First Report of a 16SrII-C Phytoplasma Associated with Asymptomatic Acid Lime (Citrus aurantifolia) in Brazil

Abstract

At present, the principal bacterial disease of citrus in Brazil is Huanglongbing, caused by the alpha-proteobacterium ‘Candidatus Liberibacter spp.’ (although a phytoplasma of the 16SrIX group is also associated with this disease [4]). While there is a wide diversity of phytoplasmas in crop species in Brazil (3), there have been no reports of symptoms associated with phytoplasma in Brazilian citrus. Asymptomatic infections of citrus cannot be excluded as a possibility and such plants could serve as a reservoir of phytoplasma inoculum. The aim of this study was to assess the presence of phytoplasma in asymptomatic Citrus aurantifolia (acid lime) in Brazil. Thirty-three leaf samples (young leaves from the upper canopies) were randomly collected from different plants in the states of Minas Gerais (n = 23), Santa Catarina (n = 2), and São Paulo (n = 8). Two additional samples of C. limonia (‘Rangpur’ lime) and one of C. latifolia (‘Persian’ or ‘Tahiti’ lime) were collected in Minas Gerais. Total DNA extraction was performed using NucleoSpin Plant II Kit (Macherey-Nagel) according to the manufacturer's recommendations. PCR was carried out with a universal P1/P7 primer set followed by nested primers R16F2n/R16R2 (2). Additionally, direct PCR was performed using primers specific for phytoplasma immune-dominant membrane protein IMP3F/IMP3R (1). ‘Rangpur’ and ‘Tahiti’ lime were not infected with phytoplasma. Of the C. aurantifolia samples, 52% were positive for phytoplasma in the direct and nested PCR assays. The numbers of positive samples in Minas Gerais, Santa Catarina, and São Paulo states were 12, 1, and 4, respectively. Of these, five were selected for DNA purification and 1,246-bp fragments were ligated to the pGEM T-easy vector (Promega) and partial 16Sr DNA was sequenced. Nucleotide sequences of Brazilian phytoplasma strains BR:MG:FNS10:2011, BR:MG:FNS53:2011, BR:SP:FNS73:2011, BR:SC:FNS86:2011, and BR:MG:FNS126:2012 (GenBank Accession Nos. KJ158173, KJ158174, KJ158175, KJ158176, and KJ158177, respectively) were subjected to RFLP analyses. The 16S rDNA RFLP in silico patterns for the five strains were identical to each other and to Cactus witches'-broom phytoplasma (16SrII-C subgroup, AJ293216). In addition, the highest similarity coefficient (5) and nucleotide sequence identity of Brazilian phytoplasma strains were 0.99 and 99%, respectively, with Cactus witches'-broom phytoplasma. PCR-RFLP analyses using the enzymes Bstu I, EcoR I, and Hpa II were consistent with RFLP in silico results, showing the same pattern as the 16SrII-C subgroup. Phylogenetic analyses based on 16S rDNA sequences (1,246 bp) demonstrated that all the Brazilian strains grouped in the same clade with other representative sequences from the 16S rDNAII group. To confirm the absence of any macroscopic symptoms, morphological characteristics of 10 uninfected and 10 phytoplasma-infected plants randomly selected from a single field in Minas Gerais were analyzed. There were no significant differences in leaf area, stalk diameter, or numbers of leaves, flowers, or fruits per branch. To our knowledge, this is the first report of the 16SrII-C subgroup phytoplasma associated with C. aurantifolia in Brazil, and the first report of asymptomatic citrus plants infected with phytoplasma. References: (1) N. Askari et al. J. Microbiol. Biotechnol. 21:81, 2011. (2) I. M. Lee et al. Phytopathology 84:559, 1994. (3) H. G. Montano et al. Bull. Insectol. 60:129, 2007. (4) D. C. Teixeira et al. Phytopathology 98:977, 2008. (5) Y. Zhao et al. Meth. Mol. Biol. 938:329, 2013.

Expression of phytoplasma‐induced witches’ broom disease symptoms in acid lime (Citrus aurantifolia) trees is affected by climatic conditions

“…However, little attention has been given to acid lime grown in areas with no apparent WBD symptoms. A recent study from Brazil indicated the association of phytoplasma from the 16SrII-C group with lime trees developing no typical symptoms of WBD (Da Silva et al, 2014). Due to the lack of previous studies on the association between climatic conditions and symptom development due to phytoplasma, the present study focused on characterizing the development of WBD symptoms in districts with different climatic conditions in Oman and the UAE.…”

Invasive mutualisms between a plant pathogen and insect vectors in the Middle East and Brazil

“…This was tested with the invasive insect D. citri ( H. phycitis was not used as it was not possible to establish) in Oman, where the phytoplasma causes WBDL symptoms, and then in Brazil, where infections are symptomless [24]. We studied various vector life-history traits measured as insects were reared on infected and uninfected lime plants and compare the fitness based on these traits.…”

“…Recently, an asymptomatic infection of lime by ‘ Ca . Phytoplasma aurantifolia’ was reported from São Paulo State, Brazil [24]. Brazil represents a location potentially vulnerable to novel biological invasions or, perhaps more subtly, novel plant-pathogen–vector associations.…”

Asymptomatic Phytoplasma Reveal a Novel and Troublesome Infection

“…Asymptomatic ("silent") infections have recently been detected in lime trees in Brazil [11] and Oman [12]. This silent infection was observed through molecular testing of plant material, yet the host plants themselves show no obvious visible symptoms.…”

“…As "Ca. P. aurantifolia" and other asymptomatic plant pathogens like it spread to novel sites of infection globally [31], and as these infections become more difficult to detect [11], new rapid detection methods will be required in order to effectively detect sources of pathogen and monitor its evolution. This study has presented both the difficulties in monitoring "silent" infections using PCR based methods, but has also identified target genes that behave consistently and distinctly during infection by this Phytoplasma.…”