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Clover yellow mosaic virus infection of seedling roots of Pisum sativum
Physiological
Plant Pathology
(1974)
4, 2 19-228
Clover yellow mosaic virus infection of seedling roots of Pisum sativumt H. E. MOLINE$ Botany
and R.E.
and Plant Pathology
(Accepdfm
Publication
FORD
Department,
November
Iowa State University,
Ames, Iowa 50010,
U.S.A.
1973)
Clover yellow mosaic virus (CYMV) multiplication was studied in roots of Pisum sativum (garden pea). Mechanical inoculation of pea roots with CYMV resulted in systemic infection of shoots. CYMV translocation from roots was directly related to temperature. CYMV was purified from pea roots with minimal alterations of existing schedules; yields were comparable to those from leaves (45 mg/kg, roots; 50 mg/kg, leaves). Electron-microscopic observations of thin-sectioned CYMV-infected root-tip tissue revealed large numbers of viral inclusions in meristematic root initials. Nitrogen and carbohydrate levels were significantly altered in roots and shoots of infected seedlings.
INTRODUCTION
This, the third in a series of papers devoted to the study of viruses in plant roots [18, 191 considers a member of the Potexvirus group, clover yellow mosaic virus (CYMV) . Several previous studies have shown that the rates of movement of viruses within plants, especially to and from roots, vary considerably [5, 6, 24, 2.51. Meristem tip cultures have been used in many instances to free vegetatively propagated crops from viruses [14]. This technique is not universally effective for all plant-virus combinations, however, because a number of viruses are capable of invading meristematic tissue [2, 7, 8, 26, 271. A portion of this study has been devoted to the examination of root meristems of infected seedlings. Since virus replication entails the synthesis of virus-specific proteins, disturbances in nitrogen metabolism of infected plants is a normal consequence of virus infection [4, 9, 13, 231. S ome viruses seem to have little effect on carbohydrates, however, while others do alter their rate of synthesis and (or) rate of translocation [3, 12, 15, 17, 281. The three viruses used in this study have shown considerable differences in effects on nitrogen and carbohydrate metabolism of infected hosts [18, 191. A preliminary study with CYMV suggests that roots may provide certain advantages over leaves for virus purification [IO]. We also examined the effects of CYMV infections on Pisum sativum (garden pea) root physiology, cytology via electron microscopy and viral translocation. t Journal Ames, Iowa. degree, Iowa $ Present
Paper No. J-7619 of the Iowa Agriculture and Home Economics Experiment Station, Project No. 1878. This study is a portion of a thesis by the senior author for the Ph.D. State University, and was supported in part by the Iowa Experiment Station. address: ARS-USDA, AMRI-HCML, ARC-West, Beltsville, Md 20705.
220
H. E. Molinemd
R. E. Ford
MATERIALS AND METHODS Virus replication and tramlocation Pratt’s B strain of CYMV [ZO] was maintained in the greenhouse on Inca rosea L. Periodic transfers were made to garden pea and Gomphrena globosa L. G. globasa was used for all local-lesion assays; “Wilt Resistant Perfection” pea was the source of inoculum and the host for all experiments. Pea seedlings used for leaf inoculation were grown in a steamed soil mixture [18]. For root inoculations pea seedlings were germinated on autoclaved germinating papers. The primary root of each seedling was inoculated 6 to 7 days after germination. Five inoculation techniques [18] were tested. Control plants were treated in the same manner, substituting healthy sap for inoculum. After inoculation, roots were rinsed with tap water, and four seedlings were planted in each 4-in clay pot containing vermiculite. The pots were placed in plastic trays, eight pots/tray, and watered from beneath to maintain proper moisture conditions. Plants were supplemented with half strength Hoagland’s solution [16] twice weekly. Seedling roots and leaves were assayed at predetermined intervals to detect when the virus had replicated in the roots and (or) had moved to the shoots. In the first group of experiments, at least 10 seedlings were assayed onto G. globosa at each interval. The opposite-leaf assay technique [25] was used with a purified frozen preparation of CYMV serving as the standard reference from which aliquots were removed for each assay. Tissue was diluted on a weight/volume basis with O-01 M-phosphate buffer (pH 7.0 to 7.2) and inoculated onto eight leaves. After we established that CYMV increased in roots and moved to shoots, relative virus concentrations in root and shoot were determined. Lesions were counted 10 days after inoculation. Lesion counts were made from all leaves having fewer than 300 lesions; greater numbers were estimated. Physiology of qfected plants Fourteen pans of “Wilt Resistant Perfection” peas were seeded with 45 seeds per pan. Pea seedlings in six pans were inoculated with CYMV. The other eight were rubbed with healthy sap and served as controls. Pea seedlings were harvested and treated as described previously [18]. Th e seedlings from each pan served as one treatment. These plants were counted and separated into plant parts: roots, shoots and seeds. Fresh weights and number of plants were recorded, and all parts were placed in 95% ethanol for storage. Pea seedlings infected with CYMV were treated as seedlings in previous physiology studies [18, 191. No significant differences existed among gram fresh weight, gram dry weight and per-plant basis; therefore, data are presented on a gram-fresh weight basis. Pur&ation CYMV was purified from roots and shoots of infected pea seedlings. Seedlings were grown in either vermiculite or soil and were inoculated 2 to 3 days after emergence. Plants were rinsed with distilled water and placed in a greenhouse for 12 to 16 days.
