Occurrence of 16SrIV Subgroup A Phytoplasmas in Roystonea regia and Acrocomia mexicana Palms with Lethal Yellowing‐like Syndromes in Yucatán,Mexico

The lethal yellowing (LY) disease and LY‐type syndromes affecting several palm species are associated with 16SrIV phytoplasmas in the Americas. In Mexico, palms of the species Roystonea regia and the native Acrocomia mexicana were found to exhibit LY‐type symptoms, including leaf decay, starting with mature leaves, necrosis and atrophy of inflorescences. DNA extracts obtained from these palms could be amplified by nested‐PCR using phytoplasma‐universal primer pair P1/P7 followed by LY‐group‐specific primer pair LY16Sr/LY16Sf. Blast analysis of the sequences obtained revealed an identity of 100% for R. regia and 99.27% for A. mexicana with 16SrIV‐A strain associated with LY in Florida, USA (Acc. AF498309 ). Computer‐simulated RFLP analysis showed that the patterns for the phytoplasma DNA of the two palm species were highly similar to that for 16SrIV subgroup A strain. A neighbour‐joining tree was constructed, and the sequences of the two palm species clustered in the same clade of group 16SrIV subgroup A. The results therefore support that LY‐type syndromes observed in palms of R. regia and A. mexicana in the Yucatan region of Mexico are associated with 16SrIV subgroup A phytoplasmas.     

16S rRNA interoperon sequence heterogeneity distinguishes strain populations of palm lethal yellowing phytoplasma in the Caribbean region

DNA of phytoplasmas in lethal yellowing (LY)‐diseased palms was detected by a nested polymerase chain reaction (PCR) assay employing rRNA primer pair P1/P7 followed by primer pair LY16Sf/ LY16‐23Sr. Polymorphisms revealed by Hinfl endonuclease digestion of rDNA products differentiated coconut‐infecting phytoplasmas in Jamaica from those detected in palms in Florida, Honduras and Mexico. A three fragment profile was generated for rDNA from phytoplasmas infecting all 21 Jamaican palms whereas a five fragment profile was evident for phytoplasmas infecting the majority of Florida (20 of 21), Honduran (13 of 14) and Mexican (5 of 5) palms. The RFLP profile indicative of Florida LY phytoplasma was resolved by cloning into two patterns, one of three bands and the other of four bands, that together constituted the five fragment profile. The two patterns were attributed to presence of two sequence heterogeneous rRNA operons, rrnA and rrnB, in most phytoplasmas composing Florida, Honduran and Mexican LY strain populations. Unique three and four fragment RFLP profiles indicative of LY phytoplasmas infecting Howea forsteriana and coconut palm in Florida and Honduras, respectively, were also observed. By comparison, the Jamaican LY phytoplasma population uniformly contained one or possibly two identical rRNA operons. No correlation between rRNA interoperon heterogeneity and strain variation in virulence of the LY agent was evident from this study.     

Molecular study of two distinct phytoplasma species associated with streak yellows of date palm in Iran

A disease with symptoms similar to palm lethal yellowing was noticed in the early 2013 in Khuzestan Province (Iran) in date palm (Phoenix dactylifera). Infected trees displaying symptoms of streak yellows and varied in the incidence and severity of yellowing. A study was initiated to determine whether phytoplasma was the causal agent. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods using universal phytoplasma primers pairs R16mF1/mR1 and M1/M2 were employed to detect putative phytoplasma(s) associated with date palm trees. Nested PCR using universal primers revealed that 40 out of 53 trees were positive for phytoplasma while asymptomatic date palms from another location (controls) tested negative. RFLP analyses and DNA sequencing of 16S rDNA indicated that the presence of two different phytoplasmas most closely related to clover proliferation (CP) phytoplasma (group 16SrVI) and ash yellows (AY) phytoplasma (group 16SrVII). Sequence analysis confirmed that palm streak yellows phytoplasmas in each group were uniform and to be phylogenetically closest to “CandidatusP. fraxini” (MF374755) and “Ca. P. trifolii” isolate Rus‐CP361Fc1 (KX773529). Result of RFLP analysis of secA gene of positive samples using TruI and TaqI endonuclease is in agreement with rDNA analysis. On this basis, both strains were classified as members of subgroups 16SrVI‐A and 16SrVII‐A. This is the first report of a phytoplasma related to CP and AY phytoplasma causing date palm yellows disease symptoms.     

