Study Records North America’s First Phytoplasma in Common Florida Weed

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Florida’s beautiful palm nurseries face a serious problem called Lethal Bronzing disease. This sickness, caused by a tiny organism named ‘Candidatus Phytoplasma aculeata’ (also known as 16SrIV-D), started harming palm trees around 2006. Since then, it has caused significant economic losses for the nursery industry.

Florida Palm Disease Economic Threat

The disease spreads mainly through a small insect called the planthopper, Haplaxius crudus. This insect is widespread and abundant all over Florida and the southeastern United States. Unfortunately, it’s also very hard to control because of its complicated life cycle and habits.

While the adult insects feed on palm leaves high up in the trees, their young, called nymphs, live down on the ground. Research has shown these nymphs live on over 40 different kinds of grasses (plants like lawn grass or corn, in the Poaceae family) and sedges (similar to grasses but with solid stems, in the Cyperaceae family).

Because the nymphs spend so much time on these common plants, often seen as weeds, scientists began to wonder: Could these grasses and sedges be acting as secret hiding places, storing the dangerous Lethal Bronzing phytoplasma and helping it spread?

To answer this important question, researchers from the University of Florida’s Department of Entomology and Nematology at the Fort Lauderdale Research and Education Center carried out a detailed survey between 2020 and 2021.

They chose a palm nursery near Fort Pierce, Florida, for their study because this location had heavy disease pressure from Lethal Bronzing and also had a large population of the H. crudus planthopper.

In this environment, the team focused on the three most common weed species that consistently supported populations of the planthopper nymphs. These species were:

1. Guineagrass (Urochloa maxima [Jacq.] R. Webster) – A robust, invasive grass (Poaceae).
2. Smut grass (Sporobolus indicus [L.] R. Br.) – Another common weedy grass (Poaceae).
3. Globe sedge (Cyperus globulosus L.) – A widespread sedge (Cyperaceae).

The researchers collected a total of 108 plant samples using a careful, random method. They took exactly 36 samples from each of the three weed species.

Knowing that phytoplasmas live inside the plant’s food-carrying tubes (phloem), they collected specific parts from each plant to maximize their chance of finding the pathogen. For each individual sample, the collected tissue comprised:

• One cluster of leaves.
• The base (thatch layer) where leaves connect to the stem.
• The roots directly connected to that sampled section of the plant.

This thorough approach made sure they checked the tissues most likely to contain phytoplasmas if they were present.

Weed DNA Phytoplasma Detection Process

Moving from plant samples to confirming the presence of phytoplasmas required sophisticated laboratory work. The first step involved extracting all the DNA from each of the 108 plant samples.

Scientists placed each plant tissue sample into a special bag containing 5 milliliters of a powerful solution called guanidine buffer.

This solution, made with specific chemicals like guanidine thiocyanate, sodium acetate, EDTA, and PVP-40, helps break open plant cells and protect the genetic material inside. They then used a machine called a HOMEX6 tissue homogenizer to grind the tissue into a fine liquid mixture called a lysate.

From this lysate, they carefully measured out 400 microliters and transferred it to a small tube. Next, they used a commercial kit called the DNeasy Plant Mini Kit to purify the DNA, following the kit’s instructions exactly. This cleaning step removed substances from the plant that could interfere with later tests, leaving pure DNA ready for analysis.

Because phytoplasmas can be present in very low amounts, especially in plants that might not show any signs of sickness, the researchers needed an extremely sensitive detection method.

They chose a technique called nested Polymerase Chain Reaction (PCR), which involves two rounds of DNA copying to amplify tiny traces of the pathogen’s genetic signature. The first round, known as primary PCR, screened all 108 DNA samples using special tools called universal primers P1 and P7.

These primers are short pieces of DNA designed to latch onto parts of a gene (the 16S ribosomal RNA gene) that are almost identical in all known phytoplasmas. Think of them as hooks that only grab phytoplasma DNA. The primary PCR reaction was meticulously assembled in a total volume of 25 µl per sample, containing:

• 8.8 µl of ultrapure water
• 5 µl of 5× GoTaq Flexi Buffer
• 2.5 µl of MgCl₂ (25 mM)
• 0.5 µl of dNTPs (10 mM each)
• 0.5 µl of forward primer P1 (10 µM)
• 0.5 µl of reverse primer P7 (10 µM)
• 5 µl of PVP-40 (10%)
• 0.2 µl of GoTaq G2 Flexi DNA Polymerase
• 2 µl of template DNA (tDNA)

This mixture went into a machine called a thermocycler, programmed to heat and cool repeatedly, making millions of copies of any phytoplasma DNA present if it matched the primers.

