Katie Helms

Novel virus-like particles containing circular single-stranded DNAs associated with subterranean clover stunt disease.

Novel virus-like particles, 17-19 nm in diameter, have been isolated from subterranean clover and pea plants infected with the pathogen of subterranean clover stunt disease (SCSD). The structure and genetic organization of these particles suggest that the pathogen of SCSD is representative of a new group of plant DNA viruses. SCS virus-like particles (SCSV) are isometric and band as a single component with buoyant densities of 1.24 g/ml in Cs2SO4 and 1.34 g/ml in CsCl. The A260 nm/A280 nm is about 1.35, which is consistent with an estimated nucleic acid content of 17%. Molecular calculations suggest that the particles have a T = 1 capsid structure containing 60 polypeptide subunits each with Mr of 19,000. Nucleic acid analysis including restriction enzyme digestions of double-stranded cDNAs suggests that SCSV have a divided genome composed of multiple species of circular, single-stranded DNA molecules each of approximately 850-880 nucleotides and that each is encapsidated in a separate particle. Linear and aggregated forms of these DNAs are also detected by gel electrophoresis. Evidence suggests that these virus-like particles are the pathogen of SCSD.

Studies on size of lesions of tobacco mosaic virus on Pinto bean

Abstract Strains U1 and U2 of tobacco mosaic virus produced both macroscopic (macro) and microscopic (micro) necrotic lesions on leaves of Phaseolus vulgaris L. var. Pinto. The lesions were recognizable also as starch lesions on leaves bleached with alcohol and stained with iodine. On green leaves, lesions of the two strains were distinguishable by color and size. Infectivity tests showed that virus from macro and micro lesions was biologically indistinguishable. In addition, the population distribution patterns of lesion size indicated that macro and micro lesions together constituted a unimodal, positively skew population. Therefore (1) the division of the lesion population into macro and micro lesions was an arbitrary division of lesions into two size groups; (2) the ratio of numbers of macro to starch lesions (at a magnification of 10×) provided a measure of lesion size; and (3) counts of starch lesions provided a more accurate measure of numbers of infections than counts of macro lesions. The ratio of numbers of macro lesions to numbers of starch lesions ranged from 45% to 75% for strain U1 and from 1% to 20% for strain U2. Size of lesions of strains U1 and U2 was increased by 80% and 76%, respectively, when ribonucleic acid rather than complete virus was used as inoculum. Size of lesions of strain U1 was increased with increase in virus concentration; the rate of increase in the ratio of macro to starch lesions per doubling of virus concentration was approximately 1%. For strain U1 there was heterogeneity in respect to lesion size between leaves of the same or different age and between areas within leaves.

Light-induced susceptibility of Phaseolus vulgaris L. to tobacco mosaic virus infection I. Effects of light intensity, temperature, and the length of the preinoculation dark period

Abstract When dark-treated plants of Phaseolus vulgaris L. var. Pinto were exposed to light for 2–123 minutes before they were inoculated, their susceptibility to tobacco mosaic virus increased up to 5-fold. After the dark treatment, the rate of increase in susceptibility was at first greater at a light intensity of 4000 ft.c. than at 640 ft.c., and greater at 30° than at 25°. Plants were more susceptible at the end of a dark treatment of 24 hours than at the end of a dark treatment of 48 hours. But when plants that were dark-treated for 24 hours, and plants that were dark-treated for 48 hours, were exposed to light of 640 ft.c. for about 90 minutes, they became equally susceptible. It is suggested that a substance directly or indirectly concerned with the establishment of infection, is depleted or inactivated during darkness and is synthesized or made available in response to photosynthesis or another photo response. The data indicate that the common procedure of dark-treating plants before they are used for local lesion assay can cause a significant experimental error unless the assay is designed to eliminate the effect of changing susceptibility.

