Murraya paniculata and Related Species as Potential Hosts and Inoculum Reservoirs of 'Candidatus Liberibacter asiaticus', Causal Agent of Huanglongbing.

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of 'Candidatus Liberibacter asiaticus' from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted 'Ca. L. asiaticus' into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of 'Ca. L. asiaticus' from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of 'Ca. L. asiaticus' in infected Murraya.

Cultivation of 'Candidatus Liberibacter asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus' associated with huanglongbing.

A new medium designated Liber A has been designed and used to successfully cultivate all three 'Candidatus Liberibacter spp.,' the suspect causative agents of huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of 'Ca. Liberibacter spp.' for four or five single-colony transfers before viability declined. Colonies, positive for 'Ca. L. asiaticus' by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex, and 0.1 to 0.3 mm after 3 to 4 days. Suspect 'Ca. L. asiaticus' and 'Ca. L. americanus' cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped, 0.3 to 0.4 by 0.5 to 2.0 microm, often with fimbriae-like appendages. Two strains of 'Ca. L. asiaticus' and one of 'Ca. L. americanus' grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of 'Ca. L. asiaticus' and 'Ca. L. americanus' associated with symptoms of HLB.

Identification of bacterial plant pathogens using multilocus polymerase chain reaction/electrospray ionization-mass spectrometry.

Polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as "TIGER") utilizes PCR with broad-range primers to amplify products from a wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses allows for calculations of base compositions for the broad-range PCR products, which can then be compared to a database for identification. PCR/ESI-MS has the benefits of PCR in sensitivity and high-throughput capacity, but also has the distinct advantage of being able to detect and identify organisms with no prior characterization or sequence data. Existing broad range PCR primers, designed with an emphasis on human pathogens, were tested for their ability to amplify DNA of well characterized phytobacterial strains, as well as to populate the existing PCR/ESI-MS bacterial database with base counts. In a blinded panel study, PCR/ESI-MS successfully identified 93% of unknown bacterial DNAs to the genus level and 73% to the species/subspecies level. Additionally, PCR/ESI-MS was capable of detecting and identifying multiple bacteria within the same sample. The sensitivity of PCR/ESI-MS was consistent with other PCR based assays, and the specificity varied depending on the bacterial species. Preliminary tests with real life samples demonstrate a high potential for using PCR/ESI-MS systems for agricultural diagnostic applications.

First Report of Crown Gall Caused by an Agrobacterium sp. on Diffuse Knapweed (Centaurea diffusa).

Diffuse knapweed (DK) plants were discovered in Mosier, Wasco County, OR (45.6842°N, 121.4021°W) with crown gall-like symptoms near the soil line. Specimens were collected on 27 July 2004 and sent to the USDA-ARS at Ft. Detrick, MD for identification of disease and pathogen. Pure culture of a bacterium was obtained on potato dextrose agar, and hyperplasia and hypertrophy developed on carrot disks and tomato stems after wound inoculation with a needle contaminated by the agar culture. The same bacterium was reisolated from the galls on DK, thus fulfilling Koch's postulates. Pathogenicity tests involving needle inoculations of stems and petioles resulted in gall formation on Acroptilon repens, Carthamus tinctorius, Centaurea solstitialis, C. maculosa, C. cyanus, Crupina vulgaris, Helianthus annuus, and Rubus armeniacus. In biochemical tests typically used for identification of Agrobacterium species (3), the DK strain grew on D1M agar but not on 2% NaCl medium, produced acid from erythritol but not from melezitose, converted malonic acid to base, and turned litmus milk alkaline. These results are characteristic of Agrobacterium rhizogenes (= Biovar 2), except for the litmus milk reaction. Using 16S rRNA cluster analysis by unweighted pair group method with arithmetic mean (UPGMA, 500 replicates) and basic local alignment search tool (BLAST), the DK strain clustered most closely with A. rubi (GenBank Accession Nos. D12787 and AM181759). The DK strain differed from A. larrymoorei (GenBank Accession No. Z30542), A. tumefaciens (GenBank Accession No. AJ389896), A. rhizogenes (GenBank Accession No. AB247607), and A. vitis (GenBank Accession No. AB247599) on the basis of 16S rRNA sequence cluster analysis. The DK strain differed from A. rubi on the basis of differential reactions with erythritol, litmus milk, and 2% NaCl medium (2,4); and the 16S rRNA sequence of the DK strain differed from that of A. rubi by 11 bp (99.2% similarity). Comparisons also were made between the DK strain and two strains (83A and 135A) of A. tumefaciens (= Biovar 1), described from New Mexico on A. repens (1), a plant species in the same tribe and subtribe of the Asteraceae as DK. Host range reported for the two A. repens strains after artificial greenhouse inoculations was similar to that of the DK strain and it included diffuse knapweed (1). However, 16S sequencing, which confirmed identification of both A. repens strains as A. tumefaciens, showed they differed from the DK strain. The DK strain belongs in the genus Agrobacterium, but it could not be assigned to any known species on the basis of data from phenotypic or 16S sequence comparisons. To our knowledge, this is the first report of crown gall on diffuse knapweed in the field. This strain has been deposited into the International Collection of Phytopathogenic Bacteria at Fort Detrick (Accession No. 60099), and the 16S rRNA sequence has been deposited into the GenBank database (Accession No. EF687663). References: (1) A. J. Caesar, Plant Dis. 78:796, 1994. (2) B. Holmes and P. Roberts, J. Appl. Bacteriol. 50:443, 1981. (3) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (4) K. Ophel and A. Kerr, Int. J. Syst. Bacteriol. 40:236, 1990.

