Whole genome sequences of two Xylella fastidiosa strains (M12 and M23) causing almond leaf scorch disease in California.

Xylella fastidiosa is a Gram-negative plant-pathogenic bacterium causing many economically important diseases, including almond leaf scorch disease (ALSD) in California. Genome information greatly facilitates research on this nutritionally fastidious organism. Here we report the complete genome sequences of two ALSD strains of this bacterium, M12 and M23.

First Report of 'Candidatus Liberibacter asiaticus' from Atalantia buxifolia in Guangdong, China.

Atalantia buxifolia (Poir.) Oliv., synonym Severinia buxifolia (Poir.) Ten. as commonly found in literature, is a common landscape plant and a popular Chinese medicinal herb known as Jiubingle or Dongfengjie. It remains unclear if this rutaceous plant could host 'Candidatus Liberibacter asiaticus', the pathogen of citrus Huanglongbing (HLB) in Guangdong, P. R. China. This information is important for HLB control in citrus because infected A. buxifolia could serve as a source of inoculum. In August of 1994, three A. buxifolia plants adjacent to a citrus experimental orchard of the South China Agricultural University at Guangzhou were found showing leaf mottle/yellowing symptoms. Two buds from each plant were grafted onto three mandarin trees (Citrus reticulata cv. Pongan) in a psyllid-proof screenhouse for indexing. By October of 1995, typical leaf mottle symptoms were observed in all three grafted trees compared with a healthy control. In March of 1996, one of the A. buxifolia plants was transferred to a screenhouse and has been maintained there. The leaf mottle/yellowing symptoms persisted but did not significantly affect plant growth. DNA was extracted from leaf samples in October 2006 by using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR using the general bacterial 16S rDNA primer set fDl/rD1 as the first round of amplification and primer set OI1/OI2c as second round amplification (1,3). After agarose gel electrophoresis and staining with ethidium bromide, an approximate 1.1-kb DNA band was detected in symptomatic samples but not healthy leaf samples of A. buxifolia and C. reticulata. XbaI digestion of the amplicons yielded approximate 500- and 600-bp fragments, characteristic of 'Ca. L. asiaticus'. Similarly, a standard PCR with primer set A5/J2 (3) yielded an approximate 700-bp DNA band characteristic of 'Ca. L. asiaticus' from symptomatic samples only. To our knowledge, this is the first report of graft transmission and PCR detection of 'Ca. L. asiaticus' from A. buxifolia in Guangdong, P. R. China. This work also confirms the findings from Taiwan (2) that A. buxifolia could serve as a source of 'Ca. L. asiaticus'. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) T.-H. Hung et al. Eur. J. Plant Pathol. 107:183, 2001. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.

Identification and Characterization of the Huanglongbing Bacterium in Pummelo from Multiple Locations in Guangdong, P. R. China.

Huanglongbing (HLB, yellow shoot disease, ex. citrus greening disease), caused by Candidatus Liberibacter spp., is highly destructive to citrus production in Asia, Africa, and South America. Although primarily affecting sweet orange and mandarin, HLB has long been observed in pummelo in Guangdong Province, People's Republic of China; however, the disease in pummelo has received little research attention. Accordingly, it remains unclear how closely related the strains of Ca. Liberibacter in pummelo are to those in other citrus species. In this study, the loci of 16S rDNA, rplAJ (ß-operon of ribosomal protein), and an outer membrane protein (omp) gene were analyzed and characterized among strains of Ca. Liberibacter in pummelo samples from six different locations in Guangdong. Sequence comparisons indicated that 'Candidatus Liberibacter asiaticus', but not 'Ca. Liberibacter africanus' or 'Ca. Liberibacter americanus', was exclusively associated with HLB symptoms in pummelo. The pummelo strains of 'Ca. Liberibacter asiaticus' from Guangdong were highly homogeneous. Analyses of single-nucleotide polymorphisms in the omp locus showed that the Guangdong pummelo strains grouped with 'Ca. Liberibacter asiaticus' strains from Thailand, Nepal, and an unspecified location in China but differed from the Philippine and China-Behai strains. Based on the sequence homogeneity at the omp locus, the history of pummelo culture and the means by which HLB is known to be spread, we believe that, likely, the pummelo strain of 'Ca. Liberibacter asiaticus' recently was spread to pummelo in the study areas from infected sweet orange or mandarin trees by insect vectors or by propagation of pummelo infected elsewhere.