Allozyme variation and structure of the Canarian endemic palm tree Phoenix canariensis (Arecaceae): implications for conservation

Electrophoretic analysis of 18 allozyme loci was used to estimate the levels and structuring of genetic variation within and among natural populations of the protected endemic palm species from the Canary Islands (Phoenix canariensis) to evaluate its genetic relationship with the widespread congener P. dactylifera, and to assess comparatively the genetic variation in the populations where the two species coexist with morphologically intermediate plants (mixed populations). Our survey revealed that the within-population component explains roughly 75% of the genetic variation levels detected in P. canariensis (A=1.59; P=41.8; He=0.158), which rank higher than those reported for other species of the Arecaceae. A Principal Component analysis (PCA) based on allele frequencies consistently separates populations of P. canariensis and P. dactylifera, and reveals a close genetic relationship between P. canariensis and the mixed populations. Reduced levels of genetic variation in P. canariensis with respect to P. dactylifera, the fact that the genetic makeup of the Canarian endemic (with no unique alleles) is a subset of that found in P. dactylifera, and the high genetic identity between both species strongly suggest that P. canariensis is recently derived from a common ancestor closely related to P. dactylifera.     

Detection and Identification of Aster Yellows Phytoplasma Associated with Lipstick Yellow Frond Disease in Malaysia

Yellowing symptoms similar to coconut yellow decline phytoplasma disease were observed on lipstick palms (Cyrtostachys renda) in Selangor state, Malaysia. Typical symptoms were yellowing, light green fronds, gradual collapse of older fronds and decline in growth. Polymerase chain reaction assay was employed to detect phytoplasma in symptomatic lipstick palms. Extracted DNA was amplified from symptomatic lipstick palms by PCR using phytoplasma‐universal primer pair P1/P7 followed by R16F2n/R16R2. Phytoplasma presence was confirmed, and the 1250 bp products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas associated with lipstick yellow frond disease were isolates of ‘Candidatus Phytoplasma asteris’ belonging to the 16SrI group. Virtual RFLP analysis of the resulting profiles revealed that these palm‐infecting phytoplasmas belong to subgroup 16SrI‐B and a possibly new 16SrI‐subgroup. This is the first report of lipstick palm as a new host of aster yellows phytoplasma (16SrI) in Malaysia and worldwide.     

Detection and Molecular Identification of Aster Yellows Phytoplasma in Date Palm in Egypt

Phytoplasma‐like symptoms were detected in date palm trees (Phoenix dactylifera L.) in Al‐Giza Governorate in Egypt. Symptoms varied from leaf chlorotic streaks, stunting and marked reduction in fruit and stalk sizes. Direct and nested PCR of symptomatic samples using P1/P7 and R16F2n/R16R2n primers, respectively, of the 16S rRNA gene, resulted in a DNA amplification product of c. 1.3 kbp. Symptomless samples collected from the same location and the healthy control produced no product upon amplification. Products were cloned into TOPO TA vector for sequencing. Data generated were deposited in the GenBank (Accession KF826615 ). A BLAST search showed that the sequence of the 16SrRNA gene shared ‘Candidatus Phytoplasma asteris’ (16SrI group) with other isolates. Phylogenetic analysis revealed that the isolate clustered with the date palm phytoplasma causing Al‐Wijam disease in Saudi Arabia.     

Detection and variability of the lethal yellowing group (16Sr IV) phytoplasmas in the Cedusa sp. (Hemiptera: Auchenorrhyncha: Derbidae) in Jamaica

Lethal yellowing (LY) group phytoplasma was detected in members of the Cedusa species of Derbids from Jamaica by nested PCR assay employing the rRNA primer pairs P1/P7 and LY16Sf/LY16Sr. A 1400‐bp product was obtained from 13/43 Derbids that were analysed. Restriction fragment length polymorphism analysis of the nested PCR product revealed variations in 6/13 Derbids analysed, suggesting that the planthoppers could be infected with different strains of the LY group phytoplasma. Sequence analysis of the 16S rDNA gene determined that the percentage of similarity of the phytoplasma strains in the Derbids ranged from 97–98% to the phytoplasma strain found in coconuts in Florida (LYFL‐C2) and Jamaica (LYJ‐C8). The phytoplasma strains in the Derbids clustered together with the Jamaican and Florida coconut LY phytoplasma, as well as other characterised strains composing the LY phytoplasma (16Sr IV) group.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

Wine produced from date palm (Phoenix dactylifera L.) fruits using Saccharomyces cerevisiae X01 isolated from Nigerian locally fermented beverages

Archives of Microbiology - The study focused on the production of wine from date palm fruits (Phoenix dactylifera L.) using a strain of yeast isolated from selected Nigerian locally fermented...     

