Occurrence of Mosaic Mottle, a Viroid Disease of Pigeonpea (Cajanus cajan) in India

During investigations of viroid diseases of food crops in Delhi, India, an infectious low-molecular-weight RNA (M_r 1.3 × 10⁵) was isolated from pigeonpea (Cajanus cajan) plants showing mosaic mottling and reduced size of leaves, stunting and no flowering. This RNA infected mechanically inoculated C. cajan, Chenopodium amaranticolor, Nicotiana glutinosa, and N. clevelandii. The causal agent, for which the name pigeonpea mosaic mottle viroid (PMMVd) is proposed, differed from N. glutinosa stunt viroid (NgSVd), the only viroid reported to infect legumes, in size and symptom expression on the original host species.     

Detection of coconut cadang-cadang viroid-like sequences in oil and coconut palm and other monocotyledons in the south-west Pacific

Fractionation, electroblotting and molecular hybridisation of nucleic acids extracted from tissue of African oil palm and coconut palm and some other monocotyledonous species, collected in several areas of the south-west Pacific region, demonstrated the presence of small nucleic acids with nucleotide sequences and secondary structure similar to coconut cadang-cadang viroid (CCCVd). The oil palms which contained CCCVd-related molecules showed orange leaf spots resembling those described for oil palm naturally infected with CCCVd in the Philippines, and also characteristic of a condition known as "genetic orange spotting" (GOS). We provide preliminary evidence that GOS is an infectious disorder caused by a viroid. The coconut palms did not show symptoms typical of cadang-cadang disease, but sometimes were chlorotic, stunted, or had a reduced yield. The possibility that the isolates represent variants of CCCVd is discussed. The data suggest that viroids with nucleotide sequences similar to CCCVd occur widely in palms and other monocotyledons outside the Philippines.     

Fatal Yellowing of Oil Palms: Search for Viroids and Double-Stranded RNA

Fatal yellowing is a serious disease of still unknown origin affecting oil palms in several regions of Central and South America. In this study a search for viroids and viroid-like RNAs in oil palms was performed using two-dimensional gel electrophoresis and return gel electrophoresis of nucleic acid extracts. Although RNAs showing viroid-like gel-electrophoretic properties were detected, the presence of the known viroids was excluded by hybridization experiments using probes specific for potato spindle tuber viroid (PSTVd), coconut cadang-cadang viroid (CCCVd), or Coleus blumei viroid 1 (CbVd1). By using double-stranded RNA (dsRNA) specific monoclonal antibodies, which do not react with viroid RNA, we were able to show that oil palm RNAs, migrating like viroids are double-stranded RNA species. Since the same dsRNA pattern was found in extracts from diseased as well as from healthy oil palms, the dsRNAs can neither be part of the causative agent of fatal yellowing, nor are they associated with the disease. Their possible origin is discussed. In addition to the standard electrophoretic methods, which have been used for identification of viroids and viroid-like RNAs, we describe additional control experiments to differentiate unequivocally between circular single stranded and linear dsRNA.     

A novel aspect of the information content of viroids

Viroids were found to exhibit a structural periodicity characterized by repeat units of a length of 11 or 12 (potato spindle tuber viroid group and coconut cadang-cadang viroid), 60 (apple scar skin viroid) and 80 (avocado sunblotch viroid) nucleotide residues, respectively. It is suggested that structural periodicity of viroids is an indication of their protein-binding ability.     

Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid — host cell interaction

Photosynthetically active callus and cell suspension cultures were established from uninfected Lycopersicon peruvianum plants and from uninfected and potato spindle tuber viroid (PSTVd) infected plants of Lycopersicon esculentum cv. Rutgers. Viroid infection was maintained in photoheterotrophic culture on media containing 3% sucrose, but during continuous photo-mixotrophic culture in low sucrose media (1% sucrose), the level of PSTVd accumulation decreased. Photoautotrophic cell suspensions could be established with uninfected, but not with viroid infected tomato cells. As compared to uninfected cells, PSTVd infected cells grew slowly, were morphologically different in size and shape, and formed tight cell aggregates. Electronmicroscopy showed that starch accumulation in chloroplasts, deformation of the chloroplast envelope and irregular plasmalemmasomes at the cell membrane were associated with PSTVd infection.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - CEVd citrus exocortis viroid - CSVd chrysanthemum stunt viroid - PSTVd potato spindle tuber viroid - TMV tobacco mosaic virus - phc photoheterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture     

In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus

Viroids, small single-stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin-labelled RNA probes were used with subsequent detection by gold-labelled monoclonal anti-biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.     

