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Masters Theses 1911 - February 2014
1973
Occurrence and importance of maize dwarf mosaic virus in Massachusetts.
Robert A. Schall University of Massachusetts Amherst
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OCCURRENCE AND IMPORTANCE OF MAIZE DWARF
MOSAIC VIRUS IN MASSACHUSETTS
ROBERT A. SCHALL
B. A.
Rutgers University Newark, New Jersey
Thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
University of Massachusetts
Department of Plant Pathology
Amherst, Massachusetts
July, 1973 OCCURRENCE AND IMPORTANCE OF MAIZE DWARF
MOSAIC VIRUS IN MASSACHUSETTS
A Thesis by Robert A. Schall
Approved as to style and content by:
(Member)
(Month) ; (Year) ACKNOWLEDGEMENTS
I am deeply grateful to my advisor Dr. George N. Agrios for his constant encouragement and advice throughout this work and for his assistance in the preparation of this manuscript.
I am also indebted to Professors Cecil L. Thomson and
William H. Lachman for assisting me in this work with their comprehensive knowledge of corn culture and production.
Also, I wish to thank Messrs. Dominic Marini, Walter
Melnick, and Alden Miller who, in their capacity as Exten¬ sion Specialists, helped me survey for the virus.
Finally, I thank all who have aided and encouraged me in numerous ways in this endeavor. CONTENTS
« Page
INTRODUCTION... 1
LITERATURE REVIEW. 3
MATERIALS AND METHODS.,. 15
The Virus. 15 The Plants . 15 Mechanical inoculation. 17 Properties of virus in crude sap. 17 Dilution end point Longevity i_n vi tro Thermal inactiva tion point Host range studies. 18 Purification and electron microscopy. 19 Serology. 21 Resistance tests on sweet corn varieties. 22 Field survey procedures . 22
RESULTS. 24
The disease. 24 Mechanical inoculation. 24 Dilution end point. 25 Longevity i_n vitro. 30 Thermal inactivation point. 30 Host range studies. 30 Purification and electron microscopy. 35 Serol ogy. 35 Resistence to sweet corn varieties. 38 Fieldsurvey. 41
DISCUSSION . 43
SUMMARY 50
LITERATURE CITED . 52 INTRODUCTION
In early September 1971, sweet corn plants in a field in Leominster, Massachusetts were found affected with a disease that appeared to be caused by a virus. On many leaves, mosaic patterns consisting of dark green and light green patches were observed. Later on in the season, many of the ears were unmarketable due to the failure of kernels to develop properly, especially at the base of the ear. When material showing the mosaic pattern was used in mechanical inoculations, inoculated sweet corn plants developed the mosaic pattern. The appearance of the mosaic pattern, the
kernel skip and the results of the mechanical inoculation suggested that the disease might be caused by maize dwarf mosaic virus, a virus that had not previously been reported in Massachusetts. Maize dwarf mosaic virus is known to have caused severe losses on field corn, and even more severe losses on sweet corn in the states from which it has been reported.
However, several other mechanically transmissible viruses, such as wheat streak mosaic virus, barley stripe mosaic virus, cucumber mosaic virus, and Iowa soybean mosaic virus, are known to produce mosaic symptoms on corn. It was therefore considered most important that this newly detected virus in Massachusetts be identified and its properties studied if any plans for control of the disease were to be attempted.
The potential economic impact of a serious virus
1 2
disease of corn could be very great in Massachusetts be¬ cause sweet corn, planted in about 9,000 acres annually is the major vegetable grown in the state and field corn, planted in about 31,000 acres, is extensively grown for silage. The extent of this disease in Massachusetts, its pattern of distribution and spread, the severity of the disease in various locations, the possible means of over¬ wintering of the virus, and the resistance or suscepti¬
bility to this virus of presently used sweet corn varieties
were considered important aspects of the disease and,
along with the identification of the virus, were included
in the present study. LITERATURE REVIEW
Several viruses and mycoplasmas cause mosaic or mosaic-like symptoms on corn. Certain of these, such as maize mosaic 1, maize streak disease virus and maize streak virus, do not occur within the continental United States
(Smith, 1957). Maize leaf fleck virus is found only in
California and causes pale spots in the interveinal area of the corn leaf and marginal burn (Stoner, 1952). Corn stunt, now known to be caused by a mycoplasma, causes chlorotic, green and white striping on the leaves and stunted plants; this disease occurs mostly in the southern United States
(Kunkel, 1948; Chen and Granados, 1970). Wheat streak mosaic virus, also called maize mosaic virus 2 or sweet corn mosaic virus, causes broken or continuous, interveinal chlorotic streaks on sweet corn (Finley, 1957; Stoner, 1968).
Cucumber mosaic virus produces mottling consisting of light- colored spots of various lengths parallel with the leaf veins (Price, 1935). Barley stripe mosaic virus and soybean mosaic virus also produce mosaic patterns on corn (Stoner,
1968). Finally, sugarcane mosaic virus (Brandes and Klap- haak, 1923) and maize dwarf mosaic ■ virus (Janson and Ellett,
1963) are two important and widespread viruses that infect corn and produce similar mosaic symptoms. Wheat streak mosaic virus, cucumber mosaic virus, barley stripe mosaic virus, Iowa soybean mosaic virus, sugarcane mosaic virus and maize dw&rf mosaic virus are mechanically transmissible,
3 4
corn stunt and maize leaf fleck are not.
Sugarcane mosaic virus (SCMV) and maize dwarf mosaic
virus (MDMV) are almost identical in most of their pro¬
perties and particle characteristics (Bond and Piror.e, 1971);
Krass and Ford, 1969; Tosic and Ford, 1971; Williams and
Alexander, 1965), but they differ slightly in their host
range and serological properties (Shepherd, 1965; Snazelle
et al, 1971; Tosic and Ford, 1972).
A mosaic disease of corn very similar to the one af¬
fecting sweet corn in Massachusetts was observed in 1963
by Janson and Ellett in southern Ohio. In some fields
losses were estimated to be more than half of the expected
yield per acre. Some late plantings of sweet corn were
almost a total loss. The symptoms were described as mosaic
leaf patterns, dwarfing, shortened internodes, ears with
kernels missing, numerous small ears and/or complete lack
of ear formation. The disease was shown to be caused by
an aphid-borne virus that could be easily transmitter
mechanically. The disease was later called maize dwarf
mosaic (MDM) and the virus was called maize dwarf mosaic
virus (MDMV) (Williams and Alexander, 1965).
By 1964 the disease had spread throughout Ohio. That
year the corn yield loss in southern Ohio was estimated to
be as high as 30 percent, while in northern counties the
loss was less than one percent. Losses in individua fie1 ^ 5
throughout the state varied from a trace to nearly 100 percent (Ellett, Janson and Williams, 1965). MDM is now known to be present throughout the Corn Belt and the South.
