Big Bud Research Articles

Comparison and Characterization of Tomato Big Bud‐ and Sweet Potato Little Leaf‐Group Phytoplasmas

Abstract EcoRI or HindIII‐restricted DNA from phytoplasmas associated with tomato big bud (TBB) and sweet potato little leaf (SPLL‐V4) was cloned into pBluescript and the recombinant plasmids were screened by reciprocal DNA hybridization with TBB‐ and SPLL‐V4 DNA. Most of the recombinant plasmids hybridized to DNA of both phytoplasmas. Southern hybridization analysis of EcoRI‐ or HindIII‐restricted TBB‐ and SPLL‐V4 DNA with two TBB probes (pTBB32 and pTBB87) gave identical restriction patterns. Southern hybridizations of HindIII‐ or EcoRI digested DNA from 10 phytoplasma‐positive field samples revealed a different restriction fragment pattern in two phytoplasmas from sweet potato. Three TBB probes which did not hybridize to SPLL‐V4 were selected for Southern blot analysis of samples. The pTBB42 and pTBB78 hybridized to nine of the 10 TBB‐symptomatic field samples producing identical patterns. pTBB88 contained extrachromosomal DNA and hybridized with two phytoplasmas from diseased sweet potato and perennial phlox. The SPLL‐V4‐specific probe pH 21 hybridized to five field samples generating different restriction patterns. About 80 field samples were subjected to the polymerase chain reaction using primers deduced from pH 21, pTBB42 and pTBB88. These phytoplasmas had been previously characterized and grouped by their 16S/23S spacer region restriction fragment pattern but this classification was in most cases different to the results obtained with these primers.

Phylogenetic positions of phytoplasmas associated with dieback, yellow crinkle and mosaic diseases of papaya, and their proposed inclusion in 'Candidatus Phytoplasma australiense' and a new taxon, 'Candidatus Phytoplasma australasia'.

DNA extracted from three papaya (Carica papaya L.) plants, individually affected by dieback, yellow crinkle or mosaic diseases, was subjected to PCR using phytoplasma-specific primers to amplify the 16S rRNA gene plus 16S-23S rRNA intergenic spacer region. Near-complete DNA sequences obtained for the three PCR amplimers were subjected to phylogenetic analyses and direct sequence comparison with other phytoplasma 16S rDNA and 16S-23S spacer region DNA sequences. The papaya yellow crinkle (PpYC) and papaya mosaic (PpM) sequences were identical to each other, but distinctly different from the papaya dieback (PpDB) sequence, showing 90.3% identity in the he 16S rDNA and 87.8% identity in the 16S-23S spacer region DNA sequences. A phylogenetic tree based on 16S rDNA sequences was calculated, in which PpYC and PpM are most closely related to the tomato big bud phytoplasma (TBB; 99.7% 16S rDNA sequence identity) from Australia, within subclade iii. This subclade consists of strains only reported occurring in the Southern Asian region and Australia, which indicates an Asian/Australasian origin. PpDB is most closely related to the Phormium yellow leaf phytoplasma from new Zealand (PYL; 99.9% identity) and the Australian grapevine yellows phytoplasma (AGY; 99.7% identity). These three phytoplasma strains form a distinct clade within subclade xii, which also includes the European strains STOL and VK as another distinct clade. The origin of the closely related but geographically separated AGY-like strains and STOL-like strains of subclade xii is unclear. It is proposed that phytoplasma strains PpDB, PYL and AGY be included in the previously described taxon 'Candidatus Phytoplasma australiense', and that PbYC, PpM and TBB be assigned to a new taxon, "Candidatus Phytoplasma australasia'.

MORPHOLOGY, ONTOGENY, AND INTRASPECIFIC VARIABILITY OF THE YEW BIG BUD MITE,CECIDOPHYOPSIS PSILASPIS(ACARI: ERIOPHYIDAE)

AbstractAll ontogenetic stases of the yew big bud mite,Cecidophyopsis psilaspis(Nalepa), from British Columbia were studied by scanning electron and light microscopy and were compared with an English population of this species. The species was redescribed from the two populations, which were found to be quite similar in general appearance and in most of the morphometric data analyzed. Spermatophores in this species are similar to those observed in other Eriophyoidea. Details of a male spermatophoric organ are described and illustrated for the first time in an eriophyoid mite. Moulting inC.psilaspisis shown to be prodehiscent. The data reported here neither support nor reject the alternative hypothesis that the first active stase is larval versus nymphal. However, to account for the ontogenetic evidence, the first active stase is herein considered larval.

