Studies on Taxonomic Relationships of Mycoplasma–like Organisms by Southern blot Analysis

Chromosomal DNA fragments from the mycoplasma-like organisms (MLOs) associated with American aster yellows, apple proliferation, clover phyllody, and vaccinium witches' broom were cloned. Several MLO-specific fragments from each of these four isolates and a sequence from the 16S rRNA gene of an aster yellows MLO were used in Southern blot hybridizations to investigate the taxonomic relationships of 26 pathologically and geographically diverse MLOs. These MLOs were divided into four categories according to the symptoms induced in periwinkle. Genotypically, these isolates represented four groups (16S RFLP groups) of a classification based on restriction fragment length polymorphisms (RFLP) and sequencing data of the 16S rRNA gene. Probes from three isolates of one symptom category hybridized with isolates from all symptom categories. This result indicates that classification of MLOs by symptomatology does often not coincide with genetic relationships. The hybridization results confirmed the findings, of the 16S RFLP classification that most MLOs from herbaceous plants, especially those inducing virescence in periwinkle, are interrelated. These isolates, which were assigned to one 16S RFLP group, could be further differentiated in this study. Itcould be shown that aster yellows, clover phyllody, stolbur, and safflower phyllody and sandal spike are caused by distinct MLOs. The MLOs associated with apple proliferation, vaccinium witches' broom, and witches' broom of lime as well as two isolatesfrom, stone fruits could also be recognized as distinct organisms.     

Production and characteristics of antisera to Spiroplasma citri and clover phyllody-associated antigens derived from plants

Antisera were produced to clover phyllody- and Spiroplasma citri-associated antigens partially purified from infected Vinca rosea plants. Separate antisera were made to ‘membrane fraction’ (MF) preparations comprising the resuspended pellet obtained by high speed centrifugation, and to ‘soluble fraction’ (SA) preparations, comprising the supernatant from high speed centrifugation concentrated by freeze-drying. All antisera showed considerable activity against normal plant antigens but after cross-absorption with extracts of healthy plants the MF antisera were used in F(ab')₂based ELISA tests to detect S. citri- or clover phyllody-associated antigens in infected plants. The ‘clover phyllody’ antiserum also reacted specifically with extracts of clover plants with phyllody, and with naturally-infected strawberry plants showing symptoms of green petal disease. Both the ‘clover phyllody’ and S. citri antisera were specific for their respective homologous antigens. No cross-reactions were observed in heterologous tests or between either antiserum and extracts of V. rosea infected with various MLOs obtained from different host plants.     

Resolution of strawberry virus complexes; the isolation and some properties of virus 3

Aphids ( Capitophorus fragariae Theob.) allowed to feed for several days on a strawberry plant with severe crinkle transmitted two viruses. The isolation and properties of one (virus I) have already been described. The other (virus 3) was separated by transferring the aphids to fresh indicators after 24 hr.
Virus 3 was transmitted by aphids which had been allowed to feed on an infected plant for 6 days or more and persisted in the vector for several days. There was some evidence that the virus has a latent period in the vector. The symptoms produced by virus 3 on Fragaria vesca and Royal Sovereign strawberry are described.
On Royal Sovereign, viruses 1 and 3 together produced symptoms of severe crinkle and viruses z and 3 together produced yellow-edge. A form of severe crinkle is thus shown to be caused by a virus complex which can be resolved by means of the vector, and severe crinkle is shown to be etiologically distinct from mild crinkle.     