Improved Bioassay of Xylella fastidiosa Using Nicotiana tabacum Cultivar SR1.

Readily transformable Nicotiana tabacum cv. SR1 (Petite Havana) was evaluated as a host for the bioassay of Xylella fastidiosa strains. Plant growing conditions and inoculation methods were optimized to enhance symptom expression 4 to 6 weeks post inoculation. Tobacco plants were inoculated with X. fastidiosa strains associated with almond leaf scorch disease (ALSD) and Pierce's disease (PD) of grapevine in California. All PD strains and the ALSD strain Dixon caused characteristic leaf scorch symptoms, whereas two other ALSD-associated strains (M12 and M23) caused severe leaf chlorosis followed by necrosis, leaf death, and drooping of older leaves. Symptoms began to develop 10 to 14 days post inoculation and proceeded to resemble those of X. fastidiosa-infected grape and almond. The presence of X. fastidiosa in affected plants was confirmed by reisolation of the pathogen, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction (QPCR), and observation of X. fastidiosa cells by transmission and scanning electron microscopy, as well as by confocal laser scanning microscopy, in the xylem cells of inoculated plants. The pathogenicity of selected reisolated strains was confirmed by inoculation of grape plants in the greenhouse. The average levels of X. fastidiosa cells/g of tissue, estimated by QPCR, were higher for PD strains than for ALSD strains and reflected the relative titers of these strains in economic hosts. No symptoms were observed and bacteria were not detected in untreated tobacco or in tobacco inoculated with Xanthomonas campestris pv. campestris or water. Symptoms induced by Xylella fastidiosa in this bioassay were fully expressed within 2 months following inoculation. The described bioassay, under optimized environmental conditions, provides a useful system for studying X. fastidiosa strains (e.g., confirmation of pathogenicity and differentiation of PD and ALSD pathotypes) and for investigating X. fastidiosa-host interactions. N. tabacum cv. SR1 tobacco was a better bioassay host for X. fastidiosa than N. tabacum cvs. Havana, RP1, and TNN described previously.

Nested-PCR Detection and Sequence Confirmation of 'Candidatus Liberibacter asiaticus' from Murraya paniculata in Guangdong, China.

Murraya paniculata (orange jasmine) is a popular ornamental rutaceaous plant and is known to be a preferred host for the Asian citrus psyllid, Diaphorina citri (Kuwayana), the primary vector of 'Candidatus Liberibacter spp.' that causes citrus Huanglongbing (HLB). HLB is a highly destructive citrus disease worldwide. However, the presence of 'Ca. Liberibacter spp.' in M. paniculata remains uncertain (2). Clarification of M. paniculata as a host of 'Ca. Liberibacter spp.' has direct impact on HLB control programs. During June of 2006, we identified three M. paniculata trees near a mandarin orchard affected by HLB in Luoding City and two trees from Guangzhou City, Guangdong Province, People's Republic of China. All trees had leaves showing yellowing and mottling symptoms characteristic of HLB. Both symptomatic and asymptomatic leaves were collected. DNA was extracted using the CTAB (cetyltrimethylammoniumbromide) method and assayed by nested-PCR. The general bacterial 16S rDNA primer set fDl/rD1 (3) was used for the first round of amplification. Amplification was conducted as previously described (1), and 2 µl of PCR reaction product were used for a second round of amplification using the same procedure but with 35 PCR cycles with primer set OI1/OI2c (3,4). After agarose gel electrophoresis and staining with ethidium bromide, a 1.1-kb DNA band was unambiguously associated with symptomatic but not asymptomatic leaf samples. Nonnested-PCR using primer set OI1/OI2c alone did not yield a target DNA band or yielded a very weak DNA band. XbaI digestion of the nested-PCR DNA product yielded two fragments, 520 and 640 bp long, characteristic of 'Ca. L. asiaticus'. PCR amplicons were sequenced and were 1,095 bp long. This sequence shared >98% similarity to sequences of 'Ca. L. asiaticus' in the GenBank database. We observed that nested-PCR is necessary for consistent amplification of DNA from 'Ca. L. asiaticus' from M. paniculata. We excluded the possible nonspecific amplification associated with nested-PCR by XbaI restriction enzyme digestion and by nucleotide sequence analysis. Our data indicate that M. paniculata is a host of 'Ca. L. asiaticus' but the bacterial titer might be low. References: (1) X. Deng et al. Online publication. doi:10.1094/PHP-2007-0419-01-BR. Plant Health Progress, 2007. (2) M. Garnier and J. Bove. Huanglongbing (Greening). Page 46 in: Compendium of Citrus Diseases. 2nd ed. L. W. Timmer et al., eds. The American Phytopathological Society, St. Paul, MN, 2000. (3) S. Jagoueix et al. Int. J. Syst. Bacteriol. 44:379, 1994. (4) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996.

