Ash Yellows Phytoplasmas Research Articles

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

AbstractA 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.

Incidence and characterization of the presence of phytoplasmas in Fraxinus uhdei related with Ash Yellow disease in Colombia

For a long time, Urapan trees (Ash) have been a popular plant in parks and avenues in the principal cities of the Andes in Colombia, but now more than 53,000 trees are infected and many of them died. In 2004, the presence of phytoplasmas in the phloem of some trees with dieback symptoms was reported. In this study, the phytoplasma’s presence has been detected in all the tested Fraxinus uhdei (Wenz.) (common name, Urapan), using microscopy techniques like optical microscopy, transmission electron microscopy, and DAPI fluorescent microscopy, which indicated a further spread of the disease. The presence of phytoplasmas in the phloem of the leaves of Urapan trees was evident and related with the symptoms of the disease “Ash Yellows”. Here the relation phytoplasmas–disease symptoms have been confirmed by reproducing the symptoms through phytoplasmas transfection in Periwinkle (Catharanthus roseus) by using the ectoparasite Cuscuta campestris. The molecular analysis using PCR with universal primers of the region 16S-23S rDNA (P1/Timt, R16mF2/R16mR1, fB1/P7, fPD/Tint, F1/P7, Phytuf22/Phytuf24) confirmed the parasite’s presence and its identification as Ash Yellows phytoplasma or Candidatus Phytoplasma fraxini which belongs to the phytoplasma group 16SrVII and subgroup 16SrVII-A. These results permit to suggest a possible treatment with antibiotics that would avoid the species extinction throughout the Andes cities.

A novel subgroup 16SrVII-D phytoplasma identified in association with Erigeron witches' broom.

Erigeron sp. plants showing symptoms of witches' broom and stunting were found near orchards of passion fruit in São Paulo state, Brazil. These symptoms were indicative of infection by phytoplasmas. Thus, the aim of this study was to detect and identify possible phytoplasmas associated with diseased plants. Total DNA was extracted from symptomatic and asymptomatic plants and used in nested PCR conducted with the primer pairs P1/Tint and R16F2n/16R2. Amplification of genomic fragments of 1.2 kb from the 16S rRNA gene confirmed the presence of phytoplasma in all symptomatic samples. The sequence identity scores between the 16S rRNA gene of the phytoplasma strain identified in the current study and those of previously reported 'Candidatus Phytoplasma fraxini'-related strains ranged from 98% to 99% indicating the phytoplasma to be a strain affiliated with 'Candidatus Phytoplasma fraxini'. The results from a phylogenetic analysis and virtual RFLP analysis of the 16S rRNA gene sequence with 17 restriction enzymes revealed that the phytoplasma strain belongs to the ash yellows phytoplasma group (16SrVII); the similarity coefficient of RFLP patterns further suggested that the phytoplasma represents a novel subgroup, designated 16SrVII-D. The representative of this new subgroup was named EboWB phytoplasma (Erigeron bonariensis Witches' Broom).

Herbal Drug Quality and Phytochemical Composition of Hypericum perforatum L. Affected by Ash Yellows Phytoplasma Infection

Qualitative/quantitative phytochemical variations were observed in dried flowering tops of cultivated Hypericum perforatum L. cv. Zorzi infected by phytoplasmas of the "ash yellows" class, identified by direct and nested polymerase chain reaction (PCR); this is the first report of ribosomial group 16SrVII phytoplasmas in St. John's Wort. Methanolic extracts of healthy and infected plants were separated by reversed phase high-performance liquid chromatography to quantify naphthodianthrones and flavonoids, while essential oils were analyzed by means of gas chromatography (GC)-GC/MS. The affected plants exhibited decreased amounts of rutin (1.96 +/- 0.23 vs 4.96 +/- 0.02 mg/g), hyperoside (2.38 +/- 0.21 vs 3.04 +/- 0.05 mg/g), isoquercitrin (1.47 +/- 0.04 vs 3.50 +/- 0.08 mg/g), amentoflavone (0.12 +/- 0.01 vs 0.39 +/- 0.02 mg/g), and pseudohypericin (1.41 +/- 0.23 vs 2.29 +/- 0.07 mg/g), whereas the chlorogenic acid content was doubled (1.56 +/- 0.11 vs 0.77 +/- 0.02 mg/g). Hypericin, quercitrin, and quercetin contents were not severely affected. The essential oil yield was drastically reduced in infected material (0.11 vs 0.75% in healthy material) and revealed an increased abundance of sesquiterpenes (beta-caryophyllene, delta-elemene, and germacrene D, in particular) and a matching decrease in monoterpene hydrocarbons and aliphatics. The consequences that the phytopathological condition of cultivated H. perforatum plants has on the commercial quality, market value, and therapeutic efficacy are outlined.