Phytoplasma distribution in coconut palms affected by lethal yellowing disease

Lethal yellowing (LY), the most devastating disease affecting the coconut palm in America, is caused by phytoplasmas known to be distributed in different parts of infected plants. However, no comprehensive reports exist on the phytoplasma distribution. This study refers to the detection of LY phytoplasma DNA using PCR in different coconut plant parts, throughout the development of the disease. Sample analysis of positive palms taken at different stages of disease development (either symptomatic or symptomless) showed differences in the percentage of LY detection between plant parts. Some parts showed a very high level of LY DNA (stem, young leaves, inflorescences, stem apex and root apex), low levels were found in the intermediate leaves and roots without apex, whereas no LY phytoplasma DNA was detected in mature leaves. The detection percentage of LY phytoplasma DNA was lowest in symptomless‐infected palms for all parts, except the stem, where phytoplasma accumulations were consistently detected. This pattern of detection among parts is consistent with the hypothesis that phytoplasmas move from photosynthate source tissues to sink tissues via the phloem mass flow process. The accumulations in the (lower) stem, prior to the appearance of symptoms, suggest that this part of the palm is where phytoplasmas first move from leaves after foliar feeding by vectors and in which they probably multiply and distribute to other palm parts, including roots. Embryos from infected palms were analysed by nested‐PCR and 28% of 394 embryos were positive. Phytoplasma DNA was detected in embryos from fruit on any of the fruiting bunches regardless the age, but no pattern of quantitative distribution throughout the bunch developmental stages was observed. Germination of seeds from LY‐positive symptomatic palms was 58% and from LY‐negative symptomless palms were 71%. No phytoplasma was detected in seedlings tested from both symptomatic and non‐symptomatic palms. Seedlings tested after 2 years did not develop LY symptoms or eventually died.     

Detection and differentiation of African coconut phytoplasmas: RFLP analysis of PCR-amplified 16S rDNA and DNA hybridisation

Phytoplasmas associated with lethal decline diseases of the coconut palm (Cocos nucifera) in west and east Africa were detected by the specific amplification of their 16S rRNA genes. The primers used were based on conserved mollicute-specific and coconut-phytoplasma 16S rRNA gene sequences. Phytoplasma 16S rDNA was amplified from all African decline affected palms, some periwinkle maintained phytoplasmas, but not from healthy palms, infected palms from Florida or from the Cocos spiroplasma and Acholeplasma sp. mollicutes. African phytoplasmas were also detected by DNA hybridisation using two probes from the palm lethal yellowing phytoplasma from Florida. Probes hybridised at moderate stringency to dot blots of lethal decline affected palms from Africa, indicating possible genetic relationships between different coconut phytoplasmas. RFLP analysis of rDNA fragments (length c. 1.45 kbp) detected polymorphisms, indicating that the pathogens found in west and east Africa are not identical. This provides a useful tool for further epidemiological studies of African coconut phytoplasmal diseases.     

Molecular identification of ‘Candidatus Phytoplasma palmicola’ associated with coconut lethal yellowing in Equatorial Guinea

During the past two decades, a high mortality of coconut palms was observed in the coastal areas of Equatorial Guinea. Reportedly, the palm population has been reduced by 60%–70%, and coconut production has decreased accordingly. To identify the cause of the mortality, a survey was carried out in April 2021 in various localities of the coconut belt. Molecular analyses carried out on 16S rRNA and secA genes detected phytoplasma presence in the majority of the samples. Sequencing and BLAST search of the 16S rRNA gene sequences showed >99% identity of the detected phytoplasmas to ‘Candidatus Phytoplasma palmicola’. The RFLP analyses of 16S ribosomal gene using Tru1I and TaqI enzymes led to assign these phytoplasmas to subgroup 16SrXXII-A. In all samples that tested positive, including one from a hybrid coconut palm and two from oil palm the same phytoplasma was identified. The phylogenetic analyses of 16S rRNA and secA genes confirmed respectively 99.98%–100% and 97.94%–100% identity to ‘Ca. P. palmicola’. RFLP analyses using MboII enzyme on the secA gene amplicon differentiated the phytoplasma found in Equatorial Guinea from those present in Ghana and Ivory Coast. The Equatorial Guinean phytoplasma strain resulted to be identical to the strains from Mozambique, confirming the presence of a geographic differentiation among phytoplasma strains in the coastal areas of Western and Central Africa. The identified phytoplasma is different from the ‘Ca. P. palmicola’ strains found in Ghana and Ivory Coast and represents the first identification a 16SrXXII-A strain in Equatorial Guinea and in Central Africa. Strict monitoring and surveillance procedures for early detection of the pathogen are strongly recommended to reduce its impact and further spread in the country and permit the recovery of coconut plantations.     

Detection of somaclonal variations in tissue cultured date palm (Phoenix dactylifera L.) using transposable element-based markers

Plant Cell, Tissue and Organ Culture (PCTOC) - In vitro regeneration of date palm (Phoenix dactylifera L.) plants through somatic embryogenesis leads to the generation of somaclonal variants. The...     