Accumulation of potato virus Y is enhanced in Solatium brevidens also infected with tobacco mosaic virus or potato spindle tuber viroid

The accumulation of potato virus Y?(PVY?) and potato leaf roll virus (PLRV) was studied in plants of Solanum brevidens co-infected with each of six viruses or a viroid. Virus could not be detected by ELISA in plants of S. brevidens infected solely with PVY. However, accumulation of PVY was increased c. 1000-fold in plants doubly infected with tobacco mosaic virus or potato spindle tuber viroid (PSTVd). PVY titres in doubly infected plants of S. brevidens were between 1% and 0.1% of those found in the PVY-susceptible interspecific Solanum hybrid DTO-33. Double infections of 5. brevidens by PVY and alfalfa mosaic virus or potato viruses M, S, T or X did not significantly enhance PVY accumulation. Accumulation of PLRV was not enhanced in plants co-infected with any of the six viruses or PSTVd.     

Response of Cucumber Cultivars and Other Cucurbitaceous Species to Infection by Hop Stunt Viroid1

The response of 8 different species within the Cucurbitaceae to hop stunt viroid (HSV) was examined. Some of these plants developed several kinds of systemic symptoms depending upon species, variety or cultivar infected. Among 12 out of 26 cucumber cultivars become infected severely, so visual symptoms are reliable for diagnosis because symptom severity is correlated with viroid content. Other cucurbitaceous species except Citrullus lanatus, Cucurbita maxima and Cucurbita moschata were also susceptible. Among them, Benincasa hispida was found to be a more suitable bioassay plant than Suuyou cucumber which has been hitherto used.     

Effects of sacbrood virus on adult honey-bees

Much sacbrood virus accumulates in the brains of infected bees, especially of drones, without causing symptoms. However, infected individuals fly earlier in life than healthy bees and most infected foragers fail to collect pollen, as do bees briefly anaesthetized with CO₂. The few infected bees that gather pollen contaminate their loads with much sacbrood virus. Infection does not shorten the lives of drones, or of workers that have been deprived of pollen, but it much shortens the prolonged lives of workers that have eaten pollen. Infected workers, healthy workers deprived of pollen, and senile individuals are unable to maintain the usual metabolic rates of bees at temperatures below 35^oC, or to resist chilling.     

Transmission of Three Viroids Through Seed and Pollen of Tomato Plants

The severe strain of potato spindle tuber viroid (s-PSTV) as well as chrysanthemum stunt (CSV) and cucumber pale fruit (CPFV) viroids were found to be transmitted through seed and pollen of the tomato cvs. Rutgers and Najwcze?niejszy. Plants pollinated with a pollen infected with any of these three viroids became systematically infected. Plant, fruit and seed symptoms of viroid infection were noted on sap- and pollen-inoculated plants and the yield of these plants was reduced. Tomato cv. Rutgers plants grown from infected seeds were symptomless although all three viroids were detected in these plants by bioassay and by electrophoresis on 5% polyacrylamide gel. When DNA complementary to s-PSTV RNA was used for a direct viroid detection in seed samples by spot hybridization technique it hybridized not only with s-PSTV RNA but also with CSV RNA as well as with CPFV RNA.     

Survival of Hop Stunt Viroid in the Hop Garden1)

A lot of 105 specimens from 25 families including weeds or wild plants which had grown naturally in the severely infested hop garden were tested for detecting reservoir plants for hop stunt viroid (HSV). HSV was detected in hop plants only. Susceptibility tests with various cultivated plants including 14 families indicated that hop and Humulus japonicus developed visible symptoms, while tomato was symptomless. When infected hop plant residues, leaves and cones, were left to be weather-beaten, infectivity of HSV was completely lost within 3 months. No transmission through the pollen or the ovule was demonstrated. HSV could survice in root systems of hop plants during the winter months. Based on these results, the route of HSV survival in the hop garden was discussed.     

