Candidatus Phytoplasma palmae is known to be vectored by the planthopper Haplaxius (syn. Myndus) crudus.

Other Haplaxius sp. and Cedusa sp. are suspected vectors of the lethal yellowing (LY) phytoplasma.

Method	Instructions	NAPIS Survey Method
Visual	Collect symptomatic plant material.	3031 - General Visual Observation

For FY 2014 and 2015 surveys: Follow instructions in Phytoplasma sample submission for Cooperative Agricultural Pest Survey (CAPS) Program and Farm Bill Goal 1 surveys FY 2014 and 2015

For FY2016 surveys: Follow instructions in Phytoplasma sample submission for Cooperative Agricultural Pest Survey (CAPS) Program and Farm Bill Goal 1 surveys FY 2016

For FY2017 surveys: Follow instructions in Phytoplasma sample submission for Cooperative Agricultural Pest Survey (CAPS) Program and Farm Bill Goal 1 surveys FY 2017

If you have taken the hands-on phytoplasma specific training at CPHST Beltsville, you can screen your own phytoplasma samples. Note: You will still have to follow the protocol in the linked document for confirmations.

Palm samples from immature field-grown palms with symptoms suggestive of phytoplasma disease should be received as freshly harvested leaflets (pinnate species) or leaflet lamina and midvein tissues (palmate species) taken from the youngest leaf (i.e., spear).

For mature palms, tissue samples can be removed as stem borings.

• Prior to sampling each palm, the bit should be flame sterilized using a portable propane torch and cooled by rinsing with water.



• Stem samples are removed by boring a hole (10 to 15 cm in length) into the palm stem (trunk) using a portable electric drill and 5/16 in. (ca. 7.8 mm) diameter bit.



• Begin sampling by drilling a shallow pilot hole in the lower stem to remove the outermost layer of pseudobark (discard these tissues).

• Resume drilling incrementally through the pilot hole into the interior stem to the final depth of ~15 cm using a back and forth motion to dislodge shavings.



• Tissue borings from the stem are collected directly into a clean sealable plastic bag.



• Once the sampling is complete, the stem can be sealed (if necessary) by tapping a wooden dowel into the hole to prevent sap bleeding and to provide a barrier to invasion by pests (see Harrison et al., 2013 for more details).


Each freshly harvested sample will be packaged separately, labeled, documented, and shipped for laboratory testing by overnight express service according to APHIS regulations. Each sample, in a separate plastic bag, will be labeled according to cultivar (when known) and plant species, date sample was collected, location of plant sampled, and name and institution of sample collector; each plastic bag should contain samples collected from only a single plant. For extended transport, stem tissues will be shipped after first drying each sample at 37 degrees (99 degrees F).

No specific signs are present.

No single symptom associated with lethal yellowing is diagnostic of the disease as each symptom may vary according to the particular species and cultivar of palm affected and by a variety of other causes. Rather it is the sequential and progressive development of symptoms (syndrome) that identify LY and help to distinguish it from other diseases and disorders that induce similar but isolated symptoms.

For mature, bearing coconut and other mature palm species infected by "Ca. Phytoplasma palmae", the earliest visible symptom is a premature shedding of most or all fruit regardless of developmental stage. Fruit that are shed from coconut often develop a blackened, or water soaked appearance at the calyx end. Necrosis of newly emergent inflorescences accompanies or follows fruit drop. Flower spikelets, which are normally light yellow to creamy white in color, appear partially or totally blackened. Fruit and flower symptoms are followed shortly thereafter by foliar discoloration. On the Atlantic tall coconut ecotype, leaves turn a golden yellow color. Discoloration begins on the lowermost (oldest) leaves and progresses to successively younger leaves in the upper part of the crown. Discolored leaves typically remain turgid for some time before turning brown, drying and hanging downward around the stem for a few days before falling to the ground. The newest unopened leaf (spear) collapses once foliar discoloration is advanced. Death of the apical meristem occurs at this stage after which the remaining crown withers and topples away leaving just a bare trunk standing.

While premature fruit drop and inflorescence necrosis are common to all palms with lethal yellowing, leaves turn reddish brown rather than yellow on many coconut ecotypes and most other palm species. On date palms, death of the spear leaf and underlying apical meristem occurs shortly after leaves first begin to discolor. Most affected palms die within 3 to 5 months after the onset of symptoms.

For preliminary field diagnosis of disease, symptoms on palms induced by "Ca. Phytoplasma palmae"-related strains (i.e. subgroups 16SrIV-B, 16SrIV-D, 16SrIV-E and 16SrIV-F strains) are not sufficiently distinct in appearance to distinguish them from those attributed to "Ca. Phytoplasma palmae". For example, on Phoenix sylvestris, symptoms of Texas Phoenix palm decline (TPPD) attributed to subgroup 16SrIV-D phytoplasmas, foliar discoloration begins on the lowermost (oldest leaves), which turn reddish brown, starting at the tips and intensifies to involve successfully younger leaves in the mid-crown and upper crown. Shedding of most or all fruits as inflorescences wither and die prematurely is accompanied by collapse and death of the newest (spear) early in the foliar discoloration phase. Once discoloration has progressed to leaves of the mid-crown, mature roots of palms at or near the soil are unusually soft in texture and easily severed. Palms that have undergone these adverse changes can be easily pushed back and forth. However, loss of the structural integrity of the root system has not been demonstrated for any other palm species affected by subgroup 16SrIV-D phytoplasmas.

ID/Diagnostic: Molecular

Molecular: For FY 2014 and 2015 surveys: Follow instructions in Phytoplasma sample submission for Cooperative Agricultural Pest Survey (CAPS) Program and Farm Bill Goal 1 surveys FY 2014 and 2015 For FY2016 surveys: Follow instructions in Phytoplasma sample submission for Cooperative Agricultural Pest Survey (CAPS) Program and Farm Bill Goal 1 surveys FY 2016 For FY2017 surveys: Follow instructions in Phytoplasma sample submission for Cooperative Agricultural Pest Survey (CAPS) Program and Farm Bill Goal 1 surveys FY 2017 If you have taken the hands-on phytoplasma specific training at CPHST Beltsville, you can screen your own phytoplasma samples. Note: You will still have to follow the protocol in the linked document for confirmations.

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.

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 �m 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).

Molecular:
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