Use of Heteroduplex Mobility Analysis (HMA) for Differentiating Phytoplasma Isolates Causing Witches’ Broom Disease on Populus nigra cv Italica and Stolbur or Big Bud Symptoms on Tomato

AbstractHeteroduplex mobility analysis (HMA) was performed to distinguish French and German isolates of phytoplasmas from Populus nigra cv Italica witches broom. The French isolate was similar to the phytoplasma responsible for the European aster yellows while the German isolate was different but closely related to it. When phytoplasmas inducing similar “stolbur” symptoms in tomato were compared to HMA, a high degree of genetic differences was observed among the reference stolbur C (StC) isolate, the European aster yellows and the other phytoplasmas inducing stolbur or big bud symptoms in tomato. Pseudo‐stolbur B and D from Brazil were differentiated from the reference St C using this method.

Phytoplasma associated with virescence in an epiphytic orchid in Australia

The orchid Sarcochilus hartmanii × Sarcochilus falcatus showing flower virescence with some evidence of stunting was found during an orchid virus survey of south-east Queensland. Phytoplasma bodies were detected by thinsection electron microscopy and phytoplasma DNA was detected using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis was used to compare this phytoplasma with those found in dicotyledonous plants. RFLP analysis demonstrated that this orchid phytoplasma was indistinguishable from the tomato big bud phytoplasma.

ORIGIN AND DISTRIBUTION OF THE YEW BIG BUD MITE,CECIDOPHYOPSIS PSILASPIS(NALEPA), IN BRITISH COLUMBIA

AbstractThe distribution of the yew big bud mite,Cecidophyopsis psilaspis(Nalepa), a pest causing severe damage to Pacific yew,Taxus brevifoliaNutt., was determined within British Columbia. From 1992 to 1994, vegetative and reproductive buds on branch samples collected from 38 interior and 43 coastal populations of Pacific yew were examined for the occurrence of the mite. Mite damage was characterized by marked swelling and browning of both vegetative and reproductive buds. Interior Pacific yew populations were found to be mite free, whereas coastal yew populations were mite infested, except at three high-elevation (>700 m) coastal locations which were apparently mite free. At three other sites, individual trees without symptoms of mite attack did harbour low-density, nondamaging mite populations. The hypothesis that this mite is indigenous is less supported than the hypothesis that it was introduced to British Columbia on English yew,Taxus baccataL., and is limited to its present distribution by climatic and mountain barriers.

Detection and Differentiation of Phytoplasmas Associated with Diseases of Strawberry in Florida.

Strawberry (Fragaria × ananassa Duchesne) plants with symptoms suggestive of phytoplasmal disease were identified in commercial fields and a breeder's plot in west central Florida during the 1995 to 1996 winter growing season. Affected plants were all conspicuously stunted and unproductive. Primary symptoms on cvs. Rosa Linda and Carlsbad and on a breeder's accession resembled those of strawberry green petal (SGP). Plants displayed sparse clusters of virescent flowers with enlarged sepals and phylloid receptacles that failed to develop fully into fleshy structures or redden on ripening. Symptoms on cv. Oso Grande were more typical of multiplier disease and included a proliferation of branch crowns producing numerous small leaves with spindly petioles. Oso Grande and Carlsbad originated as transplants from a nursery in Montreal, Canada, whereas Rosa Linda transplants were from Nova Scotia. Plants were assessed for phytoplasma infection by polymerase chain reaction with total DNAs from leaves and petioles as template and phytoplasma-specific ribosomal RNA primers P1 and P7 (3), or mollicute-specific ribosomal protein (rp) gene primers rpF1 and rpR4 (2). Amplification of a 1.8-kb rDNA or 1.2-kb rp gene product, respectively, confirmed infection of Rosa Linda (7 of 7 plants), Carlsbad (3 of 7), Oso Grande (4 of 4), and a single breeder's accession. No products were amplified from DNAs of healthy plants. Restriction fragment length polymorphism patterns of rDNA digested with AluI, EcoRI, HaeIII, HhaI, HpaII, KpnI, ScaI, or Tru9I endonucleases, or of rp gene products digested with AluI, DraI, RsaI, TaqI, or Tru9I, revealed no differences among phytoplasma strains affecting both Rosa Linda and Carlsbad. Collectively, patterns were comparable to those of clover phyllody and SGP phytoplasmas, two Canadian strains previously classified as members of phytoplasma 16S rRNA (rr)-ribosomal protein (rp) group 16S rI, subgroup C (16S rI-C (rr-rp)) (1). Similarly, no differences were evident among phytoplasmas associated with all four diseased Oso Grande plants. Both rDNA and rp fragment profiles associated with this cultivar were characteristic of strains such as tomato big bud and eastern aster yellows delineated as 16S rI-A (rr-rp) subgroup members (1). However, AluI rDNA and TaqI rp fragment patterns were unique, identifying Oso Grande-infecting strains as representatives of a new subgroup within the larger 16S rI (rr-rp) group. Cumulative rDNA and rp fragment profiles of the phytoplasma associated with the breeder's accession matched those of the Mexican periwinkle virescence phytoplasma, identifying this strain as a 16S rI-I (rr-rp) subgroup member (1) and a second possible etiological agent of SGP. This is the first report of phytoplasmas infecting strawberry in Florida.

