Barley mild mosaic virus inside its fungal vector, Polymyxa graminis

In an electron microscope study to investigate the association of barley mild mosaic virus (BaMMV) with its fungal vector, Polymyxa graminis, thin sections were made of zoospores of the vector and of barley roots containing different stages in the life cycle of the fungus. Immunogold labelling was used to identify the virus in sections. Labelled bundles of presumed virus particles were seen in c. 1% of zoospores liberated from plant roots and in zoospores inside zoosporangia. A few zoosporangial plasmodia had localised labelling but no bundles were seen. No virus particles were seen in sections of resting spores.     

Confirmation of the transmission of barley yellow mosaic virus (BaYMV) by the fungus Polymyxa graminis

Resting spores (cystosori) of Polymyxa graminis, selected from roots of barley plants infected with barley yellow mosaic virus (BaYMV), were used to start mono-fungal sand cultures. Out of 20 attempts using over 800 cystosori, P. graminis became established in 12, and in two of these BaYMV symptoms also occurred. BaYMV was detected by ELISA in extracts of dried roots heavily infected with cystosori and in zoospores of P. graminis. Calculations suggested that, on average, each zoospore carried less than 100 virus particles. In two virus acquisition experiments, non-viruliferous isolates of P. graminis failed to acquire BaYMV from roots of mechanically-inoculated plants. In two further experiments, non-viruliferous isolates were grown on rooted tillers produced from healthy plants and those infected with BaYMV by either vector or mechanical inoculation. Zoospores and cystosori of P. graminis subsequently transmitted the virus, but only from plants where it had been introduced by the vector. Repeated mechanical transmission appeared to have selected a strain of virus that could not be acquired and/or transmitted by the vector. The results provide convincing evidence that P. graminis is a vector of BaYMV but suggest that, in natural populations, only a small proportion of spores may be viruliferous.     

The effect of cultivar used as host for Polymyxa gmminis on the multiplication and transmission of barley yellow mosaic virus (BaYMV)

A viruliferous isolate of the fungal vector Polymyxa graminis was grown on roots of barley cultivars immune or susceptible to barley yellow mosaic virus (BaYMV). Zoospores or resting spores of the vector produced on different cultivars were then inoculated to a virus-susceptible test cultivar. Although the vector established in all treatments, transmission of BaYMV was rare and usually nil from immune cultivars; amounts of virus detected serologically in their roots were very low, thus showing that resistance was to virus multiplication. If immune cultivars decrease the virus content of vector populations in the field, this would have important implications for disease control.     

MORPHOLOGY AND THERMAL DEATH POINT OF OLPIDIUM BRASSICAE

The morphology of single-sporangial isolates of lettuce, tomato, mustard, and oat Olpidium brassicae (Wor.) Dang. growing in their respective hosts as well as in cowpea were compared in situ and after extraction from the roots. The sporangia, zoospores, and resting spores of all isolates were within the established limits of the species. Single exit tubes or pores predominated which means that these isolates should not be transferred to the genus Pleotrachelus. A satisfactory assay for the presence of resting spores was developed by air-drying of the roots for a week or longer. This treatment killed zoospores and vegetative sporangia, but not resting spores. Factors affecting resting spore formation were investigated unsuccessfully. The thermal death point of zoospores of mustard isolates that did not form resting spores was between 40 and 45 C for 10 min.     

MORPHOLOGICAL AND ECOLOGICAL STUDY OF PHYSODERMA DULICHII

A new species of aquatic Phycomycete, Physoderma dulichii Johns, parasitic on the aquatic sedge Dulichium arundinaceum (L.) Britt., is described from northern Michigan. This parasite infects and kills the upper epidermal cells of the host leaves. Macroscopically, infection by P. dulichii is indicated by striking brown bands with irregular margins, at intervals on the upper surfaces of the leaves. Like other species of Physoderma, this organism's development includes two distinct phases, an epibiotic monocentric phase producing asexual zoospores and an endobiotic polycentric phase bearing thick-walled resting spores that germinate after an extensive period of maturation at low temperature to form zoospores. The morphology and development of the two phases and of resting spore germination are reported in detail. Only the immature leaves of the host are susceptible to infection, which may be initiated by the introduction of mature resting spores, zoospores from germinated resting spores, or zoospores from epibiotic sporangia. Resting-spore zoospores may also produce the endobiotic stage directly. Initiation of infection in nature requires that the terminal cluster of immature leaves on the host plant be submerged, but infection of subsequently formed leaves of emergent culms can be accomplished through the agency of zoospores from epibiotic sporangia on older leaves. The relation of infected stands of hosts to their environment is discussed and the importance of standing water to infection noted. The geographical distribution of the parasite shows correlation with the drainage basins of the Great Lakes, the St. Lawrence River, and the northern Atlantic Coastal Plain     