Rapid PCR-based Detection of Phytoplasmas from Infected Plants

Five simplified DNA preparation procedures for polymerase chain reaction (PCR) amplification were tested for detection of phytoplasmas from infected herbaceous and woody plants. Thin freehand cross-sections made from infected plant tissues and stored in acetone were used as sources for DNA preparation. The tissue sections were treated by: 1) grinding in sodium hydroxide; 2) sonicating in water; 3) microwaving in water; 4) boiling in sodium hydroxide; or 5) placing directly in PCR tube. PCR amplification was performed with a universal phytoplasma-specific primer pair in a reaction buffer containing 0.5% (v/v) Triton X-100, 1.5 mm magnesium chloride, and 10 mm Tris-HCl. All five procedures provided phytoplasmal template DNA for successful PCR amplification from infected herbaceous plants {periwinkle [Catharanthus roseus (L.) G. Don (periwinkle)], carrot (Daucus carota L.), maize (Zea mays L.)}, while the grinding, microwaving, and boiling procedures also allowed positive amplification from a woody plant [green ash (Fraxinus pennsylvanica Marsh.)]. The quality of the resulting DNA was adequate for subsequent identification of the aster yellows and ash yellows phytoplasmas through nested-PCR using phytoplasma group-specific primer pairs. These methods provide remarkable savings in labor and materials, making disease testing and indexing of plant materials much more attractive.

Erigeron Witches'-Broom Phytoplasma in Brazil Represents New Subgroup VII-B in 16S rRNA Gene Group VII, the Ash Yellows Phytoplasma Group.

A previously undescribed phytoplasma, Erigeron witches'-broom phytoplasma, was detected in diseased plants of Erigeron sp. and Catharanthus roseus exhibiting symptoms of witches'-broom and chlorosis in the state of São Paulo, Brazil. On the basis of restriction fragment length polymorphism (RFLP) analysis of 16S rDNA amplified in the polymerase chain reaction (PCR), Erigeron witches'-broom phytoplasma was classified in group 16SrVII (ash yellows phytoplasma group), new subgroup VII-B. Phylogenetic analysis of 16S rDNA sequences indicated that this phytoplasma represents a new lineage that is distinct from that of described strains of ash yellows phytoplasma. Erigeron witches'-broom phytoplasma is the first member of the ash yellows phytoplasma group to be recorded in Brazil.

Occurrence, Associated Symptoms, and Potential Insect Vectors of the Ash Yellows Phytoplasma In Iowa, U.S.

The ash yellows (AshY) phytoplasma has been reported widely across the United States as an organism associated with ash decline in urban communities. Prior surveys conducted in Iowa communities indicated that AshY was found in up to 20% of the urban green ash (Fraxinus pennsylvanica Marsh.) when using the DAPI (4' ,6-diamidino-2-pheyIindole'2HCl) staining method. A new survey of trees in nine Iowa communities used polymerase chain reaction (PCR.) to detect the AshY phytoplasma. PCR analyses of tree tissue were positive for AshY in 4% of the 240 trees surveyed. Although 145 of the sampled trees had decline symptoms similar to those exhibited by trees infected with the AshY phytoplasma, the phytoplasma was detected in only 11 trees. Because Homoptera have been shown to vector phytoplasmas, insect populations associated with ash trees in two Iowa communities were also sampled to help determine whether the insects were carrying phytoplasmas. A total of 471 insects, representing 34 species, were collected from green ash trees; of these, 396 were assayed for the presence of phytoplasmas using PCR. Three leafhopper species were found that were reported to vector phytoplasma diseases, but phytoplasmas were not detected in any of the insects assayed.

