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.     

Use of Polyclonal Antibodies to Identify Mycoplasma-like organisms (MLOs) From the Sudan and from Thailand

Rabbit polyclonal antibodies prepared against faba bean phyllody MLO from the Sudan reacted with its homologous antigen and with extracts of Catharanthus roseus experimentally infected with the same or a related MLO from Crotalaria saltiana showing symptoms of phyllody disease, as well as with extracts of naturally MLO-infected C. saltiana growing in the field in the Sudan. The antibodies also reacted positively with extracts of C. roseus experimentally infected with Crotalaria juncea phyllody MLO and soybean phyllody MLO from Thailand. Polyclonal antibodies prepared against an MLO associated with witches' broom disease in C. juncea reacted positively in ELISA tests with homologous antigen extracts from naturally infected C. juncea as well as with extracts from experimentally infected C. roseus and with extracts prepared from Sesamum indicum plants with phyllody symptoms growing in Thailand. There was no reaction between these antibodies and extracts from C. roseus plants infected with the MLOs associated with C. juncea phyllody or with soybean phyllody. No cross reactions were observed among the antigens and antibodies of the two MLO groups by immunoflorescence, ELISA or western blotting. However, the molecular weight of the principal protein antigen, determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting was the same for both types of MLO. Serologically-similar MLOs thus occur in the Sudan and in Thailand, where they are associated with phyllody symptoms in C. saltiana and faba bean and with C. juncea and soybean, respectively. A second, serologically distinct MLO group was also found infecting C. juncea and S. indicum in Thailand but MLOs from this group have not yet been identified in crops from the Sudan.     

Use of Polyclonal Antibodies to Identify Mycoplasma-like organisms (MLOs) From the Sudan and from Thailand

Rabbit polyclonal antibodies prepared against faba bean phyllody MLO from the Sudan reacted with its homologous antigen and with extracts of Catharanthus roseus experimentally infected with the same or a related MLO from Crotalaria saltiana showing symptoms of phyllody disease, as well as with extracts of naturally MLO-infected C. saltiana growing in the field in the Sudan. The antibodies also reacted positively with extracts of C. roseus experimentally infected with Crotalaria juncea phyllody MLO and soybean phyllody MLO from Thailand. Polyclonal antibodies prepared against an MLO associated with witches' broom disease in C. juncea reacted positively in ELISA tests with homologous antigen extracts from naturally infected C. juncea as well as with extracts from experimentally infected C. roseus and with extracts prepared from Sesamum indicum plants with phyllody symptoms growing in Thailand. There was no reaction between these antibodies and extracts from C. roseus plants infected with the MLOs associated with C. juncea phyllody or with soybean phyllody. No cross reactions were observed among the antigens and antibodies of the two MLO groups by immunoflorescence, ELISA or western blotting. However, the molecular weight of the principal protein antigen, determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting was the same for both types of MLO. Serologically-similar MLOs thus occur in the Sudan and in Thailand, where they are associated with phyllody symptoms in C. saltiana and faba bean and with C. juncea and soybean, respectively. A second, serologically distinct MLO group was also found infecting C. juncea and S. indicum in Thailand but MLOs from this group have not yet been identified in crops from the Sudan.     

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.     

Investigations on Transmission of Grapevine Leafroll, Yellow Speckle and Fleck Diseases by Dodder

Attempts were made to transmit the infective agents of leafroll, yellow speckle, and fleck diseases from infected grapevmes to several herbaceous species and to healthy vines using dodder (Cuscuta campestris). Dodder transmitted leafroll to all and fleck to half of the respective receptor vines, but none of the three dieases were transmitted to herbaceous plants including Chenopodium, Gomphrena and Nicotiana species, Cowpea, Bean, Cucumber, Squash and Petunia. Suggested transmission of yellow speckle to vine was inconclusive because this disease may have been spread naturally.     

First report of parasite dodder (Cuscuta campestris) on jujube trees (Ziziphus jujuba) from Iran

Jujube (Zizyphus jujuba Mill.) as a naturally host for dodder (Cuscuta campestris Yunck.) is newly recorded from Southern Khorasan province of Iran. This is the first report of C. campestris, a stem parasite plant, naturally infecting jujube trees worldwide.     

<Emphasis Type="Italic">Catharanthus roseus</Emphasis> L. plants and explants infected with phytoplasmas: alkaloid production and structural observations

Summary. The results of several experiments concerning the presence and composition of alkaloids in different tissues (stems, leaves, roots) of Catharanthus roseus L. plants and explants, healthy and infected by clover phyllody phytoplasmas, are reported. The alkaloids extracted and determined by the reverse phase high-pressure liquid chromatography were vindoline, ajmalicine, serpentine, vinblastine, and vincristine. The total alkaloid concentration was higher in infected plants than in the controls, in particular the increase of vinblastine in infected roots was very significant. The ultrastructural observations of infected roots showed alterations of the cell walls and of the nuclei. These results demonstrate that phytoplasmas, detected in all infected tissues by light fluorescence and transmission electron microscopy, play an important role on secondary metabolism of the diseased plants, modifying both the total content of alkaloids and their ratio.Correspondence and reprints: Dipartimento di Biologia Evolutiva e Funzionale, Universitá degli Studi di Parma, Parco Area delle Scienze 11A, 43100 Parma, Italy.     