Further evidence on the nature of the dependence of carrot mottle virus on carrot red leaf virus for transmission by aphids

Preparations were made from chervil plants doubly infected with carrot mottle virus (CMotV) and its helper virus, carrot red leaf (CRLV), on which it depends for transmission by the aphid Cavariella aegopodii, by the procedure developed previously for CRLV. The preparations contained 25 nm isometric particles which were indistinguishable from those of CRLV but possessed aphid-transmissible infectivity of both viruses and manually transmissible infectivity of CMotV. Only one sedimenting and buoyant density component was detected. The manually transmissible CMotV infectivity was resistant to freezing and to organic solvents, treatments that destroyed the CMotV infectivity in extracts from singly infected plants. The aphid-transmissible CMotV infectivity in preparations from CRLV/ CMotV-infected plants, and that in extracts from CRLV/CMotV-carrying C. aegopodii, was abolished by treatment with CRLV antiserum but not with normal serum. These results show that transmission of CMotV by C. aegopodii is dependent on the packaging of its RNA in coats composed partially or entirely of CRLV particle protein. The aphid Myzus persicae does not transmit CRLV or CMotV from plants mixedly or singly infected with these viruses but it is a vector of beet western yellows virus (BWYV) and potato leafroll virus (PLRV) and it transmitted CMotV from plants that also contained either of these viruses. This suggests that the coat proteins of BWYV and PLRV can substitute for that of CRLV in packaging CMotV nucleic acid and thereby confer on it their own vector specificities.     

Identification of specific cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment

Cucumber necrosis virus (CNV) is naturally transmitted in the soil by zoospores of the fungal vector Olpidium bornovanus. Successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. Mechanically passaged CNV was screened for mutants deficient in fungus transmission. We found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (WT) CNV. Results of in vitro virus-zoospore binding assays show that each mutant binds to zoospores less efficiently than WT CNV (21 to 68%), suggesting that defects in transmission for these mutants are at least partially due to inefficient zoospore binding. Analysis of the structure of the CNV coat protein subunit and trimer indicates that affected amino acids in all of the mutants are located in the shell or protruding domain and that five of six of them are potentially exposed on the surface of the virus particle. In addition, several of the mutated sites, along with a previously identified site in a region of subunit-subunit interaction in the coat protein shell domain (M. A. Robbins, R. D. Reade, and D. M. Rochon, Virology 234:138-146, 1997), are located on the particle quasi-threefold axis, suggesting that this region of the capsid may be important in recognition of a putative zoospore receptor. The individual sites may directly affect attachment to a receptor or could indirectly affect attachment via changes in virion conformation.     

A shift of vector specificity acquired by a begomovirus through natural homologous recombination

Recombination is common in plant viruses such as geminiviruses, but the ecological and pathogenic consequences have been explored only in a few cases. Here, we found that a new begomovirus, tomato yellow leaf curl Shuangbai virus (TYLCSbV), probably originated from the recombination of Ageratum yellow vein China virus (AYVCNV) and tobacco curl shoot virus (TbCSV). Agrobacterium-mediated inoculation showed that TYLCSbV and AYVCNV have similar levels of infectivity on tomato and tobacco plants. However, the two viruses exhibit contrasting specificities for vector transmission, that is, TYLCSbV was efficiently transmitted by the whitefly Bemisia tabaci Mediterranean (MED) rather than by the whitefly B. tabaci Middle East-Asia Minor 1 (MEAM1), whereas AYVCNV was more efficiently transmitted by MEAM1. We also showed that the transmission efficiencies of TYLCSbV and AYVCNV are positively correlated with the accumulation of the viruses in whitefly whole bodies and organs/tissues. The key coat protein amino acids that determine their accumulation are between positions 147 and 256. Moreover, field surveys suggest that MED has displaced MEAM1 in some regions where TYLCSbV was collected. Viral competition assays indicated that TYLCSbV outcompeted AYVCNV when transmitted by MED, while the outcome was the opposite when transmitted by MEAM1. Our findings suggest that recombination has resulted in a shift of vector specificity that could provide TYLCSbV with a potential selective transmission advantage, and the population shift of whitefly cryptic species could have influenced virus evolution towards an extended trajectory of transmission.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XIV. PHYSODERMA CALAMI

Physoderma calami, a chytrid (Phycomycetes), has not been studied or apparently even collected in Europe since 1895 and has never been recorded in this hemisphere. Material widely distributed in herbaria indicated it was a Physoderma but no details save color, shape, and size of resting spores were known. A study of living material from Vermont indicates resting spore germination occurs by dehiscence of a lid and production of a sporangium with zoospores. The latter may form either an epibiotic or “ephemeral” sporangial stage and zoospores, or an endobiotic one, with extensive polycentric thallus bearing turbinate cells along the rhizoids, and, as outgrowths of these turbinate cells, straw-colored, thick-walled resting spores. The latter stage produces dark-brown spots on infected parts of the host.     

