The spread of mycoplasma in tobacco (Nicotiana glauca GRAH.) -a symptomless carrier of potato witches’ broom disease

The spread of mycoplasma causing potato witches’ broom disease from infectious grafts of both tomato and tobacco plants intoNicotiana glauca Grah. plants is much slower than that into tomato plants. An earlier infection inN. glauca stock plants appears when infectiousN. glauca grafts are used. On the other hand, the process of infection proceeds very slowly when using infectious tomato grafts and in some cases the infection is not realized even after three months of graft symbiosis. The authors suppose that mycoplasma has a preference for tomato plants.     

Mycoplasma-like bodies inSolanum laciniatum plants infected with potato witches’ broom disease

Mycoplasma-like organisms (MLO) spread from the infectious grafts intoSolanum laciniatum Ait. stock plants relatively slowly. MLO were present in all sprouts ofS. laciniatum four weeks after grafting, but the infected plants remained under glasshouse conditions mostly symptomless and flowered normally and formed fruits like healthy plants. The growth of plants with infectious tomato grafts was identical with the controls but that of plants with infectious tobacco (Nicotiana glauca Grah.) grafts was expressively stimulated. The first flower symptoms appeared onS. laciniatum plants with tomato grafts after five and half months and on.S. laciniatum plants with tobacco grafts after seven months of graft symbiosis. Electron micrographs of ultrathin sections showed the presence of MLO in sieve tubes of potiols and midribs of the infected but symptomless plants. In the phloem parenchyma cells of the witches’ broom diseased plants, highly ordered crystals were occasionally found lying in a microbody surrounded by a membrane. The possible reasons of the disease latency are discussed.     

Persistence and spread of mycoplasma in axenic callus tissue cultures of tobacco (Nicotiana glauca Grah.) in the presence of kinetin and IAA in nutrient medium

A technique is described for the establishment of infectious axenic callus cultures originated from stems of tobacco plants suffering from a mycoplasma disease, potato witches' broom. When stem pieces from a diseased tobacco plant (Nicotiana glauca Grah.) are culture on a modified nutrient medium according toMurashige andSkoog (1962) with added kinetin (0.64 mg or 2.56 mg 1 000 ml^?1) and IAA (2 mg or 4 mg 1 000 ml^?1), the pathogen persists and spreads in the newly formed callus tissue. The presence of the pathogen was proved by implantation of callus slices into stems of tomato plants which indicate the disease very reliably. Reconstituted tobacco plants too recovered the infectious agent of potato witches' broom; 27 plants from the 35 reconstituted plants were diseased. Similar results were obtained with Crimean yellows. Calli were cultured and subcultured from 1 to 18 monthsin vitro and some infectious cultures are maintained and their investigation goes on. We suppose that kinetin plays a very important role for infectivity preservation in callus cultures.     

The effect of kinetin on the organogenesis in tobacco (Nicotiana glauca Grah.) callus cultures derived from healthy and mycoplasma-infected plants

In contradiction to Paulet’s (1965) data, we found that kinetin/IAA strongly affected organogenesis in callus tissue derived from the stem ofNicotiana glauca Grah. plants both in primary expiants and in subcultured calli. The effect of these substances was higher in the subcultured calli derived from mycoplasma-infected plants. Evidence of the absence of the infectious agent in de novo-formed plants in subcultured calli was given by grafting and by the electron micrograph.     

Contribution to the problem of mycoplasma-like organism transmigration in plants

Anatomical-histological investigations of slide series revealed that the secondary vascular formations appearing scattered in callus tissue culture derived from a stem piece containing mycoplasma-like organisms (MLO) (potato witches’ broom agent) have no connection with the vascular tissues of the original stem piece. 77% of reconstituted tobacco plants (Nicotiana glauca Grah.) from these callus cultures were infected with MLO. When diseasedNicotiana glauca scions are grafted on healthy tomato plants as stocks, MLO transmigration into the stocks is realized in all cases ten days after grafting; at that time, the scions and stocks are united only by a zone of a few layers of parenchymatous cells without any indication of vascular tissue union between the two components. In both cases, the MLO transmigration could not be realized by passage through sieve pores in sieve tubes. Our results bring indirect evidence of MLO transmigration through parenchymatous cells and thus support the hypothesis of MLO passage through the plasmodesmata.     

Metabolic disturbances innicotiana glauca,a symptomless

Metabolic disturbances inNicotiana glauca, a symptomless carrier of potato witches’ broom were studied. The dry weight content of leaves of diseased plants was slightly decreased, that of the stems increased. The ash content in the dry weight of the whole diseased plant was decreased by 11%. Contrary to tomato plants which had shown considerable disproportions in the distribution of metabolites among the individual organs investigated (ULRYCHOVÁ, LIMBERK 1964), the content of the total nitrogen and of the individual nitrogen fractions of both leaves and stems of diseasedNicotiana glauca were decreased. The content of glutamine was increased in both organs in spite of the decrease of ammonia nitrogen by nearly 30% in both cases. The most striking disturbance found in the diseased plants ofNicotiana glauca was the 67% increase of a low-molecular phosphorus fraction (P extractable with 0-2N HCIO₄) in the leaves. This result agrees with that found in diseased tomato plants and may be primarily associated with the virus infection. The content of all other phosphorus fractions of both leaves and stems was decreased.     