Responses of 11 Fraxinus Cultivars to Ash Yellows Phytoplasma Strains of Differing Aggressiveness.

Five cultivars of Fraxinus americana (white ash) and five of F. pennsylvanica (green ash) were graft-inoculated with three strains of ash yellows phytoplasmas at Ames, IA, and with thrsee other strains at Ithaca, NY. A sixth green ash cultivar was tested only in New York. Trees were allowed to grow in field plots for 3 years. Infection was detected via the DAPI (4', 6-diamidino-2-phenylindole 2HCl) fluorescence test. Incidence of witches'-brooms on infected trees was greater on white ash than green ash and varied significantly among phytoplasma strain treatments at both locations. Volume growth of infected ash, averaged across cultivars over 2 years in Iowa and 3 years in New York, was 49 and 59%, respectively, as great as that of noninfected trees. Foliar greenness was reduced significantly by infection at both locations, and this reduction was positively correlated with growth reduction. Cultivars at each location varied significantly in growth of noninfected trees and in growth of diseased trees relative to that of nonin-fected trees (a measure of phytoplasma tolerance), but cultivar means for these variables in Iowa were not significantly correlated with those in New York. Green ash cvs. Bergeson, Dakota Centennial, and Patmore and white ash cv. Autumn Applause were above average in tolerance at both locations. Phytoplasma strains at each location varied significantly in aggressiveness as indicated by host growth suppression.

Condition of Green Ash, Incidence of Ash Yellows Phytoplasmas, and Their Association in the Great Plains and Rocky Mountain Regions of North America.

About 50% of 1,057 green ash (Fraxinus pennsylvanica) systematically sampled in the Great Plains and Rocky Mountain regions had substantial dieback (>10% of crown branches with dieback), and the average growth ring width during the last 20 years was 2.9 mm. The overall condition of the population was rated fair. Ash yellows phytoplasmas were identified at 102 of 106 sites throughout six U.S. states (North Dakota, South Dakota, Wyoming, Nebraska, Colorado, Kansas) and three Canadian provinces (Alberta, Saskatchewan, Manitoba). These phytoplasmas had not previously been known in Alberta, Saskatchewan, Manitoba, Wyoming, Colorado, or Kansas. Incidence of phytoplasmal detection ranged from 16% in Wyoming to 71% in South Dakota. Incidence varied in the range 41 to 67% across site types and crown dieback classes. Incidence was highest in rural plantings, in trees with the most crown dieback, and in larger diameter trees. No significant relationships were detected between presence of ash yellows phytoplasmas and radial growth rates of trees.

Variation in Aggressiveness of Ash Yellows Phytoplasmas

Twelve strains of phytoplasmas belonging to the ash yellows (AshY) group, from across the known range of AshY and representing six host species, were assessed for differences in ability to suppress growth and cause chlorosis in graft-inoculated Fraxinus pennsylvanica (green ash) and Catharanthus roseus (periwinkle). In each of two experiments with ash and one with periwinkle, different strains caused significantly different degrees of growth suppression and loss of foliar greenness. These growth and color impacts were positively and significantly correlated among experiments and between ash and periwinkle, indicating strain variation in aggressiveness. After two strains that differed in aggressiveness were coinoculated to periwinkle plants, polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) assays of DNA from leaves remote from the inoculation sites revealed the presence of the aggressive strain sooner and more frequently than that of the less aggressive strain. Thus, aggressiveness was associated with more rapid multiplication and/or movement than was achieved by the less aggressive strain. When either strain was inoculated 11 weeks before the other into the same plant, only the initial strain could be detected after a further 12 weeks of incubation. Thus, the initial strain or its effect on the host may have interfered with multiplication and/or long-distance movement of the second strain. A concept of preemptive dominance is proposed to account for detection by primary PCR of only single phytoplasma strains in plants that may harbor two or more strains.