Cecidophyopsis ribis . [Distribution map

Abstract A new distribution map is provided for Cecidophyopsis ribis (Westwood). Acarina: Eriophyidae (black currant gall mite, black currant big bud mite, currant bud mite). Attacks Ribes spp. especially black currant. Information is given on the geographical distribution in Europe, Austria, Belgium, Bulgaria, Czech republic, Denmark, Finland, France, Germany, Hungary, Malta, Netherlands, Norway, Poland, Slovakia, Spain, Sweden, Switzerland, United Kingdom, Yugoslavia, CIS (formerly USSR), Georgia, Latvia, Lithuania, Ukraine, Russia, Altai area, Karelia, Kursk, Leningrad region, Moscow, Murmansk, Primorskiy Kray, Sakhalin, Siberia, Asia, China, Heilongjiang, Australasia, Australia, Tasmania, New Zealand, North America, Canada, British Columbia.

Pre- and post-embedding immunogold labeling and electron microscopy in plant host tissues of three antigenically unrelated MLOs: primula yellows, tomato big bud and bermudagrass white leaf

Methods are described for pre- and post-embedding immunogold labeling of mycoplasmalike organisms (MLOs) in thin sections of infected plants. Antisera against primula yellows (PY), tomato big bud (TBB) and bermudagrass white leaf (BGWL) MLOs, and a monoclonal antibody (mab) against PY were tested with the three serologically unrelated MLOs. Labeling was specific for each MLO and was localized to the outer surface of the MLOs. The antisera performed well in both pre- and post-embedding experiments; the mab reacted well in pre-embedding conditions but gave no labeling with post-embedding. Glutaraldehyde fixation reduced levels of labeling in post-embedding conditions. The results show that these techniques can be used to differentiate MLOs reliably, and extend the usefulness of electron microscopy in this area.

Anthony David Lees, 27 February 1917 - 3 October 1992

Tony Lees was a third-generation scientist and academic. His paternal grandfather, David Bridge Lees, graduated from Cambridge in classics, divinity and mathematics, but retrained for the medical profession at Guy’s Hospital before joining the staffs of the Great Ormond Street Hospital for Children, and later, St Mary’s. Here he carried out research into rheumatism, pneumonia and tuberculosis. His youngest son, Alan Henry Lees, who became Tony’s father, also went up to Cambridge to read Zoology and Botany. After graduation he joined the advisory service of the Ministry of Agriculture in the Vale of Evesham. In 1912 he was appointed plant pathologist at the newly founded Long Ashton Research Station near Bristol, where he conducted research into the control of the ‘big bud’ mite affecting blackcurrants, and later on apple and pear cultivation. Tony’s mother, Mary Hughes Bomford, came from a large farming family near Evesham. After marriage, the couple settled in Long Ashton where their only child, Tony, was born. His mother died prematurely when Tony was only eight years old.

