Lethal yellowing symptoms can be confused with those caused by Ganoderma butt rot, boron deficiency, and potassium deficiency on palms. Ganoderma butt rot, caused by the fungus G. zonatum, is a basal stem rot that leads to canopy wilting, early death of lower leaves, and spear leaf. Potassium deficiency will lead to discoloration and early death of the lowest leaves in the canopy. Boron deficiency will lead to early nut fall in coconut. These nuts will not be discolored, nor will they have water-soaked appearance at the calyx of the nut.
In Progress / Literature-based Diagnostics:
Culture: The phytoplasma that causes lethal yellowing is an obligate parasite and cannot be cultured on microbiological growth media.
Electron Microscopy: Phloem restricted phytoplasmas can be detected by transmission electron microscopy (Thomas and Norris, 1980). In coconut, nonfilamentous forms average 295 nm in diameter and filamentous forms average 142 nm in diameter and at least 16 ï¿½m in length (Waters and Hunt, 1980).
Fluorescence Microscopy: For large scale diagnosis, the DAPI (4", 6"-diamidino-2-phenilindole, 2HCl) staining method (Andrade and Arismendi, 2013) can be used, although the percentage of false negative detections can reach high levels, especially in palms. False negatives generally occur when phytoplasma colonization of plants is poor or uneven. This test detects fluorescence of DNA-containing phytoplasma cells in the sieve tubes of the leaf veins.
Transmission electron microscopy and fluorescence microscopy only reveal the presence of phytoplasma. Neither is specific to which phytoplasma might be affecting the host (Harrison et al., 1999).
DNA dot hybridization assays have been used to detect the lethal yellowing phytoplasma by using random fragments of phytoplasma genomic DNA cloned from LY-diseased Manila palm (Adonidia (Veitchia ) merrillii ) or windmill palm (Trachycarpus fortunei ) as probes (Harrison et al., 1992; Harrison and Oropeza, 2008). These probes, however, have been shown to vary in detection sensitivity and specificity (Harrison et al., 2008).
Southern blot hybridization analysis of phytoplasma DNA restriction profiles with cloned probes has been used to provide a measure of genetic variability among closely related phytoplasma strains (Harrison et al., 1992; 2008).
A "universal" PCR assay has been developed that enables amplification of the 16S rRNA genes of phytoplasmas. These assays readily amplify rDNA of most, or all, phytoplasmas. Digestion of the PCR products with selected restriction enzymes, a process known as restriction fragment length polymorphism (RFLP), provides a DNA fingerprint in the form of 16S rDNA fragment patterns that can be used to determine phytoplasma identity when resolved on agarose or by polyacrylamide gel electrophoresis (PAGE). These primers, however, have also identified non-phytoplasma target sequences. The latter PCR products are similar in size to PCR products from phytoplasmas, so the phytoplasma identity is not known (Harrison et al., 1999). Profiles resolved by PAGE after separate digestion of products with AluI, HinfI, TaqI or Tru9I endonucleases are especially useful for identification of group 16SrIV phytoplasmas (Harrison et al., 1999).
Group or subgroup-specific detection of phytoplasmas by utilizing primers for PCR based upon variable regions of the 16S rRNA gene or the 16-23S intergenic spacer region (SR) sequences of the phytoplasma genome reportedly permit selective amplification of rRNA gene sequences of "Ca. Phytoplasma palmae" and related strains in a group-specific manner. Primers 503f and LY16Sr derived from the 16S rRNA gene of the LY phytoplasma selectively amplify a 928 bp rDNA product from the LY phytoplasma strains infecting coconut and Pandanus and from the YLD (Yucatan coconut lethal decline) and CPY (Carludovica palmata yellows) phytoplasmas (Harrison et al., 1999; Cordova et al., 2000). Strains can be further differentiated by AluI digestion of the resulting amplification products.
LY16Sf and LY16Sr also selectively amplify 16SrRNA gene sequences of the LY agent from mixtures with host palm DNA (Harrison and Oropeza, 2008). When used to reamplify products obtained by PCR employing universal primer pair P1 and P7, LY16Sf/LY16Sr amplifies of rDNA from the LY phytoplasma and related strains in a group (16SrIV)-specific manner (Harrison et al., 2002a). Polymorphisms revealed by HinfI endonuclease digestion of the rDNA products differentiated coconut-infecting phytoplasmas in Jamaica from those detected in Florida, Honduras, and Mexico (Harrison et al., 2002a).
Exclusive detection of 16SrIV-A subgroup strains is possible by a PCR assay employing nonribosomal primer pair LYF1/LYR1 permitting unequivocal identification of "Ca. Phytoplasma palmae" (i.e. subgroup 16SrIV-A) in palms, Pandanus utilis , and the vector Haplaxius crudus (Harrison et al., 1994; Llauger et al., 2002).
Analysis of less conserved secA gene sequences has also been used to distinguish species and subgroups of phytoplasmas (Hodgetts et al., 2008).
There are five related strains/subgroups that vary in host range, symptomatology, and distribution - see CPHST pest datasheet for specific information