Plant Science

Mandarin (Citrus reticulata) is the most important cash crop in Bhutan and plantations total approximately 1.8 million trees (Ministry of Agriculture, Thimphu, Bhutan, 2000). Most trees are a local mandarin variety. Seedlings trees are produced by local farmers or supplied by Druk Seed Nursery. Mandarin seedlings have also been introduced from India. In the mid-1990s, mandarin trees growing in Punakha Valley and Wangdue districts began showing symptoms of decline that included sparse yellow foliage and shoot die-back. After initial surveys in 2000, huanglongbing (HLB) was suspected as the cause of declining trees based on symptomatology and presence of the psyllid vector Diaphorina citri, but no confirmatory tests were carried out. In August 2002, we surveyed eight locations in the valley from Rimchu (North) to Kamichu (South). HLB-like leaf mottle symptoms were observed on declining mandarin trees at all locations at altitudes ranging from 700 to 1,450 m. Orchards around Punakha (1,350m) in the center of the valley were more severely affected. Symptoms were also observed on Mexican lime (Citrus aurantifolia), citron (Citrus medica), and on tangelo trees (Minneola, Seminola, and Iyo) introduced originally as certified HLB-free budwoods from Corsica, France and grafted onto Rangpur lime at the Wangdue Research Center (1,300m). Leaves were collected from symptomatic trees and three declining mandarin trees without characteristic leaf mottle symptoms. Two specific polymerase chain reaction (PCR) tests for the detection of HLB Liberibacter species (1,2) were carried out on 16 DNA samples extracted from leaf mid-veins of 10 mandarins, two Mexican limes, three tangelos, and one citron tree. “Candidatus Liberibacter asiaticus” was readily detected by both PCR assays in all but two samples (one mandarin with noncharacteristic symptoms and citron) and all sampled orchards. The presence in the Wangdue Research Center of liberibacter infected trees, propagated from certified HLB-free budwoods, suggests that natural spread of the HLB by D. citri is occuring, as the psyllid had been identified previously in the Punakha area by Bhutanese Entomologists. It is likely that the disease was originally introduced as infected planting material although its source has not been determined. References: (1) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (2) S. Jagoueix et al. Mol. Cell.Probes 10:43,1996.

Catharanthus roseus (L.) G. Don (periwinkle) is well known as an experimental host for diverse phytoplasmas that are artificially transmitted to it through the use of dodder (Cuscuta sp.), laboratory vector insects, or grafting. However, few phytoplasma taxa have been reported in natural infections of C. roseus, and the role of C. roseus in phytoplasma dissemination and natural disease spread is not clear. In this study, naturally diseased plants of C. roseus exhibiting yellowing and witches' broom symptoms indicative of phytoplasma infection were observed throughout the year in the state of Rio de Janeiro, Brazil. Shoots and leaves of four diseased plants were assayed for the presence of phytoplasma DNA sequences by nested polymerase chain reactions (PCR) as previously described (2,3). Phytoplasma rDNA was amplified from diseased periwinkle plants in PCR primed by primer pair P1/P7 and was reamplified in nested PCR primed by primer pair R16F2n/R16R2 (F2n/R2). The results indicated the presence of phytoplasma in all four diseased plants. Phytoplasma identification was accomplished by restriction fragment length polymorphism (RFLP) analysis, using 11 restriction enzymes, of 16S rDNA amplified in PCR primed by F2n/R2. Phytoplasmas were classified according to the system of Lee et al. (1). On the basis of collective RFLP patterns of 16S rDNA, the phytoplasma infections in the four periwinkle plants could not be distinguished from one another. Furthermore, the collective RFLP patterns were indistinguishable from those reported previously for hibiscus witches' broom phytoplasma, “Candidatus Phytoplasma brasiliense” (2). The phytoplasma found in C. roseus, designated strain HibWB-Cr, was classified in group 16SrXV (hibiscus witches' broom phytoplasma group). HibWB-Cr is tentatively considered a new strain of “Ca. P. brasiliense”. C. roseus is the first known, naturally diseased alternate plant host of “Ca. P. brasiliense”. The present study identified strain HibWB-Cr in Rio de Janeiro State, where hibiscus witches' broom disease is prevalent (2). How this economically important disease of hibiscus spreads is not known. Our findings raise the possibility that a polyphagous insect vector is involved in the natural transmission of “Ca. P. brasiliense” and that C. roseus or other plant species serve as reservoirs for the spread of this phytoplasma taxon. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) H. G. Montano et al. Int. J. Syst. Evol. Microbiol. 51:1109, 2001. (3) H. G. Montano et al. Plant Dis. 84:429, 1999.

The identity of phytoplasmas detected in strawberry plants with green petal (SGP) and lethal yellows (SLY) diseases was determined by RFLP analysis of the 16S rRNA gene and adjacent spacer region (SR). RFLP and sequence comparisons indicated that the phytoplasmas associated with SGP and SLY were indistinguishable and were most closely related to ‘ Candidatus Phytoplasma australiense’, the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases. This taxon lies within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify only members of the AY strain cluster, amplified a DNA product from the SGP and SLY phytoplasmas. Primers deduced from the 16S rRNA/SR of P. australiense that amplify only members of this taxon amplified rDNA sequences from the SGP and SLY phytoplasmas. Primers that selectively amplify members of the faba bean phyllody (FBP) phytoplasma group, the most commonly occurring phytoplasma group in Australia, did not amplify rDNA from the SGP and SLY phytoplasmas.