Citrus Stubborn Disease: Current Insights on an Enigmatic Problem Prevailing in Citrus Orchards

Citrus stubborn was initially observed in California in 1915 and was later proven as a graft-transmissible disease in 1942. In the field, diseased citrus trees have compressed and stunted appearances, and yield poor-quality fruits with little market value. The disease is caused by Spiroplasma citri, a phloem-restricted pathogenic mollicute, which belongs to the Spiroplasmataceae family (Mollicutes). S. citri has the largest genome of any Mollicutes investigated, with a genome size of roughly 1780 Kbp. It is a helical, motile mollicute that lacks a cell wall and peptidoglycan. Several quick and sensitive molecular-based and immuno-enzymatic pathogen detection technologies are available. Infected weeds are the primary source of transmission to citrus, with only a minor percentage of transmission from infected citrus to citrus. Several phloem-feeding leafhopper species (Cicadellidae, Hemiptera) support the natural spread of S. citri in a persistent, propagative manner. S. citri-free buds are used in new orchard plantings and bud certification, and indexing initiatives have been launched. Further, a quarantine system for newly introduced types has been implemented to limit citrus stubborn disease (CSD). The present state of knowledge about CSD around the world is summarized in this overview, where recent advances in S. citri detection, characterization, control and eradication were highlighted to prevent or limit disease spread through the adoption of best practices.

Improved Real-Time PCR Diagnosis of Citrus Stubborn Disease by Targeting Prophage Genes of Spiroplasma citri

Spiroplasma citri is a phloem-limited bacterium causing citrus stubborn disease (CSD). Isolation and culturing of S. citri is technically demanding and time consuming. S. citri is typically low in titer and unevenly distributed in citrus, making reliable detection challenging. The current preferred detection method is polymerase chain reaction (PCR) assays with primers developed from sequences of S. citri housekeeping genes. Recent genome sequencing of S. citri revealed that the bacterium harbors multiple copies of prophage genes. Therefore, targeting multicopy prophage genes was hypothesized to improve sensitivity of PCR detection. Two primer sets, Php-orf1 and Php-orf3, were developed from conserved prophage sequences in the S. citri genome. These primer sets were used to evaluate detection sensitivity in SYBR Green-based quantitative PCR (qPCR) assays with 18 S. citri in cultures isolated from different hosts and locations. Prophage primer set Php-orf1 increased detection sensitivity by 4.91 and 3.65 cycle threshold (Cq) units compared with housekeeping gene primers for spiralin and P58 putative adhesin gene, respectively. Detection was slightly less sensitive for the Php-orf3 primer set at 3.02 and 1.76 Cq units, respectively, over the same housekeeping gene primers. The prophage primer sets were validated for qPCR detection with field samples from three citrus orchards in California's San Joaquin Valley collected from 2007 to 2013. The data showed that S. citri prophage sequences improved sensitivity for qPCR detection of S. citri-infected trees at least 10-fold and reduced the number of false-negative results. No false-positive samples were detected with any of the primer sets. The enhanced sensitivity resulted from the higher copy number of prophage genes in the S. citri genome and, thus, improved CSD diagnosis from field samples.

Novel Diagnosis for Citrus Stubborn Disease by Detection of a Spiroplasma citri-Secreted Protein

Citrus stubborn disease (CSD), first identified in California, is a widespread bacterial disease found in most arid citrus-producing regions in the United States and the Mediterranean Region. The disease is caused by Spiroplasma citri, an insect-transmitted and phloem-colonizing bacterium. CSD causes significant tree damage resulting in loss of fruit production and quality. Detection of CSD is challenging due to low and fluctuating titer and sporadic distribution of the pathogen in infected trees. In this study, we report the development of a novel diagnostic method for CSD using an S. citri-secreted protein as the detection marker. Microbial pathogens secrete a variety of proteins during infection that can potentially disperse systemically in infected plants with the vascular flow. Therefore, their distribution may not be restricted to the pathogen infection sites and could be used as a biological marker for infection. Using mass spectrometry analysis, we identified a unique secreted protein from S. citri that is highly expressed in the presence of citrus phloem extract. ScCCPP1, an antibody generated against this protein, was able to distinguish S. citri-infected citrus and periwinkle from healthy plants. In addition, the antiserum could be used to detect CSD using a simple direct tissue print assay without the need for sample processing or specialized lab equipment and may be suitable for field surveys. This study provides proof of a novel concept of using pathogen-secreted protein as a marker for diagnosis of a citrus bacterial disease and can probably be applied to other plant diseases.