Host Effect on Acquisition and Transmission of Tobacco ... · Cucumber (4 days) then cucumber (3 wk) 11 / 34 et al. (1). This lack of effectiveness is a paradox, since the Cucumber

Vector Relations

Host Effect on Acquisition and Transmission of TobaccoRingspot Virus by Xiphinema americanum

J. M. McGuire and L. B. Douthit

Professor and Research Assistant, respectively, Department of Plant Pathology, University of Arkansas,Fayetteville, AR 72701.

Published with the approval of the Director, Arkansas Agricultural Experiment Station.Accepted for publication 29 August 1977.

ABSTRACT

MC GUIRE, J. M., and L. B. DOUTHIT. 1978. Host effect on acquisition and transmission of tobacco ringspot virus by Xiphinemaamericanum. Phytopathology 68: 457-459.

Xiphinema americanum acquired tobacco ringspot virus transmit TRSV to soybean. There was more TRSV in the(TRSV) from soybean as efficiently as from cucumber, but stomatodeum of nematodes after acquisition access totransmission to soybean was much less than to cucumber. soybean or postacquisition access to cucumber than inTransmission to soybean was not affected by virus isolate or nematodes that had postacquisition access to soybean.age of bait plants. Postacquisition feeding access to healthy Tobacco ringspot virus was recovered from roots of soybeancucumber did not improve the ability of X. americanum to 32 days after mechanical inoculation.

Additional key words: electron microscopy, virus replication.

Xiphinema americanum Cobb was demonstrated to be Unless otherwise indicated, test plants were grown inan efficient vector of tobacco ringspot virus (TRSV) to autoclaved, fine river sand (7) in 250-ml plastic cups. Ifcertain hosts in laboratory and greenhouse studies (7, and nematodes were to be recovered from the sand, it wasL. Douthit and J. McGuire, unpublished). Bergeson et al. sifted through a sieve with 420-/im openings. Nutrients(1) reported, however, that it was an inefficient vector of a were provided by watering twice weekly with a completebud-blighting strain of TRSV to soybean and cucumber fertilizer solution.in greenhouse tests. Xiphinema americanum also has Acquisition and transmission.--Nematodes werebeen associated with field occurrence of TRSV in handled for virus acquisition and transmission aswatermelon (2), grape (3), blueberry (5), and spearmint previously described (7). Acquisition access on soybean(13). or cucumber was for 10 days. After acquisition, single

The objectives of this investigation were: (i) to nematodes or groups of 10 or 100 nematodes were washeddetermine the comparative ability of X. americanum to into sand around the roots of 1- or 2-wk-old soybean andacquire TRSV from soybean and cucumber, (ii) to cucumber bait plants for 3 wk of transmission access. Incompare levels of transmission of TRSV by X. some tests, two bait plants were grown in each cup toamericanum to soybean and cucumber, and (iii) to determine whether presence of cucumber affectedattempt to find explanations for low levels of nematode transmission to soybean. After acquisition, singletransmission of TRSV to soybean. A preliminary report nematodes were washed into sand in which bait plantshas been made (8). were two cucumber, two soybean, or a cucumber and a

soybean in each pot.MATERIALS AND METHODS Virus symptoms were recorded, and roots of all bait

plants were indexed onto cowpea (Vigna unguiculata L.A watermelon isolate of tobacco ringspot virus [TRSV: 'Monarch' or 'Early Ramshorn') and cucumber.