Immunosorbent electron microscopy and gold label antibody decoration of MLOs from crude preparations of infected plants and vector insects

A simple method is given for immunotrapping, gold labelling and electron microscopy of mycoplasmalike organisms (MLOs) from crude preparations of infected plants or insects, enabling MLOs to be confidently distinguished from host materials in negative stain preparations. The appearance of the MLOs is described. MLOs were trapped from extracts of infected periwinkle plants (Catharanthus roseus) and infected vector leaf hoppers (Macrosteles quadripunctulatus), using electron microscope grids coated with the F(ab)2 portion of specific rabbit IgGs. The MLOs were then decorated with the intact igG and labelled with goat anti‐rabbit IgG conjugated to gold particles 5 or 15 nm in diameter. Preparations were negatively stained in 0.5% ammonium molybdate or left unstained. The MLOs used were European aster yellows (EAY) from plant and vector, and Australian tomato big bud (TBB) from the plant. Preparation time was 4 h; large numbers of MLOs and MLO fragments could be trapped and identified, and EAY could be confidently distinguished from the serologically unrelated TBB. Morphologically, EAY and TBB were indistinguishable, both presenting spheroidal bodies, tubular forms and apparently budding chains of cocci. Bacilliform virus‐like particles were sometimes associated with EAY MLOs, especially those from the vector.

Genetic Differentiation of Phytoplasma Isolates Determined by a DNA Heteroduplex Mobility Assay.

Regions representing about 80% of the 16S rDNA sequences of nine phytoplasma isolates were amplified by polymerase chain reaction (PCR). These partial 16S rDNA sequences amplified from the various phytoplasmas were used in DNA heteroduplex mobility assays (HMA). Based on DNA distances derived from HMA analysis, the nine phytoplasma isolates may be classified into four distinct groups: group I, paulownia witches'-broom (China), potato purple top (France); group II, tomato stolbur (France); group III, eastern aster yellows (U. S.A.), faba bean phyllody (Sudan), vinca phyllody (Sudan), tomato big bud (Australia); group IV, crotalaria witches'-broom (Thailand), clover proliferation (Canada).

Association of mycoplasma‐like organisms with tomato big bud disease in Greece

The big bud disease of tomato is described in Greece for the first time. Its identity and aetiology were ascertained by electron microscopy, fluorescence and bright‐field microscopy, transmission tests and antibiotic treatments. Pleomorphic mycoplasma‐like organisms (MLOs) were detected in the sieve tubes of infected leaf tissues by thin‐section electron microscopy and also by fluorescence and bright‐field microscopy after staining with the DNA‐binding fluorescent stain Hoechst 33258, and with toluidine blue. Infected tomato plants showed remission of symptoms when treated with tetracycline‐HCl, but not with benzylpenicillin, while healthy plants developed big bud symptoms when graft inoculated with diseased tomato shoots. The occurrence of natural spread of big bud disease in Greece indicates that one or more vector species are present in the country.

The Beet Leafhopper-Transmitted Virescence Agent Causes Tomato Big Bud Disease in California

Mycoplasmalike organisms (MLOs) are associated with big bud disease of tomatoes (Lycopersicon esculentum) in many parts of the world. In only a few cases, however, have the MLOs associated with the disease been characterized. We used biological and genetic data to establish that the causal agent of tomato big bud (TBB) disease in California is the beet leafhopper-transmitted virescence agent (BLTVA) MLO. Healthy Circulifer tenellus leafhoppers acquired the BLTVA MLO from field-collected, symptomatic tomato plants and transmitted it to healthy tomato plants, which developed typical big bud symptoms. Healthy tomatoes inoculated with the BLTVA type line (FC-83-13) also developed the floral gigantism and virescence characteristic of the disease (.)

Differentiation of Strains in the Aster Yellows Mycoplasmalike Organism Strain Cluster by Serological Assay with Monoclonal Antibodies

Monoclonal antibodies (MAbs) raised against the tomato big bud (BB) mycoplasmalike organism (MLO), a member of the aster yellows (AY) MLO strain cluster, were employed in dot immunobinding assays. The MAbs reacted only with strains in the AY MLO cluster, and not with any of several other MLOs not affiliated with the AY MLO strain cluster. However, reactions with MLOs in the AY cluster varied by strain. All MAbs reacted with BB and several MLO strains previously termed «aster yellows,» including NAY (eastern AY), OKAY1 (Oklahoma strain), NJAY (New Jersey strain), and AY27 (Alberta strain); but none of the MAbs reacted with certain other strains of AY MLO, including strains termed SAYS (western AY), OKAYS (Oklahoma strain), NYAY (New York strain), and MNAY (Minnesota strain)