Two Xylella fastidiosa Genotypes Associated with Almond Leaf Scorch Disease on the Same Location in California.

ABSTRACT Almond leaf scorch disease (ALSD) has recently reemerged in the San Joaquin Valley of California threatening almond production. ALSD is caused by Xylella fastidiosa, a nutritionally fastidious bacterium. Single nucleotide polymorphisms (SNPs) in the 16S rRNA gene (16S rDNA) of X. fastidiosa strains were identified to characterize the bacterial population in infected trees. Genotype-specific SNPs were used to design primers for multiplex polymerase chain reaction assays of early passage cultures. Two genotypically distinct types of X. fastidiosa strains, G-type and A-type, coexist simultaneously in the same infected almond orchard. This was substantiated by restriction fragment length polymorphism analysis of a different genetic locus, RST31-RST33, which has previously been used to identify and differentiate X. fastidiosa strains. Furthermore, unique bacterial colony morphology was consistently associated with the A-type X. fastidiosa strains. To our knowledge, this is the first report of a mixed genotype infection of X. fastidiosa disease on the same location under natural environmental conditions. The concept of mixed genotype infection could affect the current epidemiological study based on the assumption that one genotype causes ALSD on one location and, therefore, the disease management strategy.

Spatial Analysis of Almond Leaf Scorch Disease in the San Joaquin Valley of California: Factors Affecting Pathogen Distribution and Spread.

Almond leaf scorch (ALS) disease has emerged as a serious threat to almond (Prunus amygdalus) production areas throughout California's San Joaquin Valley. This disease is caused by the xylem-limited bacterium Xylella fastidiosa, and this pathogen is transmitted by xylophagous insects including sharpshooter leafhoppers (Hemiptera: Cicadellidae) and spittlebugs (Hemiptera: Cercopidae). Among four orchards surveyed, enzyme-linked immunosorbent assay (ELISA) and bacterial isolation followed by polymerase chain reaction (PCR) were equally effective in detecting X. fastidiosa from ALS-symptomatic trees. Disease incidence varied among almond cultivars in each orchard, with the highest mean incidence and most severe symptoms frequently encountered in 'Sonora'. X. fastidiosa isolates consisted of mixtures of grape or "G-genotype" and almond or "A-genotype" strains present in surveyed orchards. The X. fastidiosa G-genotypes characterized from each orchard were associated with the most severely affected 'Sonora' trees in three of the four orchards. Both ordinary runs and simple randomization analyses revealed aggregations of ALS in three of the four orchards. Clusters of ALSaffected trees frequently occurred in the outermost orchard rows. Plots of semivariance in ALS incidence over distance varied in shape and magnitude among cultivars. Semivariance increased over distance in 'Sonora' and 'Carmel', indicating spatial dependence or aggregations of incidence best fit by a combination of spherical and linear models. These results document both random and aggregate patterns of ALS spatial distribution in selected orchards and further illustrate how cultivar susceptibility influences the distribution patterns of ALS incidence. Following the recent introduction and establishment of the glassy-winged sharpshooter, Homalodisca coagulata, the impact upon the epidemiology and spread of ALS is unknown.

Analysis of the Spatial Patterns of Pierce's Disease Incidence in the Lower San Joaquin Valley in California.

ABSTRACT The incidence of Pierce's disease (PD), caused by Xylella fastidiosa, was monitored in 11 naturally infested commercial vineyards to determine the presence of an X. fastidiosa vector, Homalodisca coagulata (glassy-winged sharpshooter [GWSS]), to examine the spatial patterns of the disease and elucidate possible influences of surrounding environments. Disease incidence ranged from <1 to 65% among vineyards in 2001. Our efforts to trap or capture potential disease vectors have indicated that the GWSS is the most likely vector. Disease incidence doubled in most vineyards during the 2002 production season. Spatial patterns of symptomatic vines in 2001 and 2002, as determined by ordinary runs analysis, showed strong evidence for within- and across-row aggregation of infected vines. In most fields, they were no disease gradients observed relative to GWSS source (e.g., citrus). Within fields, however, disease incidence displayed strong spatial dependence and a high degree of anisotropy, indicating strongly aggregated patterns of disease with distinct directional orientation. The within-row (0 degrees ) and across-row (90 degrees ) orientations generally were the predominant directions of increased disease incidence, consistent with vine-to-vine spread of X. fastidiosa. We concluded that the distribution of PD in vineyards reflected the feeding pattern of vectors carrying X. fastidiosa. Based on these results, effective PD management is likely to be based on practices that reduce significant insect vector populations and remove infected vines as soon as identified and on the use of resistant cultivars.