PV-125 ATCC, serologically homologous to strain NC- Appearance of necrotic local lesions on cowpea and a72 (5)] which is transmitted by X. americanum (7) was systemic chlorotic mottle in cucumber was evidence ofused in most tests. It was purified (12) from infected presence of TRSV.cucumber (Cucumus sativus L. 'Model'), standardized Comparison of TRSV isolates.-Two isolates ofat approximately 1.5 mg/ml in 0.05 M phosphate buffer, TRSV from soybean, a bud-blighting isolate designatedpH 7.0, and frozen. The suspension was thawed and 70 A, and a mottling isolate designated 85 which isdiluted 1:100 and used to mechanically inoculate serologically homologous to Gooding's NC-38 (5), werecucumber and soybean (Glycine max L. 'Lee') acquisition compared with PV-125 for transmission to soybean by X.plants, or as described later for other tests. americanum. Nematodes were given 10 days of

The population of X. americanum was obtained from a acquisition access to soybean infected with isolate 70 A,soybean field in Eastern Arkansas and maintained in a 30- 85, or PV-125 TRSV, followed by transmission access tocm diameter clay pot continuously cropped with Lee cucumber and soybean bait plants.soybean in the greenhouse. Post-acquisition feeding access.--Following

00032-949X/78/000 076$03.00/0 acquisition access, groups of nematodes were given 4 or 7Copyright © 1978 The American Phytopathological Society, 3340 days of feeding access to healthy cucumber. Incidence ofPilot Knob Road, St. Paul, MN 55121. All rights reserved, transmission to soybean or cucumber by single

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458 PHYTOPATHOLOGY [Vol. 68

nematodes from these groups were compared with RESULTStransmission by nematodes allowed access to soybeanimmediately after acquisition. Acquisition and transmission.--Soybean and

Anterior portions of nematodes, which included the cucumber were good sources of TRSV for acquisition bystomatodeum, were fixed and stained (9) for electron X. americanum (Table 1). The amounts of transmissionmicroscopic examination either after 14 days of to cucumber bait plants by single nematodes were 32%acquisition access to TRSV-infected soybean, or 7 days and 43%, respectively, when cucumber and soybean weretransmission access to soybean or cucumber after the acquisition hosts. Nearly all cucumber bait plantsacquisition access to soybean. Specimens were embedded were infected when 10 nematodes had feeding access toin Spurr's medium (11) and sectioned at 60-80 nm. each plant. However, single nematodes from the sameSections were mounted on 200- or 300-mesh grids, stained sources did not transmit TRSV to soybean which were Iwith uranyl acetate and lead citrate (10) and examined. wk old when nematodes were added, and only 8%Quantitative estimates of TRSV in the stomatodeum transmission was obtained in each case with 10were made according to the index in Table 3. nematodes per bait plant (Table 1). The level of

Tobacco ringspot virus (TRSV) in roots of transmission to soybean increased to 60% when 100mechanically inoculated soybean.--Primary leaves of nematodes had transmission access to each plant. Also,Lee soybean were mechanically inoculated with a 1:30 there was no transmission to soybean plants which were 2dilution of purified TRSV. For root inoculations, plants wk old when single nematodes were added forwere grown in autoclaved coarse sand until the seedlings transmission access.were in the primary leaf stage. Roots of these plants were Transmission of isolates.-The amount ofwashed, blotted, immersed in a 1:10 dilution of purified transmission to cucumber with single nematodes per baitTRSV and pricked and scratched several times with a no. plant was I1 / 29, 10/30, and 9/30 for 70 A, 85, and PV-1 dental root-canal file (6). Each seedling then was 125, respectively. None of the isolates was transmitted totransplanted into fine sand and kept at 28 C in a sandbed soybean by single nematodes.in the greenhouse. Roots were indexed after 8 or 10, 14, Postacquisition feeding access.--There was no increase21, and 32 days onto half-leaves of cowpea. The opposite in transmission of TRSV to soybean when nematodeshalf-leaves were inoculated with a 1:100 dilution of were given 4 or 7 days of feeding access to healthypurified TRSV for comparison, cucumber between acquisition access and transmission

access (Table 2).Particles of TRSV were evident in the lumina of the

TABLE 1. Comparison of soybean and cucumber as hosts for odontophores and/or the esophagi of 4/4, 3/4, and 2/4acquisition and transmission of tobacco ringspot virus (TRSV) X. americanum following acquisiton access to soybean orby Xiphinema americanum after postacquisition feeding access for 7 days to

cucumber or soybean, respectively. There seemed to beAcquisition Transmission Nematode density more virus present in more locations within the

source host 1 10 100 stomatodeum of nematodes that had postacquisitionCucumber Cucumber 24/76a 8/12 ...b access to cucumber than in nematodes from the other twoCucumber Soybean 0/25 1/12 6/10 groups (Table 3). Fewest particles were present inSoybean Cucumber 32/75a 12/12 .. nematodes that had 7 days of postacquisition access toSoybean Soybean 0/90 3/36a ... soybean.