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.     

Studies of Polyclonal Antibodies for the Detection of MLOs Associated with Faba Bean (Vicia faba L.) Using Different ELISA Methods and Dot-blot

Polyclonal antibodies were raised in rabbits against MLO associated with faba bean (Vicia. faba L.) phyllody which exists in the Sudan. Two indirect ELISA methods were able to detect the MLO antigens. In the former, the whole antigen was directly coated onto plates, while in the second, only the F(ab')₂, fragments of the IgG were used to coat the ELISA plates. Higher detectable efficiency was obtained when the F(ab')₂ method was used. Moreover the obtainable antiserum was found to exhibit a high degree of specificity through which the MLO associated with faba bean phyllody in the Sudan, are serologically differentiated from other isolates of MLO existing in the Sudan as well as European MLO isolates maintained at Versailles, and Spiroplasma citri, causal agent of Citrus Stubborn Disease. The positive reactions obtained with this antiserum against MLO phyllody naturally existing in the Sudan on Crotalaria saltiana and some Catharanthus roseus demonstrate that these plants are potential reservoirs of the disease in the Sudan. The same antiserum was used in order to distinguish healthy and diseased plant preparations using the membrane ELISA method (dot-blot).     

Auxin‐treatment induces recovery of phytoplasma‐infected periwinkle

Aims: To test the effect of auxin‐treatment on plant pathogenic phytoplasmas and phytoplasma‐infected host. Methods and Results: In vitro grown periwinkle shoots infected with different ‘Candidatus Phytoplasma’ species were treated with indole‐3‐acetic acid (IAA) or indole‐3‐butyric acid (IBA). Both auxins induced recovery of phytoplasma‐infected periwinkle shoots, but IBA was more effective. The time period and concentration of the auxin needed to induce recovery was dependent on the ‘Candidatus Phytoplasma’ species and the type of auxin. Two ‘Candidatus Phytoplasma’ species, ‘Ca. P. pruni’ (strain KVI, clover phyllody from Italy) and ‘Ca. P. asteris’ (strain HYDB, hydrangea phyllody), were susceptible to auxin‐treatment and undetected by nested PCR or detected only in the second nested PCR in the host tissue. ‘Ca. P. solani’ (strain SA‐I, grapevine yellows) persisted in the host tissue despite the obvious recovery of the host plant and was always detected in the direct PCR. Conclusions: Both auxins induced recovery of phytoplasma‐infected plants and affected tested ‘Candidatus Phytoplasma’ species in the same manner, implying that the mechanism involved in phytoplasma elimination/survival is common to both, IAA and IBA. Significance and Impact of the Study: The results imply that in the case of some ‘Candidatus Phytoplasma’ species, IBA‐treatment could be used to eliminate phytoplasmas from in vitro grown Catharanthus roseus shoots.     

Cross-reactive Antigens between Xanthomonas campestris pv. citri Pathotypes and Citrus Species

Cross-reactive antigens were detected in crude and semi-purified preparations from acetone powder of Citrus aurantifolia and Citrus sinensis leaves with antisera to Xanthomonas campestris pv. citri pathotypes A and C by DAS-ELISA. Antiserum to X. campestris pv. citri pathotype C revealed an antigenic disparity between C. aurantifolia (susceptible host to pathotype C) and C. sinensis (resistant host to pathotype C) whereas antiserum to X, campestris pv. citri pathotype A did not reveal any antigenic disparity between these hosts, both susceptible to pathotype A. The occurrence of “key” cross-reactive antigens in Citrus species and X. campestris pv. citri and their possible involvement in such interaction are discussed.     

