Plant Disease

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, while the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

Evidence from a preliminary survey highlighted that ‘Candidatus Phytoplasma solani’, the etiological agent of bois noir (BN) disease of grapevine, infects grapevine varieties in Georgia, a country of the South Caucasus. In this study, field surveys were carried out to investigate the BN symptom severity in international and Georgian native varieties. ‘Ca. P. solani’ was detected and identified by polymerase chain reaction-based amplification and restriction fragment length polymorphism analysis of 16S ribosomal DNA, and further characterized by multiple gene typing analysis (vmp1 and stamp genes). Obtained data highlighted that the majority of Georgian grapevine varieties showed moderate and mild symptoms, whereas international cultivars exhibited severe symptoms. Molecular characterization of ‘Ca. P. solani’ from grapevine revealed the presence of 11 distinct phytoplasma types. Only one type (VmGe12/StGe7) was identical to a strain previously reported in periwinkle from Lebanon; the other ‘Ca. P. solani’ types are described here for the first time. Phylogenetic analyses of vmp1 and stamp gene concatenated nucleotide sequences showed that ‘Ca. P. solani’ strains in Georgia are associated mainly with the bindweed-related BN host system. Moreover, the fact that ‘Ca. P. solani’ strains are distributed in grapevine cultivars showing a range of symptom intensity suggests a different susceptibility of such local cultivars to BN.

‘Candidatus Phytoplasma cynodontis’ is widespread in bermudagrass and has only been found in monocotyledonous plants. Molecular studies carried out on strains collected in Italy, Serbia, and Albania enabled verification of molecular variability in the 16S ribosomal RNA (rRNA) gene. Based on restriction fragment length polymorphism and sequence analyses, the strains from Serbia were clearly differentiated from all others and assigned to a new ribosomal DNA (rDNA) subgroup designated as 16SrXIV-C. A system for amplification of fragments containing the ‘Ca. P. cynodontis’ groEL gene was developed to enable study of its variability in related strains belonging to different 16SrXIV subgroups. Despite the fact that the groEL gene exhibited a greater sequence variation than 16S rRNA, the phylogenetic tree based on groEL gene sequence analysis was highly congruent with the 16S rDNA-based tree. The groEL gene analyses supported differentiation of the Serbian strains and definition of the new subgroup 16SrXIV-C. Phylogenetic analyses of both genes confirmed distinct phylogenetic lineages for strains belonging to 16SrXIV subgroups. Furthermore, groEL is the only nonribosomal marker developed for characterization of ‘Ca. P. cynodontis’ thus far, and its application in molecular surveys should provide better insight into the relationships among these phytoplasmas and correlation between strain differentiation and their geographical distribution.

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.