Clover yellow mosaic virus infection of seedling
roots
221
Then they were harvested and divided into roots and shoots for comparative purification. Soil or vermiculite was removed from roots by gently rinsing them in running water [18]. Tissue was either purified immediately or stored at - 14 “C. CYMV was purified by grinding fresh or frozen tissue in 1 vol. of 0.01 Mphosphate buffer, pH 7.0, with a blendor. The tissue was then expressed through four layers of cheesecloth, and the filtrate collected. The brei was discarded. The filtrate was centrifuged at 3000 g for 10 min, the supernatant was saved and the green pellet was discarded. Attempts were made to remove chlorophyll and plant proteins by (i) heating the supernatant at 40 “C for 10 to 30 min and (ii) after the first low-speed centrifugation, by blending chloroform in the supernatant to final concentrations of 3, 10, 25, or 50% (v/v) for 2 min. The 25% chloroform treatment proved superior and was used for all subsequent purifications. Then, the mixture was centrifuged at 3000 g for 10 min; the upper supernatant was collected for further treatment, and the solid pellet and green lower layer were discarded. The supernatant was centrifuged for 120 min at 78 000 g. The supernatant was discarded, and one-twentieth of the original volume of buffer added to the pellets. Pellets were either allowed to resuspend overnight or were resuspended immediately by homogenizing in a glass homogenizer. The solution was then centrifuged at 3000 g for 10 min, and the pellet discarded. The supernatant was centrifuged at 234 000 g for 60 min. Then, the supernatant was discarded after assay; the pellet was resuspended in 1 to 2 ml of buffer and centrifuged at 3000 g for 10 min. The supernatant was layered on 10 to 50% sucrose or 3 M-CsCl gradients. Sucrose gradients were run for 4 h in a Beckman SW 25.1 rotor. CsCl gradients were run for 24 h in a Beckman SW 50.1 rotor. After centrifugation, the density-gradient tubes were fractionated with the aid of an ISCO density-gradient fractionater and U.V. analyzer, the fractions collected and additional U.V. scans made on all fractions with a Beckman DBG spectrophotometer. Virus-containing zones were dialyzed against phosphate buffer for infectivity assay. Yields of virus were based on infectivity assays and U.V. scans, which could be interpreted as to yield in mg/lOO ml. Electron
microscopy
Pea root tips and portions of mature root tissue were examined for the presence of viral inclusions. Infected roots were fixed 8, 12, 16 and 24 days after inoculation and prepared for observation as described previously [28]. RESULTS Translocation
Although mechanical inoculation techniques with CYMV on pea roots resulted in infection (Table l), rubbing silicon carbide (600 mesh) dusted roots between thumb and forefinger, which had been dipped in inoculum, gave most consistent results (Table 1) ; therefore, this technique was used in all subsequent studies. CYMV detected in roots 3 days after inoculation, increased rapidly in titer to a maximum 1 x lob3 8 days after inoculation. CYMV detected in leaves 6 days after root inoculation reached a maximum titer (2.5 x lOA) 26 days after inoculation
I-L E. Moline
222 TABLE
Comparatiz
transmission
of CYMV
Treatmenta
R. E. Ford
1
to pea hoots after inoculatin~~ the wets by jioe techniques Test
Experiment
and
Inoculated
plants --. Infected”
Check -_-.-____Inoculated
plants Infected
1
A B C D E
66 96 96 96 96
65 94 90 9.5 89
66 64 64 64 64
0 0 0 0 0
2
A B c D E
96 96 96 96 96
96 90 93 96 85
64 64 64 64 64
0 0 0 0 0
3
A B C D E
96 96 96 96 96
92 93 96 94 73
64 64 64 64 64
0 0 0 0 0
B Five treatments employed: (A) roots were dusted with silicon carbide, then rubbed between forefinger and thumb which had been dipped in inoculum; (B) roots were dusted and inoculum was applied with a camel’s hair brush that had been dipped in inoculum; (C) roots were dusted, rubbed between forefinger and thumb to injure them then immediately dipped in inoculum; (D) inoculum was injected into the Steele of the root with a hypodermic syringe; and (E) inoculum was mixed with silicon carbide and sprayed onto roots with an artist’s air brush. h Symptoms were observed on shoots of inoculated seedlings 15 to 20 days after inoculation.
(Fig. 1). Infected plants were necrotic 20 days after inoculation and were usually dead 40 days after inoculation. Roots of inoculated seedlings showed severe browning 15 days after inoculation. The translocation rate of virus from roots to leaves was directly correlated with an increase in temperature (Fig. 2). When root-inoculated seedlings were placed at 26, 20, 15 and 10 “C in controlled-environment chambers, CYMV was recovered from leaves, respectively, 4, 5, 8 and 9 days after root inoculation. Virus was recovered from roots 3 days after inoculation at all four temperatures tested (Fig. 2). Physiology of a$ected plants Nitrogen content. CYMV affects both residue and soluble nitrogen levels similarly in infected pea shoots (Table 2). Although lower 4 days after inoculation, nitrogen levels then increased 8 and 12 days after inoculation, resulting in an accumulation of total nitrogen (Table 2). Nitrogen content of infected roots decreased at each sampling date. Four days after inoculation, residue nitrogen content decreased IS:&, and soluble nitrogen content decreased 23 %. This trend was also observed 8 and 12 days after inoculation, leading to a total reduction of nitrogen in all infected roots (Table 2). Carbohydrates. Sucrose levels in infected pea shoots were greater at 4, 8 and 12 days after inoculation (Table 3). Reducing-sugar content rapidly increased 4 days after
Clover
yellow
mosaic
virus
infection
of seedling
roots
223
inoculation, was comparable with healthy shoots 8 days after inoculation and decreased 12 days after inoculation resulting in no net change between healthy and infected shoots 12 days after inoculation. 100000
:
2.
,! 10000
., l \
a-.;.’
-
.
\
l‘\\sh$ot
9
9.
B 1000
-
I I 100
1. \ #, .-. root
5
5 .L 2
1.
2’
z
z
\
1 .a-, Lf’
-j
Y,.
-
: ( I
B -1 IO -. I I I ’
I 0
I IO Days
ofler
I 20 moculolion
I 30
FIG. 1. CYMV concentration in pea shoots and roots following root inoculation. Virus titer based on wt/vol dilution of crude sap in 0.01 M-phosphate buffer (pH 7.0). Infected seedlings were assayed at 24-h intervals for the first 12 days, then every other day for the remainder of the experiment. Values are means of 10 assays onto G. globosa. TABLE
Nitrogen Factor
measured
levels in CTMV-infected HO(CO)O
H4
3.1e 37.05 33.6 0.639
3.50 89.32 14.2 0.639
1.3 18.91 47.6 0.666
1.5 29.25 26.6 0.666
2
and healthy pea seedlings (per g fresh c4
weight)
H8
C8
HI2
Cl2
4.1” 69.01 8.3 0.831
4.1c 81.11 12*5** 0.950
5.7c 59.38 13.1 2.02
5.7= 81.58 11.3* 1.24*
2.0 28.16 24.8 0.820
1.9 23.60 30.1** 1.08*
3.2 27.94 24.3 0.972
2.4** 21.85 28-O** 1.32**
Shoot Res N/Sol Total N FW/DWC FW/plantf
Nb
Res N/Sol Total N FW/DW FW/plant
N
2.gc**a
76.46 16.0* 0.655 Root 2:.:3 33.0** 0.858*
(C) seedlings; a Abbreviations refer to date of harvest of healthy (H) and CYMV-infected HO(C0) date of inoculation, H4 and C4 4 days after, H8 and C8 8 days after and H12 and Cl2 12 days after inoculation. b Res N/Sol N = ratio of residue to soluble nitrogen concentration. G Each datum point represents the mean of three experiments replicated three times. d * = infected mean significantly different from healthy at 5% probability level. ** = infected mean significantly different from healthy at 1 o/0 probability level. Split plot analysis of variance. e FW/DW = ratio of fresh weight to dry weight. f FW/plant = total fresh weight per plant part (g).
