuta, regularly tested positive for X. fastidiosa using this technique. These insects are therefore potential vectors of X. fastidiosa. Using immunocapture and nested PCR, it was possible to detect as few as five bacteria per sample.