Publications

4862

Huanglongbing (HLB) is considered one of the most destructive diseases of citrus because the plants rapidly become unproductive, enter a decline, and eventually die. HLB is caused by the phloem-limited bacterium ‘Candidatus Liberibacter’ spp. The objective of this study was to evaluate seasonal variation of the in planta population of ‘Ca. Liberibacter asiaticus’ in the foliage of citrus trees in Brazil using real-time polymerase chain reaction (qPCR). Eleven plants (naturally infected, then screened) in the field with very mild and localized symptoms of HLB were confirmed to be ‘Ca. L. asiaticus’ infected by conventional PCR, and the canopies were divided into four quadrants. The bacterial population in the trees was tested on a monthly basis for up to 20 months by quantifying ‘Ca. L. asiaticus’ DNA using qPCR ‘Ca. L. asiaticus’-specific primers (As84F/As180R). The average cycle threshold (Ct) values, which relate to ‘Ca. L. asiaticus’ titer, were analyzed using a mixed model. Significant differences were observed in Ct values between seasons (F = 8.77, P = 0.0004), and abrupt changes were observed in Ct values in different quadrants of the trees. Autumn had the lowest Ct values, indicating the highest ‘Ca. L. asiaticus’ titer, and, thus, is considered the best period to detect ‘Ca. L. asiaticus’ infection in foliage of citrus trees in southern Brazil. In addition to the seasonal changes in Ct values, there was an initial decline in the Ct value in the months following initial detection, the rate of decline slowing with time. Concomitant with the increase of the bacterial population in the host, there was an increase in severity of HLB symptoms in the trees over time (Spearman’s rank correlation, r = −0.4083, P < 0.0001). The results identify the optimal season to sample foliage for ‘Ca. L. asiaticus’ in southern Brazil (autumn) and confirm the importance of sample timing to maximize detection of ‘Ca. L. asiaticus’ and, thus, contribute to the search for effective measures to manage HLB.

North American grapevine yellows (NAGY) disease has sometimes been attributed to infection of Vitis vinifera L. by Prunus X-disease phytoplasma (‘Candidatus Phytoplasma pruni’) but this attribution may not be fully adequate. In this study, phytoplasma strains related to ‘Ca. Phytoplasma pruni’ were found in NAGY-diseased grapevines in Maryland, Pennsylvania, Virginia, Ohio, Missouri, and New York State. Based on restriction fragment length polymorphism analysis of 16S ribosomal RNA gene (16S rDNA) sequences, the strains (termed NAGYIII strains) were classified in group 16SrIII (X-disease group) but they contained a recognition site for the restriction endonuclease MseI that is not present in the 16S rDNA of ‘Ca. Phytoplasma pruni’. The 16S rDNA of the strains differed by three or four nucleotides from that of ‘Ca. Phytoplasma pruni’, indicating that they belonged to two novel 16S rDNA sequevars, designated NAGYIIIα and NAGYIIIβ. Both sequevars differed from ‘Ca. Phytoplasma pruni’ by a single base in each of three regions corresponding to species-unique (signature) sequences described for ‘Ca. Phytoplasma pruni’. Phylogenetic analyses of 16S rRNA genes and SecY proteins, and single-nucleotide polymorphism analyses of secY and ribosomal protein genes, further distinguished the two grapevine sequevar lineages from one another and from ‘Ca. Phytoplasma pruni’. The NAGYIIIα and NAGYIIIβ sequevars also differed from ‘Ca. Phytoplasma pruni’ in regions of the folded SecY protein that are predicted to be near or exposed at the outer surface of the phytoplasma membrane. No evidence indicated that diseased grapevines contained any phytoplasma strain conforming to ‘Ca. Phytoplasma pruni’ sensu stricto. Because the NAGYIII sequevars have not been reported in X-disease, a question is raised as to whether NAGYIII and Prunus X-disease are caused by different phytoplasma genotypes.

“Candidatus Liberibacter europaeus” (Leu) is one of six currently known Liberibacter species. It is known primarily from pear and related species across Europe, and from Scotch broom and its associated psyllids in New Zealand (introduced from Britain). The psyllids were introduced to New Zealand as a biocontrol agent for broom and it is thought the bacterium may have been introduced as an endosymbiont of the psyllids. No symptoms in apple or pear trees have been reported, but mild symptoms can occur in broom. 16S and 16S–23S intergenic spacer region DNA sequences of this species are available from GenBank. Analysis of these sequences and associated ecological descriptions indicates the occurrence of two haplotypes, LeuA and LeuB, defined not only genetically but also by geographic range as well as by plant/psyllid host species composition. Liberibacter species are new to science and haplotype identifications enable historical accounts of relationships and spread to be generated.

AbstractHere we employed quantitative real-time PCR (qPCR) assays for polyphosphate kinase 1 (ppk1) and 16S rRNA genes to assess relative abundances of dominant clades of Candidatus Accumulibacter phosphatis (referred to Accumulibacter) in 18 globally distributed full-scale wastewater treatment plants (WWTPs) from six countries. Accumulibacter were not only detected in the 6 WWTPs performing biological phosphorus removal, but also inhabited in the other 11 WWTPs employing conventional activated sludge (AS) with abundances ranging from 0.02% to 7.0%. Among the AS samples, clades IIC and IID were found to be dominant among the five Accumulibacter clades. The relative abundance of each clade in the Accumulibacter lineage significantly correlated (p < 0.05) with the influent total phosphorus and chemical oxygen demand instead of geographical factors (e.g. latitude), which showed that the local wastewater characteristics and WWTPs configurations could be more significant to determine the proliferation of Accumulibacter clades in full-scale WWTPs rather than the geographical location. Moreover, two novel Accumulibacter clades (IIH and II-I) which had not been previously detected were discovered in two enhanced biological phosphorus removal (EBPR) WWTPs. The results deepened our understanding of the Accumulibacter diversity in environmental samples.