H. E. Moline
224
!7 !I !
10,000
1000
Tfmm ,a root / 1 ,i ’ /shoot
and
R. E. Ford
/
100
-2 2 ‘;;L &u
IO 15 “C
5 Y) :. ‘S :
9 (cl
10,000
r;o t/ .I
IO00
/~ .2--=m.‘.
/
‘shoot
Im
:* n
4: I/ .m
I
I-
100
IO
4
8
12 Days
FIG. 2. CYMV translocation (c) 21 “C, (d) 26 “C.
to shoots
4 after
8
12
inoculation
at different
temperatures:
(a)
10 “C,
(b)
15 ‘c,
Starch content was always greatest in infected shoot tissue, with maximum accumulation 8 days after inoculation (Table 3). The large accumulation of starch in infected shoots offset the decline in soluble carbohydrates, resulting in a greater amount of total carbohydrates on all sampling dates (Table 3). Infected roots contained more soluble carbohydrates, but less starch, than did healthy roots (Table 3). Sucrose content of infected roots was three to four times more than that extracted from healthy roots. Reducing-sugar content of infected roots also was higher than that of comparable healthy roots 8 and 12 days after inoculation. Starch content was lower on all sampling dates and, because starch is the major carbohydrate component in roots, this reflects total carbohydrate reduction on all sampling dates after inoculation (Table 3).
Clover yellow mosaic virus infection of seedling
roots
TABLE Carbohydrate Factor
measured
levels in CYMV-infected HO(
H4
225
3
and healthy pea seedlings c4
H8
(per g fresh
ca
weight) H12
Cl2
Shoot Red. sugar/sucroseb Starch/sol carb’ Total sol carb (pmol) Total carb (mg) Red. sugar/sucrose Starch/sol carb Total sol carb (pn-101) Total carb (mg)
0.8” 4.5 15.12 15.11
0.9 5.7 8.23 9.95
0.8cWd
lO.lf 5.2 23.10 26.03
1*7** 101.23** 48.93**
5*3c 2.5 52.52 33.55
2.2 8.4 9-72 16.43
Root 0.3** 3*3** 15*99** 11*78*
4,3 6.6 16.58 22.69
1 .oc**
2.2 92.28’* 53.83**
11.6** 1.5** 36*61** 16.66*
4.7c 1.6 83.83 38.90 6.3 4.8 21.99 22.92
1.2** 1.5 79.42 45*12*
3*9** 0.8** 56*84** 18.36
o Abbreviations refer to date of harvest of healthy (H) and CYMV-infected (C) seedlings; HO(C0) date of inoculation, H4 and C4 4 days after, H8 and C8 8 days after and H12 and Cl2 12 days after inoculation. b Red. sugar/sucrose = ratio of reducing sugar to sucrose concentration. c Each datum point represents the mean of three experiments replicated three times. d * = infected mean significantly different from healthy at 5% probability level. ** = infected mean significantly different from healthy at 1 o/0 probability level. Split-plot analysis of variance. s Starch/sol carb = ratio of starch to soluble carbohydrate concentration.
Purijkation
Purification of CYMV from pea roots required modification of previously employed techniques [1, 201. Host protein was the major contaminant encountered. Chloroform treatment worked well for removing host protein and also removed chlorophyll, which was a major contaminant of pellets obtained from shoot tissue. Heating was not successful for clarifying virus preparations because the virus was too sensitive to heating to enhance separation of host protein from virus protein. Virus yields from infected pea roots were consistently as high as those obtained from shoot tissue in following our modified clarification techniques. A comparison of dry and fresh weights of roots and shoots was made to more nearly equate virus yields. Because large amounts of water were used for rinsing roots, they were consistently heavier than accompanying shoots. Dry weights of shoots averaged 6.4% of fresh weights, and dry weights of roots averaged only 4.4%. This meant that less buffer could be used for clarification of root tissue than for shoot tissue. Electron mixroscopy
Aggregates of CYMV particles were abundant in root tips fixed 8 days after shoot inoculation. Virus aggregates often occupied a major portion of cells (Plate 1). The aggregates were not uniform in size or shape. Individual particles were quite easy to define when cut either in the longitudinal or transverse plane (Plate 2). Viral aggregates were numerous in differentiated xylem and phloem elements. These bundles were not as densely packed as were those in younger parenchyma cells. As xylem vessels matured, vacuolation increased, and viral particles became appressed to cell walls. 15
226
Cytological changes accompanying viral infection mitotic activity, accompanied by changes in mitochondrial were smaller, more rounded and had highly invaginated Other than these changes, there seemed to be minimal inclusion-bearing cells.