Transmission of tomato leaf curl geminiviruses by Bemisia tabaci: effects of virus isolate and vector biotype

Cultures of Bemisia tabaci from Ivory Coast (IC), Pakistan (PK) and USA (US B-type) were compared for the frequency with which they transmitted three tomato geminivirus isolates: Indian tomato leaf curl virus from Bangalore (ITmLCV), and tomato yellow leaf curl viruses from Nigeria (TYLCV-Nig) and Senegal (TYLCV-Sen). Frequency of transmission from tomato to tomato depended both on the whitefly culture and the virus isolate. US B-type and IC whiteflies transmitted TYLCV-Sen more frequently than ITmLCV whereas PK whiteflies transmitted ITmLCV more frequently than TYLCV-Sen. US B-type whiteflies transmitted both viruses four to nine times more frequently than IC whiteflies. TYLCV-Nig was transmitted rarely by US B-type and not at all by IC whiteflies. Previous work indicates that the geminivirus coat protein controls vector transmissibility. The differential adaptation of TYLCV-Sen to transmission by US B-type whiteflies and of ITmLCV to PK whiteflies was associated with a large difference in epitope profile of the coat proteins of the two viruses. Also, the readily transmissible TYLCV-Sen differed appreciably in epitope profile from the poorly transmissible TYLCV-Nig, which reached a consistently greater concentration in source tissues but lacked epitope 18. However, the lack of epitope 18 in ITmLCV did not prevent its transmission by US B-type whiteflies. Differences in frequency and specificity of geminivirus transmission by whitefly cultures from different countries therefore were associated with differences among epitope profiles of the coat proteins of the viruses, but the structural features of the proteins that control transmission remain to be determined.     

Observations on chytridiaceous parasites of Phanerogams

Summary A reexamination of Physoderma maculare Wallroth the type species of the genus, on Alisma, confirms Clinton's (1902) account of the production of an epibiotic stage from resting spore zoospores. The latter on mature host tissue may also give rise to the endobiotic stage which bears resting spores. On seedlings, however, resting spore zoospores produce only epibiotic sporangia. The fungus could not successfully infect Sagittaria, a closely related host.Contribution No. 1152     

Ultrastructure of chytridiomycete and oomycete zoospores using spray-freeze fixation

Summary The ultrastructure of zoospores of several zoosporic fungi was examined using a modified cryofixation technique. An atomizer was used to spray a zoospore suspension into the cold propane reservoir of a conventional plunge freeze-substitution apparatus. Spray-freeze fixation and freeze-substitution of zoospores porvided better fixation of vacuolar structures, membranes and the extracellular coat than that obtained with chemical fixation. The overall shape of cryofixed spores was closer to that seen in living zoospores. Two types of vacuoles were seen in cryofixed zoospores ofMonoblepharella andChytridium. One type of vacuole contained electron-opaque material within the lumen while the other type had no visible internal material in the lumen and appeared to be part of the water expulsion vacuole complex. Coated pits and coated vesicles were observed associated with both the water expulsion vacuoles and the plasma membrane inMonoblepharella andPhytophthora, suggesting that endocytosis of the plasma membrane and expulsion vacuoles is part of membrane recycling during osmoregulatory events. An extracellular coat was seen on the outer surface of cryofixed zoospores ofMonoblepharella sp.,Chytridium confervae andPhytophthora palmivora without the use of carbohydrate-specific stains. The spray-freeze method gave good and reproducible fixation of the wall-less spores in quantities greater than those obtained in previously described zoospore cryofixation studies. The technique is potentially useful for cell suspensions in that freeze damage from excess water is limited.Abbreviations ddH₂O deionized distilled water - PME Pipes/MgCl2/EGTA buffer - WEV water expulsion vacuole     

The Partial Characterization of a Badnavirus Infecting the Greater Asiatic or Water Yam (Dioscorea alata)

A bacilliform virus from Dioscorea alata, designated Dioscorea alata bacilliform virus (DaBV), from Barbados and West Africa and from other Dioscorea spp. from West African, Carribean, Asian and South American countries, has been characterized. The virus was transmitted by the mealybug, Planococcus citri and by mechanical transmission of partially purified preparations to several Dioscorea spp. DaBV was serologically related to a distinct bacilliform virus from Dioscorea bulbifera, to one isolate of sugarcane bacilliform badnavirus and two isolates of banana streak badnavirus (BSV) but was not related to another isolate of BSV or to Kalanchoe top spotting or cacao swollen shoot badnaviruses. The coat protein of DaBV was about 56 kDa and the nucleic acid was double-stranded DNA of about 7.5 kbp, part of which showed distant homology with other badnaviruses. Thus, DaBV is a distinct hitherto uncharacterized badnavirus.     