Stimulation effect of carrageenan on enzymatic defense system of sweet basil against Cuscuta campestris infection

Sweet basil is an important medicinal plant used especially for therapeutical potentials. Sweet basil is a common host for Cuscuta campestris, which has a negative effect on infected plants. Therefore, natural friendly control of C. campestris seems to be useful. It has been shown that carrageenans can act as elicitors of plant defense responses. In this work, the effect of κ-carrageenans on protection against C. campestris and suppression of its invasion in basils were studied. Basils were sprayed with a solution of κ-carrageenan (1?g?L^?1), once a week, three times in total. Infection of basils with C. campestris was performed two days after the last carrageenan treatment. C. campestris stem and the leaves of basils were collected two weeks after C. campestris inoculation for biochemical studies. Treatment with carrageenan significantly increased shoot length and leaf area of basil and decreased C. campestris infestation by about 26%. The content of malondialdehyde, other aldehydes, hydrogen peroxide and lipoxygenase (LOX) activity increased significantly in basils parasitized by C. campestris. There were significant differences in phenylalanine ammonia lyase (PAL), catalase (CAT), superoxide dismutase (SOD) and peroxidase activity of parasitized basils by C. campestris compared with healthy basils. Carrageenan treatment of basils caused a significant increase in H₂O₂ content and the activity of PAL, CAT and SOD, but not of malondialdehyde, other aldehydes content and LOX, polyphenol oxidase (PPO) and peroxidases activity. The activated enzymatic defense system (PAL, PPO, CAT, SOD and peroxidase) in carrageenan-treated basils have a vital role in alleviating oxidative stress damage in infected plants, by removing excess reactive oxygen species and inhibiting LOX activity and lipid peroxidation that was observed in this study. Our results showed that the application of κ-carrageenan-induced beneficial effects in plants, with regard to growth stimulation and the activation of enzymatic defense system. Thus, carrageenan was recommended as a natural biostimulator for protection of plants against C. campestris.     

Role of Seed in Survival and Transmission of Xanthomonas campestris pv. oryzae Causing Bacterial Blight of Rice

Survival period and possibility of seed transmission of X. campestris pv. oryzae were studied. The bacterium survived for longer (170–180 days) in kharif than rabi (120–130 days) harvested seed. The percentage of infected seeds was higher in kharif than rabi. The infected seed when sown failed to produce the symptoms on respective seedlings due to the low number of bacterial population. Present studies indicated that infected seed though may not produce symptoms on the seedlings directly but serve as a source of inoculum form season to season.     

Some properties of chrysanthemum stunt, a virus with the characteristics of an uncoated ribonucleic acid

Grafting to virus-free Mistletoe chrysanthemums was the most reliable method of detecting the chrysanthemum stunt agent, but specific light and temperature conditions were required for the diagnostic ‘measles’ symptoms to develop. Although stunt agent was highly infectious, leaf-rubbing inoculations with chrysanthemum sap gave erratic results. Colorimetric and electrophoretic tests were unreliable for indexing chrysanthemums. Stunt agent infected eight of twenty-nine species in the family Compositae, but none of 116 species in forty-seven other families. Stunt spread rapidly by foliage contact and by handling plants, but dipping the hands in 2% trisodium orthophosphate when handling plants increased the amount of spread. Stunt agent was not transmitted by four species of aphids, the glasshouse redspider mite, dodder (Cuscuta campestris) or through chrysanthemum seed. Stunt agent withstood 10 min at c. 98 °C and dilution to 10^-4, was not pelleted by ultracentrifugation, and was inactivated by RNase in weak, but not strong, buffer, suggestive of an uncoated RNA ‘viroid’. Partially purified preparations were made by homogenizing frozen chrysanthemum leaves in 0.5 m phosphate buffer with antioxidant at c. 2 °C, and clarification by n-but-anol and chloroform, followed by centrifugation. Highly infective RNA was precipitated from the supernant fluid by 2.5 vol. cold ethanol, and resuspended in a small volume of buffer. The u.v. absorption spectra of infective preparations and the u.v. absorbance profiles of density-gradients, were very similar to those of preparations from healthy chrysanthemum. Infective partially purified preparations of stunt agent withstood exposure to 2% formaldehyde or tri-sodium orthophosphate, u.v. irradiation, and sonication. Stunt preparations contained no virus particles recognizable by electron microscopy, gave no distinct peak on analytical ultracentrifugation, and did not consistently contain any specific antigen. Although similar to the ‘viroids’ potato spindle tuber and citrus exocortis, stunt agent did not infect Citrus limon, Gynura aurantiaca or tomato.     

Occurrence,Molecular Characterization and Vector Transmission of a Phytoplasma Associated with Rapeseed Phyllody in Iran

Symptoms of rapeseed phyllody were observed in rapeseed fields of Fars, Ghazvin, Isfahan, Kerman and Yazd provinces in Iran. Circulifer haematoceps leafhoppers testing positive for phytoplasma in polymerase chain reaction (PCR) successfully transmitted a rapeseed phyllody phytoplasma isolate from Zarghan (Fars province) to healthy rapeseed plants directly after collection in the field or after acquisition feeding on infected rapeseed in the greenhouse. The disease agent was transmitted by the same leafhopper from rape to periwinkle, sesame, stock, mustard, radish and rocket plants causing phytoplasma‐type symptoms in these plants. PCR assays using phytoplasma‐specific primer pair P1/P7 or nested PCR using primers P1/P7 followed by R16F2n/R2, amplified products of expected size (1.8 and 1.2 kbp, respectively) from symptomatic rapeseed plants and C. haematoceps specimens. Restriction fragment length polymorphism analysis of amplification products of nested PCR and putative restriction site analysis of 16S rRNA gene indicated the presence of aster yellows‐related phytoplasmas (16SrI‐B) in naturally and experimentally infected rapeseed plants and in samples of C. haematoceps collected in affected rapeseed fields. Sequence homology and phylogenetic analysis of 16S rRNA gene confirmed that the associated phytoplasma detected in Zarghan rapeseed plant is closer to the members of the subgroup 16SrI‐B than to other members of the AY group. This is the first report of natural occurrence and characterization of rapeseed phyllody phytoplasma, including its vector identification, in Iran.     