H. E. Moline
and
R. E. Ford
included a reduction ot structure, Mitochondria inner membrane systems morphological changes in
DISCUSSION Pea roots were as easily inoculated with CYMV as were leaves, and virus titer inwas creased markedly in roots before infectivity could be detected in shoots. CYMV Electronrapidly translocated via the vascular system throughout infected seedlings. microscopic examination of vascular elements revealed that xylem and phloem elements contained large amounts of virus. The high virus titer in roots, as determined by local-lesion assays, was supported by observations of numerous virus aggregates in root tips examined with the electron microscope. The large number of viral inclusions in very young root tip cells is indicative that CYMV is capable of infecting meristematic tissues. Inclusions were more numerous in cells of the terminal 1 mm of root tip sectioned than they were in more mature cells. This may have been the result of decreased viral synthesis as cells matured and became highly vacuolated. Virus aggregates were found in all cell types and in various arrangements. Many cells contained inclusions larger than nuclei in cross-section. Some cells were completely filled with loosely packed viral aggregates. Pea root tips may prove an invaluable tool in the study of early phases of viral infection. A consistently high percentage of roots were infected, and many cells observed in thin-sectioned material contained inclusions. Labelled antibodies may enable us to visualize early sites of viral synthesis and follow viral replication as root cells mature further back from the meristem. CYMV was easier to purify from pea roots than from shoots, The product obtained following this schedule was of much higher purity than obtained by any previously reported purification schedules. The decrease of soluble and residue nitrogen observed in pea shoots 4 days after inoculation is difficult to explain on the basis of other reports of nitrogen metabolism in infected seedlings [II, 211. The decrease of soluble nitrogen, however, follows the same pattern observed in sugarcane mosaic virus (SCMV) and bromegrass mosaic virus (BMV) infected corn shoots. The decrease of soluble nitrogen in early stages of infection may indicate a shock symptom in which nitrogen metabolism is depressed in response to infection. Eight and 12 days after inoculation, the accumulation of soluble and residue nitrogen reported elsewhere are noted [II, 211. The accumulation of nitrogen correlates with the severity of the disease on the seedlings. The metabolic processes are severely disrupted by the infection, eventually leading to chlorosis, necrosis and death of infected seedlings. Nitrogen is accumulated at the expense of other products in the infected shoot. In an extreme case, protein incorporated into virus at the expense of the host has been reported as high as 30% without changes being noted in total nitrogen content [4]. Nitrogen levels in infected roots decreased on all sampling dates, with the exception of soluble nitrogen 12 days after inoculation. This may indicate that viral
PLATE
with many (N) nucleus.
1. CYMV-infected viral inclusions (Bar = 1 pm).
root tip 12 days after leaf inoculation. Meristematic derivativrs (V) filling ceil. (W) cell wall, (P) plastids, (M) mitochondria.
PLATE 2. CYMV-infected pea root tip 12 days after leaf inoculation. Viral inclusion (V) in meristermatic derivatives in terminal millimeter of pea root tip. (M) mitochondria, (P) plastid. (Bar = 250 nm.1
Clover
yellow
mosaic
virus
infection
of seedling
roots
227
replication is not taxing root metabolism to the extent that it is of shoot metabolism. Ultrastructural changes in leaves [ZZ] seem more severe than has been observed in any of the root tips examined. Carbohydrate balances in infected seedlings were severely affected by CYMV. Sucrose accumulation was noted in shoots at all sampling dates. The initial large accumulation of reducing sugars 4 days after inoculation, with a reduction of reducing-sugar content 12 days after inoculation, suggests that translocation of carbohydrates may be interrupted in early stages of infection. Starch accumulated in shoots while starch content of roots decreased, supporting the assumption that translocation of carbohydrates was interrupted. The accumulation of starch 4 days after inoculation was much greater than observed 8 and 12 days after inoculation, again supporting the conclusion that translocation of carbohydrates was interrupted. Carbohydrate and nitrogen levels of infected seedlings supported previous observations concerning the differences in severity of the three virus diseases. SCMV, which was present in low concentration in corn [18], caused less alteration of carbohydrate and nitrogen metabolism than did BMV [19], which eventually killed infected corn seedlings. CYMV interrupted translocation of carbohydrates from pea leaves to roots, as evidenced by accumulation of sucrose and starch in infected shoots, accompanied by reductions of starch content in infected roots. This virus also eventually killed infected pea seedlings. These observations show how difficult it is to compare the effects of different viruses on their hosts, except where the same plant has served as host for more than one virus, as with SCMV and BMV in corn. They further reflect the changes in host metabolism caused by a virus at different stages of infection. REFERENCES 1. AGRAWAL,
Nature, 2. APPIANO,
potato 3. BAWDEN, Mass.
N. W. (1946). The virus content of plants suffering from tobacco mosaic. 3oumd of Experimental Pathology 27, 81-90. BENNETT, C. W. (1940). Relation of food translocation to movement of virus to tobacco mosaic. 3ournal of Agricultural Research 60, 361-390. CRAFTS, A. S. (1951). Movement of assimilates, ViNSeS, growth regulators, and chemical indicators in plants. Botany Review 17, 203-284. CROWLEY, N. C., DAVISON, E. M., FRANCKI, R. I. B. & Owusu, G. K. (1969). Infection of bean root meristems by tobacco ringspot virus. Virology 39, 322-330. DAVISON, E. M. (1969). Cell to cell movement of tobacco ringspot virus. Virology 37, 694-696. Annual Review of Phytopathology I, DIENER, T. 0. (1963). Physiology of virus-infected plants. 197-218. FORD, R. E. (1973). Concentration and purification of clover yellow mosaic virus from pea roots and leaves. Phytopatholoa 63, 926930. FORD, R. E. & Tu, J. C. (1969). Free amino acid contents in corn infected with maize dwarf mosaic virus and sugarcane mosaic virus. Phytopathology 31, 575-598. GOODMAN, P. J., WATSON, M. A. & HILL, A. R. C. (1965). Sugar and fructosan accumulation in virus-infected plants: rapid testing by circular-paper chromatography. Annals of Applied Btilogy 56, 56-72. HOLDEN, M. & TFLACEY, M. V. (1948). The effect of infection with tobacco mosaic virus on the levels of nitrogen, phosphorus, protease, and pectase in tobacco leaves and on their response to fertilizers. Biochemical Journal 43, 151-156.
4. BA~DEN,
British
5. 6. 7. 8. 9. 10. 11. 12.
13.