After this cycling, the scientists checked the results by running the products through a jelly-like substance called a 1.5% agarose gel, stained with a dye called GelRed to make the DNA visible under UV light. Samples showing a DNA band of the expected size were considered possible positives.

However, to be absolutely sure about these possible positives and to make the test even more sensitive and specific, a second round of PCR was essential. This is called nested PCR. Scientists took a small amount of the diluted product from the first PCR and used it as the starting material for a new reaction.

This time, they used different primers named Rt6F2n and Rt6R2. These primers bind inside the DNA region copied in the first round, hence the name “nested.” This two-step process drastically reduces mistakes and makes the results much more reliable.

The nested PCR reaction was also done in 25 microliters with the same ingredients as the first round, using specific temperature cycles. Again, the final products were run on a 1.5% agarose gel and checked for a band of the correct size. Only samples producing a clear band at this stage were considered strong candidates for having phytoplasma.

Confirming Pathogen Identity Sequencing Methods

Samples that tested positive in the nested PCR needed final confirmation. The amplified DNA pieces, called amplicons, were first cleaned using a special reagent called ExoSAP-IT to remove any leftover primers or enzymes.

The clean DNA was then sent to a specialized lab (Eurofins Genomics) for Sanger sequencing. This process determines the exact order of the chemical letters (A, T, C, G) that make up the DNA fragment.

The sequence data came back as raw information. Scientists used computer software called DNA Baser (version 4.36) to assemble these pieces into one accurate, continuous sequence for each positive sample.

The next crucial step was identification. They used a powerful online tool called BLAST to compare their assembled sequences against the enormous GenBank database, which contains DNA sequences from millions of organisms worldwide. BLAST searches for the closest matches in the database, revealing the most likely identity of the organism the DNA came from.

To definitively figure out how any detected phytoplasma was related to known species and strains, the researchers performed a phylogenetic analysis. They used advanced software called MEGA11.

This involved lining up the sequences from their positive samples alongside reference sequences from various well-known phytoplasmas obtained from GenBank. They then built a maximum likelihood phylogenetic tree. This type of tree is a statistical estimate of how the sequences are evolutionarily related to each other.

To check how reliable the branches of the tree were, they used 1,000 bootstrap replicates. This is a statistical method that tests how well the patterns hold up if the data is resampled; values above 70% are generally considered strong support. The resulting tree visually shows how closely related different phytoplasmas are, grouping them like branches on a family tree.

New Phytoplasma Discovery North America

The results of this detailed molecular investigation provided clear answers, but not the ones the scientists initially expected about the Lethal Bronzing reservoir:

• Guineagrass (Urochloa maxima): Every single one of the 36 samples tested completely negative for any phytoplasma in both rounds of PCR.
• Smut Grass (Sporobolus indicus): Similarly, all 36 samples also tested completely negative.

This finding was important: in this specific nursery, under heavy pressure from Lethal Bronzing disease, these two common grasses showed no evidence of harboring the lethal bronzing phytoplasma (16SrIV-D). They were not acting as hiding places for the disease in this location.

However, the analysis of the Globe Sedge (Cyperus globulosus) samples revealed a startling and groundbreaking result:

• Out of the 36 sedge samples tested, three (3) gave clear positive results in the nested PCR and the following sequencing.
• The piece of DNA amplified from these three infected globe sedge plants was 1,172 base pairs (bp) long.
• When they compared this DNA sequence against the GenBank database using BLAST, they found an exceptionally close match: 99.8% identical to the DNA of ‘Candidatus Phytoplasma brasiliense’ (specifically, the sequence stored under GenBank number MH428963).

This near-perfect genetic match provided definitive proof: the phytoplasma infecting the Florida globe sedge was not the Lethal Bronzing pathogen they were hunting for. Instead, it was a completely different species called ‘Candidatus Phytoplasma brasiliense’, which belongs to the 16SrXV group.

Florida Phytoplasma Strain Genetic Analysis

The maximum likelihood phylogenetic tree helped the scientists understand more about this Florida discovery. The DNA sequences from the three positive globe sedge samples clustered very closely together on the tree, supported by strong statistical numbers, meaning they are very similar variants of the same phytoplasma strain.

This group clearly belonged within the broader ‘Candidatus Phytoplasma brasiliense’ family (16SrXV group). Importantly, while these Florida sequences were closest to known types within the 16SrXV-B subgroup (especially one found previously in a tree called Guazuma ulmifolia in Costa Rica, GenBank HQ258882, and the reference MH428963), they formed their own distinct branch.

The analysis showed that even though related to 16SrXV-B, the Florida samples represent a genetically distinct strain from other known strains of ‘Ca. P. brasiliense’.

This strongly suggests they might represent a new subgroup within this phytoplasma species. This genetic difference makes sense biologically. Florida is geographically far from South America, where ‘Ca. P. brasiliense’ is normally found.