Effect of phytoplasmal infection on concentration and translocation of carbohydrates and amino acids in periwinkle and tobacco

Source and sink tissue of Catharanthus roseus (periwinkle) infected with the grapevine yellows (GY), apple proliferation (AP), or ash yellows (ASHY) phytoplasma and of Nicotiana tabacum (tobacco) infected with the AP phytoplasma, were examined for concentrations of glucose, fructose, sucrose, starch and total amino acids. Phytoplasmal infection caused growth inhibition and severe yellowing in both plants, the GY phytoplasma being more virulent than the AP and ASHY agents in periwinkle. Leaf size in periwinkle was greatly reduced by the ASHY phytoplasma with little or no effect caused by either the AP or GY agents. Both phytoplasma strain and host plant had an influence on the observed results. In source leaves of periwinkle and tobacco, infection mostly led to a marked increase in soluble carbohydrates and starch. Increase was smallest in periwinkle affected by ASHY. During a dark period of 42 h, carbohydrate levels tended to decrease in healthy plants whereas they remained high in diseased plants. In sink leaves of periwinkle, infections by the GY and AP phytoplasmas led to a marked decrease in sugar whereas starch levels were either only slightly or not affected at all. Infection by the ASHY phytoplasma led to a smaller decrease of sugars and an increase in starch. In tobacco sink leaves, sugars remained largely unchanged whereas starch increased after infection with the AP phytoplasma. In periwinkle roots, soluble carbohydrates were not markedly altered but starch was considerably reduced, in the GY and AP phytoplasmas in particular. In tobacco roots, there was a general decrease in soluble carbohydrates and starch following infection by the AP phytoplasma. The amino acid concentrations were not altered by infection, except that source and sink leaves of ASHY-infected periwinkles and source leaves of AP-infected tobacco showed higher levels of amino acids than healthy plants. The results are discussed with respect to the effect of phytoplasmas on phloem transport, source leaf phenotype and sink growth.

Heritable tolerance of ash yellows phytoplasmas in green ash

Heritable tolerance of ash yellows phytoplasmas in green ash

Heritable tolerance of ash yellows phytoplasmas in green ash

Variation in response of green ash (Fraxinus pennsylvanica Marsh.) to infection by ash yellows (AshY) phytoplasmas was assessed. Twenty seedlings of each of 35 half-sib families from 15 sources (37n50∞N, 70n98∞W) were planted in pairs (two-tree plots) in a randomized complete block design at Ithaca, N.Y. One plant of each pair was inoculated at age 2 years, and some of these plants again at age 3 years, with New York strains of AshY phytoplasmas by bark-patch grafting. DAPI tests revealed 99% of inoculated trees and 2% of controls were diseased 4 years after first inoculation. Incidence of dieback and mortality associated with winter damage and transplanting shock was significantly higher in diseased trees than in noninfected controls. Foliage of many diseased trees appeared lighter green than foliage of noninfected controls. Foliar color, measured with a Minolta SPAD-502 chlorophyll meter 4 years after first inoculation, varied significantly (p < 0.005) between diseased trees and controls, among families, and in response to family × treatment interaction. Analyses 5 years after first inoculation revealed significant (p < 0.01) phytoplasma-associated suppression of growth in height, stem diameter, and volume index (diam. 2 × height × 10 n2 ). Average increase in volume index of diseased trees in years 4 + 5, as a proportion of the increase in noninfected controls, was 0.44 and varied among families from 0.13 to 0.84. Family × treatment interaction on volume growth was significant (p < 0.001). Heritability of volume growth response to phytoplasmal infection was 0.60 ± 0.17 (mean ± SE) for families and 0.52 for individual trees. These findings indicate potential for selection or breeding of AshY-tolerant green ash.