aNumerator is number of plants to which transmission Tobacco ringspot virus (TRSV) in roots of soybeanoccurred; denominator is total plants. Composite of three tests. following mechanical inoculation.-TRSV wasBait plants were I wk old when nematodes were added. recovered from roots of soybean up to 32 days after"One nematode per bait plant transmitted TRSV to 30% of the mechanical inoculation of roots or primary leaves (thecucumber in the test of transmission by 100 nematodes to longest time tested). The average number of lesions persoybean. half-leaf of cowpea generally decreased with time in both

treatments. Virus was recovered from roots of all plants(20 per treatment) indexed 8-10 and 14 days after

TABLE 2. Effect of feeding access of viruliferous Xiphinema inoculation and from 13/20 and 14/20 leaf- and root-americanum to cucumber on subsequent transmission of inoculated plants, respectively, at both 21 and 32 daystobacco ringspot virus (TRSV) to soybean by single nematodes after inoculation.

TRSV DISCUSSIONFeeding access hosts (time) transmissionFeedinacu esh ( t) tran n These experiments confirm that X. americanum is aCucumber (3 wk) 9/30- poor vector of TRSV to soybean, as reported by BergesonCucumber (4 days) then cucumber (3 wk) 11 / 34 et al. (1). This lack of effectiveness is a paradox, since theCucumber (4 days) then soybean (3 wk) 1/60 nematode transmits the virus efficiently to a number ofCucumber (3 wk) 16/30 other hosts, including cucumber (7, and L. Douthit and J.Soybean (3 wk) 0/30 McGuire, unpublished). Xiphinema americanum feedsCucumber (7 days) then cucumber (3 wk) 6/36 on soybean, since it is maintained on this host in theCucumber (7 days) then soybean (3 wk) 0/60 greenhouse and can acquire TRSV from infected soybean

"Numerator is number of soybean plants to which plants. Inasmuch as TRSV replicated in soybean rootstransmission occurred: the denominator is total plants. following mechanical inoculation, it also would be

March 1978] MC GUIRE AND DOUTHIT: TRSV/XIPHINEMA/TOBACCO 459

TABLE 3. Relative quantities of tobacco ringspot virus (TRSV) within the stomatodeum of Xiphinema americanum followingvarious types of feeding access

TRSV in lumena (range)Anterior Esophageal

Feeding access Odontophore esophagus bulb Total"

14 days to TRSV-infected soybean 0.75(0-2)c 0.25(0-1) 1.00(0-2) 2.00(0-2)

7 days to cucumber after 14 days toTRSV-infected soybean 0.75(0-2) 1.25(0-2) 1.50(0-3) 3.50(0-3)

7 days to soybean after 14 days toTRSV-infected soybean 0.25(0-1) 0.50(0-1) 0.50(0-1) 1.25(0-1)

'Estimates of relative amounts of TRSV: 0= none; 1 = scattered particles along wall of lumen; 2 = part of circumference of lumenwall lined with particles, locations along length of lumen scattered; 3 = circumference of lumen wall lined with particles, sometimesmore than one row, at many locations along length of lumen.