Furthermore, discovering it in a fundamentally different kind of host plant – a monocot sedge, compared to all previously known dicot hosts – strongly indicates that this Florida population is an evolutionarily unique lineage.

This distinct group may have adapted to its new environment and host over time, or it could be the result of a separate introduction event. The GenBank database now permanently stores the sequences of these three historic Florida finds under the numbers ON000494, ON000495, and ON000496.

New Plant Pathogen Florida Risks

Finding ‘Candidatus Phytoplasma brasiliense’ in Florida, and therefore in North America for the first time, is a discovery of major importance for both science and agriculture. Its significance comes from several critical points:

1. First Report in North America: This is the first time ever this specific phytoplasma species has been found and confirmed anywhere in North America. Before this discovery, ‘Ca. P. brasiliense’ was only definitively known from South America (where it was first identified) and had one report from Azerbaijan.

Finding it in Florida suddenly expands the known geographic range of this pathogen by thousands of miles, bringing it right into a major agricultural region with a climate that favors many plant diseases.

2. A Biologically Important New Host: Globe sedge (Cyperus globulosus) is a completely new type of host for ‘Ca. P. brasiliense’. All other known hosts are dicot plants. Important previously known hosts include Hibiscus (causing “hibiscus witches’ broom” in Brazil), Papaya (causing “papaya bunchy top” in Peru), and Peach (found infected in Azerbaijan).

Globe sedge, however, is a monocot. Monocots and dicots are the two main groups of flowering plants, with fundamental differences in their structure and how they grow. Finding ‘Ca. P. brasiliense’ infecting a monocot shows a remarkable and unexpected ability for this pathogen to jump to very different kinds of plants.

This breaks the previous idea that it could only infect dicots, massively widening the number of plants that could potentially get sick and raising worries about infection in other important monocots like true grasses (including crops like corn or rice), other sedges, or even palm trees.

3. Sign of Wider Undetected Spread: Finding ‘Ca. P. brasiliense’ in places as far apart as South America, Azerbaijan, and now Florida strongly suggests this phytoplasma is much more widespread around the world than scientists previously knew or reported. This pattern points to two main ways it could be spreading:

Natural Spread: Insects carrying the pathogen might be blown long distances by wind or travel on migratory birds, although jumping continents this way is less likely.

Human-Caused Spread: The far more likely explanation is the frequent, often unnoticed, movement of infected plants through international trade in plants and garden materials.

Florida, as a major global center for importing and exporting ornamental plants, and with strong connections to tropical regions in the Americas, is especially at risk for these kinds of introductions. This discovery highlights the constant danger posed by the global movement of plants and the pathogens they might carry.

4. Immediate Risks for Florida’s Farms and Gardens: While the study didn’t find the Lethal Bronzing phytoplasma in the weeds they tested, discovering ‘Ca. P. brasiliense’ introduces a new and potentially severe threat to Florida’s plant life and agriculture. The dangers are real and multi-faceted:

Threat to Key Crops: Two of its known hosts, hibiscus and papaya, are not just common in Florida; they are economically vital. Hibiscus is a hugely important ornamental plant, central to Florida’s multi-billion dollar nursery and landscaping industry.

Papaya is a significant tropical fruit crop, with Florida being a major producer in the mainland United States. The big question is whether the hibiscus and papaya varieties grown commercially in Florida are susceptible to this specific Florida strain of ‘Ca. P. brasiliense’.

Right now, this is completely unknown. The diseases it causes elsewhere are devastating: “witches’ broom” in hibiscus makes plants grow abnormally bushy and stunted, ruining them for sale; “bunchy top” in papaya severely stunts the plant, yellows the leaves, and stops fruit production. If this new strain can make Florida hibiscus or papaya sick, the economic damage could be huge.

Potential Risk to Peaches: Florida also has a peach industry. The report from Azerbaijan showing ‘Ca. P. brasiliense’ infecting peach trees means there’s concern this new Florida strain could potentially threaten peach crops in the state too.

The Reservoir Problem – Globe Sedge: Finding globe sedge infected is particularly worrying because this plant is extremely common and widespread all across Florida.

It grows easily in disturbed areas, along roadsides, in farm fields, gardens, and nurseries. Its presence as an infected host creates a large, persistent source of the pathogen across the landscape. This common weed could help the pathogen survive, multiply, and spread to important crops, making control efforts very difficult.

Unknown Range of Host Plants: The fact that it jumped to infect a monocot sedge suggests the potential range of plants it can infect in Florida’s diverse environment could be much larger than we currently know. Other sedges, grasses (including pasture or lawn grasses), ornamental monocots, or even other crops could potentially be vulnerable.