Some morphological peculiarities ofMycoplasma bodies occurring in plants infected with potato witches' broom disease

Our paper describes some morphological peculiarities ofMycoplasma bodies located in vascular bundles ofNicotiana glauca Grah. and tomato infected with potato witches' broom disease. The influence of postfixation on the density of bodies and possible development of artifacts was studied in particular. It was found that bodies of adjoining cells may have a different shape. Also elementary bodies, approximately of uniform diameter, may occur in masses in close proximity to the cells containing “adult”Mycoplasma bodies. Deformed large bodies giving in a certain position the impression of “vacuolization” are evidently degenerated saucershaped forms. In addition toMycoplasma some other bodies with different inner structure were observed in the same cells.     

Somatic hybrids of plants by fusion of protoplasts

Summary Fusions of protoplasts from Nicotiana langsdorffii and Nicotiana glauca were induced using polyethylene glycol. Parasexual hybrid colonies were selected for their ability to grow without growth substances. Hybrid plants, regenerated after grafting, were all tumorous and exhibited morphological and chromosome number variations. Out of 48 colonies selected in vitro only 6 regenerated flowering plants. Two of these plants had 42 chromosomes and were morphologically identical to the sexual amphidiploid Nicotiana glaucaxlangsdorffii.     

Effect of cholesterol and stigmasterol on symptom manifestation of potato witches broom —A disease which is supposed to be caused byMycoplasma

Evidence for the effect of sterols on plant mycoplasma is presented. Two essential sterolsi.e. cholesterol and stigmasterol were applied in the following way: grafts from tomato plants infected with potato witches'broom, wedge-shaped at the base were dusted on the cut surfaces with the corresponding sterol in the amount of 4 mg to one graft, inserted in freshly cut wedges of stock plants and firmly tightened with rubber tubes. Both sterols stimulated growth of diseased grafts and delayed symptom manifestation. The stimulation was more evident with stigmasterol and the delay in symptom manifestation and intensity was more distinct with cholesterol. Supplying tomato plants with sterols externally probably enables additional multiplication of mycoplasma in already infected grafts, and the plants need not provide the grafts with essential metabolites which they themselves need for the development of reproductive organs. Stigmasterol, a metabolite peculiar to the tomato plant, is probably more rapidly consumed than cholesterol which may remain in the plant as a metabolic pool of sterols for mycoplasma propagation.     

Elimination of mycoplasma in tobacco callus tissues (Nicotiana glauca Grah.) culturedin vitro in the presence of 2,4-D in nutrient medium

Callus tissue cultures were established from stems of tobacco plants (N. glauca Grah.) both healthy and mycoplasma (potato witches' broom disease) infected on a modified nutrient medium (with a lower content of mineral salts) according toMurashige andSkoog (1962) in the presence of 2,4-D (1 mg l^?1) as a growth regulator. No differences were observed in the growth and development of both tissues. Organogenesis appeared on a nutrient medium (Petr? et al. 1972) supplemented with kinetin (0.64 mg or 2.56 mg l^?1) and IAA (2 or 4 mg l^?1). Callus derived from mycoplasma diseased plants started to form numerous buds after three months whereas organogenesis in callus from healthy controls appeared only after six months. We suppose that the reason of this difference is the fact that an expressively higher content of 2,4-D was found in the calli from healthy plants in comparison with the corresponding tissue from mycoplasma diseased ones. Reconstituted plants were isolated, rooted and transferred in the soil. The infectivity of these plants was assayed by grafting their stem tips on tomato plants which indicate very reliably and sensitively this mycoplasma disease. 31 reconstituted plants were obtained in the whole from calli isolated from mycoplasma infected plants and all of them were healthy. It was established that mycoplasma failed in the presence of 2,4-Din vitro. Stem pieces from diseased plants in which mycoplasma presence was proved, lose their infectivity after 4 weeks of cultivation on nutrient medium with this growth regulator. On the contrary 2,4-D which spreads and acts especially through phloem (Smith et al. 1947) does not kill mycoplasmain vivo even in doses evoking strong symptoms of 2,4-D effect on experimental plants.     