Structural transitions in viroid-like RNAs associated with cadang-cadang disease, velvet tobacco mottle virus, and Solanum nodiflorum mottle virus.

The conformational transitions of viroid-like RNAs associated with cadang-cadang disease, velvet tobacco mottle virus, and solanum nodiflorum mottle virus were studied by melting analysis and fast temperature jump technique in 1 mM sodium-cacodylate, 10 mM NaCl, 0.1 mM EDTA, pH 6.8. The 4 circular RNAs of cadang-cadang show a highly cooperative transition between 45 and 49 degrees C, respectively, and a second transition of less hypochromicity at about 10 degrees C higher temperatures. The data are interpreted quantitatively on the basis of the sequences and secondary structure models. A very similar scheme for the structure and structural transitions as derived earlier for other viroids applies to the cadang-cadang RNAs. In the main transition the total native secondary structure is disrupted and a stable hairpin consisting of 9 base pairs is newly formed which dissociates in the second transition. The thermal denaturation of the circular RNAs from the viruses mentioned above is clearly distinct from viroid RNA in respect to stability and cooperativity. The results on cadang-cadang RNA are discussed in the light of recent hypotheses about the interference of viroids with the splicing process of the host cell.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs.

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.     

The effect of cherry leaf roll virus infection on the performance of birch pollen and studies on virus replication in germinating pollen

The rates of germ tube elongation in vitro of pollen from cherry leaf roll virus (CLRV)-infected birch (Betula pendula) did not differ significantly from those of pollen from virus-free trees. Differences in percentage germination of pollen collected from trees at different sites were significant but percentages of germination of pollen from virus-infected and virus-free trees did not differ greatly from one another. In vivo, pollen from infected trees germinated on healthy and CLRV-infected stigmas and callose plugs formed in both types of tissues. However, the extent of callose plug formation was greater in the pollen tubes of virus-free grains germinating in infected stigmas than in reciprocal crosses. CLRV coat antigen was detected by ELISA in stigmatic tissue, from healthy trees, on which virus-carrying pollen grains had germinated. Using fluorescein isothiocyanate conjugated to CLRV-specific γ-globulin, viral coat antigen was detected throughout germ tubes from virus-carrying but not virus-free pollen germinating in vitro. Protoplasts released following Driselase digestion of pollen germ tubes from virus-infected trees contained CLRV antigen detectable by ELISA. During germination of virus-infected pollen there was little synthesis of viral coat protein or nucleic acid but, following inoculation with purified virus particles, protoplasts made from healthy germinating pollen produced increasing amounts of CLRV-specific antigen implying that CLRV replicated in this system.     

In vitro system for studying interactions between Citrus exocortis viroid and Gynura aurantiaca (Blume) DC. metabolism and growing conditions

Citrus exocortis viroid (CEVd) is an economically important plant pathogen, which infects a broad range of hosts. In order to investigate complex interactions among viroid, host-growing conditions, and plant secondary metabolism, we setup an in vitro system for the cultivation of CEVd-infected and viroid-free Gynura aurantiaca (Blume) DC. shoots. Both basal Murashige and Skoog (MS) medium and MS medium supplemented with cytokinines supported the growth of shoots, but viroid-free shoots performed better than infected ones. The addition of cytokinines induced an increase in the concentration of purple-reddish pigment, cyanidin tetra-glucoside (GAA), which did not depend on the presence of viroid. The differences in single strand conformation polymorphism patterns between CEVd sequence variants obtained from plants grown on medium supplemented with cytokinines and those obtained from plants grown on basal MS medium or in the greenhouse, indicated that cytokinines may influence not only the plant metabolism, but also the viroid population structure.     