Furthermore, the Lethal Bronzing insect vector, Haplaxius crudus, whose young feed on sedges like Globe sedge, is now a prime suspect as a potential carrier for this new phytoplasma too, creating a possible overlap in how both diseases might spread.

Urgent Florida Pathogen Research Needed

Finding ‘Candidatus Phytoplasma brasiliense’ in Florida, hosted by a common and widespread sedge, is not just a scientific curiosity. It represents a significant new threat to plant health that demands immediate attention and coordinated action.

While the study answered its original question – showing that Guineagrass and Smut grass were not reservoirs for Lethal Bronzing in that specific nursery at that time – it uncovered a potentially larger, more complex, and more widespread problem.

The ability of this pathogen to infect a monocot host, combined with Florida’s warm climate and its huge role in both domestic and international plant trade, creates a perfect situation for the disease to take hold. ‘Ca. P. brasiliense’ could become firmly established in Florida’s environment and farms, threatening multiple agricultural industries.

Right now, we simply don’t know enough to fully understand the risk or to figure out how to manage it effectively. Proactive, thorough, and well-funded research is now urgently needed, focusing on several key areas:

First, scientists need to map how widespread ‘Ca. P. brasiliense’ is within Florida. The current finding comes from just one nursery near Fort Pierce. Systematic surveys are required across many different places:

• commercial nurseries,
• agricultural fields (especially papaya,
• hibiscus, and peach farms),
• natural areas,
• roadsides, and
• home landscapes.

These surveys must target globe sedge and other potential host plants, both monocots and dicots, using the same highly sensitive nested PCR techniques that led to the initial discovery. Knowing where the pathogen currently exists is the essential first step for trying to contain it.

Second, researchers must determine the full range of plants that this Florida strain can infect. Crucially, this requires artificial inoculation studies, where healthy plants are deliberately exposed to the pathogen (using grafting or insect vectors) to see if they get sick and show symptoms.  The highest priority plants to test include major Florida crops like hibiscus, papaya, and peach varieties.

Other potential targets should include other fruit crops (like citrus, mango, or avocado, though these are less likely hosts), other popular ornamental plants (especially those related to hibiscus or other potential monocot ornamentals), and common weeds (like other sedges and grasses). Given the context, testing a few key palm species would also be a sensible precaution.

Third, identifying the insect vector(s) responsible for spreading ‘Ca. P. brasiliense’ in Florida is absolutely essential. Is Haplaxius crudus, the Lethal Bronzing vector that was abundant in the nursery where the pathogen was found and whose nymphs feed on sedges like Globe sedge, capable of transmitting this new phytoplasma too?

Or are other insects, like different planthoppers, leafhoppers, or psyllids, involved? Finding the primary spreader is non-negotiable for developing targeted control methods.

This research involves collecting potential insect vectors near infected globe sedge plants, using nested PCR to screen these insects for the phytoplasma, trying to prove transmission in controlled lab experiments if possible, and studying the insect’s life cycle and movement patterns in relation to the disease.

Fourth, we need a much better understanding of the pathogen itself and the actual threat it poses. This involves further genetic sequencing of the Florida strain, looking at other genes beyond the 16S rRNA (like ribosomal protein genes or the SecY gene), to confirm if it truly is a new subgroup and to trace its possible origins by comparing it to strains from other countries.

Scientists also need to document the specific symptoms this Florida strain causes in known hosts like hibiscus and papaya under local growing conditions. How fast does the disease develop?

What are the potential yield losses or plant death rates? Understanding how severe the disease is and how it spreads (its epidemiology) under Florida’s unique environment is vital for assessing the real economic risk and knowing when and how to intervene.

Fifth, developing and implementing sensitive diagnostic protocols for state and federal plant diagnostic labs is crucial. Labs need standardized methods, like the nested PCR used in this discovery, to routinely check suspect plants and potential insect vectors for ‘Ca. P. brasiliense’. Early detection is key to managing any new disease outbreak.

The discovery reported in this research paper is a vital first step, but it is only the beginning of the story. The silent arrival and establishment of ‘Candidatus Phytoplasma brasiliense’ in Florida, using a common sedge as its foothold, presents a clear and present danger to the state’s agricultural economy and its rich horticultural landscape.

Constant vigilance, dedicated research funding, and strong coordination among research institutions (like the University of Florida’s IFAS), state and federal agricultural agencies (like FDACS and USDA-APHIS), plant diagnostic labs, and the horticulture and farming industries are essential.

The goal must be to thoroughly understand the biology, spread, and danger level of this pathogen before it escalates from a concerning finding into a full-blown, expensive agricultural disaster. The hidden invader has been discovered; the race to understand its potential and stop its spread is now underway.