Plant Science

While performing a routine field survey on 2-year-old canes of Rubus fruticosus (cv. Evergreen Thornless) in the region of Plovdiv (central southern Bulgaria), severe stunting of single or grouped plants (3 to 4 in a row) was found in late August of 2009. It was noteworthy that the leaves of these plants were curved upwards and stayed green until the end of the season. The bushy aspect of the diseased plants led to the assumption of a phytoplasma origin; therefore, specific PCR and sequence based identification methods were applied on leaves, petioles, and stems from three infected Rubus plants grown in different rows of the field (midsummer, nine samples in total) and the same number of asymptomatic samples. Partial amplification of the 16S ribosomal RNA gene with generic phytoplasma primer pairs P1/P7 and fu5/ru3 (3), followed by a nested PCR specific for all members of the Phytoplasma stolbur subgroup by means of the stol11 primers (1), and an RFLP analysis of the tuf gene (elongation factor Tu) fragment produced with PCR primers tufAY/r tufAY (3), were used for the identification and characterization of the pathogen. All target amplicons were also sequenced by Macrogen (Seoul, South Korea) following gel purification (Nucleospoin Plant II, Macher-Nagel). Identical sequences were obtained from each of the P1/P7-derived amplicons (100% homology between samples) and a consensus 1,142 bp sequence was delineated and submitted to NCBI GenBank with accession no. JF293091. It had the highest similarity (99 to 100%) to sequences of ‘Bois noir’ phytoplasma (e.g. HQ589193; Candidatus Phytoplasma solani, position 29 to 1,171). The fu5/ru3 amplicons produced sequences that showed 99.5% homology to the Ca. Phytoplasma solani strains of a southern Russian and Romanian phytoplasma survey on different hosts (potato, tomato, Convolvulus) (GenBank Accession No. HM449999 to HM4450002). The stolbur specific primers also produced an amplicon in all samples and again the consensus sequence was identified (100% homology between the samples) and deposited in GenBank (JN561701). RFLP analysis of the tuf gene with the enzymes HindIII, HinfI, HpaII, and TaqI (Fermentas) produced the same profile types for the different samples and clearly allocated the phytoplasma in the tuf type-b (VKII), according to (2). This type is commonly reported as associated with bindweed (Convolvulus arvensis). Additionally, the sequenced tufAY fragment also confirmed a 100% correspondence with the submitted Tu elongation factor fragments of Ca. Phytoplasma solani strains in GenBank. No phytoplasma was detected in symptomless blackberry plants that were sampled from the same plot. In the molecular identification tests, a stolbur phytoplasma control (potato isolate), a Rubus stunt (EY subgroup, 16SrV) and an apple proliferation phytoplasma (AP subgroup, 16SrX) were used as controls. Based on the symptoms and the laboratory results, we concluded that the Rubus plants were infected by Ca. Phytoplasma solani, a species belonging to the stolbur subgroup (16SrXII-A). To our knowledge, this is the first report of Ca. Phytoplasma solani on Rubus fruticosus in Bulgaria. The disease is not likely to be an isolated case in the future because of the pathogen's spread on other hosts and the expected increase in blackberry fields. References: (1) X. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (2) M. Langer and M. Maixner. Vitis 43:191, 2004. (3) K.-H. Lorenz et al. Phytopathology 85:771, 1995.

Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The three known causal agents of HLB are species of α-proteobacteria: ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’. Previous studies have found distinct variations in temperature sensitivity and tolerance among these species. Here, we describe the use of controlled heat treatments to cure HLB caused by ‘Ca. L. asiaticus’, the most prevalent and heat-tolerant species. Using temperature-controlled growth chambers, we evaluated the time duration and temperature required to suppress or eliminate the ‘Ca. L. asiaticus’ bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrate significant decreases in the ‘Ca. L. asiaticus’ bacterial titer, combined with healthy vigorous growth by all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of ‘Ca. L. asiaticus’, while untreated control plants remained highly infected. Continuous thermal exposure to 40 to 42°C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate ‘Ca. L. asiaticus’ bacteria entirely in HLB-affected citrus seedlings. This method may be useful for the control of ‘Ca. Liberibacter’-infected plants in nursery and greenhouse settings.

With diseases caused by vector-borne plant pathogens, acquisition and inoculation are two primary stages of the transmission, which can determine vector efficiency in spreading the pathogen. The present study was initiated to quantify acquisition and inoculation successes of ‘Candidatus Liberibacter solanacearum’, the etiological agent of zebra chip disease of potato, by its psyllid vector, Bactericera cockerelli (Hemiptera: Triozidae). Acquisition success was evaluated in relation to feeding site on the host plant as well as the acquisition access period. Inoculation success was evaluated in relation to vector number (1 and 4) on the plants. Acquisition success was influenced by the feeding site on the plant. The highest acquisition success occurred when insects had access to the whole plant. The results of the inoculation study indicated that the rate of successfully inoculated plants increased with the vector number. Plants inoculated with multiple psyllids had higher bacterial titer at the point of inoculation. Although disease incubation period was significantly shorter in plants inoculated with multiple psyllids, this effect was heterogeneous across experimental blocks, and was independent of pathogen quantity detected in the leaflets 3 days postinoculation. Disease progress was not affected by bacterial quantity injected or psyllid numbers.