First Report of Citrus Canker Caused by Xanthomonas citri in Somalia.

Xanthomonas citri (synonym = Xanthomonas axonopodis pv. citri) (3) has been reported in several countries in Africa (1) but not Somalia. During 2006 and 2007, hyperplasia-type lesions, often surrounded by a water-soaked margin and yellow halo, typical of citrus canker caused by X. citri were found on 8- to 10-year-old lime (Citrus limetta) and grapefruit (Citrus × paradisi Macfed.) trees in northern and southern Somalia, respectively. Ten leaf samples diagnosed presumptively as citrus canker by Xac ImmunoStrip test kits (Agdia, Elkhart, IN) were mailed to the USDA Foreign Disease-Weed Science Research Unit at Ft. Detrick, MD. To confirm the identification of X. citri, isolations were made from several lesions from each sample onto yeast-dextrose-CaCO3 (YDC) agar (2). Yellow, xanthomonad-like mucoid, convex colonies were purified and stored on YDC slants. Phenotypic tests were done as described (2), and real-time PCR assays were done using primers XCit8F and XCit5R with probe XCitP2 (N. W. Schaad, unpublished). For pathogenicity tests, cultures were grown overnight in liquid nutrient broth-yeast (4) medium adjusted to contain 1 × 105 CFU/ml and inoculated into leaves of lime seedlings with the blunt end of a 2-ml syringe. After 21 to 30 days in a lighted dew chamber (Model I-60DLM; Percival Scientific, Inc. Perry, IA) at 30/23°C day/night, symptoms were recorded. Cultures of sample S-1 (northern Somalia) from lime were phenotypically atypical of X. citri, PCR negative, and nonpathogenic. However, cultures of samples 3 to 7 (southern Somalia) from grapefruit were typical of X. citri and PCR positive; cultures 3 and 4 were tested for pathogenicity and produced erumpent lesions on lime. Isolations onto YDC agar resulted in typical mucoid, convex, yellow, PCR-positive colonies. To our knowledge, this is the first report of X. citri on citrus plants in Somalia. Strains S3 and S4 have been deposited in ICPB at Ft. Detrick, MD as ICPB 11650 and 11651, respectively. References: (1) J. F. Bradbury. Guide to Plant Pathogenic Bacteria. CAB International, Egham, UK, 1986. (2) N. W. Schaad et al. Xanthomonas. Page 175 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al. eds. American Phytopathological Society, St. Paul. MN. 2001. (3) N. W. Schaad et al. Syst. Appl. Microbiol. 29:690, 2006. (4) A. K. Vidaver. Appl. Microbiol. 15:1523, 1967.

Genetic Diversity in Populations of Xanthomonas campestris pv. campestris in Cruciferous Weeds in Central Coastal California.

ABSTRACT Xanthomonas campestris pv. campestris (X. campestris) infects a large number of cruciferous plants, including weeds. California has one of the largest and most diverse populations of wild cruciferous plants in the world. Although considerable information is available on the genetic diversity of X. campestris in commercial crop plants, nothing is known about the diversity in strains infecting weeds. To assess the genetic diversity among strains of X. campestris in weeds in noncultivated and cultivated areas, strains of the pathogen were isolated from populations of cruciferous weeds growing in coastal valley crop-production sites and from remote nonproduction sites along the California central coast. Results of fingerprinting over 68 strains using amplified fragment length polymorphism along with representative strains by sequence analysis showed the presence of seven genotypes. Genotypes A and B were limited to coastal sites; genotypes C, D, and E were from inland cultivated sites; and genotypes F and G were present in both coastal noncultivated and inland cultivated sites. Crop strains were grouped outside any weed strain group and were separated from the weed strains and other pathovars of X. campestris. These results revealed, for the first time, that strains of X. campestris present in noncultivated coastal weed populations generally were unique to a site and genetically distinct from strains present in populations of weeds in crop-production areas located nearby.

Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by BIO-PCR and an Automated Real-Time Fluorescence Detection System.

Ring rot of potato, caused by Clavibacter michiganensis subsp. sepedonicus, is one of the most regulated diseases of potatoes world wide. The organism is often difficult to detect in symptomless tubers because of low populations and slow competitive growth on available media. Polymerase chain reaction (PCR) primers and a fluorescent probe for use in the Perkin Elmer 7700 automated real time PCR detection system (TaqMan) were designed from a C. michiganensis subsp. sepedonicus-specific genomic DNA fragment for development of a BIO-PCR assay for C. michiganensis subsp. sepedonicus in potato tubers. Results of screening the primers with strains of C. michiganensis subsp. sepedonicus and other bacteria showed the primers to be specific. A total of 30 naturally infected ring rot suspect tubers were sampled by the core extract, shaker incubation procedure and assayed by (i) plating aliquots onto agar media, (ii) classical PCR, and (iii) BIO-PCR. In all, 4 tubers were positive by agar plating and pathogenicity tests, 8 by classical TaqMan PCR, and 26 by TaqMan BIO-PCR. We conclude that BIO-PCR combined with the TaqMan automated closed detection system is a rapid, reliable method of assaying large numbers of potato tuber extracts for C. michiganensis subsp. sepedonicus. Furthermore, for a large central laboratory running large numbers of PCR assays, the high-throughput TaqMan system can reduce costs per sample over the more labor-intensive classical PCR.