In the Northeast, MDM was found in Pennsylvania and New
Jersey in 1 965 (Stoner, 1 968). In New York, MDM was ob¬ served in 1967 and 1968 but caused little or no apparent loss; however, in 1969 MDM was widespread and damaging to sweet corn in the Hudson Valley (Boothroyd and Larsen, 1970).
A reddish discoloration of foliage was mentioned as a symptom of the disease in several early reports on MDM
(Janson and Ellett, 1 963 ; Janson et al , 1 965), but others noted that leaf reddening was seldom found associated with
MDMV-infected corn (Ford, Buchholtz and Lambe, 1967; Wernham et al , 1 966). It was later shown that leaf reddening was associated with a phosphorus deficiency or a high potas¬ sium level combined with a magnesium deficiency, and not with the MDMV infection (Tu and Ford, 1968b). The severity of certain symptoms, such as dwarfing, shortened internodes and kernel abortion, depends on the time of infection; the earlier the infection, the more severe the symptoms (Ford,
Buchholtz and Lambe, 1 967; Janson et al , 1 965). Plants in¬ fected late in the season show only mottling and, occasicn- any, shortening of the upper internodes (Josephson et a I ,
1969) . In a comparison of early-.and later inoculations, a 6
field inoculated early, when plants were at the 3 and 4 leaf stage, yielded 76 bu/acre; another field inoculated later
in the season when plants were knee-high yielded 86 bu/acre;
the control, an uninoculated field, yielded 98 bu/acre
(Scheifele, 1969). In a study on sweet corn in Texas the
percentage of plants infected increased from 83% at 50 days
after seeding to 100% at harvest (Tober et al , 1 970). In
Mississippi the percentage of mosaic-infected dent corn plants
increased from 6% at 25 days after planting, to 54% at 51 days
after planting; and when the ear weights were compared 84 days
after planting, healthy plants yielded 23% more than mosaic-
infected plants (Pitre, 1967).
Maize dwarf mosaic virus has several strains; two, A
and B, seem to be the most common (Boothroyd and Larsen, 1970);
Halisky and DeBlois, 1968). The A strain, MDMV-A infects
Johnson grass. Sorghum halepense, while the B strain, MDMV-B,
does not (Mackenzie and Wernham, 1967). Closely related to
MDMV-A, which produces a common mosaic symptom, are strains
C, D, E, and F which produce chevron, ring, mild mosaic and
fleck symptoms, respectively. Like MDMV-A, they also infect
Johnson grass (Louie and Knoke, 1970). Each strain may con¬
tain isolates causing different reactions to certain varieties.
For example, field corn inbreds reported as resistant to a
MDMV-A isolate from Mississippi were susceptible to a MDMV-A
isolate from Ohio. Also, near-isogenic corn inbreds, grown
in different states, respond in various ways to a single 7
MDMV strain. Thus, inbred Oh 07 grown from seed obtained
from Illinois but not from Indiana was susceptible to sys¬
temic infection by MDMV-D (Louie, Knoke and Findley, 1972).
Besides host range studies, serology for strain identi¬
fication has been advocated (Snazelle et al , 1971), but
varying virus concentrations may complicate the serological
test (Bancroft et al, 1966). Certain isolates are degraded by certain purification schemes (Snazelle et al , 1971), and
this is a third method for strain identification. Strain
identification by differences in dilution end point, longe¬ vity in vitro or thermal inactivation point is not feasible
(Tosic and Ford, 1971).
Mosaic symptoms are more severe in MDMV-A infected than in MDMV-B infected corn (Ford and Tu, 1968). When three field corn hybrids, one resistant and two susceptible to each of the strains A and B of MDMV, were inoculated with each of these strains the total plant and ear weight of the resistant hybrid were not affected by either MDMV-A or MDMV-B.
Inoculation of all three hybrids, considered as a croup, with strain A reduced green weight by 12.5%, while inoculation with strain B reduced green weight by 7.5%. Strain A re¬ duced grain yields of the three hybrids by 19%, while strain
B reduced yields by 3% (Cole et al , 1 969 ).
Because inbred lines resistant to strain A are often susceptible to strain B, it is believed that different genetic systems control resistance to strains A and B 8
(Scheifele and Wernham, 1969; Wernham and Scheifele, 1968).
It was believed earlier that breeding for resistance to one isolate would result in resistance to another isolate of the same strain of the virus (Scott, Rosenkranz and Nelson,
1969), but it is now known that resistance to one isolate does not guarantee resistance to another isolate of the same strain (Louis, Knoke and Findley, 1972).
MDMV infection of corn causes the formation of intra¬ cellular microinclusion bodies of unknown function as well as numerous individual virus rods, bundles and pinwheels
(Krass and Ford, 1969). When infected with MDMV, tolerant varieties of sorghum contain bundles and pinwheels in about one of fifty cells with up to 15 pinwheel-type inclusions per cell, while susceptible varieties contain viral inclu¬ sions in about one of ten cells with up to 20 pinwheel-type inclusions per cell (Snow and Toler, 1970). MDMV infection also caused severe cellular disruption, characterized by the presence of numerous vesicles and chloroplasts with fewer grana (Krass and Ford, 1 969). Diseased leaves have
18% fewer chloroplasts than healthy leaves. While healthy leaves have 17, 74 and 9 percent small, medium and large chloroplasts, respectively, the respective percentages are
45, 51, and 4 in diseased leaves. Not only are there fewer and smaller chloroplasts in diseased leaves, but the amounts of chlorophyll a and chlorophyll b_ are reduced in diseased leaves as compared to heal thy'-1 eaves (iu, i-ord and Krass, * ^ °) • 9
In corn leaves infected systemically with MDMV, photo¬ synthesis is reduced by as much as 30 percent, while respira¬
tion is at first increased by about 12 percent and then
returns to a level near that of healthy leaves (Gates and
Gudaukas, 1969). In another study of leaves systemically
infected with MDMV, respiration increased by 32 percent at
the time of appearance of symptoms but it later declined to a level slightly higher than that in healthy leaves; photo¬
synthesis declined by 25% because of MDMV infection (Tu and
Ford, 1968a).
The concentration of nitrogen containing compounds in
the plant is also influenced by MDMV infection. In one
study, twelve amino acids increased, three varied, and
three decreased in infected corn as compared to healthy corn. All ammonium compounds and amides increased in in¬ fected corn (Ford and Tu, 1968). Changes in amino acid concentration were greater in corn infected with strain A of MDMV than with strain B (Ford and Tu, 1969). With Hl-A, a field corn host of MDMV in which only the inoculated leaves become infected, there is a slight change in amino acid concentration in leaves emerging after inoculation and no infectivity is detected by assay from these leaves. With
Seneca Chief, a systemically infected sweet corn, leaves emerging after inoculation show increased amino acid con¬ centration when compared with healthy leaves (Tu and ford,
1970). 10
MDMV infection also affects the concentration of several chemical elements in corn leaves. In one field corn hybrid infected with MDMV there was an increase in Zn (+37%) and
A1 (+44%), and a decrease in Mg (-13%) and Sr (-15%). Other elements showed slight shifts (Cole et a 1 , 1969). In another study, infected leaves contained greater amounts of Fe, P, B,
Al, and Cu (MacKenzie and Cole, 1968).
