Publications

4514

Australian lucerne yellows (ALuY), a phytoplasma-associated disease, is a major problem in Australia that causes the pasture seed industry millions of dollars in losses annually (3). Samples were collected from lucerne (Medicago sativa L.) plants exhibiting symptoms indicative of ALuY (4) in a seed lucerne paddock (cv CW 5558) at Griffith, southwestern New South Wales (NSW), Australia, in November 2005 and again in January 2006. Samples were kept at 4°C and processed within 36 h of collection. Total DNA was extracted from approximately 0.3 g of leaf midribs and petioles of each plant sample and used as template in a nested PCR assay with phytoplasma universal primer pairs P1/P7 and fU5/m23sr. PCR products resulting from the first amplification were diluted (1:30) with sterile distilled water (SDW) before reamplification with fU5/m23sr. DNA of Australian tomato big bud (TBB) phytoplasma and SDW were used as positive and negative assay controls, respectively. Ten of fifteen plant samples collected in November tested positive for phytoplasma DNA. Restriction digestion profiles of nested PCR amplicons with HpaII endonuclease were the same for all symptomatic plants but differed from the control. Phytoplasma identity was determined by sequencing two nested PCR products that yielded identical sequences. One was deposited in the GenBank database (Accession No. DQ786394). BLAST analysis of the latter sequence revealed a >99.6% similarity with “Candidatus Phytoplasma australiense” (L76865) and related strains papaya dieback (Y10095), phormium yellow leaf (U43570), strawberry green petal (AJ243044), and strawberry lethal yellows (AJ243045). Direct PCR with primers FP 5′-GCATGTCGCGGTGAATAC-3′ and RY 5′-TGAGCTATAGGCCCTTAATC-3′ designed to specifically amplify DNA of “Ca. P. australiense” detected the phytoplasma in 8 of 40 samples collected in January. Whether this phytoplasma is the etiological agent solely responsible for ALuY is currently under investigation. “Ca. P. asteris” and stolbur group (16SrXII) phytoplasmas have been reported in lucerne in the United States (2) and Italy (1), respectively. Within the stolbur group 16SrXII, “Ca. P. australiense” and stolbur phytoplasma are regarded as separate species and both are distinct from “Ca. P. asteris”, a group 16SrI strain. To our knowledge, this is the first report of a “Ca. P. australiense” related strain in lucerne. References: (1) C. Marzachi et al. J. Plant Pathol. 82:201, 2000. (2) R. D. Peters et al. Plant Dis. 83:488, 1999. (3) L. J. Pilkington et al. Australas. Plant Pathol. 28:253, 1999. (4) L. J. Pilkington et al. First report of a phytoplasma associated with ‘Australian lucerne yellows’ disease. New Disease Report. Online publication at http://www.bspp.org.uk/ndr/jan2002/2001-46.asp .

Summary Studies were conducted in opposing gradients of oxygen and sulfide in microslide capillaries to (i) characterize the chemical microenvironment preferred by Candidatus Arcobacter sulfidicus, a highly motile, sulfur‐oxidizing bacterium that produces sulfur in filamentous form, and (ii) to develop a model describing the mechanism of filamentous‐sulfur formation. The highly motile microorganisms are microaerophilic, with swarms effectively aggregating within oxic‐anoxic interfaces by exhibiting a chemotactic response. The position of the band was found to be largely independent of the sulfide concentration as it always formed at the oxic‐anoxic interface. Flux calculations based on steady state gradients of oxygen and sulfide indicate that sulfide is incompletely oxidized to sulfur, in line with the formation of filamentous sulfur by these organisms. It is proposed that Candidatus Arcobacter sulfidicus effectively competes with other sulfur‐oxidizing bacteria in the environment by being able to tolerate higher concentrations of hydrogen sulfide (1–2 mM) and by possessing the ability to grow at very low oxygen concentrations (1–10 μM). The formation of mat‐like structures from filamentous sulfur appears to be a population mediated effort allowing these organisms to effectively colonize environments characterized by high sulfide, low oxygen and dynamic fluid movement.

Citrus huanglongbing (HLB), also known as citrus greening or citrus yellow shoot, is considered the most serious disease of citrus worldwide. The disease has Asian, African, and American forms caused by “Candidatus Liberibacter asiaticus”, “Ca. L. africanus”, and “Ca. L. americanus”, respectively, which can be spread efficiently by the psyllid vectors Diaphorina citri and Trioza erytreae and through contaminated plant materials. Infected citrus groves are usually destroyed or become unproductive in 5 to 8 years. The presumed low concentration and uneven distribution of the pathogens in citrus plants and vector insects make the phloem-limited bacterium difficult to detect consistently. In this study, we compared and validated four conventional polymerase chain reaction (PCR)-based protocols, one loop-mediated isothermal amplification (LAMP) protocol, and three TaqMan real-time PCR protocols. The detection sensitivity of the validated conventional PCR assays reported are improved compared with the original protocols. All of the validated conventional and the newly developed real-time methods were reliable for confirmatory tests for the presence of “Ca. Liberibacter spp.” in symptomatic samples. There were no differences in assay specificity among the standard format PCR-based methods evaluated. The TaqMan real-time PCR was 10- to 100-fold more sensitive than conventional PCR and LAMP, showing the potential to become a valuable tool for early detection and identification of “Ca. Liberibacter spp.” prior to the appearance of disease symptoms. The methods validated in this study will be very useful for regulatory response, effective management of infected trees, and development of a “Ca. Liberibacter spp.”-free nursery system.

ABSTRACT Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantii T (90.1%). The spirochete, here named “ Candidatus Treponema suis, ” was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa.