Protocol for the Acclimatization of Coconut Vitro-Plants Under Ex Vitro Conditions.

The coconut tree is a crop widely distributed in more than 90 countries worldwide. It has a high economic value derived from the large number of products obtained from the plant, with fast-growing global markets for some of them. Unfortunately, coconut production is decreasing mainly due to the old age of the plants and devastating pests and diseases, such as phytoplasma disease lethal yellowing (LY). Massive replanting is required with phytoplasma-resistant and high-yielding selected coconut plants to keep up with the market demand for fruit. For this purpose, an efficient micropropagation technology via somatic embryogenesis has been established at CICY, yielding fully developed vitro-plants grown within an in vitro environment. Hence, the last stage of the micropropagation process is the acclimatization of the vitro-plants, which are gradually adapted to live in external conditions outside the glass container and the growth room. A protocol has been developed at CICY to acclimate the coconut vitro-plants, and close to 80% survival can be obtained. This protocol is described here.

Development of a TaqMan Assay for the Detection of 'Candidatus Phytoplasma brasiliense' and Assessment by High-Resolution Melt Curve Analysis.

'Candidatus Phytoplasma brasiliense' (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp., where it causes witches broom symptoms, and papaya, where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study, two quantitative PCR assays are developed: one using high-resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMan assay successfully amplified two of the three isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII, and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting melting point (Tm) products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.

Leptodelphax maculigera (Hemiptera: Delphacidae) Harbors the Corn Stunt Complex Pathogens.

The African planthopper Leptodelphax maculigera (Hemiptera: Delphacidae) has been recently reported in many places in Brazil in association with maize. Its occurrence in maize production fields in Brazil has brought concerns to the corn production chain regarding the possibility of this planthopper to be a vector for maize bushy stunt phytoplasma (MBSP), corn stunt spiroplasma (Spiroplasma kunkelii), maize rayado fino virus (MRFV), and maize striate mosaic virus (MSMV). The phytoplasma and spiroplasma, which are bacteria belonging to the class Mollicutes, and the two viruses are associated with the corn stunt disease complex. Given the presence of the African planthopper species and the corn stunt complex in Brazil, we further investigated the abundance of this planthopper species in the State of Santa Catarina, Brazil, and whether the planthopper can carry the four pathogens. We inspected 12 maize production fields in different municipalities in the state for 20 weeks, using two yellow sticky traps for each maize field. The sticky traps were replaced weekly. A total of 130 specimens of L. maculigera were captured, with a great discrepancy in quantity among locations and weeks. We detected the mollicute MBSP and the viruses MRFV and MSMV in L. maculigera, whereas S. kunkelii was absent in the assessed African planthopper samples. The molecular detection of the phytoplasma and the viruses in field-collected African planthoppers is strong evidence that this insect species has the ability to acquire those pathogens through feeding from the phloem of diseased maize plants. Nonetheless, transmission capacity needs to be experimentally proven to assert L. maculigera as a vector for the corn-stunting pathogens.

Insect Vector and Reservoir Plant of 'Fragaria × ananassa' Phyllody Phytoplasma (16SrXIII-F) in Central Region of Chile.

Strawberry phyllody has emerged as a prevalent disease affecting Chilean strawberry in recent years. The causal pathogen, 'Fragaria × ananassa' phyllody phytoplasma (StrPh), is categorized within the 16S ribosomal group XIII that is exclusively found in the Americas. In the context of economically significant crops, hemipteran insect vectors and alternative host plants play a pivotal role in their natural dissemination. This study comprehensively examined the key epidemiological facets of StrPh in the central region of Chile: the insect vector and alternative hosts. Through field surveys, we identified an abundance of an insect species, Cixiosoma sp., in an StrPh-infected strawberry field and confirmed its role as a vector of this phytoplasma through subsequent transmission assays. Moreover, we found a spontaneous weed species, Galega officinalis, to be infected with StrPh, raising the possibility of it being a potential alternative host plant for this phytoplasma. StrPh was also detected in cold-stored strawberry runners purchased from a nursery that supplies the local strawberry cultivation, suggesting a potential source of this phytoplasma in Chile. Collectively, these findings provide a significant epidemiological source of StrPh dissemination in central Chile.

Ceratobasidium sp. is associated with cassava witches' broom disease, a re-emerging threat to cassava cultivation in Southeast Asia.

Cassava witches' broom disease (CWBD) is a devastating disease of cassava in Southeast Asia (SEA), of unknown etiology. Affected plants show reduced internodal length, proliferation of leaves and weakening of stems. This results in poor germination of infected stem cuttings (i.e., planting material) and significant reductions in fresh root yields and starch content, causing economic losses for farmers and processors. Using a metagenomic approach, we identified a fungus belonging to the Ceratobasidium genus, sharing more than 98.3-99.7% nucleotide identity at the Internal Transcribed Spacer (ITS), with Ceratobasidium theobromae a pathogen causing similar symptoms in cacao. Microscopy analysis confirmed the identity of the fungus and specific designed PCR tests readily showed (1) Ceratobasidium sp. of cassava is strongly associated with CWBD symptoms, (2) the fungus is present in diseased samples collected since the first recorded CWBD outbreaks in SEA and (3) the fungus is transmissible by grafting. No phytoplasma sequences were detected in diseased plants. Current disease management efforts include adjustment of quarantine protocols and guarantee the production and distribution of Ceratobasidium-free planting material. Implications of related Ceratobasidium fungi, infecting cassava, and cacao in SEA and in other potential risk areas are discussed.

Distribution of Dalbulus maidis (DeLong) (Hemiptera: Cicadellidae) and incidence of maize rayado fino virus and Candidatus Phytoplasma asteris in corn succession planting systems.

BACKGROUND: Corn is one of the main crops grown globally to produce food for human consumption and animal feed, including raw materials for bioenergy. Effective pest management is critical for the economic viability of corn production. The leafhopper Dalbulus maidis and the diseases transmitted by it have become relevant to corn production. Our study aimed to determine environmental parameters that affect D. maidis populations and the impacts of pathogen dispersion on corn productivity under different rotation systems and sowing seasons. RESULTS: The population density of leafhoppers found in the studied crops was low but capable of establishing the diseases and spreading them widely in the crops. The leafhopper's highest occurrence was in the corn vegetative development stage, and its population peaks were earlier in the corn off-season. The incidence of maize rayado fino virus and maize bushy stunt phytoplasma were higher in corn off-season than in the growing season. The incidence of diseases was higher in the final stages of the cultivation cycle. Yield losses were significantly higher for maize bushy stunt phytoplasma and not significant for maize rayado fino virus. CONCLUSION: Our study observed that corn's physiological stage was the main factor influencing D. maidis dynamics. The occurrence of D. maidis at low densities was sufficient to ensure the efficient transmission and dissemination of maize rayado fino virus and maize bushy stunt phytoplasma, which had a higher incidence in the reproductive stage and the corn sowed off-season. © 2023 Society of Chemical Industry.

Detection of 16SrIV-A phytoplasma DNA in Colpoptera sp. (Hemiptera: Nogodinidae) insects in Yucatan Peninsula, Mexico.

Lethal yellowing (LY) is a disease that affects coconut and other palm species. It is associated to phytoplasmas of the group 16SrIV and the only reported insect vector for this pathogen so far is Haplaxius crudus. H. crudus is present in Mexico and has been associated to 16SrIV phytoplasmas, however, it was not detectable during a LY outbreak in the coast of Yucatan, Mexico, suggesting the existence of other vector species. To test this hypothesis a survey of insects was carried out and a total of 3074 insects were captured during a year of monthly sampling. Ten taxonomic orders were identified in this sample, Hemiptera being the most abundant (N=2094), and these were classified into nine families. The leafhopper Colpoptera sp. from to the Nogodinidae family was de most abundant representing 56% of the total number of insects sampled and 23% of these samples resulted positive for LY phytoplasma by PCR detection. The BLAST comparison, virtual RFLP and phylogenetic analyses of the sequenced amplicons relate the detected phytoplasma to the subgroup 16SrIV-A. The findings presented herein suggest that Colpoptera sp. could be considered as a new putative vector of the LY-causing phytoplasmas in Mexico and a candidate for further research.