Temperature affects MDMV infection, incubation and multi¬ plication. With increasing temperature, either before or after inoculation, the percentage of corn plants infected increases. At temperatures of 26.5, 21, and 15.5°C symptoms appear on infected plants after 3, 4, and 9 days, respectively.
Virus concentration increases with increasing temperature
(Tu and Ford, 1969a). Movement of the virus within the plant is also temperature-dependent. The time for movement from infected leaves is shortened with increasing tempera¬ ture. At 26.5°C the virus moves out of an infected leaf in
2 days from inoculation, at 10°C in 5 or more days (Tu and
Ford , 1 969b).
The commonly used method of MDMV purification involves a chloroform extraction of homogenized material followed by low and high speed centrifugations (Bancroft et al , 1 966 ;
Kuhn, 1 968; Shepherd, 1 965 ; Snazelle et al , 1971), After the centrifugations the partially purified material is often further purified by sucrose density gradient centrifuga¬ tion (Bancroft et al, 1 966 ; Kuhn, 1 968; Sehgal , 1 968; 11
Shepherd, 1965). In place of the chloroform extraction
Sehgal in 1968 recommended acidification to pH 5.3 followed by a DEAE-cel1ulose filtration. This pH is very near the lower pH limit of the virus wnich is 5.0 (Sehgal, 1966).
Since it is known that infectlvity decreases gradually with decreasing pH of the sap from neutral (Tu and Ford, 1969c), the chloroform clarification is often retained. Instead of differential centrifugation, the virus can also be purified by polyethylene glycol precipitation. The final purification step, the sucrose density gradient, may be replaced by an equilibrium centrifugation in cesium chloride
(Hill et al, 1972).
The particle size of MDMV has been given as 750-800 nm long by 15 nm in diameter (Bancroft et al, 1966; Bond and
Pirone, 1971; Kuhn, 1968; Sehgal, 1966; Williams and Alex¬ ander, 1965). The molecular weight of the virus is esti¬ mated to be 40-45 million of which 5-6% is ribonucletic acid (RNA) and the rest protein (Sehgal, 1968). The mole¬ cular weight of the protein subunits is 35-36,000 daltons
(Hill et al, 1 972) .
MDMV is transmitted in nature .by several species of aphids (Bancroft et al , 1 966 ; Niblett and Edmunds, 1971).
When eleven aphid species in eight genera were tested, all were found to transmit MDMV. Single aphids of different species vary in their ability to transmit the virus. Thus, single aphids of Dactynotus sp. transmit the virus 60% of 12
the time, while single aphids of Rhopalosiphum maidis trans¬ mit it less than 10% of the time (Bancroft et al, 1966).
The virus is nonpers i stent or stylet-borne in its aphid vectors. The acquisition feeding period is 10 to 20 seconds, and the retention time is 15 to 20 minutes. The inoculation feeding period is one minute (Messieha, 1967).
MDMV was at first thought to be seed-borne in both
field and sweet corn, but the percentage of seed trar.s-
♦ mission demonstrated was very low, about 0.4% or less
(Shepherd and Holdeman, 1965). In a seed transmission test
using seed of which the male parent, the female parent, or
both were MDMV-infected, there was no seed transmission.
This test involved over 22,000 seedlings of both field and
sweet corn varieties (Sehgal, 1966). When over 29,000 seed¬
lings of 15 sweet corn, 11 popcorn and numerous dent corn
varieties were grown from seed harvested from MDMV-infected
plants, only two seedlings were infected with the virus.
This level of seed transmission, even if it really occurs,
would not be economically significant (Williams et al, 1968).
MDMV rarely infected corn when the roots were inoculated
by rubbing and other means. It appears, therefore, that root
grafts are not an important factor in the spread of this
disease (Moline, 1971).
Corn seedlings infected with MDMV are more susceptible
to root rots, stalk rots and seedling blights. Fusarium,
Pi piodia , Pythium, and Helminthosporium caused more severe 13
root rots in MDMV-infected than in virus-free seedlings.
MDMV increased the occurrence of seedling blight caused by
Penici11ium. Stalk rot in the greenhouse as well as in the field increased with MDMV infection (Mwanza and Williams,
1966). MDMV causes increased root leakage which allows root rot pathogens, such as Helminthosporium and Gibberella, to reach high densities. Since the corn is also internally weakened by MDMV, severe root rots often result (Tu and
Ford , 1971a).
Besides infecting corn, MDMV causes an economically
important disease of grain sorghum (Niblett and Edmunds,
1971) .
In the Gramineae, the grass family, there are numerous
hosts for MDMV. This is the only family susceptible to
MDMV (Ford, 1967a and b; Roane and Tolin, 1969; Tosic and
Ford, 1972). Perennial weeds, especially Johnson grass,
Sorghum halepense, are overwintering hosts for MDMV (Ford,
1967b).
Although all sweet corn varieties are susceptible to
MDMV (Hall et al, 1966), certain sweet corn varieties pos¬
sess limited tolerance to this virus (Janson and Deema,
1965 ; Johnson et al , 1 972; Toler et al , 1 970). Sweet corn
inbred lines also show some tolerance (Josephson and Hilty,
1965). At present, partial control of MDM is possible
through the use of tolerant sweet corn varieties (Johnson et al, 1972). Tolerance to MDMV is also characteristic of 14
numerous field corn varieties (Janson et al, 1965; Jellum and Kuhn, 1970).
Removal of Johnson grass, the perennial host in which the virus overwinters, has reduced the loss caused by MDMV
(Johnson et al , 1 972),
Control of the disease by pesticide control of the aphid vector is only partially effective or ineffective
(Jellum and Kuhn, 1969; Kuhn and Jellum, 1970). MATERIALS AND METHODS
The Virus
Two isolates of the virus collected in two sweet corn fields in widely separated locations were used. These two isolates were maintained by mechanical inoculation on
'Golden Cross Bantam' sweet corn. One isolate, which was later shown to be strain A of maize dwarf mosaic virus, was also maintained on Johnson grass. Sorghum halepense.
The other isolate, which was later shown to be strain B of
MDMV, was used to test the resistance or susceptibility of several sweet corn varieties and to determine the proper¬ ties of the virus in crude sap, such as, dilution end point, longevity i_n vitro and thermal inactivation point. The A strain was used in the serological work. Both strains were used in the host range study.