Nonradioactive Screening Method for Isolation of Disease-Specific Probes To Diagnose Plant Diseases Caused by Mycoplasmalike Organisms

DNA fragments of tomato big bud (BB) mycoplasmalike organism (MLO) in diseased periwinkle plants (Catharanthus roseus L.) were cloned to pSP6 plasmid vectors and amplified in Escherichia coli JM83. A nonradioactive method was developed and used to screen for MLO-specific recombinants. Cloned DNA probes were prepared by nick translation of the MLO recombinant plasmids by using biotinylated nucleotides. The probes all hybridized with nucleic acid from BB MLO-infected, but not healthy, plants. Results from dot hybridization analyses indicated that several MLOs, e.g., those of Italian tomato big bud, periwinkle little leaf, and clover phyllody, are closely related to BB MLO. The Maryland strain of aster yellows and maize bushy stunt MLOs are also related to BB MLO. Among the remaining MLOs used in this study, Vinca virescence and elm yellows MLOs may be very distantly related, if at all, to BB MLO. Potato witches' broom, clover proliferation, ash yellows, western X, and Canada X MLOs are distantly related to BB MLO. Southern hybridization analyses revealed that BB MLO contains extrachromosomal DNA that shares sequence homologies with extrachromosomal DNAs from aster yellows and periwinkle little leaf MLOs.     

Preliminary studies on sap-transmissible viruses of red clover (Trifolium pratense L.) in England and Wales

Red clover plants, collected from nine widely separated permanent pastures in England and Wales, were tested for sap-transmissible viruses. Viruses were identified by the symptoms they caused in test plants, by electron microscopy, and by serological tests. Of the 265 plants tested 14% were infected. Only pea mosaic virus was common and widespread; it was found in 8% of the plants, and in seven of the fields. Other viruses isolated were arabis mosaic, bean yellow mosaic, red clover mottle, and red clover vein mosaic; only red clover mottle virus produced diagnostic symptoms in red clover. No viruses were detected in seedlings grown from seed from eighty-nine commercial seed crops. Attempts to transmit red clover mottle virus by the Collembolan Sminthurus viridis L., which is common on red clover, failed.     

Development of Cloned DNA Probes for Phytoplasma Associated with Loofah Witches' Broom

DNA was isolated from periwinkle ( Catharanihus roseus ) infected with a phytoplasma that originated in loofah witches' broom affected by loofah. Cloned DNA inserts from six LfWB-phytoplasma specific recombinant plasmids were not only labelled with digoxigenin, but also used as probes. Probes hybridized with DNA derived from LfWB-phytoplasma affected periwinkle and loofah, but not with DNA from healthy plants or plants infected with phytoplasmas associated with elm yellows, red bird cactus, peanut witches' broom, paulownia witches' broom, Ipomoea obscura witches' broom, aster yellows (two isolates), and sweet potato witches' broom obtained with DNA from different phytoplasmas experimentally maintained in periwinkle. The probes could detect LfWB-phytoplasma DNA with as little as 16 ng and 32 ng of DNA from periwinkle and loofah, respectively. The method proposed herein provides a means for specifically detecting and identifying of loofah witches' broom phytoplasma, as well as confirming the notion that this phytoplasma represents a distinct strain cluster,     

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.     

The Genetic Relationship Between Mycoplasma-like Organisms Causing Diseases in the Sudan and Different Continents Revealed by Polymerase Chain Reaction (PCR) Amplification of the 16S rRNA Gene Followed by Restriction Fragment Length Polymorphism Analysis

The genetic relationship between faba bean (Vicia faba L.) phyllody and other mycoplasma-like organism (MLO) diseases has been studied by amplification of the conserved region of the 16S rRNA gene followed by restriction fragment length polymorphism (RFLP) analysis using Alu I restriction endonuclease. The restriction patterns produced by faba bean phyllody MLO were smilar to that of Crotalaria saltiana phyllody MLO which persists throughout the year in the Sudan. These, and serological results clearly confirmed that C. saltiana is a reservoir of faba bean phyllody MLO in the Sudan. Moreover, restriction patterns have also shown that MLOs of other diseases have the same RFLP fragment pattern as faba bean phyllody MLO, including C. juncea witches'broom (Thailand) and tomato big-bud (Australia), which differs from the other selected MLO diseases (Gladiolus aster yellow, clover phyllody and yellow decline of lavender, aqll from France). Fragment patterns also revealed the existence of genetically diverse MLO strains in the Sudan. Faba bean phyllody may be placed in group III including WX, apricot chlorotic leaf roll, golden flaveswcence dorée of grapevine, plum leptonecrosis of Prunus salciana, peachy yellow leaf roll, sunnhemp phyllody from Thailand, and blueberry witches' broom.     

