Genipa americana and Ageratina anisochroma, two new hosts of Candidatus Phytoplasma asteris in Costa Rica
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We report two new plant species hosts for Candidatus Phytoplasma asteris: Genipa americana (Rubiaceae) and Ageratina anisochroma (Asteraceae). Phytoplasma infections were detected by real-time loop-mediated isothermal amplification and nested PCR. Consensus sequences from both hosts share 99% identity to the 16SrI-B subgroup using BLAST; however, potential new subgroups are suggested due to unique RFLP patterns of the 16S rDNA F2nR2 fragment.
KeywordsNested PCR Real-time LAMP Jagua tree Guaitil tree
Phytoplasmas are pleomorphic prokaryotes that lack a cell wall (class Mollicutes) and live in plant phloem tissue and insect vectors (mainly species of Cicadellidae). Phytoplasmas cause plant diseases worldwide and have a negative impact on crops of economic interest as well as natural ecosystems (Hogenhout et al. 2008; Weintraub and Beanland 2006).
The second native plant species that we noticed displaying phytoplasma-like symptoms was Ageratina anisochroma (Klatt) R.M. King & H. Robinson (Asteraceae). During a field trip (2017) to different locations at Bellavista Mountain, few patches of A. anisochroma plants showing axillary proliferation, little leaves and dwarfing were observed (Fig. 1b). Six symptomatic plants (per locality) were collected at La Cangreja (9°47′52.05” N, 83°57′55.07” W, 1889 masl, Cartago province) and at La Sierra (9°44′39.59” N, 83°58′36.05” W, 2024 masl, San Jose province), additionally two asymptomatic plants were also collected in each location.
Ageratina anisochroma (formerly Eupatorium anisochromum Klatt) is an endemic flowering shrub, up to 2 m tall known from Nicaragua, Costa Rica, and western Panama (King and Robinson 1970, 1972; Woodson et al. 1975), usually found above 1500 masl. This plant is a frequent pioneer species during secondary forest succession; therefore, the sampled patches were distributed along the sides of roads (sites of constant disturbance of the vegetation). Tamayo-Castillo et al. (1988) found germacranolides and several thymol derivatives in this species. This kind of compounds may have potential pharmacological activities, i.e. anti-inflamatory, antiseptic, antiviral, or antimycotic action.
To determine if phytoplasmas were associated with symptoms observed in both wild species, DNA was extracted from each sample using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. As a first screening, the symptomatic Genipa tree and six of the symptomatic Ageratina plants, plus one asymptomatic sample of each plant species were evaluated by Real-time Loop-Mediated Isothermal Amplification (RT-LAMP) on a Genie II instrument, using the 2× Isothermal Master Mix (OptiGene), the three pairs of universal primers: UNIF3/ UNIB3, UNIFIP/ UNIBIP, and UNIFL/UNIBL, and protocols described in Dickinson (2015). A negative control consisted of water as template, and DNA from E. poeppigiana infected with phytoplasmas of 16SrI group (Saborío-R et al. 2007) was used as a positive control. The DNA samples of symptomatic Genipa tree and Ageratina, as well as the 16SrI group positive control, produced a clear fluorescence signal curve, whereas non symptomatic samples and the water control showed a flat line (no fluorescence). RT-LAMP results indicated the presence of phytoplasmas; therefore, all the collected samples were tested by nested PCR using universal phytoplasma primers P1/P7 (Deng and Hiruki 1991; Duduk et al. 2013) followed by R16F2n/R16R2 (Gundersen and Lee 1996) in a total volume of 27 μL using 2× DreamTaq PCR Master Mix (Thermo Scientific), according to Villalobos et al. (2011). The expected amplification product (1200 bp) was detected only from symptomatic samples of Genipa (n = 1) and Ageratina (n = 12), and the positive control. The Genipa tree sample was tested throughout 5 years (once per year) resulting in a consistent association of the symptomatic tissue with phytoplasmas detection through time. Likewise, consistent association for 16 different symptomatic plants was observed in the case of A. anisochroma. The PCR products obtained from the jagua tree and two Ageratina samples were directly sequenced in both directions (Macrogen Inc., Korea). Contig sequences were assembled using BioEdit (Hall 1999), and a BLASTn search was done (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phytoplasmas detected in both species share 99% identity to the 16SrI-B phytoplasma subgroup.
Semi-nested PCR reactions were run using primers P1/R16S-SR and P1A/R16S-SR (Lee et al. 2004). Amplifications were performed with a PCR Gradient Palm Cycler (Corbett Research Model CG1–96, Australia) in 25 μL reactions containing 200 μM of each of the four dNTPs, 0.4 μM of each primer, 1.5 mM MgCl2, 0.625 units of DreamTaq DNA polymerase (Thermo Fisher Scientific Inc. USA), and 1 μL of diluted DNA. Amplicons (ca. 1.5 kb) were purified, cloned (TA system, Macrogen Inc., Korea) and sequenced in both directions for the symptomatic jagua tree, one Ageratina sample and E. poeppigiana. The resulting nucleotide sequences from three clones for each species were edited and one consensus sequence per species: Ageratina anisochroma, Genipa americana and E. poeppigiana, was obtained and deposited in GenBank (GB, Accession numbers: MH011345, MH011346 and MH011347, respectively).
After 8 years of our first observation of the Genipa symptomatic tree, no more symptomatic trees were observed in the area. This observation suggests that this species tree may be an example of an incidental or dead-end host plant for this phytoplasma (Alma et al. 2000; Weintraub and Beanland 2006). Conversely, a putative different epidemiological scenario seems to occur with Ageratina: a patch with a proportion of symptomatic plants was found at two locations during 2017, and an additional patch near to La Sierra was found recently (February 2018). No symptomatic A. anisochroma plants were noticed in previous surveys to the Bellavista Mountain area. A gross estimation of symptomatic plants incidence per patch was near 4%; therefore suggesting a possible new plant host-phytoplasma interaction. Lee et al. (1998) speculated how distinct phytoplasma strains evolved. Those authors proposed that occasionally a polyphagous insect vector may feed on a non-host plant for the insect species. If the non-host plant is susceptible to the phytoplasma strain carried by the insect a new infection is stablished. Afterwards, there is opportunity for an insect species that regularly feeds on the newly infected plant to acquire and transmit the phytoplasma. In this way, other individuals of the new host species or alternative species may become infected.
The record of natural host plants for ´Ca. Phytoplasma asteris´ worldwide increases each year. Its occurrence in Costa Rica was already known (Saborío-R et al. 2007), herein we report two new wild hosts: Genipa americana and Ageratina anisochroma. Both plant species were infected with different and potentially new 16SrI subgroups as suggested by comparison of 16S rDNA sequences and virtual RFLP’s. It is of scientific and economic interest to register the establishment of phytoplasmas in new hosts in order to understand their ecology. Phytoplasmas spreading to new hosts and their diversification (Lee et al. 1998) may affect the survival of infected wild plants and the yield and/or economic value of cultivated plant species. To our knowledge, this is the first report of infection of Genipa americana and Ageratina anisochroma by ´Ca. Phytoplasma asteris´ or any other phytoplasma strain worldwide.
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