Spatial-temporal distribution of Dalbulus maidis (Hemiptera: Cicadellidae) and factors affecting its abundance in Brazil corn.

BACKGROUND: Since the last decade, Dalbulus maidis has become the primary pest in cornfields, particularly due to its ability to transmit plant pathogens. Dalbulus maidis is the main vector of the corn stunt spiroplasma and maize bushy stunt phytoplasma. However, there is little information available on this pest. Understanding its spatial dynamics may allow us to determine how its infestations begin and to identify its colonization patterns, dispersal, and the role of landscape structure on D. maidis dynamics. Thus, this study aimed to investigate within-field spatial distribution and the factors associated with D. maidis abundance in five commercial fields. RESULTS: In all fields, higher infestations occurred at the boundaries of the central pivot, showing a clear edge-biased distribution. Ranges varied from 100.4 to 611.8 m, and our models' overall fit indicated strong to moderate spatial dependency. Additionally, correlation analyses indicated a positive effect of air temperature on the population of D. maidis. Conversely, rainfall negatively affected D. maidis. CONCLUSION: This study provides essential guidance for improving D. maidis integrated pest management at regional and local scales. Based on its high dispersal ability, our study suggests the need for a legislative or regulatory method of control for D. maidis, especially in regions where corn has more than one growing season. © 2022 Society of Chemical Industry.

Insecticide seed treatment against corn leafhopper: helping protect grain yield in critical plant growth stages.

BACKGROUND: The corn leafhopper, Dalbulus maidis (Hemiptera: Cicadellidae), spreads maize stunt pathogens and requires timely and effective crop protection. We determined the interaction between maize phenology and the vector feeding/infection period by stunt pathogens with the residual efficacy of neonicotinoid insecticidal seed treatments. Greenhouse- and field-grown maize plants, seed-treated with clothianidin or imidacloprid insecticides, were infested during seven growth stages with corn leafhoppers reared under controlled conditions on maize plants displaying infection symptoms by both spiroplasma (corn stunt spiroplasma, Spiroplasma kunkelii) and phytoplasma (maize bushy phytoplasma) pathogens. RESULTS: In the greenhouse and field settings, seed treatment reduced the stunt disease symptoms and corn yield loss during the VE-V4 maize growth stages and caused no phytotoxicity. The neonicotinoid seed treatment reduced 20-60% of the yield losses from the corn stunt disease until the V4 growth stage. Infestation by infective corn leafhoppers in the V12 maize growth stage caused a 25-30% yield loss irrespective of seed treatment, yet no stunt disease symptom was evident. Nonetheless, corn yield losses and visual stunt symptoms as rated by a nine-category ordinal scale were strongly correlated (r = 0.79, P < 0.01). CONCLUSION: These results reinforce that maize plants are more susceptible to leafhopper stunt disease during the VE-V4 growth stages (emergence to the fourth-leaf stage). Seed treatment helps reduce the damage in the early growth stages (VE-V2), although supplemental control measures depending on leafhopper population density may be needed from VE-V12 to protect yield losses from the maize stunt condition. © 2021 Society of Chemical Industry.

Eight Decades of Dalbulus maidis (DeLong & Wolcott) (Hemiptera, Cicadellidae) in Brazil: What We Know and What We Need to Know.