H. O., CHESSIN, M. & Bos, L. (1962). Purification of clover yellow mosaic virus. London 194, 408-409. A. & PENNAZIO, S. (1972). Electron microscopy of potato meristem tips infected with virus X. Journal of General Virology 11, 273-276. F. C. (1950). Plant Viruses and Virus Diseases, 3rd ed. Chronica Botanica Co., M’altham, F. C. & PIRIE,
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E. Ford
14. HOLLINGS, M. (1965). Disease control through virus-free stock. Annual Review r~f Phytopatholop 3, 367-396. 15. -TENSEN, S. G. (1972). Differential effects of barley yellow dwarf virus on the physiology -. of tissues of hard red spring wheat. Phytopathology 62, 290-293. 16. TOHNSON. C. M.. STOUT. P. R.. BROYER. T. C. & CARL.TO?I. A. B. (1957). Comnarative chlorine requirements of different plant species. Plant and Soil 8, 337-353. 17. MATTHEWS, R. E. F. (1970). Plant Virology. Academic Press, New York. 18. MOLINE, H. E. & FORD, R. E. (1974a). Sugarcane mosaic virus infection of seedling roots of zea mays and Sorghum halepense. Physiological Plant Pathology 4, 197-207. 19. MOLINE, H. E. & FORD, R. E. (197413). Bromegrass mosaic virus infection of seedling roots of +a mays, Triticum aestivum and Hordeum uulgare. Physiological Plant Pathology 4, 209-2 17. 20. PRATT, M. J. & REICHMANN M. E. (1963). The stability of white clover mosaic and clover yellow mosaic virus particles. Proceedings of the Canadian Phytopathology Society 30, 16 (Abstr.). 21. PROLL, E. (1967). Untersuchungen tiber die Vermehrung und Ausbreitung des Trespenmosaikvirus (bromegrass mosaic virus) in Gerste. III. Virusvermehrung bei Lichtmangel und virusbedinget Ver%derungen im N&offwechsel. Phytopathologische
lY
~
,
I
29,276-284. 23. 24.
M. S. &r. MATTHEWS, R. E. F. (1966). On the origin mosaic virus. Virolos), 28, 563-570. ROBERTS, F. M. (1950). The infection of plants by viruses
REID,
of the mosaic through
roots.
induced Annals
by turnip
yellow
of Applied
Bioloa
37,385-396. 25.
ROCHOW, W. F., Ross, A. F., & SIEGEL, B. M. (1955). Comparison of local-lesion and microscope particle-count methods for assay of potato virus X from plants doubly by potato viruses X and Y. Virology 1, 28-39. 26. SMITH, F. H. & MCWHORTER, F. P. (1957). Anatomical effects of tomato ringspot virus faba. American Journal of Botany 44, 470-477. 27. WALKEY, D. G. A. & WEBB M. J. W. (1968). Virus in plant apical meristems. Journal Virolopy 3, 311-313. 28. WYND, F. L. (1943). Metabolic phenomena associated with virus infection in plants. Review 9, 395-465. 29. YARWOOD, C. E. (1959). Virus increase in seedling roots. Phytopatholosy 49, 220-223.
electron infected in
Vicia
of General Botanical
Plant Pathology
(1974)
4, 2 19-228
Clover yellow mosaic virus infection of seedling roots of Pisum sativumt H. E. MOLINE$ Botany
and R.E.
and Plant Pathology
(Accepdfm
Publication
FORD
Department,
November
Iowa State University,
Ames, Iowa 50010,
U.S.A.
1973)
Clover yellow mosaic virus (CYMV) multiplication was studied in roots of Pisum sativum (garden pea). Mechanical inoculation of pea roots with CYMV resulted in systemic infection of shoots. CYMV translocation from roots was directly related to temperature. CYMV was purified from pea roots with minimal alterations of existing schedules; yields were comparable to those from leaves (45 mg/kg, roots; 50 mg/kg, leaves). Electron-microscopic observations of thin-sectioned CYMV-infected root-tip tissue revealed large numbers of viral inclusions in meristematic root initials. Nitrogen and carbohydrate levels were significantly altered in roots and shoots of infected seedlings.
INTRODUCTION
This, the third in a series of papers devoted to the study of viruses in plant roots [18, 191 considers a member of the Potexvirus group, clover yellow mosaic virus (CYMV) . Several previous studies have shown that the rates of movement of viruses within plants, especially to and from roots, vary considerably [5, 6, 24, 2.51. Meristem tip cultures have been used in many instances to free vegetatively propagated crops from viruses [14]. This technique is not universally effective for all plant-virus combinations, however, because a number of viruses are capable of invading meristematic tissue [2, 7, 8, 26, 271. A portion of this study has been devoted to the examination of root meristems of infected seedlings. Since virus replication entails the synthesis of virus-specific proteins, disturbances in nitrogen metabolism of infected plants is a normal consequence of virus infection [4, 9, 13, 231. S ome viruses seem to have little effect on carbohydrates, however, while others do alter their rate of synthesis and (or) rate of translocation [3, 12, 15, 17, 281. The three viruses used in this study have shown considerable differences in effects on nitrogen and carbohydrate metabolism of infected hosts [18, 191. A preliminary study with CYMV suggests that roots may provide certain advantages over leaves for virus purification [IO]. We also examined the effects of CYMV infections on Pisum sativum (garden pea) root physiology, cytology via electron microscopy and viral translocation. t Journal Ames, Iowa. degree, Iowa $ Present
Paper No. J-7619 of the Iowa Agriculture and Home Economics Experiment Station, Project No. 1878. This study is a portion of a thesis by the senior author for the Ph.D. State University, and was supported in part by the Iowa Experiment Station. address: ARS-USDA, AMRI-HCML, ARC-West, Beltsville, Md 20705.