Sensitive detection and identification of mycoplasma-like organisms in plants by polymerase chain reactions

DNA amplification by polymerase chain reactions (PCR) was employed to detect host plant infection by several mycoplasmalike organisms (MLOs), including the aster yellows (AY), dwarf aster yellows (DAY), and periwinkle little leaf (0-1) MLOs. For PCR, two pairs of oligonucleotide primers, designated AY18pm and AY19pm, respectively, were synthesized on the basis of partial sequences of cloned AY MLO DNA fragments AY18 and AY19. Reaction mixtures containing primer pair AY18pm yielded a DNA product of 1.6Kbp, when template consisted of DNA extracted from AY MLO- or DAY MLO-infected Catharanthus roseus (periwinkle). A DNA product of 1.0Kbp was obtained with primer pair AY19pm, when template consisted of DNA extracted from C. roseus infected by AY MLO, DAY MLO, or periwinkle little leaf (strain O-1) MLO. MLO-specific bands were observed when reaction mixtures contained as little as 5 pg total nucleic acid from infected plants. No PCR product was observed when reaction mixtures contained only DNA from healthy plants or DNA from plants infected by western X MLO or by tomato big bud MLO. The findings indicated that the PCR system is useful for sensitive detection and differentiation of MLOs in infected hosts.

Mycoplasma-like Organisms Associated with Proliferation and Big Bud of Solanum lycocarpum St. Hil.

Mycoplasma-like organisms (MLOs) were found in sieve tubes of Solanum lycocarpum affected with prolferation and big bud. The MLOs were graft-transmitted to tomato plants which developed similar symptoms. The association of MLOs with affected but not with symptomless S. lycocarpum and tomato suggests that the abnormality is induced by MLOs. The possible role of S. lycocarpum as a reservoir of a strain of the tomato big bud is discussed.

A Genotype-Based System for Identification and Classification of Mycoplasmalike Organisms (MLOs) in the Aster Yellows MLO Strain Cluster

Fifteen mycoplasmalike organism (MLO) strains from North America and Europe, including aster yellows (AY), tomato big bud (BB), clover phyllody (CPh), chrysanthemum yellows (CY), and unknown MLOs, were studied. These MLOs are among those previously identified, on the basis of dot hybridizations, as members of the AY MLO strain cluster. Restriction fragment length polymorphism (RFLP) analyses revealed that all 15 strains can be classified into three genomic types: type I (typified by eastern AY MLOs), type II (e.g., western AY and CY MLOs), and type III (e.g., CPh MLO) (...)

Apparent Immunity and Tolerance to Tomato Big Bud Disease inLycopersicon peruvianumand in Two of Its Tomato Hybrids

Apparent Immunity and Tolerance to Tomato Big Bud Disease in<i>Lycopersicon peruvianum</i>and in Two of Its Tomato Hybrids

Genetic Interrelatedness among Clover Proliferation Mycoplasmalike Organisms (MLOs) and Other MLOs Investigated by Nucleic Acid Hybridization and Restriction Fragment Length Polymorphism Analyses

DNA was isolated from clover proliferation (CP) mycoplasmalike organism (MLO)-diseased periwinkle plants (Catharanthus roseus (L.) G. Don.) and cloned into pSP6 plasmid vectors. CP MLO-specific recombinant DNA clones were biotin labeled and used as probes in dot hybridization and restriction fragment length polymorphism analyses to study the genetic interrelatedness among CP MLO and other MLOs, including potato witches'-broom (PWB) MLO. Results from dot hybridization analyses indicated that both a Maryland strain of aster yellows and a California strain of aster yellows are distantly related to CP MLO. Elm yellows, paulownia witches'-broom, peanut witches'-broom, loofah witches'-broom, and sweet potato witches'-broom may be very distantly related, if at all, to CP MLO. A new Jersey strain of aster yellows MLO, tomato big bud MLO, clover phyllody MLO, beet leafhopper-transmitted virescence MLO, and ash yellows MLO are related to CP MLO, but PWB MLO is the most closely related. Similarity coefficients derived from restriction fragment length polymorphism analyses revealed that PWB and CP MLOs are closely related strains and thus provided direct evidence of their relatedness in contrast to reliance solely on biological characterization.