The corn leafhopper Dalbulus maidis (DeLong & Wolcott) is one of the most important maize (Zea mays L.) pests in Latin America because of its ability to efficiently transmit pathogens [maize bushy stunt phytoplasma (MBSP) and corn stunt spiroplasma-Spiroplasma kunkelli Whitcomb et al. (CSS)] associated with corn stunt disease complex and maize rayado fino virus (MRFV). This leafhopper species, considered a secondary pest until a few years ago, was first reported in Brazil in 1938. Since 2015, corn stunt diseases have been the main phytosanitary threat to corn production in Brazil, and D. maidis has assumed the status of a key pest of the crop. In this study, we gathered pertinent information about the corn leafhopper, from the time it was first recorded in Brazil. Aspects such as origin, association with maize, bioecology, geographical distribution in the Americas, and its congeners are addressed. We present a history of studies performed with this species in the country, its importance as a pest, host plants, and survival strategies during the maize off-season. Based on the available scientific knowledge, the main management strategies for insect vectors and diseases are discussed. Finally, the main knowledge gaps for this insect vector and the prospects for future studies and actions to mitigate the damage caused by insect vectors in maize crops in Brazil are presented and discussed.

Diversity of the 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma fraxini' isolates that infect urban trees in Bogotá, Colombia.

Phytoplasmas have been associated with a disease that affects trees of at least 11 species from different botanic families in Bogotá, Colombia. 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma fraxini' are the major groups of phytoplasma in the area of Bogotá. In this study, the genetic diversity within 'Ca. P. asteris' and 'Ca. P. fraxini' was studied in five urban tree species: Croton species (Euphorbiaceae), Fraxinus uhdei (Oleaceae), Magnolia grandiflora (Magnoliaceae), Populus nigra (Salicaceae) and Quercus humboldtii (Fagaceae). Analyses of the 16S rRNA gene using nested PCR, RFLP and sequencing showed that phytoplasmas of 'Ca. P. asteris' could be assigned to: subgroup 16SrI-B; a new subgroup named 16SrI-AF, with a restriction pattern similar to that of 16SrI-B; and a new subgroup named 16SrI-AG, with a restriction pattern similar to that of 16SrI-K and 16SrI-AH with a restriction pattern similar to that of 16SrI-AC. 'Ca. P. fraxini' isolates belonged to a new subgroup named 16SrVII-G, with a restriction pattern similar to that of 16SrVII-A. To complement the identification of the phytoplasma strains, we amplified nonribosomal genes such as leuS and secA. Unexpectedly, it was observed that in 16 trees in which 16S rRNA gene analysis showed the presence of 'Ca. P. fraxini' only, the leuS or secA primers amplified sequences exclusively affiliated to 'Ca. P. asteris. In those plants, sequences belonging to 'Ca. P. fraxini' leuS or secA genes were not amplified. The present work contributes to the identification of novel strains of both species in Colombia, and supports previous suggestions that phytoplasmas in South America are highly variable.

The glutathione peroxidase family of Theobroma cacao: Involvement in the oxidative stress during witches' broom disease.

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system inhibiting the ROS-induced damages of membranes and proteins. In cacao (Theobroma cacao L.) genome, five GPX genes were identified. Cysteine insertion codons (UGU) were found in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and tryptophan insertion codon (UGG) in TcGPX8. Multiple alignments revealed conserved domains between TcGPXs and other plants and human GPXs. Homology modeling was performed using the Populus trichocarpa GPX5 structure as template, and the molecular modeling showed that TcGPXs have affinity with selenometionine in their active site. In silico analysis of the TcGPXs promoter region revealed the presence of conserved cis-elements related to biotic stresses and hormone responsiveness. The expression analysis of TcGPXs in cacao plantlet meristems infected by M. perniciosa showed that TcGPXs are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that TcGPXs may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from T. cacao.

Haplaxius crudus (Hemiptera: Cixiidae) Transmits the Lethal Yellowing Phytoplasmas, 16SrIV, to Pritchardia pacifica Seem. & H.Wendl (Arecaceae) in Yucatan, Mexico.