Light-induced susceptibility of Phaseolus vulgaris L. to tobacco mosaic virus infection II. Daily variation in susceptibility

Abstract Pinto bean plants grown under a photoperiod of 8 hours of light and 16 hours of darkness, showed a daily variation in susceptibility to TMV. Fewer lesions formed in plants inoculated at the beginning of the light period (zero time) than in plants exposed to light for 6 minutes before they were inoculated. The number of lesions reached a maximum when plants received light of 4000 ft-c for 54 minutes before inoculation, or light of 640 ft-c for 105 minutes. With further exposure of plants to preinoculation light, the number of lesions decreased, and when plants received 6–8 hours of light before they were inoculated, lesion number did not differ significantly from that in plants inoculated at zero time. In plants inoculated during the 16 hours of darkness that followed the 8-hour photoperiod, the number of lesions remained essentially constant if the light intensity during the preceding light period was 4000 ft-c, but decreased if the light intensity during the preceding light period was 640 ft-c. In plants inoculated after 24 hours of continuous light at either 4000 ft-c or 640 ft-c, the number of lesions did not differ significantly from that in plants inoculated at zero time.

Translocation of tobacco mosaic virus and photosynthetic assimilate in Nicotiana glutinosa

Abstract Translocation of tobacco mosaic virus and photosynthetic assimilate were examined in partially defoliated plants of Nicotiana glutinosa kept under a constant temperature of 36 °C and constant light of 64 W m −2 (400–700 nm). Virus multiplied prior to long distance movement, when inoculated by rubbing or by jet-injection. The earliest detection of movement of virus was at 18 h after inoculation. Movement of virus was detected earlier in relatively young plants than in relatively mature plants. At 18 or 24 h after inoculation, virus was detected in more segments of the petiole and stem when an upper, almost-expanded leaf rather than a lower, older leaf was inoculated, and this was associated with earlier multiplication in an upper than a lower leaf. At 0 to 24 h after exposure of plants to constant temperature and light, export of assimilate measured as dry matter was greater from an upper than a lower leaf. At an early stage of infection, the distribution of virus in adjacent segments of the petiole and stem was discontinuous. The highest estimated mean speed of movement of virus (2·3 cm h −1 was lower than that of 14 C labelled photosynthetic assimilate (43 cm h −1 or 32 P applied as orthophosphate (92 ± 20 cm h −1 ).

Masking of local lesions and the biology of infections of tobacco mosaic virus on pinto bean.

Abstract On leaves of Pinto bean ( Phaseolus vulgaris L.) maintained at 25°C, numbers of macroscopically identifiable necrotic lesions (macro lesions) of tobacco mosaic virus strain U2 (TMV-U2), but not of TMV-U1, decreased between 2 and 6 days after inoculation. Throughout this period macro lesions of both strains remained detectable as starch lesions on bleached, iodine-stained leaves. The inoculated leaves expanded during the course of the experiments. Areas of starch lesions of TMV-U2 reached a maximum at 2 days, whereas those of TMV-U1 continued to increase in size during a period of 5 days. The maximum mean lesion area for TMV-U2 and TMV-U1 was 0.91 × 10 −2 and 4.58 × 10 −2 mm 2 , respectively. Macro lesions of TMV-U2 and TMV-U2 RNA (ribonucleic acid) appeared earlier and more synchronously than those of TMV-U1 and TMV-U1 RNA. The mean time of appearance of 50% of the macro lesions of TMV-U2 and TMV-U2 RNA was 29.4 and 27.5 hours, respectively; for TMV-U1 and TMV-U1 RNA it was 47.5 and 34.4 hours, respectively. The mean time of appearance of 50% of the starch lesions (measured at a magnification of 10×) was later than for macro lesions. It was concluded that the time-course decrease in numbers of macro lesions of TMV-U2 was due to masking of lesions caused by postinoculation expansion of leaves in association with rapid but limited lesion growth.