Phyllody Disease of Crotalaria saltiana in the Sudan: Transmission to Catharanthus roseus and Detection of MLOs by Fluorescence and Electron Microscopy

Phyllody disease of Crotalaria saltiana Andr. first noted in the Sudan in 1962, was recently observed in many localities in the Gezira province in the central region of the country. Diseased plants generally exhibited stunting and excessive proliteration of lateral shoots (witches' broom growth) with small and chlorotic leaves. Morphological transformations of flowers were the most striking symptoms. Floral segments showed various stages of virescence and phyllody as a part of a complete transformation of floral buds into leafy branches. The Crotalaria phyllody agent was transmitted by grafting to faba bean (Vicia faba L.) and with dodder from the latter to periwinkle (Catharanthus roseus). The symptoms reproduced in C. roseus resembled those induced in it by the faba bean phyllody MLO (mycoplasma-like organism), suggesting a close relationship between the two agents. Fluorescence and electron microscopy were used to detect and characterize MLO in diseased plants. Fluorescence reactions in sieve tube elements were observed in sections stained with the DNA-binding fluorochrome Bisbenzimid H 33258. Electron microscope observations in corresponding zones permitted the visualization of wall-less pleiomorphic MLOs confined to sieve tube elements of the phloem tissues of diseased plants.     

Molecular Cloning, Detection of Chromosomal DNA of the Mycoplasmalike Organism (MLO) Associated with Faba Bean (Vicia faba L.) Phyllody by Southern Blot Hybridization and the Polymerase Chain Reaction (PCR)

DNA from faba bean (Vicia faba L.) phyllody-diseased periwinle plants was separated from host plant DNA by bisbenzimid-CsCl buoyant-density gradient centrifugation. The mycoplasmalike organism (MLO) DNA was used for the construction of DNA probes. Two probes, 1.45 and 1.35 kbp, were selected and used for the detection of MLO DNA associated with faba pean (FBP) and for assessing the genetic relatedness of FBP-MLO with other mollicutes. The 1.45 kbp DNA probe hybridized with all MLO strains and, with Spiroplasma citri. The 1.35 kbp DNA probe specifically detected the MLO associated with FBP. Moreover, a specific primer pair (E1 and E2) selected from the partially sequenced 1.35 kbp probe allowed amplification of the 1.35 kbp fragment. DNA amplification was obtained also with Crotaltiana saltiana phyllody (Sudan), C. juncea, witches' broom (Thailand), and tomato big-bud (Australia), but no amplification was obtained in the cases of the healthy control, C. roseus phyllody (isolate n⁰) from Sudan, clover phyllody, Gladiolus aster yellow and yellow decline of lavender from France. The very strong signal observed in the case of FBP and C. saltiana phyllody agrees with previous results indicating that FBP and C. saltiana phyllody are caused by an identical MLO, and hence, C. saltiana acts as a reservoir of FBP-MLO in the Sudan. The weak signal obtained in the case of C. juncea witches' broom and tomáto big-bud indicates partial nucleotide homology. The major interest of this primer pair is the low quantity (as little as 100 pg) of the total DNA of diseased plant required for the detection of the FBP disease and the possibility of detecting genetic relatedness with other MLOs.     

The elimination of clover diseases by shoot tip culture

Shoot tip culture was used to eliminate white clover mosaic virus (WCMV) and red clover necrotic mosaic virus (RCNMV) from red clover, and clover phyllody disease (CP) and clover red leaf disease (CRL) from white clover. Shoot tips up to 2.4 mm (in some cases 3 mm) could regenerate plants free from the pathogens, but the efficiency of elimination, at least for WCMV and CRL, tended to decrease with increasing shoot tip size. The efficiency of plant regeneration from shoot tips generally improved with increasing tip size.     