Reproductive interactions between hybridizing irises: analyses of pollen-tube growth and fertilization success

Pollen-tube growth and seed siring ability were measured in crosses between the Louisiana iris species Iris fulva and Iris hexagona and their F₁ and F₂ hybrids. Flowers of the parental species were pollinated with self, outcross intraspecific, and interspecific pollen. Pollen-tube lengths were similar for all three pollen types in I. fulva, but in I. hexagona interspecific pollen tubes were longer than intraspecific pollen tubes. Pollen-tube lengths also differed for F₁ and F₂ flowers pollinated with I. fulva, I. hexagona, and hybrid pollen. For both hybrid classes I. fulva pollen tubes were the shortest while pollen tubes from I. hexagona and hybrids grew the furthest. Mixtures of genetically marked pollen were used to assess the seed siring ability of intra- and interspecific pollen in the parental species by varying the proportion of each pollen type in a replacement series design. For both species, the observed proportions of hybrid seeds were lower than the expected based on the frequency of each pollen type in the mixtures across all treatments. Flowers of I. fulva produced less than 10% hybrid progeny even when 75% of the pollen applied to stigmas was derived from interspecific flowers. The frequency of hybrid seed formation was somewhat greater in I. hexagona, but was still significantly lower than expected across all mixture treatments. Seed set per fruit remained constant across the mixture treatments for both species, but in I. fulva fruit set decreased with an increase in the proportion of interspecific pollen. The data indicate that both pre- and postfertilization processes contribute to discrimination against hybrid seed formation.     

Detection of Coconut cadang-cadang viroid sequences in oil and coconut palm by ribonuclease protection assay

A ribonuclease protection assay (RPA) has been developed for detecting Coconut cadang-cadang viroid (CCCVd) sequences. An RNA probe complementary to full-length CCCVd₂₄₆ was used, terminating at nucleotide 65 in the upper conserved region, and linked to a non-viroid 5' sequence, which acted as an internal control for ribonuclease activity. Extracts from CCCVd-infected coconut ( Cocos nucifera ) and African oil ( Elaeis guineensis ) palms protected three major fragments of approximately 250, 125 and 50 nt and a variable number of minor fragments. Extracts of healthy coconut palms, Potato spindle tuber viroid -infected tomato and transfer RNA did not protect the probe. The approximately 250 nt fragment is predicted to indicate the presence of monomers and dimers of circular CCCVd₂₄₆, linear CCCVd₂₄₆ with the same termini as the probe and point mutants of these forms. The origin of smaller protected fragments is discussed. RPA-detected CCCVd sequences in 13 of 18 oil palms surveyed in a commercial plantation in Malaysia. Signal intensity varied between the positive oil palms and was generally lower than in coconut palms infected with CCCVd. An infection phenotype was implied but not confirmed by the observation that in a group of 10 oil palms with orange leaf spotting, 9 contained CCCVd, whereas in a group of 8 palms without orange spotting, the viroid was detected in 4. Of four coconut palms in Sri Lanka shown by dot-blot assay to contain CCCVd-related RNA, one was shown by RPA to be positive for the CCCVd₂₄₆ sequence. RPA is therefore a robust and sensitive test for CCCVd sequences, and our results show that sequences closely related to CCCVd₂₄₆ are not confined to the Philippines.     

Does natural deposition of pine pollen affect the ovipositional behavior of Frankliniella occidentalis and Frankliniella fusca?

Tree pollen, especially Pinus spp. (Pinaceae), is shed in large quantities every spring in North America. Pine pollen deposition onto leaves was found to significantly influence the ovipositional behaviors of certain thrips species (Thysanoptera: Thripidae) in peanut and tomato leaf choice and no‐choice tests. Pine pollen (Pinus elliottii Engelm.) increased the oviposition rate 2.9‐fold for Frankliniella occidentalis (Pergande) (western flower thrips) and 1.6‐fold for Frankliniella fusca (Hinds) (tobacco thrips) in choice tests averaged over both plant species. These results support the idea that pollen has a greater impact on F. occidentalis behavior than on F. fusca behavior. The most dramatic increase was in peanut, where F. occidentalis only oviposited on leaves dusted with pollen, suggesting that the addition of pollen stimulated this flower thrips to lay eggs on a poor host‐plant part. The impact of pollen on the rate of oviposition by thrips is important because it is the early‐instar nymphs that acquire tomato spotted wilt virus (TSWV), which these two thrips species vector. In a laboratory bioassay, the addition of pine pollen to TSWV‐infected peanut foliage increased the percentage of infected F. fusca after one generation.