A mitochondrial molecular marker of resistance to Bayoud disease in date palm

We have previously shown that two circular plasmid-like DNAs (the S and the R plasmids) can be found in the mitochondria of date palm (Phoenix dactylifera L.), a dioecious monocotyledonous tree. The two plasmids differ essentially by the absence, in the R plasmid, of a 109-bp DNA segment. Using 36 date palm varieties and employing a PCR-based approach, we show that the simultaneous presence of the R plasmid and absence of the S plasmid can be considered as a reliable molecular marker of resistance to a vascular wilt (Bayoud disease) caused by the fungus Fusarium oxysporum f. sp. albedinis. Conversely, the simultaneous presence of the S plasmid and absence of the R plasmid is correlated to Bayoud disease susceptibility The availibility of this diagnostic tool for plasmid characterization should subsequently allow simple, rapid and efficient selection of Bayoud-resistant individuals from the large number of date palms obtained by natural crosses which display good date quality. Received: 14 August 2000 / Accepted: 10 November 2000     

2,4-D induction of somaclonal variations in in vitro grown date palm (Phoenix dactylifera L. cv Barhee)

Plant Cell, Tissue and Organ Culture (PCTOC) - The present study is a part of a program designed at improving the date palm, Phoenix dactylifera L. cv. Barhee, through induced somaclonal variation....     

Mycobiota of the date palm phylloplane: description and interactions

We have analysed the mycobiota of date palm (Phoenix dactylifera, L.) leaves using several techniques. Profusely sporulating fungi (Penicillium spp. and Cladosporium spp.) developed when plating leaf fragments and leaf washings. Fusarium oxysporum, was particularly abundant in leaves infested with the red scale insect Phoenicococcus marlatti Cockerell, 1899, but an undescribed Lecanicillium cf. psalliotae was also found. Dual and overlay cultures showed interactions between palm pathogens, entomopathogenic and saprotrophic fungi. The most significant was the strong inhibition of the palm pathogen Penicillium vermoesenii caused by the entomopathogen Beauveria bassiana. No symptoms developed when F. oxysporum isolated from scale insects or the entomopathogens B. bassiana or Lecanicillium dimorphum were wound-inoculated on P. dactylifera petioles.     

Date Palm DNA Mini-Preparation without Liquid Nitrogen

We have developed a new mini-procedure for isolation of total cellular DNA from date palm (Phoenix dactylifera L.). The procedure, which does not use liquid nitrogen, has proved useful due to temporary disruptions in supplies of liquid nitrogen that occur in countries where date palm trees are cultivated. DNA suitable for RFLP and PCR analyses is obtained.     

Cloning and Sequencing of Phytoplasma Ribosomal DNA (rONA) Associated with Kerala Wilt Disease of Coconut Palms

Using the polymerase chain reaction (PCR) the 165 rRNA gene of phytoplasma associated with Kerala wilt disease of coconut palm (Cocos nucifera L) was amplified from infected leaf samples. Within the three universal primer pairs P1/P6, P1/P7and P41 P7, the primer pair P4/P7 only showed an amplification of 650 bp DNA fragment. 5ince P4/P7 amplifies the 16S-23S intergenic spacer region of 165 rRNA gene, the PCR product 650 bp of Kerala wilt disease palm indicates the phytoplasma DNA. The amplified fragment was sequenced and deposited in Genbank data library (Accession No. AY158660). The absence of restriction sites for Bcll and Rsa/l in 650 bp indicates phytoplasmic nature of DNA and its strain difference. A comparison of the 650 bp sequence with other phytoplasmas and its restriction profile indicates Kerala wilt disease phytoplasma as a separate 165 rRNA group in the classification of phytoplasmas. To our knowledge, this report records the first finding of the phytoplasma DNA using universal primers and its sequence analysis in coconut palms of Kerala, south India.     

Identification of Phoenix dactylifera L. varieties based on amplified fragment length polymorphism (AFLP) markers

The amplified fragment length polymorphism (AFLP) technique was applied to identify palm varieties. Fluorescence labelled primers were used in selective amplifications and the amplified fragments were detected on capillary gel electrophoresis using an automated DNA sequencer with the analysis fragment option. This is a rapid and efficient technique for detecting a large number of DNA markers on the date palm. Phoenix dactylifera L. varieties Bou-Fegous, Medjool, and E-528 from Estación Phoenix (Elche), Spain, were analysed, yielding a total of 310 AFLP fragments derived from five primer combinations. The process for regenerating the date palm cultivars from in vitro tissue culture should yield individuals phenotypically and genetically identical to the explant they are derived from. The AFLP markers obtained were successfully used for comparing and identifying vitroplants of palm.