The Plants
Seed of the Sorghum bico1or varieties 'AKS 614' and
1AK 3003* was furnished by Dr. J. York of the Agronomy De¬ partment, University of Arkansas, Fayetteville, Arkansas.
Seed of the Sorghum bicolor varieties used for strain iden¬ tification was provided by Mr. D.T. Rosenow, Texas A & M,
University Agricultural Research and Extension Center,
Lubbock, Texas. Dr. Joseph Troll of the University of
Massachusetts supplied seed of the common turf grasses; red top, Agrostis alba; velvet bent, A, canina; creeping
15 16
bent, A_. p a 1 u s t r i s ; Rhode Island bent, A_. teni us ; red fescue,
Festuca rubra; timothy, Phleum pratense; Canada bluegrass,
PoQ compress a ; Kentucky bluegrass, p r a t e n sis; and meadow grass, P_. t r i v i a 1 i s . Seed of several sweet corn, Zea mays, varieties evaluated for resistance to MDMV was donated by
Professors Cecil L. Thomson and William H. Lachman, Depart¬ ment of Plant and Soil Science, University of Massachusetts.
Dr, Martin Weeks, also of the Department of Plant and Soil
Science at the University of Massachusetts contributed seed of the following: oats, Avena satina; annual rye-grass,
Lolium mult if lor um; perennial rye-grass, L_. Perenne; rye,
Secale cereale; wheat, Triticum vulgare; 'Iroquois' alfalfa;
Medicago sativa; 'Cayuga' alfalfa, M. sativa; and Ladino clover, Trifolium repens.
Seed of crabgrass, Digitaria sanguinalis, barnyard grass, Echinochloa crusgal li , stirikgrass, Eragrostis sp. , witchgrass, Panicum capillare, fall panicum, P. d i c ho t o- florum, foxtail, Setaria glauca, quackgrass, Agroj3y_ron repens , and orchard grass, Dactyl i s gl omera_ta_, was gathered from weeds growing naturally in various parts oi the state.
Seed of tobacco, Nicotiana tabacum, and lamb's quarter,
Chenopodium amaranti col or and C_. q u i n o a , was made available by the Department of Plant Pathology, University of Massa¬ chusetts. Seed of vegetables and flowers was purchased from commercial seed companies. 17
All the plants were grown in a greenhouse maintained at about 26°C during the winter. In the summer the green¬ house temperature was 26°C or higher. All plants were grown in a sterilized mixture of peat moss, sand and soil in a
1:1:2 ratio. During the winter, artificial lights were used to supplement the natural light.
Mechanical Inoculation
Newly emerged sweet corn leaves showing obvious virus symptoms were collected and used as virus source for inocu¬ lation experiments. The symptomatic areas of the leaves were cut out and immediately ground by pestle in a mortar containing glass beads and 2 ml of 0.01 M neutral phosphate buffer per gram of infected tissue. After straining the ground tissue through several layers of cheesecloth, inocu¬ lations were made by gently rubbing leaves of test plants dusted with carborundum with a small piece of cheesecloth moistened with the crude sap from infected leaves.
Properti es of the Virus i n Crude Sap
Dilution End Point. One gram of virus-infected tissue was ground in 9 parts of 0.01 M neutral phosphate buffer and then strained through cheesecloth. This was considered to be a 1/10 dilution. The 1/10 dilution was further diluted with the same buffer to give serial 10-fold dilutions. Both
'Golden Cross Bantam', a systemic sweet corn host, and sor¬ ghum 'AKS 614', a local lesion host, were used in these experiments. Corn was inoculated at or after the ^hree leaf 18
stage, sorghum when the leaf blade was about half an inch wi de.
Longevity in vitro. The inoculum was prepared by grinding one part of infected tissue in two parts of 0.01
M pH 7 phosphate buffer. The ground tissue was then strained through cheesecloth and inoculations were per¬ formed at various intervals on 'Golden Cross Bantam' sweet corn and/or 'AKS 614' sorghum.
Thermal Inactivation Point. Infected leaves were ground with equal parts (w/v) of 0.01 M pH 7 phosphate buffer. The sap was strained and placed in 7.4 cm x 1.0 cm tubes. The tips of the tubes were quickly flamed to eli¬ minate contamination. The tubes containing the sap were heated for 10 minutes in a water bath preset at selected temperatures ranging from 45 to 65°C. After ten minutes the tubes were removed from the water bath and placed in ice cold water to cool. The heated sap was then used to inoculate local lesion sorghum plants.
Host Range Studies
Plants belonging to several species were mechanically inoculated with virus preparations from infected corn to determine the host range and identity of the virus. The indicator plants were selected to represent several families of monocots and dicots, although the greatest emphasis was placed on the grass family, Gramineae, which includes corn 19
and other hosts susceptible to MDMV. To differentiate be¬ tween the several other mechanically transmissible viruses known to produce mosaic symptoms in sweet corn, hosts were included which were susceptible to these viruses but not to MDMV. Two isolates of the virus infecting corn in
Massachusetts were used to determine the host range of the virus and also to mechanically inoculate selected sorghum varieties. The response of the sorghum varieties is used to distinguish between strains of SCMV and MDMV (Snazelle
0 et al , 1971).
Purification and Electron Microscopy
The procedure used for purification of the virus from corn (Table 1) was a modification of that used by Kuhn (1968) for MDMV. Because the pellets of the low speed centrifuga¬ tions often failed to remain firm when the supernatant was decanted, the time of the low speed centrifugation was in¬ creased at first from 10 to 15 minutes ar.d later to 20 minutes. Throughout the purification all equipment and solutions were kept at 4°C to insure maximum stability of the virus.
For electron microscopy, a drop of purified virus pre¬ paration was placed on a 300 mesh grid coated with a carbon- stabilized formvar film. To this, a drop of 1% PTA (Phos- photungstic acid) at pH 7.0 was added for negative staining.
The drop was allowed to stand for 45 seconds before the ex¬ cess liquid was removed with filter paper. The grid was 20
TABLE I PROCEDURE FOR PURIFICATION OF THE VIRUS FROM CORN
1. Young, symptomatic corn leaves were collected and cut
into small pieces by electric knife.
2. Two ml of 0,05 M sodium citrate in 0.5% mercaptoethanol
were added per g of tissue, '.
3. The tissue was homogenized in a Waring blender and
strained through cheesecloth.
4. One ml of chloroform was added per 3 ml of sap and the
mixture was stirred thoroughly,
5. The mixture was centrifuged at 5,000 g for 20 min., and
the supernatant (aqueous phase) was saved.
6. The supernatant was centrifuged at 100,000 g for 1.5 hrs.
7. Each pellet from the ultracentrifugation was suspended
in 2 ml of 0.01 M pH 7 phosphate buffer and homogenized
in a glass homogenizer.