Interference between two strains of tobacco mosaic virus in leaves of pinto bean

Abstract In leaves of Phaseolus vulgaris L. var. Pinto inoculated with mixed inocula containing strain U1 and increasing concentrations of strain U2 of tobacco mosaic virus, there were fewer macroscopic lesions than in leaves inoculated with U1 alone. This was an expression of the reduced size of U1 lesions. The total number of lesions detectable at a magnification of 10 × (in bleached, iodine-stained leaves), increased with increase in concentration of U2. It is suggested that the early multiplication of U2 caused metabolic changes which inhibited the multiplication of U1.

HEAT-INDUCED VARIATIONS IN NUMBER AND SIZE OF LESIONS OF TOBACCO MOSAIC VIRUS ON PINTO BEAN.

Abstract The effects of short preinoculation and post-inoculation heat treatments (49°–50°C for 30 seconds) on number and size of lesions were examined on Pinto bean ( Phaseolus vulgaris L.) leaves inoculated with complete virus and with ribonucleic acid of strains Ul and U2 tobacco mosaic virus (TMV-Ul, TMV-U1 RNA, TMV-U2, and TMV-U2 RNA). Assays of starch lesions provided a more valid measure of the effects of heat on lesion development than did assays of macroscopic lesions. With all fofr inocula, there were five recognizable trends in the time-cofrse patterns for numbers of starch lesions in heated leaves: (1) in leaves heated 4 and 6 hofrs before inoculation the ratio between the number of lesions in heated leaves and the number of lesions in unheated leaves ( t/c ) was greater than unity with all inocula except TMV-U1; (2) as the interval between heat treatment and inoculation was decreased from 6 hofrs to 5 minutes, t/c dropped below unity with the shorter intervals; (3) leaves inoculated within 20 seconds after heating was finished developed more lesions than did those inoculated 5 minutes after heating; (4) when heating was started within 20 seconds after inoculation, t/c was less than unity and the value of t/c increased progressively toward unity as the interval between inoculation and heating was increased to aboft 5 hofrs; (5) in leaves heated 5 hofrs or more after inoculation, t/c exceeded unity with TMV-U2 and with TMV-U1 and was approximately equal to unity with the other 2 inocula. With all fofr inocula, lesions in heated leaves usually were larger than those in unheated leaves. Variations among inocula in the number (and size) of lesions in heated leaves relative to that in unheated leaves were correlated with known differences in the characteristics of infection of the inocula. The time-cofrse patterns for number and size of lesions were interpreted to indicate that heated cells pass throfgh several physiological conditions which are recognizable by a changing capacity to support the initiation and development of infections. Excluding the effect of heat applied within a few seconds before or after inoculation, which was attributed to an ephemeral effect on virus entry into cells, this sequence of changing susceptibility cofld be accofnted for simply, if heated cells pass from a condition initially unfavofrable to virus multiplication, to one which is more favofrable than normal.

Assay of tobacco mosaic virus in bean leaves expanded under far-red light

Abstract Detached, etiolated leaves of Phaseolus vulgaris L. cv. Pinto, expanded at constant temperature under continuous far-red light, formed local lesions in response to infection with tobacco mosaic virus. Numbers of lesions increased when seedlings grown in vermiculite were watered with nutrient rather than with demineralised water, when leaves were inoculated 6, 7, or 8 days after seeds were sown rather than after 5 days, and when leaves received far-red light after inoculation rather than darkness. A preinoculation dark treatment of 24 hr decreased numbers of lesions in leaves inoculated 5 days after seeds were sown but did not significantly affect those in older leaves. Leaves could be used for virus assay, since there was a linear relationship between the number of lesions formed and the concentration of virus inoculated (from 0.25 to 1.00 μg/ml). For assay purposes, satisfactory lesions developed in leaves of 7-day-old seedlings grown under continuous far-red light and incubated after inoculation in darkness for 48 hr. Advantages of the system are noted.