'Sum of odontophore and esophageal index values.'Mean and range of four X. americanum.

expected to replicate if nematode transmission occurred. 54:723-728.It should be recoverable during indexing 3 wk after 2. FULTON, J. P. 1962. A soil-borne virus in Arkansas.nematodes were added, since virus was recovered from Arkansas Farm Res. 11:3.

soybean roots more than 1 mo after mechanical 3. GILMER, R. M., J. K. UYEMOTO, and L. J. KELTS.

inoculation. Also, the presence of fewer particles within 1970. A new grapevine disease induced by tobaccoringspot virus. Phytopathology 60:619-627.

the stomatodeum of X. americanum after postacquisition 4. GOODING, G. V., JR. 1970. Natural serological strains ofaccess to soybean suggested that the virus is released from tobacco ringspot virus. Phytopathology 60:708-713.the nematode during feeding. There was no evidence that 5. GRIFFIN, G. D., J. E. HUGUELET, and J. W. NELSON.feeding on cucumber induced the release of TRSV. 1963. Xiphinema americanum as a vector of necrotic

Transmission of TRSV to roots of soybean may require ringspot virus of blueberry. Plant Dis. Rep. 47:703-704.

introduction of virus into specific cells or tissues not 6. HALK, E. L., and J. M. MC GUIRE. 1973. Translocation of

frequently reached in the feeding of X. americanum. The tobacco ringspot virus in soybean. Phytopathology

higher level of transmission when increased numbers of 63:1291-1300.

nematodes had feeding access to a soybean plant could be 7. MC GUIRE, J. M. 1964. Efficiency of Xiphinemaamericanum as a vector of tobacco ringspot virus.

explained by this theory. An occasional nematode might Phytopathology 54:799-801.introduce virus into a location where infection could 8. MC GUIRE, J. M., and L. B. DOUTHIT. 1975. Acquisitionoccur, and the likelihood of this would increase if greater and transmission of tobacco ringspot virus to soybean bynumbers of nematodes had feeding access to the plant. Xiphinema americanum. Proc. Am. Phytopathol. Soc.Although some observations of feeding by other 2:43. (Abstr.)Xiphinema spp. have been reported (14, 15, 16), little is 9. MC GUIRE, J. M., K. S. KIM, and L. B. DOUTHIT. 1970.

known about feeding by X. americanum, and we have Tobacco ringspot virus in the nematode Xiphinema

been unsuccessful in attempts to observe feeding by this americanum. Virology 42:212-216.

species. Tobacco ringspot virus is readily transmitted to 10. REYNOLDS, E. S. 1963. The use of lead citrate at high pHas an electron-opaque stain in electron microscopy. J.

roots of soybean by mechanical inoculation, but it is not Cell Biol. 17:208-212.known which cells become infected by this procedure. 11. SPURR, A. R. 1969. A low-viscosity epoxy resin embedding

Necrosis caused by nematode feeding also could medium for electron microscopy. J. Ultrastruct. Res.explain the poor transmission to soybean. If cells became 26:31-43.necrotic before the virus replicated sufficiently to move 12. STEERE, R. L. 1956. Purification and properties of tobaccointo other parts of the root, virus introduced by the ringspot virus. Phytopathology 46:60-69.nematode would remain localized and probably would 13. STONE, W. J., G. I. MINK, and G. B. BERGESON. 1962. A

not be recovered by indexing. This could account for new disease of American spearmint caused by tobacco

acquisition and transmission differences in soybean, since 1 ringspot virus. Plant Dis. Rep. 46:623-624.viruisitionulnd beracquissn beffrenecrsis soyrsi 14. TRUDGILL, D. L. 1976. Observations on the feeding ofvirus could be acquired before necrosis occurs. Xiphinema diversicaudatum. Nematologica 22:417-423.

15. WEISCHER, B., and U. WYSS. 1976. Feeding behavior andLITERATURE CITED pathogenicity of Xiphinema index on grapevine roots.

Nematologica 22:319-325.I. BERGESON, G. B., K. L. ATHOW, F. A. LAVIOLETTE, 16. WYSS, U. 1975. Feeding of Trichodorus, Longidorus and

and SISTER MARY THOMASINE. 1964. Xiphinema. Pages 203-221 in F. Lamberti, C. E. Taylor,Transmission, movement, and vector relationships of and J. W. Seinhorst, eds. Nematode Vectors of Planttobacco ringspot virus in soybean. Phytopathology Viruses. Plenum Press, N. Y. 460 p.