Resolution of Strawberry Virus Complexes

Aphids ( Capitophorus fragariae Theob.) allowed to feed for several days on a strawberry plant infected with yellow-edge transmitted two virus fractions. The isolation and properties of one (virus 1) have been described previously. The other (virus 2) was separated by transferring the aphids to fresh indicators after 24 hr.
Virus 2 was retransmitted after infection feeding periods of 24 hr. or more and persisted in the vector for several days. There is some evidence that it is itself a complex of viruses which can be separated further. On Fragaria vesca virus 2 produced chlorotic spotting, slight marginal chlorosis of the leaves and slight cupping of the leaflets. On Royal Sovereign strawberry it produced slight chlorosis of the young leaves.
On Royal Sovereign viruses 1 and 2 together produced symptoms of yellow-edge which is thus shown to be caused by a virus complex which can be resolved by means of the aphis vector.     

Phytoplasmas: bacteria that manipulate plants and insects

TAXONOMY: Superkingdom Prokaryota; Kingdom Monera; Domain Bacteria; Phylum Firmicutes (low-G+C, Gram-positive eubacteria); Class Mollicutes; Candidatus (Ca.) genus Phytoplasma. HOST RANGE: Ca. Phytoplasma comprises approximately 30 distinct clades based on 16S rRNA gene sequence analyses of approximately 200 phytoplasmas. Phytoplasmas are mostly dependent on insect transmission for their spread and survival. The phytoplasma life cycle involves replication in insects and plants. They infect the insect but are phloem-limited in plants. Members of Ca. Phytoplasma asteris (16SrI group phytoplasmas) are found in 80 monocot and dicot plant species in most parts of the world. Experimentally, they can be transmitted by approximately 30, frequently polyphagous insect species, to 200 diverse plant species. DISEASE SYMPTOMS: In plants, phytoplasmas induce symptoms that suggest interference with plant development. Typical symptoms include: witches' broom (clustering of branches) of developing tissues; phyllody (retrograde metamorphosis of the floral organs to the condition of leaves); virescence (green coloration of non-green flower parts); bolting (growth of elongated stalks); formation of bunchy fibrous secondary roots; reddening of leaves and stems; generalized yellowing, decline and stunting of plants; and phloem necrosis. Phytoplasmas can be pathogenic to some insect hosts, but generally do not negatively affect the fitness of their major insect vector(s). In fact, phytoplasmas can increase fecundity and survival of insect vectors, and may influence flight behaviour and plant host preference of their insect hosts. DISEASE CONTROL: The most common practices are the spraying of various insecticides to control insect vectors, and removal of symptomatic plants. Phytoplasma-resistant cultivars are not available for the vast majority of affected crops.     

The isolation and identification of rhubarb viruses occurring in Britain

Virus-like symptoms were common in British crops of rhubarb. All plants tested of the three main varieties, ‘Timperley Early’, ‘Prince Albert’ and ‘Victoria’, were virus-infected. Turnip mosaic virus and a severe isolate of arabis mosaic virus (AMV) were obtained from ‘Timperley Early’; and ‘Prince Albert’ contained turnip mosaic virus, cherry leaf roll virus (CLRV), a mild isolate of AMV and, infrequently, cucumber mosaic virus (CMV). The main commercial variety ‘Victoria’ contained turnip mosaic virus, CLRV, a mild isolate of AMV and, infrequently, strawberry latent ringspot virus (SLRV). All the viruses were identified serologically. The rhubarb isolates did not differ markedly from other isolates of these viruses in herbaceous host reactions, properties in vitro or particle size and shape. A rhubarb isolate of CLRV was distinguished serologically from a cherry isolate of the virus. Turnip mosaic virus, CLRV and SLRV, were transmitted with difficulty, but AMV isolates were readily transmitted by mechanical inoculation. Turnip mosaic virus was also transmitted to rhubarb by Myzus persicae and Aphis fabae. CLRV was transmitted in 6–8% of the seed of infected ‘Prince Albert’ and ‘Victoria’ rhubarb and in 72% of the seed of infected Chenopodium amaranticolor. Mild isolates of AMV were also transmitted in 10–24% of the seed of infected ‘Prince Albert’ and ‘Victoria’ plants.     