220
H. E. Molinemd
R. E. Ford
MATERIALS AND METHODS Virus replication and tramlocation Pratt’s B strain of CYMV [ZO] was maintained in the greenhouse on Inca rosea L. Periodic transfers were made to garden pea and Gomphrena globosa L. G. globasa was used for all local-lesion assays; “Wilt Resistant Perfection” pea was the source of inoculum and the host for all experiments. Pea seedlings used for leaf inoculation were grown in a steamed soil mixture [18]. For root inoculations pea seedlings were germinated on autoclaved germinating papers. The primary root of each seedling was inoculated 6 to 7 days after germination. Five inoculation techniques [18] were tested. Control plants were treated in the same manner, substituting healthy sap for inoculum. After inoculation, roots were rinsed with tap water, and four seedlings were planted in each 4-in clay pot containing vermiculite. The pots were placed in plastic trays, eight pots/tray, and watered from beneath to maintain proper moisture conditions. Plants were supplemented with half strength Hoagland’s solution [16] twice weekly. Seedling roots and leaves were assayed at predetermined intervals to detect when the virus had replicated in the roots and (or) had moved to the shoots. In the first group of experiments, at least 10 seedlings were assayed onto G. globosa at each interval. The opposite-leaf assay technique [25] was used with a purified frozen preparation of CYMV serving as the standard reference from which aliquots were removed for each assay. Tissue was diluted on a weight/volume basis with O-01 M-phosphate buffer (pH 7.0 to 7.2) and inoculated onto eight leaves. After we established that CYMV increased in roots and moved to shoots, relative virus concentrations in root and shoot were determined. Lesions were counted 10 days after inoculation. Lesion counts were made from all leaves having fewer than 300 lesions; greater numbers were estimated. Physiology of qfected plants Fourteen pans of “Wilt Resistant Perfection” peas were seeded with 45 seeds per pan. Pea seedlings in six pans were inoculated with CYMV. The other eight were rubbed with healthy sap and served as controls. Pea seedlings were harvested and treated as described previously [18]. Th e seedlings from each pan served as one treatment. These plants were counted and separated into plant parts: roots, shoots and seeds. Fresh weights and number of plants were recorded, and all parts were placed in 95% ethanol for storage. Pea seedlings infected with CYMV were treated as seedlings in previous physiology studies [18, 191. No significant differences existed among gram fresh weight, gram dry weight and per-plant basis; therefore, data are presented on a gram-fresh weight basis. Pur&ation CYMV was purified from roots and shoots of infected pea seedlings. Seedlings were grown in either vermiculite or soil and were inoculated 2 to 3 days after emergence. Plants were rinsed with distilled water and placed in a greenhouse for 12 to 16 days.
Clover yellow mosaic virus infection of seedling
roots
221
Then they were harvested and divided into roots and shoots for comparative purification. Soil or vermiculite was removed from roots by gently rinsing them in running water [18]. Tissue was either purified immediately or stored at - 14 “C. CYMV was purified by grinding fresh or frozen tissue in 1 vol. of 0.01 Mphosphate buffer, pH 7.0, with a blendor. The tissue was then expressed through four layers of cheesecloth, and the filtrate collected. The brei was discarded. The filtrate was centrifuged at 3000 g for 10 min, the supernatant was saved and the green pellet was discarded. Attempts were made to remove chlorophyll and plant proteins by (i) heating the supernatant at 40 “C for 10 to 30 min and (ii) after the first low-speed centrifugation, by blending chloroform in the supernatant to final concentrations of 3, 10, 25, or 50% (v/v) for 2 min. The 25% chloroform treatment proved superior and was used for all subsequent purifications. Then, the mixture was centrifuged at 3000 g for 10 min; the upper supernatant was collected for further treatment, and the solid pellet and green lower layer were discarded. The supernatant was centrifuged for 120 min at 78 000 g. The supernatant was discarded, and one-twentieth of the original volume of buffer added to the pellets. Pellets were either allowed to resuspend overnight or were resuspended immediately by homogenizing in a glass homogenizer. The solution was then centrifuged at 3000 g for 10 min, and the pellet discarded. The supernatant was centrifuged at 234 000 g for 60 min. Then, the supernatant was discarded after assay; the pellet was resuspended in 1 to 2 ml of buffer and centrifuged at 3000 g for 10 min. The supernatant was layered on 10 to 50% sucrose or 3 M-CsCl gradients. Sucrose gradients were run for 4 h in a Beckman SW 25.1 rotor. CsCl gradients were run for 24 h in a Beckman SW 50.1 rotor. After centrifugation, the density-gradient tubes were fractionated with the aid of an ISCO density-gradient fractionater and U.V. analyzer, the fractions collected and additional U.V. scans made on all fractions with a Beckman DBG spectrophotometer. Virus-containing zones were dialyzed against phosphate buffer for infectivity assay. Yields of virus were based on infectivity assays and U.V. scans, which could be interpreted as to yield in mg/lOO ml. Electron
microscopy
Pea root tips and portions of mature root tissue were examined for the presence of viral inclusions. Infected roots were fixed 8, 12, 16 and 24 days after inoculation and prepared for observation as described previously [28]. RESULTS Translocation
Although mechanical inoculation techniques with CYMV on pea roots resulted in infection (Table l), rubbing silicon carbide (600 mesh) dusted roots between thumb and forefinger, which had been dipped in inoculum, gave most consistent results (Table 1) ; therefore, this technique was used in all subsequent studies. CYMV detected in roots 3 days after inoculation, increased rapidly in titer to a maximum 1 x lob3 8 days after inoculation. CYMV detected in leaves 6 days after root inoculation reached a maximum titer (2.5 x lOA) 26 days after inoculation
I-L E. Moline
222 TABLE
Comparatiz
transmission
of CYMV
Treatmenta
R. E. Ford
1
to pea hoots after inoculatin~~ the wets by jioe techniques Test
Experiment
and
Inoculated
plants --. Infected”
Check -_-.-____Inoculated
plants Infected
1
A B C D E
66 96 96 96 96
65 94 90 9.5 89
66 64 64 64 64
0 0 0 0 0
2
A B c D E
96 96 96 96 96
96 90 93 96 85
64 64 64 64 64
0 0 0 0 0
3
A B C D E
96 96 96 96 96
92 93 96 94 73
64 64 64 64 64
0 0 0 0 0
B Five treatments employed: (A) roots were dusted with silicon carbide, then rubbed between forefinger and thumb which had been dipped in inoculum; (B) roots were dusted and inoculum was applied with a camel’s hair brush that had been dipped in inoculum; (C) roots were dusted, rubbed between forefinger and thumb to injure them then immediately dipped in inoculum; (D) inoculum was injected into the Steele of the root with a hypodermic syringe; and (E) inoculum was mixed with silicon carbide and sprayed onto roots with an artist’s air brush. h Symptoms were observed on shoots of inoculated seedlings 15 to 20 days after inoculation.