Factors restraining parasitism of the invasive vine Mikania micrantha by the holoparasitic plant Cuscuta campestris

Mikania micrantha (Asteraceae) is one of the 10 most invasive weeds in the world and has caused tremendous economic and environmental losses in southern China. The dodder Cuscuta campestris (Convolvulaceae) is a native holoparasite that can parasitize and suppress M. micrantha, and thus is recommended as an effective control agent. However, the natural growth of dodder lags behind that of M. micrantha and fails to exert direct year-round suppression. To verify the effective parasitic distance, we placed a dodder seedling at a designated distance from the stem of M. micrantha and monitored coiling, haustorium formation, and survival. To verify suitable host stems for the parasite, we grew M. micrantha for more than 6 months to form stems of different sizes, taped dodder seedlings to the stems, and monitored. We used various temperatures to determine the effect on dodder seed germination and M. micrantha sprouting. The results showed that a dodder–host distance of 4 cm decreased the probability of successful parasitism to 0; dodder seedlings cannot parasitize M. micrantha stem diameters ≥0.3 cm; and the temperatures for the highest dodder seed germination and M. micrantha sprouting are 26 and 30 °C, respectively. We conclude that the lack of suitable M. micrantha parts within the dodder’s effective parasitism distance is the major cause of restricted dodder parasitism and that the lower temperature for the highest dodder germination compared to that for the highest M. micrantha sprouting may decrease the possibility of parasitism. To acquire aggressive parasitism, dodder should be manually assisted by dispersing its vegetative form.     

Cyclamen (Cyclamen persicum L.): a dead-end host species for 16Sr-IB and -IC subgroup phytoplasmas

Phytoplasmas belonging to the 16S rDNA subgroups IB and IC were found in five cyclamen (Cyclamen persicum L.) plants showing virescence and yellow stunted leaves and one plant showing phyllody, rolled and thickened leaves, respectively. Two cyclamens, representing the two syndromes, were chosen as source plants for transmission trials in which three leafhopper species, known as vectors of IB and IC subgroup phytoplasmas, were used to inoculate cyclamen and periwinkle [Catharanthus roseus (L.) G. Don] test plants. Out of 366 tested plants only one periwinkle exposed to Euscelis incisus was found harbouring a 16Sr‐IB phytoplasma. Out of 60 tested vector insects, only one adult of Macrosteles quadripunctulatus and two of E. incisus fed on 16Sr‐IB source cyclamen gave a positive amplification signal in nested PCR. This extremely low level of transmission to both cyclamen and the very susceptible periwinkle strongly suggests that cyclamen, commonly found infected in crops, is an unsuitable species for phytoplasma acquisition and can be regarded as a dead‐end host plant for phytoplasmas belonging to both IB and IC subgroups. Indications for glasshouse management are drawn from these findings. Among the leafhoppers investigated E. incisus falls most under suspicion since it feeds better than the others on cyclamen, was able to transmit the disease to one periwinkle plant, and IB phytoplasmas were detected in two individuals.     

TRANSMISSION AND HOST-RANGE STUDIES OF STRAWBERRY GREEN-PETAL VIRUS

The virus causing phyllody (virescence) in clover flowers was transferred by Cuscuta subinclusa to Fragaria vesca and Duchesnia indica plants which then produced symptoms of strawberry green-petal disease.
The jassid Euscelis plebejus (Fall.) in several forms, including E. lineolatus Brullé, transmitted green-petal virus from clover to clover, to and from a wide range of other hosts, and from but not to strawberry. Two viruses (or strains) were distinguished, one causing phyllody and the other witches' broom on clover; both were retained for more than 2 months by the vector, in which both had a latent period of about 30 days. Macrosteles viridigriseus (Edwards) also transmitted both viruses.
Variation in symptoms on strawberry plants infected naturally, and experimentally through dodder, suggested that two diseases have previously been grouped under the name 'green petal". It is proposed to distinguish these as ( a ) green petal caused by the virus inducing phyllody in clover, and ( b ) bronze leaf wilt caused by the clover witches' broom virus.