Characterization of oncogene-silenced transgenic plants: implications for Agrobacterium biology and post-transcriptional gene silencing.

SUMMARY Agrobacterium tumefaciens tumorigenesis is initiated by the horizontal transfer of a suite of oncogenes that alter hormone synthesis and sensitivity in infected plant cells. Transgenic plants silenced for the iaaM and ipt oncogenes are highly recalcitrant to tumorigenesis, and present a unique resource to elucidate fundamental questions related to Agrobacterium biology and post-transcriptional gene silencing (PTGS). The oncogene-silenced transgenic tomato line 01/6 was used to characterize A. tumefaciens growth in planta and to screen for iaaM and ipt sequence variants. Even in the absence of macroscopic and microscopic indications of tumorigenesis, A. tumefaciens is capable of long-term survival in the hypocotyl tissues of the 01/6 line. A. tumefaciens growth, however, is significantly reduced in the 01/6 line, with populations decreased by 96% relative to wild-type at 52 days post-inoculation. In addition, the 01/6 line displayed suppression of tumorigenesis against all 35 tested strains of A. tumefaciens. High target homology is an absolute requirement of PTGS, therefore this result suggests that regions of the iaaM and ipt oncogenes are very highly conserved across most A. tumefaciens strains. Finally, graft transmissibility of oncogene silencing was assessed by grafting various non-silenced tomato genotypes on to the 01/6 line. Phenotypic and molecular evidence (tumorigenesis and absence of small interfering RNAs, respectively) suggest that oncogene silencing is not graft-transmissible, at least to wild-type and antisense iaaM-over-expressing genotypes.

RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis.

Crown gall disease, caused by the soil bacterium Agrobacterium tumefaciens, results in significant economic losses in perennial crops worldwide. A. tumefaciens is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate de novo auxin and cytokinin biosynthesis to generate tumors. Specifically, the iaaM and ipt oncogenes, which show approximately 90% DNA sequence identity across studied A. tumefaciens strains, are required for tumor formation. By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of iaaM and ipt, we generated transgenic Arabidopsis thaliana and Lycopersicon esculentum plants that are highly resistant to crown gall disease development. In in vitro root inoculation bioassays with two biovar I strains of A. tumefaciens, transgenic Arabidopsis lines averaged 0.0-1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis. Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of A. tumefaciens displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis. This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor-ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable. If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks.

Evaluation of the Biolog Substrate Utilization System To Identify and Assess Metabolic Variation among Strains of Xanthomonas campestris pv. Citri.

Metabolic fingerprints of 148 strains of Xanthomonas campestris pv. citri originating from 24 countries and associated with various forms of citrus bacterial canker disease (CBCD) were obtained by using the Biolog substrate utilization system. Metabolic profiles were used to attempt strain identification. Only 6.8% of the studied strains were correctly identified when the commercial Microlog 2N data base was used alone. When the data base was supplemented with data from 54 strains of X. campestris pv. citri (40 CBCD-A strains, 8 CBCD-B strains, and 6 CBCD-C strains) and data from 43 strains of X. campestris associated with citrus bacterial spot disease, the percentage of correct identifications was 70%. Thus, it is recommended that users supplement the commercial data base with additional data prior to using the program for identification purposes. The utilization of Tween 40 in conjunction with other tests can help to differentiate strains associated with CBCD and citrus bacterial spot disease. These results confirmed the separation of X. campestris pv. citri into different subgroups (strains associated with Asiatic citrus canker [CBCD-A], cancrosis B [CBCD-B], and Mexican lime canker [CBCD-C]). The utilization of l-fucose, d-galactose, and alaninamide can be used as markers to differentiate strains associated with these groups. A single strain associated with bacteriosis of Mexican lime in Mexico (CBCD-D) was closely similar to CBCD-B strains.