8. The homogenized suspension was centrifuged at 10,000 g
for 20 min.
9. The supernantant was saved and centrifuged at 100,000 g
for 1 . 5 hours .
10. Each pellet was again suspended in 2 ml of 0.01 M pH 7
phosphate buffer and homogenized in a glass homogenizer.
11. This suspension was centrifuged at 1 0,000 g for 20 mm;
the supernatant was the purified vir^s preparation in
buffer, 21
then examined with a Phillips EM 200 electron microscope.
Serology
The serological relationship of the virus isolated from corn with antisera to MDMV and sugarcane mosaic virus
(SCMV) was studied by the micropreci pti ti n and the agar gel double-diffusion tests (Ball, 1961). Antisera to MDMV strain
A and SCMV strain H were kindly provided by Dr. R.M. Lister of Purdue University. When preliminary tests showed that the agar gel double-diffusion test was unsatisfactory for this virus, the microprecipitin test was used exclusively.
For agar gel double-diffusion tests, 1 or 2 percent agarose gel, to which 1 ml of a 1:1000 dilution of merthiolate was added, was poured into plastic petri plates. One central well about 5 mm in diameter was opened and this was sur¬ rounded by four peripheral wells about 4-5 mm from the central one. Antiserum to MDMV or SCMV was placed in the central well and purified sap from healthy or virus in¬ fected corn leaves was placed in the peripheral wells, The plates then were kept at room temperature and were observed for appearance of the typical serological reaction lines.
The microprecipitin test was carried out in disposable petri dishes. A grid of squares was drawn with a wax pencil at the inside of the bottom of the plate and single drop¬ lets of two-fold dilutions of the antigen and the antiserum were placed and mixed in each square. The plates were then 22
kept moist by placing a saturated paper towel below the petri dish cover. The plates were examined after 2-3 hours incubation at room temperature and were then placed in the refrigerator overnight before being examined again.
Resistance Tests on Sweet Corn Varieties
A greenhouse study was conducted to determine the de¬ gree of resistance to this virus in eighteen sweet corn varieties commonly used in Massachusetts and in two new varieties claimed to be resistant to MDMV. The evaluation of resistance in the various varieties was made by using the "disease index" developed by Kuhn and Jell urn (1970) for MDMV. To obtain the rating of resistance of each variety to MDMV these workers used the following formula:
MDMV disease index = (Wx4) + (Xx3) + (Yx2) + (Zxl), where
W = percent of plants with symptoms 6 days after inoculation
X = percent of plants with symptoms 11 days after inoculation
Y = percent of plants with symptoms 16 days after inoculation
Z = percent of plants with symptoms 23 days after inoculation
The higher the disease index of a variety the more susceptible is the variety. Thus, a rating of 901-1000 means a highly susceptible variety; 701-900, a susceptible variety; 301-700, a moderately resistant variety; 101-300, a resistant variety; and 1-100, a highly resistant variety.
Field Survey Procedures
Sweet corn and field corn fields in each ^ounty in 23
Massachusetts were visited at least once to determine the extent and the severity of virus infections of the corn plants. Since the virus is usually first found at the peri¬ phery of a field (Janson et al, 1965), sweet corn and field corn plantings were surveyed by examining the corn plants near their periphery. Samples of corn leaves showing virus¬ like symptoms were collected and used to inoculate sweet corn and/or sorghum indicator hosts in the greenhouse. Ob¬ servations of the severity of the disease in the field were also used to give an indication of the susceptibility of various sweet corn varieties to this virus. RESULTS
The Disease
Sweet corn and field corn plants in fields throughout
Massachusetts often exhibit a mosaic pattern of dark and
light green areas on their leaves. Many infected plants, especially of certain varieties, appear stunted. In some plants ear kernels were aborted, and the ears were smaller or completely lacking. Occasionally there were numerous nubbins in place of a single large ear. Even when a single ear did develop, the kernel skip was often severe enough to render the ear unmarketable.
The appearance of the diseased plants indicated that the disease was probably caused by a virus. This was further reinforced when efforts to isolate cellular pathogens (bac¬ teria, fungi, nematodes) from diseased corn plants proved negative. The virus etiology of the disease was proven when sap from diseased leaves inoculated on healthy corn leaves could produce the disease.
Mechanical Inoculation
The virus obtained from sweet corn grown in Massachu¬ setts fields was mechanically transmitted to a high percen¬ tage of the plants inoculated in the greenhouse. Generally, about 85% of the inoculated ’Golden Cross Bantam' plants be¬ came infected. Only on a few occasions was 100% transmis¬ sion achieved.
24 25
On 'Golden Cross Bantam' the symptoms produced were the typical mosaic pattern on the leaves (Fig. 1). Dwarfing and kernel skip (Fig. 2) were not observed in the greenhouse since the plants were grown there for only one month. Symp¬ toms were more prominent on the younger leaves (Fig. 3).
On individual leaves mosaic symptoms were more prominent towards the base of the blade rather than the apex (Fig. 4).
On sweet corn, mosaic symptoms appeared as early as five days after inoculation; usually, symptom appearance occurred about a week after inoculation. The sorghum local lesion hosts produced symptoms as early as three days after inoculation. All sorghum local lesion hosts except one pro¬ duced dark purple lesions, similar to those produced on sorghum 'AKS 614' (Fig. 5); the single exception was sorghum
1SA 394' which developed small, light brown, necrotic le¬ sions on the inoculated leaf (Fig. 6) and systemic necrosis in the new leaves. If local lesions were not produced after inoculation with this virus, the sorghum variety either remained non-susceptible or it became systemically infected, as did e.g., s TX 412 ‘ (Fig. 7).
Dilution End Point
With 0.01 M neutral phosphate buffer as the dilution medium, the dilution end point of the virus from corn was - 3 - 4 found to be between 10 and 10 Fig. 1 . The dark green-light green mosaic pattern
appearing on leaves of 'Golden Cross Bantam
sweet corn infected with the virus isolated
from sweet corn in Massachusetts.
Fig. 2 Kernel skip on an ear of 'Butter and Sugar'
sweet corn taken from a plant infected with
the virus in the field. 26 Fig. 3 Symptoms on the younger leaves of 'Golden
Cross Bantam' sweet corn plants systemi-
cally infected with the virus from corn.
Fig. 4 On the younger leaves of 'Golden Cross
Bantam' sweet corn the mosaic patterns
are more prominent near the base of the
1eaf blade . 27 Fig, 5, Dark purple local lesions produced on
Sorghum bicolor 1AKS 614* by mechanical
inoculation with a partially purified
preparation of the virus from sweet
corn.
Fig, 6. Light brown local lesions produced on
inoculated leaves of Sorghum bico1or
' SA 394'. The lesions precede sys¬
temic necrosis in the newly emerged
1 eaves.