Phytoplasmas of the 16SrVII group in ornamental Fraxinus uhdei trees (1) growing in different cities of the Colombian Andes have been reported (2). In surveys made in Bogotá during March and May 2011, symptoms suggestive of phytoplasma infection were observed in ornamental woody species: Croton spp. (Euphorbiaceae), Pittosporum undulatum (Pittosporaceae) and Populus nigra (Salicaceae) trees, growing close to infected F. uhdei (Oleaceae). Symptoms included witches' broom, yellowing, dieback, epicormic sprouts, tufted foliage, abnormal elongation or shortening of internodes, and deliquescent branching leading to dramatic changes in crown architecture. P. undulatum and F. uhdei are introduced species representing the second and third most abundant trees in the city. P. nigra is an introduced species and Croton spp. is an Andean genus. In order to screen for the presence of phytoplasmas in Croton spp., P. undulatum, and P. nigra, four individuals of each species and two F. uhdei trees were sampled. For DNA extraction, 1 g of vascular tissue from young stems was used. Samples were tested by nested PCR with primers P1A/P7A (4) followed by R16F2n/R16R2 (3). The frequency of phytoplasma detection varied among species; P. undulatum and Croton spp. had three positives each, while P. nigra had one positive. Both F. uhdei were positive. Sequences from the amplicons (three reads) were aligned. BLAST analysis of 16S rDNA sequences from the four species tested had 99.2 to 99.7% similarity to 16SrI group sequences. Phylogenetic analysis further confirmed this relationship. Virtual sequence analysis using the iPhyclassifier tool ( http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi ) showed that the sequence derived from P. undulatum (JQ730861) produced an identical RFLP pattern to group 16SrI-B (reference sequence NC_005303). RFLP similarity coefficients of the phytoplasmas from F. uhdei, Croton spp., and P. nigra (JQ730859, JQ730859 and JQ730861) were less than 0.97, suggesting the presence of a new subgroup within group 16SrI. The vectors of phytoplasmas are unknown in the region. Phytoplasma hosts previously reported in Colombia are: Solanum quitoense (16SrIII), Manihot esculenta (16SrIII), Liquidambar styraciflua (16SrI and 16SrVII), Elaeis guineensis (16SrI and 16SrIII), Coffea arabica (16SrIII), Cordia alliodora (16SrIII), Solanum tuberosum (16SrV and 16SrXII), and Zea mays (16SrI). To our knowledge, this is the first report of Croton spp. and P. undulatum as phytoplasma hosts. Phytoplasmas of group 16SrI are known to infect more than 100 species of different families worldwide. Detection of this group in several tree species and the observation of similar symptoms in other trees species raises concerns about a possible epidemic affecting plants in the Andean region. Implications are at several levels: i) epidemiological, with infected trees representing a potential inoculum source for other ornamental plants or crops growing in the agricultural surrounding areas; ii) economic, since eventually it will be necessary to replace diseased plants; and iii) environmental, because of the negative impact on the services provided by trees and green areas. References: (1) J. J. Filgueira et al. Plant Pathology 53:520, 2004. (2) L. Franco-Lara et al. Fitopatología Colombiana 29:32, 2005. (3) D. E Gundersen et al. Phytopathol. Mediterr. 35:144, 1996. (4) I-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004.

This study reports the development of a loop-mediated isothermal amplification procedure (LAMP) for polymerase chain reaction (PCR)-based detection of ‘Candidatus Liberibacter solanacearum’, the bacterial causal agent of potato zebra chip (ZC) disease. The 16S rDNA gene of ‘Ca. Liberibacter solanacearum’ was used to design a set of six primers for LAMP PCR detection of the bacterial pathogen in potato plants and the psyllid vector. The advantage of the LAMP method is that it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Positive LAMP results can be visualized directly as a precipitate. The LAMP strategy reported here reliably detected ‘Ca. Liberibacter solanacearum’ and the closely related species ‘Ca. Liberibacter asiaticus’, the causative agent of huanglongbing disease of citrus, in plant DNA extracts. Although not as sensitive as quantitative real-time PCR, LAMP detection was equivalent to conventional PCR in tests of ZC-infected potato plants from the field. Thus, the LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting ‘Ca. Liberibacter’ pathogens in psyllids and field-grown potato plants and tubers.