(Fig. 1). Infected plants were necrotic 20 days after inoculation and were usually dead 40 days after inoculation. Roots of inoculated seedlings showed severe browning 15 days after inoculation. The translocation rate of virus from roots to leaves was directly correlated with an increase in temperature (Fig. 2). When root-inoculated seedlings were placed at 26, 20, 15 and 10 “C in controlled-environment chambers, CYMV was recovered from leaves, respectively, 4, 5, 8 and 9 days after root inoculation. Virus was recovered from roots 3 days after inoculation at all four temperatures tested (Fig. 2). Physiology of a$ected plants Nitrogen content. CYMV affects both residue and soluble nitrogen levels similarly in infected pea shoots (Table 2). Although lower 4 days after inoculation, nitrogen levels then increased 8 and 12 days after inoculation, resulting in an accumulation of total nitrogen (Table 2). Nitrogen content of infected roots decreased at each sampling date. Four days after inoculation, residue nitrogen content decreased IS:&, and soluble nitrogen content decreased 23 %. This trend was also observed 8 and 12 days after inoculation, leading to a total reduction of nitrogen in all infected roots (Table 2). Carbohydrates. Sucrose levels in infected pea shoots were greater at 4, 8 and 12 days after inoculation (Table 3). Reducing-sugar content rapidly increased 4 days after
Clover
yellow
mosaic
virus
infection
of seedling
roots
223
inoculation, was comparable with healthy shoots 8 days after inoculation and decreased 12 days after inoculation resulting in no net change between healthy and infected shoots 12 days after inoculation. 100000
:
2.
,! 10000
., l \
a-.;.’
-
.
\
l‘\\sh$ot
9
9.
B 1000
-
I I 100
1. \ #, .-. root
5
5 .L 2
1.
2’
z
z
\
1 .a-, Lf’
-j
Y,.
-
: ( I
B -1 IO -. I I I ’
I 0
I IO Days
ofler
I 20 moculolion
I 30
FIG. 1. CYMV concentration in pea shoots and roots following root inoculation. Virus titer based on wt/vol dilution of crude sap in 0.01 M-phosphate buffer (pH 7.0). Infected seedlings were assayed at 24-h intervals for the first 12 days, then every other day for the remainder of the experiment. Values are means of 10 assays onto G. globosa. TABLE
Nitrogen Factor
measured
levels in CTMV-infected HO(CO)O
H4
3.1e 37.05 33.6 0.639
3.50 89.32 14.2 0.639
1.3 18.91 47.6 0.666
1.5 29.25 26.6 0.666
2
and healthy pea seedlings (per g fresh c4
weight)
H8
C8
HI2
Cl2
4.1” 69.01 8.3 0.831
4.1c 81.11 12*5** 0.950
5.7c 59.38 13.1 2.02
5.7= 81.58 11.3* 1.24*
2.0 28.16 24.8 0.820
1.9 23.60 30.1** 1.08*
3.2 27.94 24.3 0.972
2.4** 21.85 28-O** 1.32**
Shoot Res N/Sol Total N FW/DWC FW/plantf
Nb
Res N/Sol Total N FW/DW FW/plant
N
2.gc**a
76.46 16.0* 0.655 Root 2:.:3 33.0** 0.858*
(C) seedlings; a Abbreviations refer to date of harvest of healthy (H) and CYMV-infected HO(C0) date of inoculation, H4 and C4 4 days after, H8 and C8 8 days after and H12 and Cl2 12 days after inoculation. b Res N/Sol N = ratio of residue to soluble nitrogen concentration. G Each datum point represents the mean of three experiments replicated three times. d * = infected mean significantly different from healthy at 5% probability level. ** = infected mean significantly different from healthy at 1 o/0 probability level. Split plot analysis of variance. e FW/DW = ratio of fresh weight to dry weight. f FW/plant = total fresh weight per plant part (g).
H. E. Moline
224
!7 !I !
10,000
1000
Tfmm ,a root / 1 ,i ’ /shoot
and
R. E. Ford
/
100
-2 2 ‘;;L &u
IO 15 “C
5 Y) :. ‘S :
9 (cl
10,000
r;o t/ .I
IO00
/~ .2--=m.‘.
/
‘shoot
Im
:* n
4: I/ .m
I
I-
100
IO
4
8
12 Days
FIG. 2. CYMV translocation (c) 21 “C, (d) 26 “C.
to shoots
4 after
8
12
inoculation
at different
temperatures:
(a)
10 “C,
(b)
15 ‘c,
Starch content was always greatest in infected shoot tissue, with maximum accumulation 8 days after inoculation (Table 3). The large accumulation of starch in infected shoots offset the decline in soluble carbohydrates, resulting in a greater amount of total carbohydrates on all sampling dates (Table 3). Infected roots contained more soluble carbohydrates, but less starch, than did healthy roots (Table 3). Sucrose content of infected roots was three to four times more than that extracted from healthy roots. Reducing-sugar content of infected roots also was higher than that of comparable healthy roots 8 and 12 days after inoculation. Starch content was lower on all sampling dates and, because starch is the major carbohydrate component in roots, this reflects total carbohydrate reduction on all sampling dates after inoculation (Table 3).
Clover yellow mosaic virus infection of seedling
roots
TABLE Carbohydrate Factor
measured
levels in CYMV-infected HO(
H4
225
3
and healthy pea seedlings c4
H8
(per g fresh
ca
weight) H12
Cl2
Shoot Red. sugar/sucroseb Starch/sol carb’ Total sol carb (pmol) Total carb (mg) Red. sugar/sucrose Starch/sol carb Total sol carb (pn-101) Total carb (mg)
0.8” 4.5 15.12 15.11
0.9 5.7 8.23 9.95
0.8cWd
lO.lf 5.2 23.10 26.03
1*7** 101.23** 48.93**
5*3c 2.5 52.52 33.55
2.2 8.4 9-72 16.43
Root 0.3** 3*3** 15*99** 11*78*
4,3 6.6 16.58 22.69
1 .oc**
2.2 92.28’* 53.83**
11.6** 1.5** 36*61** 16.66*
4.7c 1.6 83.83 38.90 6.3 4.8 21.99 22.92
1.2** 1.5 79.42 45*12*
3*9** 0.8** 56*84** 18.36
o Abbreviations refer to date of harvest of healthy (H) and CYMV-infected (C) seedlings; HO(C0) date of inoculation, H4 and C4 4 days after, H8 and C8 8 days after and H12 and Cl2 12 days after inoculation. b Red. sugar/sucrose = ratio of reducing sugar to sucrose concentration. c Each datum point represents the mean of three experiments replicated three times. d * = infected mean significantly different from healthy at 5% probability level. ** = infected mean significantly different from healthy at 1 o/0 probability level. Split-plot analysis of variance. s Starch/sol carb = ratio of starch to soluble carbohydrate concentration.