1
Fig. 7, Systemic symptoms on Sorghum bicolor
1TX 412' inoculated with the virus
isolate from sweet corn. 29 30
Longevity in vitro
The longevity of the virus at room temperature, about
26°C, in 0,01 M pH 7 phosphate buffer was between 24 and 48 hours. In four experiments performed, the virus failed to infect sorghum, a local lesion host, 36, 48, 24, and 48 hours after the start of the experiment. Infectivity was found to decline rapidly in the buffered sap kept at room temperature.
More than 90% of the infectivity was lost after 12 hours at room temperature; the remaining infectivity declined slowly
until no infectivity could be detected after 24 to 48 hours
from the start of the experiment (Fig. 8).
Thermal Inactivation Point
Heating of the virus in buffered crude sap for 10 minutes
at 55°C did not result in its inactivation, but the virus was
inactivated after heating for 10 minutes at 60°C. The same
results were obtained in three replications of the experi¬
ment.
Host Range Studi es The host range studies not only helped identify the
unknown virus from sweet corn as MDMV, but they also estab¬
lished that there were two groups of isolates corresponding
to the two strains of MDMV in Massachusetts. By use of sor¬
ghum varieties as indicators, the two isolate groups were
shown to be strain A and strain B of MDMV (Table il).
Table HI shows that the host range of the virus isolated Fig. 8. Results of four experiments on the
longevity vitro of the virus
isolated from sweet corn. Infected
tissue was ground in 0.01 M phosphate
buffer pH 7 and kept at room tempera¬
ture (26°C) for varying periods of
time before inoculated onto 1AK 3003'
and 'AKS 614' sorghum. 31
I HOURS FROM GRINDING OF VIRU5-LNFEC'FED TISSUE
m mipjdos hhh sNoisan toot ^o #om Table II. Comparison of Reactions of Selected Sorghum Varieties to Inocultion with Strains of MDMV and SCMV and the Corn Virus Isolates from Massachusetts SCMV SCMV SCMV STM\7 SCMV RDMV MDMV Isolate Isolate -A* -B* -D* -E* -H* -B* -A* I 11 O + o 0+ + 0 +000 o +ooooooo CO + CO CO + ooo. o o r^. o I— >< o co r- x CO 00 cr> 1— CD X cr> o h- X CO 2e; Z X CD CD • i— CM •z? X 0 +00 0 +00 i— CO cn h- CD X *3- CO CT> < CO 00 CO to O CO ►-H ■Cl. • • co CO o Y- CD X Or + o CO +> 4- O n3 to to o E to II q; ro * CD fO N CD c CD O s- to to CD ii +-> -O +> O to to E rO >> Cl o E to +> to CD CD O 32 33
* i/> 4-> 4-5 CD CD CD 3 4-> CO co o r—- _J | _| _1 _| _I _I I I CO CO I I co CO I I I I I I 03 o CO CO CO CO CO (/) 03
aj CD 2 co
C 03 o "—" *r— S- c I— c <<<<<< a. C
4—O
co oo oo CD o •i— oo o - 00 ■u - X3 - cj r-* 00 OO 13
from sweet corn seems to be confined to the Gramineae, the grass family.
Purification and Electron Microscopy
The final supernatant containing the purified virus was analyzed by UV spectrophotometer. The readings showed a high virus ribonucleoprotein ratio with the most common 260/280 ratios being about 1.20, When the purified material was used to inoculate sorghum 1AKS 614’, a local lesion host, there was an increase in the number of local lesions per leaf by a factor of five or more (Fig. 5).
Examination of the purified virus preparation with the electron microscope revealed the presence of long, semi- flexuous virus particles. Although particles of longer and shorter lengths were present in the preparations, the most common length of the virus particles was approximately 760nm with a diameter of 15 nm (Fig. 9). Particle fragmentation and end-to-end aggregation were common in the purified pre¬ para ti ons .
Serology Purified virus preparations consistently formed a preci¬ pitate when reacted with antiserum to MDMV-A. The antiserum produced a visible precipitate even when it was reacted with a 1:128 dilution of antigen (Fig. 10). No precipitate formed when healthy material was reacted with MDMV-A antiserum.
When the purified virus was reacted with antiserum v,o SCMV strain H, no precipitate formed. Fig. 9. Electron micrograph of partially purified
virus from systemically infected 'Golden
Cross Bantam' leaves. Some 760 nm long,
semi-flexous virus particles (arrows)
are evident, mixed with particles of
longer or shorter lengths and cellular
debris. 36 Fig. 10. Results of a typical microprecipitin
test between a purified preparation
of the virus isolated from sweet corn
(strain A) and antiserum to strain A
of maize dwarf mosaic virus. Observa¬
tions were made two hours after the
antigen and antiserum were mixed. VIRUS ANTIGEN DILUTION oo C\J CO •r— LO c CM CO •r— CO cm co ■'3- CO CO 4-> -P co S- C CD E 3 3 o c 4- + + 4- 4- 4* 4- 4- 4- 4- 4- 4- 4- 1 1 00 + 4- 4* 4- 4- 4- 4- 4* + 4* 4- 4- 4- 1 l l 1— co 4- 4- 4- 4- + 4- 4- 4- 4- 1 1 1 1 CO CM 4* 4- 4- 4* 4- 4- 4- 4- 4- 4* 1 1 1 1 CO <3- 4- 4- 4- 4* 4- 1 1 1 1 1 1 . “*• CXI co 1 1 1 1 1 1 1 1 cm LO CO 1 1 1 1 1 1 1 1 < o 1 1 1 1 1 1 1 1 *o 4- +-> •i— ra -P -p c£ 2; -P CD s- ra c co CD
Resistance to Sweet Corn Varieties
Table IV presents the results of inoculation tests of sweet corn varieties with the virus isolated from corn. The resistance or susceptibility of sweet corn varieties to this virus in the greenhouse was expressed in terms of "disease
index" numbers obtained as described under "Materials and
Methods".
According to these greenhouse tests, 'New Golden Splen¬
dor', 'Earlibel1e' , and 'Early Golden Giant' are apparently
the most susceptible of the sweet corn varieties tested to
this virus, while two varieties 'Gold Crown' and 'Southern
Belle' sold as resistant to MDMV, are apparently the most
resistant to the virus isolated from corn in Massachusetts.
'Golden Cross Bantam', which is known to be very sus¬
ceptible to MDMV, had a disease index of 608, which was above
the mean of 501. 'Butter and Sugar', which was found to be
extremely susceptible to the virus in the field, had a dis¬
ease index of 392, which was lower than the mean.