Plant infection by two different viruses induce contrasting changes of vectors fitness and behavior

Insect-vectored plant viruses can induce changes in plant phenotypes,thus influencing plant-vector interactions in a way that may promote their dispersal according to their mode of transmission (i.e.,circulative vs.noncirculative).This indirect vector manipulation requires host-virus-vector coevolution and would thus be effective solely in very specific plant-virus-vector species associations.Some studies suggest this manipulation may depend on multiple factors relative to various intrinsic characteristics of vectors such as transmission efficiency.In anintegrative study,we tested the effects of infection of the Brassicaceae Camelina sativa with the noncirculative Cauliflower mosaic virus (CaMV)or the circulative Turnip yellows virus (TuYV)on the host-plant colonization of two aphid species differing in their virus transmission efficiency:the polyphagous Myzus persicae,efficient vector of both viruses,and the Brassicaceae specialist Brevicoryne brassicae,poor vector of TuYV and efficient vector of CaMV.Results confirmed the important role of virus mode of transmission as plant-mediated effects of CaMV on the two aphid species induced negative alterations of feeding behavior (i.e.,decreased phloem sap ingestion)and performance that were both conducive for virus fitness by promoting dispersion after a rapid acquisition.In addition,virus transmission efficiency may also play a role in vector manipulation by viruses as only the responses of the efficient vector to plant-mediated effects of TuYV,that is,enhanced feeding behavior and performances,were favorable to their acquisition and further dispersal.Altogether,this work demonstrated that vector transmission efficiency also has to be considered when studying the mechanisms underlying vector manipulation by viruses.Our results also re- inforce the idea that vector manipulation requires coevolution between plant,virus and vector.     

A Bemisia tabaci midgut protein interacts with begomoviruses and plays a role in virus transmission

Begomoviruses are a major group of plant viruses, transmitted exclusively by Bemisia tabaci (Gennadius) in a persistent circulative non‐propagative manner. The information regarding molecular and cellular basis underlying Begomovirus – whitefly interaction is very scarce. Evidences have suggested that the insect gut possesses some crucial protein receptors that allow specific entry of virus into the insect haemolymph. We have performed yeast two hybrid gut cDNA expression library screening against coat protein of Tomato leaf curl New Delhi virus (ToLCV) and Cotton leaf curl Rajasthan virus (CLCuV) as bait. Midgut protein (MGP) was the common protein found interacting with both ToLCV and CLCuV. MGP was localized in whole mount B. tabaci as well as in dissected guts through confocal microscopy. Pull down and dot blot assays confirmed in vitro interaction between ToLCV/CLCuV coat protein and MGP. Immunolocalization analysis also showed colocalization of ToLCV/CLCuV particles and MGP within insect's gut. Finally, anti‐MGP antibody fed B. tabaci, exhibited 70% reduction in ToLCV transmission, suggesting a supportive role for MGP in virus transmission.     

A novel method for detection of viable zoospores of Pythium in irrigation water

A membrane filtration test has been developed for the detection of viable zoospores of Pythium species. Zoospore suspensions were filtered through 5 (m nitrocellulose membranes and the membranes incubated overnight in 0.07 m glucose, rifamycin (30 mg litre^-1) and pimaricin (100 mg litre^-1). Zoospore germlings were detected using a polyclonal antiserum, raised to mycelial surface washings of five Pythium spp., and visualised with Sigma fast red. The assay gave positive results for all Pythium spp. tested and also to zoospores of Phytophthora cryptogea. Of 10 fungal species isolated from commercial irrigation water, two were detected by the polyclonal antiserum in ELISA tests but only one produced detectable zoospore germlings. The latter isolate was later identified as a Pythium sp. Irrigation water samples collected from commercial UK nurseries yielded zoospores of both Pythium and Phytophthora spp. which, using the assay, were positively identified. Results indicated greater sensitivity than was seen with conventional plating methods. This is a test which could be adapted for on-site use in commercial nurseries.     

CotL,a new morphogenetic spore coat protein of Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σ^E and σ^K. CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.