RESUMO
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.
RESUMO
The vascularization of the pedicel in Marisol clementine (Citrus clementina Hort. ex Tanaka) has been characterized in relation to fruit growth. Phloem and xylem formation occurred during the first half of the period of fruit growth. Phloem cross-sectional area reached its maximum value by the end of fruitlet abscission, 78 d after anthesis (DAA), shortly after the rate of accumulation of dry matter in fruitlets reached its maximum value. Secondary xylem formation occurred until day 93, well after the end of fruitlet abscission. At fruit maturity, xylem accounted for 42-46 % of the cross-section of the pedicel. Vessels differentiated in this late-formed xylem. Formation of phloem and early xylem was directly related to fruitlet size (and growth rate). Differences in the rate of formation of conductive tissues in the pedicel of the developing fruitlets followed rather than preceded the differences in growth rate. Specific mass transfer (SMT) in the phloem was highest in the fastest growing fruitlets, and peaked during the late stages of fruitlet abscission (72-78 DAA) and during the main period of fruit growth (107-121 DAA). Application of a synthetic auxin to developing fruits, either at the end of flowering (2,4-D) or by day 64 after flowering (2,4-DP), increased the growth rate of the fruit and fruit size at maturity (8-13 % increase in fruit diameter at maturity). These auxin applications also enhanced the formation of conductive tissues in the pedicel, with a specific effect on phloem formation. Applying auxin at flowering resulted in a reduction in the phloem SMT by days 72-78, whereas auxin application on day 64 increased this parameter. Despite this difference in behaviour, which resulted from the different time-course of the growth response of the fruit to auxin applications, these applications increased fruit size to a similar extent. Severing 37 % of the phloem of the pedicel during the main period of fruit growth resulted in an increase in the specific mass transfer in the phloem but had no influence on fruit growth. These observations demonstrate that the transport capacity in the phloem of the pedicel does not limit fruit growth and, within the limits of our experiments, an increase in demand by the fruit appeared to be matched by an increase in SMT. The dependence of late xylem formation (after the period of fruitlet abscission) on fruitlet growth was demonstrated in Salustiana orange [Citrus sinensis (L.) Osbeck] by means of controlling fruit growth through the manipulation of leaf area. Fruit growth at this time was more closely related to leaf area than to carbohydrate levels, suggesting that it may be limited by current photosynthesis.