The disease index values listed in Table IV were ob¬
tained from tests performed during the winter and were much
lower than expected. When a similar test was carried out
during the summer, symptoms appeared earlier, and the disease
index values were much higher. Thus, Seneca Chief had
disease index values of 526 and 375 when tested in the winter
and early spring. When tested during the summer, Seneca
Chief' had disease index values of 892 and 900 (Fig. 11). 39
Table IV. Disease index values of selected sweet corn varieties inoculated with the virus isolated from sweet corn in Massachusetts
'New Golden Splendor' 762
'Earlibelle' 690
i 'Early Golden Giant' 686
'Golden Jubilee' 633
'Golden Cross Bantam' 608
'Silver Queen' 608
'MorningSun' 563
'Golden Beauty' 560
'North Star' 556
'Sprite' 533
'Iochief' 498
'Earliking' 484
'GoldWinner' 479
'Seneca Chief' 450
'Gold Cup' 442
'Agway XP420 441
'Butter and Sugar’ 392
'Barbecue' 356
'Gold Crown' 149
'Southern Belle' 148 Fig. 11 Seasonal influence on the rate of symptom
appearance on sweet corn plants of 'Seneca
Chief' following inoculations with the
virus isolates from corn. 40
U"\ CM
8
tf\ H
O
i—1 DAYS AFTER INOCULATION
SWOIdWXS ONIMQKS SiNVTd £.0 IMO'cSd 41
'Butter and Sugar' reacted like 'Seneca Chief' when tested in the summer and in the winter.
Two varieties, 'Gold Crown' and 'Southern Belle', claimed to be resistant to MDMV had low disease index values whether tested in the winter or the summer. 'Gold Crown' had an aver¬ age disease index value of 149, and 'Southern Belle', 148.
Field Survey Eleven of the fourteen counties in Massachusetts were surveyed and the virus disease of sweet corn was found in eight of the eleven counties (Fig. 12). The virus disease of corn, which was shown to be MDMV, was found in the counties Franklin, Hampshire, and Hampden, which contain the Connecticut River Valley in Massachusetts.
In Hampshire county, in September, in a field of 'Stylepak', many of the corn stalks were barren and many of the ears had missing kernels. In a field of late 'Butter and Sugar' in
Hampshire county, the stalks were dwarfed and the leaves showed mosaic symptoms. The disease was also found in Worces¬ ter county on 'Seneca Chief' which produced many unmarketable ears. In the eastern portion of the state, the disease was found in Essex, Middlesex, Norfolk and Plymouth counties. Dis¬ eased 'Butter and Sugar' in Essex county showed severe stunting and its yield was reduced to a few bushels per acre. 'Early
Fortune', on the other hand, seemed to show some resistance.
In Middlesex and Norfolk counties the disease did not appear
to cause appreciable losses. In Plymouth county there was severe damage to 'Butter and Sugar'; in contrast, 'Seneca Scout' on the same farm seemed to be completely unaffected. Fig, 12. Results of field survey on the occurrence
of the virus disease of corn in the various
Massachusetts counties. *3 DISCUSSION
The results of the various experiments with the unknown virus from sweet corn revealed it to be MDMV. All proper¬
ties of the unknown virus, as determined by experiments on mechanical inoculation, dilution end point, longevity i_n
vitro, thermal inactivation point, host range, electron
microscopy, and serology, are identical to those of MDMV.
The percent of the 'Golden Cross Bantam1 plants in¬
fected after mechanical inoculation, 85%, is similar to the
75% reported by Janson and Deema in 1965. Kuhn and Jellum
(1970) stated that the sweet corn variety 'Golden Cross
Bantam1 is so susceptible that, with few exceptions, 100%
of the plants became infected after inoculation with highly
infectious MDMV. In our experiments, however, 100% infec¬
tion was only occasionally achieved.
The minimum time elapsed before mosaic symptoms ap¬
peared on sweet corn was 5 to 7 days which is in agreement
with the results of others (Dale, 1965; Lambe and Dunlevy,
1965); however, Tu and Ford (1969a) and Williams and Alex¬
ander (1965) have reported that at high temperatures (26.5 C
symptoms may show as early as three days after inoculation.
The dilution end point of MDMV is variable because of
differences in procedure. Estimates range from 10~2 in undi¬
luted sap (Shepherd, 1965) to 1(T5 in sap diluted with a buf¬
fer containing a reducing agent (Jones and Tolin, 1 972b); Vh 1-
Hams and Alexander, 1965). In the present work the dilution end
43 44
point was between 10 ^ and when 10 ^ when 0.01 pH 7 phosphate buffer was used, and it agrees with that reported in the literature (Tu and Ford, 1971b, 1970, 1969a, 1969c, and 1968b).
The longevity of MDMV i_n vi tro in unbuffered sap was reported as 1 to 2 days (Shepherd, 1965) or as short as 12 to 14 hours (Tosic and Ford, 1971). When the sap is diluted by a buffer containing a reducing agent, the virus survives in vitro for 5 or 6 days (Sehgal, 1966). Longevity at 40°C is 4 to 5 days without a reducing agent in the buffer (Sehgal,
1966), and 8 days with a reducing agent in the buffer
(Williams and Alexander, 1965). In the present work, in which the virus infected tissue was ground in phosphate buffer and kept at room temperature, the longevity of the virus was 1 to 2 days. This agrees with the reports of Dale
(1965), Sehgal (1966), and Tosic and Ford (1971) who reported the longevity of MDMV to be 1 to 2 days when the test is done at room temperature with phosphate buffer.
Several studies have found the thermal inactivation point of MDMV to be between 50 and 55°C (Dale, 1965; Shepherd,
1965; Williams and Alexander, 1965). Sehgal (1966) reports the thermal inactivation point to be between 53 and 56°C.
DifferenCeS in MDMV-A isolates were reported by Tosic and
Ford (1971) who found that five isolates were infective at
54°C and three at 56°C« Zurnmo and Gordon s (1971) findings support the existence of differences in MDMV-A isolates since
the thermal inactivation point of their isolates varied from 45
54°C to 58°C. The virus isolated from corn in Massachu¬ setts was infective at 55°C but not at 60°C; this is within the range of thermal inactivation points reported by others for MDMV.
Host range studies were used to compare the virus iso¬ lated from corn in Massachusetts with MDMV and the other mechanically transmissible viruses that produce mosaic symp- toms on corn. The host range of the latter viruses differ considerably from that of MDMV. Thus, wheat streak mosaic virus (WSMV) and barley stripe mosaic virus (BSMV) infect cereals; cucumber mosaic virus (CMV) infects cucurbits, and
Iowa soybean mosaic virus (SMV) infects soybeans (Stoner,
1968). Since cereal, cucurbit and soybean hosts were not infected when inoculated mechanically with the virus isolates from corn, this virus was different from WSMV, BSMV, CMV, and
Iowa SMV. This virus, however, appeared to have the same host range as MDMV and SCMV, the other mechanically trans¬ missible viruses causing mosaic symptoms on corn. It was concluded, therefore, from the host range studies that the unknown virus was either MDMV or SCMV.