Immunochemical comparison of glutamine synthetases from some solanaceae plants

The presence of different glutamine synthetase isoenzymes in different Solanaceae plants and their relative antigenicities against antiglutamine synthetase from tomato leaf serum were studied. All the plants tested showed one glutamine synthetase isoenzyme except for Mandragora autumnalis, which showed two, after discontinuous polyacrylamide gel electrophoresis and specific in situ assay. Antigenicities were compared by the double immunodiffusion technique. The Nicotiana glauca enzyme showed equal reactivity to that of Lycopersicon esculentum, but its antigenicity was higher than Withania frutescens, Datura stramonium, and Hyoscyamus niger. The study of relative antigenicities permitted differentiation of the glutamine synthetase enzymes from uncultivated species of Solanaceae.     

Mycoplasma-like bodies in plants infected with potato witches' broom disease and the response of plants to tetracycline treatment

The virus origin of a Czechoslovak isolate of potato witches' broom disease is discounted: electron micrographs of ultrathin section of phloem tissues from plants infected with potato witches' broom disease demonstratedMycoplasma-like bodies, spherical or elongated showing binary fission and fragmentation. The minute corpuscles have a diameter of about 50–60 nm, the largest bodies of 1000 nm. The width of elongated filamentous structures is about 200 nm, most oval bodies have a diameter of 250 nm. A weak tetracycline treatment of diseased plants causes a delay of symptom development; a strong dose of tetracycline (applied by means of the wick method into the stem) inhibits symptom appearance completely. Tetracycline produces a phytotoxic effect inhibiting the growth of tomato plants and causing (at higher concentrations) necrosis and death of these plants. There is a note in the paper dealing with the term “mycoplasma”. The word mycoplasma in the sense ofEriksson (1897) or ofMereschkowsky (1910) does not correspond to the genus nameMycoplasma Nowak (1929).     

Dominant lethal assay in female mice after oral dosing with dichlorvos or exposure to atmospheres containing dichlorovos.

The effect of a synthetic auxin-like substance (2,4-D) and a synthetic cell division factor (kinetin) on the induction of chromosome aberrations was studied on tissue cultures of Nicotiana glauca and the tumorous amphidiploid hybrid Nicotiana glauca × Nicotiana langsdorffii.The aberration frequencies in normal Nicotiana glauca tissue were proportional to the length of time of culture in the presence of 2,4-D. Moreover, both 2,4-D and kinetin increased chromosome breakage in the habiatouated Nicotiana glauca tissue but not in the amphidiploid hybrid tissue.The data are discussed in terms of genotype-hormone equilibria in long-term development of plant tissue culture.     

Two TOBAMOVIRUS MULTIPLICATION 2A homologs in tobacco control asymptomatic response to tobacco mosaic virus

The most common response of a host to pathogens is arguably the asymptomatic response. However, the genetic and molecular mechanisms responsible for asymptomatic responses to pathogens are poorly understood. Here we report on the genetic cloning of two genes controlling the asymptomatic response to tobacco mosaic virus (TMV) in cultivated tobacco (Nicotiana tabacum). These two genes are homologous to tobamovirus multiplication 2A (TOM2A) from Arabidopsis, which was shown to be critical for the accumulation of TMV. Expression analysis indicates that the TOM2A genes might play fundamental roles in plant development or in responses to stresses. Consistent with this hypothesis, a null allele of the TOM2A ortholog in tomato (Solanum lycopersicum) led to the development of bent branches and a high tolerance to both TMV and tomato mosaic virus (ToMV). However, the TOM2A ortholog in Nicotiana glauca did not account for the asymptomatic response to TMV in N. glauca. We showed that TOM2A family is plant-specific and originated from Chlorophyte, and the biological functions of TOM2A orthologs to promote TMV accumulation are highly conserved in the plant kingdom—in both TMV host and nonhost species. In addition, we showed that the interaction between tobacco TOM1 and TOM2A orthologs in plant species is conserved, suggesting a conserved nature of TOM1–TOM2A module in promoting TMV multiplication in plants. The tradeoff between host development, the resistance of hosts to pathogens, and their influence on gene evolution are discussed. Our results shed light on mechanisms that contribute to asymptomatic responses to viruses in plants and provide approaches for developing TMV/ToMV-resistant crops.

Tobacco TOBAMOVIRUS MULTIPLICATION 2A homologs control the asymptomatic response to tobacco mosaic virus and have highly conserved biological functions related to virus multiplication.     

Pepper veinal mottle virus associated with a streak diseaseof tomato in Nigeria

A destructive streak disease of tomato (Lycopersicon esculentum) was observed on the University of Ife farm. The disease killed many plants and greatly diminished the quantity and quality of fruit produced by the other plants. A virus that is readily transmitted by mechanical inoculation, by the green peach aphid (Myzus persicae) and by grafting was isolated consistently from diseased plants. The virus was propagated in Nicotiana occidentalis and assayed in Physalis angulata. It was infective after dilution to io^-3 but not io^-4; after iomin at 55 but not 60^oC; or after 5 days but not 7 days at 20–26^oC. Electron microscope examination of sap from infected N. occidentalis leaves revealed flexuous rods with a modal length of about 780 nm. Based on the host range and symptomatology, particle morphology and size, properties in vitro and serology, the virus is shown to be related to, and possibly indistinguishable from, pepper veinal mottle virus.     

