qPCR Quantification of PathogenicGuignardia citricarpaand NonpathogenicG. mangiferaein Citrus

Abstract

Citrus black spot, a major citrus disease caused by Guignardia citricarpa, was recently introduced in Florida. The nonpathogenic fungal endophyte G. mangiferae is commonly found in the same citrus tissues as G. citricarpa. Quantitative polymerase chain reaction (qPCR) assays based on internal transcribed spacer (ITS)-1 genes were developed to detect, quantify, and distinguish between these morphologically similar organisms in environmental samples. The primer/probe sets GCITS and GMITS were more than 95% efficient in single-set reactions in complex environmental DNA samples. Detection of 10 fg of G. citricarpa and G. mangiferae DNA was possible. Pycnidiospore disruption resulted in detection of single pycnidiospores with 78 (59 to 102; 95% confidence interval [CI]) and 112 (92 to 136; 95% CI) ITS copies for G. citricarpa and G. mangiferae, respectively. Detection was from partially decomposed leaves where fruiting bodies cannot be morphologically distinguished. Temperature and wetting period have significant effects on Guignardia spp. pseudothecia production in leaf litter. Based on relative biomass or the proportion of nuclei detected, G. citricarpa and G. mangiferae respond more strongly to wetting period than temperature. This qPCR assay will provide additional epidemiological data on black spot in tissues where G. citricarpa and G. mangiferae are not easily distinguished.

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…With the exception of one study investigating a fungal pathogen (Guignardia sp.) of citrus (Hu et al 2014), all investigations were undertaken in aquatic environments. A majority of these studies investigated the presence of exotic fish such as common carp (Cyprinus carpio; Eichmiller et al 2014;Takahara et al 2015), silver and bighead carp (Hypophthalmichthys molitrix and nobilis; Jerde et al 2013;Farrington et al 2015), African jewelfish (Hemichromis lifalili; Moyer et al 2014), bluegill sunfish (Lepomis macrochirus; Takahara & Minimoto 2013) and zebrafish (Danio rerio; Collins et al 2013).…”

MOLECULAR DIAGNOSIS OF Guignardia citricarpa IN ASYMPTOMATIC SWEET ORANGE TISSUE

“…Real-time PCR was suitable for detecting G. citricarpa DNA at a minimum concentration of 232 DNA copies, equivalent to 0.00001 ng of DNA. The number of copies was estimated as proposed by Hu et al (2014). In addition to validating the in-house technique, these results indicate that the method is robust and more sensitive than conventional PCR, which may allow the phytopathogen to be detected in asymptomatic plant tissue, that is, before symptoms emerge.…”

“…citri and Diaporthe citri. The existence of an endophytic species (G. mangiferae) that is very similar to the pathogen and the occurrence of false negatives when using morphological markers (BAAYEN et al, 2002;WANG et al, 2012;HU et al, 2014) confirm the need for specific and efficient early diagnosis of the disease.…”

“…All varieties of sweet orange (Citrus sinensis) and species such as C. limon, C. paradisi, C. reticulata and C. deliciosa are susceptible to the pathogen (HU et al, 2014). It was first reported in Australia in 1895 (SUTTON and WATERSTON, 1966) and has since spread to other citrus growing regions, such as southern and central Africa, South America and Asia, becoming a major phytosanitary problem in citrus crops (HINCAPIE et al, 2014;MARTÍNEZ-MINAYA et al, 2015).…”

“…This dormant period F. de S. FAGANELLO et al is interrupted by fruit maturation and climate conditions that favor the pathogen. The occurrence of long periods of latent infection demonstrates the importance of early detection, before symptoms emerge (HU et al, 2014).…”