Purijkation
Purification of CYMV from pea roots required modification of previously employed techniques [1, 201. Host protein was the major contaminant encountered. Chloroform treatment worked well for removing host protein and also removed chlorophyll, which was a major contaminant of pellets obtained from shoot tissue. Heating was not successful for clarifying virus preparations because the virus was too sensitive to heating to enhance separation of host protein from virus protein. Virus yields from infected pea roots were consistently as high as those obtained from shoot tissue in following our modified clarification techniques. A comparison of dry and fresh weights of roots and shoots was made to more nearly equate virus yields. Because large amounts of water were used for rinsing roots, they were consistently heavier than accompanying shoots. Dry weights of shoots averaged 6.4% of fresh weights, and dry weights of roots averaged only 4.4%. This meant that less buffer could be used for clarification of root tissue than for shoot tissue. Electron mixroscopy
Aggregates of CYMV particles were abundant in root tips fixed 8 days after shoot inoculation. Virus aggregates often occupied a major portion of cells (Plate 1). The aggregates were not uniform in size or shape. Individual particles were quite easy to define when cut either in the longitudinal or transverse plane (Plate 2). Viral aggregates were numerous in differentiated xylem and phloem elements. These bundles were not as densely packed as were those in younger parenchyma cells. As xylem vessels matured, vacuolation increased, and viral particles became appressed to cell walls. 15
226
Cytological changes accompanying viral infection mitotic activity, accompanied by changes in mitochondrial were smaller, more rounded and had highly invaginated Other than these changes, there seemed to be minimal inclusion-bearing cells.
H. E. Moline
and
R. E. Ford
included a reduction ot structure, Mitochondria inner membrane systems morphological changes in
DISCUSSION Pea roots were as easily inoculated with CYMV as were leaves, and virus titer inwas creased markedly in roots before infectivity could be detected in shoots. CYMV Electronrapidly translocated via the vascular system throughout infected seedlings. microscopic examination of vascular elements revealed that xylem and phloem elements contained large amounts of virus. The high virus titer in roots, as determined by local-lesion assays, was supported by observations of numerous virus aggregates in root tips examined with the electron microscope. The large number of viral inclusions in very young root tip cells is indicative that CYMV is capable of infecting meristematic tissues. Inclusions were more numerous in cells of the terminal 1 mm of root tip sectioned than they were in more mature cells. This may have been the result of decreased viral synthesis as cells matured and became highly vacuolated. Virus aggregates were found in all cell types and in various arrangements. Many cells contained inclusions larger than nuclei in cross-section. Some cells were completely filled with loosely packed viral aggregates. Pea root tips may prove an invaluable tool in the study of early phases of viral infection. A consistently high percentage of roots were infected, and many cells observed in thin-sectioned material contained inclusions. Labelled antibodies may enable us to visualize early sites of viral synthesis and follow viral replication as root cells mature further back from the meristem. CYMV was easier to purify from pea roots than from shoots, The product obtained following this schedule was of much higher purity than obtained by any previously reported purification schedules. The decrease of soluble and residue nitrogen observed in pea shoots 4 days after inoculation is difficult to explain on the basis of other reports of nitrogen metabolism in infected seedlings [II, 211. The decrease of soluble nitrogen, however, follows the same pattern observed in sugarcane mosaic virus (SCMV) and bromegrass mosaic virus (BMV) infected corn shoots. The decrease of soluble nitrogen in early stages of infection may indicate a shock symptom in which nitrogen metabolism is depressed in response to infection. Eight and 12 days after inoculation, the accumulation of soluble and residue nitrogen reported elsewhere are noted [II, 211. The accumulation of nitrogen correlates with the severity of the disease on the seedlings. The metabolic processes are severely disrupted by the infection, eventually leading to chlorosis, necrosis and death of infected seedlings. Nitrogen is accumulated at the expense of other products in the infected shoot. In an extreme case, protein incorporated into virus at the expense of the host has been reported as high as 30% without changes being noted in total nitrogen content [4]. Nitrogen levels in infected roots decreased on all sampling dates, with the exception of soluble nitrogen 12 days after inoculation. This may indicate that viral
PLATE
with many (N) nucleus.
1. CYMV-infected viral inclusions (Bar = 1 pm).
root tip 12 days after leaf inoculation. Meristematic derivativrs (V) filling ceil. (W) cell wall, (P) plastids, (M) mitochondria.
PLATE 2. CYMV-infected pea root tip 12 days after leaf inoculation. Viral inclusion (V) in meristermatic derivatives in terminal millimeter of pea root tip. (M) mitochondria, (P) plastid. (Bar = 250 nm.1
Clover
yellow
mosaic
virus
infection
of seedling
roots
227
replication is not taxing root metabolism to the extent that it is of shoot metabolism. Ultrastructural changes in leaves [ZZ] seem more severe than has been observed in any of the root tips examined. Carbohydrate balances in infected seedlings were severely affected by CYMV. Sucrose accumulation was noted in shoots at all sampling dates. The initial large accumulation of reducing sugars 4 days after inoculation, with a reduction of reducing-sugar content 12 days after inoculation, suggests that translocation of carbohydrates may be interrupted in early stages of infection. Starch accumulated in shoots while starch content of roots decreased, supporting the assumption that translocation of carbohydrates was interrupted. The accumulation of starch 4 days after inoculation was much greater than observed 8 and 12 days after inoculation, again supporting the conclusion that translocation of carbohydrates was interrupted. Carbohydrate and nitrogen levels of infected seedlings supported previous observations concerning the differences in severity of the three virus diseases. SCMV, which was present in low concentration in corn [18], caused less alteration of carbohydrate and nitrogen metabolism than did BMV [19], which eventually killed infected corn seedlings. CYMV interrupted translocation of carbohydrates from pea leaves to roots, as evidenced by accumulation of sucrose and starch in infected shoots, accompanied by reductions of starch content in infected roots. This virus also eventually killed infected pea seedlings. These observations show how difficult it is to compare the effects of different viruses on their hosts, except where the same plant has served as host for more than one virus, as with SCMV and BMV in corn. They further reflect the changes in host metabolism caused by a virus at different stages of infection. REFERENCES 1. AGRAWAL,
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electron infected in
Vicia
of General Botanical