Gene regulatory networks for compatible versus incompatible grafts identify a role for SlWOX4 during junction formation

Grafting has been adopted for a wide range of crops to enhance productivity and resilience; for example, grafting of Solanaceous crops couples disease-resistant rootstocks with scions that produce high-quality fruit. However, incompatibility severely limits the application of grafting and graft incompatibility remains poorly understood. In grafts, immediate incompatibility results in rapid death, but delayed incompatibility can take months or even years to manifest, creating a significant economic burden for perennial crop production. To gain insight into the genetic mechanisms underlying this phenomenon, we developed a model system using heterografting of tomato (Solanum lycopersicum) and pepper (Capsicum annuum). These grafted plants express signs of anatomical junction failure within the first week of grafting. By generating a detailed timeline for junction formation, we were able to pinpoint the cellular basis for this delayed incompatibility. Furthermore, we inferred gene regulatory networks for compatible self-grafts and incompatible heterografts based on these key anatomical events, which predict core regulators for grafting. Finally, we examined the role of vascular development in graft formation and uncovered SlWOX4 as a potential regulator of graft compatibility. Following this predicted regulator up with functional analysis, we show that Slwox4 homografts fail to form xylem bridges across the junction, demonstrating that indeed, SlWOX4 is essential for vascular reconnection during grafting, and may function as an early indicator of graft failure.

Gene regulatory networks for compatible self-grafted and incompatible heterografted pepper/tomato plants identify a role for SlWOX4 during junction formation.     

A Graft Mimic Strategy for Verticillium Resistance in Tomato

Grafting vegetables for disease resistance has increased greatly in popularity over the past 10 years. Verticillium wilt of tomato is commonly controlled through grafting of commercial varieties on resistant rootstocks expressing the Ve1 R-gene. To mimic the grafted plant, proteomic analyses in tomato were used to identify a suitable root-specific promoter (TMVi), which was used to express the Ve1-allele in susceptible Craigella (Cs) tomato plants. The results indicate that when infected with Verticillim dahliae, race 1, the transformed plants are comparable to resistant cultivars (Cr) or grafted plants.     

Virp1 is a host protein with a major role in Potato spindle tuber viroid infection in Nicotiana plants

Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1-suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1-suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1-suppressed plants is likely the result of cell-autonomous events.     

Siderophore and chitinase producing isolates from the rhizosphere of Nicotiana glauca Graham enhance growth and induce systemic resistance in Solanum lycopersicum L.

A screening for Plant Growth Promoting Rhizobacteria (PGPR) was carried out in the rhizosphere of wild populations of Nicotiana glauca Graham in south-eastern Spain. Nine hundred and sixty strains were isolated and grouped in four parataxonomic groups: Gram positive endospore forming bacilli, Gram positive non-endospore forming bacilli, Gram negative bacilli and others. Two groups were selected to continue the study: Gram negative bacilli since it was the most abundant, and Gram positive sporulated bacilli, seeking their sporulating capacity as an advantage for inoculants formulation. The ability of these to release siderophores and chitinases in vitro was evaluated. Ninety six isolates were siderophore producers, and 56 of them were also able to produce chitinases. Fifty percent of these were tested for growth promotion in tomato. The best results were obtained with 5 Gram negative bacilli and one Gram positive sporulated bacilli; 5 strains increased all growth parameters while one of them, N21.4, severely compromised plant growth. The ability of these 6 strains to induce systemic resistance against the leaf pathogen Xanthomonas campestris in tomato was evaluated. Five of them effectively reduced disease symptoms (up to 50%). The six strains were identified by 16s rDNA sequencing resulting in 3 Pseudomonas, 1 Bacillus and 2 Stenotrophomonas; it’s striking that 2 Pseudomonas protected up to 50% while the other increased disease incidence. This indicates that systemic induction is strain specific and not necessarily related to production of siderophores and chitinases.     

T-DNA analysis of plants regenerated from hairy root tumors

Summary Plants regenerated from hairy root tumors induced on Nicotiana glauca and Nicotiana tabacum by Agrobacterium rhizogenes strain A4 were examined for the presence of T-DNA. Regenerated N. tabacum plants contained intact copies of both TL-DNA and TR-DNA. However, plants regenerated from N. glauca tumors did not contain the TR-DNA region corresponding to the tms (auxin synthesis) genes. Some of the regenerants exhibited an abnormal phenotype which is characterized by severe leaf wrinkling. This phenotype is correlated with the presence of TL-DNA, but not TR-DNA.