Dodder Transmission of Pear Decline, European Stone Fruit Yellows, Rubus Stunt, Picris echioides Yellows and Cotton Phyllody Phytoplasmas to Periwinkle

The pear decline, European stone fruit yellows and rubus stunt agents as well as the phytoplasmas causing Picris echioides (bristly oxtongue) yellows and cotton (Gossypium hirsutum) phyllody, respectively, were transmitted from naturally infected plants to the experimental host Catharanthus roseus (periwinkle) via dodder (Cuscuta spp.) bridges. The identities of the dodder-transmitted phytoplasmas were confirmed by restriction length fragment polymorphism analysis of polymerase chain reaction-amplified ribosomal DNA. On the basis of restriction profiles the cotton phyllody agent could be differentiated from the phytoplasma causing faba bean phyllody, a disease previously thought to be induced by the same organism as cotton phyllody.     

Occurrence and Characterization of a 16SrII‐D Subgroup Phytoplasma Associated with Parsley Witches’ Broom Disease in Iran

During 2010–2013 surveys for the presence of phytoplasma diseases in Yazd province (Iran), a parsley witches’ broom (PrWB) disease was observed. Characteristic symptoms were excessive development of short spindly shoots from crown buds, little leaf, yellowing, witches’ broom, stunting, flower virescence and phyllody. The disease causative agent was dodder transmitted from symptomatic parsley to periwinkle and from periwinkle to periwinkle by grafting inducing phytoplasma‐type symptoms. Expected length DNA fragments of nearly 1800 and 1250 bp were, respectively, amplified from naturally infected parsley and experimentally inoculated periwinkle plants in direct polymerase chain reaction (PCR) using phytoplasma primer pair P1/P7 or nested PCR using the same primer pair followed by R16F2n/R16R2 primers. Restriction fragment length polymorphism and phylogenetic analyses of 16S rRNA gene sequences showed that the phytoplasma associated with PrWB disease in Yazd province belong to 16SrII‐D phytoplasma subgroup. This is the first report of association of a 16SrII‐related phytoplasma with PrWB disease in Iran.     

Identification,occurrence, incidence and transmission of phytoplasma associated with Petunia violacea witches’ broom in Iran

A petunia witches’ broom (PvWB) disease, characterized by phyllody, virescence, witches’ broom, little leaf and yellowing, was observed in municipal lands and parks in Bandar Abbas, Hormozgan province, Iran. The disease was present with an average incidence of 20%. PCR and sequencing analysis carried out on selected samples from symptomatic plants showed the presence of a phytoplasma associated with the disease. The molecular comparison of the 16S ribosomal gene indicated 99% sequence identity with the one of “Candidatus Phytoplasma australasia”. This phytoplasma was transmitted to healthy petunia plants under experimental conditions by the leafhopper Orosius albicinctus that was then demonstrated to be a vector of this phytoplasma.     

The behaviour of some naturally occurring strains of potato virus Y

Potato virus Y was obtained from field crops of potatoes in many strains which differed widely in virulence and caused diseases in the variety Majestic ranging from severe leaf-drop streak to mild mosaic. The symptoms caused by these strains in seven potato varieties and tobacco are described and compared with those caused by the serologically related potato virus C. No changes were noted in the behaviour of any of the strains over three years, during which they were transmitted to many different plants.
Potato virus C was not transmitted by Myzus persicae , the most efficient vector of other strains of virus Y. Nor was virus C transmitted by eleven other species of aphides, eight of which transmitted virus Y. The efficiency with which different species acted as vectors of virus Y varied greatly, and it is suggested that in some species only occasional individuals can transmit.
Possible mechanisms for the evolution of viruses C and Y are indicated, and the effects of changes in virus, vector, and host on the prevalence of insect-transmitted viruses are discussed.