Lethal yellowing (LY) affects several palm species in the Americas. It is caused by 16SrIV group phytoplasmas. In Florida (USA), LY was shown to be transmitted by the planthopper Haplaxius crudus ( Van Duzee ) (Hemiptera, Cixiidae) to different palm species, including Pritchardia pacifica Seem . & H. Wendl . (Arecaceae) in insect-proof cage experiments in the 1980s, a result that had never been reproduced later. LY has destroyed many coconut plantations as well as other palm species in the Caribbean and Mexico. In order to evaluate if H. crudus is a vector of LY phytoplasmas in Mexico, experiments were carried out in Yucatan (Mexico). Several H. crudus from palms infected by LY in the field were introduced into cages containing young P. pacifica palms. These insects were able to transmit 16SrIV group phytoplasmas to P. pacifica palms. According to DNA sequences comparative analysis, virtual restriction fragment length polymorphism, and phylogenetic analysis, the phytoplasmas detected in these infected P. pacifica were of subgroups A and D. All of ten P. pacifica palms infected with the subgroup D phytoplasmas developed symptoms of LY and died, whereas only one of two palms infected with subgroup A developed LY symptoms and died. This is the first time, more than 30 years later, that the role of H. crudus as a vector of LY is confirmed.

Detection and Identification of Phytoplasmas in the 16SrIV-A, -B, and -D Subgroups in Palms in Tabasco, Mexico.

The 16SrIV-A phytoplasmas are associated with the devastating disease lethal yellowing (LY) of palms. In Tabasco (Mexico), the death of Cocos nucifera, Adonidia merrillii, and Attalea butyracea palms have been suspected to be associated with LY based on symptomatology. Samples from the trunk of both symptomatic and nonsymptomatic palms were collected in three different environments: two species of palms within a rural zone and the other within an urban zone. DNA was extracted to perform a nested PCR with phytoplasma primers P1/P7-LY16SF/R16R2. A 1,345-bp fragment was amplified from the DNA extracted from each of the 29 LY-symptomatic palms sampled. Phytoplasma identification was achieved by amplicon sequencing and virtual restriction fragment length polymorphism analyses. Three 16SrIV phytoplasma subgroups were detected: 16SrIV-A in C. nucifera, 16SrIV-B in A. merrillii, and 16SrIV-D in C. nucifera, A. merrillii, and A. butyracea. Phylogenetic analysis showed also that the three phytoplasma strains found in the palm species clustered with phytoplasmas reported in the literature in the three subgroups identified. This is the first report of phytoplasmas associated with these palm species in Tabasco.

Derbid Planthoppers (Hemiptera: Fulgoroidea: Derbidae) Associated with Coconut and Oil Palm in Brazil.

We present surveys of derbid planthoppers associated with coconut (Cocos nucifera L.) and oil palm (Elaeis guineensis Jacq.) collected in Northeastern (Sergipe) and North (Pará and Roraima) Brazil. The surveys were intended to contribute to our knowledge of possible vectors of phytoplasmas or other phloem-restricted plant pathogens. Eight derbid taxa were found, two in the subfamily Cedusinae, tribe Cedusini (Cedusa yipara Kramer and C. yowza Kramer) and six in the subfamily Derbinae, tribe Cenchreini: Herpis sp., Persis pugnax Stål, Omolicna anastomosa (Caldwell), O. nigripennis (Caldwell), and two new species in the genus Agoo Bahder & Bartlett are described here. Genus-level features between Omolicna and Agoo are discussed and a key to the species of Agoo is provided.

Revealing the 1H NMR metabolome of mirasol chili peppers (Capsicum annuum) infected by Candidatus Phytoplasma trifolii.

The infection of Capsicum annuum cv. mirasol by Candidatus Phytoplasma trifolii (16SrVI) causes devastating crop losses in northern Mexico. This study addresses the metabolomics profiling of mirasol chili peppers (Capsicum annuum cv. mirasol) infected by Candidatus Phytoplasma trifolii. For this study, 25 diseased fruits and 25 healthy fruits were used. Principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) revealed dramatic changes in the content of 42 metabolites which were identified in diseased and healthy mirasol chili peppers. The endogenous levels of fructose, glucose and formic acid were substantially decreased in the diseased chili peppers. In the same group of samples, high concentrations of alanine, asparagine, fumaric acid, sucrose and threonine were observed. The content of Choline didnt present a significant difference. This evidence supports the fact that Candidatus Phytoplasma trifolii infection reduces de CO2 fixation into carbohydrates, decreases invertase activity, and inhibits glycolysis in the diseased plant tissues. The levels of ascorbic acid, capsaicin and dihydrocapsaicinin in diseased fruits were dramatically decreased, suggesting that Candidatus Phytoplasma trifolii can reduce the pungency and the nutraceutical value of mirasol chili peppers.