To distinguish these two related viruses and their strains, the sorghum variety test was used (Snazelle et al,
1971). From the sorghum test results it was determined that the unknown virus isolated from corn was MDMV and that one isolate (from Danvers) was MDMV-A and another isolate (from
Leominster) was MDMV-B. Strain identification was confirmed 46
by inoculation on Johnson grass, Sorghum ha 1 opense. As ex¬ pected, the isolate from Danvers, strain A, did infect
Johnson grass, while the isolate from Leominster, strain B, did not.
Purification procedures using chloroform extraction of tissue have been extensively used (Kuhn, 1968; Shepherd,
1965; Snazelle et al, 1971). By use of a purification sequence involving a chloroform extraction, partially purified material with a fairly high virus titer, as determined by ultraviolet spectrophotometry, local lesion assay, and electron micro¬ scopy, was obtained. When examined under the electron micro¬ scope cellular debris was seen to be present along with the virus particles. For completely purified virus the second high speed centrifugation was replaced with a sucrose density gradient centrifugation.
There was no reaction in the agar gel diffusion plates when antiserum to MOMV strain A was reacted with purified
MDMV. This was probably due to the fact that antigens con¬ sisting of long, flexible virus particles, such as WSMV and
MDMV, diffuse poorly or not at all in immunodiffusion plates
(Langenberg and Ball, 1972). In the microprecipitin test, the purified corn virus reacted with antiserum to the A strain of MDMV but not with antiserum to the H strain of
SCMV, The greenhouse test of sweet corn varieties produced no conclusive evidence on resistance or susceptibility that 47
compares with field data. For example, 'Butter and Sugar' which is extremely susceptible in the field would be rated as resistant according to the results of the greenhouse
test. 'Seneca Chief' and 'Gold Crown' are also resistant
according to the greenhouse test, but susceptible in the
field.
Observations in the field during the state-wide survey
allow formulation of rough estimates on the response of
several varieties to MDMV. In Massachusetts, 'Butter and
Sugar' is very susceptible; 'Seneca Chief' is moderately
susceptible; 'Stylepak' is intermediate; 'Sweet Sue' is
moderately resistant; while 'Seneca Scout' is resistant.
Similar results with these varieties are also reported for
the state by Mr. Alden Miller, regional vegetable specialist,
who also lists, 'Wonderful', 'Gold Crown', 'WK 199', ' NC X 425
1 Ml 69.71 ' , and ' Northern Belle' as resistant. 'Seneca Scout' was among the most resistant seven of 40 varieties tested in
New York in 1970 and in 1971 (Boothroyd, personal communica¬
tion). The other six promising varieties were: 'Earlibelle'
'Imperial', 'Queen Anne', 'Yukon', 'Supreme Golden' and 'Sum¬
mer Treat'. In California, 'Stylepak' was found to be suscep
tible rather than intermediate, while 'VCX 200', '70-2109 ,
'Sunshine State', 'Goldie', 'Bonanza' and 'Jubilee' showed
good tolerance (Johnson et al , 1972).
At the present time the most promising means of control
of this disease seems to be the use of resistant varieties. 48
Use of very susceptible varieties, such as 'Butter and Sugar' and 'Seneca Chief', will probably have to be abandoned. In an experiment by Professor R. Young of the Suburban Experi¬ ment Station at Waltham, Massachusetts, three commonly used varieties, 'Seneca Chief1, 'Golden Cross Bantam' and 'Won¬ derful had 80, 52, and 95% of their ears marketable, respec¬ tively. Of two varieties sold as resistant, 'Gold Crown' had no marketable ears, while 'Southern Belle' had all its ears marketable (private correspondence). It is possible that the failure of the resistant variety 'Gold Crown' was due to the different virulence of the virus isolate in
Massachusetts (Louie et al, 1972).
None of the perennial grasses tested were found to be susceptible to MDMV in the host range study. However, others have found perennial rye-grass, ' L o1iu m perenne' to be sus¬ ceptible (Leisy and Toler, 1969; Tosic and Ford, 1972).
Perennial rye-grass was found to be nonsusceptib1e by Roane and Troutman (1965) and Shepherd (1965). The practice of seeding corn fields with grasses as a winter cover may have to be abandoned because the perennial grass may serve as an overwintering reservoir for the virus.
A measure that could aid in controlling the disease is the eradication of perennial weed hosts which serve as the alternate host for the virus during the winter. In California, removal of Johnson grass, a perennial grass which serves as an overwintering host of MDMV, reduces the losses caused by MDMV
(Johnson et al , 1 972), 49
These two measures, control of perennial weeds and non¬ use of grass as a winter cover could reduce the initial inoculum level of the virus in the spring. Another measure of still uncertain value would be to surround the susceptible sweet corn variety with a non-susceptible field corn variety, such as Pa 54 x Pa 11. This variety is totally resistant to
MDMV in the field (MacKenzie and Cole, 1968) and may serve as a buffer zone on which aphids would cease to retain the virus after they come from infected perennial weed hosts.
Although strains A and B have been positively identified in certain counties in the state, it is likely that both strains are present throughout the state. SUMMARY
A mosaic disease of sweet corn was observed for the first time in central Massachusetts, Mechanical inocula¬ tions using diseased corn leaves as the source of inoculum produced systemic symptoms on sweet corn five to seven days later. Dilution end point of the virus in 0.01 M pH 7 phos¬ phate buffer was between 10"^ and 10"^. The virus was inacti¬ vated when heated for 10 minutes at 60°C but not at 55°C, and it survived jjn vitro at room temperature for 1 to 2 days.
EM studies of partially purified material showed long, flexu- ous rod-shaped particles, 760 x 15 nm. When mechanically transmitted, the virus produced systemic symptoms on sweet corn, crabgrass, barnyard grass, stinkgrass, witchgrass, fall panicum, foxtail and some sorghum varieties. Certain sorghums, when inoculated, produced local lesions or were monsusceptible. In microprecipitin tests partially purified material reacted with MDMV-A antiserum but not SCMV-H anti- serur^. All the properties of the virus isolated from corn
in Massachusetts are identical to those of maize dwarf mosaic virus (MDMV). This then is the first report of the
presence of MDMV in Massachusetts. The virus seems to exist
in two groups of isolates corresponding to strain A and B of MDMV. Greenhouse tests of commonly used sweet corn
varieties gave no conclusive indication of the resistance or susceptibility of the variety to the virus; results were
not always in agreement with the reaction of the variety to
50 51
the virus in the field. Eleven of fourteen counties were surveyed and MDMV was found in eight of the eleven. Damage on individual farms was occasionally severe, especially on late planted ’Butter and Sugar' and 'Seneca Chief'.
» 52
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t