Phytoplasmas diversity and identification of new aster yellows subgroup (16SrI) associated with weed species in Argentina.

Symptoms of phytoplasma infection were observed in different weed species, Bidens subalternans, Conyza bonariensis, Heterosperma ovatifolium and Conium maculatum, collected from diverse geographical regions in Argentina. To confirm the association of phytoplasma infection with symptomatic plants, PCR, RFLP and phylogenetic analyses based on 16S rRNA-encoding sequences were performed. In this work, we report the presence of phytoplasmas from group 16SrVII (subgroup 16VII-B) infecting C. bonariensis and B. subalternans and from group 16SrIII (subgroup 16SrIII-X) B. subalternans, H. ovatifolium, and C. maculatum. Phytoplasmas from the aster yellows group were detected infecting C. bonariensis and B. subalternans. Analysis of 16S rRNA-encoding genes revealed the presence of two distinct operons, rrnB (16SrI-B) and newly described rrnA, which is different from the reference RFLP patterns of all previously established 16SrI-subgroups. A single rp operon sequence analysis reveals the presence of simple infection and confirms a description of a novel subgroup. On the basis of these results we propose a designation of new subgroup 16SrI-(B/AJ) AJ (rp-AJ). To our knowledge, this is the first report of phytoplasmas infecting Bidens subalternans¸ Heterosperma ovatifolium and Conium maculatum.

Occurrence in Brazil of Haplaxius crudus (Hemiptera: Cixiidae), Vector of Coconut Lethal Yellowing.

The lethal yellowing (LY) is a major phytoplasma causing disease seriously threatening coconut plantations worldwide, with imminent risk of entering Brazil. The LY phytoplasma is restricted to the phloem and transmitted by the planthopper Haplaxius crudus (Van Duzee) (Cixiidae). In this study, Auchenorrhyncha were collected on the leaves of Dwarf vs. Dwarf hybrids and Brazilian Green Dwarf Jiqui in the Brazilian northern state of Para using yellow adhesive traps in May of 2016. The planthopper H. crudus was found in coconut plantations of Brazilian Green Dwarf Jiqui, accounting for 87% of the individuals captured. This is the first report of vector H. crudus in Brazilian coconut plantations. These findings are of great scientific relevance since H. crudus could negatively impact the Brazilian coconut industry and this knowledge could be used in contingency measures in the case of LY be introduced in the country.

Molecular Characterization and Detection of 16SrIII Group Phytoplasma Associated with Huanglongbing Symptoms.

When huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' was infecting most of the trees while 'Ca. L. asiaticus' was present in a minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus-growing regions in Brazil. A phytoplasma from the 16SrIX group was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter spp. In this report, HLB samples testing negative for 'Ca. L. asiaticus', 'Ca. L. americanus', and 16SrIX phytoplasma were infected with 16SrIII phytoplasmas. Coinfection with 'Ca. L. asiaticus' and 16SrIII was also found. The 16S ribosomal RNA (rRNA) gene sequences from 22 samples were obtained and sequenced, confirming that the 16SrIII group phytoplasma is associated with HLB symptoms in SP and MG States. Ten single-nucleotide polymorphisms (SNPs) were found in the 1,427-bp 16S rRNA gene sequences from 16SrIII phytoplasmas from citrus, whereas none was detected in 16S rRNA gene sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group-specific primers, sequenced from a subset of nine samples, and assembled into three groups based on eight SNPs. SNPs in 16S rRNA gene and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB-associated microbiomes.